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Sample records for cellulase

  1. Engineering Cellulases for Biorefinery

    Energy Technology Data Exchange (ETDEWEB)

    Manoj Kumar, PhD

    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.

  2. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Manoj Kumar, PhD

    2010-03-24

    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. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Manoj Kumar, PhD

    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.

  4. 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.

  5. 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.

  6. 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.

  7. Bioprospecting thermophiles for cellulase production: a review

    OpenAIRE

    Somen Acharya; Anita Chaudhary

    2012-01-01

    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...

  8. Exo-endo cellulase fusion protein

    Science.gov (United States)

    Bower, Benjamin S.; Larenas, Edmund A.; Mitchinson, Colin

    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.

  9. 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.

  10. Cellulase hydrolysis of unsorted MSW

    DEFF Research Database (Denmark)

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

    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...

  11. 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...

  12. Synergy in Trichoderma Reesei cellulases

    OpenAIRE

    Jepsen Kudal, Eva Rose; Ejaz, Rooshanie Nadia; Poniewierska, Julia Alicja

    2016-01-01

    Synergy has been observed between the various cellulolytic enzymes of the fungus Trichoderma Reesei. Termed cellulases, these enzymes come together to form a potent cocktail of biomass degrading enzymes. The biofuel industry has seen a recent surge partly due to the advances in attaining a more applicable understanding of the mechanisms of synergy that occur when said enzymes are used to degrade biomass into fermentable sugar. To that end, this review summarizes the various theories postulati...

  13. 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

  14. Reaction mechanism of dicofol removal by cellulase.

    Science.gov (United States)

    Wang, Ziyuan; Yang, Ting; Zhai, Zihan; Zhang, Boya; Zhang, Jianbo

    2015-10-01

    It remains unclear whether dicofol should be defined as a persistent organic pollutant. Its environmental persistence has gained attention. This study focused on its degradation by cellulase. Cellulase was separated using a gel chromatogram, and its degradation activity towards dicofol involved its endoglucanase activity. By analyzing the kinetic parameters of cellulase reacting with mixed substrates, it was shown that cellulase reacted on dicofol and carboxyl methyl cellulose through two different active centers. Thus, the degradation of dicofol was shown to be an oxidative process by cellulase. Next, by comparing the impacts of tert-butyl alcohol (a typical OH free-radical inhibitor) on the removal efficiencies of dicofol under both cellulase and Fenton reagent systems, it was shown that the removal of dicofol was initiated by OH free radicals produced by cellulase. Finally, 4,4'-dichloro-dibenzophenone and chloride were detected using gas chromatography mass spectrometry and ion chromatography analysis, which supported our hypothesis. The reaction mechanism was analyzed and involved an attack by OH free radicals at the orthocarbon of dicofol, resulting in the degradation product 4,4'-dichloro-dibenzophenone. PMID:26456602

  15. Outlook for cellulase improvement: Screening and selection strategies

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yiheng P [ORNL; Himmel, Michael [ORNL; Mielenz, Jonathan R [ORNL

    2006-03-01

    Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. 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. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.

  16. Hidden cellulases in termites: revision of an old hypothesis

    OpenAIRE

    Tokuda, Gaku; Watanabe, Hirofumi

    2007-01-01

    The intestinal flagellates of termites produce cellulases that contribute to cellulose digestion of their host termites. However, 75% of all termite species do not harbour the cellulolytic flagellates; the endogenous cellulase secreted from the midgut tissue has been considered a sole source of cellulases in these termites. Using the xylophagous flagellate-free termites Nasutitermes takasagoensis and Nasutitermes walkeri, we successfully solubilized cellulases present in the hindgut pellets. ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenjuan [Department of Machine Intelligence and Systems Engineering, Faculty of Systems Engineering, Akita Prefectural University, Akita 015-0055 (Japan); Qiu, Jianhui, E-mail: qiu@akita-pu.ac.jp [Department of Machine Intelligence and Systems Engineering, Faculty of Systems Engineering, Akita Prefectural University, Akita 015-0055 (Japan); Feng, Huixia [College of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou 730050 (China); Zang, Limin; Sakai, Eiichi [Department of Machine Intelligence and Systems Engineering, Faculty of Systems Engineering, Akita Prefectural University, Akita 015-0055 (Japan)

    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. - Highlights: • Three Amino-silane modified magnetic nanospheres were prepared. • Cellulase immobilized AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than free cellulase. • The potential of biofuel production using this immobilized cellulase.

  18. 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...... CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3), for their sensitivity to the products glucose and cellobiose. The strongest inhibition was found for Cel7A, which showed a 50% activity-loss in 19 mM cellobiose (IC50 = 19 mM). The other exoglucanase, Cel6A, was much less inhibited by...

  19. Kinetic Studies on Trichoderna Viride Cellulase

    International Nuclear Information System (INIS)

    Studies on cellulase enzyme (EC 3.2.1.4), which catalyzes the hydrolysis of. cellulose to yield glucose, were made. Cellulase from a fungus source, Trichoderma viride was cultivated on Czapek's agar medium and enzyme production broth medium was employed for parameter tests. The microscopic examination and cellulase hydrolysis test on subcultured fungi were applied to confirm the T. viride species. A calibration curve for standard glucose was plotted by using visible spectroscopy. Dinitrosalicylic acid was used as enzyme reaction inhibitor and the colour intensity was measured in a UV-visible spectrophotometer at a λmax of 570 nm. The parameters such as optimum pH, optimum temperature, effect of substrate concentration, effect, of enzyme concentration, enzyme unit (EU), reaction order (n), maximum velocity (Vmax), Michaelis-Menten constant (Km) using various substrates, viz., carboxy methylcellulose, cotton fibre and filter paper determined. (author)

  20. 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...... 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...

  1. Lactose enhances cellulase production by the filamentous fungus Acremonium cellulolyticus.

    Science.gov (United States)

    Fang, Xu; Yano, Shinichi; Inoue, Hiroyuki; Sawayama, Shigeki

    2008-08-01

    Acremonium cellulolyticus is a fungus that produces cellulase and has been exploited by enzyme industry. To promote cellulase production by A. cellulolyticus strain C-1, we evaluated the effects of the saccharides: Solka Floc (cellulose), soluble soybean polysaccharide (SSPS), pullulan, lactose, trehalose, sophorose, cellobiose, galactose, sorbose, lactobionic acid, and mixtures as carbon sources for cellulase production. Solka Floc with SSPS enhanced cellulase production. Lactose as the sole carbon source induced cellulase synthesis in this fungus, and the synergistic effects between lactose and Solka Floc was observed. Various enzyme activities and the protein composition of crude enzyme produced by cultures with or without addition of lactose were analyzed. The results showed that lactose addition greatly improves the production of various proteins with cellulase activity by A. cellulolyticus. To our knowledge, this is the first report on production of cellulases by lactose in the A. cellulolyticus. PMID:18804052

  2. A steady state theory for processive cellulases

    DEFF Research Database (Denmark)

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

    2013-01-01

    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....... 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....

  3. Continous monitoring of cellulase action on microcrystalline cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Kremer, S.M.; Wood, P.M. (Bristol Univ. (United Kingdom). Dept. of Biochemistry)

    1992-09-01

    Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for cellobiose oxidase, in [mu]mol.min[sup -1]. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K[sub m] of 4.8 [+-] 1.0 (g cellulose).1[sup -1], which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375 [+-] 25 [mu]mol.(g cellulase)[sup -1].min[sup -1] in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6 [+-] 1.3 [mu]mol.(g cellulose)[sup -1].min[sup -1]. (orig.).

  4. 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.

  5. HYDROLYSIS OF PAPER-DISHWARE WASTES BY CELLULASE

    Institute of Scientific and Technical Information of China (English)

    JieLu; ShulanShi; RunanYang; FuzhengLiang

    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.

  6. Cellulase production and activity in a species ofCladosporium.

    Science.gov (United States)

    Abrha, B; Gashe, B A

    1992-03-01

    ACladosporium species produced large amounts of cellulase enzyme components when grown in shake-culture with medium containing carboxymethylcellulose. There was significantly less activity when Avicel, filter paper or cotton were used as substrates. KNO3 was better than NH4Cl or urea for the production of cellulase. Tween 80 at 0.1% (w/v) increased the production of cellulase by 1.5 to 4.5-fold. All the cellulase components were optimally active in the assay at pH 5.0 and 60°C. PMID:24425401

  7. Production of cellulase by Trichoderma reesei from dairy manure.

    Science.gov (United States)

    Wen, Zhiyou; Liao, Wei; Chen, Shulin

    2005-03-01

    Cellulase production by the fungi Trichoderma reesei was studied using dairy manure as a substrate. Data showed that T. reesei RUT-C30 had higher cellulase production than T. reesei QM 9414 and that a homogenized manure, treated by a blender to reduce fiber size, led to higher cellulase production. The cellulase production was further optimized by growing T. reesei RUT-C30 on homogenized manure. The effects of manure concentration, pH, and temperature on cellulase production were investigated with optimal parameter values determined to be 10 g/l manure (dry basis), 25.5 degrees C, and pH 5.7, respectively. Elimination of CaCl2, MgSO4, nitrogen sources (NH4+ and urea) and trace elements (Fe2+, Zn2+, Co2+ and Mn2+) from the original salt solution had no negative influence on the cellulase production, while phosphate elimination did reduce cellulase production. Based on above results, the final medium composition was simplified with manure additives being KH2PO4, tween-80 and CoCl2 only. Using this medium composition and a reaction time of 6-8 days, a maximum cellulase production activity of 1.74 IU/ml of filter paper activity, 12.22 IU/ml of CMCase activity, and 0.0978 IU/ml of beta-glucosidase was obtained. This filter paper activity is the highest ever reported in cellulase production from agricultural wastes. PMID:15491832

  8. Xyloglucan oligosaccharides promote growth and activate cellulase: Evidence for a role of cellulase in cell expansion

    International Nuclear Information System (INIS)

    Oligosaccharides produced by the action of fungal cellulase on xyloglucans promoted the elongation of etiolated pea (Pisum sativum L.) stem segments in a straight-growth bioassay designed for the determination of auxins. The oligosaccharides were most active at about 1 micromolar. We tested the relative growth-promoting activities of four HPLC-purified oligosaccharides which shared a common glucose4·xylose3 (XG7) core. The substituted oligosaccharides XG8 (glucose4·xylose3·galactose) and XG9n (glucose4·xylose3·galactose2) were more effective than XG7 itself and XG9 (glucose4·xylose3·galactose·fucose). The same oligosaccharides also promoted the degradation, assayed viscometrically, of xyloglucan by an acidic cellulase from bean (Phaseolus vulgaris L.) leaves. The oligosaccharides were highly active at 10-4 molar, causing up to a fourfold increase in activity, but the effect was still detectable at 1 micromolar. Those oligosaccharides (XG8 and XG9n) which best promoted growth, stimulated cellulase activity to the greatest extent. The oligosaccharides did not stimulate the action of the cellulase in an assay based on the conversion of [3H]xyloglucan to ethanol-soluble fragments. This suggests that the oligosaccharides enhanced the midchain hydrolysis of xyloglucan molecules (which would rapidly reduce the viscosity of the solution), at the expense of cleavage near the termini (which would yield ethanol-soluble products)

  9. Production Of Cellulase In Plastids Of Transgenic Plants

    Science.gov (United States)

    Lamppa, Gayle

    2002-08-06

    A genetic construct encoding a fusion protein including endogluconase E1 and a transit peptide is used to transform plants. The plants produce cellulase by expressing the genetic construct. The cellulase is targeted to plastids and can be collected and purified.

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

    Science.gov (United States)

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

    2014-01-01

    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. PMID:24291747

  11. 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.

  12. 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.

  13. Regulation of Biosynthesis of Individual Cellulases in Thermomonospora fusca

    OpenAIRE

    Spiridonov, Nikolay A.; Wilson, David B.

    1998-01-01

    Regulation of the biosynthesis of the six cellulases comprising the cellulolytic system of the thermophilic soil bacterium Thermomonospora fusca ER1 was studied. The levels of the individual enzymes produced on different noninducing and inducing carbon sources were determined. The lowest level of cellulase synthesis (3 nM) was observed with xylose as a carbon source, and the highest level (247 to 1,670 nM for different enzymes) was found in cultures grown on microcrystalline cellulose. Endoce...

  14. Rheology of carboxymethyl cellulose solutions treated with cellulases

    OpenAIRE

    Lee, J. M.; Heitmann, J. A.; Pawlak, J. J.

    2007-01-01

    The effect of cellulase treatments on the rheology of carboxymethyl cellulose (CMC) solutions was studied using a rotational viscometer. The rheological behaviors of CMC solutions of different molecular mass and degrees of substitution where studied as a function of time after various treatments. These solutions were subjected to active and heat-denatured cellulase, a cationic polyelectrolyte (C-PAM), as well as different shear rates. A complex protein-polymer interaction was observed, lea...

  15. Microbial cellulases and their applications in textile processing

    OpenAIRE

    Mojsov, Kiro

    2012-01-01

    Basic and applied research on microbial cellulases has not only generated significant scientific knowledge but has also revealed their enormous potential in biotechnology.At present, cellulases and related enzymes are used in food, brewery and wine, animal feed, textile and laundry, pulp and paper industries, as well as in agriculture and for research purposes. Textile processing is a growing industry that traditionally has used a lot of water, energy and harsh chemicals. Due to the ever-grow...

  16. Modelling of amorphous cellulose depolymerisation by cellulases, parametric studies and optimisation

    OpenAIRE

    Niu, Hongxing; Shah, Nilay; Kontoravdi, Cleo

    2016-01-01

    Highlights • A mechanistic model for heterogeneous cellulose hydrolysis by cellulases. • A modeling framework for uncertainty analysis, model reduction and refinement. • The parameters were estimated. • Composition of cellulases cocktail was optimized using the model.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    OpenAIRE

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

    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...

  19. 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.

  20. 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... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Cellulase enzyme preparation derived from....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase...

  1. The catalytic inactivation of cellulase enzyme components by palladium complexes

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J.; Gooch, M.G.; Shultz, M.D.

    1993-10-01

    It has been discovered that sodium hexachloropalladate is a strong inhibitor of cellobiohydrolase I (CBH I) from Trichoderma reesei having an I{sub 50} of <50 {mu}M with p-nitrophenylcellobioside (PNPC) as the substrate. Similar complexes of the metals platinum, osmium, iridium, and rhodium have little effect on CBH I. Other cellulase activities (Avicelase, {beta}-glucanase) are also inhibited by the palladium complex, suggesting that inhibition of two major types of catalytic activity in cellulase are affected. Preliminary data on the kinetics of inhibition of CBH I by sodium hexachloropalladate indicate that the inhibition is reversible and, possibly, uncompetitive. It is anticipated that sodium hexachloropalladate and other palladium complexes will be useful for determining the effect of the binding of catalytically inactivated CBH I and other cellulase components on the structure of cellulose fibers.

  2. Factors influencing the production of cellulases by Sporotrichum thermophile.

    Science.gov (United States)

    Coutts, A D; Smith, R E

    1976-06-01

    Cellulase production and growth of a strain of Sporotrichum thermophile were studied by using a mineral salts medium supplemented with yeast extract and insoluble cellulose. The effects of cultural conditions, such as pH, nitrogen source, substrate concentration, and temperature, were examined. Maximum production of C1 and CX cellulases occurred at 45 C in 2 to 4 days, in the presence of 1% Solka/Floc as substrate, when NaNO3 or urea used as sources of nitrogen. Under these conditions, cellulolytic activity of culture filtrates appeared to be similar to that reported for Trichoderma viride grown in a favorable environment. However, comparable yields of cellulase were produced by S. thermophile in less than one-quarter the time required by mesophilic fungi. PMID:7194

  3. 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.

  4. Enhancement of Cellulase Production by Cellulomonas Fimi and Bacillus Subtilis

    International Nuclear Information System (INIS)

    Two bacterial strains identified as Cellulomonas fimi and Baciliius subtilus are cosidered as highly active cellulytic bacteria. Trials for maximizing the cellulolytic activites of the two strains were conducted. A maximum cellulase production was achieved at 1 and 1.5%carboxy methyl cellulose as carbon source, sodium nitrate and yeast as nitrogen source for Cellulomonas fimi and Bacillus subtilis, respectively. Incubation temprature at 30 and 45 degree C, ph at 6 and 7 achieved the highest activity of cellulase for Cellulomonas fimi and bacillus subtilis, respectively

  5. Regulation of Laccase and Cellulase Genes Transcription in Agaricus bisporus

    OpenAIRE

    Ohga, Shoji; Wood, David A.

    1998-01-01

    A time course for laccase and cellulase genes transcription of Agaricus bisporus compost culture are examined. The results of assays for laccase gene leel show that the expression of this gene increased in the compost until pinning stage of development. In the fruiting cultures the amount of leel declined rapidly over a 4-5 d period immediately. Cellulase gene celS expression contrasted sharply appeared with leel expression by remaining at a low level until after the pins were seen. The cel3...

  6. Cellulase production by halophytic fungi. Pt. 2. The role of some cultural conditions on cellulase production by Ulocladium chartarum

    Energy Technology Data Exchange (ETDEWEB)

    Sallam, L.A.R.; EL-Refai, A.M.H.; El-Sayed, S.M.

    1988-01-01

    Cellulase activities of Ulocladium chartarum were found to be influenced by the mode of incubation as well as by the age of the fungal culture. Maximal cellulase activities were, however, maintained after 3 weeks using the surface culture mode of incubation. U. chartarum was grown at different temperatures, where maximal CMCase and FPase activities were achieved at 30/sup 0/C. The cellulase activities were also tested using initially adjusted pH as well as buffered media. The highest CMCase and FPase activities (0.289 and 0.043 unit/ml respectively) were obtained with pH 6.5 and the phosphate buffer gave better results as compared with the acetate buffer.

  7. Properties of cellulase as template molecule on chitosan—methyl methacrylate membrane

    Science.gov (United States)

    Lian, Qi; Zheng, Xuefang; Wu, Haixia; Song, Shitao; Wang, Dongjun

    2015-12-01

    In this study, a novel molecular imprinting membrane made of chitosan and methyl methacrylate (MMA) was fabricated with cellulase as template molecule and the thermal response to cellulase was characterized. The film was characterized by infrared spectroscopy (IR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and the permeation experiment. The results showed that the space structure of the film was as similar as the cellulase. Moreover, the membrane had advanced molecular imprinting capability to cellulase comparing to pepsin and pectinase at any temperature and the film had excellent ability to identify specific template molecule (cellulase) at the synthesis temperature compared to other temperatures.

  8. 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. PMID:27099940

  9. 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.

  10. CBH1 homologs and variant CBH1 cellulases

    Energy Technology Data Exchange (ETDEWEB)

    Goedegebuur, Frits (Rozenlaan, NL); Gualfetti, Peter (San Francisco, CA); Mitchinson, Colin (Half Moon Bay, CA); Neefe, Paulien (Zoetermeer, NL)

    2011-05-31

    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.

  11. 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.

  12. Tropical Soil Fungi Producing Cellulase and Related Enzymes in Biodegradation

    Directory of Open Access Journals (Sweden)

    Wattanachai Pathomsiriwong

    2012-01-01

    Full Text Available The objective of this study was to screen, identify and characterize of cellulolytic fungi from various soil management fields; organic, young organic, semi-chemical and chemical soil from Surin rice fields, Thailand. Fungi from various type of soils were isolated by dilution plating technique on Reese minimal medium supplemented with cellulose powder and rice straw then incubated at 25°C for 3-7 days. The isolated fungi were screened and identified using slide culture technique. The enzymatic activities were assessed by qualitative method for cellulase, xylanase, peroxidase and laccase activities. Two-hundred and fifty-eight fungi isolates found in Surin rice fields belonging to the genus Penicillium (5 species, Paecilomyces (4 species, Aspergillus (3 species, Acremonium (2 species, Chaetomium (2 species, Alternaria (1 species, Bipolaris (1 species, Curvularia (1 species, Fusarium (1 species, Humicola (1 species, Mucor (1 species, Nigrospora (1 species, Phoma (1 species, Pyrenochaeta (1 species, Pythium (1 species, Rhizopus (1 species, Sporotrichum (1 species and Trichoderma (1 species. Out of 29 fungal species clearly showed different in enzymatic activities. Most tropical soil fungi had ability to produce cellulase, xylanase, laccase and peroxidase, respectively. The highest capacity was found only in cellulase. Aspergillus niger, Aspergillus sp., Chaetomium murorum and Trichoderma sp. showed the highest potential to produce cellulase. Eleven species of soil fungi showed high capacity in xylanase activity. For laccase and peroxidase activity, there were found in 2 species. The results also revealed that only ten showed highest carboxymethyl cellulase, xylanase, peroxidase and laccase activities by qualitative screening method for enzymatic assay. They were identified as Aspergillus niger, Acremonium sp., Aspergillus sp., Chaetomium murorum, Humicola grisea, Mucor sp., Paecilomyces victoriae, Penicillium janthinellum, Penicillium lanosum and

  13. Efficient cellulase production by the filamentous fungus Acremonium cellulolyticus.

    Science.gov (United States)

    Ikeda, Yuko; Hayashi, Hiroyuki; Okuda, Naoyuki; Park, Enock Y

    2007-01-01

    Cellulase production was investigated in a culture of a strain of Acremonium cellulolyticus. The medium components were optimized for the improvement of cellulase production. The maximum production of cellulolytic enzymes was obtained in a medium containing (grams per liter) 50 Solka Floc, 5 (NH4)2SO4, 24 KH2PO4, 4.7 potassium tartrate hemihydrate, 1.2 MgSO4.7H2O, 1 Tween 80, 4 urea, 0.01 ZnSO4.7H2O, 0.01 MnSO4.6H2O, and 0.01 CuSO4.7H2O, with a pH of 4.0. In the flask culture, 15.5 filter paper units (FPU)/mL of maximum cellulase activity was obtained, 17.42 FPU/mL in a 7-L bioreactor, and 13.08 FPU/mL in a 50-L scale bioreactor for 4-8 d at 30 degrees C. Average production rates were 1.94 FPU/mL.d in flasks, 2.86 FPU/mL.d in the 7-L bioreactor, and 2.56 FPU/mL.d in the 50-L bioreactor. Cellulase production on a small scale was successfully reproduced in the 50-L pilot scale bioreactor. Saccharification activity from A. cellulolyticus was compared with cellulolytic enzymes produced by other strains. The A. cellulolyticus culture broth had a comparable saccharification yield in comparison with those of other Trichoderma enzymes (GC220 or Cellulosin T2) under the same total cellulase activity. Its saccharification yield (percent of released reducing sugar to used dried substrate) was 60%, and its glucose content was 83%. PMID:17253723

  14. Immobilized Microbial Cellulases in Organic-Inorganic Hybrid Materials

    Directory of Open Access Journals (Sweden)

    Monica Dragomirescu

    2011-05-01

    Full Text Available The Aspergillus niger cellulase was immobilized by entrapment in natrium alginate gels and in natrium alginate/silica gel hybrid materials. The silica gels were obtained using two different precursors, tetraethoxysilane (TEOS and tetramethoxysilane (TMOS, by sol-gel method. Comparing the enzymatic activities of the immobilized products, it was noticed that the CMCase activities obtained in natrium alginate were 1.12-1.17 times higher than those obtained by entrapment in mixed organic – inorganic gels, for similar enzyme loadings. The operational stability was tested in the substrate presence. The cellulase entrapped in the three types of organic-inorganic gel matrices mentioned above retains about 13% of its activity after 4 cycles. After one hour of storage at 37ºC, pH 3.0, the relative activity of the immobilized Aspergillus niger CMCase was more than 98% of initial.

  15. Optimization of cultivation conditions for the cellulase producer aspergillus terreus

    Energy Technology Data Exchange (ETDEWEB)

    Svistova, I.D.; Zherebtsov, N.A.

    1984-01-01

    Aspergillus terreus was able to synthesize C1-cellulase, Cx-cellulase, endoglucanase and cellobiase in a synthetic minimal medium containing agricultural wastes (sawdust, straw, sugar beet pulp, barley husks, etc) as a sole source of C. The highest enzyme activity was observed with sugar beet pulp whereas the lowest activity occurrred with sawdust. Of various N sources tested, NaNO3 and peptone were most effective. Addition of corn meal, malt sprout, or protein-vitamin concentration (PVC) significantly stimulated enzyme biosynthesis. The optimum culture medium, determined by the mathematical method of experimental factor design was composed of sugar beet pulp 30, KH2PO4 1-4, NaNO3 7.5, and PVC 15 g/L.

  16. Molecular cloning of cellulase genes from indigenous bacterial isolates

    International Nuclear Information System (INIS)

    Indigenous cellulolytic bacterial isolates having high activities in degrading carboxymethyl cellulose (CMC) were isolated from local environments. Identification of these isolates were performed by molecular techniques. By using polymerase chain reaction (PCR) techniques, PCR products encoding cellulase gene were amplified from the total genomic DNAs. Purified PCR product was successfully cloned and expressed in Escherichia coli host system. The complete nucleotide sequences of the cellulase genes determined. The analysis of amino acid sequences deduced from the genes indicated that the cloned DNA fragments show high homology to those of endoglucanase genes of family GH5. All cloned genes consist of an N-terminal signal peptide, a catalytic domain of family 5 glycosyl hydrolase and a cellulose-binding domain of family III. (Author)

  17. Enhanced Cellulose Degradation Using Cellulase-Nanosphere Complexes

    OpenAIRE

    Blanchette, Craig; Lacayo, Catherine I.; Fischer, Nicholas O.; Hwang, Mona; Thelen, Michael P.

    2012-01-01

    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 cellu...

  18. Inhibition of lignin-derived phenolic compounds to cellulase

    OpenAIRE

    Qin, Lei; Li, Wen-Chao; Liu, Li; Zhu, Jia-Qing; Li, Xia; Li, Bing-zhi; Yuan, Ying-Jin

    2016-01-01

    Background Lignin-derived phenolic compounds are universal in the hydrolysate of pretreated lignocellulosic biomass. The phenolics reduce the efficiency of enzymatic hydrolysis and increase the cost of ethanol production. We investigated inhibition of phenolics on cellulase during enzymatic hydrolysis using vanillin as one of the typical lignin-derived phenolics and Avicel as cellulose substrate. Results As vanillin concentration increased from 0 to 10 mg/mL, cellulose conversion after 72-h e...

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

    OpenAIRE

    Gupta Munishwar; Sharma Aparna; Dalal Sohel

    2007-01-01

    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 characterize...

  20. Effects of lignin and surfactant on adsorption and hydrolysis of cellulases on cellulose

    OpenAIRE

    Yanfei LI; Sun, Zongping; Ge, Xiaoyan; Zhang, Junhua

    2016-01-01

    Background Considerable works have been reported concerning the obstruction of enzymatic hydrolysis efficiency by lignin. However, there is a lack of information about the influence of lignin on the adsorption of cellulases on cellulose, along with the hydrolytic activity of the cellulases adsorbed on lignin. In addition, limited discovery has been reported about the influence of additives on cellulase desorption from lignin and lignocellulosic materials. In this work, the effects of lignin o...

  1. Aspergillus nidulans protein kinase A plays an important role in cellulase production

    OpenAIRE

    de Assis, Leandro José; Ries, Laure Nicolas Annick; Savoldi, Marcela; dos Reis, Thaila Fernanda; Brown, Neil Andrew; Goldman, Gustavo Henrique

    2015-01-01

    Background The production of bioethanol from lignocellulosic feedstocks is dependent on lignocellulosic biomass degradation by hydrolytic enzymes. The main component of lignocellulose is cellulose and different types of organisms are able to secrete cellulases. The filamentous fungus Aspergillus nidulans serves as a model organism to study cellulase production and the available tools allow exploring more in depth the mechanisms governing cellulase production and carbon catabolite repression. ...

  2. Exogenous Cellulase Contributes to Mycoherbicidal Activity of Fusarium arthrosporioides on Orobanche aegyptiaca

    OpenAIRE

    Babalola, Olubukola O.

    2010-01-01

    This paper investigates an association between the tubercle size of Orobanche aegyptiaca, tubercle death, and days to tubercle death in relation to cellulase-assisted mycoherbicide. Fusarium arthrosporioides killed 56% of tubercles when applied with cellulase compared to 35% when no cellulase was added. Death was inversely correlated with days over the two fungal treatment types. O. aegyptiaca tubercle size significantly correlated with the two other infection parameters studied. For F. arthr...

  3. Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414

    OpenAIRE

    Ng, Thomas K.; Zeikus, J. G.

    1981-01-01

    The crude extracellular cellulase of Clostridium thermocellum LQRI (virgin strain) was very active and solubilized microcrystalline cellulose at one-half the rate observed for the extracellular cellulase of Trichoderma reesei QM9414 (mutant strain). C. thermocellum cellulase activity differed considerably from that of T. reesei as follows: higher endoglucanase/exoglucanase activity ratio; absence of extracellular cellobiase or β-xylosidase activity; long-chain oligosaccharides instead of shor...

  4. Production of Resveratrol by Piceid Deglycosylation Using Cellulase

    Directory of Open Access Journals (Sweden)

    Chia-Hung Kuo

    2016-02-01

    Full Text Available Resveratrol is a dietary polyphenolic compound widely used in medicine, food, and cosmetic products. The glycoside form of resveratrol, piceid, is also present in several plant materials but is less bioavailable. In this study, enzymatic transformation of piceid into resveratrol using inexpensive cellulase was investigated. Response surface methodology was used to evaluate the effect of reaction parameters, including reaction temperature, reaction time, enzyme amount and pH. The optimal conditions for biotransformation of piceid to resveratrol are: a reaction temperature of 50 °C, reaction time of 4.75 h, enzyme amount of 2.5 fungal β-glucanase (FBG units and pH of 4.3. In addition, the extracts from Polygonum cuspidatum root contained high amounts of piceid were treated with cellulase in order to deglycosylation that increased resveratrol yield. After treatment, the resveratrol yield significantly increased from 2.72 to 9.49 mg/g, while the piceid contents decreased from 8.60 to 0 mg/g. The result provides an efficient method to convert piceid in the extracts of P. cuspidatum root into resveratrol by cellulase.

  5. Biomining active cellulases from a mining bioremediation system.

    Science.gov (United States)

    Mewis, Keith; Armstrong, Zachary; Song, Young C; Baldwin, Susan A; Withers, Stephen G; Hallam, Steven J

    2013-09-20

    Functional metagenomics has emerged as a powerful method for gene model validation and enzyme discovery from natural and human engineered ecosystems. Here we report development of a high-throughput functional metagenomic screen incorporating bioinformatic and biochemical analyses features. A fosmid library containing 6144 clones sourced from a mining bioremediation system was screened for cellulase activity using 2,4-dinitrophenyl β-cellobioside, a previously proven cellulose model substrate. Fifteen active clones were recovered and fully sequenced revealing 9 unique clones with the ability to hydrolyse 1,4-β-D-glucosidic linkages. Transposon mutagenesis identified genes belonging to glycoside hydrolase (GH) 1, 3, or 5 as necessary for mediating this activity. Reference trees for GH 1, 3, and 5 families were generated from sequences in the CAZy database for automated phylogenetic analysis of fosmid end and active clone sequences revealing known and novel cellulase encoding genes. Active cellulase genes recovered in functional screens were subcloned into inducible high copy plasmids, expressed and purified to determine enzymatic properties including thermostability, pH optima, and substrate specificity. The workflow described here provides a general paradigm for recovery and characterization of microbially derived genes and gene products based on genetic logic and contemporary screening technologies developed for model organismal systems. PMID:23906845

  6. Cellulase-assisted extraction of oligosaccharides from defatted rice bran.

    Science.gov (United States)

    Patindol, J; Wang, L; Wang, Y-J

    2007-11-01

    Defatted rice bran was subjected to cellulase treatment in order to increase its extractable oligosaccharides. Various combinations of enzyme concentration (0%, 0.5%, 1.0%, and 2.0%), temperature (room, 30, 40, and 50 degrees C), and time (1, 3, 5, and 16 h) were tested to identify the optimum extraction conditions. The saccharide content and composition of the extracts were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Digestibility was assayed in vitro with human salivary and porcine pancreatic alpha-amylases. Extraction yield ranged from 13.4% (without cellulase) to 39.9% (with 2% cellulase). Total carbohydrates, reducing sugars, and crude protein of the dried extracts ranged from 69.2% to 87.2%, 18.7% to 62.3%, and 7.1% to 22.3%, respectively. Mono- and disaccharides constituted more than 50% of the total carbohydrates in the extracts. Inherent oligosaccharides and those produced by cellulolysis made up less than 25%. The in vitro digestibility of the extracts by alpha-amylases was lower compared with that of the original rice bran sample and potato dextrin, which could be attributed to the increased concentrations of oligosaccharides and reducing sugars. PMID:18034713

  7. Cellulase induction in Trichoderma reesei by cellulose requires its own basal expression.

    Science.gov (United States)

    Carle-Urioste, J C; Escobar-Vera, J; El-Gogary, S; Henrique-Silva, F; Torigoi, E; Crivellaro, O; Herrera-Estrella, A; El-Dorry, H

    1997-04-11

    The induction of cellulases by cellulose, an insoluble polymer, in the filamentous fungus Trichoderma reesei is puzzling. We previously proposed a mechanism that is based on the presence of low levels of cellulase in the uninduced fungus; this basal cellulase activity would digest cellulose-releasing oligosaccharides that could enter the cell and trigger expression of cellulases. We now present experiments that lend further support to this model. We show here that transcripts of two members of the cellulase system, cbh1 and egl1, are present in uninduced T. reesei cells. These transcripts are induced at least 1100-fold in the presence of cellulose. We also show that a construct containing the hygromycin B resistance-encoding gene driven by the cbh1 promoter confers hygromycin B resistance to T. reesei cells grown in the absence of cellulose. Moreover, cellulose-induced production of the cbh1 transcript was suppressed when antisense RNA against three members of the cellulase system was expressed in vivo. Experiments are presented indicating that extracellular cellulase activity is the rate-limiting event in induction of synthesis of the cellulase transcripts by cellulose. The results reveal a critical requirement for basal expression of the cellulase system for induction of synthesis of its own transcripts by cellulose. PMID:9092563

  8. Xyloglucan oligosaccharides promote growth and activate cellulase: Evidence for a role of cellulase in cell expansion. [Pisum sativum L

    Energy Technology Data Exchange (ETDEWEB)

    McDougall, G.J.; Fry, S.C. (Univ. of Edinburgh (England))

    1990-07-01

    Oligosaccharides produced by the action of fungal cellulase on xyloglucans promoted the elongation of etiolated pea (Pisum sativum L.) stem segments in a straight-growth bioassay designed for the determination of auxins. The oligosaccharides were most active at about 1 micromolar. We tested the relative growth-promoting activities of four HPLC-purified oligosaccharides which shared a common glucose{sub 4} {center dot} xylose{sub 3} (XG7) core. The substituted oligosaccharides XG8 (glucose{sub 4} {center dot} xylose{sub 3} {center dot} galactose) and XG9n (glucose{sub 4} {center dot} xylose{sub 3} {center dot} galactose{sub 2}) were more effective than XG7 itself and XG9 (glucose{sub 4} {center dot} xylose{sub 3} {center dot} galactose {center dot} fucose). The same oligosaccharides also promoted the degradation, assayed viscometrically, of xyloglucan by an acidic cellulase from bean (Phaseolus vulgaris L.) leaves. The oligosaccharides were highly active at 10{sup {minus}4} molar, causing up to a fourfold increase in activity, but the effect was still detectable at 1 micromolar. Those oligosaccharides (XG8 and XG9n) which best promoted growth, stimulated cellulase activity to the greatest extent. The oligosaccharides did not stimulate the action of the cellulase in an assay based on the conversion of ({sup 3}H)xyloglucan to ethanol-soluble fragments. This suggests that the oligosaccharides enhanced the midchain hydrolysis of xyloglucan molecules (which would rapidly reduce the viscosity of the solution), at the expense of cleavage near the termini (which would yield ethanol-soluble products).

  9. 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.

  10. Nucleosome transactions on the Hypocrea jecorina (Trichoderma reesei) cellulase promoter cbh2 associated with cellulase induction.

    Science.gov (United States)

    Zeilinger, S; Schmoll, M; Pail, M; Mach, R L; Kubicek, C P

    2003-10-01

    The 5' regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes -1 and -2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome -1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5' regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing

  11. Adsorption and mechanism of cellulase enzymes onto lignin isolated from corn stover pretreated with liquid hot water

    OpenAIRE

    Lu, Xianqin; Zheng, Xiaoju; Li, Xuezhi; Zhao, Jian

    2016-01-01

    Background In the bioconversion of lignocellulosic substrates, the adsorption behavior of cellulase onto lignin has a negative effect on enzymatic hydrolysis of cellulose, decreasing glucose production during enzymatic hydrolysis, thus decreasing the yield of fermentation and the production of useful products. Understanding the interaction between lignin and cellulase is necessary to optimize the components of cellulase mixture, genetically engineer high-efficiency cellulase, and reduce cost ...

  12. Charge engineering of cellulases improves ionic liquid tolerance and reduces lignin inhibition.

    Science.gov (United States)

    Nordwald, Erik M; Brunecky, Roman; Himmel, Michael E; Beckham, Gregg T; Kaar, Joel L

    2014-08-01

    We report a novel approach to concurrently improve the tolerance to ionic liquids (ILs) as well as reduce lignin inhibition of Trichoderma reesei cellulase via engineering enzyme charge. Succinylation of the cellulase enzymes led to a nearly twofold enhancement in cellulose conversion in 15% (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). The improvement in activity upon succinylation correlated with the apparent preferential exclusion of the [Cl] anion in fluorescence quenching assays. Additionally, modeling analysis of progress curves of Avicel hydrolysis in buffer indicated that succinylation had a negligible impact on the apparent KM of cellulase. As evidence of reducing lignin inhibition of T. reesei cellulase, succinylation resulted in a greater than twofold increase in Avicel conversion after 170 h in buffer with 1 wt% lignin. The impact of succinylation on lignin inhibition of cellulase further led to the reduction in apparent KM of the enzyme cocktail for Avicel by 2.7-fold. These results provide evidence that naturally evolved cellulases with highly negative surface charge densities may similarly repel lignin, resulting in improved cellulase activity. Ultimately, these results underscore the potential of rational charge engineering as a means of enhancing cellulase function and thus conversion of whole biomass in ILs. PMID:24522957

  13. 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

    cellulases to lignin. In the current work we address this hypothesis using tannic acid (TAN) as a general poly-phenolic model compound (for lignin and soluble phenolics) and measure the mutual interactions of cellulases (CBHI, CBHII, EGI, EGII and BG), TAN and NIS (Triton X-100) using isothermal titration...

  14. Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass.

    Science.gov (United States)

    Sun, Fubao Fuebiol; Hong, Jiapeng; Hu, Jinguang; Saddler, Jack N; Fang, Xu; Zhang, Zhenyu; Shen, Song

    2015-11-01

    The potential of cellulase enzymes in the developing and ongoing "biorefinery" industry has provided a great motivation to develop an efficient cellulase mixture. Recent work has shown how important the role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study, three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cellulase preparations were found to display significantly different hydrolytic performances irrelevant with the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diversified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase preparations was finally verified by supplementation with β-glucosidase, xylanase and lytic polysaccharide monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which can further provide a useful reference for the rational customization of cellulase cocktails in order to realize an efficient conversion of natural lignocellulosic substrates. PMID:26320713

  15. 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.

  16. Regulation of the cellulolytic system in Trichoderma reesei by sophorose: induction of cellulase and repression of beta-glucosidase.

    OpenAIRE

    Sternberg, D; Mandels, G. R.

    1980-01-01

    Sophorose has two regulatory roles in the production of cellulase enzymes in Trichoderma reesei: beta-glucosidase repression and cellulase induction. Sophorose also is hydrolyzed by the mycelial-associated beta-glucosidase. Repression of beta-glucosidase reduces sophorose hydrolysis and thus may increase cellulase induction.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    Cellulases and hemicellulases are two classes of enzymes produced by filamentous fungi and secreted into the cultivation medium. Both classes of enzymes consist of a subset of classes of which the fungi produce several enzymes with varying molecular mass and pI but similar enzymatic activities...... silica capillary at pH values close to neutral. The improvement of the separation of these six proteins by the addition of alpha, omega-diaminoalkanes with chain lengths from three to seven carbon units was investigated. Dynamically coating the capillary with 1,3-diaminopropane resulted in separation of...

  18. Carboxymethyl cellulase activity in the myxomycete Physarum polycephalum

    Energy Technology Data Exchange (ETDEWEB)

    Koevenig, J.L.; Liu, E.H.

    1981-11-01

    Carboxymethyl cellulase (CMCase) activity at different life cycle stages in Physarum polycephalum was measured viscometrically in homogenized axenic cultures incubated with CMC. Protein concentrations were determined using the Bio-Rad (Coomassie Blue) assay. Mean activity for several dilutions of the homogenized samples ranged from 318 CMCase activity units/hr/mg protein in old plasmodia to 2423 CMCase unit/hr/mg protein in dry spores. Activity in spores and sclerotia was higher than in a vegetative plasmodium. This CMCase activity level is comparable to that found in some true fungi and suggests that myxomycetes may utilize cellulose and play a role in the breakdown of plant products. (Refs. 18).

  19. 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.

  20. 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...... silica capillary at pH values close to neutral. The improvement of the separation of these six proteins by the addition of alpha, omega-diaminoalkanes with chain lengths from three to seven carbon units was investigated. Dynamically coating the capillary with 1,3-diaminopropane resulted in separation of...

  1. 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.

  2. 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. PMID:26672443

  3. Effect of exogenous cellulase enzyme on feed digestibility in lamb

    International Nuclear Information System (INIS)

    The aim of this study was to determine the effect of exogenous enzyme on digestibility and N retention in lamb. Eight lambs were randomly allocated to 2 experiment group in group comparison design trial. Experimental treatments were: 1) CTL (No enzyme) and 2 50NZ (Mixed enzyme with high cellulase at 50g/100kg.feed). The digestibility study showed that Exogenous enzyme increased (P<0.05) dry matter and crude protein digestibility of treated lamb compared to those of control. A similar trend (P=0.11) was observed for the NDF digestibility. Mean values for dry matter digestibility were 57.86 and 69.83% and for protein digestibility were 64.76 and 73.38%, for CTL and 50NZ, respectively). The N intake was similar among treatment, averaging 22.57g/head/day. Percent N retained of 50 NZ treated lambs was higher (P<.05) than those of CTL group (mean value were 47.74 and 59.07 for CTC and 50NZ, respectively). Feed efficiency or feed conversion ratio was numerically improved for enzyme-treated groups. Overall, the results of this study provide evidence that mixed cellulase enzyme can be used to improver performance of lambs as compare to non-enzyme diet.

  4. Microbial Cellulases Immobilized in/on Porous Supports

    Directory of Open Access Journals (Sweden)

    Monica Dragomirescu

    2010-05-01

    Full Text Available Biodegradation of cellulose by enzymatic hydrolysis using cellulases has an important value in biotechnology and the immobilization of enzyme on inorganic materials is very useful in practical applications. Enzymatic preparations with cellulase and cellobiase activities from Trichoderma viride were liophylized from the culture medium and immobilized in/on porous matrices. The methods used for immobilization were physical adsorption on ceramics and entrapment in glass sol-gel matrices using as alkoxysilane precursors tetraethoxysilane (TEOS and tetramethoxysilane (TMOS. The immobilization efficiency of the solid enzymatic preparations was about 60%. The immobilized enzymatic preparations were used for hydrolysis of carboxymethyl cellulose (CMC and cellobiose at different temperature and pH values. The resulted immobilized enzymes had the same optimum pH of 4.0 in the case of cellobiase substrate and a shifted optimum pH towards the less acid side (pH 5.0 in the hydrolysis of CMC. The optimum temperature of entrapped enzyme against CMC was shifted to a lower temperature (40°C in comparison with the native one (60°C.

  5. 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. PMID:23274988

  6. Rheology of carboxymethyl cellulose solutions treated with cellulases

    Directory of Open Access Journals (Sweden)

    Lee, J. M.

    2007-02-01

    Full Text Available The effect of cellulase treatments on the rheology of carboxymethyl cellulose (CMC solutions was studied using a rotational viscometer. The rheological behaviors of CMC solutions of different molecular mass and degrees of substitution where studied as a function of time after various treatments. These solutions were subjected to active and heat-denatured cellulase, a cationic polyelectrolyte (C-PAM, as well as different shear rates. A complex protein-polymer interaction was observed, leading to a potential error source in the measurement of enzymatic activity by changes in the intrinsic viscosity. The interaction was termed a polymeric effect and defined as a reduction in viscosity of the substrate solution without significant formation of reducing sugars from enzymatic hydrolysis. The cause of the reduction in viscosity appears to be related to the interaction between the enzymes as amphipathic particles and the soluble CMC. Thus, the polymeric effect may cause a considerable experimental error in the measurement of enzymatic activity by viscometric methods.

  7. Cellulase-lignin interactions in the enzymatic hydrolysis of lignocellulose

    Energy Technology Data Exchange (ETDEWEB)

    Rahikainen, J.

    2013-11-01

    Today, the production of transportation fuels and chemicals is heavily dependent on fossil carbon sources, such as oil and natural gas. Their limited availability and the environmental concerns arising from their use have driven the search for renewable alternatives. Lignocellulosic plant biomass is the most abundant, but currently underutilised, renewable carbon-rich resource for fuel and chemical production. Enzymatic degradation of structural polysaccharides in lignocellulose produces soluble carbohydrates that serve as ideal precursors for the production of a vast amount of different chemical compounds. The difficulty in full exploitation of lignocellulose for fuel and chemical production lies in the complex and recalcitrant structure of the raw material. Lignocellulose is mainly composed of structural polysaccharides, cellulose and hemicellulose, but also of lignin, which is an aromatic polymer. Enzymatic degradation of cellulose and hemicellulose is restricted by several substrate- and enzyme-related factors, among which lignin is considered as one of the most problematic issues. Lignin restricts the action of hydrolytic enzymes and enzyme binding onto lignin has been identified as a major inhibitory mechanism preventing efficient hydrolysis of lignocellulosic feedstocks. In this thesis, the interactions between cellulase enzymes and lignin-rich compounds were studied in detail and the findings reported in this work have the potential to help in controlling the harmful cellulase-lignin interactions, and thus improve the biochemical processing route from lignocellulose to fuels and chemicals.

  8. A single molecule study of cellulase hydrolysis of crystalline cellulose

    Science.gov (United States)

    Liu, Yu-San; Luo, Yonghua; Baker, John O.; Zeng, Yining; Himmel, Michael E.; Smith, Steve; Ding, Shi-You

    2010-02-01

    Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate β-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.

  9. 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.

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    Qing Song

    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.

  12. 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.

  13. 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

  14. Developing improved MD codes for understanding processive cellulases

    International Nuclear Information System (INIS)

    The mechanism of action of cellulose-degrading enzymes is illuminated through a multidisciplinary collaboration that uses molecular dynamics (MD) simulations and expands the capabilities of MD codes to allow simulations of enzymes and substrates on petascale computational facilities. There is a class of glycoside hydrolase enzymes called cellulases that are thought to decrystallize and processively depolymerize cellulose using biochemical processes that are largely not understood. Understanding the mechanisms involved and improving the efficiency of this hydrolysis process through computational models and protein engineering presents a compelling grand challenge. A detailed understanding of cellulose structure, dynamics and enzyme function at the molecular level is required to direct protein engineers to the right modifications or to understand if natural thermodynamic or kinetic limits are in play. Much can be learned about processivity by conducting carefully designed molecular dynamics (MD) simulations of the binding and catalytic domains of cellulases with various substrate configurations, solvation models and thermodynamic protocols. Most of these numerical experiments, however, will require significant modification of existing code and algorithms in order to efficiently use current (terascale) and future (petascale) hardware to the degree of parallelism necessary to simulate a system of the size proposed here. This work will develop MD codes that can efficiently use terascale and petascale systems, not just for simple classical MD simulations, but also for more advanced methods, including umbrella sampling with complex restraints and reaction coordinates, transition path sampling, steered molecular dynamics, and quantum mechanical/molecular mechanical simulations of systems the size of cellulose degrading enzymes acting on cellulose

  15. 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

  16. 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.

  17. 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.

  18. Exogenous Cellulase Contributes to Mycoherbicidal Activity of Fusarium arthrosporioides on Orobanche aegyptiaca

    Directory of Open Access Journals (Sweden)

    Olubukola O. Babalola

    2010-01-01

    Full Text Available This paper investigates an association between the tubercle size of Orobanche aegyptiaca, tubercle death, and days to tubercle death in relation to cellulase-assisted mycoherbicide. Fusarium arthrosporioides killed 56% of tubercles when applied with cellulase compared to 35% when no cellulase was added. Death was inversely correlated with days over the two fungal treatment types. O. aegyptiaca tubercle size significantly correlated with the two other infection parameters studied. For F. arthrosporioides, only 9% (2 of the variation in days to death was explained by variation in tubercle size, whereas with cellulase it reaches 14%. In this study, mycelia of F. arthrosporioides did not show apparent damage to the tomato roots.

  19. Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

    Energy Technology Data Exchange (ETDEWEB)

    Acebal, C.; Castillon, M.P.; Estrada, P.; Mata, I.; Costa, E.; Aguado, J.; Romero, D.; Jimenez, F.

    1986-06-01

    Trichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.

  20. Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting celluloytic complexes

    NARCIS (Netherlands)

    Mingardon, F.; Chanal, A.; Lopez Contreras, A.M.; Dray, C.; Bayer, E.A.; Fierobe, H.P.

    2007-01-01

    Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostrid

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

    DEFF Research Database (Denmark)

    Alftrén, Johan

    and hemicellulose) can be enzymatically hydrolyzed into monomers and subsequently fermented to bioethanol or another desirable biochemical. One of the main costs and obstacles in making the bioprocess economically viable is the costs of cellulases which catalyze the hydrolysis of cellulose into...... lignocellulosic 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...... immobilized enzyme and separate the magnetic particles from residual cellulose in pilot scale, before using them in 3 subsequent 20 L hydrolysis cycles. The results in this thesis thus demonstrate that cheap magnetic immobilized cellulases can be used for repeated hydrolysis cycles at pilot scale and...

  2. Metabolic engineering strategies for the improvement of cellulase production by Hypocrea jecorina

    Directory of Open Access Journals (Sweden)

    Schmoll Monika

    2009-09-01

    Full Text Available Abstract Hypocrea jecorina (= Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerise plant biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. Cellulases are formed adaptively, and several positive (XYR1, ACE2, HAP2/3/5 and negative (ACE1, CRE1 components involved in this regulation are now known. In addition, its complete genome sequence has been recently published, thus making the organism susceptible to targeted improvement by metabolic engineering. In this review, we summarise current knowledge about how cellulase biosynthesis is regulated, and outline recent approaches and suitable strategies for facilitating the targeted improvement of cellulase production by genetic engineering.

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

    OpenAIRE

    C. W. Kan; Wong, W. Y.; Song, L. J.; M. C. Law

    2013-01-01

    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...

  4. Character of Cellulase Activity in the Guts of Flagellate-Free Termites with Different Feeding Habits

    OpenAIRE

    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...

  5. Adsorption of cellulases onto sugar beet shreds and modeling of the experimental data

    OpenAIRE

    Ivetić Darjana Ž.; Omorjan Radovan P.; Antov Mirjana G.

    2014-01-01

    This study investigated the adsorption of cellulases onto sugar beet shreds. The experiments were carried out using untreated, as well as dried and not dried dilute acid and steam pretreated sugar beet shreds at different initial enzyme loads. Both dilute acid and steam pretreatment were beneficial in respect of cellulases adsorption providing 8 and 9 times higher amounts of adsorbed proteins, respectively, in comparison to the results obtained with the unt...

  6. 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. PMID:19997787

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

    OpenAIRE

    Karin Vega; Gretty K. Villena; Sarmiento, Victor H.; Yvette Ludeña; Nadia Vera; Marcel Gutiérrez-Correa

    2012-01-01

    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 ap...

  8. Efficient leaching of cellulases produced by Trichoderma harzianum in solid state fermentation

    OpenAIRE

    Roussos, Sevastianos; Raimbault, Maurice; Saucedo-Castaneda, G.; Lonsane, B.K.

    1992-01-01

    Recovery of cellulases from solid state culture of #Trichoderma harzianum$ was efficiently achieved by hydraulic pressing. Pressing of fermented solids yielded carboxymethyl-cellulase (CMCase) extraction efficiency of 71% and a ratio of leachate to fermented solids of 0.58 (v/w). Addition of water to pressed solids and second pressing improved the efficiency (95%) with simultaneous increase in the ratio to 1.16 (v/w). The overall extraction of filter paper activity was lower (85%) than that o...

  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. Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum

    OpenAIRE

    Zhonghai Li; Guangshan Yao; Ruimei Wu; Liwei Gao; Qinbiao Kan; Meng Liu; Piao Yang; Guodong Liu; Yuqi Qin; Xin Song; Yaohua Zhong; Xu Fang; Yinbo Qu

    2015-01-01

    Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these...

  11. 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

    In the present study whole cellulase mixtures were covalently immobilized on non-porous magnetic particles to enable enzyme reuse. It was shown that CellicCTec2 immobilized on magnetic particles activated with cyanuric chloride gave the highest bead activity measured by mass of reducing sugar pro...... immobilized cellulases and the results confirmed the potential of hydrolyzing real lignocellulosic substrate and enabling enzyme reuse for biorefinery and biomass derived ethanol applications....

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

    OpenAIRE

    Md. Z. Alam; Nurdina Muhammad; Mohd E. Mahmat

    2005-01-01

    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 ...

  13. Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions

    OpenAIRE

    Jourdier, Etienne; Cohen, Céline; Poughon, Laurent; Larroche, Christian; Monot, Frédéric; Ben Chaabane, Fadhel

    2013-01-01

    Background: On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions. Results: The effect of sugar mixtu...

  14. A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background

    OpenAIRE

    Uzbaş, Fatma; Uzbas, Fatma; Sezerman, Uğur; Sezerman, Ugur; Hartl, Lukas; Kubicek, Christian P.; Seiboth, Bernhard

    2012-01-01

    Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, ...

  15. Effects of gamma-ray irradiation on cellulase secretion of Trichoderma reesei

    International Nuclear Information System (INIS)

    Trichoderma reesei was irradiated with gamma rays to investigate the effects of different dosages on cellulase production. Doses above 0.7 kGy induced cell lysis. Cell growth began to be obstructed at 2.0 kGy. As a result, the cells irradiated at 2.0 kGy secreted 1.8 times as much cellulase as the untreated cells

  16. Effects of different dietary cation-anion balance and cellulase on blood biochemical indexes in sheep

    International Nuclear Information System (INIS)

    Five sheep with average live weight of 38 kg were selected and fitted with permanent ruminal fistulae for the study of the influence of DCAB and cellulase on blood biochemical indexes in sheep. With the design of 5 x 5 Latin square, the goats were undergone four groups of treatments. As sheep in control group were only fed with basic feed, other treatments were fed with basic feed and additives. Those additives were: treatment 1 with cellulase (0.2% of dietary), treatment 2 with DCAB1 (20 mEq/100g DM), treatment 3 with cellulase (0.2% of dietary) and DCAB1 (20 mEq/100g DM) and treatment 4 with cellulase (0.2% of dietary) and DCAB2 (40 mEq/100 g DM). The results show that different DCAB and cellulase had no apparent influence on the concentrations of T3 and T4 (P>0.05), but they had significant influence on the concentration of insulin (P0.05). The study indicated that addition of 0.2% cellulase to dietary while adjusting DCAB to 0.2-40 mEq/100 g DM respectively did not destroy tissues and organs of sheep. (authors)

  17. 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.

  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. Synergistic effect of cellulase and xylanase during hydrolysis of natural lignocellulosic substrates.

    Science.gov (United States)

    Song, Hui-Ting; Gao, Yuan; Yang, Yi-Min; Xiao, Wen-Jing; Liu, Shi-Hui; Xia, Wu-Cheng; Liu, Zi-Lu; Yi, Li; Jiang, Zheng-Bing

    2016-11-01

    Synergistic combination of cellulase and xylanase has been performed on pre-treated substrates in many previous studies, while few on natural substrates. In this study, three unpretreated lignocellulosic substrates were studied, including corncob, corn stover, and rice straw. The results indicated that when the mixed cellulase and xylanase were applied, reducing sugar concentrations were calculated as 19.53, 15.56, and 17.35mg/ml, respectively, based on the 3,5 dinitrosalicylic acid (DNS) method. Compared to the treatment with only cellulose, the hydrolysis yields caused by mixed cellulase and xylanase were improved by 133%, 164%, and 545%, respectively. In addition, the conversion yield of corncob, corn stover, and rice straw by cellulase-xylanase co-treatment reached 43.9%, 48.5%, and 40.2%, respectively, based on HPLC analysis, which confirmed the synergistic effect of cellulase-xylanase that was much higher than either of the single enzyme treatment. The substrate morphology was also evaluated to explore the synergistic mechanism of cellulase-xylanase. PMID:27560367

  20. A Newly Isolated Penicillium oxalicum 16 Cellulase with High Efficient Synergism and High Tolerance of Monosaccharide.

    Science.gov (United States)

    Zhao, Xi-Hua; Wang, Wei; Tong, Bin; Zhang, Su-Ping; Wei, Dong-Zhi

    2016-01-01

    Compared to Trichoderma reesei RUT-C30 cellulase (Trcel), Penicillium oxalicum 16 cellulase (P16cel) from the fermentation supernatant produced a 2-fold higher glucose yield when degrading microcrystalline cellulose (MCC), possessed a 10-fold higher β-glucosidase (BGL) activity, but obtained somewhat lower other cellulase component activities. The optimal temperature and pH of β-1,4-endoglucanase, cellobiohydrolase, and filter paperase from P16cel were 50-60 °C and 4-5, respectively, but those of BGL reached 70 °C and 5. The cellulase cocktail of P16cel and Trcel had a high synergism when solubilizing MCC and generated 1.7-fold and 6.2-fold higher glucose yields than P16cel and Trcel at the same filter paperase loading, respectively. Additional low concentration of fructose enhanced the glucose yield during enzymatic hydrolysis of MCC; however, additional high concentration of monosaccharide (especially glucose) reduced cellulase activities and gave a stronger monosaccharide inhibition on Trcel. These results indicate that P16cel is a more excellent cellulase than Trcel. PMID:26410224

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  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. Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum.

    Science.gov (United States)

    Li, Zhonghai; Yao, Guangshan; Wu, Ruimei; Gao, Liwei; Kan, Qinbiao; Liu, Meng; Yang, Piao; Liu, Guodong; Qin, Yuqi; Song, Xin; Zhong, Yaohua; Fang, Xu; Qu, Yinbo

    2015-09-01

    Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular β-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a "seesaw model" in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors. PMID:26360497

  5. 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.

  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. Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance.

    Science.gov (United States)

    Mhlongo, Sizwe I; den Haan, Riaan; Viljoen-Bloom, Marinda; van Zyl, Willem H

    2015-12-01

    In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme-inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility. PMID:26453468

  8. Ruminococcus flavefaciens 007C cellulosomes and cellulase consortium

    Directory of Open Access Journals (Sweden)

    Maša VODOVNIK

    2015-11-01

    Full Text Available Ruminococcus flavefaciens is among the most important cellulolytic bacterial species in rumen and gastrointestinal tract of monogastric herbivorous animals. Its efficiency in degradation of (hemicellulosic substrates is associated with the production of remarkably intricate extracellular multienzyme complexes, named cellulosomes. In the present work we investigated the cellulolytic system of 007C. The bioinformatic analysis of the draft genome sequence revealed identical organization of sca gene cluster as has previously been found in four other strains of R. flavefaciens. The cluster consists of five genes in the following order: scaC-scaA-scaB-cttA-scaE. The cellulases of R. flavefaciens 007C belong to four families of glycoside hydrolases, namely GH48, GH44, GH9 in GH5. Majority of these enzymes are putative endoglucanases, belonging to families GH5 and GH9, whereas only one gene encoding GH44 and GH48 was found. Apart from catalytic domains, most of these proteins also contain dockerins – signature sequences, which indicate their attachement to cellulosomes. On the other hand, carbohydrate-binding modules were only found coupled to GH9 catalytic domains. Zymogram analysis showed that larger endoglucanases were mostly constitutively expressed, wheras smaller enzymes were only detected in later phases of Avicel-grown cultures.

  9. Cellulase production by Aspergillus niger in biofilm, solid-state, and submerged fermentations.

    Science.gov (United States)

    Gamarra, Norma N; Villena, Gretty K; Gutiérrez-Correa, Marcel

    2010-06-01

    Cellulase production by Aspergillus niger was compared in three different culture systems: biofilm, solid-state, and submerged fermentation. Biofilm and solid-state fermentations were carried out on perlite as inert support, and lactose was used as a carbon source in the three culture systems. In cryo-scanning electron microscopy, biofilm and solid-state cultures gave similar morphological patterns and confirmed that both spore first attachment and hyphal adhered growth are helped by the production of an adhesive extracellular matrix. Biofilm cultures produced higher cellulase activities than those in submerged and solid-state cultures (1,768, 1,165, and 1,174 U l(-1), respectively). Although biofilm cultures grew less than the other cultures, they produced significantly higher cellulase yields (370, 212, and 217 U g(-1) lactose, respectively) and volumetric productivities (24, 16, and 16 U l(-1) h(-1), respectively). Likewise, endoglucanase and xylanase activities were higher in biofilm cultures. Under the conditions tested, it seems that fungal attached growth on perlite may favor better enzyme production. Biofilms are efficient systems for cellulase production and may replace solid-state fermentation. Biofilm fermentation holds promise for further optimization and development. The results of this work reveal that fungal biofilms may be used for the commercial production of cellulase employing the technology developed for submerged fermentation at high cell densities. PMID:20354693

  10. 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.

  11. Optimization Studies on Cellulase Production from Bacillus Anthracis and Ochrobactrum Anthropic (YZ1 Isolated from Soil

    Directory of Open Access Journals (Sweden)

    Mohammad Badrud Duza

    2015-06-01

    Full Text Available The present study was carried out to demonstrate the optimization of growth conditions of bacteria with high cellulase activity. Cellulose degrading bacteria were isolated from soil samples collected from different areas of Guntur district, A.P. The bacteria were isolated using serial dilution and pour plate methods. The isolated bacteria were identified by morphological, biochemical and molecular procedures. The isolated bacterial species were screened for cellulase production in sub-merged fermentation process. The two tested bacterial species showed maximum yield for cellulase production. These two bacteria were identified as Bacillus anthracis and Ochrobactrum anthropi (YZ1. Supplementation of glucose, peptone, tyrosine and EDTA to the fermentation medium is favoured enzyme secretion. The optimum pH and temperature for the activity of crude enzyme was 8 and 45°C, respectively for Ochrobactrum anthropi (YZ1 while for Bacillus anthracis, it was 8 and 4°C, respectively.14% of inoculum level and 96 h of incubation period showed the maximum yield by both the species bacteria for cellulase production. The results of present study indicated that favorable fermentation conditions and the selection of a suitable growth medium played a key role in the production of cellulase from newly isolated Bacillus anthracis and Ochrobactrum anthropi (YZ1.

  12. Efficient production of cellulase in the culture of Acremonium cellulolyticus using untreated waste paper sludge.

    Science.gov (United States)

    Prasetyo, Joni; Zhu, Jing; Kato, Tatsuya; Park, Enoch Y

    2011-01-01

    Cellulase was produced by Acremonium cellulolyticus using untreated waste paper sludge (PS) as the carbon source. The clay present in PS did not show any inhibitory effect on cellulase production but did alter the pH during fermentation. On the flask scale, the maleate buffer concentration and pH were key factors that affected the efficiency of cellulase production from PS cellulose. Optimum cellulase production in a 3-L fermentor of working volume 1.5 L was achieved by controlling the pH value at 6.0 using 2 M NaOH and 2 M maleic acid, and the productivity reached 8.18 FPU/mL. When 40.89 g/L PS cellulose, 2.2 g/L (NH(4) )(2) SO(4) , and 4.4 g/L urea were added to a 48-h culture, the cellulase activity was 9.31 FPU/mL at the flask scale and 10.96 FPU/mL in the 3-L fermentor. These values are ∼80% of those obtained when pure cellulose is used as the carbon source. The method developed here presents a new route for the utilization of PS. PMID:21312360

  13. Production, Optimization, and Characterization of Organic Solvent Tolerant Cellulases from a Lignocellulosic Waste-Degrading Actinobacterium, Promicromonospora sp. VP111.

    Science.gov (United States)

    Thomas, Lebin; Ram, Hari; Kumar, Alok; Singh, Ved Pal

    2016-07-01

    High costs of natural cellulose utilization and cellulase production are an industrial challenge. In view of this, an isolated soil actinobacterium identified as Promicromonospora sp. VP111 showed potential for production of major cellulases (CMCase, FPase, and β-glucosidase) utilizing untreated agricultural lignocellulosic wastes. Extensive disintegration of microcrystalline cellulose and adherence on it during fermentation divulged true cellulolytic efficiency of the strain. Conventional optimization resulted in increased cellulase yield in a cost-effective medium, and the central composite design (CCD) analysis revealed cellulase production to be limited by cellulose and ammonium sulfate. Cellulase activities were enhanced by Co(+2) (1 mM) and retained up to 60 °C and pH 9.0, indicating thermo-alkaline tolerance. Cellulases showed stability in organic solvents (25 % v/v) with log P ow  ≥ 1.24. Untreated wheat straw during submerged fermentation was particularly degraded and yielded about twofold higher levels of cellulases than with commercial cellulose (Na-CMC and avicel) which is especially economical. Thus, this is the first detailed report on cellulases from an efficient strain of Promicromonospora that was non-hemolytic, alkali-halotolerant, antibiotic (erythromycin, kanamycin, rifampicin, cefaclor, ceftazidime) resistant, multiple heavy metal (Mo(+6) = W(+6) > Pb(+2) > Mn(+2) > Cr(+3) > Sn(+2)), and organic solvent (n-hexane, isooctane) tolerant, which is industrially and environmentally valuable. PMID:26956574

  14. 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.

    2016-08-01

    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 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.

  15. Recyclable thermoresponsive polymer-cellulase bioconjugates for biomass depolymerization.

    Science.gov (United States)

    Mackenzie, Katherine J; Francis, Matthew B

    2013-01-01

    -derived cellulases or to the separation of multiple species using polymers with different recovery temperatures. PMID:23270527

  16. 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. PMID:27470141

  17. Structural and functional analysis of a bacterial cellulase by proteolysis.

    Science.gov (United States)

    Gilkes, N R; Kilburn, D G; Miller, R C; Warren, R A

    1989-10-25

    CenA is an endo-beta 1,4-glucanase from the cellulolytic bacterium Cellulomonas fimi. It is a bifunctional enzyme comprising an amino-terminal cellulose-binding domain and a carboxyl-terminal catalytic domain joined by a short sequence of prolyl and threonyl residues (the Pro-Thr box). Additional structural and functional information was revealed by a detailed analysis of the products generated by proteolytic cleavage of a nonglycosylated form of CenA. An extracellular C. fimi protease attacked nonglycosylated CenA at the junctions between the Pro-Thr box and the two functional domains. A stable "core" peptide (p30), corresponding to the catalytic domain, remained after extensive proteolysis. p30 was resistant to further attack even in the presence of 2-mercaptoethanol plus urea or dithiothreitol, but treatment in the presence of sodium dodecyl sulfate allowed complete fragmentation to small peptides. Stable peptides, identical, or closely related to p30, were generated by alpha-chymotrypsin or papain. These results indicated that the catalytic domain adopts a tightly folded conformation affording protection from proteolytic attack. In contrast, the cellulose-binding domain showed a relatively loose conformation. Progressive proteolytic truncation from the amino terminus was apparent during incubation with alpha-chymotrypsin or papain, or with C. fimi protease under reducing conditions. Affinity for cellulose was retained by products missing up to 64 amino-terminal amino acids. The remaining carboxyl-proximal region of the cellulose-binding domain with affinity (47 amino acids) contained sequences highly conserved in analogous domains from other bacterial endo-beta 1,4-glucanases. By analogy with other systems, the properties of the Pro-Thr box are consistent with an elongated conformation. The results of this investigation suggest that CenA has a tertiary structure which resembles that of certain fungal cellulases. PMID:2681184

  18. 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

    studies revealed that two of the cellulases were acting as cellobiohydrolases by being active on only microcrystalline cellulose (Avicel). Three of the cellulases were active on both Avicel and carboxymethyl cellulose indicating endoglucanase activity. Two of these showed furthermore mannanase activity......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...... by being able to hydrolyze galactomannan (locust bean gum). Adsorption studies revealed that the smaller of the two enzymes was not able to bind to cellulose. Similarity in molecular mass, pI and hydrolytic properties suggested that these two enzymes were identical, but the smaller one was lacking...

  19. Deciphering the molecular mechanisms behind cellulase production in Trichoderma reesei, the hyper-cellulolytic filamentous fungus.

    Science.gov (United States)

    Shida, Yosuke; Furukawa, Takanori; Ogasawara, Wataru

    2016-09-01

    The filamentous fungus Trichoderma reesei is a potent cellulase producer and the best-studied cellulolytic fungus. A lot of investigations not only on glycoside hydrolases produced by T. reesei, but also on the machinery controlling gene expression of these enzyme have made this fungus a model organism for cellulolytic fungi. We have investigated the T. reesei strain including mutants developed in Japan in detail to understand the molecular mechanisms that control the cellulase gene expression, the biochemical and morphological aspects that could favor this phenotype, and have attempted to generate novel strains that may be appropriate for industrial use. Subsequently, we developed recombinant strains by combination of these insights and the heterologous-efficient saccharifing enzymes. Resulting enzyme preparations were highly effective for saccharification of various biomass. In this review, we present some of the salient findings from the recent biochemical, morphological, and molecular analyses of this remarkable cellulase hyper-producing fungus. PMID:27075508

  20. 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.

  1. Protection of Aspergillus niger cellulases by urea during growth on glucose or glycerol supplemented media.

    Science.gov (United States)

    Gokhale, D V; Patil, S G; Bastawde, K B

    1992-10-01

    The cellulase enzymes of Aspergillus niger were found to undergo catabolite repression in the presence of glucose and glycerol accompanied by sudden drop in pH of the fermentation medium below 2.0. This sudden drop in pH caused inactivation of cellulolytic enzymes produced by Aspergillus niger. The supplementation of nitrogen sources, especially urea, protects A. niger cellulases from inactivation caused by a sudden drop in pH, since urea helped to maintain the pH of the fermentation medium between 3.5 and 4.5. The role of urea in the protection of cellulase was more prominent when it was used in combination with glycerol (5%). PMID:1288413

  2. 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.

  3. Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei.

    Science.gov (United States)

    Ilmén, M; Saloheimo, A; Onnela, M L; Penttilä, M E

    1997-04-01

    Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression. PMID:9097427

  4. Evaluation of ten wild nigerian mushrooms for amylase and cellulase activities.

    Science.gov (United States)

    Jonathan, Segun Gbolagade; Adeoyo, Olusegun Richard

    2011-06-01

    Amylases and cellulases are important enzymes that can be utilized for various biological activities. Ten different wild Nigerian mushrooms (Agaricus blazei, Agaricus sp., Corilopsis occidentalis, Coriolus versicolor, Termitomyces clypeatus, Termitomyces globulus, Pleurotus tuber-regium, Podoscypha bolleana, Pogonomyces hydnoides, and Nothopanus hygrophanus) were assayed for production of these secondary metabolites. The results revealed that most of the tested wild fungi demonstrated very good amylase and cellulase activities. With the incorporation of carboxymethyl-cellulose (a carbon source) into the culture medium, Agaricus blazei had the highest amylolytic activity of 0.60 unit/mL (at 25℃, pH 6.8). This was followed in order by P. tuber-regium and Agaricus sp. with 0.42 and 0.39 unit/mL, respectively (p ≤ 0.05). Maltose and sucrose supplementation into the submerged liquid medium made N. hygrophanus and P. hydnoides to exhibit very low amylase activities of 0.09 and 0.11 unit/mL, respectively. Introducing peptone (an organic nitrogen source) into the basal medium enhanced the ability of C. versicolor to produce a cellulase value of 0.74 unit/mL. Other organic nitrogen sources that supported good cellulase activities were yeast extract and urea. Sodium nitrate (inorganic nitrogen source) generally inhibited cellulase production in all mushrooms. The best carbon source was carboxymethyl-cellulose, which promoted very high cellulase activity of 0.67 unit/mL in C. versicolor, which was followed in order by P. tuber-regium, T. chypeatus, and C. occidentalis (p ≤ 0.05). Sucrose was the poorest carbon compound, supporting the lowest values of 0.01, 0.01, and 0.14 unit/mL in P. hydnoides, A. blazei, and Agaricus sp., respectively. PMID:22783085

  5. CELLULASES FROM PENICILLIUM JANTHINELLUM MUTANTS: SOLID-STATE PRODUCTION AND THEIR STABILITY IN IONIC LIQUIDS

    OpenAIRE

    Mukund G. Adsul; Asawari P. Terwadkar; Anjani J. Varma; Digambar V. Gokhale

    2009-01-01

    The cellulase production by P. janthinellum mutants on lignocellulosic material such as cellulose or steam exploded bagasse (SEB) in combination with wheat bran was studied in solid state fermentation (SSF). One of the mutants, EU2D21, produced the highest levels of endoglucanase (3710 IU g-1 carbon source) and β-glucosidase (155 IU g-1 carbon source). Ionic liquids are so-called green solvents that have become attractive for biocatalysis. Stability of mutant cellulases was tested in 10-50% o...

  6. Improvement of Fungal Cellulase Production by Mutation and Optimization of Solid State Fermentation

    OpenAIRE

    Vu, Van Hanh; Pham, Tuan Anh; Kim, Keun

    2011-01-01

    Spores of Aspergillus sp. SU14 were treated repeatedly and sequentially with Co60 γ-rays, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine. One selected mutant strain, Aspergillus sp. SU14-M15, produced cellulase in a yield 2.2-fold exceeding that of the wild type. Optimal conditions for the production of cellulase by the mutant fungal strain using solid-state fermentation were examined. The medium consisted of wheat-bran supplemented with 1% (w/w) urea or NH4Cl, 1% (w/w) ric...

  7. Coexpression of cellulases in Pichia pastoris as a self-processing protein fusion

    OpenAIRE

    de Amorim Araújo, Juliana; Ferreira, Túlio César; Rubini, Marciano Régis; Duran, Ana Gilhema Gomez; De Marco, Janice Lisboa; Moraes, Lidia Maria Pepe de; Torres, Fernando Araripe Gonçalves

    2015-01-01

    The term cellulase refers to any component of the enzymatic complex produced by some fungi, bacteria and protozoans which act serially or synergistically to catalyze the cleavage of cellulosic materials. Cellulases have been widely used in many industrial applications ranging from food industry to the production of second generation ethanol. In an effort to develop new strategies to minimize the costs of enzyme production we describe the development of a Pichia pastoris strain able to coprodu...

  8. Study of thermal and chemical effects on cellulase enzymes: Viscosity measurements

    Energy Technology Data Exchange (ETDEWEB)

    Ghaouar, N., E-mail: naoufel-ghaouar@lycos.co [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Institut National des Sciences Appliquees et de Technologie, INSAT, Centre Urbain Nord, BP. 676, Tunis (Tunisia); Aschi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Belbahri, L. [Agronomy Department, School of Engineering of Lullier, University of Applied Sciences of Western Switzerland, 150, Route de Presinge, 1254 Jussy (Switzerland); Trabelsi, S.; Gharbi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia)

    2009-11-15

    The behaviour of cellulase enzymes in phosphate saline buffer has been studied over a wide range of temperatures and enzyme concentrations by using viscosity measurements. To characterize the conformation change of cellulase versus temperature and chemical denaturants, such as guanidinium chloride (GdmCl) and urea, the information about the intrinsic viscosity and the hydrodynamic radius are necessary. The dependence of the intrinsic viscosity and the hydrodynamic radius in its random coil conformation on temperature and denaturant concentration were studied. Our results and discussions are limited to the dilute regime of concentration because of abnormalities in conformation observed in the very dilute regime due to the presence of capillary absorption effects.

  9. Study of thermal and chemical effects on cellulase enzymes: Viscosity measurements

    International Nuclear Information System (INIS)

    The behaviour of cellulase enzymes in phosphate saline buffer has been studied over a wide range of temperatures and enzyme concentrations by using viscosity measurements. To characterize the conformation change of cellulase versus temperature and chemical denaturants, such as guanidinium chloride (GdmCl) and urea, the information about the intrinsic viscosity and the hydrodynamic radius are necessary. The dependence of the intrinsic viscosity and the hydrodynamic radius in its random coil conformation on temperature and denaturant concentration were studied. Our results and discussions are limited to the dilute regime of concentration because of abnormalities in conformation observed in the very dilute regime due to the presence of capillary absorption effects.

  10. Study of thermal and chemical effects on cellulase enzymes: Viscosity measurements

    Science.gov (United States)

    Ghaouar, N.; Aschi, A.; Belbahri, L.; Trabelsi, S.; Gharbi, A.

    2009-11-01

    The behaviour of cellulase enzymes in phosphate saline buffer has been studied over a wide range of temperatures and enzyme concentrations by using viscosity measurements. To characterize the conformation change of cellulase versus temperature and chemical denaturants, such as guanidinium chloride (GdmCl) and urea, the information about the intrinsic viscosity and the hydrodynamic radius are necessary. The dependence of the intrinsic viscosity and the hydrodynamic radius in its random coil conformation on temperature and denaturant concentration were studied. Our results and discussions are limited to the dilute regime of concentration because of abnormalities in conformation observed in the very dilute regime due to the presence of capillary absorption effects.

  11. 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.

  12. Cellulases and hemicellulases from endophytic Acremonium species and its application on sugarcane bagasse hydrolysis

    Science.gov (United States)

    The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing L-arabinose, D-xylose, oat spelt xylan, sugarcane bagasse, or...

  13. Enhanced soluble expression of a thermostable cellulase from Clostridium thermocellum in Escherichia coli.

    Science.gov (United States)

    Peng, Jingjing; Wang, Weiwei; Jiang, Yuyao; Liu, Mingjie; Zhang, Hui; Shao, Weilan

    2011-12-01

    In this study, the cellulase gene celD from Clostridium thermocellum was cloned into expression vectors pET-20b(+) and pHsh. While high expression can be achieved by means of both these expression systems, only the pHsh expression system gives soluble proteins. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of CelD was increased from 4.1 ± 0.3 to 6.4 ± 0.4 U ml(-1) in LB medium. Recombinant CelD was purified by heat treatment followed by Ni-NTA affinity. The purified CelD exhibited the highest activity at pH 5.4 and 60°C, and retained more than 50% activity after incubation at 70°C for 1 h. The cellulase activity of CelD was significantly enhanced by Ca(2+) but inhibited by EDTA. The favorable properties of CelD offer the potential for genetic modification of strains for biomass degradation. Presently, one of the major bottlenecks for industrial cellulase users is the high cost of enzyme production. The high level expression of soluble enzymes from the pHsh expression system offers a novel approach for the production of cellulases to be used in various agro-industrial processes such as chemical, food and textile. PMID:21938523

  14. Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes▿

    OpenAIRE

    2007-01-01

    Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-bor...

  15. Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting celluloytic complexes

    OpenAIRE

    Mingardon, F.; Chanal, A.; Lopez Contreras, A.M.; Dray, C.; Bayer, E A; Fierobe, H P

    2007-01-01

    Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-bor...

  16. 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...

  17. 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

    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...

  18. 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...

  19. Evaluation of Ten Wild Nigerian Mushrooms for Amylase and Cellulase Activities

    OpenAIRE

    Jonathan, Segun Gbolagade; Adeoyo, Olusegun Richard

    2011-01-01

    Amylases and cellulases are important enzymes that can be utilized for various biological activities. Ten different wild Nigerian mushrooms (Agaricus blazei, Agaricus sp., Corilopsis occidentalis, Coriolus versicolor, Termitomyces clypeatus, Termitomyces globulus, Pleurotus tuber-regium, Podoscypha bolleana, Pogonomyces hydnoides, and Nothopanus hygrophanus) were assayed for production of these secondary metabolites. The results revealed that most of the tested wild fungi demonstrated very go...

  20. Plant carbohydrate binding module enhances activity of hybrid microbial cellulase enzyme

    Directory of Open Access Journals (Sweden)

    Caitlin Siobhan Byrt

    2012-11-01

    Full Text Available A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM to a synthetic glycosyl hydrolase (GH improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the carbohydrate-binding module (CBM of the tomato (Solanum lycopersicum SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using CMC, MUC and native crystalline cellulose assays. The presence of the CBM substantially improved the endo-glucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.

  1. 21 CFR 173.120 - Carbohydrase and cellulase derived from Aspergillus niger.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Carbohydrase and cellulase derived from Aspergillus niger. 173.120 Section 173.120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...) Aspergillus niger is classified as follows: Class, Deuteromycetes; order, Moniliales; family,...

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

    OpenAIRE

    Vinit; Ashutosh; Amit; Sonia

    2015-01-01

    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 - glu...

  4. Coexpression of cellulases in Pichia pastoris as a self-processing protein fusion.

    Science.gov (United States)

    de Amorim Araújo, Juliana; Ferreira, Túlio César; Rubini, Marciano Régis; Duran, Ana Gilhema Gomez; De Marco, Janice Lisboa; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

    2015-12-01

    The term cellulase refers to any component of the enzymatic complex produced by some fungi, bacteria and protozoans which act serially or synergistically to catalyze the cleavage of cellulosic materials. Cellulases have been widely used in many industrial applications ranging from food industry to the production of second generation ethanol. In an effort to develop new strategies to minimize the costs of enzyme production we describe the development of a Pichia pastoris strain able to coproduce two different cellulases. For that purpose the eglII (endoglucanase II) and cbhII (cellobiohydrolase II) genes from Trichoderma reesei were fused in-frame separated by the self-processing 2A peptide sequence from the foot-and-mouth disease virus. The protein fusion construct was placed under the control of the strong inducible AOX1 promoter. Analysis of culture supernatants from methanol-induced yeast transformants showed that the protein fusion was effectively processed. Enzymatic assay showed that the processed enzymes were fully functional with the same catalytic properties of the individual enzymes produced separately. Furthermore, when combined both enzymes acted synergistically on filter paper to produce cellobiose as the main end-product. Based on these results we propose that P. pastoris should be considered as an alternative platform for the production of cellulases at competitive costs. PMID:26698316

  5. The production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles.

    Science.gov (United States)

    Webb, C; Fukuda, H; Atkinson, B

    1986-01-01

    Continuous cellulase production by Trichoderma viride QM 9123, immobilized in 6 mm diameter, spherical, stainless steel biomass support particles, has been achieved using a medium containing glucose as the main carbon source. Experiments were carried out in a 10-L spouted bed fermentor. In this type of reactor-recycled broth is used to create a jet at the base of a bed of particles, causing the particles to spout and circulate. During the circulation, particles pass through a region of high shear near the jet inlet. This effectively prevents a buildup of excess biomass and thus enables steady-state conditions to be achieved during continuous operation. Continuous production of cellulase was achieved at significantly higher yield and productivity than in conventional systems. At a dilution rate of 0.15 h(-1) (nominal washout rate for freely suspended cells is 0.012 h(-1)), the yield of cellulase on glucose was 31% higher than that measured during batch operation, while the volumetric productivity (31.5 FPA U/L. h) was 53% greater than in the batch system. The specific cellulase productivity of the immobilized cells was more than 3 times that of freely suspended cells, showing that diffusional limitations can be beneficial. This offers significant opportunity for the further development of biomass support particles and associated bioreactors. PMID:18553840

  6. Some characteristics and partial purification of the Ganoderma lucidum cellulase system.

    Science.gov (United States)

    Jakucs, E; Rácz, I; Lásztity, D

    1994-01-01

    The extracellular cellulase system of the white-rotting basidiomycete Ganoderma lucidum was characterised while growing in cellulose-containing shaken liquid culture. The protein content of the culture filtrate reached its maximum after 36 days and cellulase activity at about 60 days. Different cellulase activities (endoglucanase, cellobiohydrolase and beta-glucosidase) were determined in a range of pH extending from 6 to 2. All of the three enzyme activities have at least three peaks between pH 6 and 2, although optimum points of the different enzymes are slightly different, showing that the enzyme complex consists of a number of enzymes and isozymes. Partial purification of the enzyme complex was carried out by DEAE-cellulose column chromatography. Using 0-3 M linear urea gradient, protein was eluted in one sharp peak corresponding mainly to beta-glucosidase activity. Comparing crude extracellular protein with that of purified by the column using PAGE indicated that this method was suitable for the separation and partial purification of one type of Ganoderma cellulases. PMID:7921848

  7. Genome Sequence and Annotation of Trichoderma parareesei, the Ancestor of the Cellulase Producer Trichoderma reesei

    OpenAIRE

    Yang, Dongqing; Pomraning, Kyle; Kopchinskiy, Alexey; Karimi Aghcheh, Razieh; Atanasova, Lea; Chenthamara, Komal; Baker, Scott E.; Zhang, Ruifu; Shen, Qirong; Freitag, Michael; Kubicek, Christian P.; Druzhinina, Irina S.

    2015-01-01

    The filamentous fungus Trichoderma parareesei is the asexually reproducing ancestor of Trichoderma reesei, the holomorphic industrial producer of cellulase and hemicellulase. Here, we present the genome sequence of the T. parareesei type strain CBS 125925, which contains genes for 9,318 proteins.

  8. Genome Sequence and Annotation of Trichoderma parareesei, the Ancestor of the Cellulase Producer Trichoderma reesei.

    Science.gov (United States)

    Yang, Dongqing; Pomraning, Kyle; Kopchinskiy, Alexey; Karimi Aghcheh, Razieh; Atanasova, Lea; Chenthamara, Komal; Baker, Scott E; Zhang, Ruifu; Shen, Qirong; Freitag, Michael; Kubicek, Christian P; Druzhinina, Irina S

    2015-01-01

    The filamentous fungus Trichoderma parareesei is the asexually reproducing ancestor of Trichoderma reesei, the holomorphic industrial producer of cellulase and hemicellulase. Here, we present the genome sequence of the T. parareesei type strain CBS 125925, which contains genes for 9,318 proteins. PMID:26272569

  9. 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. PMID:26481618

  10. 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.

  11. Molecular imprinting and immobilization of cellulase onto magnetic Fe3O4@SiO2 nanoparticles.

    Science.gov (United States)

    Li, Yue; Wang, Xiang-Yu; Zhang, Rui-Zhuo; Zhang, Xiao-Yun; Liu, Wei; Xu, Xi-Ming; Zhang, Ye-Wang

    2014-04-01

    Supermagnetic Fe3O4@SiO2 nanoparticles were molecular-imprinted prepared with cellulase as the template. The molecular imprinted nanoparticles were used as support to immobilization of cellulase. The transmission electron microscopy confirmed the core-shell structure and revealed that the size of the nanoparticles was around 10 nm. It was observed that cellulase was immobilized on the nanoparticles successfully from the Fourier transform infrared spectra. The adsorption of cellulase on the nanoparticles was specific and rapid. A high immobilization efficiency of 95% was achieved after the optimization. At 70 degrees C, the half-life of the immobilized cellulase was 3.3-fold of the free enzyme. Compared with the free enzyme, the immobilized cellulase has the same optimal pH, higher optimal temperature, better thermal stability and higher catalytic efficiency. The results strongly suggest that the immobilized cellulase on molecular imprinted Fe3O4@SiO2 has the potential applications in the production of bioethanol, paper and pulp industry, and pharmaceutical industry. PMID:24734713

  12. Horizontal gene transfer of microbial cellulases into nematode genomes is associated with functional assimilation and gene turnover

    Directory of Open Access Journals (Sweden)

    Dieterich Christoph

    2011-01-01

    Full Text Available Abstract Background Natural acquisition of novel genes from other organisms by horizontal or lateral gene transfer is well established for microorganisms. There is now growing evidence that horizontal gene transfer also plays important roles in the evolution of eukaryotes. Genome-sequencing and EST projects of plant and animal associated nematodes such as Brugia, Meloidogyne, Bursaphelenchus and Pristionchus indicate horizontal gene transfer as a key adaptation towards parasitism and pathogenicity. However, little is known about the functional activity and evolutionary longevity of genes acquired by horizontal gene transfer and the mechanisms favoring such processes. Results We examine the transfer of cellulase genes to the free-living and beetle-associated nematode Pristionchus pacificus, for which detailed phylogenetic knowledge is available, to address predictions by evolutionary theory for successful gene transfer. We used transcriptomics in seven Pristionchus species and three other related diplogastrid nematodes with a well-defined phylogenetic framework to study the evolution of ancestral cellulase genes acquired by horizontal gene transfer. We performed intra-species, inter-species and inter-genic analysis by comparing the transcriptomes of these ten species and tested for cellulase activity in each species. Species with cellulase genes in their transcriptome always exhibited cellulase activity indicating functional integration into the host's genome and biology. The phylogenetic profile of cellulase genes was congruent with the species phylogeny demonstrating gene longevity. Cellulase genes show notable turnover with elevated birth and death rates. Comparison by sequencing of three selected cellulase genes in 24 natural isolates of Pristionchus pacificus suggests these high evolutionary dynamics to be associated with copy number variations and positive selection. Conclusion We could demonstrate functional integration of acquired

  13. Production of xylanases and cellulases by aspergillus fumigatus ms16 using crude lignocellulosic substrates

    International Nuclear Information System (INIS)

    Xylanolytic and cellulolytic potential of a soil isolate, Aspergillus fumigatus (MS16) was studied by growing it on a variety of lignocellulosics, purified cellulose and xylan supplemented media. It was noted that carboxymethyl cellulose, salicin and xylan induce the -glucosidase and xylanase, respectively production of endoglucanase. The study revealed that Aspergillus fumigatus (MS16) co-secretes xylanase and cellulase in the presence of xylan; the ratio of the two enzymes was influenced by the initial pH of the medium. The maximum titers of xylanase and cellulase were noted at initial pH of 5.0. Relatively higher titers of both the enzymes were obtained when the fungus was cultivated at 35 degree C. Whereas, cellulase production was not detected when the fungus was cultivated at 40 degree C. The volumetric productivity (Qp) of xylanase was much higher than cellulases. The organism produced 2-3 folds higher titers of xylanase when grown on lignocellulosic materials in submerged cultivation than under solid-state cultivation, suggesting a different pattern of enzyme production in presence and in absence of free water. The partial characterization of enzymes showed that xylanase from this organism has -glucosidase. The higher melting temperature than endoglucanase and optimum temperature for activity was higher for xylanases than cellulases, whereas the optimum pH differed slightly i.e. in the range of 4.0-5.0. Enzyme preparation from this organism was loaded on some crude substrates and it showed that the enzyme preparation can be used to hydrolyze a variety of vegetable and agricultural waste materials. (author)

  14. 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.

  15. 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.

  16. Evaluating leachate recirculation with cellulase addition to enhance waste biostabilisation and landfill gas production.

    Science.gov (United States)

    Frank, R R; Davies, S; Wagland, S T; Villa, R; Trois, C; Coulon, F

    2016-09-01

    The effect of leachate recirculation with cellulase augmentation on municipal solid waste (MSW) biostabilisation and landfill gas production was investigated using batch bioreactors to determine the optimal conditions of moisture content, temperature and nutrients. Experimentation was thereafter scaled-up in 7L bioreactors. Three conditions were tested including (1) leachate recirculation only, (2) leachate recirculation with enzyme augmentation and (3) no leachate recirculation (control). Cumulative biogas production of the batch tests indicated that there was little difference between the leachate and control test conditions, producing on average 0.043m(3)biogaskg(-1) waste. However the addition of cellulase at 15×10(6)Utonne(-1) waste doubled the biogas production (0.074m(3)biogaskg(-1) waste). Similar trend was observed with the bioreactors. Cellulase addition also resulted in the highest COD reduction in both the waste and the leachate samples (47% and 42% COD reduction, respectively). In both cases, the quantity of biogas produced was closer to the lower value of theoretical and data-based biogas prediction indicators (0.05-0.4m(3)biogaskg(-1) waste). This was likely due to a high concentration of heavy metals present in the leachate, in particular Cr and Mn, which are known to be toxic to methanogens. The cost-benefit analysis (CBA) based on the settings of the study (cellulase concentration of 15×10(6)Utonne(-1) waste) showed that leachate bioaugmentation using cellulase is economically viable, with a net benefit of approximately €12.1million on a 5Mt mixed waste landfill. PMID:27397800

  17. Investigation of newly developed solid state fermenter on carboxymethyl cellulase production

    Directory of Open Access Journals (Sweden)

    Lee, C. K.

    2013-01-01

    Full Text Available Aims: Enzyme (cellulase contributes 10% to overall cost in bioethanol production from lignocellulosic biomass. This means that the cost for bioethanol production will be reduced if cellulase can be produced using cheaper method. Compared with submerged fermentation, it is recognized that the cost for cellulase production using solid state fermentation (SSF process is much cheaper. The present study aimed to optimize cellulase production via SSF process using agro-industrial residual as substrate.Methodology and result: Newly developed solid state bioreactor, FERMSOSTAT had been evaluated in cellulase production using local isolate Aspergillus niger USM AI 1 grown on sugarcane baggase and palm kernel cake as substrates at 1:1 (w/w ratio. Under optimized SSF conditions of 0.5 kg substrate; 70% (w/w moisture content; 30 °C; aeration at 4 L/h.g fermented substrate for 5 min and mixing at 0.5 rpm for 5 min, about 62.6 U/g of CMCase activity obtained. At the same time, comparative studies of the enzyme production under the same SSF conditions indicated that CMCase produced by Trichoderma reesei was about 9% lower compared with A. niger USM AI 1.Conclusion, significance and impact of study: It can be concluded that the performance of newly developed SSF fermenter is good since it can used to produce CMCase enzyme with reasonable good title (863% increased in CMCase production after optimization. Thus, this newly developed SSF bioreactor has highly potential be used as prototype for larger scale bioreactor design.

  18. 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

  19. 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.

    under solid state fermentation (SSF) processes. In both the agro wastes used, an increase in cellulases (beta-endoglucanase, beta-Glucosidase, and beta-exoglucanase) production was observed with increase in pH. This enhanced carbohydrate...

  20. 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. PMID:27268435

  1. Purification and characterization of an extracellular cellulase from Anoxybacillus gonensis O9 isolated from geothermal area in Turkey.

    Science.gov (United States)

    Genc, Berna; Nadaroglu, Hayrunnisa; Adiguzel, Ahmet; Baltaci, Ozkan

    2015-11-01

    In the present study, cellulase was purified and characterized from Anoxybacillus gonensis (Gen bank Number: KM596794) which was isolated and characterized from Agri Diyadin Hot spring. It was found to synthesize cellulase which had a wide range of industrial applications. Twenty four-hour-cultured bacteria induced cellulase production and specific activities during the purification steps were 1.47, 81.06 and 109.4 EU mg(-1) protein at crude extract, ammonium sulphate precipitated and DEAE-Sephadex purification steps. The highest enzyme activity was observed at 50°C and the optimum range of pH was 3-10. Molecular weight of enzyme was determined approximately 40kDa. The kinetic parameters of cellulase against carboxymethylcellulose (CMC) were 153.4 pmol min(-1) mg for Vmax and 0.46mM for Km. Among effectors of the enzyme, Zn2+, Ca2+, Co2+ and EDTA decreased enzyme activity. PMID:26688967

  2. Optimization of process parameters for cellulase production from Bacillus sp. JS14 in solid substrate fermentation using response surface methodology

    Directory of Open Access Journals (Sweden)

    Jagdish Singh

    2012-08-01

    Full Text Available The aim of this work was to isolate the potent bacterial strains for the production of cellulose enzyme. A total 30 bacterial isolates showed positive results for the cellulase production but highest enzyme activity was shown by isolate JS 14. From the morphological and biochemical reactions, the isolate was identified as Bacillus sp. Cellulase production was studied by this strain using response surface methodology (RSM. A central composite design (CCD quadratic response surface was applied to explicate the parameters that significantly affected cellulase production in solid substrate fermentation (SSF. The wheat bran concentration and incubation period were significant factors. The process parameters optimized with response surface methodology was wheat bran concentration 400 g/L; pH, 6.5; temperature, 400C and incubation period 5 days when inoculum 10 % (1x107 cells/ ml was used for cellulase production in SSF. Supplementation of lactose and CMC to the wheat bran medium favored the enzyme formation.

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

    OpenAIRE

    Gong Xia; Gruninger Robert J; Qi Meng; Paterson Lyn; Forster Robert J; Teather Ron M; McAllister Tim A

    2012-01-01

    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...

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

    OpenAIRE

    O.O. Olaniyi; Fabunmi, A. O.; Akinyele, J. B.

    2013-01-01

    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...

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

    OpenAIRE

    Denton Jai A; Kelly Joan M

    2011-01-01

    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 functi...

  6. 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.

  7. PEA PEEL WASTE: A LIGNOCELLULOSIC WASTE AND ITS UTILITY IN CELLULASE PRODUCTION BY Trichoderma reesei UNDER SOLID STATE CULTIVATION

    Directory of Open Access Journals (Sweden)

    Nitin Verma

    2011-03-01

    Full Text Available A wide variety of waste bioresources are available on our planet for conversion into bioproducts. In the biological systems, microorganisms are used to utilize waste as an energy source for the synthesis of valuable products such as biomass proteins and enzymes. The large quantities of byproducts generated during the processing of plant food involve an economic and environmental problem due to their high volumes and elimination costs. After isolation of the main constituent, there are abundant remains which represent an inexpensive material that has been undervalued until now. Pea peel waste is one of the undervalued, unused sources of energy that can serve as a potential source for cellulase production. Batch experiments have been performed, using pea peel waste as a carbon source for cellulase production under solid state cultivation by Trichoderma reesei. It was observed that 30 oC temperature and pH 5.0 are the most favorable conditions for cellulase production by T. reesei. FPase activity significantly increases by incorporation of whey as well as wheat starch hydrolysate in the basal salt media used in the production study. The present study describes the utility of pea peel waste, whey as well as wheat starch hydrolysate in cellulase production by T. reesei. The utilization of economically cheap, pea peel waste for cellulase production could be a novel, cost effective, and valuable approach in cellulase production as well as in solid waste management.

  8. Production and characterization of cellulase-free xylanase from Trichoderma inhamatum.

    Science.gov (United States)

    de Oliveira da Silva, Leonor Alves; Carmona, Eleonora Cano

    2008-08-01

    The production of extracellular cellulase-free xylanase from Trichoderma inhamatum was evaluated in liquid Vogel medium with different carbon sources as natural substrates and agricultural or agro-industrial wastes. Optimal production of 244.02 U/mL was obtained with xylan as carbon source, pH 6.0 at 25 degrees C, 120 rpm, and 60-h time culture. Optimal conditions for enzyme activity were 50 degrees C and pH 5.5. Thermal stability of T. inhamatum xylanolytic complex expressed as T1/2 was 2.2 h at 40 degrees C and 2 min at 50 degrees C. The pH stability was high from 4.0 to 11.0. These results indicate possible employment of such enzymatic complex in some industrial processes which require activity in acid pH, wide-ranging pH stability, and cellulase activity absence. PMID:18607546

  9. 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.

  10. Production of nanotubes in delignified porous cellulosic materials after hydrolysis with cellulase.

    Science.gov (United States)

    Koutinas, Αthanasios Α; Papafotopoulou-Patrinou, Evgenia; Gialleli, Angelika-Ioanna; Petsi, Theano; Bekatorou, Argyro; Kanellaki, Maria

    2016-08-01

    In this study, tubular cellulose (TC), a porous cellulosic material produced by delignification of sawdust, was treated with a Trichoderma reesei cellulase in order to increase the proportion of nano-tubes. The effect of enzyme concentration and treatment duration on surface characteristics was studied and the samples were analyzed with BET, SEM and XRD. Also, a composite material of gelatinized starch and TC underwent enzymatic treatment in combination with amylase (320U) and cellulase (320U) enzymes. For TC, the optimum enzyme concentration (640U) led to significant increase of TC specific surface area and pore volume along with the reduction of pore diameter. It was also shown that the enzymatic treatment did not result to a significant change of cellulose crystallinity index. The produced nano-tubular cellulose shows potential for application to drug and chemical preservative delivery systems. PMID:26996258

  11. 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. PMID:27289054

  12. [Molecular engineering of cellulase catalytic domain based on glycoside hydrolase family].

    Science.gov (United States)

    Zhang, Xiaomei; Li, Dandan; Wang, Lushan; Zhao, Yue; Chen, Guanjun

    2013-04-01

    Molecular engineering of cellulases can improve enzymatic activity and efficiency. Recently, the Carbohydrate-Active enZYmes Database (CAZy), including glycoside hydrolase (GH) families, has been established with the development of Omics and structural measurement technologies. Molecular engineering based on GH families can obviously decrease the probing space of target sequences and structures, and increase the odds of experimental success. Besides, the study of cellulase active-site architecture paves the way toward the explanation of catalytic mechanism. This review focuses on the main GH families and the latest progresses in molecular engineering of catalytic domain. Based on the combination of analysis of a large amount of data in the same GH family and their conservative active-site architecture information, rational design will be an important direction for molecular engineering and promote the rapid development of the conversion of biomass. PMID:23894816

  13. 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.

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

    DEFF Research Database (Denmark)

    Rosgaard, Lisa; Pedersen, Sven; Langston, J; Akerhielm, D; Cherry, JR; Meyer, Anne S.

    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...... cellobiohydrolases (CBHs) and endo-1,4-beta-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the...... cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational...

  15. 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.

  16. Pretreatment of brewers' spent grains for cellulases production by Aspergillus niger van Tieghem

    OpenAIRE

    Michelin, Michele; Maria de Lourdes T. M. Polizeli; Ruzene, Denise S.; Silva, Daniel Pereira da; Vicente, A. A.; Teixeira, J. A.

    2011-01-01

    Successful utilization of cellulosic materials as a renewable carbon source is dependent on the development of economically feasible process technologies both for the production of biomass-degrading enzymes, and for the enzymatic hydrolysis of cellulosic materials to low molecular weight products. Significant cost reduction is required in order to enhance the commercial viability of cellulase production technology and biomass pretreatment can be an essential processing step for this purpose. ...

  17. Fermentation of wort from a starch-containing raw material prepared using cellulases

    Energy Technology Data Exchange (ETDEWEB)

    Marinchenko, V.A.; Nosik, S.V.; Tiunova, N.A.

    1980-01-01

    Wort was prepared from powdered barley by treatment with cellulase from Geotrichum candidum or the enzyme preparation cellocandin G3X. After fermentation the alcohol concentration increased and the amount of nonmetabolized starch significantly decreased. The use of cellulolytic enzymes in the production of beer not only reduces the requirements for amylolytic enzymes but also increases the EtOh yield by 2.0-3.5%.

  18. A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

    OpenAIRE

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

    2011-01-01

    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...

  19. Precipitation of Trichoderma reesei commercial cellulase preparations under standard enzymatic hydrolysis conditions for lignocelluloses

    OpenAIRE

    Chylenski, Piotr; Felby, Claus; Haven, Mai Østergaard; Gama, F. M.; Selig, Michael J

    2012-01-01

    Comparative studies between commercial Trichoderma reesei cellulase preparations show that, depending on the preparation and loading, total protein precipitation can be as high as 30 % under standard hydrolysis conditions used for lignocellulosic materials. ATR-IR and SDS-PAGE data verify precipitates are protein-based and contain key cell wall hydrolyzing enzymes. Precipitation increased considerably with incubation temperature; roughly 50–150 % increase from 40 to 50 °C and 800 % greater at...

  20. APPLICATION OF SOLID-STATE FERMENTATION FOR CELLULASE ENZYME PRODUCTION USING TRICHODERMA VIRIDE

    OpenAIRE

    Kiro Mojsov

    2010-01-01

    The Solid-state fermentation (SSF) is alternative to submerged fermentation for production antibiotics, single cell protein, enzymes, organic acids, biofuel, etc. However, the advantages of SSF in various processes are found to be greater than in submerged fermentation. This technique not only decreases the cost of the process but also makes product cheaper for consumers. The paper describes experimental application of SSF on wheat straw for production of mycelia protein and cellulase enzym...

  1. 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)

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

    OpenAIRE

    Ping Sheng; Yushan Li; Sean D. G. Marshall; Hongyu Zhang

    2015-01-01

    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...

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

    OpenAIRE

    Salunke, Bipinchandra K.; Sawant, Shailesh S.; Tae Koo Kang; Deok Yun Seo; Youngjong Cha; Sun A. Moon; Bassam Alkotaini; Ezhaveni Sathiyamoorthi; Beom Soo Kim

    2015-01-01

    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 Maximowic...

  4. Evaluating leachate recirculation with cellulase addition to enhance waste biostabilisation and landfill gas production

    OpenAIRE

    Frank, R. R.; S. Davies; Wagland, Stuart T.; Villa, Raffaella; Trois, C.; Coulon, Frederic

    2016-01-01

    The effect of leachate recirculation with cellulase augmentation on municipal solid waste (MSW) biostabilisation and landfill gas production was investigated using batch bioreactors to determine the optimal conditions of moisture content, temperature and nutrients. Experimentation was thereafter scaled-up in 7 L bioreactors. Three conditions were tested including (1) leachate recirculation only, (2) leachate recirculation with enzyme augmentation and (3) no leachate recirculation (control). C...

  5. Ultrasounds pretreatment of olive pomace to improve xylanase and cellulase production by solid-state fermentation

    OpenAIRE

    Leite, A; Salgado, José Manuel; Venâncio, Armando; Domínguez, José Manuel; Belo, Isabel

    2016-01-01

    Abstract Olive mills generate a large amount of waste that can be revaluated. This work aim to improve the production lignocellulolytic enzymes by solid-state fermentation using ultrasounds pretreated olive mill wastes. The composition of olive mill wastes (crude and exhausted olive pomace) was compared and several physicochemical characteristics were significantly different. The use of both wastes in SSF was evaluated and a screening of fungi for xylanase and cellulase production was carried...

  6. 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 .

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

    OpenAIRE

    Cruys-Bagger, Nicolaj; Badino, Silke Flindt; Tokin, Radina Naytchova; Gontsarik, Mark; Fathalinejad, Samin; Jensen, Kenneth; Toscano, Miguel Duarte; Sørensen, Trine Holst; Borch, Kim; Tatsumi, Hirosuke; Väljamäe, Priit; Westh, Peter

    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 ...

  8. 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.

  9. Exo and endo-glucanolytic activity of cellulases purified from Trichoderma reesei

    OpenAIRE

    Gama, F. M.; Vilanova, Manuel; Mota, M

    1998-01-01

    Four cellulases, produced by Trichoderma reesei, have been purified by preparative isoelectric focusing (Rotofor), size exclusion (Sephacryl 100 HR), anionic (Mono Q) and cationic (Mono S) chromatography and chromatofocusing (Mono P). Enzymatic activity with a large number of substrates allowed the proteins to be classified as: cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II. The exo- or endo- glucanase character of these enzymes was analysed by using a techniq...

  10. 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

  11. Effect of Colloidal Materials on Cellulase Production by Trichoderma reesei Rut-C30

    OpenAIRE

    Duff, Sheldon J.B.; Cooper, David G.; Fuller, O. Maynard

    1985-01-01

    The addition of positively charged colloidal materials to the growth medium markedly increased the concentration of cellulase enzymes produced by Trichoderma reesei Rut-C30. Filter paper activities of up to 4 and 13 IU/ml have been achieved by the addition of colloidal materials, using 3% lactose and 3% cellulose, respectively, as a substrate. The particles exert their effect by binding soluble sugars and slowing their uptake by the organism.

  12. Production of a high-efficiency cellulase complex via β-glucosidase engineering in Penicillium oxalicum

    OpenAIRE

    Yao, Guangshan; Wu, Ruimei; Kan, Qinbiao; Gao, Liwei; Liu, Meng; Yang, Piao; Du, Jian; Li, Zhonghai; Qu, Yinbo

    2016-01-01

    Background Trichoderma reesei is a widely used model cellulolytic fungus, supplying a highly effective cellulase production system. Recently, the biofuel industry discovered filamentous fungi from the Penicillium genus as a promising alternative to T. reesei. Results In our study, we present a systematic over-expression analysis of nine β-glucosidase encoding genes in the wild-type strain 114-2 of Penicillium oxalicum. We found that the over-expression of BGL1, BGL4, or BGL5 significantly enh...

  13. Enhanced cellulase production by Trichoderma harzianum by cultivation on glycerol followed by induction on cellulosic substrates.

    Science.gov (United States)

    da Silva Delabona, Priscila; Lima, Deise Juliana; Robl, Diogo; Rabelo, Sarita Cândida; Farinas, Cristiane Sanchez; da Cruz Pradella, José Geraldo

    2016-05-01

    The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of Trichoderma harzianum followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector. PMID:26883662

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

    OpenAIRE

    Rui Sergio F. da Silva; Chigurupati S. Rao

    1980-01-01

    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 wh...

  15. 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

    OpenAIRE

    Nurul Kartini Abu Bakar,; Zuraidah Zanirun; Suraini Abd-Aziz; Farinazleen Mohd Ghazali; Mohd Ali Hassan

    2012-01-01

    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...

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

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    Sumiati

    2012-06-01

    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 hen aged 25-30 weeks. The Jatropha curcas meal was fermented using Rizhopus oligosporus. In this study 200 laying hens were used and distributed to 5 treatments and 4 replications in Completely Randomized Design. The diet treatments were: R0 = control diet (without JCM, R1; diet contained fermented JCM 7.5%, R2; diet contained fermented JCM 7.5% + celullase 200 g/ton, R3; diet contained fermented JCM 7.5% + phytase 200 g/ton and R4; diet contained fermented JCM 7.5% + cellulase 200 g/ton + phytase 200 g/ton. The parameters observed were feed consumption, hen day egg production, egg mass production, 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 feed consumption, hen day egg and egg mass production. However, the treatments did not influence the egg weight. Supplementation of cellulase (R2 or phytase (R3 improved the feed conversion ratio with the value as same as the R0 diet.

  17. Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines.

    Science.gov (United States)

    Okumura, Fumihiko; Kameda, Hiroyuki; Ojima, Takao; Hatakeyama, Shigetsugu

    2010-05-01

    We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism. PMID:20381456

  18. 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. PMID:22052232

  19. In vitro flow cytometry-based screening platform for cellulase engineering.

    Science.gov (United States)

    Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

    2016-01-01

    Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 10(7) events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

  20. Ultrasounds pretreatment of olive pomace to improve xylanase and cellulase production by solid-state fermentation.

    Science.gov (United States)

    Leite, Paulina; Salgado, José Manuel; Venâncio, Armando; Domínguez, José Manuel; Belo, Isabel

    2016-08-01

    Olive mills generate a large amount of waste that can be revaluated. This work aim to improve the production lignocellulolytic enzymes by solid-state fermentation using ultrasounds pretreated olive mill wastes. The composition of olive mill wastes (crude and exhausted olive pomace) was compared and several physicochemical characteristics were significantly different. The use of both wastes in SSF was evaluated and a screening of fungi for xylanase and cellulase production was carried out. After screening, the use of exhausted olive pomace and Aspergillus niger led to the highest enzyme activities, so that they were used in the study of ultrasounds pre-treatment. The results showed that the sonication led to a 3-fold increase of xylanase activity and a decrease of cellulase activity. Moreover, the liquid fraction obtained from ultrasounds treatment was used to adjust the moisture of solid and a positive effect on xylanase (3.6-fold increase) and cellulase (1.2-fold increase) production was obtained. PMID:27209456

  1. In vitro flow cytometry-based screening platform for cellulase engineering

    Science.gov (United States)

    Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

    2016-01-01

    Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

  2. Application of solid-state fermentation for cellulase enzyme production using Trichoderma viride

    Directory of Open Access Journals (Sweden)

    Kiro Mojsov

    2010-06-01

    Full Text Available The Solid-state fermentation (SSF is alternative to submerged fermentation for production antibiotics, single cell protein, enzymes, organic acids, biofuel, etc. However, the advantages of SSF in various processes are found to be greater than in submerged fermentation. This technique not only decreases the cost of the process but also makes product cheaper for consumers. The paper describes experimental application of SSF on wheat straw for production of mycelia protein and cellulase enzymes by Trichoderma viride. This actual waste from agriculture industry was used as a nourishing base by Trichoderma viride in SSF for cellulase enzyme production. Growth and enzymes production by Trichoderma viride were evaluated on wheat straw and alkali treatment wheat straw (wet processing. The growth of the microorganism (biomass content shows maximum (123.44 mg/petri dish on alkali treatment wheat straw compared (96.36 mg/petri dish on wheat straw during of 240 hours. The results obtained demonstrate that the wheat straw waste from agriculture industry can be used as inexpensive base (carbon source for industrial production on cellulase enzymes by Trichoderma viride.

  3. 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.

  4. Improvement of fungal cellulase production by mutation and optimization of solid state fermentation.

    Science.gov (United States)

    Vu, Van Hanh; Pham, Tuan Anh; Kim, Keun

    2011-03-01

    Spores of Aspergillus sp. SU14 were treated repeatedly and sequentially with Co(60) γ-rays, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine. One selected mutant strain, Aspergillus sp. SU14-M15, produced cellulase in a yield 2.2-fold exceeding that of the wild type. Optimal conditions for the production of cellulase by the mutant fungal strain using solid-state fermentation were examined. The medium consisted of wheat-bran supplemented with 1% (w/w) urea or NH(4)Cl, 1% (w/w) rice starch, 2.5 mM MgCl(2), and 0.05% (v/w) Tween 80. Optimal moisture content and initial pH was 50% (v/w) and 3.5, respectively, and optimal aeration area was 3/100 (inoculated wheat bran/container). The medium was inoculated with 25% 48 hr seeding culture and fermented at 35℃ for 3 days. The resulting cellulase yield was 8.5-fold more than that of the wild type strain grown on the basal wheat bran medium. PMID:22783068

  5. Optimization of Cellulase Production by Trichoderma reesei HY07 Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Shikai Wang

    2013-05-01

    Full Text Available Response Surface Methodology (RSM based on a three-level, three-variable Box and Behnken Factorial Design (BBFD was used to evaluate the interactive effects of corn stalk: bran ratio, tween80 and temperature on the cellulase production by solid fermentation. The optimum conditions derived via RSM were: corn stalk: bran ratio 1.29:1, Tween80 11.05 &muL and temperature 31 °C for carboxymethyl cellulase (CMCase and corn stalk: bran ratio 0.77:1, Tween80 12.54&muL and temperature 32 °C for Filter Paper Activity (FPA. The actual experimental yield was 406.42 U/g for CMCase and 93.62 U/g for FPA under optimum condition, which compared well to the maximum predicted value of 405.67 U/g and 91.29 U/g. The cellulase yield under optimal conditions was 1.45 fold for CMCase and 1.33 fold for FPA to the control.

  6. Mutagenesis and evaluation of cellulase properties and cellulose hydrolysis of Talaromyces piceus.

    Science.gov (United States)

    He, Ronglin; Cai, Pengli; Wu, Gaihong; Zhang, Can; Zhang, Dongyuan; Chen, Shulin

    2015-11-01

    A fungal species with a high yield of β-glucosidase was isolated and identified as Talaromyces piceus 9-3 (anamorph: Penicillium piceum) by morphological and molecular characterization. Through dimethyl sulphate mutagenesis, the cellulase over-producing strain T. piceus H16 was obtained. The FPase activity and β-glucosidase activity of T. piceus H16 were 5.83 and 53.12 IU ml(-1) respectively--a 5.34- and 4.43-times improvement from the parent strain T. piceus 9-3. The optimum pH and temperature for enzyme activity were pH 5.0 and 50 °C for FPase activity and pH 5.0 and 55 °C for β-glucosidase activity, respectively. The cellulase were quite stable at 37 °C, only losing Trichoderma reesei RUT C30 on hydrolyzing different substrates due to the high β-glucosidase activity of T. piceus H16. These data suggest that T. piceus H16 can be used as a potential cellulase producer with good prospects. PMID:26330062

  7. Effet de la pectolyase Y-23 et de la cellulase Onozuka RS sur le rendement en protoplastes viables de Prunus cerasus L.

    Directory of Open Access Journals (Sweden)

    Mehri-Kamoun R.

    2001-01-01

    Full Text Available Effect of pectolyase Y-23 and cellulase Onozuka RS on the yield of viable protoplasts of Prunus cerasus L. ""Montmorency"". To isolate leaf mesophyll, leaf and root callus protoplasts of Prunus cerasus L. ""Montmorency"", we have determined the optimum enzymatic mixtures to be used, and characterized the specific activity of these enzymes. The analysis of the specific activities of enzymes allows to compare the different cellulases and pectinases used to obtain protoplasts in relation with the tissue sources. This analysis concerned the FPase (degradation of filter paper and CMCase activities for cellulases Onozuka RS and R-10, and the PME (pectinmethylesterase, PL (pectate lyase and PG (polygalacturonase activities for the pectinases Macerozyme R-10 and Pectolyase Y-23. The results show that the digestion of leaf mesophyll tissues need cellulase Onozuka RS and Pectolyase Y-23 while callus protoplasts of the same material, can be isolated with cellulase Onozuka R-10 and Macerozyme R-10. The enzymes cellulase Onozuka RS and Pectolyase Y-23 (as pectinase improved significantly the yield and the viability of leaf mesophyll protoplasts compared to cellulase Onozuka R-10 and Macerozyme R-10. These results were correlated to the specific activities of the enzymes. Significant differences between the 2 pectinases are observed for PME, PL and PG activities and between the 2 cellulases for CMCase activity. From callus, the maximum amount of viable protoplasts was obtained with cellulase Onozuka R-10 (low CMCase activity and Macerozyme R-10 (low PG activity.

  8. 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

  9. A GHF7 cellulase from the protist symbiont community of Reticulitermes flavipes enables more efficient lignocellulose processing by host enzymes.

    Science.gov (United States)

    Sethi, Amit; Kovaleva, Elena S; Slack, Jeffrey M; Brown, Susan; Buchman, George W; Scharf, Michael E

    2013-12-01

    Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7-3, GHF7-5, and GHF7-6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full-length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus-insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7-3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and β-glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell-1), beta-glucosidase (β-glu), and laccase (LacA). GHF7-3 was the only GHF7 to enhance glucose release by Cell-1 and β-glu. Finally, GHF7-3, Cell-1, and β-glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only β-glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7-3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases. PMID:24186432

  10. 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.

  11. The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1.

    Science.gov (United States)

    Xu, Xinxin; Li, Jinyang; Shi, Pengjun; Ji, Wangli; Liu, Bo; Zhang, Yuhong; Yao, Bin; Fan, Yunliu; Zhang, Wei

    2016-01-01

    Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/10(6) conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens. PMID:27506519

  12. Chitinolytic and chitosanolytic activities from crude cellulase extract produced by A. niger grown on apple pomace through Koji fermentation.

    Science.gov (United States)

    Dhillon, Gurpreet Singh; Brar, Satinder Kaur; Kaur, Surinder; Valero, Jose R; Verma, Mausam

    2011-12-01

    Enzyme extracts of cellulase [filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase)], chitinase, and chitosanase produced by Aspergillus niger NRRL-567 were evaluated. The interactive effects of initial moisture and different inducers for FP cellulase and CMCase production were optimized using response surface methodology. Higher enzyme activities [FPase 79.24+/- 4.22 IU/gram fermented substrate (gfs) and CMCase 124.04+/-7.78 IU/gfs] were achieved after 48 h fermentation in solid-state medium containing apple pomace supplemented with rice husk [1% (w/w)] under optimized conditions [pH 4.5, moisture 55% (v/w), and inducers veratryl alcohol (2 mM/kg), copper sulfate (1.5 mM/kg), and lactose 2% (w/w)] (pchitosan substrates with different physicochemical characteristics, such as degree of deacetylation, molecular weight, and viscosity. Higher chitinase and chitosanase activities of 70.28+/-3.34 IU/gfs and 60.18+/-3.82 to 64.20+/-4.12 IU/gfs, respectively, were achieved. Moreover, the enzyme was stable and retained 92-94% activity even after one month. Cellulase enzyme extract obtained from A. niger with chitinolytic and chitosanolytic activities could be potentially used for making low-molecular-weight chitin and chitosan oligomers, having promising applications in biomedicine, pharmaceuticals, food, and agricultural industries, and in biocontrol formulations. PMID:22210619

  13. The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1

    Science.gov (United States)

    Xu, Xinxin; Li, Jinyang; Shi, Pengjun; Ji, Wangli; Liu, Bo; Zhang, Yuhong; Yao, Bin; Fan, Yunliu; Zhang, Wei

    2016-01-01

    Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/106 conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens. PMID:27506519

  14. Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase A from Cellulomonas fimi.

    Science.gov (United States)

    Cockburn, Darrell W; Vandenende, Chris; Clarke, Anthony J

    2010-03-01

    Cellulase A (CenA) from Cellulomonas fimi is an inverting glycoside hydrolase and a member of family 6 of the CAZy database classification system. We replaced its putative catalytic base aspartyl residues, Aps392 and Asp216, with cysteinesulfinate using a combination of site-directed mutagenesis and chemical modification to investigate the applicability of this approach for the modulation of enzymatic properties. The substituted cysteinyl residues were oxidized to cysteinesulfinic acid with hydrogen peroxide, and the resulting protein products were demonstrated to retain their native structure. Oxidation of the Asp392Cys mutant enzyme restored 52% of wild-type activity when assessed at pH 7.5, whereas Asp216Cys CenA remained inactive. This suggests that Asp216 is not the catalytic base and provides further support for Asp392 performing this role. Similar substitution of the catalytic acid residue Asp252 or the catalytic nucleophile of the retaining enzyme Cel5A from Thermobifida fusca failed to produce active enzymes. This indicates a potential utility of this approach for uniquely identifying catalytic base residues. The replacement of Asp392 with cysteinesulfinate induced an acidic shift in the pH profile of the enzyme such that this enzyme derivative was more active than wild-type CenA below pH 5.5. These data demonstrate the potential of combining site-directed mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH. PMID:20136145

  15. 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

  16. Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from coptotermes formosanus and expressed in E. coli

    Science.gov (United States)

    The endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in E.coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrate...

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

    Science.gov (United States)

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

    2011-01-01

    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. PMID:21307835

  18. pH-dependent stability of EGX, a multi-functional cellulase from mollusca, Ampullaria crossean.

    Science.gov (United States)

    Li, Wen-Ying; Wang, Ji; Li, Yan-Hong; Ding, Ming; Xu, Gen-Jun; Liu, Lan-Ying; Zhao, Fu-Kun

    2004-09-01

    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 con-formation 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 alpha-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 alpha-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. PMID:15346197

  19. 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...... jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate....... Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use....

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

    OpenAIRE

    Sumiati . .; R. Mutia; A. Darmansyah

    2012-01-01

    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 hen aged 25-30 weeks. The Jatropha curcas meal was fermented using Rizhopus oligosporus. In this study 200 laying hens were used and distributed to 5 treatments and 4 replications in Completely Randomized Design. The diet treatments were: R0 = control diet (without JCM), R1; diet contained fermented JCM 7.5%,...

  1. Cellulase production by two mutant strain of Trichoderma longibrachiatum Qm9414 and Rut C30

    International Nuclear Information System (INIS)

    Native or pretreated biomass from Onopordum nervosum boiss, has been examined as candidate feedstock for cellulase production by two mutant strain of trichoderma longibrachiatum 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. longibrachiatum Rut C30 on 55% (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 fermentor.(author)

  2. Characterization of Actinomycetes and Trichoderma spp. for cellulase production utilizing crude substrates by response surface methodology

    OpenAIRE

    Pirzadah, Tanveer; Garg, Shashank; Singh, Joginder; Vyas, Ashish; Kumar, Manish; Gaur, Naseem; Bala, Madhu; Rehman, Reiaz; Varma, Ajit; Kumar, Vivek; Kumar, Manoj

    2014-01-01

    Laboratory bench scaling was done and an average of 1.85 fold increase by Response Surface Methodology (RSM) optimization was obtained. It was found that the predicted value (4.96 IU/ml) obtained by RSM is in close accordance with observed activity 5.14 IU/ml. Endoglucanases are mainly induced by CMC while Wheat bran (natural substrate) exoglucanase is more active when induced by avicel and cellulose. Addition of substrate beyond a level caused inhibition of cellulase production. The molecula...

  3. 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.

  4. Interkalation der Enzyme Papain und Cellulase sowie von Graphenoxid in Hydrotalcit- und Hydrocalumit-artige Schichtstrukturen (LDHs)

    OpenAIRE

    Zou, Nan

    2014-01-01

    In dieser Arbeit wurden der Einbau, die kristallchemischen und thermischen Eigenschaften von Interkalationsverbindungen der Enzyme Papain und Cellulase in Hydrotalcit-artige sowie von Graphenoxid (GO) in Hydrocalumit-artige Layered Double Hydroxides (LDHs) untersucht. Bei der Interkalationsverbindung Mg2Al-Papain-LDH wurde ein Schichtabstand von 5,3 nm gefunden, und für Mg2Al-Cellulase-LDH ein Wert von 5,0 nm. Mittels Anionenaustausch mit Sulfat können die Enzyme aus der Interkalationsverbind...

  5. Ethanol production from non-detoxified whole slurry of sulfite-pretreated empty fruit bunches at a low cellulase loading.

    Science.gov (United States)

    Cheng, Jinlan; Leu, Shao-Yuan; Zhu, J Y; Jeffries, Thomas W

    2014-07-01

    Sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) was applied to an empty fruit bunches (EFB) for ethanol production. SPORL facilitated delignification through lignin sulfonation and dissolution of xylan to result in a highly digestible substrate. The pretreated whole slurry was enzymatically saccharified at a solids loading of 18% using a relatively low cellulase loading of 15 FPU/g glucan and simultaneously fermented without detoxification using Saccharomyces cerevisiae of YRH400. An ethanol yield of 217 L/tonne EFB was achieved at titer of 32 g/L. Compared with literature studies, SPORL produced high ethanol yield and titer with much lower cellulase loading without detoxification. PMID:24874873

  6. 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.

  7. Rather than by direct acquisition via lateral gene transfer, GHF5 cellulases were passed on from early Pratylenchidae to root-knot and cyst nematodes

    Directory of Open Access Journals (Sweden)

    Rybarczyk-Mydłowska Katarzyna

    2012-11-01

    Full Text Available Abstract Background Plant parasitic nematodes are unusual Metazoans as they are equipped with genes that allow for symbiont-independent degradation of plant cell walls. Among the cell wall-degrading enzymes, glycoside hydrolase family 5 (GHF5 cellulases are relatively well characterized, especially for high impact parasites such as root-knot and cyst nematodes. Interestingly, ancestors of extant nematodes most likely acquired these GHF5 cellulases from a prokaryote donor by one or multiple lateral gene transfer events. To obtain insight into the origin of GHF5 cellulases among evolutionary advanced members of the order Tylenchida, cellulase biodiversity data from less distal family members were collected and analyzed. Results Single nematodes were used to obtain (partial genomic sequences of cellulases from representatives of the genera Meloidogyne, Pratylenchus, Hirschmanniella and Globodera. Combined Bayesian analysis of ≈ 100 cellulase sequences revealed three types of catalytic domains (A, B, and C. Represented by 84 sequences, type B is numerically dominant, and the overall topology of the catalytic domain type shows remarkable resemblance with trees based on neutral (= pathogenicity-unrelated small subunit ribosomal DNA sequences. Bayesian analysis further suggested a sister relationship between the lesion nematode Pratylenchus thornei and all type B cellulases from root-knot nematodes. Yet, the relationship between the three catalytic domain types remained unclear. Superposition of intron data onto the cellulase tree suggests that types B and C are related, and together distinct from type A that is characterized by two unique introns. Conclusions All Tylenchida members investigated here harbored one or multiple GHF5 cellulases. Three types of catalytic domains are distinguished, and the presence of at least two types is relatively common among plant parasitic Tylenchida. Analysis of coding sequences of cellulases suggests that root

  8. Solid state fermentation for production of microbial cellulases: Recent advances and improvement strategies.

    Science.gov (United States)

    Behera, Sudhanshu S; Ray, Ramesh C

    2016-05-01

    Lignocellulose is the most plentiful non-food biomass and one of the most inexhaustible renewable resources on the planet, which is an alternative sustainable energy source for the production of second generation biofuels. Lignocelluloses are composed of cellulose, hemicellulose and lignin, in which the sugar polymers account for a large portion of the biomass. Cellulases belong to the glycoside hydrolase family and catalyze the hydrolysis of glyosidic linkages depolymerizing cellulose to fermentable sugars. They are multi-enzymatic complex proteins and require the synergistic action of three key enzymes: endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.176) (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21) for the depolymerization of cellulose to glucose. Solid state fermentation, which holds growth of microorganisms on moist solid substrates in the absence of free flowing water, has gained considerable attention of late due its several advantages over submerged fermentation. The review summarizes the critical analysis of recent literature covering production of cellulase in solid state fermentation using advance technologies such as consolidated bioprocessing, metabolic engineering and strain improvement, and circumscribes the strategies to improve the enzyme yield. PMID:26601764

  9. 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.

  10. Mutant strain screening by 60Co γ-rays irradiation and its cellulase enzyme produce condition

    International Nuclear Information System (INIS)

    A mutant strain A50 with high cellulase activity was induced and isolated by using 60Co γ-rays irradiation from the initial Penicillium decumbens A10. The optimum fermentation conditions of A50 were investigated through orthogonal designing experiment, the major carbon resource 5%, the ratio between wheat bran and corn straw 1:1, the concentration of glucose as supplemental carbon 0.1%, the concentration of (NH4)2HPO4 as supplemental nitrogen resource 0.2%, the initial pH of liquid medium 5.0, the inoculated amount for fermentation 10% and the concentration of Tween-80 0.1%, 30 ml initial media filled in the 300 ml flask with culture condition of 32 degree C and 200 r/min. Under the optimum conditions mentioned above, the highest activities of cellulase and filter paper enzyme were 27.28 and 1.98IU/ml at 60 h fermentation, respectively, which was 33.2% and 45.59% higher than those of the initial strain. (authors)

  11. 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.

  12. 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. PMID:24045195

  13. 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 10h 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. PMID:26917391

  14. 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. PMID:27318817

  15. 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.

  16. An expansin-like protein from Hahella chejuensis binds cellulose and enhances cellulase activity.

    Science.gov (United States)

    Lee, Hee Jin; Lee, Saeyoung; Ko, Hyeok-Jin; Kim, Kyoung Heon; Choi, In-Geol

    2010-04-01

    Molecular function of the expansin superfamily has been highlighted for cellulosic biomass conversion. In this report, we identified a new bacterial expansin subfamily by analysis of related bacterial sequences and biochemically examined a member of this new subfamily from Hahella chejuensis (HcEXLX2). Among the various complex polysaccharides tested, HcEXLX2 bound most efficiently to cellulose. The relative binding constant (K( r )) against Avicel was 2.1 L g(-1) at pH 6.0 and 4 degrees C. HcEXLX2 enhanced the activity of cellulase, producing about 4.6 times more hydrolysis product after a 36 h reaction relative to when only cellulase was used. The extension strength test on filter paper indicated that HcEXLX2 has a texture loosening effect on filter paper, which was 53% of that observed for 8 M urea treatment. These activities, compared with a cellulose binding domain from Clostridium thermocellum, implied that the synergistic effect of HcEXLX2 comes from not only binding to cellulose but also disrupting the hydrogen bonds in cellulose. Based on these results, we suggest that the new bacterial expansin subfamily functions by binding to cell wall polysaccharides and increasing the accessibility of cell wall degrading enzymes. PMID:20213317

  17. Influence of different cultural conditions on cellulase production by Nectria catalinensis.

    Science.gov (United States)

    Pardo, A G; Forchiassin, F

    1998-01-01

    The production of the extracellular cellulolytic enzyme system (endoglucanase, exoglucanase and cellobiase) of N. catalinensis was tested with different nitrogen sources, inorganic and organic ones, in liquid culture medium with microcrystalline cellulose. The nitrogen compounds used were: potassium nitrate, sodium nitrate, ammonium nitrate, ammonium phosphate, ammonium sulphate, ammonium chloride, ammonium carbonate, ammonium acetate, ammonium tartrate, urea, casamino acids, glycine, L-alanine, L-leucine, L-proline, L-lysine, L-aspartic acid, L-glutamic acid, L-asparagine, L-glutamine, L-phenylalanine, L-tyrosine, L-tryptophan, L-methionine and L-cysteine. Among these, ammonium nitrate and ammonium tartrate gave the highest yields of cellulases in 20-day-old cultures at a concentration equivalent to 0.75 g N/l in both cases. Optimal temperature for cellulase production, growth and cellulose degradation was 23 degrees C. On the other hand, an initial pH of 6.5 gave the highest yields of endoglucanase and cellobiase. In the same way, at pH 6.5, maximal growth and cellulose degradation were achieved. However, maximal exoglucanase production and glycogen content were reached at pH 7.5. PMID:9629604

  18. Filamentous fungi and media for cellulase production in solid state cultures

    Directory of Open Access Journals (Sweden)

    B.V. Kilikian

    2014-01-01

    Full Text Available Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414, two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821 and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77. Solid state cultures were performed using sugarcane bagasse (C, wheat bran (W and/or soybean bran (S. The highest FPA was 10.6 U/gdm for M77 in SC (10:90 at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60 was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80 was 2.3 times relative to WC (20:80 for M77.

  19. Effect of Pre-harvest Treatments on the Cellulase Activity and Quality of Ber Fruit Under Cold Storage Conditions

    Directory of Open Access Journals (Sweden)

    Sukhjit Kaur JAWANDHA

    2009-12-01

    Full Text Available Studies were carried out to find out the effect of various pre-harvest treatments such as CaCl2 (@ 0.5%, 1.0% and 2.0%, Ca(NO32 (@0.5%, 1.0% and 2.0%, GA3 (@ 20, 40 and 60 ppm and Bavistin (@ 0.1% on the cellulase activity and quality of 'Umran' ber fruits during cold storage. Marked trees were sprayed at colour break stage with the test chemicals. Fruits were packed in CFB boxes and placed in cold storage (3-5 oC and 85 � 90 % RH for 30 days. The fruits were evaluated after 10, 20 and 30 days interval for various parameters such as cellulase activity, phenolics content, palatability rating and rotting percentage. Cellulase activity registered a gradual increase upto 20 days of storage thereafter a decline was noted in all the treatments. The palatability rating increased up to 10 days of storage in all the treatments, except control but subsequently it decreased with an advancement in storage period. Among the various pre-harvest treatments CaCl2 (2% recorded minimum cellulase activity and rotting percentage and have also registered high palatability rating and phenolics content during cold storage conditions. Studies showed that pre-harvest application of CaCl2 (2% maintained very good fruit quality and prolonged shelf-life for 20 days under cold storage conditions.

  20. 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.

  1. 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)

  2. Hydrolysis of cellular tissue of starch-containing raw material in the presence of cellulases under alcohol production conditions

    Energy Technology Data Exchange (ETDEWEB)

    Nosik, S.V.; Marinchenko, V.A.; Tiunova, N.A.

    1980-01-01

    Treatment of barley flour (cellulose content 4.72%) or brewers' mash (cellulose content, 19.06%) with C1-cellulase from Geotrichum candidum or the enzyme preparation cellocandin G3X hydrolyzed 40-43% of the cellulose content. Fermentation of wort prepared from the hydrolyzed raw materials increased the EtOH content of beer by 3.76%.

  3. The effects of cellulase on capsaicin production in freely suspended cells and immobilized cell cultures of capsicum annuum

    International Nuclear Information System (INIS)

    The effect of different concentrations of cellulase on the production of capsaicin in freely suspended cell and immobilized cell cultures of Kahramanmara pepper seeds (Capsicum annuum L.) were studied. Calluses were obtained from in vitro germinated hypocotyl explants of pepper seedlings and cell suspensions were prepared from these calluses. Immobilized cell suspension cultures with calcium alginate and free cell suspension cultures were obtained by using cell suspensions. Elicitor such as cellulase (5-30 micro g/ml), was applied both for the free and immobilized cell suspensions and control group without elicitor was prepared. The concentration of capsaicin in freely suspended cells, immobilized cells and their filtrates were identified by HPLC after extraction with ethyl acetate. It was found that the immobilization process had an increasing effect on the capsaicin accumulation. The concentration of capsaicin in the immobilized cells for both control groups and elicitor added samples was higher than the free cells. In general, capsaicin concentration in the filtrate for free cells was higher than the immobilized cells. When all the cellulase and the sampling hours were compared, the highest capsaicin concentration for the immobilized cells was determined as 362,91 micro g/ml f.w. at the 24th hour for 30 micro g/ml cellulase applied samples. (author)

  4. Utilization of spent coffee grounds for isolation and stabilization of Paenibacillus chitinolyticus CKS1 cellulase by immobilization.

    Science.gov (United States)

    Buntić, Aneta V; Pavlović, Marija D; Antonović, Dušan G; Šiler-Marinković, Slavica S; Dimitrijević-Branković, Suzana I

    2016-08-01

    This study has explored the feasibility of using spent coffee grounds as a good supporting material for the Paenibacillus chitinolyticus CKS1 cellulase immobilization. An optimal operational conditions in a batch-adsorption system were found to be: carrier mass of 12 g/L, under the temperature of 45 °C and no pH adjustments. The immobilization yield reached about 71%. An equilibrium establishment between the cellulase and the carrier surface occurred within 45 min, whereas the process kinetics may be predicted by the pseudo-second-order model. An immobilized cellulase preparation expressed very good avicelase activity, this reached up to 2.67 U/g, and revealed an improved storage stability property, compared to free enzyme sample counterpart. The addition of metal ions, such as K(+) and Mg(2+) did not affect positively immobilization yield results, but on the contrary, contributed to an improved bio-activities of the immobilized cellulase, thus may be employed before each enzyme application. The method developed in this study offers a cheap and effective alternative for immediate enzyme isolation from the production medium and its stabilization, compared to other carriers used for the immobilization. PMID:27626091

  5. Development and selection of fungal and bacterial mutants using ionizing radiation and radioisotopes for improved enzyme production (cellulase and coagulase)

    International Nuclear Information System (INIS)

    Ultraviolet and gamma radiations, chemical mutagens, and combinations of chemical and physical mutagens were used in order to obtain mutants of Bacillus mesentericus and Trichoderma viridae with a higher production of coagulase and cellulase, respectively. It was possible to isolate mutant strains, with enzyme activity increased by a factor of 2 and 3

  6. Neurospora crassa tox-1 Gene Encodes a pH- and Temperature-Tolerant Mini-Cellulase.

    Science.gov (United States)

    Xiao, Yue; Zhang, Qiongsi; Luo, Yiquan; Zhang, Ying; Luo, Xi; Wang, Yuchuan; Cao, Weiguo; Pinto, Vito De; Liu, Qiuyun; Li, Gang

    2016-06-15

    Cellulases that endure extreme conditions are essential in various industrial sectors. This study reports a mini-cellulase gene tox-1 from Neurospora crassa. The gene tox-1 was cloned in Escherichia coli after chimerization with the YebF gene and substitutions of certain isoleucine and valine with leucine residues. The yeast transformants could grow on rice straw-agar medium. The 44-amino acid peptide and its two mutant variants displayed potent cellulase activities in Congo Red assay and enzymatic assays. Conservative replacements with leucine have substantially increased the stabilities and half-lives of the peptides at alkaline pH and low and high temperatures and also the tolerance to organic solvents and surfactants, on the basis of activities toward cellose. The small size of the mini-cellulase would allow for commercially viable automatic chemical peptide synthesis. This work suggests that conservative leucine replacements may serve as a general strategy in the engineering of more robust enzymes with special features with little loss of activities. PMID:27229865

  7. Synergistic action of recombinant accessory hemicellulolytic and pectinolytic enzymes to Trichoderma reesei cellulase on rice straw degradation.

    Science.gov (United States)

    Laothanachareon, Thanaporn; Bunterngsook, Benjarat; Suwannarangsee, Surisa; Eurwilaichitr, Lily; Champreda, Verawat

    2015-12-01

    Synergism between core cellulases and accessory hydrolytic/non-hydrolytic enzymes is the basis of efficient hydrolysis of lignocelluloses. In this study, the synergistic action of three recombinant accessory enzymes, namely GH62 α-l-arabinofuranosidase (ARA), CE8 pectin esterase (PET), and GH10 endo-1,4-beta-xylanase (XYL) from Aspergillus aculeatus expressed in Pichia pastoris to a commercial Trichoderma reesei cellulase (Accellerase® 1500; ACR) on hydrolysis of alkaline pretreated rice straw was studied using a mixture design approach. Applying the full cubic model, the optimal ratio of quaternary enzyme mixture was predicted to be ACR:ARA:PET:XYL of 0.171:0.079:0.100:0.150, which showed a glucose releasing efficiency of 0.173 gglc/FPU, higher than the binary ACR:XYL mixture (0.122 gglc/FPU) and ACR alone (0.081 gglc/FPU) leading to a 47.3% increase in glucose yield compared with that from ACR at the same cellulase dosage. The result demonstrates the varying degree of synergism of accessory enzymes to cellulases useful for developing tailor-made enzyme systems for bio-industry. PMID:26433794

  8. Screening and characterization of a novel cellulase gene from the gut microflora of Hermetia illucens using metagenomic library.

    Science.gov (United States)

    Lee, Chang-Muk; Lee, Young-Seok; Seo, So-Hyeon; Yoon, Sang-Hong; Kim, Soo-Jin; Hahn, Bum-Soo; Sim, Joon-Soo; Koo, Bon-Sung

    2014-09-01

    A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50°C and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20~50°C and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thinlayer chromatography suggested that CS10 is an endo-β-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes. PMID:25022521

  9. Screening, cloning and expression analysis of a cellulase derived from the causative agent of hypertrophy sorosis scleroteniosis, Ciboria shiraiana.

    Science.gov (United States)

    Lü, Ruihua; Zhao, Aichun; Li, Jun; Liu, Changying; Wang, Chuanhong; Wang, Xiling; Wang, Xiaohong; Pei, Ruichao; Lu, Cheng; Yu, Maode

    2015-07-10

    A cellulase gene (KJ700939, CsCelA) from Ciboria shiraiana that is highly expressed during the infection of mulberry fruit was screened by quantitative real-time PCR (qRT-PCR). Using cDNA isolated from infected mulberry fruits as template, the full-length 1170-bp sequence of CsCelA was obtained, which encodes a 390-amino acid protein with a putative signal peptide of 24 amino acids. The 998-bp fragment encoding the mature peptide of CsCelA was cloned into the multiple cloning site of the pPIC9K vector and overexpressed as an active protein of 55.3kDa in the methylotrophic yeast Pichia pastoris. The specific activity of induced supernatants of the recombinant cellulase (CsCelA) was 17.44U/ml and 135U/g for freeze-dried powder. The Kmax and Vmax of CsCelA for sodium carboxymethylcellulose (CMC) were 4.6mg/ml and 107.2U/mg, respectively. The supernatant and freeze-dried powder of the recombinant cellulase exhibited stable activity from pH4.0 to 9.0, and at temperatures ranging from 30°C to 55°C. Finally, the activity of the recombinant cellulase was assessed by enzymatic hydrolysis of the cell walls of mulberry leaves. CsCelA showed an endo-cellulase mode of cleavage, as assessed by thin layer chromatography (TLC). PMID:25865297

  10. Effect of cellulase producing fungi on plant residues degradation used as organic fertilizer

    International Nuclear Information System (INIS)

    Series of laboratory and field experiments were conducted at Soil microbiology Unit and Farm of soil and Water research department, Nuclear Research Center, Atomic Energy Authority, Egypt. Laboratory experiments revealed that between nine fungal strain, A. niger was the most potent cellulolytic fungus able to degrade many cellulosic sources (CP, CMC, and FP). Study the effect of cellulolytic fungi on degradation of plant residues used as organic fertilizer in addition to nitrogen fixing bacteria (symbiotically) on lupine growth, yield and nutrients uptake (Field experiment) had been carried out. This objective aims to recycling different plant residues in soil which is consistent with (sustainable development) and utilization of these organic residues as a single carbon source for cellulolytic fungi.Application of 15N- tracer technique gave us the chance and opportunity to quantify the exact amounts of N derived from the different sources of nitrogen available to lupine plant under the effect of cellulolytic fungi on different plant residues.The obtained results could be summarized as following:I.Laboratory Technique Selection of the most potent cellulolytic fungi 1-Nine fungal strains of Aspergillus niger; Penicillium oxalicum; Trichoderma longibranchiatum; Aspergillus terreus; Aspergillus flavus; Alterrnaria sp.; Trichderma harzianum ; Rhizopus sp. and Syncephalastrum sp. obtained from different sources and tested for their cellulolytic activity. 2-Aspergillus niger and Pencillium oxalicum exhibited the highest cellulase productivity followed by Trichoderma longibranchiatum and Aspergillus terreus.3- fungal mixtures of the most potent four genera Aspergillus niger; Penicillium oxalicum; Trichoderma longibranchiatum and Aspergillus terreus found to have a lower cellulolytic activities for all substrates compared with single inoculation with A. niger.4-Highest FPase activities were exhibited by A. niger when filter paper (FP) used as a carbon source.5-A. niger is

  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. 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.

  13. Produção de celulases por Aspergillus niger e cinética da desativação celulásica=Cellulases production by Aspergillus niger and cellulase deactivation kinetic

    Directory of Open Access Journals (Sweden)

    Caroline Mariana de Aguiar

    2011-10-01

    Full Text Available O presente trabalho teve como objetivo a avaliação da cinética de produção de enzimas celulases pelo fungo Aspergillus niger e da cinética de desativação das celulases. Foi utilizado bagaço de cana-de-açúcar pré-tratado como fonte de carbono na fermentação para a produção do complexo celulásico e também como substrato da hidrólise enzimática. A. niger foi cultivado em três bateladas, cada uma contendo 10, 50 e 100 g L-1 de bagaço pré-tratado com NaOH 4% (m v-1. A cinética da produção das celulases foi obtida determinando-se a atividade enzimática das amostras coletadas ao longo do tempo. As variações do pH também foram determinadas. A deativação enzimática foi avaliada determinando-se periodicamente a atividade das amostras armazenadas nas condições de resfriamento (4°C e de congelamento (-18ºC. Conclui-se que o A. niger produz celulases quando cultivado em meio de cultivo contendo bagaço de cana-de-açúcar pré-tratado e que o tempo ideal para coleta do caldo enzimático foi de aproximadamente sete dias, com produtividade máxima de 0,0013 U mL-1∙h para a batelada com 10 g L-1 e 0,0018 U mL-1∙h para as bateladas com 50 e 100 g L-1. O complexo celulásico não sofre desativação se armazenado à temperatura de -18°C por 43 dias, mas perde cerca de 40% da sua atividade após 48h se armazenado a 4°C.This work aimed to evaluate the kinetic for the cellulase production by Aspergillus niger and the deactivation kinetic of the cellulase enzymes. Cellulase were produced in three different batches using NaOH 4% (w v-1 pre-treated sugarcane bagasse as the carbon source in the fermentation broth. The amount of the bagasse in each batch was 10, 50 and 100 g L-1. The kinetic of the cellulase production was accomplished by periodically determining the cellulasic activity of the fermentation broth using pre-treated bagasse as the hydrolysis substrate. Changes in the pH also were determined. The cellulase

  14. Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei.

    Science.gov (United States)

    Bischof, Robert H; Ramoni, Jonas; Seiboth, Bernhard

    2016-01-01

    More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei. PMID:27287427

  15. Cellulose chain binding free energy drives the processive move of cellulases on the cellulose surface.

    Science.gov (United States)

    Wang, Yefei; Zhang, Shujun; Song, Xiangfei; Yao, Lishan

    2016-09-01

    Processivity is essential for cellulases in their catalysis of cellulose hydrolysis. But what drives the processive move is not well understood. In this work, we use Trichoderma reesei Cel7B as a model system and show that its processivity is directly correlated to the binding free energy difference of a cellulose chain occupying the binding sites -7 to +2 and that occupying sites -7 to -1. Several mutants that have stronger interactions with glycosyl units in sites +1 and +2 than the wild type enzyme show higher processivity. The results suggest that after the release of the product cellobiose located in sites +1 and +2, the enzyme pulls the cellulose chain to fill the vacant sites, which propels its processive move on the cellulose surface. Biotechnol. Bioeng. 2016;113: 1873-1880. © 2016 Wiley Periodicals, Inc. PMID:26928155

  16. Enhanced cellulase and β-glucosidase production by a mutant of Alternaria alternata

    International Nuclear Information System (INIS)

    The cellulolytic activity of the wild type and a mutant strain of A. alternata was investigated. Mutants were induced by gamma radiation. A suspension of about 105 condidia/mL in 0.05M phosphate buffer pH 5 were irradiated in a gamma-cell-type (Cammacell 220, Atomic Energy of Canada Limited, Ottawa, Canada) 60Co source with a dose rate of 2.5 krad/min. The amount of radiation given was 70 krad which resulted in about 10% survival level. The stock culture was maintained on a sterile growth medium supplemented with 1% cellulose 123 and 0.3% agar. Following the incubation period, the fungal biomass was harvested by centrifugation (5000g for 10 min) and the clarified supernatant was used as the source of cellulase and β-glucosidase

  17. Cellulase production by halophytic fungi. Pt. 1. Screening experiments and salt relations

    Energy Technology Data Exchange (ETDEWEB)

    Sallam, L.A.R.; EL-Refai, A.M.H.; El-Sayed, S.M.

    1988-01-01

    The potentiality of 3 local halophytic fungi, isolated from the salt marshes at Burg El-Arab area in the Western Mediterranean Coast of Egypt, to produce cellulose-hydrolysing enzymes has been studied under static culture conditions using 4 different media charged with avicel as a sole carbon source. Ulocladium chartarum was selected as the most potent for cellulase production. This fungus has not been proviously reported to possess cellulolytic activities. Dispensing the basal medium 2 mm depth and incubation of U. chartarum for 3 weeks were the most favourable conditions for best cellulolytic activities. The production of cellulolytic enzymes was markedly affected by the salt supplied and its concentration. Best CMCase and FPase activities (0.289 and 0.043 unit/ml respectively) were noticed on using 60 g Na/sub 2/SO/sub 4//l.

  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

    were incubated for 48 h at 39 °C in the cellulase solution (celullase Onozuka R10), in the jars of DaisyII Incubator (ANKOM Technology Corporation). After incubation the bags with residues were extracted in a neutral detergent (ND) for 1 h at 100 °C in Ankom220 Fiber Analyzer (ANKOM Technology...... calculated for whole crop corn herbages, grass silages and clover silages. It can be concluded that the in vitro method presented in this study is a simple alternative for existing methods in which buffered rumen fluid is used. Using a standard enzyme available commercially worldwide may decrease variation...... 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. Cellulase production by two mutant strain of Trichoderma longibranchiatum QM 9414 and Rut C30

    International Nuclear Information System (INIS)

    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

  20. 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.

  1. The Activity of Cellulase from Thermophilic Fungi Isolated from CaneBagasses

    International Nuclear Information System (INIS)

    The activity of cellulase from thermophilic fungi isolated from canebagasses has been measured. This wild strain, named fungal strain PJ-2,secreted a large amount of cellulolytic enzyme components consisting of 0.46units of avicelase, 0.8 units of carboxymethyl cellulose hydrolizing enzyme(CMCase) and 0.5 units of β-glucosidase per ml of culture broth oncultivation in Mandels Reese medium for 7 days at 500 oC. These cellulasesproduction was lower than that of Trichoderma reesei NRRL 3653 producing 0.5units/ml avicelase, 1.6 units/ml CMCase and 0.4 units/ml ofβ-glucosidase cultivated in the same medium at 30 oC. However,thermophilic fungi may be potential to be exploited in lignocellulosedegradation at the tropical areas as the process usually needs temperature ofabove 50 oC. (author)

  2. Cellulase hydrolysis of rice straw and inactivation of endoglucanase in urea solution.

    Science.gov (United States)

    Tao, Yi-Ming; Xu, Xin-Qi; Ma, Su-Juan; Liang, Ge; Wu, Xiao-Bing; Long, Min-Nan; Chen, Qing-Xi

    2011-10-26

    In order to optimize the cellulase (from Aspergillus glaucus) hydrolysis of pretreated rice straw, the effects of varying enzyme concentration, temperature, and pH were studied. The best experimental conditions found to degrade the pretreated rice straws were 24 h of incubation at 55 °C and pH 5.0, with an enzyme concentration of 48 mg/L. Urea is one of the important nitrogen sources used in fungi culture, but it is also a denaturant. The model of denaturation of endoglucanase (EG) in urea solutions was established. The denaturation was a slow, reversible reaction. Determination of microscopic rate constants showed k(+0) > k'(+0), indicating that EG was protected by the substrate to a certain extent during denaturation. Comparison with the results from fluorescence emission spectroscopy revealed that the inactivation of EG occurred before the marked conformational changes could be detected. PMID:21919515

  3. 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.

  4. 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

    2010-01-01

    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. PMID:20497524

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

    Directory of Open Access Journals (Sweden)

    Parilla Philip A

    2010-05-01

    Full Text Available Abstract 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.

  6. Monocentric and polycentric anaerobic fungi produce structrally related cellulases and xylanases

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xin-Liang; Chen, Huizhong; Ljungdahl, L.G. [Univ. of Georgia, Athens, GA (United States)

    1997-02-01

    Cellulase and xylanase cDNAs were isolated from a cDNA library of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 constructed in Escherichia coli. The cellulase cDNA (celB) was 1.8 kb long with an open reading frame (ORF) coding for a polypeptide of 471 amino acids, and the xylanase cDNA (xynA) was 1.2 kb long with an ORF encoding a polypeptide of 362 amino acids. Single transcripts of 1.9 kb for celB and 1.5 kb for xynA were detected in total RNA of Orpinomyces grown on Avicel. Genomic DNA regions coding for CelA and XynA were devoid of introns. The enzymes were highly homologous (80 to 85% identity) to the corresponding enzymes of the monocentric anaerobic fungus Neocallimastix patriciarum and, like those, contained in addition to a catalytic domain, a noncatalytic repeated peptide domain (NCRPD). The Orpinomyces xylanase contained one catalytic domain and thus differed from the Neocallimastix xylanase, which had two similar catalytic domains. Two peptides corresponding to the catalytic domain and the NCRPD of XynA were synthesized, and antibodies against them were raised and affinity column purified. The antibodies against the catalytic domain peptide reacted specifically with the xylanases of Orpinomyces and Neocallimastix, while the antibodies against the NCRPD reacted with many (at least eight) extracellular proteins of Orpinomyces and Neocallimastix, suggesting that the NCRPD is present in a number of polypeptides. 36 refs., 8 figs., 2 tabs.

  7. Biochemical Characterization of a Protease Involved in the Processing of a Streptomyces reticuli Cellulase (Avicelase).

    Science.gov (United States)

    Moormann, M; Schlochtermeier, A; Schrempf, H

    1993-05-01

    A 36-kDa protease from Streptomyces reticuli had recently been shown to be responsible for the in vivo and in vitro processing of the 82-kDa cellulase (Avicelase) Cel-1 from S. reticuli to a 42-kDa truncated enzyme. It was induced only in the presence of Avicel, hydroxyethylcellulose, and xylan. The addition of the nonionic detergent Tween 80 to the culture medium containing Avicel as the carbon source led to a 10-fold increase in extracellular proteolytic activity. The protease, which has an isoelectric point of 3.9, was purified to homogeneity from the culture filtrate by a combination of anion-exchange and hydrophobic-interaction chromatographies and was characterized biochemically. The enzyme hydrolyzed gelatin and the chromogenic substrates Azocoll, Azocasein, and Azoalbumin. Its highest activity was determined between pH 7.0 and 7.7 and at 55 degrees C. The proteolytic activity was inhibited by 1,10-phenanthroline and EDTA; however, no metal ions were detected to be associated with the protein. The protease was stable in the presence of 1 M urea and 0.01 M sodium dodecyl sulfate. The inhibitory effect of alpha-2-macroglobulin indicated an endo-mode of proteolytic cleavage. Studies with lectins and sugar analysis by mass spectroscopy indicated that the cellulase (Avicelase) Cel-1 was neither N nor O glycosylated. Its processing by the protease occurred at temperatures ranging from 30 to 55 degrees C, pH 7.5, in the presence of 2 mM dithiothreitol. PMID:16348937

  8. Genome sequencing of the Trichoderma reesei QM9136 mutant identifies a truncation of the transcriptional regulator XYR1 as the cause for its cellulase-negative phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Lichius, Alexander; Bidard, Frederique; Buchholz, Franziska; Le Crom, Stphane; Martin, Joel X.; Schackwitz, Wendy; Austerlitz, Tina; Grigoriev, Igor V.; Baker, Scott E.; Margeot, Antoine; Seiboth, Bernhard; Kubicek, Christian P.

    2015-12-01

    Background: Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a. Results: In QM9136, we detected a surprisingly low number of mutagenic events in the promoter and coding regions of genes, i.e. only eight indels and six single nucleotide variants. One of these indels led to a frame-shift in the Zn2Cys6 transcription factor XYR1, the general regulator of cellulase and xylanase expression, and resulted in its C-terminal truncation by 140 amino acids. Retransformation of strain QM9136 with the wild-type xyr1 allele fully recovered the ability to produce cellulases, and is thus the reason for the cellulase-negative phenotype. Introduction of an engineered xyr1 allele containing the truncating point mutation into the moderate producer T. reesei QM9414 rendered this strain also cellulase-negative. The correspondingly truncated XYR1 protein was still able to enter the nucleus, but failed to be expressed over the basal constitutive level. Conclusion: The missing 140 C-terminal amino acids of XYR1 are therefore responsible for its previously observed auto-regulation which is essential for cellulases to be expressed. Our data present a working example of the use of genome sequencing leading to a functional explanation of the QM9136 cellulase-negative phenotype.

  9. 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.

  10. ROLE OF ALKALINE-TOLERANT FUNGAL CELLULASES IN RELEASE OF TOTAL ANTIOXIDANTS FROM AGRO-WASTES UNDER SOLID STATE FERMENTATION

    Directory of Open Access Journals (Sweden)

    Chinnarajan Ravindran

    2011-07-01

    Full Text Available The alkaline-tolerant marine-derived fungus Chaetomium globosum was tested for the production of enhanced levels of cellulases and free phenolics under highly alkaline conditions using agro wastes (cotton seed, sugar cane bagasse as substrates under solid state fermentation (SSF processes. In both the agro wastes used, an increase in cellulases (β-endoglucanase, β-Glucosidase, and β-exoglucanase production was observed with increase in pH. This enhanced carbohydrate-hydrolyzing enzymes (β-endoglucanase, β-Glucosidase and β-exoglucanase and thereby enriched the total phenolic release from agro-wastes under SSF conditions of higher pH. A linear correlation was observed between released total phenolic contents of agro-wastes and total antioxidant property. The increased antioxidant activity on free radical scavenging was also observed with the increase in pH. Thus, the present study makes it possible to produce nutraceutical ingredients cost-effectively from agricultural wastes.

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

    OpenAIRE

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

    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 centr...

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

    OpenAIRE

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

    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...

  13. Thermostability Mechanism for the Hyperthermophilicity of Extremophile Cellulase TmCel12A: Implied from Molecular Dynamics Simulation.

    Science.gov (United States)

    Lian, Peng; Yuan, Congmin; Xu, Qin; Fu, Wei

    2016-08-01

    Thermostability is of considerable importance for the application of cellulase in cellulosic ethanol production. The cellulase 12A from the hyperthermophile Thermotoga maritima (TmCel12A) is an ideal candidate to study thermostability of cellulases. Optimal temperature of the wild-type enzyme is 85 °C. Recently, it has been observed that surface loop mutation Y61G not only accelerates the hydrolysis rate but also extends the half-life of the enzyme at high temperature. However, the mechanism of how Y61G enhances thermostability of TmCel12A has not been revealed. Here, molecular dynamics simulation together with dynamic correlation network analysis was used to explore thermostability mechanism of TmCel12A. A hydrophobic cluster constructed by Y61, W176, V62, and L144 in the binding pocket was found to play a pivotal role in modulating thermostability as well as catalytic capability of TmCel12A. It stabilizes the apoenzyme at high temperature; however, it impedes the substrate binding. Y61G mutation disturbs the hydrophobic cluster as the counterpart amino acid W176 forms a cation-π interaction with R60 instead of the π-π interaction with Y61 in WT. Moreover, Y61G mutation makes the enzyme more rigid and more extended via altering the amino acid communities at the hinge part of the enzyme. An earlier hypothesis proposed from crystallographic observation that Y61G may accelerate the products releasing has been also confirmed by our simulations. These findings may provide a new direction for both theoretical and experimental scientists to improve the thermostability of other cellulases that can be potentially applied in biofuel industry. PMID:27384708

  14. Kinetic transcriptome analysis reveals an essentially intact induction system in a cellulase hyper-producer Trichoderma reesei strain

    OpenAIRE

    Poggi-Parodi, Dante; Bidard, Frédérique; Pirayre, Aurélie; Portnoy, Thomas; Blugeon, Corinne; Seiboth, Bernhard; Kubicek, Christian P.; Le Crom, Stéphane; Margeot, Antoine

    2014-01-01

    Background The filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can ...

  15. Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes

    OpenAIRE

    Eman Zakaria Gomaa

    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, re...

  16. Sulphur metabolism and cellulase gene expression are connected processes in the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei

    Directory of Open Access Journals (Sweden)

    Schmoll Monika

    2008-10-01

    Full Text Available Abstract Background Sulphur compounds like cysteine, methionine and S-adenosylmethionine are essential for the viability of most cells. Thus many organisms have developed a complex regulatory circuit that governs the expression of enzymes involved in sulphur assimilation and metabolism. In the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei little is known about the participants in this circuit. Results Analyses of proteins binding to the cellulase activating element (CAE within the promotor of the cellobiohydrolase cbh2 gene led to the identification of a putative E3 ubiquitin ligase protein named LIMPET (LIM1, which is an orthologue of the sulphur regulators SCON-2 of Neurospora crassa and Met30p of Saccharomyces cerevisiae. Transcription of lim1 is specifically up-regulated upon sulphur limitation and responds to cellulase inducing conditions. In addition, light dependent stimulation/shut down of cellulase gene transcription by methionine in the presence of sulphate was observed. Further, lim1 transcriptionally reacts to a switch from constant darkness to constant light and is subject to regulation by the light regulatory protein ENVOY. Thus lim1, despite its function in sulphur metabolite repression, responds both to light as well as sulphur- and carbon source. Upon growth on cellulose, the uptake of sulphate is dependent on the light status and essential for growth in light. Unlike other fungi, growth of H. jecorina is not inhibited by selenate under low sulphur conditions, suggesting altered regulation of sulphur metabolism. Phylogenetic analysis of the five sulphate permeases found in the genome of H. jecorina revealed that the predominantly mycelial sulphate permease is lacking, thus supporting this hypothesis. Conclusion Our data indicate that the significance of the sulphate/methionine-related signal with respect to cellulase gene expression is dependent on the light status and reaches beyond detection of sulphur

  17. Isolation and Characterization of Thermophilic Cellulase-Producing Bacteria from Empty Fruit Bunches-Palm Oil Mill Effluent Compost

    OpenAIRE

    Azhari S. Baharuddin; Mohamad N.A. Razak; Lim S. Hock; Mohd N. Ahmad; , Suraini Abd-Aziz,; Nor A.A. Rahman; Umi K.M Shah; Mohd A. Hassan; Kenji Sakai; Yoshihito Shirai

    2010-01-01

    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 ...

  18. Enhancement of fermentable sugar yield by competitive adsorption of non-enzymatic substances from yeast and cellulase on lignin

    OpenAIRE

    Tang, Yong; Lei, Fuhou; Cristhian, Carrasco; Liu, Zuguang; Yu, Hailong; Jiang, Jianxin

    2014-01-01

    Background Enhancement of enzymatic digestibility by some supplementations could reduce enzyme loading and cost, which is still too high to realize economical production of lignocellulosic biofuels. A recent study indicates that yeast hydrolysates (YH) have improved the efficiency of cellulases on digestibility of furfural residues (FR). In the current work, the components of YH were separated by centrifugation and size exclusion chromatography and finally characterized in order to better und...

  19. The VELVET A orthologue VEL1 of Trichoderma reesei regulates fungal development and is essential for cellulase gene expression

    OpenAIRE

    Karimi Aghcheh, Razieh; Németh Zoltán (1987-) (biomérnök); Atanasova, Lea; Fekete Erzsébet (1975-) (biotechnológus); Paholcsek Melinda (1984-) (molekuláris biológus, genetikus); Sándor Erzsébet; Aquino, Benigno; Druzhinina, Irina S.; Karaffa Levente; Kubicek, Christian P.

    2014-01-01

    Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. ...

  20. The correlation between mannanase and cellulase activities towards fibre content of palm oil sludge fermented with Aspergillus niger

    Directory of Open Access Journals (Sweden)

    T. Purwadaria

    1998-12-01

    Full Text Available Enzyme (mannanase and cellulase activities and fibre (hemicellulose, cellulose and lignin contents were determined during the fermentation course of palm oil sludge with Aspergillus niger TL (wild type and A. niger ES I (an asporogenous mutant. The analyses were carried out at the incubation time of 3 and 4 days of aerobic fennentation and at 2 days of anaerobic fermentation afterward. The correlations between mamlanase activity with hemicellulose content and cellulose activity with cellulose content were calculated by linear regression . The activities of matutanase and cellulase are increasing during the aerobic fennentation, while in the anaerobic fennentation the enzyme activities are decreasing due to instability of the enzymes. The enzyme activities of ESI are higher than the TL. The regression coefficient is highly significant for correlation between mamlanase and hemicellulose content of fermented product by ESI (r = 0.83; P0 .05 . Marutanase and cellulase activities were also detected after the fermented product dried at 60°C which indicated the enzymes are quite stable .

  1. 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. PMID:24839153

  2. Correlation between Agar Plate Screening and Solid-State Fermentation for the Prediction of Cellulase Production by Trichoderma Strains

    Directory of Open Access Journals (Sweden)

    Camila Florencio

    2012-01-01

    Full Text Available The viability of converting biomass into biofuels and chemicals still requires further development towards the reduction of the enzyme production costs. Thus, there is a growing demand for the development of efficient procedures for selection of cellulase-producing microorganisms. This work correlates qualitative screening using agar plate assays with quantitative measurements of cellulase production during cultivation under solid-state fermentation (SSF. The initial screening step consisted of observation of the growth of 78 preselected strains of the genus Trichoderma on plates, using microcrystalline cellulose as carbon source. The 49 strains that were able to grow on this substrate were then subjected to a second screening step using the Congo red test. From this test it was possible to select 10 strains that presented the highest enzymatic indices (EI, with values ranging from 1.51 to 1.90. SSF cultivations using sugarcane bagasse and wheat bran as substrates were performed using selected strains. The CG 104NH strain presented the highest EGase activity (25.93 UI·g−1. The EI results obtained in the screening procedure using plates were compared with cellulase production under SSF. A correlation coefficient (R2 of 0.977 was obtained between the Congo red test and SSF, demonstrating that the two methodologies were in good agreement.

  3. 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. PMID:27297903

  4. 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. PMID:26724278

  5. 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 %. PMID:24052336

  6. Nitrogen source optimization for cellulase production by Penicillium funiculosum, using a sequential experimental design methodology and the desirability function.

    Science.gov (United States)

    Maeda, Roberto Nobuyuki; da Silva, Mariana Mello Pereira; Santa Anna, Lídia Maria Melo; Pereira, Nei

    2010-05-01

    The present study aimed at maximizing cellulase production by Penicillium funiculosum using sequential experimental design methodology for optimizing the concentrations of nitrogen sources. Three sequential experimental designs were performed. The first and the second series of experiments consisted of a 2(4) and a 2(3) factorial designs, respectively, and in the third one, a central composite rotational design was used for better visualizing the optimum conditions. The following nitrogen sources were evaluated: urea, ammonium sulfate, peptone, and yeast extract. Peptone and ammonium sulfate were removed from the medium optimization since they did not present significant statistical effect on cellulase production. The optimal concentrations of urea and yeast extract predicted by the model were 0.97 and 0.36 g/L, respectively, which were validated experimentally. By the use of the desirability function, it was possible to maximize the three main enzyme activities simultaneously, which resulted in values for FPase of 227 U/L, for CMCase of 6,917 U/L, and for beta-glucosidase of 1,375 U/L. These values corresponded to increases of 3.3-, 3.2-, and 6.7-folds, respectively, when compared to those obtained in the first experimental design. The results showed that the use of sequential experimental designs associated to the use of the desirability function can be used satisfactorily to maximize cellulase production by P. funiculosum. PMID:20013074

  7. Soluble inhibitors generated during hydrothermal pretreatment of oil palm mesocarp fiber suppressed the catalytic activity of Acremonium cellulase.

    Science.gov (United States)

    Zakaria, Mohd Rafein; Hirata, Satoshi; Fujimoto, Shinji; Ibrahim, Izzudin; Hassan, Mohd Ali

    2016-01-01

    Oil palm mesocarp fiber was subjected to hydrothermal pretreatment under isothermal and non-isothermal conditions. The pretreated slurries were separated by filtration, pretreated liquids and solids were characterized. An enzymatic digestibility study was performed for both pretreated slurries and solids to understand the effect of soluble inhibitors generated during the pretreatment process. The highest glucose yield obtained from pretreated slurries was 70.1%, and gradually decreased with higher pretreatment severities. The highest glucose yield obtained in pretreated solids was 100%, after pretreatment at 210°C for 20min. In order to study the inhibitory effects of compounds generated during pretreatment with cellulase, technical grade solutions that mimic the pretreated liquid were prepared and their effect on Acremonium cellulase activity was monitored using Avicel. Xylo-oligomers and tannic acid were identified as powerful inhibitors of Acremonium cellulase, and the lowest hydrolysis rate of Avicel of 0.18g/g-glucose released/L/h was obtained from tannic acid. PMID:26524253

  8. Composition and microstructure alteration of triticale grain surface after processing by enzymes of cellulase complex

    Directory of Open Access Journals (Sweden)

    Elena Kuznetsova

    2016-01-01

    Full Text Available It is found that the pericarp tissue of grain have considerable strength and stiffness, that has an adverse effect on quality of whole-grain bread. Thereby, there exists the need for preliminary chemical and biochemical processing of durable cell walls before industrial use. Increasingly used in the production of bread finds an artificial hybrid of the traditional grain crops of wheat and rye - triticale, grain which has high nutritional value. The purpose of this research was to evaluate the influence of cellulose complex (Penicillium canescens enzymes on composition and microstructure alteration of triticale grain surface, for grain used in baking. Triticale grain was processed by cellulolytic enzyme preparations with different composition (producer is Penicillium canescens. During experiment it is found that triticale grain processing by enzymes of cellulase complex leads to an increase in the content of water-soluble pentosans by 36.3 - 39.2%. The total amount of low molecular sugars increased by 3.8 - 10.5 %. Studies show that under the influence of enzymes the microstructure of the triticale grain surface is changing. Microphotographs characterizing grain surface structure alteration in dynamic (every 2 hours during 10 hours of substrate hydrolysis are shown. It is found that the depth and direction of destruction process for non-starch polysaccharides of grain integument are determined by the composition of the enzyme complex preparation and duration of exposure. It is found, that xylanase involved in the modification of hemicelluloses fiber having both longitudinal and radial orientation. Hydrolysis of non-starch polysaccharides from grain shells led to increase of antioxidant activity. Ferulic acid was identified in alcoholic extract of triticale grain after enzymatic hydrolysis under the influence of complex preparation containing cellulase, xylanase and β-glucanase. Grain processing by independent enzymes containing in complex

  9. 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. PMID:26256022

  10. Statistical optimization of cellulases production by Penicillium chrysogenum QML-2 under solid-state fermentation and primary application to chitosan hydrolysis.

    Science.gov (United States)

    Zhang, Hui; Sang, Qing

    2012-03-01

    Solid-state fermentation conditions for cellulases production by a newly isolated Penicillium chrysogenum QML-2 were investigated using statistical methods. At first, significant variables for cellulases production including (NH(4))(2)SO(4), initial pH and inoculum size were screened by using Plackett-Burman Design. Then the optimal regions of the significant variables were investigated by using the method of steepest ascent. Finally, central composite design and response surface analysis were adopted to determine the optimal values of the significant variables and investigate the combined effects of each variable's pair on cellulases production. The results showed that the optimal ranges of (NH(4))(2)SO(4) concentration, initial pH and inoculum size for three types of cellulases activities were 1.97-2.15 g, pH 4.32-4.41 and 13.3-13.7% (v/w), respectively. Using the mixture of corn stover powder and wheat bran (CSP/WB, 1/1) as carbon source, the optimization resulted in 370.15, 101.76 and 321.56 U/g for maximal endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. Compared with maximum values of cellulases activities (endoglucanase activity 85.21 U/g, filter paper activity 16.62 U/g and β-glucosidase activity 67.68 U/g) obtained under unoptimized conditions, the optimization resulted in 3.34, 5.12 and 3.75 folds improvement for endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. For chitosan hydrolysis, the crude cellulases had the optimal temperature of 55°C, pH of 4.4 and exhibited Michaelis constant (K (m)) value of 8.34 mg/ml and maximum velocity (V (max)) of 2.21 μmol glucosamine/min by 1 ml of the crude cellulases. PMID:22805837

  11. 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. PMID:21898149

  12. Dramatic performance of Clostridium thermocellum explained by its wide range of cellulase modalities.

    Science.gov (United States)

    Xu, Qi; Resch, Michael G; Podkaminer, Kara; Yang, Shihui; Baker, John O; Donohoe, Bryon S; Wilson, Charlotte; Klingeman, Dawn M; Olson, Daniel G; Decker, Stephen R; Giannone, Richard J; Hettich, Robert L; Brown, Steven D; Lynd, Lee R; Bayer, Edward A; Himmel, Michael E; Bomble, Yannick J

    2016-02-01

    Clostridium thermocellum is the most efficient microorganism for solubilizing lignocellulosic biomass known to date. Its high cellulose digestion capability is attributed to efficient cellulases consisting of both a free-enzyme system and a tethered cellulosomal system wherein carbohydrate active enzymes (CAZymes) are organized by primary and secondary scaffoldin proteins to generate large protein complexes attached to the bacterial cell wall. This study demonstrates that C. thermocellum also uses a type of cellulosomal system not bound to the bacterial cell wall, called the "cell-free" cellulosomal system. The cell-free cellulosome complex can be seen as a "long range cellulosome" because it can diffuse away from the cell and degrade polysaccharide substrates remotely from the bacterial cell. The contribution of these two types of cellulosomal systems in C. thermocellum was elucidated by characterization of mutants with different combinations of scaffoldin gene deletions. The primary scaffoldin, CipA, was found to play the most important role in cellulose degradation by C. thermocellum, whereas the secondary scaffoldins have less important roles. Additionally, the distinct and efficient mode of action of the C. thermocellum exoproteome, wherein the cellulosomes splay or divide biomass particles, changes when either the primary or secondary scaffolds are removed, showing that the intact wild-type cellulosomal system is necessary for this essential mode of action. This new transcriptional and proteomic evidence shows that a functional primary scaffoldin plays a more important role compared to secondary scaffoldins in the proper regulation of CAZyme genes, cellodextrin transport, and other cellular functions. PMID:26989779

  13. 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.

  14. Effect of culture medium composition on Trichoderma reesei's morphology and cellulase production.

    Science.gov (United States)

    Ahamed, Aftab; Vermette, Patrick

    2009-12-01

    The objective of this study was to determine how fungal morphology influences the volumetric cellulase productivity of Trichoderma reesei cultured in four media with lactose and lactobionic acid as fed-batch in a 7 L stirred tank bioreactor. The use of a cellulose-yeast extract culture medium yielded the highest enzyme production with a volumetric enzyme activity of 69.8 U L(-1) h(-1), and a maximum fungal biomass of 14.7 g L(-1). These findings were associated with the following morphological characteristics of the fungus: total mycelia was 98% of total mean projected area, mean hyphae length of 10 mm, mean hyphae volume of 45.1 mm(3), mean hyphae diameter of 7.9 microm, number of branches 9, and number of tips per hypha 29. A positive correlation was found between the total mycelia, the number of tips and the volumetric enzyme productivity, indicating the weight of these variables on the enzyme productivity. PMID:19592237

  15. [Screening and Enzyme Production Characteristics of Thermophilic Cellulase-producing Strains].

    Science.gov (United States)

    Feng, Hong-mei; Qin, Yong-sheng; Li, Xiao-fan; Zhou, Jin-xing; Peng, Xia-wei

    2016-04-15

    A total of 6 thermophilic cellulase-producing strainswere isolated from organic garden waste mixed chicken composting at thermophilic period. These isolates were identified as Streptomyces thermoviolaceus, S. thermodiastaticus, S. thermocarboxydus, S. albidoflavus, S. thermovulgaris and Brevibacillus borstelensis through 16S rRNA gene sequence alignment and phylogenetic tree analysis. The cellulose-degrading microbial community has been investigated in few researches so far both at home and abroad. In this study, the mixed strains M-1 was made up of the 6 cellulose-decomposing microorganisms. The CMCase activity of the mixed strains M- 1 was stronger than any of the 6 single strains. Production of CMCase from mixed strains M-1 was studied by optimizing different physico-chemical parameters. The Maximum CMCase production (135.9 U · mL⁻¹) of strains M-1 was achieved at 45 °C in a liquid medium (pH 4) inoculated with 1% (volume fraction), containing a mixture of wheat bran and starch, corn flour and KNO₃. After optimization of separation conditions, CMCase production capacity was improved by 1.8 times. PMID:27548981

  16. Research on Extraction and Characterization of Cellulase from Commercial Enzyme Preparation

    Directory of Open Access Journals (Sweden)

    Guowei Shu

    2013-07-01

    Full Text Available The extraction of cellulase from commercial enzyme preparation by ammonium sulfate precipitation and its enzymatic characterization were studied. The results were as follows: the conditions of precipitation was 60% saturation of ammonium sulfate, the recovery rate of carboxymethylcellulase (CMCase and Filter Paper enzyme Activity (FPA were 73.8 and 71.4%, respectively. The enzyme has optimal temperature of 55°C, optimal pH of 4.8, the ions of Co2+, Mn2+ in the buffer lowered the activity of CMCase, but the Cu2+ in low concentration activated the CMCase. The ions of Co2+, Mn2+ and Al2+ in the buffer lowered the activity of FPA, but the Cu2+ and K+ in low concentration activated the FPA; Km and Vmax of the enzyme were 2.22×10-3 g/mL and 1.11×104 U/h using Lineweaver-Burk graphic method, respectively.

  17. Investigating Commercial Cellulase Performances Toward Specific Biomass Recalcitrance Factors Using Reference Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Xiaohui; Bowden, Mark E.; Engelhard, Mark H.; Zhang, Xiao

    2014-04-01

    Three commercial cellulase preparations, Novozymes Cellic® Ctec2, Dupont Accellerase® 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulose enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulose performance. While the degree of polymerization of cellulose in the reference substrates did not present a major recalcitrance factor to Novozymes Cellic® Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Cellulases and hemicellulases from endophytic Acremonium species and its application on sugarcane bagasse hydrolysis.

    Science.gov (United States)

    de Almeida, Maíra Nicolau; Guimarães, Valéria Monteze; Bischoff, Kenneth M; Falkoski, Daniel Luciano; Pereira, Olinto Liparini; Gonçalves, Dayelle S P O; de Rezende, Sebastião Tavares

    2011-09-01

    The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing L -arabinose, D -xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced by Acremonium sp. EA0810 cultivated in SC using D -xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase, β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion into glucose. PMID:21573756

  20. PRODUCTION AND CHARACTERIZATION OF CELLULASE BY LOCAL FUNGAL ISOLATE OF INDIA USING WATER HYACINTH AS CARBON SOURCE AND REUSE OF FUNGAL BIOMASS FOR DYE DEGRADATION

    Directory of Open Access Journals (Sweden)

    SACHIN TALEKAR,

    2011-04-01

    Full Text Available The production of cellulase using Eichhornia crassipes (water hyacinth as a carbon source and dye degradation potential of local fungal isolate of India was studied. The basal medium supplemented with water hyacinth blend in the proportion of 1:05(V/V as carbon source and pH 5.0 showed maximum cellulase production after 6 days of incubation at 30º C with agitation speed of 150 rpm in rotary shaker.Effect on enzyme activity was investigated at different temperatures and pH. The optimum temperature and pH for the cellulase activity was 40ºC and 5.0. Kinetic investigations showed that KM and Vmax of cellulase were 4.7 mg/ml and 58.3μmol/ml/min, respectively. The reuse of fungal biomass after cellulase production for de-colorization of methylene blue was studied. Methylene blue was completely decolorizedwithin 5 days of incubation at temperature 30º C and pH 5.0 with agitation speed of 150 rpm. This demonstrates reuse of fungal biomass for dye degradation after enzyme production.

  1. Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers.

    Science.gov (United States)

    Li, Ao; Wang, Ruyi; Li, Xianliang; Liu, Mingyong; Fan, Jian; Guo, Kai; Luo, Bing; Chen, Tingting; Feng, Shengqiu; Wang, Yanting; Wang, Bingrui; Peng, Liangcai; Xia, Tao

    2016-01-01

    Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction. PMID:27192945

  2. 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. PMID:26400494

  3. Two-stage statistical medium optimization for augmented cellulase production via solid-state fermentation by newly isolated Aspergillus niger HN-1 and application of crude cellulase consortium in hydrolysis of rice straw.

    Science.gov (United States)

    Sandhu, Simranjeet Kaur; Oberoi, Harinder Singh; Babbar, Neha; Miglani, Kanupriya; Chadha, Bhupinder Singh; Nanda, Dhiraj Kumar

    2013-12-26

    Cellulolytic enzyme production by newly isolated Aspergillus niger HN-1 was statistically optimized using Plackett-Burman and central composite design (CCD). Optimum concentrations of 2, 0.40, 0.01, and 0.60 g L (-1) for KH2PO4, urea, trace elements solution, and CaCl2·2H2O, respectively, were suggested by Design-Expert software. The two-stage optimization process led to a 3- and 2-fold increases in the filter paper cellulase (FP) and β-glucosidase activities, respectively. FP, β-glucosidase, endoglucanase, exopolygalaturonase, cellobiohydrolase, xylanase, α-l-arabinofuranosidase, β-xylosidase, and xylan esterase activities of 36.7 ± 1.54 FPU gds(-1), 252.3 ± 7.4 IU gds(-1), 416.3 ± 22.8 IU gds(-1), 111.2 ± 5.4 IU gds(-1), 8.9 ± 0.50 IU gds(-1), 2593.5 ± 78.9 IU gds(-1), 79.4 ± 4.3 IU gds(-1), 180.8 ± 9.3 IU gds(-1), and 288.7 ± 11.8 IU gds(-1), respectively, were obtained through solid-state fermentation during the validation studies. Hydrolysis of alkali-treated rice straw with crude cellulases resulted in about 84% glucan to glucose, 89% xylan to xylose, and 91% arabinan to arabinose conversions, indicating potential for biomass hydrolysis by the crude cellulase consortium obtained in this study. PMID:24328069

  4. 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.

  5. Metagenomic cellulases highly tolerant towards the presence of ionic liquids--linking thermostability and halotolerance.

    Science.gov (United States)

    Ilmberger, Nele; Meske, Diana; Juergensen, Julia; Schulte, Michael; Barthen, Peter; Rabausch, Ulrich; Angelov, Angel; Mientus, Markus; Liebl, Wolfgang; Schmitz, Ruth A; Streit, Wolfgang R

    2012-07-01

    Cellulose is an important renewable resource for the production of bioethanol and other valuable compounds. Several ionic liquids (ILs) have been described to dissolve water-insoluble cellulose and/or wood. Therefore, ILs would provide a suitable reaction medium for the enzymatic hydrolysis of cellulose if cellulases were active and stable in the presence of high IL concentrations. For the discovery of novel bacterial enzymes with elevated stability in ILs, metagenomic libraries from three different hydrolytic communities (i.e. an enrichment culture inoculated with an extract of the shipworm Teredo navalis, a biogas plant sample and elephant faeces) were constructed and screened. Altogether, 14 cellulolytic clones were identified and subsequently assayed in the presence of six different ILs. The most promising enzymes, CelA2, CelA3 (both derived from the biogas plant) and CelA84 (derived from elephant faeces), showed high activities (up to 6.4 U/mg) in the presence of 30% (v/v) ILs. As these enzymes were moderately thermophilic and halotolerant, they retained 40% to 80% relative activity after 34 days in 4 M NaCl, and they were benchmarked with two thermostable enzymes, CelA from Thermotoga maritima and Cel5K from a metagenome library derived from Avachinsky crater in Kamchatka. These enzymes also exhibited high activity (up to 11.1 U/mg) in aqueous IL solutions (30% (v/v)). Some of the enzymes furthermore exhibited remarkable stability in 60% (v/v) IL. After 4 days, CelA3 and Cel5K retained up to 79% and 100% of their activity, respectively. Altogether, the obtained data suggest that IL tolerance appears to correlate with thermophilicity and halotolerance. PMID:22143172

  6. 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.

  7. Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum.

    Science.gov (United States)

    Lamed, R; Setter, E; Bayer, E A

    1983-11-01

    The isolation and biochemical characterization of the extracellular form of a cellulose-binding factor (CBF) from Clostridium thermocellum is described. The CBF was isolated from the culture supernatant by a two-step procedure which included affinity chromatography on cellulose and gel filtration on Sepharose 4B. The isolated CBF was homogeneous as determined by immunoelectrophoresis, polyacrylamide gel electrophoresis, gel filtration, and analytical ultracentrifugation analysis. The CBF was found to form a complex which exhibited a molecular weight estimated at 2.1 million. Electron microscopic analysis of negatively stained preparations of the isolated CBF revealed a particulate, multisubunit entity of complicated quaternary structure. The molecule appeared to be about 18 nm in size. Although urea failed to break the complex into its component parts, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolved the CBF complex into 14 polypeptide bands. Immunoprecipitation experiments confirmed that these polypeptides indeed formed part of the same complex. Interestingly, by using the whole-cell immunization procedure described in the accompanying article (Bayer et al., J. Bacteriol., 156:818-827, 1983) only one CBF subunit (Mr = 210,000) was found to be antigenically active. By using a gel-overlay assay technique, at least eight of the remaining CBF-associated polypeptide components were shown to exhibit cellulolytic activity. The results are consistent with the contention that the CBF comprises a discrete, multisubunit complex or group of closely related complexes which exhibit separate antigenic and multiple cellulase activities in addition to the property of cellulose binding. It appears that the CBF is not only responsible for the adherence of the cells to cellulose but also constitutes a major part of the cellulolytic apparatus of this organism. PMID:6195146

  8. 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

    Directory of Open Access Journals (Sweden)

    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.

  9. Simultaneous Cellulase Production, Saccharification and Detoxification Using Dilute Acid Hydrolysate of S. spontaneum with Trichoderma reesei NCIM 992 and Aspergillus niger.

    Science.gov (United States)

    Sateesh, Lanka; Rodhe, Adivikatla Vimala; Naseeruddin, Shaik; Yadav, Kothagauni Srilekha; Prasad, Yenumulagerard; Rao, Linga Venkateswar

    2012-06-01

    Bioethanol production from lignocellulosic materials has several limitations. One aspect is the high production cost of cellulases used for saccharification of substrate and inhibition of fermenting yeast due to inhibitors released in acid hydrolysis. In the present work we have made an attempt to achieve simultaneous cellulases production, saccharification and detoxification using dilute acid hydrolysate of Saccharum spontaneum with and without addition of nutrients, supplemented with acid hydrolyzed biomass prior to inoculation in one set and after 3 days of inoculation in another set. Organisms used were T. reesei NCIM 992, and Aspergillus niger isolated in our laboratory. Cellulase yield obtained was 0.8 IU/ml on fourth day with T. reesei. Sugars were found to increase from fourth to fifth day, when hydrolysate was supplemented with nutrients and acid hydrolyzed biomass followed by inoculation with T. reesei. Phenolics were also found to decrease by 67%. PMID:23729891

  10. Enzyme-based lignocellulose hydrolyzation – Brief data survey for cellulase performance characterization on behalf of the Sauter mean diameter of raw material particles

    Directory of Open Access Journals (Sweden)

    Robert Glaser

    2015-12-01

    Full Text Available The data presented here supports the informational background of enzyme-based lignocellulose hydrolyzation, cellulase characterization, and sugar yield prediction for the work “Enzyme-based lignocellulose hydrolyzation – Sauter mean diameter of raw materials as a basis for cellulase performance characterization and yield prediction” by Glaser [1]. Glucose yields from the enzymatic hydrolysis of the raw materials were shown as a function of cellulase enzyme loading as well as of particle size with different solid loading. The data for the proposed methods of the determination of enzyme activity in inhomogeneous samples of lignocellulosic raw materials are presented. The data of the empirical model that was developed for the prediction of hydrolysis yields for different enzyme concentrations, substrate specific particle size, and solid loadings, are given. Data are also given in relation of terms of scale-up opportunities.

  11. Application of ZnO Nanoparticles for Improving the Thermal and pH Stability of Crude Cellulase Obtained from Aspergillus fumigatus AA001

    Science.gov (United States)

    Srivastava, Neha; Srivastava, Manish; Mishra, P. K.; Ramteke, Pramod W.

    2016-01-01

    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, ultraviolet-visible spectroscope, field emission scanning electron microscope and high resolution scanning electron microscope. The crude thermostable cellulase has been obtained from the Aspergillus fumigatus AA001 and treated with ZnO nanoparticle which shows thermal stability at 65°C 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. PMID:27148203

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Thermodynamics of adsorption/desorption of cellulases NS 50013 on /from Avicel PH 101 and Protobind 1000

    Directory of Open Access Journals (Sweden)

    Khurram Shahzad Baig

    2016-06-01

    Full Text Available Insight from thermodynamic parameters of enthalpy (ΔH, entropy (ΔS and Gibbs free energy (ΔG was used to predict conditions for desorption of cellulases from wheat straw. The analogues of the cellulose and lignin components of wheat straw used were Avicel PH 101 and Protobind 1000, respectively. The ΔHa for adsorption on Avicelat pH 5 was -16.10kJmol-1 , ΔSa was -50.10 Jmol-1K -1 which indicated an increase in order due to adsorption and ΔGa was negative only from 298K to 323K. Results of adsorption on Protobind 1000 were pretty much the inverse over the same range of temperature, proving the great affinity of cellulases for lignin. Over 298K to 333K, desorption from Avicel resulted in a ΔHdincrease from 17.50 to 26.20 kJmol-1 , and a ΔSdincrease from 46.89 to 75.65Jmol-1K -1when pH increased from 6 to 9indicating an enthalpy-driven, nonspontaneous desorption. For Protobind, the positive ΔHd (9.65 to 6.90 kJmol-1 with small positive ΔSd (21.70 to 9.03 Jmol-1K -1 indicated that disorder was less than that of Avicel. For both the substrates ΔGd decreased with rise in temperature in given temperature range. The minimum ΔGdof Avicel than that of Protobind proved that it was more difficult to desorb cellulases from Protobind.For bioethanol producing industries, using lignocellulosic material (e.g., wheat straw where cellulose is embedded in lignin, removal of lignin is recommend along with adsorption/hydrolysis to be conducted at 323 K and desorption from used material at 333 K and pH 9.

  15. 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.

  16. Effect of temperature on the production of cellulases, xylanases and lytic enzymes by selected Trichoderma reesei mutants

    OpenAIRE

    Piotr Janas; Zdzisław Targoński

    2014-01-01

    The effect of temperature in the rangę of 26-38°C on the production of cellulases, xylanases and lytic enzymes by four mutant strains of Trichoderma reesei was analysed. On the basis of these investigations three thermosensitive strains (M-7. RUT C 30 and VTT-D-78085) which showed reduced excretion of the above mentioned enzymes as well as protein and a thermoresistant mutant (VTT-D-79I24) which grew within a temperature range of 26-34°C were characterized. Higher temperature caused an increa...

  17. ENHANCED PRODUCTION OF CELLULASE-FREE XYLANASE BY ALKALOPHILIC BACILLUS SUBTILIS ASH AND ITS APPLICATION IN BIOBLEACHING OF KRAFT PULP

    OpenAIRE

    Ashwani Sanghi; Neelam Garg; Kalika Kuhar; Kuhad, Ramesh C.; Gupta, Vijay K

    2009-01-01

    This paper reports high level production of a cellulase-free xylanase using wheat bran, a cost-effective substrate, under submerged fermentation by alkalophilic Bacillus subtilis ASH. Production of xylanase was observed even at alkaline pH up to 11.0 and temperature 60 °C, although the highest enzyme titer was recorded at neutral pH and 37 °C. The enzyme production under optimized fermentation was 1.5-fold greater than under unoptimized conditions. Pre-treatment of unbleached pulp of 10% cons...

  18. 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; Olsen, Johan Pelck; Badino, Silke Flindt; Windahl, Michael Skovbo; Colussi, Francieli; Cruys-Bagger, Nicolaj; Borch, Kim; Westh, Peter

    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...... 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 in...

  19. One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

    OpenAIRE

    Sanghi, Ashwani; Garg, Neelam; Gupta, V. K.; Mittal, Ashwani; R.C. Kuhad

    2010-01-01

    The present study describes the one-step purification and characterization of an extracellular cellulase-free xylanase from a newly isolated alkalophilic and moderately thermophilic strain of Bacillus subtilis ASH. Xylanase was purified to homogeneity by 10.5-fold with ~43% recovery using ion-exchange chromatography through CM-Sephadex C-50. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular mass of 23 kDa. It showed an optimum pH at 7.0 and was stable over the pH ran...

  20. Use of inedible wheat residues from the KSC-CELSS breadboard facility for production of fungal cellulase

    Science.gov (United States)

    Strayer, R. F.; Brannon, M. A.; Garland, J. L.

    1990-01-01

    Cellulose and xylan (a hemicellulose) comprise 50 percent of inedible wheat residue (which is 60 percent of total wheat biomass) produced in the Kennedy Space Center Closed Ecological Life Support System (CELSS) Breadboard Biomass Production Chamber (BPC). These polysaccharides can be converted by enzymatic hydrolysis into useful monosaccharides, thus maximizing the use of BPC volume and energy, and minimizing waste material to be treated. The evaluation of CELSS-derived wheat residues for production for cellulase enzyme complex by Trichoderma reesei and supplemental beta-glucosidase by Aspergillus phoenicis is in progress. Results to date are given.

  1. Effects of dietary addition of cellulase and a Saccharomyces cerevisiae fermentation product on nutrient digestibility, rumen fermentation and enteric methane emissions in growing goats.

    Science.gov (United States)

    Lu, Qi; Wu, Jian; Wang, Min; Zhou, Chuanshe; Han, Xuefeng; Odongo, Edwin Nicholas; Tan, Zhiliang; Tang, Shaoxun

    2016-01-01

    This study was designed to assess the effectiveness of dietary cellulase (243 U/g, derived from Neocallimastix patriciarum) and a Saccharomyces cerevisiae fermentation product (yeast product) on ruminal fermentation characteristics, enteric methane (CH4) emissions and methanogenic community in growing goats. The experiment was conducted in a 5 × 5 Latin square design using five Xiangdong black wether goats. The treatments included a Control and two levels of cellulase (0.8 g and 1.6 g/kg dry matter intake (DMI), i.e. 194 U/kg and 389 U/kg DMI, respectively) crossed over with two levels (6 g or 12 g/kg DMI) of the yeast product. There were no significant differences regarding feed intake, apparent digestibility of organic matter, neutral detergent fibre and acid detergent fibre among all the treatments. In comparison with the Control, the ruminal ammonia N concentration was decreased (p = 0.001) by cellulase and yeast product addition. The activities of carboxymethylcellulase and xylanase were decreased after cellulase addition. Moreover, dietary cellulase and yeast product addition led to a significant reduction (p < 0.05) of enteric CH4 emissions although the diversity and copy numbers of methanogens among treatments were not dissimilar. The present results indicate that the combination of cellulase and yeast fermentation product can reduce the production of CH4 energy and mitigate the enteric CH4 emissions to a certain degree. PMID:27032031

  2. Modulation of cellulase activity by charged lipid bilayers with different acyl chain properties for efficient hydrolysis of ionic liquid-pretreated cellulose.

    Science.gov (United States)

    Mihono, Kai; Ohtsu, Takeshi; Ohtani, Mai; Yoshimoto, Makoto; Kamimura, Akio

    2016-10-01

    The stability of cellulase activity in the presence of ionic liquids (ILs) is critical for the enzymatic hydrolysis of insoluble cellulose pretreated with ILs. In this work, cellulase was incorporated in the liposomes composed of negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and zwitterionic phosphatidylcholines (PCs) with different length and degree of unsaturation of the acyl chains. The liposomal cellulase-catalyzed reaction was performed at 45°C in the acetate buffer solution (pH 4.8) with 2.0g/L CC31 as cellulosic substrate. The crystallinity of CC31 was reduced by treating with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) at 120°C for 30min. The liposomal cellulase continuously catalyzed hydrolysis of the pretreated CC31 for 48h producing glucose in the presence of 15wt% [Bmim]Cl. The charged lipid membranes were interactive with [Bmim](+), as elucidated by the [Bmim]Cl-induced alterations in fluorescence polarization of the membrane-embedded 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules. The charged membranes offered the microenvironment where inhibitory effects of [Bmim]Cl on the cellulase activity was relieved. The maximum glucose productivity GP of 10.8 mmol-glucose/(hmol-lipid) was obtained at the reaction time of 48h with the cellulase incorporated in the liposomes ([lipid]=5.0mM) composed of 50mol% POPG and 1,2-dilauroyl-sn-glycero-3-phosohocholine (DLPC) with relatively short and saturated acyl chains. PMID:27318965

  3. Effects of Additives on the Thermostability of Cellulase%添加剂对纤维素酶耐温性的影响

    Institute of Scientific and Technical Information of China (English)

    姚秀清; 孙丽萍; 周丹丹

    2011-01-01

    In order to improve the thermostability of cellulase under higher temperature, the effects of five additives including polyethylene glycol (PEG) , glycerol, xylitol, calcium chloride and xanthan gum on cellulose activities were studied. The results showed that protective effect was found in the presence of polyethylene glycol, glycerol, xylitol and xanthan gum under the condition of 58 ℃ and preserved 24 h. The cellulase activities with the addition of additives were higher than that without additives. Among the four additives, protective effect of xylitol on the cellulase activities was most evident. Relative cellulase activity was increased from 0.59 to 0.94. While inhibiting effect of calcium chloride on the cellulase activities was existed with relative cellulase activity decreased from 0.59 to 0.49.%为提高纤维素酶在较高温度条件下的耐温性,考察5种添加剂(聚乙二醇、甘油、木糖醇、氯化钙、黄原胶)对纤维素酶酶活的影响.结果表明,在58℃、保存24h条件下,聚乙二醇、甘油、木糖醇、黄原胶对纤维素酶酶活有保护效应,较未添加时的相对酶活有所增加.木糖醇的保护效应最为显著,相对酶活由0.59提高到0.94;而氯化钙对酶活有抑制效应,相对酶活由0.59降低为0.49.

  4. The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images.

    Science.gov (United States)

    Hidayat, Budi J; Weisskopf, Carmen; Felby, Claus; Johansen, Katja S; Thygesen, Lisbeth G

    2015-12-01

    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 or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen 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 degradation at these locations. PMID:26626331

  5. Structural and molecular basis of cellulase Cel48F by computational modeling: Insight into catalytic and product release mechanism.

    Science.gov (United States)

    Qian, Mengdan; Guan, Shanshan; Shan, Yaming; Zhang, Hao; Wang, Song

    2016-06-01

    As a processive cellulase, Cel48F from Clostridium cellulolyticum plays a crucial role in cellulose fiber degradation. It has been confirmed in experiment that residue Glu44 will greatly affect the catalytic activity but the mechanism is still unknown. In this study, conventional molecular dynamics, steered molecular dynamics and free energy calculation were integrated to simulate the hydrolysis and product release process to gain insights into the factors that influence catalytic activity. Analysis of simulation results indicated that Glu44 could maintain the proper conformation of its substrate to ensure successful cleavage reaction or serve as a base required in the inverting mechanism in hydrolysis. After hydrolysis is completed, residues Glu44, Asp494, Trp611 and Glu55 participate in hydrogen bond rearrangement during product releasing process. This rearrangement can reduce the sliding barrier and stimulate the product to move toward the exit in the initial release stage. Dependent on the rearrangement, the product moves toward the exit and is exposed to an increasing amount of solvent molecules, which makes solvent effect more and more notable. With the assistance of solvent interaction, product can get rid of the enzyme more easily. However, the subsequent release process remains uncertain because of the disordered motion of solvent molecules. This work provides theoretical data as a basis of cellulase modification or mutation. PMID:26993462

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

    Directory of Open Access Journals (Sweden)

    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. Performance and efficiency of old newspaper deinking by combining cellulase/hemicellulase with laccase-violuric acid system

    International Nuclear Information System (INIS)

    Performance and efficiency of old newspaper (ONP) deinking by combining cellulase/hemicellulase with laccase-violuric acid system (LVS) were investigated in this study. Brightness, effective residual ink concentration (ERIC) and physical properties were evaluated for the deinked pulp. Fiber length, coarseness, specific surface area and specific volume were also tested. The changes of dissolved lignin during the deinking processes were measured with UV spectroscopy. The fiber morphology was observed with environmental scanning electronic microscopy (ESEM). Experimental results showed that, compared to the pulp deinked with each individual enzyme, ERIC was lower for the cellulase/hemicellulase-LVS-deinked pulp. This indicated that a synergy existed in ONP deinking using a combination of enzymes. After being bleached by H2O2, enzyme-combining deinked pulp gave higher brightness and better strength properties. Compared with individual enzyme deinked pulp, average fiber length and coarseness decreased a little for the enzyme-combining deinked pulps. A higher specific surface area and specific volume of the pulp fibers were achieved. UV analysis proved that more lignin was released during the enzyme-combining deinking process. ESEM images showed that more fibrillation was observed on the fiber surface due to synergistic treatment

  8. Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin (Budorcas taxicolor Bedfordi)

    International Nuclear Information System (INIS)

    A bacterial strain with high cellulase activity was isolated of feces sample of Golden Takin (Budorcas taxicolor Bedfordi). The bacterium was classified and designated Bacillus subtilis LN by morphological and 16SrDNA gene sequence analysis. Two putative cellulase genes, CelL15 and CelL73, were simultaneously cloned from the isolated strain by PCR. The putative gene CelL15 consisted of an open reading frame (ORF) of 1470 nucleotides and encoded a protein of 490 amino acids with a molecular weight of 54 kDa. The CelL73 gene consisted of an open reading frame (ORF) of 741 nucleotides and encoded a protein of 247 amino acids with a molecular weight of 27 kDa. Both genes were purified and cloned into pET-28a for expression in Escherichia coli BL21 (DE3). The ability of E. coli to degrade cellulose was enhanced when the two recombinants were cultured together.

  9. 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. PMID:25057412

  10. A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains

    Directory of Open Access Journals (Sweden)

    Jourdier Etienne

    2012-05-01

    Full Text Available Abstract Background During bioprocess development, secondary screening is a key step at the boundary between laboratory and industrial conditions. To ensure an effective high-throughput screening, miniaturized laboratory conditions must mimic industrial conditions, especially for oxygen transfer, feeding capacity and pH stabilization. Results A feeding strategy has been applied to develop a simple screening procedure, in which a stoichiometric study is combined with a standard miniaturization procedure. Actually, the knowledge of all nutriments and base or acid requirements leads to a great simplification of pH stabilization issue of miniaturized fed-batch cultures. Applied to cellulase production by Trichoderma reesei, this strategy resulted in a stoichiometric mixed feed of carbon and nitrogen sources. While keeping the pH between shake flask and stirred bioreactor comparable, the developed shake flask protocol reproduced the strain behaviour under stirred bioreactor conditions. Compared to a an already existing miniaturized shake flasks protocol, the cellulase concentration was increased 5-fold, reaching about 10 g L-1. Applied to the secondary screening of several clones, the newly developed protocol succeeded in selecting a clone with a high industrial potential. Conclusions The understanding of a bioprocess stoichiometry contributed to define a simpler and more effective miniaturization. The suggested strategy can potentially be applied to other fed-batch processes, for the screening of either strain collections or experimental conditions.

  11. 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.

  12. 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.

  13. Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus –production of extracellular enzymes and characterization of the major cellulases

    Czech Academy of Sciences Publication Activity Database

    Valášková, Vendula; Baldrian, Petr

    2006-01-01

    Roč. 152, - (2006), s. 3613-3622. ISSN 0026-2617 R&D Projects: GA ČR(CZ) GP204/02/P100 Institutional research plan: CEZ:AV0Z50200510 Keywords : degradation * piptoporus betulinus * cellulases Subject RIV: EE - Microbiology, Virology Impact factor: 0.543, year: 2006

  14. Characterization of the Ca(2+) -responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut-C30.

    Science.gov (United States)

    Chen, Ling; Zou, Gen; Wang, Jingzhi; Wang, Jin; Liu, Rui; Jiang, Yanping; Zhao, Guoping; Zhou, Zhihua

    2016-05-01

    Calcium signaling plays pivotal roles in the hyphal growth, conidiation, and osmosis sensitivity of fungi through the Ca(2+) /calmodulin-calcineurin-dependent pathway. This study found that an appropriate extracellular Ca(2+) concentration markedly stimulated the hyphal growth, cellulase production, and total protein secretion of the cellulase hyper-producing strain, Trichoderma reesei Rut-C30. Transcription analysis revealed upregulation of not only encoding genes of cellulases and the transcriptional activator XYR1 but also several genes encoding endoplasmic reticulum-chaperones after Ca(2+) addition. The function of CRZ1, T. reesei calcineurin-responsive zinc finger transcription factor 1, was further characterized by gene disruption. Electrophoretic mobility shift assays (EMSAs) in combination with chromatin immunoprecipitation (ChIP) verified that CRZ1 could bind directly to the upstream regions of xyr1 and cbh1 (cellobiohydrolase I-encoding gene) in response to Ca(2+) . A DNase I footprinting assay identified its putative binding consensus site (5'-[T/G]GGCG-3' or 5'-GGGC[G/T]-3'). EMSAs confirmed that CRZ1 competed for occupancy of the xyr1 promoter with another transcription factor, ACE1. These results revealed putative signaling pathways downstream of calcineurin in response to extracellular Ca(2+) involved in upregulation of cellulose degradation-related genes, reflecting progress in the study of Ca(2+) signaling in filamentous fungi. This study also provides insight that will facilitate further improvement of (hemi-)cellulase production by T. reesei. PMID:27109892

  15. Overexpression of an endo-1,4-β-glucanase V gene (EGV) from Trichoderma reesei leads to the accumulation of cellulase activity in transgenic rice.

    Science.gov (United States)

    Li, X Y; Liu, F; Hu, Y F; Xia, M; Cheng, B J; Zhu, S W; Ma, Q

    2015-01-01

    The ectopic expression of cellulase in biomass can reduce the cost of biofuel conversion. This trait modification technique is highly beneficial for biofuel production. In this study, we isolated an endo-1,4-beta-glucanase gene (EGV) from Trichoderma reesei and inserted this gene downstream of a fragment encoding the signal peptide Apo-SP in a modified pCAMBIA1301 vector to obtain an Apo-SP and AsRed fusion protein. Transient expression of this fusion protein in onion epidermal cells showed that the Apo-SP signal was localized to the plastids. EGV transgenic rice plants that did not carry screening marker genes were obtained through overexpression of the pDTB double T-DNA vector. Western blotting showed that EGV was expressed in the dry straw of T0 generation transgenic rice plants and in fresh leaves of the T1 generation. More importantly, our results also showed that the peptide product of EGV in the transgenic plants folded correctly and was capable of digesting the cellulase substrate CMC. Additionally, cellulase activity remained stable in the straw that had been dried at room temperature for three months. This study presents an important technical approach for the development of transgenic rice straw that has stable cellulase activity and can be used for biofuel conversion. PMID:26782396

  16. [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%. PMID:26859961

  17. 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. PMID:24158534

  18. 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. PMID:22942683

  19. Effect of temperature on the production of cellulases, xylanases and lytic enzymes by selected Trichoderma reesei mutants

    Directory of Open Access Journals (Sweden)

    Piotr Janas

    2014-08-01

    Full Text Available The effect of temperature in the rangę of 26-38°C on the production of cellulases, xylanases and lytic enzymes by four mutant strains of Trichoderma reesei was analysed. On the basis of these investigations three thermosensitive strains (M-7. RUT C 30 and VTT-D-78085 which showed reduced excretion of the above mentioned enzymes as well as protein and a thermoresistant mutant (VTT-D-79I24 which grew within a temperature range of 26-34°C were characterized. Higher temperature caused an increase in the level of xylanolytic enzymes produced by the four mutants. In addition. it effected the complex composition of cellulolytic enzymes secreted by VTT-D-79l 24 (i.c. increased and reduced excertion of (β-glucosidase and β-1,4-endoglucanase respectively.

  20. Effect of Pre-harvest Treatments on the Cellulase Activity and Quality of Ber Fruit Under Cold Storage Conditions

    OpenAIRE

    Sukhjit Kaur JAWANDHA; Mahajan, B. V. C.; Parmpal Singh GILL

    2009-01-01

    Studies were carried out to find out the effect of various pre-harvest treatments such as CaCl2 (@ 0.5%, 1.0% and 2.0%), Ca(NO3)2 (@0.5%, 1.0% and 2.0%), GA3 (@ 20, 40 and 60 ppm) and Bavistin (@ 0.1%) on the cellulase activity and quality of 'Umran' ber fruits during cold storage. Marked trees were sprayed at colour break stage with the test chemicals. Fruits were packed in CFB boxes and placed in cold storage (3-5 oC and 85 � 90 % RH) for 30 days. The fruits were evaluated after 10, 20 and...

  1. Partial purification and properties of cellulase-free alkaline xylanase produced by Rhizopus stolonifer in solid-state fermentation

    OpenAIRE

    Antonio José Goulart; Eleonora Cano Carmona; Rubens Monti

    2005-01-01

    Rhizopus stolonifer was cultivated in wheat bran to produce a cellulase-free alkaline xylanase. The purified enzyme obtained after molecular exclusion chromatography in Sephacryl S-200 HR showed optimum temperature as 45º C and hydrolysis pHs optima as pH 6.0 and 9.0. Xylanase presented higher Vmax at pH 9.0 (0.87 µmol/mg protein) than at pH 6.0 and minor Km at pH 6.0 (7.42 mg/mL) than at pH 9.0.Rhizopus stolonifer foi cultivado em meio de farelo de trigo para produzir uma xilanase alcalina c...

  2. Degradation of corn stover by fungal cellulase cocktail for production of polyhydroxyalkanoates by moderate halophile Paracoccus sp. LL1.

    Science.gov (United States)

    Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

    2015-10-01

    Bioprocessing of lignocellulose as a renewable resource for fuels, chemicals or value added products is a necessity to fulfil demands of petroleum products. This study aims to convert corn stover to polyhydroxyalkanoates (PHA). Corn stover was hydrolyzed to crude sugars by an on-site prepared cellulase cocktail from co-culture of Trichoderma reesei and Aspergillus niger. The potent PHA producer, Paracoccus sp. LL1, was isolated from Lonar Lake, India and could accumulate PHA up to 72.4% of its dry cell weight. PHA production reached 9.71 g/L from corn stover hydrolysate containing 40 g/L sugar mixture. The PHA synthase gene (phaC) sequence of the isolate showed 79% identity with the phaC gene of Paracoccus seriniphilus (E71) strain from the NCBI database. The nature/type of PHA was found to be poly(3-hydroxybutyrate) by Fourier transform infrared spectroscopy. PMID:26207871

  3. 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.

  4. 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.

  5. One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

    Directory of Open Access Journals (Sweden)

    Ashwani Sanghi

    2010-06-01

    Full Text Available The present study describes the one-step purification and characterization of an extracellular cellulase-free xylanase from a newly isolated alkalophilic and moderately thermophilic strain of Bacillus subtilis ASH. Xylanase was purified to homogeneity by 10.5-fold with ~43% recovery using ion-exchange chromatography through CM-Sephadex C-50. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular mass of 23 kDa. It showed an optimum pH at 7.0 and was stable over the pH range 6.0-9.0. The optimum temperature for enzyme activity was 55 ºC. The purified xylanase did not lose any activity up to 45 ºC, however, it retained 80% and 51% of its activity after pre-incubation at 55 ºC and 60 ºC, respectively. The enzyme obeyed Michaelis-Menton kinetics towards birch wood xylan with apparent Km 3.33 mg/ml and Vmax 100 IU/ml. The enzyme was strongly inhibited by Hg2+ and Cu2+ while enhanced by Co2+ and Mn2+. The purified enzyme could be stored at 4 ºC for six weeks without any loss of catalytic activity. The faster and economical purification of the cellulase-free xylanase from B. subtilis ASH by one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.

  6. AMINO ACIDS PROFILE OF THE LIGNOCELLULOSIC FEED TREATED WITH CELLULASE-FREE LIGNOLYTIC MUTANTS OF PLEUROTUS OSTREATUS

    Directory of Open Access Journals (Sweden)

    Vijaya Lakshmi Chalamcherla

    2010-02-01

    Full Text Available Defining and quantifying amino acid requirements will become an important consideration in the next generation of feeding schemes for dairy cattle beyond the current emphasis on identification of limiting amino acids. In this context different amino acid profiles of untreated, urea treated, fungal treated, and urea plus fungal treated lignocellulosic feed by both P. ostreatus wild and its two cellulase-minus/ less lignolytic mutants were analyzed. Cellulase-free mutant strains were obtained after 20 minutes of exposure to UV light and 0.4 seconds to X-rays. A UV mutant of P. ostreatus (POM1 exhibited better performance than the X-ray mutant (POM2 in terms of production of less cellulolytic and more lignolytic enzymes. Urea treatment of straw enhanced the total amino acid content by less than a factor of two, while the fungal treatment improved it by 13-14 times. Fungal treatment of urea-treated straw improved the total amino acid content by a factor of 15, indicating the importance of urea in the straw. Further, the fungal treatment of urea-treated straw enhanced the quantity of amino acids such as glutamine, glycine, aspergine, etc. by 15-20 times. The quantity of limiting amino acids such as methionine, lysine, and histidine was also enhanced by 8 to 10 times through the fungal treatment. Maximum amounts of all the amino acids were found in urea plus fungal (POM1 treated paddy straw than in only fungal treated and only urea treated paddy straws.

  7. 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.

  8. Effect of feeding alfalfa hay or Tifton 85 bermudagrass haylage with or without a cellulase enzyme on performance of Holstein cows.

    Science.gov (United States)

    Bernard, J K; Castro, J J; Mullis, N A; Adesogan, A T; West, J W; Morantes, G

    2010-11-01

    Forty-four lactating Holstein cows (173±30 DIM, 42.5±6.8 kg of milk, 4.03±0.69% fat, 674±78 kg of body weight) were used in an 8-wk, completely randomized trial with a 2 × 2 factorial arrangement of treatments to determine the effect of forage source and supplemental cellulase enzyme on performance. Treatments included 2 forage combinations (corn silage plus 12.2% dry matter, DM, from either alfalfa hay or Tifton 85 bermudagrass haylage) with or without a commercial cellulase enzyme applied to the total mixed ration at the rate of 4 g/head per day (Promote N.E.T.-L, Cargill Animal Nutrition, Minneapolis, MN). Experimental diets were formulated to provide similar concentrations of protein (16.5% of DM), energy (1.63 Mcal of net energy for lactation/kg of DM), and neutral detergent fiber (41.7% of DM) and were fed once daily as a total mixed ration behind Calan doors for ad libitum intake. The cellulase enzyme provided 1,200 cellulase units of activity/g of product and was applied to the total mixed ration and allowed to mix for 5min before feeding. Before beginning the trial, all cows were trained to use Calan (American Calan, Northwood, NH) doors and then fed the alfalfa hay-based diet for 2 wk. Data collected during wk 2 were used as a covariate in the statistical analysis. At the beginning of the 6-wk experimental period, cows were assigned randomly to 1 of the 4 experimental diets. No interactions were observed between forage and enzyme for any measures. Daily DM intake; milk yield; concentrations of milk fat, true protein, lactose, and solids not fat; energy-corrected milk yield; and dairy efficiency were not different among alfalfa or Tifton 85 bermudagrass rations with or without cellulase enzyme supplementation. The results of this trial indicate that Tifton 85 bermudagrass haylage can replace alfalfa hay in diets fed to high-producing, lactating dairy cows without depressing DM intake or milk yield when rations are balanced for NDF. Although

  9. SCREENING OF HARDWOOD AND SOFTWOOD SPECIES AS BEST SUBSTRATE FOR CELLULASE AND XYLANASE PRODUCTION USING CONSORTIUM OF POTENTIAL ISOLATES BACILLUS COAGULANS B30 + PAENIBACILLUS MUCILAGINOUS B5 + BACILLUS SP. UNDER SSF

    OpenAIRE

    Richa Kaushal*, Nivedita Sharma and Divya Tandon

    2014-01-01

    The lignocellulosic biomass is known to be an excellent carbon source for microbial enzyme production. In this paper, the cellulase and xylanase production from lignocellulosic materials using consortia of potential hydrolytic bacteria i.e. Bacillus coagulans B30 + Paenibacillus mucilaginous B5 + Bacillus sp. B21 isolated from forest soil under solid state fermentation (SSF) was investigated. The maximum cellulase activity of 97.84 U/g and xylanase activity of 67.06 U/g were obtained with wat...

  10. 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.

  11. 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. PMID:24085387

  12. 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

  13. Production of cellulase-free xylanase by Trichoderma reesei SAF3 Produção de xilanase livre de celulase por Trichoderma reesei SAF3

    OpenAIRE

    Sanjay Kar; Asish Mandal; Das Mohapatra, Pradeep K.; Mondal, Keshab C.; Bikash R. Pati

    2006-01-01

    A xylanase producing fungi has been isolated from soil and identified as Trichoderma reesei SAF3. Maximum growth of the organism was found at 48 h under submerged condition in xylan containing enriched medium, whereas highest enzyme production (4.75U/mL) was recorded at 72 h. No detectable cellulase activity was noted during whole cultivation period. The partially purified enzyme hydrolyzed xylan into xylopentose and xylose. All these properties of xylanase highlighten its promising uses in i...

  14. 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. PMID:24316485

  15. CELLULASES PRODUCTION UNDER SOLID STATE FERMENTATION USING AGRO WASTE AS A SUBSTRATE AND ITS APPLICATION IN SACCHARIFICATION BY TRAMETES HIRSUTA NCIM

    Directory of Open Access Journals (Sweden)

    Bhaumik R. Dave

    2014-12-01

    Full Text Available Food and energy crisis are the biggest constraint all over the world which has focused lights on need of utilizing renewable resources to meet the future demand. A promising strategy is efficient utilization of lignocellulosic waste and fermentation of the resulting sugars for production of desired metabolites or biofuel. Production of all the cellulase enzymes on wheat bran and different parameters regulating it like pH, moisture ratio (substrate: liquid, temperature and inoculum size has been optimized which found to be 4.5, 1:3, 30°C and 108 spores respectively. Salient feature of partially purified enzyme with stability in the range of 30-50°C under acidic pH range was found to be prominent for industrial applications, moreover in this study, Trametes hirsuta, an efficient cellulase producer, was observed to be an effective species for saccharification of wheat straw to enhance the sugar yield. Enzymatic hydrolysis of wheat straw with 15 FPU of cellulase from the species showed 73% yield in 20 hrs. It may prove to be a suitable choice for the industrial saccharification of lignocellulosic biomasses.

  16. 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

  17. 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.

  18. 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.

  19. ENHANCED PRODUCTION OF CELLULASE-FREE XYLANASE BY ALKALOPHILIC BACILLUS SUBTILIS ASH AND ITS APPLICATION IN BIOBLEACHING OF KRAFT PULP

    Directory of Open Access Journals (Sweden)

    Ashwani Sanghi

    2009-08-01

    Full Text Available This paper reports high level production of a cellulase-free xylanase using wheat bran, a cost-effective substrate, under submerged fermentation by alkalophilic Bacillus subtilis ASH. Production of xylanase was observed even at alkaline pH up to 11.0 and temperature 60 °C, although the highest enzyme titer was recorded at neutral pH and 37 °C. The enzyme production under optimized fermentation was 1.5-fold greater than under unoptimized conditions. Pre-treatment of unbleached pulp of 10% consistency with crude xylanase (6 IU/g o.d. pulp at 60 ºC for 2 h increased the final brightness by 4.9%. The enzyme treatment reduced the chlorine consumption by 28.6% with the same brightness as in the control. A reduction in kappa number and increase in viscosity was observed after enzyme pre-treatment. Scanning electron microscopy revealed loosening and swelling of pulp fibers. The strength properties viz. grammage, fiber thickness, beating degree, tensile index, breaking length, tear index and double fold of the treated pulp were improved as compared to the control pulp. This study reveals the potential of B. subtilis ASH xylanase as a biobleaching agent for the paper and pulp industry.

  20. Molecular cloning, expression, purification and characterization of a novel cellulase gene (Bh-EGaseI) in the beetle Batocera horsfieldi.

    Science.gov (United States)

    Mei, Hui-Zhen; Xia, Ding-Guo; Zhao, Qiao-Ling; Zhang, Guo-Zheng; Qiu, Zhi-Yong; Qian, Ping; Lu, Cheng

    2016-01-15

    Wood-feeding insects depend heavily on the secretion of a combination of cellulases, mainly endoglucanases and other glucanases such as exoglucanases and xylanases, to achieve efficient digestion of the cellulose of cellulosic materials. In this paper, we report a novel cellulose Bh-EGaseI belonging to the glycoside hydrolase family 45(gh45-1) obtained from the beetle Batocera horsfieldi. The Bh-EGaseI gene spans 714 bp and consists of three exons coding 237 amino acid residues. The cDNA encoding Bh-EGaseI was expressed as 25 KDa in baculovirus-infected Bombyx mori larvae. The expression products of Bh-EGaseI from larval hemolymph showed a specific enzymatic activity of approximately 1030.87 IU per mg. The enzyme was active over a wide range of pH and temperatures; optimal activity was observed at 40 °C and pH 4.0. The effects of ions on Bh-EGaseI activity were also studied, and results indicated that activity decreased to different extents upon addition of ions. Investigations on Bh-EGaseI facilitate their potential application in the production of bioenergy and biomaterials from cellulosic biomass in the future. PMID:26410410

  1. Production, purification, and characterization of a cellulase-free thermostable endo-xylanase from Thermoanaerobacterium thermosaccharolyticum DSM 571.

    Science.gov (United States)

    Li, Xun; Shi, Hao; Ding, Huaihai; Zhang, Yu; Wang, Fei

    2014-12-01

    This is the first report describing the cloning, expression, and characterization of a putative thermostable, cellulase-free xylanase (XYN) from the thermophilic bacterium Thermoanaerobacterium thermosaccharolyticum DSM 571. The temperature and pH values for optimal enzyme activity of XYN were found to be 65 °C and pH 6.5, respectively. The XYN activity was apparently enhanced by Co(2+), Mn(2+), and Tween 60 and significantly inactivated by Al(3+), Cu(2+), Zn(2+), and SDS. The K m and V max values of XYN for the hydrolysis of beechwood xylan were 2.1 mg/ml and 222.1 U/mg, respectively. The k cat values of XYN for beechwood xylan at the optimal temperature and pH values were 481.4 s(-1). XYN represents an attractive candidate for use in the large-scale production of xylooligosaccharides (XOs) from forest residues because it is an endo-xylanase capable of degrading xylan. PMID:25261357

  2. Effect of commercial cellulases and refining on kraft pulp properties: correlations between treatment impacts and enzymatic activity components.

    Science.gov (United States)

    Cui, Li; Meddeb-Mouelhi, Fatma; Laframboise, François; Beauregard, Marc

    2015-01-22

    The importance of enzymes as biotechnological catalysts for paper industry is now recognized. In this study, five cellulase formulations were used for fibre modification. The number of PFI revolutions decreased by about 50% while achieving the same freeness value (decrease in CSF by 200 mL) with the enzymatic pretreatment. The physical properties of handsheets were modified after enzymatic pretreatment followed by PFI refining. A slight decrease in tear strength was observed with enzymes C1 and C4 at pH 7 while the most decrease in tear was observed after C2, C3, C5 treatments. C1 and C4 which had xylanase activity improved paper properties, while other enzymes had a negative impact. Therefore, the intricate balance between cellulolytic and hemicellulolytic activity is the key to optimizing biorefining and paper properties. It was also observed that C1 impact was pH dependent, which supports the importance of pH in developing an enzymatic strategy for refining energy reduction. PMID:25439885

  3. Yeasts in mixed deciduous forest areas of Phujong Nayoy National Park and their ability to produce xylanase and carboxymethyl cellulase

    Directory of Open Access Journals (Sweden)

    Jantaporn Thongekkaew,

    2012-04-01

    Full Text Available A total of 61 yeast strains were obtained from 132 samples collected from various sources such as soil, mushroom,flowers, fruits, tree barks and insect frass in the mixed deciduous forest areas of Phujong Nayoy National Park, Thailand.Based on D1/D2 region at the 5 end of the large subunit ribosomal RNA gene (rRNA gene region D1/D2 analysis, 39 strainswere identified as ascomycetous yeasts and distributed to 7 genera i.e. Blastobotrys, Candida, Debaryomyces, Dipodascus,Kodamaea, Pichia and Torulaspora. Twenty strains were identified as basidiomycetous yeasts which belonged to the generaAsterotremella, Cryptococcus, Sporidiobolus and Trichosporon. Another two strains of yeast-like fungi were belonged togenus Aureobasidium. The predominant genus was Candida with a 31.14% contribution. For testing of xylanase and carboxymethylcellulase production of the 61 strains of yeasts and yeast-like fungi, Candida glabrata and Aureobasidiumpullulans showed xylanase activity of 0.91 and 0.52 UmL-1, respectively, and carboxymethyl cellulase activity of 0.38 and0.44 UmL-1, respectively.

  4. Optimization of cellulase production by a brown rot fungus Fomitopsis sp. RCK2010 under solid state fermentation.

    Science.gov (United States)

    Deswal, Deepa; Khasa, Yogender Pal; Kuhad, Ramesh Chander

    2011-05-01

    Culture conditions for enhanced cellulase production from a newly isolated brown rot fungus, Fomitopsis sp. RCK2010 were optimized under solid state fermentation. An initial pH of 5.5 and moisture ratio of 1:3.5 (solid:liquid) were found to be optimal for maximum enzyme production. Of the different carbon sources tested wheat bran gave the maximum production of CMCase (71.526 IU/g), FPase (3.268 IU/g), and β-glucosidase (50.696 IU/g). Among the nitrogen sources, urea caused maximum production of CMCase (81.832 IU/g), where as casein and soyabean meal gave the highest FPase (4.682 IU/g) and β-glucosidase (69.083 IU/g) production, respectively. Among amino acids tested glutamic acid gave the highest production for CMCase (84.127I U/g); however 4-hydroxy-l-proline stimulated maximum FPase production (6.762 IU/g). Saccharification of pretreated rice straw and wheat straw by crude enzyme extract from Fomitopsis sp. RCK2010 resulted in release of 157.160 and 214.044 mg/g of reducing sugar, respectively. PMID:21470856

  5. Effect of nutritional factors on cellulase enzyme and microbial protein production by Aspergillus terreus and its evaluation.

    Science.gov (United States)

    Garg, S K; Neelakantan, S

    1982-01-01

    The biomass yield, cellulolytic activity, and protein recovery using Aspergillus terreus GN1 with alkali-treated sugarcane bagasse was studied using different levels (250-600 mg of N/L of broth) of organic and inorganic nitrogen sources. e.g., cattle urine, urea, cornsteep liquor, ammonium sulfate, ammonium nitrate, ammonium iron sulfate, ammonium chloride, and sodium nitrate. Among different levels of alkali-treated bagasse substrate concentrations (0.5-4.0% w/v) tested, 1.0% substrate yielded the highest crude protein content, protein recovery, and cellulolytic activity. The biomass recovery with 1.0% substrate ranged from 290-380 mg/500 mg bagasse substrate in a 50-mL broth with a nitrogen level of 250-600 mg of N/L in all the sources except ammonium iron sulfate, which yielded 402-439 mg/500 mg bagasse substrate. However, crude protein content of biomass obtained with an ammonium iron sulfate nitrogen source was the lowest. Cornsteep liquor nitrogen source at the rate of 600 mg of N/L yielded the maximum crude protein of 32.9%, protein recovery of 22.2 g/100 g of bagasse, and carboxymethyl cellulase and filter paper enzyme activities of 1.1 and 0.09 units/mL, among the organic and inorganic nitrogen sources studied. In general, the organic nitrogen sources and inorganic nonammonium nitrogen sources were utilized preferentially for protein production over the inorganic ammonium nitrogen sources. The fermentation time required under optimum cultural and nutritional conditions for A. terreus GN1 was also evaluated. The crude protein content of the biomass increased gradually up to the seventh day of fermentation, but the protein recovery rate was high up to two or three days. It was observed that the cellulose utilization rate increased after an initial lag of one day up to the third day and gradually increased further, which corresponded positively with protein content, biomass protein recovery, and cellulase enzyme activity. On the seventh day of fermentation

  6. 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.

  7. 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. PMID:24294252

  8. 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

  9. 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.

  10. In vitro and in situ activity of carboxymethyl cellulase and glutamate dehydrogenase according to supplementation with different nitrogenous compounds

    Directory of Open Access Journals (Sweden)

    Isabela Pena Carvalho de Carvalho

    2012-03-01

    Full Text Available Two experiments were carried out to evaluate the effect of supplementation with different nitrogenous compounds on the activities of carboxymethil cellulase (CMCase and glutamate dehydrogenase (GDH. In the first experiment, four treatments were evaluated in vitro: cellulose, cellulose with casein, cellulose with urea, and cellulose with casamino acids. After 6, 12 and 24 hours of incubation, CMCase and GDH activity, pH, and concentrations of ammonia nitrogen (AN and microbial protein were measured. In the three incubation periods, the concentration of AN was higher when urea was used as a supplemental source of nitrogen. The activity of CMCase was higher with the addition of urea and casamino acids when compared with the control and the casein treatment. Supplementation with casamino acids provided higher GDH activity when compared with the control at 6 hours of incubation. At 12 hours of incubation, the GHD activity was also stimulated by casein. At 24 hours, there was no difference in GHD activity among treatments. In the second experiment, three rumen-fistulated bulls were used for in situ evaluation. Animals were fed Tifton hay (Cynodon sp. ad libitum. The treatments consisted of control (no supplementation, supplementation with non-protein nitrogenous compounds (urea and ammonium sulphate, 9:1 and supplementation with protein (albumin. In treatments with nitrogenous compound supplementation, 1 g of crude protein/kg of body weight was supplied. The experiment was conducted in a 3 × 3 Latin square design. The measurements were performed at 6, 12 and 24 hours after supplementation. No difference in GDH activity was observed among treatments. The control treatment showed higher CMCase activity when compared with the treatments containing supplemental sources of nitrogen. However, urea supplementation provided higher CMCase activity compared to albumin.

  11. Enzymatic saccharification of sugar cane bagasse by continuous xylanase and cellulase production from cellulomonas flavigena PR-22.

    Science.gov (United States)

    Rojas-Rejón, Óscar A; Poggi-Varaldo, Héctor M; Ramos-Valdivia, Ana C; Ponce-Noyola, Teresa; Cristiani-Urbina, Eliseo; Martínez, Alfredo; de la Torre, Mayra

    2016-03-01

    Cellulase (CMCase) and xylanase enzyme production and saccharification of sugar cane bagasse were coupled into two stages and named enzyme production and sugar cane bagasse saccharification. The performance of Cellulomonas flavigena (Cf) PR-22 cultured in a bubble column reactor (BCR) was compared to that in a stirred tank reactor (STR). Cells cultured in the BCR presented higher yields and productivity of both CMCase and xylanase activities than those grown in the STR configuration. A continuous culture with Cf PR-22 was run in the BCR using 1% alkali-pretreated sugar cane bagasse and mineral media, at dilution rates ranging from 0.04 to 0.22 1/h. The highest enzymatic productivity values were found at 0.08 1/h with 1846.4 ± 126.4 and 101.6 ± 5.6 U/L·h for xylanase and CMCase, respectively. Effluent from the BCR in steady state was transferred to an enzymatic reactor operated in fed-batch mode with an initial load of 75 g of pretreated sugar cane bagasse; saccharification was then performed in an STR at 55°C and 300 rpm for 90 h. The constant addition of fresh enzyme as well as the increase in time of contact with the substrate increased the total soluble sugar concentration 83% compared to the value obtained in a batch enzymatic reactor. This advantageous strategy may be used for industrial enzyme pretreatment and saccharification of lignocellulosic wastes to be used in bioethanol and chemicals production from lignocellulose. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:321-326, 2016. PMID:26701152

  12. Isolation and Characterization of Thermophilic Cellulase-Producing Bacteria from Empty Fruit Bunches-Palm Oil Mill Effluent Compost

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    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.

  13. Optimization of covalent immobilization of Trichoderma reesei cellulase onto modified ReliZyme HA403 and Sepabeads EC-EP supports for cellulose hydrolysis, in buffer and ionic liquids/buffer media.

    Science.gov (United States)

    Bilgin, Ramazan; Yalcin, M Serkan; Yildirim, Deniz

    2016-08-01

    The covalent immobilization of Trichoderma reesei cellulase onto modified ReliZyme HA403 and Sepabeads EC-EP supports were carried out. The optimal immobilization conditions were determined using response surface methodology. The hydrolysis of cellulose using the free and immobilized cellulase preparations in ionic liquids (IL) using cosolvents was investigated. The hydrolytic activities in buffer medium containing 25% (v/v) of 1-butyl-3-methylimidazolium hexafluorophosphate were around 2.6-, 1.6-, and 5.5-fold higher than the activities in buffer medium. The retained initial activities were 32% and 57%, respectively for cellulase preparations immobilized onto Sepabeads EC-EP support and onto modified ReliZyme HA403 support after 5 reuses. PMID:25811997

  14. Design and characterizations of two novel cellulases through single-gene shuffling of Cel12A (EG3) gene from Trichoderma reseei.

    Science.gov (United States)

    Yenenler, Aslı; Sezerman, Osman Uğur

    2016-06-01

    Cellulases have great potential to be widely used for industrial applications. In general, naturally occurring cellulases are not optimized and limited to meet the industrial needs. These limitations lead to demand for novel cellulases with enhanced enzymatic properties. Here, we describe the enzymatic and structural properties of two novel enzymes, EG3_S1 and EG3_S2, obtained through the single-gene shuffling approach of Cel12A(EG3) gene from Trichoderma reseei EG3_S1 and EG3_S2 shuffled enzymes display 59 and 75% identity in protein sequence with respect to native, respectively. Toward 4-MUC, the minimum activity of EG3_S1 was reported as 5.9-fold decrease in native at 35°C, whereas the maximum activity of EG3_S2 was reported as 15.4-fold increase in native activity at 40°C. Also, the diminished enzyme activity of EG3_S1 was reported within range of 0.6- to 0.8-fold of native and within range of 0.5- to 0.7-fold of native toward CMC and Na-CMC, respectively. For EG3_S2 enzyme, the improved enzymatic activities within range of 1.1- to 1.4-fold of native and within range of 1.1- to 1.6-fold of native were reported toward CMC and Na-CMC, respectively. Moreover, we have reported 6.5-fold increase in the kcat/Km ratio of EG3_S2 with respect to native and suggested EG3_S2 enzyme as more efficient catalysis for hydrolysis reactions than its native counterpart. PMID:27129352

  15. Use of a Cellulase-Derepressed Mutant of Cellulomonas in the Production of a Single-Cell Protein Product from Cellulose †

    OpenAIRE

    Hitchner, E. V.; Leatherwood, J. M.

    1980-01-01

    A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30°C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 8...

  16. Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

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    Shu-Yang Wang

    Full Text Available 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.

  17. Thermal stability of Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase having a cellulose binding domain from a fungal exoglucanase: Comparison to recombinant CSCMCase

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    Jantaporn Thongekkaew

    2009-10-01

    Full Text Available The Cryptococcus sp. S-2 carboxymethyl cellulase gene (CSCMCase containing 1023 nucleotides and fused to acellulose-binding domain (CBD which originated from cellobiohydrolase I of Trichoderma reesei (CSCMCase-CBD wascloned and expressed in the methylotrophic yeast, Pichia pastoris. The fusion of CBD to CSCMCase resulted in a greatlyenhanced binding toward cellulose for CSCMCase-CBD compared with that for CSCMCase. This was useful for enzymepurification and enzyme immobilization. Furthermore, the recombinant CSCMCase-CBD showed high thermal stability afterincubating at 90oC for 3 h, whereas the recombinant CSCMCase rapidly lost its activity after incubating at 90oC for 1 h.

  18. BIOCONVERSION OF SUGARCANE BAGASSE INTO SECOND GENERATION BIOETHANOL AFTER ENZYMATIC HYDROLYSIS WITH IN-HOUSE PRODUCED CELLULASES FROM Aspergillus sp. S4B2F

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    Sanjeev Kumar Soni

    2010-05-01

    Full Text Available An integrated approach was studied for in-house cellulase production, pretreatment, and enzymatic conversion of sugarcane bagasse into glucose followed by the production of second generation bioethanol. Solid state cultures of Aspergillus sp. S4B2F produced significant levels of cellulase complex on wheat bran, supplemented with 1% (w/w soyabean meal, moistened with 1.5 parts of distilled water after 96 h of incubation at 30oC. The highest productivities of endoglucanase, exoglucanase, and β-glucosidase were 66, 60, and 26 IU/g of fermented dry bran, respectively. The enzyme components had a temperature and pH optima at 50oC and 4.0, respectively and revealed high thermostability at 50oC, retaining 66, 54, and 84% residual activities after 72 h. Pretreatment with 2% alkali in combination with steam was the most efficient pre-hydrolysis method for enzymatic bioconversion and fermentation of cellulosic residue of sugarcane bagasse, which produced the highest cellulose conversion (67%, with glucose and alcohol yields of 323 mg and 175 l respectively per dry gram of bagasse.

  19. Optimization of Enzyme Production Conditions for High-yield Cellulase Strains%纤维素酶高产菌株的产酶条件优化

    Institute of Scientific and Technical Information of China (English)

    张铁涛; 徐云升

    2016-01-01

    CMC-Congo red stain is used to screen the cellulase-degrading strain A2 with high cellulase activity and good stability from lodgy or felled banana plant near the humus soil sample.Then the strain mentioned above is identified as fungus by its morphology.The results of fermentation test show that the initial pH of medium is 5.5,the content of peptone is 0.60%,the content of carbon source is 2.40%,and the maximal activity of FPA is 2.069 IU/g.%采用刚果红鉴别培养基,从倒伏或被砍伐的香蕉株附近的腐质土样中,通过筛选得到一株产纤维素酶活力高且稳定性良好的菌株 A2,镜检初步断定菌株 A2为真菌,确定最佳产酶条件为培养基初始pH 5.5,蛋白胨含量0.60%,碳源含量2.40%,此时滤纸片酶活法(FPA)测定酶活力最大为2.069 IU/g。

  20. Molecular characterization of SCO0765 as a cellotriose releasing endo-β-1,4-cellulase from Streptomyces coelicolor A(3).

    Science.gov (United States)

    Hong, Joo-Bin; Dhakshnamoorthy, Vijayalakshmi; Lee, Chang-Ro

    2016-09-01

    The sco0765 gene was annotated as a glycosyl hydrolase family 5 endoglucanase from the genomic sequence of Streptomyces coelicolor A3(2) and consisted of 2,241 bp encoding a polypeptide of 747 amino acids (molecular weight of 80.5 kDa) with a 29-amino acid signal peptide for secretion. The SCO0765 recombinant protein was heterogeneously over-expressed in Streptomyces lividans TK24 under the control of a strong ermE* promoter. The purified SCO0765 protein showed the expected molecular weight of the mature form (718 aa, 77.6 kDa) on sodium dodecyl sulfate-polyacryl amide gel electrophoresis. SCO0765 showed high activity toward β-glucan and carboxymethyl cellulose (CMC) and negligible activity to Avicel, xylan, and xyloglucan. The SCO0765 cellulase had a maximum activity at pH 6.0 and 40°C toward CMC and at pH 9.0 and 50-60°C toward β-glucan. Thin layer chromatography of the hydrolyzed products of CMC and β-glucan by SCO0765 gave cellotriose as the major product and cellotetraose, cellopentaose, and longer oligosaccharides as the minor products. These results clearly demonstrate that SCO0765 is an endo-β-1,4-cellulase, hydrolyzing the β-1,4 glycosidic bond of cellulose into cellotriose. PMID:27572512

  1. Effect of gamma irradiation and environmental factors on the production of extracellular cellulase enzyme by trichoderma Spp. using banana waste under solid state bio processing

    International Nuclear Information System (INIS)

    Fungal strains were isolated from degraded banana waste including leaves, pseudo stems and skins. Many isolated strains showed cellulolytic activities using the plate screening medium. The hyper cellulolytic isolates were selected on the basis of the diameter of the hydrolysis zone surrounding the colonies and identified to the genus level. The identified strains were found to belong to one of the genera Trichoderma, Aspergillus, Pleurotus or Penicillium. The strain with the larger diameter of the hydrolysis zone was found to belong to the genus Trichoderma. It was further identified to be Trichoderma harzianum, which was selected to be studied. Banana waste including leaves and pseudo stems were inoculated by the selected fungus and the production of the carboxymethyl cellulase (CMCase) and filter paper cellulase (FPCase) was followed during changes of the growth conditions under solid state fermentation. It was found that the two enzymes shared the same incubation temperature (25 degree C) and incubation period (18 days) for the maximum enzyme production. The gamma radiation dose of 1.5 KGy increased the production of CMCase produced on leaves by 4.0% and on pseudo stems by 5.6% and the production of FPCase produced on leaves by 2.4% and on pseudo stems by 2.3%. The results also suggest that FPCase and CMCase enzymes produced on leaves were higher than those produced from pseudo stems and the level of CMCase enzyme produced was higher than that of FPCase

  2. Evaluation of different lignocellulosic substrates for the production of cellulases and xylanases by the basidiomycete fungi Bjerkandera adusta and Pycnoporus sanguineus.

    Science.gov (United States)

    Quiroz-Castañeda, Rosa Estela; Pérez-Mejía, Nancy; Martínez-Anaya, Claudia; Acosta-Urdapilleta, Lourdes; Folch-Mallol, Jorge

    2011-06-01

    Agricultural waste products are potential resources for the production of a number of industrial compounds, including biofuels. Basidiomycete fungi display a battery of hydrolytic enzymes with prospective use in lignocellulosic biomass transformation, however little work has been done regarding the characterization of such activities. Growth in several lignocellulosic substrates (oak and cedar sawdust, rice husk, corn stubble, wheat straw and Jatropha seed husk) and the production of cellulases and xylanases by two basidiomycete fungi: Bjerkandera adusta and Pycnoporus sanguineus were analyzed. Growth for P. sanguineus was best in rice husk while corn stubble supported the highest growth rate for B. adusta. Among the substrates tested, cedar sawdust produced the highest cellulolytic activities in both fungal species, followed by oak sawdust and wheat straw. Xylanolytic activity was best in oak and cedar sawdust for both species. We found no correlation between growth and enzyme production. Zymogram analysis of xylanases and cellulases showed that growth in different substrates produced particular combinations of protein bands with hydrolytic activity. PMID:20963471

  3. Partial purification and properties of cellulase-free alkaline xylanase produced by Rhizopus stolonifer in solid-state fermentation

    Directory of Open Access Journals (Sweden)

    Antonio José Goulart

    2005-05-01

    Full Text Available Rhizopus stolonifer was cultivated in wheat bran to produce a cellulase-free alkaline xylanase. The purified enzyme obtained after molecular exclusion chromatography in Sephacryl S-200 HR showed optimum temperature as 45º C and hydrolysis pHs optima as pH 6.0 and 9.0. Xylanase presented higher Vmax at pH 9.0 (0.87 µmol/mg protein than at pH 6.0 and minor Km at pH 6.0 (7.42 mg/mL than at pH 9.0.Rhizopus stolonifer foi cultivado em meio de farelo de trigo para produzir uma xilanase alcalina celulase-free. Uma amostra parcialmente purificada desta enzima foi obtida após cromatografia de exclusão molecular em Sephacryl S-200 HR. A temperatura ótima de hidrólise determinada (45º C está dentro do intervalo citado na literatura (45º C a 60º C para xilanases microbianas. Quanto ao pH ótimo, a amostra obtida apresentou atividades máximas em pH 6,0 e 9,0. Estes dados diferem da literatura, uma vez que o pH ótimo citado para a maioria das xilanases estudadas varia entre 4,0 e 5,5. De acordo com os estudos cinéticos realizados, a xilanase apresentou maior Vmax em pH 9,0 (0,87 µmol/mg proteína e menor Km em pH 6,0 (7,42 mg/mL. Os dois pHs ótimos determinados podem indicar a presença de isoformas desta enzima. Estes dados são interessantes pelo fato de que enzimas xilanolíticas alcalinas celulase-free podem ser utilizadas para o biobranqueamento da polpa na indústria de papel.

  4. Engineering cellulase mixtures by varying the mole fraction of Thermomonospora fusca E[sub 5] and E[sub 3], Trichoderma reesei CBHI, and Caldocellum saccharolyticum [beta]-glucosidase

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-11-05

    In this study, different mole fractions of pure Thermomonospora fusca E[sub 5] and E[sub 3], plus Trichoderma reesei CBHI were studied for reducing sugar production at 2 h, degree of synergism, and cellulose binding. In addition, the effects of introducing the Caldocellum saccharolyticum [Beta]-glucosidase into this cellulase system were investigated. The cellulases used were purified to homogeneity. Avicel PH 102 was the substrate. Reactions were run at 50 C for 2 h using total cellulase concentrations of 8.3 or 12.2 [mu]M. A bimixture of T. fusca E[sub 3] and T. reesei CBHI was very effective in hydrolyzing microcrystalline cellulose. The addition of endoglucanase E[sub 5] to the mixture only increased conversion to 9.8%. However, when both E[sub 5] and [Beta]-glucosidase were added, conversion increased to 14%. It was also observed that increasing total cellulase concentration beyond 8.3 [mu]M did little to increase percent conversion of cellulose into glucose. The results of the binding studies indicate no competition for binding sites between the endo- and exocellulases.

  5. SCREENING OF HARDWOOD AND SOFTWOOD SPECIES AS BEST SUBSTRATE FOR CELLULASE AND XYLANASE PRODUCTION USING CONSORTIUM OF POTENTIAL ISOLATES BACILLUS COAGULANS B30 + PAENIBACILLUS MUCILAGINOUS B5 + BACILLUS SP. UNDER SSF

    Directory of Open Access Journals (Sweden)

    Richa Kaushal*, Nivedita Sharma and Divya Tandon

    2014-05-01

    Full Text Available The lignocellulosic biomass is known to be an excellent carbon source for microbial enzyme production. In this paper, the cellulase and xylanase production from lignocellulosic materials using consortia of potential hydrolytic bacteria i.e. Bacillus coagulans B30 + Paenibacillus mucilaginous B5 + Bacillus sp. B21 isolated from forest soil under solid state fermentation (SSF was investigated. The maximum cellulase activity of 97.84 U/g and xylanase activity of 67.06 U/g were obtained with water containing pretreated Celtis australis. Additional nutrients of inorganic salts were added in the form of modified basal salt medium (BSM which resulted in increased enzyme production i.e. cellulase activity of 177.14 U/g and xylanase activity of 115.42 U/g using pretreated C. australis under solid-state fermentation (SSF. Results indicate the excellent scope of utilizing C. australis as solid substrate for commercial production of cellulase and xylanase employing consortium of Bacillus spp.

  6. Structure and stability of metagenome-derived glycoside hydrolase family 12 cellulase (LC-CelA a homolog of Cel12A from Rhodothermus marinus

    Directory of Open Access Journals (Sweden)

    Hiroyuki Okano

    2014-01-01

    Full Text Available Ten genes encoding novel cellulases with putative signal peptides at the N-terminus, termed pre-LC-CelA–J, were isolated from a fosmid library of a leaf–branch compost metagenome by functional screening using agar plates containing carboxymethyl cellulose and trypan blue. All the cellulases except pre-LC-CelG have a 14–29 residue long flexible linker (FL between the signal peptide and the catalytic domain. LC-CelA without a signal peptide (residues 20–261, which shows 76% amino acid sequence identity to Cel12A from Rhodothermus marinus (RmCel12A, was overproduced in Escherichia coli, purified and characterized. LC-CelA exhibited its highest activity across a broad pH range (pH 5–9 and at 90 °C, indicating that LC-CelA is a highly thermostable cellulase, like RmCel12A. The crystal structure of LC-CelA was determined at 1.85 Å resolution and is nearly identical to that of RmCel12A determined in a form without the FL. Both proteins contain two disulfide bonds. LC-CelA has a 16-residue FL (residues 20–35, most of which is not visible in the electron density map, probably due to structural disorder. However, Glu34 and Pro35 form hydrogen bonds with the central region of the protein. ΔFL-LC-CelA (residues 36–261 and E34A-LC-CelA with a single Glu34 → Ala mutation were therefore constructed and characterized. ΔFL-LC-CelA and E34A-LC-CelA had lower melting temperatures (Tm than LC-CelA by 14.7 and 12.0 °C respectively. The Tm of LC-CelA was also decreased by 28.0 °C in the presence of dithiothreitol. These results suggest that Glu34-mediated hydrogen bonds and the two disulfide bonds contribute to the stabilization of LC-CelA.

  7. Cellulase, Clostridia, and Ethanol†

    OpenAIRE

    Demain, Arnold L.; Newcomb, Michael; Wu, J. H. David

    2005-01-01

    Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulas...

  8. Cellulase production by Talaromyces emersonii CBS 814. 70 and a mutant UV7 during growth on cellulose, lactose and glucose containing media

    Energy Technology Data Exchange (ETDEWEB)

    Morrison, J.; McCarthy, U.; McHale, A.P.

    1987-07-01

    Recently it has been reported that Talaromyces emersonii CBS 814.70 is capable of growth on lactose containing media. During growth on such media large amounts of beta-glucosidase are secreted into the medium. In addition, low levels of endoglucanase activity have been detected. In order to enhance endoglucanase production, u.v. irradiation and a modified selection procedure yielded a number of mutants. One of these, UV7, was capable of increased cellulase production during growth on cellulose, lactose and glucose containing media. Comparative studies between the wild-type organism and the mutant have shown that the former apparently produces constitutive levels of both endoclucanase and beta-glucosidase. The form of beta-glucosidase that appears to be constitutive is that form previously named BG-1. (Refs. 12).

  9. Production and Optimization of Cellulase Enzyme Using Aspergillus niger USM AI 1 and Comparison with Trichoderma reesei via Solid State Fermentation System

    OpenAIRE

    Ibrahim, C. O.; Darah, I.; Lee, C. K.

    2011-01-01

    Novel design solid state bioreactor, FERMSOSTAT, had been evaluated in cellulase production studies using local isolate Aspergillus niger USM AI 1 grown on sugarcane bagasse and palm kernel cake at 1 : 1 (w/w) ratio. Under optimised SSF conditions of 0.5 kg substrate; 70% (w/w) moisture content; 30°C; aeration at 4 L/h · g fermented substrate for 5 min and mixing at 0.5 rpm for 5 min, about 3.4 U/g of Filter paper activity (FPase) was obtained. At the same time, comparative studies of the enz...

  10. Study on Isolation and Characterization of Bacteria Producing Cellulase%纤维素酶产生菌的筛选及酶学性质研究

    Institute of Scientific and Technical Information of China (English)

    孙玉宇

    2014-01-01

    Isolated Cellulose decomposing ability was the strongest from city life garbage, and extracellular enzyme production strains, tentatively identified as Bacillus ,and enzymatic properties of cellulase produced by the bacteria were investigated. The cellulase reaction, the optimum temperature is 50℃ ; the optimum pH7.0; enzyme at 40~50℃ has good thermal stability; the activity of CMCase in pH 6 ~ 7 can be maintained above 70%, the activity of FPA in pH 6~7 can be maintained more than 79%; Ca2+ and Mg2+ for the enzyme reaction were increased markedly, While Fe3+ and Mn2+ had inhibitory effect on the enzyme reaction, effect of Na+, Zn2+ and K+ on the enzyme is very small.%从城市生活垃圾中分离到一株分解纤维素能力最强,并产生胞外酶的菌株,初步确定为芽孢杆菌,并对该菌所产的纤维素酶的酶学性质进行了初步研究.该纤维素酶反应最佳温度为50℃;最适 pH7.0;酶在40~50℃热稳定性较好; CMCase活力在 pH 6.0~7.0处可保持70%以上, FPA酶活在pH 6.0~7.0处可保持79%以上; Ca2+和Mg2+对酶反应表现为明显的促进作用,而Fe3+和Mn2+对酶反应有抑制作用, Na+、Zn2+和K+对酶的影响很小.

  11. Molecular cloning and characterization of a glycosyl hydrolase family 9 cellulase distributed throughout the digestive tract of the cricket Teleogryllus emma.

    Science.gov (United States)

    Kim, Namjung; Choo, Young Moo; Lee, Kwang Sik; Hong, Seong Jin; Seol, Kwang Youl; Je, Yeon Ho; Sohn, Hung Dae; Jin, Byung Rae

    2008-08-01

    A novel endogenous beta-1,4-endoglucanase (EG) gene belonging to the glycosyl hydrolase family 9 (GHF 9) that is distributed throughout the digestive tract of the cricket Teleogryllus emma was cloned and characterized. This gene, named TeEG-I, consists of eight exons encoding 453 amino acid residues and exists as a single copy in the T. emma genome. TeEG-I possesses all the features, including signature motifs and catalytic domains, of GHF 9 members, sharing high levels of identity with the termite, Mastotermes darwiniensis (64% protein sequence identity), and the cockroach, Panesthia cribrata (62%), GHF 9 cellulases. Recombinant TeEG-I, which is expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells, showed an optimal pH and temperature of pH 5.0 and 40 degrees C. The K(m) and V(max) values for digestion of carboxymethyl cellulose were 5.4 mg/ml and 3118.4 U/mg, respectively. Northern and Western blot analyses revealed that TeEG-I is present throughout the digestive tract, which correlated with the TeEG-I distribution and cellulase activity in the digestive tract as assayed by immunofluorescence staining and enzyme activity assay, respectively. These results indicate that TeEG-I is distributed throughout the entire digestive tract of T. emma, suggesting a functional role of endogenous TeEG-I in a sequential cellulose digestion process throughout the T. emma digestion tract. PMID:18514003

  12. Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hydrolysis of cellulose in presence of xylans.

    Science.gov (United States)

    Dondelinger, Eve; Aubry, Nathalie; Ben Chaabane, Fadhel; Cohen, Céline; Tayeb, Jean; Rémond, Caroline

    2016-03-01

    Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemicellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes developed in the context of lignocellulosic biorefinery. PMID:27001439

  13. Production of Cellulases, Xylanase, Pectinase, alpha-amylase and Protease Enzymes Cocktail by Bacillus spp. and Their Mixed Cultures with Candida tropicalis and Rhodotorula glutinis under Solid State Fermentation

    International Nuclear Information System (INIS)

    A group of twelve locally isolated Bacillus species, B.megaterium (MAI and MA II), B.licheniformis (MLI and ML II); B. circulans, B. stearothermophilis, B.cereus, B.sphaericus, B. pumilus, B. laterosporus, B. coagulans and B. pantothenticus, were examined for the production of cellulases, xylanase, pectinase, alpha-amylase and protease enzymes cocktail on wheat bran under solid state fermentation (SSF). All species were found to be potent hydrolyzing enzymes producers and the superior producing species were B. megaterium MAI and B. licheniformis. On the other hand, both of them still produced highest enzyme titres when mixed with Candida tropicalis or Rhodotorula glutinis, yeast strains. The two superior bacterial strains produced the highest enzymatic activities when coculturing with C. tropicalis compared with coculturing with R. glutinis only or with both C. tropicalis and R. glutinis in combination. The inferior activities of cocultures (B. megaterinm MAI and R. glutinis) were enhanced in carboxymethyl cellulase (CMCase), filter paper cellulase (FPase), avecilase, xylanase, pectinase, -amylase and protease by gamma irradiation at dose 1.0 kGy with percent increase 8 %, 20 %, 10 %, 4 %, 31 %, 22 % and 34 %, respectively as compared with un-irradiated cocultures

  14. 一株产纤维素酶的甲醇利用细菌的鉴定及其纤维素降解条件优化%Identification and optimal degradation conditions for cellulase-degrading enzyme of a methanol-utilizing and cellulase-producing bacterium

    Institute of Scientific and Technical Information of China (English)

    高健; 肖丹青; 刘喜平; 王能强; 张大为

    2012-01-01

    采用刚果红染色法,从废弃矿山周边土壤中筛选出一株产纤维素酶的甲醇利用细菌,命名为xt - 04.形态特征、生理试验及16S rDNA序列和gyrB序列分析表明,该菌株属于Bacillus methylotrophicus.为提高该菌所产纤维素酶的降解能力,首先通过单因子实验考察了底物CMC -Na浓度、反应温度及缓冲液pH值对纤维素酶活力的影响;然后采用响应面分析法对影响纤维素酶活力的3个单因子进行了优化.结果表明,单因素实验得出的适宜反应温度、缓冲液pH和底物浓度分别为70℃、5.0和2% (20 mg/mL);响应面法得出的最高酶活力条件:反应温度、pH和底物浓度分别为66.1℃、4.81和19.01mg/mL.在最优条件下,酶活力达到17.85 U/mL,比优化前的酶活力12.84 U/mL提高了39.01%.因此,鉴于这种纤维素酶能耐受较高温度和酸性条件,该菌株所产纤维素酶可能在工业中具有良好的应用前景.%A methanol-utilizing and cellulase-producing bacteria, designated strain xt-04, was isolated from the soil of abandoned mine lands of Hunan Province by Congo red staining test. This strain was identified as the species Bacillus methylotrophicus based on the morphological, physiological characteristics and 16S rDNA and gyrB gene sequences analysis. In order to improve the catalytic ability of the cellulase produced by this strain, influences of the concentration of CMC-Na, temperature and pH on the activity of cellulose-decomposing enzyme were investigated by single factor experiment. Then, response surface analysis was used to optimize the influences of three factors on the cellulase activity. The results indicated that the appropriate concentrations of CMC-Na, temperature and pH, based on single-factor experiments, were 20 mg/mL, 70 ℃ , and 5.0, respectively. And the optimal concentrations of CMC-Na, temperature and pH of cellulase-producing, from response surface analysis, were 19.01 mg/mL, 66.1 ℃ and 4

  15. Use of a cellulase-derepressed mutant of cellulomonas in the production of a single-cell protein product from cellulose.

    Science.gov (United States)

    Hitchner, E V; Leatherwood, J M

    1980-02-01

    A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30 degrees C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 85% of the cells with the flotation procedure. Cellulomonas was 68% true protein and had an essential amino acid profile featuring a high lysine content (6.5% of protein). The Cellulomonas product was evaluated nutritionally with weanling rats. The net protein utilization value for the protein supplemented with methionine was 50.4% Weight gain of rats on the Cellulomonas diet was similar to that of rats fed a casein diet. PMID:16345511

  16. 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.

  17. 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)

    Guido Zacchi

    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.

  18. Purification and characterization of cellulase-free low molecular weight endo β-1,4 xylanase from an alkalophilic Cellulosimicrobium cellulans CKMX1 isolated from mushroom compost.

    Science.gov (United States)

    Walia, Abhishek; Mehta, Preeti; Chauhan, Anjali; Kulshrestha, Saurabh; Shirkot, C K

    2014-10-01

    Alkalophilic Cellulosimicrobium cellulans CKMX1 isolated from mushroom compost is first report on actinomycetes that has the ability to produce thermostable cellulase-free xylanase, which is an important industrial enzyme used in the pulp and paper industry. Strain CKMX1 was characterized by metabolic fingerprinting, whole-cell fatty acids methyl ester analysis and 16Sr DNA and found to be C. cellulans CKMX1.The enzyme was purified by gel permeation and anion exchange chromatography and had a molecular mass of 29 kDa. Xylanase activity was optimum at pH 8.0 and 55 °C. The enzyme was somewhat thermostable, retaining 50 % of the original activity after incubation at 50 °C for 30 min. The xylanase had K m and V max values of 2.64 mg/ml and 2,000 µmol/min/mg protein in oat spelt xylan, respectively. All metal ions except HgCl2, CoCl2 as well as CdCl2 were well tolerated and did not adversely affect xylanase activity. The deduced internal amino acid sequence of C. cellulans CKMX1 xylanase by matrix assisted laser desorption ionization-time of flight mass spectrometry resembled the sequence of β-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation could be useful for pulp and paper biobleaching are discussed in this manuscript. PMID:24908422

  19. A 24.7-kDa copper-containing oxidase, secreted by Thermobifida fusca, significantly increasing the xylanase/cellulase-catalyzed hydrolysis of sugarcane bagasse.

    Science.gov (United States)

    Chen, Cheng-Yu; Hsieh, Zhi-Shen; Cheepudom, Jatuporn; Yang, Chao-Hsun; Meng, Menghsiao

    2013-10-01

    Thermobifida fusca is a moderately thermophilic soil bacterium belonging to Actinobacteria. It has been known for its capability to degrade plant cell wall polymers except lignin and pectin. To know whether it can produce enzymes to facilitate lignin degradation, the extracellular proteins bound to sugarcane bagasse were harvested and identified by liquid chromatography tandem mass spectrometry. Among the identified proteins, a putative copper-containing polyphenol oxidase of 241 amino acids, encoded by the locus Tfu_1114, was thought to presumably play a role in lignin degradation. This protein (Tfu1114) was thus expressed in E. coli and characterized. Similarly to common laccases, Tfu1114 is able to catalyze the oxidation reaction of phenolic and nonphenolic lignin related compounds such as 2,6-dimethoxyphenol and veratryl alcohol. More interestingly, it can significantly enhance the enzymatic hydrolysis of bagasse by xylanase and cellulase. Tfu1114 is stable against heat, with a half-life of 4.7 h at 90 °C, and organic solvents. It is sensitive to ethylenediaminetetraacetic acid and reducing agents but resistant to sodium azide, a potent inhibitor of laccases. Atomic absorption spectroscopy indicated that the ratio of copper to the protein monomer is 1, instead of 4, a feature of classical laccases. All these data suggest that Tfu1114 is a novel oxidase with laccase-like activity, potentially useful in biotechnology application. PMID:23377789

  20. Isolation, Screening, and Identification of Cellulolytic Bacteria from Natural Reserves in the Subtropical Region of China and Optimization of Cellulase Production by Paenibacillus terrae ME27-1

    Directory of Open Access Journals (Sweden)

    Yan-Ling Liang

    2014-01-01

    Full Text Available From different natural reserves in the subtropical region of China, a total of 245 aerobic bacterial strains were isolated on agar plates containing sugarcane bagasse pulp as the sole carbon source. Of the 245 strains, 22 showed hydrolyzing zones on agar plates containing carboxymethyl cellulose after Congo-red staining. Molecular identification showed that the 22 strains belonged to 10 different genera, with the Burkholderia genus exhibiting the highest strain diversity and accounting for 36.36% of all the 22 strains. Three isolates among the 22 strains showed higher carboxymethyl cellulase (CMCase activity, and isolate ME27-1 exhibited the highest CMCase activity in liquid culture. The strain ME27-1 was identified as Paenibacillus terrae on the basis of 16S rRNA gene sequence analysis as well as physiological and biochemical properties. The optimum pH and temperature for CMCase activity produced by the strain ME27-1 were 5.5 and 50°C, respectively, and the enzyme was stable at a wide pH range of 5.0–9.5. A 12-fold improvement in the CMCase activity (2.08 U/mL of ME27-1 was obtained under optimal conditions for CMCase production. Thus, this study provided further information about the diversity of cellulose-degrading bacteria in the subtropical region of China and found P. terrae ME27-1 to be highly cellulolytic.

  1. Production of cellulase-free xylanase by Trichoderma reesei SAF3 Produção de xilanase livre de celulase por Trichoderma reesei SAF3

    Directory of Open Access Journals (Sweden)

    Sanjay Kar

    2006-12-01

    Full Text Available A xylanase producing fungi has been isolated from soil and identified as Trichoderma reesei SAF3. Maximum growth of the organism was found at 48 h under submerged condition in xylan containing enriched medium, whereas highest enzyme production (4.75U/mL was recorded at 72 h. No detectable cellulase activity was noted during whole cultivation period. The partially purified enzyme hydrolyzed xylan into xylopentose and xylose. All these properties of xylanase highlighten its promising uses in industrial scale.A partir de solo, isolou-se uma cepa de fungos produtos de xilanase, posteriormente identificado como Trichoderma reesei SAF3. O crescimento máximo do fungo foi obtido após 48h em condições submersas em meio de cultura contendo xilano, enquanto produção máxima de enzima (4,75U/mL ocorreu em 72h. Durante o período de cultivo, não foi detectada atividade celulásica. A enzima parcialmente purificada hidrolizou xilano a xilopentose e xilose. Essas propriedades da xilanase destacam seu uso promissor em escala industrial.

  2. 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.

  3. Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis

    International Nuclear Information System (INIS)

    The crystallization-phase-diagram-guided method is effective for growing large protein crystals for neutron protein crystallography. Neutron protein crystallography (NPC) is a powerful tool for determining the hydrogen position and water orientation in proteins, but a much larger protein crystal is needed for NPC than for X-ray crystallography, and thus crystal preparation is a bottleneck. To obtain large protein crystals, it is necessary to know the properties of the target protein in the crystallization solution. Here, a crystal preparation method of fungal cellulase PcCel45A is reported, guided by the phase diagram. Nucleation and precipitation conditions were determined by sitting-drop vapor diffusion. Saturation and unsaturation conditions were evaluated by monitoring crystal dissolution, and a crystallization phase diagram was obtained. To obtain a large crystal, crystallization solution was prepared on a sitting bridge (diameter = 5 mm). Initial crystallization conditions were 40 µl of crystallization solution (40 mg ml−1 protein with 30.5% 3-methyl-1,5-pentanediol in 50 mM tris-HCl pH 8.0) with a 1000 µl reservoir (61% 3-methyl-1,5,-pentanediol in 50 mM tris-HCl pH 8.0) at 293 K. After the first crystal appeared, the concentration of precipitant in the reservoir solution was reduced to 60% to prevent formation of further crystals. Finally, we obtained a crystal of 6 mm3 volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction

  4. Modification in the properties of paper by using cellulase-free xylanase produced from alkalophilic Cellulosimicrobium cellulans CKMX1 in biobleaching of wheat straw pulp.

    Science.gov (United States)

    Walia, Abhishek; Mehta, Preeti; Guleria, Shiwani; Shirkot, Chand Karan

    2015-09-01

    Alkalophilic Cellulosimicrobium cellulans CKMX1 isolated from mushroom compost is an actinomycete that produces industrially important and environmentally safer thermostable cellulase-free xylanase, which is used in the pulp and paper industry as an alternative to the use of toxic chlorinated compounds. Strain CKMX1 was previously characterized by metabolic fingerprinting, whole-cell fatty acids methyl ester analysis, and 16S rDNA and was found to be C. cellulans CKMX1. Crude enzyme (1027.65 U/g DBP) produced by C. cellulans CKMX1, having pH and temperature optima of 8.0 and 60 °C, respectively, in solid state fermentation of apple pomace, was used in the production of bleached wheat straw pulp. Pretreatment with xylanase at a dose of 5 U/g after pulping decreased pulp kappa points by 1.4 as compared with the control. Prebleaching with a xylanase dose of 5 U/g pulp reduced the chlorine charge by 12.5%, increased the final brightness points by approximately 1.42% ISO, and improved the pulp strength properties. Xylanase could be substituted for alkali extraction in C-Ep-D sequence and used for treating chemically bleached pulp, resulting in bleached pulp with higher strength properties. Modification of bleached pulp with 5 U of enzyme/g increased pulp whiteness and breaking length by 1.03% and 60 m, respectively; decreased tear factor of pulp by 7.29%; increased bulk weight by 3.99%, as compared with the original pulp. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. Cellulosimicrobium cellulans CKMX1 has a potential application in the pulp and paper industries. PMID:26220821

  5. Selection of Bacillus spp. for Cellulase and Xylanase Production as Direct-Fed Microbials to Reduce Digesta Viscosity and Clostridium perfringens Proliferation Using an in vitro Digestive Model in Different Poultry Diets

    Science.gov (United States)

    Latorre, Juan D.; Hernandez-Velasco, Xochitl; Kuttappan, Vivek A.; Wolfenden, Ross E.; Vicente, Jose L.; Wolfenden, Amanda D.; Bielke, Lisa R.; Prado-Rebolledo, Omar F.; Morales, Eduardo; Hargis, Billy M.; Tellez, Guillermo

    2015-01-01

    Previously, our laboratory has screened and identified Bacillus spp. isolates as direct-fed microbials (DFM). The purpose of the present study was to evaluate the cellulase and xylanase production of these isolates and select the most appropriate Bacillus spp. candidates for DFM. Furthermore, an in vitro digestive model, simulating different compartments of the gastrointestinal tract, was used to determine the effect of these selected candidates on digesta viscosity and Clostridium perfringens proliferation in different poultry diets. Production of cellulase and xylanase were based on their relative enzyme activity. Analysis of 16S rRNA sequence classified two strains as Bacillus amyloliquefaciens and one of the strains as Bacillus subtilis. The DFM was included at a concentration of 108 spores/g of feed in five different sterile soybean-based diets containing corn, wheat, rye, barley, or oat. After digestion time, supernatants from different diets were collected to measure viscosity, and C. perfringens proliferation. Additionally, from each in vitro simulated compartment, samples were taken to enumerate viable Bacillus spores using a plate count method after heat-treatment. Significant (P < 0.05) DFM-associated reductions in supernatant viscosity and C. perfringens proliferation were observed for all non-corn diets. These results suggest that antinutritional factors, such as non-starch polysaccharides from different cereals, can enhance viscosity and C. perfringens growth. Remarkably, dietary inclusion of the DFM that produce cellulase and xylanase reduced both viscosity and C. perfringens proliferation compared with control diets. Regardless of diet composition, 90% of the DFM spores germinated during the first 30 min in the crop compartment of the digestion model, followed by a noteworthy increased in the intestine compartment by ~2log10, suggesting a full-life cycle development. Further studies to evaluate in vivo necrotic enteritis effects are in

  6. EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

    OpenAIRE

    Rincón, Marco T.; McCrae, Sheila I.; Kirby, James; Scott, Karen P.; Flint, Harry J.

    2001-01-01

    The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino aci...

  7. 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

  8. 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

  9. Isolation of a Cotton Stalk Cellulose Decomposing Fungus and Its cellulase Properties%一株棉秆纤维素分解真菌的分离筛选及酶学性质研究

    Institute of Scientific and Technical Information of China (English)

    白宝伟; 张琴; 滕立平; 杨瑛; 李艳宾

    2012-01-01

    [Objective] The purpose of this project was to study cellulase properties of a cellulose decomposing fungus SJ - 1 isolated from cotton stalk decomposing products were studied. [Method] Effects of temperature, pH value and metal ions on cellulase activity were studied, and Michaelis constant Km of enzymatic reaction and the maximum reaction rate Vmax were examined by Lineweaver - Burk method. [ Result]The best reaction temperature to CMCase and FPase was ranged from 50 to 60℃, and the best pH value was 7. 0, which showed the tolerance to high temperature and alkali. K+ and Fe2+ activated the cellulase activity significantly, while Cu2 + , Mg2 + , Ca2+ and Al3 + showed certain inhibition, the inhibition of Al3+ exhibited much more strongly, Cu2+ inhibited FPase strongly as well, Mn2+ showed activation to FPase but inhibition to CMCase, Zn2 + had no influence on cellulase activity. When CMC - Na was taken as substrate, Km and Vmax of the enzymatic reaction were 2.69 mg/mL and 0.53 mg/( mL·min) respectively. [Conclusion] The strain showed good properties of cellulase activity, and provided references for further breeding and alteration of this fungus.%[目的]对从棉秆腐解物中筛选得到的高效棉秆分解真菌SJ-1进行纤维素酶酶学性质的研究.[方法]研究温度、pH、金属离子对纤维素酶活力的影响,并以Lineweaver -Burk作图法测定酶促反应米氏常数Km及最大反应速率Vmax.[结果]该菌株CMCase和FPase的最适反应温度在50~60℃,最适pH为7.0,有较好的耐高温及耐碱能力.K+、Fe2对酶活有显著的激活作用,而Cu2+、Mg2+、Ca2+、Al3+等有一定的抑制作用,其中Al3的抑制作用较为强烈,Cu2+对FPase抑制较强,Mn2-对FPase有激活作用而对CMCase有抑制作用,Zn2对酶活性无明显影响.以CMC - Na做底物时酶反应的Km为2.69 mg/mL,Vmax为0.53 mg/( mL·min).[结论]菌株SJ-1的纤维素酶性质较为优良,为进一步进行菌株的选育与改造提供参考依据.

  10. 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

  11. Atividade da celulase e β-galactosidase no estudo da firmeza da polpa de mamões 'golden' e 'gran golden' Cellulase and β-galactosidase activities in 'golden' and 'gran golden' papaya softening

    Directory of Open Access Journals (Sweden)

    Camilla Zanotti Gallon

    2009-12-01

    Full Text Available O objetivo desse trabalho foi avaliar a ação das enzimas celulase e β-galactosidase em relação à perda de firmeza dessas cultivares de mamões 'Gran Golden' e 'Golden' devido a relatos de uma perda de firmeza diferenciada entre as cvs. Os frutos foram armazenados a 25ºC e analisados diariamente quanto à firmeza da polpa e à atividade enzimática da celulase e β-galactosidase durante 8 dias. Os resultados de firmeza da polpa e atividade enzimática foram submetidos às análises de correlação e regressão. No 4º dia pós-colheita os mamões 'Golden' apresentaram firmeza média de 60,6 N e os 'Gran Golden' 31,1 N e a um aumento da atividade da celulase e da β-galactosidase. Os dados gerados neste trabalho sugerem que as enzimas celulase e β-galactosidase atuam diferentemente no processo de perda de firmeza dos frutos das cultivares Goldene Gran Golden. Aantecipaçãonaperdade firmezade 'Gran Golden' pode estar relacionada com a maior atividade dessas enzimas.It has been reported by orchards from the north of Espírito Santo state that 'Gran Golden' papaya loses firmness faster than 'Golden'. The goal of this work was to evaluate the action of cellulase and β-galactosidase related to the softening on papaya. The fruits have been stored at 25ºC and firmness and enzymes activities were daily analyzed during 8 days. The results were submitted to correlation and regression analysis. The activity of cellulase and β-galactosidase had increased for both cultivars. The 4th postharvest day showed that 'Golden' firmness was 6.18 while 'Gran Golden' was 31.1 N. Fruit softening in 'Gran Golden' was intense and the fruit was very soft at ripe stage. These works show that hydrolytic enzymes cellulase and β-galactosidase act differently in the softening process in 'Golden'and 'Gran Golden'papaya. The flesh firmness on 'Gran Golden' is related to the increased activity of these enzymes. These results can help to choose which cultivar to produce

  12. Effects of acids, pH and temperature on acid cellulase activity%酸的种类、pH和温度对酸性纤维素酶活力的影响

    Institute of Scientific and Technical Information of China (English)

    刘幸乐; 姚继明; 吴远明

    2012-01-01

    以8种酸(甲酸、冰醋酸、羟基乙酸、乳酸、柠檬酸、酒石酸、氨基乙酸和氨基磺酸)配制成pH=5.0的缓冲液,测定酸性纤维素酶在各缓冲液中的活力.同时以冰醋酸作为pH调节剂,采用中心合成设计法,分析和优化pH和温度对酶活力的影响,得出线性回归方程和优化值.结果表明,在pH=5.0的条件下,乳酸和酒石酸为酸剂的酶活力高于冰醋酸,羟基乙酸、甲酸和柠檬酸比冰醋酸略低,而氨基乙酸和氨基磺酸明显低于冰醋酸;采用冰醋酸调节pH,酶活力随着温度的升高和pH的降低而增加,pH影响的显著性要大于温度.酸性纤维素酶在49.8℃,pH=4.8可以实现最佳的活力.%Eight kinds of acids (formic acid, acetic acid, glycoiic acid, lactic acid, citric acid, tartaric acid; glycine and aminosulphonic acid) were used as buffer solutions with Ph=5.0. The acid cellulase activities were measured individually in these buffer solutions. Meanwhile using acetic acid as Ph regulator, the effects of Ph and temperature on cellulose activity were analyzed and optimized by central composite design to obtain linear regression equatian and optimal factors. The results showed that tartaric acid and lactic acid exhibited higher activity than acetic acid at Ph 5.0, glycoiic acid, formic acid and citric acid gave little lower activity compared with acetic acid, while the glycine, aminosulphonic acid gave much lower cellulase activity than acetic acid. Applying acetic acid to adjust Ph, cellulase activity increased with the rising of temperature and the decline of Ph. Ph showed more significance than temperature, the optimum performance of activity was at 49.8 ℃,Ph= 4.8.

  13. Engineering Cellulase Enzymes for Bioenergy

    OpenAIRE

    Atreya, Meera Elizabeth

    2015-01-01

    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 polysacch...

  14. 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.

  15. Kinetics of Cellulase Based on the Model of Second-order Enzymatic Deactivation%基于二级酶失活模型的纤维素酶反应动力学

    Institute of Scientific and Technical Information of China (English)

    张宇; 许敬亮; 余强; 袁振宏; 刘云云; 亓伟

    2011-01-01

    酶催化、失活机制的模糊以及影响异相体系因素的大量存在,使得纤维素水解的酶催化过程高度复杂,很难为之建立机理模型.假定纤维素酶失活模型为二级反应,由准稳态理论推导出最终产物葡萄糖浓度与时间,初始酶浓度关系的半经验半理论模型.该模型只含两个参数,能对试验数据很好的拟合,相关系数R2均在0.98以上.用Bailey改进的米式方程进一步拟合不同初始酶浓度下的初速度关系,相关系数R2=0.977 3,求得最大反应速度为2.7425 g/(L·h),半饱和常数为3.010 6 g/L.该模型还表明酶失活速率常数随着初始酶浓度的增加而减小,呈线性关系.失活速度随着初始酶浓度的增加而增加.%Enzymatic hydrolysis of cellulose was extremely complex because of the unclear enzymatic hydrolysis , deactivation mechanisms and many factors that affect the heterogeneous system. Therefore, it is difficult to build a mechanistic model to study cellulose hydrolysis by cellulase. Under some assumed conditions such as the second-order cellulase deactivation and quasi-steady state theory, a semi mechanistic and empirical model describing the relationship between product concentration and time, the initial enzyme concentration was deduced. The mathematic model was a simple mathematic function that contained only two parameters. The experimental result was in accordance with the deduced mathematic model, where the correlation coefficients (R2) were above 0. 98. The relationship between initial enzyme concentration and initial hydrolysis rate calculated from the mathematic model showed good agreement with another type of Henri-Michaelis-Menten equation proposed by Bailey, where the R2 was 0. 977 3. The maximum hydrolysis rate and half saturation constant was 2. 742 4 g/ (L · h) and 3. 013 0 g/L, respectively. It was shown from the model that the rate constant of cellulase deactivation decreased when initial enzyme concentration increased

  16. 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

  17. Study on Optimal Conditions of Degradation of Wheat Straw by Cellulase and Analysis of Kinetics%纤维素酶降解小麦秸秆最适条件的研究及其动力学分析

    Institute of Scientific and Technical Information of China (English)

    田萍; 王浩菊; 马齐; 陈坤奇; 周婷

    2012-01-01

    以小麦秸秆为原料,通过正交实验对纤维素酶降解秸秆纤维的影响因素进行了研究.结果表明,影响小麦秸秆降解的因素依次为:酶量>酶解时间>料液比>反应温度,其最适条件是:加酶量为40 u/g,酶解时间为10h,反应温度为40℃,料液比为1∶3,总糖含量达到43.24%.以米氏方程为基础,建立起最适酶解条件下总纤维素降解的动力学模型.%The influence factors of degradation of straw fiber by cellulase were studied using wheat straw as the raw material through the orthogonal experiments. The results showed that the factors of influence on enzymatic hydrolysis of wheat straw were successively: the amount of enzyme>the time of hydrolysis>the ratio of material and liquid> reaction temperature. The optimal conditions of enzymatic hydrolysis were that the amount of cellulase was 40 u/g, the time of hydrolysis was 10 h, the reaction temperature was 40 C and the ratio of material to liquid was 1:3. The total sugar contents could reach 43. 24%. Under the optimal conditions, the kinetics model of degradation of the total cellulose was established based on Michaelis-Menten equation.

  18. Optimization of Solid State Fermentation(SSF) Conditions with Orange Peels for Production of Cellulase by Rhizopus oryzae%米根霉固态发酵橘皮产纤维素酶工艺的优化

    Institute of Scientific and Technical Information of China (English)

    张帅; 董基; 吴紫俊

    2012-01-01

    With orange peels as raw materials and Rhizopus oryzae as production strains, Cellulase was producted by solid state fermentation(SSF). Three important factors of water content of medium, inoculums size and culture time on cellulose activities were researched respectively by single factor test. Based on it, the enzyme production conditions were optimized by Box-Behnken Design, regression and analysis of variance of experimental data was made by Design Expert. The optimum conditions of enzyme production was determined finally, water content of SSF medium was 12.24 mL, inoculum size was 10.76%, culture time was 72.64 h. Activity of cellulase producted on the above conditions were 464.33 U/g.%以橘皮为原料,以米根霉为生产菌株,采用固态发酵法生产纤维素酶。通过单因子试验分别考察了发酵培养基水分含量、接种量及培养时间三个重要因子对纤维素酶活力的影响,在此基础上,采用Box-Behnken设计对产酶工艺进行优化,利用Design Expert软件对试验数据进行回归拟合和方差分析,最终确定产酶最优工艺条件为:发酵培养基水分含量12.24 mL,接种量10.76%,培养时间72.64 h,在最优条件下所得纤维素酶的酶活力为464.33 U/g。

  19. 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

    OpenAIRE

    Juan Reyes; Patricio Peralta-Zamora; Nelson Durán

    1998-01-01

    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 structu...

  20. 纤维素酶法提取竹叶黄酮的传质动力学%Mass Transfer Kinetics of Flavonoids Extraction from Baantant Leaves with Cellulase-Assisted Aqueous Extractant

    Institute of Scientific and Technical Information of China (English)

    魏凤玉; 陈玮; 方菊; 宝呼和

    2013-01-01

    采用纤维素酶法提取竹叶中的总黄酮类化合物,讨论了温度、原料尺寸、转速及纤维素酶用量对传质速率的影响,并用扫描电镜观察了水提和酶提取后样品的细胞结构变化。结果表明,竹叶黄酮的提取动力学符合平板型Fick 第二定律,提取过程主要受内扩散控制;纤维素酶不改变传质动力学机理,它只破坏细胞壁,从而降低扩散阻力使溶质的传质速率和表观扩散系数D′提高。实验还得到了D′与提取温度、竹叶几何尺寸之间的关系,在30~60℃,酶提时的 D′为1.95×10-13~6.85×10-13 m2×s-1,水提时的 D′为1.73×10-13~6.05×10-13 m2×s-1;酶提时的活化能 Ea 为11.853 kJ×mol-1,比水提时降低了9.59%。该研究为酶法提取竹叶黄酮的工艺设计及工业化应用提供了理论依据。%In this study, cellulase-assisted aqueous extractant (CAE) and aqueous extractant (AE) were adopted separatively for the extraction of the total flavonoid compounds from bamboo leaves. The influence of temperature, raw material size, rotation speed of stirrer and cellulase dosage on the mass transfer rates of the flavonoid compounds was examined. In addition, the micro-structural changes of bamboo leaf cells after the CAE or AE process were observed by scanning electron microscopy (SEM). Results show that the Fick’s second law fits well with the experimental data and the extraction rate is controlled mainly by the inner diffusion process. The presence of cellulase has nearly no influence on the mass transfer mechanism, and can only damage the cell walls and reduce the resistance of diffusion. The mass transfer rate and apparent diffusion constant D′ are increased accordingly. The relationship of D′with the extraction temperature and the bamboo leaf size was deduced. D′ varies from 1.95×10-13 to 6.85×10-13 m2×s-1 for CAE and from 1.73×10-13 to 6.05×10-13 m2×s-1 for AE in a temperature range between 30

  1. Development of malfunction diagnosis system for fermentation based on fuzzy inference and its application to industrial scale production of alkaline cellulase; Fuzzy suiron ni motozuku baiyo ijo shindan shisutemu no kaihatsu to arukarisei seruraze kogyo seisan eno tekiyo

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, T. [Kao Corp., Tokyo (Japan). Processing Development Research Lab.; Osaka Univ., Osaka (Japan). Graduate School of Engineering Science; Takeuchi, K. [Kao Corp., Tokyo (Japan). Processing Development Research Lab.; Taya, M. [Osaka Univ., Osaka (Japan). Graduate School of Engineering Science

    2000-09-10

    A malfunction diagnosis system based on fuzzy inference was developed for industrial scale fermentation with an alkaliphilic cellulase-producing bacterium. The index J indicating the degree of malfunction in the fermentation process is calculated from the deviations of actual values from the standard values with respect to three kinds of process data obtained from on-line sensors. The diagnosis system proposed in this study is as follows. The fermentation process is automatically categorized into one of three states (normal, intermediate and abnormal) according to computation of the J value. Only when the fermentation process is categorized to an 'intermediate' state, an operator should supervise the process by means of direct observation of process data. The diagnosis system was applied to 100 runs of batch fermentations conducted on an industrial scale. In all cases, it was confirmed that this system worked to offer the correct diagnosis results. The introduction of the system resulted in the reduction of over 99 % in the operators' diagnosing labor because their supervising tasks could be limited to the intermediate cases. (author)

  2. EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

    Science.gov (United States)

    Rincón, Marco T.; McCrae, Sheila I.; Kirby, James; Scott, Karen P.; Flint, Harry J.

    2001-01-01

    The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945–1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex. PMID:11571138

  3. 纤维素酶—乙醇法提取苦丁茶中总黄酮%Extraction of Total Flavonoids from the Leaves of Ilex latifolia by Cellulase-Ethanol Method

    Institute of Scientific and Technical Information of China (English)

    黄靖

    2012-01-01

    Total flavonoids were extracted from leaves of Ilex latifolia (Kuding tea) by cellulase-ethanol method. The effects of cellulose dose, enzymolysis time, temperature, pH, volume fraction of ethanol, and dose of ethanol on yield of total flavonoids were studied by single factor tests so as to optimize the extraction conditions. The results showed that the optimum extraction conditions were obtained as follows, dose of cellulose enzyme, 1.0 mg per 5.0 g Kuding tea flour; enzymolysis time, 2.0 h; enzymolysis temperature, 55℃; pH, 4.5; volume fraction of ethanol, 60%; dose of ethanol, 35 mL per 5.0 g Kuding tea flour. The yield of flavonoids was up to 7.80% under these conditions.%采用纤维素酶酶解预处理与乙醇浸提法相结合从苦丁茶中提取总黄酮.通过单因素试验考察酶用量、酶解时间、酶解温度、pH、乙醇体积分数和乙醇用量对总黄酮提取率的影响,优化提取工艺条件.结果表明,优化的提取工艺条件为0.5 g苦丁茶粉末中加入纤维素酶1.0mg、酶解时间2.0 h、酶解温度55℃、酶解pH 4.5、体积分数60%的乙醇用量35 mL,最佳提取工艺条件下苦丁茶中总黄酮提取率达7.80%.

  4. 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. PMID:26073313

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 甲酸与纤维素酶和木聚糖酶对多花黑麦草与白三叶混合青贮料发酵品质的影响%Effects of formic acid, cellulase and xylanase on fermentation quality of Lolium multiflorum and Trifolium repens mixture silage during ensiling

    Institute of Scientific and Technical Information of China (English)

    庄苏; 丁立人; 周建国; 王恬

    2013-01-01

    To evaluate the effects of formic acid, cellulase and xylanase on fermentation quality of mixture silage during ensiling, 2 000 g chopped Lolium multijlorum(80% ) and Trifolium repens(20% ) mixtures were ensiled in laboratory plastic bag either untreated (control) or treated with formic acid, cellulase, formic acid plus cellulase, xylanase, formic acid plus xylanase, cellulase plus xylanase and formic acid plus cellulase plus xylanase, respectively. Triplicate bags were opened at 0 d,2d,4d,6d,8d and 30 d of ensiling for chemical analyses. The pH value in all treated silages was lower (P<0. 05) than control at the end of ensiling. The formic acid, enzyme or formic acid plus enzyme treatments enhanced (P<0. 05) water soluble carbohydrate content significantly compared with control at all ensiling periods. The lactic acid content and acetic acid content were higher (P<0. 05) in the enzyme treatment than those in the formic acid-contained treatments and control, respectively. However, the acetic acid content was lower ( P<0. 05 ) in formic acid-contained treatments than that in enzymes treated silages. Relative to control, all treatments had lower (P< 0. 05 ) ammonia-N concentrations during ensiling. The enzyme treatments effectively (P<0. 05) decreased neutral detergent fiber and acid detergent fiber contents in the silages. The results suggested that the addition of formic acid and enzymes improved the Lolium multiflorum and Trifolium repens mixture silage quality, and the enzyme treatments were better than formic acid treatments during ensiling.%为评价甲酸与纤维素酶和木聚糖酶处理后多花黑麦草与白三叶混合青贮料发酵品质的变化,试验将混合青贮料分为对照组(未处理)、甲酸添加组、纤维素酶添加组、甲酸+纤维素酶添加组、木聚糖酶添加组、甲酸+木聚糖酶添加组、纤维素酶+木聚糖酶添加组、甲酸+纤维素酶+木聚糖酶添加组共8组,每组3个重复.青贮原料按80

  12. Efeito da celulase sobre a decomposição do pergaminho e sua influência na velocidade e porcentagem de germinação de sementes de cafeeiro Effect of the cellulase about the decomposition of the parchment and speed of germination of coffee seeds

    Directory of Open Access Journals (Sweden)

    Juliana de Fátima Sales

    2005-12-01

    Full Text Available Com este trabalho, objetivou-se verificar a ação da celulase sobre a decomposição do pergaminho das sementes de cafeeiro. Foram implantados dois experimentos, sendo o primeiro, realizado em condições in vitro. Constituído por três ensaios para avaliar os efeitos das concentrações da enzima, do substrato e sacarificação enzimática do pergaminho. O segundo, foi realizado em condições in vivo, com imersão das sementes de cafeeiro em solução de celulase na concentração de 1,6 g.L-1 em tampão citrato de potássio 0,05 mol.L-1. Observou-se que a quantidade de açúcar liberado in vitro foi diretamente relacionada com a concentração de enzima, e que houve aumento expressivo e linear na quantidade de açúcares redutores liberados nas duas concentrações (1,6 e 3,2 g.L-1 à medida que se aumentou o tempo de incubação das amostras de substrato (pergaminho com a solução enzimática. Foi verificado também, nos testes de germinação e emergência, que as sementes sem pergaminho apresentam maiores índices de velocidade e porcentagem de germinação e emergência comparado às sementes com pergaminho e que a celulase nesta concentração ainda não proporciona aumento na velocidade de germinação das sementes, necessitando-se de concentrações mais elevadas.This paper aimed to verify the action of cellulase on the decomposition of parchment of coffee seeds. For this purpose two experiments were conducted and the first, was realized in vitro conditions, containing of three assays (effect of the concentration of the enzyme, effect of the concentration of the substrate and enzyme scarification of the parchment. The second was made in vitro conditions soaking of coffee seeds in a cellulase solution at the concentration of 1.6 g.L-1 in 0.05 M potassium citrate buffer. It was found that the amount of sugar released was directly related with the enzyme concentration and that there was a linear and marked increase in the amount of

  13. A Novel Approach to Degrading Plant Cellulose: Continual Adding Materials and Cycling Utilization of Acids and Cellulase%原料连续添加和酸酶循环利用法(CACU法)降解植物纤维素新技术

    Institute of Scientific and Technical Information of China (English)

    王卫国; 赵永亮

    2002-01-01

    A novel approach to degrading plant cellulose--Continual Adding Materials and Cycling Utilization of Acids and Cellulase(CACU) is developed on the basis of the optimum results of degrading cellulose with single acid, double or multiple acids and cellulase-lyses for short time by orthogonal experiments. The schematic flow diagram for continual adding materials and cycling utilization of acids and enzymes for the production of glucose from cellulose was designed, drawn and described. The experimental results show that the CACU method is an effective way of degrading cellulose, which possesses the properties of common conditions, simple process, lower cost and a short period. The whole system consists of five or six sets of equipment, including three sets of reaction equipment and two sets of plant containers. There are totally twelve steps of operation in the whole process. The final transformation ratio of glucose to cellulose and the final concentration of glucose in the end-residue can increase up to 95.34% and 3.21%, respectively, with CH3COOH, HCl and cellulase at 100℃, 1 kg/cm2, and for 15 h by the CACU method. It consumes a quarter of acetic acid and half of HCl compared with the traditional way. The CACU method can decrease the cost of production of glucose from cellulose greatly. Thus, the CACU method is worthy to be developed and spread because of its excellent properties.%以乙酸、草酸、盐酸、硫酸等单酸、二酸、三酸混和及纤维素酶降解植物纤维素的正交试验得出的最佳工艺条件为基础,进一步研究出一种原料连续添加和酸酶循环利用法降解植物纤维素新技术(CACU法).该技术的整个过程只需要12步操作,5或6套设备,包括3~4套反应釜和2套贮罐.在常压、温度100℃、反应15 h的条件下,以CH3COOH,HCl和纤维素酶为反应剂,按照该工艺技术能使纤维素转化成葡萄糖的转化率达95.34%,反应终液中的葡萄糖浓度达3.21%.与常规

  14. 限饲与营养补偿对小尾寒羊生长性能、消化代谢和瘤胃液纤维素酶活性的影响%Effects of Dietary Restriction and Realimentation on Growth Performance, Digestion, Metabolism and Cellulase Activity in Ruminal Fluid of Small Tail Han Sheep

    Institute of Scientific and Technical Information of China (English)

    陈军强; 丁路明; 高强; 龙瑞军; 安吾; 刘培培; 张丽莉

    2015-01-01

    本试验旨在研究不同限饲水平与营养补偿对小尾寒羊生长性能、消化代谢以和瘤胃液纤维素酶活性的影响. 选择体重相近的3月龄小尾寒羊公羊40只,采用单因素4水平随机设计,每组10只,正试期90 d. 对照(Ⅰ)组全期自由采食;30 d限饲期内,Ⅱ、Ⅲ和Ⅳ组分别按照NRC(2007)推荐的日增重500、400和300 g/d所需要的代谢能(ME)和粗蛋白质(CP)的量配制饲粮,限饲结束后,试验组自由采食60 d为营养补偿期. 在24~30 d和84~90 d进行消化代谢试验. 结果表明:1 )限饲期,限饲水平对结束体重和平均日增重( ADG )有显著影响( P0.05);Ⅱ、Ⅲ和Ⅳ组体重补偿百分比分别为对照组的104.36%、101.71%、99.61%;限饲水平对ADG有显著影响( P0.05). 营养补偿期,组间营养物质的表观消化率差异不显著( P>0.05). 3)限饲期,限饲水平对食入氮、氮沉积、氮沉积率有显著影响( P0.05);粪氮和尿氮的变化不显著(P>0.05). 营养补偿期,组间氮代谢指标差异不显著(P>0.05). 4)1~30 d,试验组瘤胃液纤维素酶活性呈降低趋势,31~90 d,呈升高趋势,在90 d,试验组均高于对照组. 综上所述,对小尾寒羊采用短期适度限饲,经过营养补偿可以使其生长性能、消化代谢不受影响.%The objective of this study was to explore the effects of dietary restriction level and realimentation on growth performance, digestion, metabolism and cellulase activity in ruminal fluid of small tail Han sheep. For-ty 3-month old small tail Han sheep with average body weight (BW) of (19.77±1.34) kg were randomly split into four groups with ten sheep in each group by a single factor four levels design. The study lasted for 90 days. Control group ( group Ⅰ) was fed ad libitum during the whole period; during 30-day dietary restriction peri-od, three experimental groups were fed diets with different metabolizable energy ( ME ) and crude protein ( CP) levels, which were designed to meet

  15. Recyclable Thermoresponsive Polymer-Cellulase Bioconjugates for Biomass Depolymerization

    OpenAIRE

    Mackenzie, Katherine J.; Francis, Matthew B.

    2012-01-01

    Here we report the construction and characterization of a recoverable, thermoresponsive polymer-endoglucanase bioconjugate that matches the activity of unmodified enzymes on insoluble cellulose substrates. Two copolymers exhibiting a thermoresponsive lower critical solution temperature (LCST) were created through the copolymerization of an aminooxy-bearing methacrylamide with N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMa). The aminooxy group provided a handle through which...

  16. A kinetic model for the burst phase of processive cellulases

    DEFF Research Database (Denmark)

    Præstgaard, Eigil; Olsen, Jens Elmerdahl; Murphy, Leigh;

    2011-01-01

    negligible. Here, we propose an explicit kinetic model for this behavior, which uses classical burst phase theory as the starting point. The model is tested against calorimetric measurements of the activity of the cellobiohydrolase Cel7A from Trichoderma reesei on amorphous cellulose. A simple version of the...

  17. The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex

    NARCIS (Netherlands)

    Vain, T.; Crowell, E.F.; Timpano, H.; Biot, E.; Desprez, T.; Mansoori Zangir, N.; Trindade, L.M.; Pagant, S.; Robert, S.; Hofte, H.; Gonneau, M.; Vernhettes, S.

    2014-01-01

    Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis

  18. 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 to...

  19. 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.

  20. Engineering yeast for the expression and secretion of cellulase cocktails

    Science.gov (United States)

    Enzyme systems that digest the cellulose in plant cell walls have potential value in the biorefining of renewable feedstocks such as crop residues, straws, and grasses to biofuels and other bioproducts. The bacterium Clostridium cellulovorans is a useful source of biomass-degrading enzymes because ...

  1. 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...

  2. Engineering cellulases for enhanced degradation of insoluble substrates

    OpenAIRE

    Reyes-Ortiz, Vimalier

    2013-01-01

    There is tremendous social and political interest in the production of sustainable and carbon neutral liquid fuels that can meet our transportation energy demands. Second-generation biofuels, derived from cellulosic non-edible plant matter (biomass), represent a possible solution to this issue. The US Department of Energy and the Department of Agriculture estimate that the US can produce the energy equivalent 42 percent of the total US annual transportation consumption from cellulosic biomass...

  3. Production of Trichoderma reesei cellulases on glucose-containing media.

    OpenAIRE

    Nakari-Setälä, T; Penttilä, M

    1995-01-01

    The filamentous fungus Trichoderma reesei was shown to secrete active cellobiohydrolase I and the endoglucanase I catalytic core domain into the culture medium when the fungus was grown on glucose-containing medium. The expression of the proteins was driven by the promoters of the elongation factor 1 alpha, tef1, and the unidentified gene for cDNA1. The cDNA1 promoter gave the best yields. The highest amounts of cellobiohydrolase I and the endoglucanase I core, being 50 to 100 mg/liter, accou...

  4. 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. PMID:26819239

  5. Systems biological approaches towards understanding cellulase production by Trichoderma reesei

    OpenAIRE

    Kubicek, Christian P.

    2013-01-01

    Recent progress and improvement in “-omics” technologies has made it possible to study the physiology of organisms by integrated and genome-wide approaches. This bears the advantage that the global response, rather than isolated pathways and circuits within an organism, can be investigated (“systems biology”). The sequencing of the genome of Trichoderma reesei (teleomorph Hypocrea jecorina), a fungus that serves as a major producer of biomass-degrading enzymes for the use of renewable lignoce...

  6. 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.

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

    OpenAIRE

    Alftrén, Johan; Hobley, Timothy John

    2014-01-01

    Der er vigtigt at erstatte oliebaserede brændstoffer og kemikalier med mere miljøvenlige alternativer, da olie bidrager til en netto produktion af drivhusgasser og er en begrænset resurse. Biomasse fra lignocellulose er et meget interessant råmateriale for produktion af biokemikalier og biobrændstof på grund af den store overflod af råmaterialet og det faktum at det er spildmateriale og ikke direkte konkurrerer med fødevareproduktion. Lignocellulose består blandt andet af cellulose og hemicel...

  8. 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.

  9. Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose

    DEFF Research Database (Denmark)

    Rosgaard, L.; Pedersen, S.; Meyer, Anne Boye Strunge

    2006-01-01

    the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence......This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile...

  10. 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)

  11. Biobleaching application of cellulase poor and alkali stable xylanase from Bacillus pumilus SV-85S

    OpenAIRE

    Nagar, Sushil; Jain, R. K.; Thakur, Vasanta Vadde; Gupta, Vijay Kumar

    2012-01-01

    The potential of extracellular alkali stable and thermo tolerant xylanase produced by Bacillus pumilus SV-85S through solid state fermentation was investigated in pulp bleaching in association with conventional bleaching using chlorine and chlorine dioxide. The biobleaching of kraft pulp with xylanase was the most effective at an enzyme dose of 10 IU/g oven dried pulp, pH 9.0 and 120 min incubation at 55 °C. Under the optimized conditions, xylanase pretreatment reduced Kappa number by 1.6 poi...

  12. NOVEL SOURCES OF FUNGAL CELLULASES OF THERMOPHILIC / THERMOTOLERANT FOR EFFICIENT DEINKING OF COMPOSITE PAPER WASTE

    Directory of Open Access Journals (Sweden)

    Rohit Soni

    2008-02-01

    Full Text Available Twenty thermophilic/thermotolerant fungal strains were isolated from compositing soils and screened for production of different enzymes (Endoglucanases, β-glucosidase, Fpase and xylanases to assess their deinking efficiency. Three isolates, Aspergillus sp. AMA, Aspergillus terreus AN1, and Myceliophthora fergusii T4I, identified on the basis of morphological and sequencing of amplified ITS1-5.8S-ITS2 rDNA region, showed significant deinking of composite waste paper (70% magazine and 30% Xerox copier/ laser print paper waste as well as improved properties (brightness, tensile strength, tear index of recycled paper sheets. The chosen strains Aspergillus sp. AMA, Aspergillus terreus AN1 and Myceliophthora fergusii T4I, showed 53, 52.7, and 40.32% deinking with increase in brightness by 4.32, 3.56, and 3.01 % ISO, respectively. These cultures were found to produce multiple endoglucanases and were characterized to lack a cellulose binding module (CBD, which may be responsible for their better deinking efficiency.

  13. The cellulases and their application in degrading agro-industrial waste

    OpenAIRE

    Schwarz, Wolfgang H.

    2011-01-01

    A huge amount of lignocellulosic biomass is available which can be used to produce storable energy and basic material for the chemical industry. Its use is especially beneficial for a country's economy if it is waste material, which can be obtained at almost no cost and which presents an environmental burden. However, the polysaccharides present in biomass are difficult to degrade due to their heterogeneity and crystalline structure. This article addresses the enzymatic hydrolysis of cellulos...

  14. Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency*

    OpenAIRE

    Eibinger, Manuel; Ganner, Thomas; Bubner, Patricia; Rošker, Stephanie; Kracher, Daniel; Haltrich, Dietmar; Ludwig, Roland; Plank, Harald; Nidetzky, Bernd

    2014-01-01

    Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulos...

  15. 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...

  16. Characterization and Phylogenetic Diversity of Carboxymethyl Cellulase Producing Bacillus Species from a Landfill Ecosystem

    OpenAIRE

    Korpole, Suresh; Sharma, Rashmi; Verma, Dipti

    2011-01-01

    Total population of cellulose degrading bacteria was studied in a landfill ecosystem as a part of microbial diversity study. Samples were obtained from 3 and 5 feet depth of a local landfill being operated for past 10 years. Among many isolates, 22 bacterial strains were selected based on their capability to decompose carboxymethyl cellulose (CMC). These isolates were cultivated on agar medium with CMC as the carbon source. All isolates were Gram positive, endospore forming and alkalophilic b...

  17. 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...

  18. 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...

  19. Application of cellulase and pectinase from fungal origin for the liquefaction and saccharification of biomass

    Energy Technology Data Exchange (ETDEWEB)

    Beldman, G.; Rombouts, F.M.; Voragen, A.G.J.; Pilnik, W.

    1984-01-01

    Commercialcellulase from Trichoderma viride and pectinase from Aspergillus niger were applied to produce fermentation syrups from sugar beet pulp and potato fiber. Cellulosic, hemicellulosic, and pectic polysaccharides of these substrates were hydrolyzed extensively. Recovery of enzymes was investigated in a packed-column reactor, connected with a hollow-fiber ultrafiltration unit. Enzymes appeared to be stable in this type of reactor, although part of the enzyme activity was lost, especially by adsorption onto the substrate residue.

  20. 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...... that they exhibit different substrate specificities. Therefore, a small library of derivatives of 2,4-dinitrophenyl cellobioside (2,4-DNPC) and 3,4-dinitrophenyl cellobioside (3,4-DNPC) was prepared. These derivatives contained a series of substituents (X and Y) located at the O4' and O6' position. Inspection...... chemistry or by amine coupling. The kinetic parameters of the library of compounds are to be determined....

  1. Biotechnological valorisation of agro-industrial wastes for the production of cellulases

    OpenAIRE

    DAMATO, GIUSEPPE

    2012-01-01

    This experimental work is embedded in a wider European-funded project named Etoile (FP7/2007-2013, Project n° 222331). The aim of Etoile project was to develop a new integrated process where the two main wastes coming from olive oil traditional three-phase production process, the solid lignocellulosic olive pomace (OP) and the liquid olive oil mill waste water (OOMW), are exploited for the production of cellulolytic enzymes and bioethanol.More specifically, this experimental work was focus...

  2. 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

    2016-01-01

    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). PMID:27347917

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

    OpenAIRE

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

    2010-01-01

    Abstract 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 di...

  4. Simple, fast and high‐efficiency transformation system for directed evolution of cellulase in Bacillus subtilis

    OpenAIRE

    Zhang, Xiao‐Zhou; Zhang, Y.‐H. Percival

    2010-01-01

    Summary Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme‐, phosphatase‐ and ligas...

  5. Mechanisms employed by cellulase systems to gain access through the complex architecture of lignocellulosic substrates.

    Science.gov (United States)

    Donohoe, Bryon S; Resch, Michael G

    2015-12-01

    To improve the deconstruction of biomass, the most abundant terrestrial source of carbon polymers, en route to renewable fuels, chemicals, and materials more knowledge is needed into the mechanistic interplay between thermochemical pretreatment and enzymatic hydrolysis. In this review we highlight recent progress in advanced imaging techniques that have been used to elucidate the effects of thermochemical pretreatment on plant cell walls across a range of spatial scales and the relationship between the substrate structure and the function of various glycoside hydrolase components. The details of substrate and enzyme interactions are not yet fully understood and the challenges of characterizing plant cell wall architecture, how it dictates recalcitrance, and how it relates to enzyme-substrate interactions is the focus for many research groups in the field. Better understanding of how to match pretreatments with improved enzyme mixtures will lead to lower costs for industrial biorefining. PMID:26529490

  6. 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...

  7. Hydrolysis behavior of various crystalline celluloses treated by cellulase of Tricoderma viride

    OpenAIRE

    Abdullah, Rosnah; Saka, Shiro

    2014-01-01

    Cellobiose and glucose are valuable products that can be obtained from enzymatic hydrolysis of cellulose. This study discusses changes in the crystalline form of celluloses to enhance the production of sugars and examines the effect on structural properties during enzymatic hydrolysis. Various crystalline celluloses consisting of group I (cell I, cell III[I], cell IV[I]) and group II (cell II, cell III[II], cell IV[II]) of similar DPs were prepared as starting materials. The similar DP values...

  8. 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

    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...... of a variety of cellulolytic enzymes. The aim of this work is to discover new thermostable and robust cellulolytic enzymes for improved enzymatic hydrolysis of biomass. For this purpose two screening methods are applied in different fungal strains with high cellulolytic activities: an expression...

  9. Pseudomonas coleopterorum sp nov., a cellulase-producing bacterium isolated from the bark beetle Hylesinus fraxini

    Czech Academy of Sciences Publication Activity Database

    Menéndez, E.; Ramírez-Bahena, M.H.; Fabryová, A.; Igual, J.M.; Benada, Oldřich

    2015-01-01

    Roč. 65, SEP 2015 (2015), s. 2852-2858. ISSN 1466-5026 R&D Projects: GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : CURCULIONIDAE SCOLYTINAE * NUCLEOTIDE-SEQUENCES * DENDROCTONUS-RHIZOPHAGUS Subject RIV: EE - Microbiology, Virology Impact factor: 2.511, year: 2014

  10. Folding and stability of endoglucanase III, a single-domain cellulase from Trichoderma reesei.

    Science.gov (United States)

    Arunachalam, U; Kellis, J T

    1996-09-01

    The reversible folding of an endoglucanase (EGIII) from the filamentous fungus Trichoderma reesei was investigated by activity, tryptophan fluorescence, and peptide CD measurements. Equilibrium stability was determined by urea denaturation at various pH and temperature values. Unfolding and refolding rates were measured over a range of urea concentrations. The data from the equilibrium and kinetic studies fit a simple two-state model, except at lower urea concentrations, where the folding kinetics indicate a transient intermediate. Unfolding is very slow, with a half-life of about 2 h in 8 M urea at pH 5.5 and 25 degrees C. Comparison of the urea dependence of the folding kinetics and equilibrium indicates the protein undergoes 93% of its total change in solvent exposure on going from the unfolded state to the transition state. Thus, the transition state is quite compact. The presence of dithiothreitol destabilized the protein by 7 kcal/mol, indicating the presence of an unusually strong disulfide linkage between the two cysteines in the molecule. Protein stability is dramatically reduced at alkaline pH values; this can be attributed to a titratable shift (pKa = 7.8) in the slope of the urea dependence of unfolding. PMID:8784193

  11. Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli.

    Science.gov (United States)

    O'Neill, G P; Kilburn, D G; Warren, R A; Miller, R C

    1986-10-01

    A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism. PMID:3096205

  12. Studies on cellulase production by a mutant-penicillium funiculosum uv-49.

    Science.gov (United States)

    Joglekar, A V; Karanth, N G

    1984-09-01

    In search of hypercellulolytic microorganisms, ultraviolet irradiation carried out with Penicillium funiculosum has yielded a superior mutant. The investigations reported in this article are shake flask studies on some important nutritional requirements of the mutant, namely, nitrogen source, carbon source, and inducers. The mutant shows an ability to metabolize inorganic nitrogen sources like urea and sodium nitrate both for growth and enzyme production. A comparison of the long-term saccharification ability and the utilization efficiency of the mutant enzyme with those reported in the literature is also carried out, showing the superior performance of the mutant enzyme. PMID:18553529

  13. 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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

  15. Produção de celulases por Aspergillus niger e cinética da desativação celulásica=Cellulases production by Aspergillus niger and cellulase deactivation kinetic

    OpenAIRE

    Caroline Mariana de Aguiar; Sérgio Luiz de Lucena

    2011-01-01

    O presente trabalho teve como objetivo a avaliação da cinética de produção de enzimas celulases pelo fungo Aspergillus niger e da cinética de desativação das celulases. Foi utilizado bagaço de cana-de-açúcar pré-tratado como fonte de carbono na fermentação para a produção do complexo celulásico e também como substrato da hidrólise enzimática. A. niger foi cultivado em três bateladas, cada uma contendo 10, 50 e 100 g L-1 de bagaço pré-tratado com NaOH 4% (m v-1). A cinética da produção das cel...

  16. Cellulase activity of a Lentinula edodes (Berk. Pegl. strain grown in media containing Carboximetilcellulose or microcrystalline cellulose

    Directory of Open Access Journals (Sweden)

    José Antônio de Sousa Pereira Júnior

    2003-06-01

    Full Text Available Endoglucanase and exocellobiohydrolase produced b Lentinula edodes (Berk. Pegl. strain thatt was cultivated in carboxymetilcellulose (CMC or microcrystalline cellulose (Avicel liquid media. The concentration and type of cellulose influenced the enzyme activity and production. Extra-cellular cellobiase activity was not detected in CMC or Avicel media. This enzyme was detected in mycelial extracts only. With 1.7% Avicel liquid medium, the strain did not produce exocellobiohydrolase, but 74 µmol RBBR/mg protein/min was detected with 0.5% Avicel. The substitution of Avicel by 0.5% CMC reduced this activity. Endoglucanase also had maximum activity in 0.5% Avicel medium (approximately 820 UI/mg protein after 96 h incubation. In supernatants from 0.5% CMC, the maximum activity attained was 200 UI/mg protein only.Neste trabalho foram estudadas as atividades de endoglucanase, exocelobiohidrolase e celobiase em uma linhagem de Lentinula edodes (Berk. Pegl. cultivada em meio líquido contendo carboximetilcelulose (CMC ou celulose microcristalina (Avicel. Foram detectadas as atividades de endoglucanase e exocelobiohidrolase no sobrenadante das culturas crescidas tanto em meios contendo CMC como nos meios contendo Avicel, sendo observada a influência da concentração e do tipo de celulose. Não foi detectada atividade de celobiase nos sobrenadantes, sendo a mesma detectada somente no extrato micelial. Com uma concentração de 1,7% de Avicel, a linhagem estudada não demonstrou atividade de exocelobiohidrolase. Porém, à concentração de 0,5% obteve-se uma atividade de 74 µmol de RBBR/mg de proteína/min. Com a substituição de Avicel por CMC a 0,5%, a atividade de exocelobiohidrolase foi reduzida a menos de 50%. A máxima atividade de endoglucanase em sobrenadantes obtidos em meio com Avicel a 0,5% foi em torno de 800 UI/mg de proteína, após 96 horas de cultivo. Em sobrenadantes obtidos de meio com CMC, a atividade desta enzima foi de apenas 200 UI/mg de proteína.

  17. 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

    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...

  18. Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies

    OpenAIRE

    Fong, Monica; Berrin, Jean-Guy

    2016-01-01

    Background Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant su...

  19. Neutron Reflectometry and QCM-D Study of the Interaction of Cellulase Enzymes with Films of Amorphous Cellulose

    International Nuclear Information System (INIS)

    Improving the efficiency of enzymatic hydrolysis of cellulose is one of the key technological hurdles to reduce the cost of producing ethanol and other transportation fuels from lignocellulosic material. A better understanding of how soluble enzymes interact with insoluble cellulose will aid in the design of more efficient enzyme systems. We report a study involving neutron reflectometry (NR) and quartz crystal microbalance with dissipation (QCM-D) of the interaction of a commercial fungal enzyme extract (T. viride), two purified endoglucanses from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima), and a mesophilic fungal endoglucanase (Cel45A from H. insolens) with amorphous cellulose films. The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. NR reveals the profile of water through the film at nm resolution, while QCM-D provides changes in mass and film stiffness. At 20 C and 0.3 mg/ml, the T. viride cocktail rapidly digested the entire film, beginning from the surface followed by activity throughout the bulk of the film. For similar conditions, Cel9A and Cel5A were active for only a short period of time and only at the surface of the film, with Cel9A releasing 40 from the ∼ 700 film and Cel5A resulting in only a slight roughening/swelling effect at the surface. Subsequent elevation of the temperature to the Topt in each case resulted in a very limited increase in activity, corresponding to the loss of an additional 60 from the film for Cel9A and 20 from the film for Cel5A, and very weak penetration into and digestion within the bulk of the film, before the activity again ceased. The results for Cel9A and Cel5A contrast sharply with results for Cel45A where very rapid and extensive penetration and digestion within the bulk of the film was observed at 20 C. We speculate that the large differences are due to the use of the thermophilic enzymes far below their optimal temperatures and also the presence of a cellulose binding module (CBM) on Cel45A while the thermophilic enzymes lack a CBM.

  20. 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...