Determination of the cellulase activity distribution in Clostridium thermocellum and Caldicellulosiruptor obsidiansis cultures using a fluorescent substrate

DOE PAGES

Morrell-Falvey, Jennifer L.; Elkins, James G.; Wang, Zhi-Wu

2015-05-30

This study took advantage of resorufin cellobioside as a fluorescent substrate to determine the distribution of cellulase activity in cellulosic biomass fermentation systems. Cellulolytic biofilms were found to express nearly four orders greater cellulase activity compared to planktonic cultures of Clostridium thermocellum and Caldicellulosiruptor obsidiansis, which can be primarily attributed to the high cell concentration and surface attachment. The formation of biofilms results in cellulases being secreted close to their substrates, which appears to be an energetically favorable stategy for insoluble substrate utilization. For the same reason, cellulases should be closely associated with the surfaces of suspended cell in solublemore » substrate-fed culture, which has been verified with cellobiose-fed cultures of C. thermocellum and C. obsidiansis. This study addressed the importance of cellulase activity distribution in cellulosic biomass fermentation, and provided theoretical foundation for the leading role of biofilm in cellulose degradation. System optimization and reactor designs that promote biofilmformation in cellulosic biomass hydrolysismay promise an improved cellulosic biofuel process.« less

Strong cellulase inhibitors from the hydrothermal pretreatment of wheat straw

PubMed Central

2013-01-01

Background The use of the enzymatic hydrolysis of lignocellulose with subsequent fermentation to ethanol provides a green alternative for the production of transportation fuels. Because of its recalcitrant nature, the lignocellulosic biomass must be pretreated before enzymatic hydrolysis. However, the pretreatment often results in the formation of compounds that are inhibitory for the enzymes or fermenting organism. Although well recognized, little quantitative information on the inhibition of individual cellulase components by identified inhibitors is available. Results Strong cellulase inhibitors were separated from the liquid fraction of the hydrothermal pretreatment of wheat straw. HPLC and mass-spectroscopy analyses confirmed that the inhibitors were oligosaccharides (inhibitory oligosaccharides, IOS) with a degree of polymerization from 7 to 16. The IOS are composed of a mixture of xylo- (XOS) and gluco-oligosaccharides (GOS). We propose that XOS and GOS are the fragments of the xylan backbone and mixed-linkage β-glucans, respectively. The IOS were approximately 100 times stronger inhibitors for Trichoderma reesei cellobiohydrolases (CBHs) than cellobiose, which is one of the strongest inhibitors of these enzymes reported to date. Inhibition of endoglucanases (EGs) by IOS was weaker than that of CBHs. Most of the tested cellulases and hemicellulases were able to slowly degrade IOS and reduce the inhibitory power of the liquid fraction to some extent. The most efficient single enzyme component here was T. reesei EG TrCel7B. Although reduced by the enzyme treatment, the residual inhibitory power of IOS and the liquid fraction was strong enough to silence the major component of the T. reesei cellulase system, CBH TrCel7A. Conclusions The cellulase inhibitors described here may be responsible for the poor yields from the enzymatic conversion of the whole slurries from lignocellulose pretreatment under conditions that do not favor complete degradation of hemicellulose. Identification of the inhibitory compounds helps to design better enzyme mixtures for their degradation and to optimize the pretreatment regimes to minimize their formation. PMID:24053778

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

PubMed Central

Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

2015-01-01

Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains

PubMed Central

Herpoël-Gimbert, Isabelle; Margeot, Antoine; Dolla, Alain; Jan, Gwénaël; Mollé, Daniel; Lignon, Sabrina; Mathis, Hughes; Sigoillot, Jean-Claude; Monot, Frédéric; Asther, Marcel

2008-01-01

Background Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions. Results The protein composition of the secretome produced by industrial T. reesei (strain CL847) grown on a medium promoting the production of both cellulases and hemicellulases was explored using two-dimensional electrophoresis and MALDI-TOF or LC-MS/MS protein identification. A total of 22 protein species were identified. As expected, most of them are potentially involved in biomass degradation. The 2D map obtained was then used to compare the secretomes produced by CL847 and another efficient cellulolytic T. reesei strain, Rut-C30, the reference cellulase-overproducing strain using lactose as carbon source and inducer of cellulases. Conclusion This study provides the most complete mapping of the proteins secreted by T. reesei to date. We report on the first use of proteomics to compare secretome composition between two cellulase-overproducing strains Rut-C30 and CL847 grown under similar conditions. Comparison of protein patterns in both strains highlighted many unexpected differences between cellulase cocktails. The results demonstrate that 2D electrophoresis is a promising tool for studying cellulase production profiles, whether for industrial characterization of an entire secretome or for a more fundamental study on cellulase expression at genome-wide scale. PMID:19105830

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

PubMed Central

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

2015-01-01

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

The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa.

PubMed

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

2015-01-09

Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Reaction mechanism of dicofol removal by cellulase.

PubMed

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. Copyright © 2015. Published by Elsevier B.V.

The production and activity test of cellulases using bagasse substrate on Aspergillus niger isolated from Clove field, Kare, Madiun

NASA Astrophysics Data System (ADS)

Ardhi, Muh. Waskito; Sulistyarsi, Ani; Pujiati

2017-06-01

Aspergillus sp is a microorganism which has a high ability to produce cellulase enzymes. In producing Cellulase enzymes requires appropriate concentration and incubation time to obtain optimum enzyme activity. This study aimed to determine the effect of inoculum concentration and incubation time towards production and activity of cellulases from Aspergillus sp substrate bagasse. This research used experiments method; completely randomized design with 2 factorial repeated 2 times. The treatment study include differences inoculum (K) 5% (K1), 15% (K2) 25%, (K3) and incubation time (F) that is 3 days (F1), 6 days (F2), 9 days (F3), 12 days (F4). The data taken from the treatment are glucose reduction and protein levels of crude cellulase enzyme activity that use Nelson Somogyi and Biuret methods. Analysis of variance ANOVA data used two paths with significance level of 5% then continued with LSD test. The results showed that: Fhit>Ftab. Thus, there is effect of inoculum concentrations and incubation time toward activity of crude cellulases of Aspergillus sp. The highest glucose reduction of treatment is K3F4 (concentration of inoculum is 25% with 12 days incubation time) amount 12.834 g / ml and the highest protein content is K3F4 (concentration of inoculum is 25% with with 12 days incubation time) amount 0.740 g / ml.

The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

PubMed Central

Seiboth, Bernhard; Karimi, Razieh Aghcheh; Phatale, Pallavi A; Linke, Rita; Hartl, Lukas; Sauer, Dominik G; Smith, Kristina M; Baker, Scott E; Freitag, Michael; Kubicek, Christian P

2012-01-01

Summary Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified. PMID:22554051

[Optimization of process of icraiin be hydrolyzed to Baohuoside I by cellulase based on Plackett-Burman design combined with CCD response surface methodology].

PubMed

Song, Chuan-xia; Chen, Hong-mei; Dai, Yu; Kang, Min; Hu, Jia; Deng, Yun

2014-11-01

To optimize the process of Icraiin be hydrolyzed to Baohuoside I by cellulase by Plackett-Burman design combined with Central Composite Design (CCD) response surface methodology. To select the main influencing factors by Plackett-Burman design, using CCD response surface methodology to optimize the process of Icraiin be hydrolyzed to Baohuoside I by cellulase. Taking substrate concentration, the pH of buffer and reaction time as independent variables, with conversion rate of icariin as dependent variable,using regression fitting of completely quadratic response surface between independent variable and dependent variable,the optimum process of Icraiin be hydrolyzed to Baohuoside I by cellulase was intuitively analyzed by 3D surface chart, and taking verification tests and predictive analysis. The best enzymatic hydrolytic process was as following: substrate concentration 8. 23 mg/mL, pH 5. 12 of buffer,reaction time 35. 34 h. The optimum process of Icraiin be hydrolyzed to Baohuoside I by cellulase is determined by Plackett-Burman design combined with CCD response surface methodology. The optimized enzymatic hydrolytic process is simple, convenient, accurate, reproducible and predictable.

MICROBIAL FERMENTATION OF ABUNDANT BIOPOLYMERS: CELLULOSE AND CHITIN

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leschine, Susan

Our research has dealt with seven major areas of investigation: i) characterization of cellulolytic members of microbial consortia, with special attention recently given to Clostridium phytofermentans, a bacterium that decomposes cellulose and produces uncommonly large amounts of ethanol, ii) investigations of the chitinase system of Cellulomonas uda; including the purification and characterization of ChiA, the major component of this enzyme system, iii) molecular cloning, sequence and structural analysis of the gene that encodes ChiA in C. uda, iv) biofilm formation by C. uda on nutritive surfaces, v) investigations of the effects of humic substances on cellulose degradation by anaerobic cellulolyticmore » microbes, vi) studies of nitrogen metabolism in cellulolytic anaerobes, and vii) understanding the molecular architecture of the multicomplex cellulase-xylanase system of Clostridium papyrosolvens. Also, progress toward completing the research of more recent projects is briefly summarized. Major accomplishments include: 1. Characterization of Clostridium phytofermentans, a cellulose-fermenting, ethanol-producing bacterium from forest soil. The characterization of a new cellulolytic species isolated from a cellulose-decomposing microbial consortium from forest soil was completed. This bacterium is remarkable for the high concentrations of ethanol produced during cellulose fermentation, typically more than twice the concentration produced by other species of cellulolytic clostridia. 2. Examination of the use of chitin as a source of carbon and nitrogen by cellulolytic microbes. We discovered that many cellulolytic anaerobes and facultative aerobes are able to use chitin as a source of both carbon and nitrogen. This major discovery expands our understanding of the biology of cellulose-fermenting bacteria and may lead to new applications for these microbes. 3. Comparative studies of the cellulase and chitinase systems of Cellulomonas uda. Results of these studies indicate that the chitinase and cellulase systems of this bacterium are distinct in terms of the proteins involved and the regulation of their production. 4. Characterization of the chitinase system of C. uda. A 70,000-Mr endochitinase, designated ChiA, was purified from C. uda culture supernatant fluids and characterized. 5. Analysis of chiA, which codes for the major enzymatic component of the chitinase system of C. uda. The gene encoding the endochitinase ChiA in C. uda was cloned, its complete nucleotide sequence was determined and its implications were investigated. 6. Formation of biofilms by C. uda on cellulose and chitin. Microscopic observations indicated that, under conditions of nitrogen limitation, C. uda cells grew as a biofilm attached tightly to the surface of cellulose or chitin. 7. Development of tools for a genetic approach to studies of cellulose fermentation by cellulolytic clostridia. We have explored the potential of various techniques, and obtained evidence indicating that Tn916 mutagenesis may be particularly effective in this regard. As part of this research, we identified the presence of a plasmid in one strain, which was cloned, sequenced, and analyzed for its utility in the development of vectors for genetic studies. 8. Effects of humic substances on cellulose degradation by anaerobic cellulolytic microbes. We determined that humic substances play an important role in the anaerobic cellulose decomposition and in the physiology of cellulose-fermenting soil bacteria. 9. Nitrogenases of cellulolytic clostridia. We described a nitrogenase gene from a cellulolytic clostridium and presented evidence, based on sequence analyses and conserved gene order, for lateral gene transfer between this bacterium and a methanogenic archaeon. 10. Characterization of Clostridium hungatei, a new N2-fixing cellulolytic species isolated from a methanogenic consortium from soil. 11. Understanding the molecular architecture of the multicomplex cellulase-xylanase system of Clostridium papyrosolvens. We discovered that C. papyrosolvens produces a multiprotein, multicomplex cellulase-xylanase enzyme system that hydrolyzes crystalline cellulose, and we have described this system in detail.« less

Genome Sequence and Analysis of the Soil Cellulolytic ActinomyceteThermobifida fusca

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lykidis, Athanasios; Mavromatis, Konstantinos; Ivanova, Natalia

Thermobifida fusca is a moderately thermophilic soilbacterium that belongs to Actinobacteria. 3 It is a major degrader ofplant cell walls and has been used as a model organism for the study of 4secreted, thermostable cellulases. The complete genome sequence showedthat T. fusca has a 5 single circular chromosome of 3642249 bp predictedto encode 3117 proteins and 65 RNA6 species with a coding densityof 85percent. Genome analysis revealed the existence of 29 putative 7glycoside hydrolases in addition to the previously identified cellulasesand xylanases. The 8 glycosyl hydrolases include enzymes predicted toexhibit mainly dextran/starch and xylan 9 degrading functions. T. fuscapossesses twomore » protein secretion systems: the sec general secretion 10system and the twin-arginine translocation system. Several of thesecreted cellulases have 11 sequence signatures indicating theirsecretion may be mediated by the twin-arginine12 translocation system. T.fusca has extensive transport systems for import of carbohydrates 13coupled to transcriptional regulators controlling the expression of thetransporters and14 glycosylhydrolases. In addition to providing anoverview of the physiology of a soil 15 actinomycete, this study presentsinsights on the transcriptional regulation and secretion of16 cellulaseswhich may facilitate the industrial exploitation of thesesystems.« less

Assessing cellulolysis in passive treatment systems for mine drainage: a modified enzyme assay.

PubMed

McDonald, Corina M; Gould, W Douglas; Lindsay, Matthew B J; Blowes, David W; Ptacek, Carol J; Condon, Peter D

2013-01-01

A modified cellulase enzyme assay was developed to monitor organic matter degradation in passive treatment systems for mine drainage. This fluorogenic substrate method facilitates assessment of exo-(1,4)-β-D-glucanase, endo-(1,4)-β-D-glucanase, and β-glucosidase, which compose an important cellulase enzyme system. The modified method was developed and refined using samples of organic carbon-amended mine tailings from field experiments where sulfate reduction was induced as a strategy for managing water quality. Sample masses (3 g) and the number of replicates ( ≥ 3) were optimized. Matrix interferences within these metal-rich samples were found to be insignificant. Application of this modified cellulase assay method provided insight into the availability and degradation of organic carbon within the amended tailings. Results of this study indicate that cellulase enzyme assays can be applied to passive treatment systems for mine drainage, which commonly contain elevated concentrations of metals. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Improvement of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic Saccharomyces cerevisiae.

PubMed

Liu, Zhuo; Inokuma, Kentaro; Ho, Shih-Hsin; den Haan, Riaan; van Zyl, Willem H; Hasunuma, Tomohisa; Kondo, Akihiko

2017-06-01

Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surface display system. This work provides key insights in engineering the cellulase cocktail in a consolidated bioprocessing yeast strain. Biotechnol. Bioeng. 2017;114: 1201-1207. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A liquid crystal-based sensor for the simple and sensitive detection of cellulase and cysteine.

PubMed

Wang, Yi; Hu, Qiongzheng; Tian, Tongtong; Gao, Yan'an; Yu, Li

2016-11-01

A liquid crystal (LC)-based sensor, which is capable of monitoring enzymatic activity at the aqueous/LC interface and detecting cellulase and cysteine (Cys), was herein reported. When functionalized with a surfactant, dodecyl β-d-glucopyranoside, the 4-cyano-4'-pentylbiphenyl (5CB) displays a dark-to-bright transition in the optical appearance for cellulase. We attribute this change to the orientational transition of LCs, as a result of enzymatic hydrolysis between cellulase and surfactant. Furthermore, by adding cellulase and Cu(2+), our surfactant-LCs system performs an interesting ability to detect Cys, even though Cys could not interact with surfactant or LC directly. Alternatively, through the strong binding between Cys and Cu(2+), cellulase was able to hydrolyze surfactant in the presence of Cu(2+), leading to the transition of LCs from dark to bright. The detection limit of the LC sensor was around 1×10(-5)mg/mL and 82.5μM for cellulase and Cys, respectively. The LC-based sensor may contribute to the development of low-cost, expedient, and label-free detection for cellulase and Cys and the design strategy may also provide a novel way for detecting multiple analytes. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2014-01-01

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

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

PubMed Central

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

2014-01-01

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

Methods for Discovery of Novel Cellulosomal Cellulases Using Genomics and Biochemical Tools.

PubMed

Ben-David, Yonit; Dassa, Bareket; Bensoussan, Lizi; Bayer, Edward A; Moraïs, Sarah

2018-01-01

Cell wall degradation by cellulases is extensively explored owing to its potential contribution to biofuel production. The cellulosome is an extracellular multienzyme complex that can degrade the plant cell wall very efficiently, and cellulosomal enzymes are therefore of great interest. The cellulosomal cellulases are defined as enzymes that contain a dockerin module, which can interact with a cohesin module contained in multiple copies in a noncatalytic protein, termed scaffoldin. The assembly of the cellulosomal cellulases into the cellulosomal complex occurs via specific protein-protein interactions. Cellulosome systems have been described initially only in several anaerobic cellulolytic bacteria. However, owing to ongoing genome sequencing and metagenomic projects, the discovery of novel cellulosome-producing bacteria and the description of their cellulosomal genes have dramatically increased in the recent years. In this chapter, methods for discovery of novel cellulosomal cellulases from a DNA sequence by bioinformatics and biochemical tools are described. Their biochemical characterization is also described, including both the enzymatic activity of the putative cellulases and their assembly into mature designer cellulosomes.

Combined strategy of transcription factor manipulation and β-glucosidase gene overexpression in Trichoderma reesei and its application in lignocellulose bioconversion.

PubMed

Xia, Ying; Yang, Lirong; Xia, Liming

2018-06-16

The industrial application of Trichoderma reesei has been greatly limited by insufficient β-glucosidase activity in its cellulase system. In this study, a novel β-glucosidase expression cassette was constructed and integrated at the target site in T. reesei ZU-02, which achieved the overexpression of β-glucosidase gene and in situ disruption of the cellulase transcriptional repressor ACE1. The resulting transformants showed significant increase in both β-glucosidase activity (BGA) and filter paper activity (FPA). The BGA and FPA increased to 25.13 IU/mL and 20.06 FPU/mL, respectively, 167- and 2.45-fold higher than that of the host strain. Meanwhile, the obtained cellulase system exhibited improved ratio of BGA to FPA, leading to better synergistic effect between cellulase components. Furthermore, submerged fermentation of the transformant was established in 50 m 3 fermenter yielding 112.2 IU/mL β-glucosidase and 89.76 FPU/mL total cellulase. The newly constructed T. reesei transformant achieved improved hydrolysis yield (90.6%) with reduced enzyme loading (15 FPU/g substrate).

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

PubMed

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

2014-12-01

Fungi grow over a relatively wide pH range and adapt to extracellular pH through a genetic regulatory system mediated by a key component PacC, which is a pH transcription regulator. The cellulase production of the filamentous fungi Trichoderma reesei is sensitive to ambient pH. To investigate the connection between cellulase expression regulation and ambient pH, an ortholog of Aspergillus nidulans pacC, Trpac1, was identified and functionally characterized using a target gene deletion strategy. Deleting Trpac1 dramatically increased the cellulase production and the transcription levels of the major cellulase genes at neutral pH, which suggested Trpac1 is involved in the regulation of cellulase production. It was further observed that the expression levels of transcription factors xyr1 and ace2 also increased in the ΔTrpac1 mutant at neutral pH. In addition, the ΔTrpac1 mutant exhibited conidiation defects under neutral and alkaline pH. These results implied that Trpac1 in involved in growth and development process and cellulase gene expression in T. reesei. Copyright © 2014 Elsevier Inc. All rights reserved.

Synergetic effect of yeast cell-surface expression of cellulase and expansin-like protein on direct ethanol production from cellulose

PubMed Central

2013-01-01

Background Numerous studies have examined the direct fermentation of cellulosic materials by cellulase-expressing yeast; however, ethanol productivity in these systems has not yet reached an industrial level. Certain microorganisms, such as the cellulolytic fungus Trichoderma reesei, produce expansin-like proteins, which have a cellulose-loosening effect that may increase the breakdown of cellulose. Here, to improve the direct conversion of cellulose to ethanol, yeast Saccharomyces cerevisiae co-displaying cellulase and expansin-like protein on the cell surface were constructed and examined for direct ethanol fermentation performance. Results The cellulase and expansin-like protein co-expressing strain showed 246 mU/g-wet cell of phosphoric acid swollen cellulose (PASC) degradation activity, which corresponded to 2.9-fold higher activity than that of a cellulase-expressing strain. This result clearly demonstrated that yeast cell-surface expressed cellulase and expansin-like protein act synergistically to breakdown cellulose. In fermentation experiments examining direct ethanol production from PASC, the cellulase and expansin-like protein co-expressing strain produced 3.4 g/L ethanol after 96 h of fermentation, a concentration that was 1.4-fold higher than that achieved by the cellulase-expressing strain (2.5 g/L). Conclusions The PASC degradation and fermentation ability of an engineered yeast strain was markedly improved by co-expressing cellulase and expansin-like protein on the cell surface. To our knowledge, this is the first report to demonstrate the synergetic effect of co-expressing cellulase and expansin-like protein on a yeast cell surface, which may be a promising strategy for constructing direct ethanol fermenting yeast from cellulose. PMID:23835302

Insights from the genome of a high alkaline cellulase producing Aspergillus fumigatus strain obtained from Peruvian Amazon rainforest.

PubMed

Paul, Sujay; Zhang, Angel; Ludeña, Yvette; Villena, Gretty K; Yu, Fengan; Sherman, David H; Gutiérrez-Correa, Marcel

2017-06-10

Here, we report the complete genome sequence of a high alkaline cellulase producing Aspergillus fumigatus strain LMB-35Aa isolated from soil of Peruvian Amazon rainforest. The genome is ∼27.5mb in size, comprises of 228 scaffolds with an average GC content of 50%, and is predicted to contain a total of 8660 protein-coding genes. Of which, 6156 are with known function; it codes for 607 putative CAZymes families potentially involved in carbohydrate metabolism. Several important cellulose degrading genes, such as endoglucanase A, endoglucanase B, endoglucanase D and beta-glucosidase, are also identified. The genome of A. fumigatus strain LMB-35Aa represents the first whole sequenced genome of non-clinical, high cellulase producing A. fumigatus strain isolated from forest soil. Copyright © 2017 Elsevier B.V. All rights reserved.

The identification of and relief from Fe3+ inhibition for both cellulose and cellulase in cellulose saccharification catalyzed by cellulases from Penicillium decumbens.

PubMed

Wang, Mingyu; Mu, Ziming; Wang, Junli; Hou, Shaoli; Han, Lijuan; Dong, Yanmei; Xiao, Lin; Xia, Ruirui; Fang, Xu

2013-04-01

Lignocellulosic biomass is an underutilized, renewable resource that can be converted to biofuels. The key step in this conversion is cellulose saccharification catalyzed by cellulase. In this work, the effect of metal ions on cellulose hydrolysis by cellulases from Penicillium decumbens was reported for the first time. Fe(3+) and Cu(2+) were shown to be inhibitory. Further studies on Fe(3+) inhibition showed the inhibition takes place on both enzyme and substrate levels. Fe(3+) treatment damages cellulases' capability to degrade cellulose and inhibits all major cellulase activities. Fe(3+) treatment also reduces the digestibility of cellulose, due to its oxidation. Treatment of Fe(3+)-treated cellulose with DTT and supplementation of EDTA to saccharification systems partially relieved Fe(3+) inhibition. It was concluded that Fe(3+) inhibition in cellulose degradation is a complicated process in which multiple inhibition events occur, and that relief from Fe(3+) inhibition can be achieved by the supplementation of reducing or chelating agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

Conversion of rice straw to bio-based chemicals: an integrated process using Lactobacillus brevis.

PubMed

Kim, Jae-Han; Block, David E; Shoemaker, Sharon P; Mills, David A

2010-05-01

Commercialization of lignocellulosic biomass as a feedstock for bio-based chemical production is problematic due to the high processing costs of pretreatment and saccharifying enzymes combined with low product yields. Such low product yield can be attributed, in large part, to the incomplete utilization of the various carbohydrate sugars found in the lignocellulosic biomass. In this study, we demonstrate that Lactobacillus brevis is able to simultaneously metabolize all fermentable carbohydrates in acid pre-processed rice straw hydrolysate, thereby allowing complete utilization of all released sugars. Inhibitors present in rice straw hydrolysate did not affect lactic acid production. Moreover, the activity of exogenously added cellulases was not reduced in the presence of growing cultures of L. brevis. These factors enabled the use of L. brevis in a process termed simultaneous saccharification and mixed sugar fermentation (SSMSF). In SSMSF with L. brevis, sugars present in rice straw hydrolysate were completely utilized while the cellulase maintained its maximum activity due to the lack of feedback inhibition from glucose and/or cellobiose. By comparison to a sequential hydrolysis and fermentation process, SSMSF reduced operation time and the amount of cellulase enzyme necessary to produce the same amount of lactic acid.

Screening method for inhibitors against formosan subterranean termite beta-glucosidases in vivo.

PubMed

Zhu, Betty C R; Henderson, Gregg; Laine, Roger A

2005-02-01

Cellulose, a main structural constituent of plants, is the major nutritional component for wood-feeding termites. Enzymatic hydrolysis of cellulose to glucose occurs by the action of cellulases, a mixture of the three major classes of enzymes including endo-1,4-beta-glucanases, exo-1,4-beta-glucanases, and beta-glucosidase. Lower termites, such as the Formosan subterranean termite, Coptotermes formosanus Shiraki, require cellulolytic protozoa to efficiently digest cellulose for survival. Inhibitors developed against any of these cellulase system enzymes would be a potential termite treatment avenue. Our effort was to develop a screening system to determine whether termites could be controlled by administration of cellulase system inhibitors. Some reported compounds such as gluconolactone, conduritol B epoxide, and 1-deoxynojirimycin are potential beta-glucosidase inhibitors, but they have only been tested in vitro. We describe an in vivo method to test the inhibitory ability of the designated chemicals to act on beta-1,4-glucosidases, one member of the cellulase system that is the key step that releases glucose for use as an energy and carbon source for termites. Inhibition in releasing glucose from cellooligosaccharides might be sufficient to starve termites. Fluorescein di-beta-D-glucopyranoside was used as the artificial enzyme substrate and the fluorescent intensity of the reaction product (fluorescein) quantified with an automated fluorescence plate reader. Several known in vitro beta-1,4-glucosidase inhibitors were tested in vivo, and their inhibitory potential was determined. Endogenous and protozoan cellulase activities are both assumed to play a role.

Comprehensive Enzymatic Analysis of the Cellulolytic System in Digestive Fluid of the Sea Hare Aplysia kurodai. Efficient Glucose Release from Sea Lettuce by Synergistic Action of 45 kDa Endoglucanase and 210 kDa ß-Glucosidase

PubMed Central

Tsuji, Akihiko; Tominaga, Keiko; Nishiyama, Nami; Yuasa, Keizo

2013-01-01

Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 ß-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU–ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed. PMID:23762366

Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.

PubMed

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

2011-09-02

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

Development of a low-cost cellulase production process using Trichoderma reesei for Brazilian biorefineries.

PubMed

Ellilä, Simo; Fonseca, Lucas; Uchima, Cristiane; Cota, Junio; Goldman, Gustavo Henrique; Saloheimo, Markku; Sacon, Vera; Siika-Aho, Matti

2017-01-01

During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery. Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a Trichoderma reesei strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous β-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from Aspegillus niger into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw. In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using T. reesei . With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases.

Engineering towards a complete heterologous cellulase secretome in Yarrowia lipolytica reveals its potential for consolidated bioprocessing

DOE PAGES

Wei, Hui; Wang, Wei; Alahuhta, Markus; ...

2014-10-16

Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichodermamore » reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient degradation of cellulosic substrates. In Conclusion: Taken together, this work demonstrates the first case of successful expression of a chimeric CBHI with essentially full native activity in Y. lipolytica, and supports the notion that Y. lipolytica strains can be genetically engineered, ultimately by heterologous expression of fungal cellulases and other enzymes, to directly convert lignocellulosic substrates to biofuels.« less

Biochemical Characterization of Extracellular Cellulase from Tuber maculatum Mycelium Produced Under Submerged Fermentation.

PubMed

Bedade, Dattatray K; Singhal, Rekha S; Turunen, Ossi; Deska, Jan; Shamekh, Salem

2017-02-01

Interaction of truffle mycelium with the host plant involves the excretion of extracellular enzymes. The ability of Tuber maculatum mycelium to produce an extracellular cellulase during submerged fermentation was demonstrated for the first time. T. maculatum mycelia were isolated and tested for extracellular cellulase production at variable pH on solid agar medium, and the highest activity was observed at pH 7.0. Furthermore, T. maculatum was subjected to submerged fermentation in basal salt medium for cellulase production. Under optimized conditions using sodium carboxymethyl cellulose (0.5 % w/v) as carbon source and an initial pH of 7.0, the enzyme production yielded 1.70 U/mL of cellulase in the cell-free supernatant after 7 days of incubation time. The optimum of the obtained cellulase's activity was at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 99 % of its maximal activity over an incubation time of 100 min. The cellulase activity was inhibited by Fe 2+ and slightly activated by Mn 2+ and Cu 2+ at 1 mM concentration. The results indicated that truffle mycelium is utilizing cellulosic energy source in the root system, and the optimal conditions are those existing in the acidic Finnish soil.

Cellular automata modeling depicts degradation of cellulosic material by a cellulase system with single-molecule resolution.

PubMed

Eibinger, Manuel; Zahel, Thomas; Ganner, Thomas; Plank, Harald; Nidetzky, Bernd

2016-01-01

Enzymatic hydrolysis of cellulose involves the spatiotemporally correlated action of distinct polysaccharide chain cleaving activities confined to the surface of an insoluble substrate. Because cellulases differ in preference for attacking crystalline compared to amorphous cellulose, the spatial distribution of structural order across the cellulose surface imposes additional constraints on the dynamic interplay between the enzymes. Reconstruction of total system behavior from single-molecule activity parameters is a longstanding key goal in the field. We have developed a stochastic, cellular automata-based modeling approach to describe degradation of cellulosic material by a cellulase system at single-molecule resolution. Substrate morphology was modeled to represent the amorphous and crystalline phases as well as the different spatial orientations of the polysaccharide chains. The enzyme system model consisted of an internally chain-cleaving endoglucanase (EG) as well as two processively acting, reducing and non-reducing chain end-cleaving cellobiohydrolases (CBHs). Substrate preference (amorphous: EG, CBH II; crystalline: CBH I) and characteristic frequencies for chain cleavage, processive movement, and dissociation were assigned from biochemical data. Once adsorbed, enzymes were allowed to reach surface-exposed substrate sites through "random-walk" lateral diffusion or processive motion. Simulations revealed that slow dissociation of processive enzymes at obstacles obstructing further movement resulted in local jamming of the cellulases, with consequent delay in the degradation of the surface area affected. Exploiting validation against evidence from atomic force microscopy imaging as a unique opportunity opened up by the modeling approach, we show that spatiotemporal characteristics of cellulose surface degradation by the system of synergizing cellulases were reproduced quantitatively at the nanometer resolution of the experimental data. This in turn gave useful prediction of the soluble sugar release rate. Salient dynamic features of cellulose surface degradation by different cellulases acting in synergy were reproduced in simulations in good agreement with evidence from high-resolution visualization experiments. Due to the single-molecule resolution of the modeling approach, the utility of the presented model lies not only in predicting system behavior but also in elucidating inherently complex (e.g., stochastic) phenomena involved in enzymatic cellulose degradation. Thus, it creates synergy with experiment to advance the mechanistic understanding for improved application.

Ultrasonic hyperactivation of cellulase immobilized on magnetic nanoparticles.

PubMed

Ladole, Mayur Ramrao; Mevada, Jayesh Sevantilal; Pandit, Aniruddha Bhalchandra

2017-09-01

In the present work, effect of low power, low frequency ultrasound on cellulase immobilized magnetic nanoparticles (cellulase@MNPs) was studied. To gain maximum activity recovery in cellulase@MNPs various parameters viz. ratio of MNPs:cellulase, concentration of glutaraldehyde and cross-linking time were optimized. The influence of ultrasonic power on cellulase@MNPs was studied. Under ultrasonic conditions at 24kHz, 6W power, and 6min of incubation time there was almost 3.6 fold increased in the catalytic activity of immobilized cellulase over the control. Results also indicated that there was improvement in pH and temperature stability of cellulase@MNPs. Furthermore, thermal deactivation energy required was more in cellulase@MNPs than that of the free cellulase. Secondary structural analysis revealed that there were conformational changes in free cellulase and cellulase@MNPs before and after sonication which might be responsible for enhanced activity after ultrasonication. Finally, the influence of ultrasound and cellulase@MNPs for biomass hydrolysis was studied. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolasemore » activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific glycosyl hydrolase activities. It remains unclear whether loss of endocellulase activity to lignin binding is problematic for biomass conversion.« less

Bioprospecting thermophiles for cellulase production: a review

PubMed Central

Acharya, Somen; Chaudhary, Anita

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 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. PMID:24031898

Bioprospecting thermophiles for cellulase production: a review.

PubMed

Acharya, Somen; Chaudhary, Anita

2012-07-01

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

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

PubMed Central

Huang, Po-Jung; Chang, Ken-Lin; Chen, Shui-Tein

2015-01-01

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. PMID:25874210

Chimeric enzymes with improved cellulase activities

DOEpatents

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.

Engineering Cellulase Enzymes for Bioenergy

NASA Astrophysics Data System (ADS)

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 methods. Protein engineering targets to improve cellulases include reducing enzyme inhibition, improving inter-enzyme synergy, and increasing enzyme thermotolerance. Ameliorating enzyme inhibition could improve catalytic activity and thus the speed of conversion from biomass to fermentable sugars. Improved enzyme synergy could reduce the enzyme loading required to achieve equivalent biomass conversion. Finally, thermostable enzymes could enable more biomass to be processed at a time, due to high temperatures decreasing the viscosity of biomass slurries. A high-temperature enzyme saccharification reaction could also decrease the risk of contamination in the resulting concentrated sugar solution. Throughout my PhD, I have explored research projects broadly across all of these topics, with the most success in addressing the issue of enzyme inhibition. Cellulase enzyme Cel7A is the most abundant cellulase employed by natural systems for cellulose hydrolysis. Cellobiohydrolase enzymes like Cel7A break down cellulose into cellobiose (two glucose molecules). Unfortunately, upon cleavage, this product molecule interferes with continued hydrolysis activity of Cel7A; the strong binding of cellobiose in the active site can obstruct the enzyme from processing down the cellulase chain. This phenomenon, known as product inhibition, is a bottleneck to efficient biomass breakdown. Using insights from computational protein modeling studies, I experimentally generated and tested mutant Cel7A enzymes for improved tolerance to cellobiose. Indeed, this strategy yielded Cel7A enzymes exhibiting reduced product inhibition, including some mutants completely impervious to cellobiose. The improvements in tolerance to cellobiose, however, resulted in an overall reduction of enzyme activity for the mutants tested. Nevertheless, my findings substantiated computational reports with experimental evidence and pinpointed an amino acid residue in the Cel7A product binding site that is of interest for follow-up mutational studies. My goal was to improve the effective catalytic activity of cellulase enzymes in industrially-relevant conditions (such as in the presence of high concentrations of cellobiose or at elevated temperatures). The insights gained from my work on enzyme inhibition may inform future efforts to address this important issue. More efficient enzymes should reduce the amount of these proteins needed to break down cellulose to glucose. This, in turn, should decrease the price of the resulting biofuel making it more cost-competitive with fossil fuels and thus encouraging adoption of renewable transportation fuels that reduce our greenhouse gas emissions.

On-site cellulase production and efficient saccharification of corn stover employing cbh2 overexpressing Trichoderma reesei with novel induction system.

PubMed

Li, Yonghao; Zhang, Xiaoyue; Xiong, Liang; Mehmood, Muhammad Aamer; Zhao, Xinqing; Bai, Fengwu

2017-08-01

Although on-site cellulase production offers cost-effective saccharification of lignocellulosic biomass, low enzyme titer is still a barrier for achieving robustness. In the present study, a strain of T. reesei was developed for enhanced production of cellulase via overexpression of Cellobiohydrolase II. Furthermore, optimum enzyme production was achieved using a novel inducer mixture containing synthesized glucose-sophorose (MGD) and alkali pre-treated corn stover (APCS). Within 60h, a remarkably higher cellulase productivity and activity were achieved in the fed-batch fermentation using the optimized ratio of MGD and APCS in the inducer mixture, compared to those reported using cellulosic biomass as the sole inducer. After the enzyme production, APCS was added directly into the fermentation broth at 20% solid loading, which produced 122.5g/L glucose and 40.21g/L xylose, leading to the highest yield reported so far. The improved enzyme titers during on-site cellulase production would benefit cost-competitive saccharification of lignocellulosic biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering Cellulases for Biorefinery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

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 reconstitutionmore » 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.« less

Thermostable Cellulases: Why & How?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

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 reconstitutionmore » 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.« less

Cellulase and cell differentiation in Acer pseudoplatanus.

PubMed

Sheldrake, A R

1970-06-01

Homogenates of differentiating xylem and phloem tissue have higher cellulase activities than cambial samples; the highest activity is always found in phloem. Callus tissue, in which no vascular differentiation occurs, contains only low cellulase activity. The results suggest that cellulase is involved in vascular differentiation. Different pH optima of cellulase activity were found: in cambium, xylem and phloem tissue, cellulase activity with an optimum at about pH 5.9 is predominantly membrane-bound; it is sedimentable at 100,000 g and releasable by Triton X-100. The same may be true of activity with an optimum at pH 5.3. Phloem tissue also contains a soluble, cytoplasmic cellulase of high activity at pH 7.1, and xylem tissue contains cytoplasmic cellulase with an optimum at pH 6.5. Low cellulase activity with a pH optimum similar to that of xylem homogenates was found in xylem sap. Cellulase activity in abscission zones increases greatly just before leaf abscission. Abscission zone cellulase has two pH optima, et 5.3 and 5.9; both activities are increased by Triton treatment of homogenates. The possible existence of several different cellulases forming part of a cellulase complex, and the rôle of the enzymes in hydrolysing wall material during cell differentiation are discussed.

Proteomic and functional analysis of the cellulase system expressed by Postia placenta during brown rot of solid wood

Treesearch

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

2011-01-01

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...

Biomining active cellulases from a mining bioremediation system.

PubMed

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. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Thermostable Cellulases: Why & How?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-03-24

These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » 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.« less

Highly Efficient Thermostable DSM Cellulases: Why & How?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-04-26

These are the slides from this presentation. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase componentmore » 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.« less

Process for converting cellulosic materials into fuels and chemicals

DOEpatents

Scott, Charles D.; Faison, Brendlyn D.; Davison, Brian H.; Woodward, Jonathan

1994-01-01

A process for converting cellulosic materials, such as waste paper, into fuels and chemicals utilizing enzymatic hydrolysis of the major constituent of paper, cellulose. A waste paper slurry is contacted by cellulase in an agitated hydrolyzer. The cellulase is produced from a continuous, columnar, fluidized-bed bioreactor utilizing immobilized microorganisms. An attritor and a cellobiase reactor are coupled to the agitated hydrolyzer to improve reaction efficiency. The cellulase is recycled by an adsorption process. The resulting crude sugars are converted to dilute product in a fluidized-bed bioreactor utilizing microorganisms. The dilute product is concentrated and purified by utilizing distillation and/or a biparticle fluidized-bed bioreactor system.

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

DOEpatents

Aehle, Wolfgang; Bott, Richard R; Bower, Benjamin; Caspi, Jonathan; Estell, David A; Goedegebuur, Frits; Hommes, Ronaldus W.J.; Kaper, Thijs; Kelemen, Bradley; Kralj, Slavko; Van Lieshout, Johan; Nikolaev, Igor; Van Stigt Thans, Sander; Wallace, Louise; Vogtentanz, Gudrun; Sandgren, Mats

2014-03-25

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

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

DOEpatents

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

2016-12-20

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

Enhanced cellulase production by Penicillium oxalicum for bio-ethanol application.

PubMed

Saini, Reetu; Saini, Jitendra Kumar; Adsul, Mukund; Patel, Anil Kumar; Mathur, Anshu; Tuli, Deepak; Singhania, Reeta Rani

2015-01-01

Present study was focused on cellulase production from an indigenously isolated filamentous fungal strain, identified as Penicillium oxalicum. Initially, cellulase production under submerged fermentation in shake flasks resulted in cellulase activity of 0.7 FPU/mL. Optimization of process parameters enhanced cellulase production by 1.7-fold and resulted in maximum cellulase activity of 1.2 FPU/mL in 8 days. Cellulase production was successfully scaled-up to 7 L fermenter under controlled conditions and incubation time was reduced from 8 days to 4 days for achieving similar cellulase titer. Optimum pH and temperature for activity of the crude enzyme were pH 5 and 50 °C, respectively. At 50 °C the produced cellulase retained approximately 50% and 26% of its activity at 48 h and 72 h, respectively. Hydrolytic efficiency of P. oxalicum was comparable to commercial cellulase preparations which indicate its great potential for application in the lignocellulose hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Cellulase enzyme preparation derived from....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase enzyme.... reesei). The enzyme, cellulase, catalyzes the endohydrolysis of 1,4-beta-glycosidic linkages in cellulose...

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

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Cellulase enzyme preparation derived from....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase enzyme.... reesei). The enzyme, cellulase, catalyzes the endohydrolysis of 1,4-beta-glycosidic linkages in cellulose...

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

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Cellulase enzyme preparation derived from....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase enzyme.... reesei). The enzyme, cellulase, catalyzes the endohydrolysis of 1,4-beta-glycosidic linkages in cellulose...

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

Code of Federal Regulations, 2010 CFR

2010-04-01

... 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 enzyme.... reesei). The enzyme, cellulase, catalyzes the endohydrolysis of 1,4-beta-glycosidic linkages in cellulose...

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

PubMed

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.

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

PubMed

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Improved cellulase production by Botryosphaeria rhodina from OPEFB at low level moisture condition through statistical optimization.

PubMed

Bahrin, E K; Ibrahim, M F; Abd Razak, M N; Abd-Aziz, S; Shah, U K Md; Alitheen, N; Salleh, M Md

2012-01-01

The response surface method was applied in this study to improve cellulase production from oil palm empty fruit bunch (OPEFB) by Botryosphaeria rhodina. An experimental design based on a two-level factorial was employed to screen the significant environmental factors for cellulase production. The locally isolated fungus Botryosphaeria rhodina was cultivated on OPEFB under solid-state fermentation (SSF). From the analysis of variance (ANOVA), the initial moisture content, amount of substrate, and initial pH of nutrient supplied in the SSF system significantly influenced cellulase production. Then the optimization of the variables was done using the response surface method according to central composite design (CCD). Botryosphaeria rhodina exhibited its best performance with a high predicted value of FPase enzyme production (17.95 U/g) when the initial moisture content was at 24.32%, initial pH of nutrient was 5.96, and 3.98 g of substrate was present. The statistical optimization from actual experiment resulted in a significant increment of FPase production from 3.26 to 17.91 U/g (5.49-fold). High cellulase production at low moisture content is a very rare condition for fungi cultured in solid-state fermentation.

Bioprospecting of functional cellulases from metagenome for second generation biofuel production: a review.

PubMed

Tiwari, Rameshwar; Nain, Lata; Labrou, Nikolaos E; Shukla, Pratyoosh

2018-03-01

Second generation biofuel production has been appeared as a sustainable and alternative energy option. The ultimate aim is the development of an industrially feasible and economic conversion process of lignocellulosic biomass into biofuel molecules. Since, cellulose is the most abundant biopolymer and also represented as the photosynthetically fixed form of carbon, the efficient hydrolysis of cellulose is the most important step towards the development of a sustainable biofuel production process. The enzymatic hydrolysis of cellulose by suites of hydrolytic enzymes underlines the importance of cellulase enzyme system in whole hydrolysis process. However, the selection of the suitable cellulolytic enzymes with enhanced activities remains a challenge for the biorefinery industry to obtain efficient enzymatic hydrolysis of biomass. The present review focuses on deciphering the novel and effective cellulases from different environmental niches by unculturable metagenomic approaches. Furthermore, a comprehensive functional aspect of cellulases is also presented and evaluated by assessing the structural and catalytic properties as well as sequence identities and expression patterns. This review summarizes the recent development in metagenomics based approaches for identifying and exploring novel cellulases which open new avenues for their successful application in biorefineries.

Heterologous expression of Talaromyces emersonii cellobiohydrolase Cel7A in Trichoderma reesei increases the efficiency of corncob residues saccharification.

PubMed

Sun, Ningning; Qian, Yuanchao; Wang, Weiwei; Zhong, Yaohua; Dai, Meixue

2018-07-01

Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii. Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65 °C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction. The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.

In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

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

An extracellular enzyme synthesizes narrow-sized silver nanoparticles in both water and methanol

NASA Astrophysics Data System (ADS)

Rai, Tripti; Panda, Debashis

2015-03-01

Cellulase reduces silver ions in both aqueous and methanolic media yielding stable narrow-sized silver nanoparticles (Ag-NP) at room temperature. The synthesized nanoparticles have been characterized by various spectroscopic, microscopic methods. The redox potentials of tyrosine residues and protein backbone play an instrumental role to reduce the metal ions. The average size of nanoparticles formed in aqueous medium is of 5.04 ± 3.50 nm. Post-synthesis of Ag-NP secondary structure of enzyme is completely lost whereas upon incubation with chemically synthesized Ag-NP a significant gain in secondary structure is observed. Cellulase as a capping ligand stabilizes the silver nanoparticles even in methanol.

Engineering Aspergillus oryzae A-4 through the Chromosomal Insertion of Foreign Cellulase Expression Cassette to Improve Conversion of Cellulosic Biomass into Lipids

PubMed Central

Shen, Qi; Ma, Junwei; Fu, Jianrong; Zhao, Yuhua

2014-01-01

A genetic modification scheme was designed for Aspergillus oryzae A-4, a natural cellulosic lipids producer, to enhance its lipid production from biomass by putting the spotlight on improving cellulase secretion. Four cellulase genes were separately expressed in A-4 under the control of hlyA promoter, with the help of the successful development of a chromosomal genetic manipulation system. Comparison of cellulase activities of PCR-positive transformants showed that these transformants integrated with celA gene and with celC gene had significantly (p<0.05) higher average FPAase activities than those strains integrated with celB gene and with celD gene. Through the assessment of cellulosic lipids accumulating abilities, celA transformant A2-2 and celC transformant D1-B1 were isolated as promising candidates, which could yield 101%–133% and 35.22%–59.57% higher amount of lipids than the reference strain A-4 (WT) under submerged (SmF) conditions and solid-state (SSF) conditions, respectively. Variability in metabolism associated to the introduction of cellulase gene in A2-2 and D1-B1 was subsequently investigated. It was noted that cellulase expression repressed biomass formation but enhanced lipid accumulation; whereas the inhibitory effect on cell growth would be shielded during cellulosic lipids production owing to the essential role of cellulase in substrate utilization. Different metabolic profiles also existed between A2-2 and D1-B1, which could be attributed to not only different transgene but also biological impacts of different integration. Overall, both simultaneous saccharification and lipid accumulation were enhanced in A2-2 and D1-B1, resulting in efficient conversion of cellulose into lipids. A regulation of cellulase secretion in natural cellulosic lipids producers could be a possible strategy to enhance its lipid production from lignocellulosic biomass. PMID:25251435

Engineering Aspergillus oryzae A-4 through the chromosomal insertion of foreign cellulase expression cassette to improve conversion of cellulosic biomass into lipids.

PubMed

Lin, Hui; Wang, Qun; Shen, Qi; Ma, Junwei; Fu, Jianrong; Zhao, Yuhua

2014-01-01

A genetic modification scheme was designed for Aspergillus oryzae A-4, a natural cellulosic lipids producer, to enhance its lipid production from biomass by putting the spotlight on improving cellulase secretion. Four cellulase genes were separately expressed in A-4 under the control of hlyA promoter, with the help of the successful development of a chromosomal genetic manipulation system. Comparison of cellulase activities of PCR-positive transformants showed that these transformants integrated with celA gene and with celC gene had significantly (p<0.05) higher average FPAase activities than those strains integrated with celB gene and with celD gene. Through the assessment of cellulosic lipids accumulating abilities, celA transformant A2-2 and celC transformant D1-B1 were isolated as promising candidates, which could yield 101%-133% and 35.22%-59.57% higher amount of lipids than the reference strain A-4 (WT) under submerged (SmF) conditions and solid-state (SSF) conditions, respectively. Variability in metabolism associated to the introduction of cellulase gene in A2-2 and D1-B1 was subsequently investigated. It was noted that cellulase expression repressed biomass formation but enhanced lipid accumulation; whereas the inhibitory effect on cell growth would be shielded during cellulosic lipids production owing to the essential role of cellulase in substrate utilization. Different metabolic profiles also existed between A2-2 and D1-B1, which could be attributed to not only different transgene but also biological impacts of different integration. Overall, both simultaneous saccharification and lipid accumulation were enhanced in A2-2 and D1-B1, resulting in efficient conversion of cellulose into lipids. A regulation of cellulase secretion in natural cellulosic lipids producers could be a possible strategy to enhance its lipid production from lignocellulosic biomass.

Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

DOEpatents

Cascao-Pereira, Luis; Kaper, Thijs; Kelemen, Bradley R.; Liu, Amy D.

2017-07-04

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

DOEpatents

Cascao-Pereira, Luis G; Kaper, Thijs; Kelemen, Bradley R; Liu, Amy D

2015-04-07

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

DOEpatents

Cascao-Pereira, Luis G.; Kaper, Thijs; Kelemen, Bradley R; Liu, Amy D.

2012-08-07

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

Integrated cellulosic enzymes hydrolysis and fermentative advanced yeast bioconversion solution ready for biomass biorefineries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-05-04

These are slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme,more » 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.« less

Fully Integrated Lignocellulosic Biorefinery with Onsite Production of Enzymes and Yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-06-14

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 reconstitutionmore » 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.« less

Cellulase, Coca-Cola®, pancreatin and ursodeoxycholic acid in the dissolution of gastric bezoars: why not all together?

PubMed

Cerezo Ruiz, Antonio; Domínguez Jiménez, José Luis; Uceda-Vaño, Antonio

2018-06-14

Two cases of a chemical dissolution of gastric phytobezoars are presented. The novel approach of that management is the pharmacological mixture than completely made disappear the bezoars in patients fated to surgery removal.

Increase in stability of cellulase immobilized on functionalized magnetic nanospheres

NASA Astrophysics Data System (ADS)

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

2015-02-01

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

Process for converting cellulosic materials into fuels and chemicals

DOEpatents

Scott, C.D.; Faison, B.D.; Davison, B.H.; Woodward, J.

1994-09-20

A process is described for converting cellulosic materials, such as waste paper, into fuels and chemicals utilizing enzymatic hydrolysis of the major constituent of paper, cellulose. A waste paper slurry is contacted by cellulase in an agitated hydrolyzer. The cellulase is produced from a continuous, columnar, fluidized-bed bioreactor utilizing immobilized microorganisms. An attrition mill and a cellobiase reactor are coupled to the agitated hydrolyzer to improve reaction efficiency. The cellulase is recycled by an adsorption process. The resulting crude sugars are converted to dilute product in a fluidized-bed bioreactor utilizing microorganisms. The dilute product is concentrated and purified by utilizing distillation and/or a biparticle fluidized-bed bioreactor system. 1 fig.

Ionic liquid-tolerant cellulase enzymes

DOEpatents

Gladden, John; Park, Joshua; Singer, Steven; Simmons, Blake; Sale, Ken

2017-10-31

The present invention provides ionic liquid-tolerant cellulases and method of producing and using such cellulases. The cellulases of the invention are useful in saccharification reactions using ionic liquid treated biomass.

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

PubMed

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 supplemental cellulase enzymes have been shown to improve ration digestibility and animal performance in previous trials, no advantages were observed in the current trial. Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Vertical Integration of Biomass Saccharification of Enzymes for Sustainable Cellulosic Biofuel Production in a Biorefinery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-05-09

These are a set of slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » 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.« less

Cellulases and coding sequences

DOEpatents

Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

2001-02-20

The present invention provides three fungal cellulases, their coding sequences, recombinant DNA molecules comprising the cellulase coding sequences, recombinant host cells and methods for producing same. The present cellulases are from Orpinomyces PC-2.

Cellulases and coding sequences

DOEpatents

Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

2001-01-01

The present invention provides three fungal cellulases, their coding sequences, recombinant DNA molecules comprising the cellulase coding sequences, recombinant host cells and methods for producing same. The present cellulases are from Orpinomyces PC-2.

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

PubMed

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

2016-02-01

A cellulase producing strain was newly isolated from soil samples and identified as Paenibacillus sp. LLZ1. A novel aqueous-dimethyl sulfoxide (DMSO)/1-ethyl-3-methylimidazolium diethyl phosphate ([Emin]DEP)-cellulase system was designed and optimized. In the pretreatment, DMSO was found to be a low-cost substitute of up to 70% ionic liquid to enhance the cellulose dissolution. In the enzymatic saccharification, the optimum pH and temperature of the Paenibacillus sp. LLZ1 cellulase were identified as 6.0 and 40°C, respectively. Under the optimized reaction condition, the conversion of microcrystalline cellulose and bagasse cellulose increased by 39.3% and 37.6%, compared with unpretreated cellulose. Compared to current methods of saccharification, this new approach has several advantages including lower operating temperature, milder pH, and less usage of ionic liquid, indicating a marked progress in environmental friendly hydrolysis of biomass-based materials. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification and characterization of a cellulolytic multienzyme complex produced by Neocallimastix patriciarum J11.

PubMed

Wang, Hui-Chang; Chen, Yo-Chia; Hseu, Ruey-Shyang

2014-08-22

Understanding the roles of the components of the multienzyme complex of the anaerobial cellulase system, acting on complex substrates, is crucial to the development of efficient cellulase systems for industrial applications such as converting lignocellulose to sugars for bioethanol production. In this study, we purified the multienzyme complex of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The multienzyme complex is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight of these constituents have demonstrated β-glucanase activity on zymogram analysis. The multienzyme complex contained scaffoldings that respond to the gathering of the cellulolytic components. The levels and subunit ratio of the multienzyme complex from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the complexes were consistent. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through liquid chromatography/mass spectrometry, revealing that at least three types of cellulase, including one endoglucanase and two exoglucanases, could be found in the multienzyme complex of N. patriciarum J11. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the multienzyme complex produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase. Copyright © 2014 Elsevier Inc. All rights reserved.

[Induction and regulation of cellulase expression in filamentous fungi: a review].

PubMed

Zhang, Fei; Bai, Fengwu; Zhao, Xinqing

2016-11-25

Production of bioenergy and bio-based chemicals by using fermentable sugars released from low-cost renewable lignocellulosic biomass has received great attention. Efficient cellulolytic enzymes are crucial for lignocellulose bioconversion, but high cellulase production cost is limiting the bioconversion efficiency of cellulosic biomass and industrial applications of lignocellulose biorefinery. Studies on induction and regulation of cellulase in filamentous fungi will help to further develop superior fungal strains for efficient cellulase production and reduce cellulase production cost. With the advances in high-throughput sequencing and gene manipulation technology using fungal strains, an in-depth understanding of cellulase induction and regulation mechanisms of enzyme expression has been achieved. We reviewed recent progresses in the induction and regulation of cellulase expression in several model filamentous fungi, emphasizing sugar transporters, transcription factors and chromatin remodeling. Future prospects in application of artificial zinc finger proteins for cellulase induction and regulation in filamentous fungi were discussed.

Poly(acrylic acid) nanogel as a substrate for cellulase immobilization for hydrolysis of cellulose.

PubMed

Ahmed, Ibrahim Nasser; Chang, Ray; Tsai, Wei-Bor

2017-04-01

Cellulase was adsorbed onto poly(acrylic acid), PAA, nanogel, that was fabricated via inverse-phase microemulsion polymerization. The PAA nanogel was around 150nm in diameter and enriched with carboxyl groups. The surface charge of PAA nanogel depended on the pHs of the environment and affected the adsorption of cellulase. The temperature stability of the immobilized cellulase was greatly enhanced in comparison to the free enzyme, especially at high temperature. At 80°C, the immobilized cellulase remained ∼75% of hydrolytic activity, in comparison to ∼55% for the free cellulase. Furthermore, the immobilized cellulase was more active than the free enzyme in acidic buffers. The immobilized cellulase could be recovered via centrifugation and can be used repeatedly, although the recovery ratio needs further improvement. In conclusion, PAA nanogel has the potential in the application of enzyme immobilization for biochemical processes. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Exo-endo cellulase fusion protein

DOEpatents

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

2012-01-17

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

Strain improvement of Trichoderma viride for increased cellulase production by irradiation of electron and (12)C(6+)-ion beams.

PubMed

Li, Zhaozhou; Chen, Xiujin; Li, Zhili; Li, Daomin; Wang, Yao; Gao, Hongli; Cao, Li; Hou, Yuze; Li, Songbiao; Liang, Jianping

2016-06-01

To improve cellulase production and activity, Trichoderma viride GSICC 62010 was subjected to mutation involving irradiation with an electron beam and subsequently with a (12)C(6+)-ion beam. Mutant CIT 626 was the most promising cellulase producer after preliminary and secondary screening. Soluble protein production and cellulase activities were increased mutifold. The optimum temperature, pH and culture time for the maximum cellulase production of the selected mutant were 35 °C, pH 5 and 6 days. The highest cellulase production was obtained using wheat bran. The prepared cellulases from T. viride CIT 626 had twice the hydrolytic performance with sawdust (83 %) than that from the parent strain (42.5 %). Furthermore, molecular studies demonstrated that there were some key mutation sites suggesting that some amino acid changes in the protein caused by base mutations had led to the enhanced cellulase production and activity. Mutagenesis with electron and (12)C(6+)-ion beams could be developed as an effective tool for improvement of cellulase producing strains.

Kinetic studies on batch cultivation of Trichoderma reesei and application to enhance cellulase production by fed-batch fermentation.

PubMed

Ma, Lijuan; Li, Chen; Yang, Zhenhua; Jia, Wendi; Zhang, Dongyuan; Chen, Shulin

2013-07-20

Reducing the production cost of cellulase as the key enzyme for cellulose hydrolysis to fermentable sugars remains a major challenge for biofuel production. Because of the complexity of cellulase production, kinetic modeling and mass balance calculation can be used as effective tools for process design and optimization. In this study, kinetic models for cell growth, substrate consumption and cellulase production in batch fermentation were developed, and then applied in fed-batch fermentation to enhance cellulase production. Inhibition effect of substrate was considered and a modified Luedeking-Piret model was developed for cellulase production and substrate consumption according to the growth characteristics of Trichoderma reesei. The model predictions fit well with the experimental data. Simulation results showed that higher initial substrate concentration led to decrease of cellulase production rate. Mass balance and kinetic simulation results were applied to determine the feeding strategy. Cellulase production and its corresponding productivity increased by 82.13% after employing the proper feeding strategy in fed-batch fermentation. This method combining mathematics and chemometrics by kinetic modeling and mass balance can not only improve cellulase fermentation process, but also help to better understand the cellulase fermentation process. The model development can also provide insight to other similar fermentation processes. Copyright © 2013 Elsevier B.V. All rights reserved.

Feasibility test of utilizing Saccharophagus degradans 2-40(T) as the source of crude enzyme for the saccharification of lignocellulose.

PubMed

Jung, Young Hoon; Kim, Hyun Kyung; Song, Du-Sup; Choi, In-Geol; Yang, Taek Ho; Lee, Hee Jong; Seung, Doyoung; Kim, Kyoung Heon

2014-04-01

In the conversion of lignocellulose into high-value products, including fuels and chemicals, the production of cellulase and the enzymatic hydrolysis for producing fermentable sugar are the largest contributors to the cost of production of the final products. The marine bacterium Saccharophagus degradans 2-40(T) can degrade more than ten different complex polysaccharides found in the ocean, including cellulose and xylan. Accordingly, S. degradans has been actively considered as a practical source of crude enzymes needed for the saccharification of lignocellulose to produce ethanol by others including a leading commercial company. However, the overall enzyme system of S. degradans for hydrolyzing cellulose and hemicellulose has not been quantitatively evaluated yet in comparison with commercial enzymes. In this study, the inductions and activities of cellulase and xylanase of cell-free lysate of S. degradans were investigated. The growth of S. degradans cells and the activities of cellulase and xylanase were promoted by adding 2 % of cellulose and xylan mixture (cellulose:xylan = 4:3 in mass ratio) to the aquarium salt medium supplemented with 0.2 % glucose. The specific cellulase activity of the cell-free lysate of S. degradans, as determined by the filter paper activity assay, was approximately 70 times lower than those of commercial cellulases, including Celluclast 1.5 L and Accellerase 1000. These results imply that significant improvement in the cellulase activity of S. degradans is needed for the industrial uses of S. degradans as the enzyme source.

Bioconversion potential of Trichoderma viride HN1 cellulase for a lignocellulosic biomass Saccharum spontaneum.

PubMed

Iqtedar, Mehwish; Nadeem, Mohammad; Naeem, Hira; Abdullah, Roheena; Naz, Shagufta; Qurat ul Ain Syed; Kaleem, Afshan

2015-01-01

The industrialisation of lignocellulose conversion is impeded by expensive cellulase enzymes required for saccharification in bioethanol production. Current research undertakes cellulase production from pretreated Saccharum spontaneum through Trichoderma viride HN1 under submerged fermentation conditions. Pretreatment of substrate with 2% NaOH resulted in 88% delignification. Maximum cellulase production (2603 ± 16.39 U/mL/min carboxymethyl cellulase and 1393 ± 25.55 U/mL/min FPase) was achieved at 6% substrate at pH 5.0, with 5% inoculum, incubated at 35°C for 120 h of fermentation period. Addition of surfactant, Tween 80 and metal ion Mn(+2), significantly enhanced cellulase yield. This study accounts proficient cellulase yield through process optimisation by exploiting cheaper substrate to escalate their commercial endeavour.

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

DOEpatents

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.

Cytochemical localization of cellulases in decayed and nondecayed wood

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1987-01-01

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

Cationic polyacrylamide enhancing cellulase treatment efficiency of hardwood kraft-based dissolving pulp.

PubMed

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

2015-05-01

Cellulase treatment for decreasing viscosity and increasing Fock reactivity of dissolving pulp is a promising approach to reduce the use of toxic chemicals, such as hypochlorite in the dissolving pulp manufacturing process in the industry. Improving the cellulase treatment efficiency during the process is of practical interest. In the present study, the concept of using cationic polyacrylamide (CPAM) to enhance the cellulase treatment efficiency was demonstrated. This was mainly attributed to the increased cellulase adsorption onto cellulose fibers based on the patching/bridging mechanism. Results showed that the cellulase adsorption was increased by about 20% with the addition of 250 ppm of CPAM under the same conditions as those of the control. It was found that the viscosity decrease and Fock reactivity increase for the cellulase treatment was enhanced from using CPAM. The CPAM-assisted cellulase treatment concept may provide a practical alternative to the present hypochlorite-based technology for viscosity control in the industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification.

PubMed

Xue, Yiyun; Wang, Xiahui; Chen, Xingxuan; Hu, Jiajun; Gao, Min-Tian; Li, Jixiang

2017-06-01

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification were investigated in this study. All cellulases tested increased the contents of phenolic acids during saccharification. However, few free phenolic acids were detected, as they were present in conjugated form after saccharification when the cellulases from Trichoderma reesei, Trichoderma viride and Aspergillus niger were used. On the other hand, phenolic acids were present in free form when the Acremonium cellulolyticus cellulase was used. Assays of enzyme activity showed that, besides high cellulase activity, the A. cellulolyticus cellulase exhibited high feruloyl esterase (FAE) activity. A synergistic interaction between FAE and cellulase led to the increase in free phenolic acids, and thus an increase in antioxidative and antiradical activities of the phenolic acids. Moreover, a cost estimation demonstrated the feasibility of phenolic acids as value-added products to reduce the total production cost of ethanol. Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimization of Cellulase Production from Bacteria Isolated from Soil

PubMed Central

Sethi, Sonia; Datta, Aparna; Gupta, B. Lal; Gupta, Saksham

2013-01-01

Cellulase-producing bacteria were isolated from soil and identified as Pseudomonas fluorescens, Bacillus subtilIs, E. coli, and Serratia marcescens. Optimization of the fermentation medium for maximum cellulase production was carried out. The culture conditions like pH, temperature, carbon sources, and nitrogen sources were optimized. The optimum conditions found for cellulase production were 40°C at pH 10 with glucose as carbon source and ammonium sulphate as nitrogen source, and coconut cake stimulates the production of cellulase. Among bacteria, Pseudomonas fluorescens is the best cellulase producer among the four followed by Bacillus subtilis, E. coli, and Serratia marscens. PMID:25937986

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

NASA Astrophysics Data System (ADS)

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.

A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

DOE PAGES

Linger, Jeffrey G.; Taylor, II, Larry E.; Baker, John O.; ...

2015-03-18

One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated amore » native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity. In conclusion, he elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.« less

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

Code of Federal Regulations, 2013 CFR

2013-04-01

... PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.120 Carbohydrase and cellulase derived from Aspergillus niger. Carbohydrase and cellulase enzyme preparation derived from... Aspergillus niger from the carbohydrase and cellulase enzyme product. (d) The additive is used or intended for...

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

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Cellulase enzyme preparation derived from... SAFE Listing of Specific Substances Affirmed as GRAS § 184.1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase enzyme preparation is derived from a nonpathogenic...

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

Code of Federal Regulations, 2012 CFR

2012-04-01

... PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.120 Carbohydrase and cellulase derived from Aspergillus niger. Carbohydrase and cellulase enzyme preparation derived from... Aspergillus niger from the carbohydrase and cellulase enzyme product. (d) The additive is used or intended for...

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

Code of Federal Regulations, 2014 CFR

2014-04-01

... Enzyme Preparations and Microorganisms § 173.120 Carbohydrase and cellulase derived from Aspergillus niger. Carbohydrase and cellulase enzyme preparation derived from Aspergillus niger may be safely used... the carbohydrase and cellulase enzyme product. (d) The additive is used or intended for use as follows...

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

Code of Federal Regulations, 2011 CFR

2011-04-01

... PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.120 Carbohydrase and cellulase derived from Aspergillus niger. Carbohydrase and cellulase enzyme preparation derived from... Aspergillus niger from the carbohydrase and cellulase enzyme product. (d) The additive is used or intended for...

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

Code of Federal Regulations, 2010 CFR

2010-04-01

... PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.120 Carbohydrase and cellulase derived from Aspergillus niger. Carbohydrase and cellulase enzyme preparation derived from... Aspergillus niger from the carbohydrase and cellulase enzyme product. (d) The additive is used or intended for...

Construction of cellulose-utilizing Escherichia coli based on a secretable cellulase.

PubMed

Gao, Dongfang; Luan, Yaqi; Wang, Qian; Liang, Quanfeng; Qi, Qingsheng

2015-10-09

The microbial conversion of plant biomass into value added products is an attractive option to address the impacts of petroleum dependency. The Gram-negative bacterium Escherichia coli is commonly used as host for the industrial production of various chemical products with a variety of sugars as carbon sources. However, this strain neither produces endogenous cellulose degradation enzymes nor secrets heterologous cellulases for its poor secretory capacity. Thus, a cellulolytic E. coli strain capable of growth on plant biomass would be the first step towards producing chemicals and fuels. We previously identified the catalytic domain of a cellulase (Cel-CD) and its N-terminal sequence (N20) that can serve as carriers for the efficient extracellular production of target enzymes. This finding suggested that cellulose-utilizing E. coli can be engineered with minimal heterologous enzymes. In this study, a β-glucosidase (Tfu0937) was fused to Cel-CD and its N-terminal sequence respectively to obtain E. coli strains that were able to hydrolyze the cellulose. Recombinant strains were confirmed to use the amorphous cellulose as well as cellobiose as the sole carbon source for growth. Furthermore, both strains were engineered with poly (3-hydroxybutyrate) (PHB) synthesis pathway to demonstrate the production of biodegradable polyesters directly from cellulose materials without exogenously added cellulases. The yield of PHB reached 2.57-8.23 wt% content of cell dry weight directly from amorphous cellulose/cellobiose. Moreover, we found the Cel-CD and N20 secretion system can also be used for the extracellular production of other hydrolytic enzymes. This study suggested that a cellulose-utilizing E. coli was created based on a heterologous cellulase secretion system and can be used to produce biofuels and biochemicals directly from cellulose. This system also offers a platform for conversion of other abundant renewable biomass to biofuels and biorefinery products.

Biochemistry and genetics of actinomycete cellulases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilson, D.B.

1992-01-01

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionationmore » of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from T. curvata. The T. fusca cellulase genes are expressed at a low level in Escherichia coli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. 73 refs., 8 figs., 4 tabs.« less

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

PubMed

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

2010-05-07

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. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Optimization of renewable pinene production from the conversion of macroalgae Saccharina latissima.

PubMed

Scullin, Chessa; Stavila, Vitalie; Skarstad, Anita; Keasling, Jay D; Simmons, Blake A; Singh, Seema

2015-05-01

Enzymatic hydrolysis of Saccharina latissima with laminarinase was compared to hydrolysis with different combinations of cellulase and hemicellulase enzyme mixtures. The hemicellulase mixture resulted in similar release of glucose, while the cellulase mixture released 40% more glucose than laminarinase alone. The combination of a laminarinase augmented with a cellulase mixture resulted in a 53% increase of glucose release from S. latissima than laminarinase. Increasing biomass loading above 4% (w/v) reduced the sugar yield. Resulting macroalgae hydrolysates were used as a carbon source for the production of pinene, making use of a novel two plasmid Escherichia coli system. The macroalgal hydrolysates were suitable for the novel microbial production of pinene with no further treatment and/or purification. Copyright © 2015. Published by Elsevier Ltd.

Thermostable cellulase from a thermomonospora gene

DOEpatents

Wilson, David B.; Walker, Larry P.; Zhang, Sheng

1997-10-14

The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity.

Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering.

PubMed

Liu, Qian; Gao, Ranran; Li, Jingen; Lin, Liangcai; Zhao, Junqi; Sun, Wenliang; Tian, Chaoguang

2017-01-01

Over the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila , an important thermophilic biomass-degrading fungus, has attracted industrial interest for the production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology. In this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica . This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase production pathway, including cre - 1 , res - 1 , gh1 - 1, and alp - 1 , were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple strains exhibiting pronounced hyper-cellulase production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental strain. A genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate strain engineering of thermophilic fungi for production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.

Pulsed laser deposition and characterization of cellulase thin films

NASA Astrophysics Data System (ADS)

Cicco, N.; Morone, A.; Verrastro, M.; Viggiano, V.

2013-08-01

Thin films of cellulase were obtained by pulsed laser deposition (PLD) on an appropriate substrate. Glycoside hydrolase cellulase has received our attention because it emerges among the antifouling enzymes (enzymes being able to remove and prevent the formation of micro-organism biofilms) used in industry and medicine field. Pressed cellulase pellets, used as target material, were ablated with pulses of a Nd-YAG laser working at wavelength of 532 nm. In this work, we evaluated the impact of PLD technique both on molecular structure and hydrolytic activity of cellulase. Characteristic chemical bonds and morphology of deposited layers were investigated by FTIR spectroscopy and SEM respectively. The hydrolytic activity of cellulase thin films was detected by a colorimetric assay.

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

PubMed

Li, Yun; Qi, Benkun; Wan, Yinhua

2014-09-01

Pretreatment of lignocellulosic material produces a wide variety of inhibitory compounds, which strongly inhibit the following enzymatic hydrolysis of cellulosic biomass. Vanillin is a kind of phenolics derived from degradation of lignin. The effect of vanillin on cellulase activity for the hydrolysis of cellulose was investigated in detail. The results clearly showed that vanillin can reversibly and non-competitively inhibit the cellulase activity at appropriate concentrations and the value of IC50 was estimated to be 30 g/L. The inhibition kinetics of cellulase by vanillin was studied using HCH-1 model and inhibition constants were determined. Moreover, investigation of three compounds with similar structure of vanillin on cellulase activity demonstrated that aldehyde group and phenolic hydroxyl groups of vanillin had inhibitory effect on cellulase. These results provide valuable and detailed information for understanding the inhibition of lignin derived phenolics on cellulase. Copyright © 2014 Elsevier Ltd. All rights reserved.

Conversion of cellulosic materials to sugar

DOEpatents

Wilke, Charles R.; Mitra, Gautam

1976-08-03

A process for the production of sugar, mainly glucose, by the enzymatic degradation of cellulosic materials, particularly cellulosic wastes, which comprises hydrolyzing the cellulosic material in the presence of cellulase enzyme to produce a sugar solution and recovering from the hydrolysis products a major proportion of the cellulase enzyme used in the hydrolysis reaction for re-use. At least a portion of the required makeup cellulase enzyme is produced in a two-stage operation wherein, in the first stage, a portion of the output sugar solution is utilized to grow a cellulase-secreting microorganism, and, in the second stage, cellulase enzyme formation is induced in the microorganism-containing culture medium by the addition of an appropriate inducer, such as a cellulosic material. Cellulase enzyme is precipitated from the culture liquid by the addition of an organic solvent material, such as a low molecular weight alkyl ketone or alcohol, and the cellulase precipitate is then fed to the hydrolysis reaction.

Abscisic acid and ethephon regulation of cellulase in the endosperm cap and radicle during lettuce seed germination.

PubMed

Chen, Bingxian; Ma, Jun; Xu, Zhenjiang; Wang, Xiaofeng

2016-10-01

The purpose of this study was to investigate the role of cellulase in endosperm cap weakening and radicle elongation during lettuce (Lactuca sativa L.) seed germination. The application of abscisic acid (ABA) or ethephon inhibits or promotes germination, respectively, by affecting endosperm cap weakening and radicle elongation. Cellulase activities, and related protein and transcript abundances of two lettuce cellulase genes, LsCEL1 and LsCEL2, increase in the endosperm cap and radicle prior to radicle protrusion following imbibition in water. ABA or ethephon reduce or elevate, respectively, cellulase activity, and related protein and transcript abundances in the endosperm cap. Taken together, these observations suggest that cellulase plays a role in endosperm cap weakening and radicle elongation during lettuce seed germination, and that the regulation of cellulase in the endosperm cap by ABA and ethephon play a role in endosperm cap weakening. However, the influence of ABA and ethephon on radicle elongation may not be through their effects on cellulase. © 2016 Institute of Botany, Chinese Academy of Sciences.

Using temperature-responsive zwitterionic surfactant to enhance the enzymatic hydrolysis of lignocelluloses and recover cellulase by cooling.

PubMed

Cai, Cheng; Pang, Yuxia; Zhan, Xuejuan; Zeng, Meijun; Lou, Hongming; Qian, Yong; Yang, Dongjie; Qiu, Xueqing

2017-11-01

Some zwitterionic surfactants exhibit upper critical solution temperature (UCST) in aqueous solutions. For the zwitterionic surfactant solution mixed with cellulase, when its temperature is below UCST, the cellulase can be recovered by coprecipitation with zwitterionic surfactant. In this work, 3-(Hexadecyldimethylammonio) propanesulfonate (SB3-16) was selected to enhance the enzymatic hydrolysis of lignocelluloses and recover the cellulase. After adding 2mmol/L of SB3-16, the enzymatic digestibility of eucalyptus pretreated by dilute acid (Eu-DA) and by sulfite (Eu-SPORL) increased from 27.9% and 35.1% to 72.6% and 89.7%, respectively. The results showed that SB3-16 could reduce the non-productive adsorption of cellulase on hydrophobic interface, while it did not significantly inhibit the activity of cellulase. For the solution contained 1wt% SB3-16 and 200mg protein/L CTec2 cellulase, 55.2% of protein could be recovered by cooling. The filter paper activity of the recovered cellulase was 1.93FPU/mg protein, which was 95.8% of its initial activity. Copyright © 2017. Published by Elsevier Ltd.

An Ime2-like mitogen-activated protein kinase is involved in cellulase expression in the filamentous fungus Trichoderma reesei.

PubMed

Chen, Fei; Chen, Xiu-Zhen; Su, Xiao-Yun; Qin, Li-Na; Huang, Zhen-Bang; Tao, Yong; Dong, Zhi-Yang

2015-10-01

Eukaryotic mitogen-activated protein kinases (MAPKs) play crucial roles in transducing environmental and developmental signals inside the cell and regulating gene expression, however, the roles of MAPKs remain largely unknown in Trichoderma reesei. T. reesei ime2 (TrIme2) encodes an Ime2-like MAPK in T. reesei. The deletion of the TrIme2 gene led to 90% increase in cellulase activity against filter paper during earlier period time of cellulase induction as well as the extracellular protein production. Compared to the parent strain, the transcriptional levels of the three major cellulase genes cbh1,cbh2, egl1 were increased by about 9 times, 4 times, 2 times, respectively, at 8 h after cellulase induction in the ΔTrIme2 mutant. In addition, the disruption of TrIme2 caused over 50% reduction of the transcript levels of cellulase transcriptional regulators cre1 and xyr1. TrIme2 functions in regulation of the expression of cellulase gene in T.reesei, and is a good candidate for genetically engineering of T. reesei for higher cellulase production.

Characterization of a thermophilic cellulase from Geobacillus sp. HTA426, an efficient cellulase-producer on alkali pretreated of lignocellulosic biomass.

PubMed

Potprommanee, Laddawan; Wang, Xiao-Qin; Han, Ye-Ju; Nyobe, Didonc; Peng, Yen-Ping; Huang, Qing; Liu, Jing-Yong; Liao, Yu-Ling; Chang, Ken-Lin

2017-01-01

A themophilic cellulase-producing bacterium was isolated from a hot spring district and identified as Geobacillus sp. HTA426. The cellulase enzyme produced by the Geobacillus sp. HTA426 was purified through ammonium sulfate precipitation and ion exchange chromatography, with the recovery yield and fold purification of 10.14% and 5.12, respectively. The purified cellulase has a molecular weight of 40 kDa. The optimum temperature and pH for carboxymethyl cellulase (CMCase) activity of the purified cellulase were 60°C and pH 7.0, respectively. The enzyme was also stable over a wide temperature range of 50°C to 70°C after 5 h of incubation. Moreover, the strain HTA426 was able to grow and produce cellulase on alkali-treated sugarcane bagasse, rice straw and water hyacinth as carbon sources. Enzymatic hydrolysis of sugarcane bagasse, which was regarded as the most effective carbon source for cellulase production (CMCase activity = 103.67 U/mL), followed by rice straw (74.70 U/mL) and water hyacinth (51.10 U/mL). This strain producing an efficient thermostable cellulose is a potential candidate for developing a more efficient and cost-effective process for converting lignocellulosic biomass into biofuel and other industrial process.

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

PubMed

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. Copyright © 2015 Elsevier Inc. All rights reserved.

High-throughput selection for cellulase catalysts using chemical complementation.

PubMed

Peralta-Yahya, Pamela; Carter, Brian T; Lin, Hening; Tao, Haiyan; Cornish, Virginia W

2008-12-24

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.

Characterization of a thermophilic cellulase from Geobacillus sp. HTA426, an efficient cellulase-producer on alkali pretreated of lignocellulosic biomass

PubMed Central

Potprommanee, Laddawan; Wang, Xiao-Qin; Han, Ye-Ju; Nyobe, Didonc; Peng, Yen-Ping; Huang, Qing; Liu, Jing-yong; Liao, Yu-Ling; Chang, Ken-Lin

2017-01-01

A themophilic cellulase-producing bacterium was isolated from a hot spring district and identified as Geobacillus sp. HTA426. The cellulase enzyme produced by the Geobacillus sp. HTA426 was purified through ammonium sulfate precipitation and ion exchange chromatography, with the recovery yield and fold purification of 10.14% and 5.12, respectively. The purified cellulase has a molecular weight of 40 kDa. The optimum temperature and pH for carboxymethyl cellulase (CMCase) activity of the purified cellulase were 60°C and pH 7.0, respectively. The enzyme was also stable over a wide temperature range of 50°C to 70°C after 5 h of incubation. Moreover, the strain HTA426 was able to grow and produce cellulase on alkali-treated sugarcane bagasse, rice straw and water hyacinth as carbon sources. Enzymatic hydrolysis of sugarcane bagasse, which was regarded as the most effective carbon source for cellulase production (CMCase activity = 103.67 U/mL), followed by rice straw (74.70 U/mL) and water hyacinth (51.10 U/mL). This strain producing an efficient thermostable cellulose is a potential candidate for developing a more efficient and cost-effective process for converting lignocellulosic biomass into biofuel and other industrial process. PMID:28406925

A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation

PubMed Central

Peralta-Yahya, Pamela; Carter, Brian T.; Lin, Hening; Tao, Haiyan; Cornish, Virginia W.

2010-01-01

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases however is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Due to the large number of enzyme variants selections can test compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity. PMID:19053460

Strong cellulase inhibition by Mannan polysaccharides in cellulose conversion to sugars.

PubMed

Kumar, Rajeev; Wyman, Charles E

2014-07-01

Cellulase enzymes contribute a major fraction of the total cost for biological conversion of lignocellulosic biomass to fuels and chemicals. Although a several fold reduction in cellulase production costs and enhancement of cellulase activity and stability have been reported in recent years, sugar yields are still lower at low enzyme doses than desired commercially. We recently reported that hemicellulose xylan and its oligomers strongly inhibit cellulase and that supplementation of cellulase with xylanase and β-xylosidase would significantly reduce such inhibition. In this study, mannan polysaccharides and their enzymatically prepared hydrolyzates were discovered to be strongly inhibitory to fungal cellulase in cellulose conversion (>50% drop in % relative conversion), even at a small concentration of 0.1 g/L, and inhibition was much greater than experienced by other known inhibitors such as cellobiose, xylooligomers, and furfural. Furthermore, cellulase inhibition dramatically increased with heteromannan loading and mannan substitution with galactose side units. In general, enzymatically prepared hydrolyzates were less inhibitory than their respective mannan polysaccharides except highly substituted ones. Supplementation of cellulase with commercial accessory enzymes such as xylanase, pectinase, and β-glucosidase was effective in greatly relieving inhibition but only for less substituted heteromannans. However, cellulase supplementation with purified heteromannan specific enzymes relieved inhibition by these more substituted heteromannans as well, suggesting that commercial preparations need to have higher amounts of such activities to realize high sugar yields at the low enzyme protein loadings needed for low cost fuels production. © 2014 Wiley Periodicals, Inc.

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

DOE PAGES

Yarbrough, John. M.; Zhang, Ruoran; Mittal, Ashutosh; ...

2017-03-07

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yarbrough, John. M.; Zhang, Ruoran; Mittal, Ashutosh

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

Intake, digestibility, and composition of orchardgrass and alfalfa silages treated with cellulase, inoculant, and formic acid fed to lambs.

PubMed

Nadeau, E M; Russell, J R; Buxton, D R

2000-11-01

The objectives of this study were to determine the effect of a cellulase (from Trichoderma longibrachiatum) alone or combined with a bacterial inoculant (Lactobacillus plantarum and Pediococcus cerevisiae) or formic acid on composition, intake, and digestibility of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages. Orchardgrass and alfalfa were harvested at the early heading stage and at the early bloom stage of maturity and wilted to approximately 22 and 32% DM, respectively. Forages were then ensiled in 100-L sealed barrels for at least 60 d before they were fed to lambs. Silage treated with cellulase had lower (P < .001) pH and lower (P < .001) acetic acid and NH3 N concentrations than untreated silage of both plant species and a higher (P = .004) lactic acid concentration than the control treatment of alfalfa silage. Fermentation characteristics of cellulase-treated silages, especially of alfalfa, were further enhanced by use of inoculant. Formic acid addition increased (P < .001), reducing sugar concentration of cellulase-treated orchardgrass and alfalfa silage by 90 and 154%, respectively, and decreased (P < .001) NH3 N concentration of cellulase-treated alfalfa silage by 19%. Averaged across plant species, cellulase, combined with inoculant or formic acid, resulted in 8 and 13% greater (P = .03) DMI, respectively, than the control silage. Extensive enzymatic cell-wall degradation during ensiling decreased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 25% and decreased (P = .001) cellulose intake by 23%, when averaged across plant species. Addition of formic acid increased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 19%. Averaged across species, cellulase application decreased (P < .05) silage NDF digestibility by 18%. Greater sugar and lower acetic acid, NH3 N, and NDF concentrations resulted in greater DMI of cellulase-treated silage than of control silage, when cellulase was combined with formic acid or inoculant.

Thermostable cellulase from a thermomonospora gene

DOEpatents

Wilson, D.B.; Walker, L.P.; Zhang, S.

1997-10-14

The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity. 3 figs.

Role of carbon source in the shift from oxidative to hydrolytic wood decomposition by Postia placenta.

PubMed

Zhang, Jiwei; Schilling, Jonathan S

2017-09-01

Brown rot fungi initiate wood decay using oxidative pretreatments to improve access for cellulolytic enzymes. These pretreatments are incompatible with enzymes, and we recently showed that Postia placenta overcomes this issue by delaying glycoside hydrolase (GH) gene upregulation briefly (<48h) until expression of oxidoreductases (ORs) is repressed. This implies an inducible cellulase system rather than a constitutive system, as often reported, and it remains unclear what cues this transition. To address this, we grew P. placenta along wood wafers and spatially mapped expression (via quantitative PCR) of twelve ORs and GHs targeted using functional genomics analyses. By layering expression patterns over solubilized sugar data (via HPLC) from wood, we observed solubilization of wood glucose, cellobiose, mannose, and xylose coincident with the OR-GH transition. We then tested effects of these soluble sugars, plus polymeric carbon sources (spruce powder, cellulose), on P. placenta gene expression in liquid cultures. Expression of ORs was strictly (aox1, cro5) or progressively repressed over time (qrd1, lcc1) by all soluble sugars, including cellobiose, but not by polymeric sources. Simple sugars repressed hemicellulase gene expression over time, but these sugars did not repress cellulases. Cellulase genes were upregulated, however, along with hemicellulases in the presence of soluble cellobiose and in the presence of polymeric carbon sources, relative to starvation (carbon-free). This verifies an inducible cellulase system in P. placenta that lacks carbon catabolite repression (CCR), and it suggests that brown rot fungi use soluble sugars, particularly cellobiose, to cue a critical oxidative-hydrolytic transition. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun-Ki; Himmel, Michael E.; Bomble, Yannick J.

Members of the genusCaldicellulosiruptorhave the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species,Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass.C. besciicontains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase from Thermotoga maritimaimproves the phosphorolytic pathway inC. besciiand results in synergistic activity with endogenous enzymes, includingmore » CelA, to increase cellulolytic activity and growth on crystalline cellulose. CelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. Here, this work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.« less

Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity

DOE PAGES

Kim, Sun-Ki; Himmel, Michael E.; Bomble, Yannick J.; ...

2017-11-03

Members of the genusCaldicellulosiruptorhave the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species,Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass.C. besciicontains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase from Thermotoga maritimaimproves the phosphorolytic pathway inC. besciiand results in synergistic activity with endogenous enzymes, includingmore » CelA, to increase cellulolytic activity and growth on crystalline cellulose. CelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. Here, this work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.« less

Modeling the Effect of pH and Temperature for Cellulases Immobilized on Enzymogel Nanoparticles.

PubMed

Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

2015-06-01

Production costs of cellulosic biofuels can be lowered if cellulases are recovered and reused using particulate carriers that can be extracted after biomass hydrolysis. Such enzyme recovery was recently demonstrated using enzymogel nanoparticles with grafted polymer brushes loaded with cellulases. In this work, cellulase (NS50013) and β-glucosidase (Novozyme 188) were immobilized on enzymogels made of poly(acrylic acid) polymer brushes grafted to the surface of silica nanoparticles. Response surface methodology was used to model effects of pH and temperature on hydrolysis and recovery of free and attached enzymes. Hydrolysis yields using both enzymogels and free cellulase and β-glucosidase were highest at the maximum temperature tested, 50 °C. The optimal pH for cellulase enzymogels and free enzyme was 5.0 and 4.4, respectively, while both free β-glucosidase and enzymogels had an optimal pH near 4.4. Highest hydrolysis sugar concentrations with cellulase and β-glucosidase enzymogels were 69 and 53 % of those with free enzymes, respectively. Enzyme recovery using enzymogels decreased with increasing pH, but cellulase recovery remained greater than 88 % throughout the operating range of pH values less than 5.0 and was greater than 95 % at pH values below 4.3. Recovery of β-glucosidase enzymogels was not affected by temperature and had little impact on cellulase recovery.

Preparation, characterisation and use for antioxidant oligosaccharides of a cellulase from abalone (Haliotis discus hannai) viscera.

PubMed

Tao, Zhi-Peng; Sun, Le-Chang; Qiu, Xu-Jian; Cai, Qiu-Feng; Liu, Guang-Ming; Su, Wen-Jin; Cao, Min-Jie

2016-07-01

In China, abalone (Haliotis discus hannai) production is growing annually. During industrial processing, the viscera, which are abundant of cellulase, are usually discarded or processed into low-value feedstuff. Thus, it is of interest to obtain cellulase from abalone viscera and investigate its application for preparation of functional oligosaccharides. A cellulase was purified from the hepatopancreas of abalone by ammonium sulfate precipitation and two-steps column chromatography. The molecular weight of the cellulase was 45 kDa on SDS-PAGE. Peptide mass fingerprinting analysis yielded 103 amino acid residues, which were identical to cellulases from other species of abalone. Substrate specificity analysis indicated that the cellulase is an endo-1,4-β-glucanase. Hydrolysis of seaweed Porphyra haitanensis polysaccharides by the enzyme produced oligosaccharides with degree of polymerisation of two to four, whose monosaccharide composition was 58% galactose, 4% glucose and 38% xylose. The oligosaccharides revealed 2,2'-diphenyl-1-picrylhydrazyl free radical as well as hydrogen peroxide scavenging activity. It is feasible and meaningful to utilise cellulase from the viscera of abalone for preparation of functional oligosaccharides. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Enhanced cellulose degradation using cellulase-nanosphere complexes.

PubMed

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 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.

Enhanced Cellulose Degradation Using Cellulase-Nanosphere Complexes

PubMed Central

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 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. PMID:22870287

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

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of rhamnolipid on the cellulase and xylanase in hydrolysis of wheat straw.

PubMed

Wang, Hong-Yuan; Fan, Bing-Quan; Li, Chun-Hua; Liu, Shuang; Li, Min

2011-06-01

The effects of biosurfactant rhamnolipid (RL) and chemical surfactant Triton X-100 on the production of cellulases and xylanase from Penicillium expansum (P. expansum) in untreated, acid- and alkali-pretreated wheat straw submerged fermentations were studied, and the influences on the activity and stability of Cellulase R-10 were also investigated. The results showed that RL and Triton X-100 enhanced the activities of cellulases and xylanase to different extents and the stimulatory effects of RL were superior to those of Triton X-100. During the peak enzyme production phase, RL (60 RE mg/l) increased cellulases activities by 25.5-102.9%, in which the raise of the same enzyme in acid-pretreated straw broths was the most. It was found that the reducing sugars by hydrolyzing wheat straw with Cellulase R-100 were not visibly increased after adding RL. However, it distinctly protected Cellulase R-10 from degradation or inactivation, keeping the reducing sugars yield at about 17%. Copyright © 2011. Published by Elsevier Ltd.

Genome sequencing and transcriptome analysis of Trichoderma reesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction.

PubMed

Ivanova, Christa; Ramoni, Jonas; Aouam, Thiziri; Frischmann, Alexa; Seiboth, Bernhard; Baker, Scott E; Le Crom, Stéphane; Lemoine, Sophie; Margeot, Antoine; Bidard, Frédérique

2017-01-01

The hydrolysis of biomass to simple sugars used for the production of biofuels in biorefineries requires the action of cellulolytic enzyme mixtures. During the last 50 years, the ascomycete Trichoderma reesei , the main source of industrial cellulase and hemicellulase cocktails, has been subjected to several rounds of classical mutagenesis with the aim to obtain higher production levels. During these random genetic events, strains unable to produce cellulases were generated. Here, whole genome sequencing and transcriptomic analyses of the cellulase-negative strain QM9978 were used for the identification of mutations underlying this cellulase-negative phenotype. Sequence comparison of the cellulase-negative strain QM9978 to the reference strain QM6a identified a total of 43 mutations, of which 33 were located either close to or in coding regions. From those, we identified 23 single-nucleotide variants, nine InDels, and one translocation. The translocation occurred between chromosomes V and VII, is located upstream of the putative transcription factor vib1 , and abolishes its expression in QM9978 as detected during the transcriptomic analyses. Ectopic expression of vib1 under the control of its native promoter as well as overexpression of vib1 under the control of a strong constitutive promoter restored cellulase expression in QM9978, thus confirming that the translocation event is the reason for the cellulase-negative phenotype. Gene deletion of vib1 in the moderate producer strain QM9414 and in the high producer strain Rut-C30 reduced cellulase expression in both cases. Overexpression of vib1 in QM9414 and Rut-C30 had no effect on cellulase production, most likely because vib1 is already expressed at an optimal level under normal conditions. We were able to establish a link between a chromosomal translocation in QM9978 and the cellulase-negative phenotype of the strain. We identified the transcription factor vib1 as a key regulator of cellulases in T. reesei whose expression is absent in QM9978. We propose that in T. reesei , as in Neurospora crassa , vib1 is involved in cellulase induction, although the exact mechanism remains to be elucidated. The data presented here show an example of a combined genome sequencing and transcriptomic approach to explain a specific trait, in this case the QM9978 cellulase-negative phenotype, and how it helps to better understand the mechanisms during cellulase gene regulation. When focusing on mutations on the single base-pair level, changes on the chromosome level can be easily overlooked and through this work we provide an example that stresses the importance of the big picture of the genomic landscape during analysis of sequencing data.

Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

PubMed Central

2011-01-01

Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX) and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy. PMID:21702938

Processing of cellulosic material by a cellulase-containing cell-free fermentate produced from cellulase-producing bacteria, ATCC 55702

DOEpatents

Dees, H.C.

1998-08-04

Bacteria which produce large amounts of a cellulase-containing cell-free fermentate, have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase degrading bacterium ATCC 55702, which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic materials. 5 figs.

Processing of cellulosic material by a cellulase-containing cell-free fermentate produced from cellulase-producing bacteria, ATCC 55702

DOEpatents

Dees, H. Craig

1998-01-01

Bacteria which produce large amounts of a cellulase-containing cell-free fermentate, have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase degrading bacterium ATCC 55702, which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic materials.

Genetically enhanced cellulase production in Pseudomonas cellulosa using recombinant DNA technology

DOEpatents

Dees, H. Craig

1999-01-01

An enhanced strain of Pseudomonas celllulosa was obtained by introducing a recombinant genetic construct comprising a heterologous cellulase gene operably connected to a promoter into ATCC 55702, mutagenizing the transformants by treatment with MNNG, and selecting a high cellulase producing transformant. The transformant, designated Pseudomonas cellulosa ATCC XXXX, exhibits enhanced levels of cellulase production relative to the untransformed Pseudomonas cellulosa strain #142 ATCC 55702.

Discovery of new cellulases from the metagenome by a metagenomics-guided strategy.

PubMed

Yang, Chao; Xia, Yu; Qu, Hong; Li, An-Dong; Liu, Ruihua; Wang, Yubo; Zhang, Tong

2016-01-01

Energy shortage has become a global problem. Production of biofuels from renewable biomass resources is an inevitable trend of sustainable development. Cellulose is the most abundant and renewable resource in nature. Lack of new cellulases with unique properties has become the bottleneck of the efficient utilization of cellulose. Environmental metagenomes are regarded as huge reservoirs for a variety of cellulases. However, new cellulases cannot be obtained easily by functional screening of metagenomic libraries. In this work, a metagenomics-guided strategy for obtaining new cellulases from the metagenome was proposed. Metagenomic sequences of DNA extracted from the anaerobic beer lees converting consortium enriched at thermophilic conditions were assembled, and 23 glycoside hydrolase (GH) sequences affiliated with the GH family 5 were identified. Among the 23 GH sequences, three target sequences (designated as cel7482, cel3623 and cel36) showing low identity with those known GHs were chosen as the putative cellulase genes to be functionally expressed in Escherichia coli after PCR cloning. The three cellulases were classified into endo-β-1,4-glucanases by product pattern analysis. The recombinant cellulases were more active at pH 5.5 and within a temperature range of 60-70 °C. Computer-assisted 3D structure modeling indicated that the active residues in the active site of the recombinant cellulases were more similar to each other compared with non-active site residues. The recombinant cel7482 was extremely tolerant to 2 M NaCl, suggesting that cel7482 may be a halotolerant cellulase. Moreover, the recombinant cel7482 was shown to have an ability to resist three ionic liquids (ILs), which are widely used for cellulose pretreatment. Furthermore, active cel7482 was secreted by the twin-arginine translocation (Tat) pathway of Bacillus subtilis 168 into the culture medium, which facilitates the subsequent purification and reduces the formation of inclusion body in the context of overexpression. This study demonstrated a simple and efficient method for direct cloning of new cellulase genes from environmental metagenomes. In the future, the metagenomics-guided strategy may be applied to the high-throughput screening of new cellulases from environmental metagenomes.

Cellulase and xylanase production at pilot scale by solid-state fermentation from coffee husk using specialized consortia: The consistency of the process and the microbial communities involved.

PubMed

Cerda, Alejandra; Mejías, Laura; Gea, Teresa; Sánchez, Antoni

2017-11-01

Solid state fermentation is a promising technology however rising concerns related to scale up and reproducibility in a productive process. Coffee husk and a specialized inoculum were used in a 4.5L and then in 50L reactors to assess the reproducibility of a cellulase and hemicellulase production system. Fermentations were consistent in terms of cellulase production and microbial communities. The higher temperatures achieved when operating at 50L generated a shift on the microbial communities and a reduction of nearly 50% on cellulase production at pilot scale. In spite, an overall enzymatic production of 3.1±0.5FPUg -1 DM and 48±4Ug -1 DM for FPase and Xyl activities was obtained, respectively, with low deviation coefficients of 16 and 19% for FPase and Xyl production. Gaseous emissions assessment revealed an emission factor of 2.6·10 -3 kg volatile organic compounds per Mg of coffee husk and negligible NH 3 , CH 4 and N 2 O emissions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chemical and structural changes of pretreated empty fruit bunch (EFB) in ionic liquid-cellulase compatible system for fermentability to bioethanol.

PubMed

Elgharbawy, Amal A; Alam, Md Zahangir; Moniruzzaman, Muhammad; Kabbashi, Nassereldeen Ahmad; Jamal, Parveen

2018-05-01

The pretreatment of empty fruit bunch (EFB) was conducted using an integrated system of IL and cellulases (IL-E), with simultaneous fermentation in one vessel. The cellulase mixture ( PKC-Cel ) was derived from Trichoderma reesei by solid-state fermentation. Choline acetate [Cho]OAc was utilized for the pretreatment due to its biocompatibility and biodegradability. The treated EFB and its hydrolysate were characterized by the Fourier transform infrared spectroscopy, scanning electron microscopy, and chemical analysis. The results showed that there were significant structural changes in EFB after the treatment in IL-E system. The sugar yield after enzymatic hydrolysis by the PKC-Cel was increased from 0.058 g/g of EFB in the crude sample (untreated) to 0.283 and 0.62 ± 06 g/g in IL-E system after 24 and 48 h of treatment, respectively. The EFB hydrolysate showed the eligibility for ethanol production without any supplements where ethanol yield was 0.275 g ethanol/g EFB in the presence of the IL, while lower yield obtained without IL-pretreatment. Moreover, it was demonstrated that furfural and phenolic compounds were not at the level of suppressing the fermentation process.

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

PubMed

Rybarczyk-Mydłowska, Katarzyna; Maboreke, Hazel Ruvimbo; van Megen, Hanny; van den Elsen, Sven; Mooyman, Paul; Smant, Geert; Bakker, Jaap; Helder, Johannes

2012-11-21

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. 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. 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-knot and cyst nematodes did not acquire this gene directly by lateral genes transfer. More likely, these genes were passed on by ancestors of a family nowadays known as the Pratylenchidae.

Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis, Biophysical Measurements, and Molecular Simulation

PubMed Central

Sammond, Deanne W.; Payne, Christina M.; Brunecky, Roman; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions. PMID:23139804

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

PubMed

Gao, Dahai; Haarmeyer, Carolyn; Balan, Venkatesh; Whitehead, Timothy A; Dale, Bruce E; Chundawat, Shishir Ps

2014-01-01

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification. In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion(a) (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin. Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

Cloning of cellulase genes from acidothermus cellulolyticus

DOEpatents

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

1996-01-01

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

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

PubMed

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Disruption of non-anchored cell wall protein NCW-1 promotes cellulase production by increasing cellobiose uptake in Neurospora crassa.

PubMed

Lin, Liangcai; Chen, Yong; Li, Jingen; Wang, Shanshan; Sun, Wenliang; Tian, Chaoguang

2017-04-01

To elucidate the mechanism of cellulase signal transduction in filamentous fungi including the components of the cellulase induction pathway. Neurospora crassa ncw-1 encodes a non-anchored cell wall protein. The absence of ncw-1 increased cellulase gene expression and this is not due to relieving carbon catabolite repression mediated by the cre-1 pathway. A mutant lacking genes encoding both three major β-glucosidase enzymes and NCW-1 (Δ3βGΔncw-1) was constructed. Transcriptome analysis of the quadruple mutant demonstrated enhanced expression of cellodextrin transporters after ncw-1 deletion, indicating that ncw-1 affects cellulase expression and production by inhibiting the uptake of the cellodextrin. NCW-1 is a novel component that plays a critical role in the cellulase induction signaling pathway.

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

PubMed

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.

Gene Cloning and Expression of Cellulase of Bacillus amyloliquefaciens Isolated from the Cecum of Goose.

PubMed

Sun, Linghong; Cao, Jiangyan; Liu, Ying; Wang, Junjie; Guo, Panpan; Wang, Zaigui

2017-01-02

A kind of bacteria secreting cellulase and showing probiotic attributes was isolated from the cecum of goose and identified as Bacillus amyloliquefaciens by analysis of 16S rRNA gene sequence and named as B. amyloliquefaciens S1. In vitro assays, the enzymatic activity of the strain was determined by the reducing-sugar method, and the proper culture conditions of producing cellulase and some properties of the cellulase were investigated. The cultural mixture of the bacteria had a high cellulase activity of 1.25 U/mL. In order to improve the utilization rate of the cellulase, some properties of the cellulase were studied. The best reaction pH of the enzymes was 7.0 and the optimum reaction temperature was 60°C. The enzyme was a kind of neutral cellulase that possessing strong resistance against heat and acidity. It showed high activity to absorbent cotton, soybean meal, and filter paper. Meanwhile, a gene encoding a kind of cellulase was cloned and prokaryotic expressed in Escherichia coli. The gene had 1500 bp in length, encoding a protein of 55 kDa, which was confirmed by SDS-PAGE and Western blotting. This study explored the possibility of degrading ability of bacteria with its probiotic attributes to enhance digestibility of the feed and gut health of animal. It also provided some basis for its further functional analysis and practical application as a microbial preparation for the breeding.

Lignocellulosic Fermentation of Wild Grass Employing Recombinant Hydrolytic Enzymes and Fermentative Microbes with Effective Bioethanol Recovery

PubMed Central

Das, Saprativ P.; Ghosh, Arabinda; Gupta, Ashutosh; Das, Debasish

2013-01-01

Simultaneous saccharification and fermentation (SSF) studies of steam exploded and alkali pretreated different leafy biomass were accomplished by recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for bioethanol production. The recombinant C. thermocellum GH5 cellulase and GH43 hemicellulase genes expressed in Escherichia coli cells were grown in repetitive batch mode, with the aim of enhancing the cell biomass production and enzyme activity. In batch mode, the cell biomass (A 600 nm) of E. coli cells and enzyme activities of GH5 cellulase and GH43 hemicellulase were 1.4 and 1.6 with 2.8 and 2.2 U·mg−1, which were augmented to 2.8 and 2.9 with 5.6 and 3.8 U·mg−1 in repetitive batch mode, respectively. Steam exploded wild grass (Achnatherum hymenoides) provided the best ethanol titres as compared to other biomasses. Mixed enzyme (GH5 cellulase, GH43 hemicellulase) mixed culture (Saccharomyces cerevisiae, Candida shehatae) system gave 2-fold higher ethanol titre than single enzyme (GH5 cellulase) single culture (Saccharomyces cerevisiae) system employing 1% (w/v) pretreated substrate. 5% (w/v) substrate gave 11.2 g·L−1 of ethanol at shake flask level which on scaling up to 2 L bioreactor resulted in 23 g·L−1 ethanol. 91.6% (v/v) ethanol was recovered by rotary evaporator with 21.2% purification efficiency. PMID:24089676

Microbial Cellulose Utilization: Fundamentals and Biotechnology

PubMed Central

Lynd, Lee R.; Weimer, Paul J.; van Zyl, Willem H.; Pretorius, Isak S.

2002-01-01

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts. PMID:12209002

Cellulase assisted synthesis of nano-silver and gold: Application as immobilization matrix for biocatalysis.

PubMed

Mishra, Abhijeet; Sardar, Meryam

2015-01-01

In the present study, we report in vitro synthesis of silver and gold nanoparticles (NPs) using cellulase enzyme in a single step reaction. Synthesized nanoparticles were characterized by UV-VIS spectroscopy, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD), Circular Dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR). UV-visible studies shows absorption band at 415nm and 520nm for silver and gold NPs respectively due to surface plasmon resonance. Sizes of NPs as shown by TEM are 5-25nm for silver and 5-20nm for gold. XRD peaks confirmed about phase purity and crystallinity of silver and gold NPs. FTIR data shows presence of amide I peak on both the NPs. The cellulase assisted synthesized NPs were further exploited as immobilization matrix for cellulase enzyme. Thermal stability analysis reveals that the immobilized cellulase on synthesized NPs retained 77-80% activity as compared to free enzyme. While reusability data suggests immobilized cellulase can be efficiently used up to sixth cycles with minimum loss of enzyme activity. The secondary structural analysis of cellulase enzyme during the synthesis of NPs and also after immobilization of cellulase on these NPs was carried out by CD spectroscopy. Copyright © 2015 Elsevier B.V. All rights reserved.

Thermophilic Bacillus coagulans requires less cellulases for simultaneous saccharification and fermentation of cellulose to products than mesophilic microbial biocatalysts.

PubMed

Ou, Mark S; Mohammed, Nazimuddin; Ingram, L O; Shanmugam, K T

2009-05-01

Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g(-1) cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g(-1) cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.

Cloning of cellulase genes from Acidothermus cellulolyticus

DOEpatents

Lastick, S.M.; Tucker, M.P.; Grohmann, K.

1996-05-07

A process is described for moving fragments that code for cellulase activity from the genome of A. cellulolyticus to several plasmid vectors and the subsequent expression of active cellulase activity in E. coli. 5 figs.

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

NASA Astrophysics Data System (ADS)

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

2016-11-01

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

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

PubMed

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.

Intercalation of cellulase enzyme into a hydrotalcite layer structure

NASA Astrophysics Data System (ADS)

Zou, N.; Plank, J.

2015-01-01

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

Enhancement in ionic liquid tolerance of cellulase immobilized on PEGylated graphene oxide nanosheets: Application in saccharification of lignocellulose.

PubMed

Xu, Jiaxing; Sheng, Zhenhuan; Wang, Xinfeng; Liu, Xiaoyan; Xia, Jun; Xiong, Peng; He, Bingfang

2016-01-01

The objective of the present work was to improve ionic liquid (IL) tolerance of cellulase based on the exploration of functional nanoscale carriers for potential application in lignocellulosic biorefinery. PEGylated graphene oxide (GO) composite was successfully fabricated by chemical binding of 4-arm-PEG-NH2 and GO and applied to the immobilization of cellulase. The PEGylated GO-Cellulase retained 61% of the initial activity in 25% (w/v) 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) while free cellulase only retained 2%. The IL stability was enhanced more than 30 times. The relatively minor change in Km value (from 2.7 to 3.2mgmL(-1)) after the immobilization suggested that PEGylated GO-Cellulase was capable of closely mimicking the performance of free enzyme. After treating rice straw with [Bmim][Cl] and dilution to a final IL concentration of 15% (w/v), the slurry was directly hydrolyzed using PEGylated GO-Cellulase without IL removing and a high hydrolysis rate of 87% was achieved. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

2012-09-01

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels. Copyright © 2012 Elsevier Ltd. All rights reserved.

Kinetic study of Escherichia coli BPPTCC-EgRK2 to produce recombinant cellulase for ethanol production from oil palm empty fruit bunch

NASA Astrophysics Data System (ADS)

Limoes, S.; Rahman, S. F.; Setyahadi, S.; Gozan, M.

2018-03-01

Oil Palm Empty Fruit Bunch (OPEFB) is an abundant biomass resource in Indonesia, which contains 46,77% (w/w) of cellulose. The high cellulose content of OPEFB can be used as a substrate for bacteria cultivation to produce cellulase. By using OPEFB as an alternative substrate, the production cost of cellulase in industrial scale can be suppressed. However, currently there are no available research that simulate a cellulase production plant design. Prior to simulating the cellulase plant design, kinetic studies of bacteria used in cultivation are needed to create an accurate simulation. In this research, kinetic studies of E. coli BPPTCC-EgRK2 growth were examined with the Monod approach to get the Monod constant (Ks) and maximum specific growth rate (μmax). This study found that E. coli BPPTCC-EgRK2 have μmax and Ks of 1.581 and 0.0709 respectively. BPPTCC-EgRK2 produced intracellular cellulase, thus gave linear correlation between cell concentration and cellulase production.

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

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

Saravanan, P; Muthuvelayudham, R; Viruthagiri, T

2012-01-01

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

Application of Molecular Imprinted Magnetic Fe3O4@SiO2 Nanoparticles for Selective Immobilization of Cellulase.

PubMed

Tao, Qing-Lan; Li, Yue; Shi, Ying; Liu, Rui-Jiang; Zhang, Ye-Wang; Guo, Jianyong

2016-06-01

Magnetic Fe3O4@SiO2 nanoparticles were prepared with molecular imprinting method using cellulase as the template. And the surface of the nanoparticles was chemically modified with arginine. The prepared nanoparticles were used as support for specific immobilization of cellulase. SDS-PAGE results indicated that the adsorption of cellulase onto the modified imprinted nanoparticles was selective. The immobilization yield and efficiency were obtained more than 70% after the optimization. Characterization of the immobilized cellulase revealed that the immobilization didn't change the optimal pH and temperature. The half-life of the immobilized cellulase was 2-fold higher than that of the free enzyme at 50 degrees C. After 7 cycles reusing, the immobilized enzyme still retained 77% of the original activity. These results suggest that the prepared imprinted nanoparticles have the potential industrial applications for the purification or immobilization of enzymes.

Immobilization of cellulase on a silica gel substrate modified using a 3-APTES self-assembled monolayer

DOE PAGES

Zhang, Dezhi; Hegab, Hisham E.; Lvov, Yuri; ...

2016-01-20

Cellulase was immobilized onto silica gel surfaces pretreated with (3-aminopropyl) triethoxy-silane (3-APTES), and glutaraldehyde (GA) was used as a cross-linker. A carboxymethyl cellulose sodium salt (CMC) solution was used for activity experiments. Protein assay was performed to determine the mass immobilized and compare with free enzyme. Cellulase was successfully demonstrated to be immobilized on the modified silica gel surface, and no detectable amount of enzyme was stripped off during the hydrolysis of the CMC solution. The specific activity of the immobilized cellulase is 7 ± 2 % compared to the similar amount of free cellulase. Significant activity over multiple reusesmore » was observed. The seventh batch achieved 82 % activity of the initial batch, and the fifteenth batch retained 31 %. Lastly, it was observed that the immobilized cellulase retained 48 % of its initial activity after 4 days, and 22 % even after 14 days.« less

A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production.

PubMed

Li, Chengcheng; Lin, Fengming; Li, Yizhen; Wei, Wei; Wang, Hongyin; Qin, Lei; Zhou, Zhihua; Li, Bingzhi; Wu, Fugen; Chen, Zhan

2016-09-01

The conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low β-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial application. Thus, there are ongoing interests in research to develop methods to overcome this insufficiency. Moreover, although β-glucosidases have been demonstrated to influence cellulase production and participate in the regulation of cellulase production, the underlying mechanism remains unclear. The T. reesei recombinant strain TRB1 was constructed from T. reesei RUT-C30 by the T-DNA-based mutagenesis. Compared to RUT-C30, TRB1 displays a significant enhancement of extracellular β-glucosidase (BGL1) activity with 17-fold increase, a moderate increase of both the endoglucanase (EG) activity and the exoglucanase (CBH) activity, a minor improvement of the total filter paper activity, and a faster cellulase induction. This superiority of TRB1 over RUT-C30 is independent on carbon sources and improves the saccharification ability of TRB1 cellulase on pretreated corn stover. Furthermore, TRB1 shows better resistance to carbon catabolite repression than RUT-C30. Secretome characterization of TRB1 shows that the amount of CBH, EG and BGL in the supernatant of T. reesei TRB1 was indeed increased along with the enhanced activities of these three enzymes. Surprisingly, qRT-PCR and gene cloning showed that in TRB1 β-glucosidase cel3D was mutated through the random insertion by AMT and was not expressed. The T. reesei recombinant strain TRB1 constructed in this study is more desirable for industrial application than the parental strain RUT-C30, showing extracellular β-glucosidase hyper production, high cellulase production within a shorter time and a better resistance to carbon catabolite repression. Disruption of β-glucosidase cel3D in TRB1 was identified, which might contribute to the superiority of TRB1 over RUT-C30 and might play a role in the cellulase production. These results laid a foundation for future investigations to further improve cellulase enzymatic efficiency and reduce cost for T. reesei cellulase production.

A coarse-grained model for synergistic action of multiple enzymes on cellulose

DOE PAGES

Asztalos, Andrea; Daniels, Marcus; Sethi, Anurag; ...

2012-08-01

In this study, degradation of cellulose to glucose requires the cooperative action of three classes of enzymes, collectively known as cellulases. Endoglucanases randomly bind to cellulose surfaces and generate new chain ends by hydrolyzing -1,4-D-glycosidic bonds. Exoglucanases bind to free chain ends and hydrolyze glycosidic bonds in a processive manner releasing cellobiose units. Then, -glucosidases hydrolyze soluble cellobiose to glucose. Optimal synergistic action of these enzymes is essential for efficient digestion of cellulose. Experiments show that as hydrolysis proceeds and the cellulose substrate becomes more heterogeneous, the overall degradation slows down. As catalysis occurs on the surface of crystalline cellulose,more » several factors affect the overall hydrolysis. Therefore, spatial models of cellulose degradation must capture effects such as enzyme crowding and surface heterogeneity, which have been shown to lead to a reduction in hydrolysis rates. As a result, we present a coarse-grained stochastic model for capturing the key events associated with the enzymatic degradation of cellulose at the mesoscopic level. This functional model accounts for the mobility and action of a single cellulase enzyme as well as the synergy of multiple endo- and exo-cellulases on a cellulose surface. The quantitative description of cellulose degradation is calculated on a spatial model by including free and bound states of both endo- and exo-cellulases with explicit reactive surface terms (e.g., hydrogen bond breaking, covalent bond cleavages) and corresponding reaction rates. The dynamical evolution of the system is simulated by including physical interactions between cellulases and cellulose. In conclusion, our coarse-grained model reproduces the qualitative behavior of endoglucanases and exoglucanases by accounting for the spatial heterogeneity of the cellulose surface as well as other spatial factors such as enzyme crowding. Importantly, it captures the endo-exo synergism of cellulase enzyme cocktails. This model constitutes a critical step towards testing hypotheses and understanding approaches for maximizing synergy and substrate properties with a goal of cost effective enzymatic hydrolysis.« less

Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region.

PubMed

Costa, M G S; Silva, Y F; Batista, P R

2018-03-14

Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

Effect of some variable in cellulase production by Aspergillus niger ITBCC L74 using solid state fermentation

NASA Astrophysics Data System (ADS)

Abdullah, B.; Maftukhah, S.; Listyaningrum, E.; Faradhiba, F.

2018-03-01

Cellulase is a very important enzyme for ethanol production, food, papper, etc, from lignocellulose and others. Rice straw and corn cob are the largest agricultural waste in Indonesia, while the water hyacinth weed is a plant that has not been used optimally. The content of cellulose is high enough on rice straw, water hyacinth and corn corb so it can be used as a substrate in the production of cellulase to increase the economic value of the rice straw, hyacinth, and corncob. As for the purpose of this study is to use the rice straw, water hyacinth, and corn cob as substrates of cellulase enzyme, determine the effect type of substrates, moisture content and fermentation time in production of cellulase enzyme and also determining the optimum conditions for production of cellulase enzymes. The method is solid fermentation system and using fungi Aspergillus niger ITBCC L74 as inoculum. The variable used were fermentation time is 2, 4, 6, 8 and 10 days, moisture content is 50, 60, 70, and 80%, as well as the type of substrate is rice straw, water hyacinth, and corn cob. The results showed that the highest protein content in the crude enzyme of the rice straw, water hyacinth and corncobs @ is 0.0153 mg/ml, 0.0194 mg/ml and 0. 0146 mg/ml, respectively. The optimum enzyme activity were for the rice straw, water hyacinth and corn cobs @ 2.569 U/ml, 1.606 U/ml and 1.302 U/ml, respectively. The optimum moisture content were obtain for rice straw, water hyacinth and corn cob respectively 80%, 70% and 60%. And the optimum fermentation time for rice straw, corn cob, and water hyacinth is on the sixth day. In this study showed the highest enzyme activity on the type of rice straw substrate with a water content of 80% and fermentation time 6 day.

Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

PubMed

Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

2016-10-01

We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.

Nucleo-cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulase and xylanase gene expression in Trichoderma reesei

PubMed Central

Lichius, Alexander; Seidl-Seiboth, Verena; Seiboth, Bernhard; Kubicek, Christian P

2014-01-01

Trichoderma reesei is a model for investigating the regulation of (hemi-)cellulase gene expression. Cellulases are formed adaptively, and the transcriptional activator XYR1 and the carbon catabolite repressor CRE1 are main regulators of their expression. We quantified the nucleo-cytoplasmic shuttling dynamics of GFP-fusion proteins of both transcription factors under cellulase and xylanase inducing conditions, and correlated their nuclear presence/absence with transcriptional changes. We also compared their subcellular localization in conidial germlings and mature hyphae. We show that cellulase gene expression requires de novo biosynthesis of XYR1 and its simultaneous nuclear import, whereas carbon catabolite repression is regulated through preformed CRE1 imported from the cytoplasmic pool. Termination of induction immediately stopped cellulase gene transcription and was accompanied by rapid nuclear degradation of XYR1. In contrast, nuclear CRE1 rapidly decreased upon glucose depletion, and became recycled into the cytoplasm. In mature hyphae, nuclei containing activated XYR1 were concentrated in the colony center, indicating that this is the main region of XYR1 synthesis and cellulase transcription. CRE1 was found to be evenly distributed throughout the entire mycelium. Taken together, our data revealed novel aspects of the dynamic shuttling and spatial bias of the major regulator of (hemi-)cellulase gene expression, XYR1, in T. reesei. PMID:25302561

Evaluation of nanoparticle-immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lupoi, Jason; Smith, Emily

2011-12-01

Ethanol yields were 2.1 (P = 0.06) to 2.3 (P = 0.01) times higher in simultaneous saccharification and fermentation (SSF) reactions of microcrystalline cellulose when cellulase was physisorbed on silica nanoparticles compared to enzyme in solution. In SSF reactions, cellulose is hydrolyzed to glucose by cellulase while yeast simultaneously ferments glucose to ethanol. The 35 C temperature and the presence of ethanol in SSF reactions are not optimal conditions for cellulase. Immobilization onto solid supports can stabilize the enzyme and promote activity at non-optimum reaction conditions. Mock SSF reactions that did not contain yeast were used to measure saccharification productsmore » and identify the mechanism for the improved ethanol yield using immobilized cellulase. Cellulase adsorbed to 40 nm silica nanoparticles produced 1.6 times (P = 0.01) more glucose than cellulase in solution in 96 h at pH 4.8 and 35 C. There was no significant accumulation (<250 {mu}g) of soluble cellooligomers in either the solution or immobilized enzyme reactions. This suggests that the mechanism for the immobilized enzyme's improved glucose yield compared to solution enzyme is the increased conversion of insoluble cellulose hydrolysis products to soluble cellooligomers at 35 C and in the presence of ethanol. The results show that silica-immobilized cellulase can be used to produce increased ethanol yields in the conversion of lignocellulosic materials by SSF.« less

Production Of Cellulase In Plastids Of Transgenic Plants

DOEpatents

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.

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

PubMed Central

2011-01-01

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 cellulase genes into the nematode's biology as predicted by theory. Thus, functional assimilation, remarkable gene turnover and selection might represent key features of horizontal gene transfer events in nematodes. PMID:21232122

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

PubMed

Mayer, Werner E; Schuster, Lisa N; Bartelmes, Gabi; Dieterich, Christoph; Sommer, Ralf J

2011-01-13

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. 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. We could demonstrate functional integration of acquired cellulase genes into the nematode's biology as predicted by theory. Thus, functional assimilation, remarkable gene turnover and selection might represent key features of horizontal gene transfer events in nematodes.

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

PubMed Central

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

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 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. PMID:23213539

Accelerating effects of cellulase in the removal of denture adhesives from acrylic denture bases.

PubMed

Harada-Hada, Kae; Mimura, Sumiyo; Hong, Guang; Hashida, Tatsumi; Abekura, Hitoshi; Murata, Hiroshi; Nishimura, Masahiro; Nikawa, Hiroki

2017-04-01

Studies of effective methods for the easy removal of denture adhesives from a denture base are not well represented in the literature. We previously assessed the removability of denture adhesives by immersing within denture cleaners, showing that some cleaners have a weak effect, insufficiently effective in daily use. In this study, we prepared a cellulase, as a potential component for denture adhesive removers, and we examined whether the addition of cellulase to denture cleaners is effective in the removal of cream denture adhesives. We prepared the cellulase Meicelase as one component for the liquefaction of denture adhesives. We used two denture cleaners and two cream adhesives. After the immersion of plates in sample solutions, we evaluated the area of the sample plate still covered with adhesives. Biofilm removal assay was also performed using denture cleaners containing cellulase. The addition of cellulase accelerated the removal of cream adhesives in immersion experiments to a rate faster than that of water and denture cleaners. However, it did not influence the removability of Candida albicans biofilms from acrylic resin specimens. Cellulase hastened the liquefaction of cream adhesives. Copyright © 2016 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

Novel Magnetic Cross-Linked Cellulase Aggregates with a Potential Application in Lignocellulosic Biomass Bioconversion.

PubMed

Jia, Junqi; Zhang, Weiwei; Yang, Zengjie; Yang, Xianling; Wang, Na; Yu, Xiaoqi

2017-02-10

The utilization of renewable biomass resources to produce high-value chemicals by enzymatic processes is beneficial for alternative energy production, due to the accelerating depletion of fossil fuels. As immobilization techniques can improve enzyme stability and reusability, a novel magnetic cross-linked cellulase aggregate has been developed and applied for biomass bioconversion. The crosslinked aggregates could purify and immobilize enzymes in a single operation, and could then be combined with magnetic nanoparticles (MNPs), which provides easy separation of the materials. The immobilized cellulase showed a better activity at a wider temperature range and pH values than that of the free cellulase. After six cycles of consecutive reuse, the immobilized cellulase performed successful magnetic separation and retained 74% of its initial activity when carboxylmethyl cellulose (CMC) was used as the model substrate. Furthermore, the structure and morphology of the immobilized cellulase were studied by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Moreover, the immobilized cellulase was shown to hydrolyze bamboo biomass with a yield of 21%, and was re-used in biomass conversion up to four cycles with 38% activity retention, which indicated that the immobilized enzyme has good potential for biomass applications.

Method of saccharifying cellulose

DOEpatents

Johnson, Eric A.; Demain, Arnold L.; Madia, Ashwin

1985-09-10

A method of saccharifying cellulose by incubation with the cellulase of Clostridium thermocellum in a broth containing an efficacious amount of a reducing agent. Other incubation parameters which may be advantageously controlled to stimulate saccharification include the concentration of alkaline earth salts, pH, temperature, and duration. By the method of the invention, even native crystalline cellulose such as that found in cotton may be completely saccharified.

On the conflicting findings of Role of Cellulose-Crystallinity in Enzume Hydrolysis of Biomass

Treesearch

Umesh Agarwal; Sally Ralph

2014-01-01

In the field of conversion of biomass to ethanol, an important area of research is the enzymatic hydrolysis of cellulose. Once cellulose is converted to glucose, it can be easily fermented to ethanol. As the cellulosic ethanol technology stands now, costly pretreatments and high dosages of cellulases are needed to achieve complete hydrolysis of the cellulose fraction...

Recovery and reuse of cellulase catalyst in an exzymatic cellulose hydrolysis process

DOEpatents

Woodward, Jonathan

1989-01-01

A process for recovering cellulase from the hydrolysis of cellulose, and reusing it in subsequent hydrolyois procedures. The process utilizes a commercial adsorbent that efficiently removes cellulase from reaction products which can be easily removed by simple decantation.

A possible water-soluble inducer for synthesis of cellulase in Aspergillus niger.

PubMed

Zhang, Jian-Guo; Li, Qi-Meng; Thakur, Kiran; Faisal, Shah; Wei, Zhao-Jun

2017-02-01

The synthesis of cellulase in filamentous fungi can be triggered by several inducers. In this study, a bamboo-shoot shell pretreated with Pleurotus ostreatus could promote the formation of cellulases in Aspergillus niger. Further identification, including UPLC-TOF-MS, ultrafiltration, and FT-IR, denoted that the soluble inducer was not a traditional disaccharide but a type of modified lignin polymer. This revelation may result in incipient strategies to ameliorate cellulase productivity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Production of β-xylosidase from Trichoderma asperellum KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase.

PubMed

Inoue, Hiroyuki; Kitao, Chiaki; Yano, Shinichi; Sawayama, Shigeki

2016-11-01

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of β-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high β-xylosidase dominance. β-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 β-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48 h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.

Roles of Protein Kinase A and Adenylate Cyclase in Light-Modulated Cellulase Regulation in Trichoderma reesei

PubMed Central

Schuster, André; Tisch, Doris; Seidl-Seiboth, Verena; Kubicek, Christian P.

2012-01-01

The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process. PMID:22286997

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2013-01-01

Cellulose digestion in termites (Isoptera) is highly important for ecological reasons and applications in biofuel conversion. The speciose Termitidae family has lost flagellates in the hindgut and developed diverse feeding habits. To address the response of cellulase activity to the differentiation of feeding habits, a comparative study of the activity and distribution of composite cellulases, endo-β-1, 4-glucanase, and β-glucosidase was performed in seven common flagellate-free termites with three feeding habits: the humus-feeding termites Sinocapritermes mushae (Oshima et Maki), Malaysiocapritermes zhangfengensis Zhu, Yang et Huang and Pericapritermes jiangtsekiangensis (Kemner); the fungus-growing termites Macrotermes barneyi Light and Odontotermes formosanus (Shiraki); and the wood-feeding termites Nasutitermes parvonasutus (Shiraki) and Havilanditermes orthonasus (Tsai et Chen). The results showed that in diverse feeding groups, the wood-feeding group had the highest total composite cellulase and endo-β-1, 4-glucanase activities, while the fungus-growing group had the highest β-glucosidase activity. In terms of the distribution of cellulase activity in the alimentary canals, the cellulase activities in wood-feeding termites were concentrated in the midgut, but there was no significant difference between all gut segments in humus-feeding termites. As for the fungus-growing termites, the main site of composite cellulase activity was in the midgut. The endo-β-1, 4-glucanase activity was restricted to the midgut, but the primary site of β-glucosidase activity was in the foregut and the midgut (Mac. barneyi). The functions of the gut segments apparently differentiated between feeding groups. The results suggest that the differentiation of feeding habits in flagellate-free termites was characterized by the distribution of cellulases in the gut rather than by variations in cellulase activity. PMID:23895662

Ethanol production from ensiled rice straw and whole-crop silage by the simultaneous enzymatic saccharification and fermentation process.

PubMed

Shinozaki, Yukiko; Kitamoto, Hiroko K

2011-03-01

Silage production from rice straw and whole-plant forage paddy rice is increasing in Japan because of decrease in rice consumption. One potential use for this silage is bioethanol production. In this study, we analyzed the effectiveness of three different commercially available cellulases at saccharification of sun-dried rice straw, ensiled rice straw, and rice whole-crop silage (WCS). Furthermore, the ethanol productivity of the simultaneous saccharification and ethanol fermentation process (SSF) from the same plant substrates was analyzed. Among the three kinds of cellulases tested (Novozymes NS50013, Genencor GC220, and Acremonium cellulase), Acremonium cellulase showed the highest ethanol production for the three plant substrates, and the WCS produced the highest ethanol level. Analysis of the enzymatic degradation activity of the cellulases revealed that Acremonium cellulase contained remarkably high glucoamylase and pectinase side activities relative to the other cellulase preparations. The addition of glucoamylase and pectinase to the other two cellulases significantly increased ethanol productivity to levels observed for the Acremonium cellulase preparation, which showed little enhanced performance with the addition of the same enzymes. Finally, we tested whether milling and sterilization had an effect on ethanol production and found that sterilized silage produced higher ethanol levels but that the milling process had no significant effect. These results show that (i) silage made from whole-plant rice can be used for bioethanol production and (ii) the proper selection and combination of commercially available enzymes can make SSF more cost efficient by removing the need for a pre-treatment step. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Reliable simultaneous zymographic method of characterization of cellulolytic enzymes from fungal cellulase complex.

PubMed

Dojnov, Biljana; Grujić, Marica; Vujčić, Zoran

2015-08-01

A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4-methylumbelliferyl-β-d-cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed Central

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

2012-01-01

Optimization of the culture medium for cellulase production using Trichoderma reesei was carried out. The optimization of cellulase production using mango peel as substrate was performed with statistical methodology based on experimental designs. The screening of nine nutrients for their influence on cellulase production is achieved using Plackett-Burman design. Avicel, soybean cake flour, KH2PO4, and CoCl2 ·6H2O were selected based on their positive influence on cellulase production. The 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. PMID:23304453

Measurement of filter paper activities of cellulase with microplate-based assay.

PubMed

Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

2016-01-01

It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC.

Measurement of filter paper activities of cellulase with microplate-based assay

PubMed Central

Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

2015-01-01

It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC. PMID:26858572

Lignin-based polyoxyethylene ether enhanced enzymatic hydrolysis of lignocelluloses by dispersing cellulase aggregates.

PubMed

Lin, Xuliang; Qiu, Xueqing; Yuan, Long; Li, Zihao; Lou, Hongming; Zhou, Mingsong; Yang, Dongjie

2015-06-01

Water-soluble lignin-based polyoxyethylene ether (EHL-PEG), prepared from enzymatic hydrolysis lignin (EHL) and polyethylene glycol (PEG1000), was used to improve enzymatic hydrolysis efficiency of corn stover. The glucose yield of corn stover at 72h was increased from 16.7% to 70.1% by EHL-PEG, while increase in yield with PEG4600 alone was 52.3%. With the increase of lignin content, EHL-PEG improved enzymatic hydrolysis of microcrystalline cellulose more obvious than PEG4600. EHL-PEG could reduce at least 88% of the adsorption of cellulase on the lignin film measured by quartz crystal microbalance with dissipation monitoring (QCM-D), while reduction with PEG4600 was 43%. Cellulase aggregated at 1220nm in acetate buffer analyzed by dynamic light scattering. EHL-PEG dispersed cellulase aggregates and formed smaller aggregates with cellulase, thereby, reduced significantly nonproductive adsorption of cellulase on lignin and enhanced enzymatic hydrolysis of lignocelluloses. Copyright © 2015 Elsevier Ltd. All rights reserved.

Undefined cellulase formulations hinder scientific reproducibility

DOE PAGES

Himmel, Michael E.; Abbas, Charles A.; Baker, John O.; ...

2017-11-28

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparationsmore » may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.« less

Benefits from additives and xylanase during enzymatic hydrolysis of bamboo shoot and mature bamboo.

PubMed

Li, Kena; Wang, Xiao; Wang, Jingfeng; Zhang, Junhua

2015-09-01

Effects of additives (BSA, PEG 6000, and Tween 80) on enzymatic hydrolysis of bamboo shoot and mature bamboo fractions (bamboo green, bamboo timber, bamboo yellow, bamboo node, and bamboo branches) by cellulases and/or xylanase were evaluated. The addition of additives was comparable to the increase of cellulase loadings in the conversion of cellulose and xylan in bamboo fractions. Supplementation of xylanase (1 mg/g DM) with cellulases (10 FPU/g DM) in the hydrolysis of bamboo fractions was more efficient than addition of additives in the production of glucose and xylose. Moreover, addition of additives could further increase the glucose release from different bamboo fractions by cellulases and xylanase. Bamboo green exhibited the lowest hydrolyzability. Almost all of the polysaccharides in pretreated bamboo shoot fractions were hydrolyzed by cellulases with the addition of additives or xylanase. Additives and xylanase showed great potential for reducing cellulase requirement in the hydrolysis of bamboo. Copyright © 2015 Elsevier Ltd. All rights reserved.

Optimization of a natural medium for cellulase by a marine Aspergillus niger using response surface methodology.

PubMed

Xue, Dong-Sheng; Chen, Hui-Yin; Lin, Dong-Qiang; Guan, Yi-Xin; Yao, Shan-Jing

2012-08-01

The components of a natural medium were optimized to produce cellulase from a marine Aspergillus niger under solid state fermentation conditions by response surface methodology. Eichhornia crassipes and natural seawater were used as a major substrate and a source of mineral salts, respectively. Mineral salts of natural seawater could increase cellulase production. Raw corn cob and raw rice straw showed a significant positive effect on cellulase production. The optimum natural medium consisted of 76.9 % E. crassipes (w/w), 8.9 % raw corn cob (w/w), 3.5 % raw rice straw (w/w), 10.7 % raw wheat bran (w/w), and natural seawater (2.33 times the weight of the dry substrates). Incubation for 96 h in the natural medium increased the biomass to the maximum. The cellulase production was 17.80 U/g the dry weight of substrates after incubation for 144 h. The natural medium avoided supplying chemicals and pretreating substrates. It is promising for future practical fermentation of environment-friendly producing cellulase.

High consistency cellulase treatment of hardwood prehydrolysis kraft based dissolving pulp.

PubMed

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

2015-01-01

For enzymatic treatment of dissolving pulp, there is a need to improve the process to facilitate its commercialization. For this purpose, the high consistency cellulase treatment was conducted based on the hypothesis that a high cellulose concentration would favor the interactions of cellulase and cellulose, thus improves the cellulase efficiency while decreasing the water usage. The results showed that compared with a low consistency of 3%, the high consistency of 20% led to 24% increases of cellulase adsorption ratio. As a result, the viscosity decrease and Fock reactivity increase at consistency of 20% were enhanced from 510 mL/g and 70.3% to 471 mL/g and 77.6%, respectively, compared with low consistency of 3% at 24h. The results on other properties such as alpha cellulose, alkali solubility and molecular weight distribution also supported the conclusion that a high consistency of cellulase treatment was more effective than a low pulp consistency process. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

2017-01-01

The effect of sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) on composition, structure, enzymatic hydrolysis and cellulase adsorption profiles of sugarcane bagasse (SCB) was investigated. SPORL gave a higher SCB hydrolysis yield (85.33%) compared to dilute acid pretreatment (DA) (64.39%). The SEM pictures showed that SPORL SCB structure became more disordered and looser, suggesting SPORL SCB was more accessible to cellulase. The zeta potential of SPORL SCB suspension (-21.89mV) was significantly different from that of DA SCB (-12.87mV), which demonstrated the lignin in SPORL SCB was more hydrophilic. With regard to cellulase adsorption profiles, SPORL SCB had a lower non-productive adsorption (14.87mg/glignin) and a higher productive adsorption (37.67 mg/gcarbohydrate) compared with DA SCB (17.05mg/glignin; 25.79mg/gcarbohydrate). These results indicated that SPORL SCB had better accessibility to cellulase and the higher productive cellulase adsorption of SPORL SCB had improved hydrolysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Process for recovering hydrocarbons from hydrocarbon-containing biomass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dzadzic, P.M.; Price, M.C.; Shih, C.J.

1982-07-06

A process is disclosed for enzymatically converting whole plant biomass containing hydrocarbon-containing laticifers to soluble sugars and recovering hydrocarbons in increased yields. The process comprises hydrolyzing whole plant cellulosic material in the presence of enzymes, particularly cellulase, hemicellulase, and pectinase, to produce a hydrocarbon product and recovering from the hydrolysis products a major proportion of the cellulase, hemicellulase and pectinase enzymes for reuse. At least some portion of the required make-up of cellulase, hemicellulase and pectinase enzymes is produced in a two-stage operation wherein, in the first stage, a portion of the output sugar solution is used to grow enzymemore » secreting microorganisms selected from the group consisting of cellulase-secreting microorganisms, hemicellulase-secreting microorganisms, pectinase-secreting microorganisms, and mixtures thereof, and in the second stage, cellulase, hemicellulase and pectinase enzyme formation is induced in the microorganism-containing culture medium by the addition of an appropriate inducer such as biomass. The cellulase, hemicellulase and pectinase enzymes are then recycled for use in the hydrolysis reaction.« less

Bioethanol production from wheat straw via enzymatic route employing Penicillium janthinellum cellulases.

PubMed

Singhania, Reeta Rani; Saini, Jitendra Kumar; Saini, Reetu; Adsul, Mukund; Mathur, Anshu; Gupta, Ravi; Tuli, Deepak Kumar

2014-10-01

This study concerns in-house development of cellulases from a mutant Penicillium janthinellum EMS-UV-8 and its application in separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes for bioethanol production from pre-treated wheat straw. In a 5L fermentor, the above strain could produce cellulases having activity of 3.1 FPU/mL and a specific activity of 0.83 FPU/mg of protein. In-house developed cellulase worked more efficiently in case of SSF as ethanol concentration of 21.6g/L and yield of 54.4% were obtained which were higher in comparison to SHF (ethanol concentration 12 g/L and 30.2% yield). This enzyme preparation when compared with commercial cellulase for hydrolysis of pre-treated wheat straw was found competitive. This study demonstrates that P. janthinellum EMS-UV-8 is a potential fungus for future large-scale production of cellulases. Copyright © 2014 Elsevier Ltd. All rights reserved.

Undefined cellulase formulations hinder scientific reproducibility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Himmel, Michael E.; Abbas, Charles A.; Baker, John O.

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparationsmore » may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.« less

Dehydrogenase GRD1 Represents a Novel Component of the Cellulase Regulon in Trichoderma reesei (Hypocrea jecorina) ▿ † §

PubMed Central

Schuster, André; Kubicek, Christian P.; Schmoll, Monika

2011-01-01

Trichoderma reesei (Hypocrea jecorina) is nowadays the most important industrial producer of cellulase and hemicellulase enzymes, which are used for pretreatment of cellulosic biomass for biofuel production. In this study, we introduce a novel component, GRD1 (glucose-ribitol dehydrogenase 1), which shows enzymatic activity on cellobiose and positively influences cellulase gene transcription, expression, and extracellular endo-1,4-β-d-glucanase activity. grd1 is differentially transcribed upon growth on cellulose and the induction of cellulase gene expression by sophorose. The transcription of grd1 is coregulated with that of cel7a (cbh1) under inducing conditions. GRD1 is further involved in carbon source utilization on several carbon sources, such as those involved in lactose and d-galactose catabolism, in several cases in a light-dependent manner. We conclude that GRD1 represents a novel enhancer of cellulase gene expression, which by coregulation with the major cellulase may act via optimization of inducing mechanisms. PMID:21602376

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

PubMed

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

2017-08-01

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

Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose

PubMed Central

Zhou, Qingxin; Xu, Jintao; Kou, Yanbo; Lv, Xinxing; Zhang, Xi; Zhao, Guolei; Zhang, Weixin; Chen, Guanjun

2012-01-01

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular β-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular β-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three β-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three β-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals. PMID:23002106

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies.

PubMed

Kumar, Rajeev; Wyman, Charles E

2009-09-01

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO(2)) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with beta-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and beta-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and beta-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase-xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.

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

PubMed

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

2016-12-01

Natural lactic acid bacteria (LAB) populations in tropical grasses and their fermentation characteristics on silage prepared with cellulase enzyme and LAB inoculants were studied. A commercial inoculant Lactobacillus plantarum Chikuso 1 (CH), a local selected strain Lactobacillus casei TH14 (TH14), and 2 cellulases, Acremonium cellulase (AC) and Maicelase (MC; Meiji Seika Pharma Co. Ltd., Tokyo, Japan), were used as additives to silage preparation with fresh and wilted (6 h) Guinea grass and Napier grass. Silage was prepared using a laboratory-scale fermentation system. Treatments were CH, TH14, AC at 0.01% fresh matter, AC 0.1%, MC 0.01%, MC 0.1%, CH+AC 0.01%, CH+AC 0.1%, CH+MC 0.01%, CH+MC 0.1%, TH14+AC 0.1%, TH14+AC 0.01%, TH14+MC 0.1%, and TH14+MC 0.01%. Microorganism counts of Guinea grass and Napier grass before ensiling were 10 2 LAB and 10 6 aerobic bacteria; these increased during wilting. Based on morphological and biochemical characteristics, and 16S rRNA gene sequence analysis, natural strains from both grasses were identified as L. plantarum, L. casei, Lactobacillus acidipiscis, Leuconostoc pseudomesenteroides, Leuconostoc garlicum, Weissella confusa, and Lactococcus lactis. Lactobacillus plantarum and L. casei are the dominant species and could grow at lower pH and produce more lactic acid than the other isolates. Crude protein and neutral detergent fiber were 5.8 and 83.7% of dry matter (DM) for Guinea grass, and 7.5 and 77.1% of DM for Napier grass. Guinea grass had a low level of water-soluble carbohydrates (0.39% of DM). Guinea grass silage treated with cellulase had a lower pH and higher lactic acid content than control and LAB treatments. The 0.1% AC and MC treatments had the best result for fermentation quality. All high water-soluble carbohydrate (2.38% DM) Napier grass silages showed good fermentation quality. Compared with control and LAB-inoculated silage, the cellulase-treated silages had significantly higher crude protein content and lower neutral detergent fiber and acid detergent fiber contents. The results confirmed that cellulase could improve tropical silage quality, inhibiting protein degradation and promoting fiber degradation. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Recovery and reuse of cellulase catalyst in an enzymatic cellulose hydrolysis process

DOEpatents

Woodward, J.

1987-09-18

A process for recovering cellulase from the hydrolysis of cellulose, and reusing it in subsequent hydrolyois procedures. The process utilizes a commercial adsorbent that efficiently removes cellulase from reaction products which can be easily removed by simple decantation. 1 fig., 4 tabs.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1979-01-01

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

Bioethanol potentials of corn cob hydrolysed using cellulases of Aspergillus niger and Penicillium decumbens.

PubMed

Saliu, Bolanle Kudirat; Sani, Alhassan

2012-01-01

Corn cob is a major component of agricultural and domestic waste in many parts of the world. It is composed mainly of cellulose which can be converted to energy in form of bioethanol as an efficient and effective means of waste management. Production of cellulolytic enzymes were induced in the fungi Aspergillus niger and Penicillium decumbens by growing them in mineral salt medium containing alkali pre-treated and untreated corn cobs. The cellulases were characterized and partially purified. Alkali pre-treated corn cobs were hydrolysed with the partially purified cellulases and the product of hydrolysis was fermented using the yeast saccharomyces cerevisae to ethanol. Cellulases of A. niger produced higher endoglucanase and exoglucanase activity (0.1698 IU ml(-1) and 0.0461 FPU ml(-1)) compared to that produced by P. decumbens (0.1111 IU ml(-1) and 0.153 FPU ml(-1)). Alkali pre-treated corn cob hydrolysed by cellulases of A. niger yielded 7.63 mg ml(-1) sugar which produced 2.67 % (v/v) ethanol on fermentation. Ethanol yield of the hydrolysates of corn cob by cellulases of P. decumbens was much lower at 0.56 % (v/v). Alkali pre-treated corn cob, hydrolysed with cellulases of A. niger is established as suitable feedstock for bioethanol production.

The effect of leachate recirculation with enzyme cellulase addition on waste stability in landfill bioreactor

NASA Astrophysics Data System (ADS)

Saffira, N.; Kristanto, G. A.

2018-01-01

Landfill bioreactor with leachate recirculation is known to enhance waste stabilization. However, the composition of waste in Indonesia is comprised by organic waste which is lignocellulosic materials that considered take a long time to degrade under anaerobic condition. To accelerate the degradation process, enzyme addition is ought to do. Cellulase is an enzyme that can catalyse cellulose and other polysaccharide decomposition processes. Therefore, operation of waste degradation using leachate recirculation with a cellulase addition to enhance waste stabilization was investigated using anaerobic bioreactor landfill. The experiment was performed on 2 conditions; leachate recirculation with cellulase addition and recirculation only as a control. The addition of cellulase is reported to be significant in decreasing organic content, represented by volatile solid parameter. The volatile solid reduction in the cellulase augmented reactor and control reactor was 17.86% and 7.90%, respectively. Cellulase addition also resulted in the highest cellulose reduction. Settlement of the landfill in a bioreactor with enzyme addition (32.67%) was reported to be higher than the control (19.33%). Stabilization of landfill review by the decreasing rate constant of the cellulose and lignin ratio parameter was more rapidly achieved by the enzyme addition (0.014 day-1) compared to control (0.002 day-1).

Impact of enzyme loading on the efficacy and recovery of cellulolytic enzymes immobilized on enzymogel nanoparticles.

PubMed

Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

2015-03-01

Cellulase and β-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 μg (protein) mg(-1) (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53%, respectively, of concentrations when using free cellulase. β-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38% of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for β-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and β-glucosidases present in cellulase mixtures. When loading β-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96% of cellulase and 66 % of β-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.

Fluorescent cellulose microfibrils as substrate for the detection of cellulase activity.

PubMed

Helbert, William; Chanzy, Henri; Husum, Tommy Lykke; Schülein, Martin; Ernst, Steffen

2003-01-01

To devise a sensitive cellulase assay based on substrates having most of the physical characteristics of native cellulose, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was used as a grafting agent to prepare suspensions of fluorescent microfibrils from bacterial cellulose. These suspensions were digested by a series of commercially relevant cellulases from Humicola insolens origin: cloned Cel6B and Cel 45A as well as crude H. insolens complex. The digestion induced the release of fluorescent cellodextrins as well as reducing sugars. After adequate centrifugation, these soluble products were analyzed as a function of grafting content, digestion time, and cellulase characteristics. The resulting data allowed the grafting conditions to be optimized in order to maximize the quantity of soluble products and therefore to increase the sensitivity of the detection. A comparison between the amount of released fluorescence and that of released reducing sugar allowed the differentiation between processive exo and endo cellulase activities. The casting of films of DTAF-grafted microfibrils at the bottom of the microwell titer plates also led to sensitive cellulase detection. As these films kept their integrity and remained firmly glued to the well bottom during the digestion time, they are tailored made for a full automation of the cellulases testing.

Salicylic Acid and Ethylene Pathways Are Differentially Activated in Melon Cotyledons by Active or Heat-Denatured Cellulase from Trichoderma longibrachiatum

PubMed Central

Martinez, Christelle; Blanc, Frédéric; Le Claire, Emilie; Besnard, Olivier; Nicole, Michel; Baccou, Jean-Claude

2001-01-01

Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Co-infiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease. PMID:11553761

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

NASA Technical Reports Server (NTRS)

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.

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

NASA Astrophysics Data System (ADS)

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.

Method of saccharifying cellulose

DOEpatents

Johnson, E.A.; Demain, A.L.; Madia, A.

1983-05-13

A method is disclosed of saccharifying cellulose by incubation with the cellulase of Clostridium thermocellum in a broth containing an efficacious amount of thiol reducing agent. Other incubation parameters which may be advantageously controlled to stimulate saccharification include the concentration of alkaline earth salts, pH, temperature, and duration. By the method of the invention, even native crystalline cellulose such as that found in cotton may be completely saccharified.

Lipopeptide produced from Bacillus sp. W112 improves the hydrolysis of lignocellulose by specifically reducing non-productive binding of cellulases with and without CBMs.

PubMed

Liu, Jiawen; Zhu, Ning; Yang, Jinshui; Yang, Yi; Wang, Ruonan; Liu, Liang; Yuan, Hongli

2017-01-01

Surfactants have attracted increasing interest for their capability to improve the enzymatic hydrolysis of lignocellulosic biomass. Compared to chemical surfactants, biosurfactants have a broader prospect for industrial applications because they are more environmentally friendly and more effective in some researches. Commercial cellulase preparations are mainly composed of endoglucanases (EGs) and cellobiohydrolases (CBHs) that possess carbohydrate-binding modules (CBMs). However, the effects of lipopeptide-type biosurfactants on enzymatic saccharification of lignocellulose and adsorption behaviors of cellulases with CBMs remain unclear. In this study, we found that Bacillus sp. W112 could produce a lipopeptide-type biosurfactant from untreated biomass, such as wheat bran and Jerusalem artichoke tuber. The lipopeptide could enhance the enzymatic hydrolysis of dilute acid pretreated Giant Juncao grass (DA-GJG) by fungal and bacterial enzymes. The enhancement increased over a range of temperatures from 30 to 50 °C. Lipopeptide was shown to be more effective in promoting DA-GJG saccharification than chemical surfactants at low dosages, with a best stimulatory degree of 20.8% at 2% loading of the substrates (w/w). Lipopeptide increased the thermostability of EG and CBH in commercial cellulase cocktails. Moreover, the dual effects of lipopeptide on the adsorption behaviors of cellulases were found. It specifically lowered the non-productive binding of cellulases to lignin and increased the binding of cellulases to cellulose. In addition, we investigated the influence of lipopeptide on the adsorption behaviors of CBHs with CBMs for the first time. Our results showed that lipopeptide reduced the adsorption of CBM-deleted CBH to DA-GJG to a greater extent than that of intact CBH while the non-productive binding of intact CBH to lignin was reduced more, indicating that lipopeptide decreased the binding of CBMs onto lignin but not their combination with cellulose. In this study, we found that lipopeptide from Bacillus sp. W112 promoted the enzymatic hydrolysis of DA-GJG at relative low loadings. The stimulatory effect could be attributed to increasing the cellulase thermostability, reducing non-productive adsorption of cellulases with CBMs caused by lignin and enhancing the binding of cellulases to cellulose.

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

NASA Astrophysics Data System (ADS)

Rahman, Mohammad; Inoue, Akira; Ojima, Takao

2014-08-01

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

Process relevant screening of cellulolytic organisms for consolidated bioprocessing.

PubMed

Antonov, Elena; Schlembach, Ivan; Regestein, Lars; Rosenbaum, Miriam A; Büchs, Jochen

2017-01-01

Although the biocatalytic conversion of cellulosic biomass could replace fossil oil for the production of various compounds, it is often not economically viable due to the high costs of cellulolytic enzymes. One possibility to reduce costs is consolidated bioprocessing (CBP), integrating cellulase production, hydrolysis of cellulose, and the fermentation of the released sugars to the desired product into one process step. To establish such a process, the most suitable cellulase-producing organism has to be identified. Thereby, it is crucial to evaluate the candidates under target process conditions. In this work, the chosen model process was the conversion of cellulose to the platform chemical itaconic acid by a mixed culture of a cellulolytic fungus with Aspergillus terreus as itaconic acid producer. Various cellulase producers were analyzed by the introduced freeze assay that measures the initial carbon release rate, quantifying initial cellulase activity under target process conditions. Promising candidates were then characterized online by monitoring their respiration activity metabolizing cellulose to assess the growth and enzyme production dynamics. The screening of five different cellulase producers with the freeze assay identified Trichoderma   reesei and Penicillium   verruculosum as most promising. The measurement of the respiration activity revealed a retarded induction of cellulase production for P.   verruculosum but a similar cellulase production rate afterwards, compared to T.   reesei . The freeze assay measurement depicted that P.   verruculosum reaches the highest initial carbon release rate among all investigated cellulase producers. After a modification of the cultivation procedure, these results were confirmed by the respiration activity measurement. To compare both methods, a correlation between the measured respiration activity and the initial carbon release rate of the freeze assay was introduced. The analysis revealed that the different initial enzyme/cellulose ratios as well as a discrepancy in cellulose digestibility are the main differences between the two approaches. With two complementary methods to quantify cellulase activity and the dynamics of cellulase production for CBP applications, T.   reesei and P.   verruculosum were identified as compatible candidates for the chosen model process. The presented methods can easily be adapted to screen for suitable cellulose degrading organisms for various other applications.

Enhanced cellulase recovery without β-glucosidase supplementation for cellulosic ethanol production using an engineered strain and surfactant.

PubMed

Huang, Renliang; Guo, Hong; Su, Rongxin; Qi, Wei; He, Zhimin

2017-03-01

Recycling cellulases by substrate adsorption is a promising strategy for reducing the enzyme cost of cellulosic ethanol production. However, β-glucosidase has no carbohydrate-binding module (CBM). Thus, additional enzymes are required in each cycle to achieve a high ethanol yield. In this study, we report a new method of recycling cellulases without β-glucosidase supplementation using lignocellulosic substrate, an engineered strain expressing β-glucosidase and Tween 80. The cellulases and Tween 80 were added to an aqueous suspension of diluted sulfuric acid/ammonia-treated corncobs in a simultaneous saccharification and fermentation (SSF) process for ethanol production. Subsequently, the addition of fresh pretreated corncobs to the fermentation liquor and remaining solid residue provided substrates with absorbed cellulases for the next SSF cycle. This method provided excellent ethanol production in three successive SSF cycles without requiring the addition of new cellulases. For a 10% (w/v) solid loading, a cellulase dosage of 30 filter paper units (FPU)/g cellulose, 0.5% Tween 80, and 2 g/L of the engineered strain, approximately 90% of the initial ethanol concentration from the first SSF process was obtained in the next two SSF processes, with a total ethanol production of 306.27 g/kg corncobs and an enzyme productivity of 0.044 g/FPU. Tween 80 played an important role in enhancing cellulase recovery. This new enzyme recycling method is more efficient and practical than other reported methods. Biotechnol. Bioeng. 2017;114: 543-551. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

PubMed

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-07-29

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.

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

PubMed Central

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-01-01

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. PMID:19680472

Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

Treesearch

Abduvali Valiev; Zumrut B. Ogel; Dier D. Klepzig

2009-01-01

In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase....

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

PubMed

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

2017-03-01

Medium optimization and repeated fed-batch fermentation were performed to improve the cellulase productivity by P. oxalicum RE-10 in submerged fermentation. First, Plackett-Burman design (PBD) and central composite design (CCD) were used to optimize the medium for cellulase production. PBD demonstrated wheat bran and NaNO 3 had significant influences on cellulase production. The CCD results showed the maximum filter paper activity (FPA) production of 8.61U/mL could be achieved in Erlenmeyer flasks. The maximal FPA reached 12.69U/mL by submerged batch fermentation in a 7.5-L stirred tank, 1.76-fold higher than that on the original medium. Then, the repeated fed-batch fermentation strategy was performed successfully for increasing the cellulase productivity from 105.75U/L/h in batch fermentation to 158.38U/L/h. The cellulase activity and the glucan conversion of delignined corn cob residue hydrolysis had no significant difference between the enzymes sampled from different cycles of the repeated fed-batch fermentation and that from batch culture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune.

PubMed

Metreveli, Eka; Kachlishvili, Eva; Singer, Steven W; Elisashvili, Vladimir

2017-10-01

Mono and dual cultures of four white-rot basidiomycete species were evaluated for cellulase and xylanase activity under submerged fermentation conditions. Co-cultivation of Pycnoporus coccineus or Trametes hirsuta with Schizophyllum commune displayed antagonistic interactions resulting in the decrease of endoglucanase and total cellulase activities. In contrast, increases in cellulase and xylanase activity were revealed through the compatible interactions of Irpex lacteus with S. commune. Co-cultivation conditions were optimized for maximum enzyme production by I. lacteus and S. commune, the best producers of cellulase/xylanase and β-glucosidase, respectively. An optimized medium for the target enzyme production by the mixed culture was established in a laboratory fermenter yielding 7U/mL total cellulase, 142U/mL endoglucanase, 104U/mL xylanase, and 5.2U/mL β-glucosidase. The dual culture approach resulted in an enzymatic mixture with 11% improved lignocellulose saccharification potential compared to enzymes from a monoculture of I. lacteus. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthetic Gene Network with Positive Feedback Loop Amplifies Cellulase Gene Expression in Neurospora crassa.

PubMed

Matsu-Ura, Toru; Dovzhenok, Andrey A; Coradetti, Samuel T; Subramanian, Krithika R; Meyer, Daniel R; Kwon, Jaesang J; Kim, Caleb; Salomonis, Nathan; Glass, N Louise; Lim, Sookkyung; Hong, Christian I

2018-05-18

Second-generation or lignocellulosic biofuels are a tangible source of renewable energy, which is critical to combat climate change by reducing the carbon footprint. Filamentous fungi secrete cellulose-degrading enzymes called cellulases, which are used for production of lignocellulosic biofuels. However, inefficient production of cellulases is a major obstacle for industrial-scale production of second-generation biofuels. We used computational simulations to design and implement synthetic positive feedback loops to increase gene expression of a key transcription factor, CLR-2, that activates a large number of cellulases in a filamentous fungus, Neurospora crassa. Overexpression of CLR-2 reveals previously unappreciated roles of CLR-2 in lignocellulosic gene network, which enabled simultaneous induction of approximately 50% of 78 lignocellulosic degradation-related genes in our engineered Neurospora strains. This engineering results in dramatically increased cellulase activity due to cooperative orchestration of multiple enzymes involved in the cellulose degradation pathway. Our work provides a proof of principle in utilizing mathematical modeling and synthetic biology to improve the efficiency of cellulase synthesis for second-generation biofuel production.

Synergism of cellulase, xylanase, and pectinase on hydrolyzing sugarcane bagasse resulting from different pretreatment technologies.

PubMed

Li, Jingbo; Zhou, Pengfei; Liu, Hongmei; Xiong, Chunjiang; Lin, Jianghai; Xiao, Wenjuan; Gong, Yingxue; Liu, Zehuan

2014-03-01

Sugarcane bagasse (SCB) resulting from different pretreatments was hydrolyzed by enzyme cocktails based on replacement of cellulase (Celluclast 1.5 L:Novozym 188=1FPU:4pNPGU) by xylanase or pectinase at different proportions. Lignin content of NaOH pretreated SCB and hemicellulose content of H2SO4 pretreated SCB were the lowest. NaOH pretreatment showed the best for monosaccharide production among the four pretreatments. Synergism was apparently observed between cellulase and xylanase for monosaccharide production from steam exploded SCB (SESB), NaOH, and H2O2 pretreated SCB. No synergism was observed between cellulase and pectinase for producing glucose. Additionally, no synergism was present when H2SO4 pretreated SCB was used. Replacement of 20% of the cellulase by xylanase enhanced the glucose yield by 6.6%, 8.8%, and 9.5% from SESB, NaOH, and H2O2 pretreated SCB, respectively. Degree of synergism between cellulase and xylanase had positive relationship with xylan content and was affected by hydrolysis time. Copyright © 2014 Elsevier Ltd. All rights reserved.

Chimeric cellulase matrix for investigating intramolecular synergism between non-hydrolytic disruptive functions of carbohydrate-binding modules and catalytic hydrolysis.

PubMed

Wang, Yuguo; Tang, Rentao; Tao, Jin; Wang, Xiaonan; Zheng, Baisong; Feng, Yan

2012-08-24

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.

Characterization of thermostable cellulase produced by Bacillus strains isolated from solid waste of carrageenan

NASA Astrophysics Data System (ADS)

Listyaningrum, N. P.; Sutrisno, A.; Wardani, A. K.

2018-03-01

Cellulase-producing bacteria was isolated from solid waste of carrageenan and identified as Bacillus licheniformis C55 by 16S rRNA sequencing. The optimum condition for cellulase production was obtained at pH and temperature of 8.0 and 50°C, respectively in a medium containing glucose as carbon source and 1.0% carboxymethyl cellulose (CMC) to stimulate the cellulase production. Most remarkably, the enzyme retained its relative activity over 50% after incubation at 50°C for 90 minutes. Substrate specificity suggested that the enzyme is an endoglucanase. The molecular mass of Bacillus licheniformis C55 crude cellulase was found about 18 kDa by SDS-PAGE analysis. This thermostable enzyme would facilitate development of more efficient and cost-effective forms of the process to convert lignocellulosic biomass into high-value products.

Analysis of Enzymatic Degradation of Cellulose Microfibrils using Quantitative Surface Plasmon Resonance Imaging

NASA Astrophysics Data System (ADS)

Reiter, Kyle; Raegen, Adam; Allen, Scott; Quirk, Amanda; Clarke, Anthony; Lipkowski, Jacek; Dutcher, John

2013-03-01

Cellulose is the largest component of biomass on Earth and, as a result, is a significant potential energy source. The production of cellulosic ethanol as a fuel source requires conversion of cellulose fibers into fermentable sugars. Increasing our understanding of the action of cellulose enzymes (cellulases) on cellulose microfibrils is an important step in developing more efficient industrial processes for the production of cellulosic ethanol. We have used a custom designed Surface Plasmon Resonance imaging (SPRi) device to study the action of cellulases from the Hypocrea jecorinasecretome on bacterial cellulose microfibrils. This has allowed us to determine the rates of action and extent of degradation of cellulose microfibrils on exposure to both individual cellulases and combinations of different classes of cellulases, which has allowed us to investigate synergistic interactions between the cellulases.

Enhanced production and application of acidothermophilic Streptomyces cellulase.

PubMed

Budihal, Saikumar R; Agsar, Dayanand; Patil, Sarvamangala R

2016-01-01

An efficient cellulolytic and acidothermophilic actinobacterium was isolated from soil, adhered to decomposing tree bark and was identified as Streptomyces DSK59. Screening of synthetic media and the media components identified that, a medium based on starch casein minerals containing carboxy methyl cellulose (CMC) and beef extract (BE) could support enhanced cellulase production by the organism. CMC, BE, NaCl, temperature and pH were accounted as significant for cellulase production and these were optimized using a response surface central composite design (CCD). Optimization of cellulase production resulted in an enhancement of endoglucanase activity to 27IUml(-1). Acidothermophillic Streptomyces cellulase was found to be efficient for hydrolysis of pretreated sorghum stover and liberated 0.413gg(-1) of total reducing sugars which was higher than previously reported sugar yields obtained using fungal enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed Central

Chen, Ji-Hong; Li, Wen-Jian; Liu, Jing; Hu, Wei; Xiao, Guo-Qing; Dong, Miao-Yin; Wang, Yu-Chen

2015-01-01

The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and β-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme. PMID:26656155

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

PubMed

Carrasco, Mario; Villarreal, Pablo; Barahona, Salvador; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

2016-02-19

Amylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose, and their potential extracellular amylases and cellulases. All tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30-37 °C, except for Rhodotorula glacialis that showed elevated activity at 10-22 °C. In general, cellulase activity was high until pH 6.2 and between 22-37 °C, while the sample from Mrakia blollopis showed high activity at 4-22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi. Almost all yeast species showed extracellular amylase or cellulase activity, and an inducing effect by the respective substrate was observed in a minor number of yeasts. These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively. However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C. In this sense, these yeasts are interesting candidates for industrial processes that require lower temperatures.

Isolation and characterization of Bacillus subtilis strain BY-3, a thermophilic and efficient cellulase-producing bacterium on untreated plant biomass.

PubMed

Meng, F; Ma, L; Ji, S; Yang, W; Cao, B

2014-09-01

Bioconversion of biomass, particularly crop wastes, into biofuels is being developed as an alternative approach in meeting the high energy demand. In this study, a thermophilic bacterial strain BY-3 that exhibits cellulolytic potential was isolated from faecal samples of Tibetan pigs; this strain was identified as Bacillus subtilis. The strain can produce cellulase when grown on various substrates, including carboxymethyl cellulose, rice straw, corn stover, soluble starch and wheat bran. The maximum cellulase activity of the strain was up to 4·323 ± 0·065 U ml(-1) when cultivated in the medium containing corn stover (30 g l(-1) ) for 24 h. The results demonstrated that corn stover is the most suitable substrate for cellulase production by the strain BY-3. The crude cellulase of strain BY-3 was most active at pH 5·5 and 60°C, and the enzyme in acetate buffer (50 mmol l(-1) ) demonstrated a good stability at 60°C for at least 1 h. The crude cellulase exhibited a strong antibacterial activity against Staphylococcus aureus. The strain can be used in cost-efficient cellulase production for bioconversion of agricultural residual biomass into biofuels. The increased consumption of fossil fuels has caused serious energy crisis and environmental problem. Thus, an alternative energy source is necessary. Bioconversion of biomass, particularly agricultural residuals, into value-added bioproducts, such as biofuels and chemical solvents, has received considerable attention. In this study, the newly isolated thermophilic Bacillus subtilis strain BY-3 produces cellulase efficiently with the use of untreated corn stover as a sole carbon source. This strain possesses the thermostable cellulase that is active with diverse crop wastes with a broad pH range and is a highly promising candidate for agricultural waste management. © 2014 The Society for Applied Microbiology.

Cellulase applied to the leaves of sweet pepper (Capsicum annuum L. var. grossum) upregulates the production of salicylic and azelaic acids.

PubMed

Sato, Chizuru; Oka, Norikuni; Nabeta, Kensuke; Matsuura, Hideyuki

2011-01-01

Treating the leaves of sweet pepper (Capsicum annuum L. var. grossum) with an aqueous solution of cellulase resulted in a four-fold increase in the salicylic acid level compared to a control plant. The level of endogenous azelaic acid was also elevated by the cellulase treatment. Azelaic acid has recently been reported to act as a mobile "priming" agent to arm plants against pathogenic attack. Our results are consistent with this and that the cellulase treatment enhanced the ability of sweet pepper to withstand viral attack.

Successive construction of cellulase hyperproducers of Trichoderma using hyperpolyploids.

PubMed

Toyama, H; Toyama, N

2000-01-01

When the swollen conidia of Trichoderma reesei QM 6a are treated with 0.1% (w/v) colchicine solution, huge autopolyploid nuclei can be formed in those swollen conidia. When a mycelial mat derived from such a conidum is treated with a haploidizing reagent, benomyl, many fan-shaped sectors are produced from the colony, and cellulase hyperproducers are selected from conidia on the colony. When colchicine and benomyl treatments are repeated on cellulase hyperproducers, new hyperproducers can be constructed successively and systematically. Moreover, when conidia derived from autopolyploids are treated with ethylmethanesulfonate solution, another type of cellulase hyperproducers (polyploids) can be obtained.

Comparison of Three Ionic Liquid-Tolerant Cellulases by Molecular Dynamics

PubMed Central

Jaeger, Vance; Burney, Patrick; Pfaendtner, Jim

2015-01-01

We have employed molecular dynamics to investigate the differences in ionic liquid tolerance among three distinct family 5 cellulases from Trichoderma viride, Thermogata maritima, and Pyrococcus horikoshii. Simulations of the three cellulases were conducted at a range of temperatures in various binary mixtures of the ionic liquid 1-ethyl-3-methyl-imidazolium acetate with water. Our analysis demonstrates that the effects of ionic liquids on the enzymes vary in each individual case from local structural disturbances to loss of much of one of the enzyme’s secondary structure. Enzymes with more negatively charged surfaces tend to resist destabilization by ionic liquids. Specific and unique structural changes in the enzymes are induced by the presence of ionic liquids. Disruption of the secondary structure, changes in dynamical motion, and local changes in the binding pocket are observed in less tolerant enzymes. Ionic-liquid-induced denaturation of one of the enzymes is indicated over the 500 ns timescale. In contrast, the most tolerant cellulase behaves similarly in water and in ionic-liquid-containing mixtures. Unlike the heuristic approaches that attempt to predict enzyme stability using macroscopic properties, molecular dynamics allows us to predict specific atomic-level structural and dynamical changes in an enzyme’s behavior induced by ionic liquids and other mixed solvents. Using these insights, we propose specific experimentally testable hypotheses regarding the origin of activity loss for each of the systems investigated in this study. PMID:25692593

Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.

PubMed

Zhang, Fei; Bai, Fengwu; Zhao, Xinqing

2016-10-01

Trichoderma reesei Rut-C30 is a well-known cellulase producer, and improvement of its cellulase production is of great interest. An artificial zinc finger protein (AZFP) library is constructed for expression in T. reesei Rut-C30, and a mutant strain T. reesei U3 is selected based on its enhanced cellulase production. The U3 mutant shows a 55% rise in filter paper activity and 8.1-fold increased β-glucosidase activity, when compared to the native strain T. reesei Rut-C30. It is demonstrated that enhanced β-glucosidase activity was due to elevated transcription level of β-glucosidase gene in the U3 mutant. Moreover, significant elevation in transcription levels of several putative Azfp-U3 target genes is detected in the U3 mutant, including genes encoding hypothetical transcription factors and a putative glycoside hydrolase. Furthermore, U3 cellulase shows 115% higher glucose yield from pretreated corn stover, when compared to the cellulase of T. reesei Rut-C30. These results demonstrate that AZFP can be used to improve cellulase production in T. reesei Rut-C30. Our current work offers the establishment of an alternative strategy to develop fungal cell factories for improved production of high value industrial products. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Co-fermentation using Recombinant Saccharomyces cerevisiae Yeast Strains Hyper-secreting Different Cellulases for the Production of Cellulosic Bioethanol.

PubMed

Lee, Cho-Ryong; Sung, Bong Hyun; Lim, Kwang-Mook; Kim, Mi-Jin; Sohn, Min Jeong; Bae, Jung-Hoon; Sohn, Jung-Hoon

2017-06-30

To realize the economical production of ethanol and other bio-based chemicals from lignocellulosic biomass by consolidated bioprocessing (CBP), various cellulases from different sources were tested to improve the level of cellulase secretion in the yeast Saccharomyces cerevisiae by screening an optimal translational fusion partner (TFP) as both a secretion signal and fusion partner. Among them, four indispensable cellulases for cellulose hydrolysis, including Chaetomium thermophilum cellobiohydrolase (CtCBH1), Chrysosporium lucknowense cellobiohydrolase (ClCBH2), Trichoderma reesei endoglucanase (TrEGL2), and Saccharomycopsis fibuligera β-glucosidase (SfBGL1), were identified to be highly secreted in active form in yeast. Despite variability in the enzyme levels produced, each recombinant yeast could secrete approximately 0.6-2.0 g/L of cellulases into the fermentation broth. The synergistic effect of the mixed culture of the four strains expressing the essential cellulases with the insoluble substrate Avicel and several types of cellulosic biomass was demonstrated to be effective. Co-fermentation of these yeast strains produced approximately 14 g/L ethanol from the pre-treated rice straw containing 35 g/L glucan with 3-fold higher productivity than that of wild type yeast using a reduced amount of commercial cellulases. This process will contribute to the cost-effective production of bioenergy such as bioethanol and biochemicals from cellulosic biomass.

Effects of metal ions on the catalytic degradation of dicofol by cellulase.

PubMed

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

2015-07-01

A new technique whereby cellulase immobilized on aminated silica was applied to catalyze the degradation of dicofol, an organochlorine pesticide. In order to evaluate the performance of free and immobilized cellulase, experiments were carried out to measure the degradation efficiency. The Michaelis constant, Km, of the reaction catalyzed by immobilized cellulase was 9.16 mg/L, and the maximum reaction rate, Vmax, was 0.40 mg/L/min, while that of free cellulase was Km=8.18 mg/L, and Vmax=0.79 mg/L/min, respectively. The kinetic constants of catalytic degradation were calculated to estimate substrate affinity. Considering that metal ions may affect enzyme activity, the effects of different metal ions on the catalytic degradation efficiency were explored. The results showed that the substrate affinity decreased after immobilization. Monovalent metal ions had no effect on the reaction, while divalent metal ions had either positive or inhibitory effects, including activation by Mn2+, reversible competition with Cd2+, and irreversible inhibition by Pb2+. Ca2+ promoted the catalytic degradation of dicofol at low concentrations, but inhibited it at high concentrations. Compared with free cellulase, immobilized cellulase was affected less by metal ions. This work provided a basis for further studies on the co-occurrence of endocrine-disrupting chemicals and heavy metal ions in the environment. Copyright © 2015. Published by Elsevier B.V.

Silage fermentation and ruminal degradation of stylo prepared with lactic acid bacteria and cellulase.

PubMed

Li, Mao; Zhou, Hanlin; Zi, Xuejuan; Cai, Yimin

2017-10-01

In order to improve the silage fermentation of stylo (Stylosanthes guianensis) in tropical areas, stylo silages were prepared with commercial additives Lactobacillus plantarum Chikuso-1 (CH1), L. rhamnasus Snow Lact L (SN), Acremonium cellulase (CE) and their combination as SN+CE or CH1 + CE, and the fermentation quality, chemical composition and ruminal degradation of these silages were studied. Stylo silages treated with lactic acid bacteria (LAB) or cellulase, the pH value and NH 3 -N ⁄ total-N were significantly (P < 0.05) decreased while the ruminal degradability of dry matter (DM), crude protein (CP), neutral detergent fiber (aNDFom) and acid detergent fiber (ADFom) were significantly (P < 0.05) increased compared to control. Compared to LAB or cellulase-treated silages, the DM, CP contents and relative feed value (RFV), and the ruminal degradability in LAB plus cellulase-treated silages were significantly (P < 0.05) higher, but the aNDFom content was significantly (P < 0.05) lower. CH1 + CE treatment was more effective in silage fermentation and ruminal degradation than SN+CE treatment. The results confirmed that LAB or LAB plus cellulase treatment could improve the fermentation quality, chemical composition and ruminal degradation of stylo silage. Moreover, the combined treatment with LAB and cellulase may have beneficial synergistic effects on ruminal degradation. © 2017 Japanese Society of Animal Science.

In situ, rapid, and temporally resolved measurements of cellulase adsorption onto lignocellulosic substrates by UV-vis spectrophotometry

Treesearch

Hao Liu; J. Y. Zhu; X. S. Chai

2011-01-01

This study demonstrated two in situ UV-vis spectrophotometric methods for rapid and temporally resolved measurements of cellulase adsorption onto cellulosic and lignocellulosic substrates during enzymatic hydrolysis. The cellulase protein absorption peak at 280 nm was used for quantification. The spectral interferences from light scattering by small fibers (fines) and...

One-pot bioethanol production from cellulose by co-culture of Acremonium cellulolyticus and Saccharomyces cerevisiae

PubMed Central

2012-01-01

Background While the ethanol production from biomass by consolidated bioprocess (CBP) is considered to be the most ideal process, simultaneous saccharification and fermentation (SSF) is the most appropriate strategy in practice. In this study, one-pot bioethanol production, including cellulase production, saccharification of cellulose, and ethanol production, was investigated for the conversion of biomass to biofuel by co-culture of two different microorganisms such as a hyper cellulase producer, Acremonium cellulolyticus C-1 and an ethanol producer Saccharomyces cerevisiae. Furthermore, the operational conditions of the one-pot process were evaluated for maximizing ethanol concentration from cellulose in a single reactor. Results Ethanol production from cellulose was carried out in one-pot bioethanol production process. A. cellulolyticus C-1 and S. cerevisiae were co-cultured in a single reactor. Cellulase producing-medium supplemented with 2.5 g/l of yeast extract was used for productions of both cellulase and ethanol. Cellulase production was achieved by A. cellulolyticus C-1 using Solka-Floc (SF) as a cellulase-inducing substrate. Subsequently, ethanol was produced with addition of both 10%(v/v) of S. cerevisiae inoculum and SF at the culture time of 60 h. Dissolved oxygen levels were adjusted at higher than 20% during cellulase producing phase and at lower than 10% during ethanol producing phase. Cellulase activity remained 8–12 FPU/ml throughout the one-pot process. When 50–300 g SF/l was used in 500 ml Erlenmeyer flask scale, the ethanol concentration and yield based on initial SF were as 8.7–46.3 g/l and 0.15–0.18 (g ethanol/g SF), respectively. In 3-l fermentor with 50–300 g SF/l, the ethanol concentration and yield were 9.5–35.1 g/l with their yields of 0.12–0.19 (g/g) respectively, demonstrating that the one-pot bioethanol production is a reproducible process in a scale-up bioconversion of cellulose to ethanol. Conclusion A. cellulolyticus cells produce cellulase using SF. Subsequently, the produced cellulase saccharifies the SF, and then liberated reducing sugars are converted to ethanol by S. cerevisiae. These reactions were carried out in the one-pot process with two different microorganisms in a single reactor, which does require neither an addition of extraneous cellulase nor any pretreatment of cellulose. Collectively, the one-pot bioethanol production process with two different microorganisms could be an alternative strategy for a practical bioethanol production using biomass. PMID:22938388

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

PubMed

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.

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

PubMed Central

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

2015-01-01

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 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

Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis.

PubMed

Knutsen, Jeffrey S; Davis, Robert H

2004-01-01

Technologies suitable for the separation and reuse of cellulase enzymes during the enzymatic saccharification of pretreated corn stover are investigated to examine the economic and technical viability of processes that promote cellulase reuse while removing inhibitory reaction products such as glucose and cellobiose. The simplest and most suitable separation is a filter with relatively large pores on the order of 20-25 mm that retains residual corn stover solids while passing reaction products such as glucose and cellobiose to form a sugar stream for a variety of end uses. Such a simple separation is effective because cellulase remains bound to the residual solids. Ultrafiltration using 50-kDa polyethersulfone membranes to recover cellulase enzymes in solution was shown not to enhance further the saccharification rate or overall conversion. Instead, it appears that the necessary cellulase enzymes, including beta-glucosidase, are tightly bound to the substrate; when fresh corn stover is contacted with highly washed residual solids, without the addition of fresh enzymes, glucose is generated at a high rate. When filtration was applied multiple times, the concentration of inhibitory reaction products such as glucose and cellobiose was reduced from 70 to 10 g/L. However, an enhanced saccharification performance was not observed, most likely because the concentration of the inhibitory products remained too high. Further reduction in the product concentration was not investigated, because it would make the reaction unnecessarily complex and result in a product stream that is much too dilute to be useful. Finally, an economic analysis shows that reuse of cellulase can reduce glucose production costs, especially when the enzyme price is high. The most economic performance is shown to occur when the cellulase enzyme is reused and a small amount of fresh enzyme is added after each separation step to replace lost or deactivated enzyme.

Covalent Immobilization of Cellulase Using Magnetic Poly(ionic liquid) Support: Improvement of the Enzyme Activity and Stability.

PubMed

Hosseini, Seyed Hassan; Hosseini, Seyedeh Ameneh; Zohreh, Nasrin; Yaghoubi, Mahshid; Pourjavadi, Ali

2018-01-31

A magnetic nanocomposite was prepared by entrapment of Fe 3 O 4 nanoparticles into the cross-linked ionic liquid/epoxy type polymer. The resulting support was used for covalent immobilization of cellulase through the reaction with epoxy groups. The ionic surface of the support improved the adsorption of enzyme, and a large amount of enzyme (106.1 mg/g) was loaded onto the support surface. The effect of the presence of ionic monomer and covalent binding of enzyme was also investigated. The structure of support was characterized by various instruments such as FT-IR, TGA, VSM, XRD, TEM, SEM, and DLS. The activity and stability of immobilized cellulase were investigated in the prepared support. The results showed that the ionic surface and covalent binding of enzyme onto the support improved the activity, thermal stability, and reusability of cellulase compared to free cellulase.

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

PubMed

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.

Aerobic and anaerobic cellulase production by Cellulomonas uda.

PubMed

Poulsen, Henrik Vestergaard; Willink, Fillip Wolfgang; Ingvorsen, Kjeld

2016-10-01

Cellulomonas uda (DSM 20108/ATCC 21399) is one of the few described cellulolytic facultative anaerobes. Based on these characteristics, we initiated a physiological study of C. uda with the aim to exploit it for cellulase production in simple bioreactors with no or sporadic aeration. Growth, cellulase activity and fermentation product formation were evaluated in different media under both aerobic and anaerobic conditions and in experiments where C. uda was exposed to alternating aerobic/anaerobic growth conditions. Here we show that C. uda behaves as a true facultative anaerobe when cultivated on soluble substrates such as glucose and cellobiose, but for reasons unknown cellulase activity is only induced under aerobic conditions on insoluble cellulosic substrates and not under anaerobic conditions. These findings enhance knowledge on the limited number of described facultative cellulolytic anaerobes, and in addition it greatly limits the utility of C. uda as an 'easy to handle' cellulase producer with low aeration demands.

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

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cellulase immobilization on magnetic nanoparticles encapsulated in polymer nanospheres.

PubMed

Lima, Janaina S; Araújo, Pedro H H; Sayer, Claudia; Souza, Antonio A U; Viegas, Alexandre C; de Oliveira, Débora

2017-04-01

Immobilization of cellulases on magnetic nanoparticles, especially magnetite nanoparticles, has been the main approach studied to make this enzyme, economically and industrially, more attractive. However, magnetite nanoparticles tend to agglomerate, are very reactive and easily oxidized in air, which has strong impact on their useful life. Thus, it is very important to provide proper surface coating to avoid the mentioned problems. This study aimed to investigate the immobilization of cellulase on magnetic nanoparticles encapsulated in polymeric nanospheres. The support was characterized in terms of morphology, average diameter, magnetic behavior and thermal decomposition analyses. The polymer nanospheres containing encapsulated magnetic nanoparticles showed superparamagnetic behavior and intensity average diameter about 150 nm. Immobilized cellulase exhibited broader temperature stability than in the free form and great reusability capacity, 69% of the initial enzyme activity was maintained after eight cycles of use. The magnetic support showed potential for cellulase immobilization and allowed fast and easy biocatalyst recovery through a single magnet.

The TcEG1 beetle (Tribolium castaneum) cellulase produced in transgenic switchgrass is active at alkaline pH and auto-hydrolyzes biomass for increased cellobiose release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willis, Jonathan D.; Grant, Joshua N.; Mazarei, Mitra

Genetically engineered biofuel crops, such as switchgrass (Panicum virgatum L.), that produce their own cell wall-digesting cellulase enzymes would reduce costs of cellulosic biofuel production. To date, non-bioenergy plant models have been used in nearly all studies assessing the synthesis and activity of plant-produced fungal and bacterial cellulases. One potential source for cellulolytic enzyme genes is herbivorous insects adapted to digest plant cell walls. Here we examine the potential of transgenic switchgrass-produced TcEG1 cellulase from Tribolium castaneum (red flour beetle). This enzyme, when overproduced in Escherichia coli and Saccharomyces cerevisiae, efficiently digests cellulose at optima of 50 °C and pHmore » 12.0. TcEG1 that was produced in green transgenic switchgrass tissue had a range of endoglucanase activity of 0.16–0.05 units (µM glucose release/min/mg) at 50 °C and pH 12.0. TcEG1 activity from air-dried leaves was unchanged from that from green tissue, but when tissue was dried in a desiccant oven (46 °C), specific enzyme activity decreased by 60%. When transgenic biomass was “dropped-in” into an alkaline buffer (pH 12.0) and allowed to incubate at 50 °C, cellobiose release was increased up to 77% over non-transgenic biomass. Saccharification was increased in one transgenic event by 28%, which had a concurrent decrease in lignin content of 9%. Histological analysis revealed an increase in cell wall thickness with no change to cell area or perimeter. Transgenic plants produced more, albeit narrower, tillers with equivalent dry biomass as the control. This work describes the first study in which an insect cellulase has been produced in transgenic plants; in this case, the dedicated bioenergy crop switchgrass. Switchgrass overexpressing the TcEG1 gene appeared to be morphologically similar to its non-transgenic control and produced equivalent dry biomass. Therefore, we propose TcEG1 transgenics could be bred with other transgenic germplasm (e.g., low-lignin lines) to yield new switchgrass with synergistically reduced recalcitrance to biofuel production. In addition, transgenes for other cell wall degrading enzymes may be stacked with TcEG1 in switchgrass to yield complementary cell wall digestion features and complete auto-hydrolysis.« less

The TcEG1 beetle (Tribolium castaneum) cellulase produced in transgenic switchgrass is active at alkaline pH and auto-hydrolyzes biomass for increased cellobiose release

DOE PAGES

Willis, Jonathan D.; Grant, Joshua N.; Mazarei, Mitra; ...

2017-11-30

Genetically engineered biofuel crops, such as switchgrass (Panicum virgatum L.), that produce their own cell wall-digesting cellulase enzymes would reduce costs of cellulosic biofuel production. To date, non-bioenergy plant models have been used in nearly all studies assessing the synthesis and activity of plant-produced fungal and bacterial cellulases. One potential source for cellulolytic enzyme genes is herbivorous insects adapted to digest plant cell walls. Here we examine the potential of transgenic switchgrass-produced TcEG1 cellulase from Tribolium castaneum (red flour beetle). This enzyme, when overproduced in Escherichia coli and Saccharomyces cerevisiae, efficiently digests cellulose at optima of 50 °C and pHmore » 12.0. TcEG1 that was produced in green transgenic switchgrass tissue had a range of endoglucanase activity of 0.16–0.05 units (µM glucose release/min/mg) at 50 °C and pH 12.0. TcEG1 activity from air-dried leaves was unchanged from that from green tissue, but when tissue was dried in a desiccant oven (46 °C), specific enzyme activity decreased by 60%. When transgenic biomass was “dropped-in” into an alkaline buffer (pH 12.0) and allowed to incubate at 50 °C, cellobiose release was increased up to 77% over non-transgenic biomass. Saccharification was increased in one transgenic event by 28%, which had a concurrent decrease in lignin content of 9%. Histological analysis revealed an increase in cell wall thickness with no change to cell area or perimeter. Transgenic plants produced more, albeit narrower, tillers with equivalent dry biomass as the control. This work describes the first study in which an insect cellulase has been produced in transgenic plants; in this case, the dedicated bioenergy crop switchgrass. Switchgrass overexpressing the TcEG1 gene appeared to be morphologically similar to its non-transgenic control and produced equivalent dry biomass. Therefore, we propose TcEG1 transgenics could be bred with other transgenic germplasm (e.g., low-lignin lines) to yield new switchgrass with synergistically reduced recalcitrance to biofuel production. In addition, transgenes for other cell wall degrading enzymes may be stacked with TcEG1 in switchgrass to yield complementary cell wall digestion features and complete auto-hydrolysis.« less

Lignases and aldo-keto reductases for conversion of lignin-containing materials to fermentable products

DOEpatents

Scharf, Michael; Sethi, Amit

2016-09-13

Termites have specialized digestive systems that overcome the lignin barrier in wood to release fermentable simple sugars. Using the termite Reticulitermes flavipes and its gut symbionts, high-throughput titanium pyrosequencing and proteomics approaches experimentally compared the effects of lignin-containing diets on host-symbiont digestome composition. Proteomic investigations and functional digestive studies with recombinant lignocellulases conducted in parallel provided strong evidence of congruence at the transcription and translational levels and provide enzymatic strategies for overcoming recalcitrant lignin barriers in biofuel feedstocks. Briefly described, therefore, the disclosure provides a system for generating a fermentable product from a lignified plant material, the system comprising a cooperating series of at least two catalytically active polypeptides, where said catalytically active polypeptides are selected from the group consisting of: cellulase Cell-1, .beta.-glu cellulase, an aldo-keto-reductase, a catalase, a laccase, and an endo-xylanase.

Assembly of synthetic cellulose I.

PubMed

Lee, J H; Brown, R M; Kuga, S; Shoda, S; Kobayashi, S

1994-08-02

Cellulose microfibrils with an electron diffraction pattern characteristic of crystalline native cellulose I have been assembled abiotically by means of a cellulase-catalyzed polymerization of beta-cellobiosyl fluoride substrate monomer in acetonitrile/acetate buffer. Substantial purification of the Trichoderma viride cellulase enzyme was found to be essential for the formation of the synthetic cellulose I allomorph. Assembly of synthetic cellulose I appears to be a result of a micellar aggregation of the partially purified enzyme and the substrate in an organic/aqueous solvent system favoring the alignment of glucan chains with the same polarity and extended chain conformation, resulting in crystallization to form the metastable cellulose I allomorph.

Characterization of cellulolytic activity from digestive fluids of Dissosteira carolina (Orthoptera: Acrididae).

PubMed

Willis, Jonathan D; Klingeman, William E; Oppert, Cris; Oppert, Brenda; Jurat-Fuentes, Juan L

2010-11-01

Previous screening of head-derived and gut fluid extracts of Carolina grasshoppers, Dissosteira carolina (L.) revealed relatively high activity against cellulase substrates when compared to other insect groups. In this work we report on the characterization and identification of enzymes involved in cellulolytic activity in digestive fluids of D. carolina. In zymograms using carboxymethylcellulose (CMC) as substrate, we detected four distinct cellulolytic protein bands in D. carolina gut fluids, common to all developmental stages. These cellulolytic enzymes were localized to foregut and midgut regions of the D. carolina digestive tract. Cellulases were purified from D. carolina head and gut fluid extracts by liquid chromatography to obtain N-terminal amino acid sequence tags. Database searches with sequence tags from head fluids indicated high similarity with invertebrate, bacterial and plant beta1,4-endoglucanases, while no homologues were identified for the gut-derived protein. Our data demonstrate the presence of cellulolytic activity in the digestive system of D. carolina and suggest that cellulases of endogenous origin are present in this organism. Considering that this grasshopper species is a pest of grasses, including switchgrass that has been suggested bioethanol feedstock, characterization of insect cellulolytic systems may aid in developing applications for plant biomass biodegradation for biofuel production. Copyright 2010 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Qinghua; Fu Yingjuan; Gao Yang

2009-05-15

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 inmore » ONP deinking using a combination of enzymes. After being bleached by H{sub 2}O{sub 2}, 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.« less

Thermal tolerant cellulase from Acidothermus cellulolyticus

DOEpatents

Ding, Shi-You; Adney, William S.; Vinzant, Todd B.; Himmel, Michael E.; Decker, Stephen R.

2006-06-13

The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as GuxA. GuxA has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified GuxA. Methods of making and using GuxA polypeptides, including fusions, variants, and derivatives, are also disclosed.

Thermal Tolerant Cellulase from Acidothermus Cellulolyticus

DOEpatents

Ding, S. Y.; Adney, W. S.; Vinzant, T. B.; Himmel, M. E.; Decker, S. R.

2006-06-13

The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as GuxA. GuxA has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified GuxA. Methods of making and using GuxA polypeptides, including fusions, variants, and derivatives, are also disclosed.

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

USDA-ARS?s Scientific Manuscript database

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...

The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brunecky, Roman; Donohoe, Bryon S.; Yarbrough, John M.

The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systemsmore » employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Furthermore, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.« less

Cellulase production in a new mutant strain of Penicillium decumbens ML-017 by solid state fermentation with rice bran.

PubMed

Liu, Yun-Tao; Luo, Ze-Yu; Long, Chuan-Nan; Wang, Hai-Dong; Long, Min-Nan; Hu, Zhong

2011-10-01

To produce cellulolytic enzyme efficiently, Penicillium decumbens strain L-06 was used to prepare mutants with ethyl methane sulfonate (EMS) and UV-irradiation. A mutant strain ML-017 is shown to have a higher cellulase activity than others. Box-Behnken's design (BBD) and response surface methodology (RSM) were adopted to optimize the conditions of cellulase (filter paper activity, FPA) production in strain ML-017 by solid-state fermentation (SSF) with rice bran as the substrate. And the result shows that the initial pH, moisture content and culture temperature all have significant effect on the production of cellulase. The optimized condition shall be initial pH 5.7, moisture content 72% and culture temperature 30°C. The maximum cellulase (FPA) production was obtained under the optimized condition, which is 5.76 IU g(-1), increased by 44.12% to its original strain. It corresponded well with the calculated results (5.15 IU g(-1)) by model prediction. The result shows that both BBD and RSM are the cellulase optimization methods with good prospects. Copyright © 2011 Elsevier B.V. All rights reserved.

The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose

DOE PAGES

Brunecky, Roman; Donohoe, Bryon S.; Yarbrough, John M.; ...

2017-08-29

The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systemsmore » employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Furthermore, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.« less

Rce1, a novel transcriptional repressor, regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei.

PubMed

Cao, Yanli; Zheng, Fanglin; Wang, Lei; Zhao, Guolei; Chen, Guanjun; Zhang, Weixin; Liu, Weifeng

2017-07-01

Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei. © 2017 John Wiley & Sons Ltd.

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis*

PubMed Central

Wang, Yuguo; Tang, Rentao; Tao, Jin; Wang, Xiaonan; Zheng, Baisong; Feng, Yan

2012-01-01

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose. PMID:22778256

Evaluation of Bacterial Expansin EXLX1 as a Cellulase Synergist for the Saccharification of Lignocellulosic Agro-Industrial Wastes

PubMed Central

Lin, Hui; Shen, Qi; Zhan, Ju-Mei; Wang, Qun; Zhao, Yu-Hua

2013-01-01

Various types of lignocellulosic wastes extensively used in biofuel production were provided to assess the potential of EXLX1 as a cellulase synergist. Enzymatic hydrolysis of natural wheat straw showed that all the treatments using mixtures of cellulase and an optimized amount of EXLX1, released greater quantities of sugars than those using cellulase alone, regardless of cellulase dosage and incubation time. EXLX1 exhibited different synergism and binding characteristics for different wastes, but this can be related to their lignocellulosic components. The cellulose proportion could be one of the important factors. However, when the cellulose proportion of different biomass samples exhibited no remarkable differences, a higher synergism of EXLX1 is prone to occur on these materials, with a high proportion of hemicellulose and a low proportion of lignin. The information could be favorable to assess whether EXLX1 is effective as a cellulase synergist for the hydrolysis of the used materials. Binding assay experiments further suggested that EXLX1 bound preferentially to alkali pretreated materials, as opposed to acid pretreated materials under the assay condition and the binding preference would be affected by incubation temperature. PMID:24086425

Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli.

PubMed

Ko, Chun-Han; Tsai, Chung-Hung; Lin, Po-Heng; Chang, Ko-Cheng; Tu, Jenn; Wang, Ya-Nang; Yang, Chien-Ying

2010-10-01

The Cel-BL11 gene from Paenibacillus campinasensis BL11 was cloned and expressed in Escherichia coli as a His-tag fusion protein. Zymographic analysis of the recombinant protein revealed cellulase activity corresponding to a protein with a 38-kDa molecular weight. The optimum temperature and pH for purified cellulase were 60 °C and pH 7.0, respectively. The enzyme retained more than 80% activity after 8h at 60 °C at pH 6 and 7. The cellulase has a Km of 11.25 mg/ml and a Vmax of 1250 μmol/min/mg with carboxylmethyl cellulose (CMC). Then enzyme was active on Avicel, swollen Avicel, CMC, barley β-glucan, laminarin in the presence of 100 mM acetate buffer. It was inhibited by Hg²⁺, Cu²⁺ and Zn²⁺. Significant kraft pulp refining energy saving, 10%, was exhibited by the pretreatment of this cellulase applied at 2 IU per gram of oven-dried pulp. Broad pH and temperature stability render this cellulase a convenient applicability toward current mainstream biomass conversion and other industrial processes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase.

PubMed

El-Ghonemy, Dina Helmy; Ali, Thanaa Hamed; El-Bondkly, Ahmed Mohamed; Moharam, Maysa El-Sayed; Talkhan, Fatma Nabeeh

2014-11-01

Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH4Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.

Expression of three Trichoderma reesei cellulase genes in Saccharomyces pastorianus for the development of a two-step process of hydrolysis and fermentation of cellulose.

PubMed

Fitzpatrick, J; Kricka, W; James, T C; Bond, U

2014-07-01

To compare the production of recombinant cellulase enzymes in two Saccharomyces species so as to ascertain the most suitable heterologous host for the degradation of cellulose-based biomass and its conversion into bioethanol. cDNA copies of genes representing the three major classes of cellulases (Endoglucanases, Cellobiohydrolases and β-glucosidases) from Trichoderma reesei were expressed in Saccharomyces pastorianus and Saccharomyces cerevisiae. The recombinant enzymes were secreted by the yeast hosts into the medium and were shown to act in synergy to hydrolyse cellulose. The conditions required to achieve maximum release of glucose from cellulose by the recombinant enzymes were defined and the activity of the recombinant enzymes was compared to a commercial cocktail of T. reesei cellulases. We demonstrate that significantly higher levels of cellulase activity were achieved by expression of the genes in S. pastorianus compared to S. cerevisiae. Hydrolysis of cellulose by the combined activity of the recombinant enzymes was significantly better at 50°C than at 30°C, the temperature used for mesophilic yeast fermentations, reflecting the known temperature profiles of the native enzymes. The results demonstrate that host choice is important for the heterologous production of cellulases. On the basis of the low activity of the T. reesei recombinant enzymes at fermentation temperatures, we propose a two-step process for the hydrolysis of cellulose and its fermentation into alcohol using cellulases produced in situ. © 2014 The Society for Applied Microbiology.

Cellulose digestion in primitive hexapods: Effect of ingested antibiotics on gut microbial populations and gut cellulase levels in the firebrat,Thermobia domestica (Zygentoma, Lepismatidae).

PubMed

Treves, D S; Martin, M M

1994-08-01

Antibiotic feeding studies were conducted on the firebrat,Thermobia domestica (Zygentoma, Lepismatidae) to determine if the insect's gut cellulases were of insect or microbial origin. Firebrats were fed diets containing either nystatin, metronidazole, streptomycin, tetracycline, or an antibiotic cocktail consisting of all four antibiotics, and then their gut microbial populations and gut cellulase levels were monitored and compared with the gut microbial populations and gut cellulase levels in firebrats feeding on antibiotic-free diets. Each antibiotic significantly reduced the firebrat's gut micro-flora. Nystatin reduced the firebrat's viable gut fungi by 89%. Tetracycline and the antibiotic cocktail reduced the firebrat's viable gut bacteria by 81% and 67%, respectively, and metronidazole, streptomycin, tetracycline, and the antibiotic cocktail reduced the firebrat's total gut flora by 35%, 32%, 55%, and 64%, respectively. Although antibiotics significantly reduced the firebrat's viable and total gut flora, gut cellulase levels in firebrats fed antibiotics were not significantly different from those in firebrats on an antibiotic-free diet. Furthermore, microbial populations in the firebrat's gut decreased significantly over time, even in firebrats feeding on the antibiotic-free diet, without corresponding decreases in gut cellulase levels. Based on this evidence, we conclude that the gut cellulases of firebrats are of insect origin. This conclusion implies that symbiont-independent cellulose digestion is a primitive trait in insects and that symbiont-mediated cellulose digestion is a derived condition.

Effects of controlled gas environments in microbial enhancement of plant protein recovery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mudgett, R.E.; Bajracharya, R.

Controlled gas environments were maintained by a novel aeration system in solid substrate fermentations for enhanced protein recovery from pressed alfalfa residues. High O/sub 2/ pressures stimulated biomass and enzyme production by an Aspergillus species, isolated from alfalfa, which produced cellulase and pectinase activities in growth-associated metabolism. High CO/sub 2/ pressures also stimulated enzyme production, but had less effect on biomass production, as established from the dissimilation of plant solids. Cellulase and pectinase activities were generally related to protein recoveries. Recoveries were greater than or equal to 50% higher than those obtained by mechanical extraction, with maximum recoveries of greatermore » than or equal to 70% of crude protein contents. Protein not recovered at high cellulase and pectinase activities were believed to be in structurally bound forms not amenable to recovery by nonproteolytic enzymes. Buffering at pH 8 and autoclaving of residues prior to fermentation had little effect on protein recoveries. Controlled gas environments are seen to offer an interesting potential for optimizing industrial fermentation processes for the production of microbial enzymes.« less

Study of cellulase enzymes self-assembly in aqueous-acetonitrile solvent: Viscosity measurements

NASA Astrophysics Data System (ADS)

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

2009-11-01

The present study extends the viscosity measurements performed by Ghaouar et al. [Physica B, submitted for publication.] to study the conformational change of the cellulase enzymes in aqueous-acetonitrile mixture. We aim to investigate: (i) the denaturation process by measuring the specific viscosity for temperatures varying between 25 and 65 °C and acetonitrile concentrations between 0% and 50%, (ii) the enzyme-enzyme interaction by calculating the Huggins coefficient and (iii) the enzyme sizes by following the hydrodynamic radius for various temperatures. The precipitation of cellulases versus acetonitrile concentration is also considered. We show from all physical quantities measured in this work that the precipitation and the denaturation processes of cellulase enzymes exist together.

The effect of cellulases on the biodegradation and morphology of naturally colored cotton fibers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evans, B.R.; Lee, I.; Woodward, J.

We have investigated the effect of cellulases on the biodegradation and structure of natural colored cotton (Foxfibre{reg_sign}). Compared to the white cotton and palo verde (sage green) varieties, buffalo (mocha brown) and coyote (reddish brown) varieties were quite resistant to hydrolysis by Trichoderma reesei celluclast and purified cellobiohydrolase I (CBH I) under the conditions of the assay, but binding of CBH I to buffalo cotton was unaffected. Sodium hydroxide extracts of all the colored cotton varieties were found to be strong inhibitors of cellulase activity and the buffalo cotton was labile in that the inhibitory effect decreased over time inmore » the presence of cellulase; incubation of {beta}-glucosidase with the extract also decreased the inhibition. The chemical composition of the inhibitor is currently under investigation. Atomic force microscopy of the colored cotton fibers with bound cellulase components should prove useful in the context of elucidating the effect of binding on the morphology of cellulose fibers.« less

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

PubMed

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.

Development of cellulase-nanoconjugates with enhanced ionic liquid and thermal stability for in situ lignocellulose saccharification.

PubMed

Grewal, Jasneet; Ahmad, Razi; Khare, S K

2017-10-01

The present work aimed to improve catalytic efficiency of Trichoderma reesei cellulase for enhanced saccharification. The cellulase was immobilized on two nanomatrices i.e. magnetic and silica nanoparticles with immobilization efficiency of 85% and 76% respectively. The nanobioconjugates exhibited increase in V max , temperature optimum, pH and thermal stability as compared with free enzyme. These could be efficiently reused for five repeated cycles and were stable in 1-ethyl-3-methylimidazoliumacetate [EMIM][Ac], an ionic liquid. Ionic liquids (IL) are used as green solvents to dissolve lignocellulosic biomass and facilitate better saccharification. The cellulase immobilized on magnetic nanoparticles was used for in situ saccharification of [EMIM][Ac] pretreated sugarcane bagasse and wheat straw for two cycles. The structural deconstruction and decrease in biomass crystallinity was confirmed by SEM, XRD and FTIR. The high hydrolysis yields (∼89%) obtained in this one-pot process coupled with IL stability and recycled use of immobilized cellulase, potentiates its usefulness in biorefineries. Copyright © 2017 Elsevier Ltd. All rights reserved.

Thermostable, haloalkaline cellulase from Bacillus halodurans CAS 1 by conversion of lignocellulosic wastes.

PubMed

Annamalai, Neelamegam; Rajeswari, Mayavan Veeramuthu; Elayaraja, Sivaramasamy; Balasubramanian, Thangavel

2013-04-15

An extracellular thermostable, haloalkaline cellulase by bioconversion of lignocellulosic wastes from Bacillus halodurans CAS 1 was purified to homogeneity with recovery of 12.54% and purity fold 7.96 with the molecular weight of 44 kDa. The optimum temperature, pH and NaCl for enzyme activity was determined as 60°C, 9.0 and 30% and it retained 80% of activity even at 80°C, 12 and 35% respectively. The activity was greatly inhibited by EDTA, indicating that it was a metalloenzyme and significant inhibition by PMSF revealed that serine residue was essential for catalytic activity. The purified cellulase hydrolyzed CMC, cellobiose and xylan, but not avicel, cellulose and PNPG. Furthermore, the cellulase was highly stable in the presence of detergents and organic solvents such as acetone, n-hexane and acetonitrile. Thus, the purified cellulase from B. halodurans utilizing lignocellulosic biomass could be greatly useful to develop industrial processes. Published by Elsevier Ltd.

Secretion of clostridium cellulase by E. coli

DOEpatents

Yu, Ida Kuo

1998-01-01

A gene, encoding an endocellulase from a newly isolated mesophilic Clostridium strain IY-2 which can digest bamboo fibers, cellulose, rice straw, and sawdust, was isolated by shotgun cloning in an E. coli expression plasmid pLC2833. E. coli positive clones were selected based on their ability to hydrolyze milled bamboo fibers and cellulose present in agar plates. One clone contained a 2.8 kb DNA fragment that was responsible for cellulase activity. Western blot analyses indicated that the positive clone produced a secreted cellulase with a mass of about 58,000 daltons that was identical in size to the subunit of one of the three major Clostridium cellulases. The products of cellulose digestion by this cloned cellulase were cellotetraose and soluble higher polymers. The cloned DNA contained signal sequences capable of directing the secretion of heterologous proteins from an E. coli host. The invention describes a bioprocess for the treatment of cellulosic plant materials to produce cellular growth substrates and fermentation end products suitable for production of liquid fuels, solvents, and acids.

DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca.

PubMed Central

Lao, G; Ghangas, G S; Jung, E D; Wilson, D B

1991-01-01

The DNA sequences of the Thermomonospora fusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined. Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon. There were no significant homologies between the coding regions of the three genes. The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes. E2 belongs to a family of cellulases that includes celA from M. bispora, cenA from Cellulomonas fimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei. E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family. There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C. fimi and Pseudomonas fluorescens cellulose binding domains. PMID:1904434

Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain.

PubMed

Kaur, Baljit; Oberoi, H S; Chadha, B S

2014-03-01

A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant '64', when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed Central

2011-01-01

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. PMID:22070776

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

PubMed

Mohapatra, Bidyut R

2017-05-01

In an effort to screen out the alginolytic and cellulolytic bacteria from the putrefying invasive seaweed Sargassum species accumulated off Barbados' coast, a potent bacterial strain was isolated. This bacterium, which simultaneously produced alginate lyase and cellulase, was identified as Exiguobacterium sp. Alg-S5 via the phylogenetic approach targeting the 16S rRNA gene. The co-produced alginate lyase and cellulase exhibited maximal enzymatic activity at pH 7.5 and at 40°C and 45°C, respectively. The K m and V max values recorded as 0.91mg/mL and 21.8U/mg-protein, respectively, for alginate lyase, and 10.9mg/mL and 74.6U/mg-protein, respectively, for cellulase. First order kinetic analysis of the thermal denaturation of the co-produced alginate lyase and cellulase in the temperature range from 40°C to 55°C revealed that both the enzymes were thermodynamically efficient by displaying higher activation energy and enthalpy of denaturation. These enzymatic properties indicate the potential industrial importance of this bacterium in algal biomass conversion. This appears to be the first report on assessing the efficacy of a bacterium for the co-production of alginate lyase and cellulase. Copyright © 2017 Elsevier B.V. All rights reserved.

Use of spectroscopic and imaging techniques to evaluate pretreated sugarcane bagasse as a substrate for cellulase production under solid-state fermentation.

PubMed

Rodríguez-Zúñiga, Ursula Fabiola; Bertucci Neto, Victor; Couri, Sonia; Crestana, Silvio; Farinas, Cristiane Sanchez

2014-03-01

The enzymatic cocktail of cellulases is one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels and other chemicals. Here, the influence of liquid hot water, dilute acid, alkali, and combined acid/alkali pretreatments on sugarcane bagasse (SCB) used for cellulase production was investigated by means of spectroscopic and imaging techniques. Chemical composition and structural characteristics, such as crystallinity (determined by X-ray diffraction), functional groups (Fourier transform infrared spectroscopy), and microstructure (scanning electron microscopy), were used to correlate SCB pretreatments with enzymatic biosynthesis by a strain of the filamentous fungus Aspergillus niger under solid-state fermentation. The combined acid/alkali pretreatment resulted in a SCB with higher cellulose content (86.7%). However, the high crystallinity (74%) of the resulting biomass was detrimental to microbial uptake and enzyme production. SCB pretreated with liquid hot water yielded the highest filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and xylanase activities (0.4, 14.9, and 26.1 U g(-1), respectively). The results showed that a suitable pretreatment for SCB to be used as a substrate for cellulase production should avoid severe conditions in order to preserve amorphous cellulose and to enhance the physical properties that assist microbial access.

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

PubMed

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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.

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

PubMed

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)] (p<0.05). Koji fermentation in trays was carried out and higher enzyme activities (FPase 96.67+/-4.18 IU/gfs and CMCase 146.50+/-11.92 IU/gfs) were achieved. The nonspecific chitinase and chitosanase activities of cellulase enzyme extract were analyzed using chitin and chitosan 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.

Enhanced cellulase production in Trichoderma reesei RUT C30 via constitution of minimal transcriptional activators.

PubMed

Zhang, Jiajia; Zhang, Guoxiu; Wang, Wei; Wang, Wei; Wei, Dongzhi

2018-05-17

Cellulase can convert lignocellulosic feedstocks into fermentable sugars, which can be used for the industrial production of biofuels and chemicals. The high cost of cellulase production remains a challenge for lignocellulose breakdown. Trichoderma reesei RUT C30 serves as a well-known industrial workhorse for cellulase production. Therefore, the enhancement of cellulase production by T. reesei RUT C30 is of great importance. Two sets of novel minimal transcriptional activators (DBD ace2 -VP16 and DBD cre1 -VP16) were designed and expressed in T. reesei RUT C30. Expression of DBD ace2 -VP16 and DBD cre1 -VP16 improved cellulase production under induction (avicel or lactose) and repression (glucose) conditions, respectively. The strain T MTA66 under avicel and T MTA139 under glucose with the highest cellulase activities outperformed other transformants and the parental strain under the corresponding conditions. For T MTA66 strains, the highest FPase activity was approximately 1.3-fold greater than that of the parental strain RUT C30 at 120 h of cultivation in a shake flask using avicel as the sole carbon source. The FPase activity (U/mg biomass) in T MTA139 strains was approximately 26.5-fold higher than that of the parental strain RUT C30 at 72 h of cultivation in a shake flask using glucose as the sole carbon source. Furthermore, the crude enzymes produced in the 7-L fermenter from T MTA66 and T MTA139 supplemented with commercial β-glucosidase hydrolyzed pretreated corn stover effectively. These results show that replacing natural transcription factors with minimal transcriptional activators is a powerful strategy to enhance cellulase production in T. reesei. Our current study also offers an alternative genetic engineering strategy for the enhanced production of industrial products by other fungi.

Cellulase and Xylanase Production from Three Isolates of Indigenous Endophytic Fungi

NASA Astrophysics Data System (ADS)

Yopi; Tasia, W.; Melliawati, R.

2017-12-01

Cellulases and hemicellulases have good potential to be used in energy production, in pulp, paper, textile industries, as well as in animal feed industries. Moreover, its utilization in food industries also cannot be ignored, among others, cellulase and xylanase roles in bakery, wine, and fruit and vegetables juice production. One of the potential enzyme source is endophytic fungi. Object of this study is to explore the potency of endophytic fungi isolated from medicinal plants as source of cellulolytic and xylanolytic enzymes. HL.47F.216 is endophytic fungi isolated from traditional medicinal plants ironwood tree was determined as xylanase producer. HL.51F.235 from pin-flower tree is cellulase producer, while CBN.6F.29 which produces both xylanase and cellulase is originated from Madagascar periwinkle. HL.47F.216 showed 2.5 cm in clear zone diameter and its xylanase activity was 0.262 U/mL with optimum condition pH 7 at 50°C. HL.51F.235 showed 2.4 cm clear zone diameter and 0.239 U/mL of cellulase activity at pH 5 and 70°C. CBN.6F.29 showed 2.8 cm and 0.394 U/mL (pH 5, 40°C) for its cellulase activity, while 2.3 cm and 0.439 U/mL (pH 8, 70°C) for its xylanase activity. Xylanase from HL.47F.216 and CBN.6F.29 showed low molecular masses of 20 kDa and 37-50 kDa, respectively. Molecular masses for cellulases from HL.51F.235 and CBN.6F.29 were 25 and 50 kDa for HL.51F.235 and 100 kDa for CBN.6F.29. Based on macroscopic and microscopic identification, fungal isolate CBN.6F.29 is a member of Class Coelomycetes, while HL.47F.216 was Acremonium sp. and HL.51F.235 was Aspergillus nigri.

Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes.

PubMed

Duan, Cheng-Jie; Huang, Ming-Yue; Pang, Hao; Zhao, Jing; Wu, Chao-Xing; Feng, Jia-Xun

2017-07-01

In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V max and catalytic efficiency (k cat /K M ) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.

Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.

PubMed

Xue, Xianli; Wu, Yilan; Qin, Xing; Ma, Rui; Luo, Huiying; Su, Xiaoyun; Yao, Bin

2016-07-11

The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. The thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. We report herein the successful overexpression of a thermophilic N. fischeri β-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved β-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.

Improving biogas production from microalgae by enzymatic pretreatment.

PubMed

Passos, Fabiana; Hom-Diaz, Andrea; Blanquez, Paqui; Vicent, Teresa; Ferrer, Ivet

2016-01-01

In this study, enzymatic pretreatment of microalgal biomass was investigated under different conditions and evaluated using biochemical methane potential (BMP) tests. Cellulase, glucohydrolase and an enzyme mix composed of cellulase, glucohydrolase and xylanase were selected based on the microalgae cell wall composition (cellulose, hemicellulose, pectin and glycoprotein). All of them increased organic matter solubilisation, obtaining high values already after 6h of pretreatment with an enzyme dose of 1% for cellulase and the enzyme mix. BMP tests with pretreated microalgae showed a methane yield increase of 8 and 15% for cellulase and the enzyme mix, respectively. Prospective research should evaluate enzymatic pretreatments in continuous anaerobic reactors so as to estimate the energy balance and economic cost of the process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Saccharification of rice straw by cellulase from a local Trichoderma harzianum SNRS3 for biobutanol production.

PubMed

Rahnama, Nooshin; Foo, Hooi Ling; Abdul Rahman, Nor Aini; Ariff, Arbakariya; Md Shah, Umi Kalsom

2014-12-12

Rice straw has shown to be a promising agricultural by-product in the bioconversion of biomass to value-added products. Hydrolysis of cellulose, a main constituent of lignocellulosic biomass, is a requirement for fermentable sugar production and its subsequent bioconversion to biofuels such as biobutanol. The high cost of commercial enzymes is a major impediment to the industrial application of cellulases. Therefore, the use of local microbial enzymes has been suggested. Trichoderma harzianum strains are potential CMCase and β-glucosidase producers. However, few researches have been reported on cellulase production by T. harzianum and the subsequent use of the crude cellulase for cellulose enzymatic hydrolysis. For cellulose hydrolysis to be efficiently performed, the presence of the whole set of cellulase components including exoglucanase, endoglucanase, and β-glucosidase at a considerable concentration is required. Biomass recalcitrance is also a bottleneck in the bioconversion of agricultural residues to value-added products. An effective pretreatment could be of central significance in the bioconversion of biomass to biofuels. Rice straw pretreated using various concentrations of NaOH was subjected to enzymatic hydrolysis. The saccharification of rice straw pretreated with 2% (w/v) NaOH using crude cellulase from local T. harzianum SNRS3 resulted in the production of 29.87 g/L reducing sugar and a yield of 0.6 g/g substrate. The use of rice straw hydrolysate as carbon source for biobutanol fermentation by Clostridium acetobutylicum ATCC 824 resulted in an ABE yield, ABE productivity, and biobutanol yield of 0.27 g/g glucose, 0.04 g/L/h and 0.16 g/g glucose, respectively. As a potential β-glucosidase producer, T. harzianum SNRS3 used in this study was able to produce β-glucosidase at the activity of 173.71 U/g substrate. However, for cellulose hydrolysis to be efficient, Filter Paper Activity at a considerable concentration is also required to initiate the hydrolytic reaction. According to the results of our study, FPase is a major component of cellulose hydrolytic enzyme complex system and the reducing sugar rate-limiting enzyme. Our study revealed that rice straw hydrolysate served as a potential substrate for biobutanol production and FPase is a rate-limiting enzyme in saccharification.

A paradigm shift in biomass technology from complete to partial cellulose hydrolysis: lessons learned from nature.

PubMed

Chen, Rachel

2015-01-01

A key characteristic of current biomass technology is the requirement for complete hydrolysis of cellulose and hemicellulose, which stems from the inability of microbial strains to use partially hydrolyzed cellulose, or cellodextrin. The complete hydrolysis paradigm has been practiced over the past 4 decades with major enzyme companies perfecting their cellulase mix for maximal yield of monosaccharides, with corresponding efforts in strain development focus almost solely on the conversion of monosaccharides, not cellodextrin, to products. While still in its nascent infancy, a new paradigm requiring only partial hydrolysis has begun to take hold, promising a shift in the biomass technology at its fundamental core. The new paradigm has the potential to reduce the requirement for cellulase enzymes in the hydrolysis step and provides new strategies for metabolic engineers, synthetic biologists and alike in engineering fermenting organisms. Several recent publications reveal that microorganisms engineered to metabolize cellodextrins, rather than monomer glucose, can reap significant energy gains in both uptake and subsequent phosphorylation. These energetic benefits can in turn be directed for enhanced robustness and increased productivity of a bioprocess. Furthermore, the new cellodextrin metabolism endows the biocatalyst the ability to evade catabolite repression, a cellular regulatory mechanism that is hampering rapid conversion of biomass sugars to products. Together, the new paradigm offers significant advantages over the old and promises to overcome several critical barriers in biomass technology. More research, however, is needed to realize these promises, especially in discovery and engineering of cellodextrin transporters, in developing a cost-effective method for cellodextrin generation, and in better integration of cellodextrin metabolism to endogenous glycolysis.

Effect of ferrous chloride on biogas production and enzymatic activities during anaerobic fermentation of cow dung and Phragmites straw.

PubMed

Zhang, Huayong; Tian, Yonglan; Wang, Lijun; Mi, Xueyue; Chai, Yang

2016-06-01

The effect of ferrous (added as FeCl2) on the anaerobic co-digestion of Phragmites straw and cow dung was studied by investigating the biogas properties, pH values, organic matter degradation (COD) and enzyme activities (cellulase, protease and dehydrogenase) at different stages of mesophilic fermentation. The results showed that Fe(2+) addition increased the cumulative biogas yields by 18.1 % by extending the peak period with high daily biogas yields. Meanwhile, the methane (CH4) contents in the Fe(2+) added groups were generally higher than the control group before the 15th day. The pH values were not significantly impacted by Fe(2+) concentrations during the fermentation process. The COD concentrations, cellulase, protease and dehydrogenase activities varied with the added Fe(2+) concentrations and the stages of the fermentation process. At the beginning stage of fermentation (4th day), Fe(2+) addition increased the biogas production by improving the cellulase and dehydrogenase activities which caused a decline in COD. At the peak stage of fermentation (8th day), Fe(2+) addition enhanced the cellulase and protease activities, and resulted in lower COD contents than the control group. When the biogas yields decreased again (13th day), the COD contents varied similar with the protease and dehydrogenase activities, whilst cellulase activities were not sensitive to Fe(2+) concentrations. At the end of fermentation (26th day), Fe(2+) addition decreased the cellulase activities, led to lower COD contents and finally resulted the lower biogas yields than the control group. Taking the whole fermentation process into account, the promoting effect of Fe(2+) addition on biogas yields was mainly attributed to the extension of the gas production peak stage and the improvement of cellulase activities.

Evaluation of Ten Wild Nigerian Mushrooms for Amylase and Cellulase Activities

PubMed Central

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 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

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

PubMed

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

2011-07-01

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

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

NASA Astrophysics Data System (ADS)

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

2015-01-01

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

Comparative performance of precommercial cellulases hydrolyzing pretreated corn stover

PubMed Central

2011-01-01

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 information about the performance of four precommercial cellulase preparations. Though test conditions were not necessarily optimal for some of the enzymes, all were able to effectively saccharify PCS cellulose. Large differences in the estimated enzyme dosage requirements depending on the assay used to measure protein concentration highlight the need for better consensus methods to quantify enzyme protein. PMID:21899748

A solid state fungal fermentation-based strategy for the hydrolysis of wheat straw.

PubMed

Pensupa, Nattha; Jin, Meng; Kokolski, Matt; Archer, David B; Du, Chenyu

2013-12-01

This paper reports a solid-state fungal fermentation-based pre-treatment strategy to convert wheat straw into a fermentable hydrolysate. Aspergillus niger was firstly cultured on wheat straw for production of cellulolytic enzymes and then the wheat straw was hydrolyzed by the enzyme solution into a fermentable hydrolysate. The optimum moisture content and three wheat straw modification methods were explored to improve cellulase production. At a moisture content of 89.5%, 10.2 ± 0.13 U/g cellulase activity was obtained using dilute acid modified wheat straw. The addition of yeast extract (0.5% w/v) and minerals significantly improved the cellulase production, to 24.0 ± 1.76 U/g. The hydrolysis of the fermented wheat straw using the fungal culture filtrate or commercial cellulase Ctec2 was performed, resulting in 4.34 and 3.13 g/L glucose respectively. It indicated that the fungal filtrate harvested from the fungal fermentation of wheat straw contained a more suitable enzyme mixture than the commercial cellulase. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Improving the enzymatic hydrolysis of dilute acid pretreated wheat straw by metal ion blocking of non-productive cellulase adsorption on lignin.

PubMed

Akimkulova, Ardak; Zhou, Yan; Zhao, Xuebing; Liu, Dehua

2016-05-01

Eleven salts were selected to screen the possible metal ions for blocking the non-productive adsorption of cellulase onto the lignin of dilute acid pretreated wheat straw. Mg(2+) was screened finally as the promising candidate. The optimal concentration of MgCl2 was 1 mM, but the beneficial action was also dependent on pH, hydrolysis time and cellulase loading. Significant improvement of glucan conversion (19.3%) was observed at low cellulase loading (5 FPU/g solid). Addition of isolated lignins, tannic acid and lignin model compounds to pure cellulose hydrolysis demonstrated that phenolic hydroxyl group (Ph-OH) was the main active site blocked by Mg(2+). The interaction between Mg(2+) and Ph-OH of lignin monomeric moieties followed an order of p-hydroxyphenyl (H)>guaiacyl (G)>syringyl (S). Mg(2+) blocking made the lignin surface less negatively charged, which might weaken the hydrogen bonding and electrostatically attractive interaction between lignin and cellulase enzymes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Catalytic performance of corn stover hydrolysis by a new isolate Penicillium sp. ECU0913 producing both cellulase and xylanase.

PubMed

Shi, Qian-Qian; Sun, Jie; Yu, Hui-Lei; Li, Chun-Xiu; Bao, Jie; Xu, Jian-He

2011-07-01

A fungal strain, marked as ECU0913, producing high activities of both cellulase and xylanase was newly isolated from soil sample collected near decaying straw and identified as Penicillium sp. based on internal transcribed spacer sequence homology. The cultivation of this fungus produced both cellulase (2.40 FPU/ml) and xylanase (241 IU/ml) on a stepwisely optimized medium at 30 °C for 144 h. The cellulase and xylanase from Penicillium sp. ECU0913 was stable at an ambient temperature with half-lives of 28 and 12 days, respectively. Addition of 3 M sorbitol greatly improved the thermostability of the two enzymes, with half-lives increased by 2.3 and 188-folds, respectively. Catalytic performance of the Penicillium cellulase and xylanase was evaluated by the hydrolysis of corn stover pretreated by steam explosion. With an enzyme dosage of 50 FPU/g dry substrate, the conversions of cellulose and hemicellulose reached 77.2% and 47.5%, respectively, without adding any accessory enzyme.

Cell recycle batch fermentation of high-solid lignocellulose using a recombinant cellulase-displaying yeast strain for high yield ethanol production in consolidated bioprocessing.

PubMed

Matano, Yuki; Hasunuma, Tomohisa; Kondo, Akihiko

2013-05-01

The aim of this study is to develop a scheme of cell recycle batch fermentation (CRBF) of high-solid lignocellulosic materials. Two-phase separation consisting of rough removal of lignocellulosic residues by low-speed centrifugation and solid-liquid separation enabled effective collection of Saccharomyces cerevisiae cells with decreased lignin and ash. Five consecutive batch fermentation of 200 g/L rice straw hydrothermally pretreated led to an average ethanol titer of 34.5 g/L. Moreover, the display of cellulases on the recombinant yeast cell surface increased ethanol titer to 42.2 g/L. After, five-cycle fermentation, only 3.3 g/L sugar was retained in the fermentation medium, because cellulase displayed on the cell surface hydrolyzed cellulose that was not hydrolyzed by commercial cellulases or free secreted cellulases. Fermentation ability of the recombinant strain was successfully kept during a five-cycle repeated batch fermentation with 86.3% of theoretical yield based on starting biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.

Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library.

PubMed

Song, Yun-Hee; Lee, Kyung-Tai; Baek, Jin-Young; Kim, Min-Ju; Kwon, Mi-Ra; Kim, Young-Joo; Park, Mi-Rim; Ko, Haesu; Lee, Jin-Sung; Kim, Keun-Sung

2017-05-01

This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-β-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20-50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20-40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.

One-step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme.

PubMed

Cano-Ramírez, Claudia; Santiago-Hernández, Alejandro; Rivera-Orduña, Flor Nohemí; Pineda-Mendoza, Rosa María; Zúñiga, Gerardo; Hidalgo-Lara, María Eugenia

2017-02-01

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The cellulose binding region in Trichoderma reesei cellobiohydrolase I has a higher capacity in improving crystalline cellulose degradation than that of Penicillium oxalicum.

PubMed

Du, Jian; Zhang, Xiu; Li, Xuezhi; Zhao, Jian; Liu, Guodong; Gao, Baoyu; Qu, Yinbo

2018-06-19

Commercial cellulase preparations for lignocellulose bioconversion are mainly produced by the fungus Trichoderma reesei. The maximum cellulose conversion of T. reesei cellulase mixture was 15%-20% higher than that of Penicillium oxalicum in the hydrolysis of corncob residue and Avicel. Nevertheless, both preparations hydrolyzed more than 92% of cellulose in NaOH-mercerized Avicel. When added to Avicel hydrolysis residue that was less reactive to P. oxalicum cellulases, cellobiohydrolase I (CBH I) from T. reesei resulted in a higher cellulose conversion than its homologous proteins from P. oxalicum and Aspergillus niger at the same protein loadings. Further domain exchange experiment attributed the high hydrolytic efficiency of T. reesei CBH I to its inter-domain linker and cellulose-binding domain. The results in part explained the superior performance of T. reesei cellulases on the degradation of native crystalline cellulose, and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulases. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

PubMed

Chen, Hongzhang; Shao, Meixue; Li, Hongqiang

2014-03-05

The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r=0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation. Copyright © 2013 Elsevier Inc. All rights reserved.

Cellulase with high β-glucosidase activity by Penicillium oxalicum under solid state fermentation and its use in hydrolysis of cassava residue.

PubMed

Su, Lin-Hui; Zhao, Shuai; Jiang, Sui-Xin; Liao, Xu-Zhong; Duan, Cheng-Jie; Feng, Jia-Xun

2017-02-01

In this study, we investigated cellulase production by Penicillium oxalicum EU2106 under solid-state fermentation (SSF) and its hydrolysis efficiency toward NaOH-H 2 O 2 -pretreated cassava residue (NHCR) produced after bioethanol fermentation. Optimization of SSF cultivation conditions for P. oxalicum EU2106 using a Box-behnken design-based response-surface methodology resulted in maximal cellulase activity of 34.0 ± 2.8 filter-paper units/g dry substrate, exhibiting a ~ twofold increase relative to activities obtained under non-optimized conditions. Furthermore, SSF-derived cellulase converted 94.3 ± 1.5% of NHCR cellulose into glucose within 96 h. Interestingly, P. oxalicum EU2106 produced higher β-glucosidase activity under SSF conditions than that under submerged-state fermentation conditions, resulting in the elimination of cellobiose inhibition during the early stages of NHCR cellulose hydrolysis. Overall, this work provided an alternative for a potential cellulase source and a preferred option for cassava residue biotechnological application.

A solid state fungal fermentation-based strategy for the hydrolysis of wheat straw☆

PubMed Central

Pensupa, Nattha; Jin, Meng; Kokolski, Matt; Archer, David B.; Du, Chenyu

2013-01-01

This paper reports a solid-state fungal fermentation-based pre-treatment strategy to convert wheat straw into a fermentable hydrolysate. Aspergillus niger was firstly cultured on wheat straw for production of cellulolytic enzymes and then the wheat straw was hydrolyzed by the enzyme solution into a fermentable hydrolysate. The optimum moisture content and three wheat straw modification methods were explored to improve cellulase production. At a moisture content of 89.5%, 10.2 ± 0.13 U/g cellulase activity was obtained using dilute acid modified wheat straw. The addition of yeast extract (0.5% w/v) and minerals significantly improved the cellulase production, to 24.0 ± 1.76 U/g. The hydrolysis of the fermented wheat straw using the fungal culture filtrate or commercial cellulase Ctec2 was performed, resulting in 4.34 and 3.13 g/L glucose respectively. It indicated that the fungal filtrate harvested from the fungal fermentation of wheat straw contained a more suitable enzyme mixture than the commercial cellulase. PMID:24121367

Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

NASA Astrophysics Data System (ADS)

Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.

Production of cellulose by Aspergillus niger under submerged and solid state fermentation using coir waste as a substrate

PubMed Central

Mrudula, Soma; Murugammal, Rangasamy

2011-01-01

Aspergillus niger was used for cellulase production in submerged (SmF) and solid state fermentation (SSF). The maximum production of cellulase was obtained after 72 h of incubation in SSF and 96 h in Smf. The CMCase and FPase activities recorded in SSF were 8.89 and 3.56 U per g of dry mycelial bran (DBM), respectively. Where as in Smf the CMase & FPase activities were found to be 3.29 and 2.3 U per ml culture broth, respectively. The productivity of extracellular cellulase in SSF was 14.6 fold higher than in SmF. The physical and nutritional parameters of fermentation like pH, temperature, substrate, carbon and nitrogen sources were optimized. The optimal conditions for maximum biosynthesis of cellulase by A. niger were shown to be at pH 6, temperature 30 °C. The additives like lactose, peptone and coir waste as substrate increased the productivity both in SmF and SSF. The moisture ratio of 1:2 (w/v) was observed for optimum production of cellulase in SSF. PMID:24031730

Solid-state fermentation for cellulase production by Pestalotiopsis versicolor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rao, M.N.A.; Mithal, B.M.; Thakkur, R.N.

1983-03-01

Solid-state fermentation (SSF) refers to the fermentation process on solid substrate without the presence of free liquid. It is found to be ideal when the organism is a fungus and the substrate is insoluble, like cellulose. Production of cellulase by SSF has been studied in detail by Toyama and Ogawa. It has been found that more concentrated enzyme preparations can be obtained by SSF than in liquid type since the enzyme gets diluted in the whole medium in liquid culture. In the present study, a plant pathogenic fungus Pestalotiopsis versicolor has been grown on various solid cultures of cellulosic substancesmore » and production of cellulase has been studied. Earlier, we had studied the production of cellulase by P. versicolor in liquid culture. (Refs. 7).« less

Fractionation and cellulase treatment for enhancing the properties of kraft-based dissolving pulp.

PubMed

Duan, Chao; Wang, Xinqi; Zhang, YanLing; Xu, Yongjian; Ni, Yonghao

2017-01-01

The aim of this study was to investigate a combined process involving pulp fractionation and cellulase treatment of each fraction for improving the molecular weight distribution (MWD) and reactivity of a kraft-based dissolving pulp. Three pulp fractions, namely long-fiber, mid-fiber and short-fiber fractions (LF, MF and SF, respectively), were used as the substrates. The results showed that the SF had the highest accessibility, lowest viscosity, and highest cellulase adsorption capacity, while the opposite was true for the LF. At a given viscosity, the combined process led to a lower polydispersity index (3.71 vs 4.98) and a higher Fock reactivity (85.6% vs 76.3%), in comparison to the conventional single-stage cellulase treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Two Major Facilitator Superfamily Sugar Transporters from Trichoderma reesei and Their Roles in Induction of Cellulase Biosynthesis*

PubMed Central

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

2013-01-01

Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement. PMID:24085297

Heterogeneous Expression and Functional Characterization of Cellulose-Degrading Enzymes from Aspergillus niger for Enzymatic Hydrolysis of Alkali Pretreated Bamboo Biomass.

PubMed

Ali, Nasir; Ting, Zhang; Li, Hailong; Xue, Yong; Gan, Lihui; Liu, Jian; Long, Minnan

2015-09-01

Enzymatic hydrolysis of cellulosic biomass has caught much attention because of modest reaction conditions and environment friendly conditions. To reduce the cost and to achieve good quantity of cellulases, a heterologous expression system is highly favored. In this study, cellulose-degrading enzymes, GH3 family β-glucosidase (BGL), GH7 family-related cellobiohydrolases (CBHs), and endoglucanase (EG) from a newly isolated Aspergillus niger BE-2 are highly expressed in Pichia pastoris GS115. The strain produced EG, CBHs, and BGL enzymatic concentration of 0.56, 0.11, and 22 IU/mL, respectively. Mode of actions of the recombinant enzymes for substrate specificity and end product analysis are verified and found specific for cellulose degradation. Bamboo biomass saccharification with A. niger cellulase released a high level of fermentable sugars. Hydrolysis parameters are optimized to obtain reducing sugars level of 3.18 g/L. To obtain reducing sugars from a cellulosic biomass, A. niger could be a good candidate for enzymes resource of cellulase to produce reducing sugars from a cellulosic biomass. This study also facilitates the development of highly efficient enzyme cocktails for the bioconversion of lignocellulosic biomass into monosaccharides and oligosaccharides.

Characterization and Strain Improvement of a Hypercellulytic Variant, Trichoderma reesei SN1, by Genetic Engineering for Optimized Cellulase Production in Biomass Conversion Improvement.

PubMed

Qian, Yuanchao; Zhong, Lixia; Hou, Yunhua; Qu, Yinbo; Zhong, Yaohua

2016-01-01

The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG) activity but lower β-glucosidase (BGL) activity than those of the others. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30.0% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65.0% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.

Enzymatic hydrolysis of beer brewers' spent grain and the influence of pretreatments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beldman, G.; Hennekam, J.; Voragen, A.G.J.

1987-01-01

The enzymatic saccharification of plant material has been shown to be of interest in various fields, such as the production of fruit juices and the utilization of biomass. A combination of cellulase, pectinase, and hemicellulases is usually used because of the chemical composition of the matrix of plant cell walls. For apples, beet pulp, and potato fiber, almost a complete hydrolysis of polysaccharides is obtained by combining cellulase and pectinase. For nonparenchymatic tissue, the situation is somewhat different: pectin is a minor component and the hemicellulose content is much higher. Enzyme action is restricted by the lignin barrier and bymore » the high crystallinity of cellulose in this material. For such materials, mechanical, thermal, or chemical pretreatments are necessary to achieve efficient hydrolysis. This communication describes various enzymatic treatments and chemical and physical pretreatment, using brewers' spent grain as substrate. Spent grain is the residue of malt and grain which remains in the mash-kettle after the liquefied and saccharified starch has been removed by filtration. (Refs. 15).« less

Cloning of novel cellulases from cellulolytic fungi: heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris.

PubMed

Salinas, Alejandro; Vega, Marcela; Lienqueo, María Elena; Garcia, Alejandro; Carmona, Rene; Salazar, Oriana

2011-12-10

Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation. Copyright © 2011 Elsevier Inc. All rights reserved.

Ethanol from Agriculture for Arkansas and America

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hood, Elizabeth E

The purpose of this project was to develop technology that would facilitate production of sugars from agricultural residues to enable biofuels and biobased product manufacturing. Our primary technology is to use genetic engineering to put bacterial and fungal cellulase genes into corn kernels, using the grain as the production system for the enzymes. At the beginning of this DoE funded program, we were producing two cellulases—E1 endocellulase from a bacterium found in a hot spring at Yellowstone National Park, and CBH I exocellulase from a wood rot fungus. Our team developed several new regulatory sequences (promoters) that increased enzyme proteinmore » accumulation in two kernel compartments (embryo and endosperm). We were also able to capitalize on the diverse genetics of corn to increase protein accumulation. High oil germplasm in particular was instrumental in this increase. A second task in the program was to produce enzymes and proteins that enhanced the activity of the E1 and CBH I enzymes. Our team produced CBH II, from the same wood rot fungus at a level that enabled highly enhanced deconstruction activity of E1 and CBH I in a synergistic manner. We analyzed an additional protein, expansin from cucumber that was expressed in the maize grain expression system. This protein had been previously shown to enhance cellulase activity (D. Cosgrove, Penn State University), and required a large-scale production platform. Our team showed that the corn production system allows industrial amounts of active expansin to be harvested from the grain. One of the challenges of any new production system is to maximize recovery of active ingredient from the raw materials at a cost compatible with its final use. Our team showed that low pH extraction of grain solubilized the enzymes without contamination of native corn protein and active product could be concentrated through ultrafiltration. The final outcomes of this project were the following: 3 cellulase enzymes and the synergistic protein expansin produced at high levels in corn grain, new promoters and combinations of promoters to enhance protein accumulation in grain, application of unique germplasm pools to enhance protein accumulation, and highly efficient processing enabling cost-effective production of cellulases that are highly active in biomass deconstruction.« less

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

PubMed Central

2012-01-01

Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases. PMID:22998985

The crystallization and structural analysis of cellulases (and other glycoside hydrolases): strategies and tactics.

PubMed

Roberts, Shirley M; Davies, Gideon J

2012-01-01

The three-dimensional (3-D) structures of cellulases, and other glycoside hydrolases, are a central feature of research in carbohydrate chemistry and biochemistry. 3-D structure is used to inform protein engineering campaigns, both academic and industrial, which are typically used to improve the stability or activity of an enzyme. Examples of classical protein engineering goals include higher thermal stability, reduced metal-ion dependency, detergent and protease resistance, decreased product inhibition, and altered specificity. 3-D structure may also be used to interpret the behavior of enzyme variants that are derived from screening or random mutagenesis approaches, with a view to establishing an iterative design process. In other areas, 3-D structure is used as one of the many tools to probe enzymatic catalysis, typically dovetailing with physical organic chemistry approaches to provide complete reaction mechanisms for enzymes by visualizing catalytic site interactions at different stages of the reaction. Such mechanistic insight is not only fundamentally important, impacting on inhibitor and drug design approaches with ramifications way beyond cellulose hydrolysis, but also provides the framework for the design of enzyme variants to use as biocatalysts for the synthesis of bespoke oligosaccharides. Here we review some of the strategies and tactics that may be applied to the X-ray structure solution of cellulases (and other carbohydrate-active enzymes). The general approach is first to decide why you are doing the work, then to establish correct domain boundaries for truncated constructs (typically the catalytic domain only), and finally to pursue crystallization of pure, homogeneous, and monodisperse protein with appropriate ligand and additive combinations. Cellulase-specific strategies are important for the delineation of domain boundaries, while glycoside hydrolases generally also present challenges and opportunities for the selection and optimization of ligands to both aid crystallization, and also provide structural and mechanistic insight. As the many roles for plant cell wall degrading enzymes increase, so does the need for rapid high-quality structure determination to provide a sound structural foundation for understanding mechanism and specificity, and for future protein engineering strategies. Copyright © 2012 Elsevier Inc. All rights reserved.

Preparation of cellulase concoction using differential adsorption phenomenon.

PubMed

Birhade, Sachinkumar; Pednekar, Mukesh; Sagwal, Shilpa; Odaneth, Annamma; Lali, Arvind

2017-05-28

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.

Enhancement of Cellulase and Xylanase Production Using pH-Shift and Dissolved Oxygen Control Strategy with Streptomyces griseorubens JSD-1.

PubMed

Zhang, Dan; Luo, Yanqing; Chu, Shaohua; Zhi, Yuee; Wang, Bin; Zhou, Pei

2016-01-01

In this study, the production of cellulase and xylanase by Streptomyces griseorubens JSD-1 was improved by integrating the pH-shift and dissolved oxygen (DO)-constant control strategies. The pH-shift control strategy was carried out by analyzing the specific cell growth rate (μ) and specific enzyme formation rate (Q p) of S. griseorubens JSD-1. The pH was controlled at 8.0 during the first 48 h to maintain high cell growth, which then shifted to 7.5 after 48 h to improve the production of cellulase and xylanase. Using this method, the maximum activities of cellulase, xylanase, and filter paper enzyme (FPase) increased by 47.9, 29.5, and 113.6 %, respectively, compared to that obtained without pH control. On the basis of pH-shift control, the influence of DO concentrations on biomass and enzyme production was further investigated. The maximum production of cellulase, xylanase, and FPase reached 114.38 ± 0.96 U mL(-1), 330.57 ± 2.54 U mL(-1), and 40.11 ± 0.38 U mL(-1), which were about 1.6-fold, 0.6-fold, and 3.2-fold higher than that of neutral pH without DO control conditions. These results supplied a functional approach for improving cellulase and xylanase production.

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

PubMed

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). Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2016-05-19

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.

Effects of cellulase supplementation to corn soybean meal-based diet on the performance of sows and their piglets.

PubMed

Upadhaya, Santi D; Lee, Sang In; Kim, In Ho

2016-07-01

A total of 15 primiparous sows (Landrace × Yorkshire) and their litters were used in the current study to evaluate the efficacy of cellulase supplementation on the production performance of sows and piglets. Pigs were randomly allocated into one of three treatments with five replicates per treatment. The dietary treatments were as follows: (i) CON (corn-soybean meal-based control); (ii) EZ1 (CON + 0.05% cellulase); and (iii) EZ2 (CON + 0.10% cellulase). The supplementation of cellulase had no effect (P > 0.05) on body weight and feed intake of lactating sows. At weaning, back fat thickness loss decreased (P = 0.04) linearly in EZ1 and EZ2 treatments. The average daily gain (ADG) of piglets increased (linear P = 0.06, quadratic P = 0.04)) during days 14 to 21 as well as at days 21 to 25 (linear P = 0.03 and quadratic P = 0.01) with the increase in the level of supplemented enzyme. Dry matter and nitrogen digestibility increased (linear P = 0.01) in lactating sows fed EZ1 and EZ2 diet compared with CON. In conclusion, it is suggested that cellulase supplementation to corn-soybean meal based diet exerts beneficial effects to sows in reducing their back fat thickness loss at weaning and also helps to improve nutrient digestibility. It also helped to improve the ADG of piglets. © 2015 Japanese Society of Animal Science.

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

PubMed

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

2013-07-01

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

Production and Characterization of Organic Solvent-Tolerant Cellulase from Bacillus amyloliquefaciens AK9 Isolated from Hot Spring.

PubMed

Irfan, Muhammad; Tayyab, Ammara; Hasan, Fariha; Khan, Samiullah; Badshah, Malik; Shah, Aamer Ali

2017-08-01

A cellulase-producing bacterium, designated as strain AK9, was isolated from a hot spring of Tatta Pani, Azad Kashmir, Pakistan. The bacterium was identified as Bacillus amyloliquefaciens through 16S rRNA sequencing. Cellulase from strain AK9 was able to liberate glucose from soluble cellulose and carboxymethyl cellulose (CMC). Enzyme was purified through size exclusion chromatography and a single band of ∼47 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified with recovery of 35.5%, 3.6-fold purity with specific activity of 31 U mg -1 . The purified cellulase retained its activity over a wide range of temperature (50-70 °C) and pH (3-7) with maximum stability at 60 °C and pH 5.0. The activity inhibited by ethylenediaminetetraacetic acid (EDTA), suggested that it was metalloenzyme. Diethyl pyrocarbonate (DEPC) and β-mercaptoethanol significantly inhibited cellulase activity that revealed the essentiality of histidine residues and disulfide bonds for its catalytic function. It was stable in non-ionic surfactants, in the presence of various metal ions, and in water-insoluble organic solvents. Approximately 9.1% of reducing sugar was released after enzymatic saccharification of DAP-pretreated agro-residue, compared to a very low percentage by autohydrolysis treatment. Hence, it is concluded that cellulase from B. amyloliquefaciens AK9 can potentially be used in bioconversion of lignocellulosic biomass to fermentable sugars.

An Optimized Microplate Assay System for Quantitative Evaluation of Plant Cell Wall Degrading Enzyme Activity of Fungal Culture Extracts

USDA-ARS?s Scientific Manuscript database

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospe...

Lignocellulose-Adapted Endo-Cellulase Producing Streptomyces Strains for Bioconversion of Cellulose-Based Materials.

PubMed

Ventorino, Valeria; Ionata, Elena; Birolo, Leila; Montella, Salvatore; Marcolongo, Loredana; de Chiaro, Addolorata; Espresso, Francesco; Faraco, Vincenza; Pepe, Olimpia

2016-01-01

Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces ( S .) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens ) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies , and Streptomyces flavofuscus ), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus , and Streptomyces ambofaciens . Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers ( S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and xylanase preparations from Genencore (Accellerase BG and Accellerase XY). Cellulose and xylan conversion, when conducted using commercial (hemi)cellulases, gave glucose and xylose yields of 30.17 and 68.9%, respectively. The replacement of the cellulolytic preparation from Genencor (Accellerase 1500), with the endo-cellulase from S. argenteolus AE58P resulted in almost 76% of the glucose yield obtained in the presence of the commercial counterpart. Due to the promising results obtained by using the enzymatic crude extracts from S. argenteolus AE58P in the pretreated A. donax saccharification experiments, the proteins putatively responsible for endo-cellulase activity in this strain were identified by proteomics. Several proteins were confidently identified in different Streptomyces spp., eight of which belong to the class of Carbohydrate active enzymes. Overall results highlighted the biotechnological potential of S. argenteolus AE58P being an interesting candidate biocatalyst-producing bacterium for lignocellulose conversion and production of biochemicals and bioenergy.

Characterization of Lignocellulolytic Activities from a Moderate Halophile Strain of Aspergillus caesiellus Isolated from a Sugarcane Bagasse Fermentation

PubMed Central

Miranda-Miranda, Estefan; Sánchez-Reyes, Ayixón; Cuervo-Soto, Laura; Aceves-Zamudio, Denise; Atriztán-Hernández, Karina; Morales-Herrera, Catalina; Rodríguez-Hernández, Rocío; Folch-Mallol, Jorge

2014-01-01

A moderate halophile and thermotolerant fungal strain was isolated from a sugarcane bagasse fermentation in the presence of 2 M NaCl that was set in the laboratory. This strain was identified by polyphasic criteria as Aspergillus caesiellus. The fungus showed an optimal growth rate in media containing 1 M NaCl at 28°C and could grow in media added with up to 2 M NaCl. This strain was able to grow at 37 and 42°C, with or without NaCl. A. caesiellus H1 produced cellulases, xylanases, manganese peroxidase (MnP) and esterases. No laccase activity was detected in the conditions we tested. The cellulase activity was thermostable, halostable, and no differential expression of cellulases was observed in media with different salt concentrations. However, differential band patterns for cellulase and xylanase activities were detected in zymograms when the fungus was grown in different lignocellulosic substrates such as wheat straw, maize stover, agave fibres, sugarcane bagasse and sawdust. Optimal temperature and pH were similar to other cellulases previously described. These results support the potential of this fungus to degrade lignocellulosic materials and its possible use in biotechnological applications. PMID:25162614

Electrotransformation and expression of cellulase genes in wild-type Lactobacillus reuteri.

PubMed

Li, Wang; Yang, Ming-Ming; Zhang, Guang-Qin; He, Wan-Ling; Li, Yuan-Xiao; Chen, Yu-Lin

2012-01-01

Two cellulase genes, Cel15 and Cel73, were amplified from Bacillus subtilis genome DNA in a previous study. Two integrative vectors, pLEM4153 and pLEM4154, containing the genes Cel15 and Cel73, respectively, were constructed and successfully electroporated into the wild-type Lactobacillus reuteri which was isolated from chick guts through an optimized procedure. Two recombinant L. reuteri were selected from a Man, Rogosa, and Sharp (MRS) plate with 10 µg/ml erythromycin, and named L. reuteri XNY-Cel15 and L. reuteri XNY-Cel73, respectively. To verify the transcription and expression of the two cellulase genes in the recombinant L. reuteri strains, the mRNA relative quantity (RQ) and the cellulase activity were determined. The mRNA RQ of Cel15 in L. reuteri XNY-Cel15 is 1,8849.5, and that of Cel73 in L. reuteri XNY-Cel73 is 1,388, and the cellulase activity of the modified MRS broth cultured with L. reuteri XNY-Cel15 was 0.158 U/ml, whereas that with L. reuteri XNY-Cel73 was 0.15 U/ml. Copyright © 2012 S. Karger AG, Basel.

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

PubMed

Liu, Gang; Zhang, Jian; Bao, Jie

2016-01-01

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

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

PubMed

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 <10% of their initial activity after 24 h of incubation. Hydrolysis analysis results showed that a highly efficient synergistic effect was achieved by combining cellulase from T. piceus H16 with that from 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.

Hemicellulolytic Enzymes from the Maize Endophyte Acremonium zeae

USDA-ARS?s Scientific Manuscript database

Despite recent advances in cellulase development, there is still a need for efficient enzyme systems for the economical depolymerization of lignocellulosic feedstocks to fermentable sugars. In particular, the varying and heterogeneous structure of the hemicellulose fraction requires a complex suite...

Cellulase producing microorganism ATCC 55702

DOEpatents

Dees, H. Craig

1997-01-01

Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

DOEpatents

Dees, H. Craig

1997-12-16

Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

Cellulase producing microorganism ATCC 55702

DOEpatents

Dees, H.C.

1997-12-30

Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

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

NASA Astrophysics Data System (ADS)

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 mimic pretreatment hydrolyzates. No group has yet attempted to elucidate the inhibitory action of compounds isolated from pretreatment hydrolyzates. Elucidating the inhibition of cellulases using actual biomass hydrolyzates would offer insights as to which inhibitors, formed during a pretreatment, are key in causing inhibition. Knowing the key inhibitor(s) would allow for the development of processing conditions that minimize their production or of their removal through hydrolyzate detoxification methods. This research has characterized various chemical compounds released during dilute acid and hot water pretreatment of rice straw and has evaluated their inhibitory effects on endo-cellulase, exo-cellulase and beta-glucosidase enzymes. The hot water pretreatment hydrolyzate, generated at 220°C and 52 min, was found to be particularly inhibitory to exo- and endo-cellulases, and was chosen for further evaluation. This hot water hydrolyzate was fractionated using centrifugal partition chromatography (CPC) and grouped into furans, organic acids, phenolics, monomeric and oligomeric sugars. When these fractions were incubated with exo-cellulase, it was determined that fractions containing acetic acid and phenolics were highly inhibitory, resulting in 92% and 87% inhibition of initial hydrolysis rates, respectively. This study proposes a new approach for identifying key inhibitory compounds in biomass prehydrolyzates, eventually paving the way for developing strategies to the improve the enzymatic saccharification efficiency of lignocellulosic biomass.

Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

PubMed Central

Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

2012-01-01

Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the powerful competitive ability of plant cell wall degrading fungi in nature. PMID:23152805

Polyphenolic Contents and Antioxidant Activities of Underutilized Grape (Vitis vinifera L.) Pomace Extracts.

PubMed

Kabir, Faisal; Sultana, Mosammad Shahin; Kurnianta, Heri

2015-09-01

Grape pomace is an abundant source of underutilized winery by-products. Polyphenols were extracted from grape pomace using cellulase and gluco-amylase enzymes. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu's assays were used to measure antioxidant activity and total polyphenolic contents. Both cellulase, and gluco-amylase digested grape pomace showed efficient radical scavenging activity. In addition, the total polyphenolic contents of cellulase digested grape pomace showed lower concentrations were effective compared to higher concentrations, whereas gluco-amylase enzyme did not show remarkable variations. The DPPH radical scavenging activity and total polyphenolic contents were significantly higher in the cellulase digested grape pomace compared to the gluco-amylase digested and the not digested grape pomace. It is notable that enzymatic digestions were efficient for extracting polyphenols from grape pomace. The underutilized grape pomace polyphenols can be further used for food safety as a natural antioxidant.

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

PubMed Central

Zarafeta, Dimitra; Kissas, Dimitrios; Sayer, Christopher; Gudbergsdottir, Sóley R.; Ladoukakis, Efthymios; Isupov, Michail N.; Chatziioannou, Aristotelis; Peng, Xu; Littlechild, Jennifer A.; Skretas, Georgios; Kolisis, Fragiskos N.

2016-01-01

With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics. PMID:26741138

Polyphenolic Contents and Antioxidant Activities of Underutilized Grape (Vitis vinifera L.) Pomace Extracts

PubMed Central

Kabir, Faisal; Sultana, Mosammad Shahin; Kurnianta, Heri

2015-01-01

Grape pomace is an abundant source of underutilized winery by-products. Polyphenols were extracted from grape pomace using cellulase and gluco-amylase enzymes. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu’s assays were used to measure antioxidant activity and total polyphenolic contents. Both cellulase, and gluco-amylase digested grape pomace showed efficient radical scavenging activity. In addition, the total polyphenolic contents of cellulase digested grape pomace showed lower concentrations were effective compared to higher concentrations, whereas gluco-amylase enzyme did not show remarkable variations. The DPPH radical scavenging activity and total polyphenolic contents were significantly higher in the cellulase digested grape pomace compared to the gluco-amylase digested and the not digested grape pomace. It is notable that enzymatic digestions were efficient for extracting polyphenols from grape pomace. The underutilized grape pomace polyphenols can be further used for food safety as a natural antioxidant. PMID:26451359

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

PubMed

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.

A Constitutive Expression System for Cellulase Secretion in Escherichia coli and Its Use in Bioethanol Production

PubMed Central

Munjal, Neha; Jawed, Kamran; Wajid, Saima; Yazdani, Syed Shams

2015-01-01

The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol. PMID:25768292

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

PubMed

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

2011-02-01

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

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

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel bZIP transcription factor ClrC positively regulates multiple stress responses, conidiation and cellulase expression in Penicillium oxalicum.

PubMed

Lei, Yunfeng; Liu, Guodong; Yao, Guangshan; Li, Zhonghai; Qin, Yuqi; Qu, Yinbo

2016-06-01

Cellulase production in filamentous fungi is largely regulated at the transcriptional level, and several transcription factors have been reported to be involved in this process. In this study, we identified ClrC, a novel transcription factor in cellulase production in Penicillium oxalicum. ClrC and its orthologs have a highly conserved basic leucine zipper (bZIP) DNA binding domain, and their biological functions have not been explored. Deletion of clrC resulted in pleiotropic effects, including altered growth, reduced conidiation and increased sensitivity to oxidative and cell wall stresses. In particular, the clrC deletion mutant ΔclrC showed 46.1% ± 8.1% and 58.0% ± 8.7% decreases in production of filter paper enzyme and xylanase activities in cellulose medium, respectively. In contrast, 57.4% ± 10.0% and 70.9% ± 19.4% increased production of filter paper enzyme, and xylanase was observed in the clrC overexpressing strain, respectively. The transcription levels of major cellulase genes, as well as two cellulase transcriptional activator genes, clrB and xlnR, were significantly downregulated in ΔclrC, but substantially upregulated in clrC overexpressing strains. Furthermore, we observed that the absence of ClrC reduced full induction of cellulase expression even in the clrB overexpressing strain. These results indicated that ClrC is a novel and efficient engineering target for improving cellulolytic enzyme production in filamentous fungi. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility.

PubMed

Zhu, Zhiguang; Sathitsuksanoh, Noppadon; Vinzant, Todd; Schell, Daniel J; McMillan, James D; Zhang, Y-H Percival

2009-07-01

Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF-pretreated corn stover but reached only approximately 60% for the DA-pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were measured for the first time. The COSLIF-pretreated corn stover had a CAC of 11.57 m(2)/g, nearly twice that of the DA-pretreated biomass (5.89 m(2)/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA- and COSLIF-pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility. (c) 2009 Wiley Periodicals, Inc.

Acetylation of loofa (Luffa cylindrica) sponge as immobilization carrier for bioprocesses involving cellulase.

PubMed

Hideno, Akihiro; Ogbonna, James C; Aoyagi, Hideki; Tanaka, Hideo

2007-04-01

The feasibility of using loofa sponge for immobilization of cellulase-producing microorganisms was investigated by acetylating loofa sponge. Acetylation was achieved by autoclaving process of loofa sponge immersed in acetic anhydride at various temperatures for various times. The degree of acetylation, as inferred by the weight percentage gain (WPG), was enhanced by increasing both temperature and the duration of acetylation. The acetylation of a piece of loofa sponge in an autoclave at 120 degrees C for 20 min resulted in a WPG of about 8%, which was sufficient to protect the loofa sponge against cellulose degradation. The acetylated loofa sponge prepared under this condition was not decomposed by commercial cellulase and its structure was maintained for more than 720 h during repeated-batch treatments with commercial cellulase. A flocculating yeast (Saccharomyces cerevisiae IR-2) and a fungus (Trichoderma reesei QM9414) were successfully immobilized in the acetylated loofa sponge. In each case, the percentage of immobilized cells was as high as that obtained using nonacetylated loofa sponge. Acetylation had no adverse effects on cell growth and immobilization of T. reesei QM9414, as well as on cell growth and ethanol production by S. cerevisiae IR-2. T. reesei QM9414 immobilized on an acetylated loofa sponge was successfully used for repeated-batch cellulase production from commercial cellulose powder. Although the acetylated loofa sponge showed a slight weight loss, it was not disintegrated by activated sludge. The results obtained in this study showed that acetylated loofa sponge is suitable as an immobilization carrier for bioprocesses involving cellulase.

The optimization of fermentation conditions for producing cellulase of Bacillus amyloliquefaciens and its application to goose feed

PubMed Central

Ye, Miao; Sun, Linghong; Yang, Ru; Qi, KeZong

2017-01-01

The proper culture conditions for producing cellulase of Bacillus amyloliquefaciens S1, isolated from the cecum of goose was optimized by single-factor experiment combined with orthogonal test. The properties of the cellulase were investigated by DNS method. The appropriate doses of B. amyloliquefaciens S1 were obtained by adding them to goose feed. It indicated that the suitable culture conditions of producing cellulase were the culture temperature of 37°C, the initial pH of 7.0, the incubation time of 72 h and the loaded liquid volume of 75 ml per 250 ml. The effects of each factor on producing cellulase by B. amyloliquefaciens S1 were as follows: initial pH > incubation time = culture temperature > loaded liquid volume. The optimum reaction temperature and pH were 50°C and 7.0, respectively. This enzyme is a kind of neutral cellulase that possesses resistance to heat and acidity. It showed high activity to absorbent cotton, soya bean meal and filter paper. By adding different doses of B. amyloliquefaciens S1 to the goose feed, it was found that the egg production, average egg weight, fertilization rate and the hatching rate were promoted both in experiment 1 (1.5 g kg−1) and experiment 2 (3 g kg−1). Also the difference of egg production, fertilization rate and hatching rate between experiment 1 and control group was obvious (p < 0.05), and the average egg weight was significantly increased in experiment 2 (p < 0.05). PMID:29134097

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDougall, G.J.; Fry, S.C.

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{submore » 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).« less

Intracellular β-Glucosidases CEL1a and CEL1b Are Essential for Cellulase Induction on Lactose in Trichoderma reesei

PubMed Central

Xu, Jintao; Zhao, Guolei; Kou, Yanbo; Zhang, Weixin; Zhou, Qingxin; Chen, Guanjun

2014-01-01

Lactose (1,4-O-β-d-galacto-pyranosyl-d-glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular β-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei. We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular β-glucosidases abolished cbh1 gene expression on lactose. d-Galactose restored the efficient cellulase gene induction in the Δcel1a strain independently of its reductive metabolism, but not in the Δcel1a Δcel1b strain. A further comparison of the transcriptional responses of the Δcel1a Δcel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the Δcel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis β-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular β-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes. PMID:24879125

Nanomechanical Sensing of Biological Interfacial Interactions

NASA Astrophysics Data System (ADS)

Du, Wenjian

Cellulose is the most abundant biopolymer on earth. Cellulase is an enzyme capable of converting insoluble cellulose into soluble sugars. Cellulosic biofuel produced from such fermentable simple sugars is a promising substitute as an energy source. However, its economic feasibility is limited by the low efficiency of the enzymatic hydrolysis of cellulose by cellulase. Cellulose is insoluble and resistant to enzymatic degradation, not only because the beta-1,4-glycosidic bonds are strong covalent bonds, but also because cellulose microfibrils are packed into tightly bound, crystalline lattices. Enzymatic hydrolysis of cellulose by cellulase involves three steps--initial binding, decrystallization, and hydrolytic cleavage. Currently, the mechanism for the decrystallization has not yet been elucidated, though it is speculated to be the rate-limiting step of the overall enzymatic activity. The major technical challenge limiting the understanding of the decrystallization is the lack of an effective experimental approach capable of examining the decrystallization, an interfacial enzymatic activity on solid substrates. The work presented develops a nanomechanical sensing approach to investigate both the decrystallization and enzymatic hydrolytic cleavage of cellulose. The first experimental evidence of the decrystallization is obtained by comparing the results from native cellulase and non-hydrolytic cellulase. Surface topography has been applied to examine the activities of native cellulase and non-hydrolytic cellulase on cellulose substrate. The study demonstrates additional experimental evidence of the decrystallization in the hydrolysis of cellulose. By combining simulation and monitoring technology, the current study also investigates the structural changes of cellulose at a molecular level. In particular, the study employs cellulose nanoparticles with a bilayer structure on mica sheets. By comparing results from a molecular dynamic simulation and the distance between cellulose layers monitored by means of the atomic force microscopy (AFM), the current study shows that water molecules can efficiently reduce the energy required for separating two layers of cellulose bilayers during hydration of cellulose bilayer nanoparticles. The findings of the study contribute to explicating the mechanism of cellulose the decrystallization, a free-energetically unfavorable process, through enzymatic hydrolysis of cellulase. The study also investigates the application of a cell-based microcantilever sensor to monitor the real-time ligand-induced response of living cells. These nanomechanical approaches offer unique perspectives on the interfacial activities of biological molecules.

Lignosulfonate and elevated pH can enhance enzymatic saccharification of lignocelluloses

PubMed Central

2013-01-01

Background Nonspecific (nonproductive) binding (adsorption) of cellulase by lignin has been identified as a key barrier to reduce cellulase loading for economical sugar and biofuel production from lignocellulosic biomass. Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) is a relatively new process, but demonstrated robust performance for sugar and biofuel production from woody biomass especially softwoods in terms of yields and energy efficiencies. This study demonstrated the role of lignin sulfonation in enhancing enzymatic saccharification of lignocelluloses – lignosulfonate from SPORL can improve enzymatic hydrolysis of lignocelluloses, contrary to the conventional belief that lignin inhibits enzymatic hydrolysis due to nonspecific binding of cellulase. Results The study found that lignosulfonate from SPORL pretreatment and from a commercial source inhibits enzymatic hydrolysis of pure cellulosic substrates at low concentrations due to nonspecific binding of cellulase. Surprisingly, the reduction in enzymatic saccharification efficiency of a lignocellulosic substrate was fully recovered as the concentrations of these two lignosulfonates increased. We hypothesize that lignosulfonate serves as a surfactant to enhance enzymatic hydrolysis at higher concentrations and that this enhancement offsets its inhibitive effect from nonspecific binding of cellulase, when lignosulfonate is applied to lignocellulosic solid substrates. Lignosulfonate can block nonspecific binding of cellulase by bound lignin on the solid substrates, in the same manner as a nonionic surfactant, to significantly enhance enzymatic saccharification. This enhancement is linearly proportional to the amount of lignosulfonate applied which is very important to practical applications. For a SPORL-pretreated lodgepole pine solid, 90% cellulose saccharification was achieved at cellulase loading of 13 FPU/g glucan with the application of its corresponding pretreatment hydrolysate coupled with increasing hydrolysis pH to above 5.5 compared with only 51% for the control run without lignosulfonate at pH 5.0. The pH-induced lignin surface modification at pH 5.5 further reduced nonspecific binding of cellulase by lignosulfonate. Conclusions The results reported in this study suggest significant advantages for SPORL-pretreatment in terms of reducing water usage and enzyme dosage, and simplifying process integration, i.e., it should eliminate washing of SPORL solid fraction for direct simultaneous enzymatic saccharification and combined fermentation of enzymatic and pretreatment hydrolysates (SSCombF). Elevated pH 5.5 or higher, rather than the commonly believed optimal and widely practiced pH 4.8-5.0, should be used in conducting enzymatic saccharification of lignocelluloses. PMID:23356796

Purification and characterization of an endoglucanase from the marine rotifer, Brachionus plicatilis.

PubMed

Chun, C Z; Hur, S B; Kim, Y T

1997-10-01

The marine rotifer, Brachionus plicatilis, is able to digest Chlorella efficiently, suggesting that the rotifer contains a powerful cellulolytic enzyme system. A multi-component cellulolytic complex, including endoglucanase (CM-cellulase), cellobiohydrolase and beta-glucosidase, was found in Brachionus plicatilis. Endoglucanase (endo-beta-1,4 glucanase) was purified to homogeneity from rotifer homogenates using a sequential chromatographic method. The purified enzyme exhibits a strong hydrolytic activity with carboxymethyl(CM)-cellulose. The optimum temperature and pH for the endoglucanase activity were 37 degrees C and 7.0, respectively. 80% of the CM-cellulase activity was retained in salt mixture that ranged from 150 to 500 mM NaCl equivalent. The purified protein was isolated with a molecular weight of approximately 62 kDa estimated by SDS-polyacrylamide gel electrophoresis.

Optimization of fed-batch enzymatic hydrolysis from alkali-pretreated sugarcane bagasse for high-concentration sugar production.

PubMed

Gao, Yueshu; Xu, Jingliang; Yuan, Zhenhong; Zhang, Yu; Liu, Yunyun; Liang, Cuiyi

2014-09-01

Fed-batch enzymatic hydrolysis process from alkali-pretreated sugarcane bagasse was investigated to increase solids loading, produce high-concentration fermentable sugar and finally to reduce the cost of the production process. The optimal initial solids loading, feeding time and quantities were examined. The hydrolysis system was initiated with 12% (w/v) solids loading in flasks, where 7% fresh solids were fed consecutively at 6h, 12h, 24h to get a final solids loading of 33%. All the requested cellulase loading (10 FPU/g substrate) was added completely at the beginning of hydrolysis reaction. After 120 h of hydrolysis, the maximal concentrations of cellobiose, glucose and xylose obtained were 9.376 g/L, 129.50 g/L, 56.03 g/L, respectively. The final total glucan conversion rate attained to 60% from this fed-batch process. Copyright © 2014. Published by Elsevier Ltd.

Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass

USDA-ARS?s Scientific Manuscript database

Critical cellulase and hemicellulase activities are identified for hydrolysis of ionic liquid (IL) pretreated poplar and switchgrass; hemicellulase rich substrates with amorphous cellulose. Enzymes from Aspergillus nidulans were expressed and purified: an endoglucanase (EG) a cellobiohydrolase (CBH)...

Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

DOEpatents

Dees, H. Craig

1998-01-01

Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

Evaluation of alkali treatment for biodegradation of corn cobs by Aspergillus niger.

PubMed

Singh, A; Abidi, A B; Agrawal, A K; Darmwal, N S

1989-01-01

Effect of NaOH pretreatment on the biodegradation of corn cobs for the production of cellulase and protein was studied using Aspergillus niger. Delignification of cobs with NaOH remarkably increased the production of cellulase and protein. Treatment of cobs with 2% NaOH was found to be the best with respect to their susceptibility to biodegradation for maximum production of cellulose 1,4-beta-cellobiosidase, cellulase, beta-glucosidase soluble protein and crude protein; this also led to the highest protein recovery, maximum cellulose utilization and also for the maximum degradation of substrate.

Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

DOEpatents

Dees, H.C.

1997-12-16

Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

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

PubMed

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.

Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse

PubMed Central

2011-01-01

Background Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. Aspergillus niger has been shown to produce a wide spectrum of polysaccharide hydrolytic enzymes. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse (SEB). Results Herein we report the main cellulase- and hemicellulase-encoding genes with increased expression during growth on SEB. We also sought to determine whether the mRNA accumulation of several SEB-induced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 (58% of A. niger predicted cellulases) and 21 (58% of A. niger predicted hemicellulases) cellulase- and hemicellulase-encoding genes, respectively, that were highly expressed during growth on SEB. Conclusions Degradation of sugarcane bagasse requires production of many different enzymes which are regulated by the type and complexity of the available substrate. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol. PMID:22008461

A new earthworm cellulase and its possible role in the innate immunity.

PubMed

Park, In Yong; Cha, Ju Roung; Ok, Suk-Mi; Shin, Chuog; Kim, Jin-Se; Kwak, Hee-Jin; Yu, Yun-Sang; Kim, Yu-Kyung; Medina, Brenda; Cho, Sung-Jin; Park, Soon Cheol

2017-02-01

A new endogenous cellulase (Ean-EG) from the earthworm, Eisenia andrei and its expression pattern are demonstrated. Based on a deduced amino acid sequence, the open reading frame (ORF) of Ean-EG consisted of 1368 bps corresponding to a polypeptide of 456 amino acid residues in which is contained the conserved region specific to GHF9 that has the essential amino acid residues for enzyme activity. In multiple alignments and phylogenetic analysis, the deduced amino acid sequence of Ean- EG showed the highest sequence similarity (about 79%) to that of an annelid (Pheretima hilgendorfi) and could be clustered together with other GHF9 cellulases, indicating that Ean-EG could be categorized as a member of the GHF9 to which most animal cellulases belong. The histological expression pattern of Ean-EG mRNA using in situ hybridization revealed that the most distinct expression was observed in epithelial cells with positive hybridization signal in epidermis, chloragogen tissue cells, coelomic cell-aggregate, and even blood vessel, which could strongly support the fact that at least in the earthworm, Eisenia andrei, cellulase function must not be limited to digestive process but be possibly extended to the innate immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Utilization of radiation technique on the saccharification and fermentation of biomass

NASA Astrophysics Data System (ADS)

Kaetsu, I.; Kumakura, M.; Fujimura, T.; Yoshii, F.; Kojima, T.; Tamada, M.

The application of irradiation technique to the process of saccharification and subsequent fermentation of cellulosic wastes such as chaff and rice straw to obtain ethanol, was investigated. It was found that when waste raw materials were irradiated by ?-ray or electron beam, they became accessible to the subsequent enzymatic saccharification reaction. Irradiation of 10 7-10 8 Rad was enough for this effect. Some kind of additives reduced necessary dosage for this pretreatment. Cellulase, Trichoderma reesei which produce cellulase, and yeast were immobilized as biocatalysts for biomass conversion by radiation-induced polymerization of glass-forming monomer at low temperature. The immobilized cellulase showed almost same activity of glucose production as the native cellulase. Continuous saccharification reaction was carried out by using the immobilized cellulase. The immobilized Trichoderma reesei and the immobilized yeast showed almost same activity as the intact biocatalysts. It was concluded that the continuous saccharification and subsequent fermentation could be carried out effectively by using the immobilized biocatalysts. Spinach chloroplasts were immobilized by the same method as the first step for the conversion of water into hydrogen gas using solar energy. The immobilized chloroplasts kept the O 2 evolution activity in storage more than 30 days at 4°C. Thermostatility of chloroplasts was also improved greatly by the immobilization.

The unique GH5 cellulase member in the extreme halotolerant fungus Aspergillus glaucus CCHA is an endoglucanase with multiple tolerance to salt, alkali and heat: prospects for straw degradation applications.

PubMed

Li, Zhengqun; Pei, Xue; Zhang, Ziyu; Wei, Yi; Song, Yanyue; Chen, Lina; Liu, Shouan; Zhang, Shi-Hong

2018-07-01

In a halotolerant fungus Aspergillus glaucus CCHA, several functional proteins with stress-tolerant activity have been studied, but no secretory enzymes have been identified yet. The unique GH5 cellulase candidate from A. glaucus, an endoglucanase termed as AgCMCase, was cloned, expressed in the Pichia pastoris system and the purified enzyme was characterized. A large amount of recombinant enzyme secreted by the P. pastoris GS115 strain was purified to homogeneity. The molecular weight of the purified endoglucanase is about 55.0 kDa. The AgCMCase exhibited optimum catalytic activity at pH 5.0 and 55 °C. However, it remained relatively stable at temperatures ranging from 45 to 80 °C and pH ranging from 4.0 to 9.0. In addition, it showed higher activity at extreme NaCl concentrations from 1.0 to 4.0 M, suggesting it is an enzyme highly stable under heat, acid, alkaline and saline conditions. To evaluate the catalytic activity of AgCMCase, the hydrolysis products of rice and corn straws were successfully studied. In conclusion, the AgCMCase is a thermostable and salt-tolerant cellulase with potential for industrial application.

A microplate assay for quantitative evaluation of plant cell wall degrading enzymes

USDA-ARS?s Scientific Manuscript database

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Plant pathogenic fungi are a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass convers...

Effects of Oligosaccharides Isolated From Pinewood Hot Water Pre-hydrolyzates on Recombinant Cellulases.

PubMed

Fang, Hong; Kandhola, Gurshagan; Rajan, Kalavathy; Djioleu, Angele; Carrier, Danielle Julie; Hood, Kendall R; Hood, Elizabeth E

2018-01-01

Loblolly pine residues have enormous potential to be the raw material for advanced biofuel production due to extensive sources and high cellulose content. Hot water (HW) pretreatment, while being a relatively economical and clean technology for the deconstruction of lignocellulosic biomass, could also inhibit the ensuing enzymatic hydrolysis process because of the production of inhibitors. In this study, we investigated the effect of oligosaccharide fractions purified from HW pre-hydrolyzate of pinewood using centrifugal partition chromatography (CPC) on three recombinant cellulolytic enzymes (E1, CBHI and CBHII), which were expressed in the transgenic corn grain system. The efficiency of recombinant enzymes was measured using either a 4-methylumbelliferyl-β-D-cellobioside (MUC) or a cellulose-dinitrosalicylic acid (DNS) assay system. The results showed that HW pre-hydrolyzate CPC fractions contain phenolics, furans, and monomeric and oligomeric sugars. Among CPC fractions, oligomers composed of xylan, galactan, and mannan were inhibitory to the three recombinant enzymes and to the commercial cellulase cocktail, reducing the enzymatic efficiency to as low as 10%.

Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass

DOE PAGES

Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.; ...

2014-10-09

Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysismore » of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. In Conclusion: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized. The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.« less

Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.

Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysismore » of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. In Conclusion: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized. The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.« less

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger

DOE PAGES

Amaike Campen, Saori; Lynn, Jed; Sibert, Stephanie J.; ...

2017-12-27

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme productionmore » host before they could be considered a viable alternative to current commercial cellulases. Aspergillus Niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. Niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. Niger and Escherichia coli. Finally, this comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. Niger is equivalent, suggesting that A. Niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.« less

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amaike Campen, Saori; Lynn, Jed; Sibert, Stephanie J.

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme productionmore » host before they could be considered a viable alternative to current commercial cellulases. Aspergillus Niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. Niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. Niger and Escherichia coli. Finally, this comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. Niger is equivalent, suggesting that A. Niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.« less

The productive cellulase binding capacity of cellulosic substrates.

PubMed

Karuna, Nardrapee; Jeoh, Tina

2017-03-01

Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases. Productive cellulase binding to cellulose results in hydrolysis and can be quantified by measuring hydrolysis rates. In this study, we measured the productive Trichoderma reesei Cel7A (TrCel7A) binding capacity of five cellulosic substrates from different sources and processing histories. Swollen filter paper and bacterial cellulose had higher productive binding capacities of ∼6 µmol/g while filter paper, microcrystalline cellulose, and algal cellulose had lower productive binding capacities of ∼3 µmol/g. Swelling and regenerating filter paper using phosphoric acid increased the initial accessibility of the reducing ends to TrCel7A from 4 to 6 µmol/g. Moreover, this increase in initial productive binding capacity accounted in large part for the difference in the overall digestibility between filter paper and swollen filter paper. We further demonstrated that an understanding of how the productive binding capacity declines over the course of the hydrolysis reaction has the potential to predict overall saccharification time courses. Biotechnol. Bioeng. 2017;114: 533-542. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Tao; Datta, Supratim; Eichler, Jerry

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 halophilicmore » 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.« less

The use of thermostable bacterial hemicellulases improves the conversion of lignocellulosic biomass to valuable molecules.

PubMed

Rakotoarivonina, Harivony; Revol, Pierre-Vincent; Aubry, Nathalie; Rémond, Caroline

2016-09-01

The hydrolysis of xylans, one of the main classes of carbohydrates that constitute lignocellulosic biomass, requires the synergistic action of several enzymes. The development of efficient enzymatic strategies for hydrolysis remains a challenge in the pursuit of viable biorefineries, particularly with respect to the valorisation of pentoses. The approach developed in this work is based on obtaining and characterising hemicellulasic cocktails from Thermobacillus xylanilyticus after culturing this bacterium on the hemicellulose-rich substrates wheat bran and wheat straw, which differ in their chemistries. The two obtained cocktails (WSC and WBC, for cocktails obtained from wheat straw and wheat bran, respectively) were resistant to a broad range of temperature and pH conditions. At 60 °C, both cocktails efficiently liberated pentoses and phenolic acids from wheat bran (liberating more than 60, 30 and 40 % of the total xylose, arabinose and ferulic acid in wheat bran, respectively). They acted to a lesser extent on the more recalcitrant wheat straw, with hydrolytic yields of more than 30 % of the total arabinose and xylose content and 22 % of the ferulic acid content. Hydrolysis is associated with a high rate of sugar monomerisation. When associated with cellulases, high quantities of glucose were also obtained. On wheat bran, total glucose yields were improved by 70 % compared to the action of cellulases alone. This improvement was obtained by cellulase complementation either with WSC or with WBC. On wheat straw, similar levels of total glucose were obtained for cellulases alone or complemented with WSC or WBC. Interestingly, the complementation of cellulases with WSC or WBC induced an increase in the monomeric glucose yield of more than 20 % compared to cellulases alone.

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger

PubMed Central

Lynn, Jed; Sibert, Stephanie J.; Srikrishnan, Sneha; Phatale, Pallavi; Feldman, Taya; Guenther, Joel M.; Hiras, Jennifer; Tran, Yvette Thuy An; Singer, Steven W.; Adams, Paul D.; Sale, Kenneth L.; Simmons, Blake A.; Baker, Scott E.; Magnuson, Jon K.; Gladden, John M.

2017-01-01

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry. PMID:29281693

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger.

PubMed

Amaike Campen, Saori; Lynn, Jed; Sibert, Stephanie J; Srikrishnan, Sneha; Phatale, Pallavi; Feldman, Taya; Guenther, Joel M; Hiras, Jennifer; Tran, Yvette Thuy An; Singer, Steven W; Adams, Paul D; Sale, Kenneth L; Simmons, Blake A; Baker, Scott E; Magnuson, Jon K; Gladden, John M

2017-01-01

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.

Engineering yeast for the expression and secretion of cellulase cocktails

USDA-ARS?s Scientific Manuscript database

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 ...

Enhanced processive cellulases

DOEpatents

Adney, William S.; Beckham, Gregg T.; Jarvis, Eric; Himmel, Michael E.; Decker, Stephen R.; Linger, Jeffrey G.; Podkaminer, Kara; Baker, John O.; Taylor, II, Larry; Xu, Qi; Singh, Arjun

2017-06-20

Nucleic acid sequences encoding chimeric polypeptides that exhibit enhanced cellulase activities are disclosed herein. These nucleic acids may be expressed in hosts such as fungi, which in turn may be cultured to produce chimeric polypeptides. Also disclosed are chimeric polypeptides and their use in the degradation of cellulosic materials.

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

NASA Astrophysics Data System (ADS)

Donaldson, Lloyd; Vaidya, Alankar

2017-03-01

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

The effect of nonenzymatic protein on lignocellulose enzymatic hydrolysis and simultaneous saccharification and fermentation.

PubMed

Wang, Hui; Kobayashi, Shinichi; Hiraide, Hatsue; Cui, Zongjun; Mochidzuki, Kazuhiro

2015-01-01

Nonenzymatic protein was added to cellulase hydrolysis and simultaneous saccharification and fermentation (SSF) of different biomass materials. Adding bovine serum albumin (BSA) and corn steep before cellulase enhanced enzyme activity in solution and increased cellulose and xylose conversion rates. The cellulose conversion rate of filter paper hydrolysis was increased by 32.5 % with BSA treatment. When BSA was added before cellulase, the remaining activity in the solution was higher than that in a control without BSA pretreatment. During SSF with pretreated rice straw as the substrate, adding 1.0 mg/mL BSA increased the ethanol yield by 13.6 % and final xylose yield by 42.6 %. The results indicated that lignin interaction is not the only mechanism responsible for the positive BSA effect. BSA had a stabilizing effect on cellulase and relieved cumulative sugar inhibition of enzymatic hydrolysis of biomass materials. Thus, nonenzymatic protein addition represents a promising strategy in the biorefining of lignocellulose materials.

Towards efficient bioethanol production from agricultural and forestry residues: Exploration of unique natural microorganisms in combination with advanced strain engineering.

PubMed

Zhao, Xinqing; Xiong, Liang; Zhang, Mingming; Bai, Fengwu

2016-09-01

Production of fuel ethanol from lignocellulosic feedstocks such as agricultural and forestry residues is receiving increasing attention due to the unsustainable supply of fossil fuels. Three key challenges include high cellulase production cost, toxicity of the cellulosic hydrolysate to microbial strains, and poor ability of fermenting microorganisms to utilize certain fermentable sugars in the hydrolysate. In this article, studies on searching of natural microbial strains for production of unique cellulase for biorefinery of agricultural and forestry wastes, as well as development of strains for improved cellulase production were reviewed. In addition, progress in the construction of yeast strains with improved stress tolerance and the capability to fully utilize xylose and glucose in the cellulosic hydrolysate was also summarized. With the superior microbial strains for high titer cellulase production and efficient utilization of all fermentable sugars in the hydrolysate, economic biofuels production from agricultural residues and forestry wastes can be realized. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microplate-based filter paper assay to measure total cellulase activity.

PubMed

Xiao, Zhizhuang; Storms, Reginald; Tsang, Adrian

2004-12-30

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%. 2004 Wiley Periodicals, Inc.

Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator.

PubMed

Zhang, Xiaoyue; Li, Yonghao; Zhao, Xinqing; Bai, Fengwu

2017-01-01

The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved. Copyright © 2016. Published by Elsevier Ltd.

Synthesis and Characterization of Carboxymethylcellulose-Methacrylate Hydrogel Cell Scaffolds

PubMed Central

Reeves, Robert; Ribeiro, Andreia; Lombardo, Leonard; Boyer, Richard; Leach, Jennie B.

2012-01-01

Many carbohydrates pose advantages for tissue engineering applications due to their hydrophilicity, degradability, and availability of chemical groups for modification. For example, carboxymethylcellulose (CMC) is a water-soluble cellulose derivative that is degradable by cellulase. Though this enzyme is not synthesized by mammalian cells, cellulase and the fragments derived from CMC degradation are biocompatible. With this in mind, we created biocompatible, selectively degradable CMC-based hydrogels that are stable in routine culture, but degrade when exposed to exogenous cellulase. Solutions of CMC-methacrylate and polyethylene glycol dimethacrylate (PEG-DM) were co-crosslinked to form stable hydrogels; we found that greater CMC-methacrylate content resulted in increased gel swelling, protein diffusion and rates of degradation by cellulase, as well as decreased gel shear modulus. CMC-methacrylate/PEG-DM gels modified with the adhesive peptide RGD supported fibroblast adhesion and viability. We conclude that hydrogels based on CMC-methacrylate are suitable for bioengineering applications where selective degradability may be favorable, such as cell scaffolds or controlled release devices. PMID:22708058

Temperature and pH influence adsorption of cellobiohydrolase onto lignin by changing the protein properties.

PubMed

Lu, Xianqin; Wang, Can; Li, Xuezhi; Zhao, Jian

2017-12-01

Non-productive adsorption of cellulase onto lignin restricted the movement of cellulase and also hindered the cellulase recycling in bioconversion of lignocellulose. In this study, effect of temperature and pH on adsorption and desorption of cellobiohydrolase (CBH) on lignin and its possible mechanism were discussed. It found that pH value and temperature influenced the adsorption and desorption behaviors of CBH on lignin. Different thermodynamic models suggested that the action between lignin and CBH was physical action. More CBH was adsorbed onto lignin, but lower initial adsorption velocity was detected at 50°C comparing with 4°C. Elevating pH value could improve desorption of cellulase from lignin. The changes of hydrophobicity and electric potential on protein surface may partially explain the impact of environmental conditions on the adsorption and desorption behaviors of CBH on lignin, and comparing to electrical interaction, the hydrophobicity may be the dominating factor influencing the behaviors. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Improvement of halophilic cellulase production from locally isolated fungal strain.

PubMed

Gunny, Ahmad Anas Nagoor; Arbain, Dachyar; Jamal, Parveen; Gumba, Rizo Edwin

2015-07-01

Halophilic cellulases from the newly isolated fungus, Aspergillus terreus UniMAP AA-6 were found to be useful for in situ saccharification of ionic liquids treated lignocelluloses. Efforts have been taken to improve the enzyme production through statistical optimization approach namely Plackett-Burman design and the Face Centered Central Composite Design (FCCCD). Plackett-Burman experimental design was used to screen the medium components and process conditions. It was found that carboxymethylcellulose (CMC), FeSO4·7H2O, NaCl, MgSO4·7H2O, peptone, agitation speed and inoculum size significantly influence the production of halophilic cellulase. On the other hand, KH2PO4, KOH, yeast extract and temperature had a negative effect on enzyme production. Further optimization through FCCCD revealed that the optimization approach improved halophilic cellulase production from 0.029 U/ml to 0.0625 U/ml, which was approximately 2.2-times greater than before optimization.

Improvement of halophilic cellulase production from locally isolated fungal strain

PubMed Central

Gunny, Ahmad Anas Nagoor; Arbain, Dachyar; Jamal, Parveen; Gumba, Rizo Edwin

2014-01-01

Halophilic cellulases from the newly isolated fungus, Aspergillus terreus UniMAP AA-6 were found to be useful for in situ saccharification of ionic liquids treated lignocelluloses. Efforts have been taken to improve the enzyme production through statistical optimization approach namely Plackett–Burman design and the Face Centered Central Composite Design (FCCCD). Plackett–Burman experimental design was used to screen the medium components and process conditions. It was found that carboxymethylcellulose (CMC), FeSO4·7H2O, NaCl, MgSO4·7H2O, peptone, agitation speed and inoculum size significantly influence the production of halophilic cellulase. On the other hand, KH2PO4, KOH, yeast extract and temperature had a negative effect on enzyme production. Further optimization through FCCCD revealed that the optimization approach improved halophilic cellulase production from 0.029 U/ml to 0.0625 U/ml, which was approximately 2.2-times greater than before optimization. PMID:26150755

Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07.

PubMed

Gaur, Rajeeva; Tiwari, Soni

2015-03-19

The rising concerns about the scarcity of fossil fuels, the emission of green house gasses and air pollution by incomplete combustion of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermo-solvent tolerant cellulase producing bacterium from natural resources, and then applied for purification and characterization. The purified enzyme was to be accessible for the bioethanol production as well as industrial exploitation (discuss in our next study). It is the first instance when thermo-solvent tolerant cellulase producing bacterium was isolated from soil sample. The culture was identified as Bacillus vallismortis RG-07 by 16S rDNA sequence analysis. Bacillus vallismortis RG-07 reported maximum cellulase production from sugarcane baggase (4105 U ml(-1)) used as agro-waste carbon source. The cellulase enzyme produced by the Bacillus sp. was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with overall recovery of 28.8%. The molecular weight of purified cellulase was 80 kDa as revealed by SDS-PAGE and activity gel analysis. The optimum temperature and pH for enzyme activity was determined as 65°C and 7.0 and it retained 95 and 75% of activity even at 95°C, and 9.0 respectively. The enzyme activity was enhanced in the presence of organic solvents (30%) n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, and cyclohexane, after prolonged incubation (7 days). The enzyme activity was also stimulated by Ca(2+), mercaptoethanol, Tween-60, and Sodium hypochloride whereas strongly inhibited by Hg. Kinetic analysis of purified enzyme showed the Km and Vmax to be 1.923 mg ml(-1) and 769.230 μg ml(-1) min(-1), respectively. The unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for current mainstream biomass conversion into fuel and other industrial process.

A Mitogen-Activated Protein Kinase Tmk3 Participates in High Osmolarity Resistance, Cell Wall Integrity Maintenance and Cellulase Production Regulation in Trichoderma reesei

PubMed Central

Wang, Mingyu; Zhao, Qiushuang; Yang, Jinghua; Jiang, Baojie; Wang, Fangzhong; Liu, Kuimei; Fang, Xu

2013-01-01

The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, ‘budded’ hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei is evolved to favor solid state growth, bringing up the proposal that the submerged condition normally used during investigations on fungal physiology might be misleading. PMID:23991059

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

DOEpatents

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

2013-04-23

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

Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

DOEpatents

Dees, H.C.

1998-07-14

Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

Lignin-blocking treatment of biomass and uses thereof

DOEpatents

Yang, Bin [Hanover, NH; Wyman, Charles E [Norwich, VT

2009-10-20

Disclosed is a method for converting cellulose in a lignocellulosic biomass. The method provides for a lignin-blocking polypeptide and/or protein treatment of high lignin solids. The treatment enhances cellulase availability in cellulose conversion. Cellulase efficiencies are improved by the protein or polypeptide treatment. The treatment may be used in combination with steam explosion and acid prehydrolysis techniques. Hydrolysis yields from lignin containing biomass are enhanced 5-20%, and enzyme utilization is increased from 10% to 50%. Thus, a more efficient and economical method of processing lignin containing biomass materials utilizes a polypeptide/protein treatment step that effectively blocks lignin binding of cellulase.

Screening for Cellulase Encoding Clones in Metagenomic Libraries.

PubMed

Ilmberger, Nele; Streit, Wolfgang R

2017-01-01

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

Compounds inhibiting the bioconversion of hydrothermally pretreated lignocellulose.

PubMed

Ko, Ja Kyong; Um, Youngsoon; Park, Yong-Cheol; Seo, Jin-Ho; Kim, Kyoung Heon

2015-05-01

Hydrothermal pretreatment using liquid hot water, steam explosion, or dilute acids enhances the enzymatic digestibility of cellulose by altering the chemical and/or physical structures of lignocellulosic biomass. However, compounds that inhibit both enzymes and microbial activity, including lignin-derived phenolics, soluble sugars, furan aldehydes, and weak acids, are also generated during pretreatment. Insoluble lignin, which predominantly remains within the pretreated solids, also acts as a significant inhibitor of cellulases during hydrolysis of cellulose. Exposed lignin, which is modified to be more recalcitrant to enzymes during pretreatment, adsorbs cellulase nonproductively and reduces the availability of active cellulase for hydrolysis of cellulose. Similarly, lignin-derived phenolics inhibit or deactivate cellulase and β-glucosidase via irreversible binding or precipitation. Meanwhile, the performance of fermenting microorganisms is negatively affected by phenolics, sugar degradation products, and weak acids. This review describes the current knowledge regarding the contributions of inhibitors present in whole pretreatment slurries to the enzymatic hydrolysis of cellulose and fermentation. Furthermore, we discuss various biological strategies to mitigate the effects of these inhibitors on enzymatic and microbial activity to improve the lignocellulose-to-biofuel process robustness. While the inhibitory effect of lignin on enzymes can be relieved through the use of lignin blockers and by genetically engineering the structure of lignin or of cellulase itself, soluble inhibitors, including phenolics, furan aldehydes, and weak acids, can be detoxified by microorganisms or laccase.

The effect of lignin removal by alkaline peroxide pretreatment on the susceptibility of corn stover to purified cellulolytic and xylanolytic enzymes.

PubMed

Selig, Michael J; Vinzant, Todd B; Himmel, Michael E; Decker, Stephen R

2009-05-01

Pretreatment of corn stover with alkaline peroxide (AP) at pH 11.5 resulted in reduction of lignin content in the residual solids as a function of increasing batch temperature. Scanning electron microscopy of these materials revealed notably more textured surfaces on the plant cell walls as a result of the delignifying pretreatment. As expected, digestion of the delignified samples with commercial cellulase preparations showed an inverse relationship between the content of lignin present in the residual solids after pretreatment and the extent of both glucan and xylan conversion achievable. Digestions with purified enzymes revealed that decreased lignin content in the pretreated solids did not significantly impact the extent of glucan conversion achievable by cellulases alone. Not until purified xylanolytic activities were included with the cellulases were significant improvements in glucan conversion realized. In addition, an inverse relationship was observed between lignin content after pretreatment and the extent of xylan conversion achievable in a 24-h period with the xylanolytic enzymes in the absence of the cellulases. This observation, coupled with the direct relationship between enzymatic xylan and glucan conversion observed in a number of cases, suggests that the presence of lignins may not directly occlude cellulose present in lignocelluloses but rather impact cellulase action indirectly by its association with xylan.

Cellulase enzyme: Homology modeling, binding site identification and molecular docking

NASA Astrophysics Data System (ADS)

Selvam, K.; Senbagam, D.; Selvankumar, T.; Sudhakar, C.; Kamala-Kannan, S.; Senthilkumar, B.; Govarthanan, M.

2017-12-01

Cellulase is an enzyme that degrades the linear polysaccharide like cellulose into glucose by breaking the β-1,4- glycosidic bonds. These enzymes are the third largest enzymes with a great potential towards the ethanol production and play a vital role in degrading the biomass. The production of ethanol depends upon the ability of the cellulose to utilize the wide range of substrates. In this study, the 3D structure of cellulase from Acinetobacter sp. was modeled by using Modeler 9v9 and validated by Ramachandran plot. The accuracy of the predicted 3D structure was checked using Ramachandran plot analysis showed that 81.1% in the favored region, compatibility of an atomic model (3D) with amino acid sequence (1D) for the model was observed as 78.21% and 49.395% for Verify 3D and ERRAT at SAVES server. As the binding efficacy with the substrate might suggests the choice of the substrate as carbon and nitrogen sources, the cellobiose, cellotetraose, cellotetriose and laminaribiose were employed in the docking studies. The docking of cellobiose, cellotetraose, cellotetriose and laminaribiose with cellulase exhibited the binding energy of -6.1523 kJ/mol, -7.8759 kJ/mol,-6.1590 kJ/mol and -6.7185 kJ/mol, respectively. These docking studies revealed that cellulase has the greater potential towards the cellotetraose as a substrate for the high yield of ethanol.

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

PubMed

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

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.

Biological pretreatment of rice straw with Streptomyces griseorubens JSD-1 and its optimized production of cellulase and xylanase for improved enzymatic saccharification efficiency.

PubMed

Zhang, Dan; Luo, Yanqing; Chu, Shaohua; Zhi, Yuee; Wang, Bin; Zhou, Pei

2016-08-17

Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L(-1) for rice straw, wheat bran, peptone, and CaCO3, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL(-1) were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.

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

PubMed

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

2015-12-21

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.

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

PubMed Central

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

2017-01-01

Objective In order to improve fermentation quality of natural grasses, their silage preparation and fermentation quality in meadow steppe (MS) and typical steppe (TS) were studied. Methods The small-scale silages and round bale silages of mixed natural grasses in both steppes were prepared using the commercial lactic acid bacteria (LAB) inoculants Chikuso-1 (CH, Lactobacillus plantarum) and cellulase enzyme (AC, Acremonium cellulase) as additives. Results MS and TS contained 33 and 9 species of natural grasses, respectively. Stipa baicalensis in MS and Stipa grandi in TS were the dominant grasses with the highest dry matter (DM) yield. The crude protein (CP), neutral detergent fiber and water-soluble carbohydrate of the mixed natural grasses in both steppes were 8.02% to 9.03%, 66.75% to 69.47%, and 2.02% to 2.20% on a DM basis, respectively. All silages treated with LAB and cellulase were well preserved with lower pH, butyric acid and ammonia-N content, and higher lactic acid and CP content than those of control in four kinds of silages. Compared with CH- or AC-treated silages, the CH+ AC-treated silages had higher lactic acid content. Conclusion The results confirmed that combination with LAB and cellulase may result in beneficial effects by improving the natural grass silage fermentation in both grasslands. PMID:27703133

The O-Glycosylated Linker from the Trichoderma reesei Family 7 Cellulase Is a Flexible, Disordered Protein

PubMed Central

Beckham, Gregg T.; Bomble, Yannick J.; Matthews, James F.; Taylor, Courtney B.; Resch, Michael G.; Yarbrough, John M.; Decker, Steve R.; Bu, Lintao; Zhao, Xiongce; McCabe, Clare; Wohlert, Jakob; Bergenstråhle, Malin; Brady, John W.; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.

2010-01-01

Fungi and bacteria secrete glycoprotein cocktails to deconstruct cellulose. Cellulose-degrading enzymes (cellulases) are often modular, with catalytic domains for cellulose hydrolysis and carbohydrate-binding modules connected by linkers rich in serine and threonine with O-glycosylation. Few studies have probed the role that the linker and O-glycans play in catalysis. Since different expression and growth conditions produce different glycosylation patterns that affect enzyme activity, the structure-function relationships that glycosylation imparts to linkers are relevant for understanding cellulase mechanisms. Here, the linker of the Trichoderma reesei Family 7 cellobiohydrolase (Cel7A) is examined by simulation. Our results suggest that the Cel7A linker is an intrinsically disordered protein with and without glycosylation. Contrary to the predominant view, the O-glycosylation does not change the stiffness of the linker, as measured by the relative fluctuations in the end-to-end distance; rather, it provides a 16 Å extension, thus expanding the operating range of Cel7A. We explain observations from previous biochemical experiments in the light of results obtained here, and compare the Cel7A linker with linkers from other cellulases with sequence-based tools to predict disorder. This preliminary screen indicates that linkers from Family 7 enzymes from other genera and other cellulases within T. reesei may not be as disordered, warranting further study. PMID:21112302

Genome sequence and annotation of Trichoderma parareesei, the ancestor of the cellulase producer Trichoderma reesei

DOE PAGES

Yang, Dongqing; Pomraning, Kyle; Kopchinskiy, Alexey; ...

2015-08-13

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.

Thermal tolerant avicelase from Acidothermus cellulolyticus

DOEpatents

Ding, Shi-You [Golden, CO; Adney, William S [Golden, CO; Vinzant, Todd B [Golden, CO; Himmel, Michael E [Littleton, CO

2008-04-29

The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized from Acidothermus cellulolyticus and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

[Derepression of cellulase synthesis in Trichoderma lignorum during limitation of consumption of readily available carbon sources].

PubMed

Lobanok, A G; Pavlovskaia Zhi

1975-01-01

The synthesis of Cx-cellulase was de-repressed in Trichoderma lignorum growing on various easily metabolized carbon sources when their assimilation was limited. A reverse correlation has been established between the growth rate and the rate of the enzyme synthesis in the fungus.

Thermal tolerant avicelase from Acidothermus cellulolyticus

DOEpatents

Ding, Shi-You [Golden, CO; Adney, William S [Golden, CO; Vinzant, Todd B [Golden, CO; Himmel, Michael E [Littleton, CO

2009-05-26

The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized from Acidothermus cellulolyticus and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

Enhanced Learning of Biotechnology Students by an Inquiry-Based Cellulase Laboratory

ERIC Educational Resources Information Center

Ketpichainarong, Watcharee; Panijpan, Bhinyo; Ruenwongsa, Pintip

2010-01-01

This study explored the effectiveness of an inquiry-based cellulase laboratory unit in promoting inquiry in undergraduate students in biotechnology. The following tools were used to assess the students' achievements and attitude: conceptual understanding test, concept mapping, students' documents, CLES questionnaire, students' self reflection, and…

Towards complete hydrolysis of soy flour carbohydrates by enzyme mixtures for protein enrichment: A modeling approach.

PubMed

Loman, Abdullah Al; Ju, Lu-Kwang

2016-05-01

Soy protein is a well-known nutritional supplement in proteinaceous food and animal feed. However, soybeans contain complex carbohydrate. Selective carbohydrate removal by enzymes could increase the protein content and remove the indigestibility of soy products for inclusion in animal feed. Complete hydrolysis of soy flour carbohydrates is challenging due to the presence of proteins and different types of non-structural polysaccharides. This study is designed to guide complex enzyme mixture required for hydrolysis of all types of soy flour carbohydrates. Enzyme broths from Aspergillus niger, Aspergillus aculeatus and Trichoderma reesei fermentations were evaluated in this study for soy carbohydrate hydrolysis. The resultant hydrolysate was measured for solubilized carbohydrate by both total carbohydrate and reducing sugar analyses. Conversion data attained after 48h hydrolysis were first fitted with models to determine the maximum fractions of carbohydrate hydrolyzable by each enzyme group, i.e., cellulase, xylanase, pectinase and α-galactosidase. Kinetic models were then developed to describe the increasing conversions over time under different enzyme activities and process conditions. The models showed high fidelity in predicting soy carbohydrate hydrolysis over broad ranges of soy flour loading (5-25%) and enzyme activities: per g soy flour, cellulase, 0.04-30 FPU; xylanase, 3.5-618U; pectinase, 0.03-120U; and α-galactosidase, 0.01-60U. The models are valuable in guiding the development and production of optimal enzyme mixtures toward hydrolysis of all types of carbohydrates present in soy flour and in optimizing the design and operation of hydrolysis reactor and process. Copyright © 2016 Elsevier Inc. All rights reserved.

Occupational asthma caused by cellulase and lipase in the detergent industry.

PubMed

Brant, A; Hole, A; Cannon, J; Helm, J; Swales, C; Welch, J; Taylor, A Newman; Cullinan, P

2004-09-01

Three employees from two different detergent companies were investigated for occupational asthma, using skin prick tests, serum specific IgE, and specific bronchial challenge. Two were challenged with lipase and one with cellulase. All three cases had immunological evidence of sensitisation to the detergent enzymes with which they worked. Bronchial challenge in each provoked a reproducible dual asthmatic response, which reproduced their work related symptoms. These are the first reported cases of occupational asthma attributable to cellulase and lipase in the detergent industry. Four of the most common enzymes used in this industry have now been reported to cause occupational asthma; continued vigilance and caution are needed when working with these or other enzymes.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lichius, Alexander; Bidard, Frédérique; Buchholz, Franziska

2015-04-20

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.

The isolation and functional identification on producing cellulase of Pseudomonas mendocina

PubMed Central

Zhang, Jianfeng; Hou, Hongyan; Chen, Guang; Wang, Shusheng; Zhang, Jiejing

2016-01-01

ABSTRACT The straw can be degraded efficiently into humus by powerful enzymes from microorganisms, resulting in the accelerated circulation of N,P,K and other effective elements in ecological system. We isolated a strain through screening the straw degradation strains from natural humic straw in the low temperature area in northeast of china, which can produce cellulase efficiently. The strain was identified as Pseudomonas mendocina by using morphological, physiological, biochemical test, and molecular biological test, with the functional clarification on producing cellulase for Pseudomonas mendocina for the first time. The enzyme force constant Km and the maximum reaction rate (Vmax) of the strain were 0.3261 g/L and 0.1525 mg/(min.L) through the enzyme activity detection, and the molecular weight of the enzyme produced by the strain were 42.4 kD and 20.4 kD based on SDS-PAGE. The effects of various ecological factors such as temperature, pH and nematodes on the enzyme produced by the strain in the micro ecosystem in plant roots were evaluated. The result showed that the optimum temperature was 28°C, and the best pH was 7.4∼7.8, the impact heavy metal was Pb2+ and the enzyme activity and biomass of Pseudomonas mendocina increased the movement and predation of nematodes. PMID:27710430

Advanced evolutionary molecular engineering to produce thermostable cellulase by using a small but efficient library.

PubMed

Ito, Y; Ikeuchi, A; Imamura, C

2013-01-01

We aimed at constructing thermostable cellulase variants of cellobiohydrolase II, derived from the mesophilic fungus Phanerochaete chrysosporium, by using an advanced evolutionary molecular engineering method. By aligning the amino acid sequences of the catalytic domains of five thermophilic fungal CBH2 and PcCBH2 proteins, we identified 45 positions where the PcCBH2 genes differ from the consensus sequence of two to five thermophilic fungal CBH2s. PcCBH2 variants with the consensus mutations were obtained by a cell-free translation system that was chosen for easy evaluation of thermostability. From the small library of consensus mutations, advantageous mutations for improving thermostability were found to occur with much higher frequency relative to a random library. To further improve thermostability, advantageous mutations were accumulated within the wild-type gene. Finally, we obtained the most thermostable variant Mall4, which contained all 15 advantageous mutations found in this study. This variant had the same specific cellulase activity as the wild type and retained sufficient activity at 50°C for >72 h, whereas wild-type PcCBH2 retained much less activity under the same conditions. The history of the accumulation process indicated that evolution of PcCBH2 toward improved thermostability was ideally and rapidly accomplished through the evolutionary process employed in this study.

Development of crosslinked methylcellulose hydrogels for soft tissue augmentation using an ammonium persulfate-ascorbic acid redox system.

PubMed

Gold, Gittel T; Varma, Devika M; Taub, Peter J; Nicoll, Steven B

2015-12-10

Hydrogels composed of methylcellulose are candidate materials for soft tissue reconstruction. Although photocrosslinked methylcellulose hydrogels have shown promise for such applications, gels crosslinked using reduction-oxidation (redox) initiators may be more clinically viable. In this study, methylcellulose modified with functional methacrylate groups was polymerized using an ammonium persulfate (APS)-ascorbic acid (AA) redox initiation system to produce injectable hydrogels with tunable properties. By varying macromer concentration from 2% to 4% (w/v), the equilibrium moduli of the hydrogels ranged from 1.47 ± 0.33 to 5.31 ± 0.71 kPa, on par with human adipose tissue. Gelation time was found to conform to the ISO standard for injectable materials. Cellulase treatment resulted in complete degradation of the hydrogels within 24h, providing a reversible corrective feature. Co-culture with human dermal fibroblasts confirmed the cytocompatibility of the gels based on DNA measurements and Live/Dead imaging. Taken together, this evidence indicates that APS-AA redox-polymerized methylcellulose hydrogels possess properties beneficial for use as soft tissue fillers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genomic organization of a cellulase gene family in Phanerochaete chrysosporium

Treesearch

Sarah F. Covert; Jennifer Bolduc; Daniel Cullen

1992-01-01

Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene (cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were...

Methods of using thermal tolerant avicelase from Acidothermus cellulolyticus

DOEpatents

Adney, William S [Golden, CO; Vinzant, Todd B [Golden, CO; Ding, Shih-You [Golden, CO; Himmel, Michael E [Golden, CO

2011-04-26

The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized from Acidothermus cellulolyticus, and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

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

USDA-ARS?s Scientific Manuscript database

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...

Lignosulfonate and elevated pH can enhance enzymatic saccharification of lignocelluloses

Treesearch

ZJ Wang; TQ Lan; JY Zhu

2013-01-01

Nonspecific (nonproductive) binding (adsorption) of cellulase by lignin has been identified as a key barrier to reduce cellulase loading for economical sugar and biofuel production from lignocellulosic biomass. Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) is a relatively new process, but demonstrated robust performance for sugar and biofuel...

Enhancement of β-xylosidase productivity in cellulase producing fungus Acremonium cellulolyticus

PubMed Central

2011-01-01

Enzymatic hydrolysis is one of the most important processes in bioethanol production from lignocellulosic biomass. Acremonium cellulolyticus is a filamentous fungus with high cellulase production but productivity of hemicellulase, especially β-xylosidase, is lower than other filamentous fungi. We identified 2.4 Kb β-xylosidase gene in the A. cellulolyticus genome sequence information and it encoded 798 amino acids without introns. To enhance hemicellulase productivity in A. cellulolyticus, we transformed this fungus with the identified β-xylosidase gene driven by the cellobiohydrolase Ι (cbh1) promoter, using the protoplast-polyethyleneglycol (PEG) method, and obtained a transformant, YKX1. Hydrolysis rate of xylooligosaccharides was more than 50-fold higher using culture supernatant from YKX1 than that from the parental strain, Y-94. Total cellulase activity (measured by filter paper assay) in YKX1 was not affected by the cbh1 promoter used for expression of β-xylosidase, and induced by cellulose. Since YKX1 can produce larger amount of β-xylosidase without affecting cellulase productivity, it is considered to be beneficial for practical monosaccharide recoveries from lignocellulosic biomass. PMID:21906369

Enhancement of β-xylosidase productivity in cellulase producing fungus Acremonium cellulolyticus.

PubMed

Kanna, Machi; Yano, Shinichi; Inoue, Hiroyuki; Fujii, Tatsuya; Sawayama, Shigeki

2011-06-30

Enzymatic hydrolysis is one of the most important processes in bioethanol production from lignocellulosic biomass. Acremonium cellulolyticus is a filamentous fungus with high cellulase production but productivity of hemicellulase, especially β-xylosidase, is lower than other filamentous fungi. We identified 2.4 Kb β-xylosidase gene in the A. cellulolyticus genome sequence information and it encoded 798 amino acids without introns. To enhance hemicellulase productivity in A. cellulolyticus, we transformed this fungus with the identified β-xylosidase gene driven by the cellobiohydrolase Ι (cbh1) promoter, using the protoplast-polyethyleneglycol (PEG) method, and obtained a transformant, YKX1. Hydrolysis rate of xylooligosaccharides was more than 50-fold higher using culture supernatant from YKX1 than that from the parental strain, Y-94. Total cellulase activity (measured by filter paper assay) in YKX1 was not affected by the cbh1 promoter used for expression of β-xylosidase, and induced by cellulose. Since YKX1 can produce larger amount of β-xylosidase without affecting cellulase productivity, it is considered to be beneficial for practical monosaccharide recoveries from lignocellulosic biomass.

The inhibition of hemicellulosic sugars on cellulose hydrolysis are highly dependant on the cellulase productive binding, processivity, and substrate surface charges.

PubMed

Zhai, Rui; Hu, Jinguang; Saddler, Jack N

2018-06-01

In this study, the influence of major hemicellulosic sugars (mannose and xylose) on cellulose hydrolysis and major enzyme activities were evaluated by using both commercial enzyme cocktail and purified cellulase monocomponents over a "library" of cellulosic substrates. Surprisingly, the results showed that unlike glucose, mannose/xylose did not inhibit individual cellulase activities but significantly decreased their hydrolytic performance on cellulose substrates. When various enzyme-substrate interactions (e.g. adsorption/desorption, productive binding, and processive moving) were evaluated, it appeared that these hemicellulosic sugars significantly reduced the productive binding and processivity of Cel7A, which in turn limited cellulase hydrolytic efficacy. Among a range of major cellulose characteristics (e.g. crystallinity, degree of polymerization, accessibility, and surface charges), the acid group content of the cellulosic substrates seemed to be the main driver that determined the extent of hemicellulosic sugar inhibition. Our results provided new insights for better understanding the sugar inhibition mechanisms of cellulose hydrolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Conversion of woody biomass into fermentable sugars by cellulase from Agaricus arvensis.

PubMed

Jeya, Marimuthu; Nguyen, Ngoc-Phuong-Thao; Moon, Hee-Jung; Kim, Sang-Hwan; Lee, Jung-Kul

2010-11-01

Agaricus arvensis, a newly isolated basidiomycetous fungus, was found to secrete efficient cellulases. The strain produced the highest endoglucanase (EG), cellobiohydrolase (CBH) and beta-glucosidase (BGL) activities of 0.3, 3.2 and 8U/mg-protein, respectively, with rice straw as the carbon source. Saccharification of the woody biomass with A. arvensis cellulase as the enzyme source released a high level of fermentable sugars. Enzymatic hydrolysis of the poplar biomass was optimized using the response surface methodology in order to study the influence of the variables (pH, temperature, cellulases concentration and substrate concentration). The enzyme and substrate concentrations were identified as the limiting factors for the saccharification of poplar wood biomass. A total reducing sugar level of 29g/L (293mg/g-substrate) was obtained at an enzyme concentration of 65FPU/g-substrate after optimization of the hydrolysis parameters. The model validation showed a good agreement between the experimental results and the predicted responses. A. arvensis could be a good candidate for the production of reducing sugars from a cellulosic biomass.

Co-immobilization of cellulase and lysozyme on amino-functionalized magnetic nanoparticles: An activity-tunable biocatalyst for extraction of lipids from microalgae.

PubMed

Chen, Qingtai; Liu, Dong; Wu, Chongchong; Yao, Kaisheng; Li, Zhiheng; Shi, Nan; Wen, Fushan; Gates, Ian D

2018-05-03

An activity-tunable biocatalyst for Nannochloropsis sp. cell-walls degradation was prepared by co-immobilization of cellulase and lysozyme on the surface of amino-functionalized magnetic nanoparticles (MNPs) employing glutaraldehyde. The competition between cellulase and lysozyme during immobilization was caused by the limited active sites of the MNPs. The maximum recovery of activities (cellulase: 78.9% and lysozyme: 69.6%) were achieved due to synergistic effects during dual-enzyme co-immobilization. The thermal stability in terms of half-life of the co-immobilized enzymes was three times higher than that in free form and had higher catalytic efficiency for hydrolysis of cell walls. Moreover, the co-immobilized enzymes showed greater thermal stability and wider pH tolerance than free enzymes under harsh conditions. Furthermore, the co-immobilized enzymes retained up to 60% of the residual activity after being recycled 6 times. This study provides a feasible approach for the industrialization of enzyme during cell-walls disruption and lipids extraction from Nannochloropsis sp. Copyright © 2018. Published by Elsevier Ltd.

Physicochemical and functional properties of coconut (Cocos nucifera L) cake dietary fibres: Effects of cellulase hydrolysis, acid treatment and particle size distribution.

PubMed

Zheng, Yajun; Li, Yan

2018-08-15

Effects of cellulase hydrolysis, acid treatment and particle size distribution on the structure, physicochemical and functional properties of coconut cake dietary fiber (DCCDF) were studied. Results showed that both the cellulase hydrolysis and acid treatment contributed to the structural modification of DCCDF as evident from XRD, FT-IR and SEM analysis. Moreover, the cellulase hydrolysis enhanced soluble carbohydrate content, water holding capacity (WHC) and swelling capacity (WSC), α-amylase inhibition activity (α-AAIR), glucose dialysis retardation index (GDRI) and cation-exchange capacity (CEC) of DCCDF; but it had undesirable effects on colour, oil holding capacity (OHC) and emulsifying capacity (EC). On other hand, acid treatment decreased the WHC, WSC and GDRI, but improved the colour, CEC, OHC and emulsion stability of DCCDF. Furthermore, the WHC, WSC and EC of DCCDF increased as the particle size reduced from 250 to 167 μm, while the GDRI, OHC, α-AAIR and emulsion stability decreased with decreasing particle size. Copyright © 2018 Elsevier Ltd. All rights reserved.

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed.

PubMed

Facchini, Fernanda Dell Antonio; Vici, Ana Claudia; Reis, Victor Ricardo Amin; Jorge, João Atilio; Terenzi, Héctor Francisco; Reis, Ricardo Andrade; Polizeli, Maria de Lourdes Teixeira de Moraes

2011-03-01

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 °C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 °C and 50-55 °C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 °C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu++ and Mn++ increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

Mitochondrial functions mediate cellulase gene expression in Trichoderma reesei.

PubMed

Abrahão-Neto, J; Rossini, C H; el-Gogary, S; Henrique-Silva, F; Crivellaro, O; el-Dorry, H

1995-08-22

We examined the effects of inhibition of mitochondrial functions on the expression of two nuclear genes encoding the extracellular cellobiohydrolase I (cbh1) and endoglucanase I (egl1) of the cellulase system of the filamentous fungus Trichoderma reesei. The cbh1 and egl1 transcripts are repressed at a low oxygen tension, and by glucose at a concentration known to repress mitochondrial respiration. The transcripts are also down-regulated by chemical agents known to dissipate the proton electrochemical gradient of the inner mitochondrial membrane and blocking of the electron-transport chain, such as DNP and KCN, respectively. These results suggest that expression of those transcripts is influenced by the physiological state of the mitochondria. In addition, heterologous gene fusion shows that the sensitivity of the expression of those transcripts to the functional state of the mitochondria is transcriptionally controlled through the 5'-flanking DNA sequence of those genes.

Characterization of cellulolytic extract from Pycnoporus sanguineus PF-2 and its application in biomass saccharification.

PubMed

Falkoski, Daniel Luciano; Guimarães, Valéria Monteze; de Almeida, Maíra Nicolau; Alfenas, Acelino Couto; Colodette, Jorge Luiz; de Rezende, Sebastião Tavares

2012-03-01

The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, β-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase activities. Cellulase activities were characterized in relation to pH and temperature. β-Glucosidase and FPase activities were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5-4.0. All cellulase activities were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and 64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification.

Xylose induces cellulase production in Thermoascus aurantiacus.

PubMed

Schuerg, Timo; Prahl, Jan-Philip; Gabriel, Raphael; Harth, Simon; Tachea, Firehiwot; Chen, Chyi-Shin; Miller, Matthew; Masson, Fabrice; He, Qian; Brown, Sarah; Mirshiaghi, Mona; Liang, Ling; Tom, Lauren M; Tanjore, Deepti; Sun, Ning; Pray, Todd R; Singer, Steven W

2017-01-01

Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus . Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted to produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. Xylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.

Suppression of Cellulase and Polygalacturonase and Induction of Alcohol Dehydrogenase Isoenzymes in Avocado Fruit Mesocarp Subjected to Low Oxygen Stress

PubMed Central

Kanellis, Angelos K.; Solomos, Theophanes; Roubelakis-Angelakis, Kalliopi A.

1991-01-01

Expression of polygalacturonase and cellulase, two hydrolytic enzymes of avocado (Persea americana, cv Hass) fruit which are synthesized de novo during ripening, and alcohol dehydrogenase, a known anaerobic protein, were studied under different O2 regimes. Low O2 concentrations (2.5-5.5%) diminished the accumulation of polygalacturonase and cellulase proteins and the expression of their isoenzymes. This pattern of change in cellulase protein was also reflected in the steady-state amount of its mRNA. In contrast, 7.5 and 10% O2 did not alter the changes observed in fruits ripened in air. On the other hand, alcohol dehydrogenase was induced in 2.5, 3.5, and 5.5% O2 but not in 7.5 or 10% O2. The recovery from the hypoxic stress upon returning the fruits back to air for 24 hours, was also a function of O2 tensions under which the fruits were kept. Thus, the synthesis of polygalacturonase and cellulase was directly related to O2 levels, while the activity of the isoenzymes of alcohol dehydrogenase was inversely related to O2 levels. The results indicate that hypoxia exerts both negative and positive effects on the expression of certain genes and that these effects are initiated at the same levels of O2. ImagesFigure 2Figure 3Figure 4Figure 5Figure 6Figure 7 PMID:16668163

Central carbon metabolism influences cellulase production in Bacillus licheniformis.

PubMed

Wang, J; Liu, S; Li, Y; Wang, H; Xiao, S; Li, C; Liu, B

2018-01-01

Bacillus licheniformis that can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for 13 C metabolic flux analysis. A significant change was observed in metabolic fluxes after 13 C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering. Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering. © 2017 The Society for Applied Microbiology.

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.

PubMed

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.

Discovery and characterization of ionic liquid-tolerant thermophilic cellulases from a switchgrass-adapted microbial community.

PubMed

Gladden, John M; Park, Joshua I; Bergmann, Jessica; Reyes-Ortiz, Vimalier; D'haeseleer, Patrik; Quirino, Betania F; Sale, Kenneth L; Simmons, Blake A; Singer, Steven W

2014-01-29

The development of advanced biofuels from lignocellulosic biomass will require the use of both efficient pretreatment methods and new biomass-deconstructing enzyme cocktails to generate sugars from lignocellulosic substrates. Certain ionic liquids (ILs) have emerged as a promising class of compounds for biomass pretreatment and have been demonstrated to reduce the recalcitrance of biomass for enzymatic hydrolysis. However, current commercial cellulase cocktails are strongly inhibited by most of the ILs that are effective biomass pretreatment solvents. Fortunately, recent research has shown that IL-tolerant cocktails can be formulated and are functional on lignocellulosic biomass. This study sought to expand the list of known IL-tolerant cellulases to further enable IL-tolerant cocktail development by developing a combined in vitro/in vivo screening pipeline for metagenome-derived genes. Thirty-seven predicted cellulases derived from a thermophilic switchgrass-adapted microbial community were screened in this study. Eighteen of the twenty-one enzymes that expressed well in E. coli were active in the presence of the IL 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) concentrations of at least 10% (v/v), with several retaining activity in the presence of 40% (v/v), which is currently the highest reported tolerance to [C2mim][OAc] for any cellulase. In addition, the optimum temperatures of the enzymes ranged from 45 to 95°C and the pH optimum ranged from 5.5 to 7.5, indicating these enzymes can be used to construct cellulase cocktails that function under a broad range of temperature, pH and IL concentrations. This study characterized in detail twenty-one cellulose-degrading enzymes derived from a thermophilic microbial community and found that 70% of them were [C2mim][OAc]-tolerant. A comparison of optimum temperature and [C2mim][OAc]-tolerance demonstrates that a positive correlation exists between these properties for those enzymes with a optimum temperature >70°C, further strengthening the link between thermotolerance and IL-tolerance for lignocelluolytic glycoside hydrolases.

Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

PubMed

Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

2016-11-14

This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli.

PubMed

Gao, Dongfang; Wang, Shengjun; Li, Haoran; Yu, Huili; Qi, Qingsheng

2015-04-10

The Gram-negative bacterium Escherichia coli has been widely used as a cell factory for the production of proteins and specialty chemicals because it is the best characterized host with many available expression and regulation systems. However, recombinant proteins produced in Escherichia coli are generally intracellular and often found in the form of inclusion bodies. Extracellular production of proteins is advantageous compared with intracellular production because extracellular proteins can be purified more easily and can avoid protease attack, which results in higher product quality. In this study, we found a catalytic domain of a cellulase (Cel-CD) and its N-terminus can be employed as carriers for extracellular production of recombinant proteins. In this report, we identified the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. that can be secreted into the medium from recombinant E. coli BL21 (DE3) in large quantities without its native signal peptide. By subcellular location analysis, we proved that the secretion was a two-step process and the N-terminal sequence of the full length Cel-CD played a crucial function in secretion. Both the Cel-CD and its N-terminal sequence can serve as carriers for efficient extracellular production of select target proteins. Fusion of heterologous proteins with N20 from Cel-CD can carry the target proteins out of the cells with a concentration from 101 to 691 mg/L in flask cultivation. The extracellular recombinant proteins with a relative high purity. The results suggested that this system has a potential application in plant biomass conversion and industrial production of enzymes and therapeutic proteins.

Effects of Oligosaccharides Isolated From Pinewood Hot Water Pre-hydrolyzates on Recombinant Cellulases

PubMed Central

Fang, Hong; Kandhola, Gurshagan; Rajan, Kalavathy; Djioleu, Angele; Carrier, Danielle Julie; Hood, Kendall R.; Hood, Elizabeth E.

2018-01-01

Loblolly pine residues have enormous potential to be the raw material for advanced biofuel production due to extensive sources and high cellulose content. Hot water (HW) pretreatment, while being a relatively economical and clean technology for the deconstruction of lignocellulosic biomass, could also inhibit the ensuing enzymatic hydrolysis process because of the production of inhibitors. In this study, we investigated the effect of oligosaccharide fractions purified from HW pre-hydrolyzate of pinewood using centrifugal partition chromatography (CPC) on three recombinant cellulolytic enzymes (E1, CBHI and CBHII), which were expressed in the transgenic corn grain system. The efficiency of recombinant enzymes was measured using either a 4-methylumbelliferyl-β-D-cellobioside (MUC) or a cellulose-dinitrosalicylic acid (DNS) assay system. The results showed that HW pre-hydrolyzate CPC fractions contain phenolics, furans, and monomeric and oligomeric sugars. Among CPC fractions, oligomers composed of xylan, galactan, and mannan were inhibitory to the three recombinant enzymes and to the commercial cellulase cocktail, reducing the enzymatic efficiency to as low as 10%. PMID:29868572

Thermophilic Gram-Positive Biocatalysts for Biomass Conversion to Ethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shanmugam, K.T.; Ingram, L.O.; Maupin-Furlow, J.A.

2003-12-01

Production of energy from renewable sources is receiving increased attention due to the finite nature of fossil fuels and the environmental impact associated with the continued large scale use of fossil energy sources. Biomass, a CO2-neutral abundant resource, is an attractive alternate source of energy. Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals. Extracellular cellulases produced by fungi are commercially developed for depolymerization of cellulose in biomass to glucose for fermentation by appropriate biocatalysts in a simultaneous saccharification and fermentation (SSF) process. Due to the differences in themore » optimum conditions for the activity of the fungal cellulases and the growth and fermentation characteristics of the current industrial biocatalysts, SSF of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity leading to higher than required cost of cellulase in SSF. We have isolated bacterial biocatalysts whose growth and fermentation requirements match the optimum conditions for commercial fungal cellulase activity (pH 5.0 and 50 deg. C). These isolates fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to L(+)-lactic acid. Xylose was metabolized through the pentose-phosphate pathway by these organisms as evidenced by the fermentation profile and analysis of the fermentation products of 13C1-xylose by NMR. As expected for the metabolism of xylose by the pentose-phosphate pathway, 13C-lactate accounted for more than 90% of the total 13C-labeled products. All three strains fermented crystalline cellulose to lactic acid with the addition of fungal cellulase (Spezyme CE) (SSF) at an optimum of about 10 FPU/g cellulose. These isolates also fermented cellulose and sugar cane bagasse hemicellulose acid hydrolysate simultaneously. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans although B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. For successful production of ethanol from pyruvate, both pyruvate decarboxylase (PDC) and alcohol dehydrogenase (AHD) need to be produced at optimal levels in these biocatalysts. A plasmid containing the S. ventriculi pdc gene and the adh gene from geobacillus stearothermophilus was constructed using plasmid pWH1520 that was successfully used for expression of pdc in B. megaterium. The resulting portable ethanol (PET) plasmid, pJAM423, was transformed into B. megaterium. After xylose induction, a significant fraction of cell cytoplasm was composed of the S. ventriculi PDC and G. stearothermophilus ADH proteins. In preliminary experiments, the amount of ethanol produced by b. megaterium with plasmid pJAM423 was about twice (20 mM) of the bacterium without the plasmid. These results show that the PET operon is functional in B. megaterium but high level ethanol production needs further genetic and metabolic engineering. A genetic transfer system for the second generation biocatalysts needs to be developed for transferring the plasmid pJAM423 and its derivatives for engineering these organisms for ethanol production from biomass derived sugars and cellulose to ethanol. One of the new biocatalysts, strain P4-102B was found to be transformable with plasmids and the method for introducing plasmid pJAM423 into this strain and expression of the encoded DNA is being optimized. These new second generation biocatalysts have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource for production of fuels and chemicals.« less

Biochemical changes in mango after infection with Rhizoctonia bataticola.

PubMed

Vyas, H G; Chhatpar, H S

1980-04-15

Rhizoctonia bataticola is responsible for the spoilage of mango fruits (Mangifera india) during post-harvest preservation and storage. Culture of R. bataticola exhibited significant pectinase and cellulase activity. In Rhizoctonia-infected fruits an increase of protease and cellulase activity, and a decrease in certain enzymes of carbohydrate metabolism, were observed in comparison to healthy fruits.

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

PubMed

Wang, Hong-Bin

2014-03-15

In the present study, we investigated the cellulase-assisted extraction and antibacterial activity of water-soluble polysaccharides from the dandelion Taraxacum officinale. The extraction conditions, optimized for improving yield, were as follows: time, 46.11 min; temperature, 54.87 °C; pH, 4.51 and cellulase enzyme, 4000 U/g. Under these conditions, the yield of polysaccharides from dandelion (PD) reached 20.67% (w/w). The sugar content of PD was 95.6% (w/w), and it displayed high antibacterial activity at a concentration of 100mg/mL against Escherichia coli, Bacillus subtilis and Staphylococcus aureus. These results indicate that PD may be a viable option for use as a food preservative. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vermaas, Josh V.; Crowley, Michael F.; Beckham, Gregg T.

In nature, polysaccharide glycosidic bonds are cleaved by hydrolytic enzymes for a vast array of biological functions. Recently, a new class of enzymes that utilize an oxidative mechanism to cleave glycosidic linkages was discovered; these enzymes are called lytic polysaccharide monooxygenases (LPMO). These oxidative enzymes are synergistic with cocktails of hydrolytic enzymes and are thought to act primarily on crystalline regions, in turn providing new sites of productive attachment and detachment for processive hydrolytic enzymes. In the case of cellulose, the homopolymer of ..beta..-1,4-d-glucose, enzymatic oxidation occurs at either the reducing end or the nonreducing end of glucose, depending onmore » enzymatic specificity, and results in the generation of oxidized chemical substituents at polymer chain ends. LPMO oxidation of cellulose is thought to produce either a lactone at the reducing end of glucose that can spontaneously or enzymatically convert to aldonic acid or 4-keto-aldose at the nonreducing end that may further oxidize to a geminal diol. Here, we use molecular simulation to examine the effect of oxidation on the structure of crystalline cellulose. The simulations highlight variations in behaviors depending on the chemical identity of the oxidized species and its location within the cellulose fibril, as different oxidized species introduce steric effects that disrupt local crystallinity and in some cases reduce the work needed for polymer decrystallization. Reducing-end oxidations are easiest to decrystallize when located at the end of the fibril, whereas nonreducing end oxidations readily decrystallize from internal cleavage sites despite their lower solvent accessibility. The differential in decrystallization free energy suggests a molecular mechanism consistent with experimentally observed LPMO/cellobiohydrolase synergy. Additionally, the soluble oxidized cellobiose products released by hydrolytic cellulases may bind to the active sites of cellulases with different affinities relative to cellobiose itself, which potentially affects hydrolytic turnover through product inhibition. To examine the effect of oxidation on cello-oligomer binding, we use thermodynamic integration to compute the relative change in binding free energy between the hydrolyzed and oxidized products in the active site of Family 7 and Family 6 processive glycoside hydrolases, Trichoderma reesei Cel7A and Cel6A, which are key industrial cellulases and commonly used model systems for fungal cellulases. Our results suggest that the equilibrium between the two reducing end oxidized products, favoring the linear aldonic acid, may increase product inhibition, which would in turn reduce processive substrate turnover. In the case of LMPO action at the nonreducing end, oxidation appears to lower affinity with the nonreducing end specific cellulase, reducing product inhibition and potentially promoting processive cellulose turnover. Overall, this suggests that oxidation of recalcitrant polysaccharides by LPMOs accelerates degradation not only by increasing the concentration of chain termini but also by reducing decrystallization work, and that product inhibition may be somewhat reduced as a result.« less

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

PubMed Central

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 cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization. PMID:25361767

Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice.

PubMed

Tayi, Lavanya; Maku, Roshan V; Patel, Hitendra Kumar; Sonti, Ramesh V

2016-01-01

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.

Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice

PubMed Central

Tayi, Lavanya; Maku, Roshan V.; Patel, Hitendra Kumar; Sonti, Ramesh V.

2016-01-01

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo. PMID:27907079

Evidence for transceptor function of cellodextrin transporters in Neurospora crassa.

PubMed

Znameroski, Elizabeth A; Li, Xin; Tsai, Jordan C; Galazka, Jonathan M; Glass, N Louise; Cate, Jamie H D

2014-01-31

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as "transceptors" with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.

Effect of bovine serum albumin (BSA) on enzymatic cellulose hydrolysis.

PubMed

Wang, Hui; Mochidzuki, Kazuhiro; Kobayashi, Shinichi; Hiraide, Hatsue; Wang, Xiaofen; Cui, Zongjun

2013-06-01

Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ≥90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.

Xylose induces cellulase production in Thermoascus aurantiacus

DOE PAGES

Schuerg, Timo; Prahl, Jan -Philip; Gabriel, Raphael; ...

2017-11-15

Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus. Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted tomore » produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. CXylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.« less

Xylose induces cellulase production in Thermoascus aurantiacus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schuerg, Timo; Prahl, Jan -Philip; Gabriel, Raphael

Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus. Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted tomore » produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. CXylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.« less

Identification of novel biomass-degrading enzymes from genomic dark matter: Populating genomic sequence space with functional annotation.

PubMed

Piao, Hailan; Froula, Jeff; Du, Changbin; Kim, Tae-Wan; Hawley, Erik R; Bauer, Stefan; Wang, Zhong; Ivanova, Nathalia; Clark, Douglas S; Klenk, Hans-Peter; Hess, Matthias

2014-08-01

Although recent nucleotide sequencing technologies have significantly enhanced our understanding of microbial genomes, the function of ∼35% of genes identified in a genome currently remains unknown. To improve the understanding of microbial genomes and consequently of microbial processes it will be crucial to assign a function to this "genomic dark matter." Due to the urgent need for additional carbohydrate-active enzymes for improved production of transportation fuels from lignocellulosic biomass, we screened the genomes of more than 5,500 microorganisms for hypothetical proteins that are located in the proximity of already known cellulases. We identified, synthesized and expressed a total of 17 putative cellulase genes with insufficient sequence similarity to currently known cellulases to be identified as such using traditional sequence annotation techniques that rely on significant sequence similarity. The recombinant proteins of the newly identified putative cellulases were subjected to enzymatic activity assays to verify their hydrolytic activity towards cellulose and lignocellulosic biomass. Eleven (65%) of the tested enzymes had significant activity towards at least one of the substrates. This high success rate highlights that a gene context-based approach can be used to assign function to genes that are otherwise categorized as "genomic dark matter" and to identify biomass-degrading enzymes that have little sequence similarity to already known cellulases. The ability to assign function to genes that have no related sequence representatives with functional annotation will be important to enhance our understanding of microbial processes and to identify microbial proteins for a wide range of applications. © 2014 Wiley Periodicals, Inc.

Inhibition of cellulase-catalyzed lignocellulosic hydrolysis by iron and oxidative metal ions and complexes.

PubMed

Tejirian, Ani; Xu, Feng

2010-12-01

Enzymatic lignocellulose hydrolysis plays a key role in microbially driven carbon cycling and energy conversion and holds promise for bio-based energy and chemical industries. Cellulases (key lignocellulose-active enzymes) are prone to interference from various noncellulosic substances (e.g., metal ions). During natural cellulolysis, these substances may arise from other microbial activities or abiotic events, and during industrial cellulolysis, they may be derived from biomass feedstocks or upstream treatments. Knowledge about cellulolysis-inhibiting reactions is of importance for the microbiology of natural biomass degradation and the development of biomass conversion technology. Different metal ions, including those native to microbial activity or employed for biomass pretreatments, are often tested for enzymatic cellulolysis. Only a few metal ions act as inhibitors of cellulases, which include ferrous and ferric ions as well as cupric ion. In this study, we showed inhibition by ferrous/ferric ions as part of a more general effect from oxidative (or redox-active) metal ions and their complexes. The correlation between inhibition and oxidation potential indicated the oxidative nature of the inhibition, and the dependence on air established the catalytic role that iron ions played in mediating the dioxygen inhibition of cellulolysis. Individual cellulases showed different susceptibilities to inhibition. It is likely that the inhibition exerted its effect more on cellulose than on cellulase. Strong iron ion chelators and polyethylene glycols could mitigate the inhibition. Potential microbiological and industrial implications of the observed effect of redox-active metal ions on enzymatic cellulolysis, as well as the prevention and mitigation of this effect in industrial biomass conversion, are discussed.

LPMOs in cellulase mixtures affect fermentation strategies for lactic acid production from lignocellulosic biomass.

PubMed

Müller, Gerdt; Kalyani, Dayanand Chandrahas; Horn, Svein Jarle

2017-03-01

Enzymatic catalysis plays a key role in the conversion of lignocellulosic biomass to fuels and chemicals such as lactic acid. In the last decade, the efficiency of commercial cellulase cocktails has increased significantly, in part due to the inclusion of lytic polysaccharide monooxygenases (LPMOs). However, the LPMOs' need for molecular oxygen to break down cellulose demands reinvestigations of process conditions. In this study, we evaluate the efficiency of lactic acid production from steam-exploded birch using an LPMO-containing cellulase cocktail in combination with lactic acid bacteria, investigating both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). While the SSF set up generally has been considered to be more efficient because it avoids sugar accumulation which may inhibit the cellulases, the SHF set up in our study yielded 26-32% more lactic acid than the SSF. This was mainly due to competition for oxygen between LPMOs and the fermenting organisms in the SSF process, which resulted in reduced LPMO activity and thus less efficient saccharification of the lignocellulosic substrate. By means of aeration it was possible to activate the LPMOs in the SSF, but less lactic acid was produced due to a shift in metabolic pathways toward production of acetic acid. Overall, this study shows that lactic acid can be produced efficiently from lignocellulosic biomass, but that the use of LPMO-containing cellulase cocktails in fermentation processes demands re-thinking of traditional process set ups due to the requirement of oxygen in the saccharification step. Biotechnol. Bioeng. 2017;114: 552-559. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Reducing Enzyme Costs Increases the Market Potential of Biofuels (Fact Sheet)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

Cellulosic ethanol prices depend heavily on the cost of the cellulase enzymes used to break down the biomass into fermentable sugars. To reduce these costs, NREL partnered with two leading enzyme companies, Novozymes and Genencor, to engineer new cellulase enzymes that are exceptionally good at breaking down cellulose. Genencor is now part of DuPont Industrial Biosciences.

CBH1 homologs and varian CBH1 cellulase

DOEpatents

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.

Thermal tolerant exoglucanase from Acidothermus cellulolyticus

DOEpatents

Adney, William S [Golden, CO; Ding, Shi-You [Golden, CO; Vinzant, Todd B [Golden, CO; Himmel, Michael E [Littleton, CO; Decker, Stephen R [Berthoud, CO; McCarter, Suzanne Lantz [Denver, CO

2008-07-01

The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as Gux1. Gux1 has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified Gux1. Methods of making and using Gux1 polypeptides, including fusions, variants, and derivatives, are also disclosed.

CBH1 homologs and variant CBH1 cellulases

DOEpatents

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.

Effects of phytase, cellulase, and dehulling treatments on iron and zinc in vitro solubility in faba bean (Vicia faba L.) Flour and Legume Fractions.

PubMed

Luo, Yu-Wei; Xie, Wei-Hua; Cui, Qun-Xiang

2010-02-24

Simulations of gastrointestinal digestion were used to try to identify the nature of the complexes between antinutritional factors and iron and zinc in faba bean and legume fractions. In digestible residue of raw faba bean flour, simultaneous action of cellulase and phytases made it possible to release about 28% units more iron than that released with the treatment without enzymes. About 49.8% of iron in raw faba bean flour was solubilized after in vitro digestion and simultaneous action of cellulase and phytase. In the hull fraction, the action of phytases and the simultaneous action of cellulase and phytase allowed about 7 and 35% units of additional zinc to be solubilized, respectively. Single enzymatic degradation of phytates from dehulled faba bean allowed solubilization from 65 to 93% of zinc, depending upon the treatment. In dehulled faba bean, iron was chelated by phytates and by fibers, whereas zinc was almost exclusively chelated by phytates. In the hull of faba bean, a high proportion of iron was chelated by iron-tannins, while the rest of iron as well as the majority of zinc were chelated in complexes between phytates and fibers.

Bioprocessing of wheat bran for the production of lignocellulolytic enzyme cocktail by Cotylidia pannosa under submerged conditions.

PubMed

Sharma, Deepika; Garlapat, Vijay Kumar; Goel, Gunjan

2016-04-02

Characterization and production of efficient lignocellulytic enzyme cocktails for biomass conversion is the need for biofuel industry. The present investigation reports the modeling and optimization studies of lignocellulolytic enzyme cocktail production by Cotylidia pannosa under submerged conditions. The predominant enzyme activities of cellulase, xylanase and laccase were produced in the cocktail through submerged conditions using wheat bran as a substrate. A central composite design approach was utilized to model the production process using temperature, pH, incubation time and agitation as input variables with the goal of optimizing the output variables namely cellulase, xylanase and laccase activities. The effect of individual, square and interaction terms on cellulase, xylanase and laccase activities were depicted through the non-linear regression equations with significant R(2) and P-values. An optimized value of 20 U/ml, 17 U/ml and 13 U/ml of cellulase, xylanase and laccase activities, respectively, were obtained with a media pH of 5.0 in 77 h at 31C, 140 rpm using wheatbran as a substrate. Overall, the present study introduces a fungal strain, capable of producing lignocellulolytic enzyme cocktail for subsequent applications in biofuel industry.

Improving genetic immobilization of a cellulase on yeast cell surface for bioethanol production using cellulose.

PubMed

Yang, Jinying; Dang, Hongyue; Lu, Jian Ren

2013-04-01

In this study, Saccharomyces cerevisiae was genetically engineered to harbor the capability of utilizing celluloses for bioethanol production by displaying active cellulolytic enzymes on the cell surface. An endo-1,4-β-glucanase gene egX was cloned from Bacillus pumilus C-9 and its expression products, the EGX cellulases, were displayed on the cell surface of S. cerevisiae by fusing egX with aga2 that encodes the binding subunit of the S. cerevisiae cell wall protein α-agglutinin. To achieve high gene copies and stability, multicopy integration was obtained by integrating the fusion aga2-egX gene into the rDNA region of the S. cerevisiae chromosome. To achieve high expression and surface display efficiency, the aga2-egX gene was expressed under the control of a strong promoter. The presence of the enzymatically active cellulase fusion proteins on the S. cerevisiae cell surface was verified by carboxymethyl cellulase activity assay and immunofluorescence microscopy. This work presented a promising strategy to genetically engineer yeasts to perform efficient fermentation of cellulosic materials for bioethanol production. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioprocessing of wheat bran for the production of lignocellulolytic enzyme cocktail by Cotylidia pannosa under submerged conditions

PubMed Central

Sharma, Deepika; Garlapat, Vijay Kumar; Goel, Gunjan

2016-01-01

ABSTRACT Characterization and production of efficient lignocellulytic enzyme cocktails for biomass conversion is the need for biofuel industry. The present investigation reports the modeling and optimization studies of lignocellulolytic enzyme cocktail production by Cotylidia pannosa under submerged conditions. The predominant enzyme activities of cellulase, xylanase and laccase were produced in the cocktail through submerged conditions using wheat bran as a substrate. A central composite design approach was utilized to model the production process using temperature, pH, incubation time and agitation as input variables with the goal of optimizing the output variables namely cellulase, xylanase and laccase activities. The effect of individual, square and interaction terms on cellulase, xylanase and laccase activities were depicted through the non-linear regression equations with significant R2 and P-values. An optimized value of 20 U/ml, 17 U/ml and 13 U/ml of cellulase, xylanase and laccase activities, respectively, were obtained with a media pH of 5.0 in 77 h at 31C, 140 rpm using wheatbran as a substrate. Overall, the present study introduces a fungal strain, capable of producing lignocellulolytic enzyme cocktail for subsequent applications in biofuel industry. PMID:26941214

Ethanol production from sunflower meal biomass by simultaneous saccharification and fermentation (SSF) with Kluyveromyces marxianus ATCC 36907.

PubMed

Camargo, Danielle; Gomes, Simone D; Sene, Luciane

2014-11-01

The lignocellulosic materials are considered promising renewable resources for ethanol production, but improvements in the processes should be studied to reduce operating costs. Thus, the appropriate enzyme loading for cellulose saccharification is critical for process economics. This study aimed at evaluating the concentration of cellulase and β-glucosidase in the production of bioethanol by simultaneous saccharification and fermentation (SSF) of sunflower meal biomass. The sunflower biomass was pretreated with 6% H2SO4 (w/v), at 121 °C, for 20 min, for hemicellulose removal and delignificated with 1% NaOH. SSF was performed with Kluyveromyces marxianus ATCC 36907, at 38 °C, 150 rpm, for 72 h, with different enzyme concentrations (Cellulase Complex NS22086-10, 15 and 20 FPU/gsubstrate and β-Glucosidase NS22118, with a cellulase to β-glucosidase ratio of 1.5:1; 2:1 and 3:1). The best condition for ethanol production was cellulase 20 FPU/gsubstrate and β-glucosidase 13.3 CBU/gsubstrate, resulting in 27.88 g/L ethanol, yield of 0.47 g/g and productivity of 0.38 g/L h. Under this condition the highest enzymatic conversion of cellulose to glucose was attained (87.06%).

Preliminary plant design of Escherichia coli BPPTCC-EgRK2 cell culture for recombinant cellulase production using Oil Palm Empty Fruit Bunch (OPEFB) as substrate

NASA Astrophysics Data System (ADS)

Surya, E. A.; Rahman, S. F.; Zulamraini, S.; Gozan, M.

2018-03-01

An economic analysis of recombinant cellulase production from E. coli BPPTCC Eg-RK2 was conducted to support the fulfilling of Indonesia’s energy roadmap for ethanol production. The plant use oil palm empty fruit bunch (OPEFB) as primary substrate in cellulase production, with the expected lifetime of 12 years. The plant is assumed to be built in Indonesia and will fulfill 1% of total market demand. The effect of different pretreatment process (alkaline, steam explosion, and sequential acid-alkaline) on the economic value was also studied. A simulation using SuperPro Designer was used to calculate the mass and energy balance based on the kinetic parameter of E. coli BPPTCC-EgRK2. Technology evaluation show that alkaline pretreatment gave the highest yield with no known inhibitors formed. The steam explosion show the lowest lignin and hemicellulose removal and known to form known fermentation inhibitors. The net present value of alkaline, steam explosion, and sequential acid-alkaline pretreatment were USD 7,118,000; - USD 73,411,000 and USD -114,013,000 respectively, which mean alkaline pretreatment is the only economically feasible pretreatment method for recombinant cellulase production.

Investigating commercial cellulase performances toward specific biomass recalcitrance factors using reference substrates.

PubMed

Ju, Xiaohui; Bowden, Mark; Engelhard, Mark; Zhang, Xiao

2014-05-01

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 cellulase enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulase 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.

Exogenous Cellulase Switches Cell Interdigitation to Cell Elongation in an RIC1-dependent Manner in Arabidopsis thaliana Cotyledon Pavement Cells.

PubMed

Higaki, Takumi; Takigawa-Imamura, Hisako; Akita, Kae; Kutsuna, Natsumaro; Kobayashi, Ryo; Hasezawa, Seiichiro; Miura, Takashi

2017-01-01

Pavement cells in cotyledons and true leaves exhibit a jigsaw puzzle-like morphology in most dicotyledonous plants. Among the molecular mechanisms mediating cell morphogenesis, two antagonistic Rho-like GTPases regulate local cell outgrowth via cytoskeletal rearrangements. Analyses of several cell wall-related mutants suggest the importance of cell wall mechanics in the formation of interdigitated patterns. However, how these factors are integrated is unknown. In this study, we observed that the application of exogenous cellulase to hydroponically grown Arabidopsis thaliana cotyledons switched the interdigitation of pavement cells to the production of smoothly elongated cells. The cellulase-induced inhibition of cell interdigitation was not observed in a RIC1 knockout mutant. This gene encodes a Rho-like GTPase-interacting protein important for localized cell growth suppression via microtubule bundling on concave cell interfaces. Additionally, to characterize pavement cell morphologies, we developed a mathematical model that considers the balance between cell and cell wall growth, restricted global cell growth orientation, and regulation of local cell outgrowth mediated by a Rho-like GTPase-cytoskeleton system. Our computational simulations fully support our experimental observations, and suggest that interdigitated patterns form because of mechanical buckling in the absence of Rho-like GTPase-dependent regulation of local cell outgrowth. Our model clarifies the cell wall mechanics influencing pavement cell morphogenesis. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Cellulase-assisted extraction and antioxidant activity of polysaccharides from Rhizoma imperata.

PubMed

Jiang, Long-Fa

2014-08-08

In this study, the cellulase-assisted extraction and antioxidant activity of the polysaccharides from Rhizoma imperata were investigated. To improve the yield of R. Imperata polysaccharides (RPs), the extraction conditions were optimized as follows: time, 69.48 min; temperature, 45.36°C; pH, 4.58; cellulase amount, 1,200 U/g. Under these optimum conditions, the yield of RPs reached 0.67% (w/w), and was higher than that of the traditionally aqueous extraction method. The sugar content in the RPs product reached up to 93.25% (w/w). The RPs product has high antioxidant activity including hydroxyl radical scavenging activity and 2,2-diphenyl-β-picrylhydrazyl radical scavenging activity at the concentration of 100mg/mL. Copyright © 2014 Elsevier Ltd. All rights reserved.

Regulation of Catabolic Enzyme Biosynthesis in Thermomonospora curvata

DTIC Science & Technology

1988-03-31

biomass conversion processes. 3) At the level of exoenzyme secretion, we have shown that the surfactant, Tween-80, effects a component- specific... cellulase genes in the actinomycetesl. Our paper is the first to describe PDE in any thermophilic actinomtrfcete; further- more, it is the first paper...stimulation of cellulase release. Only components active against crystalline cellulose (as opposed to those a tive against soluble cellulose derivatives

Regulation of plant cell wall degradation by light in Trichoderma.

PubMed

Schmoll, Monika

2018-01-01

Trichoderma reesei (syn. Hypocrea jecorina ) is the model organism for industrial production of plant cell wall degradating enzymes. The integration of light and nutrient signals for adaptation of enzyme production in T. reesei emerged as an important regulatory mechanism to be tackled for strain improvement. Gene regulation specific for cellulase inducing conditions is different in light and darkness with substantial regulation by photoreceptors. Genes regulated by light are clustered in the genome, with several of the clusters overlapping with CAZyme clusters. Major cellulase transcription factor genes and at least 75% of glycoside hydrolase encoding genes show the potential of light dependent regulation. Accordingly, light dependent protein complex formation occurs within the promoters of cellulases and their regulators. Additionally growth on diverse carbon sources is different between light and darkness and dependent on the presence of photoreceptors in several cases. Thereby, also light intensity plays a regulatory role, with cellulase levels dropping at higher light intensities dependent in the strain background. The heterotrimeric G-protein pathway is the most important nutrient signaling pathway in the connection with light response and triggers posttranscriptional regulation of cellulase expression. All G-protein alpha subunits impact cellulase regulation in a light dependent manner. The downstream cAMP pathway is involved in light dependent regulation as well. Connections between the regulatory pathways are mainly established via the photoreceptor ENV1. The effect of photoreceptors on plant cell wall degradation also occurs in the model filamentous fungus Neurospora crassa . In the currently proposed model, T. reesei senses the presence of plant biomass in its environment by detection of building blocks of cellulose and hemicellulose. Interpretation of the respective signals is subsequently adjusted to the requirements in light and darkness (or on the surface versus within the substrate) by an interconnection of nutrient signaling with light response. This review provides an overview on the importance of light, photoreceptors and related signaling pathways for formation of plant cell wall degrading enzymes in T. reesei . Additionally, the relevance of light dependent gene regulation for industrial fermentations with Trichoderma as well as strategies for exploitation of the observed effects are discussed.

Purification and characterization of a salt-tolerant cellulase from the mangrove oyster, Crassostrea rivularis.

PubMed

An, Tianchen; Dong, Zhu; Lv, Junchen; Liu, Yujun; Wang, Manchuriga; Wei, Shuangshuang; Song, Yanting; Zhang, Yingxia; Deng, Shiming

2015-04-01

A cellulase with wide range of pH resistance and high salt tolerance was isolated from the digestive gland of the oyster Crassostrea rivularis living in mangrove forests. The 27 kDa cellulase named as CrCel was purified 40.6 folds by anion exchange chromatography and extraction from the gel after non-reducing sodium dodecylsufate-polyacrylamide gel electrophoresis. The specific activity of the purified cellulase was 23.4 U/mg against carboxymethyl cellulose (CMC). The N-terminal amino acid sequence of CrCel was determined to be NQKCQANSRV. CrCel preferably hydrolyzes β-1,4-glucosidic bonds in the amorphous parts of cellulose materials and displays degradation activity toward xylan. The Km and Vmax values of CrCel for CMC were determined to be 2.1% ± 0.4% and 73.5 ± 3.3 U mg(-1), respectively. The optimal pH value and temperature of CrCel were 5.5 and 40°C, respectively. The enzyme was stable in a wide range of pH, retaining over 60% activity after incubation for 80 min in the pH range of 3.0-9.0. In addition, CrCel showed remarkable tolerance to salt and remained active at high NaCl concentrations, but also retained over 70% activity after incubation in 0.5-2 M NaCl for up to 24 h. On the basis of the N-terminal sequence alignment and its similar properties to other animal cellulases, CrCel was regarded as a member of glycosyl hydrolase family 45 β-1,4-glucanases. CrCel is the first reported cellulase isolated from mangrove invertebrates, which suggests that it may participate in the assimilation of cellulolytic materials derived from the food sources of the oyster and contribute to the consumption of mangrove primary production. The unique properties of this enzyme make it a potential candidate for further industrial application. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Cellulose and hemicellulose decomposition by forest soil bacteria proceeds by the action of structurally variable enzymatic systems

PubMed Central

López-Mondéjar, Rubén; Zühlke, Daniela; Becher, Dörte; Riedel, Katharina; Baldrian, Petr

2016-01-01

Evidence shows that bacteria contribute actively to the decomposition of cellulose and hemicellulose in forest soil; however, their role in this process is still unclear. Here we performed the screening and identification of bacteria showing potential cellulolytic activity from litter and organic soil of a temperate oak forest. The genomes of three cellulolytic isolates previously described as abundant in this ecosystem were sequenced and their proteomes were characterized during the growth on plant biomass and on microcrystalline cellulose. Pedobacter and Mucilaginibacter showed complex enzymatic systems containing highly diverse carbohydrate-active enzymes for the degradation of cellulose and hemicellulose, which were functionally redundant for endoglucanases, β-glucosidases, endoxylanases, β-xylosidases, mannosidases and carbohydrate-binding modules. Luteibacter did not express any glycosyl hydrolases traditionally recognized as cellulases. Instead, cellulose decomposition was likely performed by an expressed GH23 family protein containing a cellulose-binding domain. Interestingly, the presence of plant lignocellulose as well as crystalline cellulose both trigger the production of a wide set of hydrolytic proteins including cellulases, hemicellulases and other glycosyl hydrolases. Our findings highlight the extensive and unexplored structural diversity of enzymatic systems in cellulolytic soil bacteria and indicate the roles of multiple abundant bacterial taxa in the decomposition of cellulose and other plant polysaccharides. PMID:27125755

Ethanol and anaerobic conditions reversibly inhibit commercial cellulase activity in thermophilic simultaneous saccharification and fermentation (tSSF)

PubMed Central

2012-01-01

Background A previously developed mathematical model of low solids thermophilic simultaneous saccharification and fermentation (tSSF) with Avicel was unable to predict performance at high solids using a commercial cellulase preparation (Spezyme CP) and the high ethanol yield Thermoanaerobacterium saccharolyticum strain ALK2. The observed hydrolysis proceeded more slowly than predicted at solids concentrations greater than 50 g/L Avicel. Factors responsible for this inaccuracy were investigated in this study. Results Ethanol dramatically reduced cellulase activity in tSSF. At an Avicel concentration of 20 g/L, the addition of ethanol decreased conversion at 96 hours, from 75% in the absence of added ethanol down to 32% with the addition of 34 g/L initial ethanol. This decrease is much greater than expected based on hydrolysis inhibition results in the absence of a fermenting organism. The enhanced effects of ethanol were attributed to the reduced, anaerobic conditions of tSSF, which were shown to inhibit cellulase activity relative to hydrolysis under aerobic conditions. Cellulose hydrolysis in anaerobic conditions was roughly 30% slower than in the presence of air. However, this anaerobic inhibition was reversed by exposing the cellulase enzymes to air. Conclusion This work demonstrates a previously unrecognized incompatibility of enzymes secreted by an aerobic fungus with the fermentation conditions of an anaerobic bacterium and suggests that enzymes better suited to industrially relevant fermentation conditions would be valuable. The effects observed may be due to inactivation or starvation of oxygen dependent GH61 activity, and manipulation or replacement of this activity may provide an opportunity to improve biomass to fuel process efficiency. PMID:22703989

Cellulase production by Penicillium funiculosum and its application in the hydrolysis of sugar cane bagasse for second generation ethanol production by fed batch operation.

PubMed

Maeda, Roberto Nobuyuki; Barcelos, Carolina Araújo; Santa Anna, Lídia Maria Melo; Pereira, Nei

2013-01-10

This study aimed to produce a cellulase blend and to evaluate its application in a simultaneous saccharification and fermentation (SSF) process for second generation ethanol production from sugar cane bagasse. The sugar cane bagasse was subjected to pretreatments (diluted acid and alkaline), as for disorganizing the ligocellulosic complex, and making the cellulose component more amenable to enzymatic hydrolysis. The residual solid fraction was named sugar cane bagasse partially delignified cellulignin (PDC), and was used for enzyme production and ethanol fermentation. The enzyme production was performed in a bioreactor with two inoculum concentrations (5 and 10% v/v). The fermentation inoculated with higher inoculum size reduced the time for maximum enzyme production (from 72 to 48). The enzyme extract was concentrated using tangential ultrafiltration in hollow fiber membranes, and the produced cellulase blend was evaluated for its stability at 37 °C, operation temperature of the simultaneous SSF process, and at 50 °C, optimum temperature of cellulase blend activity. The cellulolytic preparation was stable for at least 300 h at both 37 °C and 50 °C. The ethanol production was carried out by PDC fed-batch SSF process, using the onsite cellulase blend. The feeding strategy circumvented the classic problems of diffusion limitations by diminishing the presence of a high solid:liquid ratio at any time, resulting in high ethanol concentration at the end of the process (100 g/L), which corresponded to a fermentation efficiency of 78% of the maximum obtainable theoretically. The experimental results led to the ratio of 380 L of ethanol per ton of sugar cane bagasse PDC. Copyright © 2012 Elsevier B.V. All rights reserved.

Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

PubMed Central

Hetzler, Stephan; Bröker, Daniel

2013-01-01

The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml−1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step. PMID:23793636

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

PubMed

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.

Cellulases: Role in Lignocellulosic Biomass Utilization.

PubMed

Soni, Sanjeev Kumar; Sharma, Amita; Soni, Raman

2018-01-01

Rapid depletion of fossil fuels worldwide presents a dire situation demanding a potential replacement to surmount the current energy crisis. Lignocellulose presents a logical candidate to be exploited at industrial scale owing to its vast availability, inexpensive and renewable nature. Microbial degradation of lignocellulosic biomass is a lucrative, sustainable, and promising approach to obtain valuable commercial commodities at gigantic scale. The enzymatic hydrolysis involving cellulases is fundamental to all the technologies needed to transform lignocellulosic biomass to valuable industry relevant products. Cellulases have enormous potential to utilize cellulosic biomass, thus reducing environmental stress in addition to production of commodity chemicals resolving the current challenge to meet the energy needs globally. The substitution of petroleum-based fuels with bio-based fuels is the subject of thorough research establishing biofuel production as the future technology to achieve a sustainable, eco-friendly society with a zero waste approach.

Cellulase production through solid-state tray fermentation, and its use for bioethanol from sorghum stover.

PubMed

Idris, Ayman Salih Omer; Pandey, Ashok; Rao, S S; Sukumaran, Rajeev K

2017-10-01

The production of cellulase by Trichoderma reesei RUT C-30 under solid-state fermentation (SSF) on wheat bran and cellulose was optimized employing a two stage statistical design of experiments. Optimization of process parameters resulted in a 3.2-fold increase in CMCase production to 959.53IU/gDS. The process was evaluated at pilot scale in tray fermenters and yielded 457IU/gDS using the lab conditions and indicating possibility for further improvement. The cellulase could effectively hydrolyze alkali pretreated sorghum stover and addition of Aspergillus niger β-glucosidase improved the hydrolytic efficiency 174%, indicating the potential to use this blend for effective saccharification of sorghum stover biomass. The enzymatic hydrolysate of sorghum stover was fermented to ethanol with ∼80% efficiency. Copyright © 2017 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vermaas, Josh V.; Petridis, Loukas; Qi, Xianghong

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process. By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose bindingmore » of TrCel7A (Y466, Y492, and Y493). In conclusion, lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.« less

Decomposition of lignin and cellobiose in relation to the enzymatic hydrolysis of cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamanaka, Y.; Carroad, P.A.; Riaz, M.

1977-02-01

Studies are reported on the use of fungal ..beta..-glucosidase in conjunction with Trichoderma viride cellulase and the search for an effective enzyme system for lignin degradation. ..beta..-glucosidase is of potential benefit in cellulose hydrolysis by catalyzing the hydrolysis of cellobiose to glucose thereby reducing product inhibition and producing a higher glucose yield. Removal of lignin from cellulosic material makes the cellulose more accessible to hydrolyzing enzymes. Hydrolysis studies on Solka Floc and newsprint were conducted with T. viride filtrates containing various proportions of B. theobromae filtrates. Significant improvement in hydrolysis rate particularly in glucose content was obtained by thus enrichingmore » the ..beta..-glucosidase content of the cellulase. In the search for a lignin degrading enzyme, major emphasis was given to the fungus Polyporous versicolor. Significant o-diphenol oxidoreductase (catecholase) activity was found in the culture filtrates. Preliminary observations of a surface culture of the fungus in a composting mode suggest that delignification may be obtained in this manner. Work is continuing on this.« less

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

PubMed Central

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

2012-01-01

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

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

PubMed

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 %.

Immobilization of Cellulase from Bacillus subtilis UniMAP-KB01 on Multi-walled Carbon Nanotubes for Biofuel Production

NASA Astrophysics Data System (ADS)

Naresh, Sandrasekaran; Hoong Shuit, Siew; Kunasundari, Balakrishnan; Hoo Peng, Yong; Qi, Hwa Ng; Teoh, Yi Peng

2018-03-01

Bacillus subtilis UniMAP-KB01, a cellulase producer was isolated from Malaysian mangrove soil. Through morphological identification it was observed that the B. subtilis appears to be in rod shaped and identified as a gram positive bacterium. Growth profile of isolated B. subtilis was established by measuring optical density (OD) at 600 nm for every 1 hour intervals. Polymath software was employed to plot the growth profile and the non-linear plot established gave the precision value of linear regression, R2 of 0.9602, root mean square deviation (RMSD) of 0.0176 and variance of 0.0025. The hydrolysis capacity testing revealed the cellulolytic index of 2.83 ± 0.46 after stained with Gram’s Iodine. The harvested crude enzyme after 24 hours incubation in carboxymethylcellulose (CMC) broth at 45°C and 100 RPM, was tested for enzyme activity. Through Filter Paper Assay (FPA), the cellulase activity was calculated to be 0.05 U/mL. The hydrolysis capacity testing and FPA shown an acceptable value for thermophilic bacterial enzyme activity. Thus, this isolated strain reasoned to be potential for producing thermostable cellulase which will be immobilized onto multi-walled carbon nanotubes and the cellulolytic activity will be characterized for biofuel production.

Michaelis kinetic analysis of extracellular cellulase and amylase excreted by Lactobacillus plantarum during cassava fermentation

NASA Astrophysics Data System (ADS)

Frediansyah, Andri; Kurniadi, Muhamad

2017-01-01

Our previous study reveal that single culture of Lactobacillus plantarum has ability to ferment cassava tuber in relation to produce modified cassava flour (mocaf). It was used to accelerate a fermentation process. L. plantarum grow well and produce some extracellular enzymes i.e. cellulase to change the structure and breakdown the cell wall of cassava tuber. Then, the starchy materials will be hydrolyzed by i.e. amylase into simple sugar and convert to organic acid. All of these process will give new characteristic of cassava i.e. lower fiber content, good flavor, taste, aroma and texture and the amount of cyanide acid is lower. Therefore this present study was to analyze Michaelis kinetics of extracellular carboxymethyl cellulase and amylase production by L. plantarum during cassava fermentation. The maximum carboxymethyl cellulase and amylase activity of 8.60 U/ml and 14.07 U/ml, respectively, were obtained from filtrate which has been incubated at 37°C for 18 h under stationary conditions. The Vmax and Km of CMCase were 0.8506 × 10-3 U/ml and 0.9594 × 10-3 g/mL, respectively. For amylase were 9.291 × 10-3 U/ml and 0.9163 × 10-3 g/ml, respectively.

Simple and efficient synthesis of copper(II)-modified uniform magnetic Fe3O4@SiO2 core/shell microspheres for immobilization of cellulase

NASA Astrophysics Data System (ADS)

Li, Shi-Kuo; Hou, Xiao-Cheng; Huang, Fang-Zhi; Li, Chuan-Hao; Kang, Wen-Juan; Xie, An-Jian; Shen, Yu-Hua

2013-11-01

In this paper, we reported a simple and efficient protocol for preparation of Cu2+-modified magnetic Fe3O4@SiO2 core/shell microspheres for immobilization of cellulase. The uniform magnetic Fe3O4@SiO2 core/shell microspheres with a thin shell of 20 nm were synthesized through a solvothermal method followed by a sol-gel process. An amino-terminated silane coupling agent of (3-aminopropyl)triethoxysilane (APTS) was then grafted on them for capturing Cu2+ ions. The reaction process is very simple, efficient, and economical. Noticeably, the content of Cu2+ ions on the magnetic core/shell microspheres can reach 4.6 Wt%, endowing them possess as high immobilization capacity as 225.5 mg/g for cellulase. And the immobilized cellulase can be retained over 90 % on the magnetic microspheres after six cycles. Meanwhile, the magnetic microspheres decorated with Cu2+ ions show a superparamagnetic character with a high magnetic saturation of 58.5 emu/g at room temperature, suggesting conveniently and rapidly recycle the enzyme from solution. This facile, recyclable, high immobilization capacity and activity strategy may find potential applications in enzyme catalytic reactions with low cost.

Mitigation of Membrane Biofouling in MBR Using a Cellulolytic Bacterium, Undibacterium sp. DM-1, Isolated from Activated Sludge.

PubMed

Nahm, Chang Hyun; Lee, Seonki; Lee, Sang Hyun; Lee, Kibaek; Lee, Jaewoo; Kwon, Hyeokpil; Choo, Kwang-Ho; Lee, Jung-Kee; Jang, Jae Young; Lee, Chung-Hak; Park, Pyung-Kyu

2017-03-28

Biofilm formation on the membrane surface results in the loss of permeability in membrane bioreactors (MBRs) for wastewater treatment. Studies have revealed that cellulose is not only produced by a number of bacterial species but also plays a key role during formation of their biofilm. Hence, in this study, cellulase was introduced to a MBR as a cellulose-induced biofilm control strategy. For practical application of cellulase to MBR, a cellulolytic ( i.e ., cellulase-producing) bacterium, Undibacterium sp. DM-1, was isolated from a lab-scale MBR for wastewater treatment. Prior to its application to MBR, it was confirmed that the cell-free supernatant of DM-1 was capable of inhibiting biofilm formation and of detaching the mature biofilm of activated sludge and cellulose-producing bacteria. This suggested that cellulase could be an effective anti-biofouling agent for MBRs used in wastewater treatment. Undibacterium sp. DM-1-entrapping beads ( i.e ., cellulolytic-beads) were applied to a continuous MBR to mitigate membrane biofouling 2.2-fold, compared with an MBR with vacant-beads as a control. Subsequent analysis of the cellulose content in the biofilm formed on the membrane surface revealed that this mitigation was associated with an approximately 30% reduction in cellulose by cellulolytic-beads in MBR.

Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

PubMed

Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

2015-12-01

Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

Analysis of casein biopolymers adsorption to lignocellulosic biomass as a potential cellulase stabilizer.

PubMed

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

2012-01-01

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

Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores.

PubMed

Hagerman, A E; Blau, D M; McClure, A L

1985-12-01

A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.

A novel biochemical route for fuels and chemicals production from cellulosic biomass.

PubMed

Fan, Zhiliang; Wu, Weihua; Hildebrand, Amanda; Kasuga, Takao; Zhang, Ruifu; Xiong, Xiaochao

2012-01-01

The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing β-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate--glucose and gluconate--can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of β-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.

A Novel Biochemical Route for Fuels and Chemicals Production from Cellulosic Biomass

PubMed Central

Fan, Zhiliang; Wu, Weihua; Hildebrand, Amanda; Kasuga, Takao; Zhang, Ruifu; Xiong, Xiaochao

2012-01-01

The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing β-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate—glucose and gluconate—can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of β-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route. PMID:22384058

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Jae San; Shary, Semarjit; Houtman, Carl J.

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 themore » 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.« less

Aquatic Plant Control Research Program. Biological Control of Hydrilla verticillata (L.f.) Royle with Lytic Enzyme-Producing Microorganisms.

DTIC Science & Technology

1985-09-01

pectinase . Lytic enzyme-positive isolates were successively subcultured on restrictive media in the laboratory to enhance enzyme production. Twenty-two...candidate microorganisms by testing isolates for produc- tion of cellulase and pectinase . c. Taxonomically characterize candidates. d. Enhance production of...present study, but could become necessary if results of this study indicate that cellulase-enhanced v ,isolates are capable of damaging hydrilla. Pectinase