WorldWideScience

Sample records for biobleaching

  1. Biobleaching of flax by degradation of lignin with laccase

    Directory of Open Access Journals (Sweden)

    Yotova, L. K.

    2007-02-01

    Full Text Available Research on lignin biodegradation has become of great interest, due to the fact that lignin is one of the most abundant renewable materials, next to cellulose. Lignin is also the substance that gives color to raw flax fibers. In order to bleach the flax and to keep its tenacity high enough for textile applications, it is necessary to remove the lignin and partially to preserve the pectin. Lignin and pectin are the main constituents of the layer which sticks the flax cells together within the multicellular technical fiber. White-rot fungi and their oxidative enzymes, laccases and peroxid-ases (lignin peroxidases and manganese peroxidases, are being applied for the biobleaching of papermaking pulp, thereby reducing the need for environmentally harmful chemicals. Some data also suggest that it is possible to use other phenolytic enzymes, such as pure laccase, for this purpose. The objective of the present work was to study the possibility of bleaching flax fibers by pure laccase and combined laccase peroxide treatment, aimed at obtaining fibers with high whiteness and well-preserved tenacity.

  2. Producing a True Lignin Depolymerase for Biobleaching Softwood Kraft Pulp

    Energy Technology Data Exchange (ETDEWEB)

    Simo Sarkanen

    2002-02-04

    This project constituted an intensive effort devoted to producing, from the white-rot fungus Tramets Cingulata, a lignin degrading enzyme (lignin depolymerase) that is directly able to biobleach or delignify softwood kraft pulp brownstock. To this end, the solutions in which T. cingulata was grown contained dissolved kraft lignin which fulfilled two functions; it behaved as a lignin deploymerase substrate and it also appeared to act as an inducer of enzyme expression. However, the lignin depolymerase isoenzymes (and other extracellular T. cingulata enzymes) interacted very strongly with both the kraft lignin components and the fungal hypae, so the isolating these proteins from the culture solutions proved to be unexpectedly difficult. Even after extensive experimentation with a variety of protein purification techniques, only one approach appeared to be capable of purifying lignin depolymerases to homogeneity. Unfortunately the procedure was extremely laborious; it involved the iso electric focusing of concentrated buffer-exchanged culture solutions followed by electro-elution of the desired protein bands from the appropriate polyacrylamide gel segments

  3. Optimization of biobleaching of paper pulp in an expanded bed bioreactor with immobilized alkali stable xylanase by using response surface methodology.

    Science.gov (United States)

    Senthilkumar, Sundar Rajan; Dempsey, Michael; Krishnan, Chandraraj; Gunasekaran, Paramasamy

    2008-11-01

    Purified alkali stable xylanase from Aspergillus fischeri was immobilized on polystyrene beads using diazotization method. An expanded bed bioreactor was developed with these immobilized beads to biobleach the paper pulp in continuous mode. Response surface methodology was applied to optimize the biobleaching conditions. Temperature (degrees C), flow rate of pulp (ml/min) and concentration of the pulp (%) were selected as variables in this study. Optimal conditions for biobleaching process were reaction temperature 60 degrees C, flow rate of 2 ml/min and 5% (w/v) of pulp. The kappa number reduced from 66 in the unbleached pulp to 20 (reduction of 87%). This system proves to be a better option for the conventional chlorine based pulp bleaching.

  4. Potential of thermo and alkali stable xylanases from Thielaviopsis basicola (MTCC-1467) in biobleaching of wood kraft pulp.

    Science.gov (United States)

    Goluguri, Baby Rani; Thulluri, Chiranjeevi; Cherupally, Madhu; Nidadavolu, Nagaraju; Achuthananda, Das; Mangamuri, Lakshmi Narasu; Addepally, Uma

    2012-08-01

    Thermo- and alkali-stable xylanases produced from Thielaviopsis basicola (MTCC-1467) on low-cost carbon source like rice straw were evaluated for their potential application in biobleaching of wood kraft pulp. Enzyme treatment at retention time of 240 min with 20 IU/gm of dried pulp resulted in ~85.2 % of reduction in kappa number. When compared to control, 110.8, 93, and 72.2 % of enhancement in brightness (percent International Organization of Standardization), whiteness, and fluorescence, respectively, were observed for enzyme-treated pulp. Spectroscopic analysis showed significant release of chromophoric compounds from enzyme-treated pulp. Furthermore, scanning electron microscope studies of unbleached and enzyme bleached pulp revealed the effectiveness of enzymatic treatment. The enzyme-treated pulp subjected to later stages of chemical bleaching resulted in 16 % decrease in chlorine consumption along with considerable reduction in chemical oxygen demand percentage (14.5 %) level of effluent. Various pulp properties like fiber length, fiber width, burst strength, burst index, tear strength, tear index, tensile strength, and breaking length were also significantly improved after enzyme treatment when compared to control.

  5. Bioprocess and biotecnology: effect of xylanase from Aspergillus niger and Aspergillus flavus on pulp biobleaching and enzyme production using agroindustrial residues as substract

    OpenAIRE

    de Alencar Guimaraes, Nelciele ; Sorgatto, Michele ; Nogueira, Simone de Peixoto; Betini, Jorge Henrique ; Zanoelo, Fabiana ; Marques, Maria ; Polizeli, Maria de Lourdes Teixeira de Moraes; Giannesi, Giovana C

    2013-01-01

    This study compares two xylanases produced by filamentous fungi such as A. niger and A. flavus using agroindustrial residues as substract and evaluated the effect of these enzymes on cellulose pulp biobleaching process. Wheat bran was the best carbon source for xylanase production by A. niger and A. flavus. The production of xylanase was 18 and 21% higher on wheat bran when we compare the xylanase production with xylan. At 50°C, the xylanase of A. niger retained over 85% activity with 2 h of ...

  6. Bioprocess and biotecnology: effect of xylanase from Aspergillus niger and Aspergillus flavus on pulp biobleaching and enzyme production using agroindustrial residues as substract.

    Science.gov (United States)

    de Alencar Guimaraes, Nelciele Cavalieri; Sorgatto, Michele; Peixoto-Nogueira, Simone de Carvalho; Betini, Jorge Henrique Almeida; Zanoelo, Fabiana Fonseca; Marques, Maria Rita; de Moraes Polizeli, Maria de Lourdes Teixeira; Giannesi, Giovana C

    2013-01-01

    This study compares two xylanases produced by filamentous fungi such as A. niger and A. flavus using agroindustrial residues as substract and evaluated the effect of these enzymes on cellulose pulp biobleaching process. Wheat bran was the best carbon source for xylanase production by A. niger and A. flavus. The production of xylanase was 18 and 21% higher on wheat bran when we compare the xylanase production with xylan. At 50°C, the xylanase of A. niger retained over 85% activity with 2 h of incubation, and A. flavus had a half-life of more than 75 minutes. At 55°C, the xylanase produced by A. niger showed more stable than from A. flavus showing a half-life of more than 45 minutes. The xylanase activity of A. niger and A. flavus were somehow protected in the presence of glycerol 5% when compared to the control (without additives). On the biobleaching assay it was observed that the xylanase from A. flavus was more effective in comparison to A. niger. The kappa efficiency corresponded to 36.32 and 25.93, respectively. That is important to emphasize that the cellulase activity was either analyzed and significant levels were not detected, which explain why the viscosity was not significantly modified.

  7. Biobleaching of industrial important dyes with peroxidase partially purified from garlic.

    Science.gov (United States)

    Osuji, Akudo Chigozirim; Eze, Sabinus Oscar O; Osayi, Emmanuel Emeka; Chilaka, Ferdinand Chiemeka

    2014-01-01

    An acidic peroxidase was extracted from garlic (Allium sativum) and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration chromatography using sephadex G-200. The specific activity of the enzyme increased from 4.89 U/mg after ammonium sulphate precipitation to 25.26 U/mg after gel filtration chromatography. The optimum temperature and pH of the enzyme were 50°C and 5.0, respectively. The Km and V max for H2O2 and o-dianisidine were 0.026 mM and 0.8 U/min, and 25 mM and 0.75 U/min, respectively. Peroxidase from garlic was effective in decolourizing Vat Yellow 2, Vat Orange 11, and Vat Black 27 better than Vat Green 9 dye. For all the parameters monitored, the decolourization was more effective at a pH range, temperature, H2O2 concentration, and enzyme concentration of 4.5-5.0, 50°C, 0.6 mM, and 0.20 U/mL, respectively. The observed properties of the enzyme together with its low cost of extraction (from local sources) show the potential of this enzyme for practical application in industrial wastewater treatment especially with hydrogen peroxide. These Vat dyes also exhibited potentials of acting as peroxidase inhibitors at alkaline pH range.

  8. Biobleaching of Industrial Important Dyes with Peroxidase Partially Purified from Garlic

    Directory of Open Access Journals (Sweden)

    Akudo Chigozirim Osuji

    2014-01-01

    Full Text Available An acidic peroxidase was extracted from garlic (Allium sativum and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration chromatography using sephadex G-200. The specific activity of the enzyme increased from 4.89 U/mg after ammonium sulphate precipitation to 25.26 U/mg after gel filtration chromatography. The optimum temperature and pH of the enzyme were 50°C and 5.0, respectively. The Km and Vmax for H2O2 and o-dianisidine were 0.026 mM and 0.8 U/min, and 25 mM and 0.75 U/min, respectively. Peroxidase from garlic was effective in decolourizing Vat Yellow 2, Vat Orange 11, and Vat Black 27 better than Vat Green 9 dye. For all the parameters monitored, the decolourization was more effective at a pH range, temperature, H2O2 concentration, and enzyme concentration of 4.5–5.0, 50°C, 0.6 mM, and 0.20 U/mL, respectively. The observed properties of the enzyme together with its low cost of extraction (from local sources show the potential of this enzyme for practical application in industrial wastewater treatment especially with hydrogen peroxide. These Vat dyes also exhibited potentials of acting as peroxidase inhibitors at alkaline pH range.

  9. Biobleaching of Paper Mulberry (Broussentia papyrifera) Pulp Using Laccase Mediator System

    OpenAIRE

    Sunita Chauhan; M. Krishna Mohan; Pradeep Bhatnagar

    2016-01-01

    In recent times, demand for cleaner production techniques in the handmade paper industry is on rise. Use of enzymes in prebleaching might be very useful in this regard. The laccase enzyme produced from Pycnoporus cinnabarinus was tested as an aid to the bleaching of paper mulberry (Broussentia papyrifera) pulp. For this, enzymatic prebleaching was carried out for two different durations i.e. 3 hours and 48 hours. The pulp was then subjected to chemical bleaching by hydrogen peroxide either...

  10. Biobleaching of Paper Mulberry (Broussentia papyrifera Pulp Using Laccase Mediator System

    Directory of Open Access Journals (Sweden)

    Sunita Chauhan

    2016-12-01

    Full Text Available In recent times, demand for cleaner production techniques in the handmade paper industry is on rise. Use of enzymes in prebleaching might be very useful in this regard. The laccase enzyme produced from Pycnoporus cinnabarinus was tested as an aid to the bleaching of paper mulberry (Broussentia papyrifera pulp. For this, enzymatic prebleaching was carried out for two different durations i.e. 3 hours and 48 hours. The pulp was then subjected to chemical bleaching by hydrogen peroxide either directly (LP sequence or after an alkaline extraction stage (LEP sequence. The synergistic effect of xylanase enzyme on laccase mediator system was also evaluated and found to be negligible. Under the defined conditions, a gain of up to ten points in brightness value of the treated pulps as compared to the control pulps could be obtained. Thus, laccase mediator system was found to be very effective in boosting the brightness of paper mulberry pulp.

  11. Application of Phanerochaete chrysosporium 1038 - Enzyme Complex and Laccase in Biobleaching of Flax Fibers

    Directory of Open Access Journals (Sweden)

    Yotova L.

    2007-12-01

    Full Text Available Bleaching processes in textile industry require to keep fibers tenacity, partially to preserve the pectin and reducing the lignin content, that gives color to row flax fibers. The use of lignocellulose-degrading enzymes from basidiomycete Phanerochaete chrysosporium 1038 strain and pure Laccase from Biocatalyst in flax fibers treatment was studied. The whiteness of enzymatically-processed fibers was significantly improved and the residual quantity of nondegraded lignin was less than obtained with chemical processing. The structural changes in the flax fibers during enzyme treatment were determined with IR spectroscopy, which confirmed the lignin degradation.

  12. Ethanol/Water Pulps From Sugar Cane Straw and Their Biobleaching With Xylanase from Bacillus pumilus

    Science.gov (United States)

    Moriya, Regina Y.; Gonçalves, Adilson R.; Duarte, Marta C. T.

    The influence of independent variables (temperature and time) on the cooking of sugar cane straw with ethanol/water mixtures was studied to determine operating conditions that obtain pulp with high cellulose contents and a low lignin content. An experimental 22 design was applied for temperatures of 185 and 215°C, and time of 1 and 2.5 h with the ethanol/water mixture concentration and constant straw-to-solvent ratio. The system was scaled-up at 200°C cooking temperature for 2 h with 50% ethanol-water concentration, and 1∶10 (w/v) straw-to-solvent ratio to obtain a pulp with 3.14 cP viscosity, 58.09 kappa-number, and the chemical composition of the pulps were 3.2% pentosan and 31.5% lignin. Xylanase from Bacillus pumilus was then applied at a loading of 5-150 IU/g dry pulp in the sugar cane straw ethanol/water pulp at 50°C for 2 and 20 h. To ethanol/water pulps, the best enzyme dosage was found to be 20 IU/g dry pulp at 20 h, and a high enzyme dosage of 150 IU/g dry pulp did not decrease the kappa-number of the pulp.

  13. Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in biobleaching of kraft pulp

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, R.; Bhosle, N.B.

    released by enzyme treatment showed a characteristic peak at 280 nm indicating the presence of lignin in the released coloring matter. Enzymatic prebleaching of kraft pulp showed 20 % reduction in kappa number of the pulp without much change in viscosity...

  14. Xylanases of marine fungi of potential use for biobleaching of paper pulp

    Digital Repository Service at National Institute of Oceanography (India)

    Raghukumar, C.; Muraleedharan, U.; Gaud, V.R.; Mishra, R.

    bleaching of sugarcane bagasse pulp by a 60 min treatment at 55oC, resulting in a decrease of 10 kappa numbers and a 30% reduction in consumption of chlorine during bleaching process. The culture filtrate showed peaks of xylanase activity at acidic pH (3...

  15. An extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4, with a potential to biobleach softwood pulp

    OpenAIRE

    Sondhi, Sonica; Sharma, Prince; George, Nancy; Chauhan, Prakram Singh; Puri, Neena; Gupta, Naveen

    2014-01-01

    Degradation of residual lignin in kraft pulp by chemical bleaching is implicated in causing environmental pollution. The use of thermo- and alkali-tolerant bacterial laccases is considered to be important biological alternative to chemical processing. Laccases from Bacillus species have shown promise in this respect but their intracellular/spore bound presence make their industrial application economically unfeasible. We report here on a novel extracellular active thermo-alkali-stable laccase...

  16. Optimization of manganese peroxidase production by the white rot fungus Bjerkandera sp. strain BOS55.

    NARCIS (Netherlands)

    Mester, T.; Field, J.A.

    1997-01-01

    Manganese dependent peroxidase (MnP) is the most ubiquitous peroxidase produced by white rot fungi. MnP is known to be involved in lignin degradation, biobleaching and in the oxidation of hazardous organopollutants. Bjerkandera sp. strain BOS55 is a nitrogen-unregulated white rot fungus which produc

  17. ENZYMATIC BLEACHING OF WHEAT STRAW SODA-AQ PULP BY LMS TREATMENT

    Institute of Scientific and Technical Information of China (English)

    Xiwen Wang; Huaiyu Zhan; Lili Xu

    2004-01-01

    Laccase has been proven that it has a good selectivity and efficiency in pulp bleaching. In this paper, the effects of LMS (Laccase Mediator System) treatment on delignification and bleaching of wheat straw pulp at various conditions, such as laccase dosage,temperature, oxygen pressure and pH, were investigated. The TCF sequence including LMS can bleach the wheat straw pulp to 81% ISO brightness with good strength. The synergetic biobleaching of LMS and xylanase of wheat straw pulp was also investigated. It was found that the final brightness and strength of synergetic biobleaching pulps increased to a certain extent and the bleaching selectivity was improved. The combination of the enzymes is feasible for the delignification and bleaching.

  18. ENZYMATIC BLEACHING OF WHEAT STRAW SODA--AQ PULP BY LMS TREATMENT

    Institute of Scientific and Technical Information of China (English)

    XiwenWang; HuaiyuZhan; LiliXu

    2004-01-01

    Laccase has been proven that it has a good selectivity and efficiency in pulp bleaching. In this paper, the effects of LMS (Laccase Mediator System) treatment on delignification and bleaching of wheat straw pulp at various conditions, such as laccase dosage, temperature, oxygen pressure and pH, were investigated. The TCF sequence including LMS can bleach the wheat straw pulp to 81% ISO brightness with good strength. The synergetic biobleaching of LMS and xylanase of wheat straw pulp was also investigated. It was found that the final brightness and strength of synergetic biobleaching pulps increased to a certain extent and the bleaching selectivity was improved. The combination of the enzymes is feasible for the delignification and bleaching.

  19. Effect of steam explosion and enzymatic pre-treatments on pulping and bleaching of Hesperaloe funifera.

    Science.gov (United States)

    Martín-Sampedro, R; Eugenio, M E; Villar, J C

    2012-05-01

    A non-wood raw material with high potential for pulp and paper applications (Hesperaloe funifera) was subjected to a steam explosion pre-treatment, and the subsequent effect of this pretreatment on biopulping and biobleaching was studied. An increase in the delignification rate, bigger than that reported for autohydrolysis and acid hydrolysis pre-treatments, and a reduction in chemical consumption were found during kraft pulping of the exploded samples. However, biopulping with the laccase-mediator system (LMS) did not lead to a reduction in the kappa number in either non-exploded or exploded unbleached pulps. On the other hand, the steam explosion pretreatment boosted the advantages of the LMS pre-treatment (decrease in kappa number and increase in brightness) favored biobleaching, with a 53.1% delignification rate and a final brightness of 67% ISO. Finally, the steam explosion pre-treatment also improved the color properties of the bleached pulp and reduced the hydrogen peroxide consumption by 24.6%.

  20. IMPROVEMENT OF TCF BLEACHING OF OLIVE TREE PRUNING RESIDUE PULP BY ADDITION OF A LACCASE AND/OR XYLANASE PRE-TREATMENT

    Directory of Open Access Journals (Sweden)

    Raquel Martín-Sampedro,

    2012-02-01

    Full Text Available This study aimed at assessing the biobleachability of soda pulps obtained from olive tree pruning residue. The enzymatic (LMS pre-treatment was applied prior to a simple totally chlorine free (TCF bleaching sequence, consisting of an alkaline extraction and a hydrogen peroxide stage. Additionally, the effect of adding xylanase jointly with or prior to LMS was evaluated. All of these enzymatic pre-treatments were associated with an enhancement of the bleaching sequence. The best results were found when both enzymes were applied in the same stage: lowest hydrogen peroxide consumption (63 percent; kappa number, 11.6; brightness, 46 percent ISO. The mechanical properties observed were similar to those reported by other authors who have studied pulps from olive tree pruning residue. Finally, bleached pulps were subjected to accelerated ageing in order to assess the evolution of brightness and colorimetric properties. Although biobleached pulps showed lower stability upon ageing, the best optical properties, even after ageing, were observed in pulps treated with both xylanase and laccase.

  1. [Biological treatments for contaminated soils: hydrocarbon contamination. Fungal applications in bioremediation treatment].

    Science.gov (United States)

    Martín Moreno, Carmen; González Becerra, Aldo; Blanco Santos, María José

    2004-09-01

    Bioremediation is a spontaneous or controlled process in which biological, mainly microbiological, methods are used to degrade or transform contaminants to non or less toxic products, reducing the environmental pollution. The most important parameters to define a contaminated site are: biodegradability, contaminant distribution, lixiviation grade, chemical reactivity of the contaminants, soil type and properties, oxygen availability and occurrence of inhibitory substances. Biological treatments of organic contaminations are based on the degradative abilities of the microorganisms. Therefore the knowledge on the physiology and ecology of the biological species or consortia involved as well as the characteristics of the polluted sites are decisive factors to select an adequate biorremediation protocol. Basidiomycetes which cause white rot decay of wood are able to degrade lignin and a variety of environmentally persistent pollutants. Thus, white rot fungi and their enzymes are thought to be useful not only in some industrial process like biopulping and biobleaching but also in bioremediation. This paper provides a review of different aspects of bioremediation technologies and recent advances on ligninolytic metabolism research.

  2. Purification and characterization of an endoxylanase from the culture broth of Bacillus cereus BSA1.

    Science.gov (United States)

    Mandal, A; Kar, S; Das Mohapatra, P K; Maity, C; Pati, B R; Mondal, K C

    2011-01-01

    An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE-cellulose chromatography and followed by gel filtration through Sephadex G-100 column. The molecular mass of the purified xylanse was about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum activity at 55 degrees C and at pH 7.0 and remained reasonably stable in a wide range ofpH (5.0-8.0) and temperature (40-65 degrees C). The Km and Vmax values were found to be 8.2 mg/ml and 181.8 micromol/(min mg), respectively. The enzyme had no apparent requirement ofcofactors, and its activity was strongly inhibited by Cu++, Hg++. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhihited xylanase activity. Since the enzyme was active over wide range ofpH, temperature and remained active in higher salt concentration, it could find potential uses in biobleaching process in paper industries.

  3. Enhanced the enzymatic hydrolysis efficiency of wheat straw after combined steam explosion and laccase pretreatment.

    Science.gov (United States)

    Qiu, Weihua; Chen, Hongzhang

    2012-08-01

    Laccase, capable of selectively degrading lignin while keeping cellulose intact, has been widely applied for the modification and bio-bleaching of pulp. In this study Sclerotium sp. laccase (MSLac) was employed in combination with steam explosion to evaluate the effect of this treatment on cellulose hydrolysis. Combined steam explosion with laccase pretreatment enhanced the cellulose conversion rate of wheat straw no matter in the case of successive (MSLac-Cel) and simultaneous (MSLac+Cel) MSLac and cellulase hydrolysis. The highest cellulose conversion rate of 84.23% was obtained when steam-exploded wheat straw (SEWS) (1.3 MPa, 5 min) was treated by MSLac+Cel at a laccase loading of 0.55 U g(-1) substrate. FT-IR and SEM analyses indicated that MSLac oxidized the phenol and changed electron configuration of the ring, which contributed to loosening the compact wrap of lignin-carbohydrate complex and consequently enhancing the enzymatic hydrolysis efficiency of cellulose. This article provided a promising method for lignocellulose bio-pretreatment.

  4. Two Decades of Laccases: Advancing Sustainability in the Chemical Industry.

    Science.gov (United States)

    Cannatelli, Mark D; Ragauskas, Arthur J

    2017-01-01

    Given the current state of environmental affairs and that our future on this planet as we know it is in jeopardy, research and development into greener and more sustainable technologies within the chemical and forest products industries is at its peak. Given the global scale of these industries, the need for environmentally benign practices is propelling new green processes. These challenges are also impacting academic research and our reagents of interest are laccases. These enzymes are employed in a variety of biotechnological applications due to their native function as catalytic oxidants. They are about as green as it gets when it comes to chemical processes, requiring O2 as their only co-substrate and producing H2 O as the sole by-product. The following account will review our twenty year journey on the use of these enzymes within our research group, from their initial use in biobleaching of kraft pulps and for fiber modification within the pulp and paper industry, to their current application as green catalytic oxidants in the field of synthetic organic chemistry.

  5. Metagenomic analysis of novel lignocellulose-degrading enzymes from higher termite guts inhabiting microbes.

    Science.gov (United States)

    Nimchua, Thidarat; Thongaram, Taksawan; Uengwetwanit, Tanaporn; Pongpattanakitshote, Somchai; Eurwilaichitr, Lily

    2012-04-01

    A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from 50 degrees C to 55 degrees C. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.

  6. Phanerochaete mutants with enhanced ligninolytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  7. Agar-agar entrapment increases the stability of endo-β-1,4-xylanase for repeated biodegradation of xylan.

    Science.gov (United States)

    Bibi, Zainab; Shahid, Faiza; Ul Qader, Shah Ali; Aman, Afsheen

    2015-04-01

    Microbial xylanases, specially endo-β-1,4-xylanase catalyzes the hydrolysis of xylan, is considered one of the most significant hydrolases. It has numerous applications but most extensively is utilized in paper and pulp industry as a bio-bleaching agent. Immobilization technique is comprehensively studied with the expectation of modifying and improving enzyme stability and characteristics for commercial purposes. Currently, matrix entrapment technique is applied to immobilize endo-β-1,4-xylanase within agar-agar gel beads produced by Geobacillus stearothermophilus KIBGE-IB29. Maximal enzyme immobilization yield was achieved at 2.5% of agar-agar concentration. Optimized conditions demonstrated an increase in the optimal reaction time from 05 min to 30 min and incubation temperature from 50 °C to 60 °C with reference to free enzyme whereas; no effect was observed for optimum pH. Entrapment technique uniquely changed the kinetic parameters of immobilized endo-β-1,4-xylanase (Km: 0.5074 mg min(-1) to 0.5230 mg min(-1) and Vmax: 4773 U min(-1) to 968 U min(-1)) as compared to free enzyme. However, immobilized enzyme displayed broad thermal stability and retained 79.0% of its initial activity at 80 °C up to 30 min whereas; free enzyme completely lost its activity at this temperature. With respect to economic feasibility, the immobilized enzyme showed impressive recycling efficiency up to six reaction cycles.

  8. Statistical Optimization of Conditions for Decolorization of Synthetic Dyes by Cordyceps militaris MTCC 3936 Using RSM

    Directory of Open Access Journals (Sweden)

    Baljinder Kaur

    2015-01-01

    Full Text Available In the present study, the biobleaching potential of white rot fungus Cordyceps militaris MTCC3936 was investigated. For preliminary screening, decolorization properties of C. militaris were comparatively studied using whole cells in agar-based and liquid culture systems. Preliminary investigation in liquid culture systems revealed 100% decolorization achieved within 3 days of incubation for reactive yellow 18, 6 days for reactive red 31, 7 days for reactive black 8, and 11 days for reactive green 19 and reactive red 74. RSM was further used to study the effect of three independent variables such as pH, incubation time, and concentration of dye on decolorization properties of cell free supernatant of C. militaris. RSM based statistical analysis revealed that dye decolorization by cell free supernatants of C. militaris is more efficient than whole cell based system. The optimized conditions for decolorization of synthetic dyes were identified as dye concentration of 300 ppm, incubation time of 48 h, and optimal pH value as 5.5, except for reactive red 31 (for which the model was nonsignificant. The maximum dye decolorizations achieved under optimized conditions for reactive yellow 18, reactive green 19, reactive red 74, and reactive black 8 were 73.07, 65.36, 55.37, and 68.59%, respectively.

  9. Statistical Optimization of Conditions for Decolorization of Synthetic Dyes by Cordyceps militaris MTCC 3936 Using RSM

    Science.gov (United States)

    Kaur, Baljinder; Kumar, Balvir; Kaur, Navneet

    2015-01-01

    In the present study, the biobleaching potential of white rot fungus Cordyceps militaris MTCC3936 was investigated. For preliminary screening, decolorization properties of C. militaris were comparatively studied using whole cells in agar-based and liquid culture systems. Preliminary investigation in liquid culture systems revealed 100% decolorization achieved within 3 days of incubation for reactive yellow 18, 6 days for reactive red 31, 7 days for reactive black 8, and 11 days for reactive green 19 and reactive red 74. RSM was further used to study the effect of three independent variables such as pH, incubation time, and concentration of dye on decolorization properties of cell free supernatant of C. militaris. RSM based statistical analysis revealed that dye decolorization by cell free supernatants of C. militaris is more efficient than whole cell based system. The optimized conditions for decolorization of synthetic dyes were identified as dye concentration of 300 ppm, incubation time of 48 h, and optimal pH value as 5.5, except for reactive red 31 (for which the model was nonsignificant). The maximum dye decolorizations achieved under optimized conditions for reactive yellow 18, reactive green 19, reactive red 74, and reactive black 8 were 73.07, 65.36, 55.37, and 68.59%, respectively. PMID:25722980

  10. Performance of an alkalophilic and halotolerant laccase from gamma-proteobacterium JB in the presence of industrial pollutants.

    Science.gov (United States)

    Singh, Gursharan; Sharma, Prince; Capalash, Neena

    2009-08-01

    An alkalophilic and halotolerant laccase from gamma-proteobacterium JB catalyzed in high concentrations of organic solvents and various salts. The enzyme retained 80-100% activity in 10% concentration of dimethylsulfoxide (DMSO), ethanol, acetone or methanol; 100, 85 and 50% activity in 20 mM MgCl(2), 5.0 mM MnCl(2) and 0.1 mM CuCl(2); 140, 120 and 110% activity in 5.0 mM MnSO(4), 10 mM MgSO(4) and 1mM CaSO(4), respectively. Sodium halides inhibited the enzyme in the order: F(-)> Br(-)> I(-)> Cl(-). In 0.5 M NaCl, pH 6.0, laccase was approximately 60% active. Decolorization of indigo carmine by laccase at pH 9.0 was not inhibited even in the presence of 0.5 M NaCl. Release of chromophoric, reducing and hydrophobic compounds during biobleaching of straw rich-soda pulp by laccase was not inhibited when the enzyme was applied in the presence of 1 M NaCl at pH 8.0. Laccase retained 50% residual activity even when incubated with 5% calcium hypochlorite for 30 min.

  11. Structure/function relationships in cellulolytic enzymes

    Institute of Scientific and Technical Information of China (English)

    Marc Claeyssens

    2004-01-01

    @@ Cellulose and hemicellulose (mostly xylan), together with lignin, are the major polymeric constituents of plant cell walls and from the largest reservoir of fixed carbon in nature. The enzymatic hydrolysis of polymeric substances by extracellular enzymes, such as cellulases, hemicellulases and laccases, is preferred to chemical depolymerisation to avoid the production of toxic by-products and waste that are expensive to treat. The monosaccharides released through enzymatic hydrolysis can subsequently be microbially converted to commercial commodities, such as bio-ethanol (fuel extender) or microbial protein as feed supplements. The individual depolymerisering enzymes used, such as cellulases,xylanases and laccases, also have industrial application in (i) biobleaching in the paper and pulp industry, (ii) improvement of animal feed (poultry and ruminants) digestibility in feed industries, and (iii) dough rheology and bread volume in the baking process, and beer viscosity and filtration velocity during brewing. The cloning of the genes, coding for several xylan degrading enzymes, and their expression in Baker' s yeast (Saccharomyces cerevisiae) and filamentous fungi (Aspergillus species)opened the possibility to study the pure enzymes, without contaminating activity.Trichoderma reesei produces several of these enzymes and detailed information on their specificity,synergies and structure/activity relationships is known. An overview will be presented.

  12. Screening metagenomic libraries for laccase activities.

    Science.gov (United States)

    Ferrer, Manuel; Beloqui, Ana; Golyshin, Peter N

    2010-01-01

    Laccases are multi-copper oxidoreductases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) able to oxidise a wide variety of phenolic and non-phenolic compounds. They are useful enzymes for a variety of applications, including bioremediation and craft pulp bio-bleaching as the most significant ones. There is a considerable interest to find new laccases through the exploration of biological diversity. Laccases have been found in plants, insects, and bacteria but predominantly in fungi: these enzymes have been documented in about 60 fungal strains. Microbial diversity constitutes a largely unexplored treasure chest with new laccases with a good potential for basic science and biotechnology. At present, due to our inability to cultivate most microbes, the only means of accessing the resources of the microbial world is to harvest genetic resources ("metagenomes"), which can further on be subjected to extensive screening programs. In this chapter, we provide an overview of screening methods to identify laccase-encoding genes from environmental resources.

  13. Production of manganese peroxidase and laccase in a solid-state bioreactor and modeling of enzyme production kinetics.

    Science.gov (United States)

    Moilanen, Ulla; Winquist, Erika; Mattila, Tuomas; Hatakka, Annele; Eerikäinen, Tero

    2015-01-01

    Lignin-modifying enzymes have various promising applications such as biobleaching, biopulping, the functionalization of lignocellulosic materials, the modification of wood fibers, the remediation of contaminated soil and effluents, as well as improvement of the enzymatic hydrolysis of lignocellulosic substrates. In this study, the production of laccase and manganese peroxidase (MnP) in solid-state cultivation was examined. Oat husks were used as an inexpensive substrate for the white-rot fungus Cerrena unicolor PM170798 (FBCC 387). The addition of a fines fraction (consisting of oat flour and finely ground husks) enhanced MnP production fivefold and laccase production almost threefold. The enzyme production was studied first on a 100 g scale, and the cultivation experiments were then repeated at a larger laboratory-scale (4 kg) in a solid-state bioreactor. High enzyme activity levels were obtained (MnP: 340 nkat g(-1) DM, laccase: 470 nkat g(-1) DM). In addition, the correlation between the CO2 evolution rate and enzyme production was mathematically modeled from the bioreactor experimental data. The model parameters could be used to predict enzyme production.

  14. FY 1999 report on the results of the project on the industrial science technology R and D. Development of utilization technology of biological resources such as bioconsortium system (Development of the petroleum substituting fuel production technology using living organisms); 1999 nendo sangyo gijutsu kenkyu kaihatsu jigyo. Fukugou seibutsukei tou seibutsu shigen riyo gijutsu kaihatsu (Seibutsu riyo sekiyu daitai nenryo seizo gijutsu no kaihatsu) seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    For the purpose of developing the technology for producing and degrading useful substances using bioconsortia, study was made of the handling technology of bioconsortia, the basic element technology, etc., and the FY 1999 results were reported. In the study of the high-grade utilization technology of lignocellulose/the components, an sample was obtained in which the effect of the bacteria culture supernatant treatment was recognized in the biobleaching by co-treatment of the bacteria culture filtrate - MnP. As to the search for control factor of lignin degrading enzyme and the utilization technology, it was found out that bisphenol A was efficiently degraded by a combination of laccase and mediator production bacteria. Concerning the utilization technology of plant symbiotic bacteria, classification/identification have been finished of approximately 60% of the stored bacteria. In the study of the production technology of the petroleum substituting useful resource, a system was constructed in which immature embryos were used for callus induction and regeneration of plantlets, and plants were regenerated at high frequency via the formation of adventitious embryos. By this, the culture cell with high propagation ability was obtained. (NEDO)

  15. STUDIES ON XYLANASE AND LACCASE ENZYMATIC PREBLEACHING TO REDUCE CHLORINE-BASED CHEMICALS DURING CEH AND ECF BLEACHING

    Directory of Open Access Journals (Sweden)

    Vasanta V. Thakur,

    2012-02-01

    Full Text Available The biobleaching efficiency of xylanase and laccase enzymes was studied on kraft pulps from wood and nonwood based raw materials employed in the Indian paper industry. Treatment of these pulps with xylanase enzyme could result in improved properties, showing 2.0% ISO gain in pulp brightness and/or reducing the demand of chlorine-based bleach chemicals by up to 15% with simultaneous reduction of 20 to 25% in AOX generation in bleach effluents. Further, mill-scale trial results revealed that enzymatic prebleaching can be successfully employed with xylanases to reach the same bleach boosting efficacy. Laccase bleaching was also studied on hardwood pulp at a pH around 8.0, where most of the pulp mills in India are operating, in contrast to earlier studies on laccase enzyme bleaching, which were conducted at acidic pHs, i.e. 4.0 to 5.0. In case of laccase bleaching, interesting results were found wherein a bleach-boosting effect was observed even at pH 8.0. Further studies carried out with HOBT as mediator in comparison to the commonly used and expensive ABTS laccase mediator system (LMS resulted in improvement of the bleaching efficiency with reduction in demand of chlorine dioxide by more than 35%. Potential for further reduction was indicated by the brightness gain, when compared with a control using the DE(pD bleach sequence.

  16. Peculiarities of Pycnoporus species for applications in biotechnology.

    Science.gov (United States)

    Lomascolo, Anne; Uzan-Boukhris, Eva; Herpoël-Gimbert, Isabelle; Sigoillot, Jean-Claude; Lesage-Meessen, Laurence

    2011-12-01

    The genus Pycnoporus forms a cosmopolitan group of four species belonging to the polyporoid white-rot fungi, the most representative group of homobasidiomycetes causing wood decay. Pycnoporus fungi are listed as food- and cosmetic-grade microorganisms and emerged in the early 1990s as a genus whose biochemistry, biodegradation and biotechnological properties have since been progressively detailed. First highlighted for their original metabolic pathways involved in the functionalization of plant cell wall aromatic compounds to yield high-value molecules, e.g. aromas and antioxidants, the Pycnoporus species were later explored for their potential to produce various enzymes of industrial interest, such as hydrolases and oxidases. However, the most noteworthy feature of the genus Pycnoporus is its ability to overproduce high redox potential laccase-a multi-copper extracellular phenoloxidase-as the predominant ligninolytic enzyme. A major potential use of the Pycnoporus fungi is thus to harness their laccases for various applications such as the bioconversion of agricultural by-products and raw plant materials into valuable products, the biopulping and biobleaching of paper pulp and the biodegradation of organopollutants, xenobiotics and industrial contaminants. All the studies performed in the last decade show the genus Pycnoporus to be a strong contender for white biotechnology. In this review, we describe the properties of Pycnoporus fungi in relation to their biotechnological applications and potential.

  17. Aspects microbiologiques de la production par fermentation solide des endo-beta-1,4-xylanases de moisissures : le cas de Penicillium canescens

    Directory of Open Access Journals (Sweden)

    Assamoi AA.

    2009-01-01

    Full Text Available Microbial aspects of endo-β-1,4-xylanase production in solid-state fermentation by Penicillia: the case of Penicillium canescens. Production of xylanases by Penicillium canescens 10-10c is the research object in Walloon Center of Industrial Biology. Previous works used submerged or liquid fermentation. The actual works are oriented more and more towards solid fermentation from agricultural or agro-alimentary residues. In addition to the valorization of these residues, solid-state fermentation reaches an increasingly significant interest in various other fields like the biological breakdown of the solid residues, the bioremediation of the organic pollutants in the grounds and the reduction of the air pollution by the biofiltration. Xylanase is an industrial enzyme used in general in extraction and clarification processes. P. canescens can produce an activity of it, particularly in its balanced forms of xylanases, beta-xylosidase and arabinosidase, and not contaminated by cellulolytic and amylolytic activities. It is a hyper producing strain of xylanase. The production rate is one of the highest in literature (535 U.ml-1 and 9,632 U.g-1 in Erlenmeyer flasks, in submerged and solid state fermentation, respectively. The biobleaching activity of the cellulose pulp by the purified enzyme is higher than a commercial preparation of xylanases from Trichoderma longibrachiatum used industrially. It has a complete hydrolysis degree of 40% (on glucuronoxylan and 35% (on arabinoxylan at 55°C and at pH of 5.9. These characteristics lead to many industrial applications of this enzyme. That is why the optimization of its production by the solid-state fermentation at the laboratory scale in order to define a policy for the industrial transposition later is carried out. This article presents a summary of the scientific literature on this subject.

  18. Xylanase production by Trichoderma strains in solid substrate fermentation

    Institute of Scientific and Technical Information of China (English)

    Krisztina Kovacs; George Szakacs; Lew Christopher

    2004-01-01

    @@ The importance of microbial enzymes in pulp and paper manufacturing has grown significantly in the last two decades. Solid substrate fermentation (SSF) holds tremendous potential for the production of microbial enzymes of commercial interest. SSF can be of special interest in those processes where the crude fermented product (whole SSF culture, in situ enzyme) may be used directly as the enzyme source. Xylanase preparations practically free of cellulase activity are especially useful for biobleaching of crude cellulose pulps. Thirty-nine Trichoderma isolates have been screened in SSF for xylanase production on hardwood oxygen-delignified soda-aq pulp as carbon source and enzyme inducer.Xylanase activities varied between 0 and 2200 IU/g dry matter (DM) of initial substrate. In most instances, the simultaneously produced cellulase levels were below 1.0 Filter Paper Unit (FPU) /g DM. The xylanase to cellulase activity ratio varied in the range of 5 to 3500. The three most promising isolates (TUB F-1647, TUB F-1658 and TUB F-1684) yielded xylanase activity of 2040,1300 and 1500 IU/g DM xylanase, respectively, and 0.64, 0.43 and 0.43 FPU/g DM cellulase with a xylanase to cellulase activity ratio of 3200, 3000 and 3500, respectively. Wild strains F-1647, F-1658 and F-1684 were isolated from tree bark of Maldives, soils of Peru (last two), respectively.Medium optimization experiments to enhance the xylanase yield and to increase the xylanase to cellulase ratio have also been performed.

  19. Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus for simultaneous biosynthesis of xylanase and laccase under solid-state fermentation.

    Science.gov (United States)

    Dwivedi, Pallavi; Vivekanand, V; Pareek, Nidhi; Sharma, Amit; Singh, Rajesh P

    2011-10-01

    Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus MTCC 1804 was evaluated for the production of xylanase-laccase mixture under solid-state fermentation (SSF) condition. Growth compatibility between mutant P. oxalicum SAU(E)-3.510 and white rot fungi (P. ostreatus MTCC 1804, Trametes hirsuta MTCC 136 and Pycnoporus sp. MTCC 137) was analyzed by growing them on potato dextrose agar plate. Extracellular enzyme activities were determined spectrophotometrically. Under derived conditions, paired culturing of mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804 resulted in 58% and 33% higher levels of xylanase and laccase production, respectively. A combination of sugarcane bagasse and black gram husk in a ratio of 3:1 was found to be the most ideal solid substrate and support for fungal colonization and enzyme production during co-cultivation. Maximum levels of xylanase (8205.31 ± 168.31 IU g(-1)) and laccase (375.53 ± 34.17 IU g(-1)) during SSF were obtained by using 4 g of solid support with 80% of moisture content. Furthermore, expressions of both xylanase and laccase were characterized during mixed culture by zymogram analysis. Improved levels of xylanase and laccase biosynthesis were achieved by co-culturing the mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804. This may be because of efficient substrate utilization as compared to their respective monocultures in the presence of lignin degradation compounds because of synergistic action of xylanase and laccase. Understanding and developing the process of co-cultivation appears productive for the development of mixed enzyme preparation with tremendous potential for biobleaching.

  20. Determination of some significant batch culture conditions affecting acetyl-xylan esterase production by Penicillium notatum NRRL-1249

    Directory of Open Access Journals (Sweden)

    Akhtar MN

    2011-05-01

    Full Text Available Abstract Background Acetyl-xylan esterase (AXE, EC 3.1.1.72 hydrolyses acetate group from the linear chain of xylopyranose residues bound by β-1,4-linkage. The enzyme finds commercial applications in bio-bleaching of wood pulp, treating animal feed to increase digestibility, processing food to increase clarification and converting lignocellulosics to feedstock and fuel. In the present study, we report on the production of an extracellular AXE from Penicillium notatum NRRL-1249 by solid state fermentation (SSF. Results Wheat bran at a level of 10 g (with 4 cm bed height was optimized as the basal substrate for AXE production. An increase in enzyme activity was observed when 7.5 ml of mineral salt solution (MSS containing 0.1% KH2PO4, 0.05% KCl, 0.05% MgSO4.7H2O, 0.3% NaNO3, 0.001% FeSO4.2H2O and 0.1% (v/w Tween-80 as an initial moisture content was used. Various nitrogen sources including ammonium sulphate, urea, peptone and yeast extract were compared for enzyme production. Maximal enzyme activity of 760 U/g was accomplished which was found to be highly significant (p ≤ 0.05. A noticeable enhancement in enzyme activity was observed when the process parameters including incubation period (48 h, initial pH (5, 0.2% (w/w urea as nitrogen source and 0.5% (v/w Tween-80 as a stimulator were further optimized using a 2-factorial Plackett-Burman design. Conclusion From the results it is clear that an overall improvement of more than 35% in terms of net enzyme activity was achieved compared to previously reported studies. This is perhaps the first report dealing with the use of P. notatum for AXE production under batch culture SSF. The Plackett-Burman model terms were found highly significant (HS, suggesting the potential commercial utility of the culture used (df = 3, LSD = 0.126.

  1. Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.

    Directory of Open Access Journals (Sweden)

    Claudia M Rivera-Hoyos

    Full Text Available Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range

  2. Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.

    Science.gov (United States)

    Rivera-Hoyos, Claudia M; Morales-Álvarez, Edwin David; Poveda-Cuevas, Sergio Alejandro; Reyes-Guzmán, Edwin Alfredo; Poutou-Piñales, Raúl A; Reyes-Montaño, Edgar Antonio; Pedroza-Rodríguez, Aura Marina; Rodríguez-Vázquez, Refugio; Cardozo-Bernal, Ángela M

    2015-01-01

    Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates

  3. Computational Analysis and Low-Scale Constitutive Expression of Laccases Synthetic Genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris

    Science.gov (United States)

    Reyes-Guzmán, Edwin Alfredo; Poutou-Piñales, Raúl A.; Reyes-Montaño, Edgar Antonio; Pedroza-Rodríguez, Aura Marina; Rodríguez-Vázquez, Refugio; Cardozo-Bernal, Ángela M.

    2015-01-01

    Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates

  4. Mill Designed Bio bleaching Technologies

    Energy Technology Data Exchange (ETDEWEB)

    Institute of Paper Science Technology

    2004-01-30

    A key finding of this research program was that Laccase Mediator Systems (LMS) treatments on high-kappa kraft could be successfully accomplished providing substantial delignification (i.e., > 50%) without detrimental impact on viscosity and significantly improved yield properties. The efficiency of the LMS was evident since most of the lignin from the pulp was removed in less than one hour at 45 degrees C. Of the mediators investigated, violuric acid was the most effective vis-a-vis delignification. A comparative study between oxygen delignification and violuric acid revealed that under relatively mild conditions, a single or a double LMS{sub VA} treatment is comparable to a single or a double O stage. Of great notability was the retention of end viscosity of LMS{sub VA} treated pulps with respect to the end viscosity of oxygen treated pulps. These pulps could then be bleached to full brightness values employing conventional ECF bleaching technologies and the final pulp physical properties were equal and/or better than those bleached in a conventional ECF manner employing an aggressively O or OO stage initially. Spectral analyses of residual lignins isolated after LMS treated high-kappa kraft pulps revealed that similar to HBT, VA and NHA preferentially attack phenolic lignin moieties. In addition, a substantial decrease in aliphatic hydroxyl groups was also noted, suggesting side chain oxidation. In all cases, an increase in carboxylic acid was observed. Of notable importance was the different selectivity of NHA, VA and HBT towards lignin functional groups, despite the common N-OH moiety. C-5 condensed phenolic lignin groups were overall resistant to an LMS{sub NHA, HBT} treatments but to a lesser extent to an LMS{sub VA}. The inactiveness of these condensed lignin moieties was not observed when low-kappa kraft pulps were biobleached, suggesting that the LMS chemistry is influenced by the extent of delignification. We have also demonstrated that the current

  5. Advanced Recombinant Manganese Peroxidase for Biosynthesis of Lignin Bioproducts, Phase I Final Report, STTR Grant #: DE-SC0007503.

    Energy Technology Data Exchange (ETDEWEB)

    Beatty, Christopher; Kitner, Joshua; Lajoie, Curtis; McClain, Sean; Potochnik, Steve

    2012-12-13

    The core purpose of this Phase I STTR was to evaluate the feasibility of a new method of producing a recombinant version of manganese peroxidase (MnP) enzyme. MnP is a potentially valuable enzyme for producing high value lignin products and also for industrial de-coloring operations such as biobleaching of pulp and color removal from textile dye effluents. This lignin-modifying enzyme is produced in small amounts by the native host, a white rot fungus. Previous work by Oregon State University developed a secreted recombinant version of the enzyme in the yeast Pichia pastoris. Unfortunately, the expression is barely moderate and the enzyme is heavily glycosylated, which inhibits purification. In this work, the gene for the enzyme is given a tag which targets production of the enzyme to the peroxisome. This is a promising approach since this location is also where heme and hydrogen peroxide are sequestered, which are both necessary cofactors for MnP. More than ten recombinant strains were constructed, verified, and expressed in the Pichia system. Constitutive (GAP) and methanol-induced promoters (AOX) were tried for peroxisomal targeted, cytosolic, and secreted versions of MnP. Only the secreted strains showed activity. The amount of expression was not significantly changed. The degree of glycosylation was lessened using the AOX (methanol) promotoer, but the resulting enzyme was still not able to be purified using immobilized metal affinity chromatography. Additional work beyond the scope of the defined Phase I project was undertaken to construct, verify, and express Pichia strains that mutated the MnP glycosylation sites to inhibit this process. These strains did not show significant activity. The cause is not known, but it is possible that these sites are important to the structure of the enzyme. Also beyond the scope proposed for our Phase I STTR, the team collaborated with AbSci, a startup with a new E. coli based expression system focused on the production of

  6. Structure and Biochemestry of Laccases from the Lignin-Degrading Basidiomycete, Ganoderma lucidum

    Energy Technology Data Exchange (ETDEWEB)

    C.A.Reddy, PI

    2005-06-30

    G. lucidum is one of the most important and widely distributed ligninolytic white rot fungi from habitats such as forest soils, agricultural soils, and tropical mangrove ecosystems and produce laccases as an important family of lignin modifying enzymes. Biochemically, laccases are blue multi copper oxidases that couple four electron reduction of molecular oxygen to water. There is a growing interest in the use of laccases for a variety of industrial applications such as bio-pulping and biobleaching as well as in their ability to detoxify a wide variety of toxic environmental pollutants. These key oxidative enzymes are found in all the three domains of life: Eukaryota. Prokarya, and Archaea. Ganoderma lucidum (strain no.103561) produces laccase with some of the highest activity (17,000 micro katals per mg of protein) reported for any laccases to date. Our results showed that this organism produces at least 11 different isoforms of laccase based on variation in mol. weight and/or PI. Our Studies showed that the presence of copper in the medium yields 15- to 20-fold greater levels of enzyme by G. lucidum. Dialysation of extra cellular fluid of G. lucidum against 10mM sodium tartrate (pH5.5) gave an additional 15 to 17 fold stimulation of activity with an observed specific activity of 17,000 {micro}katals/mg protein. Dialysis against acetate buffer gave five fold increase in activity while dialysis against glycine showed inhibition of activity. Purification by FPLC and preparative gel electrophoresis gave purified fractions that resolved into eleven isoforms as separated by isoelectric focusing, and the PI,s were 4.7, 4.6, 4.5, 4.3, 4.2, 4.1, 3.8, 3.7, 3.5, 3.4 and 3.3. Genomic clones of laccase were isolated using G. lucidum DNA as a template and using inverse PCR and forward/reverse primers corresponding to the sequences of the conserved copper binding region in the N-terminal domain of one of the laccases of this organism. Inverse PCR amplication of HindIII digested

  7. Structure and Biochemestry of Laccases from the Lignin-Degrading Basidiomycete, Ganoderma lucidum

    Energy Technology Data Exchange (ETDEWEB)

    C.A.Reddy, PI

    2005-06-30

    G. lucidum is one of the most important and widely distributed ligninolytic white rot fungi from habitats such as forest soils, agricultural soils, and tropical mangrove ecosystems and produce laccases as an important family of lignin modifying enzymes. Biochemically, laccases are blue multi copper oxidases that couple four electron reduction of molecular oxygen to water. There is a growing interest in the use of laccases for a variety of industrial applications such as bio-pulping and biobleaching as well as in their ability to detoxify a wide variety of toxic environmental pollutants. These key oxidative enzymes are found in all the three domains of life: Eukaryota. Prokarya, and Archaea. Ganoderma lucidum (strain no.103561) produces laccase with some of the highest activity (17,000 micro katals per mg of protein) reported for any laccases to date. Our results showed that this organism produces at least 11 different isoforms of laccase based on variation in mol. weight and/or PI. Our Studies showed that the presence of copper in the medium yields 15- to 20-fold greater levels of enzyme by G. lucidum. Dialysation of extra cellular fluid of G. lucidum against 10mM sodium tartrate (pH5.5) gave an additional 15 to 17 fold stimulation of activity with an observed specific activity of 17,000 {micro}katals/mg protein. Dialysis against acetate buffer gave five fold increase in activity while dialysis against glycine showed inhibition of activity. Purification by FPLC and preparative gel electrophoresis gave purified fractions that resolved into eleven isoforms as separated by isoelectric focusing, and the PI,s were 4.7, 4.6, 4.5, 4.3, 4.2, 4.1, 3.8, 3.7, 3.5, 3.4 and 3.3. Genomic clones of laccase were isolated using G. lucidum DNA as a template and using inverse PCR and forward/reverse primers corresponding to the sequences of the conserved copper binding region in the N-terminal domain of one of the laccases of this organism. Inverse PCR amplication of HindIII digested