WorldWideScience

Sample records for cellulases

  1. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [DSM Innovation, Incorporated, San Francisco, CA (United States)

    2010-04-19

    Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  2. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [Royal DSM, San Francisco, CA (United States)

    2010-03-24

    These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  3. Engineering Cellulases for Biorefinery

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [Royal DSM, San Francisco, CA (United States)

    2010-06-27

    Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  4. Measurement of saccharifying cellulase

    Directory of Open Access Journals (Sweden)

    Andreotti Raymond

    2009-09-01

    Full Text Available Abstract This article sets forth a simple cellulase assay procedure. Cellulose is variable in nature, insoluble and resistant to enzymatic attack. As a result there have been a bevy of bewildering cellulase assays published that yielded irrational results. Certain protocols focused on the rapidity of the assay while ignoring that only the most readily susceptible cellulose regions were being hydrolyzed. Other assays simplified the system by using modified soluble substrates and yielded results that bore no relationship to the real world hydrolysis of insoluble cellulose. In this study Mandels, Andreotti and Roche utilized a common substrate, Whatman filter paper. Hydrolysis of a 50 mg sample of the paper was followed to roughly 4% degradation, which circumvented the problems of attack of only the most susceptible zones. This common hydrolysis target range also resulted in some balance with regard to the interaction of the several cellulase components. The method was subsequently widely adopted. Douglas E Eveleigh

  5. Chimeric enzymes with improved cellulase activities

    Science.gov (United States)

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  6. Bioprospecting thermophiles for cellulase production: a review.

    Science.gov (United States)

    Acharya, Somen; Chaudhary, Anita

    2012-07-01

    Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

  7. Bioprospecting thermophiles for cellulase production: a review

    Directory of Open Access Journals (Sweden)

    Somen Acharya

    2012-09-01

    Full Text Available Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

  8. Cellulase hydrolysis of unsorted MSW

    DEFF Research Database (Denmark)

    Jensen, Jacob Wagner; Felby, Claus; Jørgensen, Henning

    2011-01-01

    A recent development in waste management and engineering has shown that the cellulase can be used for the liquefaction of organic fractions in household waste. The focus of this study was to optimize the enzyme hydrolysis of thermally treated municipal solid waste (MSW) by the addition of surfact......A recent development in waste management and engineering has shown that the cellulase can be used for the liquefaction of organic fractions in household waste. The focus of this study was to optimize the enzyme hydrolysis of thermally treated municipal solid waste (MSW) by the addition...... of calcium, potassium, sodium, chloride and others that may affect cellulolytic enzymes. Cellulase performance showed no effect of adding the metal ion-chelating agent EDTA to the solution. The cellulases were stable, tolerated and functioned in the presence of several contaminants....

  9. Biotechnological applications of bacterial cellulases

    Directory of Open Access Journals (Sweden)

    Esther Menendez

    2015-08-01

    Full Text Available Cellulases have numerous applications in several industries, including biofuel production, food and feed industry, brewing, pulp and paper, textile, laundry, and agriculture.Cellulose-degrading bacteria are widely spread in nature, being isolated from quite different environments. Cellulose degradation is the result of a synergic process between an endoglucanase, an exoglucanase and a,β-glucosidase. Bacterial endoglucanases degrade ß-1,4-glucan linkages of cellulose amorphous zones, meanwhile exoglucanases cleave the remaining oligosaccharide chains, originating cellobiose, which is hydrolyzed by ß-glucanases. Bacterial cellulases (EC 3.2.1.4 are comprised in fourteen Glycosil Hydrolase families. Several advantages, such as higher growth rates and genetic versatility, emphasize the suitability and advantages of bacterial cellulases over other sources for this group of enzymes. This review summarizes the main known cellulolytic bacteria and the best strategies to optimize their cellulase production, focusing on endoglucanases, as well as it reviews the main biotechnological applications of bacterial cellulases in several industries, medicine and agriculture.

  10. Cloning of cellulase genes from acidothermus cellulolyticus

    Science.gov (United States)

    Lastick, deceased, Stanley M.; Tucker, Melvin P.; Grohmann, Karel

    1996-01-01

    A process is described for moving fragments that code for cellulase activity from the genome of A. cellulolyticus to several plasmid vectors and the subsequent expression of active cellulase acitivty in E. coli.

  11. Exo-endo cellulase fusion protein

    Science.gov (United States)

    Bower, Benjamin S [Palo Alto, CA; Larenas, Edmund A [Palo Alto, CA; Mitchinson, Colin [Palo Alto, CA

    2012-01-17

    The present invention relates to a heterologous exo-endo cellulase fusion construct, which encodes a fusion protein having cellulolytic activity comprising a catalytic domain derived from a fungal exo-cellobiohydrolase and a catalytic domain derived from an endoglucanase. The invention also relates to vectors and fungal host cells comprising the heterologous exo-endo cellulase fusion construct as well as methods for producing a cellulase fusion protein and enzymatic cellulase compositions.

  12. Highly Efficient Thermostable DSM Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [DSM Innovation, Inc., San Francisco, CA (United States)

    2011-04-26

    These are the slides from this presentation. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  13. Highly Efficient Thermostable DSM Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Manoj Kumar, PhD

    2011-04-26

    Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  14. Characterizationof Crude Cellulase from Trichoderma reesei and Purification of Cellulase

    Institute of Scientific and Technical Information of China (English)

    姚善泾; 关怡新; 等

    2002-01-01

    The gel filtration was carried out for purification of cellulase.The influences of chromatographic parameters on the resolution were studied to determine the optimal conditions for purification.The purified endoglucanase was obtained by gel filtration by Superdex 75 prep grade with an activity recovery of 92.8% and the purification factor 4.2.The sample volume should be below 6% of the column bed volume and the column bed height L≥12.0 cm.The optimum catalysis temperature and pH for the enzyme were 55℃and 4.5-.0 respectively.The cellulase was stable at pH ranging from 4.0 to 6.0 and temperature below 60℃.

  15. Biotechnological applications of bacterial cellulases

    OpenAIRE

    Esther Menendez; Paula Garcia-Fraile; Raul Rivas

    2015-01-01

    Cellulases have numerous applications in several industries, including biofuel production, food and feed industry, brewing, pulp and paper, textile, laundry, and agriculture.Cellulose-degrading bacteria are widely spread in nature, being isolated from quite different environments. Cellulose degradation is the result of a synergic process between an endoglucanase, an exoglucanase and a,β-glucosidase. Bacterial endoglucanases degrade ß-1,4-glucan linkages of cellulose amorphous zones, mean...

  16. Engineering Cellulase Enzymes for Bioenergy

    Science.gov (United States)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  17. Identification of a Novel Garlic Cellulase Gene.

    Science.gov (United States)

    Kim, Aeri; Kim, Ryong Nam; Kim, Dae-Won; Choi, Sang-Haeng; Kang, Aram; Nam, Seong-Hyeuk; Park, Hong-Seog

    2010-01-01

    Genes encoding cellulase enzymes have been investigated in various plants due to the importance of cellulase enzymes in industrial applications, especially in the conversion of biomass into biofuels. Although several cellulase genes have been cloned and characterized, little is known about cellulase genes from garlic or enzyme activities of their gene products. In this study, a cellulase gene from garlic was cloned and characterized in gene and protein levels for the first time. The DNA sequence of the garlic cellulase gene showed 81% identity with the sequence of the endo-beta-1,4-glucanase of Pisum sativum. The open reading frame of this gene is 1,506 bp, which corresponds to 501 deduced amino acids. We identified the novel ORF region, which was translated into a 55.2 kDa protein using the protein expression vector, pET28a, in Escherichia coli and we confirmed that this protein has cellulase activity in vitro. Our study demonstrates that garlic is very useful, not only for the culinary and pharmaceutical industries, but also as an excellent natural source of various kinds of important genes and enzymes.

  18. [Constitutive synthesis of cellulase by Trichoderma lignorum].

    Science.gov (United States)

    Lobanok, A G; Pavlovskaia, Zh I

    1977-01-01

    The induction of cellulase synthesis by lactose was studied in the resting cells of Trichoderma lignorum OM 534. The effect depended on the concentration of lactose, pH, and the age of the mycelium. The induction of the enzyme synthesis by lactose is supressed by glucose and its metabolites. The repression by glucose is partly eliminated by Cyk 3'-5'-AMP, theophylline, and coffeine. The induction of cellulase by lactose is regarded as a derepression of the synthesis of this enzyme as a result of slow assimilation of the disaccharide. The synthesis of cellulase in T. lignorum is presumed to be constitutive.

  19. Cellulose hydrolysis by immobilized Trichoderma reesei cellulase.

    Science.gov (United States)

    Jones, Paetrice O; Vasudevan, Palligarnai T

    2010-01-01

    Cellulose hydrolysis by immobilized Trichoderma reesei cellulase in the presence of a low viscosity ionic liquid, 1-ethyl-3-methylimidazolium diethyl phosphate (EMIM-DEP), was investigated. Preparation of the carrier-free immobilized cellulase was optimized with respect to concentration of the cross-linker and the type of precipitant. The addition of 2% (v/v) EMIM-DEP during hydrolysis gave an initial reaction rate 2.7 times higher than the hydrolysis rate with no ionic liquid. The initial yield after 2 h was 0.7 g glucose/g cellulose, and the carrier-free immobilized cellulase (CFIC) was effectively re-used five times.

  20. Product inhibition of five Hypocrea jecorina cellulases

    DEFF Research Database (Denmark)

    Murphy, Leigh; Westh, Peter; Bohlin, Christina;

    2013-01-01

    Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information...... on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified...... cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly...

  1. Cellulases: Classification, Methods of Determination and Industrial Applications.

    Science.gov (United States)

    Sharma, Amita; Tewari, Rupinder; Rana, Susheel Singh; Soni, Raman; Soni, Sanjeev Kumar

    2016-08-01

    Microbial cellulases have been receiving worldwide attention, as they have enormous potential to process the most abundant cellulosic biomass on this planet and transform it into sustainable biofuels and other value added products. The synergistic action of endoglucanases, exoglucanases, and β-glucosidases is required for the depolymerization of cellulose to fermentable sugars for transformation in to useful products using suitable microorganisms. The lack of a better understanding of the mechanisms of individual cellulases and their synergistic actions is the major hurdles yet to be overcome for large-scale commercial applications of cellulases. We have reviewed various microbial cellulases with a focus on their classification with mechanistic aspects of cellulase hydrolytic action, insights into novel approaches for determining cellulase activity, and potential industrial applications of cellulases.

  2. CELLULASES FROM THE BASIDIO - MYCETES CULTURAL LIQUID

    Directory of Open Access Journals (Sweden)

    К. G. Dreval

    2013-04-01

    Full Text Available Adsorption of cellulases on substrate taking place during the cultivation process was determined. Adsorbed enzymes can be eluted by buffer solution with high ionic strength, but for determine their activity they should be transferred into the aqueous solution. On the basis of the results a method for obtaining of cellulases preparations from cultural liquids of basidiomycetes was developed. This method is the elution of cellulases from the cultivation substrate of basidiomycetes. It was found that using of the last allows to obtain enzymatic preparations with a high degree of purification in 3 stages (salting out of proteins — dialysis — gelchromatography. Cellulase preparations received original products of basidiomycetes strains К-1, А-Дон-02, Д-1 Irpex lacteus and AnSc-1 Daedaleopsis confragosa f. confragosa were obtained. They contained different proteins, enzymes with specific peaks out of column and their activity. However, common to them was a distinct maximum of outing from the column by endoglucanases or cellobiases, which may indicate that the studied cellulolytic complexes of basidiomycetes do not contain multiple forms of cellulases with different molecular mass. This method allowed to obtain preparations with different degree of purification in comparing with the original culture filtrate 7,3 for endoglucanase and 33,3 for cellobiase of strain А-Дон-02 I. lacteus; 13,1 for endoglucanase and 25,5 for cellobiase of strain Д-1 I. lacteus; 29,9 for endoglucanase and 90,1 for cellobiase of strain К-1 I. lacteus; 2,1 for endoglucanase and 30,6 for cellobiase of strain AnSc-1 D. confragosa f. confragosa.

  3. [Separation and purification of cellulase using affinity membrane].

    Science.gov (United States)

    Shi, Xiang-zhu; Guo, Chun-teng; Zhou, Jian-wu; Wang, Zhong-lai; Rao, Ping-fan

    2002-07-01

    The importance of cellulase as a means for the efficient utilization of abundant cellulose resources in the world has been well recognized. Many researchers devote themselves to studying the mechanism of the action of cellulase to cellulose so that such expensive enzyme can be used much more widely. The first step is to obtain cellulase of high purity. So purification of cellulase is the key point in this field. However, the major problem in isolation is that cellulase is a complicated enzyme system and needs too many steps for separation, and that every cellulase needs special purification processing which cannot be used for the others. A novel method for the separation of the cellulase from crude extraction of Aspergillus niger with normal qualitative filter paper processed by 5 mol/L sodium hydroxide without precipitation and desalting steps was developed. Further purification of the cellulase was achieved by using an anion-exchange column of POROS 20HQ. The cellulase purified was identified as a new endoglucanase that had relatively high endurance to pH and temperature. Its relative molecular mass was estimated to be 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme exhibited very high activity towards carboxymethyl cellulose (CMC) with specific activity of 350 U.mg-1 and the recovery of activity of 9.7%. Its optimum pH and temperature were 4.0 and 70 degrees C, respectively. This is a simple, rapid and efficient method for purifying cellulase with high activity.

  4. A steady state theory for processive cellulases

    DEFF Research Database (Denmark)

    Cruys-Bagger, Nicolaj; Olsen, Jens Elmerdahl; Præstgaard, Eigil;

    2013-01-01

    . This has significant kinetic implications, for example the maximal specific rate (Vmax/E0) for processive cellulases is much lower than the catalytic rate constant (kcat). We discuss how relationships based on this theory may be used in both comparative and mechanistic analyses of cellulases....... remains to be fully developed. In this paper, we suggest a deterministic kinetic model that relies on a processive set of enzyme reactions and a quasi steady-state assumption. It is shown that this approach is practicable in the sense that it leads to mathematically simple expressions for the steady......-state rate, and only requires data from standard assay techniques as experimental input. Specifically, it is shown that the processive reaction rate at steady state may be expressed by a hyperbolic function related to the conventional Michaelis–Menten equation. The main difference is a ‘kinetic processivity...

  5. Cellulase Inhibition by High Concentrations of Monosaccharides

    DEFF Research Database (Denmark)

    Hsieh, Chia-Wen; Cannella, David; Jørgensen, Henning;

    2014-01-01

    Biological degradation of biomass on an industrial scale culminates in high concentrations of end products. It is known that the accumulation of glucose and cellobiose, end products of hydrolysis, inhibit cellulases and decrease glucose yields. Aside from these end products, however, other...... monosaccharides such as mannose and galactose (stereoisomers of glucose) decrease glucose yields as well. NMR relaxometry measurements showed direct correlations between the initial T 2 of the liquid phase in which hydrolysis takes place and the total glucose production during cellulose hydrolysis, indicating...... that low free water availability contributes to cellulase inhibition. Of the hydrolytic enzymes involved, those acting on the cellulose substrate, that is, exo- and endoglucanases, were the most inhibited. The β -glucosidases were shown to be less sensitive to high monosaccharide concentrations except...

  6. Cellulases from Thermophilic Fungi: Recent Insights and Biotechnological Potential

    OpenAIRE

    Duo-Chuan Li; An-Na Li; Papageorgiou, Anastassios C.

    2011-01-01

    Thermophilic fungal cellulases are promising enzymes in protein engineering efforts aimed at optimizing industrial processes, such as biomass degradation and biofuel production. The cloning and expression in recent years of new cellulase genes from thermophilic fungi have led to a better understanding of cellulose degradation in these species. Moreover, crystal structures of thermophilic fungal cellulases are now available, providing insights into their function and stability. The present pap...

  7. Increase in stability of cellulase immobilized on functionalized magnetic nanospheres

    Science.gov (United States)

    Zhang, Wenjuan; Qiu, Jianhui; Feng, Huixia; Zang, Limin; Sakai, Eiichi

    2015-02-01

    Functionalized magnetic nanospheres were prepared by co-condensation of tetraethylorthosilicate with three different amino-silanes: 3-(2-aminoethylamino propyl)-triethoxysilane (AEAPTES), 3-(2-aminoethylamino propyl)-trimethoxysilane (AEAPTMES) and 3-aminopropyltriethoxysilane (APTES). Then three functionalized magnetic nanospheres were used as supports for immobilization of cellulase. The three functionalized magnetic nanospheres with core-shell morphologies exhibited higher capacity for cellulase immobilization than unfunctionalized magnetic nanospheres. The increasing of surface charge of functionalized magnetic nanospheres leads to an enhancement of the capacity of cellulase immobilization. Particularly, AEAPTMES with methoxy groups was favored to be hydrolyzed and grafted on unfunctionalized magnetic nanospheres than the others. AEAPTMES functionalized magnetic nanospheres with the highest zeta potential (29 mV) exhibited 87% activity recovery and the maximum amount of immobilized cellulase was 112 mg/g support at concentration of initial cellulase of 8 mg/mL. Immobilized cellulase on AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than other immobilized cellulases and free cellulase. In particular, it can be used in about 40 °C, demonstrating the potential of biofuel production using this immobilized cellulase.

  8. HYDROLYSIS OF PAPER-DISHWARE WASTES BY CELLULASE

    Institute of Scientific and Technical Information of China (English)

    Jie Lu; Shulan Shi; Runan Yang; Fuzheng Liang

    2004-01-01

    The optimum conditions of hydrolysis of cellulosic wastes by cellulase were studied. The results show that the optimum conditions of sulfuric acid pretreatment were sulfuric acid consistency 0.3M,pretreatment temperature 100℃, pretreatment time 4hours. After sulfuric acid pretreatment, the optimum conditions of hydrolysis by cellulase were enzymatic temperature 50℃ ,enzymatic time 48hours,pH4.8,the charge of cellulase 100IU/g and the substraste consistency 60g/l. Meanwhile this paper studies that the structural change of cellulose during sulfuric acid pretreatment and cellulase hydrolysis by analyzing the infrared spectra.

  9. Purification and characterization of a carboxymethyl cellulase from Artemia salina.

    Science.gov (United States)

    Zin, Hyun Woo; Park, Kwang-Hyun; Choi, Tae Jin

    2014-01-03

    Brine shrimp (Artemia salina) belong to a group of crustaceans that feed on microalgae and require a cellulase enzyme that can be used in ethanol production from marine algae. Protein with potential cellulase activity was purified and the activity analyzed under different conditions. After initial identification of cellulase activity by CMC cellulase, surface sterilization and PCR using 16s rRNA primers was conducted to confirm that the cellulase activity was not produced from contaminating bacteria. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. After the final purification, a 70-fold increase in specific enzyme activity was observed. SDS-PAGE results revealed that the cellulase enzyme had a molecular mass of 96 kDa. Temperature, pH, and salinity values were found to be optimal at 55 °C, pH 8.0, and 600 mM NaCl, respectively. Specifically, the enzyme showed a fivefold increase in enzyme activity in seawater compared to 600 mM NaCl in phosphate buffer. Further analysis of the purified enzyme by molecular spectrometry showed no match to known cellulases, indicating this enzyme could be a novel halophilic cellulase that can be used for the production of bioethanol from marine macroalgae.

  10. Immobilization of cellulase on functionalized cobalt ferrite nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Bohara, Raghvendra Ashok; Thorat, Nanasaheb Devappa; Pawar, Shivaji Hariba [Center for Interdisciplinary Research, D. Y. Patil University, Kolhapur (India)

    2016-01-15

    Amine functionalized cobalt ferrite (AF-CoFe{sub 2}O{sub 4}) magnetic nanoparticles (MNPs) were used for immobilization of cellulase enzyme via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS) coupling reaction. The structural, morphological and magnetic properties of AF-CoFe{sub 2}O{sub 4} were determined. TEM micrograph revealed a mean diameter of -8 nm and showed that the AF-CoFe{sub 2}O{sub 4} remain distinct with no significant change in size after binding with cellulase. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of cellulase to AF-CoFe{sub 2}O{sub 4}. The properties of immobilized cellulase were investigated by optimizing binding efficiency, pH, temperature and reusability. The results showed that the immobilized cellulase has higher thermal stability than free cellulase, which might be due to covalent interaction between cellulase and AF-CoFe{sub 2}O{sub 4} surface. The immobilized cellulase also showed good reusability after recovery. Therefore, AF-CoFe{sub 2}O{sub 4} MNPs can be considered as promising candidate for enzyme immobilization.

  11. Synergy between cellulases and pectinases in the hydrolysis of hemp.

    Science.gov (United States)

    Zhang, Junhua; Pakarinen, Annukka; Viikari, Liisa

    2013-02-01

    The impact of pectinases in the hydrolysis of fresh, steam-exploded and ensiled hemp was investigated and the synergy between cellulases, pectinases and xylanase in the hydrolysis was evaluated. About half; 59.3% and 46.1% of pectin in the steam-exploded and ensiled hemp, respectively, could be removed by a low dosage of pectinases used. Pectinases were more efficient than xylanase in the hydrolysis of fresh and ensiled hemp whereas xylanase showed higher hydrolytic efficiency than the pectinase preparation used in the hydrolysis of steam-exploded hemp. Clear synergistic action between cellulases and xylanase could be observed in the hydrolysis of steam-exploded hemp. Supplementation of pectinase resulted in clear synergism with cellulases in the hydrolysis of all hemp substrates. Highest hydrolysis yield of steam-exploded hemp was obtained in the hydrolysis with cellulases and xylanase. In the hydrolysis of ensiled hemp, the synergistic action between cellulases and pectinases was more obvious for efficient hydrolysis.

  12. Cellulase variants with improved expression, activity and stability, and use thereof

    Energy Technology Data Exchange (ETDEWEB)

    Aehle, Wolfgang; Bott, Richard R.; Bower, Benjamin S.; Caspi, Jonathan; Goedegebuur, Frits; Hommes, Ronaldus Wilhelmus Joannes; Kaper, Thijs; Kelemen, Bradley R.; Kralj, Slavko; Van Lieshout, Johannes Franciscus Thomas; Nikolaev, Igor; Wallace, Louise; Van Stigt Thans, Sander; Vogtentanz, Gudrun; Sandgren, Mats

    2016-12-20

    The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having improved expression, activity and/or stability. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, and methods of use thereof.

  13. Enhanced cellulose degradation using cellulase-nanosphere complexes.

    Directory of Open Access Journals (Sweden)

    Craig Blanchette

    Full Text Available Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC; however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.

  14. Cytochemical localization of cellulases in decayed and nondecayed wood

    Energy Technology Data Exchange (ETDEWEB)

    Murmanis, L.; Highley, T.L.; Palmer, J.G.

    1987-01-01

    Sawdust from undecayed western hemlock wood and from wood previously decayed by the brown-rot fungus Poria placenta or by the white-rot fungus Ganoderma applanatum was incubated with commercial cellulase from Trichoderma viride. Samples were treated cytochemically to locate cellulase activity and examined by TEM. Results showed that cellulase degraded undecayed wood extensively, with the attack starting on the outer border of a cell wall and progressing inside. Wood decayed by P. placenta, with or without cellulase incubation, and treated by the cytochemical test showed uniform distribution of electron dense particles throughout the cell walls. In wood decayed by G. applanatum, cellulase degradation was similar to that in undecayed wood. From measurements of particle diameter it is suggested that electron dense particles are cellulase. It is concluded that brown-rot and white-rot fungi have different effects on the microstructure of wood. The brown-rot fungus appears to open the wood microstructure so that cellulase can diffuse throughout the degraded tracheid wall.

  15. Combined Cellulase and Wrinkle-free Treatment on Cotton Fabric

    Institute of Scientific and Technical Information of China (English)

    周立明; 杨国荣; 袁进华

    2001-01-01

    1,2, 3,4 - butanetetracarboxylic acid (BTCA) offers an alternative to the conventional N-methlol compounds as a crosslinking agent for cellulose textiles. Cellulase treatment is an effective method to improve the handle of the cotton fabric. Thus it is of particular interest to treat cotton fabric using cellulase and BTCA simultaneously.In this study, BTCA was applied to the cellulase pretreated cotton fabric, and softener was also used. The results show that the treated fabric does not only have good wrinkle-free property but also good handle.

  16. Cellulases: ambiguous nonhomologous enzymes in a genomic perspective.

    Science.gov (United States)

    Sukharnikov, Leonid O; Cantwell, Brian J; Podar, Mircea; Zhulin, Igor B

    2011-10-01

    The key material for bioethanol production is cellulose, which is one of the main components of the plant cell wall. Enzymatic depolymerization of cellulose is an essential step in bioethanol production, and can be accomplished by fungal and bacterial cellulases. Most of the biochemically characterized bacterial cellulases come from only a few cellulose-degrading bacteria, thus limiting our knowledge of a range of cellulolytic activities that exist in nature. The recent explosion of genomic data offers a unique opportunity to search for novel cellulolytic activities; however, the absence of clear understanding of structural and functional features that are important for reliable computational identification of cellulases precludes their exploration in the genomic datasets. Here, we explore the diversity of cellulases and propose a genomic approach to overcome this bottleneck.

  17. Statistical analysis of cellulase production in Bacillus amyloliquefaciens UNPDV-22

    Directory of Open Access Journals (Sweden)

    Vasudeo Zambare

    2011-06-01

    Full Text Available The production of cellulase in Bacillus amyloliquefaciens UNPDV-22 was optimized usingresponse surface methodology (RSM. Central composite design (CCD was used to study the interactiveeffect of fermentation medium components (wheat bran, soybean meal, and malt dextrin on cellulaseactivity. Results suggested that wheat bran, soybean meal, and malt dextrin all have significant impacton cellulase production. The use of RSM resulted in a 70% increase in the cellulase activity over thecontrol of non-optimized basal medium. Optimum cellulase production of 11.23 U/mL was obtained in afermentation medium containing wheat bran (1.03%, w/v, soybean meal (2.43%, w/v, and maltdextrin (2.95%, w/v.

  18. Intercalation of cellulase enzyme into a hydrotalcite layer structure

    Science.gov (United States)

    Zou, N.; Plank, J.

    2015-01-01

    A new inorganic-organic hybrid material whereby cellulase enzyme is incorporated into a hydrotalcite type layered double hydroxide (LDH) structure is reported. The Mg2Al-cellulase-LDH was synthesized via co-precipitation from Mg/Al nitrate at pH=9.6. Characterization was performed using X-ray powder diffraction (XRD), small angle X-ray scattering (SAXS), elemental analysis, infrared spectroscopy (IR) and thermogravimetry (TG). From XRD and SAXS measurements, a d-value of ~5.0 nm was identified for the basal spacing of the Mg2Al-cellulase-LDH. Consequently, the cellulase enzyme (hydrodynamic diameter ~6.6 nm) attains a slightly compressed conformation when intercalated. Formation of the LDH hybrid was also confirmed via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Mg2Al-cellulase-LDH phases appear as ~20 nm thin foils which are intergrown to flower-like aggregates. Activity of the enzyme was retained after deintercalation from the Mg2Al-LDH framework using anion exchange. Accordingly, cellulase is not denatured during the intercalation process, and LDH presents a suitable host structure for time-controlled release of the biomolecule.

  19. 21 CFR 173.120 - Carbohydrase and cellulase derived from Aspergillus niger.

    Science.gov (United States)

    2010-04-01

    ... cellulase derived from Aspergillus niger. Carbohydrase and cellulase enzyme preparation derived from Aspergillus niger may be safely used in food in accordance with the following prescribed conditions: (a) Aspergillus niger is classified as follows: Class, Deuteromycetes; order, Moniliales; family,...

  20. Thermostable cellulases, and mutants thereof, capable of hydrolyzing cellulose in ionic liquid

    Science.gov (United States)

    Sapra, Rajat; Datta, Supratim; Chen, Zhiwei; Holmes, Bradley M.; Simmons, Blake A.; Blanch, Harvey W.

    2016-04-26

    The present invention provides for a composition comprising an ionic liquid and a thermostable cellulose, and a method of hydrolyzing a cellulose, comprising: (a) providing a composition comprising a solution comprising an ionic liquid and a cellulose, and (b) introducing a thermostable cellulase to the solution, such that the cellulose is hydrolyzed by the cellulase. The present invention also provides for a Thermatoga maritima thermostable cellulase mutant with increased cellulase activity.

  1. Application of Statistical Design for the Production of Cellulase by Trichoderma reesei Using Mango Peel

    OpenAIRE

    Saravanan, P.; Muthuvelayudham, R.; Viruthagiri, T.

    2012-01-01

    Optimization of the culture medium for cellulase production using Trichoderma reesei was carried out. The optimization of cellulase production using mango peel as substrate was performed with statistical methodology based on experimental designs. The screening of nine nutrients for their influence on cellulase production is achieved using Plackett-Burman design. Avicel, soybean cake flour, KH2PO4, and CoCl2 ·6H2O were selected based on their positive influence on cellulase production. The com...

  2. Ethanol from wood. Cellulase enzyme production

    Energy Technology Data Exchange (ETDEWEB)

    Szengyel, Zsolt

    2000-03-01

    Conversion of biomass to liquid fuels, such as ethanol, has been investigated during the past decades. First due to the oil crisis of the 1970s and lately because of concerns about greenhouse effect, ethanol has been found to be a suitable substitute for gasoline in transportation. Although ethanol is produced in large quantities from corn starch, the conversion of lignocellulosic biomass to ethanol is rather problematic. However, cellulosic raw materials are important as they are available in large quantities from agriculture and forestry. One of the most extensively investigated processes is the enzymatic process, in which fungal cellulolytic enzymes are used to convert the cellulose content of the biomass to glucose, which is then fermented to ethanol. In order to make the raw material accessible to biological attack, it has to be pretreated first. The most successful method, which has been evaluated for various lignocellulosic materials, is the steam pretreatment. In this thesis the utilization of steam pretreated willow (hardwood) and spruce (softwood) was examined for enzyme production using a filamentous fungus T. reesei RUT C30. Various carbon sources originating from the steam pretreated materials have been investigated. The replacement of the solid carbon source with a liquid carbon source, as well as the effect of pH, was studied. The effect of toxic compounds generated during pretreatment was also examined. Comparative study of softwood and hardwood showed that steam pretreated hardwood is a better carbon source than softwood. The hydrolytic potential of enzyme solutions produced on wood derived carbon sources was better compared to commercial cellulases. Also enzyme solutions produced on steam pretreated spruce showed less sensitivity towards toxic compounds formed during steam pretreatment.

  3. Effects of Tween 80 on cellulase stability under agitated conditions.

    Science.gov (United States)

    Okino, Shohei; Ikeo, Makoto; Ueno, Yoshiki; Taneda, Daisuke

    2013-08-01

    The mechanism of the increase in the hydrolysis rate and yield by the addition of Tween 80 to the hydrolysis reaction of filter paper was investigated under static and agitated conditions. The increase in the hydrolysis rate by addition of Tween 80 was observed under the agitated condition only. The effects of Tween 80 on the changes in the protein concentration of individual cellulase components were investigated in the absence of substrates. Agitation of the enzyme solution resulted in the drastic decrease of SDS-PAGE bands intensity of CBH2 (cellobiohydrolase 2). The addition of Tween 80 prevented this. Thus, the Tween 80 functions to stabilize instable cellulase components under the agitated condition. Moreover, addition of Tween 80 completely suppressed the decrease of CBH2 intensity by agitation at 30°C. Results suggest that Tween 80 stabilizes instable cellulase components not only during hydrolysis, but during enzyme production also.

  4. Cellulase Activity and Fruit Softening in Avocado 1

    Science.gov (United States)

    Pesis, Edna; Fuchs, Yoram; Zauberman, Giora

    1978-01-01

    Cellulase activity in detached avocado (Persea americana Mill.) fruits was found to be directly correlated with ripening processes such as climacteric rise of respiration, ethylene evolutin, and softening. This activity in the pericarp could be induced by ethylene treatment, and the more mature the fruit—the faster and the greater was the response. Only a very low cellulase activity could be detected in hard avocado fruit right after harvest. Cellulase activity was highest at the distal end of the fruit, lower in the midsection, and lowest at the proximal end. The enzyme is heat-labile and appeared to have activity of an endocellulase nature mainly. Electron micrographs of cell walls from hard and soft fruits are presented. ImagesFig. 4 PMID:16660305

  5. Production of Citric Acid from Apple Pomace Enzymolyzed by Cellulase

    Institute of Scientific and Technical Information of China (English)

    宋纪蓉; 黄洁; 徐抗震; 赵巧云

    2003-01-01

    Cellulase can evidently increase the content of glucose and has a significant effect on the production of citric acid from apple pomace by Aspergillus niger. Based on experiments, a cellulolytic enzyme named cellulase A6 was found able to produce about 170 g glucose from 1 kg dried apple pomace after 12 h reaction, with cellulase concentration of 20 U/g in the medium at 50℃, natural pH without pretreatment of alkali. Using the treated apple pomace as a liquid state substrate, Aspergillus niger-C selected out was able to produce about 256 g citric acid from 1 kg dried apple pomace at 35℃ in 3 d or 30℃ in 5 d with flask rotation speed of 210 r/min, and the conversion of citric acid could reach 80% based on the amount of sugar consumed.

  6. A Novel Cellulase Produced by a Newly Isolated Trichoderma virens

    Directory of Open Access Journals (Sweden)

    Rong Zeng

    2016-04-01

    Full Text Available Screening and obtaining a novel high activity cellulase and its producing microbe strain is the most important and essential way to improve the utilization of crop straw. In this paper, we devoted our efforts to isolating a novel microbe strain which could produce high activity cellulase. A novel strain Trichoderma virens ZY-01 was isolated from a cropland where straw is rich and decomposed, by using the soil dilution plate method with cellulose and Congo red. The strain has been licensed with a patent numbered ZL 201210295819.6. The cellulase activity in the cultivation broth could reach up to 7.4 IU/mL at a non-optimized fermentation condition with the newly isolated T. virens ZY-01. The cellulase was separated and purified from the T. virens culture broth through (NH42SO4 fractional precipitation, anion-exchange chromatography and gel filtration chromatography. With the separation process, the CMC specific activity increased from 0.88 IU/mg to 31.5 IU/mg with 35.8 purification fold and 47.04% yield. Furthermore, the enzymatic properties of the cellulase were investigated. The optimum temperature and pH is 50 °C and pH 5.0 and it has good thermal stability. Zn2+, Ca2+ and Mn2+ could remarkably promote the enzyme activity. Conversely, Cu2+ and Co2+ could inhibit the enzymatic activity. This work provides a new highly efficient T. virens strain for cellulase production and shows good prospects in practical application.

  7. Optimization of solid fermentation of cellulase from Trichoderma koningii

    Institute of Scientific and Technical Information of China (English)

    LI Pei-jun; JING De-bing; ZHOU Qi-xing; ZHANG Chun-gui

    2004-01-01

    To exploit peashrub resources in Ordos as fodders, it is very crucial to realize industrial production of cheap cellulase of high activity by optimizing culture technology, especially culture substrate. In this study, a new prescription experiment based on uniform design ideal was invented and successfully applied in the solid fermentation of Trichoderma koningii F244, which being performed with two different temperature degrees. The activities of FPA, cotton lyase, CMCase and β-glucosidase were assayed and then mathematical models of enzymatic activities, which were figured out by Unconstraint Mathematical Programming, were developed by Multivariate Regression Program of SPSS10.0. Enzymatic activities of optimized substrate prescriptions corresponding to mathematical models were forecasted to determine an ideal substrate prescription. It is revealed that in solid fermentation, Tween80 has negative effect on cellulase production. Furthermore, the ideal prescription for cellulase complex production by Trichoderma koningii F244 was straw powder 16.9%,wheat bran 26.5%, (NH4)2SO4 9.5% and water 47.1%, whose corresponding cellulase activity was expected to be at the same high level with that of Trichoderma reesei Q9414 on its own recommended substrate. Especially, goats mainly fed on peashrub tissues mixed with cellulase complex of this prescription and culture technology, got an incremental ratio of 0.3 kg/d, which brought a very promising feeding prospect for local peashrub resource. By populization of this cellulase complex, it can integrate living standard, economic construction of local residents into vegetational restoration tightly and thus this paper will be very meaningful to be use for reference for western China like Ordos to realize its sustainable development of economy, society and environment.

  8. Cellulase production by pink pigmented facultative methylotrophic strains (PPFMs).

    Science.gov (United States)

    Jayashree, Shanmugam; Lalitha, Rajendran; Vadivukkarasi, Ponnusamy; Kato, Yuko; Seshadri, Sundaram

    2011-07-01

    Pink pigmented facultative methylotrophs (PPFM) isolated from water samples of Cooum and Adyar rivers in Chennai and soil samples of forests located in various districts of Tamil Nadu, India were screened for cellulase production using carboxymethylcellulose agar (CMC agar) medium. The strains showed wide variations in the production of clearing zones around the colonies on CMC agar medium flooded with Congo red. CMCase and filter paper assays were used to quantitatively measure the cellulase activity of 13 PPFM strains. Among the strains, Methylobacterium gregans, MNW 60, MHW 109, MSF 34, and MSF 40 showed cellulolytic activity ranging from 0.73 to 1.16 U mL(-1) with wide temperature (35-65°C) and pH (5 to 8) tolerance. SDS-PAGE analysis of the crude enzyme of PPFM strain MNW 60 exhibited several protein bands, and zymogram analysis revealed two dimeric cellulase bands with molecular mass of ~92 and 42 kDa. Scanning electron microscopic studies revealed significant morphological differences between the cells grown in normal and CMC amended medium. The strain MNW 60 was identified as Methylobacterium sp. based on biochemical, physiological, and morphological analyses, and the methylotrophic nature was authenticated by the presence of mxaF gene, encoding methanol dehydrogenase as a key indicator enzyme of methylotrophs, with 99% similarity to Methylobacterium lusitanum. With the 16S ribosomal RNA sequence showing 97% similarity to M. lusitanum strain MP2, this can be proposed as a novel taxon of the genus Methylobacterium. The study forms the first detailed report on the extracellular cellulase production by pink pigmented Methylobacterium sp., and it is expected that this might be the basis for further studies on cellulase production by PPFMs to explore the molecular mechanism, strain improvement, and large-scale cellulase production for its application.

  9. ENZYMATIC DEINKING OF OLD NEWSPRINT WITH CELLULASES AND XYLANASE

    Institute of Scientific and Technical Information of China (English)

    Shoujuan Wang; Menghua Qin; Yingjuan Fu; Zhiyong Shao

    2004-01-01

    The enzymatic deinking and fiber modification of old newsprint (ONP) with several cellulases and xylanase were investigated and the suitable enzyme candidates were selected for ONP deinking in this paper. The results demonstrated that the cellulases and hemicellulases could significantly improve the deinking efficiency and fiber modification.Moreover, the synergistic effects of Novozym342 and xylanase (HC) can further enhance the deinking performance, reduce the dirt count and improve the brightness of resulting pulp. Additionally, compared to deinked pulps, obtained from conventional chemical materials, enzymatically deinked pulps had better bleachability, and the brightness of the bleached pulp reached 59.1% ISO, 9% ISO higher than the unbleached pulp.

  10. ENZYMATIC DEINKING OF OLD NEWSPRINT WITH CELLULASES AND XYLANASE

    Institute of Scientific and Technical Information of China (English)

    ShoujuanWang; MenghuaQin; YingjuanFu; ZhiyongShao

    2004-01-01

    The enzymatic deinking and fiber modification of old newsprint (ONP) with several cellulases and xylanase were investigated and the suitable enzyme candidates were selected for ONP deinking in this paper. The results demonstrated that the cellulases and hemicellulases could significantly improve the deinking efficiency and fiber modification. Moreover, the synergistic eflbcts of Novozym342 and xylanase (HC) can further enhance the deinking performance, reduce the dirt count and improve the brightness of resulting pulp. Additionally, compared to deinked pulps, obtained from conventional chemical materials, enzymatically deinked pulps had better bleachability, and the brightness of the bleached pulp reached 59.1% ISO, 9% ISO higher than the unbleached pulp.

  11. Enhancing Cellulase Commercial Performance for the Lignocellulosic Biomass Industry

    Energy Technology Data Exchange (ETDEWEB)

    Jarnigan, Alisha [Danisco, US Inc., Copenhagen (Denmark)

    2016-06-07

    Cellulase enzyme loading (Bt-G) for the economic conversion of lignocellulosic biomass to ethanol is on of the key challenges identified in the Biomass Program of DOE/EERE. The goal of Danisco’s project which ran from 2008 to 2012, was to address the technical challenge by creating more efficient enzyme that could be used at lower doses, thus reducing the enzymes’ cost contribution to the conversio process. We took the approach of protein engineering of cellulase enzymes to overcome the enzymati limitations in the system of cellulosic-hydrolyzing enzymes to improve performance in conversion o biomass, thereby creating a more effective enzyme mix.

  12. CBH1 homologs and varian CBH1 cellulase

    Science.gov (United States)

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Neefe, Paulien

    2014-07-01

    Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

  13. Separation and quantification of cellulases and hemicellulases by capillary electrophoresis

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Kutter, Jörg Peter; Olsson, Lisbeth

    2003-01-01

    . Current methods are limited in their ability to quantify all of these enzymes when all are present simultaneously in a mixture. Five different cellulases (two cellobiohydrolases and three endoglucanases) and one hemicellulase (endoxylanase) were separated using capillary electrophoresis (CE) in a fused...

  14. Recycling cellulase towards industrial application of enzyme treatment on hardwood kraft-based dissolving pulp.

    Science.gov (United States)

    Wang, Qiang; Liu, Shanshan; Yang, Guihua; Chen, Jiachuan; Ji, Xingxiang; Ni, Yonghao

    2016-07-01

    Cost-effectiveness is vital for enzymatic treatment of dissolving pulp towards industrial application. The strategy of cellulase recycling with fresh cellulase addition was demonstrated in this work to activate the dissolving pulp, i.e. decreasing viscosity and increasing Fock reactivity. Results showed that 48.8-35.1% of cellulase activity can be recovered from the filtered liquor in five recycle rounds, which can be reused for enzymatic treatment of dissolving pulp. As a result, the recycling cellulase with addition fresh cellulase of 1mg/g led to the pulp of viscosity 470mL/g and Fock reactivity 80%, which is comparable with cellulase charge of 2mg/g. Other pulp properties such as alpha-cellulose, alkaline solubility and molecular weight distribution were also determined. Additionally, a zero-release of recycling cellulase treatment was proposed to integrate into the dissolving pulp production process.

  15. A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

    Directory of Open Access Journals (Sweden)

    Gupta Munishwar

    2007-06-01

    Full Text Available Abstract Background The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Results Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. Conclusion A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1 hydrolysis of pectin, 2 hydrolysis of xylan and 3 hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.

  16. Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.

    Science.gov (United States)

    Igarashi, Kiyohiko; Uchihashi, Takayuki; Koivula, Anu; Wada, Masahisa; Kimura, Satoshi; Okamoto, Tetsuaki; Penttilä, Merja; Ando, Toshio; Samejima, Masahiro

    2011-09-02

    A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.

  17. Screening for cellulase-encoding clones in metagenomic libraries.

    Science.gov (United States)

    Ilmberger, Nele; Streit, Wolfgang R

    2010-01-01

    Modern biotechnology has the steady need to continuously identify novel enzymes for use in biotechnological applications. In industrial applications, however, enzymes often have to function under extreme and nonnatural conditions (i.e., in the presence of solvents, high temperature and/or at extreme pH values). Cellulases have many industrial applications from the generation of bioethanol, a realistic long-term energy source, to the finishing of textiles. These industrial processes require cellulolytic activity under a range of pH, temperature, and ionic conditions, and they are usually carried out by mixtures of cellulases. Investigation of the broad diversity of cellulolytic enzymes involved in the natural degradation of cellulose is necessary for optimization of these processes.

  18. Inhibitory effect of vanillin on cellulase activity in hydrolysis of cellulosic biomass.

    Science.gov (United States)

    Li, Yun; Qi, Benkun; Wan, Yinhua

    2014-09-01

    Pretreatment of lignocellulosic material produces a wide variety of inhibitory compounds, which strongly inhibit the following enzymatic hydrolysis of cellulosic biomass. Vanillin is a kind of phenolics derived from degradation of lignin. The effect of vanillin on cellulase activity for the hydrolysis of cellulose was investigated in detail. The results clearly showed that vanillin can reversibly and non-competitively inhibit the cellulase activity at appropriate concentrations and the value of IC50 was estimated to be 30 g/L. The inhibition kinetics of cellulase by vanillin was studied using HCH-1 model and inhibition constants were determined. Moreover, investigation of three compounds with similar structure of vanillin on cellulase activity demonstrated that aldehyde group and phenolic hydroxyl groups of vanillin had inhibitory effect on cellulase. These results provide valuable and detailed information for understanding the inhibition of lignin derived phenolics on cellulase.

  19. CLEAVAGE OF SOFTWOOD KRAFT PULP FIBRES BY HCL AND CELLULASES

    Directory of Open Access Journals (Sweden)

    Paul Ander

    2008-05-01

    Full Text Available A new pulp fibre testing procedure called the HCl method was used to compare different spruce and pine fibres and mixtures of these fibres to calculate number of fibre cleavages in dislocations and other weak points. This method was compared with treatment of softwood kraft pulp fibres using different cellulase mixtures. The HCl method can distinguish between mill- and laboratory-made softwood kraft pulp fibres from the same wood batch. The sugar release is characterized by xylose and other hemicellulose sugars and little glucose. This is in contrast to cellulases, which despite strong fibre cleavage, did not distinguish between mill- and laboratory-made pulp fibres and released large amounts of glucose from the fibres. Hemicellulose degradation by HCl and deep penetration of the acid into the primary and secondary fibre cell walls at 80°C seems to be of major importance for the differentiation between mill and laboratory pulp fibres. Cellulases, in contrast, act mostly on the fibre surfaces, and deep penetration only takes place in amorphous regions of dislocations.

  20. Strain Improvement of Bacillus coagulans and Geobacillus stearothermophilus for Enhanced Thermostable Cellulase Production and the Effect of Different Metal Ions on Cellulase Activity

    Directory of Open Access Journals (Sweden)

    Vikas Sharma

    2012-11-01

    Full Text Available The current study was focused on the strain improvement of Bacillus coagulans and Geobacillus stearothermophilus for thermostable cellulase production with higher enzyme activity. For strain improvement UV radiations, NTG and Sodium azide were used as mutagenic agents.NTG was found to be best mutagenic agent among all in term of highest cellulase activity. Mutant strain C11 exhibited the highest cellulase specific activity at 45 U/mg followed by C15 (39 U/mg in case of B.coagulans while Mutant strain S18 exhibited thehighest cellulase specific activity at 69 U/mg followed by S12 (62 U/mg in case of G. stearothermophilus. Specific activity of cellulase was 92 U/mg in case of B.coagulans C11 and 118 U/mg in case of G. stearothermophilus S18. Ag+, Mg+, Se2+,Ca2+,Co2+,Mn2+,K+, Zn2+ ,Fe3+, Hg2+ and Cu2+ showed positive change in specific activity while Na+, Ni2+ negative change in specific activity of cellulase with respect to specific activity of cellulase in absence of any additive in case of B.coagulans C11 and Ag+, Mg+, Se2+,Co2+,Mn2+ andHg2+ showed positive change in specific activity, Na+, K+ showed no change in specific activity while Ca2+, Zn2+, Ni2+, Fe3+ and Cu2+ showed negative change in specific activity of cellulase with respect to specific activity of cellulase in absence of any additive in case of G. stearothermophilus S18.

  1. Characterization of cellulase enzyme produced by Chaetomium sp. isolated from books and archives

    Directory of Open Access Journals (Sweden)

    Moza Mohammed AL-Kharousi

    2015-12-01

    Full Text Available Background: Cellulase is an important industrial enzyme used to degrade cellulosic biomass. The demand for cellulase enzyme is continuously increasing because of its applications in various industries. Hence, screening of cellulase producing microorganisms from different sources has gained significant importance. Material and Methods: In this study, fungi isolated from books and archives were screened for their cellulase producing abilities. Four different fungi were isolated from books and archives using potato dextrose agar. Screening of these isolates for cellulase production was carried out using carboxymethyl cellulose broth. The most efficient fungus was subjected to cellulase fermentation and enzymes produced were purified and partially characterized. Results: Four different fungi, Chaetomium sp., Aspergillus niger, Aspergillus nidulans and Penicillium sp., were isolated from books and archives. All the isolates were tested for their ability to producecellulase enzyme. During the primary screening Chaetomium sp. showed good growth and highercellulase activity (155.3±25.6 U/mL in carboxymethyl cellulose medium than the other fungi. The cellulase fermentation study was conducted with Chaetomium sp. using carboxymethyl cellulose asa substrate. During the stationary phase (144 h of the growth, the cellulase activity of Chaetomium sp. was significantly high. The maximum mycelial weight of this fungi was obtained at 168 h. Viscosity of the Chaetomium sp. inoculated fermentation medium continuously decreased until 144 h because of the degradation of carboxymethyl cellulose. During cellulase fermentation, pHincreased from the initial neutral pH to 8.5. Purified cellulase showed a specific activity of 7.3 U/mg. It exhibited maximum activity at 20°C and was stable between pH 5 and 9. Conclusions: Books and archives could be a good source for the isolation of cellulase producing fungi.

  2. Location, formation and biosynthetic regulation of cellulases in the gliding bacteria Cytophaga hutchinsonii

    Directory of Open Access Journals (Sweden)

    Elijah Johnson

    2006-01-01

    Full Text Available An analysis of the recently published genome sequence of Cytophagahutchinsonii revealed an unusual collection of genes for an organism that can attackcrystalline cellulose. Consequently, questions were being raised by cellulase scientists, as towhat mechanism this organism uses to degrade its insoluble substrates. Cellulose, being ahighly polymeric compound and insoluble in water, cannot enter the cell walls ofmicroorganisms. Cellulose-degrading enzymes have therefore to be located on the surface ofthe cell wall or released extracellularly. The location of most cellulase enzymes has beenstudied. However, basic information on C. hutchinsonii cellulases is almost non-existent. Inthe present study, the location, formation and biosynthetic regulation of cellulases in C.hutchinsonii were demonstrated on different substrates. Various fractions isolated from C.hutchinsonii after cell rupture were assayed for carboxymethyl-cellulase activity (CMC.The cellulases were found to be predominantly cell-free during active growth on solka-flok,although 30% of activity was recorded on cell-bound enzymes. Relatively little CM-cellulase was formed when cells were grown on glucose and cellobiose. Apparently glucoseor labile substrates such as cellobiose seem to repress the formation of CM-cellulase. Thesefindings should provide some insight into possible hydrolysis mechanisms by C.hutchinsonii.

  3. Cellulase in Anoplophora glabripennis adults emerging from different host tree species

    Institute of Scientific and Technical Information of China (English)

    LI Xiao-juan; YAN Xiong-fei; LUO You-qing; TIAN Gui-fang; SUN Hong

    2008-01-01

    In order to investigate different kinds of cellulase in insect pests, we selected male and female adults of Anoplophora glabripennis (Motschulsky) emerging from four different host species (Populus alba vat. pyramidalis, P. nigra vat. thevestina (Dode)Bean., P. simonii × P. pyramidliscr cv. Opera 8277 Hsu. and Salix matsudana f. lobato-glandulosa) as our research material. The enzyme activitives of three kinds of cellulase in the intestines of the adult insects were measured. The Cx-cellulase isozymes were detected with a CMC-incorporated polyacrylamide gel. The results show that: there are no statistically significant differences between the enzyme activities of males and females emerging from the different host species. The order of magnitude in activity is:Cx-cellulase >β-glycosidase > C1-cellulase. When the adults emerge from the same host species, there are no statistically significant differences between male and female enzyme activities ofβ-glycosidase and C1-cellulase, but the enzyme activity of Cx-cellulase of males is clearly higher than that of females. The patterns and migration of Cx-cellulase isozymes of males and females emerging from different hosts trees are clearly not different, and neither are they different when emerging from the same hosts.

  4. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Science.gov (United States)

    2010-04-01

    ... incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the... Trichoderma longibrachiatum. 184.1250 Section 184.1250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase...

  5. Novel Penicillium cellulases for total hydrolysis of lignocellulosics.

    Science.gov (United States)

    Marjamaa, Kaisa; Toth, Karolina; Bromann, Paul Andrew; Szakacs, George; Kruus, Kristiina

    2013-05-10

    The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35°C while at 45°C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.

  6. Useful halophilic, thermostable and ionic liquids tolerant cellulases

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

    2016-06-28

    The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

  7. Production of extremophilic bacterial cellulase enzymes in aspergillus niger.

    Energy Technology Data Exchange (ETDEWEB)

    Gladden, John Michael

    2013-09-01

    Enzymes can be used to catalyze a myriad of chemical reactions and are a cornerstone in the biotechnology industry. Enzymes have a wide range of uses, ranging from medicine with the production of pharmaceuticals to energy were they are applied to biofuel production. However, it is difficult to produce large quantities of enzymes, especially if they are non-native to the production host. Fortunately, filamentous fungi, such as Aspergillus niger, are broadly used in industry and show great potential for use a heterologous enzyme production hosts. Here, we present work outlining an effort to engineer A. niger to produce thermophilic bacterial cellulases relevant to lignocellulosic biofuel production.

  8. Optimization of cellulase production by Penicillium oxalicum using banana agrowaste as a substrate.

    Science.gov (United States)

    Shah, Shilpa P; Kalia, Kiran S; Patel, Jagdish S

    2015-01-01

    The purpose of this study was to produce a higher amount of cellulase by using an alternative carbon source, such as banana agrowaste, and to optimize the fermentation parameters for a high yield. In the present study, cellulase-producing Penicillium was isolated from a decaying wood sample. Different nutritional and environmental factors were investigated to assess their effect on cellulase production. The highest crude enzyme production was observed at a pH 6.0 and a temperature of 28°C in a medium that was supplemented with banana agrowaste as the carbon source. Pretreatment with 2N NaOH, at 7% substrate (banana agrowaste) concentration yielded the highest cellulase activity. Further to this, the effect of other parameters such as inoculum age, inoculum size, static and agitated conditions were also studied. It is concluded that Penicillium oxalicum is a powerful cellulase-producer strain under our tested experimental conditions using banana agrowaste as the carbon source.

  9. Nutrient control for stationary phase cellulase production in Trichoderma reesei Rut C-30.

    Science.gov (United States)

    Callow, Nicholas V; Ray, Christopher S; Kelbly, Matthew A; Ju, Lu-Kwang

    2016-01-01

    This work describes the use of nutrient limitations with Trichoderma reesei Rut C-30 to obtain a prolonged stationary phase cellulase production. This period of non-growth may allow for dependable cellulase production, extended fermentation periods, and the possibility to use pellet morphology for easy product separation. Phosphorus limitation was successful in halting growth and had a corresponding specific cellulase production of 5±2 FPU/g-h. Combined with the addition of Triton X-100 for fungal pellet formation and low shear conditions, a stationary phase cellulase production period in excess of 300 h was achieved, with a constant enzyme production rate of 7±1 FPU/g-h. While nitrogen limitation was also effective as a growth limiter, it, however, also prevented cellulase production.

  10. Characterization of cellulases of fungal endophytes isolated from Espeletia spp.

    Science.gov (United States)

    Cabezas, Luisa; Calderon, Carolina; Medina, Luis Miguel; Bahamon, Isabela; Cardenas, Martha; Bernal, Adriana Jimena; Gonzalez, Andrés; Restrepo, Silvia

    2012-12-01

    Endophytes are microorganisms that asymptomatically invade plant tissues. They can stimulate plant growth and/or provide defense against pathogen attacks through the production of secondary metabolites. Most endophyte species are still unknown, and because they may have several applications, the study of their metabolic capabilities is essential. We characterized 100 endophytes isolated from Espeletia spp., a genus unique to the paramo ecosystem, an extreme environment in the Andean mountain range. We evaluated the cellulolytic potential of these endophytes on the saccharification of the oil palm empty fruit bunch (OPEFB). The total cellulolytic activity was measured for each endophyte on filter paper (FPA). In addition, the specific carboxymethyl cellulase (CMCase), exoglucanase, and β-glucosidase activities were determined. We found four fungi positive for cellulases. Of these fungi, Penicillium glabrum had the highest cellulolytic activity after partial purification, with maximal CMCase, exoglucanase and β-glucosidase enzyme activities of 44.5, 48.3, and 0.45 U/ml, respectively. Our data showed that the bioprospection of fungi and the characterization of their enzymes may facilitate the process of biofuel production.

  11. The application of exogenous cellulase to improve soil fertility and plant growth due to acceleration of straw decomposition.

    Science.gov (United States)

    Han, Wei; He, Ming

    2010-05-01

    The effects of exogenous cellulase application on straw decomposition, soil fertility, and plant growth were investigated with nylon bag and pot experiments. Cellulase application promoted straw decomposition, and the decomposition rates of rice and wheat straw increased by 6.3-26.0% and 6.8-28.0%, respectively, in the nylon bag experiments. In pot experiments soil-available N and P contents, soil cellulase activity, and growth of rice seedlings increased. Soil respiration rate and microbial population were unaffected. Seventy Ug(-1) was the optimal cellulase concentration for plant growth. The exogenous cellulase persisted in soil for more than 100days. Although the data show that exogenous cellulase application can enhance soil fertility and plant growth in the short-term due to the acceleration of straw decomposition and has the potential to be an environment-friendly approach to manage straw, cellulase application to soil seems currently not economical.

  12. Investigation and Isolation of Cellulase-Producing microorganisms in the Red Sea

    KAUST Repository

    Fatani, Siham

    2016-05-01

    Cellulolytic microorganisms are considered to be key players in biorefinery, especially for the utilization of plant biomass. These organisms have been isolated from various environments. The Red Sea is one of the seas with high biodiversity and a unique environment, characterized by high water temperature and high salinity . However, there is little information regarding cellulases in Red Sea environments. The aim of the present study is to evaluate the Red Sea as a gene resource for microbial cellulase. I first surveyed microbial cellulases in the Red Sea using a method called metagenomes, and then investigated their abundance and diversity. My survey revealed that the Red Sea biome has a substantial abundance and a wide range of cellulase enzymes with substantial abundance, when compared with those in other environments. Next, I tried to isolate cellulase-active microorganisms from the Red Sea and I successfully obtained seven strains of four different taxonomic groups. These strains showed a similarity of 99% identity to Aspergillus ustus, 99% to Staphylococcus pasteuri, 99% to Bacillus aerius and 99% to Bacillus subtilis. The enzyme assay I conducted, revealed that these strains actually secreted active cellulases. These results suggest that the Red Sea environment can be, indeed, an excellent gene resource of microbial cellulases.

  13. Cellulase immobilization on superparamagnetic nanoparticles for reuse in cellulosic biomass conversion

    Directory of Open Access Journals (Sweden)

    Fernando Segato

    2016-07-01

    Full Text Available Current cellulosic biomass hydrolysis is based on the one-time use of cellulases. Cellulases immobilized on magnetic nanocarriers offer the advantages of magnetic separation and repeated use for continuous hydrolysis. Most immobilization methods focus on only one type of cellulase. Here, we report co-immobilization of two types of cellulases, β-glucosidase A (BglA and cellobiohydrolase D (CelD, on sub-20 nm superparamagnetic nanoparticles. The nanoparticles demonstrated 100% immobilization efficiency for both BglA and CelD. The total enzyme activities of immobilized BglA and CelD were up to 67.1% and 41.5% of that of the free cellulases, respectively. The immobilized BglA and CelD each retained about 85% and 43% of the initial immobilized enzyme activities after being recycled 3 and 10 times, respectively. The effects of pH and temperature on the immobilized cellulases were also investigated. Co-immobilization of BglA and CelD on MNPs is a promising strategy to promote synergistic action of cellulases while lowering enzyme consumption.

  14. Optimizing cellulase mixtures for maximum rate and extent of hydrolysis. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Walker, L.P.; Wilson, D.B. [Cornell Univ., Ithaca, NY (United States)

    1997-03-01

    Pure Thomomonospora fusca and Trichoderma reesei cellulases and their mixtures were studied to determine the optimal set of cellulases for biomass hydrolysis. The objective was to reduce the cost of cellulase in order to help lower the overall processing cost of the enzymatic conversion of biomass cellulose to sugars, which can then be fermented into fuels and other energy-intensive chemicals. No cellulase mixture was obtained that was much better than the best commercially available preparations. However, the study has greatly increased knowledge of T. fusca cellulases, synergism, and cellulose binding, and provide evidence that future work will produce cellulases with higher activity in degrading crystalline cellulose. T. fusca cellulases may have good industrial potential because: (1) they are compatible with industrial processes that operate at elevated temperatures; (2) they retain 90% of their activity under neutral or basic conditions, which provides a great deal of flexibility in reactor design and operation; and (3) tools are now available to change specific amino acid residues in their catalytic domains and to assess how these changes influence catalysis. 74 refs.

  15. Comparative performance of precommercial cellulases hydrolyzing pretreated corn stover

    Directory of Open Access Journals (Sweden)

    Mohagheghi Ali

    2011-09-01

    Full Text Available Abstract Background Cellulases and related hydrolytic enzymes represent a key cost factor for biochemical conversion of cellulosic biomass feedstocks to sugars for biofuels and chemicals production. The US Department of Energy (DOE is cost sharing projects to decrease the cost of enzymes for biomass saccharification. The performance of benchmark cellulase preparations produced by Danisco, DSM, Novozymes and Verenium to convert pretreated corn stover (PCS cellulose to glucose was evaluated under common experimental conditions and is reported here in a non-attributed manner. Results Two hydrolysis modes were examined, enzymatic hydrolysis (EH of PCS whole slurry or washed PCS solids at pH 5 and 50°C, and simultaneous saccharification and fermentation (SSF of washed PCS solids at pH 5 and 38°C. Enzymes were dosed on a total protein mass basis, with protein quantified using both the bicinchoninic acid (BCA assay and the Bradford assay. Substantial differences were observed in absolute cellulose to glucose conversion performance levels under the conditions tested. Higher cellulose conversion yields were obtained using washed solids compared to whole slurry, and estimated enzyme protein dosages required to achieve a particular cellulose conversion to glucose yield were extremely dependent on the protein assay used. All four enzyme systems achieved glucose yields of 90% of theoretical or higher in SSF mode. Glucose yields were reduced in EH mode, with all enzymes achieving glucose yields of at least 85% of theoretical on washed PCS solids and 75% in PCS whole slurry. One of the enzyme systems ('enzyme B' exhibited the best overall performance. However in attaining high conversion yields at lower total enzyme protein loadings, the relative and rank ordered performance of the enzyme systems varied significantly depending upon which hydrolysis mode and protein assay were used as the basis for comparison. Conclusions This study provides extensive

  16. Isolation, identification and expression of microbial cellulases from the gut of Odontotermes formosanus.

    Science.gov (United States)

    Zhao, Kai; Duan, Jiwei; Ma, Xueling; Zhang, Yue; Wang, Xiaohua

    2016-10-06

    Termites are destructive to agriculture, forestry and buildings, but they can also promote agro-ecosystem balance through the degradation of lignocellulose. Termite-triggered cellulose digestion may be clarified through microbial metabolism of cellulose products. In the present study, we characterized the activities of cellulase and its three components synthesized by the cellulase-producing fungal strain HDZK-BYTF620 isolated from the gut of Odontotermes formosanus. The protein components of cellulase were synthesized by strain HDZK-BYTF620, which were isolated and characterized using polyacrylamide gel electrophoresis, and the expression of cellulose was studied at the proteome level.

  17. Selection of Trichoderma mutants with enhanced cellulase production and resistant to catabolite repression

    Institute of Scientific and Technical Information of China (English)

    Szakacs G; Megyeri L; Kovacs K; Zacchi G

    2004-01-01

    @@ Due to high cost and relatively low efficiency of cellulase enzymes used for the saccharification of pretreated lignocelluloses, the improvement of cellulase secreting microorganisms is of vital importance. Trichoderma reesei QM 6a, an excellent source of cellulase was selected in the late 1960's at Natick Laboratories by its performance on pure cellulose (Solka Floc, Avicel) . QM 6a is the wild parent strain of best existing hypercellulolytic mutants such as Rut C30, VTT-D-80133,L27, CL-847 and others. Utilization of cheaper carbon sources (e. g. , pretreated wood or straw) both in enzyme production and in hydrolysis necessitates to investigate fungal species other than T. reesei.

  18. Interrelationships between cellulase activity and cellulose particle morphology

    DEFF Research Database (Denmark)

    Olsen, Johan Pelck; Donohoe, Bryon S.; Borch, Kim;

    2016-01-01

    It is well documented that the enzymatic hydrolysis of cellulose follows a reaction pattern where an initial phase of relatively high activity is followed by a gradual slow-down over the entire course of the reaction. This phenomenon is not readily explained by conventional factors like substrate...... depletion, product inhibition or enzyme instability. It has been suggested that the underlying reason for the loss of enzyme activity is connected to the heterogeneous structure of cellulose, but so far attempts to establish quantitative measures of such a correlation remain speculative. Here, we have...... on this observation we argue that cellulose structure, specifically surface area and roughness, plays a major role in the ubiquitous rate loss observed for cellulases....

  19. Possibilities for recycling cellulases after use in cotton processing: part I: Effects of end-product inhibition, thermal and mechanical deactivation, and cellulase depletion by adsorption.

    Science.gov (United States)

    Azevedo, Helena; Bishop, David; Cavaco-Paul, Artur

    2002-04-01

    Preliminary recycling experiments with cellulase enzymes after cotton treatments at 50 degrees C showed that activity remaining in the treatment liquors was reduced by about 80% after five recycling steps. The potential problems of end-product inhibition, thermal and mechanical deactivation, and the loss of some components of the cellulase complex by preferential and or irreversible adsorption to cotton substrates were studied. End-product inhibition studies showed that the build-up of cellobiose and glucose would be expected to cause no more than 40% activity loss after five textile treatment cycles. Thermal and mechanical treatments of cellulases suggested that the enzymes start to be deactivated at 60 degrees C and agitation levels similar to those used in textile processing did not cause significant enzyme deactivation. Analysis of cellulase solutions, by fast protein liquid chromatography, before and after adsorption on cotton fabrics, suggested that the cellobiohydrolase II (Cel6A) content of the cellulase complex was reduced, relative to the other components, by preferential adsorption. This would lead to a marked reduction in activity after several treatment cycles and top-up with pure cellobiohydrolase II would be necessary unless this component is easily recoverable from the treated fabric.

  20. Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling

    Directory of Open Access Journals (Sweden)

    Weiss Noah

    2013-01-01

    Full Text Available Abstract Background It is necessary to develop efficient methods to produce renewable fuels from lignocellulosic biomass. One of the main challenges to the industrialization of lignocellulose conversion processes is the large amount of cellulase enzymes used for the hydrolysis of cellulose. One method for decreasing the amount of enzyme used is to recycle the enzymes. In this study, the recycle of enzymes associated with the insoluble solid fraction after the enzymatic hydrolysis of cellulose was investigated for pretreated corn stover under a variety of recycling conditions. Results It was found that a significant amount of cellulase activity could be recovered by recycling the insoluble biomass fraction, and the enzyme dosage could be decreased by 30% to achieve the same glucose yields under the most favorable conditions. Enzyme productivity (g glucose produced/g enzyme applied increased between 30 and 50% by the recycling, depending on the reaction conditions. While increasing the amount of solids recycled increased process performance, the methods applicability was limited by its positive correlation with increasing total solids concentrations, reaction volumes, and lignin content of the insoluble residue. However, increasing amounts of lignin rich residue during the recycle did not negatively impact glucose yields. Conclusions To take advantage of this effect, the amount of solids recycled should be maximized, based on a given processes ability to deal with higher solids concentrations and volumes. Recycling of enzymes by recycling the insoluble solids fraction was thus shown to be an effective method to decrease enzyme usage, and research should be continued for its industrial application.

  1. 2009 Cellulosomes, Cellulases & Other Carbohydrate Modifying Enzymes GRC

    Energy Technology Data Exchange (ETDEWEB)

    Harry Gilbert

    2009-07-26

    The 2009 Gordon Conference on Cellulosomes, Cellulases & Other Carbohydrate Modifying Enzymes will present cutting-edge research on the enzymatic degradation of cellulose and other plant cell wall polysaccharides. The Conference will feature a wide range of topics that includes the enzymology of plant structural degradation, regulation of the degradative apparatus, the mechanism of protein complex assembly, the genomics of cell wall degrading organisms, the structure of the substrate and the industrial application of the process particularly within the biofuel arena. Indeed the deployment of plant cell wall degrading enzymes in biofuel processes will be an important feature of the meeting. It should be emphasized that the 2009 Conference will be expanded to include, in addition to cellulase research, recent advances in other plant cell wall degrading enzymes, and contributions from people working on hemicellulases and pectinases will be particularly welcome. Invited speakers represent a variety of scientific disciplines, including biochemistry, structural biology, genetics and cell biology. The interplay between fundamental research and its industrial exploitation is a particularly important aspect of the meeting, reflecting the appointment of the chair and vice-chair from academia and industry, respectively. The meeting will provide opportunities for junior scientists and graduate students to present their work in poster format and exchange ideas with more established figures in the field. Indeed, some poster presenters will be selected for short talks. The collegial atmosphere of this Conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings, provides an avenue for scientists from different disciplines to brainstorm and promotes cross-disciplinary collaborations in the various research areas represented. The Conference is likely to be heavily subscribed so we would recommend that you submit

  2. Prediction of Color Properties of Cellulase-Treated 100% Cotton Denim Fabric

    Directory of Open Access Journals (Sweden)

    C. W. Kan

    2013-01-01

    Full Text Available Artificial neural network (ANN model was used for predicting colour properties of 100% cotton denim fabrics, including colour yield (in terms of K/S value and CIE L*, a*, b*, C*, and h° values, under the influence of cellulase treatment with various combinations of cellulase processing parameters. Variables examined in the ANN model included treatment temperature, treatment time, pH, mechanical agitation, and fabric yarn twist level. The ANN model was compared with a linear regression model where the ANN model produced superior results in the prediction of colour properties of cellulase-treated 100% cotton denim fabrics. The relative importance of the examined factors influencing colour properties was also investigated. The analysis revealed that cellulase treatment processing parameters played an important role in affecting the colour properties of the treated 100% denim cotton fabrics.

  3. [Cellulase and xylanase activities of Fusarium Lk:Fr. genus fungi of different trophic groups].

    Science.gov (United States)

    Kurchenko, I M; Sokolova, O V; Zhdanova, N M; Iarynchyn, A M; Iovenko, O M

    2008-01-01

    A comparative analysis of cellulase and xylanase activities of 26 fungal strains of phytopathogenic, saprophytic and endophytic Fusarium species has been realized using the qualitative reactions. The rare of their linear growth on the media with carboxymethyl cellulose or xylane has been studied. It was shown that the fungi of genus Fusarium belonging to different trophic groups possessed low activities of investigated enzymes as a whole, but in endophytic strains their levels were lower than in phytopathogenic ones. At the same time the distinct strain dependence of cellulase and xylanase activities was fixed in the fungi of different trophic groups. As far as the cellulase and xylanase activities in phytopathogenic isolates varied from complete absence to high levels, and since the activity maximum for each of the investigated strains was observed in different growth terms the conclusion was made that the cellulase and xylanase activities could not be considered as possible markers of the fungal isolate pathogenicity on the strain level.

  4. [Cellulase and xylanase activity of phytopathogenic and endophytic fungal strains of Alternaria alternata (Fr.) Keissler].

    Science.gov (United States)

    Kurchenko, I M; Sokolova, O V; Zhdanova, N M; Iarynchyn, A M; Iovenko, O M

    2008-01-01

    A comparative analysis of cellulase and xylanase activity of 25 fungal strains of phytopathogenic and endophytic Alternaria alternata had been realized for the first time using the qualitative reactions. The rate of their linear growth on the media with carboxymethylcellulose or xylane had been studied. The cellulase and xylanase activities clearly depended on the distinct strain. The absence of distinct dependence of cellulase and xylanase activities on the species and organs of host plants was demonstrated. The majority of investigated strains of A. alternata did not possess a cellulase activity or the latter was low, but as a whole the phytopathogenic strains were more active than endophytic ones. Xylanase activity was considerable for the fungal strains of all trophyc groups. It was shown that the level of xylanase activity cannot become a biochemical marker of the A. alternata isolate pathogenicity.

  5. Recovery and reutilization of cellulases used for the hydrolysis of wood. I. Fundamentals

    Energy Technology Data Exchange (ETDEWEB)

    Fujishima, S; Yaku, F.; Koshijima, T.

    1984-01-01

    A method was developed to determine the quantity of enzyme from the CD spectrum of cellulase without interference due to coexisting lignin and saccharides in solution, and the properties of cellulase from Trichoderma viride and Aspergillus niger used for the enzymic degradation of Pinus densiflora, were investigated. The cellulase from T. viride has an intense affinity with wood, 50% of the enzyme added to the reaction system was adsorbed by the wood after 30 minutes and kept its activity in the adsorbed state, whereas the cellulase from A. niger had little affinity with wood, and activity remaining in the solution was twice as high as that in the residual wood after enzymic hydrolysis for 24 hours. The activity remaining in the solution decreased with an increase in reaction time and a decrease in pH of the solution.

  6. Immobilization of cellulases on magnetic particles to enable enzyme recycling during hydrolysis of lignocellulose

    DEFF Research Database (Denmark)

    Alftrén, Johan

    on commercial magnetic particles coated with streptavidin. The procedure enabled simultaneous purification and immobilization from crude cell lysate because of the very strong interaction and high affinity between biotin and streptavidin. A third method of immobilizing enzymes was employed in paper IV where two...... feedstocks containing insolubles. This could potentially be overcome by immobilizing the cellulases on magnetically susceptible particles. Consequently, the immobilized cellulases could be magnetically recovered and recycled for a new cycle of enzymatic hydrolysis of cellulose. The main objective...... of this thesis was to examine the possibility of immobilizing cellulases on magnetic particles in order to enable enzyme re-use. Studies at lab and pilot scale (20 L) were conducted using model and real substrates. In paper I and III beta-glucosidase or a whole cellulase mixture was covalently immobilized...

  7. Character of cellulase activity in the guts of flagellate-free termites with different feeding habits.

    Science.gov (United States)

    Li, Zhi-Qiang; Liu, Bing-Rong; Zeng, Wen-Hui; Xiao, Wei-Liang; Li, Qiu-Jian; Zhong, Jun-Hong

    2013-01-01

    Cellulose digestion in termites (Isoptera) is highly important for ecological reasons and applications in biofuel conversion. The speciose Termitidae family has lost flagellates in the hindgut and developed diverse feeding habits. To address the response of cellulase activity to the differentiation of feeding habits, a comparative study of the activity and distribution of composite cellulases, endo-β-1,4-glucanase, and β-glucosidase was performed in seven common flagellate-free termites with three feeding habits: the humus-feeding termites Sinocapritermes mushae (Oshima et Maki), Malaysiocapritermes zhangfengensis Zhu, Yang et Huang and Pericapritermes jiangtsekiangensis (Kemner); the fungus-growing termites Macrotermes barneyi Light and Odontotermes formosanus (Shiraki); and the wood-feeding termites Nasutitermes parvonasutus (Shiraki) and Havilanditermes orthonasus (Tsai et Chen). The results showed that in diverse feeding groups, the wood-feeding group had the highest total composite cellulase and endo-β-1,4-glucanase activities, while the fungus-growing group had the highest β-glucosidase activity. In terms of the distribution of cellulase activity in the alimentary canals, the cellulase activities in wood-feeding termites were concentrated in the midgut, but there was no significant difference between all gut segments in humus-feeding termites. As for the fungus-growing termites, the main site of composite cellulase activity was in the midgut. The endo-β-1,4-glucanase activity was restricted to the midgut, but the primary site of β-glucosidase activity was in the foregut and the midgut (Mac. barneyi). The functions of the gut segments apparently differentiated between feeding groups. The results suggest that the differentiation of feeding habits in flagellate-free termites was characterized by the distribution of cellulases in the gut rather than by variations in cellulase activity.

  8. Elucidation of adsorption processes of cellulases during hydrolysis of crystalline cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Mitsuo; Nakamura, Hiroko; Taniguchi, Masayuki; Morita, Takuo; Matsuno, Ryuichi; Kamikubo, Tadashi

    1986-01-01

    To elucidate the effect of adsorption of cellulases during hydrolysis of crystalline cellulose, the relationship between the rate of hydrolysis and the adsorption of crude cellulases onto crystalline cellulose was investigated under various experimental conditions. Several phases of adsorption have been proposed to explain the process of cellulose hydrolysis by these enzymes. The process of hydrolysis calculated on the basis of these phases fitted well with that obtained experimentally.

  9. Cellulase Activity in Solid State Fermentation of Palm Kernel Cake with Trichoderma sp.

    Directory of Open Access Journals (Sweden)

    Massaud, M. B. N.

    2012-01-01

    Full Text Available Aims: The effect of different types of fungal inocula to the cellulase activity measured on palm kernel cake (PKC was studied. Methodology and Results: Isolate Pro-A1 which was identified as Trichoderma sp. was selected as a potential producer of cellulase via solid state fermentation technique (SSF. Two types of PKCs were used; raw PKC (containing residual oil and defatted PKC. The PKCs were inoculated with different concentrations of conidia and varying amounts (g of solid mycelia plugs (SMP for SSF. The effect of ultrafiltered crude fungal filtrate (CFF as inocula was also being tested. The highest cellulase activity of 2.454 FPU/mL was detected with 60% (wt/wt SMP applied to the raw PKC. Conversely, 2.059 FPU/mL of cellulase activity was measured when 80% (wt/wt of SMP was applied to the defatted PKC which is 62.3% higher than the untreated defatted PKC; and more than 100% increase in enzymatic activity compared to raw PKC. The cellulase activity in the SSF inoculated with 8 x 106 conidia /mL and 12 x 106 conidia /mL were 1.704 FPU/mL for raw PKC and 1.856 FPU/mL for defatted PKC, an enhancement of about 46% from uninoculated batch. Inoculation with CFF bears corresponding maximum improvement of the cellulase activity on both PKCs of 13.58% (raw and 2.86% (defatted. Conclusion, significance and impact of study: The current study proves that Trichoderma sp. in the form of SMP can enhance the cellulase activity on PKCs effectively with more than 100% increment. Fungal conidia are also a better choice in enhancing cellulase activity of Trichoderma sp. permitted that the PKC used is devoid of oil. From this study, Trichoderma sp. holds the potential of converting lignocellulosic materials into products of commercial and industrial values such as glucose and other biofuels.

  10. Production of cellulase from kraft paper mill sludge by Trichoderma reesei rut C-30.

    Science.gov (United States)

    Wang, Wei; Kang, Li; Lee, Yoon Y

    2010-05-01

    Paper mill sludge is a solid waste material generated from pulping and papermaking operations. Because of high glucan content and its well-dispersed structure, paper mill sludges are well suited for bioconversion into value-added products. It also has high ash content originated from inorganic additives used in papermaking, which causes hindrance to bioconversion. In this study, paper mill sludges from Kraft process were de-ashed by a centrifugal cleaner and successive treatment by sulfuric acid and sodium hydroxide, and used as a substrate for cellulase production. The treated sludge was the only carbon source for cellulase production, and predominantly inorganic nutrients were used as the nitrogen source for this bioprocess. The cellulase enzyme produced from the de-ashed sludge exhibited cellulase activity of 8 filter paper unit (FPU)/mL, close to that obtainable from pure cellulosic substrates. The yield of cellulase enzyme was 307 FPU/g glucan of de-ashed sludge. Specific activity was 8.0 FPU/mg protein. In activity tests conducted against the corn stover and alpha-cellulose, the xylanse activity was found to be higher than that of a commercial cellulase. Relatively high xylan content in the sludge appears to have induced high xylanase production. Simultaneous saccharification and fermentation (SSF) was performed using partially de-ashed sludge as the feedstock for ethanol production using Sacharomyces cerevisiae and the cellulase produced in-house from the sludge. With 6% (w/v) glucan feed, ethanol yield of 72% of theoretical maximum and 24.4 g/L ethanol concentration were achieved. These results were identical to those of the SSF using commercial cellulases.

  11. Structure and Function of a Novel Cellulase 5 from Sugarcane Soil Metagenome

    OpenAIRE

    ALVAREZ, Thabata M.; Paiva, Joice H.; Ruiz, Diego M.; João Paulo L F Cairo; Pereira, Isabela O.; Paixão, Douglas A. A.; Rodrigo F. Almeida; Tonoli, Celisa C. C.; Roberto Ruller; Santos, Camila R.; Squina, Fabio M; Murakami, Mario T.

    2013-01-01

    Cellulases play a key role in enzymatic routes for degradation of plant cell-wall polysaccharides into simple and economically-relevant sugars. However, their low performance on complex substrates and reduced stability under industrial conditions remain the main obstacle for the large-scale production of cellulose-derived products and biofuels. Thus, in this study a novel cellulase with unusual catalytic properties from sugarcane soil metagenome (CelE1) was isolated and characterized. The pol...

  12. Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts

    OpenAIRE

    2016-01-01

    Background Amylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic reg...

  13. Production of Alkaline Cellulase by Fungi Isolated from an Undisturbed Rain Forest of Peru

    Directory of Open Access Journals (Sweden)

    Karin Vega

    2012-01-01

    Full Text Available Alkaline cellulase producing fungi were isolated from soils of an undisturbed rain forest of Peru. The soil dilution plate method was used for the enumeration and isolation of fast growing cellulolytic fungi on an enriched selective medium. Eleven out of 50 different morphological colonies were finally selected by using the plate clearing assay with CMC as substrate at different pH values. All 11 strains produced cellulases in liquid culture with activities at alkaline pH values without an apparent decrease of them indicating that they are true alkaline cellulase producers. Aspergillus sp. LM-HP32, Penicillium sp. LM-HP33, and Penicillium sp. LM-HP37 were the best producers of FP cellulase (>3 U mL−1 with higher specific productivities (>30 U g−1 h−1. Three strains have been found suitable for developing processes for alkaline cellulase production. Soils from Amazonian rain forests are good sources of industrial fungi with particular characteristics. The results of the present study are of commercial and biological interest. Alkaline cellulases may be used in the polishing and washing of denim processing of the textile industry.

  14. Adsorption, immobilization and activity of cellulase in soil: the impacts of maize straw and its humification

    Directory of Open Access Journals (Sweden)

    Ali Akbar Safari Sinegani

    2013-12-01

    Full Text Available The present work aimed to study some aspects of sorption and immobilization of cellulase molecules on soil components by the analysis of the reactions of cellulase in a soil treated with different levels of maize residue and incubated for 90 days. The analysis of variance showed that the effects of the treatments of maize straw, incubation time and their interaction on cellulase adsorption, desorption and immobilization were statistically significant. The adsorption and immobilization capacities of soil by application of maize straw increased significantly. However they decreased with decreasing the soil organic matter (SOM after 45 days of incubation. The desorption of adsorbed cellulase molecules from the soil by washing with distilled water depended on the SOM contents and its humification. The binding strength of cellulase molecule with fresh miaze straw was significantly stronger than that with humified maize straw. The immobilized cellulase activity, particularly its specific activity increased significantly by increasing the OC contents in the soil treated with maize straw.

  15. Cellulase Production Potentials of the Microbial Profile of Some Sugarcane Bagasse Dumping Sites in Ilorin, Nigeria

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    Kamoldeen Abiodun AJIJOLAKEWU

    2013-11-01

    Full Text Available This research work investigated cellulase production potentials of the microbial profile of three sugarcane bagasse dumping sites at Zango area, Ilorin, Nigeria. The microbial isolates were screened for cellulase production with a view to select the best organism for eventual cellulase production. Pour Plate method was used for the isolation and a total of thirteen (13 different organisms including both fungal and bacterial species were isolated and screened. Six (6 fungal isolates identified as Mucor racemosus, Aspergillus niger, Aspergillus flavus, Neurospora sitophilus, Penicillium oxalicum and Penicillium citrinum were isolated, while seven (7 different bacterial species isolated include Clostridium cellobioparum, Clostridium thermocellum,Bacillus subtilis, Bacillus pumillus, Lactobacillus spp, Pseudomonas flavescens and Serratia spp. Generally, bacterial isolates were more in abundance than fungal species. However; fungal isolates were constant and were isolated through the experimental period of three weeks. All the isolates showed cellulase production potential in varying degrees as reflected in the clearance zone around their colonies. Fungal isolates produced more cellulase than the bacterial isolates. Mucor racemosus had the highest clearance zone (75.0 mm among the fungal isolates while Clostridium cellobioparum (35.0 mm were the best producer among bacterial isolates. The least producer among fungal isolates, Penicillium citrinum (40.0 mm, is a little more than the bacterial cellulase producer (35.0 mm and is far greater than the least bacterium Serratia spp (14.0 mm.

  16. Preparation of a pH-sensitive polyacrylate amphiphilic copolymer and its application in cellulase immobilization.

    Science.gov (United States)

    Liang, Wenjuan; Cao, Xuejun

    2012-07-01

    P(MDB), a pH-sensitive and reversible water-soluble copolymer, was synthesized with methacrylic acid (MAA), 2-(dimethylamino) ethyl methacrylate (DMAEMA), and butyl methacrylate (BMA) and used as carrier for cellulase. The copolymer is insoluble between pH 2.5 and 4.1, and soluble below pH 2.5 or above 4.1. Its recovery in aqueous solution was 97.2% by adjusting its isoelectric point (pI) to 3.1. Cellulase was covalently immobilized on P(MDB) with 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide. Under optimized conditions, the activity yield of immobilized cellulase was 63.24% and its recovery was 96.8% by adjusting the pI to 3.5. Maximum activity of the immobilized cellulase was achieved at 60 °C (pH 5.0), while free cellulase exhibited maximum activity at 55 °C (pH 5.0). The immobilized cellulase retained 83.1% of its initial activity after repeated five cycles of hydrolysis reaction. P(MDB) is a promising carrier for immobilizing enzymes with high and low optimum pH due to its dissolving characteristics.

  17. Commercial cellulases and hemicellulase performance towards oil palm empty fruit bunch (OPEFB) hydrolysis

    Science.gov (United States)

    Abdul Fattah, S. S.; Mohamed, R.; Jahim, J. M.; Illias, R. M.; Abu Bakar, F. D.; Murad, A. M. A.

    2016-11-01

    In this work, commercial cellulases and hemicellulases were evaluated for their hydrolytic activity towards pretreated oil palm empty fruit bunches (OPEFB). A total of three commercial cellulase preparations, Novozyme Celluclast®, Novozyme Cellic®Ctec, Dupont Accellerase®1500, and a commercial hemicellulase preparation, Novozyme Cellic®Htec, were evaluated. The assays were performed either using the cellulase alone or cellulase in combination with the hemicellulase, formulated at different enzyme activity ratios. Among the three cellulases, the Novozyme Cellic®Ctec yielded the highest reducing sugars, whereby 32.9% hydrolysis yield of OPEFB was achieved. The addition of the commercial hemicellulase to Celluclast® and Accellerase®1500 enhanced OPEFB hydrolysis. However, the addition of the hemicellulase to Cellic®Ctec, failed to enhance the production of the reducing sugars. Nevertheless, the amount of reducing sugars produced using Cellic®Ctec alone was the highest when compared to other enzyme combinations. It can be concluded that among the three commercial cellulases evaluated, the Novozyme Cellic®Ctec is the best enzyme for OPEFB hydrolysis.

  18. Cellulase recycling after high-solids simultaneous saccharification and fermentation of combined pretreated corncob

    Directory of Open Access Journals (Sweden)

    Ruoyu eDu

    2014-06-01

    Full Text Available Despite the advantageous prospect of second-generation bioethanol, its final commercialization must overcome the primary cost impediment due to enzyme assumption. To solve this problem, this work achieves high-concentration ethanol fermentation and multi-round cellulase recycling through process integration. The optimal time and temperature of the re-adsorption process were determined by monitoring the adsorption kinetics of cellulases. Both glucose and cellobiose inhibited cellulase adsorption. After 96 h of ethanol fermentation, 40% of the initial cellulase remained in the broth, from which 62.5% of the cellulase can be recycled and reused in fresh substrate re-adsorption for 90 min. Under optimum conditions, i.e., pH 5.0, dry matter loading of 15 wt%, cellulase loading of 45 FPU/g glucan, two cycles of fermentation and re-adsorption can yield two-fold increased ethanol outputs and reduce enzyme costs by over 50%. The ethanol concentration in each cycle can be achieved at levels greater than 40 g/L.

  19. Catalysis of Rice Straw Hydrolysis by the Combination of Immobilized Cellulase from Aspergillus niger on β-Cyclodextrin-Fe3O4 Nanoparticles and Ionic Liquid

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    Po-Jung Huang

    2015-01-01

    Full Text Available Cellulase from Aspergillus niger was immobilized onto β-cyclodextrin-conjugated magnetic particles by silanization and reductive amidation. The immobilized cellulase gained supermagnetism due to the magnetic nanoparticles. Ninety percent of cellulase was immobilized, but the activity of immobilized cellulase decreased by 10%. In this study, ionic liquid (1-butyl-3-methylimidazolium chloride was introduced into the hydrolytic process because the original reaction was a solid-solid reaction. The activity of immobilized cellulase was improved from 54.87 to 59.11 U g immobilized cellulase−1 at an ionic liquid concentration of 200 mM. Using immobilized cellulase and ionic liquid in the hydrolysis of rice straw, the initial reaction rate was increased from 1.629 to 2.739 g h−1 L−1. One of the advantages of immobilized cellulase is high reusability—it was usable for a total of 16 times in this study. Compared with free cellulase, magnetized cellulase can be recycled by magnetic field and the activity of immobilized cellulase was shown to remain at 85% of free cellulase without denaturation under a high concentration of glucose (15 g L−1. Therefore, immobilized cellulase can hydrolyze rice straw continuously compared with free cellulase. The amount of harvested glucose can be up to twentyfold higher than that from the hydrolysis by free cellulase.

  20. SCREENING AND OPTIMIZATION OF CULTURE CONDITIONS FOR CELLULASE PRODUCTION BY ASPERGILLUS NIGER NSPR012 IN SUBMERGED FERMENTATION

    Directory of Open Access Journals (Sweden)

    Juliet Bamidele Akinyele

    2014-12-01

    Full Text Available This study aimed at screening of selected fungal strains and optimization of process parameters for cellulases production in submerged fermentation. Aspergillus niger NSPR012 was selected for further studies as the most potent in producing cellulase of high activity. Utilization of various agro-wastes as substitute tocarboxy methyl cellulose (CMC for cellulase production was also investigated. Among tested carbon sources, banana peels at a concentration of 5% was found to be the most effective carbon source. The cellulase production by Aspergillus niger NSPR012 in mineral salt medium attained maximum after 96 h of incubation. Maximum cellulase activity (0.466µmol/min/mL was obtained with locust beans as the best organic nitrogen source. The optimum incubation temperature and initial pH were 37°C and 5.5, respectively. With this information, banana peels could have good biotechnological potential for bio-products formation in which cellulase is one.

  1. Effect of Different Cellulase and Pectinase Enzyme Treatments on Protoplast Isolation and Viability in Lilium ledebeourii Bioss.

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    Esmaeil CHAMANI

    2012-11-01

    Full Text Available For overcoming interspecific incompatibility, protoplast combination method is a proper procedure for making a new plant withdesired traits. For this purpose, protoplast preparation is a first and important step. Hence, experiments were conducted to evaluatevarious combinations of cellulose, pectinase and their treatment times on protoplast production and protoplast viability in Liliumledebeourii Bioss. The results of experiment revealed that the protoplast yield was significantly affected by different treatment levels.Cellulase at 4% gave the highest numbers of protoplasts at 3.71×105 protoplast/g FW. Pectinase at 1% gave the highest numbers ofprotoplast. For treatment times, the highest yield of protoplast was with leaf explants treated for 24 h. Analysis of variance indicated thatconcentration, time and three-way interaction of cellulase, pectinase and time were significant at p<0.01. Cellulase at 4% and pectinase at0.2% for 24 h gave the highest viability. Interactions of cellulase × pectinase, cellulase × time, pectinase × time and cellulase × pectinase× treatment time were significant at P≤0.05 for protoplast number. The highest and lowest protoplast numbers were produced in mediacontaining 4% cellulase and 1% pectinase for 24 h (6.65×105 protoplast/g FW and 1% cellulase and 0.2% pectinase for 12 h, respectively.It’s concluded that, the best treatment for isolation of Lilium protoplast was 4% cellulase and 1% pectinase for 24 h.

  2. Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

    Energy Technology Data Exchange (ETDEWEB)

    2012-06-01

    Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

  3. The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

    Directory of Open Access Journals (Sweden)

    Miranda Maki, Kam Tin Leung, Wensheng Qin

    2009-01-01

    Full Text Available Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.

  4. Surface activation of dyed fabric for cellulase treatment.

    Science.gov (United States)

    Schimper, Christian B; Ibanescu, Constanta; Bechtold, Thomas

    2011-10-01

    Surface activation of fabric made from cellulose fibres, such as viscose, lyocell, modal fibres and cotton, can be achieved by printing of a concentrated NaOH-containing paste. From the concentration of reducing sugars formed in solution, an increase in intensity of the cellulase hydrolysis by a factor of six to eight was observed, which was mainly concentrated at the activated parts of the fabric surface. This method of local activation is of particular interest for modification of materials that have been dyed with special processes to attain an uneven distribution of dyestuff within the yarn cross-section, e.g., indigo ring-dyed denim yarn for jeans production. Fabrics made from regenerated cellulose fibres were used as model substrate to express the effects of surface activation on indigo-dyed material. Wash-down experiments on indigo-dyed denim demonstrated significant colour removal from the activated surface at low overall weight loss of 4-5%. The method is of relevance for a more eco-friendly processing of jeans in the garment industry.

  5. Cellulase-free xylanases from Bacillus and other microorganisms.

    Science.gov (United States)

    Subramaniyan, S; Prema, P

    2000-02-01

    Xylanases are used mainly in the pulp and paper industries for the pretreatment of Kraft pulp prior to bleaching to minimize use of chlorine, the conventional bleaching agent. This application has great potential as an environmentally safe method. Hydrolysis by xylanases of relocated and reprecipitated xylan on the surface of cellulose fibres formed during Kraft cooking facilitates the removal of lignin by increasing permeability to oxidising agents. Most of the xylanases reported in the literature contained significant cellulolytic activity, which make them less suitable for pulp and paper industries. The need for large quantities of xylanases which would be stable at higher temperatures and pH values and free of cellulase activity has necessitated a search for novel enzymes. We have isolated and characterised several xylanase-producing cultures, one of which (an alkalophilic Bacillus SSP-34) produced more than 100 IU ml(-1) of xylanase activity. The SSP-34 xylanases have optimum activity at 50 degrees C in a pH range 6-8, with only small amounts of cellulolytic activity (CMCase (0.4 IU ml(-1), pH 7), FPase (0.2 IU ml(-1), pH 7) and no activity at pH 9).

  6. Proteomics based compositional analysis of complex cellulase-hemicellulase mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Chundawat, Shishir P.; Lipton, Mary S.; Purvine, Samuel O.; Uppugundla, Nirmal; Gao, Dahai; Balan, Venkatesh; Dale, Bruce E.

    2011-10-07

    Efficient deconstruction of cellulosic biomass to fermentable sugars for fuel and chemical production is accomplished by a complex mixture of cellulases, hemicellulases and accessory enzymes (e.g., >50 extracellular proteins). Cellulolytic enzyme mixtures, produced industrially mostly using fungi like Trichoderma reesei, are poorly characterized in terms of their protein composition and its correlation to hydrolytic activity on cellulosic biomass. The secretomes of commercial glycosyl hydrolase producing microbes was explored using a proteomics approach with high-throughput quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we show that proteomics based spectral counting approach is a reasonably accurate and rapid analytical technique that can be used to determine protein composition of complex glycosyl hydrolase mixtures that also correlates with the specific activity of individual enzymes present within the mixture. For example, a strong linear correlation was seen between Avicelase activity and total cellobiohydrolase content. Reliable, quantitative and cheaper analytical methods that provide insight into the cellulosic biomass degrading fungal and bacterial secretomes would lead to further improvements towards commercialization of plant biomass derived fuels and chemicals.

  7. Effect of bisulfite treatment on composition, structure, enzymatic hydrolysis and cellulase adsorption profiles of sugarcane bagasse.

    Science.gov (United States)

    Liu, Z J; Lan, T Q; Li, H; Gao, X; Zhang, H

    2017-01-01

    The effect of sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) on composition, structure, enzymatic hydrolysis and cellulase adsorption profiles of sugarcane bagasse (SCB) was investigated. SPORL gave a higher SCB hydrolysis yield (85.33%) compared to dilute acid pretreatment (DA) (64.39%). The SEM pictures showed that SPORL SCB structure became more disordered and looser, suggesting SPORL SCB was more accessible to cellulase. The zeta potential of SPORL SCB suspension (-21.89mV) was significantly different from that of DA SCB (-12.87mV), which demonstrated the lignin in SPORL SCB was more hydrophilic. With regard to cellulase adsorption profiles, SPORL SCB had a lower non-productive adsorption (14.87mg/glignin) and a higher productive adsorption (37.67 mg/gcarbohydrate) compared with DA SCB (17.05mg/glignin; 25.79mg/gcarbohydrate). These results indicated that SPORL SCB had better accessibility to cellulase and the higher productive cellulase adsorption of SPORL SCB had improved hydrolysis.

  8. Antimicrobial and Antioxidant Activity of Chitosan/Hydroxypropyl Methylcellulose Film-Forming Hydrosols Hydrolyzed by Cellulase

    Directory of Open Access Journals (Sweden)

    Anna Zimoch-Korzycka

    2016-09-01

    Full Text Available The aim of this study was to evaluate the impact of cellulase (C on the biological activity of chitosan/hydroxypropyl methylcellulose (CH/HPMC film-forming hydrosols. The hydrolytic activity of cellulase in two concentrations (0.05% and 0.1% was verified by determination of the progress of polysaccharide hydrolysis, based on viscosity measurement and reducing sugar-ends assay. The 2,2-diphenyl-1-picrylhydrazyl (DPPH free radical scavenging effect, the ferric reducing antioxidant power (FRAP, and microbial reduction of Pseudomonas fluorescens, Yersinia enterocolitica, Bacillus cereus, and Staphylococcus aureus were studied. During the first 3 h of reaction, relative reducing sugar concentration increased progressively, and viscosity decreased rapidly. With increasing amount of enzyme from 0.05% to 0.1%, the reducing sugar concentration increased, and the viscosity decreased significantly. The scavenging effect of film-forming solutions was improved from 7.6% at time 0 and without enzyme to 52.1% for 0.1% cellulase after 20 h of reaction. A significant effect of cellulase addition and reaction time on antioxidant power of the tested film-forming solutions was also reported. Film-forming hydrosols with cellulase exhibited a bacteriostatic effect on all tested bacteria, causing a total reduction.

  9. Strategies to increase cellulase production with submerged fermentation using fungi isolated from the Brazilian biome

    Directory of Open Access Journals (Sweden)

    Genilton da Silva Faheina Junior

    2015-03-01

    Full Text Available Studies on new microbial sources of cellulase and accurate assessment of the steps that increase cellulase production are essential strategies to reduce costs of various processes using such enzymes. This study aimed at the selection of cellulase-producing filamentous fungi, and at the research of parameters involving cellulase production by submerged fermentation. The first test consisted of selecting the best cellulase-producing microorganisms (FPase in Erlenmeyer flasks containing 200 mL of specific growth medium. The next test was designed to further investigate the enzyme production in fermentation with four types of soluble sugars: glucose, lactose, sucrose and xylose. In bioreactor tests, three different inoculation strategies were analyzed. The best FPase activity was presented by the strain Trichoderma sp. CMIAT 041 (49.9 FPU L-1 and CMCase by the fungus Lasiodiplodia theobromae CMIAT 096 (350.0 U L-1. Sucrose proved to be the best option among the soluble sugars tested, with higher rates of FPase activity (49.9 FPU L-1 and CMCase (119.7 U L-1. The best inoculation strategy for the bioreactor was a spore suspension obtained from a semi-solid state fermentation of wheat bran for 72h.

  10. Structure of the catalytic domain of the Clostridium thermocellum cellulase CelT.

    Science.gov (United States)

    Kesavulu, Muppuru M; Tsai, Jia Yin; Lee, Hsiao Lin; Liang, Po Huang; Hsiao, Chwan Deng

    2012-03-01

    Cellulases hydrolyze cellulose, a major component of plant cell walls, to oligosaccharides and monosaccharides. Several Clostridium species secrete multi-enzyme complexes (cellulosomes) containing cellulases. C. thermocellum CelT, a family 9 cellulase, lacks the accessory module(s) necessary for activity, unlike most other family 9 cellulases. Therefore, characterization of the CelT structure is essential in order to understand its catalytic mechanism. Here, the crystal structure of free CelTΔdoc, the catalytic domain of CelT, is reported at 2.1 Å resolution. Its structure differs in several aspects from those of other family 9 cellulases. CelTΔdoc contains an additional α-helix, α-helices of increased length and two additional surface-exposed β-strands. It also contains three calcium ions instead of one as found in C. cellulolyticum Cel9M. CelTΔdoc also has two flexible loops at the open end of its active-site cleft. Movement of these loops probably allows the substrate to access the active site. CelT is stable over a wide range of pH and temperature conditions, suggesting that CelT could be used to convert cellulose biomass into biofuel.

  11. Analysis of inducers of xylanase and cellulase activities production by Ganoderma applanatum LPB MR-56.

    Science.gov (United States)

    Salmon, Denise Naomi Xavier; Spier, Michele Rigon; Soccol, Carlos Ricardo; Vandenberghe, Luciana Porto de Souza; Weingartner Montibeller, Valesca; Bier, Mário César Jucoski; Faraco, Vincenza

    2014-08-01

    This manuscript describes the analysis of the effect of cellulose, carboxymethylcellulose (CMC), xylan, and xylose as inducers of cellulase and xylanase activity production by Ganoderma applanatum MR-56 and the optimization of their production in liquid cultures by statistical methods. The Plackett-Burman screening design was applied to identify the most significant inducers of xylanase and cellulase activities production by G. applanatum MR-56. The most significant effect on xylanase and cellulase activities production was exercised by cellulose, even if xylose and CMC were also effective at some times. The combined effect of cellulose, yeast extract, and pH was analyzed by a 2(3) factorial experimental design with four central points that showed that the maximum tested cellulose (1 % w/v) and yeast extract (5 g L(-1)) concentrations gave the maximum production of xylanase (8.24 U mL(-1)) and cellulase (3.29 U mL(-1)) activity at pH 6 and 4, respectively. These values achieved for cellulase and xylanase activity represent 12-25 fold and 36 fold higher values than the maximum so far reported for other strains of G. applanatum, respectively.

  12. Improving cellulase productivity of Penicillium oxalicum RE-10 by repeated fed-batch fermentation strategy.

    Science.gov (United States)

    Han, Xiaolong; Song, Wenxia; Liu, Guodong; Li, Zhonghai; Yang, Piao; Qu, Yinbo

    2017-03-01

    Medium optimization and repeated fed-batch fermentation were performed to improve the cellulase productivity by P. oxalicum RE-10 in submerged fermentation. First, Plackett-Burman design (PBD) and central composite design (CCD) were used to optimize the medium for cellulase production. PBD demonstrated wheat bran and NaNO3 had significant influences on cellulase production. The CCD results showed the maximum filter paper activity (FPA) production of 8.61U/mL could be achieved in Erlenmeyer flasks. The maximal FPA reached 12.69U/mL by submerged batch fermentation in a 7.5-L stirred tank, 1.76-fold higher than that on the original medium. Then, the repeated fed-batch fermentation strategy was performed successfully for increasing the cellulase productivity from 105.75U/L/h in batch fermentation to 158.38U/L/h. The cellulase activity and the glucan conversion of delignined corn cob residue hydrolysis had no significant difference between the enzymes sampled from different cycles of the repeated fed-batch fermentation and that from batch culture.

  13. Application of Statistical Design for the Production of Cellulase by Trichoderma reesei Using Mango Peel

    Directory of Open Access Journals (Sweden)

    P. Saravanan

    2012-01-01

    Full Text Available Optimization of the culture medium for cellulase production using Trichoderma reesei was carried out. The optimization of cellulase production using mango peel as substrate was performed with statistical methodology based on experimental designs. The screening of nine nutrients for their influence on cellulase production is achieved using Plackett-Burman design. Avicel, soybean cake flour, KH2PO4, and CoCl2·6H2O were selected based on their positive influence on cellulase production. The composition of the selected components was optimized using Response Surface Methodology (RSM. The optimum conditions are as follows: Avicel: 25.30 g/L, Soybean cake flour: 23.53 g/L, KH2PO4: 4.90 g/L, and CoCl2·6H2O: 0.95 g/L. These conditions are validated experimentally which revealed an enhanced Cellulase activity of 7.8 IU/mL.

  14. Application of Statistical Design for the Production of Cellulase by Trichoderma reesei Using Mango Peel.

    Science.gov (United States)

    Saravanan, P; Muthuvelayudham, R; Viruthagiri, T

    2012-01-01

    Optimization of the culture medium for cellulase production using Trichoderma reesei was carried out. The optimization of cellulase production using mango peel as substrate was performed with statistical methodology based on experimental designs. The screening of nine nutrients for their influence on cellulase production is achieved using Plackett-Burman design. Avicel, soybean cake flour, KH(2)PO(4), and CoCl(2)·6H(2)O were selected based on their positive influence on cellulase production. The composition of the selected components was optimized using Response Surface Methodology (RSM). The optimum conditions are as follows: Avicel: 25.30 g/L, Soybean cake flour: 23.53 g/L, KH(2)PO(4): 4.90 g/L, and CoCl(2)·6H(2)O: 0.95 g/L. These conditions are validated experimentally which revealed an enhanced Cellulase activity of 7.8 IU/mL.

  15. Microwave pretreatment of substrates for cellulase production by solid-state fermentation.

    Science.gov (United States)

    Zhao, Xuebing; Zhou, Yujie; Zheng, Guangjian; Liu, Dehua

    2010-03-01

    The agricultural residues, wheat bran and rice hulls, were used as substrates for cellulase production with Trichoderma sp 3.2942 by solid-state fermentation. Microwave irradiation was employed to pretreat the substrates in order to increase the susceptibility. Although the highest cellulase yield was obtained by the substrates pretreated by 450 W microwave for 3 min, pretreatment time and microwave power had no significant effect on cellulase production. The initial reducing sugar content (RSC) of substrates was decreased by microwave irradiation, but more reducing sugars were produced in later fermentation. Alkali pretreatment combined with microwave pretreatment (APCMP) of rice hulls could significantly increase cellulase yields and reducing sugar. The maximum filter paper activity, carboximethylcellulase (CMC)ase, and RSC were increased by 35.2%, 21.4%, and 13%, respectively, compared with those of untreated rice hulls. The fermented residues could produce more cellulase and reducing sugars than fresh rice hulls after they were treated by APCMP. The increased accessibility of the substrates by microwave pretreatment was mainly achieved by rupture of the rigid structure of rice hulls. However, for alkali pretreatment and APCMP, delignification and removal of ash played very important roles for increasing the acceptability of substrates.

  16. Is an organic nitrogen source needed for cellulase production by Trichoderma reesei Rut-C30?

    Science.gov (United States)

    Rodriguez-Gomez, Divanery; Hobley, Timothy John

    2013-11-01

    The effect of organic and inorganic nitrogen sources on Trichoderma reesei Rut-C30 cellulase production was investigated in submerged cultivations. Stirred tank bioreactors and shake flasks, with and without pH control, respectively, were employed. The experimental design involved the addition of individual organic nitrogen sources (soy peptone, glutamate, glycine and alanine) within a basal medium containing Avicel (i.e. micro crystalline cellulose) and ammonium sulphate. It was found that in the shake flask experiments, the highest cellulase activities (~0.1 ± 0.02 FPU ml(-1)) were obtained with media containing soy peptone (3-6 g l(-1)) and glutamate (3.6 g l(-1)). However, these improvements in the cellulase titers in the presence of the organic nitrogen sources appeared to be related to smaller changes in the pH of the medium. This was confirmed using stirred tank bioreactors with pH control. No significant differences were observed in the highest cellulase titers and the protein pattern (according to the SDS-PAGE) of supernatants from pH controlled stirred tank bioreactor cultivations, when different nitrogen sources were used in the medium. Here the cellulase activities (~1.0 ± 0.2 FPU ml(-1)) were also much greater (8-150 times) than in shake flask cultivation. Consequently, the addition of ammonium sulphate as sole nitrogen source to Avicel basal medium is recommended when performing cultivations in stirred tank bioreactors with strict pH controlled conditions.

  17. (Studies of the genetic regulation of the Thermomonospora cellulase complex)

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, D.B.

    1992-01-01

    The goals of this project are to determine the molecular mechanisms regulating cellulose synthesis in the soil bacterium Thermomonosporafusca and to determine the molecular mechanism by which T.fusca cellulases degrade crystalline cellulose. We have determined a structure for the T.fusca E{sub 2} catalytic subunit (E{sub 2}-30) by x-ray crystallography. This structure is quite similar to that of T.reesei CBHU but there are a number of differences. One is that the E{sub 2} active site is in a cleft while that of CBHII is in a tunnel. This is an expected result since E{sub 2} is an endocellulase. Large amounts of homogenous E{sub 5} catalytic subunit have been prepared and attempts to crystallize it are underway. Crystals of E{sub 2}-30 were soaked in cellobiose and modified crystals detracted well, however difference Fourier analysis showed many changes, so that we could not localize cellobiose in the 3-D structure of E{sub 2}-30. This implies that binding of cellobiose causes a significant change in the structure of E{sub 2}-30. The stereochemistry of the cleavage catalyzed by E{sub l}, E{sub 2} and E{sub 5} was determined in collaboration with Dr. Stephen Withers and E{sub 1} and 2 inverted the glycoside linkage while E{sub 5} does not. The entire E{sub l} and E{sub 4} genes have been induced into Streptomyces lividans where they are expressed at a high level and the E{sub l} and E{sub 4} are completely secreted into the medium. Studies on the synergism between the exocellulase E{sub 3} and the endocellulases E{sub 2} or E{sub 5} show that both exo and endocellulase activities are stimulated when they are assayed together.

  18. Interrelationships between cellulase activity and cellulose particle morphology

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, Johan P.; Donohoe, Bryon S.; Borch, Kim; Westh, Peter; Resch, Michael G.

    2016-06-11

    It is well documented that the enzymatic hydrolysis of cellulose follows a reaction pattern where an initial phase of relatively high activity is followed by a gradual slow-down over the entire course of the reaction. This phenomenon is not readily explained by conventional factors like substrate depletion, product inhibition or enzyme instability. It has been suggested that the underlying reason for the loss of enzyme activity is connected to the heterogeneous structure of cellulose, but so far attempts to establish quantitative measures of such a correlation remain speculative. Here, we have carried out an extensive microscopy study of Avicel particles during extended hydrolysis with Hypocrea jecorina cellobiohydrolase 1 (CBH1) and endoglucanase 1 and 3 (EG1 and EG3) alone and in mixtures. We have used differential interference contrast microscopy and transmission electron microscopy to observe and quantify structural features at um and nm resolution, respectively. We implemented a semi-automatic image analysis protocol, which allowed us to analyze almost 3000 individual micrographs comprising a total of more than 300,000 particles. From this analysis we estimated the temporal development of the accessible surface area throughout the reaction. We found that the number of particles and their size as well as the surface roughness contributed to surface area, and that within the investigated degree of conversion (<30 %) this measure correlated linearly with the rate of reaction. Based on this observation we argue that cellulose structure, specifically surface area and roughness, plays a major role in the ubiquitous rate loss observed for cellulases.

  19. POTENTIAL OF THERMOSTABLE CELLULASES IN BIOPROCESSING OF SWITCHGRASS TO ETHANOL

    Directory of Open Access Journals (Sweden)

    Kasivishavanathan Muthukumarappan

    2011-04-01

    Full Text Available Switchgrass (Panicum virgatum, a perennial grass native to North America, is a promising energy crop for bioethanol production. The aim of this study was to optimize the enzymatic saccharification of thermo-mechanically pretreated switchgrass using a thermostable cellulase from Geobacillus sp. in a three-level, four-variable central composite design of response surface methodology. Different combinations of solids loadings (5 to 20%, enzyme loadings (5 to 20 FPU g-1 DM, temperature (50 to 70 oC, and time (36 to 96 h were investigated in a total of 30 experiments to model glucose release from switchgrass. All four factors had a significant impact on the cellulose conversion yields with a high coefficient of determination of 0.96. The use of higher solids loadings (20% and temperatures (70 oC during enzymatic hydrolysis proved beneficial for the significant reduction of hydrolysis times (2.67-times and enzyme loadings (4-times, with important implications for reduced capital and operating costs of ethanol production. At 20% solids, the increase of temperature of enzymatic hydrolysis from 50 oC to 70 oC increased glucose concentrations by 34%. The attained maximum glucose concentration of 23.52 g L-1 translates into a glucose recovery efficiency of 46% from the theoretical yield. Following red yeast fermentation, a maximum ethanol concentration of 11 g L-1 was obtained, accounting for a high glucose to ethanol fermentation efficiency of 92%. The overall conversion efficiency of switchgrass to ethanol was 42%.

  20. Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Davison, Steffi A; den Haan, Riaan; van Zyl, Willem Heber

    2016-09-01

    Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development.

  1. Induction of cellulases and hemicellulases by tamarind (Tamarindus indica) kernel polysaccharide

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, B.S.; Kundu, A.B.

    1980-01-01

    Tamarind kernel polysaccharide (TKP) which is available in India in abundance could be used as an excellent substrate for production of cellulases, hemicellulases, ..beta..-glucosidase and ..beta..-xylosidase. A growth medium of a known cellulytic strain, Aspergillus terreus containing TKP has been optimized to get enhanced yields of cellulase and hemicellulase for practical application in jute manufacturing units around Calcutta. Of the large number of sugars and treated and untreated cellulosics tested, TKP was found to produce the highest amounts of cellulases, hemicellulases, ..beta..-glucosidase, ..beta..-xylosidase and extra-cellular protein. The effects of the use of different inorganic nitrogenous substances, nutrients and surfactants in the optimized medium have been tested. Both initial pH of the medium and final pH of the culture filtrate have been found to have a marked effect on enzyme production, especially ..beta..-glucosidase production.

  2. Production of cellulase using a mutant strain of trichoderma reesei growing on lactose in batch culture

    Energy Technology Data Exchange (ETDEWEB)

    Chaudhuri, B.K. (Indian Inst. of Tech., New Delhi (India). Biochemical Engineering Research Centre); Sahai, V. (Indian Inst. of Tech., New Delhi (India). Biochemical Engineering Research Centre)

    1993-05-01

    The production of cellulases in batch culture was studied using a mutant strain of Trichoderma reesei C-5 growing on lactose. Growth kinetic parameters on 2% lactose were studied and the comparative results for growth and enzyme productivities at two different lactose levels are discussed. The cellulase synthesis rate depended on the lactose concentration in the medium. Although growth was favoured at a higher lactose level, the volumetric enzyme productivity did not increase in proportion and the specific enzyme productivity decreased to a certain extent, indicating that partial catabolic inhibition at higher lactose concentrations may be possible. However, it was noted that the mutant strain was highly derepressed and capable of synthesising active cellulases on lactose. (orig.)

  3. [Study on the backward extraction of cellulase in rhamnolipid reverse micelles].

    Science.gov (United States)

    Zhao, Yan-Ge; Yuan, Xing-Zhong; Huang, Hua-Jun; Cui, Kai-Long; Peng, Xin; Peng, Zi-Yuan; Zeng, Guang-Ming

    2014-02-01

    This paper studied the backward extraction of cellulase in RL/isooctane/n-hexanol reverse micelles system. Several key parameters influencing the backward extraction efficiency and activity recovery of cellulase were investigated, including stripping aqueous pH, stripping time, salt type and ionic strength, and addition of alcohols. The experiment results indicated that the optimal parameter values as follows: stripping aqueous pH 7.0, stripping time 30 min, 0.15 mol x L(-1) of KCl, dosage of n-butanol 2%. Under above optimum conditions, the backward extraction efficiency and activity recovery were up to 76.22% and 93.39%, respectively. The backward extraction of cellulase using reverse micelles based on biosurfactant RL performs well. Furthermore, RL has many advantages such as high biodegradability, low critical micelle concentration, etc. The application prospects of RL reverse micelles are extensive.

  4. IMMOBILIZATION OF GLUCOSE OXIDASE AND CELLULASE BY CHITOSAN-POLYACRYLIC ACID COMPLEX

    Institute of Scientific and Technical Information of China (English)

    WANG Lingzhi; JIANG Yingyan; ZHANG Changde; HUANG Dexiu

    1990-01-01

    This study is concerned with chitosan-polyacrylic acid complex as a carrier to immobilize glucose oxidase (GOD) and cellulase. The optimum temperature of the immobilized GOD (IG) was determined to be 60 ℃ which is higher than that of the native GOD about 40 ℃ . The optimum temperature of the immobilized cellulase (IC) was determined to be about 30 ℃ higher than that of native cellulase. Both of the optimum pH of IG and IC shifted one pH unit to acid. Immobilized enzyme may be used in more wide pH range. Their storage life are much longer compared with their native states. Both of them can be reused at least 12 times.

  5. [Xylanase and cellulase of fungus Cerrena unicolor VKM F-3196: production, properties, and applications for the saccharification of plant material].

    Science.gov (United States)

    Belova, O V; Lisov, A V; Vinokurova, N G; Kostenevich, A A; Sapunova, L I; Lobanok, A G; Leont'evskiĭ, A A

    2014-01-01

    Under the conditions of submerged cultivation in a medium containing microcrystalline cellulose, the Cerrena unicolor VKM F-3196 basidiomycete is capable of producing xylanase and cellulase. Electrophoretically homogeneous cellulase and xylanase were obtained using ion exchange and hydrophobic chromatography. The molecular weight of both cellulase and xylanase was -44 kDa. It was shown that xylanase catalyzed the hydrolysis of xylan with the production of xylose, xylobiose, and xylotetrose and it exhibited properties of endoxylanases. Cellulase hydrolyzed carboxymethylcellulose, xylan, and microcrystalline cellulose with the formation of cellotriose and cellotetraose. For both enzymes, the pH optimum was -4.0. The enzymes exhibited moderate thermostability: xylanase retained 35% of the initial activity for an hour at 60 degrees C; cellulase, 10% under the same conditions. Xylanase, cellulose, and a mixture of these enzymes saccharified plant material (wheat, rye, wheat middling, and oat), indicating the possible use of these enzymes in biotechnology.

  6. Purification and characterization of five cellulases and one xylanase from Penicillium brasilianum IBT 20888

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Eriksson, T.; Borjesson, J.;

    2003-01-01

    The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l(-1) cellulose and 10 g l(-1) xylan for 111 h and the resulting culture filtrate was used for protein purification. From the cultivation broth, five cellulases and one xylanase were purified. Hydrolysis...... the cellulose-binding domain or an essential part of it. The basic xylanase (pI > 9) was only active towards xylan. Two of the purified cellulases with endoglucanase activity were partly sequenced and based on sequence homology with known enzymes they were classified as belonging to families 5 and 12...

  7. Cellulase occurs in multiple active forms in ripe avocado fruit mesocarp.

    Science.gov (United States)

    Kanellis, A K; Kalaitzis, P

    1992-02-01

    The existence of multiple forms of avocado (Persea americana Mill. cv Hass) cellulase in crude protein extracts of ripe avocado fruit is reported. Cellulase was separated into at least 11 multiple forms by native isoelectric focusing in the pH range between 4 and 7 and visualized by both activity staining using Congo red and immunostaining. The enzyme components were acidic proteins with isoelectric points in the range of pH 5.10 to 6.80, the predominant forms having isoelectric points of 5.60, 5.80, 5.95, and 6.20. All 11 forms were immunologically related with molecular masses of 54 kilodaltons.

  8. Cellulases, nucleic acids encoding them and methods for making and using them

    Science.gov (United States)

    Blum, David; Gemsch Cuenca, Joslin; Dycaico, Mark

    2013-04-23

    This invention relates to molecular and cellular biology and biochemistry. In one aspect, the invention provides polypeptides having cellulase activity, e.g., endoglucanase, cellobiohydrolase, mannanase and/or .beta.-glucosidase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides cellulase activity, e.g., endoglucanase, cellobiohydrolase, mannanase and/or .beta.-glucosidase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts.

  9. Effect of variations in growth parameters on cellulase activity of Trichoderma viride NSPR006 cultured on different wood-dusts

    Directory of Open Access Journals (Sweden)

    Olaniyi, O. O.

    2013-01-01

    Full Text Available Aims: The biotechnology research into agro wastes has been driven by the need to screen organisms for hyper-production of novel extracellular enzymes in which cellulase plays a significant role. Therefore, the aim of the study was to pre-screen selected fungal strains and optimize cultural conditions for cellulase production by Trichoderma viride NSPR006 cultured on pretreated sawdust as lignocellulosic substrate. Methodology and results: The selected fungal isolates namely Trichoderma viride NSPR006, Botrydiplodia NSPR007 and Acremonium butyri NSPR06B obtained from the culture collection of the Nigerian Stored Products Research Institute Ilorin, Kwara State, Nigeria were screened for the production of cellulase in mineral salt medium in which carboxymethylcellulose (CMC had been incorporated as the sole carbon source. All the tested fungal isolates produced cellulase with differences in the amount of enzyme production. Of all the selected fungal isolates screened, Trichoderma viride NSPR006 was found to yield highest cellulase activity compared to the other isolates. Among tested carbon sources, Pachyslasma tessmani wood dust at 3% level proved to the best for cellulase production. Of the entire tested organic nitrogen sources, locust beans were observed to yield maximum cellulase activity (0.194 µmol/min/mL. The optimum temperature, incubation time and pH for maximum cellulase production were 28 °C, 72 h and 6.5, respectively. Conclusion, significance and impact of study: Outcome of this study shows the effectiveness of pre-treatment of wood dust as low cost system for hyper-production of cellulase for industrial application. Also, the work revealed the use of pretreated wood dust as substitute to commercial substrate known to be expensive in cellulase production.

  10. Characterization of Crude Cellulase from Trichoderma reesei and Purification of Cellulase%凝胶层析法纯化纤维素酶的研究

    Institute of Scientific and Technical Information of China (English)

    姚善泾; 关怡新; 俞丽华

    2002-01-01

    The gel filtration was carried out for purification of cellulase. The influences of chromatographic parameters on the resolution were studied to determine the optimal conditions for purification. The purified endoglucanase was obtained by gel filtration by Superdex 75 prep grade with an activity recovery of 92.8% and the purification factor 4.2. The sample volume should be below 6 % of the column bed volume and the column bed height L ≥ 12.0 cm. The optimum catalysis temperature and pH for the enzyme were 55 ℃ and 4.5-5.0, respectively. The cellulase was stable at pH ranging from 4.0 to 6.0 and temperature below 60 ℃.

  11. The Production of Fungal Mannanase, Cellulase and Xylanase Using Palm Kernel Meal as a Substrate

    Directory of Open Access Journals (Sweden)

    Nisa SAE-LEE

    2007-01-01

    Full Text Available Extracellular enzymes including mannanase, cellulase and xylanase from Aspergillus wentii TISTR 3075, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei TISTR 3080 and Penicillium sp. were investigated. The enzymes were produced in solid-state fermentation using palm kernel meal (PKM as a substrate. All fungal strains produced mainly mannanase. A maximum activity of 24.9 U/g koji was observed in A. wentii TISTR 3075 with a specific activity of 1.5 U/mg protein. During PKM fermentation, there was also found low concomitantly of cellulase and xylanase activities with high mannanase activity in all strains. The degradation of non-starch polysaccharides (NSPs in PKM by these fungal strains was indicated by the increased mannanase, cellulase and xylanase activities which correlated with the increase in reducing sugar content and pH profiles during PKM fermentation. PKM was shown to be more suitable for production of mannanase than cellulase and xylanase for all strains because of the high content of mannan as an inducer in PKM. Increases in enzyme yield might be obtained by optimizing the culture conditions.

  12. Is an organic nitrogen source needed for cellulase production by Trichoderma reesei Rut-C30?

    DEFF Research Database (Denmark)

    Rodríguez Gómez, Divanery; Hobley, Timothy John

    2013-01-01

    of individual organic nitrogen sources (soy peptone, glutamate, glycine and alanine) within a basal medium containing Avicel (i.e. micro crystalline cellulose) and ammonium sulphate. It was found that in the shake flask experiments, the highest cellulase activities (~0.1 ± 0.02 FPU ml−1) were obtained...

  13. Cooperative action of cellulase enzyme and carboxymethyl cellulose on cotton fabric cleanability from a topographical standpoint

    NARCIS (Netherlands)

    Calvimontes, A.; Lant, N.J.; Dutschk, V.

    2011-01-01

    In this study, the effect of cotton treatment with cellulose and carboxymethyl cellulose on soil release of three different types of fabric: woven plain, woven twill and knitted were systematically studied. A recent study of the effect of a cleaning cellulase enzyme on cellulose films has proven tha

  14. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

    Directory of Open Access Journals (Sweden)

    Yoichiro Ito

    Full Text Available Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

  15. Separation of hydrolytically active components of cellulase from Myrothecium verrucaria by starch gel electrophoresis

    NARCIS (Netherlands)

    Ritter, F.J.; Prins-van der Meulen, P.Y.F.; Marel, T. van der

    1968-01-01

    Using starch gel electrophoresis according to Smithies, desalted crude cellulase from Myrothecium verrucqria was separated into at least 12 protein zones. These were tested on their activity towards p-nitrophenyl-β-D-glucoside, sodium carboxymethylcellulose and α-cellulose. They were all hydrolytica

  16. Standard assays do not predict the efficiency of commercial cellulase preparations towards plant materials

    NARCIS (Netherlands)

    Kabel, M.A.; Maarel, van der M.J.E.C.; Klip, G.; Voragen, A.G.J.; Schols, H.A.

    2006-01-01

    Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex

  17. Standard Assays Do Not Predict the Efficiency of Commercial Cellulase Preparations Towards Plant Materials

    NARCIS (Netherlands)

    Kabel, Mirjam A.; Maarel, Marc J.E.C. van der; Klip, Gert; Voragen, Alphons G.J.; Schols, Henk A.

    2006-01-01

    Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex

  18. Regulation of cellulase expression, sporulation, and morphogenesis by velvet family proteins in Trichoderma reesei.

    Science.gov (United States)

    Liu, Kuimei; Dong, Yanmei; Wang, Fangzhong; Jiang, Baojie; Wang, Mingyu; Fang, Xu

    2016-01-01

    Homologs of the velvet protein family are encoded by the ve1, vel2, and vel3 genes in Trichoderma reesei. To test their regulatory functions, the velvet protein-coding genes were disrupted, generating Δve1, Δvel2, and Δvel3 strains. The phenotypic features of these strains were examined to identify their functions in morphogenesis, sporulation, and cellulase expression. The three velvet-deficient strains produced more hyphal branches, indicating that velvet family proteins participate in the morphogenesis in T. reesei. Deletion of ve1 and vel3 did not affect biomass accumulation, while deletion of vel2 led to a significantly hampered growth when cellulose was used as the sole carbon source in the medium. The deletion of either ve1 or vel2 led to the sharp decrease of sporulation as well as a global downregulation of cellulase-coding genes. In contrast, although the expression of cellulase-coding genes of the ∆vel3 strain was downregulated in the dark, their expression in light condition was unaffected. Sporulation was hampered in the ∆vel3 strain. These results suggest that Ve1 and Vel2 play major roles, whereas Vel3 plays a minor role in sporulation, morphogenesis, and cellulase expression.

  19. Evaluation of Minimal Trichoderma reesei Cellulase Mixtures on Differently Pretreated Barley Straw Substrate

    DEFF Research Database (Denmark)

    Rosgaard, Lisa; Pedersen, Sven; Langston, J;

    2007-01-01

    The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsv ae rd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably...

  20. Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated barley straw substrates

    DEFF Research Database (Denmark)

    Rosgaard, Lisa; Pedersen, Sven; Langston, Jim;

    2007-01-01

    The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsv ae rd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably...

  1. Modification Effect of Cellulase on the Physicochemical Characteristic of Polysaccharides Edible Films

    Directory of Open Access Journals (Sweden)

    Anna Zimoch-Korzycka

    2015-01-01

    Full Text Available This study was conducted to assess hydrolytic influence of cellulase (C on the physicochemical stability of chitosan (CH/hydroxypropyl methylcellulose (HPMC films in time of storage (T. Initially, nine films were physically characterized by contact angle, water vapour permeability (WVP, water activity (aw, tensile test, dynamic mechanical thermal analysis (DMTA, and thermogravimetric analysis (TGA and chemically by Fourier Transform Infrared Spectrometry (FTIR. The contact angle results varied from 53.67° to 78.33°. The presence of the enzyme and passing time reduced the WVP from 8.46E-09 to 7.41E-09 g/s·m·Pa. The enzyme treatment improved elasticity but decreased tensile strength of films. After adding cellulase Tg was shifted to a higher temperature. Thermal stability of the films decreased with addition of cellulase and after prolonging storage time. FTIR analysis proved that chemical changes in polysaccharides structure were caused by cellulase incorporation in films composition, which may be observed in appearance of 1656 cm−1 band. The aw values did not change.

  2. Cellulase production from treated oil palm empty fruit bunch degradation by locally isolated Thermobifida fusca

    Directory of Open Access Journals (Sweden)

    M. Nazli Naim

    2013-02-01

    Full Text Available The aim of this research was to evaluate the production of cellulases from locally isolated bacteria, Thermobifida fusca, using thermal and chemical treated oil palm empty fruit bunch (OPEFB as substrate in liquid-state fermentation (LSF. T. fusca was successfully isolated and was a dominant cellulase producer in OPEFB composting at the thermophilic stage. Analysis of the surface morphology of OPEFB samples using Scanning Electron Microscopy (SEM showed that the most significant changes after the combination of thermal and chemical pretreatment was the removal of silica bodies, and this observation was supported by X-ray Diffraction analysis (XRD, Fourier Transform Infrared (FTIR, and Thermogravimetric analysis (TG showing changes on the hemicelluloses, cellulose, and lignin structures throughout the pretreatment process. As a result of the pretreatment, higher cellulase production by T. fusca was obtained. The highest activity for CMCase, FPase, and β-glucosidase using optimally treated OPEFB were 0.24 U/mL, 0.34 U/mL, and 0.04 U/mL, respectively. Therefore, it can be suggested that the combination of chemical and thermal pretreatments enhances the degradation of OPEFB for subsequent use as fermentation substrate, contributing to a higher cellulases yield by T. fusca.

  3. Simultaneous production of cellulase and reducing sugar through modification of compositional and structural characteristic of sugarcane bagasse.

    Science.gov (United States)

    Yoon, Li Wan; Ngoh, Gek Cheng; Chua, Adeline Seak May

    2013-09-10

    This study examined the potential of untreated and alkali-pretreated sugarcane bagasse (SCB) in cellulase, reducing sugar (RS) and fungal biomass production via solid state fermentation (SSF) using Pycnoporus sanguineus. The impact of the composition, structure and cellulase adsorption ability of SCB on the production of cellulase, RS and fungal biomass was investigated. From the morphological and compositional analyses, untreated SCB has relatively more structural changes with a higher percentage of depolymerisation on the cellulose, hemicellulose and lignin content compared to alkali-pretreated SCB. Thus, untreated SCB favoured the production of cellulase and fungal biomass whereas alkali-pretreated SCB yielded a higher amount of RS. The composition and morphology of untreated SCB did not encourage RS production and this suggested that RS produced during SSF might be consumed in a faster rate by the more abundantly grown fungus. Besides that, alkali-pretreated SCB with higher cellulase adsorption ability could have adsorbed the cellulase produced and resulted in a lower cellulase titre. In short, the production of specific bioproducts via SSF is dependent on the structure and composition of the substrate applied.

  4. The Putative Cellodextrin Transporter-like Protein CLP1 Is Involved in Cellulase Induction in Neurospora crassa*

    Science.gov (United States)

    Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe

    2015-01-01

    Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. PMID:25398875

  5. Prospecting Agro-waste Cocktail: Supplementation for Cellulase Production by a Newly Isolated Thermophilic B. licheniformis 2D55.

    Science.gov (United States)

    Kazeem, Muinat Olanike; Shah, Umi Kalsom Md; Baharuddin, Azhari Samsu; AbdulRahman, Nor' Aini

    2017-02-07

    Bacteria isolated from thermophilic environment that can produce cellulase as well as utilise agro-waste biomass have a high potential for developing thermostable cellulase required in the biofuel industry. The cost for cellulase represents a significant challenge in converting lignocellulose to fermentable sugars for biofuel production. Among three potential bacteria examined, Bacillus licheniformis 2D55 (accession no. KT799651) was found to produce the highest cellulolytic activity (CMCase 0.33 U/mL and FPase 0.09 U/mL) at 18-24 h fermentation when grown on microcrystalline cellulose (MCC) as a carbon source in shake flask at 50 °C. Cellulase production process was further conducted on the untreated and NaOH pretreated rice straw (RS), rice husk (RH), sugarcane bagasse (BAG) and empty fruit bunch (EFB). Untreated BAG produced the highest FPase (0.160 U/mL), while the highest CMCase (0.150 U/mL) was supported on the pretreated RH. The mixture of untreated BAG and pretreated RH as agro-waste cocktail has remarkably improved CMCase (3.7- and 1.4-fold) and FPase (2.5- and 11.5-fold) compared to the untreated BAG and pretreated RH, respectively. The mechanism of cellulase production explored through SEM analysis and the location of cellulase enzymes of the isolate was also presented. Agro-waste cocktail supplementation provides an alternative method for an efficient production of cellulase.

  6. Cellulase Production by Native Bacteria Using Water Hyacinth as Substrate under Solid State Fermentation

    Directory of Open Access Journals (Sweden)

    Suresh Chandra Kurup, R.

    2005-01-01

    Full Text Available Most of the freshwater systems in tropical countries are infested with one kind of aquatic weed or the other causing serious environmental problems. All efforts to control the growth and spread of these weeds have failed miserably and hence the concept of eradication through utilization is being adopted by many researchers. Solid state fermentation, the culturing of microorganisms on moist solid substrates in the absence or near absence of free water, has generatedgreat deal of interest among researchers because of its various advantages over the submerged fermentation technique. Cellulase enzyme is used extensively in various industries, especially in textile, food and in the bioconversion of lignocellulosic wastes to alcohol. The extensive use of cellulase in industries depends on the cost of the enzyme and hence considerable research is being carried out to isolate better microbial strains and also to develop new fermentationprocesses with the aim to reduce the product cost. The objective of the present study is to determine whether water hyacinth, one of the commonly found aquatic weeds, can be used as a substrate for cellulase production, by three native bacterial isolates named WHB 3, WHB 4 and SMB 3, under the process of solid state fermentation. Results indicatethat all the three isolates produced cellulase enzyme by using water hyacinth as the solid support. Under optimized conditions of moisture, pH, temperature, incubation time and inoculum concentration, the enzyme yield increased from 16.8 to 94.8 units for SMB 3, from 25.2 to 110.4 units for WHB 3 and from 18.0 to 127.2 units for WHB 4. The addition of nitrogen and carbon sources resulted in a significant increase in cellulase yield and WHB 3 produced the maximum amount of 216 units followed by SMB 3 and WHB 4.

  7. Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination.

    Directory of Open Access Journals (Sweden)

    Chia-Jung Chang

    Full Text Available Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43% and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR, the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.

  8. Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tao; Datta, Supratim; Eichler, Jerry; Ivanova, Natalia; Axen, Seth D.; Kerfeld, Cheryl A.; Chen, Feng; Kyrpides, Nikos; Hugenholtz, Philip; Cheng, Jan-Fang; Sale, Kenneth L.; Simmons, Blake; Rubin, Eddy

    2011-02-17

    Some ionic liquids (ILs) have been shown to be very effective solvents for biomass pretreatment. It is known that some ILs can have a strong inhibitory effect on fungal cellulases, making the digestion of cellulose inefficient in the presence of ILs. The identification of IL-tolerant enzymes that could be produced as a cellulase cocktail would reduce the costs and water use requirements of the IL pretreatment process. Due to their adaptation to high salinity environments, halophilic enzymes are hypothesized to be good candidates for screening and identifying IL-resistant cellulases. Using a genome-based approach, we have identified and characterized a halophilic cellulase (Hu-CBH1) from the halophilic archaeon, Halorhabdus utahensis. Hu-CBH1 is present in a gene cluster containing multiple putative cellulolytic enzymes. Sequence and theoretical structure analysis indicate that Hu-CBH1 is highly enriched with negatively charged acidic amino acids on the surface, which may form a solvation shell that may stabilize the enzyme, through interaction with salt ions and/or water molecules. Hu-CBH1 is a heat tolerant haloalkaliphilic cellulase and is active in salt concentrations up to 5 M NaCl. In high salt buffer, Hu-CBH1 can tolerate alkali (pH 11.5) conditions and, more importantly, is tolerant to high levels (20percent w/w) of ILs, including 1-allyl-3-methylimidazolium chloride ([Amim]Cl). Interestingly, the tolerances to heat, alkali and ILs are found to be salt-dependent, suggesting that the enzyme is stabilized by the presence of salt. Our results indicate that halophilic enzymes are good candidates for the screening of IL-tolerant cellulolytic enzymes.

  9. Characterization of Cellulase Enzyme Inhibitors Formed During the Chemical Pretreatments of Rice Straw

    Science.gov (United States)

    Rajan, Kalavathy

    Production of fuels and chemicals from a renewable and inexpensive resource such as lignocellulosic biomass is a lucrative and sustainable option for the advanced biofuel and bio-based chemical platform. Agricultural residues constitute the bulk of potential feedstock available for cellulosic fuel production. On a global scale, rice straw is the largest source of agricultural residues and is therefore an ideal crop model for biomass deconstruction studies. Lignocellulosic biofuel production involves the processes of biomass conditioning, enzymatic saccharification, microbial fermentation and ethanol distillation, and one of the major factors affecting its techno-economic feasibility is the biomass recalcitrance to enzymatic saccharification. Preconditioning of lignocellulosic biomass, using chemical, physico-chemical, mechanical and biological pretreatments, is often practiced such that biomass becomes available to downstream processing. Pretreatments, such as dilute acid and hot water, are effective means of biomass conversion. However, despite their processing importance, preconditioning biomass also results in the production of carbohydrate and lignin degradation products that are inhibitory to downstream saccharification enzymes. The saccharification enzyme cocktail is made up of endo-cellulase, exo-cellulase and beta-glucosidase enzymes, whose role is to cleave cellulose polymers into glucose monomers. Specifically, endo-cellulase and exo-cellulase enzymes cleave cellulose chains in the middle and at the end, resulting in cellobiose molecules, which are hydrolyzed into glucose by beta-glucosidase. Unfortunately, degradation compounds generated during pretreatment inhibit the saccharification enzyme cocktail. Various research groups have identified specific classes of inhibitors formed during biomass pretreatment and have studied their inhibitory effect on the saccharification cocktail. These various research groups prepared surrogate solutions in an attempt to

  10. Overproduction of cellulase by Trichoderma reesei RUT C30 through batch-feeding of synthesized low-cost sugar mixture.

    Science.gov (United States)

    Li, Yonghao; Liu, Chenguang; Bai, Fengwu; Zhao, Xinqing

    2016-09-01

    Cellulase is a prerequisite for the bioconversion of lignocellulosic biomass, but its high cost presents the biggest challenge. In this article, low-cost mixture was produced from glucose through the transglycosylation reaction catalyzed by β-glucosidase for cellulase overproduction by Trichodema reesei RUT C30. As a result, cellulase titer of 90.3FPU/mL, which was more than 10 folds of that achieved with lactose as inducer, was achieved at 144h. Meanwhile, cellulase productivity was drastically increased to 627.1FPU/L/h, at least 3-5 folds higher than previously reported by the fungal species. The crude enzyme was further tested by hydrolyzing NaOH-pretreated corn stover with 15% solid loading, and 96.6g/L glucose was released with 92.6% sugar yield at 96h and 44.8g/L ethanol was obtained.

  11. Enhancement of CO/sub 2/ and ethylene production and cellulase activity by glyphosate in Phaseolus vulgaris

    Energy Technology Data Exchange (ETDEWEB)

    Abu-Irmaileh, B.E.; Jordan, L.S.; Kumamoto, J.

    1979-01-01

    The effect of glyphosate (N-(phosphonomethyl)glycine) on carbon dioxide. (CO/sub 2/) levels, ethylene production, and cellulase activity was investigated. Production of ethylene increased within 12 h and CO/sub 2/ increased within 24 h when 12-day-old bean plants (Phaseolus vulgaris L. Red Kidney) were treated with 20 mM isopropylamine salt of glyphosate. The CO/sub 2/ cycled for 3 days and then increased around treated plants. Specific activity of cellulase was increased in debladed bean seedlings that had been retreated with 20 mM isopropylamine salt of glyphosate. Cellulase enhancement was detected 2 days after the pretreated plants were debladed. Glyphosate-enhanced ethylene production may have increased the cellulase activity. 24 references, 3 figures.

  12. Random mutagenesis and media optimisation for hyperproduction of cellulase from Bacillus species using proximally analysed Saccharum spontaneum in submerged fermentation.

    Science.gov (United States)

    Abdullah, Roheena; Zafar, Wajeeha; Nadeem, Muhammad; Iqtedar, Mehwish; Naz, Shagufta; Syed, Quratulain; Butt, Zahid Ali

    2015-01-01

    This study deals with the isolation of novel mutant of Bacillus and optimisation of media for the hyperproduction of cellulase. Cellulase-producing Bacillus PC-BC6 was subjected to physical and chemical mutagenesis to enhance the cellulolytic potential. Later, mutagenesis isolates were screened both qualitatively and quantitatively. Among all the tested isolates, Bacillus N3 yielded maximum (CMCase 1250 IU/mL/min and FPase 629 IU/mL/min) activity. The Bacillus N3 strain exhibited 1.7-fold more enzyme production as compared with the parental strain. Proximate analysis of untreated and pretreated Saccharum spontaneum was carried out to improve cellulase production. Three different media were tested for the production of cellulase, among which M2 medium containing MgSO4, pretreated S. spontaneum, K2HPO4, (NH4)2SO4 and peptone was found to be the best for maximum enzyme production by mutant Bacillus N3.

  13. Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws.

    Science.gov (United States)

    Assareh, Reza; Shahbani Zahiri, Hossein; Akbari Noghabi, Kambiz; Aminzadeh, Saeed; Bakhshi Khaniki, Gholamreza

    2012-09-01

    A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels.

  14. Trpac1, a pH response transcription regulator, is involved in cellulase gene expression in Trichoderma reesei.

    Science.gov (United States)

    He, Ronglin; Ma, Lijuan; Li, Chen; Jia, Wendi; Li, Demao; Zhang, Dongyuan; Chen, Shulin

    2014-12-01

    Fungi grow over a relatively wide pH range and adapt to extracellular pH through a genetic regulatory system mediated by a key component PacC, which is a pH transcription regulator. The cellulase production of the filamentous fungi Trichoderma reesei is sensitive to ambient pH. To investigate the connection between cellulase expression regulation and ambient pH, an ortholog of Aspergillus nidulans pacC, Trpac1, was identified and functionally characterized using a target gene deletion strategy. Deleting Trpac1 dramatically increased the cellulase production and the transcription levels of the major cellulase genes at neutral pH, which suggested Trpac1 is involved in the regulation of cellulase production. It was further observed that the expression levels of transcription factors xyr1 and ace2 also increased in the ΔTrpac1 mutant at neutral pH. In addition, the ΔTrpac1 mutant exhibited conidiation defects under neutral and alkaline pH. These results implied that Trpac1 in involved in growth and development process and cellulase gene expression in T. reesei.

  15. Addressing the Recalcitrance of Cellulose Degradation through Cellulase Discovery, Nano-scale Elucidation of Molecular Mechanisms, and Kinetic Modeling

    Energy Technology Data Exchange (ETDEWEB)

    Walker, Larry P., Bergstrom, Gary; Corgie, Stephane; Craighead, Harold; Gibson, Donna; Wilson, David

    2011-06-13

    This research project was designed to play a vital role in the development of low cost sugars from cellulosic biomass and contributing to the national effort to displace fossil fuel usage in the USA transportation sector. The goal was to expand the portfolio of cell wall degrading enzymes through innovative research at the nano-scale level, prospecting for novel cellulases and building a kinetic framework for the development of more effective enzymatic conversion processes. More precisely, the goal was to elucidate the molecular mechanisms for some cellulases that are very familiar to members of our research team and to investigate what we hope are novel cellulases or new enzyme combinations from the world of plant pathogenic fungi and bacteria. Hydrolytic activities of various cellulases and cellulase cocktails were monitored at the nanoscale of cellulose fibrils and the microscale of pretreated cellulose particles, and we integrated this insight into a heterogeneous reaction framework. The over-riding approach for this research program was the application of innovative and cutting edge optical and high-throughput screening and analysis techniques for observing how cellulases hydrolyze real substrates.

  16. Cellulase-assisted extraction and antibacterial activity of polysaccharides from the dandelion Taraxacum officinale.

    Science.gov (United States)

    Wang, Hong-Bin

    2014-03-15

    In the present study, we investigated the cellulase-assisted extraction and antibacterial activity of water-soluble polysaccharides from the dandelion Taraxacum officinale. The extraction conditions, optimized for improving yield, were as follows: time, 46.11 min; temperature, 54.87 °C; pH, 4.51 and cellulase enzyme, 4000 U/g. Under these conditions, the yield of polysaccharides from dandelion (PD) reached 20.67% (w/w). The sugar content of PD was 95.6% (w/w), and it displayed high antibacterial activity at a concentration of 100mg/mL against Escherichia coli, Bacillus subtilis and Staphylococcus aureus. These results indicate that PD may be a viable option for use as a food preservative.

  17. Cellulases immobilization on chitosan-coated magnetic nanoparticles: application for Agave Atrovirens lignocellulosic biomass hydrolysis.

    Science.gov (United States)

    Sánchez-Ramírez, Jaquelina; Martínez-Hernández, José L; Segura-Ceniceros, Patricia; López, Guillermo; Saade, Hened; Medina-Morales, Miguel A; Ramos-González, Rodolfo; Aguilar, Cristóbal N; Ilyina, Anna

    2017-01-01

    In the present study, Trichoderma reesei cellulase was covalently immobilized on chitosan-coated magnetic nanoparticles using glutaraldehyde as a coupling agent. The average diameter of magnetic nanoparticles before and after enzyme immobilization was about 8 and 10 nm, respectively. The immobilized enzyme retained about 37 % of its initial activity, and also showed better thermal and storage stability than free enzyme. Immobilized cellulase retained about 80 % of its activity after 15 cycles of carboxymethylcellulose hydrolysis and was easily separated with the application of an external magnetic field. However, in this reaction, K m was increased eight times. The immobilized enzyme was able to hydrolyze lignocellulosic material from Agave atrovirens leaves with yield close to the amount detected with free enzyme and it was re-used in vegetal material conversion up to four cycles with 50 % of activity decrease. This provides an opportunity to reduce the enzyme consumption during lignocellulosic material saccharification for bioethanol production.

  18. Production of Cellulase from Oil Palm Biomass as Substrate by Solid State Bioconversion

    Directory of Open Access Journals (Sweden)

    Md. Z. Alam

    2005-01-01

    Full Text Available Solid state bioconversion (SSB of lignocellulosic material oil palm biomass (OPB generated from palm oil industries as waste was conducted by evaluating the enzyme production through filamentous fungus in lab-scale experiment. OPB in the form of empty fruit bunches (EFB was used as the solid substrate and treated with the fungus Trichoderma harzianum to produce cellulase. The results presented in this study revealed that the higher cellulase activity of 0.0413 unit was achieved at the day 3 of fermentation. While the optimum study indicated the enzyme production of 0.0433 unit with moisture content of 50%, 0.0413 unit with 5% v/w of inoculum size and 0.0413 unit with co-substrate concentration of 2% (w/w at days 9, 9 and 12 of fungal treatment, respectively. The parameters glucosamine and reducing sugar were observed to evaluate the growth and substrate utilization in the experiment.

  19. Equilibrium studies of cellulase aggregates in presence of ascorbic and boric acid.

    Science.gov (United States)

    Iram, Afshin; Amani, Samreen; Furkan, Mohammad; Naeem, Aabgeena

    2013-01-01

    The aggregate formation of cellulase was detected at 300 and 10 mM ascorbic and boric acid respectively. These aggregates showed reduced enzyme activity, loss in near-UV signal, decrease tryptophan and ANS fluorescence. They possess increase in non-native β-sheet structure as evident from far-UV CD and FTIR spectra, large hydrodynamic radii, increase thioflavin T fluorescence and shift in Congo red. Cellulase at 90 mM ascorbic acid exists as molten globule with retention of secondary structure, altered tryptophan environment, high ANS binding and loss in tertiary structure. Ascorbic acid acts as an antioxidant up to 90 mM and beyond this as a pro-oxidant.

  20. PURIFICATION AND SOME PROPERTIES OF CELLULASE FROM ODONTOTERMES FORMOSANUS (ISOPTERA: TERMITIDAE)

    Institute of Scientific and Technical Information of China (English)

    Tian-ciYang; Jian-chuMo; Jia-anCheng

    2004-01-01

    The purification of the cellulase from Odontotermes forrnosanus workers was achieved by using anion-exchange column of UNOsphere Q, BioLogic DuoFlow chromatography system. The purified cellulase was identified as an endoglucanase and some of its properties were investigated. The EGase activity was 807.5-fold as high as the initial enzyme activity using CMC as substrate and 14.4-fold using salicin as substrate. The enzyme preparations were homogeneous as judged by SDS-PAGE electrophoresis, molecular weight of which was 80 kDa and confirmed by 2-DE zymogram analysis. The enzyme was isoelectric at pH 6.4, which was active on CMC substrate.

  1. A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases

    DEFF Research Database (Denmark)

    Cruys-Bagger, Nicolaj; Badino, Silke Flindt; Tokin, Radina Naytchova

    2014-01-01

    A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface...... of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25 V (vs. Ag/AgCl). The PDH-biosensor...... was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea...

  2. Valorizing recycled paper sludge by a bioethanol production process with cellulase recycling.

    Science.gov (United States)

    Gomes, Daniel; Domingues, Lucília; Gama, Miguel

    2016-09-01

    The feasibility of cellulase recycling in the scope of bioethanol production from recycled paper sludge (RPS), an inexpensive byproduct with around 39% of carbohydrates, is analyzed. RPS was easily converted and fermented by enzymes and cells, respectively. Final enzyme partition between solid and liquid phases was investigated, the solid-bound enzymes being efficiently recovered by alkaline washing. RPS hydrolysis and fermentation was conducted over four rounds, recycling the cellulases present in both fractions. A great overall enzyme stability was observed: 71, 64 and 100% of the initial Cel7A, Cel7B and β-glucosidase activities, respectively, were recovered. Even with only 30% of fresh enzymes added on the subsequent rounds, solid conversions of 92, 83 and 71% were achieved for the round 2, 3 and 4, respectively. This strategy enabled an enzyme saving around 53-60%, while can equally contribute to a 40% reduction in RPS disposal costs.

  3. Cellulase-assisted extraction of polysaccharides from Cucurbita moschata and their antibacterial activity.

    Science.gov (United States)

    Qian, Zhi-Gang

    2014-01-30

    In this study, cellulase-assisted extraction of water soluble polysaccharides from pumpkin (Cucurbita moschata) and their antibacterial activity were investigated. The polysaccharides yield was monitored during the extraction process. The optimum extraction conditions were determined as follows: time, 40 min; temperature, 55°C; pH, 4.5; and cellulase amount, 4,000 U/g. The extracts were centrifuged, filtered, proteins removed by Sevag method, concentrated to approximately 15% (w/v), precipitated with 5 volumes of absolute ethanol, freeze-dried, and pulverized to yield a water soluble powder of pumpkin polysaccharides (PP). The sugar content of the product was 68.3%, and the yield was 17.34% (w/w), respectively. The PP had high antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli at the concentration of 100 mg/mL.

  4. Study on Preparation of the Low-Molecular-Weight Chitosan Using Cellulase

    Institute of Scientific and Technical Information of China (English)

    LI He-sheng; SUN Yu-xi; HUANG Xiao-chun; WANG Hong-fei; QIU Di-hong

    2006-01-01

    The degradation of chitosan ( DD of 72.05%) with aid of cellulase was carried out under the conditions of 45℃, pH 5.0 and a ratio of 1:15( chitosan/enzyme). The results showed cellulase could degrade chitosan efficiently. Viscosity of chitosan was decreased very quickly and reducing sugar released was increased with time during degradation. By using the membrane, the separation of the hydrolysis mixture was studied. Rejection of protein can be reached to be 99.74%.65.9% of low-molecular-weight chitosans was less than 2 kDa. Solubility of low-molecular-weight chitosan was found to be better than chitosan and transmittance could reach to be more than 95 % in entire range of pH 1 ~ 13.

  5. Statistical Optimization of Fermentation Conditions for Cellulase Production from Palm Oil Mill Effluent

    OpenAIRE

    Jamal I. Daoud; Md. Z. Alam

    2010-01-01

    Problem statement: Palm oil mill effluent discharged by the oil palm industries is considered as the mixed of high polluted effluent which is abundant (about 20 million tonnes year-1) and its effect contributes to the serious environmental problems through the pollution of water bodies. Approach: The aim of this study was to identify the potential of low cost substrate such as Palm Oil Mill Effluent (POME) for the production of cellulase enzyme by liquid state bioconversion. The filamentous f...

  6. Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi

    OpenAIRE

    Coradetti, Samuel T.; Craig, James P.; Xiong, Yi; Shock, Teresa; Tian, Chaoguang; Glass, N. Louise

    2012-01-01

    Rational engineering of filamentous fungi for improved cellulase production is hampered by our incomplete knowledge of transcriptional regulatory networks. We therefore used the model filamentous fungus Neurospora crassa to search for uncharacterized transcription factors associated with cellulose deconstruction. A screen of a N. crassa transcription factor deletion collection identified two uncharacterized zinc binuclear cluster transcription factors (clr-1 and clr-2) that were required for ...

  7. Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties

    Directory of Open Access Journals (Sweden)

    Penttilä Merja

    2010-07-01

    Full Text Available Abstract Background Trichoderma reesei is the main industrial producer of cellulases and hemicellulases that are used to depolymerize biomass in a variety of biotechnical applications. Many of the production strains currently in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in high-producing mutants of T. reesei by high-resolution array comparative genomic hybridization (aCGH. Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms underlying efficient cellulase production, and would enable targeted genetic engineering for improved production of proteins in general. Results We carried out an aCGH analysis of four high-producing strains (QM9123, QM9414, NG14 and Rut-C30 using the natural isolate QM6a as a reference. In QM9123 and QM9414 we detected a total of 44 previously undocumented mutation sites including deletions, chromosomal translocation breakpoints and single nucleotide mutations. In NG14 and Rut-C30 we detected 126 mutations of which 17 were new mutations not documented previously. Among these new mutations are the first chromosomal translocation breakpoints identified in NG14 and Rut-C30. We studied the effects of two deletions identified in Rut-C30 (a deletion of 85 kb in the scaffold 15 and a deletion in a gene encoding a transcription factor on cellulase production by constructing knock-out strains in the QM6a background. Neither the 85 kb deletion nor the deletion of the transcription factor affected cellulase production. Conclusions aCGH analysis identified dozens of mutations in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a powerful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially in studies where a large set of interesting strains is analyzed.

  8. Modeling Growth of Cellulomonas cellulans NRRL B 4567 under Substrate Inhibition During Cellulase Production

    OpenAIRE

    Agarwal, R; Mahanty, B.; Dasu, V. Venkata

    2009-01-01

    Cellulase production study was performed in shake flask and bioreactor system using Cellulomonas cellulans NRRL B 4567 for initial substrate concentration from γS0 = 2 to 12 g L–1. The growth, substrate uptake profile and enzyme activity at different initial substrate concentrations were measured. The results inferred the presence of substrate inhibition kinetics. Various substrate inhibition models were tested and parameters were estimated, using non-linear regression analysis. Han-Levenspie...

  9. Performance and Nutrient Utilization of Layers Fed Diet Supplemented with Microbial Phytase and Cellulase

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A 31-week feeding trial was conducted to investigated the effects of dietary supplementation of microbial phytase and cellulase on performance,nutrients utilization and tibia quality of laying hens fed maize and soybean meal diets.192 18-week-old Hisex layers were used in the trial A 2×2×2 factorial design was used in the experiment with three factors of two levels each:0.38% and 0.16% of dietary non-phytate P(nP).0 and 300 U*kg-1 of phytase (Ph),and 0 and 0.10% of cellulase (Ce).The results showed that supplementation of 300 U*kg-1 phytase significantly improved utilization of dietary crude ash,CP,Ca,total P and copper (P<0.05),and improved tibia breaking strength (P<0.05).No effect of phytase on performance was observed.Addition of 0.10% cellulase decreased feed intake (P<0.05),increased utilization of CF (P<0.05) and Ca(P<0.01),and decreased total tibia ash weight (P<0.05).300 U*kg-1 phytase and 0.10% cellulase exhibited obvious positive interactions to enhance utilization of dietary phytic P and copper (P<0.05).0.16% nP did not reduce performance of the layers,but improved egg shell quality at 20 wks,increased utilization of dietary total P,phytic P and Copper (P<0.01),decreased utilization of dietary CP,increased tibia breaking strength and Ca,Mn contents of tibia(P<0.01)

  10. Formation of cellulase and protein in the growth of Chaetomium cellulolyticum on cellulose-containing substrates

    Energy Technology Data Exchange (ETDEWEB)

    Faehnrich, P.; Irrgang, K.

    1982-01-01

    C. cellulolyticum was grown on glucose and different types of cellulose. The organism studied was mutant 7S/7, derived from C. cellulolyticum ATCC 32319. Cellulase formation on glucose was very slight, whereas it was much greater on cellulose, indicating that most of the activity is induced. Both cellulose and protein production were greater on newsprint than on Avicel, and greater on alkali-treated than on untreated newsprint. Crystaline structure in cellulose inhibited fermentation.

  11. Optimizing cellulase usage for improved mixing and rheological properties of acid-pretreated sugarcane bagasse.

    Science.gov (United States)

    Geddes, Claudia C; Peterson, James J; Mullinnix, Michael T; Svoronos, Spyros A; Shanmugam, K T; Ingram, Lonnie O

    2010-12-01

    Consolidation of bioprocessing steps with lignocellulose is limited by hydrolysate toxicity, the fibrous nature of suspensions, and low activity of cellulase enzymes. Combinations of enzyme dose and treatment conditions improved the flow properties and pumping of acid-pretreated sugarcane bagasse slurries (10% dry weight). Low levels of cellulase enzyme (0.1 and 0.5 FPU/g dry weight acid-pretreated bagasse) were found to reduce viscosities by 77-95% after 6 h, solubilizing 3.5% of the bagasse dry weight. Flow of slurries through small funnels was a useful predictor of success with centrifugal and diaphragm pumps. Equations were derived that describe viscosity and solubilized carbohydrates as a function of time and cellulase dosage. Blending of acid-pretreated bagasse (10% dry weight) with suspensions of acid-pretreated bagasse (10% dry weight) that had been previously digested with cellulase enzymes (low viscosity) did not increase viscosity in a linear fashion. Viscosity of these mixtures remained relatively constant until a threshold level of new fiber was reached, followed by a rapid increase with further additions. Up to 35% fresh acid-pretreated bagasse could be blended with enzyme-digested fiber (5.0 FPU/g dry weight acid-pretreated fiber; 6 h) with only a modest increase in viscosity. The smooth surfaces of enzyme-treated fiber are proposed to hinder the frequency and extent of interactions between fibrils of fresh fiber particles (acid-pretreated) until a threshold concentration is achieved, after which fiber interactions and viscosity increase dramatically. These results were used to model the viscosity in an ideal continuous stirred tank reactor (liquefaction) as a function of residence time and enzyme dosage.

  12. A comparison of plate assay methods for detecting extracellular cellulase and xylanase activity.

    Science.gov (United States)

    Meddeb-Mouelhi, Fatma; Moisan, Jessica Kelly; Beauregard, Marc

    2014-11-01

    Identification of microorganisms for the production of carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. To this end, dye-polysaccharide interactions which provide a visual indication of polymer hydrolysis (clear zones or halos) have been used for decades. For the detection of extracellular cellulase or xylanase activity many laboratories use Gram's iodine as the chromogenic dye, as it is a more rapid initial screening method compared to the use of other dyes. Here, we compared Gram's iodine and Congo red as indicators of polysaccharide hydrolysis. We attempted to detect cellulase activity using carboxymethylcellulose, and xylanase activity using birchwood xylan, in fourteen uncharacterized bacteria isolated from wood chips. Our results indicate that Gram's iodine may lead to identification of false positives in a typical screening protocol and that Congo red allows for avoidance of such pitfall. Congo red allowed detection of cellulase activity from live microbial colonies but not Gram's iodine. To confirm this, detection of enzymatic activity was also assessed using cell-free enzyme preparations. Congo red was found to be reliable in detecting cellulase activity with isolated enzymes preparations. Under the same conditions, neither of these dyes detected xylanase activity, despite independent evidence of xylanase activity for one of the preparations. We detected xylanase activity for this particular enzyme preparation using a coloured derivative of xylan (Remazol Brillant Blue R-xylan adduct) that respond to xylan hydrolysis. Our results suggest that methods that rely on interactions between a dye (Congo red or Gram's iodine) and a polymeric substrate (carboxymethylcellulose or birchwood xylan) for indirect detection of hydrolysis may require the use of relevant controls and independent confirmation of enzymatic activities.

  13. ENZYMATIC KINETICS OF CELLULASES ISOLATED FROM SOIL BACTE RIA OF DOON VALLEY , UTTARAKHAND

    Directory of Open Access Journals (Sweden)

    Vinit

    2015-05-01

    Full Text Available Cellulases refers to a suite of enzymes produced chiefly by fungi , bacteria , and protozoans that catalyze cellulolysis which is the hydrolysis of cellulose . Cellulose is the most abundant natural polymer on earth . It is the structural component of the plant cell walls which helps in the hydrolysis of 1, 4 - beta - D - glycosidic linkages in cellulose, lichenin and cereal beta - D - glucans . Cellulases are used for clarif ication of fruit juice, vegetable juice, roots, treatment of wine, extraction of oils and improving the quality of the bakery products . Eight soil samples were collected for cellulose preliminary screening from Gullarghati, Doon valley at different pH and temperatures, because maximum diversity was possible there as there was no interference by the humans . 110 colonies were isolated by the activity zone plate method containing CMC as a substrate using Congo red dye . Best twelve colonies were selected and ch ecked using DNS method at 540 A 0 . Four strains BR - 1, BR - 2, BR - 3 and BR - 4 were used on the basis of spectrophotometerically and characterized with the study of substrate . Maximum velocity (Vmax was observed for BR - 2 i . e . 170 units per mg protein with Km of 49 . 50mg/ml . Strain BR - 1 gave to pH optima at 4 . 5 and 6 . 5, strain BR - 2 gave maximum activity at 4 . 5 and 7 . 0 pH, BR - 3 strain gave maximum activity at pH 5 . 0 and 6 . 5 with the highest yield of cellulases w ere obtained at pH 4 . 5, 5 . 5 and 7 . 0 in bacterial s train BR - 4 . The results also shows the effect of temperature bacterial strain BR - 1, BR - 2 and BR - 4 with maximum cellulases activity at 45 0 C and bacterial strain BR - 3 maximum activity at 25 0 C .

  14. Statistical Optimization of Fermentation Conditions for Cellulase Production from Palm Oil Mill Effluent

    Directory of Open Access Journals (Sweden)

    Jamal I. Daoud

    2010-01-01

    Full Text Available Problem statement: Palm oil mill effluent discharged by the oil palm industries is considered as the mixed of high polluted effluent which is abundant (about 20 million tonnes year-1 and its effect contributes to the serious environmental problems through the pollution of water bodies. Approach: The aim of this study was to identify the potential of low cost substrate such as Palm Oil Mill Effluent (POME for the production of cellulase enzyme by liquid state bioconversion. The filamentous fungus Trichoderma harzianum was used for liquid state bioconversion of POME for cellulase production. Statistical optimization was carried out to evaluate the physico-chemical parameters (factors for maximum cellulase production by 2-level fractional factorial design with six central points. The polynomial regression model was developed using the experimental data including the effects of linear, quadratic and interaction of the factors. The factors involved were substrate (POME and co-substrate (wheat flour concentrations, temperature, pH, inoculum and agitation. Results: Statistical analysis showed that the optimum conditions were: Temperature of 30°C, substrate concentration of 2%, wheat flour concentration of 3%, pH of 4, inoculum of 3% and agitation of 200 rpm. Under these conditions, the model predicted the enzyme production to be about 14 FPU mL-1. Analysis Of Variance (ANOVA of the design showed a high coefficient of determination (R2 value of 0.999, thus ensuring a high satisfactory adjustment of the quadratic model with the experimental data. Conclusion/Recommendations: This study indicates a better solution for waste management through the utilization of POME for cellulase production that could be used in the industrial applications such as bioethanol production.

  15. Computer Simulations Reveal Multiple Functions for Aromatic Residues in Cellulase Enzymes (Fact Sheet)

    Energy Technology Data Exchange (ETDEWEB)

    2012-07-01

    NREL researchers use high-performance computing to demonstrate fundamental roles of aromatic residues in cellulase enzyme tunnels. National Renewable Energy Laboratory (NREL) computer simulations of a key industrial enzyme, the Trichoderma reesei Family 6 cellulase (Cel6A), predict that aromatic residues near the enzyme's active site and at the entrance and exit tunnel perform different functions in substrate binding and catalysis, depending on their location in the enzyme. These results suggest that nature employs aromatic-carbohydrate interactions with a wide variety of binding affinities for diverse functions. Outcomes also suggest that protein engineering strategies in which mutations are made around the binding sites may require tailoring specific to the enzyme family. Cellulase enzymes ubiquitously exhibit tunnels or clefts lined with aromatic residues for processing carbohydrate polymers to monomers, but the molecular-level role of these aromatic residues remains unknown. In silico mutation of the aromatic residues near the catalytic site of Cel6A has little impact on the binding affinity, but simulation suggests that these residues play a major role in the glucopyranose ring distortion necessary for cleaving glycosidic bonds to produce fermentable sugars. Removal of aromatic residues at the entrance and exit of the cellulase tunnel, however, dramatically impacts the binding affinity. This suggests that these residues play a role in acquiring cellulose chains from the cellulose crystal and stabilizing the reaction product, respectively. These results illustrate that the role of aromatic-carbohydrate interactions varies dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, the results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering

  16. Scaled-up separation of cellobiohydrolase1 from a cellulase mixture by ion-exchange chromatography.

    Science.gov (United States)

    Ye, Zhuoliang; Lane, Andrew N; Willing, Gerold A; Berson, R Eric

    2011-01-01

    Enzymatic hydrolysis of cellulose often involves cellulases produced by Trichoderma reesei, of which cellobiohydrolase1 (CBH1) is the most abundant (about 60% of total cellulases) and plays an important role in the hydrolysis of crystalline cellulose. A method for separating sufficient quantities from the bulk cellulase cocktail is highly desirable for many studies, such as those that aim to characterize binding and hydrolysis kinetics of CBH1. In this work, CBH1 was separated from other Spezyme CP cellulases by ion-exchange chromatography using an efficient modification of a smaller scale process. The ion-exchange column was connected to a vacuum manifold system to provide a steady flow through parallel columns and thus achieve scale-up for enzyme separation. With five 5-mL columns running in parallel, about 55 mg of CBH1 was separated from 145 mg of Spezyme CP in a single separation. Step elution was used to replace the continuous gradient used at smaller scale. The purified CBH1 was collected in the fraction eluted with a buffer containing 0.33 M salt and showed comparable purity and activity as the enzyme purified by a fast protein liquid chromatography system. The stability of separated CBH1 was studied for up to 2 days and good thermal stability was observed. Separated CBH1 also showed both high adsorption to bacterial microcrystalline cellulose with ~4 μmol/g maximum adsorption and a K(a) of 5.55 ± 2.34 μM(-1) , and good hydrolytic activity based on atomic force microscopy observations that show a reduction in fiber height.

  17. Screening and Characterization of the High-Cellulase-Producing Strain Aspergillus glaucus XC9

    Institute of Scientific and Technical Information of China (English)

    Xu Chang; Long Minnan; Wu Xiaobing; Xu Huijuan; Chen Zhongan; Zhang Fengzhang; Xu Liangshu

    2006-01-01

    Cellulose is a kind of renewable resource that is abundant in nature.It can be degraded by microorganisms such as mildew.A mildew strain with high cellulase activity was isolated from mildewy maize cob and classified as Aspergillus glaucus XC9 by morphological and 18S rRNA gene sequence analyses.We studied the effects of nitrogen source,initial pH,temperature,incubation time,medium composition,and surfactants on cellulase production.Maximal activities of carboxymethylcellulase (6,812 U/g dry koji) and filter paperase (172 U/g dry koji) were obtained in conditions as follows:initial pH,5.5-6.0;temperature,30℃;cultivation period,3-4 days;inoculum ratio,6% (vol/vol);sugarcane bagasse/wheat bran ratio,4:6.When bagasse was used as substrate and mixed with wet koji at a 1:1 (wt/wt) ratio,the yield of reducing sugars was 36.4%.The corresponding conversion rate of cellulose to reducing sugars went as high as 81.9%.The results suggest that A.glaucus XC9 is a preferred candidate for cellulase production.

  18. Analysis of Casein Biopolymers Adsorption to Lignocellulosic Biomass as a Potential Cellulase Stabilizer

    Science.gov (United States)

    Eckard, Anahita Dehkhoda; Muthukumarappan, Kasiviswanathan; Gibbons, William

    2012-01-01

    Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively. PMID:23118515

  19. Analysis of Casein Biopolymers Adsorption to Lignocellulosic Biomass as a Potential Cellulase Stabilizer

    Directory of Open Access Journals (Sweden)

    Anahita Dehkhoda Eckard

    2012-01-01

    Full Text Available Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM, fourier transform infrared spectroscopy (FT-IR, capillary electrophoresis (CE, and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively.

  20. Overexpression of poplar cellulase accelerates growth and disturbs the closing movements of leaves in sengon.

    Science.gov (United States)

    Hartati, Sri; Sudarmonowati, Enny; Park, Yong Woo; Kaku, Tomomi; Kaida, Rumi; Baba, Kei'ichi; Hayashi, Takahisa

    2008-06-01

    In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.

  1. Visualising recalcitrance by colocalisation of cellulase, lignin and cellulose in pretreated pine biomass using fluorescence microscopy

    Science.gov (United States)

    Donaldson, Lloyd; Vaidya, Alankar

    2017-01-01

    Mapping the location of bound cellulase enzymes provides information on the micro-scale distribution of amenable and recalcitrant sites in pretreated woody biomass for biofuel applications. The interaction of a fluorescently labelled cellulase enzyme cocktail with steam-exploded pine (SEW) was quantified using confocal microscopy. The spatial distribution of Dylight labelled cellulase was quantified relative to lignin (autofluorescence) and cellulose (Congo red staining) by measuring their colocalisation using Pearson correlations. Correlations were greater in cellulose-rich secondary cell walls compared to lignin-rich middle lamella but with significant variations among individual biomass particles. The distribution of cellulose in the pretreated biomass accounted for 30% of the variation in the distribution of enzyme after correcting for the correlation between lignin and cellulose. For the first time, colocalisation analysis was able to quantify the spatial distribution of amenable and recalcitrant sites in relation to the histochemistry of cellulose and lignin. This study will contribute to understanding the role of pretreatment in enzymatic hydrolysis of recalcitrant softwood biomass. PMID:28281670

  2. Studies on influence of natural biowastes on cellulase production by Aspergillus niger.

    Science.gov (United States)

    Kiranmayi, M Usha; Poda, Sudhakar; Vijayalakshmi, M; Krishna, P V

    2011-11-01

    The objective of this study was to determine the influence of natural biowaste substrates such as banana peel powder and coir powder at varying environmental parameters of pH (4-9) and temperature (20-50 degrees C) on the cellulase enzyme production by Aspergillus niger. The cellulase enzyme production was analyzed by measuring the amount of glucose liberated in IU ml(-1) by using the dinitrosalicylic acid assay method. The substrates were pretreated with 1% NaOH (alkaline treatment) and autoclaved. The maximum activity of the enzyme was assayed at varying pH with temperatures being constant and varying temperatures with pH being constant. The highest activity of the enzyme at varying pH was recorded at pH 6 for banana peel powder (0.068 +/- 0.002 IU ml) and coir powder (0.049 +/- 0.002 IU ml(-1)) and the maximum activity of the enzyme at varying temperature was recorded at 35 degrees C for both banana peel powder (0.072 +/- 0.001 IU ml(-1)) and coir powder (0.046 +/- 0.003 IU ml(-1)). At varying temperatures and pH the high level of enzyme production was obtained at 35 degrees C and pH 6 by using both the substrates, respectively. However among the two substrates used for the production of cellulases by Aspergillus niger banana peel powder showed maximum enzymatic activity than coir powder as substrate.

  3. PERFORMANCE OF LAYER HEN FED FERMENTED Jatropha Curcas L. MEAL SUPPLEMENTED WITH CELLULASE AND PHYTASE ENZYME

    Directory of Open Access Journals (Sweden)

    S. Sumiati

    2014-10-01

    Full Text Available The objective of the experiment was to study the effect of feeding fermented Jatropha curcas L.meal (JCM supplemented with cellulase and phytase on the performances of ISA-Brown laying henaged 25-30 weeks. The Jatropha curcas meal was fermented using Rizhopus oligosporus. In this study200 laying hens were used and distributed to 5 treatments and 4 replications in Completely RandomizedDesign. The diet treatments were: R0 = control diet (without JCM, R1; diet contained fermented JCM7.5%, R2; diet contained fermented JCM 7.5% + celullase 200 g/ton, R3; diet contained fermented JCM7.5% + phytase 200 g/ton and R4; diet contained fermented JCM 7.5% + cellulase 200 g/ton + phytase200 g/ton. The parameters observed were feed consumption, hen day egg production, egg massproduction, egg weight and feed conversion ratio. The results showed that feeding fermented JCM 7.5%,both enzyme supplemented as well as unsupplemented significantly decreased (P<0.05 the feedconsumption, hen day egg and egg mass production. However, the treatments did not influence the eggweight. Supplementation of cellulase (R2 or phytase (R3 improved the feed conversion ratio with thevalue as same as the R0 diet.

  4. Visualising recalcitrance by colocalisation of cellulase, lignin and cellulose in pretreated pine biomass using fluorescence microscopy

    Science.gov (United States)

    Donaldson, Lloyd; Vaidya, Alankar

    2017-03-01

    Mapping the location of bound cellulase enzymes provides information on the micro-scale distribution of amenable and recalcitrant sites in pretreated woody biomass for biofuel applications. The interaction of a fluorescently labelled cellulase enzyme cocktail with steam-exploded pine (SEW) was quantified using confocal microscopy. The spatial distribution of Dylight labelled cellulase was quantified relative to lignin (autofluorescence) and cellulose (Congo red staining) by measuring their colocalisation using Pearson correlations. Correlations were greater in cellulose-rich secondary cell walls compared to lignin-rich middle lamella but with significant variations among individual biomass particles. The distribution of cellulose in the pretreated biomass accounted for 30% of the variation in the distribution of enzyme after correcting for the correlation between lignin and cellulose. For the first time, colocalisation analysis was able to quantify the spatial distribution of amenable and recalcitrant sites in relation to the histochemistry of cellulose and lignin. This study will contribute to understanding the role of pretreatment in enzymatic hydrolysis of recalcitrant softwood biomass.

  5. Role of the components of the cellulase complex on hydrolysis of insoluble cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Klyosov, A.A.; Goldstein, G.C.; Kude, J.; Meyer, D.

    1986-01-01

    The kinetics of the hydrolysis of microcrystalline cellulose (MC) by a Trichoderma reesei cellulase complex and by the individual endoglucanase (pI 4.4-5.2) and cellobiohydrolase (pI 4.0-4.2) has been studied. A flow chart for the enzymatic hydrolysis of the cellulose has been revealed, which formed a basis for a computer simulation of the kinetic regularities observed. As a result of it, the values of the catalytic rate constants for the individual stages of the enzymatic degradation of MC have been calculated. Then, the synergistic behaviour of endoglucanase and cellobiohydrolase in the hydrolysis of MC has been described both quantitatively and graphically. The relative efficiency of the individual stages for the MC hydrolysis in terms of glucose and cellobiose formation for cellulase complexes of various composition has been calculated. It was quantitatively shown that cellobiohydrolase plays the key role in the MC hydrolysis by T. reesei cellulase preparations, because it gives up to 80% glucose and up to 80-90% cellobiose in the presence of endoglucanase which in turn plays a relatively minor role in a direct formation of both soluble products of the hydrolysis.

  6. Characterization of cellulase, hemicellulase and lipase and its use in deinking of laser printed paper

    Directory of Open Access Journals (Sweden)

    Che Omar, I.

    2013-01-01

    Full Text Available Aims: It is recognized that laser printed paper are difficult to deink using conventional method. This had lead to the suggestion of enzymatic approach to overcome the problem encountered by commonly employed deinking techniques. The present study aimed to investigate 7 commercially available enzymes for their suitability use in deinking of laser printed paper. Methodology and results: 3 cellulases, hemicellulases, xylanase and 2 lipases were used in enzymatic deinking of laser-printed wastepaper. Cellulase A “Amano”3 (C, Hemicellulase (H and lipase (L were selected for used in deinking because they possess either highest activity or broad pH stability compared to others enzymes. Different combination of enzymes was carried out to evaluate their effectiveness in deinking process. CH enzymes sequence was determined to be the most effective sequence in toner removal with 1.90% of brightness increment. However, only 0.95% of brightnessincrement was gained by enzyme sequence L. Highest deinking efficiency obtained was not proportional to the highest total reducing sugar produced. Conclusion, significance and impact of study: Enzyme (cellulase and hemicellulase can be used to de-ink laserprintedwastepaper, which are difficult to be deinked by conventional chemical deinking process. Thus, enzyme deinking has high possibility as alternative method to current chemical deinking process which is not environmental friendly.

  7. Cellulase-assisted extraction, characterization, and bioactivity of polysaccharides from Polygonatum odoratum.

    Science.gov (United States)

    Liu, Xin; Zhang, Miansong; Guo, Kai; Jia, Airong; Shi, Yaping; Gao, Ganglong; Sun, Zhenliang; Liu, Changheng

    2015-04-01

    Cellulase-assisted extraction of polysaccharides from Polygonatum odoratum (CPP) was optimized by response surface methodology (RSM) and the extracted CPP's preliminary chemical characteristics, as well as antioxidant and immunomodulatory activities were also investigated. The optimal extraction parameters comprised an extraction temperature of 58.21 °C, an extraction time of 3.18 h, pH value of 5.8, and cellulase amount of 6.0%. Under these conditions, the relative yield was 15.76%, higher than the yield achieved with hot water extracted polysaccharide (HPP). Chemical composition analysis demonstrated that CPP and HPP consisted of mannose, glucosamine, rhamnose, glucose, galactose, and arabinose with a molecular ratio of 7.80:1.08:1.63:65.93:3.58:1.00 and 11.22:0.23:0.23:17.59:2.73:9.10, respectively. The molecular weight distribution of CPP was lower and more homogeneous compared with HPP. CPP exhibited stronger antioxidant activities than HPP, including DPPH radical scavenging activity and reducing power. Both CPP and HPP could significantly promote the proliferation and neutral red phagocytosis of RAW 264.7 macrophage cells in vitro. These results indicate that the cellulase-assisted extraction method influenced the physicochemical characteristics, and consequently, the functional activities of polysaccharides, suggesting the cellulose-assisted method may be a viable option for extraction polysaccharides from P. odoratum.

  8. Designing novel cellulase systems through agent-based modeling and global sensitivity analysis

    Science.gov (United States)

    Apte, Advait A; Senger, Ryan S; Fong, Stephen S

    2014-01-01

    Experimental techniques allow engineering of biological systems to modify functionality; however, there still remains a need to develop tools to prioritize targets for modification. In this study, agent-based modeling (ABM) was used to build stochastic models of complexed and non-complexed cellulose hydrolysis, including enzymatic mechanisms for endoglucanase, exoglucanase, and β-glucosidase activity. Modeling results were consistent with experimental observations of higher efficiency in complexed systems than non-complexed systems and established relationships between specific cellulolytic mechanisms and overall efficiency. Global sensitivity analysis (GSA) of model results identified key parameters for improving overall cellulose hydrolysis efficiency including: (1) the cellulase half-life, (2) the exoglucanase activity, and (3) the cellulase composition. Overall, the following parameters were found to significantly influence cellulose consumption in a consolidated bioprocess (CBP): (1) the glucose uptake rate of the culture, (2) the bacterial cell concentration, and (3) the nature of the cellulase enzyme system (complexed or non-complexed). Broadly, these results demonstrate the utility of combining modeling and sensitivity analysis to identify key parameters and/or targets for experimental improvement. PMID:24830736

  9. Production of cellulase enzymes during the solid-state fermentation of empty palm fruit bunch fiber.

    Science.gov (United States)

    Kim, Seonghun; Kim, Chul Ho

    2012-01-01

    Penicillium verruculosum COKE4E is a fungal strain isolated from bituminous coal. The microorganism cultivated in a minimal medium supplemented with Avicel, carboxymethylcellulose, and oat spelt xylan produced cellulase enzymes as exhibiting carboxymethylcellulase (CMCase), Avicelase, xylanase, and cellobiosidase activities. In this study, the productivity of the extracellular enzymes in the strain was evaluated by using empty palm fruit bunch fiber (EPFBF), a lignocellulosic biomass, as a substrate for solid-state bioconversion. The highest cellulase activities were observed after 6 days of fermentation at pH 6.0 and 30 °C. The enzymes were secreted as cellulosomes for the degradation of EPFBF as a sole carbon source. Focused ion beam analysis showed that P. verruculosum COKE4E produced cellulolytic enzymes that were able to effectively biodegrade EPFBF during solid-state fermentation. In this process, 6.5 U of CMCase, 6.8 U of Avicelase, and 8.8 U of xylanase per gram of dry solid EPFBF were produced. These results demonstrate that EPFBF may be a potential raw material in solid-state fermentation for the production of cellulase enzymes to be used for biofuel production.

  10. Novel Cellulase Screening and Optimal Production from the Wood Decaying Xylariaceae: Daldinia Species.

    Science.gov (United States)

    Ng, I-Son; Chen, Po Ting; Ju, Yu-Ming; Tsai, Shau-Wei

    2010-10-21

    The highest cellulases production from Daldinia caldariorum 263 (D-263) was found among Daldinia eschscholzii and Daldinia childiae. Three cellulases, one xylanase and one β-glucosidase of the molecular weights 55, 43, 34, 30, and 105 kDa, respectively, were determined by zymographic sodium dodecyl sulfate polyacrylamide gel electrophoresis. From the N-terminal sequencing, the major cellulase CelA belonging to glycosyl hydrolase family 5 was determined. By following an orthogonal experiment design (L9), factors affecting the cultivation of D. caldariorum 263 are ranked as medium composition > temperature > pH ≥ FP (%). The optimum cultivation conditions for obtaining the best FPase (600 mU/ml) at 72 h are 150 rpm, 35 °C, pH 7, 0.2% soy peptone and 0.5% α-cellulose in minimal requirement medium. In comparison with Trichoderma reesei (ATCC26921) secreting 1,135 mU/ml of FPase after 6 days cultivation at pH 5, D. caldariorum 263 grew faster at 35 °C and produced the maximum FPase within 3 days at pH 7.

  11. fA cellular automaton model of crystalline cellulose hydrolysis by cellulases

    Directory of Open Access Journals (Sweden)

    Little Bryce A

    2011-10-01

    Full Text Available Abstract Background Cellulose from plant biomass is an abundant, renewable material which could be a major feedstock for low emissions transport fuels such as cellulosic ethanol. Cellulase enzymes that break down cellulose into fermentable sugars are composed of different types - cellobiohydrolases I and II, endoglucanase and β-glucosidase - with separate functions. They form a complex interacting network between themselves, soluble hydrolysis product molecules, solution and solid phase substrates and inhibitors. There have been many models proposed for enzymatic saccharification however none have yet employed a cellular automaton approach, which allows important phenomena, such as enzyme crowding on the surface of solid substrates, denaturation and substrate inhibition, to be considered in the model. Results The Cellulase 4D model was developed de novo taking into account the size and composition of the substrate and surface-acting enzymes were ascribed behaviors based on their movements, catalytic activities and rates, affinity for, and potential for crowding of, the cellulose surface, substrates and inhibitors, and denaturation rates. A basic case modeled on literature-derived parameters obtained from Trichoderma reesei cellulases resulted in cellulose hydrolysis curves that closely matched curves obtained from published experimental data. Scenarios were tested in the model, which included variation of enzyme loadings, adsorption strengths of surface acting enzymes and reaction periods, and the effect on saccharide production over time was assessed. The model simulations indicated an optimal enzyme loading of between 0.5 and 2 of the base case concentrations where a balance was obtained between enzyme crowding on the cellulose crystal, and that the affinities of enzymes for the cellulose surface had a large effect on cellulose hydrolysis. In addition, improvements to the cellobiohydrolase I activity period substantially improved overall

  12. Characterization of a GHF45 cellulase, AkEG21, from the common sea hare Aplysia kurodai

    Science.gov (United States)

    Rahman, Mohammad; Inoue, Akira; Ojima, Takao

    2014-08-01

    The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95 kDa, 66 kDa, 45 kDa and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., PLoS ONE, 8, e65418, 2013). The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40oC, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55oC for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic ?-1,4-glucanase (EC 3.2.1.4). A cDNA of 1,013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.

  13. Kinetic and thermodynamic properties of alginate lyase and cellulase co-produced by Exiguobacterium species Alg-S5.

    Science.gov (United States)

    Mohapatra, Bidyut R

    2017-05-01

    In an effort to screen out the alginolytic and cellulolytic bacteria from the putrefying invasive seaweed Sargassum species accumulated off Barbados' coast, a potent bacterial strain was isolated. This bacterium, which simultaneously produced alginate lyase and cellulase, was identified as Exiguobacterium sp. Alg-S5 via the phylogenetic approach targeting the 16S rRNA gene. The co-produced alginate lyase and cellulase exhibited maximal enzymatic activity at pH 7.5 and at 40°C and 45°C, respectively. The Km and Vmax values recorded as 0.91mg/mL and 21.8U/mg-protein, respectively, for alginate lyase, and 10.9mg/mL and 74.6U/mg-protein, respectively, for cellulase. First order kinetic analysis of the thermal denaturation of the co-produced alginate lyase and cellulase in the temperature range from 40°C to 55°C revealed that both the enzymes were thermodynamically efficient by displaying higher activation energy and enthalpy of denaturation. These enzymatic properties indicate the potential industrial importance of this bacterium in algal biomass conversion. This appears to be the first report on assessing the efficacy of a bacterium for the co-production of alginate lyase and cellulase.

  14. Disruption of Trichoderma reesei cre2, encoding an ubiquitin C-terminal hydrolase, results in increased cellulase activity

    Directory of Open Access Journals (Sweden)

    Denton Jai A

    2011-11-01

    Full Text Available Abstract Background The filamentous fungus Trichoderma reesei (Hypocrea jecorina is an important source of cellulases for use in the textile and alternative fuel industries. To fully understand the regulation of cellulase production in T. reesei, the role of a gene known to be involved in carbon regulation in Aspergillus nidulans, but unstudied in T. reesei, was investigated. Results The T. reesei orthologue of the A. nidulans creB gene, designated cre2, was identified and shown to be functional through heterologous complementation of a creB mutation in A. nidulans. A T. reesei strain was constructed using gene disruption techniques that contained a disrupted cre2 gene. This strain, JKTR2-6, exhibited phenotypes similar to the A. nidulans creB mutant strain both in carbon catabolite repressing, and in carbon catabolite derepressing conditions. Importantly, the disruption also led to elevated cellulase levels. Conclusions These results demonstrate that cre2 is involved in cellulase expression. Since the disruption of cre2 increases the amount of cellulase activity, without severe morphological affects, targeting creB orthologues for disruption in other industrially useful filamentous fungi, such as Aspergillus oryzae, Trichoderma harzianum or Aspergillus niger may also lead to elevated hydrolytic enzyme activity in these species.

  15. Endoglucanase and total cellulase from newly isolated Rhizopus oryzae and Trichoderma reesei: production, characterization, and thermal stability.

    Science.gov (United States)

    Kupski, Larine; Pagnussatt, Fernanda Arnhold; Buffon, Jaqueline Garda; Furlong, Eliana Badiale

    2014-01-01

    A multienzymatic complex production was evaluated, as well as endoglucanase and total cellulase characterization, during solid-state fermentation of rice industry wastes with Rhizopus oryzae CCT 7560 (newly isolated microorganism) and Trichoderma reesei QM 9414 (control). R. oryzae produced enzymes with higher activity at 15 h of fermentation (5.1 and 2.3 U g(-1) to endoglucanase and total cellulase), while T. reesei produced them at 55 h (15.3 and 2.8 U g(-1) to endoglucanase and total cellulase). The optimum temperature for total cellulase and endoglucanase was 60 °C. For Trichoderma and Rhizopus, the optimum pH was 5.0 and 6.0 for total cellulase and 6.0 and 5.0 for endoglucanase, respectively. The enzymes produced by Rhizopus presented higher stability at the temperature range evaluated (25-100 °C); the endoglucanase KM value was 20 times lower than the one found for Trichoderma. The characterization of the cellulolytic enzymes from the fungal species native of rice husk revealed that they can be more efficient than the genetically modified enzymes when rice husk and rice bran are used as substrates.

  16. The effects of adding lactic acid bacteria and cellulase in oil palm (Elais guineensis Jacq.) frond silages on fermentation quality, chemical composition and in vitro digestibility

    NARCIS (Netherlands)

    Ebrahimi, Mahdi; Rajion, Mohamed Ali; Goh, Yong Meng; Farjam, Abdoreza Soleimani; Sazili, Awis Qurni; Schonewille, Jan Thomas

    2014-01-01

    The main objective of the current study was to evaluate whether oil palm frond (OPF) can be successfully ensiled without or with the additives cellulase or lactic acid bacteria (LAB). Thus, fresh OPF was ensiled either without additives or with cellulase or LAB or their combination. Ensiling was car

  17. pH-Dependent Stability of EGX, A Multi-functional Cellulase from Mollusca, Ampullaria crossean

    Institute of Scientific and Technical Information of China (English)

    Wen-Ying LI; Ji WANG; Yan-Hong LI; Ming DING; Gen-Jun XU; Lan-Ying LIU; Fu-Kun ZHAO

    2004-01-01

    The cellulase activity and stability of EGX, a multi-functional cellulase previously purified from the mollusca Ampullaria crossean, was systematically studied under different pH. The pH induced conformation and stability change of EGX have been investigated by using the intrinsic fluorescence, ANS fluorescence and CD spectrum. It has been found that the conformation and activity of this cellulase were strongly dependent on the pH. EGX was stable for both the enzyme activity and the conformation from pH 5.6 to pH 7.4. As shown by intrinsic and ANS fluorescence, no red shift of emission maximum occurred and a negligible intensity change was observed at pH 5.6-7.4. The activity of EGX remained about 80% in pH 5.6-7.4 and obviously decreased out of side the pH range. Urea-induced changes in EGX at pH 5.4 and pH 8.0 were measured by intrinsic fluorescence and CD spectrum. At pH 5.4, a significantly red shift of emission maximum occurred when the concentration of urea was 5 M compared to the concentration was 3 M at pH 8.0. The α-helix at pH 5.4 was 40.51% in the absence of urea and 31.04% in the presence of 4 M urea. At pH 8.0 the α-helix was 7.23% in the presence of 4 M urea. The data indicated that EGX was much susceptible to urea-induced unfolding at pH 8.0 and much stable at pH 5.4. The greater pH dependent stability of EGX may allow the enzyme to adequately catalyze the hydrolysis of cellulosic materials under natural or industrial extreme conditions.

  18. A high throughput screen for biomining cellulase activity from metagenomic libraries.

    Science.gov (United States)

    Mewis, Keith; Taupp, Marcus; Hallam, Steven J

    2011-02-01

    Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

  19. Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate.

    Directory of Open Access Journals (Sweden)

    Dimitra Zarafeta

    Full Text Available With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.

  20. Effect of the ionic liquid [bmim]Cl and high pressure on the activity of cellulase

    OpenAIRE

    Salvador, Ângelo C.; Santos, Mickael Da C.; Saraiva, Jorge A.

    2010-01-01

    The effect of the ionic liquid 1-butyl-3-methylimidazolium chloride ([bmim]Cl) and of high pressure on the activity of cellulase from Aspergillus niger were studied separately and in combination. The enzyme activity decreased with increasing concentrations of [bmim]Cl, reaching 50% the value in aqueous buffer with 20% [bmim]Cl. However, when the enzyme is held in 10% [bmim]Cl and is then assayed in 1% [bmim]Cl, it showed only 8% reduction of activity. These results can be explained by the ...

  1. Fungal cellulase/xylanase production and corresponding hydrolysis using pretreated corn stover as substrates.

    Science.gov (United States)

    Zhang, Liang; Wang, Xiaoqing; Ruan, Zhenhua; Liu, Ying; Niu, Xiaorui; Yue, Zhengbo; Li, Zhimin; Liao, Wei; Liu, Yan

    2014-01-01

    Three pretreated corn stover (ammonia fiber expansion, dilute acid, and dilute alkali) were used as carbon source to culture Trichoderma reesei Rut C-30 for cellulase and xylanase production. The results indicated that the cultures on ammonia fiber expansion and alkali pretreated corn stover had better enzyme production than the acid pretreated ones. The consequent enzymatic hydrolysis was performed applying fungal enzymes on pretreated corn stover samples. Tukey's statistical comparisons exhibited that there were significant differences on enzymatic hydrolysis among different combination of fungal enzymes and pretreated corn stover. The higher sugar yields were achieved by the enzymatic hydrolysis of dilute alkali pretreated corn stover.

  2. Development of Specific Substrates for Hypocrea jecorina Cellulases

    DEFF Research Database (Denmark)

    Rasmussen, Tina Secher

    task since cellulose exists in different morphologies that can only be degraded efficiently by a battery of enzymes working together.3 One of the most efficient cellulosic systems is secreted by the fungus Hypocrea jecorina, which also has been employed in the industrial production of ethanol. However......  During the last decades a considerable amount of interest has focused on transformation of cellulosic biomass to renewable energy sources such as ethanol.1,2 Cellulases, secreted by different microorganisms, are key enzymes in this process. However, the degradation of cellulose is a difficult...

  3. In Situ Stability of Substrate-Associated Cellulases Studied by DSC

    DEFF Research Database (Denmark)

    Borch, Kim; Cruys-Bagger, Nicolaj; Badino, Silke Flindt;

    2014-01-01

    This work shows that differential scanning calorimetry (DSC) can be used to monitor the stability of substrate-adsorbed cellulases during long-term hydrolysis of insoluble cellulose. Thermal transitions of adsorbed enzyme were measured regularly in subsets of a progressing hydrolysis, and the size....... There was no correlation between the intrinsic stability, specified by the transition temperature in the DSC, and the long-term stability derived from the peak area. The results are discussed with respect to the role of enzyme denaturation for the ubiquitous slowdown observed in the enzymatic hydrolysis of cellulose....

  4. Screening Of the Association of Aspergillusfungi – Macerase and Cellulase Enzymes Producers

    Directory of Open Access Journals (Sweden)

    R.K.Bliyeva,

    2015-08-01

    Full Text Available In nature, the destruction of plant wastes takes place by not a single microorganism, butby complex of microorganisms which belong to different species and genera with complex of enzymes. Using of highly active monocultures to createfungal association which produce multifunctional enzymeswith desired properties is a promising approach to create relevant and effective crop production microorganisms with beneficial properties. Among15 industrial micro my cetes the most active association was from Aspergillusniger 355 and Aspergillusawamori 1-8 which had highmacerase and cellulase enzymes activity.

  5. Fabrication of graphene oxide decorated with Fe3O4@SiO2 for immobilization of cellulase

    Science.gov (United States)

    Li, Yue; Wang, Xiang-Yu; Jiang, Xiao-Ping; Ye, Jing-Jing; Zhang, Ye-Wang; Zhang, Xiao-Yun

    2015-01-01

    Fe3O4@SiO2-graphene oxide (GO) composites were successfully fabricated by chemical binding of functional Fe3O4@SiO2 and GO and applied to immobilization of cellulase via covalent attachment. The prepared composites were further characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. Fe3O4 nanoparticles (NPs) were monodisperse spheres with a mean diameter of 17 ± 0.2 nm. The thickness of SiO2 layer was calculated as being 6.5 ± 0.2 nm. The size of Fe3O4@SiO2 NPs was 24 ± 0.3 nm, similar to that of Fe3O4@SiO2-NH2. Fe3O4@SiO2-GO composites were synthesized by linking of Fe3O4@SiO2-NH2 NPs to GO with the catalysis of EDC and NHS. The prepared composites were used for immobilization of cellulase. A high immobilization yield and efficiency of above 90 % were obtained after the optimization. The half-life of immobilized cellulase (722 min) was 3.34-fold higher than that of free enzyme (216 min) at 50 °C. Compared with the free cellulase, the optimal temperature of the immobilized enzyme was not changed; but the optimal pH was shifted from 5.0 to 4.0, and the thermal stability was enhanced. The immobilized cellulase could be easily separated and reused under magnetic field. These results strongly indicate that the cellulase immobilized onto the Fe3O4@SiO2-GO composite has potential applications in the production of bioethanol.

  6. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

    Directory of Open Access Journals (Sweden)

    Gong Xia

    2012-10-01

    Full Text Available Abstract Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

  7. 根瘤菌中纤维素酶的研究进展%Research Progress of Cellulase from Rhizobia

    Institute of Scientific and Technical Information of China (English)

    蒋春玲; 赵洪锟; 王乾钦; 田智蕊; 李东哲; 杨美英

    2011-01-01

    基于国内外关于纤维素酶的研究成果,概述了根瘤菌中已报道的纤维素酶基因、蛋白性质、在共生中的作用及研究现状,并对进一步的研究进行展望.%Based on the research progress of cellulase at home and abroad, the reported cellulose gene, protein properties, function during symbiosis and development status were discussed. And the oncoming study of rhizobia cellulase was prospected.

  8. Controlled production of cellulases in plants for biomass conversion. Progress report, June 15, 1996--March 10, 1997

    Energy Technology Data Exchange (ETDEWEB)

    Danna, K.J.

    1997-06-01

    The goal of this project is to facilitate conversion of plant biomass to usable energy by developing transgenic plants that express genes for microbial cellulases, which can be activated after harvest of the plants. In particular, we want to determine the feasibility of targeting an endoglucanase and a cellobiohydrolase to the plant apoplast (cell wall milieu). The apoplast not only contains cellulose, the substrate for the enzymes, but also can tolerate large amounts of foreign protein. To avoid detrimental effects of cellulase expression in plants, we have chosen enzymes with high temperature optima; the genes for these enzymes are from thermophilic organisms that can use cellulose as a sole energy source.

  9. Multifunctional Cellulolytic Enzymes Outperform Processive Fungal Cellulases for Coproduction of Nanocellulose and Biofuels

    Energy Technology Data Exchange (ETDEWEB)

    Yarbrough, John M.; Zhang, Ruoran; Mittal, Ashutosh; Vander Wall, Todd; Bomble, Yannick J.; Decker, Stephen R.; Himmel, Michael E.; Ciesielski, Peter N.

    2017-03-07

    Producing fuels, chemicals, and materials from renewable resources to meet societal demands remains an important step in the transition to a sustainable, clean energy economy. The use of cellulolytic enzymes for the production of nanocellulose enables the coproduction of sugars for biofuels production in a format that is largely compatible with the process design employed by modern lignocellulosic (second generation) biorefineries. However, yields of enzymatically produced nanocellulose are typically much lower than those achieved by mineral acid production methods. In this study, we compare the capacity for coproduction of nanocellulose and fermentable sugars using two vastly different cellulase systems: the classical 'free enzyme' system of the saprophytic fungus, Trichoderma reesei (T. reesei) and the complexed, multifunctional enzymes produced by the hot springs resident, Caldicellulosiruptor bescii (C. bescii). We demonstrate by comparative digestions that the C. bescii system outperforms the fungal enzyme system in terms of total cellulose conversion, sugar production, and nanocellulose production. In addition, we show by multimodal imaging and dynamic light scattering that the nanocellulose produced by the C. bescii cellulase system is substantially more uniform than that produced by the T. reesei system. These disparities in the yields and characteristics of the nanocellulose produced by these disparate systems can be attributed to the dramatic differences in the mechanisms of action of the dominant enzymes in each system.

  10. Bioconversion of Agricultural Waste to Ethanol by SSF Using Recombinant Cellulase from Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Ruchi Mutreja

    2011-01-01

    Full Text Available The effect of different pretreatment methods, temperature, and enzyme concentration on ethanol production from 8 lignocellulosic agrowaste by simultaneous saccharification and fermentation (SSF using recombinant cellulase and Saccharomyces cerevisiae were studied. Recombinant cellulase was isolated from E. coli BL21 cells transformed with CtLic26A-Cel5-CBM11 full-length gene from Clostridium thermocellum and produced in both batch and fed-batch processes. The maximum cell OD and specific activity in batch mode were 1.6 and 1.91 U/mg, respectively, whereas in the fed-batch mode, maximum cell OD and specific activity were 3.8 and 3.5 U/mg, respectively, displaying a 2-fold increase. Eight substrates, Syzygium cumini (jamun, Azadirachta indica (neem, Saracens indica (asoka, bambusa dendrocalmus (bamboo, Populas nigra (poplar, Achnatherum hymenoides (wild grass, Eucalyptus marginata (eucalyptus, and Mangifera indica (mango, were subjected to SSF. Of three pretreatments, acid, alkali, and steam explosion, acid pretreatment Syzygium cumini (Jamun at 30°C gave maximum ethanol yield of 1.42 g/L.

  11. Influence of nutrient substrates on the expression of cellulases in Cerambyx cerdo L. (Coleoptera: Cerambycidae larvae

    Directory of Open Access Journals (Sweden)

    Pavlović R.

    2012-01-01

    Full Text Available The expression and distribution of digestive cellulases along the midgut of Cerambyx cerdo larvae were analyzed for the first time and are presented in this article. Four groups of larvae were examined: larvae developed in the wild; larvae taken from the wild and successively reared on an artificial diet based on polenta; and larvae hatched in the laboratory and reared on two different artificial diets. Seven endocellulase and seven β-D-glucosidase isoforms were detected in all midgut extracts of C. cerdo with a zymogram after native PAGE. We observed that C. cerdo larvae are capable of producing cellulase isoforms with different PAGE mobilities depending on the nutrient substrate. From our findings it can be assumed that, depending on the distribution of endocellulase and β-D-glucosidase, cellulose molecules are first fragmented in the anterior and middle midgut by endo-β-1,4-glucanase; subsequently, the obtained fragments are broken down by β-D-glucosidase mostly in middle midgut.

  12. Understanding the Role of Physical Properties of Cellulose on Its Hydrolyzability by Cellulases

    Science.gov (United States)

    O'Dell, Patrick Jonathan

    Cellulose has long been explored as a potential feedstock for biofuel, however the recalcitrance of cellulose makes its conversion into biofuel much more challenging and economically unfavorable compared to well-established processes for converting starch or sugar feedstocks into biofuel. Enzymes capable of hydrolyzing cellulose into soluble sugars, glucose and cellobiose, have been found to work processively along cellulose microfibrils starting from reducing end groups. For this study, cellulose was produced and purified in-house from Gluconacetobacter xylinum cultures, and characterized by quantifying functional groups (aldehyde, ketone, and carboxyl groups) to determine the extent of oxidation of cellulose due to the processing steps. The main goal of this study was to look at the impacts of ultrasonication on cellulose's structure and the enzymatic hydrolyzability of cellulose. A completely randomized experimental design was used to test the effect of ultrasonication time and amplitude (intensity) on changes in cellulose fibril length, degree of polymerization, and rates and extents of hydrolysis. Results indicated that sonication time does significantly impact both the fibril length and average degree of polymerization of cellulose. The impact of ultrasonication on the hydrolyzability of cellulose by commercial cellulase and beta-glucosidase preparations could not be effectively resolved due to high variability in the experimental results. These studies serve as a basis for future studies understanding the role of cellulose microstructure in the mechanism of cellulase hydrolysis of cellulose.

  13. Hydrolysis of Ammonia-pretreated Sugar Cane Bagasse with Cellulase, β-Glucosidase, and Hemicellulase Preparations

    Science.gov (United States)

    Prior, Bernard A.; Day, Donal F.

    Sugar cane bagasse consists of hemicellulose (24%) and cellulose (38%), and bioconversion of both fractions to ethanol should be considered for a viable process. We have evaluated the hydrolysis of pretreated bagasse with combinations of cellulase, β-glucosidase, and hemicellulase. Ground bagasse was pretreated either by the AFEX process (2NH3: 1 biomass, 100 °C, 30 min) or with NH4OH (0.5 g NH4OH of a 28% [v/v] per gram dry biomass; 160 °C, 60 min), and composition analysis showed that the glucan and xylan fractions remained largely intact. The enzyme activities of four commercial xylanase preparations and supernatants of four laboratory-grown fungi were determined and evaluated for their ability to boost xylan hydrolysis when added to cellulase and β-glucosidase (10 filter paper units [FPU]: 20 cellobiase units [CBU]/g glucan). At 1% glucan loading, the commercial enzyme preparations (added at 10% or 50% levels of total protein in the enzyme preparations) boosted xylan and glucan hydrolysis in both pretreated bagasse samples. Xylanase addition at 10% protein level also improved hydrolysis of xylan and glucan fractions up to 10% glucan loading (28% solids loading). Significant xylanase activity in enzyme cocktails appears to be required for improving hydrolysis of both glucan and xylan fractions of ammonia pretreated sugar cane bagasse.

  14. The Effect of Cellulose Crystal Structure and Solid-State Morphology on the Activity of Cellulases

    Energy Technology Data Exchange (ETDEWEB)

    Stipanovic, Arthur J [SUNY College of Environmental Science and Forestry

    2014-11-17

    Consistent with the US-DOE and USDA “Roadmap” objective of producing ethanol and chemicals from cellulosic feedstocks more efficiently, a three year research project entitled “The Effect of Cellulose Crystal Structure and Solid-State Morphology on the Activity of Cellulases” was initiated in early 2003 under DOE sponsorship (Project Number DE-FG02-02ER15356). A three year continuation was awarded in June 2005 for the period September 15, 2005 through September 14, 2008. The original goal of this project was to determine the effect of cellulose crystal structure, including allomorphic crystalline form (Cellulose I, II, III, IV and sub-allomorphs), relative degree of crystallinity and crystallite size, on the activity of different types of genetically engineered cellulase enzymes to provide insight into the mechanism and kinetics of cellulose digestion by “pure” enzymes rather than complex mixtures. We expected that such information would ultimately help enhance the accessibility of cellulose to enzymatic conversion processes thereby creating a more cost-effective commercial process yielding sugars for fermentation into ethanol and other chemical products. Perhaps the most significant finding of the initial project phase was that conversion of native bacterial cellulose (Cellulose I; BC-I) to the Cellulose II (BC-II) crystal form by aqueous NaOH “pretreatment” provided an increase in cellulase conversion rate approaching 2-4 fold depending on enzyme concentration and temperature, even when initial % crystallinity values were similar for both allomorphs.

  15. A minimal set of bacterial cellulases for consolidated bioprocessing of lignocellulose.

    Science.gov (United States)

    Liao, Hehuan; Zhang, Xiao-Zhou; Rollin, Joseph A; Zhang, Yi-Heng Percival

    2011-11-01

    Cost-effective release of fermentable sugars from non-food biomass through biomass pretreatment/enzymatic hydrolysis is still the largest obstacle to second-generation biorefineries. Therefore, the hydrolysis performance of 21 bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase (BsCel5), family 9 Clostridium phytofermentans processive endoglucanase (CpCel9), and family 48 C. phytofermentans cellobiohydrolase (CpCel48) was studied on partially ordered low-accessibility microcrystalline cellulose (Avicel) and disordered high-accessibility regenerated amorphous cellulose (RAC). Faster hydrolysis rates and higher digestibilities were obtained on RAC than on Avicel. The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was the most important for high cellulose digestibility regardless of substrate type. This study provides important information for the construction of a minimal set of bacterial cellulases for the consolidated bioprocessing bacteria, such as Bacillus subtilis, for converting lignocellulose to biocommodities in a single step.

  16. Endo-xylanase and endo-cellulase-assisted extraction of pectin from apple pomace.

    Science.gov (United States)

    Wikiera, Agnieszka; Mika, Magdalena; Starzyńska-Janiszewska, Anna; Stodolak, Bożena

    2016-05-20

    Pectins were extracted from apple pomace with monoactive preparation of endo-xylanase and endo-cellulase. The process was conducted for 10 h in conditions of pH 5.0 at 40 °C, with constant shaking. Endo-xylanase application resulted in the highest extraction efficiency of pectins (19.8%). The obtained polymer was characterised by a very high molecular mass, high level of neutral sugars - mainly arabinose, galactose and glucose, and very high DM (73.4). It also contained the highest level of protein and phenols. Pectin extracted with endo-cellulase had 1.5 fold lower molecular mass but contained significantly more GalA (70.5%) of a high degree of methylation (66.3%). The simultaneous application of both enzymatic preparations resulted in their cooperation, leading to a decrease of both the extraction efficiency and the molecular mass of pectin. However, this pectin was distinguished by the highest GalA (74.7%) and rhamnose contents.

  17. High Genetic Diversity of Microbial Cellulase and Hemicellulase Genes in the Hindgut of Holotrichia parallela Larvae

    Directory of Open Access Journals (Sweden)

    Ping Sheng

    2015-07-01

    Full Text Available In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95% of glycosyl hydrolase families including GH2 (24 genes, GH8 (27 genes, GH10 (19 genes, GH11 (14 genes and GH36 (17 genes families was retrieved, and certain sequences of GH2 (10.61%, GH8 (3.33%, and GH11 (18.42% families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect.

  18. The influence of sorbitol on the production of cellulases and xylanases in an airlift bioreactor.

    Science.gov (United States)

    Ritter, Carla Eliana Todero; Fontana, Roselei Claudete; Camassola, Marli; da Silveira, Maurício Moura; Dillon, Aldo José Pinheiro

    2013-11-01

    The production of cellulases and xylanases by Penicillium echinulatum in an airlift bioreactor was evaluated. In batch production, we tested media with isolated or associated cellulose and sorbitol. In fed-batch production, we tested cellulose addition at two different times, 30 h and 48 h. Higher liquid circulation velocities in the downcomer were observed in sorbitol 10 g L(-1) medium. In batch production, higher FPA (filter paper activity) and endoglucanase activities were obtained with cellulose (7.5 g L(-1)) and sorbitol (2.5 g L(-1)), 1.0 U mL(-1) (120 h) and 6.4 U m L(-1) (100 h), respectively. For xylanases, the best production condition was cellulose 10 g L(-1), which achieved 5.5 U mL(-1) in 64 h. The fed-batch process was favorable for obtaining xylanases, but not for FPA and endoglucanases, suggesting that in the case of cellulases, the inducer must be added early in the process.

  19. Cellulase and xylanase activity during the decomposition of three aquatic macrophytes in a tropical oxbow lagoon

    Directory of Open Access Journals (Sweden)

    L Sciessere

    2011-09-01

    Full Text Available Due to the connection between enzymatic activity and degradation of different fractions of organic matter, enzyme assays can be used to estimate degradation rates of particulate and dissolved organic carbon in freshwater systems. The aim of this study was to quantify and model the enzymatic degradation involving the decomposition of macrophytes, describing temporal activity of cellulases (EC 3.2.1.4 and EC 3.2.1.91 and xylanase (EC 3.2.1.8 during in situ decomposition of three aquatic macrophytes (Salvinia sp., Eichhornia azurea and Cyperus giganteus on the surface and water-sediment interface (w-s interface of an oxbow lagoon (Óleo lagoon within a natural Brazilian Savanna Reserve. Overall, the enzymatic degradation of aquatic macrophytes in Óleo lagoon occurred during the whole year and was initiated together with leaching. Xylanase production was ca. 5 times higher than cellulase values due to easy access to this compound by cellulolytic microorganisms. Enzymatic production and detritus mass decay were similar on the surface and w-s interface. Salvinia sp. was the most recalcitrant detritus, with low mass decay and enzymatic activity. E. azurea and C. giganteus decomposition rates and enzymatic production were high and similar. Due to the physicochemical homogeneity observed in the Óleo lagoon, the differences between the decay rates of each species are mostly related with detritus chemical quality.

  20. Cellulase activity and dissolved organic carbon release from lignocellulose macrophyte-derived in four trophic conditions

    Directory of Open Access Journals (Sweden)

    Flávia Bottino

    2016-06-01

    Full Text Available Abstract Considering the importance of lignocellulose macrophyte-derived for the energy flux in aquatic ecosystems and the nutrient concentrations as a function of force which influences the decomposition process, this study aims to relate the enzymatic activity and lignocellulose hydrolysis in different trophic statuses. Water samples and two macrophyte species were collected from the littoral zone of a subtropical Brazilian Reservoir. A lignocellulosic matrix was obtained using aqueous extraction of dried plant material (≈40 °C. Incubations for decomposition of the lignocellulosic matrix were prepared using lignocelluloses, inoculums and filtered water simulating different trophic statuses with the same N:P ratio. The particulate organic carbon and dissolved organic carbon (POC and DOC, respectively were quantified, the cellulase enzymatic activity was measured by releasing reducing sugars and immobilized carbon was analyzed by filtration. During the cellulose degradation indicated by the cellulase activity, the dissolved organic carbon daily rate and enzyme activity increased. It was related to a fast hydrolysable fraction of cellulose that contributed to short-term carbon immobilization (ca. 10 days. After approximately 20 days, the dissolved organic carbon and enzyme activity were inversely correlated suggesting that the respiration of microorganisms was responsible for carbon mineralization. Cellulose was an important resource in low nutrient conditions (oligotrophic. However, the detritus quality played a major role in the lignocelluloses degradation (i.e., enzyme activity and carbon release.

  1. Cellulase activity and dissolved organic carbon release from lignocellulose macrophyte-derived in four trophic conditions.

    Science.gov (United States)

    Bottino, Flávia; Cunha-Santino, Marcela Bianchessi; Bianchini, Irineu

    2016-01-01

    Considering the importance of lignocellulose macrophyte-derived for the energy flux in aquatic ecosystems and the nutrient concentrations as a function of force which influences the decomposition process, this study aims to relate the enzymatic activity and lignocellulose hydrolysis in different trophic statuses. Water samples and two macrophyte species were collected from the littoral zone of a subtropical Brazilian Reservoir. A lignocellulosic matrix was obtained using aqueous extraction of dried plant material (≈40°C). Incubations for decomposition of the lignocellulosic matrix were prepared using lignocelluloses, inoculums and filtered water simulating different trophic statuses with the same N:P ratio. The particulate organic carbon and dissolved organic carbon (POC and DOC, respectively) were quantified, the cellulase enzymatic activity was measured by releasing reducing sugars and immobilized carbon was analyzed by filtration. During the cellulose degradation indicated by the cellulase activity, the dissolved organic carbon daily rate and enzyme activity increased. It was related to a fast hydrolysable fraction of cellulose that contributed to short-term carbon immobilization (ca. 10 days). After approximately 20 days, the dissolved organic carbon and enzyme activity were inversely correlated suggesting that the respiration of microorganisms was responsible for carbon mineralization. Cellulose was an important resource in low nutrient conditions (oligotrophic). However, the detritus quality played a major role in the lignocelluloses degradation (i.e., enzyme activity) and carbon release.

  2. Entrapment of cross-linked cellulase colloids in alginate beads for hydrolysis of cellulose.

    Science.gov (United States)

    Nguyen, Le Truc; Lau, Yun Song; Yang, Kun-Lin

    2016-09-01

    Entrapment of enzymes in calcium alginate beads is a popular enzyme immobilization method. However, leaching of immobilized enzymes from the alginate beads is a common problem because enzyme molecules are much smaller than the pore size of alginate beads (∼200nm). To address this issue, we employ a millifluidic reactor to prepare cross-linked cellulase aggregate (XCA) colloids with a uniform size (∼300nm). Subsequently, these colloids are immobilized in calcium alginate beads as biocatalysts to hydrolyze cellulose substrates. By using fluorescent microscopy, we conclude that the immobilized XCA colloids distribute uniformly inside the beads and do not leach out from the beads after long-term incubation. Meanwhile, the pore size of the alginate beads is big enough for the cellulose substrates and fibers to diffuse into the beads for hydrolysis. For example, palm oil fiber and microcrystalline cellulose can be hydrolyzed within 48h and release reducing sugar concentrations up to 2.48±0.08g/l and 4.99±0.09g/l, respectively. Moreover, after 10 cycles of hydrolysis, 96.4% of the XCA colloids remain inside the alginate beads and retain 67% of the original activity. In contrast, free cellulase immobilized in the alginate beads loses its activity completely after 10 cycles. The strategy can also be used to prepare other types of cross-linked enzyme aggregates with high uniformity.

  3. Optimization of cellulase production by Enhydrobactor sp. ACCA2 and its application in biomass saccharification

    Directory of Open Access Journals (Sweden)

    Nagaiah ePremalatha

    2015-10-01

    Full Text Available Cellulase finds use in saccharification of lignocellulosic agroresidues to fermentable sugars which can be used for production of commercially important metabolites. This study reports endoglucanase (CMCase production by Enhydrobacter sp. ACCA2. The CMCase activity of the strain ACCA2 was successively improved by optimization of range of physical and nutritional parameter in a set of non-statistical and statistical experiments. Initial non-statistical selection of carbon source, incubation time, temperature and pH resulted in 1.07 fold increase of CMCase activity. In a subsequent statistical method, response surface methodology, optimization of medium components such as carboxymethylcellulose, peptone, NaCl, MgSO4•7H2O, FeSO4•7H2O, K2HPO4 and (NH42SO4 yielded further increase up to 2.39 fold CMCase activity. The cellulolytic potential was evaluated in biomass saccharification with different plant materials and the results revealed that the enzyme produced by strain may have significant commercial values for industrial saccharification process. Moreover, this is the first report of cellulase production by an Enhydrobacter spp.

  4. Cellulase and Xylanase Production by Penicillium echinulatum in Submerged Media Containing Cellulose Amended with Sorbitol

    Directory of Open Access Journals (Sweden)

    Carla Eliana Todero Ritter

    2013-01-01

    Full Text Available The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v soy bran; 0.1% (w/v wheat bran; and a solution of salts. The highest filter paper activity (FPA ( IU·mL−1 was obtained on the seventh day in the medium containing 0.5% (w/v sorbitol and 0.5% (w/v cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day ( IU·mL−1 in the medium containing 0.75% (w/v sorbitol and 0.75% (w/v cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v sorbitol and 0.25% (w/v cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems.

  5. Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

    Energy Technology Data Exchange (ETDEWEB)

    Sukumaran, Rajeev K.; Singhania, Reeta Rani; Mathew, Gincy Marina; Pandey, Ashok [Biotechnology Division, National Institute for Interdisciplinary Science and Technology, CSIR, Trivandrum-695 019 (India)

    2009-02-15

    A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of {beta}-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw. (author)

  6. Cost evaluation of cellulase enzyme for industrial-scale cellulosic ethanol production based on rigorous Aspen Plus modeling.

    Science.gov (United States)

    Liu, Gang; Zhang, Jian; Bao, Jie

    2016-01-01

    Cost reduction on cellulase enzyme usage has been the central effort in the commercialization of fuel ethanol production from lignocellulose biomass. Therefore, establishing an accurate evaluation method on cellulase enzyme cost is crucially important to support the health development of the future biorefinery industry. Currently, the cellulase cost evaluation methods were complicated and various controversial or even conflict results were presented. To give a reliable evaluation on this important topic, a rigorous analysis based on the Aspen Plus flowsheet simulation in the commercial scale ethanol plant was proposed in this study. The minimum ethanol selling price (MESP) was used as the indicator to show the impacts of varying enzyme supply modes, enzyme prices, process parameters, as well as enzyme loading on the enzyme cost. The results reveal that the enzyme cost drives the cellulosic ethanol price below the minimum profit point when the enzyme is purchased from the current industrial enzyme market. An innovative production of cellulase enzyme such as on-site enzyme production should be explored and tested in the industrial scale to yield an economically sound enzyme supply for the future cellulosic ethanol production.

  7. Production and Partial Characterization of Cellulases from Trichoderma sp. IS-05 Isolated from Sandy Coastal Plains of Northeast Brazil

    Directory of Open Access Journals (Sweden)

    Jackeline Pereira Andrade

    2011-01-01

    Full Text Available This study evaluated the production of cellulolytic enzymes by Trichoderma sp. IS-05 strain, isolated from sand dunes, according to its ability to grow on cellulose as carbon source. Wheat bran was tested as the carbon source and peptone tested as the nitrogen source. Different concentrations of carbon and nitrogen were tested using a factorial design to identify optimal cellulase activity production. The results showed that media containing wheat bran 4.0% (w/v and peptone 0.25% (w/v lead to the highest production, 564.0 U L−1 of cellulase, obtained after 2 days of fermentation. The pH and temperature profile showed optimal activity at pH 3.0 and 60∘C. As for thermostability, the cellulase was most tolerant at 60∘C, retaining more than 59.6% of maximal activity even after 4 hours of incubation. The combination of acid pH, high temperature tolerance, and production of cellulase from agro-industrial residues by Trichoderma sp. IS-05 offers possibilities condition for the biomass hydrolysis process to produce bioethanol.

  8. Optimization of alkaline cellulase production by the marine-derived fungus Chaetomium sp. using agricultural and industrial wastes as substrates

    Digital Repository Service at National Institute of Oceanography (India)

    Ravindran, C.; Naveenan, T.; Varatharajan, G.R.

    , among which Chaetomium sp. (NIOCC 36) was found to grow in a wide range of pH (between 4 and 12). This alkaline tolerant fungus was further tested for production of alkaline cellulases (Beta-endoglucanase, Beta-exoglucanase, Beta-glucosidase) using...

  9. Suppression of cellulase and polygalacturonase and induction of alcohol dehydrogenase isoenzymes in avocado fruit mesocarp subjected to low oxygen stress.

    Science.gov (United States)

    Kanellis, A K; Solomos, T; Roubelakis-Angelakis, K A

    1991-05-01

    Expression of polygalacturonase and cellulase, two hydrolytic enzymes of avocado (Persea americana, cv Hass) fruit which are synthesized de novo during ripening, and alcohol dehydrogenase, a known anaerobic protein, were studied under different O(2) regimes. Low O(2) concentrations (2.5-5.5%) diminished the accumulation of polygalacturonase and cellulase proteins and the expression of their isoenzymes. This pattern of change in cellulase protein was also reflected in the steady-state amount of its mRNA. In contrast, 7.5 and 10% O(2) did not alter the changes observed in fruits ripened in air. On the other hand, alcohol dehydrogenase was induced in 2.5, 3.5, and 5.5% O(2) but not in 7.5 or 10% O(2). The recovery from the hypoxic stress upon returning the fruits back to air for 24 hours, was also a function of O(2) tensions under which the fruits were kept. Thus, the synthesis of polygalacturonase and cellulase was directly related to O(2) levels, while the activity of the isoenzymes of alcohol dehydrogenase was inversely related to O(2) levels. The results indicate that hypoxia exerts both negative and positive effects on the expression of certain genes and that these effects are initiated at the same levels of O(2).

  10. Cellulase variants

    Energy Technology Data Exchange (ETDEWEB)

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  11. Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering

    Directory of Open Access Journals (Sweden)

    Zou Gen

    2012-02-01

    Full Text Available Abstract Background Trichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the production of heterologous enzymes is the stability of recombinant mRNA and the prevention of protein degradation within the endoplasmic reticulum, especially for the bacteria originated enzymes. In this study, the CREI binding sites within the cbh1 promoter were replaced with the binding sites of transcription activator ACEII and the HAP2/3/5 complex to improve the promoter efficiency. To further improve heterologous expression efficiency of bacterial genes within T. reesei, a flexible polyglycine linker and a rigid α-helix linker were tested in the construction of fusion genes between cbh1 from T. reesei and e1, encoding an endoglucanase from Acidothermus cellulolyticus. Results The modified promoter resulted in an increased expression level of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium. The fusion genes of cbh1 and e1 were successfully expressed in T. reesei under the control of promoter pcbh1m2. The higher enzyme activities and thermostability of the fusion protein with rigid linker indicated that the rigid linker might be more suitable for the heterologous expression system in T. reesei. Compared to the parent strain RC30-8, the FPase and CMCase activities of the secreted enzyme mixture from the corresponding transformant R1 with the rigid linker increased by 39% and 30% at 60°C, respectively, and the reduced sugar concentration in the hydrolysate of pretreated corn stover

  12. Potential of Biosynthesized Silver Nanoparticles as Nanocatalyst for Enhanced Degradation of Cellulose by Cellulase

    Directory of Open Access Journals (Sweden)

    Bipinchandra K. Salunke

    2015-01-01

    Full Text Available Silver nanoparticles (AgNPs as a result of their excellent optical and electronic properties are promising catalytic materials for various applications. In this study, we demonstrate a novel approach for enhanced degradation of cellulose using biosynthesized AgNPs in an enzyme catalyzed reaction of cellulose hydrolysis by cellulase. AgNPs were synthesized through reduction of silver nitrate by extracts of five medicinal plants (Mentha arvensis var. piperascens, Buddleja officinalis Maximowicz, Epimedium koreanum Nakai, Artemisia messer-schmidtiana Besser, and Magnolia kobus. An increase of around twofold in reducing sugar formation confirmed the catalytic activity of AgNPs as nanocatalyst. The present study suggests that immobilization of the enzyme onto the surface of the AgNPs can be useful strategy for enhanced degradation of cellulose, which can be utilized for diverse industrial applications.

  13. Cellulase-producing bacteria from Thai higher termites, Microcerotermes sp.: enzymatic activities and ionic liquid tolerance.

    Science.gov (United States)

    Taechapoempol, Kitipong; Sreethawong, Thammanoon; Rangsunvigit, Pramoch; Namprohm, Weerachart; Thamprajamchit, Bandhit; Rengpipat, Sirirat; Chavadej, Sumaeth

    2011-05-01

    The three highest hydrolysis-capacity-value isolates of Bacillus subtilis (A 002, M 015, and F 018) obtained from Thai higher termites, Microcerotermes sp., under different isolation conditions (aerobic, anaerobic, and anaerobic/aerobic) were tested for cellulase activities--FPase, endoglucanase, and β-glucosidase--at 37 °C and pH 7.2 for 24 h. Their tolerance to an ionic liquid, 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), was also investigated. The results showed that the isolate M 015 provided the highest endoglucanase activity whereas the highest FPase and β-glucosidase activities were observed for the isolate F 018. The isolate F 018 also showed the highest tolerance to [BMIM]Cl in the range of 0.1-1.0 vol.%. In contrast, the isolate A 002 exhibited growth retardation in the presence of 0.5-1.0 vol.% [BMIM]Cl.

  14. Production of cellulases and hemicellulases by an anaerobic mixed culture from lignocellulosic biomass.

    Science.gov (United States)

    Tabassum, R; Rajoka, M I; Malik, K A

    1990-03-01

    A comparison of different habitats, biogas plant, rumen fluid and sewage sludge, for cellulolytic organisms indicated sewage studge was the best source. Enrichment cultura gave a mixed culture which exhibited CMCase activity as well as extracellular Avicelase, xylanase, β-glucosidase, β-xylosidase activities and cell-bound β-glucosidase, and β-xylosidase production in a synthetic medium with eleven different cellulosic and lignocellulosic substrates. The activity of extracellular β-glucosidase and β-xylosidase production was significantly higher than endogenous activities. Hemicellulases were induced better than cellulases. The anzyme system was stable under aerobic conditions. Of the different lignocellulosic substrates, kallar grass was the best inducer of extracellular enzymes.

  15. Rate of Threading a Cellulose Chain into the Binding Tunnel of a Cellulase

    DEFF Research Database (Denmark)

    Cruys-Bagger, Nicolaj; Alasepp, Kadri; Andersen, Morten;

    2016-01-01

    Industrially important cellulase Cel7A hydrolyzes crystalline cellulose by a complex processive mechanism in which the enzyme slides along the cellulose surface with one strand of the polymeric substrate channeled through its catalytic tunnel. Each processive run must start with threading...... the tunnel with a cellulose strand and end with the opposite, that is, the dethreading process. Evidence has suggested that threading or dethreading may be rate-limiting for the overall enzyme reaction. To directly elucidate the rates of threading and dethreading, we analyzed experimental data with respect...... with the cellulose surface many times before it eventually finds a location at which it gets threaded. Moreover, it was concluded that at the quasi steady state dethreading was the main determinant of the overall hydrolytic rate under most conditions. An exception to this was at very low enzyme/substrate ratios...

  16. Molecular Dynamics and Metadynamics Simulations of the Cellulase Cel48F

    Directory of Open Access Journals (Sweden)

    Osmair Vital de Oliveira

    2014-01-01

    Full Text Available Molecular dynamics (MD and metadynamics techniques were used to study the cellulase Cel48F-sugar. Cellulase is enzyme that breaks cellulose fibers into small sugar units and is potentially useful in second generation alcohol production. In MD simulations, the overall structure of equilibrated Cel48F did not significantly change along the trajectory, retaining root mean square deviation below 0.15 nm. A set of 15 residues interacting with the sugar chains via hydrogen bonding throughout the simulation was observed. The free energy of dissociation (ΔGdiss. of the chains in the catalytic tunnel of Cel48F was determined by metadynamics. The ΔGdiss. values of the chains entering and leaving the wild-type Cel48F cavity were 13.9 and 62.1 kcal/mol, respectively. We also mutated the E542 and Q543 to alanine residue and obtained ΔGdiss. of 41.8 and 45.9 kcal/mol, respectively. These mutations were found to facilitate smooth dissociation of the sugar chain across the Cel48F tunnel. At the entry of the Cel48F tunnel, three residues were mutated to alanine: T110, T213, and L274. Contrary to the T110A-Cel48F, the mutants T213-Cel48F and L274-Cel48F prevented the sugar chain from passing across the leaving site. The present results can be a guideline in mutagenesis studies to improve processing by Cel48F.

  17. Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance.

    Science.gov (United States)

    Park, Sunkyu; Baker, John O; Himmel, Michael E; Parilla, Philip A; Johnson, David K

    2010-05-24

    Although measurements of crystallinity index (CI) have a long history, it has been found that CI varies significantly depending on the choice of measurement method. In this study, four different techniques incorporating X-ray diffraction and solid-state 13C nuclear magnetic resonance (NMR) were compared using eight different cellulose preparations. We found that the simplest method, which is also the most widely used, and which involves measurement of just two heights in the X-ray diffractogram, produced significantly higher crystallinity values than did the other methods. Data in the literature for the cellulose preparation used (Avicel PH-101) support this observation. We believe that the alternative X-ray diffraction (XRD) and NMR methods presented here, which consider the contributions from amorphous and crystalline cellulose to the entire XRD and NMR spectra, provide a more accurate measure of the crystallinity of cellulose. Although celluloses having a high amorphous content are usually more easily digested by enzymes, it is unclear, based on studies published in the literature, whether CI actually provides a clear indication of the digestibility of a cellulose sample. Cellulose accessibility should be affected by crystallinity, but is also likely to be affected by several other parameters, such as lignin/hemicellulose contents and distribution, porosity, and particle size. Given the methodological dependency of cellulose CI values and the complex nature of cellulase interactions with amorphous and crystalline celluloses, we caution against trying to correlate relatively small changes in CI with changes in cellulose digestibility. In addition, the prediction of cellulase performance based on low levels of cellulose conversion may not include sufficient digestion of the crystalline component to be meaningful.

  18. Cellulase and Xylanase Production by Penicillium echinulatum in Submerged Media Containing Cellulose Amended with Sorbitol.

    Science.gov (United States)

    Todero Ritter, Carla Eliana; Camassola, Marli; Zampieri, Denise; Silveira, Mauricio Moura; Dillon, Aldo José Pinheiro

    2013-01-01

    The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v) sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v) soy bran; 0.1% (w/v) wheat bran; and a solution of salts. The highest filter paper activity (FPA) (1.95  ±  0.04 IU·mL(-1)) was obtained on the seventh day in the medium containing 0.5% (w/v) sorbitol and 0.5% (w/v) cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day (9.99 ± 0.75 IU·mL(-1)) in the medium containing 0.75% (w/v) sorbitol and 0.75% (w/v) cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v) sorbitol and 0.25% (w/v) cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems.

  19. Natural lactic acid bacteria population of tropical grasses and their fermentation factor analysis of silage prepared with cellulase and inoculant.

    Science.gov (United States)

    Khota, Waroon; Pholsen, Suradej; Higgs, David; Cai, Yimin

    2016-12-01

    Natural lactic acid bacteria (LAB) populations in tropical grasses and their fermentation characteristics on silage prepared with cellulase enzyme and LAB inoculants were studied. A commercial inoculant Lactobacillus plantarum Chikuso 1 (CH), a local selected strain Lactobacillus casei TH14 (TH14), and 2 cellulases, Acremonium cellulase (AC) and Maicelase (MC; Meiji Seika Pharma Co. Ltd., Tokyo, Japan), were used as additives to silage preparation with fresh and wilted (6 h) Guinea grass and Napier grass. Silage was prepared using a laboratory-scale fermentation system. Treatments were CH, TH14, AC at 0.01% fresh matter, AC 0.1%, MC 0.01%, MC 0.1%, CH+AC 0.01%, CH+AC 0.1%, CH+MC 0.01%, CH+MC 0.1%, TH14+AC 0.1%, TH14+AC 0.01%, TH14+MC 0.1%, and TH14+MC 0.01%. Microorganism counts of Guinea grass and Napier grass before ensiling were 10(2) LAB and 10(6) aerobic bacteria; these increased during wilting. Based on morphological and biochemical characteristics, and 16S rRNA gene sequence analysis, natural strains from both grasses were identified as L. plantarum, L. casei, Lactobacillus acidipiscis, Leuconostoc pseudomesenteroides, Leuconostoc garlicum, Weissella confusa, and Lactococcus lactis. Lactobacillus plantarum and L. casei are the dominant species and could grow at lower pH and produce more lactic acid than the other isolates. Crude protein and neutral detergent fiber were 5.8 and 83.7% of dry matter (DM) for Guinea grass, and 7.5 and 77.1% of DM for Napier grass. Guinea grass had a low level of water-soluble carbohydrates (0.39% of DM). Guinea grass silage treated with cellulase had a lower pH and higher lactic acid content than control and LAB treatments. The 0.1% AC and MC treatments had the best result for fermentation quality. All high water-soluble carbohydrate (2.38% DM) Napier grass silages showed good fermentation quality. Compared with control and LAB-inoculated silage, the cellulase-treated silages had significantly higher crude protein content and

  20. Effect of Different Pretreatment of Sugar Cane Bagasse on Cellulase and Xylanases Production by the Mutant Penicillium echinulatum 9A02S1 Grown in Submerged Culture

    OpenAIRE

    Marli Camassola; Dillon, Aldo J.P.

    2014-01-01

    The main limitation to the industrial scale hydrolysis of cellulose is the cost of cellulase production. This study evaluated cellulase and xylanase enzyme production by the cellulolytic mutant Penicillium echinulatum 9A02S1 using pretreated sugar cane bagasse as a carbon source. Most cultures grown with pretreated bagasse showed similar enzymatic activities to or higher enzymatic activities than cultures grown with cellulose or untreated sugar cane bagasse. Higher filter paper activity (1.25...

  1. Effect of xylanase and cellulase supplementation on growth performance, volatile fatty acids and caecal bacteria of broiler chickens fed with palm kernel meal-based diet

    OpenAIRE

    2014-01-01

    In this study, the effect of xylanase and cellulase supplementation in palm kernel meal (PKM) based diet on growth performance, volatile fatty acids (VFAs) and the caecal bacterial populations of broiler chickens were investigated. Seventy five day old male Cobb broiler chicks were randomly allocated to three dietary treatment groups receiving T1 (20% PKM-based diet without enzyme), T2 (20% PKM-based diet with xylanase) and T3 (20% PKM-based diet with cellulase). Each enzyme was supplemented ...

  2. Construction of Aspergillus niger integrated with cellulase gene from Ampullaria gigas Spix for improved enzyme production and saccharification of alkaline-pretreated rice straw.

    Science.gov (United States)

    Yang, Peizhou; Zhang, Haifeng; Cao, Lili; Zheng, Zhi; Jiang, Shaotong

    2016-12-01

    Aspergillus niger is an important microorganism that has been used for decades to produce extracellular enzymes. In this study, a novel Aspergillus niger strain integrated with a eukaryotic expression vector harboring the gpd-Shi promoter of shiitake mushrooms and cellulase gene of Ampullaria gigas Spix was engineered to improve cellulase production for the achievement of highly efficient saccharification of agricultural residues. In one strain, designated ACShi27, which exhibited the highest total cellulase expression, total cellulase, endoglucanase, exoglucanase, and xylanase expression levels were 1.73, 16.23, 17.73, and 150.83 U ml(-1), respectively; these values were 14.5, 22.3, 24.6, and 17.3% higher than those of the wild-type Aspergillus niger M85 using wheat bran as an induction substrate. Production of cellulases and xylanase by solid-state fermentation followed by in situ saccharification of ACShi27 was investigated with alkaline-pretreated rice straw as a substrate. After 2 days of enzyme induction at 30 °C, followed by 48 h of saccharification at 50 °C, the conversion rate of carbon polymers into reducing sugar reached 293.2 mg g(-1), which was 1.23-fold higher than that of the wild-type strain. The expression of sestc in Aspergillus niger can improve the total cellulase and xylanase activity and synergism, thereby enhancing the lignocellulose in situ saccharification.

  3. IMPROVED CELLULASE PRODUCTION BY Aspergillus terreus USING OIL PALM EMPTY FRUIT BUNCH FIBRE AS SUBSTRATE IN A STIRRED TANK BIOREACTOR THROUGH OPTIMIZATION OF THE FERMENTATION CONDITIONS

    Directory of Open Access Journals (Sweden)

    Mahdi Shahriarinour

    2011-05-01

    Full Text Available Response surface methodology (RSM was performed to evaluate the effects of dissolved oxygen tension (DOT and initial pH on the production of carboxymethyl cellulase (CMCase, filter-paper hydrolase (FPase, and β-glucosidase by Aspergillus terreus in a 2 L stirred tank bioreactor. Delignified oil palm empty fruit bunch (OPEFB fibre was used as the main substrate under submerged fermentation. Growth of A. terreus and the production of three main components of cellulase were optimized by central composite design (CCD design. Statistical analysis of results showed that the individual terms of these two variables (DOT and pH had significant effects on growth and the production of all components of cellulase. Maximum growth (13.07 g/L and cellulase activity (CMCase = 50.33 U/mL, FPase = 2.29 U/mL and β-glucosidase = 15.98 U/ml were obtained when the DOT and initial culture pH were set at 55% and 5.5, respectively. A high proportion of β-glucosidase to FPase (8:1 in cellulase of A. terreus could be beneficial for efficient hydrolysis of cellulosic materials. The use of OPEFB as a main substrate would reduce the cost of fermentation for the production of cellulase.

  4. Lignin isolated from steam-exploded eucalyptus wood chips by phase separation and its affinity to Trichoderma reesei cellulase.

    Science.gov (United States)

    Nonaka, Hiroshi; Kobayashi, Ai; Funaoka, Masamitsu

    2013-07-01

    Steam-exploded eucalyptus wood chips were treated with p-cresol and 72% sulfuric acid at ambient temperature. Steam-exploded lignin was isolated as acetone-soluble and diethyl ether-insoluble compounds from the cresol layer. The lignin extraction yield was only 47%, and the amount of cresol grafted to lignin was much less than that in the case of eucalyptus lignin without steam explosion. Clearly, the steam explosion process depolymerized native lignin, and simultaneously, promoted polymerization via labile benzyl positions. The steam-exploded eucalyptus lignin adsorbed more Trichoderma reesei cellulase; however, its enzymatic activity was less than that of eucalyptus lignin that did not undergo steam explosion. It is evident that pretreatment potentially affects the affinity between lignin and cellulase and the resultant saccharification efficiency.

  5. A graphene screen-printed carbon electrode for real-time measurements of unoccupied active sites in a cellulase

    DEFF Research Database (Denmark)

    Cruys-Bagger, Nicolaj; Tatsumi, Hirosuke; Borch, Kim;

    2014-01-01

    Cellulases hydrolyze cellulose to soluble sugars and this process is utilized in sustainable industries based on lignocellulosic feedstock. Better analytical tools will be necessary to understand basic cellulase mechanisms, and hence deliver rational improvements of the industrial process....... In this work we describe a new electrochemical approach to the quantification of the populations of enzyme that are respectively free in the aqueous bulk, adsorbed to the insoluble substrate with an unoccupied active site or threaded with the cellulose strand in the active tunnel. Distinction of these three...... data and adsorption measuments. The approach was tested for the cellobiohydrolase Cel7A from Hypocrea jecorina acting on microcrystalline cellulose, and it was found that the threaded enzyme form dominates for this system while adsorbed enzyme with an unoccupied active site constitutes less than 5...

  6. Recycling cellulases for cellulosic ethanol production at industrial relevant conditions: potential and temperature dependency at high solid processes.

    Science.gov (United States)

    Lindedam, Jane; Haven, Mai Østergaard; Chylenski, Piotr; Jørgensen, Henning; Felby, Claus

    2013-11-01

    Different versions of two commercial cellulases were tested for their recyclability of enzymatic activity at high dry matter processes (12% or 25% DM). Recyclability was assessed by measuring remaining enzyme activity in fermentation broth and the ability of enzymes to hydrolyse fresh, pretreated wheat straw. Industrial conditions were used to study the impact of hydrolysis temperature (40 or 50°C) and residence time on recyclability. Enzyme recycling at 12% DM indicated that hydrolysis at 50°C, though ideal for ethanol yield, should be kept short or carried out at lower temperature to preserve enzymatic activity. Best results for enzyme recycling at 25% DM was 59% and 41% of original enzyme load for a Celluclast:Novozyme188 mixture and a modern cellulase preparation, respectively. However, issues with stability of enzymes and their strong adsorption to residual solids still pose a challenge for applicable methods in enzyme recycling.

  7. STATISTICAL OPTIMIZATION OF MINERAL SALT AND UREA CONCENTRATION FOR CELLULASE AND XYLANASE PRODUCTION BY Penicillium echinulatum IN SUBMERGED FERMENTATION

    OpenAIRE

    L. dos Reis; Ritter,C. E. T.; R. C. Fontana; Camassola,M.; A. J. P. Dillon

    2015-01-01

    Abstract Penicillium echinulatum S1M29 is a mutant with cellulase and xylanase production comparable to the most studied microorganisms in the literature. However, its potential to produce these enzymes has not been fully investigated. This study aimed at optimizing salt and urea concentrations in the mineral solution, employing the response surface methodology. A 25-1 Fractional Factorial Design and a 23 Central Composite Design were applied to elucidate the effect of salts and urea in enzym...

  8. Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence-assisted carbohydrate electrophoresis.

    Science.gov (United States)

    Zhang, Qing; Zhang, Xiaomei; Wang, Peipei; Li, Dandan; Chen, Guanjun; Gao, Peiji; Wang, Lushan

    2015-03-01

    Fluorescence-assisted carbohydrate electrophoresis (FACE) is a sensitive and simple method for the separation of oligosaccharides. It relies on labeling the reducing ends of oligosaccharides with a fluorophore, followed by PAGE. Concentration changes of oligosaccharides following hydrolysis of a carbohydrate polymer could be quantitatively measured continuously over time using the FACE method. Based on the quantitative analysis, we suggested that FACE was a relatively high-throughput, repeatable, and suitable method for the analysis of the action modes of cellulases. On account of the time courses of their hydrolytic profiles, the apparent processivity was used to show the different action modes of cellulases. Cellulases could be easily differentiated as exoglucanases, β-glucosidases, or endoglucanases. Moreover, endoglucanases from the same glycoside hydrolases family had a variety of apparent processivity, indicating the different modes of action. Endoglucanases with the same binding capacities and hydrolytic activities had similar oligosaccharide profiles, which aided in their classification. The hydrolytic profile of Trichoderma reesei Cel12A, an endoglucanases from T. reesei, contained glucose, cellobiose, and cellotriose, which revealed that it may have a new glucosidase activity, corresponding to that of EC 3.2.1.74. A hydrolysate study of a T. reesei Cel12A-N20A mutant demonstrated that the FACE method was sufficiently sensitive to detect the influence of a single-site mutation on enzymatic activity.

  9. Highly thermostable and pH-stable cellulases from Aspergillus niger NS-2: properties and application for cellulose hydrolysis.

    Science.gov (United States)

    Bansal, Namita; Janveja, Chetna; Tewari, Rupinder; Soni, Raman; Soni, Sanjeev Kumar

    2014-01-01

    Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9 ± 20.1 U/g, FPase 101.1 ± 3.5 U/g and β-glucosidase 99 ± 4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0-9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92-98 %.

  10. Two major facilitator superfamily sugar transporters from Trichoderma reesei and their roles in induction of cellulase biosynthesis.

    Science.gov (United States)

    Zhang, Weixin; Kou, Yanbo; Xu, Jintao; Cao, Yanli; Zhao, Guolei; Shao, Jing; Wang, Hai; Wang, Zhixing; Bao, Xiaoming; Chen, Guanjun; Liu, Weifeng

    2013-11-15

    Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement.

  11. Isolation of Homogeneous Polysaccharide Monooxygenases from Fungal Sources and Investigation of Their Synergism with Cellulases when Acting on Cellulose.

    Science.gov (United States)

    Bulakhov, A G; Gusakov, A V; Chekushina, A V; Satrutdinov, A D; Koshelev, A V; Matys, V Yu; Sinitsyn, A P

    2016-05-01

    Lytic polysaccharide monooxygenases (PMO) discovered several years ago are enzymes classified as oxidoreductases. In nature, they participate in microbial degradation of cellulose together with cellulases that belong to the hydrolytic type of enzymes (class of hydrolases). Three PMO from ascomycetes - Thielavia terrestris, Trichoderma reesei, and Myceliophthora thermophila - were isolated and purified to homogeneous state using various types of chromatography. The first two enzymes are recombinant proteins heterologously expressed by the Penicillium verruculosum fungus, while the third is a native PMO secreted by M. thermophila. When acting on microcrystalline cellulose, all these PMOs displayed synergism with the cellulase complex of the P. verruculosum fungus. Replacing 10% of cellulases (by protein concentration) with PMO in the presence of 6.25 mM gallic acid or 2.5 µM of cellobiose dehydrogenase from M. thermophila, used as electron donors for PMO, resulted in the 17-31% increase in the yield of reducing sugars after 24-48 h of the enzymatic reaction.

  12. Xylanases, Cellulases, and Acid Protease Produced by Stenocarpella maydis Grown in Solid-state and Submerged Fermentation

    Directory of Open Access Journals (Sweden)

    Edna María Hernández-Domínguez

    2014-03-01

    Full Text Available Activity levels of extracellular hydrolytic enzymes produced by Stenocarpella maydis, a fungal pathogen of maize, have so far not been reported. Production of xylanase, cellulase, and acid protease by this ascomycete using different culture media in solid-state and submerged fermentation was studied. In solid-state fermentation, polyurethane foam was used as an inert support, and corncob, corn leaves, and broken corn were used as biodegradable supports. The highest xylanase activity was produced in the medium with xylan in both fermentation systems, reaching 18,020 U/L and 19,266 U/L for submerged and solid-state fermentation, respectively. Cellulase production was observed only in the culture medium with carboxymethylcellulose, obtaining values of 7,872 U/L in submerged fermentation and 9,439 U/L in solid-state fermentation. The acid protease was produced only in minimal medium with glucose in acidic pH, reaching the highest levels of activity in SSF (806 U/L. The corncob was the best biodegradable support for the production of xylanases and acid protease. Two isoenzymes of xylanase and cellulase were observed in both fermentation systems, and three isoenzymes of xylanase were produced on the biodegradable supports.

  13. Penicillium oxalicum PoFlbC regulates fungal asexual development and is important for cellulase gene expression.

    Science.gov (United States)

    Yao, Guangshan; Li, Zhonghai; Wu, Ruimei; Qin, Yuqi; Liu, Guodong; Qu, Yinbo

    2016-01-01

    Filamentous fungi can initiate vegetative growth on complex plant polysaccharides in nature through secreting a large amount of lignocellulose-degrading enzymes. These fungi develop a large amount of asexual spores to disperse and survive under harsh conditions, such as carbon and nitrogen depletion. Numerous studies report the presence of a cross-talk between asexual development and extracellular enzyme production, especially at the regulation level. This study identified and characterized a C2H2-type transcription factor called PoFlbC, which is an Aspergillus FlbC ortholog, in cellulolytic fungus Penicillium oxalicum. Results showed that the native level of PoFlbC was crucial for the normal growth and asexual development of P. oxalicum. Importantly, deletion of the PoflbC gene substantially reduced cellulase and hemicellulase productions. Comparative transcriptome analysis by RNA sequencing revealed a global downregulation of genes encoding cellulases, hemicellulases, and other proteins with functions in lignocellulose degradation. A similar defect was also observed in the OEPoflbC strain, suggesting that the production of cellulolytic enzymes was maintained by native expression of the PoflbC. In this study, an essential activator for both fungal asexual development and cellulase production was established in P. oxalicum.

  14. An integrative process of bioconversion of oil palm empty fruit bunch fiber to ethanol with on-site cellulase production.

    Science.gov (United States)

    Zhu, Youshuang; Xin, Fengxue; Zhao, Ying; Chang, Yunkang

    2014-11-01

    The aim of this study was to efficiently convert oil palm empty fruit bunch fiber (OPEFB), one of the most commonly generated lingo-wastes in Southeast Asia, into both cellulase and bioethanol. The unprocessed cellulase crude (37.29%) produced under solid-state fermentation using OPEFB as substrate showed a better reducing sugar yield using filter paper than the commercial enzyme blend (34.61%). Organosolv pretreatment method could efficiently reduce hemicellulose (24.3-18.6%) and lignin (35.2-22.1%) content and increase cellulose content (40.5-59.3%) from OPEFB. Enzymatic hydrolysis of pretreated OPEFB using the crude cellulase with 20% solid content, enzyme loading of 15 FPU/g OPEFB at 50 °C, and pH 5.5 resulted in a OPEFB hydrolysate containing 36.01 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 17.64 g/L ethanol with 0.49 g/g yield from glucose and 0.088 g/g yield from OPEFB at 8 h using Saccharomyces cerevisiae.

  15. Cellulase production from agricultural residues by recombinant fusant strain of a fungal endophyte of the marine sponge Latrunculia corticata for production of ethanol.

    Science.gov (United States)

    El-Bondkly, Ahmed M A; El-Gendy, Mervat M A

    2012-02-01

    Several fungal endophytes of the Egyptian marine sponge Latrunculia corticata were isolated, including strains Trichoderma sp. Merv6, Penicillium sp. Merv2 and Aspergillus sp. Merv70. These fungi exhibited high cellulase activity using different lignocellulosic substrates in solid state fermentations (SSF). By applying mutagenesis and intergeneric protoplast fusion, we have obtained a recombinant strain (Tahrir-25) that overproduced cellulases (exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase) that facilitated complete cellulolysis of agricultural residues. The process parameters for cellulase production by strain Tahrir-25 were optimized in SSF. The highest cellulase recovery from fermentation slurries was achieved with 0.2% Tween 80 as leaching agent. Enzyme production was optimized under the following conditions: initial moisture content of 60% (v/w), inoculum size of 10(6) spores ml(-1), average substrate particle size of 1.0 mm, mixture of sugarcane bagasse and corncob (2:1) as the carbon source supplemented with carboxymethyl cellulose (CMC) and corn steep solids, fermentation time of 7 days, medium pH of 5.5 at 30°C. These optimized conditions yielded 450, 191, and 225 units/gram dry substrate (U gds(-1)) of carboxylmethyl cellulase, filter-paperase (FPase), and β-glucosidase, respectively. Subsequent fermentation by the yeast, Saccharomyces cerevisiae NRC2, using lignocellulose hydrolysates obtained from the optimized cellulase process produced the highest amount of ethanol (58 g l(-1)). This study has revealed the potential of exploiting marine fungi for cost-effective production of cellulases for second generation bioethanol processes.

  16. Characterization, optimization, and scale-up of cellulases production by trichoderma reesei cbs 836.91 in solid-state fermentation using agro-industrial products.

    Science.gov (United States)

    Ortiz, Gastón E; Guitart, María E; Cavalitto, Sebastián F; Albertó, Edgardo O; Fernández-Lahore, Marcelo; Blasco, Martín

    2015-11-01

    The application of cellulases in saccharification processes is restricted by its production cost. Consequently, new fungal strains able to elaborate higher cellulases titers and with special activity profiles are required to make the process economical. The aim of this investigation was to find a promising wild-type Trichoderma strain for cellulases production. The Trichoderma reesei strain 938 (CBS 836.91) was selected among twenty strains on the basis of cellulase-agar-plate screening. Evaluation of the selected strain on six solid substrates indicated the highest activities to be obtained from wheat bran. Statistical analyses of the experimental design indicated a significant effect of pH and moisture on the generation of endoglucanase (EGA) and filter-paper (FPA) activity. Furthermore, a central-composite design-based optimization revealed that pH values between 6.4 and 6.6 and moisture from 74 to 94% were optimal for cellulases production. Under these conditions, 8-10 IU gds(-1) of FPA and 15.6-17.8 IU gds(-1) of EGA were obtained. In addition, cultivation in a rotating-drum reactor under optimal conditions gave 8.2 IU gds(-1) FPA and 13.5 IU gds(-1) EGA. Biochemical characterization of T. reesei 938 cellulases indicated a substantially higher resistance to 4 mM Fe(+2) and a slightly greater tolerance to alkaline pH in comparison to Celluclast(®). These results suggest that T. reesei 938 could be a promising candidate for improved cellulases production through direct-evolution strategies.

  17. The effects of adding lactic acid bacteria and cellulase in oil palm (Elais guineensis Jacq. frond silages on fermentation quality, chemical composition and in vitro digestibility

    Directory of Open Access Journals (Sweden)

    Mahdi Ebrahimi

    2014-07-01

    Full Text Available The main objective of the current study was to evaluate whether oil palm frond (OPF can be successfully ensiled without or with the additives cellulase or lactic acid bacteria (LAB. Thus, fresh OPF was ensiled either without additives or with cellulase or LAB or their combination. Ensiling was carried out by storing 2 kg samples in airtight glass jars at 25- 30°C for 12 weeks. Thereafter, the silage samples were subjected to proximate analyses, an in vitro digestibility assay and measures on selected indices of fermentation. Fermentation of OPF without additives appeared to be unsuccessful as both pH and ammonia content were too high (4.9 and 9.9%, respectively. In contrast, the use of cellulase or LAB resulted in silages with a pH<4.5 and ammonia fractions <8.4%, but the lowest values were found when both cellulase and LAB were used, i.e. pH=4.1 and ammonia fraction=6.7%. In vitro digestibility of dry matter was significantly higher in the cellulase treated silages. The process of ensiling was associated with both a significant decrease of the fat content of OPF and a significant change of the fatty acid profile. However, the proportions of major fatty acids (C16:0 and C18:2n-6 were not affected by the process of ensiling. In conclusion, the use of cellulase additive appears a practical tool to safeguard the process of fermentation. Using a cellulase enzyme or its combination with LAB improves the fermentation profile and increases the nutritional value of the OPF silage.

  18. Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate.

    Science.gov (United States)

    Da Vinha, Fábio Nuno Marques; Gravina-Oliveira, Mônica Pires; Franco, Marcella Novaes; Macrae, Andrew; da Silva Bon, Elba Pinto; Nascimento, Rodrigo Pires; Coelho, Rosalie Reed Rodrigues

    2011-06-01

    An actinomycete strain, isolated from a soil sample under a sugar cane plantation in Brazil and identified as Streptomyces viridobrunneus SCPE-09, was selected as a promising cellulolytic strain, and tested for its ability to produce cellulases from agro-industrial residues. Sugar cane bagasse or wheat bran was tested as carbon source, and corn steep liquor tested as nitrogen source. Different concentrations of carbon and nitrogen were tested using factorial design to identify optimal cellulose production. The results showed that media containing wheat bran 2.0% (w/v) and corn steep liquid 0.19% (w/v) lead to the highest production, 2.0 U mL(-1) of CMCase, obtained on the fifth day of fermentation. The pH and temperature profile showed optimal activity at pH 4.9 and 50°C. As for thermostability, endoglucanases were most tolerant at 50°C, retaining more than 80% of maximal activity even after 2 h of incubation. Zymogram analyses using supernatant from growth under optimized conditions revealed the presence of two CMCase bands with apparent molecular masses of 37 and 119 kDa. The combination of pH tolerance and CMCase production from agro-industrial residues by S. viridobrunneus SCPE-09 offers promise for future bioethanol biotechnologies.

  19. A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels.

    Directory of Open Access Journals (Sweden)

    Joshua I Park

    Full Text Available Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.

  20. A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels.

    Science.gov (United States)

    Park, Joshua I; Steen, Eric J; Burd, Helcio; Evans, Sophia S; Redding-Johnson, Alyssa M; Batth, Tanveer; Benke, Peter I; D'haeseleer, Patrik; Sun, Ning; Sale, Kenneth L; Keasling, Jay D; Lee, Taek Soon; Petzold, Christopher J; Mukhopadhyay, Aindrila; Singer, Steven W; Simmons, Blake A; Gladden, John M

    2012-01-01

    Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.

  1. Ancestral gene duplication enabled the evolution of multifunctional cellulases in stick insects (Phasmatodea).

    Science.gov (United States)

    Shelomi, Matan; Heckel, David G; Pauchet, Yannick

    2016-04-01

    The Phasmatodea (stick insects) have multiple, endogenous, highly expressed copies of glycoside hydrolase family 9 (GH9) genes. The purpose for retaining so many was unknown. We cloned and expressed the enzymes in transfected insect cell lines, and tested the individual proteins against different plant cell wall component poly- and oligosaccharides. Nearly all isolated enzymes were active against carboxymethylcellulose, however most could also degrade glucomannan, and some also either xylan or xyloglucan. The latter two enzyme groups were each monophyletic, suggesting the evolution of these novel substrate specificities in an early ancestor of the order. Such enzymes are highly unusual for Metazoa, for which no xyloglucanases had been reported. Phasmatodea gut extracts could degrade multiple plant cell wall components fully into sugar monomers, suggesting that enzymatic breakdown of plant cell walls by the entire Phasmatodea digestome may contribute to the Phasmatodea nutritional budget. The duplication and neofunctionalization of GH9s in the ancestral Phasmatodea may have enabled them to specialize as folivores and diverge from their omnivorous ancestors. The structural changes enabling these unprecedented activities in the cellulases require further study.

  2. Functional screening of hydrolytic activities reveals an extremely thermostable cellulase from a deep-sea archaeon

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    Benedikt eLeis

    2015-07-01

    Full Text Available Extreme habitats serve as a source of enzymes which are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8 to 70 °C. Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70 °C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12 endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45 % sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92 °C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential.

  3. Probing the nature of AFEX-pretreated corn stover derived decomposition products that inhibit cellulase activity.

    Science.gov (United States)

    Humpula, James F; Uppugundla, Nirmal; Vismeh, Ramin; Sousa, Leonardo; Chundawat, Shishir P S; Jones, A Daniel; Balan, Venkatesh; Dale, Bruce E; Cheh, Albert M

    2014-01-01

    Sequential fractionation of AFEX-pretreated corn stover extracts was carried out using ultra-centrifugation, ultra-filtration, and solid phase extraction to isolate various classes of pretreatment products to evaluate their inhibitory effect on cellulases. Ultra-centrifugation removed dark brown precipitates that caused no appreciable enzyme inhibition. Ultra-filtration of ultra-centrifuged AFEX-pretreated corn stover extractives using a 10 kDa molecular weight cutoff (MWCO) membrane removed additional high molecular weight components that accounted for 24-28% of the total observed enzyme inhibition while a 3 kDa MWCO membrane removed 60-65%, suggesting significant inhibition is caused by oligomeric materials. Solid phase extraction (SPE) of AFEX-pretreated corn stover extractives after ultra-centrifugation removed 34-43% of the inhibition; ultra-filtration with a 5 kDa membrane removed 44-56% of the inhibition and when this ultra-filtrate was subjected to SPE a total of 69-70% of the inhibition were removed. Mass spectrometry found several phenolic compounds among the hydrophobic inhibition removed by SPE adsorption.

  4. Oxidoreductases and cellulases in lichens: possible roles in lichen biology and soil organic matter turnover.

    Science.gov (United States)

    Beckett, Richard P; Zavarzina, Anna G; Liers, Christiane

    2013-06-01

    Lichens are symbiotic associations of a fungus (usually an Ascomycete) with green algae and/or a cyanobacterium. They dominate on 8 % of the world's land surface, mainly in Arctic and Antarctic regions, tundra, high mountain elevations and as components of dryland crusts. In many ecosystems, lichens are the pioneers on the bare rock or soil following disturbance, presumably because of their tolerance to desiccation and high temperature. Lichens have long been recognized as agents of mineral weathering and fine-earth stabilization. Being dominant biomass producers in extreme environments they contribute to primary accumulation of soil organic matter. However, biochemical role of lichens in soil processes is unknown. Our recent research has demonstrated that Peltigeralean lichens contain redox enzymes which in free-living fungi participate in lignocellulose degradation and humification. Thus lichen enzymes may catalyse formation and degradation of soil organic matter, particularly in high-stress communities dominated by lower plants. In the present review we synthesize recently published data on lichen phenol oxidases, peroxidases, and cellulases and discuss their possible roles in lichen physiology and soil organic matter transformations.

  5. Hydrolytic properties of two cellulases of Trichoderma reesei expressed in yeast.

    Science.gov (United States)

    Bailey, M J; Siika-aho, M; Valkeajärvi, A; Penttilä, M E

    1993-02-01

    Two cellulases of the filamentous fungus Trichoderma reesei, cellobiohydrolase II (CBHII, EC 3.2.1.91) and endoglucanase I (EGI, EC 3.2.1.4), produced in recombinant strains of the yeast Saccharomyces cerevisiae, were tested in the hydrolysis of cellulose, xylan and other polymeric substrates. Both enzymes were active against unsubstituted, insoluble cellulose. CBHII had greater activity than EGI against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence for synergism was obtained when mixtures of the two enzymes were used with a constant total protein dosage. The EGI was also active against soluble substituted cellulose derivatives, whereas the activity of CBHII against these substrates was insignificant. Both enzymes were active against barley (1-->3,1-->4)-beta-glucan, but were inactive against (1-->3,1-->6)-beta-glucan (laminarin). An apparent low mannan-degrading activity of EGI against locust-bean (Ceratonia siliqua) gum galactomannan was not confirmed when homopolymeric mannan was used as substrate in a prolonged hydrolysis test. EGI exhibited considerably greater activity against insoluble, unsubstituted hardwood xylan than against amorphous cellulose. Soluble 4-O-methyl-glucuronoxylan was also attacked by EGI, although to a somewhat lesser extent than the unsubstituted xylan. By comparison with two purified xylanases of T. reesei, EGI produced xylo-oligosaccharides with a longer mean chain length when acting on both substituted and unsubstituted xylan substrates. CBHII was inactive against xylan.

  6. Accessibility of Enzymatically Delignified Bambusa bambos for Efficient Hydrolysis at Minimum Cellulase Loading: An Optimization Study

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    Arindam Kuila

    2011-01-01

    Full Text Available In the present investigation, Bambusa bambos was used for optimization of enzymatic pretreatment and saccharification. Maximum enzymatic delignification achieved was 84%, after 8 h of incubation time. Highest reducing sugar yield from enzyme-pretreated Bambusa bambos was 818.01 mg/g dry substrate after 8 h of incubation time at a low cellulase loading (endoglucanase, β-glucosidase, exoglucanase, and xylanase were 1.63 IU/mL, 1.28 IU/mL, 0.08 IU/mL, and 47.93 IU/mL, respectively. Enzyme-treated substrate of Bambusa bambos was characterized by analytical techniques such as Fourier transformed infrared spectroscopy (FTIR, X-ray diffraction (XRD, and scanning electron microscopy (SEM. The FTIR spectrum showed that the absorption peaks of several functional groups were decreased after enzymatic pretreatment. XRD analysis indicated that cellulose crystallinity of enzyme-treated samples was increased due to the removal of amorphous lignin and hemicelluloses. SEM image showed that surface structure of Bambusa bambos was distorted after enzymatic pretreatment.

  7. Purification and characterization of an acidothermophilic cellulase enzyme produced by Bacillus subtilis strain LFS3.

    Science.gov (United States)

    Rawat, Rekha; Tewari, Lakshmi

    2012-07-01

    In the present investigation, a microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated and identified as Bacillus subtilis strain LFS3 by 16S rDNA sequence analysis. The carboxymethylcellulase (CMCase) enzyme produced by the B. subtilis strain LFS3 was purified by (NH₄)₂SO₄ precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 15 %. Native-PAGE analysis of purified CMCase revealed the molecular weight of enzyme to be about 185 kDa. The activity profile of CMCase enzyme showed the optimum activity at temperature 60 °C and pH 4.0, respectively. The enzyme activity was induced by Na⁺, Mg²⁺, NH₄⁺, and EDTA, whereas strongly inhibited by Hg²⁺ and Fe³⁺. The purified enzyme hydrolyzed CMC, filter paper, and xylan, but not p-nitrophenyl β-D-glucopyranoside and cellulose. Kinetic analysis of purified enzyme showed the K(m) value of 2.2 mg/ml. Thus, acidophilic as well as thermophilic nature makes this cellulase a suitable candidate for current mainstream biomass conversion into fuel and other industrial processes.

  8. Cold adaptation of a mesophilic cellulase, EG III from Trichoderma reesei, by directed evolution

    Institute of Scientific and Technical Information of China (English)

    XIAO; Zhizhuang(肖志壮); WANG; Pan(王攀); QU; Yinbo(曲音波); GAO; Peiji(高培基); WANG; Tianhong(汪天虹)

    2002-01-01

    Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase III (EG III) from Trichoderma reesei, and obtained a cold- adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG III with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG III calculated from Arrhenius equation are 13.3 kJ@mol-1 and 26.2 kJ@mol-1, respectively. Therefore, the increased specific activity of w-3 at lower temperatures could result from increased Kcat value and decreased activation energy.

  9. Activities of cellulase and amylase in soils as influenced by insecticide interactions.

    Science.gov (United States)

    Gundi, Vijay A K B; Viswanath, Buddolla; Chandra, M Subhosh; Kumar, V Narahari; Reddy, B Rajasekhar

    2007-10-01

    Interaction effects of the insecticides monocrotophos and quinalphos (organophosphates), and cypermethrin (pyrethroid), on microbial activities in two agricultural soils-black vertisol soil and red alfinsol soil were tested for 30 days under laboratory conditions. Individual application of the three insecticides at 5, 10 and 25microg g(-1) to the soil distinctly enhanced the activities of cellulase and amylase. Insecticide combinations involving monocrotophos or quinalphos with cypermethrin yielded synergistic, antagonistic and additive interaction effects on both enzymes in the soils. At lower levels, 5 and 10microg g(-1), the insecticides in combination interacted additively or synergistically toward both enzymes. But, both combinations at the highest level of 25microg g(-1) exhibited an antagonistic interaction, with a reduction in enzyme activities to a level lower than that of the control. Interaction effects of insecticides in combinations on two enzyme activities in both soils were related to populations of cellulolytic and amylolytic organisms in soils under the impact of combination of insecticides. These interaction responses were persistent even for 30 days.

  10. Production and characterization of carboxymethyl cellulase from Paenibacillus polymyxa using mango peel as substrate.

    Science.gov (United States)

    Kumar, Devendra; Ashfaque, Mohd; Muthukumar, M; Singh, Munna; Garg, Neelima

    2012-01-01

    Mango peel, a solid mango processing waste, comprises 15-20% of total fruit weight. This, being a rich source of lignocelluloses, was used as substrate for carboxymethyl cellulase (CMCase) production using Paenibacillus polymyxa. Maximum CMCase production (7.814 U mg(-1)) was observed in a medium containing 7% mango peel (w/v) with 1.5% ammonium sulphate (w/v) at 37 degrees C and pH 5.5. Purification to an extent of 28.24 fold was achieved by affinity column chromatography. Bands corresponding to 26.5 and 34.0 kDa molecular sizes were observed on 12% denaturing Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) while of 72 kDa on 10% non-denaturing Native-PAGE, proving its heteromeric multienzyme nature. The enzyme was stable over a range of 20-60 degrees C and pH of 4.0-7.5. Michaelis-Menten equation constant (Km and Vmax) values of purified CMCase were 8.73 mg ml(-1) and 17.805 mM ml(-1) min(-1), respectively.

  11. Fermentation Optimization and Unstructured Kinetic Model for Cellulase Production by Rhizopus stolonifer var. reflexus TP-02 on Agriculture By-Products.

    Science.gov (United States)

    Li, Song; Tang, Bin; Xu, Zhongyuan; Chen, Tao; Liu, Long

    2015-12-01

    Agricultural by-products, rice straw, wheat bran juice, and soybean residue, were used as substrates for cellulase production using Rhizopus stolonifer var. reflexus TP-02. The culture medium was optimized though uniform design experimentation during shaking flask fermentation, and the ideal formulation obtained for filter paper enzyme (FPase) production was 10 % bran diffusion juice, 1 % rice straw, 0.17 % urea, 0.17 % soybean residue, 0.11 % KH2PO4, and 0.027 % Tween 80, and the maximal FPase activity in the culture supernatant was 13.16 U/mL at an incubation time of 3 days. A kinetic model for cellulase production in batch fermentation was subsequently developed. The unstructured kinetic model considered three responses, namely biomass, cellulase, and sugar. Models for the production of three types of cellulase components (i.e., endoglucanases, cellobiohydrolases, and β-glucosidases) were established to adequately describe the cellulase production pattern. It was found that the models fitted the experimental data well under pH 5.0 and 6.0, but only the avicelase production model predicted the experimental data under pH-uncontrolled conditions.

  12. EFFECT OF VARIOUS PRETREATMENTS OF OIL PALM EMPTY FRUIT BUNCH FIBRES FOR SUBSEQUENT USE AS SUBSTRATE ON THE PERFORMANCE OF CELLULASE PRODUCTION BY ASPERGILLUS TERREUS

    Directory of Open Access Journals (Sweden)

    Mahdi Shahriarinour

    2011-02-01

    Full Text Available The possibility of using treated oil palm empty fruit bunch (OPEFB fibres as substrate for cellulase production by Aspergillus terreus was studied using shaking flask fermentation. The effect of different chemical pretreatments, i.e. formic acid, acetic acid, propylamine, phosphoric acid, and n-butylamine, on the suitability of OPEFB fibres as fermentation substrate was investigated. The findings revealed that pretreatment with these chemicals significantly (P<0.05 increased the cellulose and reduced the lignin contents prior to enzymatic hydrolysis. However, fermentation using OPEFB fibres pretreated with phosphoric acid gave the highest cellulase production, which was related to high cellulose content. Further improvement in cellulase production was obtained when the chemically pretreated OPEFB fibres were subsequently treated hydrothermally (autoclaved at 160oC for 10 min and then biologically (using effective microorganisms. The final activity of the three main components of cellulase (FPase, CMCase, and β-glucosidase obtained in fermentation by A. terreus using optimally treated OPEFB fibres was (0.77 U mL−1, 8.5 U mL-1, and 6.1 U mL-1, respectively. The production of all these three major components of cellulase using pretreated OPEFB fibres (i.e. chemical, hydrothermal, and biological were about three times higher than those obtained from fermentation using untreated OPEFB fibres.

  13. In situ stability of substrate-associated cellulases studied by DSC.

    Science.gov (United States)

    Alasepp, Kadri; Borch, Kim; Cruys-Bagger, Nicolaj; Badino, Silke; Jensen, Kenneth; Sørensen, Trine H; Windahl, Michael S; Westh, Peter

    2014-06-24

    This work shows that differential scanning calorimetry (DSC) can be used to monitor the stability of substrate-adsorbed cellulases during long-term hydrolysis of insoluble cellulose. Thermal transitions of adsorbed enzyme were measured regularly in subsets of a progressing hydrolysis, and the size of the transition peak was used as a gauge of the population of native enzyme. Analogous measurements were made for enzymes in pure buffer. Investigations of two cellobiohydrolases, Cel6A and Cel7A, from Trichoderma reesei, which is an anamorph of the fungus Hypocrea jerorina, showed that these enzymes were essentially stable at 25 °C. Thus, over a 53 h experiment, Cel6A lost less than 15% of the native population and Cel7A showed no detectable loss for either the free or substrate-adsorbed state. At higher temperatures we found significant losses in the native populations, and at the highest tested temperature (49 °C) about 80% Cel6A and 35% of Cel7A was lost after 53 h of hydrolysis. The data consistently showed that Cel7A was more long-term stable than Cel6A and that substrate-associated enzyme was less long-term stable than enzyme in pure buffer stored under otherwise equal conditions. There was no correlation between the intrinsic stability, specified by the transition temperature in the DSC, and the long-term stability derived from the peak area. The results are discussed with respect to the role of enzyme denaturation for the ubiquitous slowdown observed in the enzymatic hydrolysis of cellulose.

  14. 2007 GRC on Cellulases and Cellulosomes (July 29-August 3, 2007)

    Energy Technology Data Exchange (ETDEWEB)

    Mark Morrison

    2008-09-22

    industry have made many important and valuable contributions to the success of all the Conferences. This makes the Conference a truly interactive and productive venue for all sectors interested in the fundamental and applied sciences of cellulases, cellulosomes, and other carbohydrate active enzymes.

  15. Adsorption of cellulase on cereal brans: a simple functional model from response surface methodology

    Directory of Open Access Journals (Sweden)

    Rui Sergio F. da Silva

    1980-11-01

    Full Text Available A functional model based on Langmuirian adsorption as a limiting mechanism was proposed to explain the effect of cellulase during the enzymatic pretreatment of bran, conducted prior to extraction of proteins, by wet alkaline process from wheat and buckwheat bran materials. The proposed model provides a good fit (r = 0.99 for the data generated thru predictive model taken from the response surface methodology, permitting calculation of a affinity constant (b and capacity constant (k, for wheat bran (b = 0.255 g/IU and k = 17.42% and buckwheat bran (b = 0.066g/IUand k = 78.74%.Modelo funcional baseado na adsorção de Langmuir como mecanismo limitante proposto para explicar o efeito da celulase durante o pré-tratamento enzimático de farelos, visando à extração de proteínas, através do método alcalino-úmido. O referido modelo ajusta se muito bem (r = 0,99 aos dados gerados com base em modelo preditivo obtido da metodologia da superfície de resposta. Pode-se calcular a constante de afinidade (b e a constante de capacidade (k para o farelo de trigo e farelo de trigo mourisco (sarraceno, usando uma equação análoga à isoterma de adsorção de Langmuir. Os resultados indicaram que o farelo de trigo mourisco apresenta uma capacidade mais alta para adsorver celulase e, conseqüentemente,'pode-se esperar uma resposta maior ao pré-tratamento com esta enzima.

  16. The Hadal Amphipod Hirondellea gigas possessing a unique cellulase for digesting wooden debris buried in the deepest seafloor.

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    Hideki Kobayashi

    Full Text Available The Challenger Deep in the Mariana Trench is the deepest point in the ocean (10,994 m. Certain deep-sea animals can withstand the extreme pressure at this great depth. The amphipod Hirondellea gigas is a resident of the Challenger Deep. Amphipods are common inhabitants at great depths and serve as scavengers. However, there is relatively little information available regarding the physiology of H. gigas or this organism's ecological interactions in the hadopelagic zone. To understand the feeding behavior of this scavenger in the deepest oligotrophic hadal zone, we analyzed the digestive enzymes in whole-body extracts. We describe the detection of amylase, cellulase, mannanase, xylanase, and α-glycosidase activities that are capable of digesting plant-derived polysaccharides. Our identification of glucose, maltose, and cellobiose in the H. gigas extracts indicated that these enzymes function under great pressure in situ. In fact, the glucose content of H. gigas averaged 0.4% (w/dry-w. The purified H. gigas cellulase (HGcel converted cellulose to glucose and cellobiose at an exceptional molar ratio of 2:1 and efficiently produced glucose from dried wood, a natural cellulosic biomass, at 35 °C. The enzyme activity increased under a high hydrostatic pressure of 100 MPa at 2 °C, conditions equivalent to those found in the Challenger Deep. An analysis of the amino acid sequence of HGcel supported its classification as a family 31 glycosyl hydrolase. However, none of the enzymes of this family had previously been shown to possess cellulase activity. These results strongly suggested that H. gigas adapted to its extreme oligotrophic hadal oceanic environment by evolving digestive enzymes capable of digesting sunken wooden debris.

  17. STATISTICAL OPTIMIZATION OF MINERAL SALT AND UREA CONCENTRATION FOR CELLULASE AND XYLANASE PRODUCTION BY Penicillium echinulatum IN SUBMERGED FERMENTATION

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    L. dos Reis

    2015-03-01

    Full Text Available Abstract Penicillium echinulatum S1M29 is a mutant with cellulase and xylanase production comparable to the most studied microorganisms in the literature. However, its potential to produce these enzymes has not been fully investigated. This study aimed at optimizing salt and urea concentrations in the mineral solution, employing the response surface methodology. A 25-1 Fractional Factorial Design and a 23 Central Composite Design were applied to elucidate the effect of salts and urea in enzyme production. Lower concentrations of KH2PO4 (2.0 g.L-1, (NH42SO4 (1.4 g.L-1, MgSO4.7H2O (0.375 g.L-1 and CaCl2 (0.375 g.L-1 were most suitable for the production of all enzymes evaluated. Nevertheless, higher concentrations of urea (0.525 g.L-1 gave the best results for cellulase and xylanase production. The maximum FPase (1,5 U.m.L-1, endoglucanase (7,2 U.m.L-1, xylanase (30,5 U.m.L-1 and β-glucosidase (4,0 U.m.L-1 activities obtained with the planned medium were, respectively, 87, 16, 17 and 21% higher when compared to standard medium. The experimental design contributed to adjust the concentrations of minerals and urea of the culture media for cellulase and xylanase production by P. echinulatum, avoiding waste of components in the medium.

  18. A carboxymethyl cellulase from a marine yeast ( Aureobasidium pullulans 98): Its purification, characterization, gene cloning and carboxymethyl cellulose digestion

    Science.gov (United States)

    Rong, Yanjun; Zhang, Liang; Chi, Zhenming; Wang, Xianghong

    2015-10-01

    We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase (CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U (mg protein)-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40°C, much lower than that of the CMCases from other fungi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity (pH 5.0-6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. K m and V max values of the purified enzyme were 4.7 mg mL-1 and 0.57 µmol L-1 min-1 (mg protein)-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose (CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp (EU978473). The protein deduced contained the conserved domain of cellulase superfamily (glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

  19. Unravelling the molecular basis for light modulated cellulase gene expression - the role of photoreceptors in Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Schmoll Monika

    2012-03-01

    Full Text Available Abstract Background Light represents an important environmental cue, which exerts considerable influence on the metabolism of fungi. Studies with the biotechnological fungal workhorse Trichoderma reesei (Hypocrea jecorina have revealed an interconnection between transcriptional regulation of cellulolytic enzymes and the light response. Neurospora crassa has been used as a model organism to study light and circadian rhythm biology. We therefore investigated whether light also regulates transcriptional regulation of cellulolytic enzymes in N. crassa. Results We show that the N. crassa photoreceptor genes wc-1, wc-2 and vvd are involved in regulation of cellulase gene expression, indicating that this phenomenon is conserved among filamentous fungi. The negative effect of VVD on production of cellulolytic enzymes is thereby accomplished by its role in photoadaptation and hence its function in White collar complex (WCC formation. In contrast, the induction of vvd expression by the WCC does not seem to be crucial in this process. Additionally, we found that WC-1 and WC-2 not only act as a complex, but also have individual functions upon growth on cellulose. Conclusions Genome wide transcriptome analysis of photoreceptor mutants and evaluation of results by analysis of mutant strains identified several candidate genes likely to play a role in light modulated cellulase gene expression. Genes with functions in amino acid metabolism, glycogen metabolism, energy supply and protein folding are enriched among genes with decreased expression levels in the wc-1 and wc-2 mutants. The ability to properly respond to amino acid starvation, i. e. up-regulation of the cross pathway control protein cpc-1, was found to be beneficial for cellulase gene expression. Our results further suggest a contribution of oxidative depolymerization of cellulose to plant cell wall degradation in N. crassa.

  20. Optimization of fermentation conditions for cellulases production by Bacillus licheniformis MVS1 and Bacillus sp. MVS3 isolated from Indian hot spring

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    Somen Acharya

    2012-08-01

    Full Text Available The aim of this work was to study the effect of some nutritional and environmental factors on the production of cellulases, in particular endoglucanase (CMCase and exoglucanases (FPase from Bacillus licheniformis MVS1 and Bacillus sp. MVS3 isolated from an Indian hot spring. The characterization study indicated that the optimum pH and temperature value was 6.5 to 7.0 and 50-55°C, respectively. Maximum cellulases production by both the isolates was detected after 60 h incubation period using wheat and rice straw. The combination of inorganic and organic nitrogen source was suitable for cellulases production. Overall, FPase production was much higher than CMCase production by both of the strains. Between the two thermophiles, the cellulolytic activity was more in B.licheniformis MVS1 than Bacillus sp. MVS3 in varying environmental and nutritional conditions.

  1. Isolation of Bacillus sp. strains capable of decomposing alkali lignin and their application in combination with lactic acid bacteria for enhancing cellulase performance.

    Science.gov (United States)

    Chang, Young-Cheol; Choi, Dubok; Takamizawa, Kazuhiro; Kikuchi, Shintaro

    2014-01-01

    Effective biological pretreatment method for enhancing cellulase performance was investigated. Two alkali lignin-degrading bacteria were isolated from forest soils in Japan and named CS-1 and CS-2. 16S rDNA sequence analysis indicated that CS-1 and CS-2 were Bacillus sp. Strains CS-1 and CS-2 displayed alkali lignin degradation capability. With initial concentrations of 0.05-2.0 g L(-1), at least 61% alkali lignin could be degraded within 48 h. High laccase activities were observed in crude enzyme extracts from the isolated strains. This result indicated that alkali lignin degradation was correlated with laccase activities. Judging from the net yields of sugars after enzymatic hydrolysis, the most effective pretreatment method for enhancing cellulase performance was a two-step processing procedure (pretreatment using Bacillus sp. CS-1 followed by lactic acid bacteria) at 68.6%. These results suggest that the two-step pretreatment procedure is effective at accelerating cellulase performance.

  2. Application of ZnO nanoparticles for improving the thermal and pH stability of crude cellulase obtained from Aspergillus fumigatus AA001

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    Neha eSrivastava

    2016-04-01

    Full Text Available Cellulases are the enzymes which are responsible for the hydrolysis of cellulosic biomass. In this study thermal and pH stability of crude cellulase has been investigated in the presence of zinc oxide (ZnO nanoparticles. We synthesized ZnO nanoparticle by sol-gel method and characterized through various techniques including, X-ray Diffraction (XRD, ultraviolet-visible spectroscope (UV-Vis, field emission scanning electron microscope (FE-SEM and high resolution scanning electron microscope (HR-TEM. The crude thermostable cellulase has been obtained from the Aspergillus fumigatus AA001 and treated with ZnO nanoparticle which shows thermal stability at 65oC up to 10 h whereas it showed pH stability in the alkaline pH range and retained its 53% of relative activity at pH 10.5. These findings may be promising in the area of biofuels production.

  3. Improved in situ saccharification of cellulose pretreated by dimethyl sulfoxide/ionic liquid using cellulase from a newly isolated Paenibacillus sp. LLZ1.

    Science.gov (United States)

    Hu, Dongxue; Ju, Xin; Li, Liangzhi; Hu, Cuiying; Yan, Lishi; Wu, Tianyun; Fu, Jiaolong; Qin, Ming

    2016-02-01

    A cellulase producing strain was newly isolated from soil samples and identified as Paenibacillus sp. LLZ1. A novel aqueous-dimethyl sulfoxide (DMSO)/1-ethyl-3-methylimidazolium diethyl phosphate ([Emin]DEP)-cellulase system was designed and optimized. In the pretreatment, DMSO was found to be a low-cost substitute of up to 70% ionic liquid to enhance the cellulose dissolution. In the enzymatic saccharification, the optimum pH and temperature of the Paenibacillus sp. LLZ1 cellulase were identified as 6.0 and 40°C, respectively. Under the optimized reaction condition, the conversion of microcrystalline cellulose and bagasse cellulose increased by 39.3% and 37.6%, compared with unpretreated cellulose. Compared to current methods of saccharification, this new approach has several advantages including lower operating temperature, milder pH, and less usage of ionic liquid, indicating a marked progress in environmental friendly hydrolysis of biomass-based materials.

  4. PRODUCTION OF FERMENTABLE SUGARS FROM OIL PALM EMPTY FRUIT BUNCH USING CRUDE CELLULASE COCKTAILS WITH TRICHODERMA ASPERELLUM UPM1 AND ASPERGILLUS FUMIGATUS UPM2 FOR BIOETHANOL PRODUCTION

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    Nurul Kartini Abu Bakar,

    2012-06-01

    Full Text Available Utilization of oil palm empty fruit bunch (OPEFB for bioethanol production with crude cellulase cocktails from locally isolated fungi was studied. Enzymatic saccharification of alkaline pretreated OPEFB was done using different cellulase enzyme preparations. Crude cellulase cocktails from Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 produced 8.37 g/L reducing sugars with 0.17 g/g yield. Production of bioethanol from OPEFB hydrolysate using Baker’s yeast produced approximately 0.59 g/L ethanol, corresponding to 13.8% of the theoretical yield. High reducing sugars concentration in the final fermentation samples resulted from accumulation of non-fermentable sugars such as xylose and cellobiose that were not consumed by the yeast. The results obtained support the possible utilization of OPEFB biomass for bioethanol production in the future.

  5. Optimal control strategy for the enhanced production of cellulase enzyme using the new mutant Trichoderma reesei E-12

    Energy Technology Data Exchange (ETDEWEB)

    Rakshit, S.K. (Indian Inst. of Tech., Madras (India). Dept. of Chemical Engineering); Sahai, V. (Indian Inst. of Tech., New Delhi (India). Biochemical Engineering Research Centre)

    1991-02-01

    Cellulase enzyme production was enhanced using the mutant strain Trichoderma reesei, E-12, which was shown to be partially resistant to catabolite repression. An optimal profile for pH, which was found to be the critical environmental parameter, was determined using a rigorous mathematical optimization procedure. Semi-empirical models were used to minimize complications in the computation. A 30% increase in enzyme activity and productivity was obtained using the optimal pH strategy as compared to the pH cycling strategy. (orig.).

  6. Cellulase and xylanase productions by isolated Amazon Bacillus strains using soybean industrial residue based solid-state cultivation

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    Heck Júlio X.

    2002-01-01

    Full Text Available In Brazil, a large amount of a fibrous residue is generated as result of soybean (Glycine max protein production. This material, which is rich in hemicellulose and cellulose, can be used in solid state cultivations for the production of valuable metabolites and enzymes. In this work, we studied the bioconversion of this residue by bacteria strains isolated from water and soil collected in the Amazon region. Five strains among 87 isolated bacteria selected for their ability to produce either celullases or xylanases were cultivated on the aforementioned residue. From strain BL62, identified as Bacillus subtilis, it was obtained a preparation showing the highest specific cellulase activity, 1.08 UI/mg protein within 24 hours of growth. Concerning xylanase, the isolate BL53, also identified as Bacillus subtilis, showed the highest specific activity for this enzyme, 5.19 UI/mg protein within 72 hours of cultivation. It has also been observed the production of proteases that were associated with the loss of cellulase and xylanase activities. These results indicated that the selected microorganisms, and the cultivation process, have great biotechnological potential.

  7. Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa.

    Science.gov (United States)

    Campos Antoniêto, Amanda Cristina; Ramos Pedersoli, Wellington; Dos Santos Castro, Lílian; da Silva Santos, Rodrigo; Cruz, Aline Helena da Silva; Nogueira, Karoline Maria Vieira; Silva-Rocha, Rafael; Rossi, Antonio; Silva, Roberto Nascimento

    2017-01-01

    Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0), neutral (pH 7.0), and alkaline (pH 10.0) medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry.

  8. Carboxymethyl cellulase production optimization from newly isolated thermophilic Bacillus subtilis K-18 for saccharification using response surface methodology.

    Science.gov (United States)

    Irfan, Muhammad; Mushtaq, Qudsia; Tabssum, Fouzia; Shakir, Hafiz Abdullah; Qazi, Javed Iqbal

    2017-12-01

    In this study, a novel thermophilic strain was isolated from soil and used for cellulase production in submerged fermentation using potato peel as sole carbon source. The bacterium was identified by 16S rRNA gene sequencing technology. Central composite design was applied for enhanced production using substrate concentration, inoculum size, yeast extract and pH as dependent variables. Highest enzyme titer of 3.50 ± 0.11 IU/ml was obtained at 2% substrate concentration, 2% inoculum size, 1% yeast extract, pH 5.0, incubation temperature of 50 °C for 24 h of fermentation period. The crude enzyme was characterized having optimum pH and temperature of 7.0 and 50 °C, respectively. The efficiency of enzyme was checked by enzymatic hydrolysis of acid/alkali treated pine needles which revealed that 54.389% saccharification was observed in acid treated pine needles. These results indicated that the cellulase produced by the Bacillus subtilis K-18 (KX881940) could be effectively used for industrial processes particularly for bioethanol production.

  9. Systems analysis of lactose metabolism in Trichoderma reesei identifies a lactose permease that is essential for cellulase induction.

    Directory of Open Access Journals (Sweden)

    Christa Ivanova

    Full Text Available Trichoderma reesei colonizes predecayed wood in nature and metabolizes cellulose and hemicellulose from the plant biomass. The respective enzymes are industrially produced for application in the biofuel and biorefinery industry. However, these enzymes are also induced in the presence of lactose (1,4-0-ß-d-galactopyranosyl-d-glucose, a waste product from cheese manufacture or whey processing industries. In fact, lactose is the only soluble carbon source that induces these enzymes in T. reesei on an industrial level but the reason for this unique phenomenon is not understood. To answer this question, we used systems analysis of the T. reesei transcriptome during utilization of lactose. We found that the respective CAZome encoded all glycosyl hydrolases necessary for cellulose degradation and particularly for the attack of monocotyledon xyloglucan, from which ß-galactosides could be released that may act as the inducers of T. reesei's cellulases and hemicellulases. In addition, lactose also induces a high number of putative transporters of the major facilitator superfamily. Deletion of fourteen of them identified one gene that is essential for lactose utilization and lactose uptake, and for cellulase induction by lactose (but not sophorose in pregrown mycelia of T. reesei. These data shed new light on the mechanism by which T. reesei metabolizes lactose and offers strategies for its improvement. They also illuminate the key role of ß-D-galactosides in habitat specificity of this fungus.

  10. Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa

    Science.gov (United States)

    Campos Antoniêto, Amanda Cristina; Ramos Pedersoli, Wellington; dos Santos Castro, Lílian; da Silva Santos, Rodrigo; Cruz, Aline Helena da Silva; Nogueira, Karoline Maria Vieira; Silva-Rocha, Rafael; Rossi, Antonio

    2017-01-01

    Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0), neutral (pH 7.0), and alkaline (pH 10.0) medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry. PMID:28107376

  11. Medium initial pH and carbon source stimulate differential alkaline cellulase time course production in Stachybotrys microspora.

    Science.gov (United States)

    Ben Hmad, Ines; Abdeljalil, Salma; Saibi, Walid; Amouri, Bahia; Gargouri, Ali

    2014-03-01

    The production profile of cellulases of the mutant strain A19 from the filamentous fungus Stachybotrys microspora was studied in the presence of various carbon sources (glucose, lactose, cellulose, carboxymethylcellulose (CMC), and wheat bran) and a range of medium initial pH (5, 7, and 8). Two extracellular cellulases from the Stachybotrys strain (endoglucanases and β-glucosidases) were monitored by enzymatic assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and zymogram analysis. Glucose and lactose repressed CMCase time course production while they permitted a strong β-glucosidase one. On Avicel cellulose, CMC, and wheat bran, both activities were highly produced. Wheat bran (WB) is the best carbon source with an optimum of production at days 5 and 6. The production kinetics of both activities were shown to depend on the medium initial pH, with a preference for neutral or alkaline pH in the majority of conditions. The exception concerned the β-glucosidase which was much more produced at acidic pH, on glucose and cellulose. Most interestingly, a constitutive and conditional expression of an alkaline endoglucanase was revealed on the glucose-based medium at an initial pH of 8 units. The zymogram analysis confirmed such conclusions and highlighted that carbon sources and the pH of the culture medium directed a differential induction of various endoglucanases and β-glucosidases.

  12. Optimisation of Cellulase Production by Penicillium funiculosum in a Stirred Tank Bioreactor Using Multivariate Response Surface Analysis

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    Marcelle Lins de Albuquerque de Carvalho

    2014-01-01

    Full Text Available Increasing interest in the production of second-generation ethanol necessitates the low-cost production of enzymes from the cellulolytic complex (endoglucanases, exoglucanases, and β-glucosidases, which act synergistically in cellulose breakdown. The present work aimed to optimise a bioprocess to produce these biocatalysts from the fungus Penicillium funiculosum ATCC11797. A statistical full factorial design (FFD was employed to determine the optimal conditions for cellulase production. The optimal composition of culture media using Avicel (10 g·L−1 as carbon source was determined to include urea (1.2 g·L−1, yeast extract (1.0 g·L−1, KH2PO4 (6.0 g·L−1, and MgSO4·7H2O (1.2 g·L−1. The growth process was performed in batches in a bioreactor. Using a different FFD strategy, the optimised bioreactor operational conditions of an agitation speed of 220 rpm and aeration rate of 0.6 vvm allowed the obtainment of an enzyme pool with activities of 508 U·L−1 for FPase, 9,204 U·L−1 for endoglucanase, and 2,395 U·L−1 for β-glucosidase. The sequential optimisation strategy was effective and afforded increased cellulase production in the order from 3.6 to 9.5 times higher than production using nonoptimised conditions.

  13. Optimisation of Cellulase Production by Penicillium funiculosum in a Stirred Tank Bioreactor Using Multivariate Response Surface Analysis.

    Science.gov (United States)

    de Albuquerque de Carvalho, Marcelle Lins; Carvalho, Daniele Fernandes; de Barros Gomes, Edelvio; Nobuyuki Maeda, Roberto; Melo Santa Anna, Lidia Maria; de Castro, Aline Machado; Pereira, Nei

    2014-01-01

    Increasing interest in the production of second-generation ethanol necessitates the low-cost production of enzymes from the cellulolytic complex (endoglucanases, exoglucanases, and β-glucosidases), which act synergistically in cellulose breakdown. The present work aimed to optimise a bioprocess to produce these biocatalysts from the fungus Penicillium funiculosum ATCC11797. A statistical full factorial design (FFD) was employed to determine the optimal conditions for cellulase production. The optimal composition of culture media using Avicel (10 g·L(-1)) as carbon source was determined to include urea (1.2 g·L(-1)), yeast extract (1.0 g·L(-1)), KH2PO4 (6.0 g·L(-1)), and MgSO4 ·7H2O (1.2 g·L(-1)). The growth process was performed in batches in a bioreactor. Using a different FFD strategy, the optimised bioreactor operational conditions of an agitation speed of 220 rpm and aeration rate of 0.6 vvm allowed the obtainment of an enzyme pool with activities of 508 U·L(-1) for FPase, 9,204 U·L(-1) for endoglucanase, and 2,395 U·L(-1) for β-glucosidase. The sequential optimisation strategy was effective and afforded increased cellulase production in the order from 3.6 to 9.5 times higher than production using nonoptimised conditions.

  14. G protein-cAMP signaling pathway mediated by PGA3 plays different roles in regulating the expressions of amylases and cellulases in Penicillium decumbens.

    Science.gov (United States)

    Hu, Yibo; Liu, Guodong; Li, Zhonghai; Qin, Yuqi; Qu, Yinbo; Song, Xin

    2013-01-01

    Heterotrimeric G proteins (G proteins) have been extensively investigated for their regulatory functions in morphogenesis and development in filamentous fungi. In addition, G proteins were also shown to be involved in the regulation of cellulase expression in some fungi. Here, we report the different regulatory effects of PGA3, a group III G protein α subunit, on the expressions of amylases and cellulases in Penicillium decumbens. Deletion of pga3 resulted in impaired amylase production and significantly decreased transcription of the major amylase gene amy15A. Supplementation of exogenous cAMP or its analog dibutyryl-cAMP restored amylase production in Δpga3 strain, suggesting an essential role of PGA3 in amylase synthesis via controlling cAMP level. On the other hand, the transcription of major cellulase gene cel7A-2 increased, nevertheless cellulase activity in the medium was not affected, in Δpga3. The above regulatory effects of PGA3 are carbon source-independent, and are achieved, at least, by cAMP-mediated regulation of the expression level of transcription factor AmyR. The functions of PGA3 revealed by gene deletion were partially supported by the analysis of the mutant carrying dominantly-activated PGA3. The results provided new insights into the understanding of the physiological functions of G protein-cAMP pathway in filamentous fungi.

  15. [Use of Endoglucanase IV from Trichoderma reesei to Enhance the Hydrolytic Activity of a Cellulase Complex from the Fungus Penicillium verruculosum].

    Science.gov (United States)

    Proskurina, O V; Korotkova, O G; Rozhkova, A M; Kondrat'eva, E G; Matys, V Yu; Zorov, I N; Koshelev, A V; Okunev, O N; Nemashkalov, V A; Bubnova, T V; Sinitsyn, A P

    2015-01-01

    The effect of polysaccharide monooxygenase (endoglucanase IV) from the fungus Trichoderma reesei on the hydrolysis of polysaccharide substrates by cellulases secreted by the fungus Penicillium verruculosum has been investigated. Supplementation of the enzyme complex from P. verruculosum by endoglucanase IV from T. reesei has been shown to elevate the efficiency of cellulose hydrolysis by 45%.

  16. Silage preparation and fermentation quality of natural grasses treated with lactic acid bacteria and cellulase in meadow steppe and typical steppe.

    Science.gov (United States)

    Hou, Meiling; Ge, Gentu; Liu, Tingyu; Jia, Yushan; Cai, Yimin

    2016-09-27

    Silage preparation and fermentation quality of natural grasses in meadow steppe (MS) and typical steppe (TS) were studied. MS and TS contained thirty-three and nine species of natural grasses, respectively. Stipa Baicalensis in MS and Stipa grandi in TS were the dominant grasses with the highest dry matter (DM) yield. The crude protein (CP), neutral detergent fiber (NDF) and water-soluble carbohydrate (WSC) of the mixed natural grasses in both steppes were 8.02 to 9.03, 66.75 to 69.47 and 2.02 to 2.20% on a DM basis, respectively. The small-scale silages and round bale silages of mixed natural grasses in both steppes were prepared using the commercial lactic acid bacteria (LAB) inoculants Chikuso-1 (CH, Lactobacillus plantarum) and cellulase enzyme (AC, Acremonium cellulase) as additives. All silages treated with LAB and cellulase were well preserved with lower pH, butyric acid and ammonia-N content, and higher lactic acid and CP content than those of control in four kinds of silages. Compared with CH- or AC-treated silages, the CH+AC-treated silages had higher lactic acid content. The results confirmed that combination with LAB and cellulase may result in beneficial effects by improving the natural grass silage fermentation in both grasslands.

  17. Isolation and characteristics of one marine psychrotrophic cellulase-generating bacterium Ar/w/b/75°/10/5 from Chuckchi Sea, Arctic

    Institute of Scientific and Technical Information of China (English)

    曾胤新; 陈波

    2002-01-01

    Microorganisms living in polar zones play an important part as the potential source of organic activity materials with low temperature characteristics in the biotechnological applications. A psychrotrophic bacterium (strain Ar/w/b/75°/10/5), producing cellulase at low temperatures during late-exponential and early-stationary phases of cell growth, was isolated from sea ice-covered surface water in Chuckchi Sea, Arctic. This bacterium, with rod cells, was Gram-negative, slightly halophilic. Colony growing on agar plate was in black. Optimum growth temperature was 15℃. No cell growth was observed at 35℃ or above. Optimum salt concentration for cell growth was between 2 and 3 % of sodium chloride in media. Maximal cellulase activity was detected at a temperature of 35℃ and pH8. Cellulase was irreversibly inactivated when incubated at 55℃ within 30 min. Enzyme can be kept stable at the temperature no higher than 25℃. Of special interest was that this bacterium produced various extracellular enzymes including cellulase, amylase, agar hydrolase and protease, at low or moderate temperature conditions, which is certainly of it potential value for applications.

  18. CYANOBACTERIAL BIOMASS AS N-SUPPLEMENT TO OIL PALM EMPTY FRUIT BUNCH (OPEFB FIBRE FOR IMPROVEMENT OF CELLULASE PRODUCTION BY ASPERGILLUS TERREUS IN SUBMERGED FERMENTATION

    Directory of Open Access Journals (Sweden)

    Mahdi Shahriarinour

    2011-03-01

    Full Text Available The possibility of using dry biomass of a cyanobacterium, Anacystis nidulans, as nitrogen source supplement for improvement of cellulase production by Aspergillus terreus was studied in submerged fermentation using oil palm empty fruit bunch (OPEFB fibre as a carbon source. For comparison, four other nitrogen sources (ammonium sulphate, urea, peptone, and yeast extract were also tested. Growth and cellulase production were greatly enhanced in fermentation using biomass of cyanobacterium as the nitrogen source. The use of cyanobacterial biomass as a nitrogen source also reduced the inhibitory effect of high concentrations of CaCl2 to growth of A. terreus and cellulase production. The addition of 0.3 g L-1 CaCl2 to the medium containing OPEFB fibre and cyanobacterial biomass further enhanced the cellulase production, though growth remained unchanged. The final FPase, CMCase, and β-glucosidase obtained in fermentation using 10 g L-1 OPEFB fibre and 6 g/L cynaobacterial biomass with the addition of 3 mM CaCl2 was 0.97 U mL-1, 14.1 U mL-1, and 10.4 U mL-1, respectively.

  19. Crude cellulase from oil palm empty fruit bunch by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 for fermentable sugars production.

    Science.gov (United States)

    Ibrahim, M F; Razak, M N A; Phang, L Y; Hassan, M A; Abd-Aziz, S

    2013-07-01

    Cellulase is an enzyme that converts the polymer structure of polysaccharides into fermentable sugars. The high market demand for this enzyme together with the variety of applications in the industry has brought the research on cellulase into focus. In this study, crude cellulase was produced from oil palm empty fruit bunch (OPEFB) pretreated with 2% NaOH with autoclave, which was composed of 59.7% cellulose, 21.6% hemicellulose, and 12.3% lignin using Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2. Approximately 0.8 U/ml of FPase, 24.7 U/ml of CMCase and 5.0 U/ml of β-glucosidase were produced by T. asperellum UPM1 at a temperature of 35 °C and at an initial pH of 7.0. A 1.7 U/ml of FPase, 24.2 U/ml of CMCase, and 1.1 U/ml of β-glucosidase were produced by A. fumigatus UPM2 at a temperature of 45 °C and at initial pH of 6.0. The crude cellulase was best produced at 1% of substrate concentration for both T. asperellum UPM1 and A. fumigatus UPM2. The hydrolysis percentage of pretreated OPEFB using 5% of crude cellulase concentration from T. asperellum UPM1 and A. fumigatus UPM2 were 3.33% and 19.11%, with the reducing sugars concentration of 1.47 and 8.63 g/l, respectively.

  20. Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance

    Science.gov (United States)

    Gusakov, Alexander V.; Nemashkalov, Vitaly A.; Satrutdinov, Aidar D.; Sinitsyn, Arkady P.

    2017-01-01

    Background Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases. Results Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL), and eglIV, encoding LPMO (formerly endoglucanase IV) from Trichoderma reesei (TrLPMO), were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3) varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples). The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10–43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1. Conclusions The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a

  1. Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

    Directory of Open Access Journals (Sweden)

    Del Pozo Mercedes V

    2012-09-01

    Full Text Available Abstract Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate, the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG. LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2 and 2–38000 fold higher (as compared with reported beta-glucosidases activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of

  2. Response surface optimization for enhanced production of cellulases with improved functional characteristics by newly isolated Aspergillus niger HN-2.

    Science.gov (United States)

    Oberoi, Harinder Singh; Rawat, Rekha; Chadha, Bhupinder Singh

    2014-01-01

    Fungi isolated from partially decayed wood log samples showing characteristic diversity for spore colour, colony morphology and arrangement of spores were assessed for cellulolytic enzyme production. Isolates showing a cellulolytic index of ≥2.0 were assayed for filter paper (FP) cellulase and β-glucosidase (BGL) production. Molecular characterization confirmed the identity of the selected cellulolytic isolate as a strain of Aspergillus niger (A. niger HN-2). Addition of 2 % (w/v) urea enhanced FP and BGL activity by about 20 and 60 %, respectively. Validation studies conducted at parameters (29 °C, pH 5.4, moisture content 72 % and 66 h) optimized through response surface methodology in a solid-state static tray fermentation resulted in FP, BGL, cellobiohydrolase I (CBHI), endoglucanase (EG), xylanase activity and protein content of 25.3 FPU/g ds, 750 IU/g ds, 13.2 IU/g ds, 190 IU/g ds, 2890 IU/g ds and 0.9 mg/ml, respectively. In comparison, A. niger N402 which is a model organism for growth and development studies, produced significantly lower FP, BGL, CBHI, EG, xylanase activity and protein content of 10.0 FPU/g ds, 100 IU/g ds, 2.3 IU/g ds, 50 IU/g ds, 500 IU/g ds and 0.75 mg/ml, respectively under the same process conditions as were used for A. niger HN-2. Process optimization led to nearly 1.8- and 2.2-fold increase in FP and BGL activity, respectively showing promise for cellulase production by A. niger HN-2 at a higher scale of operation. Zymogram analysis revealed two isoforms each for EG and cellobiohydrolase and three isoforms for BGL. Crude cellulase complex produced by A. niger HN-2 exhibited thermostability under acidic conditions showing potential for use in biofuel industry.

  3. Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost

    Directory of Open Access Journals (Sweden)

    Amore Antonella

    2012-12-01

    Full Text Available Abstract Background The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. Results Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose

  4. Artificial intelligence techniques to optimize the EDC/NHS-mediated immobilization of cellulase on Eudragit L-100.

    Science.gov (United States)

    Zhang, Yu; Xu, Jing-Liang; Yuan, Zhen-Hong; Qi, Wei; Liu, Yun-Yun; He, Min-Chao

    2012-01-01

    Two artificial intelligence techniques, namely artificial neural network (ANN) and genetic algorithm (GA) were combined to be used as a tool for optimizing the covalent immobilization of cellulase on a smart polymer, Eudragit L-100. 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) concentration, N-hydroxysuccinimide (NHS) concentration and coupling time were taken as independent variables, and immobilization efficiency was taken as the response. The data of the central composite design were used to train ANN by back-propagation algorithm, and the result showed that the trained ANN fitted the data accurately (correlation coefficient R(2) = 0.99). Then a maximum immobilization efficiency of 88.76% was searched by genetic algorithm at a EDC concentration of 0.44%, NHS concentration of 0.37% and a coupling time of 2.22 h, where the experimental value was 87.97 ± 6.45%. The application of ANN based optimization by GA is quite successful.

  5. Effect of pretreatment and enzymatic hydrolysis of wheat straw on cell wall composition, hydrophobicity and cellulase adsorption.

    Science.gov (United States)

    Heiss-Blanquet, Senta; Zheng, Dan; Lopes Ferreira, Nicolas; Lapierre, Catherine; Baumberger, Stéphanie

    2011-05-01

    The present study aimed to determine the impact of cell wall composition and lignin content on enzyme adsorption and degradability. Thioacidolysis analysis of residual lignins in wheat straw after steam-explosion or organosolv pretreatment revealed an increase in lignin condensation degree of 27% and 33%, respectively. Surface hydrophobicity assessed through wettability tests decreased after the pretreatments (contact angle decrease of 20-50%), but increased with enzymatic conversion (30% maximum contact angle increase) and correlatively to lignin content. Adsorption of the three major cellulases Cel7A, Cel6A and Cel7B from Trichoderma reesei decreased with increasing hydrolysis time, down to 7%, 31% and 70% on the sample with the highest lignin content, respectively. The fraction of unspecifically bound enzymes was dependent both on the enzyme and the lignin content. Adsorption and specific activity were shown to be inversely proportional to lignin content and hydrophobicity, suggesting that lignin is one of the factors restricting enzymatic hydrolysis.

  6. Identification of a laccase from Ganoderma lucidum CBS 229.93 having potential for enhancing cellulase catalyzed lignocellulose degradation

    DEFF Research Database (Denmark)

    Sitarz, Anna Katarzyna; Mikkelsen, Jørn Dalgaard; Højrup, Peter

    2013-01-01

    . Addition of the laccase-rich G. lucidum broth to lignocellulosic biomass (pretreated sugar cane bagasse) together with a state-of-the-art cellulase enzyme preparation (Cellic™CTec1) produced significantly increased cellulolytic yields, which were also better than those obtained with a T. versicolor laccase...... extract or minimal media supplemented with alkali lignin. When grown on malt extract or minimal medium supplemented with lignocellulose (sugar cane bagasse), the crude G. lucidum protein extract exhibited high laccase activity, ∼3U/mL toward syringaldazine. This activity was 13–17 fold higher than...... addition, indicating that the laccase from G. lucidum has unique properties that may be momentous in lignocellulosic biomass conversion....

  7. Reversibility of substrate adsorption for the cellulases Cel7A, Cel6A and Cel7B from H. jecorina

    DEFF Research Database (Denmark)

    Pellegrini, Vanessa de Oliveira Arnoldi; Lei, Nina; Kysaram, Madhuri;

    2014-01-01

    Adsorption of cellulases on the cellulose surface is an integral part of the catalytic mechanism, and a detailed description of the adsorption process is therefore required for a fundamental understanding of this industrially important class of enzymes. However, the mode of adsorption has proven...... cellobiohydrolases (Cel7A and Cel6A) and one endoglucanase (Cel7B) on four types of pure cellulose substrates. Specifically, we monitored dilution-induced release of adsorbed enzyme in samples that had previously been brought to a steady state (constant concentration of free enzyme). In simple dilution experiments...... that changes in the physical properties of cellulose caused by compaction of the pellet hampers subsequent release of adsorbed enzyme. This latter effect may be pertinent to both previous controversies in the literature on adsorption reversibility and the development of enzyme recycling protocols...

  8. Artificial Intelligence Techniques to Optimize the EDC/NHS-Mediated Immobilization of Cellulase on Eudragit L-100

    Science.gov (United States)

    Zhang, Yu; Xu, Jing-Liang; Yuan, Zhen-Hong; Qi, Wei; Liu, Yun-Yun; He, Min-Chao

    2012-01-01

    Two artificial intelligence techniques, namely artificial neural network (ANN) and genetic algorithm (GA) were combined to be used as a tool for optimizing the covalent immobilization of cellulase on a smart polymer, Eudragit L-100. 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) concentration, N-hydroxysuccinimide (NHS) concentration and coupling time were taken as independent variables, and immobilization efficiency was taken as the response. The data of the central composite design were used to train ANN by back-propagation algorithm, and the result showed that the trained ANN fitted the data accurately (correlation coefficient R2 = 0.99). Then a maximum immobilization efficiency of 88.76% was searched by genetic algorithm at a EDC concentration of 0.44%, NHS concentration of 0.37% and a coupling time of 2.22 h, where the experimental value was 87.97 ± 6.45%. The application of ANN based optimization by GA is quite successful. PMID:22942683

  9. Proteomic and Functional Analysis of the Cellulase System Expressed by Postia placenta during Brown Rot of Solid Wood

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Jae San; Shary, Semarjit; Houtman, Carl J.; Panisko, Ellen A.; Korripally, Premsagar; St John, Franz J.; Crooks, Casey; Siika-aho, Matti; Magnuson, Jon K.; Hammel, Ken

    2011-11-01

    Abstract Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed the presence of 34 likely glycoside hydrolases, but only four of these-two in glycoside hydrolase family 5, one in family 10, and one in family 12-have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component.

  10. Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.

    Directory of Open Access Journals (Sweden)

    Zhiwei Chen

    Full Text Available Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc] pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25-42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.

  11. Fabrication of graphene oxide decorated with Fe{sub 3}O{sub 4}@SiO{sub 2} for immobilization of cellulase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yue; Wang, Xiang-Yu; Jiang, Xiao-Ping; Ye, Jing-Jing; Zhang, Ye-Wang, E-mail: zhangyewang@ujs.edu.cn [Jiangsu University, School of Pharmacy (China); Zhang, Xiao-Yun, E-mail: zhangxiaoyungu@126.com [Jiangsu University, School of Food and Biological Engineering (China)

    2015-01-15

    Fe{sub 3}O{sub 4}@SiO{sub 2}–graphene oxide (GO) composites were successfully fabricated by chemical binding of functional Fe{sub 3}O{sub 4}@SiO{sub 2} and GO and applied to immobilization of cellulase via covalent attachment. The prepared composites were further characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. Fe{sub 3}O{sub 4} nanoparticles (NPs) were monodisperse spheres with a mean diameter of 17 ± 0.2 nm. The thickness of SiO{sub 2} layer was calculated as being 6.5 ± 0.2 nm. The size of Fe{sub 3}O{sub 4}@SiO{sub 2} NPs was 24 ± 0.3 nm, similar to that of Fe{sub 3}O{sub 4}@SiO{sub 2}–NH{sub 2}. Fe{sub 3}O{sub 4}@SiO{sub 2}–GO composites were synthesized by linking of Fe{sub 3}O{sub 4}@SiO{sub 2}–NH{sub 2} NPs to GO with the catalysis of EDC and NHS. The prepared composites were used for immobilization of cellulase. A high immobilization yield and efficiency of above 90 % were obtained after the optimization. The half-life of immobilized cellulase (722 min) was 3.34-fold higher than that of free enzyme (216 min) at 50 °C. Compared with the free cellulase, the optimal temperature of the immobilized enzyme was not changed; but the optimal pH was shifted from 5.0 to 4.0, and the thermal stability was enhanced. The immobilized cellulase could be easily separated and reused under magnetic field. These results strongly indicate that the cellulase immobilized onto the Fe{sub 3}O{sub 4}@SiO{sub 2}–GO composite has potential applications in the production of bioethanol.

  12. A mitogen-activated protein kinase Tmk3 participates in high osmolarity resistance, cell wall integrity maintenance and cellulase production regulation in Trichoderma reesei.

    Directory of Open Access Journals (Sweden)

    Mingyu Wang

    Full Text Available The mitogen-activated protein kinase (MAPK pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, 'budded' hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest

  13. Effect of lignin structure and surfactant on cellulase adsorption by lignin%木质素结构以及表面活性剂对木质素吸附纤维素酶的影响

    Institute of Scientific and Technical Information of China (English)

    姚兰; 赵建; 谢益民; 杨海涛; 曲音波

    2012-01-01

    As substitute of petrol, bioethanol production from lignocelluloses is one of hot point of research in recent years. A main obstacle of bioethanol production in commercial scale is its cost.'One of the most important methods to solve the key problem is raising yield of cellulose hydrolysis by enzyme. However, content and type of lignin in lignocellulosic substrates has a significant effect on the hydrolysis process, as lignin has double actions: as a physical barrier that decreases accessibility of cellulase to cellulose, and as an attractant that adsorbs cellulases i. e. forming non-productive binding. In this paper, structure and adsorption for cellulase of three kinds of ligin, milled wood lignin, Klasson lignin and alkaline lignin were studied, and various surfactants were screened and used for improving enzymatic hydrolysis of lignocelluloses because they have a great impact on cellulase adsorption on lignin. The results showed that there are different structure characteristics for the three kinds of lignin: the highest ability of adsorption for cellulase is Klasson lignin, and then milled wood lignin, the lowest alkaline lignin. The surfactant that can significantly increase enzymatic hydrolysis yield of lignocelluloses is PEG-8000, because it can make cellulase desorption from lignin and reactivation for hydrolysis of cellulose.

  14. Effect of physical and chemical properties of oil palm empty fruit bunch, decanter cake and sago pith residue on cellulases production by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2.

    Science.gov (United States)

    Zanirun, Zuraidah; Bahrin, Ezyana Kamal; Lai-Yee, Phang; Hassan, Mohd Ali; Abd-Aziz, Suraini

    2014-01-01

    The effect of cultivation condition of two locally isolated ascomycetes strains namely Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 were compared in submerged and solid state fermentation. Physical evaluation on water absorption index, solubility index and chemical properties of lignin, hemicellulose and cellulose content as well as the cellulose structure on crystallinity and amorphous region of treated oil palm empty fruit bunch (OPEFB) (resulted in partial removal of lignin), sago pith residues (SPR) and oil palm decanter cake towards cellulases production were determined. Submerged fermentation shows significant cellulases production for both strains in all types of substrates. Crystallinity of cellulose and its chemical composition mainly holocellulose components was found to significantly affect the total cellulase synthesis in submerged fermentation as the higher crystallinity index, and holocellulose composition will increase cellulase production. Treated OPEFB apparently induced the total cellulases from T. asperellum UPM1 and A. fumigatus UPM2 with 0.66 U/mg FPase, 53.79 U/mg CMCase, 0.92 U/mg β-glucosidase and 0.67 U/mg FPase, 47.56 U/mg and 0.14 U/mg β-glucosidase, respectively. Physical properties of water absorption and solubility for OPEFB and SPR also had shown significant correlation on the cellulases production.

  15. The impact of alkali pretreatment and post-pretreatment conditioning on the surface properties of rice straw affecting cellulose accessibility to cellulases.

    Science.gov (United States)

    Karuna, Nardrapee; Zhang, Lu; Walton, Jeffrey H; Couturier, Marie; Oztop, Mecit H; Master, Emma R; McCarthy, Michael J; Jeoh, Tina

    2014-09-01

    Rice straw was pretreated with sodium hydroxide and subsequently conditioned to reduce the pH to 5-6 by either: (1) extensive water washing or (2) acidification with hydrochloric acid then water washing. Alkali pretreatment improved the enzymatic digestibility of rice straw by increasing the cellulose accessibility to cellulases. However, acidification after pretreatment reversed the gains in cellulose accessibility to cellulases and enzymatic digestibility due to precipitation of solubilized compounds. Surface composition analyses by ToF-SIMS confirmed a reduction in surface lignin by pretreatment and water washing, and suggested that acidification precipitated a chemically modified form of lignin on the surfaces of rice straw. The spin-spin relaxation times (T2) of the samples indicated increased porosity in alkali pretreated rice straw. The acidified pretreated rice straw had reduced amounts of water in the longer T2 proton pools associated with water in the pores of the biomass likely due to back-filling by the precipitated components.

  16. Study on cellulase activity production and physiological and biochemical characteristics of cellulase producing bacteria isolated from wine fermented grains%白酒酒醅中纤维素酶产生菌的产酶活力与其生理生化特性的研究

    Institute of Scientific and Technical Information of China (English)

    郭建华; 刘冀; 郭宏文; 刘晓兰; 杜国军; 贾士儒

    2013-01-01

    以白酒酒醅中筛选出的产纤维素酶的细菌为研究对象,利用相关分析和通径分析研究了纤维素酶产生菌的生理生化特性与其产酶活力之间的关系.相关分析和通经分析结果表明:细胞长宽比和葡萄糖产酸指标与纤维素酶活力具有明显的负相关性;淀粉水解实验指标与纤维素酶活力具有明显的正相关性;细胞长宽比对细菌产纤维素酶的负向直接作用最大;葡萄糖产酸指标对纤维素酶活力有较强的负向直接作用;葡萄糖产气和酪蛋白实验指标对纤维素酶具有较强的正向直接作用;同时各个指标还通过其他指标对纤维素酶活力具有间接作用.%A correlation between the physiological and biochemical characteristics of cellulase producing bacteria isolated from wine fermented grains and cellulose enzyme activity was studied using correlation analysis and path analysis.Results of correlation analysis and path analysis showed that cell aspect ratio and acid production indicator using glucose had clear negative correlations with cellulase activity,while starch hydrolysis test indicator had positive correlation with cellulase activity.Cell aspect ratio had the largest negative direct effect on cellulase activity.Acid production indicator using glucose had strong negative direct effect on cellulase activity.Gas production indicator used glucose and casein test indicator had strong positive direct effects on cellulase activity, as well as individual indicators also had indirect effects on cellulase activity through other indicators.

  17. Cellulose hydrolysis by Trichoderma reesei cellulases: studies on adsorption, sugar production and synergism of cellobiohydrolase I,II and endoglucanase II

    Energy Technology Data Exchange (ETDEWEB)

    Medve, J.

    1997-02-01

    Three major cellulases have been purified by ion-exchange chromatography in an FPLC system. Microcrystalline cellulose (Avicel) was hydrolyzed by the single enzymes and by equimolar mixtures of CBH I-CBH II and CBH I-EG II. Enzyme adsorption was followed indirectly by selectively quantifying the enzymes in the supernatant by ion-exchange chromatography in an FPLC system. The (synergistic) production of small, soluble sugars (glucose, cellobiose and cellotriose) by the enzymes was followed by HPLC. 76 refs

  18. Effect of nickel-cobaltite nanoparticles on production and thermostability of cellulases from newly isolated thermotolerant Aspergillus fumigatus NS (class: Eurotiomycetes).

    Science.gov (United States)

    Srivastava, Neha; Rawat, Rekha; Sharma, Reetika; Oberoi, Harinder Singh; Srivastava, Manish; Singh, Jay

    2014-10-01

    In the present study, effect of nickel-cobaltite (NiCo2O4) nanoparticles (NPs) was investigated on production and thermostability of the cellulase enzyme system using newly isolated thermotolerant Aspergillus fumigatus NS belonging to the class Euratiomycetes. The NiCo2O4 NPs were synthesized via hydrothermal method assisted by post-annealing treatment and characterized through X-ray diffraction (XRD) and transmission electron microscopy (TEM) techniques. In the absence of NPs in the growth medium, filter paper cellulase (FP) activity of 18 IU/gds was achieved after 96 h, whereas 40 % higher FP activity in 72 h was observed with the addition of 1 mM concentration of NPs in the growth medium. Maximum production of endoglucanase (211 IU/gds), β-glucosidase (301 IU/gds), and xylanase (803 IU/gds) was achieved after 72 h without NPs (control), while in the presence of 1 mM concentration of NPs, endoglucanase, β-glucosidase, and xylanase activity increased by about 49, 53, and 19.8 %, respectively, after 48 h of incubation, against control, indicating a substantial increase in cellulase productivity with the addition of NiCo2O4 NPs in the growth medium. Crude enzyme was thermally stable for 7 h at 80 °C in presence of NPs, as against 4 h at the same temperature for control samples. Significant increase in the activity and improved thermal stability of cellulases in the presence of the NiCo2O4 NPs holds potential for use of NiCo2O4 NPs during enzyme production as well as hydrolysis. From the standpoint of biofuel production, these results hold enormous significance.

  19. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein: Protein Redesign to Lower Protein-lignin Binding

    Energy Technology Data Exchange (ETDEWEB)

    Haarmeyer, Carolyn N. [Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing Michigan 48824; Smith, Matthew D. [Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing Michigan 48824; Chundawat, Shishir P. S. [Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, East Lansing Michigan; Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway New Jersey; Sammond, Deanne [Biosciences Center, National Renewable Energy Laboratory, Golden Colorado; Whitehead, Timothy A. [Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing Michigan 48824; Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing Michigan 48824

    2016-11-07

    Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active

  20. 中性纤维素酶整理丝光棉织物工艺的确定%Process of neutral cellulase finishing on mercerizing cotton fabrics

    Institute of Scientific and Technical Information of China (English)

    孙洁; 贺江平; 梁金山; 赵建华

    2012-01-01

    研究中性纤维素酶整理丝光棉织物后对其性能的影响.通过实验分析酶用量、pH值、整理温度和时间对织物失重率、顶破强力、硬挺度及抗起毛起球性的影响,并最终确定最佳工艺条件.实验结果表明,中性纤维素酶整理丝光棉织物的最佳工艺为酶用量2%(o.w.f),pH值为7.5,整理温度50℃,整理时间55min.在最佳工艺条件下整理的丝光棉织物,各方面性能有不同程度的改善.并且,顶破强力控制在220N以上,仍能保持织物应有的使用性能.%The influence of neutral cellulase finishing on characters of mercerizing cotton fabrics is studied,the impact of the dosage of cellulase, pH value, treating temperature, and time on the weight-loss ratio, the bursting strength, the stiffness, and the pilling resistance are analyzed through the experiments, the optimum process of neutral cellulase finishing on characters of mercerizing cotton fabrics is determined as follows the dosage of cellulase is 2%(o. w. f), the pH value is 7. 5, the treating temperature is 50"C , and the time is 55min. The various aspects of performance of the mercerizing cotton fabrics under optimum process have improved. When the busting strength control is over 220N, it is still considered acceptable for use performance.

  1. Enhanced alkaline cellulases production by the thermohalophilic Aspergillus terreus AUMC 10138 mutated by physical and chemical mutagens using corn stover as substrate.

    Science.gov (United States)

    Isaac, George Saad; Abu-Tahon, Medhat Ahmed

    2015-01-01

    A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and β-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and β-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0-11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for β-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and β-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover.

  2. Cellulase and alcohol dehydrogenase immobilized in Langmuir and Langmuir-Blodgett films and their molecular-level effects upon contact with cellulose and ethanol.

    Science.gov (United States)

    Rodrigues, Dilmer; Camilo, Fernanda Ferraz; Caseli, Luciano

    2014-02-25

    The key challenges for producing devices based on nanostructured films with control over the molecular architecture are to preserve the catalytic activity of the immobilized biomolecules and to provide a reliable method for determining the intermolecular interactions and the accommodation of molecules at very small scales. In this work, the enzymes cellulase and alcohol dehydrogenase (ADH) were coimmobilized with dipalmitoylphosphatidylcholine (DPPC) as Langmuir-Blodgett (LB) films, and their biological activities were assayed by accommodating the structure formed in contact with cellulose. For this purpose, the polysaccharide was dissolved in an ionic liquid, 1-buthyl-3-methylimidazolium chloride (BMImCl), and dropped on the top of the hybrid cellulase-ADH-DPPC LB film. The interactions between cellulose and ethanol, which are the catalytic substrates of the enzymes as well as important elements in the production of second-generation fuels, were then investigated using polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS). Investigation of the secondary structures of the enzymes was performed using PM-IRRAS, through which the presence of ethanol and cellulose was observed to highly affect the structures of ADH and cellulase, respectively. The detection of products formed from the catalyzed reactions as well as the changes of secondary structure of the enzymes immobilization could be carried out, which opens the possibility to produce a means for producing second-generation ethanol using nanoscale arrangements.

  3. Effect of different pretreatment of sugar cane bagasse on cellulase and xylanases production by the mutant Penicillium echinulatum 9A02S1 grown in submerged culture.

    Science.gov (United States)

    Camassola, Marli; Dillon, Aldo J P

    2014-01-01

    The main limitation to the industrial scale hydrolysis of cellulose is the cost of cellulase production. This study evaluated cellulase and xylanase enzyme production by the cellulolytic mutant Penicillium echinulatum 9A02S1 using pretreated sugar cane bagasse as a carbon source. Most cultures grown with pretreated bagasse showed similar enzymatic activities to or higher enzymatic activities than cultures grown with cellulose or untreated sugar cane bagasse. Higher filter paper activity (1.253 ± 0.147 U · mL(-1)) was detected in the medium on the sixth day of cultivation when bagasse samples were pretreated with sodium hydroxide, hydrogen peroxide, and anthraquinone. Endoglucanase enzyme production was also enhanced by pretreatment of the bagasse. Nine cultures grown with bagasse possessed higher β -glucosidase activities on the sixth day than the culture grown with cellulose. The highest xylanase activity was observed in cultures with cellulose and with untreated sugar cane bagasse. These results indicate that pretreated sugar cane bagasse may be able to serve as a partial or total replacement for cellulose in submerged fermentation for cellulase production using P. echinulatum, which could potentially reduce future production costs of enzymatic complexes capable of hydrolyzing lignocellulosic residues to form fermented syrups.

  4. Effect of Different Pretreatment of Sugar Cane Bagasse on Cellulase and Xylanases Production by the Mutant Penicillium echinulatum 9A02S1 Grown in Submerged Culture

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    Marli Camassola

    2014-01-01

    Full Text Available The main limitation to the industrial scale hydrolysis of cellulose is the cost of cellulase production. This study evaluated cellulase and xylanase enzyme production by the cellulolytic mutant Penicillium echinulatum 9A02S1 using pretreated sugar cane bagasse as a carbon source. Most cultures grown with pretreated bagasse showed similar enzymatic activities to or higher enzymatic activities than cultures grown with cellulose or untreated sugar cane bagasse. Higher filter paper activity (1.253 ± 0.147 U·mL−1 was detected in the medium on the sixth day of cultivation when bagasse samples were pretreated with sodium hydroxide, hydrogen peroxide, and anthraquinone. Endoglucanase enzyme production was also enhanced by pretreatment of the bagasse. Nine cultures grown with bagasse possessed higher β-glucosidase activities on the sixth day than the culture grown with cellulose. The highest xylanase activity was observed in cultures with cellulose and with untreated sugar cane bagasse. These results indicate that pretreated sugar cane bagasse may be able to serve as a partial or total replacement for cellulose in submerged fermentation for cellulase production using P. echinulatum, which could potentially reduce future production costs of enzymatic complexes capable of hydrolyzing lignocellulosic residues to form fermented syrups.

  5. Process optimisation for the biosynthesis of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3 using submerged fermentation.

    Science.gov (United States)

    Abdullah, Roheena; Zafar, Wajeeha; Nadeem, Muhammad; Iqtedar, Mehwish; Naz, Shagufta; Syed, Quratulain; Kaleem, Afshan

    2015-01-01

    This study deals with optimisation of cultural conditions for enhanced production of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3. Influence of different variables including incubation time, temperature, inoculum size, pH, nitrogen sources and metal ions has been studied. The optimum conditions for cellulase production were incubation period of 72 h, inoculum size 4% incubation temperature 37°C, pH 7, 0.25% ammonium sulphate, 0.2% peptone as inorganic and organic nitrogen source in case of Bacillus PC-BC6. In case of mutant Bacillus N3, optimal conditions were incubation period of 48 h, incubation temperature 37°C, inoculum size 3%, pH 7, 0.2% ammonium chloride and 0.15% yeast extract. Presence of MnSO4 and CaCl2 enhances the enzyme production by Bacillus PC-BC6 and mutant Bacillus N3, respectively. This study was innovative and successful in producing cellulase economically by using cheap indigenous substrate Saccharum spontaneum.

  6. Polishing of cotton fabric with acidic cellulase%棉织物的酸性纤维素酶抛光工艺

    Institute of Scientific and Technical Information of China (English)

    刘磊; 陆必泰

    2012-01-01

    以酸性纤维素酶HT-218对棉织物的表面进行抛光整理,对影响抛光效果的各种因素(酶用量、温度、pH值、处理时间、浴比)进行了试验分析,通过对处理后棉织物的质量损失率、胀破强度及抗起毛起球性的测定和评价,确定优化的酸性纤维素酶抛光整理工艺条件为:酶用量0.6%(omf),温度50℃,pH值4.5,处理时间55min,浴比1:10.%Polishing of cotton fabric is carried out with acid cellulase HT-218. The influences of dosage of cellulase, working temperature and time, pH value, as well as bath liquor on polishing effects are investigated. The polishing process conditions are optimized by evaluating the weight loss rate, tensile strength and anti pilling property, and the processing conditions are optimized as follows.- cellulase HT-218 0.6 % (omf), working at 50℃ for 55 min with pH value 4.5 and bath ratio 1:10.

  7. [Cultivation of a novel cellulase/xylanase producer, Trichoderma longibrachiatum mutant TW 1-59-27: production of the enzyme preparation and the study of its properties].

    Science.gov (United States)

    Bekkarevicha, A O; Nemashkalov, V A; Koshelev, A V; Goryazchev, D A; Bubnova, T V; Matys, V Yu; Osipov, D O; Kondrat'eva, E G; Okunev, O N; Sinitsyn, A P

    2015-01-01

    As a result of gamma-mutagenesis of Trichoderma longibrachiatum TW1 and the subsequent selection of improved producers, a novel mutant strain, TW1-59-27, capable of efficiently secreting cellulase and xylanase was obtained. In a fed-batch cultivation, the new TW1-59-27 mutant was significantly more active compared with the original TW1 strain. For instance, the activities of cellulase (towards carboxymethylcellulose) and xylanase in the culture broth (CB) increased by 1.8 and two times, respectively, and the protein content increased by 1.47 times. The activity of these enzymes in the dry enzyme preparation derived from the CB of the TW1-59-27 mutant was 1.3-1.8 times higher than that in the preparation derived from the original TW1 strain. It was established that the cellulase from the enzyme preparation of the mutant strain demonstrated the maximum activity at 55-65 degrees C; it occurred in xylanase at 60 degrees C. The pH optima of these enzymes were pH 4.5-5.0 and pH 5.0-6.0, respectively. It was shown that the content of endoglucanases in the enzyme preparation increased from 7% to 13.5%; the effect is largely driven by the secretion of endoglucanase-1. An enzyme preparation with increased endoglucanase-1 content is promising for use as a feed additive in agriculture.

  8. Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family.

    Science.gov (United States)

    Tucker, M L; Durbin, M L; Clegg, M T; Lewis, L N

    1987-05-01

    A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.

  9. Changes in fungal population of fly ash and vinasse mixture during vermicomposting by Eudrilus eugeniae and Eisenia fetida: documentation of cellulase isozymes in vermicompost.

    Science.gov (United States)

    Pramanik, Prabhat; Chung, Young Ryun

    2011-06-01

    Fly ash (FA) and vinasse (VN), two industrial wastes, are generated in huge amounts and cause serious hazards to the environment. In this experiment, different proportions of these two wastes were used as food for two epigeic earthworms (Eisenia fetida and Eudrilus eugeniae) to standardize the recycling technique of these two wastes and to study their effect on fungal especially cellulolytic fungal population, cellulase activity and their isozyme pattern, chitin content and microbial biomass of waste mixture during vermicomposting. Increasing VN proportion from 25% to 50% or even higher, counts of both fungi and cellulolytic fungi in waste mixtures were significantly (P ≤ 0.05) increased during vermicomposting. Higher cellulase activity in treatments having 50% or more vinasse might be attributed to the significantly (P ≤ 0.05) higher concentration of group I isozyme while concentrations of other isozymes (group II and III) of cellulase were statistically at par. Higher chitin content in vinasse-enriched treatments suggested that fungal biomass and fungi-to-microbial biomass ratio in these treatments were also increased due to vermicomposting. Results revealed that Eudrilus eugeniae and Eisenia fetida had comparable effect on FA and VN mixture during vermicomposting. Periodical analysis of above-mentioned biochemical and microbial properties and nutrient content of final vermicompost samples indicated that equal proportion (1:1, w/w) of FA and VN is probably the optimum composition to obtain best quality vermicompost.

  10. The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

    Directory of Open Access Journals (Sweden)

    Pakula Tiina

    2011-05-01

    Full Text Available Abstract Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1+Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in

  11. ALKALINE PROTEASE, AMYLASE AND CELLULASE ACTIVITIES OF YELLOW RASBORA, Rasbora lateristriataBlkr., AT DIFFERENT FEEDING LEVELS

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    Untung Susilo

    2016-11-01

    Full Text Available Alkaline proteases, amylase and cellulase activities of digestive organ of yellow rasbora, Rasbora lateristriata Blkr., was evaluated with four different feeding levels of 0.34g protein+0.03g fiber, 1.01g protein+0.10g fiber, 1.69g protein+0.16g fiber and 2.36g protein+0.23g fiber/day/100 g fish biomass. A total of 280 fish with average body weight of 0.71±0.06g were used in this study. The results showed that the difference in the feeding levels resulted in a significant difference in trypsin and chymotrypsin  activities (P .05. In conclusion, protein digestion capacity increased, but not to the digestion of starch and fiber in response to different feeding levels and the optimal feeding level for yellow rasbora was 1,01g protein+0,10g fiber/day/100 g fish biomass.

  12. Biophysical characterization and activity analysis of nano-magnesium supplemented cellulase obtained from a psychrobacterium following graphene oxide immobilization.

    Science.gov (United States)

    Dutta, N; Biswas, S; Saha, M K

    2016-12-01

    Cellulase enzyme was purified from a psychrophilic strain of Bacillus subtilis obtained from east Himalayan mountains. The native enzyme showed optimum activity at 15°C and pH 8.0.The Magnesium oxide nanoparticle (MgN) supplemented enzyme when immobilized on graphene oxide nanosupport (GO), via glutaraldehyde as cross linker, showed 2.98 folds increase in enzymatic activity at 8°C and more than 3.5 folds activity increment at 90°C. The MgN-cel on graphene (GO-MgN-cel) showed a decrease in Km by 6.7 folds at 8°C and 34 folds at 90°C. GO-MgN-cel showed 5 fold and 4.7 fold increase in Vmax at 8°C and 90°C respectively than the untreated enzyme.When compared to native enzyme, GO-MgN-cel had t1/2 (half life) and Ed increased by 72.5 fold and 2.48 fold respectively at 90°C; and 41.6 fold and 2.19 fold respectively at 8°C. Enzymatic activity of GO-MgN-cel was retained even after 12 repeated uses and showed storage stability at 4°C for more than 120days. This nanoparticle assisted immobilization technique can be utilized in bioprocessing industries which require functioning at these extreme ranges of temperature.

  13. Use of recombinant cellulose-binding domains of Trichoderma reesei cellulase as a selective immunocytochemical marker for cellulose in protozoa.

    Science.gov (United States)

    Linder, Markus; Winiecka-Krusnell, Jadwiga; Linder, Ewert

    2002-05-01

    Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent beta-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.

  14. Artificial Intelligence Techniques to Optimize the EDC/NHS-Mediated Immobilization of Cellulase on Eudragit L-100

    Directory of Open Access Journals (Sweden)

    Min-Chao He

    2012-06-01

    Full Text Available Two artificial intelligence techniques, namely artificial neural network (ANN and genetic algorithm (GA were combined to be used as a tool for optimizing the covalent immobilization of cellulase on a smart polymer, Eudragit L-100. 1-Ethyl-3-(3-dimethyllaminopropyl carbodiimide (EDC concentration, N-hydroxysuccinimide (NHS concentration and coupling time were taken as independent variables, and immobilization efficiency was taken as the response. The data of the central composite design were used to train ANN by back-propagation algorithm, and the result showed that the trained ANN fitted the data accurately (correlation coefficient R2 = 0.99. Then a maximum immobilization efficiency of 88.76% was searched by genetic algorithm at a EDC concentration of 0.44%, NHS concentration of 0.37% and a coupling time of 2.22 h, where the experimental value was 87.97 ± 6.45%. The application of ANN based optimization by GA is quite successful.

  15. Novel Cellulase Profile of Trichoderma reesei Strains Constructed by cbh1 Gene Replacement with eg3 Gene Expression Cassette

    Institute of Scientific and Technical Information of China (English)

    Tian-Hong WANG; Ti LIU; Zhi-Hong WU; Shi-Li LIU; Yi LU; Yin-Bo QU

    2004-01-01

    To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbh1 gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transforments denoted as L 13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbh1 promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential industrial applications.

  16. Downregulation of cinnamyl-alcohol dehydrogenase in switchgrass by RNA silencing results in enhanced glucose release after cellulase treatment.

    Directory of Open Access Journals (Sweden)

    Aaron J Saathoff

    Full Text Available Cinnamyl alcohol dehydrogenase (CAD catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. "Alamo" with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the T-DNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol-HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin.

  17. Physiological and Biochemical Characteristics of Cellulase Producing Bacteria from Fermented Grains and Correlation with the Cellulase Activity%酒醅中纤维素酶产生菌的生理生化特性及与其产酶活力的相关性

    Institute of Scientific and Technical Information of China (English)

    郭建华; 刘冀; 郭宏文; 刘晓兰; 贾士儒

    2012-01-01

    以白酒酒醅中筛选出的产纤维素酶的细菌为研究对象,测定了这些细菌的细胞长宽比、糖发酵实验、淀粉水解实验等生理生化特性指标和它们产纤维素酶活力的大小,并利用相关分析和主成分分析研究了生理生化特性指标与纤维素酶活力之间的关系。相关分析结果表明,纤维素酶产生菌的各种生理生化特性指标之间,及这些生理生化特性指标与细菌产纤维素酶活力之间均存在着不同程度的相关性。主成分分析结果表明,纤维素酶活力与细胞长宽比和利用葡萄糖产酸之间存在着明显的负相关性,而与其淀粉水解能力、利用乳糖产酸和柠檬酸盐试验指标之间存在明显的正相关性。%Using cellulase producing bacteria from liquor fermented grains as study object, physiological and bio- chemical characteristics such as cell aspect ratio, sugar fermentation experiments, starch hydrolysis experiments, and cellulase activity of cellulase producing bacteria was measured, and a correlation between the physiological and bio- chemical characteristics and cellulose enzyme activity was studied using correlation analysis and principal component analysis. The results of correlation analysis indicated that there were different degrees of correlation among indicators of physiological and biochemical characteristics, and between cellulase activity of cellulase producing bacteria and in- dicators of physiological and biochemical characteristics. The results of principal component analysis indicated that cellulase activity had a clear negative correlation with cell aspect ratio and producing acid using glucose,while had a positive correlation with starch hydrolysis, producing acid using lactose and citrate test indicators.

  18. [Studies of the genetic regulation of the Thermomonospora cellulase complex]. Progress report, June 1, 1990--January 10, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, D.B.

    1992-08-01

    The goals of this project are to determine the molecular mechanisms regulating cellulose synthesis in the soil bacterium Thermomonosporafusca and to determine the molecular mechanism by which T.fusca cellulases degrade crystalline cellulose. We have determined a structure for the T.fusca E{sub 2} catalytic subunit (E{sub 2}-30) by x-ray crystallography. This structure is quite similar to that of T.reesei CBHU but there are a number of differences. One is that the E{sub 2} active site is in a cleft while that of CBHII is in a tunnel. This is an expected result since E{sub 2} is an endocellulase. Large amounts of homogenous E{sub 5} catalytic subunit have been prepared and attempts to crystallize it are underway. Crystals of E{sub 2}-30 were soaked in cellobiose and modified crystals detracted well, however difference Fourier analysis showed many changes, so that we could not localize cellobiose in the 3-D structure of E{sub 2}-30. This implies that binding of cellobiose causes a significant change in the structure of E{sub 2}-30. The stereochemistry of the cleavage catalyzed by E{sub l}, E{sub 2} and E{sub 5} was determined in collaboration with Dr. Stephen Withers and E{sub 1} and 2 inverted the glycoside linkage while E{sub 5} does not. The entire E{sub l} and E{sub 4} genes have been induced into Streptomyces lividans where they are expressed at a high level and the E{sub l} and E{sub 4} are completely secreted into the medium. Studies on the synergism between the exocellulase E{sub 3} and the endocellulases E{sub 2} or E{sub 5} show that both exo and endocellulase activities are stimulated when they are assayed together.

  19. Identification of a laccase from Ganoderma lucidum CBS 229.93 having potential for enhancing cellulase catalyzed lignocellulose degradation.

    Science.gov (United States)

    Sitarz, Anna K; Mikkelsen, Jørn D; Højrup, Peter; Meyer, Anne S

    2013-12-10

    Based on a differential pre-screening of 44 white-rot fungi on a lignocellulose-supplemented minimal medium, four basidiomycetes were selected for further study: Ganoderma lucidum, Polyporus brumalis, Polyporus ciliatus and Trametes versicolor. Only G. lucidum was able to grow vividly on malt extract or minimal media supplemented with alkali lignin. When grown on malt extract or minimal medium supplemented with lignocellulose (sugar cane bagasse), the crude G. lucidum protein extract exhibited high laccase activity, ∼3U/mL toward syringaldazine. This activity was 13-17 fold higher than the corresponding activities of the crude protein extracts of P. brumalis, P. ciliatus and T. versicolor. Native PAGE electrophoresis of the crude G. lucidum extract confirmed the presence of an active laccase. The G. lucidum laccase had a molecular weight of ∼62.5kDa, and a Km value of 0.107mM (determined on ABTS). A partial amino acid sequence analysis of four short de novo sequenced peptides, defined after trypsin digest analysis using MALDI-TOF MS/MS analysis, revealed 64-100% homology to sequences in related laccases in the UniProt database, but also indicated that certain sequence stretches had low homology. Addition of the laccase-rich G. lucidum broth to lignocellulosic biomass (pretreated sugar cane bagasse) together with a state-of-the-art cellulase enzyme preparation (Cellic™CTec1) produced significantly increased cellulolytic yields, which were also better than those obtained with a T. versicolor laccase addition, indicating that the laccase from G. lucidum has unique properties that may be momentous in lignocellulosic biomass conversion.

  20. A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, Venkataramanan; Schuster, Logan A.; Moore, Kyle T.; Taylor, Larry E.; Baker, John O.; Vander Wall, Todd A.; Linger, Jeffrey G.; Himmel, Michael E.; Decker, Stephen R.

    2017-02-06

    The industrial workhorse fungus, Trichoderma reesei, is typically exploited for its ability to produce cellulase enzymes, whereas use of this fungus for over-expression of other proteins (homologous and heterologous) is still very limited. Identifying transformants expressing target protein is a tedious task due to low transformation efficiency, combined with highly variable expression levels between transformants. Routine methods for identification include PCR-based analysis, western blotting, or crude activity screening, all of which are time-consuming techniques. To simplify this screening, we have adapted the 2A peptide system from the foot-and-mouth disease virus (FMDV) to T. reesei to express a readily screenable marker protein that is co-translated with a target protein. The 2A peptide sequence allows multiple independent genes to be transcribed as a single mRNA. Upon translation, the 2A peptide sequence causes a 'ribosomal skip' generating two (or more) independent gene products. When the 2A peptide is translated, the 'skip' occurs between its two C-terminal amino acids (glycine and proline), resulting in the addition of extra amino acids on the C terminus of the upstream protein and a single proline addition to the N terminus of the downstream protein. To test this approach, we have cloned two heterologous proteins on either side of a modified 2A peptide, a secreted cellobiohydrolase enzyme (Cel7A from Penicillium funiculosum) as our target protein, and an intracellular enhanced green fluorescent protein (eGFP) as our marker protein. Using straightforward monitoring of eGFP expression, we have shown that we can efficiently monitor the expression of the target Cel7A protein.

  1. Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates

    Directory of Open Access Journals (Sweden)

    Saddler Jack N

    2011-02-01

    Full Text Available Abstract A range of lignocellulosic feedstocks (including agricultural, softwood and hardwood substrates were pretreated with either sulfur dioxide-catalyzed steam or an ethanol organosolv procedure to try to establish a reliable assessment of the factors governing the minimum protein loading that could be used to achieve efficient hydrolysis. A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass (poplar, douglas fir and lodgepole pine. Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic

  2. Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes

    Directory of Open Access Journals (Sweden)

    Eman Zakaria Gomaa

    2013-01-01

    Full Text Available The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97% of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

  3. Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes.

    Science.gov (United States)

    Gomaa, Eman Zakaria

    2013-01-01

    The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg(2+), Fe2+ and Ag(+) showed a stimulatory effect on protease activity and ions of Fe(2+), Mg(2+), Ca(2+), Cu(2+) and Ag(+) caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

  4. Methane production of two roughage and total mixed ration as influenced by cellulase and xylanase enzyme addition

    Directory of Open Access Journals (Sweden)

    Belete Shenkute Gemeda

    2015-02-01

    Full Text Available In recent decades supplementation of animal feeds with exogenous fibrolytic enzymes has substantially improved digestibility and animal performance. However, information related to associated methane production is limited and inconsistent. This study evaluated the effect of cellulase and xylanase enzymes on in vitro methane production of Eragrostis curvula hay, maize (Zea mays stover and a total mixed ration (TMR at seven levels of the two enzymes. Feed samples were incubated for 2, 12, 24 and 48 h in an in vitro batch culture with buffer and rumen fluid, and fibrolytic enzymes. Gas production was measured using a pressure transducer connected to a data tracker, while methane gas was analysed using a gas chromatograph which was calibrated with standard CH4 and CO2. Increases in the level of enzyme application resulted in increases in gas volume, total volatile fatty acid (VFA production, dry matter (DM disappearance and associated increases in methane production. The linear increase in percentage and volume of methane production in tandem with increases in level of enzyme application might be due to increased fermentation, and organic matter degradability that resulted in a shift in VFA production towards acetate. Considering the efficiency of DM and neutral detergent fiber degradation and production of associated VFA with levels of enzymes, the use of 1 mg g−1 DM of enzyme can be a good option for the feeds tested. However, they cannot decrease methane production. It will be very important to consider other hydrogen sinks that can capture directly extra H+ produced by the addition of enzyme so that their supplementation could be very efficient and environmentally sound.

  5. Isolation and Characterization of Thermophilic Cellulase-Producing Bacteria from Empty Fruit Bunches-Palm Oil Mill Effluent Compost

    Directory of Open Access Journals (Sweden)

    Azhari S. Baharuddin

    2010-01-01

    Full Text Available Problems statement: Lack of information on locally isolated cellulase-producing bacterium in thermophilic compost using a mixture of Empty Fruit Bunch (EFB and Palm Oil Mill Effluent (POME as composting materials. Approach: The isolation of microbes from compost heap was conducted at day 7 of composting process where the mixture of composting materials consisted of 45.8% cellulose, 17.1% hemicellulose and 28.3% lignin content. The temperature, pH and moisture content of the composting pile at day 7 treatment were 58.3, 8.1 and 65.5°C, respectively. The morphological analysis of the isolated microbes was conducted using Scanning Electron Microscope (SEM and Gram stain method. The congo red test was conducted in order to detect 1% CMC agar degradation activities. Total genomic DNAs were extracted from approximately 1.0 g of mixed compost and amplified by using PCR primers. The PCR product was sequent to identify the nearest relatives of 16S rRNA genes. The localization of bacteria chromosomes was determined by Fluorescence In Situ Hybridization (FISH analysis. Results: Single isolated bacteria species was successfully isolated from Empty Fruit Bunch (EFB-Palm Oil Mill Effluent (POME compost at thermophilic stage. Restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs with the phylogenetic analysis showed that the isolated bacteria from EFB-POME thermophilic compost gave the highest homology (99% with similarity to Geobacillus pallidus. The strain was spore forming bacteria and able to grow at 60°C with pH 7. Conclusion: Thermophilic bacteria strain, Geobacillus pallidus was successfully isolated from Empty Fruit Bunch (EFB and Palm Oil Mil Effluent (POME compost and characterized.

  6. Controlled production of cellulases in plants for biomass conversion. Annual report, March 11, 1997--March 14, 1998

    Energy Technology Data Exchange (ETDEWEB)

    Danna, K.J.

    1998-06-01

    The goal of this project is to facilitate conversion of plant biomass to usable energy by developing transgenic plants that express genes for microbial cellulases, which can be activated after harvest of the plants. In particular, the feasibility of targeting an endoglucanase and a cellobiohydrolase to the plant apoplast (cell wall milieu) is to be determined. To avoid detrimental effects of cellulose expression in plants, enzymes with high temperature optima were chosen; the genes for these enzymes are from thermophilic organisms that can use cellulose as a sole energy source. During the past year (year 2 of the grant), efforts have been focused on testing expression of endoglucanase E{sub 1}, from Acidothermus cellulolyticus, in the apoplast of both tobacco suspension cells and Arabidopsis thaliana plants. Using the plasmids constructed during the first year, transgenic cells and plants that contain the gene for the E{sub 1} catalytic domain fused to a signal peptide sequence were obtained. This gene was constructed so that the fusion protein will be secreted into the apoplast. The enzyme is made in large quantities and is secreted into the apoplast. More importantly, it is enzymatically active when placed under optimal reaction conditions (high temperature). Moreover, the plant cells and intact plants exhibit no obvious problems with growth and development under laboratory conditions. Work has also continued to improve binary vectors for Agrobacterium-mediated transformation, to determine activity of E{sub 1} at various temperatures, and to investigate the activity of the 35S Cauliflower Mosaic Virus promoter in E. coli. 9 figs.

  7. 白蚁内源性纤维素酶基因资源研究进展%Research Advances on Endogenous Cellulase Gene Resources of Termites

    Institute of Scientific and Technical Information of China (English)

    刘小琳; 李志强; 张丹丹

    2015-01-01

    Energy shortage has become a global problem, and cellulose is the most abundant and renewable resource in nature. Termites have evolved the unique and efficient cellulose-digesting system, in which there are rich resources of cellulase and its genes. In recent years, the significance of endogenous cellulose-digesting system in termite has been recognized gradually, and the researches on the endogenous cellulase genes have been reported continuously. In order to improve the new control technologies of termite pests and to explore the cellulosic biomass for biofuels, the review provides the information for the cloning and expressions of termite endogenous cellulase genes.%能源短缺是人类关注的焦点问题之一,而纤维素是自然界最为丰富的可再生资源。白蚁已进化出了独特而高效的纤维素消化系统,具有丰富的纤维素酶及其基因资源,因而,近年来白蚁内源性纤维素消化体系的重要性被逐渐认识,不断有内源性纤维素酶基因的研究报道。为了进一步推动有害白蚁控制新技术的研发,以及纤维素生物质新能源的应用探索,综述了白蚁内源性纤维素酶基因克隆、表达等研究进展。

  8. 纤维素酶产生菌的诱变与筛选%Mutation and Screening for Cellulase Production Strain

    Institute of Scientific and Technical Information of China (English)

    王美珠; 曹月坤; 陈文艺; 姚秀清; 李妍妍

    2012-01-01

    [目的]为提高纤维素酶产生菌的酶活力.[方法]以抚顺近郊堆积腐烂秸秆的土壤为分离源,利用CMC刚果红平板培养基筛选出纤维素水解圈与菌落直径比值(Hc值)较大的菌株Y-07.以Y-07为出发菌株,在最适诱变剂量条件下,对其进行紫外线诱变和亚硝酸诱变.[结果]经过3轮复筛,紫外线诱变后的U10菌株为试验中获得的高产纤维素酶菌株,其酶活最高达到2.499 IU/ml,其活性是Y-07的3.41倍.[结论]该方法为进一步提高纤维素酶产生菌的酶活力奠定了基础.%[Objective] The research aimed to improve enzyme activity of cellulase production strain. [Method] A strain named Y-07 was isolated from rotten straw soil nearby the suburb of Fushun with culture medium containing the carboxymethylcellulose sodium and Congo Red in accordance with its larger size of ratio of hydrolysis ring to colony diameter. With the strain of Y-07 as starting Strain, the strain of Y-07 was mutagenized by ultraviolet radiation and nitrous acid mutagenesis under the most appropriate amount of mutation. [Result] After three rounds of secondary screening, the strain of U10 was achieved with the biggest cellulase activity 2. 5 IU/ml which was 3.4 times of Y-07 strain. [ Conclusion] The method provided the basis for further improvements of enzyme activity of cellulase production strain.

  9. Hydrolysis of a mixture of saccharides by cellulase from Aspergillus niger and its application for visible-light-induced hydrogen gas production system using Mg chlorophyll-a and platinum nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Amao, Yutaka; Hirakawa, Takamasa [Department of Applied Chemistry, Oita University, Dannoharu 700, Oita 870-1192 (Japan)

    2010-07-15

    Cellulase obtained from Aspergillus niger was used to hydrolyze a mixture of saccharides containing sucrose, maltose, and cellobiose; the reduced form of nicotinamide-adenine dinucleotide (NAD{sup +}), which is NADH, was produced during hydrolysis of the mixture of saccharides in the presence of NAD{sup +} and glucose dehydrogenase (GDH). We have developed a visible-light-induced enzymatic biohydrogen production system involving the combination of cellulase-mediated hydrolysis of the mixture of saccharides and hydrogen production by platinum nanoparticles using photosensitization of Mg chlorophyll-a (Mg Chl-a). Continuous production of hydrogen gas was observed when the reaction mixture containing saccharides, cellulase, GDH, NAD{sup +}, Mg Chl-a, methylviologen (MV{sup 2+}, an electron donor), and platinum nanoparticles was irradiated by visible light. After 120 min of irradiation, the amount of hydrogen produced from the mixture of saccharides was approximately 2.8 {mu}mol. (author)

  10. 纤维素酶蛋白质工程的新进展%The Recent Advances in Protein Engineering of Cellulases

    Institute of Scientific and Technical Information of China (English)

    赵喜华; 涂宗财; 魏东芝; 王玮; 唐平平

    2015-01-01

    Parent cellulases exist in some defects of a low catalytic activity,inhibition of end products,weak thermo-stability,and poor processivity,and therefore improving enzymatic properties of cellulases is a very meaningful job. To date,on the basis of the technology of evolution in vitro,it is an effective way to develop enzymatic properties of cellulases. Recent advances in protein engineering about three different regions of cellulases which consist of catalyt-ic domains,binding domains and linker regions are reviewed in the paper. The idea about systematically assembling functional structure domains which have been engineered was put forward to in the paper,which will help to improve the enzymatic properties such as catalytic activity,the ability of processive hydrolysis,resistantance to high concen-tration of product,and thermalstability,and to promote high-efficent application in energy,industry,agriculture, food,and so on.%原始纤维素酶通常存在催化活性低下、末端产物抑制、热稳定性不高、持续催化能力弱等问题,因此,改善它的酶学性质是很有意义的工作。目前,采用体外进化技术是改进纤维素酶酶学性质的一种有效途径。从纤维酶的催化域、结合域和连接区等不同区域出发,综述了纤维素酶蛋白质工程的新进展。提出了系统组装3个已改造功能结构域的思想,这有助于提高纤维素酶催化活性、持续催化能力、耐高浓度产物、热稳定性等特性,将促进纤维素酶在能源、工业、农业、食品等领域的高效应用。

  11. Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

    Science.gov (United States)

    Wang, Shu-Yang; Jiang, Bo-Ling; Zhou, Xiang; Chen, Ji-Hong; Li, Wen-Jian; Liu, Jing; Hu, Wei; Xiao, Guo-Qing; Dong, Miao-Yin; Wang, Yu-Chen

    2015-01-01

    The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and β-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.

  12. 一种来源于康氏木霉的纤维素酶增效蛋白的分离纯化%Purification of a cellulase activity enhancing protein originated from Trichoderma Koningii

    Institute of Scientific and Technical Information of China (English)

    覃益民; 高进; 刘幽燕; 唐爱星

    2011-01-01

    This research was to prove whether there is some special protein in cellulase-producing microorganism acting as a cellulase-activity-enhancing protein, thus exploring its potential application in lignocellulose hydrolysis. An extracellular protein without cellulase activity was separated and purified from culture medium of Trichoderma Koningii through a series of procedures, including ultrafiltration, ammonium sulfate precipitation, octyl-Sepharose CL-4B and Sephadex G-75 column chromatography. The molecular weight of the prtotein was estimated to be 30 X 103 by SDS-PAGE. With the addition of this protein (400μg per g filter paper) into the reaction mixture containing filter paper and cellulase (0. 06 IU per g filter paper ), the reducing sugar released during 48 h was observed to increase greatly, 104. 9% greater than that of the control hydrolysis. Moreover, it was found that this purified protein enhanced the activity of exoglucanase, one of the three components of fungi cellulase, while endoglucanase and β-glucosidase activities did not change much. Mass spectrometry analysis indicated that this purified protein was an unknown protein. Based on these results, this purified extracellular protein originated from Trichoderma Koningii could be regarded as a cellulase activity-enhancing protein, which was different from the reported plant expansin and swollenin.%引言在可持续发展、低碳经济的背景下,利用纤维素酶水解木质纤维原料生产燃料乙醇无疑是一项符合我国国情的能源战略[1].然而纤维素酶的水解效率低是制约纤维素燃料乙醇生产的最大障碍.

  13. Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production.

    Science.gov (United States)

    Delabona, Priscila da Silva; Farinas, Cristiane Sanchez; da Silva, Mateus Ribeiro; Azzoni, Sindelia Freitas; Pradella, José Geraldo da Cruz

    2012-03-01

    The on-site production of cellulases is an important strategy for the development of sustainable second-generation ethanol production processes. This study concerns the use of a specific cellulolytic enzyme complex for hydrolysis of pretreated sugar cane bagasse. Glycosyl hydrolases (FPase, xylanase, and β-glucosidase) were produced using a new strain of Trichoderma harzianum, isolated from the Amazon rainforest and cultivated under different conditions. The influence of the carbon source was first investigated using shake-flask cultures. Selected carbon sources were then further studied under different pH conditions using a stirred tank bioreactor. Enzymatic activities up to 121 FPU/g, 8000 IU/g, and 1730 IU/g of delignified steam-exploded bagasse+sucrose were achieved for cellulase, xylanase and β-glucosidase, respectively. This enzymatic complex was used to hydrolyze pretreated sugar cane bagasse. A comparative evaluation, using an enzymatic extract from Trichoderma reesei RUTC30, indicated similar performance of the T. harzianum enzyme complex, being a potential candidate for on-site production of enzymes.

  14. Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger.

    Science.gov (United States)

    Cavka, Adnan; Alriksson, Björn; Rose, Shaunita H; van Zyl, Willem H; Jönsson, Leif J

    2014-08-01

    Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.

  15. OPTIMIZATION OF CELLULASE-FREE XYLANASE PRODUCED BY A POTENTIAL THERMOALKALOPHILIC PAENIBACILLUS SP.N1 ISOLATED FROM HOT SPRINGS OF NORTHERN HIMALAYAS IN INDIA

    Directory of Open Access Journals (Sweden)

    Sanjeev Kumar Verma

    2012-08-01

    Full Text Available Hot spring bacteria are found a novel source of highly active xylanase enzyme with significant activity at high temperature. Among bacteria, Paenibacillus sp.N1 isolated from hot water spring of Manikaran, H.P., India showed highest 24.60 IU.ml-1 of cellulase-free xylanase on Reese medium. Growth conditions including medium, incubation time, pH, temperature, inoculum size, aminoacids, carbon sources, nitrogen sources and additives that affect the xylanase production by Paenibacillus sp.N1 were studied sequentially using the classical “change-one factor at a time” method. The optimal cultivation conditions predicated from canonical analysis of this model were achieved by using basal salt medium on 3rd day, pH 9.0, temperature 50ºC with inoculum size of 12.5%, phenylalanine as aminoacid, xylose as carbon source, (NH42HPO4 as nitrogen source and Tween 20 as detergent added with an approximate yield of 52.30 IU.ml-1 escalating the over level of xylanase production by 113.38%. A rare combination of all characters i.e. thermoalkalophilic nature and high units of cellulase-free xylanase produced from a new Paenibacillus sp.N1 make it of special industrial interest.

  16. Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module.

    Science.gov (United States)

    Kurokawa, J; Hemjinda, E; Arai, T; Kimura, T; Sakka, K; Ohmiya, K

    2002-08-01

    The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548-554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains. CelT devoid of the dockerin domain (CelTDeltadoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTDeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan, and low activity toward xylan. The V(max) and K(m) values were 137 micro mol min(-1) mg(-1) and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM.

  17. Gene targeting by RNAi-mediated knockdown of potent DNA ligase IV homologue in the cellulase-producing fungus Talaromyces cellulolyticus.

    Science.gov (United States)

    Hayata, Koutarou; Asada, Seiya; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Sawayama, Shigeki

    2014-11-01

    The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

  18. 丝状真菌中纤维素酶与半纤维素酶的合成调控%Regulation of cellulase and hemicellulase production in filamentous fungi

    Institute of Scientific and Technical Information of China (English)

    王方忠; 蒋艺; 刘奎美; 姜宝杰; 王明钰; 方诩

    2014-01-01

    Research advances in transcriptional regulation of cellulase and hemicellulase in filamentous fungi were reviewed.The external signal molecules ( carbon source, light ) , transcription factors and chromatin remodeling had important influence on the regulation of cellulase and hemicellulase synthesis in filamentous fungi.Understanding the regulation of the synthesis of cellulases and hemicellulases in filamentous fungi provided the theoretical basis for genetically engineering industrial strains for the improvement of cellulase and hemicellulase production.%综述了丝状真菌合成纤维素酶和半纤维素酶的相关调控研究进展。对最近研究文章分析发现:细胞外信号分子(C源、光信号),转录因子以及染色质重建等对丝状真菌合成调控纤维素酶及半纤维素酶有重要影响。同时解析丝状真菌合成纤维素酶和半纤维素酶调控网络,以期为利用基因工程改造纤维素酶和半纤维素酶生产工业菌株提供理论指导。

  19. Study on Carrot Fiber Modification with Cellulase and Hemicellulase%纤维素酶和半纤维素酶改性胡萝卜纤维的研究

    Institute of Scientific and Technical Information of China (English)

    刘欢; 贺连斌; 魏静; 马中苏

    2011-01-01

    This paper studied the influence of cellulase and hemicellulase on the length, width, sugar content and polymerization degree of carrot pomace fiber. The mechanism of synergy of cellulase and hemicellulase modified carrot fiber was analyzed. The result indicated that the complex application of cellulase and hemicellulase was more obvious effect than the single enzyme. And the synergism of cellulase and hemicellulase could improve the thin fibrosis of the fiber surface, enhance the production rate of enzymolysis fiber, and reduce the polymerization degree of the fiber.%以胡萝卜渣中的纤维为原材料,研究纤维素酶和半纤维酶对胡萝卜纤维的长度、宽度以及还原糖浓度和聚合度的影响,并对在胡萝卜纤维改性过程中纤维素酶和半纤维酶协同作用的机理进行探讨.结果表明,纤维素酶和半纤维素酶复合使用比单一酶对胡萝卜纤维改性效果明显.在两者的协同作用下,可以改善纤维表面的细纤维化,提高纤维的酶解产率,降低纤维的聚合度.

  20. Multifunctional cellulase and hemicellulase

    Energy Technology Data Exchange (ETDEWEB)

    Fox, Brian G.; Takasuka, Taichi; Bianchetti, Christopher M.

    2015-09-29

    A multifunctional polypeptide capable of hydrolyzing cellulosic materials, xylan, and mannan is disclosed. The polypeptide includes the catalytic core (cc) of Clostridium thermocellum Cthe_0797 (CelE), the cellulose-specific carbohydrate-binding module CBM3 of the cellulosome anchoring protein cohesion region (CipA) of Clostridium thermocellum (CBM3a), and a linker region interposed between the catalytic core and the cellulose-specific carbohydrate binding module. Methods of using the multifunctional polypeptide are also disclosed.

  1. 高产纤维素酶的绿色木霉菌种的诱变和筛选%Screening for a Novel Trichoderma vride Strain Highly Producing Cellulase via Ultraviolet Mutagenesis

    Institute of Scientific and Technical Information of China (English)

    屈二军; 谢展; 马孟星; 张亚飞

    2011-01-01

    [目的]诱变和筛选高产纤维素酶的绿色木霉菌种。[方法]利用紫外线对出发菌株进行诱变,经过初筛和发酵检测挑选出高产纤维素酶的菌株。[结果]筛选到高产纤维素酶的绿色木霉K6,酶活是出发菌株的1.39倍。[结论]诱变筛选得到的K6菌株高产纤维素酶,为秸秆纤维素的利用奠定了基础。%[Objective] The aim of this study was to find out a new Trichoderma vride K strain highly producing cellulase.[Method] Ultraviolet(UV) was used to induce mutagenesis on T.vride K and to select out a new Trichoderma vride strain highly producing cellulase from the first round and further selection.[Result] A new T.vride strain K6 with high yield of cellulase was obtained with the enzyme production amount of 1.39 times over that of starting strain K.This strain showed highest cellulase yield under the culture condition of 28 ℃ for 96 h.[Conclusion] The strain K6 selected out from induced mutation is endowed with better capacity of producing cellulase,which provides a new method for the utilization of straw.

  2. 产纤维素酶细菌的筛选及其紫外诱变%Screening and Ultraviolet Mutagenesis of Cellulase-Producing Bacteria

    Institute of Scientific and Technical Information of China (English)

    张学佳; 田云; 卢向阳

    2013-01-01

    采用刚果红脱色圈法初筛得到143株有纤维素酶活性的细菌,然后采用DNS法复筛得到纤维素酶酶活最高的6株细菌,进一步进行紫外诱变处理,获得酶活提高最大且具有遗传稳定性的菌株E140',酶活为0.90 IU· mL-1,较出发菌株(0.68 IU·mL-1)提高了32.35%.表明采用刚果红脱色圈法和DNS法联合筛选并结合紫外诱变,可以获得纤维素酶活性高的细菌.%Firstly, 143 strains with cellulase activity were obtained by primary screening with Congo red decolorizing ring method,and then 6 strains with the highest enzyme activity were obtained by screening with DNS method. After the furtherly UV mutagenesis treatment,a mutant strain E140' with the highest enzyme activity increasing and genetic stability was got, whose enzyme activity reached 0. 90 IU · mL-1,increasing by 32. 35% .than that of the starting strain(0. 68 IU · mL-1). It showed that cellulase-producing bacteria with high enzyme activity could be obtained by screening with both Congo red decolorizing ring method and DNS method, combining with UV mutagenesis treatment.

  3. 里氏木霉产纤维素酶的诱导和合成机理研究进展%Research progress of the mechanism of induction and synthesis of cellulase by Trichoderma reesei

    Institute of Scientific and Technical Information of China (English)

    李辉; 王义强; 陈介南; 张伟涛

    2011-01-01

    Cellulase is a kind of inducible enzyme.The inducer is required for cellulase expression.Cellulose, sophorose, lactose, cellobiose and sorbitol are common inducers.Trichoderma reesei mainly expresses three cellulases, endoglucanase, exoglucanase and β-1,4-glucosidase, in which the expression level of exoglucanase Ⅰ is highest, followed by endoglucanase and β-1,4-glucosidase.The synthesis and expression of cellulase is affected by active elements and inhibitory factors.In details, the combination of inducer and active element induces the synthesis and expression of cellulose.When the decomposition products of cellulase accumulate to a certain level, the intracellular inhibitory factor will combine with promoter to prevent the expression of cellulase.%纤维素酶是一类诱导酶,诱导剂对纤维素酶的表达是必需的.常用的诱导剂如纤维素、槐糖、乳糖、纤维二糖、山梨糖等.里氏木霉主要表达3种纤维素酶:内切葡聚糖酶、外切葡聚糖酶和β-1,4-葡萄糖苷酶,其中以外切葡聚糖苷酶Ⅰ的合成量最多,其次是内切葡聚糖苷酶和β-1,4-葡萄糖苷酶.纤维素酶的合成和表达是激活原件和抑制因子共同作用的结果:诱导物质和激活元件结合激活细胞内纤维素酶的合成和表达;当纤维素酶的分解产物达到一定水平时,细胞内的抑制因子和启动子结合,阻止纤维素酶的表达.

  4. Research on the Influential Factors of Corn Stalk's Hydrolysis by Cellulase%纤维素酶水解玉米秸秆的影响因素研究

    Institute of Scientific and Technical Information of China (English)

    王娜娜; 杨翔华; 张全; 姚秀清

    2011-01-01

    The effects of pretreatment mode, cellulase amount and initial glucose content on corn stalk's hydrolysis by cellulase were studied. The results showed that the enzymolysis yields for pretreated corn stalks were higher than that of untreated one, and the yields of the two kinds of corn stalk which had been pretreated by methods of dilute acid and steam-explosion were 97. 0% and 91. 0%, respectively. The steam-explosion pretreated corn stalk s was hydrolyzed by several cellulases, which resulted that there was no proportion relationship between the cellulase amount and filter paper activity (FPIU) of cellulase solution. Additionally, more cellulase was added, more average glucose generated rate (Gr) was achieved. At the same time, Gr was decreased with the increase of initial glucose content which demonstrated that glucose had the feed back inhibition to the hydrolysis of cellulose. This study provided a basis for optimization of production technology of fuel ethanol with corn stalk as material.%考察了预处理方式、纤维素酶加量、初始葡萄糖含量等因素对玉米秸秆酶解效果的影响。结果表明,经过稀酸和蒸汽爆破预处理玉米秸秆的酶解得率分别为97.0%和91.0%,均高于未经预处理玉米秸秆;对蒸汽爆破玉米秸秆,纤维素酶加量与酶液的滤纸酶活不成正比关系,纤维素酶加量越多,平均葡萄糖生成速率(GR)越大;葡萄糖对纤维素酶水解具有反馈抑制作用,随着初始葡萄糖含量的增大,GR呈递减趋势。为优化利用玉米秸秆为原料生产燃料乙醇的工艺提供了依据。

  5. 里氏木霉Trichoderma reesei产纤维素酶的发酵培养基碳氮源优化%Culture medium optimization for producing cellulase by Trichoderma reesei

    Institute of Scientific and Technical Information of China (English)

    祖彩霞; 李志敏; 叶勤

    2011-01-01

    The influence of carbon and nitrogen sources on cellulase production was investigated. Single factor method and response surface methodology were used to optimize the medium for cellulase production by Trichoderma reesei. Firstly, it showed that soybean powder, corn cob and corn steep liquor had positive effects on cellulase production in one factor at a time. Then, the optimal concentrations of soybean powder (32. 21 g/L) , corn cob (42. 29 g/L) and corn steep liquor (4.45 g/L) were determined by central compostion design and response surface analysis. Under these optimizations conditions, the cellulase production reached (10. 65 ±0. 50) U/mL on the flask cultivation for 7 d.%研究C、N源对里氏木霉(Trichoderma reesei)生产纤维素酶的影响,采用单因素实验方法和中心复合方法对发酵培养基进行优化.单因素实验表明:黄豆饼粉、玉米芯、玉米浆对纤维素酶的影响显著.通过响应面优化,得到最优培养基C、N源的组成:黄豆饼粉32.21g/L,玉米芯42.29g/L,玉米浆4.45g/L.优化条件下,摇瓶发酵7 d的比酶活达到(10.65±0.50)U/mL.

  6. 中性纤维素酶整理对丝光棉织物性能影响的研究%Influence of neutral cellulase finishing on properties of mercerizing cotton fabrics

    Institute of Scientific and Technical Information of China (English)

    孙洁; 贺江平; 翟锐根; 赵建华

    2013-01-01

    研究了中性纤维素酶整理丝光棉织物后对其性能的影响,通过试验分析了酶用量、pH、整理温度和时间对织物失重率、顶破强力、硬挺度及抗起毛起球性的影响,并确定最佳工艺条件:酶用量2.0%(omf),pH=7.5,整理温度50℃,整理时间55 min.在最佳工艺条件下整理的丝光棉织物,各方面性能有不同程度的改善,顶破强力控制在220 N以上.%The influences of neutral cellulase finishing on properties of mercerizing cotton fabrics were studied. The effects of the dosage of cellulase, pH, treating temperature and time on the weight loss rate, the bursting strength, the stiffness and the pilling resistance were investigated. The optimum process was determined: the dosage of cellulase was 2.0%(omf), the pH was 7.5, treated at 50 ℃ for 55 min. The various aspects of performance of the mercerizing cotton fabrics under optimum process were improved to some extent. The busting strength was controlled over 220 N.

  7. Effects of cellulase pre-treatment on properties of masson pine mechanical pulp%纤维素酶对马尾松机械浆磨浆性能影响的研究

    Institute of Scientific and Technical Information of China (English)

    罗清; 赵登; 张安龙; 李新平

    2013-01-01

    The pulp properties and paper strength by using the different amount of cellulase pretreatment on grinding silk dividing of masson pine were researched in this paper .The re-sults showed that pretreatment with cellulase before beating ,which increased the freeness of pulp ,improved filtered water ,reduced fines content and enhanced paper strength .The scan-ning electron microscopy showed that the fiber surface became rough after cellulase treat-ment ,fine fibrosis degree became larger ,the ends of fiber fibrillation increased .%用纤维素酶对搓磨分丝后的马尾松机械浆进行预处理,并研究酶用量对纸浆特性和纸张强度的影响。结果表明:经纤维素酶预处理后,纸浆的游离度上升,滤水性改善,细小纤维含量减少,纸张强度提高。原子力显微镜和扫描电镜检测表明纤维素酶处理后纤维表面粗糙度增加,细纤维化程度增大,纤维末端分丝帚化增多。

  8. 一株土壤源高产纤维素酶芽孢杆菌的分离与鉴定%Isolation and Identification of a High Yield Cellulase Bacillus Strain from Soil

    Institute of Scientific and Technical Information of China (English)

    董鹤娟; 丁轲; 恒子钤; 贾艳艳; 彭春平; 罗伟光; 李旺

    2013-01-01

    In order to obtain the high yield cellulase bacillus,58 soil samples from different areas in Henan province were collected,the bacillus producing cellulase was isolated by Congo red medium,and the cellulase activity was detected by 3,5-dinitro salicylic acid method.The high cellulase producing bacillus were screened and identified by the colony morphology,microscopic morphology in combination with the physiological and biochemical feature and 16S rDNA sequence.The results showed that the strain B.LY02 with the highest cellulase activity of 0.5351 U · mL-1 was obtained,and it was short rod,forming spores,gram strain positive,and had the ability of fermenting many sugars.The 16S rDNA sequence homology comparative analysis showed that it belonged to the genus Bacillus and most closery related to Bacillus licheniformis strain CICC10095 with 96.5% sequence simmarity.So the strain Bacillus LY02 was identified to be Bacillus licheniformis.%为了获得产纤维素酶的芽孢杆菌,从河南省不同地方采集玉米地、秸杆垛的土壤样品58份,利用刚果红平板法分离产纤维素酶的芽孢杆菌,采用3,5—二硝基水杨酸法测定纤维素酶活,结合茵落特征、显微形态、生理生化试验和16S rDNA序列进行鉴定.结果表明,从样品中分离出的42株产纤维素酶菌株中筛选出一株产纤维素酶较高的菌株B.LY02,纤维素酶活力可达0.5351 U/mL.该菌株呈短杆状,革兰氏染色阳性,能形成芽孢.基于16S rDNA序列同源性比较分析表明该菌株与Bacillus licheni ormis strain CICC10095的亲缘关系最近,基因序列的同源性为96.5%,因此鉴定该菌株为地衣芽孢杆菌.

  9. 产纤维素酶食用菌筛选及其培养基的优化%Screening for Cellulase Producing Edible Fungi and Optimization of Medium

    Institute of Scientific and Technical Information of China (English)

    丁玉萍; 张康; 叶红强; 王晓娟; 杨莎; 梅竹; 任欢欢

    2012-01-01

    利用刚果红杯碟快速筛选法对大白菇、金针菇、香菇、竹荪、磷盖红菇、杏鲍菇6种食用菌产纤维素酶能力进行比较,同时对高产纤维素酶食用菌的培养基最佳碳源和配方进行筛选、优化。结果表明:磷盖红菇出现透明圈最大,直径为20mm;对比羧甲基纤维素钠(CMC-Na)、豆渣、秸秆粉、葡萄糖4种碳源,豆渣对磷盖红菇产纤维素酶有促进作用,优化培养基为豆渣60g/L、硫酸铵2.0g/L、吐温-80 1.5mL/L、pH5.4。%The ability of six edible fungi,Russula delica Fr,Flammulina velutipe,Lentinus javanicus,Bamboo-sun,Russula lepida and Abalone mushroom,to produce cellulase were compared using the Congo red saucer rapid screening method.Meanwhile,the optimum carbon source and medium formula for cellulase production were screened and optimized.The result showed that Russula lepida produced the largest transparent rings of 20 mm in diameter,showing its high yield of cellulase.Among the four kinds of carbon source,CMC-Na,bean dregs,straw powder and glucose,the bean dregs promoted Russula lepida to produce cellulase.The optimized medium for cellulase production by Russula lepida was bean dregs 60 g/L,ammonium sulfate 2.0 g/L,Tween-80 1.5 mL/L,and pH 5.4.

  10. 纤维素酶在不同长度纤维上的吸附行为%Adsorption Behaviors of Cellulase on Cellulose Fiber Fractions with Different Fiber Length

    Institute of Scientific and Technical Information of China (English)

    吕健; 詹怀宇; 付时雨

    2011-01-01

    Adsorption of cellulase on the surfaces of cellulose fibers is the first step of cellulose enzymatic hydrolysis and saccharification. The adsorption behaviors of cellulase on cellulose fiber fractions with different fiber length were quantitatively discussed in this study. Hie adsorption equilibrium was reached in around 60 minutes, and the adsorption amount increased with increasing initial cellulase dose. The adsorption process was well described by Langmuir isotherm. The maximum adsorption amount was found on the short fiber, and there was the maximum Langmuir adsorption equilibrium constant in the adsorption on the long fiber, which indicated that cellulase shows the highest adsorption affinity on the long fiber. The calculated thermodynamics parameters indicated that adsorption of cellulase on cellulose fiber is a spontaneous, exothermic and irreversible process. The highest enthalpy change △H° and entropy change △S° were found in the adsorption on 48 mesh fraction and 28 mesh fraction respectively.%纤维素酶在纤维表面上的吸附是纤维素水解糖化的第一步,探讨了纤维素酶在不同长度纤维上的吸附行为。纤维素酶在纤维上吸附约60 min后可达到平衡,且吸附量随初始酶用量的增加而增多。吸附过程遵循Langmuir等温吸附,且纤维素酶在短纤维上具有最大的吸附量,但在长纤维上具有最大的Langmuir吸附平衡常数,说明纤维素酶在长纤维上能更快地达到吸附平衡。对吸附热力学常数的计算表明,纤维素酶吸附是自发、放热过程,且不可逆吸附。纤维素酶在48目纤维上有最大的吸附焓变,在28目纤维上有最大的吸附熵变。

  11. 硫化物对纤维素酶活及织物处理效果的影响%Effects of sulfide on cellulase activity and enzyme treatment performance of cotton fabric

    Institute of Scientific and Technical Information of China (English)

    姚继明; 窦春晓; 李倩

    2014-01-01

    1%(on the mass of acid cellulase solutions) of thiourea, sodium thiocyanate, sodium sulfite, so-dium bisulfite, sodium metabisulfite, sodium hyposulfite, sodium hydrosulfide and sodium sulfide were added to acid cellulase solutions, respectively. The filter paper enzymatic activities were measured according to cen-tral composite experiment design under 40, 50, 60 ℃ and pH 4.5, 5.0, 5.5. The effects of various sulfides on the enzymatic activities were analyzed by Minitab software. The cotton knitted fabrics were treated with cellu-lase under the same conditions, and the bursting strength, hairiness removal percentage and glucose content of residue liquid were measured to evaluate the practical performance on cotton fabrics. The results showed that the addition of sulfides could slightly enhance the cellulase activity, and widen cellulase working tempera-ture and pH range. However, except sodium sulfite and sodium hyposulfite, the addition of other six sulfides decreased the hairiness removal ability.%在酸性纤维素酶液中分别加入1%(对溶液质量)的硫脲、硫氰酸钠、亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、连二亚硫酸钠、硫氢化钠、硫化钠等8种不同的硫化物,在温度40、50、60℃,pH=4.5、5.0、5.5的条件下,根据中心复合试验设计方案测定滤纸酶活力,并用Minitab软件分析各种硫化物对纤维素酶活力的影响.在相同的条件下对纯棉针织物进行酶处理,测定处理前后织物的顶破强力、毛羽去除率、处理残液葡萄糖含量来评价对织物的实际作用效果.结果表明,硫化物的加入可以小幅提高纤维素酶活力、扩大酶的温度和pH作用区间,但除了亚硫酸钠和连二亚硫酸钠外,其他6种硫化物的加入反而降低了织物的除毛效果.

  12. Cloning of novel cellulases from cellulolytic fungi: heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris.

    Science.gov (United States)

    Salinas, Alejandro; Vega, Marcela; Lienqueo, María Elena; Garcia, Alejandro; Carmona, Rene; Salazar, Oriana

    2011-12-10

    Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation.

  13. Purification and biochemical properties of a thermostable, haloalkaline cellulase from Bacillus licheniformis AMF-07 and its application for hydrolysis of different cellulosic substrates to bioethanol production

    Science.gov (United States)

    Azadian, Fatemeh; Badoei-dalfard, Arastoo; Namaki-Shoushtari, Abdolhamid; Hassanshahian, Mehdi

    2016-01-01

    A thermophilic strain AMF-07, hydrolyzing carboxymethylcellulose (CMC) was isolated from Kerman hot spring and was identified as Bacillus licheniformis based on 16S rRNA sequence homology. The carboxymethylcellulase (CMCase) enzyme produced by the B. licheniformis was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography. The purified enzyme gave a single band on SDS- PAGE with a molecular weight of 37 kDa. The CMCase enzyme was highly active and stable over broad ranges of temperature (40-80ºC), pH (6.0-10.0) and NaCl concentration (10-25%) with an optimum at 70ºC, pH 9.0 and 20% NaCl, which showed excellent thermostable, alkali-stable and halostable properties. Moreover, it displayed high activity in the presence of cyclohexane (134%) and chloroform (120%). Saccharification of rice bran and wheat bran by the CMCase enzyme resulted in respective yields of 24 and 32 g L-1 reducing sugars. The enzymatic hydrolysates of rice bran were then used as the substrate for ethanol production by Saccharomyces cerevisiae. Fermentation of cellulosic hydrolysate using S. cerevisiae, reached maximum ethanol production about 0.125 g g-1 dry substrate (pretreated wheat bran). Thus, the purified cellulase from B. licheniformis AMF-07 utilizing lignocellulosic biomass could be greatly useful to develop industrial processes. PMID:28097168

  14. A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus sp. JB 99 suitable for paper and pulp industry: purification and characterization.

    Science.gov (United States)

    Shrinivas, Dengeti; Savitha, Gunashekaran; Raviranjan, Kumar; Naik, Gajanan Ramchandra

    2010-11-01

    A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0-10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K (m) and V (max) of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min(-1) mg(-1), respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

  15. Process Design and Economics of On-Site Cellulase Production on Various Carbon Sources in a Softwood-Based Ethanol Plant

    Directory of Open Access Journals (Sweden)

    Zsolt Barta

    2010-01-01

    Full Text Available On-site cellulase enzyme fermentation in a softwood-to-ethanol process, based on SO2-catalysed steam pretreatment followed by simultaneous saccharification and fermentation, was investigated from a techno-economic aspect using Aspen Plus© and Aspen Icarus Process Evaluator© softwares. The effect of varying the carbon source of enzyme fermentation, at constant protein and mycelium yields, was monitored through the whole process. Enzyme production step decreased the overall ethanol yield (270 L/dry tonne of raw material in the case of purchased enzymes by 5–16 L/tonne. Capital cost was found to be the main cost contributor to enzyme fermentation, constituting to 60–78% of the enzyme production cost, which was in the range of 0.42–0.53 SEK/L ethanol. The lowest minimum ethanol selling prices (4.71 and 4.82 SEK/L were obtained in those scenarios, where pretreated liquid fraction supplemented with molasses was used as carbon source. In some scenarios, on-site enzyme fermentation was found to be a feasible alternative.

  16. Composting-Like Conditions Are More Efficient for Enrichment and Diversity of Organisms Containing Cellulase-Encoding Genes than Submerged Cultures.

    Science.gov (United States)

    Heiss-Blanquet, Senta; Fayolle-Guichard, Françoise; Lombard, Vincent; Hébert, Agnès; Coutinho, Pedro M; Groppi, Alexis; Barre, Aurélien; Henrissat, Bernard

    2016-01-01

    Cost-effective biofuel production from lignocellulosic biomass depends on efficient degradation of the plant cell wall. One of the major obstacles for the development of a cost-efficient process is the lack of resistance of currently used fungal enzymes to harsh conditions such as high temperature. Adapted, thermophilic microbial communities provide a huge reservoir of potentially interesting lignocellulose-degrading enzymes for improvement of the cellulose hydrolysis step. In order to identify such enzymes, a leaf and wood chip compost was enriched on a mixture of thermo-chemically pretreated wheat straw, poplar and Miscanthus under thermophile conditions, but in two different set-ups. Unexpectedly, metagenome sequencing revealed that incubation of the lignocellulosic substrate with compost as inoculum in a suspension culture resulted in an impoverishment of putative cellulase- and hemicellulase-encoding genes. However, mimicking composting conditions without liquid phase yielded a high number and diversity of glycoside hydrolase genes and an enrichment of genes encoding cellulose binding domains. These identified genes were most closely related to species from Actinobacteria, which seem to constitute important players of lignocellulose degradation under the applied conditions. The study highlights that subtle changes in an enrichment set-up can have an important impact on composition and functions of the microcosm. Composting-like conditions were found to be the most successful method for enrichment in species with high biomass degrading capacity.

  17. Purification and biochemical properties of a thermostable, haloalkaline cellulase from Bacillus licheniformis AMF-07 and its application for hydrolysis of different cellulosic substrates to bioethanol production

    Directory of Open Access Journals (Sweden)

    Fatemeh Azadian

    2016-09-01

    Full Text Available A thermophilic strain AMF-07, hydrolyzing carboxymethylcellulose (CMC was isolated from Kerman hot spring and was identified as Bacillus licheniformis based on 16S rRNA sequence homology. The carboxymethylcellulase (CMCase enzyme produced by the B. licheniformis was purified by (NH42SO4 precipitation, ion exchange and gel filtration chromatography. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 37 kDa. The CMCase enzyme was highly active and stable over broad ranges of temperature (40-80 ºC, pH (6.0-10.0 and NaCl concentration (10-25% with an optimum at 70 ºC, pH 9.0 and 20% NaCl, which showed excellent thermostable, alkali-stable and halostable properties. Moreover, it displayed high activity in the presence of cyclohexane (134% and chloroform (120%. Saccharification of rice bran and wheat bran by the CMCase enzyme resulted in respective yields of 24 and 32 g L-1 reducing sugars. The enzymatic hydrolysates of rice bran were then used as the substrate for ethanol production by Saccharomyces cerevisiae. Fermentation of cellulosic hydrolysate using S. cerevisiae, reached maximum ethanol production about 0.125 g g-1 dry substrate (pretreated wheat bran. Thus, the purified cellulase from B. licheniformis AMF-07 utilizing lignocellulosic biomass could be greatly useful to develop industrial processes.

  18. Extracellular production of novel halotolerant, thermostable, and alkali-stable carboxymethyl cellulase by marine bacterium Marinimicrobium sp. LS-A18.

    Science.gov (United States)

    Zhao, Kun; Guo, Li-Zhong; Lu, Wei-Dong

    2012-10-01

    Cellulases which are active and stable under extreme conditions have attracted considerable attention because of their potential industrial applications. Marinimicrobium sp. LS-A18 showed high extracellular carboxymethylcellulase (CMCase) activity when grown on mineral salt medium containing carboxymethylcellulose as the sole carbon source. Maximum CMCase activity was obtained at 55°C and pH 7.0 in the absence of NaCl. Under the optimized fermentation conditions, the yield of CMCase was increased up to 2.5 U/ml, which was 3.1-fold higher than that before optimization. The enzyme retained 84 % of residual activity after incubation at 60°C for 1 h and more than 88 % of residual activity after incubation for 72 h in the presence of different pH (5-11) and NaCl concentrations (0-25 %, w/v), indicating it was halotolerant, thermostable and alkali-stable. These characteristics made the CMCase from Marinimicrobium sp. LS-A18 as a potentially novel biocatalyst in biotechnological and industrial applications.

  19. The industrial applicability of purified cellulase complex indigenously produced by Trichoderma viride through solid-state bio-processing of agro-industrial and municipal paper wastes

    Directory of Open Access Journals (Sweden)

    Muhammad Irshad

    2013-02-01

    Full Text Available An indigenous strain of Trichoderma viride produced high titers of cellulase complex in solid-state bio-processing of agro-industrial orange peel waste, which was used as the growth-supporting substrate. When the conditions of the SSF medium containing 15 g orange peel (50% w/w moisture inoculated with 5 mL of inoculum were optimal, the maximum productions of endoglucanase (655 ± 5.5 U/mL, exoglucanase (412 ± 4.3 U/mL, and β-glucosidase (515 ± 3.7 U/mL were recorded after 4 days of incubation at pH 5 and 35 °C. The enzyme with maximum activity (endoglucanase was purified by ammonium sulfate fractionation and Sephadex G-100 column gel filtration chromatographic technique. Endoglucanase was 5.5-fold purified with specific activity of 498 U/mg in comparison to the crude enzyme. The enzyme was shown to have a molecular weight of 58 kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE. The shelf life profile revealed that the enzyme could be stored at room temperature (30 °C for up to 45 days without losing much of its activity.

  20. 多菌混合固态发酵产纤维素酶研究%Cellulase production by solid-state fermentation with multi-strains

    Institute of Scientific and Technical Information of China (English)

    刘云云; 张宇; 许敬亮; 何敏超; 庄新姝; 袁振宏

    2014-01-01

    采用多菌混合发酵可以提高纤维素酶的活力,为获得高活力的纤维素酶制剂,文中以碱处理后的甘蔗渣和麸皮作为发酵产酶培养基,采用响应面法对2株纤维素酶生产菌里氏木霉CICC40359和斜卧青霉SMX固态混合发酵条件进行了优化。结果发现在发酵温度为28℃,料水比(质量体积比)1∶2.5(g/mL)的条件下,当V(青霉)∶V(木霉)为3∶1,总接种量8%(mL/g),培养基中 m(蔗渣)∶m(麸皮)为2∶1,发酵3 d 时,滤纸酶活有最大值达到101.825 FPU/g,这为后续优化工作的开展提供了依据,同时高酶活下发酵液中呈现高的糖质量分数为同步产酶发酵产乙醇提供了新思路。%Cellulase production efficiency can be improved via the mixed culture solid-state fermentation.In order to obtain high cellulase activity,alkali-treated sugarcane bagasse supplemented with wheat bran was used as enzyme fermentation substrate.The response surface method was introduced to optimize the solid-state mixed-fermentation with Trichoderma reesei CICC40359 and Penicillium decumbens SMX.The results indicate that the filter paper enzyme activity has an maximum value of 101.825 FPU/g under the following conditions:temperature is 28 ℃;mass and volume ratio of material to water is 1 ∶2.5 g/mL;V(Penicillium )∶V(Trichoderma) =3 ∶1, m(bagasse)∶m(wheat bran)=2∶1 ,inoculum dose is 0.08 g/mL and fermentation time is 3 d,which provides a basis for the next optimization work.The fermentation liquor showed high sugar content when the enzyme activity was high.These results indicate that simultaneous enzyme and ethanol production may be an effective approach for the economic production of bioethanol.

  1. Screening and Characterization of Cellulase-Producing Fungus and Its Zymological Properties%一株产纤维素酶真菌的筛选、鉴定及酶学性质初步研究

    Institute of Scientific and Technical Information of China (English)

    张苏龙; 吕淑霞; 林英; 夏杨

    2009-01-01

    经过初筛和复筛从土样中分离出1株高产纤维素酶真菌SNB9,经形态学和ITS序列分析,鉴定为黑曲霉(Aspergullus niger).生长条件的测定显示该菌生长范围偏酸.发酵后纤维素酶的最适作用pH在 4.0~5.0,最适作用温度在 45~55 ℃.滤纸酶活为 9.29 U/mL,C -1酶活为 23.69 U/mL,CMCase酶活为 38.23 U/mL,β-葡萄糖苷酶活为 65.52 U/mL.发酵液中除了纤维素酶,还发现有辅助酶,包括木聚糖酶、淀粉酶、果胶酶、蛋白酶.%A cellulase high-producing fungus strain SNB9 was isolated from soil samples after proto-screening and re-screening and characterized as Aspergillus niger after morphological and ITS sequencing analyses. Growth conditions tests indicated that it grew on meta-acidic range. And the most suitable reactive pH of the cellulase was 4.0~5.0, and temperature 45~55 ℃. The activities of FPA, C1, CMCase, and β-glycosidase were 9.29 U/mL, 23.69 U/mL, 38.23 U/mL, 65.52 U/mL respectively. Besides cellulase, it was found there were some other auxiliary enzymes in the fermented broth, including xylanase, amylase, pectinase, and protease.

  2. Screening of High-yield Cellulase-producing Strains by N + Implantation Mutation%N+离子注入诱变选育纤维素酶高产菌

    Institute of Scientific and Technical Information of China (English)

    陈阿娜; 汤斌; 刘标

    2011-01-01

    Cellulase-producing strain Rhizopus sp.TC1 was subjected to mutation involving treatment of N + implantation, UV followed by 5-Fu; successive mutant Rhizopus sp.TC1653 showed enhanced cellulase production.The FPA and CMC case of mutant TC1653 was 22.6 and 224.7 IU per gram of dry medium respectively at the end of fermentation, which was increased by 3.77 folds and 3.14 folds respectively compared to that of TC1.Ten times subculturing indicated that the mutant TC1653 had good genetic stability and character of cellulase production.TC1653 showed rapid growth on solid-state medium and its cellulase activities were highest at 96 hours.Fermentation time shortened to for 48 hours compared to that of Trichoderma virid.The results showed that mutant TC1653 had some advantages under the mutation caused by N + implantation.%纤维素酶产生菌Rhizopus sp.TCl经过N+离子注入、紫外线、5-氟尿嘧啶(5-Fu)等诱变剂诱变,选育得到突变株Rhizopus sp.TCl653,发酵终点时滤纸酶活力(FPA)和羧甲基纤维素酶活力(CMC)分别为22.6 IU/g、224.7 IU/g干物质,较出发菌株分别提高了276.7%和214.3%.突变株经传10代培养,任意2代间酶活力差值均在10%内,表明该菌株产酶性能稳定,可作为后继实验出发菌株.突变株在培养基上能够快速生长,发酵96 h即达到酶活高峰期,较绿色木霉酶活高峰期提前48 h,缩短了发酵周期.该实验结果表明,经N+离子注入等方式诱变选育的突变株具有孢子萌发率高,同步性好,代谢速度快等优点.

  3. 纤维素酶酶法改性玉米麸皮膳食纤维粉的工艺研究%Technology Research of Cellulase Enzyme Modified the Corn Bran Dietary Fiber Powder

    Institute of Scientific and Technical Information of China (English)

    王刚; 王蕾

    2012-01-01

    With corn bran as raw materials, preparing corn bran dietary fiber, through the enzymatic modification to improve the quality of dietary fiber. The following conclusions were drawn: corn dietary fiber modified process Used cellulase enzyme to obtain the soluble dietary fiber and obtain ratio was 26.45%. The optimal conditions were: cellulase dosage of 15.78 U/g, hydrolysis time 3.01 h, hydrolysis temperature 56.56 ℃ and enzyme pH 5.56.%以玉米麸皮为原料,制备膳食纤维粉,通过纤维素酶对其进行改性,提高膳食纤维品质。采用纤维素酶法,得到的可溶性膳食纤维得率为26.45%。其最适反应条件为:酶添加量15.78 U/g、酶解时间3.01 h、酶解温度50.56℃和酶解pH 5.56。

  4. On-line cut-off technique and organic modifier addition aided signal enhancement for trace analysis of carbohydrates in cellulase hydrolysate by ion exclusion chromatography-electrospray ionization mass spectrometry.

    Science.gov (United States)

    Cheng, Cheanyeh; Tsai, Hsiang-Rong; Chang, Kuo-Chung

    2006-06-30

    Paper cellulose has been hydrolyzed with calcium alginate immobilized cellulase to produce carbohydrate products and the three trace sugars, galactose, arabinose, and mannose in the cellulase hydrolysate have been analyzed by HPIEC/ESI-MS. Applying the on-line cut-off technique to the HPIEC/ESI-MS can cut the high concentration glucose off to eliminate its interference on the peaks of minor sugars and enhance their signals from 1.1- to 1.6-fold. However, the on-line post column addition of 15% ethanol to the eluate can increase the signal of the three trace sugars, galactose, arabinose, and mannose up to 17-, 23-, and 11-fold, respectively, and make the corresponding detection limits as 0.04, 0.04, and 0.03 ppm. The accuracies of the quantitative analysis for the three trace sugars with the signal enhanced HPIEC/ESI-MS by the two enhancement methods were larger than 95%. The precisions of the analytical results were also greatly improved by the assistance of the two techniques and were less than 6.5%. The quantitative analysis of the three trace sugars was performed with the internal standard method and the internal standard (IS) was sorbitol. Overall, the signal enhancement of HPIEC/ESI-MS and quantification of the three trace sugars by the on-line cut-off technique and organic modifier addition was successful.

  5. Cellulase Production from Brewer's Spent Grain by Solid-State Fermentation with Aspergillus Niger 3.316%黑曲霉3.316固态发酵啤酒糟生产纤维素酶

    Institute of Scientific and Technical Information of China (English)

    杜晓梅; 付国家; 郭庆杰; 王许云

    2012-01-01

    以啤酒糟为主要原料,采用黑曲霉3.316(Aspergillus niger 3.316)固态发酵生产纤维素酶,对培养基组成和培养条件进行优化.结果表明:啤酒糟和麸皮的质量比为8∶2、水料质量比为1.5∶1、硫酸铵质量分数为3%、发酵时间为4d时,所产纤维素酶活性最大,滤纸酶活性(FPA)和羧甲基纤维素酶活性(CMCA)分别达72.6118和692.1700nkat.%Cellulase production was carried out by Solid-State Fermentation (SSF) with Aspergillus niger 3. 316 using brewer's spent grain as the main material. The SSF conditions were studied and optimized. It indicated that the optimum conditions were as follows: the ratio of brewer's spent grain and wheat bran was 8 :2, the ratio of water to material was 1. 5 : 1. the content of the ammonium sulfate was 3%, and the fermentation time was 4 d, respectively. Under the above-mentioned conditions, the filter paper activity (FPA) and carboxymethyl cellulase activity (CMCA) can reach 72.611 8and 692. 170 0 nkat, respectively.

  6. PURIFICATION AND SOME PROPERTIES OF CELLULASE FROM ODONTOTERMES FORMOSANUS (ISOPTERA:TERMITIDAE)%黑翅土白蚁纤维素酶纯化及其特性研究

    Institute of Scientific and Technical Information of China (English)

    杨天赐; 莫建初; 程家安

    2004-01-01

    The purification of the cellulase from Odontotermes formosanus workers was achieved by using anion-exchange column of UNOsphere Q, BioLogic DuoFlow chromatography system. The purified cellulase was identified as an endoglucanase and some of its properties were investigated. The Egase activity was 807.5-fold as high as the initial enzyme activity using CMC as substrate and 14.4-fold using salicin as substrate. The enzyme preparations were homogeneous as judged by SDS-PAGE electrophoresis, molecular weight of which was 80 kDa and confirmed by 2-DE zymogram analysis. The enzyme was isoelectric at pH 6.4, which was active on CMC substrate.%利用DuoFlow层析系统阴离子交换柱UNOsphere Q,从黑翅土白蚁工蚁体内分离出内切葡聚糖酶.以羧甲基纤维素和水杨苷为底物,测得纯化酶的活性分别是原酶液的807.5和14.4倍.经变性聚丙烯酰胺凝胶电泳分析,获得单一的蛋白条带,其分子量大小为80kDa.经双向电泳图谱分析,其等电点为6.4.

  7. 酶解工艺对籽瓜浓缩汁出汁率的影响%Effect of Enzymatic Treatment with Pectinase and Cellulase on Juice Yield of Seed Melon

    Institute of Scientific and Technical Information of China (English)

    刘忆冬; 罗鹏; 刘娅; 陈国刚

    2015-01-01

    The seed melon was used as the raw material in the experiment. The application of pectinase, cellulase to increase juice yield in the production of seed melon concentrated juice was studied in this paper through a single factor and an orthogonal experiment. The results showed that the optimum enzymatic hydrolysis processing were as followed: 0.1% pectinase, cellulase 0.05%, Enzyme solution temperature 45℃, enzymolysis time 2h and enzyme solution pH=4.5. On this condition, the yield of juice was 86.3%.%以籽瓜为试验材料,利用果胶酶和纤维素酶进行酶解。通过单因素和正交试验确定籽瓜的最佳酶解工艺:果胶酶0.1%,纤维素酶0.05%,酶解温度45℃,酶解时间2h,酶解pH=4.5。在此最佳酶解条件下,出汁率达86.3%。

  8. 中度嗜盐菌HS1产纤维素酶发酵条件的研究%Study on Cellulase Production by A Moderately Halophilic Bacterium HS1

    Institute of Scientific and Technical Information of China (English)

    韩秋菊; 张倩倩

    2011-01-01

    对一株中度嗜盐菌HS1产纤维素酶的活性进行了初步研究.单因素实验和正交实验结果表明,菌株HS1产纤维素酶的最佳发酵氮源为1.5%NaNO3,优化的发酵条件为:液体种子接种量6%、培养温度35℃、初始pH值6.5、培养时间4 d.%In this paper, cellulase production by a moderately halophilic bacterium HS1 was preliminarily explored. Through single factor experiment and orthogonal experiment, it was found that the optimal nitrogen source was 1.5% NaNO3 for production of cellulase and the optimum fermentation conditions were as follows:inoculating quantity of liquid seed of 6% ,culture temperature of 35℃, initial pH value of 6.5 and culture time of 4 d.

  9. Improvement of Cellulase Producing Capacity of Aspergillus niger by Ultraviolet Mutation%紫外线诱变黑曲霉提高产酶活力的研究

    Institute of Scientific and Technical Information of China (English)

    郭艳

    2011-01-01

    [Objective] The research aimed to breed the high-yield production strain of cellulase.[Method] Aspergillus niger was used as the starting strain,and a high-yield production strain of cellulase was selected after UV mutation treatment.[Result] Under the suitable condition,the strain 2(15) with the highest CMC production capacity was selected,which nearly increased 50% than that of the starting strain.[Conclusion] The research provided the foundation for its appliation in the feed production in the future.%[目的]选育高产纤维素酶生产菌。[方法]以一株黑曲霉(Aspergillus niger)为出发菌株,经过紫外线(UV)诱变处理,选育出一株纤维素酶高产菌株生产菌。[结果]在适宜的条件下,选育得到的菌株2(15)产CMC活力最强,与出发菌株相比提高了近50%。[结论]今后应用于饲料生产中提供基础。

  10. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

    Directory of Open Access Journals (Sweden)

    Qureshi Nasib

    2006-05-01

    Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

  11. Use of Cellulases to Predict in vivo Digestible Organic Matter (D value in Pasture Silages Uso de Celulasas para Predecir el Contenido de Materia Orgánica Digestible (Valor D in vivo, en Ensilajes de Praderas

    Directory of Open Access Journals (Sweden)

    Claudia Barchiesi-Ferrari

    2011-06-01

    Full Text Available In pasture-based dairy herds where silage is a widely adopted supplement, optimized feeding requires reliable estimations of nutritional quality of this conserved forage. Metabolizable energy, an important nutritional fraction, can be predicted from digestibility-related traits, such as the digestible organic matter contained in the dry matter (D-value. The aim of the present study was to evaluate the prediction of D-value and dry matter digestibility (DMD of grass silages made from four different pastures and maturity stages, using the pepsin-cellulase method. Fungal cellulase was used, applying different enzyme concentrations, incubation times and types of final wash. The silages were prepared from permanent pasture (Dactylis glomerata L., Lolium perenne L., Bromus catharticus Vahl var. catharticus, Trifolium repens L. and Holcus lanatus L., rotation pasture (Lolium multiflorum Lam. cv. Tama, oats (Avena sativa L., and mixed pasture (L. perenne-T. repens. These were harvested at three different physiological stages (vegetative, ear emergence and dough grain. The treatment using an incubation time of 24 h, a cellulase concentration of 6.25 g L-1 and final wash with water (Treatment 3 presented the best prediction capacity of the in vivo D-value (R² = 0.78 and in vivo DMD (R² = 0.71. In vivo D-value prediction improved (R² = 0.8 when a chemical determination (crude fibre, gross energy, neutral detergent fibre, total ash or acid detergent fibre was included in addition (multiple regression to D-value obtained with cellulases (Treatment 3. Results of DMD obtained with cellulases show good precision, but underestimate in vivo values, and are closer to those obtained with ruminal fluid. Suitable equations could be used to improve accuracy.En sistemas lecheros pastoriles que utilizan ensilaje como suplemento, se requiere conocer el valor nutricional de éste para optimizar la alimentación del ganado. La energía metabolizable, importante fracci

  12. MnO2纳米粒子固载纤维素酶用于高效水解农业废弃物制备生物乙醇%Immobilization of cellulase onto MnO2 nanoparticles for bioethanol production by enhanced hydrolysis of agricultural waste

    Institute of Scientific and Technical Information of China (English)

    Elsa Cherian; Mahendradas Dharmendirakumar; Gurunathan Baskar

    2015-01-01

    Cellulase is an efficient enzymatic catalyst that hydrolyses cellulosic substances. The high costs associated with using enzymes for industrial applications can be reduced by immobilizing the cellu-lase. In the current study, cellulase produced by Aspergillus fumigatus JCF was immobilized onto MnO2 nanoparticles, which improve the activity of cellulase and offer a superior support. The sur-face characteristics of synthesized MnO2 nanoparticles and cellulase-bound MnO2 nanoparticles were investigated by scanning electron microscopy, and Fourier transform infrared spectroscopy was used to analyze the functional characteristics of the immobilized cellulase. The maximum cellu-lase binding efficiency was 75%. The properties of the immobilized cellulase, including activity, operational pH, temperature, thermal stability, and reusability were investigated and were found to be more stable than for the free enzyme. It was found that cellulase immobilized on MnO2 nanopar-ticles could be used to hydrolyze cellulosic substances over a broad range of temperature and pH. The results confirmed that cellulase immobilized on MnO2 nanoparticles was very efficient in terms of cellulolytic activity.%纤维素酶是一种有效的纤维质类物质水解催化剂,工业应用时可通过固定化纤维素酶来降低其成本。本文将烟曲霉原变种JCF产生的纤维素酶固定在MnO2纳米颗粒上。 MnO2可提高纤维素酶的活性,并充当一个更好的载体。采用扫描电镜表征了所制MnO2纳米粒子及其负载纤维素酶的表面性质,以傅里叶变换红外光谱分析了固定在MnO2纳米粒子上纤维素酶的官能团性质。纤维素酶在MnO2纳米粒子上最大的固定化效率为75%。考察了固定化纤维素酶的活性、操作pH值、温度、热稳定性和重复使用性等性质。结果表明,所制固定化酶的稳定性比游离酶更高。固定于MnO2纳米粒子上的纤维素酶可用于纤维质类物质的水解反

  13. 片烟黑曲霉菌株产纤维素酶的条件优化及酶的分离纯化%Optimization of cellulase production by Aspergillus niger separated from tobacco and its separation and purification

    Institute of Scientific and Technical Information of China (English)

    范坚强; 周彬; 宋纪真; 王金华; 陈义强; 王永泽

    2012-01-01

    为降低烟叶燃吸的刺激味、改善口感、增加香气、提高烟叶品质,从烟叶表面筛选到一株产纤维素酶活性较高的黑曲霉,应用Plackett-Burman试验确定4种显著因子,RSM法对4种显著因子进行了优化和分析,并对酶蛋白的分离纯化作了初步研究.结果显示:发酵产酶的最适碳源为玉米芯,氮源为NH4 NO3,当玉米芯用量为1.4%,NH4NO3为0.4%,温度为30.4℃,pH为5.6时产酶比活力最高,可达40.02 U· mL-.纤维素酶酶蛋白的硫酸铵盐析最佳饱和度为60%,经Sephadex G100凝胶柱层析纯化后的比活力提高了3.5倍,回收率为32.6%,达到较高水平.%In order to improve tobacco taste, decrease the smoking pungent taste, increase the fragrance and improve the smoking quality, a mold strain Aspergillus niger possessing relatively higher cellulase enzymatic activity was isolated from tobacco leaves. The Plackett-Burman design was used to choose the most important factors, then, these factors were optimized by RSM method, and the purification of enzyme was investigated. The results indicated that the optimum carbon source and nitrogen source were 1.4% corn cob and 0. 4% NH4NO3, the optimum original pH was 5.6 and the optimum cultural temperature was 30.4℃. Under these conditions, the specific enzyme activity was 40. 02 U· mL-1 . The crude cellulase was isolated and purified by means of ammonium sulfate precipitation, followed by chromatography on Sephadex G100. The specific activity of the cellulase increased to 3. 5 fold of the source enzyme with an activity recovery of 32. 6%.

  14. Effect of Cellulase on Quality of Napier Grass and Corn Straw Mixed Silage%纤维素酶对象草玉米秸秆混合青贮品质的影响

    Institute of Scientific and Technical Information of China (English)

    陈鑫珠; 张文昌; 张建国; 李国栋; 廖惠珍; 陈炎芳; 郑东升; 庄益芬

    2011-01-01

    为探明纤维素酶(cellulase,CEL)对象草(Pennisetum purpureum Schum)和甜玉米(Zea mays L.var.rugosa Bonaf.)秸秆混合青贮品质的影响.在象草:玉米秸秆为70∶ 30(鲜样质量比)的混合原料中添加CEL进行青贮调制,添加水平分别为0%(对照)、0.001%(1U/g)、0.005%(5 U/g)和0.025%(25 U/g).每个处理设3次重复.在常温条件下贮存60d,开封后测定青贮的pH、乳酸、乙酸、丙酸、丁酸,氨态氮等.结果表明,CEL能极显著减少青贮的中性洗涤纤维和半纤维素含量(P<0.01),极显著增加乳酸含量(P<0.01).在4个处理组中,0.005 %CEL组干物质和干物质回收率最高,并且该处理组的pH、乙酸、氨态氮和酸性洗涤纤维极显著(P<0.01)低于其他处理组,乳酸和可溶性碳水化合物极显著(P<0.01)高于其他处理组.上述结果表明,添加0.005 %CEL的处理效果最佳.%In order to study the influence of cellulase (CEL) additive on the quality of napier grass(Pen-nisetum purpureum) and corn straw (Zea mays 1. Var. Rugosa Bonaf. ) mixed silage, the mixed ratio of napier grass to corn straw at 70:30 was treated with cellulase at the levels of 0. 000%(control), 0. 001%(l U/g) , 0. 005%(5 U/g) and 0. 025%(25 U/g). Ensilaged for 60 days at normal temperature, the ferment-ational indexes were measured with three replicates for each treatment. The results indicated that cellulose can significantly reduce neutral detergent fiber and hemi-cellulose(P<0. 01) and increase lactic acid (P< 0. 01). The dry matter , dry matter recovery were the highest in silage with 0. 005% cellulose among the four treatments ,of which the pH, acetic acid, ammonia nitrogen and acid detergent fiber were significantly lower than other treatments while LA and WSC were higher(P<0. 01). The results showed that adding 0. 005% cellulase was the best for napier grass and corn straw mixed silage.

  15. An Applied Research on Cellulase Pre-treatment in Anaerobic Fermentation of Municipal Solid Waste%城市生活垃圾厌氧发酵中纤维素酶预处理的应用研究

    Institute of Scientific and Technical Information of China (English)

    何娟; 孙可伟; 李建昌; 尚江涛

    2011-01-01

    为解决常规厌氧发酵存在发酵速率滞后,产气量、产甲烷量低等缺点,首先利用纤维素酶对城市生活垃圾进行预处理(水解),通过单因素实验研究了纤维素酶添加量、水解温度、水解时间、水解pH和不同底物浓度(VS)对生活垃圾中纤维素水解率的影响。实验结果表明,利用纤维素酶水解城市生活垃圾的适宜条件为:纤维素酶添加量120 U/g(VS)、水解时间24 h、水解温度60℃、底物浓度为8%、水解pH5.6,此时水解率可达35.2%。随后将生活垃圾在此最佳水解条件下经过纤维素酶预处理后用于中温厌氧发酵。结果表明,经过纤维素酶预处理后用于厌氧发酵与不经过纤维素酶预处理直接进行厌氧发酵相比较,平均日产气率、VS产气量、VS产气率、平均产甲烷率、VS产甲烷量、VS产甲烷率等均显著提高;且累积产气量提高62.38%,累积产甲烷量提高87.94%。因此,采用纤维素酶预处理城市生活垃圾后应用于中温厌氧发酵,对解决目前常规厌氧发酵存在的主要问题是可行的。%To resolve the defects in conventional anaerobic fermentation process such as fermenting rate lag,low biogas production rate and so on,municipal solid waste(MSW) was pre-treated with cellulase(as hydrolysis).By using single-factor experiment technique,the effects of different cellulase doses,hydrolysis temperature and duration,hydrolysis pH level and substrate concentration(volatile solide,VS) were tested on the degree of hydrolysis for cellulose in MSW.It was indicated that optimal conditions of the MSW pre-treatment were cellulase dosage 120 units per gram of VS,hydrolysis duration 24 hours,hydrolysis temperature 60℃,substrate concentration 8%and pH value 5.6 whilst the degree of hydrolysis could reach 35.2%.Such pre-treated MSW was then taken to carry on anaerobic fermentation at moderate temperature.The results have shown that the cellulase pre

  16. Cellulase production by two mutant strain of Trichoderma longibranchiatum QM9414 and Rut C30; Produccion de celulasas a partir de dos cepas hiperproductoras de trichoderma longibranchiatum Qm9-41 4 y Rut C30

    Energy Technology Data Exchange (ETDEWEB)

    Blanco, M. J.

    1991-07-01

    Native or pretreated biomass from Onopordum nervosum Boiss, has been examined as candidate feedstock for cellulase production by two mutant strain of Trichoderma Ionqibrachiatum QM9414 and Rut C30. Batch cultivation methods were evaluated and compared with previous experiments using ball-milled, crystalline cellulose (Solka floc). Batch cultivation of T. Ionqibrachiatum Rut C30 on 5% (w/v) acid pretreated O. nervosum biomass yielded enzyme productivities and activities comparable to those obtained on Solka floc. However, the overall enzyme production performance was lower than on Solka floc at comparable cellulose concentrations. This fact may be due to the accumulation of pretreated by products and lignin in the ferment. (Author) 40 refs.

  17. Study on the Conditions for Producing Cellulase by Liquid Fermentation with Aspergillus niger HQ-1%黑曲霉HQ-1液体发酵产纤维素酶条件研究

    Institute of Scientific and Technical Information of China (English)

    张辉; 桑青

    2009-01-01

    [Objective] The study aimed to discuss the optimum condition for producing cellulase by liquid fermentation of Aspergillus niger HQ-1. [Method] With 1 strain of A. niger HQ-1 with higher activity of producing cellulase as the original srtain, the optimum conditions for liquid fermentation of A. niger HQ-1 to produce cellulase was studied primarily through the single factor experiments and orthogonal tests and the activities of CMCase and FPAase were measured. [Result] The factors influencing the production of CMCase and FPAase by liquid fermentation of A, niger HQ-1 were in order were the content of compound carbon origin, the ratio of 2 kinds of carbon, the content of nitrogen and the liquid volume in flask. The optimal medium of producing CMCase was as follows: 15‰ bran+corn stalk powder (1∶1), 2.0‰ NH4Cl, other components as same as Mandel's medium. The optimal medium of producing FPAase was as follows: 20‰ bran+corn stalk powder (1∶1), 1.5‰ NH4Cl, others were as same as Mandel's medium. For both medium, the optimum pH was 5.0 and medium volume was 50 ml/250 ml triangle flask. The activities of CMCase and FPAase reached 525.2 and 217.6 IU respectively after culturing for 4 d under 30 ℃ and 170 r/min. [Conclusion] This study provided some technique reference for the research and development of producing cellulase strain.%[目的] 探讨黑曲霉HQ-1液体发酵产纤维素酶的最适条件.[方法] 以1株产酶活力相对较高的黑曲霉HQ-1为出发菌株,采用单因子试验和正交试验对黑曲霉HQ-1液体发酵产纤维素酶的最适条件进行初步研究,并测定了CMC酶(CMCase)和滤纸酶(FPAase)的活力.[结果] 影响黑曲霉HQ-1液体发酵产CMCase和FPAase的因素依次是复合碳源的含量、2种碳源的比例、氮源含量和装液量.该菌产CMCase的最适培养基为:麸皮+玉米秸秆粉15‰(1∶1),NH4Cl 2.0‰,其他成分同Mandel's营养液;产FPAase的最适培养基为:麸皮+玉米秸秆粉20‰(1

  18. 短小芽孢杆菌产碱性纤维素酶的发酵工艺优化%Optimization of Fermentation Process of Bacillus pumilus Producing Alkalescence Cellulase

    Institute of Scientific and Technical Information of China (English)

    黄谚谚; 吴华珠; 许旭萍

    2012-01-01

    With the purpose of getting the optimum fermentation condition of alkalescence cellulase produced by B. Pumilus SI 2, the fermentation condition was optimized through the single factor and orthogonal design. The results showed that the bran and CMC-Na were the suitable carbon source and the best nitrogen source was yeast. The enzyme producing rate of organic nitrogen sources was higher than that of inorganic nitrogen sources. The optimal conditions were as follows: initial pH 8.5, culture temperature 32℃,70 mL media filled in the 250 mL triangle flask, the inoculation amount 3%(v/v), fermentation time 20 h. The alkaline cellulase activity reached 367. 9 g/mL under the optimal condition.%为了确定短小芽孢杆菌(Bacillus pumilus S12)产碱性纤维素酶的最适发酵条件,采用液体摇瓶的发酵方法,进行最适发酵条件的单因素试验和正交试验.结果表明,Bacillus pumilus S12菌株合成纤维素酶的最适碳源为CMC和麸皮,最适氮源为酵母膏,有机氮比无机氮更有利于产酶;最适产酶条件为起始pH8.5,培养温度32℃,装液量为250mL三角瓶装70mL培养液,接种量为3%,在此条件下发酵20h,酶活力可达367.9 μg/mL.

  19. 纤维素酶对废纸浆废水生化污泥脱水性能的影响%Effect of cellulase on sludge dewatering performance of recovered pulp wastewater

    Institute of Scientific and Technical Information of China (English)

    张安龙; 赵登; 景立明; 罗清

    2014-01-01

    采用纤维素酶处理废纸浆废水生化污泥,讨论了其影响因素.通过单因素实验和正交试验得到最佳工艺条件:酶用量为2.25U·g -1,温度为55℃,反应时间为75min,其处理后污泥的沉降性能得到很大程度的改善.红外光谱图和电镜扫描分析表明:经酶处理后污泥化学性质发生改变,细小颗粒污泥均匀分布,其污泥的结构紧密易实现固液分离,从而提高了污泥脱水性能.%The cellulase is applied in the treatment of biochemical sludge of recovered pulp wastewater and the influencing factors are discussed in this paper .Through the single factor experiment and orthogonal experiment to determine the optimal reaction conditions ,the re-sults show that :when dosage of enzyme is 2 .25 U · g -1 ,the temperature is 55 ℃ ,the reac-tion time is 75 min ,and the sludge settling properties has a large degree of improvement . Infrared spectrum and SEM analysis showed that cellulase treatment biochemical sludge can make sludge chemical properties change and fine granular sludge uniformly distribute ,then the compact sludge structure is easy to realize solid-liquid separation ,thereby improving the dewatering performance of sludge .

  20. 从桑树根际土壤分离的产低温碱性纤维素酶菌株BJ-XH及酶的特性研究%Isolation of Cold-active Alkaline Cellulase Producing Microbial Strain BJ-XH from Mulberry Rhizospheric Soil and Analysis of Enzymatic Property

    Institute of Scientific and Technical Information of China (English)

    李强; 季更生; 谷绪顶; 黄倩; 费娟娟; 冯圆圆

    2012-01-01

    Alkaline cellulase has shown great applicative potential in detergent, forage, textile and food industries. Aimed to obtain microbial strains producing cold-active alkaline cellulase, we isolated 16 cellulase producing microbial strains from mulberry rhizospheric soil in winter. A microbial strain BJ-XH producing high activity cellulase was obtained through isolation with cellulose Congo red selection medium and determination of cellulase activity. Based on the morphological, physiological and biochemical properties, BJ-XH was identified to be an actinomycete belonging to Nocar-dia. The optimal condition for cellulase production of BJ-XH was culture temperature 28 ℃, culture medium pH 8. 5, inoculation quantity 6%, and culture time 6 to 8 days. The major cellulase produced by this strain was endo-β-1,4-gly-canase (Cx enzyme). Activity of this enzyme could reach 5. 6 lU/mL with optimal catalytic temperature 28 ℃ and optimal catalytic pH 9.0, showing typical property of cold-active alkaline cellulases. In addition, metal ions Mn2+, Fe3+ and Co could improve its activity, while Cu2+ and Pb2+ inhibit its activity. These results indicated that microbial strain BJ-XH could yield cold-active alkaline cellulase which has great potential for further exploration and utilization.%碱性纤维素酶在洗涤、饲料、纺织和食品等领域具有较大的应用潜力.为获得产低温碱性纤维素酶的菌种,于冬季从桑树根际土壤中分离到16株纤维素酶产生菌,通过纤维素刚果红选择培养基分离及酶活性测定,筛选出1株能产酶活较高的纤维素酶的菌株BJ-XH,依据形态特征和生理生化特性初步认为该菌株为诺卡氏菌属(Nocardia)的放线菌.BJ-XH菌株的最佳产酶条件为:培养温度28℃,培养液pH 8.5,接种量6%,培养时间6~8d.该菌株产生的纤维素酶主要为内切β-1,4葡聚糖酶(Cx酶),其酶活力可达5.6 IU/mL,酶最适催化温度为28℃,最适催化pH9.0,属于低温碱性纤

  1. 一株耐冷玫红假裸囊菌HD1031的鉴定及其所产纤维素酶的研究%Identification of a psychrotroph Pseudogymnoascus roseus HD1031 and the properties of its producing cellulase

    Institute of Scientific and Technical Information of China (English)

    韩龙; 石家骥; 石磊; 王有智; 钞亚鹏; 杨敬; 张国青; 钱世钧

    2013-01-01

    [目的]以纤维素为唯一碳源,从四川省阿坝自治州黄龙沟的高山低温环境中分离筛选产纤维素酶的耐冷菌,并研究菌株的产酶特征.[方法]根据菌株的ITS序列分析及形态特征,对菌株进行鉴定.利用DNS法测定纤维素酶酶活性.[结果]从四川省阿坝自治州黄龙沟的高山腐殖土中筛选出一株产纤维素酶的耐冷菌HD1031,经鉴定该菌为玫红假裸囊菌(Pseudogymnoascus roseus).该菌可在4℃-25℃生长,最适生长温度为16℃-17℃.该菌在以微晶纤维素和玉米芯粉为碳源、硫酸铵和Tryptone为氮源的培养基中,17℃、160 r/min摇瓶发酵8d后产生纤维素酶,其中内切葡聚糖酶酶活为366.67 U/mL,滤纸酶酶活87.6 U/mL,β-葡萄糖苷酶酶活90.8 U/mL,酶最适反应pH为6.0,最适反应温度为50℃.[结论]筛选获得一株产纤维素酶的耐冷菌HD1031,此菌株所产纤维素酶在20℃-40℃下活性较高,对热敏感,具有低温纤维素酶的特点.%[Objective] To screen a psychrotolerant cellulase-producing strain from the decayed soil of Huanglong Mountain,Sichuan,China,and determine the properties of the cellulase.[Methods] The strain was identified by the morphology and ITS sequence analysis.The DNS method was used to determine the activity of cellulases.[Results] A psychrotolerant cellulase-producing strain HD1031 was screened from the decayed soil of Huanglong Mountain.It was identified as Pseudogymnoascus roseus by the morphology and ITS sequence analysis.The strain could grow between 4 ℃ and 25 ℃ and its optimum growth temperature is 16 ℃-17 ℃.In the medium containing Avicel,corn cob powder,(NH4)2SO4 and Tryptone,the strain HD1031 produced cellulases.After cultivating under the conditions of 17 ℃ and 160 r/min for 8 days,the cellulases activities in the fermentation broth were CMCase activity 366.67 U/mL,FPA 87.6 U/mL and the β-glucosidase activity 90.8 U/mL.The optimum pH value and temperature of the CMCase was pH 6

  2. 稻草生物质的预处理及其发酵产酶与酶解效果研究%Production and characterization of cellulase from straw biomass pretreated with different methods

    Institute of Scientific and Technical Information of China (English)

    沈江涛; 段学辉; 郑希帆; 张倩; 牛书操

    2015-01-01

    采用稀酸、稀碱、高温稀碱、亚硫酸盐法( SPORL法)和稀酸亚硫酸盐法(稀酸SPORL法)对粉碎稻草秸秆预处理,考察不同预处理方法对稻草基质多菌发酵产纤维素酶的影响,分析预处理前后稻草基质主要成分的变化,酶水解液中糖组分的含量。结果表明:稀酸SPORL法处理的稻草粉在固态发酵产酶和酶解糖化都具有较好的效果,所得羧甲基纤维素酶( CMCase酶)和β葡萄糖苷酶(β G)比酶活分别达到21511�22和51508�41 U/g,同时酶水解率达到84�99%。除SPORL法外,其他预处理方式所得酶活均出现了不同程度的下降。稀酸预处理对稻草基质中的半纤维素去除效果较好,含量由20�77%下降到7�34%;稀碱高温处理对木质素脱除效果较好,Klason木质素含量由12�47%下降到7�58%。通过酶解糖化实验发现,未处理稻草粉酶水解率仅为17�82%,稀碱高温法效果最好,稻酶水解率达到91�66%。稀酸和稀酸SPORL法处理后,稻草粉基质的酶解糖化液中,戊聚糖占总糖相对含量较低,分别为7�38%和6�92%。%Straw powder was pretreated by diluted acid, diluted alkali, high temperature with diluted alkali,sulfite pretreatment(SPORL),sulfite pretreatment with diluted acid(SPORL with diluted acid). The effects of pretreatment on the cellulases production by solid state fermentation and the yield of fermentable sugars hydrolyzed were studied. Results showed when the straw substrate pretreated by diluted acid with SPORL for cellulase fermentation,carboxymethyl cellulase( CMCase) and β⁃glucosidase(β⁃G) activities reached 21 511�22 U/g and 51 508�41 U/g, respectively. The hydrolyze rate of cellulases reached 84�99%. The cellulase activities decreased in different degrees excluding SPORL. The diluted acid method was better to remove hemicelluloses, and the content of hemicelluloses decreased from 20

  3. Screening of a Cellulase Producing Halotolerant Strain Marine Aspergillu sp and Its Fermentation Conditions%株耐盐纤维素酶海洋曲霉的筛选及产酶条件研究

    Institute of Scientific and Technical Information of China (English)

    刘杰凤; 薛栋升; 何可可; 陈慧英; 姚善泾

    2012-01-01

    从东海海底泥中分离到了一株耐盐和高产内切葡聚糖酶(CMCase)及β-葡萄糖苷酶(β-Gluase)的海洋曲霉ZJUBE-1,根据形态特征及18S rDNA分析,初步鉴定该菌为黑曲霉(Aspergillus niger).通过液体发酵产酶优化,获得最佳培养基组成,质量分数分别为:麸皮2.5%,氯化铵(或蛋白胨、磷酸二氢铵)1%,含NaCl 3.0%~3.6%的人工海水作无机盐液.发酵条件为100mL三角瓶装培养液30 mL,在起始pH 6.0、37℃,接种量为6%,180 r/min条件下振荡培养7d,测得发酵液中CMCase、β-Gluase.酶活及滤纸酶活(FPA)分别为5.3、5.4、0.75 U/mL.该菌在NaCl质量分数24%下仍具产酶能力,所产3种纤维素酶的最适反应温度均为65℃,最适反应pH分别为4.0、5.0、5.0;在NaCl质量分数为2%~12%(-Gluase在2%~16%)范围内,3种酶活力维持较高水平.该菌及其所产纤维素酶的高度耐盐性预示着其在废水处理、纺织业、海洋养殖、海产品加工或海藻的能源化利用等高盐环境下可能有较好的应用前景.%In this manuscript, a halotolerant marine strain Aspergillus sp ZJUBE-1 possessed the production of endoglucanase and β-Gluase with high cellulase activities was isolated from the submarine mud sample of Zhejiang inshore, and identified as Aspergillus niger by two different methods of both the morphology and 185 rDNA gene sequences. The optimal medium compositions for liquid-state fermentation for cellulase production by strain ZJUBE-1 were determined and listed as follows.- wheat bran 2. 5%, NH,Cl or (NH4)H2PO4 1%, NaCl 3~ 3. 6%. Under the condition of shaking culture for 7 days, at 37 ℃ , the initial medium pH 6. 0 and rotating speed 180 r/min, the maximum activities of carboxymethyl cellulase activity (CMCase), β-glucosidases (β-Gluase) and filter paper activity (FPA) were respectively reached at 5. 3 U/mL-1, 5. 4 U/mL-1 and 0. 75 U/mL , respectively. Moreover, the result showed that the strain still

  4. 绿液预处理玉米秸秆产纤维素酶的研究%The Research on Cellulase Production of Green Liquor Pretreated Corn Stover

    Institute of Scientific and Technical Information of China (English)

    赵士明; 陆青山; 谷峰; 余世袁

    2012-01-01

    Abstract:The effect of green liquid pretreated corn stover on cellulase production of Trichoderma reesei was investigated. Inspecting the influence of pretreatment conditions on cellulase production of corn stover at different cooking temperature, total alkali charge and sulfidity by orthogonal experiment, we could conclude from range and variance analysis that the extent of impact to cellucase production of green liquor pretreated corn stover was in turn total alkali charge, cooking temperature and sultidity. The optimum filter paper activity (FPA) reached 2.6 IU/mL. FPA productions from corn stover oretreated bv cooking temperature140℃ and 170 ℃, total alkali charge 4 %, sulfidity 0, 20 %, 30 % and 40 %, were comprehensively compared and evaluated. It was found that the optimum conditions, i. e. , cooking temperature 140℃, total alkali charge 4 %, sulfidity 0, might ensure the value of FPA on and avoid the loss of materials. The results showed that green liquid pretreatment for corn stoverwas feasible for cellulase production of Trichoderma reesei. Taking corn stover pretreated by cooking temperature 140 ℃, total alkali charge 4% and sulfidity 0 as carbon sources whose concentration was 12 g/L, FPA and β-glucosidase activity(β-GA) were up to 2.5 IU/mL and 1.3 IU/mL respectively. The enzyme production period was 5 days.%研究了绿液预处理玉米秸秆对里氏木霉产纤维素酶的影响。通过正交试验考察3个预处理条件对玉米秸秆产纤维素酶的影响,从极差和方差分析可知,对绿液预处理玉米秸秆产纤维素酶的影响程度由大到小依次是总碱量、蒸煮温度、硫化度,最大滤纸酶活(FPA)达到2.6 IU/mL。比较在蒸煮温度140℃和170℃,总碱量4%,硫化度0、20%、30%与40%下所产的FPA,经综合评定,在最优条件下蒸煮温度140℃,总碱量4%和硫化度0时可以尽量避免原料损失的前提下,保证FPA的大小,结果表明,里氏木霉利用绿液预处

  5. 绿色木霉纤维素酶AS3.3032固态发酵的研究%Production of cellulase by Trichoderma viride AS3.3032 in sloid state fermentation

    Institute of Scientific and Technical Information of China (English)

    张德强; 黄镇亚; 张志毅

    2001-01-01

    In the research, using wheat bran and steam exploded bagasse asmain substrate, cellulase is produced by solid state fermentation with Trichoderma viride AS3.3032. The effect on cellulase production is studied of nitrogen source, carbon source, surface active agent, inoculation, temperature, wate content and initial pH value of solid substrate. The results are:①With NH4SO4 as nitrogen source, FPA,CMC and β-gase all are high, 122.5 FPA U/g dried substrate, 1470.0 CMC U/g dried substrate and 119.3 β-Gase U/g dried substrate respectively;②With wheat bran and bigasse(their ratio 3 to 2) as carbon source, FPA, CMC and β-Gase get highest, 138.2 FPA U/g dried substrate, 134.6 β-Gase U/g dried substrate and 1603.1 CMC U/g dried substrate respectively;③Adding 0.1% Tween-80 to culture medium, FPA, β-Gase and CMC can increase by 230%,280% and 230% respectively, but with addition of 0.5%~0.7% washing powder, the three above indices increase by 310%,370%,300% resepctively;④The activity of produced cellulase goes up to the highest when the water amount of medium, the temperature and the initial pH are 250%,28℃ and 3.5.%该研究以麦麸和汽爆蔗渣为主要原料,采用绿色木霉AS3.3032(Trichodermaviride)固态发酵生产纤维素酶,研究了氮源、碳源、表面活性剂、接种方式、培养基含水量、培养温度、培养基起始pH值对绿色木霉产酶活力的影响.研究结果表明:①以硫酸铵为氮源,其FPA,CMC,和β-Gase酶活力均较高,每克干曲分别高达122.5FPAU/g,1470.0CMCU/g和119.3β-GaseU/g;②碳源以麸蔗比为3∶2时,FPA,β-Gase和CMC酶活力均为最高,每克干曲分别高达138.2FPAU/g,134.6β-GaseU/g和1603.1CMCU/g;③添加0.1%的Tween-80和0.5%~0.7%的洗衣粉可分别提高FPA,β-Gase和CMC为2.3倍、2.8倍、2.3倍和3.1倍、3.7倍、3.0倍;④培养基含水量、培养温度、培养起始pH值分别为250%,28℃和pH3.5,产酶活力最高.

  6. 快中子辐射诱变对绿色木霉产纤维素酶的影响%Effect of Fast Neutron Irradiation on Production of Cellulase from Trichoderma viride

    Institute of Scientific and Technical Information of China (English)

    陈光; 徐杨; 孙旸; 刘洁心; 王香琪

    2011-01-01

    The effect of deuterium ion bombardment majority targeted to produce 14Mev fast neutron irradiation, with dosage 0.6—4.8Gy, on production of cellulose from Trichoderma viride AS3.3711 was studied. The death rate was in a rising trend when irradiation dosage was 0.6—4.8 Gy. The results indicated the death rate of spores reached 80.0 % when radiation dosage was 1.2 Gy. It was shown that one positive mutant had significant difference compared to the original with paired samples test (t-test). The filter paper activity of cellulase of strain Fnl0 - 1 reached 633.63 U/mL, with 4 times enhanced, compared with the initial strain. And the CMC activity of cellulase of strain Fnl0 - 1 reached 914.4 U/mL,with 30 times enhanced, compared with the initial strain.%利用快中子对绿色木霉AS3.3711进行辐照,辐照剂量为0.6~4.8 Gy.研究不同剂量快中子对绿色木霉孢子致死率和遗传稳定性的影响,得到了提高纤维素酶活性的菌株.结果表明:辐照剂量在0.6~4.8 Gy 范围内绿色木霉的致死率呈逐渐上升趋势,辐照剂量为1.2 Gy时,绿色木霉的致死率达到80.0%,此突变株的羧甲基纤维素酶活(CMC酶活)可达914.4 U/mL,比出发菌株提高了30倍,滤纸酶活(FPA酶活)为633.63 U/mL,比出发菌株提高了4倍.

  7. Heterologously expressed Aspergillus aculeatus β-glucosidase in Saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of β-glucosidase in industrial ethanol production using Trichoderma reesei cellulases.

    Science.gov (United States)

    Treebupachatsakul, Treesukon; Nakazawa, Hikaru; Shinbo, Hideaki; Fujikawa, Hiroki; Nagaiwa, Asami; Ochiai, Nobuhiro; Kawaguchi, Takashi; Nikaido, Mitsuru; Totani, Kazuhide; Shioya, Koki; Shida, Yosuke; Morikawa, Yasushi; Ogasawara, Wataru; Okada, Hirofumi

    2016-01-01

    Trichoderma reesei is a filamentous organism that secretes enzymes capable of degrading cellulose to cellobiose. The culture supernatant of T. reesei, however, lacks sufficient activity to convert cellobiose to glucose using β-glucosidase (BGL1). In this study, we identified a BGL (Cel3B) from T. reesei (TrCel3B) and compared it with the active β-glucosidases from Aspergillus aculeatus (AaBGL1). AaBGL1 showed higher stability and conversion of sugars to ethanol compared to TrCel3B, and therefore we chose to express this recombinant protein for use in fermentation processes. We expressed the recombinant protein in the yeast Saccharomyces cerevisiae, combined it with the superb T. reesei cellulase machinery and used the combination in a simultaneous saccharification and fermentation (SSF) process, with the hope that the recombinant would supplement the BGL activity. As the sugars were processed, the yeast immediately converted them to ethanol, thereby eliminating the problem posed by end product inhibition. Recombinant AaBGL1 activity was compared with Novozyme 188, a commercially available supplement for BGL activity. Our results show that the recombinant protein is as effective as the commercial supplement and can process sugars with equal efficiency. Expression of AaBGL1 in S. cerevisiae increased ethanol production effectively. Thus, heterologous expression of AaBGL1 in S. cerevisiae is a cost-effective and efficient process for the bioconversion of ethanol from lignocellulosic biomass.

  8. 纤维素降解细菌的分离、鉴定及其系统发育分析%Isolation, Identification and Phylogenetie Analysis of the Cellulase - producing Strain

    Institute of Scientific and Technical Information of China (English)

    赵菁; 黄奋飞; 林生; 林文雄

    2011-01-01

    采用选择性培养基从不同地区土壤中筛选得到具有纤维素分解能力的菌株共65株,从中选取纤维素降解能力较高的细菌1株并对其进行表型、生理生化和分子鉴定。结果表明株菌NJ2-008属于不动杆菌属(Acinetobacter),与乙酸钙不动杆菌(Acinetobacter calcoacetirum)的序列相似度达98%。%The author screened the cellulase- producing strain from the soil of different sites by the selective medium, and then identified the strain based on their phenotype, phisiological - biochemical property and molecular identification. The result showed that strain N J2-008 is closely related to Acinetobacter calcoacetirum with 98% sequence similarity. Therefore, strain NJ2 -008 was identified as the species Acinetobacte.

  9. Research on the process of extraction pectin from orange peel using the cellulase%纤维素酶提取柑桔皮果胶工艺条件的研究

    Institute of Scientific and Technical Information of China (English)

    戴余军; 丁文; 石会军

    2011-01-01

    The research aims to investigate the best conditions for extracting pectin from orange peel. Taking orange peel as material, pectin was extracted from it using the cellulase. Results show that the maximum extracting rate of pectin could be obtained under the following conditions: the concentration of cellulose solution is 0. 5 %, the stirring time is 45 minutes, the temperature is 45 ℃, pH value is 5.6. The extracting rate of pectin is 36.56 %.%为了探索提取果胶的最佳试验条件,以柑桔皮为试验原料,采用纤维素酶法提取果胶,结果表明:在纤维素酶溶液浓度为0.5%,浸提时间为45 min,浸提温度为45℃,浸提pH值为5.6的条件下,果胶的得率最大,果胶提取率达到36.56%.

  10. Activity of cellulase from Thermoactinomycetes and Bacillus spp. isolated from Brassica waste compost Atividade de celulase de Termoactinomicetos e Bacillus spp. isolados de resíduos derivados de compostos de Brassica

    Directory of Open Access Journals (Sweden)

    Chen-Chin Chang

    2009-06-01

    Full Text Available Plant wastes present a high cellulose content, which is an ideal organic material for composting. Five strains of thermophiles from processed Brassica waste were isolated, and the hydrolytic activity on various cellulosic biomass substrata and their temperature profiles were determined. 16S rRNA sequencing identified these strains as Thermoactinomyces and Bacillus spp. Maximal cellulase activity corresponded to 2.3 U mL-1 of enzyme. The application of these strains on Brassica rapa residues demonstrates increased total nitrogen content. TA-3, a Thermoactinomycetes sp. strain, performs best among all inoculants, increasing the nitrogen content from 0.74 to 0.91%, and decreasing the carbon content from 15.4 to 12.2%, showing its high efficiency and bioactivity during compositing.Resíduos vegetais apresentam alta concentração de celulose, que é um material orgânico ideal para preparação de composto. Cinco linhagens de termófilos foram isoladas de resíduos processados de Brassica e a atividade hidrolítica em vários substratos contendo celulose e seus padrões de temperatura foram determinados. O seqüenciamento de rRNA 16S permitiu a identificação dessas isolados como Thermoactinomyces e Bacillus spp. A atividade máxima de celulase foi determinada como de 2,3 U mL-1 de enzima. O uso dessas linhagens em resíduos de Brassica rapa resultou em um aumento total do conteúdo de nitrogênio. TA-3, uma linhagem de Thermoactinomycetes sp., apresentou melhor desempenho entre os inoculantes, aumentando o conteúdo de nitrogênio de 0,74 para 0,91%, e diminuindo o conteúdo de carbono de 15,4 para 12,2%, mostrando sua alta eficiência e bioatividade durante a compostagem.

  11. Analysis of a Commensal Strain Producing Cellulase from Zingiber officinale Rhizosphere%生姜根际一株纤维素降解共生菌的分析

    Institute of Scientific and Technical Information of China (English)

    侯进慧; 王富威; 樊继强

    2012-01-01

    A Zingiber officinale rhizosphere strain SP1 with higher cellulose-decomposing activity was preliminarily analyzed. With PCR, a 1 406 bp 16S rDNA fragment of SP.I was amplified. The method of table concentrator fermentation was used to preliminarily analyze the characteristics of SP1 cellulose. The result shows, pH 7 was the best acidity, 37℃ was the best temperature, and 40 h of fermentation could reach the peak of enzyme activity. Analyze ginger commensal cellulose-decomposing strain for the first time and provided data to develop new nature cellulase.%对一株从生姜根际筛选到的纤维素降解活性较高的菌株SP1进行了初步分析。通过PCR方法获得菌株SP1的16S rDNA序列,长度为1406bp。使用摇床发酵法对菌株SP1发酵产纤维素酶的特征进行了初步分析。结果表明,该菌株产酶的最适pH为7,适宜温度为40℃,发酵40h能达到产酶高峰。试验首次分析了生姜根际纤维素降解共生菌的情况,并为开发新型天然纤维素酶提供了资料。

  12. Effects of Fermented Green Juice and Cellulase on the Fermentation Quality of Banana Leave Silage%绿汁发酵液和纤维素酶对香蕉叶青贮品质的影响

    Institute of Scientific and Technical Information of China (English)

    叶杭; 贺宇; 张国轶; 张文昌; 陈国富; 庄益芬

    2014-01-01

    To research the effect of fermented green juice and cellulase on the fermentation quality of banana leaves silage ,banana leaves single silage was set as the control group and other silages were set up into the following groups with the different additives :2 mL · kg -1 fermented green juice (FGJ) group ,0.05 ,0.15 and 0.25 g · kg -1 of cellulose groups (CEL1 ,CEL2 and CEL3) and 2 mL · kg -1 FGJ+0.05 g · kg -1 cell,2 mL · kg -1 FGJ +0.15 g · kg -1 celland 2 mL · kg -1 FGJ+ 0.25 g · kg -1 cellmixed groups (MIX1 ,MIX2 and MIX3) respectively . Then ,they were ensiled at room temperature and opened 60 days later .In the end ,the fermentation quality and the chemical composition were analyzed .The same results were obtained in all three replicates .Our results showed that the pH and GLR of the other groups are decreased (P< 0.01 or P< 0.05) ,compared with the control group . Meanwhile ,the DMR of three MIX groups were increased (P<0.01) compared with the FGJ ,and the DMR and WSC of MIX groups were increased (P<0.01) ,but the AN/TN were reduced (P<0.01) ,compared with the cellgroups with the same level′s cellulase enzyme .Furthermore ,the pH was declining on trend ,DMR and WSC were increased with the levels of cellulose in three MIX groups .Our results suggested that fermented green juice and cellulase improved the quality of banana leaves silage ,and the effect of M IX3 is the best .%为开发利用香蕉叶生产青贮饲料,研究绿汁发酵液和纤维素酶对香蕉叶青贮品质的影响,在香蕉叶青贮中以香蕉叶单独青贮为对照组,设置如下处理:添加2 mL · kg -1绿汁发酵液(FGJ)组;分别添加0.05、0.15、0.25 g · kg -1纤维素酶(CEL1、CEL2和CEL3)组;分别添加2 mL · kg -1 FGJ+0.05 g · kg -1 CEL、2 mL · kg -1 FGJ +0.15g·kg -1CEL和2mL·kg -1FGJ+0.25g·kg -1CEL混合(MIX1、MIX2和MIX3)组。每个处理重复3次。常温下贮存60 d开封,测定青贮的发酵品质和化学成分

  13. The Study on the Mechanism of Improving of P/C Fabric Soil Release Property by Cellulase Washing%纤维素酶洗涤对提高涤棉织物易去污性的作用机理

    Institute of Scientific and Technical Information of China (English)

    吴颖喆; 邵建中; 付国栋

    2012-01-01

    The aim of this article is to explain why soil release property of cotton-containing fabric can be improved after cellulase washing, and characterize the physicochemical properties of P/C fabric like wettability, abrasion resistance, crystalline and hydrophilic group content after cellulase washing by drip method, Martindale friction-SEM, XRD and copper value analysis. The result shows that after cellulase washing, the cotton fiber surface of P/C fabric becomes more brittle, and the external force can remove the brittle fibers of fabric surface easily, also remove the soil on the fibers. Meanwhile, the crystallinity of cotton fiber is decreased by cellulase, and the 1, 4-(3 glycoside bond is cut to form the aldehyde group. It can increase the hydrophilicity of cotton fiber and enhance the wettability of P/C fabric, so that soil release property of P/C fabric is improved.%为探明纤维素酶洗涤提高含棉织物易去污性的机理,采用滴水法、马丁代尔磨擦—扫描电镜联合分析法、XRD分析和铜值法等测试分析技术对涤棉织物经纤维素酶洗涤后的润湿性、耐磨性、表面形态、结晶度、亲水基团含量等理化性能进行分析表征.结果表明:经纤维素酶洗涤后,棉纤维表面发生脆损,涤棉织物的耐磨性下降,在外力作用下易使织物表面的纤维脱落从而带走污垢;同时,纤维素酶洗涤使棉纤维的结晶度有所下降,纤维素大分子的β-1,4-甙键部分切断形成醛基,从而提高了棉纤维的亲水性,增加了涤棉织物的润湿性,进而使涤棉织物的易去污性得以改善.

  14. Screening of strain producing high-yield cellulase through composite mutagenesis by N~+ implantation and ultraviolet%N~+离子注入-紫外复合诱变选育纤维素酶高产菌株

    Institute of Scientific and Technical Information of China (English)

    陈阿娜; 汤斌; 刘标

    2012-01-01

    Protoplast of Rhizopus sp.TY1 producing cellulase was mutagenized by N+ implantation and ultraviolet composite mutagenesis to screen of strain producing high-yield cellulase.The best enzymolysis time was 3h with the formation rate and regeneration rate of the protoplast as selection criteria.The best dose of N+ implantation was 2.0×1015ions/cm2 and the best irradiation time of ultraviolet was 90s with the mortality rate and positive mutation rate as selection criteria.A strain producing high-yield cellulase Rhizopus sp.TY1.2 was screened,which the FPA and CMC was 5.1U/mL and 20.9U/mL respectively at the end of liquid fermentation.The FPA and CMC were increased by 75.9% and 175.0% respectively compared to that of Rhizopus sp.TY1.Ten times subculturing showed that the mutant had good genetic stability and character of cellulase production.The results indicated that the mutation of Rhizopus sp.TY1.2 was descendible.%对产纤维素酶菌株Rhizopus sp.TY1原生质体进行N+离子注入-紫外复合诱变,以期获得高产菌株。以原生质体形成率和再生率为指标,确定最佳酶解时间3h;以致死率和正突变率为指标,确定最佳N+离子注入剂量为2.0×1015ions/cm2、最佳紫外照射时间为90s。实验筛选得到一株高产菌株Rhizopus sp.TY1.2,液态发酵终点时滤纸酶活力(FPA)和羧甲基纤维素酶活力(CMC)分别达到5.1、20.9U/mL,与出发菌株相比,FPA和CMC分别提高了75.9%和175.0%。传代实验结果显示Rhizopus sp.TY1.2产纤维素酶性能稳定,该结果表明N+离子注入-紫外复合诱变所产生的变异是可遗传的变异。

  15. 产蛋白酶和纤维素酶纳豆芽孢杆菌益生菌株的筛选及其生长特性研究%Screening of protease and cellulase producino Bacillus subtilis Natto strain and its growth characteristics

    Institute of Scientific and Technical Information of China (English)

    孙妍; 王加启; 奚晓琦; 魏宏阳; 周凌云

    2011-01-01

    According to the biological qualification of hydrolysised casein and CMC of Bacillus subtilis Natto, 10 strains were screened by pour plate method from their parent strains of Bacillus subtilis natto NB-1 and NR-1. The strain named NY-3 was selected with higher protease activity and cellulase activity through the test of protease activity and cellulase activity in 48 h fermenting medium among 10 strains. Further research of its growth characteristics showed that the best time of inoculation and fermentation were 14 and 48 h, respectively.%根据纳豆芽孢杆菌(Bacillus subtilis natto)水解酪蛋白和羧甲基纤维素的生物学特性,以菌株NB-1和NR-l为出发菌株,采用稀释涂平板法获得10株初筛纳豆芽孢杆菌,通过测定初筛菌株48 h发酵液中蛋白酶活性和纤维素酶活性,确定NY-3产蛋白酶和纤维素酶活性均相对较高.同时对该菌株生长特性进行了研究.结果表明,NY-3的最佳接种时间和最佳发酵时间分别为14和48 h.

  16. 纤维素酶催化与三液相萃取偶联制备盾叶薯蓣皂苷元%Preparation of diosgenin by integrated bioprocess of cellulase catalysis and extration in three-liquid-phase system

    Institute of Scientific and Technical Information of China (English)

    魏夺; 董悦生; 韩松; 修志龙

    2012-01-01

    Using coupling biotech of cellulase-catalysis and extrusion in three-liquid-phase system that composed of n-hexane, 1,4-dioxane and ammonium sulfate to hydrolyze dioscin in Chinese yan and glucose connected to steroidal saponins, and extration separation to collect diosgenin. The distribution of several steroidal saponins, hydrolyzed glucose, enzyme and its activity in the three-phase system were studied, and catalytic conversion by cellulase-enzyme and formation of diosgenin in three-liquid, organic and aqueous phases were compared. The results showed that diosgenin yield in the three-liquid-phase system could compare with that in organic phase and aqueous phase. The three-liquid-phase system could be suitable for simultaneous enzymatic hydrolysis of steroidal saponins and extration of diosgenin and glucose. The substrate and enzyme were held in the middle phase of 1,4-dioxane, whiles the products, diosgenin and glucose, were partitioned into the top and bottom phase, respectively. The yields of diosgenin was 69. 4% at 96 h in the three-liquid-phase system, which was twice and 27. 6 folds obtained in organic phase and aqueous phase respectively.

  17. Effect of immobilized cellulase on evaluating hydrolysis efficiency of pretreated cellulose material%固定化纤维素酶对预处理纤维素原料水解效果的影响

    Institute of Scientific and Technical Information of China (English)

    吕浩; 邢楠楠; 林增祥; 杜健; 张红漫

    2011-01-01

    Cellulase was immobilized by chitosan gel beads using the cross-linking embedding method. The immobilized enzyme was used to hydrolyze cellulic material corn stover pretreated by steam explosion , ball milling, ultrasonic, spray, and high-temperature processes. Results showed that the steam explosion pretreatment could obtain the best enzymatic hydrolysis efficiency, glucose yield reached 95% ; the ball mill was second to it, glucose yield reached 60%. Meanwhile, the cellulose structures before and after pretreatment were characterized by electron microscopy and FT-IR. It indicated that the physical structure and chemical composition of pretreated materials changed significantly. The steam explosion method and the ball milling method could make the natural structure of the material completely destroy thus increasing the surface area. The internal hydrogen bonds and functional groups of cellulose had been changed during the course of steam explosion, it caused the material more easily to be hydrolized.%以壳聚糖为载体用交联法制备固定化纤维素酶,考察固定化纤维素酶对蒸爆、球磨、超声波、喷淋、高温预处理玉米秸秆纤维素原料的酶解效果.结果表明:物料经蒸爆预处理后酶水解效率最高可以达到95%,球磨预处理水解效率次之,达到60%.用电镜和FT-IR对处理前后秸秆结构进行表征分析,证明预处理对物料的物理结构及化学组成有一定的影响.蒸爆法和球磨法可以使物料致密的天然结构彻底破坏,从而增加物料的比表面积;蒸爆预处理可以使纤维素内部氢键和官能团改变,使物料更易于酶解.

  18. Cloning and expression of cellulase XF-818 of Xylella fastidiosa in Escherichia Coli Clonagem e expressão da celulase Xf-818 de Xylella Fastidiosa em Escherichia Coli

    Directory of Open Access Journals (Sweden)

    Nelson Arno Wulff

    2003-12-01

    Full Text Available Xylella fastidiosa's genome was the first of a plant pathogen to be completely sequenced. Through comparative sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. However, the biological role of each gene should be assigned experimentally. On this regard, heterologous protein expression is a powerful tool to produce proteins from such genes, allowing their characterization. X. fastidiosa lives inside xylem vessels and eventually would degrade pit membranes from xylem cells to move radialy into the host. The identification of several putative plant cell wall degrading enzymes on X. fastidiosa genome prompted the assession of the function of such proteins. The open reading frame (ORF Xf-818 was cloned into expression vector pET20b and E. coli cells harboring such plasmid exhibited cellulase activity. Using IPTG at 0.4 mmol L-1 with a 12 h incubation at 32°C are the best conditions to produce higher amounts of heterologous protein. The enzyme degrades cellulose confirming the endoglucanase activity of Xf-818.Xylella fastidiosa foi a primeira bactéria fitopatogênica que teve seu genoma completamente seqüenciado. A identificação de diversos genes, através de similaridade de seqüências, indicou os possíveis mecanismos de patogenicidade da bactéria. Entretanto, a determinação da função de um gene requer a confirmação experimental e, neste aspecto, a expressão heteróloga é uma poderosa ferramenta. X. fastidiosa coloniza somente o xilema das plantas hospedeiras e a identificação putativa de diversos genes semelhantes a enzimas que degradam a parede celular vegetal, estimularam o presente estudo de catacterização destas enzimas. A clonagem da ORF Xf-818 de X. fastidiosa no vetor de expressão pET20b possibilitou a produção da proteína heterologamente em E. coli. O emprego de IPTG a 0,4 mmol L-1 com 12 h a 32°C, possibilitou as melhores condições para E. coli

  19. 两株高产纤维素酶细菌的筛选、鉴定及酶学特性%Isolation,identification and enzymatic characteristics of cellulose-producing strains with high cellulase activity

    Institute of Scientific and Technical Information of China (English)

    陈丽燕; 张光祥; 黄春萍; 熊艳; 李敏; 常丽梅; 张晓喻

    2011-01-01

    Two cellulose producing strains were isolated from decayed leaf and soil around it. According to morphology, biochemical and physiological characterization, and 16S rRNA sequence analysis,CT1 was identified as Bacillus licheniformis and CM2 was identified as Bacillus subtilis. Through deteminations of CMC enzyme activity and filter paper enzyme activity using liquid fermentation. The result of the CMCasae activity of CT1 is 163.3 U/mL and CM2 is 167.17 U/mL after 4 d, the FPA activity of CT1 is 211.17 U/mL after 2 d and CM2 is 207.83 U/mL after 3 d. The effect of carbon sources on the optimum cellulase-producing conditions of these strains were studied. By silver staining after SDS-polyacrylamide gel electrophoresis preliminary separation cellulose enzyme special bands. The result showed that thc utilization capabilities to different source cellulose were different.%从腐烂枯叶及附近土壤筛选分离得到2株产纤维素酶的菌株.经细菌形态观察、生理生化实验并结合16S rRNA序列分析,将其初步鉴定为地衣芽孢杆菌CT1(Bacillus licheniformis CT1)和枯草芽孢杆菌CM2(Bacillus subtilis CM2).经摇瓶发酵,测定其CMCase、FPA酶活力,结果表明CT1和CM2在液体摇瓶培养4 d后的CMC酶活最大,分别可达163.3 U/mL和167.17 U/mL;CT1摇瓶培养2 d后,FPA酶活达到了211.17 U/mL,CM2摇瓶培养3 d后,FPA酶活为207.83 U/mL.进行不同碳源对菌株产酶能力影响的试验,并通过SDS-聚丙烯酰胺凝胶电泳、银染后初步分析纤维素酶谱条带,发现菌株对不同来源纤维素的降解能力及产纤维素酶的种类均有所不同.

  20. 纤维素酶酶解法提取怀山药多糖工艺研究%Study on Extraction of Polysaccharide from Dioscorea Opposita Thunb by Enzymatic Hydrolysis of Cellulase

    Institute of Scientific and Technical Information of China (English)

    李培

    2015-01-01

    The Polysaccharide was extracted from Dioscorea opposita Thunb by enzymatic hydrolysis of Cellulase .First,the effects of the ratio of material and water ,the amount of the enzyme ,the time of enzyme hydrolysis , and the temperature of enzyme hydrolysis on the extraction rate of Polysaccharide from Di-oscorea opposita Thunb were studied by single factor experiment .Then the extraction process of Polysac-charide from Dioscorea opposita Thunb was optimized by orthogonal test .The results showed that the order of the primary and secondary factors affecting the extraction rate of Polysaccharides from Dioscorea opposi -ta Thunb was in order:the amount of the enzyme ,the time of enzyme hydrolysis ,the temperature of en-zyme hydrolysis ,the ratio of material and water .The optimal extraction conditions were as follows:the a-mount of the enzyme 3%, the time of enzyme hydrolysis 75 min,the temperature of enzyme hydrolysis 60℃,the ratio of material and water 1:30 .The Polysaccharide yield of 15.78% was achieved under these conditions .%以怀山药为材料,采用纤维素酶酶解,提取其活性成分多糖,先通过单因素实验考察了料水比、酶添加量、酶解时间,酶解温度对怀山药多糖提取得率的影响;然后通过正交试验对怀山药多糖的酶解提取工艺进行了优化,结果表明各因素对怀山药多糖提取率影响的主次顺序依次为:酶添加量、酶解时间、酶解温度、料水比;其最优提取工艺条件为:酶添加量3%,酶解时间75 min,酶解温度60 ℃,料水比1:30,在此最优工艺条件下,怀山药多糖的提取率为15.78%.

  1. Efeito de enzimas de maceração na textura do palmito (Euterpe edulis Mart Influence of cellulase, pectinase and hemicellulase on the texture of hearts of palm (Euterpe edulis Mart.

    Directory of Open Access Journals (Sweden)

    Regina Kitagawa GRIZOTTO

    1997-12-01

    Full Text Available Com a finalidade de ampliar o aproveitamento da palmeira produtora do palmito estudou-se a influência da poligalacturonase e de enzimas maceradoras na textura das partes semi-rígidas do vegetal não-comestíveis, incubando-se preparados comerciais de celulase, hemicelulase e poligalacturonase com o palmito preparado na forma de pequenos toletes (1-3 cm de comprimento e em porções de 2cm do raquis do vegetal. Embora os tratamentos com hemicelulase e mistura de hemicelulase e poligalacturonase tenham promovido ligeiro amaciamento do palmito, os resultados mostraram ,de modo geral, acréscimo na textura do palmito cortado em porções de 3,0 cm e em fatias de 1,0 cm indicando solubilização intensa das regiões suscetíveis a hidrólise com a permanência das regiões duras mais ricas em lignina. Como nos outros tecidos do palmito, no raquis fibroso, não foi comprovada estatiscamente a ação das enzimas na textura do vegetal.With the aim of amplifying the utilization of the palm tree the influence of poligalacturonase and macerating enzymes on the texture of the hard tips of hearts of palm was studied, incubating commercial preparations of cellulase , hemicellulase and poligalacturonase with the hearts of palm, prepared as small stalks of 1-3 cm length and with 2 cm portions of the rachis of the plant. Although the treatments containing hemicellulase and a mixture of hemicellulase and poligalacturonase promoted a slight softening of the hearts of palm, the results generally showed a hardening of the texture of the hearts of palm prepared as stalks of 1 to 3 cm indicating great solubilization of the parts susceptible to hydrolysis, the hard lignin rich parts remaining intact. As in the other tissue of the plant, in the rachis of the fibrous hearts of palm, the action of the enzymes was not statiscally proven.

  2. 乳酸菌和纤维素酶对青贮大豆秸秆的发酵品质和营养成分的影响%Effect of Lactobacillus and cellulase on fermentation quality and nutrition composition of ensiled soybean straw

    Institute of Scientific and Technical Information of China (English)

    顾拥建; 占今舜; 沙文锋; 朱娟; 詹康; 林淼; 张维有

    2016-01-01

    研究了乳酸菌和纤维素酶对青贮大豆秸秆发酵品质和营养成分的影响。试验分为5个处理组,分别为试验 I 组(添加20 g/t 乳酸菌)、试验 II 组(添加400 g/t 纤维素酶)、试验 III 组(添加10%麸皮+20 g/t 乳酸菌)、试验 IV 组(添加10%麸皮+400 g/t 纤维素酶)和试验 V 组(添加10%麸皮+20 g/t 乳酸菌+400 g/t 纤维素酶),每个处理组设3个重复。结果表明:试验 V 组的感官评定和 V-Score 评分分数均最高;试验 II 组的 pH 值,氨态氮、乙酸、丙酸和丁酸的含量最高;与试验 I 组相比,试验 III~V 组的粗蛋白质含量分别升高了15.66%、11.52%和10.25%,可溶性碳水化合物含量分别升高了89.01%、57.14%和21.98%,单宁含量分别升高了20.69%、51.86%和70.61%;酸性洗涤纤维含量分别下降了7.76%、10.23%和13.62%(P <0.05)。综上所述,在青贮大豆秸秆中添加麸皮、乳酸菌和纤维素酶能够影响其发酵品质和营养成分。%The effects of Lactobacillus and cellulase on fermentation quality and nutrition composition of ensiled soybean straw were studied.The experiment were divided into 5 groups for group Ⅰ (added 20 g/t Lactobacillus ),group Ⅱ (added 400 g/t cellulase),group Ⅲ (added 20 g/t Lactobacillus and 10% wheat bran),group Ⅳ (added 400 g/t cellulase and 10%wheat bran)and group Ⅴ (added 20 g/t Lactobacillus ,400 g/t cellulase and 10% wheat bran)with 3 replicates.The results showed that:according to the sensory evaluation and V-Score grade,the fermentative effect of group Ⅴwas the best.The pH, content of ammonia nitrogen,acetic acid,propionic acid and butyric acid in group Ⅱ were the highest.Compared with group Ⅰ, the crude protein,water soluble carbohydrate and tannin content of group Ⅲ,Ⅳ and Ⅴ were increased by 1 5.66%,1 1.52%, 10.25%and 89.01%,57.14%,21.98% and 20.69%,5 1.86%,70.61%,respectively.But the acid detergent fiber content of them were reduced by 7

  3. Influence of P eriodic Enzyme and Mycelia Recovery on Cellulase P roduction by Trichoderma reesei%间歇出酶/出菌丝的培养方式对里氏木霉产纤维素酶的影响

    Institute of Scientific and Technical Information of China (English)

    蒋露; 王步成; 白合超; 勇强; 余世袁

    2016-01-01

    The cellulase was produced by fed-batch cultivation of Trichoderma reesei Rut C30 with microcrystalline cellulose and starch hydrolysate as carbon source under the protection of periodic enzyme recovery. The results showed that after three days of fed-batch cultivation,cellulase was partially recovered 15%,30% or 45% at every 1,2 or 3 days. The total enzyme activites of cellulase and β-glucosidase obtained by the methods significantly increased by 26.5%-32.6% and over 46% more than that of fed-bath.Mycelia could also be removed to avoid infection.When enzymes were recovered 15% every day, the cellulase production increased by 35.4% ( without mycelia removal) and 32.5% ( with 15% mycelial removal) compared with those of normal fed-batch cultivation.A high hydrolytic ability of the produced cellulase with enzyme and mycelia recovery was also detected and the hydrolysis efficiency could reach more than 82%,far more than that of Celluclast(62.03%).%以微晶纤维素和淀粉水解液作为碳源生产纤维素酶,在里氏木霉Rut C30分批补料生产纤维素酶的过程中通过间歇取出部分酶的培养方式保护纤维素酶。实验结果显示:采用间歇出酶培养方式,在分批补料3d后,每1、2或者3天取出部分酶液。当平均每天取出15%的酶液,酶活显著增加,总纤维素酶酶活较单纯分批补料提高26.5%~32.6%,而总β-葡萄糖苷酶酶活提高超过46%;采用间歇出酶出菌丝培养方式,当每天取出15%的酶液时,纤维素酶产量比分批补料增长35.4%,去除和酶液等量的菌丝,酶活和分批补料相比增长32.5%,而且这两种培养方式所生产的纤维素酶的酶解效率高达82%,远超商品酶Celluclast(62.03%)。

  4. Inductive effects of fungal pathogens of American Ginseng and Ginseng on chitinase and cellulase of antigonistic actinomycetes%西洋参和人参病原真菌菌体对放线菌2种水解酶的诱导

    Institute of Scientific and Technical Information of China (English)

    于妍华; 薛泉宏; 唐明

    2011-01-01

    【目的】研究特定拮抗放线菌对西洋参人参土传病害病原真菌的接触抗菌机理。【方法】以5株西洋参、人参土传病害病原真菌菌体为惟一碳源,用液体培养及3,5二硝基水杨酸(DNS)法研究5株供试病原真菌对9株拮抗放线菌几丁质酶和纤维素酶合成的诱导作用;采用搭片法,观察9株拮抗放线菌与5株供试病原真菌菌丝间的相互作用。【结果】①以5株病原真菌菌体为惟一碳源时,可诱导9株拮抗放线菌合成几丁质酶和纤维素酶;9株放线菌的几丁质酶和纤维素酶活性分别为7.17~11.58和6.14~21.20 U,其中西洋参恶疫霉菌体对9株放线%【Objective】 Inhibitory mechanism of antigonistic actimomycetes fighting against fungal pathogens of soil-borne disease in American Ginseng and Ginseng was studied.【Method】 The inductiveness was assessed by the activity of chitinase and cellulase,which were induced from 9 strains of antigonistic actinomycete by using 5 dried strains of fungal pathogens of American Ginseng and Ginseng as C-source in liquid culture medium and DNS measurement,and the mutual effects were observed between antigonistic actinomycetes and fungal pathogens of American Ginseng and Ginseng through the method of building pieces on plate.【Result】 ①The activity of chitinase and cellulase,which was mainly distributed among 7.17-11.58 and 6.14-21.20 U,respectively,differed from 9 strains of antigonistic antinomycete induced from 5 strains of fungal pathogen of American Ginseng and Ginseng.The powder of Phytophthora cactorum could induce antigonistic actinomycetes to produce much more chitinase and cellulase.②The mutural effects between mycelia of Act11,Act13,Act24 and Cylindrocarpon destructans,Cylindrocarpon sp.,such as winding and decomposition,were observed distinctively.【Conclusion】 The mycelia of 5 strains of fungal pathogen of American Ginseng and Ginseng could induce 9 strains of antigonistic actinomycete

  5. Effect of adding cellulase to alfalfa meal ration on production performance, egg quality and nutrient digestibility on layers%苜蓿草粉饲粮添加纤维素酶对蛋鸡生产性能、蛋品质及养分利用率的影响

    Institute of Scientific and Technical Information of China (English)

    夏素银; 王成章; 詹发柏; 严学兵; 刘起栓; 陈艳荣; 李海艳

    2011-01-01

    .05)和4.17% (P>0.05).总的来说,在蛋鸡饲粮中添加苜蓿草粉后对生产性能、蛋品质无显著影响,但能在一定程度上改善蛋品质,蛋黄颜色得到极显著提高,高水平的苜蓿草粉组添加纤维素酶后,对粗蛋白质、钙和粗纤维的消化率有改进.%The objectives of this study were to evaluate production performance, egg quality and nutrient digest ibility of layers adding cellulase to alfalfa meal ration. An experiment was conducted using a total of 210 High land Brown Layers aged 40 weeks to study those of index. Layers were randomly divided into 7 groups, 3 repli cates for each group and 10 for each replicate. One of these groups was used as the control group, others were used as treatment groups including group I to group VI. The control group was fed the basic diet, group I to group VI were fed the diets containing alfalfa meal and cellulase in different levels (3%> 5% , 7%, 7%+0.1% cellulase, 7%+0. 2% cellulase, 7% + 0. 3% cellulase), respectively. By a series experiment of feeding and di gestion, the production performance, egg quality and nutrient digestibility of layers were measured in each group. The results showed that, 1) Layers fed diets added with alfalfa meal and cellulase had no significant difference for laying rate and egg weight (P>0. 05) than that of control group, however, average daily feed in take of the group III and feed/egg ratio of the group V were significantly decreased comparing with the control. 2)Addition of alfalfa meal and cellulase had no significant effects on egg white height, egg yolk pH, egg white pH, egg shape index, egg yolk index, and eggshell relative weight (P>0. 05), however, those of yolk color and eggshell strength, Haugh unit were significantly improved comparing with control, which egg yolk-color was significant higher (P0. 05) , but the apparent digestibility of crude protein, the crude fat, crude fiber and acid fiber in the group V increased by 14. 79% (P>0. 05), 8. 48

  6. [New cellulases efficiently hydrolyzing lignocellulose pulp].

    Science.gov (United States)

    Skomarovskiĭ, A A; Markov, A V; Gusakov, A V; Kondrat'eva, E G; Okunev, O N; Bekkerevich, A O; Matys, V Iu; Sinitsyn, A P

    2006-01-01

    Commercial and pilot enzyme preparations from fungi of the genera Penicillium and Trichoderma have been compared with regard to their action on conifer wood pretreated with acidified aqueous ethanol (organosolve). In most experiments, enzymes from the genus Penicillium allowed higher yields of reducing sugars and glucose than those from Trichoderma. High beta-glucosidase activity is essential for deep pulp hydrolysis.

  7. Enhanced cellulase production in mutants of Thermomonospora

    Energy Technology Data Exchange (ETDEWEB)

    Fennington, G.; Lupo, D.; Stutzenberger, F.

    1982-01-01

    Thermomonospora curvata, a thermophilic actinomycete, secretes multiple forms of endo-beta, 1-4-glucanase (EG) when grown on cellulose-mineral salts liquid medium. The EG activity (measured as carboxymethyl cellulose hydrolysis) was separated by ion exchange chromatography into three distinct components which differed in their kinetic properties. Exposure of T. curvata to ultraviolet light, N-nitrosoguanidine, or ethane methyl sulfonate produced mutants with enhanced EG production. Selection of colonies which cleared cellulose agar plants containing 2-deoxyglucose or glycerol yielded mutants having 1.5 to 2.6 times the extracellular EG and saccharifying activity (measured by filter-paper and cotton-fiber hydrolysis). The secretion of extracellular protein was increased proportionally in mutant cultures. (Refs. 40).

  8. 木薯纤维素乙醇发酵的纤维素酶成本评价%Evaluation of the cellulase cost during the cassava cellulose ethanol fermentation process

    Institute of Scientific and Technical Information of China (English)

    方镇宏; 邓红波; 张小希; 张建; 鲍杰

    2013-01-01

    木薯中的纤维素成分约占木薯干重的10%(W/W).文中以木薯燃料乙醇生产的木薯纤维素酒渣为原料,从纤维素酶成本角度评估了三种利用木薯纤维素组分发酵生产乙醇的方法,包括木薯纤维素酒渣的直接糖化和乙醇发酵、木薯纤维素酒渣预处理后的糖化与乙醇发酵、木薯乙醇发酵中同步淀粉与纤维素糖化以及乙醇发酵.结果表明,前两种方法的纤维素利用效率不高,酶成本分别达到13602、11659元/吨乙醇.第三种方法,即在木薯乙醇发酵过程同时加入糖化酶和纤维素酶,进行同步淀粉与纤维素糖化,进而进行乙醇发酵,木薯纤维素乙醇的收益最高.发酵结束时的乙醇浓度从101.5g/L提高到107.0g/L,纤维素酶成本为3 589元/吨乙醇.此方法利用木薯纤维素与木薯淀粉同时进行,不会带来额外的设备及操作投入,酶成本低于产品乙醇价格,可实现盈利,因此第三种方法为木薯纤维用于乙醇发酵的最适方法,本研究结果将为木薯乙醇产业深度利用木薯纤维提供依据.%Cellulose takes nearly 10% (W/W) dry weight of cassava tubers. In this study, the cellulase cost of different ethanol fermentation from cassava cellulose was evaluated. The processes include the direct saccharification and fermentation of original cassava cellulose residues, the direct saccharification and fermentation of pretreated cassava cellulose residues, and the simultaneous co-saccharification and fermentation of cassava starch and cassava cellulose. The results show that the cassava cellulose utilization in the first two processes were low with the enzyme cost of 13 602 and 11 659 RMB Yuan per tone of ethanol, respectively. In the third process, the final ethanol concentration increased from 101.5 g/L to 107.0 g/L when cassava cellulose and cassava starch were saccharified simultaneously. Comparing to the first two processes, the third one demonstrated the lowest enzyme cost at 3

  9. Comparation of Gut Cellulase Activity in Four Herbivorous Beetles%4种植食性甲虫肠道内纤维素酶活性的比较

    Institute of Scientific and Technical Information of China (English)

    李燕利; 薛怀君; 胡春祥; 杨星科

    2013-01-01

    To compare and estimate the cellulose enzyme activity in different insects, the paper presented a comprehensive analysis of enzyme activity from the gut juices of four herbivorous insect species ( larvae and adults of three leaf-eating beetles, Chrysomela populi, Ambrostoma quadriimpressum and Gallerucida bifasciata, and larva of a trunk-borer , Semanotus bifasciatus). The results showed that there were complete cellulase systems in these four beetles, and that both temperature and pH had a significant impact on the enzyme activity in the gut contents. For both larvae and adults of three leaf beetles, the optimal conditions of enzyme activity were 40 - 60℃ and pH 4 - 6, and the enzyme activity could not be detected under high temperature and high pH conditions. However, in trunk-borer of 5. bifasciatus the highest enzyme activity of exoglucanase (C1) was observed at pH 9 and 70 ℃. In addition, the enzyme activities of endoglucanase (Cx) and β-glucosidase in S. bifasciatus were lower than that of the leaf beetles, while the enzyme activity of C1 was much higher than that of these leaf beetles. The results indicated that temperature and pH had significant influence on insect cellulose enzyme activity. The different feeding habits and the phylogenetic relationship may result in different enzyme activity.%为比较和客观评价昆虫体内纤维素酶的活性,在不同温度及pH值下测定杨叶甲、二纹柱萤叶甲、紫榆叶甲成、幼虫及双条杉天牛幼虫消化道内3类纤维素酶的活性.结果表明:4种甲虫体内均有消化纤维素的完整酶系,3种叶甲成、幼虫体内各纤维素酶的最适温区为40 ~ 60℃、最适pH值范围为4~6,在高温和强碱条件下均未能检测出酶活性;双条杉天牛幼虫C1酶活性在强碱pH =9和70℃条件下活性最高,Cx酶和β-葡萄糖苷酶活性低于3种叶甲的成虫和幼虫,C1酶活性显著高于3种叶甲的成虫和幼虫.温度和pH值对昆虫体内纤维素酶

  10. 纤维素酶预处理对杨木APMP浆配抄性能的影响%Cellulase Pretreatment on Poplar APMP and Its Impact on Papermaking

    Institute of Scientific and Technical Information of China (English)

    申莉红; 杨桂花; 陈嘉川

    2014-01-01

    为了改善杨木高得率浆的配抄性能,扩大其应用范围,研究探讨了纤维素酶预处理对杨木碱性过氧化氢机械浆( APMP浆)配抄性能的影响。结果表明,与未经过酶预处理的杨木APMP浆相比,酶处理后 APMP 浆打浆能耗最大可降低19%,纤维吸水润胀程度增加,保水值增加12.6%;与不同比例针叶木漂白硫酸盐浆( BKP浆)配抄后,纸张的裂断长、耐破指数和撕裂指数均有所提高,最大可分别提高6.1%,44.6%和30.3%,纸张的松厚度、白度和不透明度稍有降低。纤维质量分析仪(FQA)、扫描电镜(SEM)和X射线衍射(XRD)分析结果显示,酶处理后纤维平均长度增加7.0个百分点,细小纤维组分减少10.0%,纤维卷曲指数降低22.4%,结晶度提高0.7%。可见,纤维素酶预处理可以改善杨木APMP浆的配抄性能,提高纸浆纤维质量,所配抄的纸张的物理强度性能指标有显著提高。%Cellulase pretreatment was utilized to treat the poplar alkaline peroxide mechanical pulp ( APMP) in present study to improve its papermaking performance and expand the application. The results showed that the beating energy consumption of the pretreated APMP decreased by 19% maximum and water retention value increased by 12. 6%, compared to the virgin APMP. Meanwhile, the breaking length, bursting index and tearing index of the papersheets, which were furnished by pretreated APMP and blended with different ratio of softwood bleached kraft pulp (BKP), increased maximum by 6. 1 %,44. 6 % and 30. 3%respectively. However, the bulk, brightness and the opacity were decreased slightly. The fiber quality analysis( FQA) , scanning electron microscope(SEM) and X-ray diffraction(XRD) were employed to characterize. The analysis results indicated that the fiber length increased by 7. 0%, the fines content decreased by 10. 0%, and the curl index decreased by 22. 4% compared with the pretreated APMP with the virgin APMP. Beside, a 0. 7% increase of the

  11. Screening and Identification of a Cellulase-Producing Bacterium Strain SWU6%一株纤维素酶产生菌SW U6菌株的筛选鉴定

    Institute of Scientific and Technical Information of China (English)

    吴晶晶; 王爱印; 周敏; 陈林; 谢洁

    2014-01-01

    为寻找高效纤维素降解菌,用于蚕沙及桑树废弃枝条等的加工处理,本研究以羧甲基纤维素钠(CMC‐Na)为唯一碳源,从西南大学校园土壤中分离纤维素降解菌。结合测定菌株在羧甲基纤维素钠平板上形成水解圈直径与其菌落直径的比值,及胞外酶活测定法(DNS法)筛选获得一株纤维素酶高产菌株SWU6。该菌株24 h发酵液上清液经DNS法检测纤维素酶活力达到136.24 U/mL。菌种鉴定结果表明SWU6菌株为革兰氏阳性芽孢杆菌;过氧化氢酶、硝酸盐还原呈阳性;能水解淀粉、液化明胶。基于16S rDNA的系统发育分析结果表明SWU6菌株与登录号为NR 116240的甲基营养型芽孢杆菌(Bacillus methylotrophicus)处于进化树的同一最小分支。综合菌体及菌落形态特征、生理生化实验结果以及基于16S rDNA的系统发育分析,将SWU6菌株鉴定为甲基营养型芽孢杆菌(Bacillus methylotrophicus)。%In order to screen out efficient cellulose‐degrading bacteria w hich can be used to process silkworm excrement and waste mulberry branches ,cellulose‐producing strains were isolated on the substrate with sodium carboxymethyl cellulose (CMC‐Na) as the sole carbon source from the campus soil of Southwest University .A highly efficient cellulose‐producing strain , SWU6 ,was obtained ,using the methods of CMC‐Nahydrolysis circle assay and extracellular enzyme activity assay (DNS ) .Determination of the fermentation supernatant of the strain with DNS method when it was cultivated 24 h showed that the cellulase activity of SWU6 was up to 136 .24 U/ml .Strain identification results showed that SWU6 was gram positive , catalase positive and nitrate reduction positive ,and could hydrolyze starch and liquefy gela‐tine .The phylogenetic analysis based on 16S rDNA sequence showed that SWU6 was in the same minimal clade with Bacillus methylotrophicus (accession number :NR116240) .In

  12. Production and properties of the cellulase-free xylanase from Thermomyces lanuginosus IOC-4145 Produção e propriedades de xilanase livre de celulase de Thermomyces lanuginosus IOC-4145

    Directory of Open Access Journals (Sweden)

    Mônica Caramez Triches Damaso

    2002-12-01

    Full Text Available In recent years, xylanases have expanded their use in many processing industries, such as pulp and paper, food and textile. Thermomyces lanuginosus IOC-4145 was able to produce a very high level of cellulase-free xylanase in shaken cultures using corncob as substrate (500 U/mL. An optimization of the medium composition in submerged fermentation was carried out aiming at a low cost medium composition for enzyme production. Statistical experiment design was employed for this purpose, pointing out corncob as the most important parameter, which affects enzyme production. Additionally, the influence of several chemicals on xylanase activity was investigated in the crude extract. A slight stimulation of the enzyme (5-15% was achieved with NaCl and urea, both at 3 and 5 mM of concentration. On the other hand, dithiothreitol and beta-mercaptoethanol at a molarity of 5mM have caused a strong stimulation of the enzyme (40-53%. The crude xylanase displayed appreciable thermostability, retaining almost 50% of activity during 24 hours of incubation at 50ºC; about 50% of activity was present at 60ºC even after 4 hours of incubation. The enzyme also exhibited good storage stability at -20ºC without any stabilizing agent.Nos últimos anos tem crescido o uso de xilanases em muitas indústrias, tais como polpa e papel, alimentos e têxtil. Thermomyces lanuginosus IOC-4145 foi capaz de produzir um alto nível de xilanase livre de celulase em culturas agitadas usando sabugo de milho como substrato (500 U/mL. Procedeu-se, inicialmente, à otimização da composição do meio de produção em fermentação submersa, com o intuito de alcançar uma composição de meio de produção de baixo custo para produção da enzima. Para este propósito, utilizou-se planejamento estatístico de experimentos. O sabugo de milho revelou-se como a mais importante variável que afeta a produção enzimática. Adicionalmente, a influência de vários reagentes na atividade xilan

  13. Effects of Cellulase and Propylene Glycol on Negative Energy Balance Related Serum Indexes of Postpartum Dairy Cows%纤维素酶和丙二醇对产后奶牛能量负平衡相关血清指标的影响

    Institute of Scientific and Technical Information of China (English)

    陈连民; 陈前岭; 王梦芝; 高健; 蔡青和; 张婵娟; 王洪荣

    2014-01-01

    本文旨在比较饲粮中添加纤维素酶和丙二醇对产后奶牛能量负平衡相关血清指标、尿液酮体浓度的影响。试验选择24头处于围产前期奶牛随机分成3组,对照组饲喂基础饲粮,另设置丙二醇组(基础饲粮+0.5%丙二醇制剂)和纤维素酶组(基础饲粮+0.1%纤维素酶制剂),测定产后第1、20、40、60和100天奶牛能量负平衡相关血清指标、尿液酮体的浓度。结果表明:在产后第1天丙二醇组血清葡萄糖和胰岛素浓度显著高于其余2组( P<0.05);在产后第1、20、40和60天,丙二醇组血清游离脂肪酸和尿液酮体浓度显著低于对照组(P<0.05),第20天显著低于纤维素酶组( P<0.05);甘油三酯等与肝脏脂肪代谢相关的血清指标试验组优于对照组,且以丙二醇组更好。综上,饲粮添加丙二醇及纤维素酶对奶牛产后能量负平衡有一定的改善作用,且以添加0.5%丙二醇制剂效果相对更好。%The objective of this study was to compare the effects of cellulase and propylene glycol on negative energy balance ( NEB) related serum indexes and urine ketone ( KET) concentration of postpartum dairy cows. Twenty four early perinatal dairy cows were randomly divided into 3 groups with 8 cows per group. Three groups were control group (basal diet), propylene glycol group (basal diet+0.5% propylene glycol prepara-tion) and cellulase group (basal diet+0.1% cellulose preparation). Serum indexes related to NEB and urine KET concentration were detected respectively on days 1, 20, 40, 60 and 100 after calving. The results showed as follows: on day 1 after calving, serum glucose and insulin concentrations of propylene glycol group were significantly higher than those of the other 2 groups ( P<0.05) . On days 1, 20, 40 and 60 after calving, serum non-esterified fatty acid and urine KET concentrations in propylene glycol group were significantly lower than those in control group (P<0.05), moreover

  14. Hidrólise enzimática de casca de arroz utilizando-se celulases: efeito de tratamentos químicos e fotoquímicos Enzymatic hydrolysis of rice hull using cellulases: effect of chemical and photochemical treatments

    Directory of Open Access Journals (Sweden)

    Juan Reyes

    1998-04-01

    Full Text Available In the present work we reported the study of rice hull enzymatic hydrolysis using a commercial cellulase preparation. The results showed that previous treatment with light and sodium chlorite inhibits the enzymatic process (31.4 and 11.8%, respectively while hydrogen peroxide and ozone favoured the enzymatic production of reducing sugars (5.9 and 54.9%, respectively. Studies performed by quimiluminescence showed that the chlorite treatment produced the most significant change in the structure of rice hull. Nevertheless, this treatment did not favour the subsequent enzymatic process. Photomicrographs obtained from rice hull hydrolysates showed that pre-treatment changed mainly the inner epidermis and parenchyma cell and that they did not change cellular organization of the hull.

  15. Avaliação da celulase e pectinase como enzimas complementares, no processo de hidrólise-sacarificação do farelo de mandioca para produção de etanol Evaluation of the cellulase and pectinase by complementary enzymes in the process of hydrolysis-saccharification of cassava fibrous waste for alcohol production

    Directory of Open Access Journals (Sweden)

    Magali LEONEL

    1999-01-01

    Full Text Available Neste trabalho objetivou-se avaliar o uso de enzimas complementares no processo enzimático de hidrólise e sacarificação para a produção de etanol a partir do resíduo fibroso das fecularias. Os resultados obtidos demonstraram que 63,42% do amido foram hidrolisados no tratamento em que não se utilizaram enzimas complementares. No tratamento com as duas enzimas complementares foram hidrolisados 89,55%, no tratamento com celulase 65,42% e no tratamento com pectinase 88,73%. A prensagem do resíduo após o processo de hidrólise e sacarificação mostrou-se eficiente, ficando 10,43% do total de açúcares obtidos retidos no resíduo fibroso final. Portanto, o tratamento em que se utilizou a pectinase como enzima complementar na hidrólise foi o melhor. A celulase não apresentou efeito significativo no rendimento do processo.This work it was proposed to evaluate the use of complementary enzymes (cellulase and pectinase in the enzymatic process of hydrolysis-saccharification of the cassava fibrous waste for alcohol production. The results indicated that 63,42% of starch was hydrolyzed in the treatment without complementary enzymes, 89,55% in the treatment with the enzymes, 65,42% with the cellulase by complementary and 88,73% in the pectinase treatment. The pressing was efficacious for sugar recuperation and 10% of total sugar was retaining in the final fibrous residue. The pectinase was the better complementary enzyme enhance the yield.

  16. 金属离子及表面活性剂对纤维素酶水解预处理玉米秸秆的影响%Influence of Metal Ions and Surfactants on the Hydrolysis of Pretreated Corn Stover by Cellulase

    Institute of Scientific and Technical Information of China (English)

    王娜娜; 姚秀清; 张全; 关浩

    2011-01-01

    Influences of metal ions and surfactants on the hydrolysis of pretreated corn stover by cellulase were researched. It was showed that some metal ions and surfactants loading in hydrolysis system, such as Cu2+ and Tween80, could enhance the activity of cellulase. In a 50 g hydrolysis system at pH 5.0, temperature 50 degrees centigrade , pretreated corn stover ( PCS) 10 wt% , enzyme loading 1 g ( 1300 from Zensun Sci & Tech Co. , Ltd) ,hydrolysis time 3 hours, the reducing sugar concentration with Tween80 (20 mg/L) loading is 21.3 percent of no Tween80 loading, and reducing sugar concentration with Cu2+ (0.13 mmol/L) loading is 138. 2 percent of no Cu2+loading.%研究了不同的金属离子及表面活性剂对纤维素酶水解的影响,试验表明添加某些金属离子及表面活性剂能够促进纤维素酶的水解,其中Cu2+和Tween80促进效果最佳.50g反应体系在pH 5.0、温度50℃、预处理玉米秸秆(PCS)10 wt%、1300纤维素酶(泽生科技)加入量1g条件水解3 h,添加20 mg/L Tween80的水解液中还原糖浓度比不加Tween80时提高21.3%;添加0.13 mmoL/LCu2+的水解液中还原糖浓度比不加金属离子时提高38.2%.

  17. The Characteristics of Cellulase and Amylase from B.amy lolique f aciens FAJT-8754 and Optimization of Fermentation Conditions%解淀粉芽胞杆菌FJAT-8754产纤维素酶和淀粉酶特性及发酵条件优化

    Institute of Scientific and Technical Information of China (English)

    王凯; 蓝江林; 刘波; 刘程程; 李安琪

    2014-01-01

    采用淀粉平板和羧甲基纤维素钠(CMC-Na)平板从选取的140株芽胞杆菌中初筛出8株具有产纤维素酶和淀粉酶复合酶芽胞杆菌,经酶活力测定解淀粉芽胞杆菌FJAT-8754(Bacillus amyloliquef aciens)具有较高的淀粉酶、纤维素酶活力。通过研究解淀粉芽胞杆菌FJAT-8754的生长、产酶曲线以及酶学特性,确定在发酵28 h后菌体生长进入稳定期,培养44 h时发酵液中活菌数达到最大为4.41×109 cf u · mL -1,在36 h时纤维素酶、淀粉酶均达到酶活最高峰,酶活分别为135.8、1543.3 U · mL -1;纤维素酶反应最适p H值为5.5、最适温度为50℃,Vmax为5.14×10-3 mg · mL -1· min-1、 Km值为7.71×10-1 mg · mL -1;淀粉酶反应最适pH值为5.5、最适温度为55℃,Vmax为3.35×10-2 mg · mL -1· min-1、 Km值为6.03×10-3 mg · mL -1。采用3因素7水平,即U 7(73)均匀设计法优化解淀粉芽胞杆菌产酶条件,确定产纤维素酶、淀粉酶的最优条件均为:初始pH值6.2、培养温度37.5℃、转速180 r · min-1,优化后解淀粉芽胞杆菌 FJAT-8754纤维素酶活力为202.9 U·mL -1、淀粉酶活力为2392.9U·mL -1。%Eight Bacillus strains which produce cellulase and amylase were screened from 140 standard Bacillus strains by using starch medium and carboxymethylcellulose sodium (CMC-Na) plate screening method .FJAT-8754 had the highest cellulase and amylase producing activities and the biological characterizes (e .g .growth curves , dynamic changes of enzyme activity and enzymatic properties ) were tested .Our results suggested that the stationary phase of FJAT-8754 was at 28 h ,the colony count was 4.41 × 109 cfu · mL -1 at 44 h and the activity of cellulase and amylase peaked at 36 h simultaneously .Meanwhile ,the optimum reaction conditions for cellulase were at pH 5.5 ,55℃ ,Vmax was 5.14 × 10-3 mg · mL -1 · min-1 and Km was 7.71 × 10-1 mg · mL -1 ;the optimal reaction conditions of

  18. 十字花科黑腐病菌中转录调控因子HpaR1调控一个纤维素酶基因的表达%The Transcriptional Regulator HpaR1 of Xanthomonas campestris pv.campe-stris (Xcc) 8004 Regulates the Expression of a Extracellular Cellulase Gene

    Institute of Scientific and Technical Information of China (English)

    薛番艳; 苏辉昭; 刘兴艳; 梁伟; 李磊; 李瑞芳; 陆光涛

    2012-01-01

    十字花科黑腐病菌(Xcc8004)中的一个转录调控因子XC_2736 (HpaR1)在致病过程中具有重要的作用.前期研究发现该转录调控因子可能调控胞外纤维素酶的合成.为了解HpaR1对纤维素酶的转录调控机理,本研究对HpaR1进行原核表达纯化,并与488 bp的包含XC_0639的启动子区DNA片段进行凝胶电泳迁移率试验,发现HpaR1与XC_0639启动子可以发生结合.将488 bp的XC_0639的启动子DNA片段与报告基因gus融合,构建XC_0639的报告质粒pGUS0639r,分别导入野生型8004菌株和缺失突变体DM2736中,分析发现在突变体背景下GUS的表达水平比野生型背景明显降低.表明HpaR1正调控XC_0639的表达.构建XC_0639的极性整合突变体PK0639,检测发现PK0639几乎丧失胞外纤维素酶的活力;通过功能反式互补构建的互补菌株CPK0639可以恢复纤维素酶活性.研究结果表明HpaR1通过调控纤维素酶基因XC_0639的表达来调控细胞的纤维素酶活性.本研究为更深入地了解HpaR1如何调控细菌生理生化功能奠定了基础.%The transcriptional regulator XC-2736 {HpaRl) of X. campestris pv. campestris (Xcc) 8004 plays important roles in regulating bacterial diverse biological processes. Previous work showed that this transcriptional regulator might play role on extracellular cellulase. To illustrate the regulatory mechanism, HpaRl was over expressed, and electrophoretic mobility shift assays (EMSA) were performed using HpaRl protein and the promoter fragment end XCA-p of XC_0639. Results showed that HpaRl could specifically bind to the promoter fragment of XC_0639. The reporter plasmid pGUS0639r was constructed by fusing the promoter of XC_0639 with gus gene, and them been introduced into wild type strain 8004 and the HpaRl mutant DM2736, respectively. The expression level of GUS decreased significantly in the mutant, compared to the wild type background, suggesting HpaRl positively regulates the expression of XC_0639

  19. BÚSQUEDA DE LAS MEJORES CONDICIONES PARA LA EXTRACCIÓN Y MEDIDA DE ACTIVIDAD DE CELULASA Y XILANASA EXTRAÍDAS DE LA CORTEZA DE PITAYA AMARILLA (Acanthocereus pitajaya Searching the Best Conditions for the Extraction and Activity Measurement of Cellulase and Xylanase Extracted from the Yellow Pitaya Fruit Peel (Acanthocereus pitajaya

    Directory of Open Access Journals (Sweden)

    YENNY MARITZA DUEÑAS GÓMEZ

    Full Text Available Para pitaya amarilla (Acanthocereus pitajaya se ha encontrado que el ablandamiento excesivo de la cáscara contribuye al deterioro del fruto, al aplicar diferentes técnicas de conservación en fresco. Dado que tanto la celulasa como la xilanasa se han vinculado con el ablandamiento de la cáscara de frutos, este trabajo se basó en la búsqueda de las mejores condiciones de extracción y medida de actividad de celulasa y xilanasa. El mejor sistema de extracción fue buffer fosfato 20 mM, NaCl 0,5 M, pH 7,0. Para la medida de actividad de celulasa es necesario incubar durante 60 min a 37 ºC, con un volumen de extracto enzimático crudo de 30 µL, empleando buffer acetato 100 mM a pH 5,0; los valores de constante aparente de Michaelis Menten (K M aparente y velocidad máxima (V MÁX fueron 0,279 mg/mL y 0,00014 nmol glucosa/min, respectivamente. Para determinar la actividad de xilanasa se establecieron 15 min de tiempo de incuba-ción, a 50 ºC, empleando 30 µL de extracto enzimático crudo a pH 4,0 (buffer acetato 100 mM; los valores de K M aparente y V MÁX para xilanasa fueron 0,073 mg/mL y 0,0011 nmol glucosa/min, respectivamente.By applying different conservation techniques on yellow pitaya fruit (Acanthocereus pitajaya it has been found that excessive softening of the peel contributes to the deterioration of the fruit. Due to that both cellulase and xylanase have been related to the softening of the fruit's peel; this work was based on the search of the best conditions not only for the extraction, but also for the activity measurement of both cellulase and xylanase. The best extraction system for both enzymes was 20 mM buffer phosphate, 0.5 M NaCl, pH 7.0. For the cellulase activity measurement it was necessary to incubate during 60 min at 37 ºC, with a volume of raw enzymatic extract of 30 µL, using buffer acetate 100 mM at pH 5,0; the values of apparent K M and V MÁX were 0.279 mg/mL and 0.00014 nmol glucose/min, respectively. To

  20. Pepsin-Cellulase Digestibility of Pasture Silages: Effects of Pasture Type, Maturity Stage, and Variations in the Enzymatic Method Digestibilidad mediante Pepsina-Celulasa de Ensilajes de Pradera: Efectos del Tipo de Pradera, Estado de Madurez y Variaciones en el Método Enzimático

    Directory of Open Access Journals (Sweden)

    Claudia Barchiesi-Ferrari

    2011-06-01

    Full Text Available Enzymatic in vitro digestibility has been studied as a method to predict energy values of forages for ruminants, although results have been affected by type of forage and methodological details of the technique. This work was performed to evaluate the effects of cellulase concentration (0.75, 1.0 and 6.25 g L-1, incubation time (24 or 48 h and type of final washing of the residue (water or acetone on the in vitro digestibility of the dry matter (DMD, organic matter (OMD and content of digestible organic matter in the DM (D value of silages made at three maturity stages from three types of pastures: a permanent pasture (Dactylis glomerata, Lolium perenne, Bromus catharticus Vahl var. catharticus, Trifolium repens and Holcus lanatus; b Italian ryegrass ley (Lolium multiflorum Lam. cv. Tama; c oats (Avena sativa L. and d mixed pasture (L. perenne-T. repens. Regression equations among cellulase results and in vitro values obtained with rumen fluid were also developed. Higher enzyme concentration, longer incubation time and final washing with acetone, resulted in a significant (P La digestibilidad enzimática in vitro ha sido estudiada para predecir el valor energético de forrajes para rumiantes, aunque el tipo de forraje y los detalles metodológicos han afectado los resultados obtenidos. Este trabajo pretende evaluar los efectos de la concentración de celulasa (0,75; 1,0 y 6,25 g L-1, tiempo de incubación (24 o 48 h y tipo de lavado final (agua o acetona del residuo, sobre la digestibilidad in vitro de la materia seca (DMD, materia orgánica (OMD y el contenido de materia orgánica digestible en la materia seca, o valor D (D value de ensilajes hechos con tres estados de madurez, de diferentes praderas: a pradera permanente (Dactylis glomerata, Lolium perenne, Bromus catharticus Vahl var. catharticus, Trifolium repens y Holcus lanatus; b ballica Italiana (Lolium multiflorum Lam. cv. Tama; c avena (Avena sativa L.; y d pradera mixta (L. perenne

  1. Cloning and Functional Expression of a Multi-Functional Cellulase Gene egx from Mollusca, Ampullaria crossean in vitro%福寿螺多功能纤维素酶基因egx的克隆及其体外功能性表达

    Institute of Scientific and Technical Information of China (English)

    黄妙容; 刘德武; 吴珍芳

    2011-01-01

    [Objective] The objective of the study is to clone the multi-functional cellulase gene from Mollusca, Ampullaria crossean, and analyze its expression in vitro. [Method] The cDNA fragment was amplified by RT-PCR from the stomach of Mollusca, Ampullaria crossean, and then cloned into pMD18-T vector. Following sequencing, the gene was subcloned into the expression vector pET-32a (+) and pcDNA3.1 (+) using EcoR I and Not I restriction sites, and the enzymatic activity was determined by DNS. [Result] Sequence analysis showed that the 1 326 bp amplicon consists of 1 185 bp coding sequence and part of flanking sequence. The DNA sequence and the putative amino acid sequence shared 99% and 100% identity with the reported sequence, respectively. The purified product from E.coli BL-21 (DE3) showed hydrolytic activities to various substrates including carboxylmethyl cellulose sodium salt (CMC-Na), 2-hydroxyethyl-cellulose, hydroxyethyl-cellulose, sigmacell and xylan with specific activities of 24.78, 15.67, 18.42, 600.91 and 175.43 U·mg-1, respectiviely, while the recombination protein expressed in PK15 showed hydrolytic activities of 0.84, 0.78, 1.01, 14.62 and 4.23 U·mL-1, respectively. [Conclusion] The multi-functional cellulase from Mollusca, Ampullaria crossean, was cloned, functional expressed in pro- and eukaryotic cells, and this could provide a foundation for further research and application of the multi-functional cellulase gene from Mollusca, Ampullaria crossean.%[目的]克隆福寿螺多功能纤维素酶基因egx,并在大肠杆菌和哺乳动物细胞中进行表达分析.[方法]通过RT-PCR的方法,从福寿螺胃组织总RNA中克隆福寿螺多功能纤维素酶基因egx序列,连接克隆载体pMD18-T,再通过EcoR Ⅰ和NotⅠ两个酶切位点分别与表达载体pET-32a(+)及pcDNA3.1(+)连接,构建原核、真核表达载体,并在E.coli BL-21 (DE3)和猪肾细胞PK15中进行表达,最后采用DNS法测定表达产物酶

  2. Single Molecule Study of Cellulase Hydrolysis of Crystalline Cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Y.-S.; Luo, Y.; Baker, J. O.; Zeng, Y.; Himmel, M. E.; Smith, S.; Ding, S.-Y.

    2009-12-01

    This report seeks to elucidate the role of cellobiohydrolase-I (CBH I) in the hydrolysis of crystalline cellulose. A single-molecule approach uses various imaging techniques to investigate the surface structure of crystalline cellulose and changes made in the structure by CBH I.

  3. Potential of Cellulases and Cellulosomes for Cellulosic Waste Management

    Energy Technology Data Exchange (ETDEWEB)

    Bayer, E. A.; Lamed, R.; Himmel, M. E.

    2007-01-01

    Lignocellulose is the most abundant plant cell wall component of the biosphere and the most voluminous waste produced by our society. Fortunately, it is not toxic or directly harmful, but our major waste disposal facilities - the landfills - are rapidly filling up with few realistic alternatives. Because cellulose is pure glucose, its conversion to fine products or fuels has remained a romantic and popular notion; however, the heterogeneous and recalcitrant nature of cellulosic waste presents a major obstacle for conventional conversion processes. One paradigm for the conversion of biomass to products in nature relies on a multienzyme complex, the cellulosome. Microbes that produce cellulosomes convert lignocelluose to microbial cell mass and products (e.g. ethanol) simultaneously. The combination of designer cellulosomes with novel production concepts could in the future provide the breakthroughs necessary for economical conversion of cellulosic biomass to biofuels.

  4. The potential of cellulases and cellulosomes for cellulosic waste management.

    Science.gov (United States)

    Bayer, Edward A; Lamed, Raphael; Himmel, Michael E

    2007-06-01

    Lignocellulose is the most abundant plant cell wall component of the biosphere and the most voluminous waste produced by our society. Fortunately, it is not toxic or directly harmful, but our major waste disposal facilities--the landfills--are rapidly filling up with few realistic alternatives. Because cellulose is pure glucose, its conversion to fine products or fuels has remained a romantic and popular notion; however, the heterogeneous and recalcitrant nature of cellulosic waste presents a major obstacle for conventional conversion processes. One paradigm for the conversion of biomass to products in nature relies on a multienzyme complex, the cellulosome. Microbes that produce cellulosomes convert lignocelluose to microbial cell mass and products (e.g. ethanol) simultaneously. The combination of designer cellulosomes with novel production concepts could in the future provide the breakthroughs necessary for economical conversion of cellulosic biomass to biofuels.

  5. Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling

    DEFF Research Database (Denmark)

    Weiss, Noah Daniel; Börjesson, Johan; Pedersen, Lars Saaby;

    2013-01-01

    To take advantage of this effect, the amount of solids recycled should be maximized, based on a given processes ability to deal with higher solids concentrations and volumes. Recycling of enzymes by recycling the insoluble solids fraction was thus shown to be an effective method to decrease enzyme...

  6. Engineering ionic liquid-tolerant cellulases for biofuels production.

    Science.gov (United States)

    Wolski, Paul W; Dana, Craig M; Clark, Douglas S; Blanch, Harvey W

    2016-04-01

    Dissolution of lignocellulosic biomass in certain ionic liquids (ILs) can provide an effective pretreatment prior to enzymatic saccharification of cellulose for biofuels production. Toward the goal of combining pretreatment and enzymatic hydrolysis, we evolved enzyme variants of Talaromyces emersonii Cel7A to be more active and stable than wild-type T. emersonii Cel7A or Trichoderma reesei Cel7A in aqueous-IL solutions (up to 43% (w/w) 1,3-dimethylimdazolium dimethylphosphate and 20% (w/w) 1-ethyl-3-methylimidazolium acetate). In general, greater enzyme stability in buffer at elevated temperature corresponded to greater stability in aqueous-ILs. Post-translational modification of the N-terminal glutamine residue to pyroglutamate via glutaminyl cyclase enhanced the stability of T. emersonii Cel7A and variants. Differential scanning calorimetry revealed an increase in melting temperature of 1.9-3.9°C for the variant 1M10 over the wild-type T. emersonii Cel7A in aqueous buffer and in an IL-aqueous mixture. We observed this increase both with and without glutaminyl cyclase treatment of the enzymes.

  7. Characterization of Thermostable Cellulases Produced by Bacillus and Geobacillus Strains

    Science.gov (United States)

    Bacterial community composition of thermophilic (60 deg C) mixed cellulose-enrichment cultures was examined by constructing a 16S rDNA clone library which demonstrated major lineages affiliated to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria. A tot...

  8. Recycling cellulases for cellulosic ethanol production at industrial relevant conditions

    DEFF Research Database (Denmark)

    Lindedam, Jane; Haven, Mai Østergaard; Chylenski, Piotr;

    2013-01-01

    wheat straw. Industrial conditions were used to study the impact of hydrolysis temperature (40 or 50. °C) and residence time on recyclability. Enzyme recycling at 12% DM indicated that hydrolysis at 50. °C, though ideal for ethanol yield, should be kept short or carried out at lower temperature...

  9. Anomeric Selectivity and Product Profile of a Processive Cellulase

    DEFF Research Database (Denmark)

    Kari, Jeppe; Kont, Riin; Borch, Kim;

    2017-01-01

    Cellobiohydrolases (CBHs) make up an important group of enzymes for both natural carbon cycling and industrial deconstruction of lignocellulosic biomass. The consecutive hydrolysis of one cellulose strand relies on an intricate pattern of enzyme–substrate interactions in the long, tunnel......-shaped binding site of the CBH. In this work, we have investigated the initial complexation mode with cellulose of the most thoroughly studied CBH, Cel7A from Hypocrea jecorina (HjCel7A). We found that HjCel7A predominantly produces glucose when it initiates a processive run on insoluble microcrystalline...... cellulose, confirming the validity of an even and odd product ratio as an estimate of processivity. Moreover, the glucose released from cellulose was predominantly α-glucose. A link between the initial binding mode of the enzyme and the reducing end configuration was investigated by inhibition studies...

  10. Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass.

    Science.gov (United States)

    Kim, Youngmi; Ximenes, Eduardo; Mosier, Nathan S; Ladisch, Michael R

    2011-04-01

    Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion.

  11. A kinetic model for the burst phase of processive cellulases

    DEFF Research Database (Denmark)

    Præstgaard, Eigil; Olsen, Jens Elmerdahl; Murphy, Leigh;

    2011-01-01

    of the model, which can be solved analytically, shows that the burst and slowdown can be explained by the relative rates of the sequential reactions in the hydrolysis process and the occurrence of obstacles for the processive movement along the cellulose strand. More specifically, the maximum enzyme activity...... reflects a balance between a rapid processive movement, on the one hand, and a slow release of enzyme which is stalled by obstacles, on the other. This model only partially accounts for the experimental data, and we therefore also test a modified version that takes into account random enzyme inactivation...

  12. Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum

    Energy Technology Data Exchange (ETDEWEB)

    Adney, William S.; Baker, John O.; Decker, Stephen R.; Chou, Yat-Chen; Himmel, Michael E.; Ding, Shi-You

    2012-10-09

    Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A)) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.

  13. Isolation, Purification and Characterization of Cellulases from Streptomyces Strains in the Gut of the Termite Odontotermesformosanus%白蚁肠道二株链霉菌株所产纤维素酶的分离、纯化及其特性

    Institute of Scientific and Technical Information of China (English)

    霍光华; 黄小晖; Shannon DILLON; 何刚强; 陈明辉; 龙昊知

    2012-01-01

    by Fe2+ and Ca2+, but inhibited by Mn2+, Cu2+, Zn2+ and Co2+. These results indicate that these cellulases originated from Streptomyce strains in O. formosanus gut are acidic, heat-tolerant enzymes and that their prosthetic groups may be Fe2+ and Ca2+. Therefore, these enzymes has potential in industrial applications for the decomposition of insoluble cellulose.

  14. 知识图谱方法科学前沿进展实证分析——以动物肠道纤维素酶基因工程研究为例%Empirical Analysis of Cutting-edge Science by Knowledge Mapping:A Case in Cellulase Genetic Engineering of Animal Intestines

    Institute of Scientific and Technical Information of China (English)

    李雅; 黄亚娟; 杨明明; 陈玉林

    2012-01-01

    为了系统、全面、准确的反映动物消化道基因工程研究演进及知识间的联系,本研究以与该专业科研人员互动为基础确定检索式,通过高级检索,采用多元统计、战略坐标、社会网络分析等知识图谱方法,在分时段分析该领域前沿热点特征的同时,增加对该研究密切相关的非热点内容的观察,结果显示:该领域研究热点从个别酶研究开始,进而是一类酶的性质研究,目前的研究热点是纤维素酶类在糖尿病、高雪病等代谢疾病的应用研究,同时观察到2007~2010年该领域研究中罗伊氏乳杆菌Pg4、肉仔鸡、瘤胃微生物、益生菌等一些低频词与β-葡聚糖酶的联系和增长态势.说明精准检索、仔细求证、量化图解分析,热点轨迹跟踪,挖掘隐含信息是科研人员多视角科学观察,准确掌握科研动态,科学决策重要参考依据.%For reflecting relationship between the evolution and knowledge of digestive tract genetic engineering studyof domestic animals systematically, comprehensively and accurately, establishment of the retrieval formula based on the communication and cooperation with the relevant researchers, senior retrieval and the knowledge mapping methods ( multivariate statistics, strategic coordinates, social network analysis and so on) , we analyzed characteristics of advancing front and hot spot in this field at different stage, also increased the observations on non-hot content. The result showed that in this field hot spot began from individual enzyme to a kind of enzyme. The present hot spot is the application of cellulase on metabolic disease, such as diabetes mellitus and Gaucher' s disease. Meanwhile we also found that relationship and increasing tendency between the low-frequency words ( such as Lactobacillus reuteri Pg4 , broilers, rumen microbial and Probiotics) and beta-Glucanase. It was suggested that exact retrieval, careful identification, quantization illustration

  15. Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering

    NARCIS (Netherlands)

    Zou, G.; Shi, S.; Jiang, Y.; van den Brink, J.; de Vries, R.P.; Chen, L.; Zhang, J.; Ma, L.; Wang, C.; Zhou, Z.

    2012-01-01

    Background A bacterial strain previously isolated from pyrite mine drainage and named BAS-10 was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III)

  16. Application of the 2-cyanoacetamide method for spectrophotometric assay of cellulase enzyme activity

    Science.gov (United States)

    Cellulose is the most abundant form of carbon on the planet. Breakdown of cellulose microfibrils in the plant cell wall is a means by which microbes gain ingress into their respective hosts. Cellulose degradation is also important for global carbon recycling and is the primary substrate for producti...

  17. Investigation of adsorption kinetics and isotherm of cellulase and B-Glucosidase on lignocellulosic substrates

    Science.gov (United States)

    Clear understanding of enzyme adsorption during enzymatic hydrolysis of lignocellulosic biomass is essential to enhance the cost-efficiency of hydrolysis. However, conclusions from literatures often contradicted each other because enzyme adsorption is enzyme, biomass/pretreatment and experimental co...

  18. The use of cellulase and filter bag technique to predict digestibility of forages

    DEFF Research Database (Denmark)

    Kowalski, Z. M.; Ludwin, J.; Górka, P.;

    2014-01-01

    Corporation). The data for all sets were used to find the best single and multiple regression equations to predict OMD. For each set the linear regressions were calculated in which OMD was the dependent variable (Y) whereas IVDMD determined by CEL48-ND was the predictor X1 and contents of nutrients (g/kg DM...... of equations was also done based on the Akaike information criterion (AIC). Mean R2 for the regression equations of OMD by CEL48-ND was 0.468 (set A), 0.581 (set B) and 0.539 (set A + B). Including the next predictor (X2, X3, X4 or X5) increased R2, and decreased RSD, MSPE and AIC, especially in the equations...... between laboratories. Further, using filter bags and Daisy Incubator decreases labour costs and use of animals. Abbreviations AIC, akaike information criterion; ADF, acid detergent fibre; aNDF, neutral detergent fibre; AFBT, ANKOM filter bag technique; CP, crude protein; CS, subset ‘clover silages’; DK...

  19. Thermodynamic study of an unusual chiral separation. Propranolol enantiomers on an immobilized cellulase

    Energy Technology Data Exchange (ETDEWEB)

    Fornstedt, T.; Sajonz, P.; Guiochon, G. [Univ. of Tennessee, Knoxville, TN (United States)]|[Oak Ridge National Lab., TN (United States)

    1997-02-12

    The thermodynamics of interaction of (R)- and (S)-propranolol between an acetic acid buffer (pH = 4.7 and 5.5) and the protein cellobiohydrolase I immobilized on silica gel was studied between 5 and 45{degree}C. The equilibrium data were fitted to a biLangmuir adsorption isotherm with excellent agreement. One of the two Langmuir contributions is the same for both enantiomers and accounts for the nonspecific interactions between these compounds and most sites on the surfaces (type-I, nonselective sites). It has a large saturation capacity. The second contribution accounts for the chiral selective interactions (type-II sites). It has a lower monolayer capacity than the first. The interaction enthalpy and entropy on type-I sites are -1.1 kcal/mol and +0.1 cal/(mol K), respectively. For type-II sites, they are -1.9 kcal/mol and -2.6 cal/(mol K), respectively, for (R)-propranolol and +1.6 kcal/mol and +11.6 cal/(mol K), respectively, for (S)-propranolol at pH = 5.5. This explains why at this pH the retention time of the less-retained R enantiomer decreases with increasing temperature, while the retention time of the S enantiomer increases, causing a large increase of the separation factor when the temperature is raised from 5 to 45{degree}C. The saturation capacity of the chiral contributions depends strongly on the pH, and the retention times of both enantiomers decrease with increasing temperature at pH = 4.7. 46 refs., 6 figs., 5 tabs.

  20. Significance of relative position of cellulases in designer cellulosomes for optimized cellulolysis.

    Directory of Open Access Journals (Sweden)

    Johanna Stern

    Full Text Available Degradation of cellulose is of major interest in the quest for alternative sources of renewable energy, for its positive effects on environment and ecology, and for use in advanced biotechnological applications. Due to its microcrystalline organization, celluose is extremely difficult to degrade, although numerous microbes have evolved that produce the appropriate enzymes. The most efficient known natural cellulolytic system is produced by anaerobic bacteria, such as C. thermocellum, that possess a multi-enzymatic complex termed the cellulosome. Our laboratory has devised and developed the designer cellulosome concept, which consists of chimaeric scaffoldins for controlled incorporation of recombinant polysaccharide-degrading enzymes. Recently, we reported the creation of a combinatorial library of four cellulosomal modules comprising a basic chimaeric scaffoldin, i.e., a CBM and 3 divergent cohesin modules. Here, we employed selected members of this library to determine whether the position of defined cellulolytic enzymes is important for optimized degradation of a microcrystalline cellulosic substrate. For this purpose, 10 chimaeric scaffoldins were used for incorporation of three recombinant Thermobifida fusca enzymes: the processive endoglucanase Cel9A, endoglucanase Cel5A and exoglucanase Cel48A. In addition, we examined whether the characteristic properties of the T. fusca enzymes as designer cellulosome components are unique to this bacterium by replacing them with parallel enzymes from Clostridium thermocellum. The results support the contention that for a given set of cellulosomal enzymes, their relative position within a scaffoldin can be critical for optimal degradation of microcrystaline cellulosic substrates.

  1. Chemical stability of a cold-active cellulase with high tolerance toward surfactants and chaotropic agent

    Directory of Open Access Journals (Sweden)

    Thaís V. Souza

    2016-03-01

    Full Text Available CelE1 is a cold-active endo-acting glucanase with high activity at a broad temperature range and under alkaline conditions. Here, we examined the effects of pH on the secondary and tertiary structures, net charge, and activity of CelE1. Although variation in pH showed a small effect in the enzyme structure, the activity was highly influenced at acidic conditions, while reached the optimum activity at pH 8. Furthermore, to estimate whether CelE1 could be used as detergent additives, CelE1 activity was evaluated in the presence of surfactants. Ionic and nonionic surfactants were not able to reduce CelE1 activity significantly. Therefore, CelE1 was found to be promising candidate for use as detergent additives. Finally, we reported a thermodynamic analysis based on the structural stability and the chemical unfolding/refolding process of CelE1. The results indicated that the chemical unfolding proceeds as a reversible two-state process. These data can be useful for biotechnological applications.

  2. Characterisation of the transcriptome of Aphelenchoides besseyi and identification of a GHF 45 cellulase

    NARCIS (Netherlands)

    Kikuchi, T.; Cock, P.J.A.; Helder, J.; Jones, J.T.

    2014-01-01

    While the majority of Aphelenchoides species are fungivorous, some species are plant parasites that have retained the ability to feed on fungi. Aphelenchoides besseyi is an important and widespread pathogen that causes ‘white tip’ disease on rice. This migratory endoparasitic nematode makes a signif

  3. [Lignocellulose degrading bacteria and their genes encoding cellulase/hemicellulase in rumen--a review].

    Science.gov (United States)

    Chen, Furong; Zhu, Yaxin; Dong, Xiuzhu; Liu, Lihua; Huang, Li; Dai, Xin

    2010-08-01

    Rumen of ruminant animals is known as a natural reactor involved in highly efficient lignocelluloses degradation. Rumen fibrolytic microbes have attracted an increasing attention for their potential value in biofuel research. Studies on rumen microbes have traditionally entailed the isolation of fibrolytic bacteria and subsequent analysis of fibrolytic enzymes. Developments in genomic and metagenomic approaches have made it possible to isolate directly genes and gene clusters encoding fibrolytic activities from rumen samples, permitting a global analysis of mechanisms of degradation of lignocellulose in rumen. Research in this field shows that lignocellulose degradation in rumen is a complex process involving a number of different microbes and is effected by a huge array of hydrolytic enzymes in a concerted fashion. This review briefly summarizes results from recent studies, especially metagenomic studies, on lignocellulose degradation in rumen.

  4. Immobilization of cellulase mixtures on magnetic particles for hydrolysis of lignocellulose and ease of recycling

    DEFF Research Database (Denmark)

    Alftrén, Johan; Hobley, Timothy John

    2014-01-01

    produced per mass of particles (2.8gkg−1min−1), when using microcrystalline cellulose as substrate. The ratio of the individual activities of endoglucanase, cellobiohydrolase and β-glucosidase changed after immobilization of CellicCTec2 and it was observed that the specific activity in units per milligram...

  5. Enzymatic hydrolysis of lignocelluloses: Identification of novel cellulase genes from filamentous fungi

    DEFF Research Database (Denmark)

    Kolasa, Marta; Ahring, Birgitte Kiær; Lübeck, Peter Stephensen;

    2010-01-01

    Lignocellulosic materials form a huge part of the plant biomass from agricultural and forestry wastes. They consist of three major components: cellulose, hemicellulose and lignin. Cellulose, the main constituent of plant cell wall, is a polymer of D–glucopyranose units linked by β-1,4-glucosidic...... bonds. Cellulose can be degraded to simple sugar components by means of enzymatic hydrolysis. However, due to its complex, crystalline structure it is difficult to break it down and the cooperative action of a variety of cellulolytic enzymes is necessary. Fungi are known to have potential in production...... using suppression subtractive hybridization and a targeted genomic screening approach using degenerate PCR. Suppression subtractive hybridization facilitates identification of genes encoding cellulolytic enzymes that are expressed when cultivating a fungal strain in medium with cellulose as the carbon...

  6. Recovery of cellulase activity after ethanol stripping in a novel pilot-scale unit

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia; Christensen, Børge Holm; Felby, Claus;

    2014-01-01

    Recycling of enzymes has a potential interest during cellulosic bioethanol production as purchasing enzymes is one of the largest expenses in the process. By recycling enzymes after distillation, loss of sugars and ethanol are avoided, but depending on the distillation temperature, there is a pot......Recycling of enzymes has a potential interest during cellulosic bioethanol production as purchasing enzymes is one of the largest expenses in the process. By recycling enzymes after distillation, loss of sugars and ethanol are avoided, but depending on the distillation temperature...

  7. A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues.

    Science.gov (United States)

    Meneses, Carlos; Silva, Bruna; Medeiros, Betsy; Serrato, Rodrigo; Johnston-Monje, David

    2016-06-25

    Many naturally-occurring cellulolytic microorganisms are not readily cultivable, demanding a culture-independent approach in order to study their cellulolytic genes. Metagenomics involves the isolation of DNA from environmental sources and can be used to identify enzymes with biotechnological potential from uncultured microbes. In this study, a gene encoding an endoglucanase was cloned from red rice crop residues using a metagenomic strategy. The amino acid identity between this gene and its closest published counterparts is lower than 70%. The endoglucanase was named EglaRR01 and was biochemically characterized. This recombinant protein showed activity on carboxymethylcellulose, indicating that EglaRR01 is an endoactive lytic enzyme. The enzymatic activity was optimal at a pH of 6.8 and at a temperature of 30 °C. Ethanol production from this recombinant enzyme was also analyzed on EglaRR01 crop residues, and resulted in conversion of cellulose from red rice into simple sugars which were further fermented by Saccharomyces cerevisiae to produce ethanol after seven days. Ethanol yield in this study was approximately 8 g/L. The gene found herein shows strong potential for use in ethanol production from cellulosic biomass (second generation ethanol).

  8. Densities, cellulases, alginate and pectin lyases of luminous and other heterotrophic bacteria associated with marine algae

    Digital Repository Service at National Institute of Oceanography (India)

    Ramaiah, N.; Chandramohan, D.

    Epiphytic luminous and non-luminous bacteria were determined quantitatively for eight intertidal algal species from rocky beaches of Goa and Lakshadweep coral reef lagoon. Luminous bacteria were present on all eight algal species and contributed 2...

  9. Cloning of a Gene Cluster from Cellvibrio mixtus which Codes for Cellulase, Chitinase, Amylase, and Pectinase

    OpenAIRE

    1986-01-01

    The soil isolate Cellvibrio mixtus UQM2294 degraded a variety of polysaccharides including microcrystalline cellulose. Among 6,000 cosmid clones carrying C. mixtus DNA, constructed in Escherichia coli with pHC79, 50 expressed the ability to degrade one or more of the following substrates: carboxymethyl cellulose, chitin, pectin (polygalacturonic acid), cellobiose, and starch. These degradative genes are encoded in a single 94.1-kilobase segment of the C. mixtus genome; a preliminary order of ...

  10. Recycling of cellulases in a continuous process for production of bioethanol

    DEFF Research Database (Denmark)

    Haven, Mai Østergaard

    The focus of the work presented in this thesis is recycling of commercial enzymes in a continuous process for production of bioethanol from biomass. To get a deeper understanding of the factors affecting the potential for enzyme recycling, the interactions between enzymes and biomass......, the adsorption and desorption as well as stability and recovery of activity was investigated. More knowledge on these factors have enabled a process adapted for enzyme recycling. The driver being that enzyme consumption remains a major cost when producing bioethanol from lignocellulosic biomass. Unlike previous...

  11. Hypolipidemic effect of the polysaccharides extracted from pumpkin by cellulase-assisted method on mice.

    Science.gov (United States)

    Zhao, Xin-Hua; Qian, Li; Yin, De-Lu; Zhou, Yi

    2014-03-01

    The fruit of pumpkin (Cucurbita moschata) is one of the most important vegetables in the world. This study was conducted to investigate the hypolipidemic effect of the polysaccharide isolated from pumpkin (PP). Male Sprague-Dawley rats were divided into three groups and treated with diets containing either high fat, PP, or normal fat. Oral administration of PP could significantly decrease the levels of plasma triacylglycerol (TG), total cholesterol (TC), and plasma low-density lipoprotein cholesterol and increase the levels of fecal fat, cholesterol, and plasma high-density lipoprotein cholesterol. Therefore, results suggest that PP had a high hypolipidemic activity and could be explored as a possible agent for hyperlipidemia.

  12. Biased clique shuffling reveals stabilizing mutations in cellulase Cel7A.

    Science.gov (United States)

    Dana, Craig M; Saija, Poonam; Kal, Sarala M; Bryan, Mara B; Blanch, Harvey W; Clark, Douglas S

    2012-11-01

    Renewable fuels produced from biomass-derived sugars are receiving increasing attention. Lignocellulose-degrading enzymes derived from fungi are attractive for saccharification of biomass because they can be produced at higher titers and at significantly less cost than those produced by bacteria or archaea. However, their properties can be suboptimal; for example, they are subject to product inhibition and are sensitive to small changes in pH. Furthermore, increased thermostability would be advantageous for saccharification as increased temperature may reduce the opportunity for microbial contamination. We have developed a mutagenesis platform to improve these properties and applied it to increase the operating temperature and thermostability of the fungal glycosyl hydrolase Cel7A. Secretion of Cel7A at titers of 26 mg/L with limited hyperglycosylation was achieved using a Saccharomyces cerevisiae strain with upregulated protein disulfide isomerase, an engineered α-factor prepro leader, and deletion of a plasma membrane ATPase. Using biased clique shuffling (BCS) of 11 Cel7A genes, we generated a small library (469) rich in activity (86% of the chimeras were active) and identified 51 chimeras with improved thermostability, many of which contained mutations in the loop networks that extend over the enzyme's active site. This BCS library was far superior as a source of active and stable chimeras compared to an equimolar library prepared from the same 11 genes.

  13. Recovery of cellulase activity after ethanol stripping in a novel pilot-scale unit.

    Science.gov (United States)

    Skovgaard, Pernille Anastasia; Christensen, Børge Holm; Felby, Claus; Jørgensen, Henning

    2014-04-01

    Recycling of enzymes has a potential interest during cellulosic bioethanol production as purchasing enzymes is one of the largest expenses in the process. By recycling enzymes after distillation, loss of sugars and ethanol are avoided, but depending on the distillation temperature, there is a potential risk of enzyme degradation. Studies of the rate of enzyme denaturation based on estimation of the denaturation constant K D was performed using a novel distillation setup allowing stripping of ethanol at 50-65 °C. Experiments were performed in a pilot-scale stripper, where the effect of temperature (55-65 °C) and exposure to gas-liquid and liquid-heat transmission interfaces were tested on a mesophilic and thermostable enzyme mixture in fiber beer and buffer. Lab-scale tests were included in addition to the pilot-scale experiments to study the effect of shear, ethanol concentration, and PEG on enzyme stability. When increasing the temperature (up to 65 °C) or ethanol content (up to 7.5 % w/v), the denaturation rate of the enzymes increased. Enzyme denaturation occurred slower when the experiments were performed in fiber beer compared to buffer only, which could be due to PEG or other stabilizing substances in fiber beer. However, at extreme conditions with high temperature (65 °C) and ethanol content (7.5 % w/v), PEG had no enzyme stabilizing effect. The novel distillation setup proved to be useful for maintaining enzyme activity during ethanol extraction.

  14. Effects of non-ionic surfactants on the interactions between cellulases and tannic acid

    DEFF Research Database (Denmark)

    Olsen, Søren Nymand; Bohlin, Christina Helena; Murphy, Leigh

    2011-01-01

    Addition of non-ionic surfactants (NIS) is known to accelerate enzymatic lignocellulose hydrolysis. The mechanism behind this accelerating effect is still not elucidated but has been hypothesized to originate from favorable NIS–lignin interactions which alleviate non-productive adsorption...

  15. A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues

    Directory of Open Access Journals (Sweden)

    Carlos Meneses

    2016-06-01

    Full Text Available Many naturally-occurring cellulolytic microorganisms are not readily cultivable, demanding a culture-independent approach in order to study their cellulolytic genes. Metagenomics involves the isolation of DNA from environmental sources and can be used to identify enzymes with biotechnological potential from uncultured microbes. In this study, a gene encoding an endoglucanase was cloned from red rice crop residues using a metagenomic strategy. The amino acid identity between this gene and its closest published counterparts is lower than 70%. The endoglucanase was named EglaRR01 and was biochemically characterized. This recombinant protein showed activity on carboxymethylcellulose, indicating that EglaRR01 is an endoactive lytic enzyme. The enzymatic activity was optimal at a pH of 6.8 and at a temperature of 30 °C. Ethanol production from this recombinant enzyme was also analyzed on EglaRR01 crop residues, and resulted in conversion of cellulose from red rice into simple sugars which were further fermented by Saccharomyces cerevisiae to produce ethanol after seven days. Ethanol yield in this study was approximately 8 g/L. The gene found herein shows strong potential for use in ethanol production from cellulosic biomass (second generation ethanol.

  16. Purification and characterization of an alkaline cellulase produced by Bacillus subtilis (AS3).

    Science.gov (United States)

    Deka, Deepmoni; Jawed, Mohammad; Goyal, Arun

    2013-01-01

    An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6-10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate.

  17. IONIC LIQUID SALT-INDUCED INACTIVATION AND UNFOLDING OF CELLULASE FROM TRICHODERMA REESEI. (R828257)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  18. Screening Of the Association of Aspergillusfungi – Macerase and Cellulase Enzymes Producers

    OpenAIRE

    R.K.Bliyeva,; Zh.B.Suleimenova

    2015-01-01

    In nature, the destruction of plant wastes takes place by not a single microorganism, butby complex of microorganisms which belong to different species and genera with complex of enzymes. Using of highly active monocultures to createfungal association which produce multifunctional enzymeswith desired properties is a promising approach to create relevant and effective crop production microorganisms with beneficial properties. Among15 industrial micro my cetes the most active associ...

  19. Pseudomonas coleopterorum sp. nov., a cellulase-producing bacterium isolated from the bark beetle Hylesinus fraxini.

    Science.gov (United States)

    Menéndez, Esther; Ramírez-Bahena, Martha H; Fabryová, Anna; Igual, José M; Benada, Oldrich; Mateos, Pedro F; Peix, Alvaro; Kolařík, Miroslav; García-Fraile, Paula

    2015-09-01

    We isolated a strain coded Esc2Am(T) during a study focused on the microbial diversity of adult specimens of the bark beetle Hylesinus fraxini. Its 16S rRNA gene sequence had 99.4% similarity with respect to its closest relative, Pseudomonas rhizosphaerae IH5(T). The analysis of partial sequences of the housekeeping genes rpoB, rpoD and gyrB confirmed that strain Esc2Am(T) formed a cluster with P. rhizosphaerae IH5(T) clearly separated from the remaining species of the genus Pseudomonas. Strain Esc2Am(T) had polar flagella and could grow at temperatures from 4 °C to 30 °C. The respiratory quinone was Q9 and the main fatty acids were C16 : 0, C18 : 1ω7c and/or C18 : 1ω6c in summed feature 8 and C16 : 1ω7c and/or C16 : 1ω6c in summed feature 3. DNA-DNA hybridization results showed 51% relatedness with respect to P. rhizosphaerae IH5(T). Oxidase, catalase and urease-positive, the arginine dihydrolase system was present but nitrate reduction and β-galactosidase production were negative. Aesculin hydrolysis was positive. Based on the results from the genotypic, phenotypic and chemotaxonomic analyses, we propose the classification of strain Esc2Am(T) as representing a novel species of the genus Pseudomonas, for which we propose the name Pseudomonas coleopterorum sp. nov. The type strain is Esc2Am(T) ( = LMG 28558(T)= CECT 8695(T)).

  20. Study on Mutation of Cellulase-Decomposing Bacteria Induced by UV to Increase Cellulase Activity%紫外线诱变提高细菌产纤维素酶活力的研究

    Institute of Scientific and Technical Information of China (English)

    陈香; 蒋立建; 韩刚; 韩冲

    2008-01-01

    从南京红山动物园土样及食草动物的粪便中分离出14种产纤维素酶菌株,其中F1表现出较高的酶活力.以F1为出发菌株,通过紫外线诱变处理,采用透明圈法初筛和摇瓶培养复筛,获得了10株高产纤维素酶的突变株Q1~Q10.经紫外线诱变处理的Q3突变株产酶活力最高,与出发菌株相比酶活力提高了15.8倍.

  1. 耐热纤维素酶产生菌的筛选、鉴定及产酶条件优化%Screening and Identification of a Thermostable Cellulase-Secreting Bacteria and Its Optimization for Cellulase Production

    Institute of Scientific and Technical Information of China (English)

    罗颖; 欧阳嘉; 许婧; 何冰芳

    2007-01-01

    从温泉热源地区采集的大量泥土和水样中,筛选出一株在60 ℃生长的纤维素酶产生菌SH2.结合菌株的生理生化特性分析与Biolog微生物自动鉴定仪的鉴定结果,命名为热葡糖苷酶地芽孢杆菌(Geobacillus thermoglucosidasius) SH2,该菌株兼性好氧,在45~60 ℃能较好地生长.对菌体生长与产酶培养条件优化表明:初始pH值为5.5,碳、氮源分别为蔗糖和玉米浆时有利于产酶,经48 h发酵后纤维素酶酶活达0.36 IU/mL.纤维素酶反应条件研究表明,该纤维素酶的最适pH值为6.0,在pH 4.0~10.0范围具有较强的耐受性;在45~65 ℃间酶活差异仅在5%之内,显示了很好的温度耐受性.

  2. Role of alkaline-tolerant fungal cellulases in release of total antioxidants from agro-wastes under solid state fermentation

    Digital Repository Service at National Institute of Oceanography (India)

    Ravindran, C.; Varatharajan, G. R.; Karthikeyan, A.

    Division, National Institute of Oceanography, Council of Scientific and Industrial Research (CSIR), Dona Paula, Goa, India-403 004. * Corresponding author: cravindran@nio.org INTRODUCTION Phenolic compounds of plant materials have numerous health... properties of synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), provide motivation to consider the use of plant materials as important possible sources of compounds with the potential to be developed...

  3. Influence of the carbon source on production of cellulases, hemicellulases and pectinases by Trichoderma reesei Rut C-30

    DEFF Research Database (Denmark)

    Olsson, Lisbeth; Christensen, T.M.I.E.; Hansen, K.P.;

    2003-01-01

    The growth and enzyme production by Trichoderma reesei Rut C-30 using different lignocellulosic materials as carbon source were investigated. Cellulose, sugar beet pulp and alkaline extracted sugar beet pulp (resulting in partial removal of hemicellulose, lignin and pectin) or mixtures thereof were...

  4. Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern

    Science.gov (United States)

    Tsay, Tung-Tsuan; Tsai, Isheng J.; Chen, Peichen J.

    2016-01-01

    Five Aphelenchoides besseyi isolates collected from bird’s-nest ferns or rice possess different parasitic capacities in bird’s-nest fern. Two different glycoside hydrolase (GH) 45 genes were identified in the fern isolates, and only one was found in the rice isolates. A Abe GH5-1 gene containing an SCP-like family domain was found only in the fern isolates. Abe GH5-1 gene has five introns suggesting a eukaryotic origin. A maximum likelihood phylogeny revealed that Abe GH5-1 is part of the nematode monophyletic group that can be clearly distinguished from those of other eukaryotic and bacterial GH5 sequences with high bootstrap support values. The fern A. besseyi isolates were the first parasitic plant nematode found to possess both GH5 and GH45 genes. Surveying the genome of the five A. besseyi isolates by Southern blotting using an 834 bp probe targeting the GH5 domain suggests the presence of at least two copies in the fern-origin isolates but none in the rice-origin isolates. The in situ hybridization shows that the Abe GH5-1 gene is expressed in the nematode ovary and testis. Our study provides insights into the diversity of GH in isolates of plant parasitic nematodes of different host origins. PMID:27391812

  5. Effect of oxygen transfer rate on cellulases production in stirred tank and internal-loop airlift bioreactors

    OpenAIRE

    Michelin, Michele; Mota,André; Silva,Daniel Pereira da; A.A. Vicente; Teixeira, J. A; Polizeli, Maria de Lourdes T. M.

    2013-01-01

    In an aerobic process, such as enzymes production by fungi, the oxygen supply into fermentation medium is an important factor in order to achieve good productivities. Oxygen has an important role in metabolism and microorganism growth, being of extreme importance the control of both the dissolved oxygen transfer rate into the bioreactor and the oxygen consumption by the microorganism [1,2]. Dissolved oxygen transfer rate can be analyzed and described by means of the mass transfer coefficient,...

  6. Use of Recombinant Cellulose-Binding Domains of Trichoderma reesei Cellulase as a Selective Immunocytochemical Marker for Cellulose in Protozoa

    OpenAIRE

    Linder, Markus; Winiecka-Krusnell, Jadwiga; Linder, Ewert

    2002-01-01

    Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent β-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellu...

  7. Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

    Institute of Scientific and Technical Information of China (English)

    LI Bi-feng; MAO Hua-ming; YANG Shu-Li; ZHU Ya-xin; GU Zhao-bing; CHEN Yuan; LENG Jing; GOU Xiao; FENG Li; LI Qing; XI Dong-mei

    2016-01-01

    Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as wel as reeds and other plant species, and grow to higher mature live weights than Yunnan Yelow cattle maintained in similar harsh environments. The aim of this study was to identify speciifc celulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38400 clones with an average insert size of 35.5 kb. TheUmcel-1 gene was isolated from this library. Investigation of the celulase activity of 24 random clones led to the identiifcation of theUmcel-1 gene, which exhibited the most potent celulase activity. Sequencing theUmcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putativegene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the celulase (GenBank accession no. YP_004310852.1) fromClostridium lentocelum DSM 5427, with 44% identity and 62% similarity. TheUmcel-1 gene was heterologously expressed inEscherichia coli BL21, and recombinant Umcel-1 was puriifed. The activity of puriifed recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl celulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the ifrst evidence for a celulase produced by bacteria in gayal rumen.

  8. The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images

    DEFF Research Database (Denmark)

    Hidayat, Budi J.; Weisskopf, Carmen; Felby, Claus

    2015-01-01

    Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations...... for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster...

  9. Cellulase from Trichoderma harzianum interacts with roots and triggers induced systemic resistance to foliar disease in maize.

    Science.gov (United States)

    Saravanakumar, Kandasamy; Fan, Lili; Fu, Kehe; Yu, Chuanjin; Wang, Meng; Xia, Hai; Sun, Jianan; Li, Yaqian; Chen, Jie

    2016-11-10

    Trichoderma harzianum is well known to exhibit induced systemic resistance (ISR) to Curvularia leaf spot. We previously reported that a C6 zinc finger protein (Thc6) is responsible for a major contribution to the ISR to the leaf disease, but the types of effectors and the signals mediated by Thc6 from Trichoderma are unclear. In this work, we demonstrated that two hydrolases, Thph1 and Thph2, from T. harzianum were regulated by Thc6. Furthermore, an electrophoretic mobility shift assay (EMSA) study revealed that Thc6 regulated mRNA expression by binding to GGCTAA and GGCTAAA in the promoters of the Thph1 and Thph2 genes, respectively. Moreover, the Thph1 and Thph2 proteins triggered the transient production of reactive oxygen species (ROS) and elevated the free cytosolic calcium levels in maize leaf. Furthermore, the genes related to the jasmonate/ethylene signaling pathway were up-regulated in the wild-type maize strain. However, the ΔThph1- or ΔThph2-deletion mutants could not activate the immune defense-related genes in maize to protect against leaf disease. Therefore, we conclude that functional Thph1 and Thph2 may be required in T. harzianum to activate ISR in maize.

  10. Systems Analysis of Lactose Metabolism in Trichoderma reesei Identifies a Lactose Permease That Is Essential for Cellulase Induction

    OpenAIRE

    Christa Ivanova; Bååth, Jenny A.; Bernhard Seiboth; Christian P Kubicek

    2013-01-01

    Trichoderma reesei colonizes predecayed wood in nature and metabolizes cellulose and hemicellulose from the plant biomass. The respective enzymes are industrially produced for application in the biofuel and biorefinery industry. However, these enzymes are also induced in the presence of lactose (1,4-0-ß-d-galactopyranosyl-d-glucose), a waste product from cheese manufacture or whey processing industries. In fact, lactose is the only soluble carbon source that induces these enzymes in T. reesei...

  11. Purification and characterization of cellulase from Trichoderma reesei%里氏木霉纤维素酶的分离纯化及酶学性质

    Institute of Scientific and Technical Information of China (English)

    朱年青; 夏文静; 勇强

    2010-01-01

    通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting凝胶色谱脱盐、Source 15 Q阴离子交换色谱技术,里氏木霉(Rut C-30)纤维素酶主要组分得以初步分开,再经过Source 15 S阳离子交换色谱、HiPrep Sephacryl S-100 HR凝胶过滤色谱、Superdex 75 PrepGrade凝胶过滤色谱进一步分离纯化,得到2个纯化的内切葡聚糖酶组分EGⅡ、EGⅠ和一个外切葡聚糖酶组分CBHⅠ;经过SDS-PAGE电泳鉴定为电泳纯,测得相对分子质量分别为5.22×104,5.62×104和6.90×104.EGⅡ的最适反应pH是5.6,最适反应温度为65 ℃;EGⅠ的最适反应pH是4.4,最适反应温度为55 ℃;以羧甲基纤维素(CMC)为底物时,EGⅠ、EGⅡ的米氏常数(Km )分别为2.20 mg/mL、3.38 mg/mL.CBHⅠ的最适反应pH是5.8,最适反应温度为60 ℃,以对硝基苯基-β-D-纤维二糖苷(PNPC)为底物时,米氏常数(Km )为0.12 mg/mL.

  12. Root cap specific expression of an endo-beta-1,4-D-glucanase (cellulase): a new marker to study root development in Arabidopsis.

    Science.gov (United States)

    del Campillo, Elena; Abdel-Aziz, Amal; Crawford, Damian; Patterson, Sara E

    2004-09-01

    The sloughing of root cap cells from the root tip is important because it assists the growing root in penetrating the soil. Using a promoter-reporter (GUS) and RT-PCR analysis, we identified an endo-beta-1,4-glucanase (AtCel5) of Arabidopsis thaliana that is expressed exclusively in root cap cells of both primary and secondary roots. Expression is inhibited by high concentrations of IAA, both exogenous and internal, as well as by ABA. AtCel5 expression begins once the mature tissue pattern is established and continues for 3 weeks. GUS staining is observed in both root cap cells that are still attached and cells that have already been shed. Using AtCel5-GUS as a marker, we observed that the root cap cells begin to separate at the sides of the tip while the cells of the central region of the tip separate last. Separation involves sequential tiers of intact cells that separate from the periphery of the root tip. A homozygous T-DNA insertion mutant that does not express AtCel5 forms the root cap and sheds root cap cells but sloughing is less efficient compared to wild type. The reduction in sloughing in the mutant does not affect the overall growth performance of the plant in loose media. The modest effect of abolishing AtCel5 expression suggests that there are multiple redundant genes regulating the process of sloughing of the root cap, including AtCel3/At1g71380, the paralog of the AtCel5 gene that is also expressed in the root cap cells. Thus, these two endo-1,4-beta-D-glucanases may have a role in the sloughing of border cells from the root tip. We propose that AtCel5, provides a new molecular marker to further analyze the process of root cap cell separation and a root cap specific promoter for targeting to the environment genes with beneficial properties for plant growth.

  13. Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and {beta}-glucosidase

    Energy Technology Data Exchange (ETDEWEB)

    Apiwatanapiwat, Waraporn; Rugthaworn, Prapassorn [Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki (Japan). Post-Harvest Science and Technology Div.; Kasetsart Univ., Bangkok (Thailand). Nanotechnology and Biotechnology Div.; Murata, Yoshinori; Kosugi, Akihiko; Arai, Takamitsu; Mori, Yutaka [Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki (Japan). Post-Harvest Science and Technology Div.; Yamada, Ryosuke; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering

    2011-04-15

    In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying {alpha}-amylase ({alpha}-AM), glucoamylase, endoglucanase, cellobiohydrase, and {beta}-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley {beta}-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes. (orig.)

  14. STUDY ON CELLULASE FERMENT PRODUCTION OF CHAETOMIUM CUPREUM%角毛壳菌产纤维素酶条件的研究

    Institute of Scientific and Technical Information of China (English)

    张海燕; 杨谦

    2006-01-01

    对角毛壳菌(Chaetomium cupreum)产纤维素酶的发酵条件进行了研究.结果表明:Mendels培养基适于角毛壳菌产纤维素酶,CMC酶活可达到15.7 U,β-葡萄糖苷酶活达到了15.8 U,滤纸酶活达到12.1 U.最佳产酶碳源是微结晶纤维素.发酵156 h是角毛壳菌产滤纸酶活的最佳时间,达到了14.4 U,发酵180 h是角毛壳菌产CMC酶活和β-葡萄糖苷酶活的最佳时间,酶活可分别达到37.6 U和38.4 U.

  15. 红树林环境产纤维素酶菌株的研究现状%Recent Progress in Cellulase-producing Strains from Mangrove Ecosystems

    Institute of Scientific and Technical Information of China (English)

    孙倩; 林海鹏

    2015-01-01

    红树林是自然分布于热带和亚热带位于陆地和海洋交界的滩涂地带,是陆地向海洋过度的特殊生态系统.由于红树林落叶等木质纤维素的沉积,产纤维素酶微生物也是该区的重要生物组成部分.文章综述了近几年来国内从红树林环境分离筛选产纤维素酶微生物的研究现状及应用前景.

  16. 纤维素酶在可再生资源利用中的作用%The role of cellulase in utilization of renewable resource

    Institute of Scientific and Technical Information of China (English)

    安登第

    2003-01-01

    @@ 纤维素是组成植物体的主要物质,是地球上最丰富的可再生资源,但这类物质大多以秸秆的形式存在,难以被降解,人类对其利用极其有限.如能将纤维类物质转化为人类食品,由于其资源量极大,因而有非常重要的意义.当前转化的主要途径是通过反刍动物.反刍动物在严酷的自然进化中获得的前胃缓慢发酵系统,使其成为哺乳动物中最有效的粗饲料消化者.但秸秆饲料中,大量结构碳水化合物被木质素保护,瘤胃微生物不能降解木质素致使这些碳水化合物不能被利用(McSweeney等,1994).

  17. Experimental and theoretical study of the adsorption behavior and mass transfer kinetics of propranolol enantiomers on cellulase protein as the selector

    Energy Technology Data Exchange (ETDEWEB)

    Fornstedt, T.; Zhong, G.; Bensetiti, Z.; Guiochon, G. [Univ. of Tennessee, Knoxville, TN (United States)]|[Oak Ridge National Lab., TN (United States)

    1996-07-15

    The thermodynamics and mass transfer kinetics of the retention of the R and S enantiomers of propranolol were investigated on a system comprising an acetic acid buffer solution as mobile phase and the protein cellobiohydrolase I immobilized on silica as the stationary phase. The bi-Langmuir isotherm model fitted best to each set of single-component isotherm data. The monolayer capacity of the nonchiral type of adsorption sites was 22.9 mM. For the chiral type of sites, it was 0.24 mM for the R enantiomer and 0.64 nM for the S enantiomer. Peak tailing was observed, even at very low concentrations allowing operation of the low-capacity chiral sites under linear conditions. This tailing can be explained on the basis of heterogeneous mass transfer kinetics. At higher concentrations, which are often used in analytical applications, the isotherms on the chiral sites no longer have a linear behavior, and peak tailing is consequently more pronounced. Under those conditions, peak tailing originates from the combined effect of heterogeneous thermodynamics and heterogeneous mass transfer kinetics. These complex phenomena are explained and modeled using the transport-dispersive model with a solid film linear driving force model modified to account for heterogeneous mass transfer kinetics. The rate coefficient of the mass transfer kinetics was found to be concentration dependent. 36 refs., 5 figs., 1 tab.

  18. Dependence on the mobile phase pH of the adsorption behavior of propranolol enantiomers on a cellulase protein used as the chiral selector

    Energy Technology Data Exchange (ETDEWEB)

    Fornstedt, T.; Goetmar, G.; Andersson, M.; Guiochon, G.

    1999-02-17

    The authors reported previously on the unusual thermodynamic characteristics of the enantioselective interactions between the enantiomers of the {beta}-blocker propranolol and the protein cellobiohydrolase I immobilized on silica. The adsorption of the more retained enantiomer, (S)-propranolol, is endothermic while that of the (R)-propranolol is exothermic. This causes a rapid increase of the selectivity factor with increasing temperature. In this work, the complex dependence of the selectivity factor on the pH of the solvent is studied. They determined the equilibrium isotherms of (R)- and (S)-propranolol in a wide concentration range (0.25 {micro}M to 1.1 mM) at six different mobile-phase pHs (4.7, 5.0, 5.2, 5.5, 5.7, and 6.0) and fitted the data obtained to the bi-Langmuir model. This gave the saturation capacity and the binding constant of the nonselective contribution for the two enantiomers. It also gave these parameters for the enantioselective contributions of each of them. The dependence of these parameters on the pH is discussed and interpreted in terms of the retention mechanism. Conclusions are in excellent agreement with recent, independent results on the structure of the protein obtained by X-ray crystallography.

  19. Cellulase Extraction Technology of Polysaccharide from Dendrobium Nobile%纤维素酶提取金钗石斛多糖工艺研究

    Institute of Scientific and Technical Information of China (English)

    张晓敏; 孙志蓉; 朱南南; 程丽丽; 杨春宁; 王晨

    2015-01-01

    ive:Objective:To optimize the parameters of the cellulose extraction of Polysaccharide from Dendrobi-um nobile .Mothods:Regard the extraction rate of polysaccharide as indexes,and use orthogonal experriments to determine the content of index in cellulose by UV Spectrophotometry.Then evaluate the merits of enzymatic parameters.Results:The optimized process conditions were solid-to-liquid ratio 1:50,pH 5.0,reflux time 170min,dosage of enzyme 4500U/g,enzymolysis temperature 55℃,and the Dendrobium nobile polysaccharide extraction rate was 7.71%,with 43.84% higher than conventional thermal reflow method.Conclusion:This method has advantages of simplicity,high extraction rate,which lays a foundation for the further study of Den-drobium nobile.It also has the potentially industrial application value in the extraction of effective ingredients of Chinese Medicine.%目的:对纤维素酶用于金钗石斛多糖提取工艺的酶解参数进行优选。方法:通过正交实验,以多糖提取率为指标,采用紫外分光光度法测定多糖含量,以此评价酶解工艺的优劣。结果:最佳酶解条件为:料液比1∶50,pH值5.0,提取时间170min,酶用量4500U/g,酶解温度55℃,此时金钗石斛多糖提取率为7.71%,比常规热回流提取方法高43.84%。结论:本方法装置简单,提取率高,为金钗石斛深入研究奠定基础,在中药有效成分的提取方面具有潜在的工业应用价值。

  20. 黑胸散白蚁纤维素酶的体外酶学特性%In vitro Characteristics of the Cellulases from Reticulitermes chinensis Snyder

    Institute of Scientific and Technical Information of China (English)

    许利霞; 徐荣; 赵焕玉; 杨红

    2012-01-01

    Reticulitermes chinensis Snyder (Isoptera: Rhinotermitidae) is a wood-feeding lower termite widely distributed in China. For understanding the mechanisms of cellulose degradation in this termite, the in vitro enzyme activities of endo-β-l,4-glucanase (EG), exo-β-1,4-glucanase (namely cellobiohydrolase, CBH), and β-l,4-glucosidase (BG) from the worker termites of R. Chinensis were studied with DNS method. The results showed that for the three enzymes, the optimum reaction time, substrate concentration, pH and temperature were 15 min, 1%, 5.6 and 35 t, respectively. Under the above conditions, the activities of EG, CBH, and BG was 71.3(±13.9) U/mg, 5.8(±0.8) U/mg and 4.1(±0.7) U/mg, respectively. Endo-β-l,4-glucanase was not stable under high temperature in vitro. The incubation of crude enzyme at above 50 X. Would result in low activity or inactivation of the enzyme. However, this enzyme was relatively stable in the pH range from 3.2 to 8.0. Native-PAGE electrophoreses showed that there were at least eight different enzyme activity bands corresponding to endo-/M,4-glucanase distributing in different gut segments in R. Chinensis, which indicated the complexity of cellulose degradation process in wood-feeding lower termites. Fig 8, Ref 17%采用DNS法研究了我国广泛分布的一种低等木食性白蚁——黑胸散白蚁纤维素酶的体外酶活特性以了解其纤维素降解机制.结果表明,内切β-1,4-葡聚糖酶(EG)、纤维二糖水解酶(CBH)和β-葡萄糖苷酶(BG)这3种酶的最佳反应时间均为15 min,最佳底物浓度为1%,最适反应pH为5.6,最适反应温度为35℃.在最适反应条件下,EG、CBH和BG的活性分别达到71.3(±13.9) U/mg、5.8(±0.8) U/mg和4.1(±0.7) U/mg.EG在体外的热稳定性较差,在50℃及更高温度酶活很低或完全失活,但该酶对pH稳定性较好,在pH 3.2~8.0范围内酶活力变化不大.Native-PAGE电泳检测到该白蚁体内至少有8种不同的EG活性条带,肠道不同部位纤维素酶活性条带种类不同.这些研究表明,木食性白蚁降解纤维素是一个复杂的过程,需要多种纤维素酶的共同作用.