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Sample records for alkaline cellulase egl-237

  1. In silico metabolic engineering of Bacillus subtilis for improved production of riboflavin, Egl-237, (R,R)-2,3-butanediol and isobutanol.

    PubMed

    Hao, Tong; Han, Binbin; Ma, Hongwu; Fu, Jing; Wang, Hui; Wang, Zhiwen; Tang, Bincai; Chen, Tao; Zhao, Xueming

    2013-08-01

    Bacillus subtilis is a Gram-positive sporiferous bacterium widely used in a variety of industrial fields as a producer of high-quality vitamins, enzymes and proteins. Many genetic modifications and evolutionary engineering optimisations aiming at obtaining a better performing strain for its products have been studied. As genome-scale metabolic network models have gained significant popularity as effective tools in metabolic phenotype studies, we reconstructed a genome-scale metabolic network of B. subtilis-iBsu1147. The accuracy of iBsu1147 is validated by growth on various carbon sources, single gene knockout and large fragment non-essential gene knockout simulations. The model is used for the in silico metabolic engineering design of reactions over/underexpressed or knockout for increasing the production of four important products of B. subtilis: riboflavin, cellulase Egl-237, (R,R)-2,3-butanediol and isobutanol. The simulation predicted candidate reactions related to the improvement of strain performance on related products. The prediction is partly supported by previously published results. Due to the complexity of the biological system, it is difficult to manually find the factors that are not directly related to the production of the target compounds. The in silico predictions provide more choices for further strain improvement for these products. PMID:23666098

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

    PubMed

    Isaac, George Saad; Abu-Tahon, Medhat Ahmed

    2015-01-01

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

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

    PubMed Central

    Isaac, George Saad; Abu-Tahon, Medhat Ahmed

    2015-01-01

    Abstract A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and β-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and β-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0–11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for β-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and β-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover. PMID:26691490

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

    PubMed Central

    Gomaa, Eman Zakaria

    2013-01-01

    The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8–10, expressed their maximum activities at pH10 and temperature range of 30–50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51–97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed. PMID:24294252

  5. A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus sp. JB 99 suitable for paper and pulp industry: purification and characterization.

    PubMed

    Shrinivas, Dengeti; Savitha, Gunashekaran; Raviranjan, Kumar; Naik, Gajanan Ramchandra

    2010-11-01

    A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0-10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K (m) and V (max) of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min(-1) mg(-1), respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

  6. Cellulases from psychrophilic microorganisms: a review.

    PubMed

    Kasana, Ramesh C; Gulati, Arvind

    2011-12-01

    Cellulases are hydrolytic enzymes that catalyze total hydrolysis of cellulose into sugars. Cellulases are produced by various groups of microorganisms and animals; however, psychrophiles are the ideal candidates for the production of enzymes active at low temperature and stable under alkaline conditions, in the presence of oxidants and detergents, which are in large demand as laundry additives. The cellulases from psychrophiles also find application in environmental bioremediation, food industry and molecular biology. Research work on cellulase has been done over the last six decades, but there is no exclusive review available on the cellulases from psychrophiles. This review is an attempt to fill this gap by providing all the relevant information exclusively for cellulases from psychrophiles, with a focus on the present status of knowledge on their activity, molecular characteristics, gene cloning, statistical experimental designs, crystal structure, and strategies for the improvement of psychrophilic cellulases.

  7. Cellulase Assays

    NASA Astrophysics Data System (ADS)

    Zhang, Y. H. Percival; Hong, Jiong; Ye, Xinhao

    Cellulose is a heterogeneous polysaccharide, and its enzymatic hydrolysis requires endoglucanase, exoglucanase (cellobiohydrolase), and β-glucosidase to work together. We summarize the most commonly used assays for individual enzymes and cellulase mixture.

  8. 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.

  9. 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.

  10. Signal peptide of cellulase.

    PubMed

    Yan, Shaomin; Wu, Guang

    2014-06-01

    Cellulase is an enzyme playing a crucial role in biotechnology industries ranging from textile to biofuel because of tremendous amount of cellulose produced in plant. In order to improve cellulase productivity, huge resource has been spent in search for good cellulases from microorganism in remote areas and in creation of ideal cellulase by engineering. However, not much attention is given to the secretion of cellulases from cell into extracellular space, where a cellulase plays its enzymatic role. In this minireview, the signal peptides, which lead secreted proteins to specific secretion systems and scatter in literature, are reviewed. The patterns of signal peptides are checked against 4,101 cellulases documented in UniProtKB, the largest protein database in the world, to determine how these cellulases are secreted. Simultaneous review on both literature and cellulases from the database not only provides updated knowledge on signal peptides but also indicates the gap in our research.

  11. Engineering Cellulases for Biorefinery

    SciTech Connect

    Manoj Kumar, PhD

    2010-06-27

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

  12. Thermostable Cellulases: Why & How?

    SciTech Connect

    Manoj Kumar, PhD

    2010-04-19

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

  13. Thermostable Cellulases: Why & How?

    SciTech Connect

    Manoj Kumar, PhD

    2010-03-24

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

  14. Development of immobilized cellulase

    SciTech Connect

    Luther, M.A.; Halbert, D.J.

    1982-08-01

    The immobilization of cellulase from the fungus Trichoderma reesei on the surface of calcium alginate gel spheres was investigated. The immobilized cellulase catalyzed the hydrolysis of cellulose to glucose. The linking agents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde, decreased free-enzyme activity by 95%, and the maximum observed retention of cellulase activity after immobilization was 2%. Leakage of enzyme from the support was observed. A fivefold increase in glucose production was seen after the addition of ..beta..-glucosidase-impregnated spheres to the cellulase spheres, suggesting that cellobiose may be accumulating during the reaction. A simple economic analysis suggested that the immobilized-enzyme activity per unit volume might have to be increased by a factor of fifty to become competitive with the free-enzyme process.

  15. Chimeric enzymes with improved cellulase activities

    SciTech Connect

    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.

  16. (Research on Trichoderma cellulases)

    SciTech Connect

    Woodward, J.

    1989-09-29

    The traveler attended TRICEL 89, the first international symposium on Trichoderma cellulases. He presented a paper summarizing his work at ORNL. He also visited Aston University in Birmingham, England, and gave a lecture describing his work at Oak Ridge National Laboratory (ORNL). In Vienna, the latest knowledge concerning Trichoderma cellulases was obtained, and two contacts were established that will lead to unclassified and nonproprietary cooperative research efforts between ORNL and these European colleagues. In Birmingham, unclassified and nonproprietary discussions were held with Professor P.E. Barker and his staff in the Department of Chemical Engineering and Applied Chemistry. As a result of these discussions, areas of research cooperation between ORNL and Aston University were identified, and it is hoped that this cooperation will attract funding from the National Science Foundation (US) and the European Economic Community (EEC).

  17. Cellulase, Clostridia, and Ethanol†

    PubMed Central

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

    2005-01-01

    Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield. PMID:15755956

  18. Cellulases Dig Deep

    PubMed Central

    Bubner, Patricia; Dohr, Judith; Plank, Harald; Mayrhofer, Claudia; Nidetzky, Bernd

    2012-01-01

    Enzymatic hydrolysis of cellulose is key for the production of second generation biofuels, which represent a long-standing leading area in the field of sustainable energy. Despite the wealth of knowledge about cellulase structure and function, the elusive mechanism by which these enzymes disintegrate the complex structure of their insoluble substrate, which is the gist of cellulose saccharification, is still unclear. We herein present a time-resolved structural characterization of the action of cellulases on a nano-flat cellulose preparation, which enabled us to overcome previous limitations, using atomic force microscopy (AFM). As a first step in substrate disintegration, elongated fissures emerge which develop into coniform cracks as disintegration continues. Detailed data analysis allowed tracing the surface evolution back to the dynamics of crack morphology. This, in turn, reflects the interplay between surface degradation inside and outside of the crack. We observed how small cracks evolved and initially increased in size. At a certain point, the crack diameter stagnated and then started decreasing again. Stagnation corresponds with a decrease in the total amount of surface which is fissured and thus leads to the conclusion that the surface hydrolysis “around” the cracks is proceeding more rapidly than inside the cracks. The mesoscopic view presented here is in good agreement with various mechanistic proposals from the past and allows a novel insight into the structural dynamics occurring on the cellulosic substrate through cellulase action. PMID:22128148

  19. Recycling cellulases by pH-triggered adsorption-desorption during the enzymatic hydrolysis of lignocellulosic biomass.

    PubMed

    Shang, Yaping; Su, Rongxin; Huang, Renliang; Yang, Yang; Qi, Wei; Li, Qiujin; He, Zhimin

    2014-06-01

    Recycling of cellulases is an effective way to reduce the cost of enzymatic hydrolysis for the production of cellulosic ethanol. In this study, we examined the adsorption and desorption behaviors of cellulase at different pH values and temperatures. Furthermore, we developed a promising way to recover both free and bound cellulases by pH-triggered adsorption-desorption. The results show that acidic pH (e.g., pH 4.8) was found to favor adsorption, whereas alkaline pH (e.g., pH 10) and low temperature (4-37 °C) favored desorption. The adsorption of cellulases reached an equilibrium within 60 min at pH 4.8 and 25 °C, leading to approximately 50 % of the added cellulases bound to the substrate. By controlling the pH of eluent (citrate buffer, 25 °C), we were able to increase the desorption efficiency of bound cellulases from 15 % at pH 4.8 to 85 % at pH 10. To recover cellulases after enzymatic hydrolysis, we employed adsorption by fresh substrate and desorption at pH 10 to recover the free cellulases in supernatant and the bound cellulases in residue, respectively. The recycling performance (based on the glucose yield) of this simple strategy could reach near 80 %. Our results provided a simple, low-cost, and effective approach for cellulase recycling during the enzymatic hydrolysis of lignocellulosic biomass.

  20. Cellulase immobilized on modified nylon for saccharification of cellulose

    SciTech Connect

    Jain, P.; Wilkins, E.S.

    1987-01-01

    The present study deals with the immobilization of cellulase on nylon and nylon incorporated with glass. The immobilized and free enzymes were compared in terms of their yields, using untreated sawdust (yellow pine wood) and carboxymethylcellulose (CMC) as substrates, at a standard pH and temperature. Also, the sawdust was pretreated with 1% alkaline hydrogen peroxide and the yield compared with the untreated sawdust hydrolysis to determine the importance of the substrate pretreatment.

  1. 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

  2. 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.

  3. 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.

  4. Cellulase recovery: Problems and potential

    SciTech Connect

    Woodward, J.; Lee, N.E.

    1989-01-01

    The enzymatic hydrolysis of cellulose may become commercially feasible if the enzyme is recovered and reused. The use of immobilized cellobiohydrolase (CBH) and endoglucanase (EG) does not appear feasible since the substrate itself is insoluble. Since the ..beta..-glucosidase (BG) component of cellulase acts upon soluble substrates it has the potential to be immobilized, used, and reused in this form. The problem of CBH and EG recovery has been tackled by examining their ability to be adsorbed to inorganically-based kieselguhr Macrosorb granules which can be used repeatedly for cellulase adsorption. The kinetics of the adsorption of Trichoderma reesei C30 cellulase protein to DEAE-Macrosorb are described and these Macrosorb particles have been used to recover cellulase from Avicel and steam-exploded aspen wood hydrolysates. The problem of ..beta..-glucosidase recovery could be achieved by immobilizing it onto an affinity material such as concanavalin A from which this enzyme is difficult to elute. Cellulase adsorbed onto lignocellulosic residues at the completion of hydrolysis can be eluted from the residue using a protein denaturant. Reactivation of the denatured enzyme may be possible. 19 refs., 1 fig., 3 tabs.

  5. 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. PMID:26956574

  6. Exo-endo cellulase fusion protein

    DOEpatents

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

    2012-01-17

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

  7. 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.

  8. 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.

  9. Microbial Cellulases and Their Industrial Applications

    PubMed Central

    Kuhad, Ramesh Chander; Gupta, Rishi; Singh, Ajay

    2011-01-01

    Microbial cellulases have shown their potential application in various industries including pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing, and agriculture. Due to the complexity of enzyme system and immense industrial potential, cellulases have been a potential candidate for research by both the academic and industrial research groups. Nowadays, significant attentions have been devoted to the current knowledge of cellulase production and the challenges in cellulase research especially in the direction of improving the process economics of various industries. Scientific and technological developments and the future prospects for application of cellulases in different industries are discussed in this paper. PMID:21912738

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

    SciTech Connect

    Manoj Kumar, PhD

    2011-04-26

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

  11. 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

  12. 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.

  13. 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.

  14. Cellulase: A key enzyme for fermentation feedstocks

    SciTech Connect

    Eveleigh, D.E.

    1988-08-01

    Biomass can be fermented to a range of useful products. For efficiency a well characterized and effective cellulase is necessary to gain fermentable sugars from cellulosics. Microbial cellulase systems are comprised of multiple components namely, endoglucanase (EG), cellobiohydrolase (CBH), and beta-glucosidase, which act synergistically. We have been clarifying the cellulase system especially that of Microbispora bispora through isolation of the individual components. As there is no direct assay for the cellobiohydrolase component, we have made monoclonal antibodies to them, and are using them to characterize the cellobiohydrolases of Trichoderma reesei and M. bispora. With T. reesei, rapid purification has been achieved through MAb affinity chromatography and also a highly sensitive MAb assay for CBH in crude cellulase broths has been developed. for M. bispora, the monoclonal antibodies towards CBH have been prepared and are being used for selection of clones from a M. bispora gene library. We have focused on the M. bispora cellulase.

  15. 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.

  16. 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

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

    PubMed Central

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

    2013-01-01

    Cellulases play a key role in enzymatic routes for degradation of plant cell-wall polysaccharides into simple and economically-relevant sugars. However, their low performance on complex substrates and reduced stability under industrial conditions remain the main obstacle for the large-scale production of cellulose-derived products and biofuels. Thus, in this study a novel cellulase with unusual catalytic properties from sugarcane soil metagenome (CelE1) was isolated and characterized. The polypeptide deduced from the celE1 gene encodes a unique glycoside hydrolase domain belonging to GH5 family. The recombinant enzyme was active on both carboxymethyl cellulose and β-glucan with an endo-acting mode according to capillary electrophoretic analysis of cleavage products. CelE1 showed optimum hydrolytic activity at pH 7.0 and 50 °C with remarkable activity at alkaline conditions that is attractive for industrial applications in which conventional acidic cellulases are not suitable. Moreover, its three-dimensional structure was determined at 1.8 Å resolution that allowed the identification of an insertion of eight residues in the β8-α8 loop of the catalytic domain of CelE1, which is not conserved in its psychrophilic orthologs. This 8-residue-long segment is a prominent and distinguishing feature of thermotolerant cellulases 5 suggesting that it might be involved with thermal stability. Based on its unconventional characteristics, CelE1 could be potentially employed in biotechnological processes that require thermotolerant and alkaline cellulases. PMID:24358302

  18. 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. PMID:26456602

  19. 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.

  20. Mining metagenomes for novel cellulase genes.

    PubMed

    Duan, Cheng-Jie; Feng, Jia-Xun

    2010-12-01

    Cellulases hydrolyze the β-1,4 linkages of cellulose and are widely used in food, brewing and wine, animal feed, textiles and laundry, and pulp and paper industries, especially for hydrolyzing cellulosic materials into sugars, which can be fermented to produce useful products such as ethanol. Metagenomics has become an alternative approach to conventional culture-dependent methods as it allows exhaustive mining of microbial genomes in their natural environments. This review covers the current state of research and challenges in mining novel cellulase genes from the metagenomes of various environments, and discusses the potential biotechnological applications of metagenome-derived cellulases.

  1. The realm of cellulases in biorefinery development.

    PubMed

    Chandel, Anuj K; Chandrasekhar, G; Silva, Messias Borges; Silvério da Silva, Silvio

    2012-09-01

    Geopolitical concerns (unstable supply of gasoline, environmental pollution, and regular price hikes), economic, and employment concerns have been prompting researchers, entrepreneurs, and policy makers to focus on harnessing the potential of lignocellulosic feedstock for fuel ethanol production and its commercialization. The carbohydrate skeleton of plant cell walls needs to be depolymerised into simpler sugars for their application in fermentation reactions as a chief carbon source of suitable ethnologic strains for ethanol production. The role of cellulolytic enzymes in the degradation of structural carbohydrates of the plant cell wall into ready-to-fermentable sugar stream is inevitable. Cellulase synergistically acts upon plant cell wall polysaccharides to release glucose into the liquid media. Cellulase predominantly dominates all the plant cell wall degrading enzymes due to their vast and diverse range of applications. Apart from the major applications of cellulases such as in detergent formulations, textile desizing, and development of monogastric feed for ruminants, their role in biorefinery is truly remarkable. This is a major area where new research tools based upon fermentation based formulations, biochemistry, and system biology to expedite the structure-function relationships of cellulases including cellulosomes and new designer enzymatic cocktails are required. In the last two decades, a considerable amount of research work has been performed on cellulases and their application in biomass saccharification. However, there are still technical and economic impediments to the development of an inexpensive commercial cellulase production process. Advancements in biotechnology such as screening of microorganisms, manipulation of novel cellulase encoding traits, site-specific mutagenesis, and modifications to the fermentation process could enhance the production of cellulases. Commercially, cheaper sources of carbohydrates and modified fermentation

  2. The realm of cellulases in biorefinery development.

    PubMed

    Chandel, Anuj K; Chandrasekhar, G; Silva, Messias Borges; Silvério da Silva, Silvio

    2012-09-01

    Geopolitical concerns (unstable supply of gasoline, environmental pollution, and regular price hikes), economic, and employment concerns have been prompting researchers, entrepreneurs, and policy makers to focus on harnessing the potential of lignocellulosic feedstock for fuel ethanol production and its commercialization. The carbohydrate skeleton of plant cell walls needs to be depolymerised into simpler sugars for their application in fermentation reactions as a chief carbon source of suitable ethnologic strains for ethanol production. The role of cellulolytic enzymes in the degradation of structural carbohydrates of the plant cell wall into ready-to-fermentable sugar stream is inevitable. Cellulase synergistically acts upon plant cell wall polysaccharides to release glucose into the liquid media. Cellulase predominantly dominates all the plant cell wall degrading enzymes due to their vast and diverse range of applications. Apart from the major applications of cellulases such as in detergent formulations, textile desizing, and development of monogastric feed for ruminants, their role in biorefinery is truly remarkable. This is a major area where new research tools based upon fermentation based formulations, biochemistry, and system biology to expedite the structure-function relationships of cellulases including cellulosomes and new designer enzymatic cocktails are required. In the last two decades, a considerable amount of research work has been performed on cellulases and their application in biomass saccharification. However, there are still technical and economic impediments to the development of an inexpensive commercial cellulase production process. Advancements in biotechnology such as screening of microorganisms, manipulation of novel cellulase encoding traits, site-specific mutagenesis, and modifications to the fermentation process could enhance the production of cellulases. Commercially, cheaper sources of carbohydrates and modified fermentation

  3. 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.

  4. Thermal tolerant cellulase from Acidothermus cellulolyticus

    SciTech Connect

    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.

  5. Comparative investigation of various cellulase assay procedures

    SciTech Connect

    Canevascini, G.; Gattlen, C.

    1981-07-01

    The cellulolytic activity of crude enzyme preparations from different cellulolytic fungi (namely Trichoderma viride, Trichoderma koningii, Fusarium solani, Sporotrichum pulverulentum, Sporotrichum thermophile) was assayed comparatively with several common analytical procedures described in the literature. The investigation was carried out with the objective of evaluating, with raw culture filtrates, the different cellulase tests in relation to their specificity for endo- and exo-cellulase action as well as to allow comparisons to be made between results from different research groups using different methods. 1) Cellulase activity was tested viscometrically as well as chemically (determination of reducing end groups) with different carboxymethylcelluloses as substrates. Essentially constant ratios between both kinds of activities were obtained, indicating that they are directly related. By estimating cellulase activity with insoluble cellulosic substrates no direct relationship could be established with the above-described activities except in the case where the cellulose was amorphous. The ratio profile between activities thus obtained and endo-cellulase activities determined viscometrically shows that some enzyme preparations (such as those from both Trichoderma sp.) are clearly more active than others against crystalline cellulose reflecting quantitative differences in enzyme composition. Nevertheless, for a biological understanding of cellulolysis, analytical procedures using crystalline celluloses are not adequate for specifically monitoring exo-cellulase activity in crude enzyme solutions for essentially two reasons: a) they are not sufficiently sensitive to detect small changes in enzyme activity during the early phase of growth, and b) exo-cellulase activity in crude enzyme solutions also depends on the endo-cellulase activity present. (Refs. 39).

  6. Cellulase and xylanase activities in higher basidiomycetes.

    PubMed

    Elisashvili, V I; Khardziani, T S; Tsiklauri, N D; Kachlishvili, E T

    1999-06-01

    Extracellular carboxymethylcellulase, xylanase, beta-glucosidase, and beta-xylosidase activities of four cultures of higher basidial fungi were studied in relation to the source of carbon in the nutrient medium. It was shown that beta-glucosidases and beta-xylosidases of all basidiomycetes and cellulases and xylanases of Pholiota aurivella IBR437 and Gloeophyllum saepiarium IBR155, the causal agents of wood brown rot, are constitutive enzymes; however, their activities depend on the source of carbon in the growth medium. Cellulases and xylanases of Coriolus pubescens IBR663 and Lentinus tigrinus IBR100 degrading wood through white rot are inducible enzymes. The synthesis of cellulases and xylanases was induced upon fungal growth on media containing crystalline cellulose and plant raw materials; carboxymethylcellulose and xylan were less effective. The induction of C. pubescens IBR663 cellulase and xylanase was observed when avicel was added to the culture growing on a mannitol-containing medium. Glucose at a concentration of 0.2-0.8% caused catabolite repression of C. pubescens IBR663 cellulase and xylanase. After utilization of glucose, leading to a decrease in its concentration below 0.1%, the synthesis of enzymes was resumed. These data indicate that the synthesis of cellulases and xylanases in the examined macromycetes is under common regulatory control. PMID:10395989

  7. Outlook for cellulase improvement: Screening and selection strategies

    SciTech Connect

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

    2006-03-01

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

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

    PubMed

    Gomes, Daniel; Domingues, Lucília; Gama, Miguel

    2016-09-01

    The feasibility of cellulase recycling in the scope of bioethanol production from recycled paper sludge (RPS), an inexpensive byproduct with around 39% of carbohydrates, is analyzed. RPS was easily converted and fermented by enzymes and cells, respectively. Final enzyme partition between solid and liquid phases was investigated, the solid-bound enzymes being efficiently recovered by alkaline washing. RPS hydrolysis and fermentation was conducted over four rounds, recycling the cellulases present in both fractions. A great overall enzyme stability was observed: 71, 64 and 100% of the initial Cel7A, Cel7B and β-glucosidase activities, respectively, were recovered. Even with only 30% of fresh enzymes added on the subsequent rounds, solid conversions of 92, 83 and 71% were achieved for the round 2, 3 and 4, respectively. This strategy enabled an enzyme saving around 53-60%, while can equally contribute to a 40% reduction in RPS disposal costs. PMID:27289054

  9. Development of effective modified cellulase for cellulose hydrolysis process

    SciTech Connect

    Park, J.W.; Kajiuchi, Toshio . Dept. of Chemical Engineering)

    1995-02-20

    Cellulase was modified with amphilic copolymers made of [alpha]-allyl-[omega]-methoxy polyoxyalkylene (POA) and maleic acid anhydride (MAA) to improve the cellulose hydrolytic reactivity and cellulase separation. Amino groups of the cellulase molecule are covalently coupled with the MAA functional groups of the copolymer. At the maximum degree of modification (DM) of 55%, the modified cellulase activity retained more than 80% of the unmodified native cellulase activity. The modified cellulase shows greater stability against temperature, pH, and organic solvents, and demonstrated greater conversion of substrate than native cellulase does. Cellulase modification is also useful for controlling strong adsorption of cellulase onto substrate. Moreover, cellulase modified with the amphiphilic copolymer displays different separation characteristics which are new. One is a reactive two-phase partition and another is solubility in organic solvents. It appears that these characteristics of modified cellulase work very effectively in the hydrolysis of cellulose as a total system, which constitutes the purification of cellulase from culture broth, hydrolysis of cellulose, and recovery of cellulase from the reaction mixture.

  10. Cellulase: A key enzyme for fermentation stocks

    SciTech Connect

    Eveleigh, D.E.; Macmillan, J.D.

    1990-03-15

    A thermophilic actinomycete Microbispora bispora was selected from many thermophilic actinomycetes as it that gave good yields of a stable, cellulase and its {beta}-glucosidase was resistant to end- product inhibition. The cellulase is comprised of endoglucanases, cellobiohydrolases and cellobiases that act synergistically. These components have been characterized to differing degrees. The initial cloning of the cellobiase and cellobiohydrolase have been also accomplished. We now propose to further characterize the cellobiohydrolase and the cellobiase with the longer term objective of being able to predict optimal proportions of these components which will result in optimal cellulolysis. The original goals of characterization of the M. bispora cellulase, developing methods to gain secretion, and the application of monoclonal antibodies to screening and for affinity purification of cellobiohydrolase have been accomplished.

  11. 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

  12. Biochemical nature of cellulases from mutants of Trichoderma reesei

    SciTech Connect

    Montenecourt, B.S.; Kelleher, T.J.; Eveleigh, D.E.; Pettersson, L.G.

    1980-01-01

    The cellulases of two new mutants of T. reesei, RUT-NG14 and RUT-C30, have been examined with respect to the separation and biochemical characterization of the various components in the cellulase complex. The cellulase of both mutants has been shown to contain enhanced proportions of a cellobiohydrolase by quantitative immune precipitation. Application of ion-exchange high-pressure liquid chromatography protein separations affords a rapid (30 min) and simple method of separating and comparing cellulase components from potential high-yielding cellulase mutants.

  13. Cellulase: A key enzyme for fermentation stocks

    SciTech Connect

    Eveleigh, D.E.; Macmillan, J.D.

    1989-07-03

    A thermophilic actinomycete Microbispora bispora was selected from many thermophilic actinomycetes as it that gave good yields of a stable, cellulase and its {beta}-glucosidase was resistant to end- product inhibition. The initial cloning of the cellobiase and cellobiohydrolase have been also accomplished. We now propose to further characterize the cellobiohydrolase and the cellobiase with the longer term objective of being able to predict optimal proportions of these components which will result in optimal cellulolysis, besides also the rationale design and genetic construction of a truly effective cellulolytic ethanologen, namely Zymomonas mobilis. This study was initiated with a further general goal of developing the application of monoclonal antibodies for studying cellulase. Monoclonal antibodies acting towards cellulases have proved most useful for developing a sensitive direct assay for cellobiohydrolase (Trichoderma reesesi) and for screening Escherichia Coli (E. coli) transformant clones containing Microbispora bispora cellobiohydrolase genes. All goals of the original proposal, characterization of the M. bispora cellulase, developing methods to gain secretion, and the application of monoclonal antibodies to screening and for affinity purification of cellobiohydrolase have been accomplished. 22 refs., 6 figs.

  14. 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.

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

    PubMed

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

    2011-11-01

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

  16. Enhanced Cellulase Production from Bacillus subtilis by Optimizing Physical Parameters for Bioethanol Production

    PubMed Central

    Deka, Deepmoni; Das, Saprativ P.; Sahoo, Naresh; Das, Debasish; Jawed, Mohammad; Goyal, Dinesh

    2013-01-01

    Effect of physical parameters such as initial pH, agitation (rpm), and temperature (°C) for cellulase production from Bacillus subtilis AS3 was investigated. Central composite design of experiments followed by multiple desirability function was applied for the optimization of cellulase activity and cell growth. The effect of the temperature and agitation was found to be significant among the three independent variables. The optimum levels of initial pH, temperature, and agitation for alkaline carboxymethylcellulase (CMCase) production predicted by the model were 7.2, 39°C, and 121 rpm, respectively. The CMCase activity with unoptimized physical parameters and previously optimized medium composition was 0.43 U/mL. The maximum activity (0.56 U/mL) and cell growth (2.01 mg/mL) predicted by the model were in consensus with values (0.57 U/mL, 2.1 mg/mL) obtained using optimized medium and optimal values of physical parameters. After optimization, 33% enhancement in CMCase activity (0.57 U/mL) was recorded. On scale-up of cellulase production process in bioreactor with all the optimized conditions, an activity of 0.75 U/mL was achieved. Consequently, the bacterial cellulase employed for bioethanol production expending (5%, w/v) NaOH-pretreated wild grass with Zymomonas mobilis yielded an utmost ethanol titre of 7.56 g/L and 11.65 g/L at shake flask and bioreactor level, respectively. PMID:25937985

  17. 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. PMID:27229865

  18. Product Binding Varies Dramatically between Processive and Nonprocessive Cellulase Enzymes

    SciTech Connect

    Bu, L.; Nimlos, M. R.; Shirts, M. R.; Stahlberg, J.; Himmel, M. E.; Crowley, M. F.; Beckham, G. T.

    2012-07-13

    Cellulases hydrolyze {beta}-1,4 glycosidic linkages in cellulose, which are among the most prevalent and stable bonds in Nature. Cellulases comprise many glycoside hydrolase families and exist as processive or nonprocessive enzymes. Product inhibition negatively impacts cellulase action, but experimental measurements of product-binding constants vary significantly, and there is little consensus on the importance of this phenomenon. To provide molecular level insights into cellulase product inhibition, we examine the impact of product binding on processive and nonprocessive cellulases by calculating the binding free energy of cellobiose to the product sites of catalytic domains of processive and nonprocessive enzymes from glycoside hydrolase families 6 and 7. The results suggest that cellobiose binds to processive cellulases much more strongly than nonprocessive cellulases. We also predict that the presence of a cellodextrin bound in the reactant site of the catalytic domain, which is present during enzymatic catalysis, has no effect on product binding in nonprocessive cellulases, whereas it significantly increases product binding to processive cellulases. This difference in product binding correlates with hydrogen bonding between the substrate-side ligand and the cellobiose product in processive cellulase tunnels and the additional stabilization from the longer tunnel-forming loops. The hydrogen bonds between the substrate- and product-side ligands are disrupted by water in nonprocessive cellulase clefts, and the lack of long tunnel-forming loops results in lower affinity of the product ligand. These findings provide new insights into the large discrepancies reported for binding constants for cellulases and suggest that product inhibition will vary significantly based on the amount of productive binding for processive cellulases on cellulose.

  19. Product Binding Varies Dramatically between Processive and Nonprocessive Cellulase Enzymes*

    PubMed Central

    Bu, Lintao; Nimlos, Mark R.; Shirts, Michael R.; Ståhlberg, Jerry; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

    2012-01-01

    Cellulases hydrolyze β-1,4 glycosidic linkages in cellulose, which are among the most prevalent and stable bonds in Nature. Cellulases comprise many glycoside hydrolase families and exist as processive or nonprocessive enzymes. Product inhibition negatively impacts cellulase action, but experimental measurements of product-binding constants vary significantly, and there is little consensus on the importance of this phenomenon. To provide molecular level insights into cellulase product inhibition, we examine the impact of product binding on processive and nonprocessive cellulases by calculating the binding free energy of cellobiose to the product sites of catalytic domains of processive and nonprocessive enzymes from glycoside hydrolase families 6 and 7. The results suggest that cellobiose binds to processive cellulases much more strongly than nonprocessive cellulases. We also predict that the presence of a cellodextrin bound in the reactant site of the catalytic domain, which is present during enzymatic catalysis, has no effect on product binding in nonprocessive cellulases, whereas it significantly increases product binding to processive cellulases. This difference in product binding correlates with hydrogen bonding between the substrate-side ligand and the cellobiose product in processive cellulase tunnels and the additional stabilization from the longer tunnel-forming loops. The hydrogen bonds between the substrate- and product-side ligands are disrupted by water in nonprocessive cellulase clefts, and the lack of long tunnel-forming loops results in lower affinity of the product ligand. These findings provide new insights into the large discrepancies reported for binding constants for cellulases and suggest that product inhibition will vary significantly based on the amount of productive binding for processive cellulases on cellulose. PMID:22648408

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

    PubMed Central

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

    2016-01-01

    Cellulases are the enzymes which are responsible for the hydrolysis of cellulosic biomass. In this study thermal and pH stability of crude cellulase has been investigated in the presence of zinc oxide (ZnO) nanoparticles. We synthesized ZnO nanoparticle by sol-gel method and characterized through various techniques including, X-ray Diffraction, ultraviolet-visible spectroscope, field emission scanning electron microscope and high resolution scanning electron microscope. The crude thermostable cellulase has been obtained from the Aspergillus fumigatus AA001 and treated with ZnO nanoparticle which shows thermal stability at 65°C up to 10 h whereas it showed pH stability in the alkaline pH range and retained its 53% of relative activity at pH 10.5. These findings may be promising in the area of biofuels production. PMID:27148203

  1. Harnessing Glycosylation to Improve Cellulase Activity

    SciTech Connect

    Beckham, G. T.; Dai, Z.; Matthews, J. F.; Momany, M.; Payne, C. M.; Adney, W. S.; Baker, S. E.; Himmel, M. E.

    2012-06-01

    Cellulases and hemicellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere, and thus form the foundation of enzyme engineering efforts in biofuels research. Many of these carbohydrate-active enzymes from filamentous fungi contain both N-linked and O-linked glycosylation, the extent and heterogeneity of which depends on growth conditions, expression host, and the presence of glycan trimming enzymes in the secretome, all of which in turn impact enzyme activity. As the roles of glycosylation in enzyme function have not been fully elucidated, here we discuss the potential roles of glycosylation on glycoside hydrolase enzyme structure and function after secretion. We posit that glycosylation, instead of hindering cellulase engineering, can be used as an additional tool to enhance enzyme activity, given deeper understanding of its molecular-level role in biomass deconstruction.

  2. Harnessing glycosylation to improve cellulase activity

    SciTech Connect

    Beckham, Gregg T.; Dai, Ziyu; Mattews, James F.; Momany, Michelle; Payne, Christina M.; Adney, William S.; Baker, Scott E.; Himmel, Michael E.

    2012-06-11

    Cellulases and hemicellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere, and thus form the foundation of enzyme engineering efforts in biofuels research. Many of these carbohydrate-active enzymes from filamentous fungi contain both N-linked and O-linked glycosylation, the extent and heterogeneity of which depends on growth conditions, expression host, and the presence of glycan trimming enzymes in the secretome, all of which in turn impacts enzyme activity. As the roles of glycosylation in enzyme function have not been fully elucidated, here we discuss the potential roles of glycosylation on glycoside hydrolase enzyme structure and function after secretion. We posit that glycosylation, instead of hindering cellulase engineering, can be used as an additional tool to enhance enzyme activity, given deeper understanding of its molecular-level role in biomass deconstruction.

  3. Multiple Cellulase System from Streptomyces antibioticus1

    PubMed Central

    Enger, M. D.; Sleeper, B. P.

    1965-01-01

    Enger, M. D. (North Dakota State University, Fargo), and B. P. Sleeper. Multiple cellulase system from Streptomyces antibioticus. J. Bacteriol. 89:23–27. 1965.—Starch-block zone electrophoresis was used to isolate five electrophoretically distinct, active cellulolytic components (I to V) from the crude extracellular cellulase system of Streptomyces antibioticus (strain C2A). Agar diffusion precipitin analyses demonstrated the immunological identity of components I, II, and III, and the nonidentity of IV and V with each other and with I to III. Kinetic studies of the purified enzymes showed a sharp decrease in the viscosity of the substrate, carboxymethylcellulose, with only a small increase in reducing sugars. These results indicated that all five enzymes are endocellulases. PMID:14255670

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

    PubMed

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

    2015-11-01

    The application of cellulases in saccharification processes is restricted by its production cost. Consequently, new fungal strains able to elaborate higher cellulases titers and with special activity profiles are required to make the process economical. The aim of this investigation was to find a promising wild-type Trichoderma strain for cellulases production. The Trichoderma reesei strain 938 (CBS 836.91) was selected among twenty strains on the basis of cellulase-agar-plate screening. Evaluation of the selected strain on six solid substrates indicated the highest activities to be obtained from wheat bran. Statistical analyses of the experimental design indicated a significant effect of pH and moisture on the generation of endoglucanase (EGA) and filter-paper (FPA) activity. Furthermore, a central-composite design-based optimization revealed that pH values between 6.4 and 6.6 and moisture from 74 to 94% were optimal for cellulases production. Under these conditions, 8-10 IU gds(-1) of FPA and 15.6-17.8 IU gds(-1) of EGA were obtained. In addition, cultivation in a rotating-drum reactor under optimal conditions gave 8.2 IU gds(-1) FPA and 13.5 IU gds(-1) EGA. Biochemical characterization of T. reesei 938 cellulases indicated a substantially higher resistance to 4 mM Fe(+2) and a slightly greater tolerance to alkaline pH in comparison to Celluclast(®). These results suggest that T. reesei 938 could be a promising candidate for improved cellulases production through direct-evolution strategies. PMID:26256022

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

    PubMed

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

    2015-11-01

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

  6. 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.

  7. Secretory pathway of cellulase: a mini-review

    PubMed Central

    2013-01-01

    Cellulase plays an important role in modern industry and holds great potential in biofuel production. Many different types of organisms produce cellulase, which go through secretory pathways to reach the extracellular space, where enzymatic reactions take place. Secretory pathways in various cells have been the focus of many research fields; however, there are few studies on secretory pathways of cellulases in the literature. It is therefore necessary and important to review the current knowledge on the secretory pathways of cellulases. In this mini-review, we address the subcellular locations of cellulases in different organisms, discuss the secretory pathways of cellulases in different organisms, and examine the secretory mechanisms of cellulases. These sections start with a description of general secreted proteins, advance to the situation of cellulases, and end with the knowledge of cellulases, as documented in UniProt Knowledgebase (UniProtKB). Finally, gaps in existing knowledge are highlighted, which may shed light on future studies for biofuel engineering. PMID:24295495

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

    PubMed Central

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

    2011-01-01

    Thermophilic fungal cellulases are promising enzymes in protein engineering efforts aimed at optimizing industrial processes, such as biomass degradation and biofuel production. The cloning and expression in recent years of new cellulase genes from thermophilic fungi have led to a better understanding of cellulose degradation in these species. Moreover, crystal structures of thermophilic fungal cellulases are now available, providing insights into their function and stability. The present paper is focused on recent progress in cloning, expression, regulation, and structure of thermophilic fungal cellulases and the current research efforts to improve their properties for better use in biotechnological applications. PMID:22145076

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

    PubMed

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

    2014-01-01

    Brine shrimp (Artemia salina) belong to a group of crustaceans that feed on microalgae and require a cellulase enzyme that can be used in ethanol production from marine algae. Protein with potential cellulase activity was purified and the activity analyzed under different conditions. After initial identification of cellulase activity by CMC cellulase, surface sterilization and PCR using 16s rRNA primers was conducted to confirm that the cellulase activity was not produced from contaminating bacteria. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. After the final purification, a 70-fold increase in specific enzyme activity was observed. SDS-PAGE results revealed that the cellulase enzyme had a molecular mass of 96 kDa. Temperature, pH, and salinity values were found to be optimal at 55 °C, pH 8.0, and 600 mM NaCl, respectively. Specifically, the enzyme showed a fivefold increase in enzyme activity in seawater compared to 600 mM NaCl in phosphate buffer. Further analysis of the purified enzyme by molecular spectrometry showed no match to known cellulases, indicating this enzyme could be a novel halophilic cellulase that can be used for the production of bioethanol from marine macroalgae. PMID:24291747

  10. 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.

  11. 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.

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

    PubMed

    Zhang, Junhua; Pakarinen, Annukka; Viikari, Liisa

    2013-02-01

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

  13. ALP (Alkaline Phosphatase) Test

    MedlinePlus

    ... known as: ALK PHOS; Alkp Formal name: Alkaline Phosphatase Related tests: AST ; ALT ; GGT ; Bilirubin ; Liver Panel ; Bone Markers ; Alkaline Phosphatase Isoenzymes; Bone Specific ALP All content on Lab ...

  14. 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.

  15. 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.

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

    SciTech Connect

    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.

  17. Optimization of pretreatment and fermentation conditions for production of extracellular cellulase complex using sugarcane bagasse

    PubMed Central

    Ashfaque, Mohammad; Solomon, Sushil; Pathak, Neelam

    2014-01-01

    Sugarcane bagasse (SCB), a lignocellulosic byproduct of juice extraction from sugarcane, is rich in cellulose (40-42%). This could be used as a substrate for the production of cellulase complex. Fermentation conditions were optimized for production of cellulase complex (CMCase, Cellulobiase and FPase) by wild type Trichoderma sp. using sugarcane bagasse as sole carbon source. Alkaline treatment (2% NaOH) of bagasse (AlSCB) was found suitable for the production of reducing sugar over the acidic pretreatment method. After 5 days of incubation period, 5% substrate concentration at pH 5.0 and 400C resulted in maximum production of CMCase (0.622 U), while maximum (3.388 U) production of cellulobiase was obtained at 300C. The CMCase was precipitated and purified to the extent of 59.06 fold by affinity chromatography with 49.09% recovery. On 12% SDS-PAGE, a single band corresponding to 33 kDa was observed. The Km and Vmax for CMCase from Trichoderma was found 507.04 mg/ml and 65.32 mM/min, respectively. The enzyme exhibited maximum activity at 300C at pH-5.0 (0.363 U) and was stable over range of 20-60°C and pH 5.0-7.5. PMID:25489168

  18. Fungal cellulase is an elicitor but its enzymatic activity is not required for its elicitor activity.

    PubMed

    Ma, Yanan; Han, Chao; Chen, Jinyin; Li, Haiyun; He, Kun; Liu, Aixin; Li, Duochuan

    2015-01-01

    Plant-pathogenic fungi produce cellulases. However, little information is available on cellulase as an elicitor in plant-pathogen interactions. Here, an endocellulase (EG1) was isolated from Rhizoctonia solani. It contains a putative protein of 227 amino acids with a signal peptide and a family-45 glycosyl hydrolase domain. Its aspartic acid (Asp) residue at position 32 was changed to alanine (Ala), resulting in full loss of its catalytic activity. Wild-type and mutated forms of the endoglucanase were expressed in yeast and purified to homogeneity. The purified wild-type and mutant forms induced cell death in maize, tobacco and Arabidopsis leaves, and the transcription of three defence marker genes in maize and tobacco and 10 genes related to defence responses in maize. Moreover, they also induced the accumulation of reactive oxygen species (ROS), medium alkalinization, Ca(2+) accumulation and ethylene biosynthesis of suspension-cultured tobacco cells. Similarly, production of the EG1 wild-type and mutated forms in tobacco induced cell death using the Potato virus X (PVX) expression system. In vivo, expression of EG1 was also related to cell death during infection of maize by R. solani. These results provide direct evidence that the endoglucanase is an elicitor, but its enzymatic activity is not required for its elicitor activity.

  19. Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae).

    PubMed

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

    2011-02-01

    The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-β-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-β-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.

  20. Solid-State Fermentation with Trichoderma reesei for Cellulase Production

    PubMed Central

    Chahal, D. S.

    1985-01-01

    Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases. PMID:16346697

  1. 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. PMID:22870287

  2. Solid-state fermentation with Trichoderma reesei for cellulase production

    SciTech Connect

    Chahal, D.S.

    1985-01-01

    Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of Trichoderma reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), ..beta..-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, ..beta..-glucosidase, and xylanases. 29 references.

  3. Cytochemical localization of cellulases in decayed and nondecayed wood

    SciTech Connect

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

    1987-01-01

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

  4. Cellulases released during the germination of Dictyostelium discoideum spores.

    PubMed Central

    Jones, T H; de Renobales, M; Pon, N

    1979-01-01

    Dormant spores of Dictyostelium discoideum contained cellulase at a specific activity of 130 to 140 U/mg of protein; when heat activated, the spores germinated, progressively releasing the cellulase activity into the extracellular medium. The cellulase release was a selective process and resulted in recovery of the cellulase activity at a specific activity of 2,000 U/mg of protein; beta-glucosidase in the spores remained completely associated with the emerging amoebae. Release of the cellulase required heat activation of the spores and occurred during the swelling stage of germination; inhibition of the emergence stage with cycloheximide had no effect on the release of the cellulase. The cellulase activity released consisted of two enzymes whose molecular weights were 136,000 and 69,000. Studies of their pH optima, heat lability, and of their sensitivity to inhibition revealed no distinctive differences between these two proteins. Analysis on diethylaminoethyl-Sephadex columns showed that the higher-molecular-weight protein could be converted into the lower-molecular-weight component in vitro. PMID:33962

  5. 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.

  6. Properties of cellulase immobilized on agarose gel with spacer

    SciTech Connect

    Chim-anage, P.; Kashiwagi, Y.; Magae, Y.; Ohta, T.; Sasaki, T.

    1986-12-01

    Cellulase produced by fungus Trichoderma viride was immobilized on agarose beads (Sepharose 4B) activated by cyanogen bromide and also on activated agarose beads that contained spacer arm (activated Ch-Sepharose 4B and Affi-Gel 15). The CMCase activity retained by immobilized cellulase on activated Sepharose containing the spacer tended to be higher than that immobilized without spacer, although the extent of protein immobilization was lower. Also, the higher substrate specificity for cellulase immobilized on beads with spacer was obtained for cellobiose, acid-swollen cellulose, or cellulose powder. The hydrolysis product from their substrates was mainly glucose. 10 references.

  7. Cellulase production by the anaerobic digestion process

    SciTech Connect

    Khan, A.W.; van den Berg, L.

    1981-01-01

    An anaerobic digestion process is described for the production of cellulolytic enzymes using a methanogenic cellulose-enrichment culture. After a heat treatment designed to destroy all but spore-forming bacteria, this culture produced cellulase from a variety of cellulosic materials as well as from cellobiose. The enzyme system contained endo- and exoglucanase, acted on filter paper, and showed cellobiase and xylanase activities. It was stable at 2/sup 0/C under aerobic conditions and showed a pH optimum at 5 and a temperature optimum at 50/sup 0/C. Endoglucanase and filter paper activities were mostly exogenic, whereas cellobiase and xylanase activities were cell associated. The cellulolytic activity produced by this mixed culture was comparable to that of commercially available fungal preparations, and the process could be useful as an alternate source for these enzymes.

  8. 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.

  9. Cellulases: ambiguous non-homologous enzymes in a genomic perspective

    PubMed Central

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

    2015-01-01

    The key material for bioethanol production is cellulose, one of the main components of the plant cell wall. Enzymatic depolymerization of cellulose, an essential step in bioethanol production, can be accomplished by fungal and bacterial cellulases. Most of the biochemically characterized bacterial cellulases come from only a few of cellulose degrading bacteria thus limiting our knowledge of a range of cellulolytic activities that exist in nature. The recent explosion of genomic data offers a unique opportunity to search for novel cellulolytic activities; however, the absence of clear understanding of structural and functional features that are important for reliable computational identification of cellulases precludes their exploration in the genomic datasets. Here we explore the diversity of cellulases and propose a genomic approach to overcome this bottleneck. PMID:21683463

  10. Secretion of cellulase - a key role in production

    SciTech Connect

    Montenecourt, B.S.; Sheir-Neiss, G.I.; Ghosh, A.; Ghosh, B.K.; Frein, E.M.; Eveleigh, D.E.

    1981-01-01

    A mutant strain, Rut-C30, was compared to wild-type Trichoderma reesei to investigate differences that might explain the observed difference in cellulase secretion between the two. 21 references, 1 figure, 1 table. (ACR)

  11. Cellulases: ambiguous nonhomologous enzymes in a genomic perspective

    SciTech Connect

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

    2011-01-01

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

  12. 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. PMID:27154570

  13. 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.

  14. Cellulase from Ruminococcus albus and mixed rumen microorganisms.

    PubMed

    Leatherwood, J M

    1965-09-01

    Cellulase in the cultural filtrates of Ruminococcus albus and cellulase extracted from mixed rumen microorganisms were investigated with acid-swollen cellulose and carboxymethylcellulose as substrates. Maximal activity occurred at approximately pH 5.8 and 47 C. Apparent Michaelis constants (K(m)) varied between 0.53 and 0.02% carboxymethylcellulose, depending on the level of activity and the method of assay. R. albus cellulase has a lower K(m) value than the enzyme extracted from mixed rumen microorganisms. Antisera from rabbits immunized with a cellulase preparation from R. albus inhibited the cellulolytic activity of both systems. Based on the relative degree of inhibition, approximately 20% of the cellulase of the mixed rumen microorganisms was immunologically similar to R. albus cellulase. Ratios of activity in different assay techniques showed the two sources of activity to be similar in the mechanisms of degradation. However, glucose is the main product of cellulose degradation by mixed rumen microorganisms, and cellobiose is the product of degradation by R. albus.

  15. Automated filter paper assay for determination of cellulase activity.

    PubMed

    Decker, Stephen R; Adney, William S; Jennings, Edward; Vinzant, Todd B; Himmel, Michael E

    2003-01-01

    Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in beta-lactamase and green fluorescent protein (GFP). Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and beta-lactamase. Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts. Furthermore, traditional cellulase assays are tedious and time-consuming. Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties. Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error. Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.

  16. Specific quantification of Trichoderma reesei cellulases in reconstituted mixtures and its application to cellulase-cellulose binding studies

    SciTech Connect

    Nidetzky, B. . Inst. of Food Technology Technical Univ. of Graz . Inst. of Biotechnology); Claeyssens, M. . Dept. of Biochemistry, Physiology, and Microbiology)

    1994-10-01

    Specific quantification of the major cellulolytic components of the Trichoderma reesei enzyme complex, i.e., endoglucanases I and III and cellobiohydrolases I and II, are described and, employing a defined mixture of these four cellulases reconstituted according to the composition of the native Trichoderma cellulase complex, used to determine the binding of each individual component onto filter paper. During substrate degradation by this enzyme mixture, the specific adsorption of each individual cellulase gradually increases and no preferential binding of one enzyme component in any particular phase of cellulose hydrolysis is found. T. reesei cellobiohydrolases I and II admixed with endoglucanases I and II represent a full-value'' cellulase system that is capable of degrading semicrystalline cellulose efficiently. In comparison with crude Trichoderma enzyme complex, almost identical adsorption properties and similar hydrolytic efficiency are found for the reconstituted mixture.

  17. 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. PMID:27309759

  18. Anodes for alkaline electrolysis

    DOEpatents

    Soloveichik, Grigorii Lev

    2011-02-01

    A method of making an anode for alkaline electrolysis cells includes adsorption of precursor material on a carbonaceous material, conversion of the precursor material to hydroxide form and conversion of precursor material from hydroxide form to oxy-hydroxide form within the alkaline electrolysis cell.

  19. Alkaline "Permanent" Paper.

    ERIC Educational Resources Information Center

    Pacey, Antony

    1991-01-01

    Discussion of paper manufacturing processes and their effects on library materials focuses on the promotion of alkaline "permanent" paper, with less acid, by Canadian library preservation specialists. Standards for paper acidity are explained; advantages of alkaline paper are described, including decreased manufacturing costs; and recyclability is…

  20. Effect of Lignin Removal by Alkaline Peroxide Pretreatment on the Susceptibility of Corn Stover to Purified Cellulolytic and Xylanolytic Enzymes

    SciTech Connect

    Selig, M. J.; Vinzant, T. B.; Himmel, M. E.; Decker, S. R.

    2009-01-01

    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.

  1. 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.

  2. Processive and nonprocessive cellulases for biofuel production--lessons from bacterial genomes and structural analysis.

    PubMed

    Wilson, David B

    2012-01-01

    Cellulases are key enzymes used in many processes for producing liquid fuels from biomass. Currently there many efforts to reduce the cost of cellulases using both structural approaches to improve the properties of individual cellulases and genomic approaches to identify new cellulases as well as other proteins that increase the activity of cellulases in degrading pretreated biomass materials. Fungal GH-61 proteins are important new enzymes that increase the activity of current commercial cellulases leading to lower total protein loading and thus lower cost. Recent work has greatly increased our knowledge of these novel enzymes that appear to be oxido-reductases that target crystalline cellulose and increase its accessibility to cellulases. They appear to carry out the C1 activity originally proposed by Dr Reese. Cellobiose dehydrogenase appears to interact with GH-61 proteins in this function, providing a role for this puzzling enzyme. Cellulase research is making considerable progress and appears to be poised for even greater advances.

  3. Competitive adsorption of cellulase components and its significance in a synergistic mechanism

    SciTech Connect

    Ryu, D.D.Y.; Kim, C.; Mandels, M.

    1984-05-01

    Some studies on the adsorption of cellulase on cellulose revealed part of the mechanisms involved in the enzymatic hydrolysis of cellulose and provided some clues to the synergistic mechanism of cellulase complex. The adsorption of cellulase was significantly affected by the reaction conditions and physical chemical characteristics of cellulose. Endoglucanase consisted of adsorbable and nonadsorbable components. Cellobiohydrolase had the strongest adsorption affinity. Each cellulase component is postulated to have distinctly different adsorption sites on cellulose, corresponding to the active sites in the hydrolysis reaction. Competitive adsorption kinetics between cellulase components were also observed during the adsorption process. The degree of competitive adsorption was most remarkable when the composition of cellulase components was nearly the same as that in the crude cellulase complex. This seems to show the optimal relative composition of cellulase components. The synergism between cellobiohydrolase and endoglucananse could be elucidated more clearly by this competitive adsorption model of the reaction mechanism.

  4. 21 CFR 173.120 - Carbohydrase and cellulase derived from Aspergillus niger.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  5. Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels.

    PubMed

    Greene, Eric R; Himmel, Michael E; Beckham, Gregg T; Tan, Zhongping

    2015-01-01

    Cellulose in plant cell walls is the largest reservoir of renewable carbon on Earth. The saccharification of cellulose from plant biomass into soluble sugars can be achieved using fungal and bacterial cellulolytic enzymes, cellulases, and further converted into fuels and chemicals. Most fungal cellulases are both N- and O-glycosylated in their native form, yet the consequences of glycosylation on activity and structure are not fully understood. Studying protein glycosylation is challenging as glycans are extremely heterogeneous, stereochemically complex, and glycosylation is not under direct genetic control. Despite these limitations, many studies have begun to unveil the role of cellulase glycosylation, especially in the industrially relevant cellobiohydrolase from Trichoderma reesei, Cel7A. Glycosylation confers many beneficial properties to cellulases including enhanced activity, thermal and proteolytic stability, and structural stabilization. However, glycosylation must be controlled carefully as such positive effects can be dampened or reversed. Encouragingly, methods for the manipulation of glycan structures have been recently reported that employ genetic tuning of glycan-active enzymes expressed from homogeneous and heterologous fungal hosts. Taken together, these studies have enabled new strategies for the exploitation of protein glycosylation for the production of enhanced cellulases for biofuel production. PMID:26613815

  6. Cellulase production by a solid state culture system

    SciTech Connect

    Kim, J.H.; Hosobuchi, M.; Kishimoto, M.; Seki, T.; Yoshida, T.; Taguchi, H.; Ryu, D.D.Y.

    1985-10-01

    Production of cellulase using solid culture systems of Trichoderma reesei QM9414 and Sporotrichum cellulophilum on wheat bran was studied. By using moisture-controlled solid culture equipment, the effect of water content of wheat bran on cell growth and cellulase production was investigated. Cellular biomass grown on solid substrate was estimated by measuring oxygen consumption rate and glucosamine content the cells. These parameters were shown to have a good linear correlation with the specific growth rate. This reliable method of estimating the cell growth rate enabled us to simulate the enzyme production in a solid culture system by means of multiple linear regression analysis which takes into account of the water content, cell mass, and the oxygen consumption rate as variables. The cell growth and cellulase production were maximized at different water content of the medium. A high water content, 57% for T. reesei and 70% for S. cellulophilum, favored mycelial growth, while the maximum cellulase activity was obtained at a lower water content such as 50% for both fungi. It was observed that cellulase production by T. reesei depended on the culture conditions that support the optimal growth rate for the maximum enzyme production. 14 references.

  7. Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels.

    PubMed

    Greene, Eric R; Himmel, Michael E; Beckham, Gregg T; Tan, Zhongping

    2015-01-01

    Cellulose in plant cell walls is the largest reservoir of renewable carbon on Earth. The saccharification of cellulose from plant biomass into soluble sugars can be achieved using fungal and bacterial cellulolytic enzymes, cellulases, and further converted into fuels and chemicals. Most fungal cellulases are both N- and O-glycosylated in their native form, yet the consequences of glycosylation on activity and structure are not fully understood. Studying protein glycosylation is challenging as glycans are extremely heterogeneous, stereochemically complex, and glycosylation is not under direct genetic control. Despite these limitations, many studies have begun to unveil the role of cellulase glycosylation, especially in the industrially relevant cellobiohydrolase from Trichoderma reesei, Cel7A. Glycosylation confers many beneficial properties to cellulases including enhanced activity, thermal and proteolytic stability, and structural stabilization. However, glycosylation must be controlled carefully as such positive effects can be dampened or reversed. Encouragingly, methods for the manipulation of glycan structures have been recently reported that employ genetic tuning of glycan-active enzymes expressed from homogeneous and heterologous fungal hosts. Taken together, these studies have enabled new strategies for the exploitation of protein glycosylation for the production of enhanced cellulases for biofuel production.

  8. Alkaline battery operational methodology

    DOEpatents

    Sholklapper, Tal; Gallaway, Joshua; Steingart, Daniel; Ingale, Nilesh; Nyce, Michael

    2016-08-16

    Methods of using specific operational charge and discharge parameters to extend the life of alkaline batteries are disclosed. The methods can be used with any commercial primary or secondary alkaline battery, as well as with newer alkaline battery designs, including batteries with flowing electrolyte. The methods include cycling batteries within a narrow operating voltage window, with minimum and maximum cut-off voltages that are set based on battery characteristics and environmental conditions. The narrow voltage window decreases available capacity but allows the batteries to be cycled for hundreds or thousands of times.

  9. 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.

  10. Possibilities for recycling cellulases after use in cotton processing: part II: Separation of cellulases from reaction products and released dyestuffs by ultrafiltration.

    PubMed

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

    2002-04-01

    The adsorption and activity of a total cellulase (Trichoderma reesei) was measured and compared on undyed and dyed cotton fabrics. Recovery of enzymes from the reaction mixture and by desorption from the cotton substrate was evaluated. About 80% of the initial protein could be recovered. The removal of released products (soluble reducing sugars and dyes) from the treatment liquor and subsequent concentration of cellulase proteins was performed using an ultrafiltration membrane. Strong protein-dye interactions made it impossible to separate efficiently the dyes from the enzyme-containing treatment liquors. The use of surfactants did not enhance cellulase desorption from cotton fabric. Although anionic surfactants have a deactivating effect on cellulases, this effect seems to be reversible, since after ultrafiltration the cellulase activity was similar to that of enzymes desorbed with buffer only. Humicola insolens cellulases were shown to be much more sensitive to anionic surfactant than T. reesei cellulases. The use of cellulases that bind reversibly to cellulose is suggested for achieving more efficient cellulase recycling and for reducing backstaining by dye-cellulase complexes.

  11. 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.

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Cellulase enzyme preparation derived from....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase...

  17. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Cellulase enzyme preparation derived from....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase...

  18. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Cellulase enzyme preparation derived from....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase...

  19. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Cellulase enzyme preparation derived from....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase...

  20. Carboxymethyl cellulase and cellobiase production by Clostridiumacetobutylicum in an industrial fermentation medium

    SciTech Connect

    Allcock, E.R.; Woods, D.R.

    1981-02-01

    The production of a carboxymethyl cellulase and a cellobiase by Clostridium acetobutylicum was demonstrated. In liquid medium the carboxymethyl cellulase was induced by molasses, and it was not repressed by glucose. Optimum carboxymethyl cellulase activity occurred at pH 4.6 and 37 degrees C. (Refs. 12).

  1. Carboxymethyl cellulase and cellobiase production by Clostridium acetobutylicum in an industrial fermentation medium

    SciTech Connect

    Allock, E.R.; Woods, D.R.

    1981-02-01

    The production of a carboxymethyl cellulase and a cellobiase by Clostridium acetobutylicum was demonstrated. In liquid medium the carboxymethyl cellulase was induced by molasses, and it was not repressed by glucose. Optimum carboxymethyl cellulase activity occurred at pH 4.6 and 37/sup 0/C.

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

    SciTech Connect

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

    1993-10-01

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

  3. Adsorption of cellulase from Trichoderma viride on cellulose

    SciTech Connect

    Ooshima, H.; Sakata, M.; Harano, Y.

    1983-12-01

    The adsorption of cellulase from Trichoderma viride (Meicelase CEP) on the surface of pure cellulose was studied. The adsorption was found to obey apparently the Langmuir isotherm. From the data concerning the effects of temperature and the crystallinity of cellulose on the Langmuir adsorption parameters, the characteristics of the adsorption of the individual cellulase components, namely CMCase (endoglucanase) and Avicelase (exoglucanase), were discussed. While beta-glucosidase also adsorbed on the surface of cellulose at 5 degrees C, it did not at 50 degrees C. (Refs. 27).

  4. 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.

  5. Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei.

    PubMed

    Wang, Min; Lu, Xuefeng

    2016-01-01

    Recent demands for the production of lignocellulose biofuels boosted research on cellulase. Hydrolysis efficiency and production cost of cellulase are two bottlenecks in "biomass to biofuels" process. The Trichoderma cellulase mixture is one of the most commonly used enzymes for cellulosic hydrolysis. During hydrolytic process cellobiose accumulation causes feedback inhibition against most cellobiohydrolases and endoglucanases. In this study, we demonstrated the synergism effects between cellobiose dehydrogenase (CDH) and cellulase both in vitro and in vivo. The CDH from Phanerochaete chrysosporium was heterologously expressed in Pichia pastoris. Supplementation of the purified CDH in Trichoderma cellulase increased the cellulase activities. Especially β-glucosidase activity was increased by 30-100% varying at different time points. On the other hand, the cdh gene was heterologously expressed in Trichoderma reesei to explore the synergism between CDH and cellulases in vivo. The analyses of gene expression and enzymatic profiles of filter paper activity, carboxymethylcellulase (CMCase) and β-glucosidase show the increased cellulase activity and the enhanced cellulase production in the cdh-expressing strains. The results elucidate a possible mechanism for diminishing the cellobiose inhibition of cellulase by CDH. These findings provide a novel perspective to make more economic enzyme cocktails for commercial application or explore alternative strategies for generating cellulase-producing strains with higher efficiency. PMID:27199949

  6. Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei.

    PubMed

    Wang, Min; Lu, Xuefeng

    2016-01-01

    Recent demands for the production of lignocellulose biofuels boosted research on cellulase. Hydrolysis efficiency and production cost of cellulase are two bottlenecks in "biomass to biofuels" process. The Trichoderma cellulase mixture is one of the most commonly used enzymes for cellulosic hydrolysis. During hydrolytic process cellobiose accumulation causes feedback inhibition against most cellobiohydrolases and endoglucanases. In this study, we demonstrated the synergism effects between cellobiose dehydrogenase (CDH) and cellulase both in vitro and in vivo. The CDH from Phanerochaete chrysosporium was heterologously expressed in Pichia pastoris. Supplementation of the purified CDH in Trichoderma cellulase increased the cellulase activities. Especially β-glucosidase activity was increased by 30-100% varying at different time points. On the other hand, the cdh gene was heterologously expressed in Trichoderma reesei to explore the synergism between CDH and cellulases in vivo. The analyses of gene expression and enzymatic profiles of filter paper activity, carboxymethylcellulase (CMCase) and β-glucosidase show the increased cellulase activity and the enhanced cellulase production in the cdh-expressing strains. The results elucidate a possible mechanism for diminishing the cellobiose inhibition of cellulase by CDH. These findings provide a novel perspective to make more economic enzyme cocktails for commercial application or explore alternative strategies for generating cellulase-producing strains with higher efficiency.

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

    PubMed

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

    2012-02-01

    Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, however, often disturbed by the concomitant production of other endogenous cellulases. As a useful alternative, we tested the production of cellulases in T. reesei in a genetic background where cellulase formation has been impaired by deletion of the major cellulase transcriptional activator gene xyr1. Three cellulase genes (cel7a, cel7b, and cel12a) were expressed under the promoter regions of the two highly expressed genes tef1 (encoding translation elongation factor 1-alpha) or cdna1 (encoding the hypothetical protein Trire2:110879). When cultivated on D: -glucose as carbon source, the Δxyr1 strain secreted all three cellulases into the medium. Related to the introduced gene copy number, the cdna1 promoter appeared to be superior to the tef1 promoter. No signs of proteolysis were detected, and the individual cellulases could be assayed over a background essentially free of other cellulases. Hence this system can be used as a vehicle for rapid and high-throughput testing of cellulase muteins in a homologous background.

  8. Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei

    PubMed Central

    Wang, Min; Lu, Xuefeng

    2016-01-01

    Recent demands for the production of lignocellulose biofuels boosted research on cellulase. Hydrolysis efficiency and production cost of cellulase are two bottlenecks in “biomass to biofuels” process. The Trichoderma cellulase mixture is one of the most commonly used enzymes for cellulosic hydrolysis. During hydrolytic process cellobiose accumulation causes feedback inhibition against most cellobiohydrolases and endoglucanases. In this study, we demonstrated the synergism effects between cellobiose dehydrogenase (CDH) and cellulase both in vitro and in vivo. The CDH from Phanerochaete chrysosporium was heterologously expressed in Pichia pastoris. Supplementation of the purified CDH in Trichoderma cellulase increased the cellulase activities. Especially β-glucosidase activity was increased by 30–100% varying at different time points. On the other hand, the cdh gene was heterologously expressed in Trichoderma reesei to explore the synergism between CDH and cellulases in vivo. The analyses of gene expression and enzymatic profiles of filter paper activity, carboxymethylcellulase (CMCase) and β-glucosidase show the increased cellulase activity and the enhanced cellulase production in the cdh-expressing strains. The results elucidate a possible mechanism for diminishing the cellobiose inhibition of cellulase by CDH. These findings provide a novel perspective to make more economic enzyme cocktails for commercial application or explore alternative strategies for generating cellulase-producing strains with higher efficiency. PMID:27199949

  9. 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.

  10. 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.

  11. CBH1 homologs and varian CBH1 cellulase

    SciTech Connect

    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.

  12. CBH1 homologs and variant CBH1 cellulases

    DOEpatents

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

    2008-11-18

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

  13. Genetics and physiology of secretion of cellulase by Trichoderma

    SciTech Connect

    Montenecourt, B.S.

    1983-01-01

    In an effort to improve the enzymatic saccharification of cellulose to glucose, a number of mutant strains of Trichoderma reesei have been isolated which show enhanced production of the cellulose complex. Two of these mutants, Rut-C30 and RL-P37, were studied in relation to their complement of cellulases and their secretory mechanisms. 22 reference, 2 figures. (ACR)

  14. CBH1 homologs and variant CBH1 cellulases

    DOEpatents

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

    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.

  15. Immobilization of cellulase and cellobiase by radiation-induced polymerization

    NASA Astrophysics Data System (ADS)

    Higa, O. Z.; del Mastro, N. L.; Castagnet, A. C.

    An application of irradiation technology for the immobilization of cellulase and cellobiase using hydrophilic glass-forming monomers was performed. Some experiments to observe the effect of additives as silicates and polyethylene glycol in the enzyme entrappment are reported. In all the cases, the enzymatic activity was maintained by more than fifteen batch enzyme rea c̵ctions.

  16. 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

  17. 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.

  18. Sequence, Structure, and Evolution of Cellulases in Glycoside Hydrolase Family 48*

    PubMed Central

    Sukharnikov, Leonid O.; Alahuhta, Markus; Brunecky, Roman; Upadhyay, Amit; Himmel, Michael E.; Lunin, Vladimir V.; Zhulin, Igor B.

    2012-01-01

    Currently, the cost of cellulase enzymes remains a key economic impediment to commercialization of biofuels (1). Enzymes from glycoside hydrolase family 48 (GH48) are a critical component of numerous natural lignocellulose-degrading systems. Although computational mining of large genomic data sets is a promising new approach for identifying novel cellulolytic activities, current computational methods are unable to distinguish between cellulases and enzymes with different substrate specificities that belong to the same protein family. We show that by using a robust computational approach supported by experimental studies, cellulases and non-cellulases can be effectively identified within a given protein family. Phylogenetic analysis of GH48 showed non-monophyletic distribution, an indication of horizontal gene transfer. Enzymatic function of GH48 proteins coded by horizontally transferred genes was verified experimentally, which confirmed that these proteins are cellulases. Computational and structural studies of GH48 enzymes identified structural elements that define cellulases and can be used to computationally distinguish them from non-cellulases. We propose that the structural element that can be used for in silico discrimination between cellulases and non-cellulases belonging to GH48 is an ω-loop located on the surface of the molecule and characterized by highly conserved rare amino acids. These markers were used to screen metagenomics data for “true” cellulases. PMID:23055526

  19. 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.

  20. 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.

  1. Structural characterization of carbohydrate attached to the glycoprotein cellulase enzymes of Trichoderma reesei QM 9414

    SciTech Connect

    de Chasteigner du Mee, C.P.R.

    1984-01-01

    Cellobiohydrolases I(D) and II were purified by ion exchange chromatography from an extracellular culture filtrate of Tricoderma reesei QM 9414. Neutral sugar composition of each was determined by gas-liquid chromatographic analysis of the peracetylated alditol and aldononitrile acetate derivatives of sugars released by either reductive US -elimination or acid hydrolysis. The alkaline borohydride-released carbohydrate was analyzed by high-pressure liquid chromatography (HPLC). The HPLC data demonstrated that each molecule of cellobiohydrolase I(D) contained 5.9% carbohydrate and that cellobiohydrolase II contained 18.9% carbohydrate. The purified oligosaccharides were shown by methylation analysis to contain (1-2) and (1-6) glycosidic linkages and the position of the 6-substituted residues was confirmed by acetolysis. The sequence and anomeric nature of the sugar residues in each oligosaccharide was determined by sequential glycosidase digestion, and all the residues were found to be -linked. Proton decoupled TC-NMR analysis suggested that for cellobiohydrolase I(D), each oligosaccharide was attached to a threonyl residue on the polypeptide and for cellobiohydrolase II, each oligosaccharide was attached to threonyl and seryl residues on the polypeptides. Coupled TC-NMR and H-NMR data to define the structures of the oligosaccharides attached to the enzyme were determined and are reported. Thus, work with the cellobiohydrolases and the endoglucanases indicated that the predominant cellulase enzymes secreted by T. reesei QM 9414 are each glycosylated with similar oligosaccharides.

  2. Biodegradation of wastepaper by cellulase from Trichoderma viride.

    PubMed

    van Wyk, J P H; Mohulatsi, M

    2003-01-01

    Environmental issues such as the depletion of non-renewable energy resources and pollution are topical. The extent of solid waste production is of global concern and development of its bioenergy potential can combine issues such as pollution control and bioproduct development, simultaneously. Various wastepaper materials, a major component of solid waste, were treated with the cellulase enzyme from Trichoderma viride, thus bioconverting their cellulose component into fermentable sugars. All wastepaper materials exhibited different susceptibilities towards the cellulase as well as the production of non-similar sugar releasing patterns when increasing amounts of paper were treated with a fixed enzyme concentration. The hydrolysis of wastepaper with changing enzyme concentrations and incubation periods also resulted in dissimilar sugar-producing tendencies. A general decline in hydrolytic efficiency was observed when increasing sugar concentrations were produced during biodegradation of all wastepaper materials. PMID:12421003

  3. Comparative activity profiles of Thielavia terrestris and Trichoderma reesei cellulases

    SciTech Connect

    Tuse, D.; Mason, B.J.; Skinner, W.A.

    1980-10-01

    The successful utilization of cellulosic materials depends on the development of economically feasible processes for the literation of low molecular weight soluble products from the polymers. These soluble products, such as hexoses and pentoses, can then be utilized as substrates for the microbial or chemical product of fuels, food, and chemical feedstocks. In the enzymatic saccharification of cellulose, one of the major roadblocks to the development of commercially attractive processes has been the instability of the cellulase complex. It is desirable, for example, to operate the conversion systems at elevated temperatures, but environments with high thermal energy can significantly shorten enzyme half life. The authors have isolated a strain of the fungus Thielavia terrestris that possesses a complete cellulase system, and its enzymes were found to have remarkable thermal stability. The author presents a comparison of the activities of the T. terrestris enzymes with those of Trichoderma reesei.

  4. Advances in improving the performance of cellulase in ionic liquids for lignocellulose biorefinery.

    PubMed

    Xu, Jiaxing; Xiong, Peng; He, Bingfang

    2016-01-01

    Ionic liquids (ILs) have been considered as a class of promising solvents that can dissolve lignocellulosic biomass and then provide enzymatic hydrolyzable holocellulose. However, most of available cellulases are completely or partially inactivated in the presence of even low concentrations of ILs. To more fully exploit the benefits of ILs to lignocellulose biorefinery, it is critical to improve the compatibility between cellulase and ILs. Various attempts have been made to screen natural IL-tolerant cellulases from different microhabitats. Several physical and chemical methods for stabilizing cellulases in ILs were also developed. Moreover, recent advances in protein engineering have greatly facilitated the rational engineering of cellulases by site-directed mutagenesis for the IL stability. This review is aimed to provide the first detailed overview of the current advances in improving the performance of cellulase in non-natural IL environments. New ideas from the most representative progresses and technical challenges will be summarized and discussed.

  5. 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.

  6. Advances in improving the performance of cellulase in ionic liquids for lignocellulose biorefinery.

    PubMed

    Xu, Jiaxing; Xiong, Peng; He, Bingfang

    2016-01-01

    Ionic liquids (ILs) have been considered as a class of promising solvents that can dissolve lignocellulosic biomass and then provide enzymatic hydrolyzable holocellulose. However, most of available cellulases are completely or partially inactivated in the presence of even low concentrations of ILs. To more fully exploit the benefits of ILs to lignocellulose biorefinery, it is critical to improve the compatibility between cellulase and ILs. Various attempts have been made to screen natural IL-tolerant cellulases from different microhabitats. Several physical and chemical methods for stabilizing cellulases in ILs were also developed. Moreover, recent advances in protein engineering have greatly facilitated the rational engineering of cellulases by site-directed mutagenesis for the IL stability. This review is aimed to provide the first detailed overview of the current advances in improving the performance of cellulase in non-natural IL environments. New ideas from the most representative progresses and technical challenges will be summarized and discussed. PMID:26602145

  7. 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.

  8. 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. PMID:25346145

  9. Alkaline quinone flow battery.

    PubMed

    Lin, Kaixiang; Chen, Qing; Gerhardt, Michael R; Tong, Liuchuan; Kim, Sang Bok; Eisenach, Louise; Valle, Alvaro W; Hardee, David; Gordon, Roy G; Aziz, Michael J; Marshak, Michael P

    2015-09-25

    Storage of photovoltaic and wind electricity in batteries could solve the mismatch problem between the intermittent supply of these renewable resources and variable demand. Flow batteries permit more economical long-duration discharge than solid-electrode batteries by using liquid electrolytes stored outside of the battery. We report an alkaline flow battery based on redox-active organic molecules that are composed entirely of Earth-abundant elements and are nontoxic, nonflammable, and safe for use in residential and commercial environments. The battery operates efficiently with high power density near room temperature. These results demonstrate the stability and performance of redox-active organic molecules in alkaline flow batteries, potentially enabling cost-effective stationary storage of renewable energy. PMID:26404834

  10. Alkaline quinone flow battery.

    PubMed

    Lin, Kaixiang; Chen, Qing; Gerhardt, Michael R; Tong, Liuchuan; Kim, Sang Bok; Eisenach, Louise; Valle, Alvaro W; Hardee, David; Gordon, Roy G; Aziz, Michael J; Marshak, Michael P

    2015-09-25

    Storage of photovoltaic and wind electricity in batteries could solve the mismatch problem between the intermittent supply of these renewable resources and variable demand. Flow batteries permit more economical long-duration discharge than solid-electrode batteries by using liquid electrolytes stored outside of the battery. We report an alkaline flow battery based on redox-active organic molecules that are composed entirely of Earth-abundant elements and are nontoxic, nonflammable, and safe for use in residential and commercial environments. The battery operates efficiently with high power density near room temperature. These results demonstrate the stability and performance of redox-active organic molecules in alkaline flow batteries, potentially enabling cost-effective stationary storage of renewable energy.

  11. Assessment of field-grown cellulase-expressing corn.

    PubMed

    Garda, Martina; Devaiah, Shivakumar P; Vicuna Requesens, Deborah; Chang, Yeun-Kyung; Dabul, Audrei; Hanson, Christy; Hood, Kendall R; Hood, Elizabeth E

    2015-04-01

    Transgenic plants in the US and abroad generated using genetic engineering technology are regulated with respect to release into the environment and inclusion into diets of humans and animals. For crops incorporating pharmaceuticals or industrial enzymes regulations are even more stringent. Notifications are not allowed for movement and release, therefore a permit is required. However, growing under permit is cumbersome and more expensive than open, non- regulated growth. Thus, when the genetically engineered pharmaceutical or industrial crop is ready for scale-up, achieving non-regulated status is critical. Regulatory compliance in the US comprises petitioning the appropriate agencies for permission for environmental release and feeding trials. For release without yearly permits, a petition for allowing non-regulated status can be filed with the United States Department of Agriculture with consultations that include the Food and Drug Administration and possibly the Environmental Protection Agency, the latter if the plant includes an incorporated pesticide. The data package should ensure that the plants are substantially equivalent in every parameter except for the engineered trait. We undertook a preliminary study on transgenic maize field-grown hybrids that express one of two cellulase genes, an exo-cellulase or an endo-cellulase. We performed field observations of whole plants and numerous in vitro analyses of grain. Although some minor differences were observed when comparing genetically engineered hybrid plants to control wild type hybrids, no significant differences were seen.

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

    PubMed

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

    2007-11-01

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

  13. Noncovalent immobilization of cellulases using the reversibly soluble polymers for biopolishing of cotton fabric.

    PubMed

    Yu, Yuanyuan; Yuan, Jiugang; Wang, Qiang; Fan, Xuerong; Wang, Ping; Cui, Li

    2015-01-01

    The hydrolytic reaction of cellulases can occur in the interior of cellulosic fibers, causing tensile strength loss of the fabrics. Cellulase immobilization is an approach to solve this problem, because enlarging the molecule size of cellulases will limit the hydrolysis to the surfaces of the fibers. In this study, commercial cellulases were noncovalently immobilized onto the reversibly soluble polymers (Eudragit S-100 and Eudragit L-100). The characteristics of cellulase-Eudragit S-100 (CES) and cellulase-Eudragit L-100 (CEL) were evaluated using Fourier transform infrared spectra, circular dichroism spectra, and fluorescence spectra. The CES showed higher stability than CEL and free cellulase, especially at higher pH and temperature. CES and CEL retained 51% and 42% of their original activities after three cycles of repeated uses, respectively. In addition, the effects of cellulase treatment on the cotton yarn and fabric have been investigated. The bending stiffness results showed that the cotton fabric samples treated with the free and immobilized cellulases were softer than untreated samples. However, less fiber damage in terms of weight loss and tensile strength of treated cotton was observed.

  14. 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.

  15. 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.

  16. 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.

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

    SciTech Connect

    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{sub 3} {center dot} galactose {center dot} fucose). The same oligosaccharides also promoted the degradation, assayed viscometrically, of xyloglucan by an acidic cellulase from bean (Phaseolus vulgaris L.) leaves. The oligosaccharides were highly active at 10{sup {minus}4} molar, causing up to a fourfold increase in activity, but the effect was still detectable at 1 micromolar. Those oligosaccharides (XG8 and XG9n) which best promoted growth, stimulated cellulase activity to the greatest extent. The oligosaccharides did not stimulate the action of the cellulase in an assay based on the conversion of ({sup 3}H)xyloglucan to ethanol-soluble fragments. This suggests that the oligosaccharides enhanced the midchain hydrolysis of xyloglucan molecules (which would rapidly reduce the viscosity of the solution), at the expense of cleavage near the termini (which would yield ethanol-soluble products).

  18. A cellular automaton model of crystalline cellulose hydrolysis by cellulases

    PubMed Central

    2011-01-01

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

  19. Isolation of cellulase-producing bacteria and characterization of the cellulase from the isolated bacterium Cellulomonas sp. YJ5.

    PubMed

    Yin, Li-Jung; Huang, Po-Shin; Lin, Hsin-Hung

    2010-09-01

    A cellulase-producing bacterium was isolated from soil and identified as Cellulomonas sp. YJ5. Maximal cellulase activity was obtained after 48 h of incubation at 30 degrees C in a medium containing 1.0% carboxymethyl cellulose (CMC), 1.0% algae powder, 1.0% peptone, 0.24% (NH4)2SO4, 0.20% K2HPO4, and 0.03% MgSO(4).7H2O. The cellulase was purified after Sephacryl S-100 chromatography twice with a recovery of 27.9% and purification fold of 17.5. It was, with N-terminal amino acids of AGTKTPVAK, stable at pH 7.5-10.5 and 20-50 degrees C with optimal pH and temperature of 7.0 and 60 degrees C, respectively. Cu2+, Fe2+, Hg2+, Cr3+, and SDS highly inhibited, but cysteine and beta-mercaptoethanol activated, its activity. Substrate specificity indicated it to be an endo-beta-1,4-glucanase.

  20. Pretreatment of cane bagasse with alkaline hydrogen peroxide for enzymatic hydrolysis of cellulose and ethanol fermentation

    SciTech Connect

    Azzam, A.M. )

    1989-01-01

    Pretreatment of the agrocellulosic waste, cane bagasse with alkaline hydrogen peroxide greatly enhances its susceptibility to enzymatic cellulolysis and thus the ethanol production from it. Various process conditions have been studied to optimize the enzymate effectiveness. These conditions include the contact time, the hydrogen peroxide concentration and the pretreatment temperature. Results obtained show, that about 50% of lignin and most of hemicellulose content of can bagasse was solubilized, by 2% alkaline hydrogen peroxide at 30{sup 0}C within 8 h. The cellulose content was consequently increased from 42% in the original cane bagasse to 75% in the oxidized pulp. Saccharification of this pulp residue with cellulase from Trichorderma viride at 45{sup 0}C for 24 h, yielded glucose with 95% efficiency. The efficiency of ethanol production from the insoluble fraction with S. cervisiae was 90% compared to about 50% for untreated cane bagasse.

  1. Alkaline galvanic cell

    SciTech Connect

    Inoue, T.; Maeda, Y.; Momose, K.; Wakahata, T.

    1983-10-04

    An alkaline galvanic cell is disclosed including a container serving for a cathode terminal, a sealing plate in the form of a layered clad plate serving for an anode terminal to be fitted into the container, and an insulating packing provided between the sealing plate and container for sealing the cell upon assembly. The cell is provided with a layer of epoxy adduct polyamide amine having amine valence in the range of 50 to 400 and disposed between the innermost copper layer of the sealing plate arranged to be readily amalgamated and the insulating packing so as to serve as a sealing agent or liquid leakage suppression agent.

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

    PubMed

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

    2003-10-01

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

  3. Alkaline fuel cells applications

    NASA Astrophysics Data System (ADS)

    Kordesch, Karl; Hacker, Viktor; Gsellmann, Josef; Cifrain, Martin; Faleschini, Gottfried; Enzinger, Peter; Fankhauser, Robert; Ortner, Markus; Muhr, Michael; Aronson, Robert R.

    On the world-wide automobile market technical developments are increasingly determined by the dramatic restriction on emissions as well as the regimentation of fuel consumption by legislation. Therefore there is an increasing chance of a completely new technology breakthrough if it offers new opportunities, meeting the requirements of resource preservation and emission restrictions. Fuel cell technology offers the possibility to excel in today's motive power techniques in terms of environmental compatibility, consumer's profit, costs of maintenance and efficiency. The key question is economy. This will be decided by the costs of fuel cell systems if they are to be used as power generators for future electric vehicles. The alkaline hydrogen-air fuel cell system with circulating KOH electrolyte and low-cost catalysed carbon electrodes could be a promising alternative. Based on the experiences of Kordesch [K. Kordesch, Brennstoffbatterien, Springer, Wien, 1984, ISBN 3-387-81819-7; K. Kordesch, City car with H 2-air fuel cell and lead-battery, SAE Paper No. 719015, 6th IECEC, 1971], who operated a city car hybrid vehicle on public roads for 3 years in the early 1970s, improved air electrodes plus new variations of the bipolar stack assembly developed in Graz are investigated. Primary fuel choice will be a major issue until such time as cost-effective, on-board hydrogen storage is developed. Ammonia is an interesting option. The whole system, ammonia dissociator plus alkaline fuel cell (AFC), is characterised by a simple design and high efficiency.

  4. 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.

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

    PubMed

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

    2014-08-01

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

  6. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ....S.C. 552(a) and 1 CFR part 51. Copies are available from the National Academy Press, 2101... 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...

  7. Effective saccharification of kraft pulp by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae.

    PubMed

    Yamada, Ryosuke; Yoshie, Toshihide; Sakai, Shoji; Wakai, Satoshi; Asai-Nakashima, Nanami; Okazaki, Fumiyoshi; Ogino, Chiaki; Hisada, Hiromoto; Tsutsumi, Hiroko; Hata, Yoji; Kondo, Akihiko

    2015-01-01

    Kraft pulp is a promising feedstock for bioproduction. The efficiency of kraft pulp saccharification was improved by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae. Application of the cellulase cocktail was demonstrated by simultaneous saccharification and fermentation, using kraft pulp and non-cellulolytic yeast. Such application would make possible to do an efficient production of other chemicals from kraft pulp.

  8. Complete genome sequence of the cellulase-producing bacterium Clavibacter michiganensis PF008.

    PubMed

    Bae, Chungyun; Oh, Eom-Ji; Lee, Han-Beoyl; Kim, Byung-Yong; Oh, Chang-Sik

    2015-11-20

    The Gram-positive Actinobacterium Clavibacter michiganensis strain PF008 produces a cellulase of biotechnological interest, which is used for degradation of cellulose, a major component of plant cell walls. Here we report the complete genome sequence of this bacterium for better understanding of cellulase production and its virulence mechanism.

  9. 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.

  10. 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. PMID:25935220

  11. 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.

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

    PubMed

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

    2014-08-01

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

  13. 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. PMID:23455222

  14. Novel Penicillium cellulases for total hydrolysis of lignocellulosics.

    PubMed

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

    2013-05-10

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

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

    SciTech Connect

    Gladden, John Michael

    2013-09-01

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

  16. Structure of CBM4 from Clostridium thermocellum cellulase K

    PubMed Central

    Alahuhta, Markus; Luo, Yonghua; Ding, Shi-You; Himmel, Michael E.; Lunin, Vladimir V.

    2011-01-01

    Here, a 2.0 Å resolution X-ray structure of Clostridium thermocellum cellulase K family 4 carbohydrate-binding module (CelK CBM4) is reported. The resulting structure was refined to an R factor of 0.212 and an R free of 0.274. Structural analysis shows that this new structure is very similar to the previously solved structure of C. thermocellum CbhA CBM4. Most importantly, these data support the previously proposed notion of an extended binding pocket using a novel tryptophan-containing loop that may be highly conserved in clostridial CBM4 proteins. PMID:21543854

  17. Adsorption and recovery of cellulases during hydrolysis of newspaper

    SciTech Connect

    Castanon, M.; Wilke, C.R.

    1980-01-01

    The adsorption of cellulases from Trichoderma viride was studied during the hydrolysis of newspaper. By measuring individual enzyme activities it was found that in the early stage of hydrolysis enzyme components showing C/sub x/A were adsorbed preferentially to those showing C/sub 1/A; afterwards this situation was inverted. Electrophoretic resolution of proteins in hydrolysates showed a continuous decrease of enzyme proteins in solution, and furthermore suggested that the enzymes once adsorbed remained immobilized on the substrate (even after extensive digestion). Experiments to recover the enzymes that had remained in solution after typical hydrolysis showed a potential saving of enzyme of up to 40%.

  18. Useful halophilic, thermostable and ionic liquids tolerant cellulases

    DOEpatents

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

    2016-06-28

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

  19. 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.

  20. Fungal Strain Improvement for Cellulase Production Using Repeated and Sequential Mutagenesis

    PubMed Central

    Vu, Van Hanh; Pham, Tuan Anh

    2009-01-01

    A fungal strain producing a high level of cellulase was selected from 320 fungal isolates and identified as Aspergillus sp. This strain was further improved for cellulase production by sequential treatments by two repeated rounds of γ-irradiation of Co60, ultraviolet treatment and four repeated rounds of treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The best mutant strain, Aspergillus sp. XTG-4, was selected after screening and the activities of carboxymethyl cellulase, filter paper cellulase and β-glucosidase of the cellulase were improved by 2.03-, 3.20-, and 1.80-fold, respectively, when compared to the wild type strain. After being subcultured 19 times, the enzyme production of the mutant Aspergillus sp. XTG-4s was stable. PMID:23983546

  1. Daily dynamics of cellulase activity in arable soils depending on management practices

    NASA Astrophysics Data System (ADS)

    Lavrent'eva, E. V.; Semenov, A. M.; Zelenev, V. V.; Chzhun, Yu.; Semenova, E. V.; Semenov, V. M.; Namsaraev, B. B.; van Bruggen, A. H. C.

    2009-08-01

    The daily dynamics of cellulase activity was studied during 27 days by the cellophane membrane method on soils managed using the conventional high-input farming system (application of mineral fertilizers and pesticides) and the biological conservation farming system (application of organic fertilizers alone) in a microfield experiment. The regular oscillatory dynamics of the cellulase activity were revealed and confirmed by the harmonic (Fourier) analysis. The oscillatory dynamics of the cellulase activity had a self-oscillatory nature and was not directly caused by the disturbing impacts of both the uncontrolled (natural) changes in the temperature and moisture (rainfall) and the controlled ones (the application of different fertilizers). The disturbing impacts affected the oscillation amplitude of the cellulase activity but not the frequency (periods) of the oscillations. The periodic oscillations of the cellulase activity were more significant in the soil under the high-input management compared to the soil under the biological farming system.

  2. 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.

  3. Assessing Cellulase Performance on Pretreated Lignocellulosic Biomass Using Saccharification and Fermentation-Based Protocols

    NASA Astrophysics Data System (ADS)

    Dowe, Nancy

    Cellulase enzyme is a key cost component in the production of fuels and chemicals from lignocellulosic biomass. Cellulolytic ability of the enzyme preparation is often measured by activity assays using model substrates such as filter paper. Using lignocellulosic biomass as the substrate to assess enzyme performance has the potential of being more process relevant. We describe two procedures that use washed pretreated cellulosic material to measure the efficacy of cellulase enzymes. First, a saccharification assay that measures glucose yield as a function of the amount of cellulase used in the process. And second, the simultaneous saccharification and fermentation (SSF) assay measures cellulase performance by the amount of ethanol produced from enzymatic hydrolysis of the cellulosic material. You can use both assays to screen cellulases under a variety of substrate types, loadings, and process conditions.

  4. Interaction of cellulase with sodium dodecyl sulfate at critical micelle concentration level.

    PubMed

    Xiang, Jin; Fan, Jun-Bao; Chen, Nan; Chen, Jie; Liang, Yi

    2006-05-01

    The interactions between Trichoderma reesei cellulase and an anionic surfactant, sodium dodecyl sulfate (SDS), at critical micelle concentration level have been investigated using isothermal titration calorimetry, fluorescence spectroscopy, and circular dichroism. SDS micelles have dual interactions with cellulase: electrostatic at first and then hydrophobic interactions. When the concentration of SDS is smaller than 45.0mM, SDS micelles cause a partial loss in the hydrolytic activity together with a steep decrease in the alpha-helical content of cellulase. With further increasing the concentration of SDS, however, a re-formation of the alpha-helical structure and a partial recovery of the hydrolytic activity of cellulase induced by SDS micelles are observed. Taken together, these results indicate that SDS micelles exert dual effects on cellulase through binding as both a denaturant and a recovery reagent.

  5. Visualization of cellulases bound to cellulose microfibrils: Evidence for endo/exo synergism

    SciTech Connect

    Nieves, R.A.; Himmel, M.E.; Ellis, R.P.

    1993-12-31

    Monoclonal antibodies (MAbs) specific for cellobiohydrolase I (CBH I) and endoglucanase I (EG I) were conjugated to 10-nm and 15-nm colloidal gold particles, respectively. The binding of CBH I and EG I to cellulose was visualized by utilizing the MAb-colloidal gold probes. The visualization procedure involved immobilization of cellulose microfibrils on copper EM grids, incubation of the cellulose-coated grids with cellulase(s), binding of MAb-colloidal gold conjugates to cellulase(s), and visualization via transmission electron microscopy (TEM). Apparent differences in the substrate binding preferences for gold-labelled CBH I and EG I were observed; however, the actual structure of the cellulose at these sites was not confirmed. Sites showing binding of both enzymes sometimes showed patterns of coordination suggestive of the {open_quotes}progressive{close_quotes} cellulase action model. The sensitivity of this technique was evident when micrographs were observed in 3-D.

  6. Rational approach to optimize cellulase mixtures for hydrolysis of regenerated cellulose containing residual ionic liquid.

    PubMed

    Engel, Philip; Krull, Susan; Seiferheld, Bianca; Spiess, Antje C

    2012-07-01

    For the efficient production of glucose for platform chemicals or biofuels, cellulosic biomass is pretreated and subsequently hydrolyzed with cellulases. Although ionic liquids (IL) are known to effectively pretreat cellulosic biomass, the hydrolysis of IL pretreated biomass has not been optimized so far. Here, we present a semi-empirical model to rationally optimize the hydrolysis of pretreated α-cellulose - regenerated from IL and containing residual IL from the pretreatment. First, the influence of the IL MMIM DMP on the individual cellulases endoglucanase I, cellobiohydrolase I and β-glucosidase was investigated. Second, an enzyme loading-dependent model was developed to describe kinetics for the individual cellulases and cellulase mixtures. Third, this model was used to optimize the cellulase mixture for the efficient hydrolysis of regenerated cellulose containing residual IL. Finally, we could significantly increase the initial hydrolysis rate in 10% (v/v) MMIM DMP by 49% and the sugar yield by 10% points. PMID:22100231

  7. 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.

  8. Increased production of cellulase of Trichoderma sp. By pH cycling and temperature profiling

    SciTech Connect

    Mukhopadhyay, S.N.; Malik, R.K.

    1980-01-01

    Cultivation of Trichoderma reesei QM 9414 on 3% cellulose medium (C/N ratio equal to 8.5) produced 4.5 IU/mL cellulase in 180 h at a cell growth of 8.0 g/L. It corresponded to an average cellulase productivity of 25.0 IU/L/h. In the same medium 9.5 g/L cell mass, 6.2 IU/mL cellulase, and 38.75 IU/L/h cellulase productivity could be obtained using pH cycling during cultivation. Cell mass, cellulase yield, and productivity were further increased to 10,0 g/L, 7.2 IU/mL, and 44.0 IU/L/h (4.5 IU/g cell/h), respectively, by simultaneous pH cycling and temperature profiling. Results are described.

  9. Dehydrogenase GRD1 represents a novel component of the cellulase regulon in Trichoderma reesei (Hypocrea jecorina).

    PubMed

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

    2011-07-01

    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.

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

    PubMed

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

    2015-01-01

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

  11. Understanding of alkaline pretreatment parameters for corn stover enzymatic saccharification

    PubMed Central

    2013-01-01

    Background Previous research on alkaline pretreatment has mainly focused on optimization of the process parameters to improve substrate digestibility. To achieve satisfactory sugar yield, extremely high chemical loading and enzyme dosages were typically used. Relatively little attention has been paid to reduction of chemical consumption and process waste management, which has proven to be an indispensable component of the bio-refineries. To indicate alkali strength, both alkali concentration in pretreatment solution (g alkali/g pretreatment liquor or g alkali/L pretreatment liquor) and alkali loading based on biomass solids (g alkali/g dry biomass) have been widely used. The dual approaches make it difficult to compare the chemical consumption in different process scenarios while evaluating the cost effectiveness of this pretreatment technology. The current work addresses these issues through pretreatment of corn stover at various combinations of pretreatment conditions. Enzymatic hydrolysis with different enzyme blends was subsequently performed to identify the effects of pretreatment parameters on substrate digestibility as well as process operational and capital costs. Results The results showed that sodium hydroxide loading is the most dominant variable for enzymatic digestibility. To reach 70% glucan conversion while avoiding extensive degradation of hemicellulose, approximately 0.08 g NaOH/g corn stover was required. It was also concluded that alkali loading based on total solids (g NaOH/g dry biomass) governs the pretreatment efficiency. Supplementing cellulase with accessory enzymes such as α-arabinofuranosidase and β-xylosidase significantly improved the conversion of the hemicellulose by 6–17%. Conclusions The current work presents the impact of alkaline pretreatment parameters on the enzymatic hydrolysis of corn stover as well as the process operational and capital investment costs. The high chemical consumption for alkaline pretreatment technology

  12. Silica in alkaline brines

    USGS Publications Warehouse

    Jones, B.F.; Rettig, S.L.; Eugster, H.P.

    1967-01-01

    Analysis of sodium carbonate-bicarbonate brines from closed basins in volcanic terranes of Oregon and Kenya reveals silica contents of up to 2700 parts per million at pH's higher than 10. These high concentrations of SiO 2 can be attributed to reaction of waters with silicates, and subsequent evaporative concentration accompanied by a rise in pH. Supersaturation with respect to amorphous silica may occur and persist for brines that are out of contact with silicate muds and undersaturated with respect to trona; correlation of SiO2 with concentration of Na and total CO2 support this interpretation. Addition of moredilute waters to alkaline brines may lower the pH and cause inorganic precipitation of substantial amounts of silica.

  13. Bifunctional alkaline oxygen electrodes

    NASA Technical Reports Server (NTRS)

    Swette, L.; Kackley, N.; Mccatty, S. A.

    1991-01-01

    The authors describe the identification and testing of electrocatalysts and supports for the positive electrode of moderate-temperature, single-unit, rechargeable alkaline fuel cells. Recent work on Na(x)Pt3O4, a potential bifunctional catalyst, is described, as well as the application of novel approaches to the development of more efficient bifunctional electrode structures. The three dual-character electrodes considered here showed similar superior performance; the Pt/RhO2 and Rh/RhO2 electrodes showed slightly better performance than the Pt/IrO2 electrode. It is concluded that Na(x)Pt3O4 continues to be a promising bifunctional oxygen electrode catalyst but requires further investigation and development.

  14. Alleviating product inhibition in cellulase enzyme Cel7A

    PubMed Central

    Atreya, Meera E.; Strobel, Kathryn L.

    2015-01-01

    ABSTRACT Enzymes that degrade cellulose into glucose are one of the most expensive components of processes for converting cellulosic biomass to fuels and chemicals. Cellulase enzyme Cel7A is the most abundant enzyme naturally employed by fungi to depolymerize cellulose, and like other cellulases is inhibited by its product, cellobiose. There is thus great economic incentive for minimizing the detrimental effects of product inhibition on Cel7A. In this work, we experimentally generated 10 previously proposed site‐directed mutant Cel7A enzymes expected to have reduced cellobiose binding energies (the majority of mutations were to alanine). We then tested their resilience to cellobiose as well as their hydrolytic activities on microcrystalline cellulose. Although every mutation tested conferred reduced product inhibition (and abolished it for some), our results confirm a trade‐off between Cel7A tolerance to cellobiose and enzymatic activity: Reduced product inhibition was accompanied by lower overall enzymatic activity on crystalline cellulose for the mutants tested. The tempering effect of mutations on inhibition was nearly constant despite relatively large differences in activities of the mutants. Our work identifies an amino acid in the Cel7A product binding site of interest for further mutational studies, and highlights both the challenge and the opportunity of enzyme engineering toward improving product tolerance in Cel7A. Biotechnol. Bioeng. 2016;113: 330–338. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26302366

  15. Alleviating product inhibition in cellulase enzyme Cel7A.

    PubMed

    Atreya, Meera E; Strobel, Kathryn L; Clark, Douglas S

    2016-02-01

    Enzymes that degrade cellulose into glucose are one of the most expensive components of processes for converting cellulosic biomass to fuels and chemicals. Cellulase enzyme Cel7A is the most abundant enzyme naturally employed by fungi to depolymerize cellulose, and like other cellulases is inhibited by its product, cellobiose. There is thus great economic incentive for minimizing the detrimental effects of product inhibition on Cel7A. In this work, we experimentally generated 10 previously proposed site-directed mutant Cel7A enzymes expected to have reduced cellobiose binding energies (the majority of mutations were to alanine). We then tested their resilience to cellobiose as well as their hydrolytic activities on microcrystalline cellulose. Although every mutation tested conferred reduced product inhibition (and abolished it for some), our results confirm a trade-off between Cel7A tolerance to cellobiose and enzymatic activity: Reduced product inhibition was accompanied by lower overall enzymatic activity on crystalline cellulose for the mutants tested. The tempering effect of mutations on inhibition was nearly constant despite relatively large differences in activities of the mutants. Our work identifies an amino acid in the Cel7A product binding site of interest for further mutational studies, and highlights both the challenge and the opportunity of enzyme engineering toward improving product tolerance in Cel7A.

  16. Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2

    PubMed Central

    Xu, Hui; Han, Dongmei; Xu, Zhaohui

    2015-01-01

    The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization. PMID:26273605

  17. Alleviating product inhibition in cellulase enzyme Cel7A.

    PubMed

    Atreya, Meera E; Strobel, Kathryn L; Clark, Douglas S

    2016-02-01

    Enzymes that degrade cellulose into glucose are one of the most expensive components of processes for converting cellulosic biomass to fuels and chemicals. Cellulase enzyme Cel7A is the most abundant enzyme naturally employed by fungi to depolymerize cellulose, and like other cellulases is inhibited by its product, cellobiose. There is thus great economic incentive for minimizing the detrimental effects of product inhibition on Cel7A. In this work, we experimentally generated 10 previously proposed site-directed mutant Cel7A enzymes expected to have reduced cellobiose binding energies (the majority of mutations were to alanine). We then tested their resilience to cellobiose as well as their hydrolytic activities on microcrystalline cellulose. Although every mutation tested conferred reduced product inhibition (and abolished it for some), our results confirm a trade-off between Cel7A tolerance to cellobiose and enzymatic activity: Reduced product inhibition was accompanied by lower overall enzymatic activity on crystalline cellulose for the mutants tested. The tempering effect of mutations on inhibition was nearly constant despite relatively large differences in activities of the mutants. Our work identifies an amino acid in the Cel7A product binding site of interest for further mutational studies, and highlights both the challenge and the opportunity of enzyme engineering toward improving product tolerance in Cel7A. PMID:26302366

  18. Chapter 6: Energy Storage in Cellulase Linker Peptides?

    SciTech Connect

    McCabe, C.; Zhao, X.; Adney, W. S.; Himmel, M. E.

    2010-01-01

    In this chapter, we discuss the use of molecular dynamics simulations and free-energy calculations to investigate the possible role the linker polypeptide, common to many cellulase enzymes, plays in the enzymatic hydrolysis of cellulose. In particular, we focus on the linker polypeptide from cellobiohydrolase I (CBH I) from Trichoderma reesei, which is one of the most active cellulase enzymes. CBH I is a multi-domain enzyme, consisting of a large catalytic domain containing an active site tunnel and a small cellulose binding module, which are joined together by a 27-amino-acid residue linker peptide. CBH I is believed to hydrolyze cellulose in a 'processive' manner; however, the exact mechanism of the depolymerization of cellulose by CBH I is not fully understood. It has been hypothesized that the flexible interdomain linker mediates a caterpillar-like motion that enables the enzyme to move along the cellodextrin strand. Although the linker polypeptide sequence is known, the spatial conformation adopted by the linker domain and its role in the hydrolysis process, if any, has yet to be determined. The simulation results obtained to date indicate that the CBH I linker's free energy is critically dependent on the existence of the cellulose substrate and the stretching/compression pathway adopted. In the presence of a cellulose surface, simulations suggest that the linker exhibits two stable states, which would support the hypothesis that the linker peptide has the capacity to store energy in a manner similar to a spring and facilitate a caterpillar-like motion.

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

    PubMed

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

    2012-12-01

    Endophytes are microorganisms that asymptomatically invade plant tissues. They can stimulate plant growth and/or provide defense against pathogen attacks through the production of secondary metabolites. Most endophyte species are still unknown, and because they may have several applications, the study of their metabolic capabilities is essential. We characterized 100 endophytes isolated from Espeletia spp., a genus unique to the paramo ecosystem, an extreme environment in the Andean mountain range. We evaluated the cellulolytic potential of these endophytes on the saccharification of the oil palm empty fruit bunch (OPEFB). The total cellulolytic activity was measured for each endophyte on filter paper (FPA). In addition, the specific carboxymethyl cellulase (CMCase), exoglucanase, and β-glucosidase activities were determined. We found four fungi positive for cellulases. Of these fungi, Penicillium glabrum had the highest cellulolytic activity after partial purification, with maximal CMCase, exoglucanase and β-glucosidase enzyme activities of 44.5, 48.3, and 0.45 U/ml, respectively. Our data showed that the bioprospection of fungi and the characterization of their enzymes may facilitate the process of biofuel production. PMID:23274988

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

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

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

  1. Deletion of the Cel48S cellulase from Clostridium thermocellum

    SciTech Connect

    Olson, Daniel G; Tripathi, Shital A.; Giannone, Richard J; Lo, Jonathan; Caiazza, Nicky; Hogsett, David A; Hettich, Robert {Bob} L; Guss, Adam M; Dubrovsky, Genia; Lynd, Lee R

    2010-01-01

    Clostridium thermocellum is a thermophilic anaerobic bacterium that rapidly solubilizes cellulose with the aid of a multienzyme cellulosome complex. Creation of knockout mutants for Cel48S (also known as CelS, SS, and S8), the most abundant cellulosome subunit, was undertaken to gain insight into its role in enzymatic and microbial cellulose solubilization. Cultures of the Cel48S deletion mutant (S mutant) were able to completely solubilize 10 g/L crystalline cellulose. The cellulose hydrolysis rate of the S mutant strain was 60% lower than the parent strain, with the S mutant strain also exhibiting a 40% reduction in cell yield. The cellulosome produced by the S mutant strain was purified by affinity digestion, characterized enzymatically, and found to have a 35% lower specific activity on Avicel. The composition of the purified cellulosome was analyzed by tandem mass spectrometry with APEX quantification and no significant changes in abundance were observed in any of the major (>1% of cellulosomal protein) enzymatic subunits. Although most cellulolytic bacteria have one family 48 cellulase, C. thermocellum has two, Cel48S and Cel48Y. Cellulose solubilization by a Cel48S and Cel48Y double knockout was essentially the same as that of the Cel48S single knockout. Our results indicate that solubilization of crystalline cellulose by C. thermocellum can proceed to completion without expression of a family 48 cellulase.

  2. Cellulase production under solid-state fermentation by Trichoderma reesei RUT C30: statistical optimization of process parameters.

    PubMed

    Mekala, Naveen Kumar; Singhania, Reeta Rani; Sukumaran, Rajeev K; Pandey, Ashok

    2008-12-01

    Sugar cane bagasse was used as substrate for cellulase production using Trichoderma reesei RUT C30, and the culture parameters were optimized for enhancing cellulase yield. The culture parameters, such as incubation temperature, duration of incubation, and inducer concentration, were optimized for enhancing cellulase yield using a Box-Behnken experimental design. The optimal level of each parameter for maximum cellulase production by the fungus was determined. Predicted results showed that cellulase production was highest (25.6 FPAase units per gram dry substrate) when the inducer concentration was 0.331 ml/gds, and the incubation temperature and time were 33 degrees C and 67 h, respectively. Crude inducer generated by cellulase action was found to be very effective in inducing cellulases. Validation of predicted results was done, and the experimental values correlated well with that of the predicted.

  3. Carbon source and pH-dependent transcriptional regulation of cellulase genes of Humicola grisea var. thermoidea grown on sugarcane bagasse.

    PubMed

    Mello-de-Sousa, Thiago Machado; Silva-Pereira, Ildinete; Poças-Fonseca, Marcio José

    2011-01-01

    Time-course expression profiles of one xylanase and eight cellulase encoding genes, as well as of two transcription factor encoding genes of Humicola grisea var. thermoidea were established in different culture media pHs and carbon sources (glucose and sugarcane bagasse). Quantitative real-time RT-PCR analysis revealed a remarkable and parallel increase in mRNA accumulation for cbh1.1, cbh1.2, egl1, egl2, egl3, bgl4 and xyn1 at alkaline pH and with sugarcane bagasse employed as the sole carbon source. Glucose utilization led to a higher creA mRNA accumulation compared to the other genes. A distinct pattern was observed for egl4, whose mRNA preferably accumulated in acidic conditions. The transcriptional profile data combined with the analysis of the in vitro binding of PacC and CreA transcription factors to the promoters support the CreA-mediated carbon repression and the PacC-related pH regulation of H. grisea cellulase and xylanase encoding genes. Moreover, EMSA analyses suggest a role for CreA on pacC transcriptional regulation. These data will be useful to H. grisea hydrolytic enzymes production improvement, as well as to the design of optimized promoters aiming industrial heterologous proteins production. PMID:22112766

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

    SciTech Connect

    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 products 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.

  5. 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.

  6. 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.

  7. Immobilization method for the recovery of cellulase used in cellulose conversion to ethanol

    SciTech Connect

    Woodward, J.; Hillman, S.K.; Vaughen, B.K.

    1984-01-01

    Mixtures of cellulase and ..beta..-D-glucosidase when added to fermentation systems containing a yeast or bacterium enable the latter to utilize cellulose if they are inherently unable to do so. It is essential that the enzymes be recovered and recycled if such a system is to have commercial potential. The major components of Trichoderma reesei C30 cellulase have been adsorbed onto concanavalin A-Sepharose (CAS) to give maximum loadings of 2.7, 44.7, and 1.5 units g/sup -1/ dry CAS at 50/sup 0/C when measured with the substrates filter paper, carboxymethylcellulose and cellobiose, respectively. Cellulase immobilized by this method desorbs from the support in the presence of insoluble cellulose and interacts with the substrate. Cellulose is thus converted to ethanol by immobilized cellulase, ad ..beta..-D-glucosidase and Z, mobilis coentrapped within calcium alginate. It is proposed that reimmobilization of cellulase will take place if all the substrate is utilized and converted to ethanol by a co-immobilized yeast or bacterium. Immobilization of cellulase and ..beta..-D-glucosidase on CAS is unaffected by 10% (w/v) ethanol. There is also little effect of 10% ethanol on the activity of cellulase and ..beta..-D-glucosidase. The enzymes could be easily recovered from an ethanol containing medium by simple filtration, and reused. 15 references, 6 figures.

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

    SciTech Connect

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

    1997-03-01

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

  9. 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. PMID:27497931

  10. Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes.

    PubMed

    Zhou, Xuguo; Smith, Joseph A; Oi, Faith M; Koehler, Philip G; Bennett, Gary W; Scharf, Michael E

    2007-06-15

    Termites have developed cellulose digestion capabilities that allow them to obtain energy and nutrition from nutritionally poor food sources, such as lignocellulosic plant material and residues derived from it (e.g., wood and humus). Lower termites, which are equipped with both endogenous (i.e., of termite origin) and symbiotic cellulases, feed primarily on wood and wood-related materials. This study investigated cellulase gene diversity, structure, and activity in the lower termite, Reticulitermes flavipes (Kollar). We initially used a metagenomics approach to identify four genes encoding one endogenous and three symbiotic cellulases, which we refer to as Cell-1, -2, -3 and -4. These four genes encode proteins that share significant sequence similarity with known endoglucanases, exoglucanases and xylanases. Phylogenetic analyses further supported these inferred relationships by showing that each of the four cellulase proteins clusters tightly with respective termite, protozoan or fungal cellulases. Gene structure studies revealed that Cell-1, -3 and -4 are intron-free, while Cell-2 contains the first intron sequence to be identified from a termite symbiont cellulase. Quantitative real-time PCR (qRT-PCR) revealed that the endogenous Cell-1 gene is expressed exclusively in the salivary gland/foregut, whereas symbiotic Cell-2, -3, and -4 are highly expressed in the hindgut (where cellulolytic protists are harbored). Cellulase activity assays mapped the distribution pattern of endoglucanase, exoglucanase and xylanase activity throughout the R. flavipes digestive tract. Cellulase gene expression correlated well with the specific types of cellulolytic activities observed in each gut region (foregut+salivary gland, midgut and hindgut). These results suggest the presence of a single unified cellulose digestion system, whereby endogenous and symbiotic cellulases work sequentially and collaboratively across the entire digestive tract of R. flavipes.

  11. Alkaline battery, separator therefore

    NASA Technical Reports Server (NTRS)

    Schmidt, George F. (Inventor)

    1980-01-01

    An improved battery separator for alkaline battery cells has low resistance to electrolyte ion transfer and high resistance to electrode ion transfer. The separator is formed by applying an improved coating to an electrolyte absorber. The absorber, preferably, is a flexible, fibrous, and porous substrate that is resistant to strong alkali and oxidation. The coating composition includes an admixture of a polymeric binder, a hydrolyzable polymeric ester and inert fillers. The coating composition is substantially free of reactive fillers and plasticizers commonly employed as porosity promoting agents in separator coatings. When the separator is immersed in electrolyte, the polymeric ester of the film coating reacts with the electrolyte forming a salt and an alcohol. The alcohol goes into solution with the electrolyte while the salt imbibes electrolyte into the coating composition. When the salt is formed, it expands the polymeric chains of the binder to provide a film coating substantially permeable to electrolyte ion transfer but relatively impermeable to electrode ion transfer during use.

  12. Evaluation of Alkaline Cleaner Materials

    NASA Technical Reports Server (NTRS)

    Partz, Earl

    1998-01-01

    Alkaline cleaners used to process aluminum substrates have contained chromium as the corrosion inhibitor. Chromium is a hazardous substance whose use and control are described by environmental laws. Replacement materials that have the characteristics of chromated alkaline cleaners need to be found that address both the cleaning requirements and environmental impacts. This report will review environmentally friendly candidates evaluated as non-chromium alkaline cleaner replacements and methods used to compare those candidates one versus another. The report will also list characteristics used to select candidates based on their declared contents. It will also describe and evaluate methods used to discriminate among the large number of prospective candidates.

  13. Cellulase activity of trichoderma reesei (RUT-C30) on municipal solid waste

    SciTech Connect

    Silva, S.; Elmore, B.B.; Huckaby, H.K.

    1995-12-31

    This work presents a preliminary investigation of Trichoderma reesei (RUT-C30) grown on municipal solid waste (MSW). Such a process offers the potential for inexpensive production of cellulase enzymes while reducing the waste stream to landfills. Cellulase enzyme activity for batch-culture growth on MSW compared favorably with growth on refined cellulosic substrates. Cellulase productivity in an initial fed-batch culture reached a maximum of 22 IFPU/L-h with a maximum activity of 1.5 IFPU/mL.

  14. Enhanced production of cellulase, hemicellulase, and β-glucosidase by Trichoderma reesei (Rut C-30)

    SciTech Connect

    Tangnu, S. Kishen; Blanch, Harvey W.; Wilke, Charles R.

    1981-08-01

    In this paper, the Production of cellulases and Hemicellulases was studied with Trichoderma reesei Rut C-30, This organism produced, together with high cellulase activities, considerable amounts of xylanases and β-glucosidase. Three cellulose concentration (1, 2.5, and 5.0%) were examined to determined the maximum levels of cellulase activity obtainable in submerged culture. Temperature and pH profiling was used to increase cell mass to maximum levels within two days and thereby enhancing fermentor productivity at higher substrate levels. Finally, the effect of temperature, pH, Tween-80 concentration, carbon sources, and substrate concentration on the ration of mycelial growth and extracellulose enzyme production are described.

  15. Enhanced production of cellulase, hemicellulase, and b-glucosidase by trichoderma reesei, (rut c-30)

    SciTech Connect

    Kishen Tangnu, S.; Blanch, H.W.; Wilke, C.R.

    1981-08-01

    The production of cellulases and hemicellulases was studied with Trichoderma reesei Rut C-30. This organism produced, together with high cellulase activities, considerable amounts of xylanases and B-glucosidase. Three cellulose concentrations (1, 2.5, and 5.0%) were examined to determine the maximum levels of cellulase activity obtainable in submerged culture. Temperature and pH profiling was used to increase cell mass to maximum levels within two days and thereby enhancing fermentor productivity at higher substrate levels. The effect of temperature, pH, Tween-80 concentration, carbon source, and substrate concentration on the ratio of mycelial growth and extracellulose enzyme production are described. (Refs. 19).

  16. Enhanced production of cellulase, hemicellulase, and beta-glucosidase by Trichoderma reesei (rut c-30)

    SciTech Connect

    Tangnu, S.K.; Blanch, H.W.; Wilke, C.R.

    1981-08-01

    The production of cellulases and hemicellulases was studied with Trichoderma reesei Rut C-30. This organism produced, together with high cellulase activities, considerable amounts of xylanases and beta-glucosidase. Three cellulose concentrations (1, 2.5, and 5.0%) were examined to determine the maximum levels of cellulase activity obtainable in submerged culture. Temperature and pH profiling was used to increase cell mass to maximum levels within two days and thereby enhancing fermentor productivity at higher substrate levels. The effect of temperature, pH, Tween-80 concentration, carbon source, and substrate concentration on the ratio of mycelial growth and extracellulose enzyme production are described. 19 refs.

  17. Fractionation of Aspergillus niger cellulases by combined ion exchange affinity chromatography

    SciTech Connect

    Boyer, R.F.; Allen, T.L.; Dykema, P.A.

    1987-02-05

    Eight chemically modified cellulose supports were tested for their ability to adsorb components of the Aspergillus niger cellulase system. At least two of the most effective adsorbents, aminoethyl cellulose and carboxymethyl cellulose, were shown to be useful for the fractionation of cellulases. These supports apparently owe their resolving capacity to both ion exchange and biospecific binding effects; however, the relative importance of each effect is unknown. These observations form the basis for a new cellulase fractionation technique, combined ion exchange-affinity chromatography. 22 references.

  18. Catalysis of rice straw hydrolysis by the combination of immobilized cellulase from Aspergillus niger on β-cyclodextrin-Fe3O4 nanoparticles and ionic liquid.

    PubMed

    Huang, Po-Jung; Chang, Ken-Lin; Hsieh, Jung-Feng; 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

  19. Catalysis of rice straw hydrolysis by the combination of immobilized cellulase from Aspergillus niger on β-cyclodextrin-Fe3O4 nanoparticles and ionic liquid.

    PubMed

    Huang, Po-Jung; Chang, Ken-Lin; Hsieh, Jung-Feng; 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.

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

    PubMed

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

    2002-04-01

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

  1. Differential regulation of the cellulase transcription factors XYR1, ACE2, and ACE1 in Trichoderma reesei strains producing high and low levels of cellulase.

    PubMed

    Portnoy, Thomas; Margeot, Antoine; Seidl-Seiboth, Verena; Le Crom, Stéphane; Ben Chaabane, Fadhel; Linke, Rita; Seiboth, Bernhard; Kubicek, Christian P

    2011-02-01

    Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly being investigated for second-generation biofuel production from lignocellulosic biomass. The induction mechanisms of T. reesei cellulases have been described recently, but the regulation of the genes involved in their transcription has not been studied thoroughly. Here we report the regulation of expression of the two activator genes xyr1 and ace2, and the corepressor gene ace1, during the induction of cellulase biosynthesis by the inducer lactose in T. reesei QM 9414, a strain producing low levels of cellulase (low producer). We show that all three genes are induced by lactose. xyr1 was also induced by d-galactose, but this induction was independent of d-galactose metabolism. Moreover, ace1 was carbon catabolite repressed, whereas full induction of xyr1 and ace2 in fact required CRE1. Significant differences in these regulatory patterns were observed in the high-producer strain RUT C30 and the hyperproducer strain T. reesei CL847. These observations suggest that a strongly elevated basal transcription level of xyr1 and reduced upregulation of ace1 by lactose may have been important for generating the hyperproducer strain and that thus, these genes are major control elements of cellulase production.

  2. Wood Extractives Promote Cellulase Activity on Cellulosic Substrates.

    PubMed

    Leskinen, Timo; Salas, Carlos; Kelley, Stephen S; Argyropoulos, Dimitris S

    2015-10-12

    Deposition of hydrophobic wood extractives and representative model compounds, on the surface of cellulose prior to enzymatic hydrolysis was found to either enhance or inhibit the action of cellulase enzymes. The effect of these compounds was correlated with their chemical structure, which may in part explain the differential effects observed between softwood and hardwood extractives. Specifically, the addition of sterol, enhanced enzymatic hydrolysis of microcrystalline cellulose by 54%, whereas the addition of a triglyceride could inhibit the hydrolysis by 49%. The effects of the different extractives' could be explained by considering their Hansen solubility parameters. The amphiphilic and/or hydrophobic character of model extractives was found to be the variable that affected the deposition of extractives on cellulose surfaces and the eventual adsorption of cellulolytic enzymes on it. The observed beneficial effects of extractives are likely related to a reduction in the irreversible binding of the enzymes on the cellulose surface.

  3. Developing Improved MD Codes for Understanding Processive Cellulases

    SciTech Connect

    Crowley, M. F.; Uberbacher, E. C.; Brooks III, C. L.; Walker, R.C.; Nimlos, M. R.; Himmel, M. E.

    2008-01-01

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

  4. 2009 Cellulosomes, Cellulases & Other Carbohydrate Modifying Enzymes GRC

    SciTech Connect

    Harry Gilbert

    2009-07-26

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

  5. Applicability evaluation of Deep Eutectic Solvents-Cellulase system for lignocellulose hydrolysis.

    PubMed

    Gunny, Ahmad Anas Nagoor; Arbain, Dachyar; Nashef, Enas Muen; Jamal, Parveen

    2015-04-01

    Deep Eutectic Solvents (DESs) have recently emerged as a new generation of ionic liquids for lignocellulose pretreatment. However, DESs contain salt components which tend to inactivate cellulase in the subsequent saccharification process. To alleviate this problem, it is necessary to evaluate the applicability of the DESs-Cellulase system. This was accomplished in the present study by first studying the stability of cellulase in the presence of selected DESs followed by applicability evaluation based on glucose production, energy consumption and kinetic performance. Results showed that the cellulase was able to retain more than 90% of its original activity in the presence of 10% (v/v) for glycerol based DES (GLY) and ethylene glycol based DES (EG). Furthermore, both DESs system exhibited higher glucose percentage enhancement and lower energy consumption as compared to diluted alkali system. Among the two DESs studied, EG showed comparatively better kinetic performance. PMID:25661309

  6. Separation of lignocresol from eucalyptus lignocresol-cellulase complex using organic solvents.

    PubMed

    Nonaka, Hiroshi; Kobayashi, Ai; Funaoka, Masamitsu

    2013-09-01

    A functional lignin-based material, lignocresol, was synthesized from eucalyptus wood meal by using phase separation treatment with p-cresol and 72% sulfuric acid. Lignocresol physically adsorbs Trichoderma reesei cellulase and functions as an immobilized cellulase. Lignocresol-immobilized cellulase was treated with acetone or ethanol to separate the enzyme and its support. Most lignocresol was dissolved in organic solvents and then precipitated in diethyl ether. The recovery yield of lignocresol using acetone was 85% and that using ethanol was 72%, with very low or negligible residual cellulase. Lignocresol is an innovative recyclable enzyme support that can be easily separated using only organic solvents after deactivation of the immobilized enzyme. PMID:23835277

  7. Applicability evaluation of Deep Eutectic Solvents-Cellulase system for lignocellulose hydrolysis.

    PubMed

    Gunny, Ahmad Anas Nagoor; Arbain, Dachyar; Nashef, Enas Muen; Jamal, Parveen

    2015-04-01

    Deep Eutectic Solvents (DESs) have recently emerged as a new generation of ionic liquids for lignocellulose pretreatment. However, DESs contain salt components which tend to inactivate cellulase in the subsequent saccharification process. To alleviate this problem, it is necessary to evaluate the applicability of the DESs-Cellulase system. This was accomplished in the present study by first studying the stability of cellulase in the presence of selected DESs followed by applicability evaluation based on glucose production, energy consumption and kinetic performance. Results showed that the cellulase was able to retain more than 90% of its original activity in the presence of 10% (v/v) for glycerol based DES (GLY) and ethylene glycol based DES (EG). Furthermore, both DESs system exhibited higher glucose percentage enhancement and lower energy consumption as compared to diluted alkali system. Among the two DESs studied, EG showed comparatively better kinetic performance.

  8. Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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)...

  9. Cellulase: a key enzyme in fermentation. Progress report, 1983-1984

    SciTech Connect

    Eveleigh, D.E.

    1984-04-27

    Progress has been achieved in three areas. N-glycosylation has been shown not to be absolutely essential for maintenance of activity of cellulase enzymes through both inhibitor (Tunicamycin) and enzyme (Endo-H) degradative studies. Thus deglycosylated cellulase may be functional as a direct transcript from mRNA. However, initial studies of in vitro synthesis of cellulase, though yielding positive cellulase antibody proteins, did not produce enzymatically active forms. These proteins produced in vitro were considerably larger than the in vivo form. Further clarification of the in vitro system is underway. Attempts to produce monoclonal antibodies have been successful with selection of clones that show antibodies specific towards cellobiohydrolase. Polyclonal antibodies in contrast do not show such specificity. The monoclonal preparations should allow development of direct assays for cellobiohydrolase, an enzyme for which no direct assay currently exists.

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

    PubMed

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

    2008-01-01

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

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

    PubMed

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

    2008-01-01

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

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

    SciTech Connect

    Evans, B.R.; Lee, I.; Woodward, J.; Fox, S.V.

    1997-12-31

    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 in 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.

  13. Molecular cloning and characterization of two novel cellulase genes from the mollusc Ampullaria crossean.

    PubMed

    Guo, Rui; Ding, Ming; Zhang, Si-Liang; Xu, Gen-Jun; Zhao, Fu-Kun

    2008-02-01

    Cellulase genes have been reported not only from fungi, bacteria and plant, but also from some invertebrate animals. Here, two cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) genes, eg27I and eg27II, were cloned from the freshwater snail Ampullaria crossean cDNA using degenerate primers. The nucleotide sequences of the two genes shared 94.5% identity. The open reading frames of both genes consisted of 588 bp, encoding 195 amino acids. Both EG27I and EG27II belong to the glycoside hydrolase family 45, and each lacks a carbohydrate-binding module. The presence of introns demonstrated a eukaryotic origin of the EG27 gene, and, in addition, successful cloning of EG27 cDNA supported endogenous production of EG27 cellulase by Ampullaria crossean. Investigation of the EG27 cDNA from A. crossean will provide further information on GHF45 cellulases.

  14. The alkaline and alkaline-carbonatite magmatism from Southern Brazil

    NASA Astrophysics Data System (ADS)

    Ruberti, E.; Gomes, C. D. B.; Comin-Chiaramonti, P.

    2015-12-01

    Early to Late Cretaceous lasting to Paleocene alkaline magmatism from southern Brazil is found associated with major extensional structural features in and around the Paraná Basin and grouped into various provinces on the basis of several data. Magmatism is variable in size, mode of occurrence and composition. The alkaline rocks are dominantly potassic, a few occurrences showing sodic affinity. The more abundant silicate rocks are evolved undersaturated to saturated in silica syenites, displaying large variation in igneous forms. Less evolved types are restricted to subvolcanic environments and outcrops of effusive suites occur rarely. Cumulatic mafic and ultramafic rock types are very common, particularly in the alkali-carbonatitic complexes. Carbonatite bodies are represented by Ca-carbonatites and Mg-carbonatites and more scarcely by Fe-carbonatites. Available radiometric ages for the alkaline rocks fit on three main chronological groups: around 130 Ma, subcoveal with the Early Cretaceous flood tholeiites of the Paraná Basin, 100-110 Ma and 80-90 Ma (Late Cretaceous). The alkaline magmatism also extends into Paleocene times, as indicated by ages from some volcanic lavas. Geochemically, alkaline potassic and sodic rock types are distinguished by their negative and positive Nb-Ta anomalies, respectively. Negative spikes in Nb-Ta are also a feature common to the associated tholeiitic rocks. Sr-Nd-Pb systematics confirm the contribution of both HIMU and EMI mantle components in the formation of the alkaline rocks. Notably, Early and Late Cretaceous carbonatites have the same isotopic Sr-Nd initial ratios of the associated alkaline rocks. C-O isotopic Sr-Nd isotopic ratios indicate typical mantle signature for some carbonatites and the influence of post-magmatic processes in others. Immiscibility of liquids of phonolitic composition, derived from mafic alkaline parental magmas, has been responsible for the origin of the carbonatites. Close association of alkaline

  15. 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.

  16. Production of cellulase from Trichoderma reesei in fed-batch fermentation from soluble carbon sources

    SciTech Connect

    Allen, A.L.; Mortensen, R.E.

    1981-11-01

    The use of a soluble carbon source in lieu of cellulose for the production of cellulase would allow greater control of the fermentation, since growth and enzyme production would no longer be dependent upon cellulose hydrolysis. Where carbon limitation is a requirement, fed-batch fermentation has proved successful. This article describes cellulase production from Trichoderma reesei using five different carbon sources, sophorose appearing to be a more likely candidate than cellobiose. (Refs. 22).

  17. Studies on the properties of cellulase enzyme from Aspergillus terreus GN1

    SciTech Connect

    Garg, S.K.; Neelakantan, S.

    1982-03-01

    The cellulase enzyme is produced by cellulolytic microorganisms and is able to degrade cellulose materials. It has assumed greater importance due to its potential use in modifying low-grade roughages, the production of syrups, powering alcohol from cellulosic wastes, and other applications such as single-cell protein production. This paper studies the properties of a cellulase enzyme system from Aspergillus terreus GN1 and the effect of various modulators on its activity. (Refs. 18).

  18. Alkaline twin-screw extrusion pretreatment for fermentable sugar production

    PubMed Central

    2013-01-01

    Background The inevitable depletion of fossil fuels has resulted in an increasing worldwide interest in exploring alternative and sustainable energy sources. Lignocellulose, which is the most abundant biomass on earth, is widely regarded as a promising raw material to produce fuel ethanol. Pretreatment is an essential step to disrupt the recalcitrance of lignocellulosic matrix for enzymatic saccharification and bioethanol production. This paper established an ATSE (alkaline twin-screw extrusion pretreatment) process using a specially designed twin-screw extruder in the presence of alkaline solution to improve the enzymatic hydrolysis efficiency of corn stover for the production of fermentable sugars. Results The ATSE pretreatment was conducted with a biomass/liquid ratio of 1/2 (w/w) at a temperature of 99°C without heating equipment. The results indicated that ATSE pretreatment is effective in improving the enzymatic digestibility of corn stover. Sodium hydroxide loading is more influential factor affecting both sugar yield and lignin degradation than heat preservation time. After ATSE pretreatment under the proper conditions (NaOH loading of 0.06 g/g biomass during ATSE and 1 hour heat preservation after extrusion), 71% lignin removal was achieved and the conversions of glucan and xylan in the pretreated biomass can reach to 83% and 89% respectively via subsequent enzymatic hydrolysis (cellulase loading of 20 FPU/g-biomass and substrate consistency of 2%). About 78% of the original polysaccharides were converted into fermentable sugars. Conclusions With the physicochemical functions in extrusion, the ATSE method can effectively overcome the recalcitrance of lignocellulose for the production of fermentable sugars from corn stover. This process can be considered as a promising pretreatment method due to its relatively low temperature (99°C), high biomass/liquid ratio (1/2) and satisfied total sugar yield (78%), despite further study is needed for process

  19. Periodical batch culture of the immobilized growing fungi Sporotrichum cellulophilum producing cellulase in the nonwoven materials

    SciTech Connect

    Tamada, M.; Kasai, N.; Kumakura, M.; Kaetsu, I.

    1986-08-01

    The thermophilic fungus Sporotrichum cellulophilum was immobilized with nonwoven materials for cellulase production. The cellulose powder concentration in the medium was an important factor controlling cellulase production. When the cellulase powder concentration in the nonwoven materials was more than 4%, cellulase production was suppressed. The growth of the immobilized fungi depended on the spaces in the nonwoven materials. Immobilized growing fungi were retained by the nonwoven materials, and the supernatant medium did not contain mycelia. The heat stability of the immobilized growing fungus was higher than that of the free fungus. The immobilized fungus gave the same FPA as the free mycelium, but the lag time for cellulase production in the immobilized fungus was longer. It was necessary for the medium to be changed in order to get the immobilized growing fungus to continue producing cellulase. In this instance there was no difference of lag time in comparsion with the free cells, and the supply of cellulose powder and polypepton was reduced to two-thirds. After 23 exchanges of the medium (2.6 mg cellulose powder/1 cubic cm nonwoven materials) FPA value was maintained. The periodic batch culture was continued for 69 days. 12 references.

  20. Evaluation of cellulases produced from four fungi cultured on furfural residues and microcrystalline cellulose.

    PubMed

    Liu, Hui-Qin; Feng, Yue; Zhao, Dan-Qing; Jiang, Jian-Xin

    2012-06-01

    Four fungal strains-Trichoderma viride, Aspergillus niger, Trichoderma koningii, and Trichoderma reesei-were selected for cellulase production using furfural residues and microcrystalline cellulose (MCC) as the substrates. The filter paper activity (FPA) of the supernatant from each fungus was measured, and the performance of the enzymes from different fungal strains was compared. Moreover, the individual activities of the three components of the cellulase system, i.e., β-glucosidase, endoglucanase, and exoglucanase were evaluated. T. koningii showed the highest activity (27.81 FPU/ml) on furfural residues, while T. viride showed an activity of 21.61 FPU/ml on MCC. The FPA of the crude enzyme supernatant from T. koningii was 30% higher on furfural residues than on MCC. T. koningii and T. viride exhibited high stability and productivity and were chosen for cellulases production. The crystallinity index (CrI) of the furfural residues varied after digested by the fungi. The results indicated differences in the functioning of the cellulase system from each fungus. In the case of T. koningii, T. reesei and T. viride, furfural residues supported a better environment for cellulase production than MCC. Moreover, the CrI of the furfural residues decreased, indicating that this material was largely digested by the fungi. Thus, our results suggest that it may be possible to use the cellulases produced from these fungi for the simultaneous saccharification and fermentation of lignocellulosic materials in ethanol production.

  1. 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.

  2. Effect of pH on cellulase production of Trichoderma reesei RUT C30.

    PubMed

    Juhász, Tamás; Szengyel, Zsolt; Szijártó, Nóra; Réczey, Kati

    2004-01-01

    Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, beta-glucosidase production was increased greatly.

  3. Cellulase production and saccharification of rice straw by the mutant strain Hypocrea koningii RSC1.

    PubMed

    Palaniyandi, Sasikumar Arunachalam; Yang, Seung Hwan; Suh, Joo-Won

    2014-01-01

    The production of cellulase using solid-state fermentation of rice straw by the mutant strain Hypocrea koningii RSC1 was studied. Optimization of culture conditions, such as the nitrogen source, pH, and temperature, resulted in a maximum filter paper cellulase activity of 44.15 U g(-1) substrate, a carboxymethylcellulase activity of 324.6 U g(-1) substrate, and a β-glucosidase activity of 7.45 U g(-1) substrate. Saccharification of untreated, 1% H(2)SO(4)-treated, and 2.5% NaOH-treated rice straw using the RSC1 cellulase resulted in 19, 17, and 34 g L(-1) of reducing sugar, respectively. Further studies on the morphological and compositional changes of rice straw upon treatment with the cellulase by scanning electron microscopy analysis and Fourier transform infrared spectroscopy revealed the disruption of the arrangement of fibers and changes in the functional groups that occur in cellulose. X-ray diffraction analysis revealed a reduction in crystallinity of the rice straw upon treatment with the cellulase. Our study shows that H. koningii RSC1 could be a good choice for the production of cellulase and reducing sugars from rice straw.

  4. 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. PMID:27560367

  5. 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. PMID:26410224

  6. Effect of different carbon sources on cellulase production by Hypocrea jecorina (Trichoderma reesei) strains

    PubMed Central

    Dashtban, Mehdi; Buchkowski, Robert; Qin, Wensheng

    2011-01-01

    The ascomycete Hypocrea jecorina, an industrial (hemi)cellulase producer, can efficiently degrade plant polysaccharides. At present, the biology underlying cellulase hyperproduction of T. reesei, and the conditions for the enzyme induction, are not completely understood. In the current study, three different strains of T. reesei, including QM6a (wild-type), and mutants QM9414 and RUT-C30, were grown on 7 soluble and 7 insoluble carbon sources, with the later group including 4 pure polysaccharides and 3 lignocelluloses. Time course experiments showed that maximum cellulase activity of QM6a and QM9414 strains, for the majority of tested carbon sources, occurred at 120 hrs, while RUT-C30 had the greatest cellulase activity around 72 hrs. Maximum cellulase production was observed to be 0.035, 0.42 and 0.33 µmol glucose equivalents using microcrystalline celluloses for QM6a, QM9414, and RUTC-30, respectively. Increased cellulase production was positively correlated in QM9414 and negatively correlated in RUT-C30 with ability to grow on microcrystalline cellulose. PMID:22003440

  7. Differential expression of cellulases and xylanases by Cellulomonas flavigena grown on different carbon sources.

    PubMed

    Sánchez-Herrera, Leticia M; Ramos-Valdivia, Ana C; de la Torre, Mayra; Salgado, Luis M; Ponce-Noyola, Teresa

    2007-12-01

    The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.

  8. 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

  9. Production of cellulase enzyme by Trichoderma reesei Cefl9 and its application in the production of bio-ethanol.

    PubMed

    Kumar, M Ravi; Kumaran, M D Bala; Balashanmugam, P

    2014-05-01

    The present study aimed at the production of cellulase enzyme from the cellulolytic fungi Trichoderma reesei CEF19 and subsequent application of the cellulase for the fermentation of ethanol. For the same, the cellulolytic fungi, Trichoderma reesei CEF19 was isolated and was allowed to produce cellulase enzyme using optimized conditions. The cellulase enzyme was extracted and purified with the help of ammonium sulphate precipitation, dialysis followed by ion exchange chromatography with DEAE-Sephadex column. The purified cellulase enzyme was characterized using SDS-PAGE analysis. The saccharification of the cellulosic substrates was done using the cellulase enzyme. The fermentation of saccharified cellulosic substrates into ethanol was carried out using Saccharomyces cerevisiae. From the results obtained, rice straw was found to be the better source for the ethanol production when compared to the other substrates.

  10. Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies.

    PubMed

    Kumar, Rajeev; Wyman, Charles E

    2009-01-01

    Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO(2)) and pure Avicel glucan was measured at 4 degrees C, as were adsorption and desorption of cellulase and adsorption of beta-glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (sigma(solids) = 195 mg/g solid) followed by dilute acid (sigma(solids) = 170.0 mg/g solid) and lime pretreated solids (sigma(solids) = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (sigma(solids) = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (sigma(cellulose) = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (sigma(lignin) = 57 mg/g lignin) followed by dilute acid lignin (sigma(lignin) = 74 mg/g lignin). AFEX lignin also had the lowest beta-glucosidase capacity (sigma(lignin) = 66.6 mg/g lignin), while lignin from SO(2) (sigma(lignin) = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO(2) followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute

  11. 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. PMID:23702163

  12. 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.

  13. The effect of alkaline pretreatment methods on cellulose structure and accessibility

    DOE PAGES

    Bali, Garima; Meng, Xianzhi; Deneff, Jacob I.; Sun, Qining; Ragauskas, Arthur J.

    2014-11-24

    The effects of different alkaline pretreatments on cellulose structural features and accessibility are compared and correlated with the enzymatic hydrolysis of Populus. The pretreatments are shown to modify polysaccharides and lignin content to enhance the accessibility for cellulase enzymes. The highest increase in the cellulose accessibility was observed in dilute sodium hydroxide, followed by methods using ammonia soaking and lime (Ca(OH)2). The biggest increase of cellulose accessibility occurs during the first 10 min of pretreatment, with further increases at a slower rate as severity increases. Low temperature ammonia soaking at longer residence times dissolved a major portion of hemicellulose andmore » exhibited higher cellulose accessibility than high temperature soaking. Moreover, the most significant reduction of degree of polymerization (DP) occurred for dilute sodium hydroxide (NaOH) and ammonia pretreated Populus samples. The study thus identifies important cellulose structural features and relevant parameters related to biomass recalcitrance.« less

  14. The effect of alkaline pretreatment methods on cellulose structure and accessibility

    SciTech Connect

    Bali, Garima; Meng, Xianzhi; Deneff, Jacob I.; Sun, Qining; Ragauskas, Arthur J.

    2014-11-24

    The effects of different alkaline pretreatments on cellulose structural features and accessibility are compared and correlated with the enzymatic hydrolysis of Populus. The pretreatments are shown to modify polysaccharides and lignin content to enhance the accessibility for cellulase enzymes. The highest increase in the cellulose accessibility was observed in dilute sodium hydroxide, followed by methods using ammonia soaking and lime (Ca(OH)2). The biggest increase of cellulose accessibility occurs during the first 10 min of pretreatment, with further increases at a slower rate as severity increases. Low temperature ammonia soaking at longer residence times dissolved a major portion of hemicellulose and exhibited higher cellulose accessibility than high temperature soaking. Moreover, the most significant reduction of degree of polymerization (DP) occurred for dilute sodium hydroxide (NaOH) and ammonia pretreated Populus samples. The study thus identifies important cellulose structural features and relevant parameters related to biomass recalcitrance.

  15. Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases.

    PubMed

    Klinger, Johannes; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

  16. Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases

    PubMed Central

    Klinger, Johannes; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host. PMID:26648951

  17. 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.

  18. Comparison of extracellular cellulase activities of ClosTridium thermocellum LQRI and trichoderma reesei QM9414

    SciTech Connect

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

    1981-08-01

    The crude extracellular cellulase of Clostridium thermocellum LQRI (virgin strain) was very active and solubilized microcrystalline cellulose at one-half the rate observed for the extracellular cellulase of Trichoderma reesei QM9414 (mutant strain). Clostridium thermocellum cellulase activity differed considerably from that of Trichoderma reesei as follows: higher endoglucanase/exoglucanase activity ratio; absence of extracellular cellobiase or beta-xylosidase activity; long-chain oligosaccharides instead of short-chain oligosaccharides as initial (15-min) hydrolytic products on microcrystalline cellulose; mainly cellobiose or xylobiose as long-term (24-h) hydrolysis products of Avicel and MN300 or xylan; and high activity and stability at 60 to 70 degrees Celcius. Under optimized reaction conditions, the kinetic properties (V max, 0.4 mu mol/min per mg of protein; energy of activation, 33 kJ; temperature coefficient, 1.8) of Clostridium thermocellum cellulose-solubilizing activity were comparable to those reported for Trichoderma reesei, except that the dyed Avicel concentration at half-maximal velocity was twofold higher (182 mu M). The cellulose-solubilizing activity of the two crude cellulases differed considerably in response to various enzyme inhibitors. Most notably, Ag/sup 2 +/ and Hg/sup 2 +/ effectively inhibited Clostridium thermocellum but not Trichoderma reesei cellulase at less than 20 mu M, whereas Ca/sup 2 +/, Mg/sup 2 +/, and Mn/sup 2 +/ inhibited Trichoderma reesei but not Clostridium thermocellum cellulase at greater than 10 mM. Both enzymes were inhibited by Cu/sup 2 +/ (greater than 20 mM), Zn2+ (greater than 10 mM), and ethylene glycol-bis (beta-aminoethyl ether)-N, N-tetraacetic acid (greater than 10 mM). The overal rates of cellooligosaccharide degradation were higher for Trichoderma reesei than for Clostridium thermocellum cellulase, except that the rates of conversion of cellohexaose to cellotrisse were equivalent.

  19. Wastepaper hydrolysate as soluble inducing substrate for cellulase production in continuous culture of trichoderma reesei

    PubMed

    Ju; Afolabi

    1999-01-01

    The enzymatic hydrolysate of wastepaper was evaluated for its cellulase-inducing capability and production characteristics in continuous culture of Trichoderma reesei RUT C30. Under the study conditions, i.e., pH 5.0, temperature 25 degreesC, and typical medium C:N ratio, the apparent cell yield constant was found to be 0. 76 (g of dry cell weight/g of reducing sugar), and the maximum specific cell growth rate was 0.26 h-1. The study on the effects of medium C:N ratio confirmed an important role of N sources in the cellulase synthesis. The cellulase production decreased significantly when the feed concentrations of N sources were reduced. An experiment at pH 7.5 with 4-fold N source concentrations also led to poorer cellulase production. When compared with cellulose, the wastepaper hydrolysate was found to have similar cellulase-inducing strength and to induce an apparently complete set of cellulase components. The hydrolysate was also concluded to be a better soluble inducer than sophorose. While comparable at a low dilution rate (0.012 h-1), the specific cellulase productivities of the hydrolysate-supported and the sophorose-induced systems exhibited opposite trends with increasing dilution rates. The specific productivity in sophorose-induced systems decreased with an increase in the dilution rate. On the other hand, with increasing dilution rate the specific productivity in the hydrolysate-supported systems increased from 2.2 FPU/g.h at D = 0.012 h-1 to 12.2 FPU/g.h at D = 0.122 h-1 before beginning to decline. The initial increasing trend was attributed to the higher concentrations of inducing oligomer intermediates at larger dilution rates.

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

    SciTech Connect

    Not Available

    2012-06-01

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

  1. 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

  2. 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.

  3. 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

  4. Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance.

    PubMed

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

    2015-12-01

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

  5. Cellulase recycling in biorefineries--is it possible?

    PubMed

    Gomes, Daniel; Rodrigues, Ana Cristina; Domingues, Lucília; Gama, Miguel

    2015-05-01

    On a near future, bio-based economy will assume a key role in our lives. Lignocellulosic materials (e.g., agroforestry residues, industrial/solid wastes) represent a cheaper and environmentally friendly option to fossil fuels. Indeed, following suitable processing, they can be metabolized by different microorganisms to produce a wide range of compounds currently obtained by chemical synthesis. However, due to the recalcitrant nature of these materials, they cannot be directly used by microorganisms, the conversion of polysaccharides into simpler sugars being thus required. This conversion, which is usually undertaken enzymatically, represents a significant part on the final cost of the process. This fact has driven intense efforts on the reduction of the enzyme cost following different strategies. Here, we describe the fundamentals of the enzyme recycling technology, more specifically, cellulase recycling. We focus on the main strategies available for the recovery of both the liquid- and solid-bound enzyme fractions and discuss the relevant operational parameters (e.g., composition, temperature, additives, and pH). Although the efforts from the industry and enzyme suppliers are primarily oriented toward the development of enzyme cocktails able to quickly and effectively process biomass, it seems clear by now that enzyme recycling is technically possible. PMID:25820600

  6. Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance.

    PubMed

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

    2015-12-01

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

  7. Studies on carboxymethyl cellulase produced by an alkalothermophilic actinomycete.

    PubMed

    George, S P; Ahmad, A; Rao, M B

    2001-04-01

    A novel alkalothermophilic actinomycete having optimum growth at pH 9 and 50 degrees C was isolated from self-heating compost from the Barabanki district of Uttar Pradesh, India. Based on its morphology, susceptibility of spores to heat and novobiocin, guaninecytosine content of chromosomal DNA and cell wall composition, the organism was classified under Thermomonospora. The alkalothermophilic actinomycete produced 23 IU/ml carboxymethyl cellulase (CMCase). The CMCase was purified by fractional ammonium sulphate precipitation followed by cellulose affinity chromatography and Sephacryl S-200 gel filtration. The CMCase had a molecular weight of 38 KD and pI of 4.1. The enzyme exhibited optimum activity at pH 5 and temperature 50 degrees C. The CMCase showed pH stability in the range 7-10. The enzyme retained 100% activity at 50 degrees C for 72 h and had half-lives of 7 and 3 h at 60 degrees C and 70 degrees C, respectively. The CMCase was stable in the presence of commercial detergents such as Ariel, Henko and Surf Excel, indicating its potential as an additive to laundry detergents.

  8. Surface activation of dyed fabric for cellulase treatment.

    PubMed

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

    2011-10-01

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

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

    SciTech Connect

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

    2011-10-07

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

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

    PubMed

    Zimoch-Korzycka, Anna; Bobak, Łukasz; Jarmoluk, Andrzej

    2016-01-01

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

  11. 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

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

    PubMed

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

    2015-01-01

    The purpose of this study was to produce a higher amount of cellulase by using an alternative carbon source, such as banana agrowaste, and to optimize the fermentation parameters for a high yield. In the present study, cellulase-producing Penicillium was isolated from a decaying wood sample. Different nutritional and environmental factors were investigated to assess their effect on cellulase production. The highest crude enzyme production was observed at a pH 6.0 and a temperature of 28°C in a medium that was supplemented with banana agrowaste as the carbon source. Pretreatment with 2N NaOH, at 7% substrate (banana agrowaste) concentration yielded the highest cellulase activity. Further to this, the effect of other parameters such as inoculum age, inoculum size, static and agitated conditions were also studied. It is concluded that Penicillium oxalicum is a powerful cellulase-producer strain under our tested experimental conditions using banana agrowaste as the carbon source. PMID:26018499

  13. 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.

  14. Genome shuffling of Aspergillus glaucus HGZ-2 for enhanced cellulase production.

    PubMed

    Zhao, Yuping; Jiang, Changxing; Yu, Hupeng; Fang, Fang; Yang, Jingzhu

    2014-10-01

    The production of cellulase from Aspergillus glaucus HGZ-2 was improved by using genome shuffling. The starting populations, obtained by UV irradiation, were subjected to recursive protoplast fusion. The optimal conditions for protoplast formation and regeneration were 7 mg/ml snailase and 5 mg/ml cellulase at 34 °C for 3.0 h using 0.7 M NaCl as an osmotic stabilizer. The protoplasts were inactivated under UV for 30 min or heated at 50 °C for 50 min, and a fusant probability of about 100 % was observed. The positive colonies were created by fusing the inactivated protoplasts. The optimal conditions for protoplast fusion were PEG6000 concentration of 35 %, CaCl2 concentration of 0.02 M, and incubation time of 12 min. After two rounds of genome shuffling, one strain (Y) was obtained. Its filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase) activity reached 71 and 70 U/ml, respectively, which were increased by 1.95-fold and 1.72-fold in comparison with that of its ancestor strain. The results indicated that genome shuffling was an efficient means for the improved production of cellulases by A. glaucus HGZ-2.

  15. Purification and characterization of a thermophilic cellulase from a novel cellulolytic strain, Paenibacillus barcinonensis.

    PubMed

    Asha, Balachandrababu Malini; Revathi, Masilamani; Yadav, Amit; Sakthivel, Natarajan

    2012-11-01

    A novel bacterial strain, MG7, with high cellulase activity was isolated and identified by morphological characteristics and molecular phylogeny analysis as Paenibacillus barcinonensis. Maximum production of cellulase by MG7 was observed at pH 7.0 and 35°C. The enzyme was purified with a specific activity of 16.88 U/mg, the cellulase activity was observed in a zymogram, and its molecular mass (58.6 kDa) was confirmed by SDS-PAGE. The purified enzyme showed maximum activity at pH 6.0 and 65°C and degraded cellulosic substrates such as carboxy methyl cellulose (CMC), Avicel, filter paper, and beta-glucan. The enzyme showed stability with 0.5% concentration of various surfactants. The K(m) and V(max) of cellulase for CMC and Avicel were found to be 0.459 mg/ml and 10.46 mg/ml/h, and 1.01 mg/ml and 10.0 mg/ml/h, respectively. The high catalytic activity and its stability to temperature, pH, surfactants, and metal ions indicated that the cellulase enzyme by MG7 is a good candidate for biotechnological applications.

  16. 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. PMID:22644643

  17. Increased production of cellulase of Trichoderma sp. by pH cycling and temperature profiling

    SciTech Connect

    Mukhopadhyay, S.N.; Malik, R.K.

    1980-11-01

    Cultivation of Trichoderma reesei QM 9414 on 3% (w/v) cellulose medium (C/N ratio equal to 8.5) produced 4.5 IU/ml cellulase in 180 hr at a cell growth of 8.0 g/liter (0.266 g cell/g cellulose). It corresponded to an average cellulase productivity 25.0 IU/liter/hr (3.5 IU/g cell/hr. In the same medium 9.5 g/liter cell mass (0.316 g cell/g cellulose), 6.2 IU/ml cellulase, and 38.75 IU/liter/hr (4.0 IU/g cell/hr) cellulase productivity could be obtained using pH cycling condition during cultivation. Cell mass, cellulase yield, and productivity were further increased to 10.0 g/liter, 7.2 IU/ml and 44.0 IU/liter/hr (4.5 IU/g cell/hr), respectively, by simultaneous pH cycling and temperature profiling strategy. Results are described.

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

    PubMed

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

    2012-03-01

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

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

    PubMed

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

    2014-08-01

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

  20. Distribution and evolution of glycoside hydrolase family 45 cellulases in nematodes and fungi

    PubMed Central

    2014-01-01

    Background Horizontal gene transfer (HGT) has been suggested as the mechanism by which various plant parasitic nematode species have obtained genes important in parasitism. In particular, cellulase genes have been acquired by plant parasitic nematodes that allow them to digest plant cell walls. Unlike the typical glycoside hydrolase (GH) family 5 cellulase genes which are found in several nematode species from the order Tylenchida, members of the GH45 cellulase have only been identified in a cluster including the families Parasitaphelenchidae (with the pinewood nematode Bursaphelenchus xylophilus) and Aphelenchoididae, and their origins remain unknown. Results In order to investigate the distribution and evolution of GH45 cellulase genes in nematodes and fungi we performed a wide ranging screen for novel putative GH45 sequences. This revealed that the sequences are widespread mainly in Ascomycetous fungi and have so far been found in a single major nematode lineage. Close relationships between the sequences from nematodes and fungi were found through our phylogenetic analyses. An intron position is shared by sequences from Bursaphelenchus nematodes and several Ascomycetous fungal species. Conclusions The close phylogenetic relationships and conserved gene structure between the sequences from nematodes and fungi strongly supports the hypothesis that nematode GH45 cellulase genes were acquired via HGT from fungi. The rapid duplication and turnover of these genes within Bursaphelenchus genomes demonstrate that useful sequences acquired via HGT can become established in the genomes of recipient organisms and may open novel niches for these organisms to exploit. PMID:24690293

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

    PubMed

    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

    2012-06-01

    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.

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

    PubMed Central

    Zimoch-Korzycka, Anna; Bobak, Łukasz; Jarmoluk, Andrzej

    2016-01-01

    The aim of this study was to evaluate the impact of cellulase (C) on the biological activity of chitosan/hydroxypropyl methylcellulose (CH/HPMC) film-forming hydrosols. The hydrolytic activity of cellulase in two concentrations (0.05% and 0.1%) was verified by determination of the progress of polysaccharide hydrolysis, based on viscosity measurement and reducing sugar-ends assay. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging effect, the ferric reducing antioxidant power (FRAP), and microbial reduction of Pseudomonas fluorescens, Yersinia enterocolitica, Bacillus cereus, and Staphylococcus aureus were studied. During the first 3 h of reaction, relative reducing sugar concentration increased progressively, and viscosity decreased rapidly. With increasing amount of enzyme from 0.05% to 0.1%, the reducing sugar concentration increased, and the viscosity decreased significantly. The scavenging effect of film-forming solutions was improved from 7.6% at time 0 and without enzyme to 52.1% for 0.1% cellulase after 20 h of reaction. A significant effect of cellulase addition and reaction time on antioxidant power of the tested film-forming solutions was also reported. Film-forming hydrosols with cellulase exhibited a bacteriostatic effect on all tested bacteria, causing a total reduction. PMID:27608008

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

    PubMed

    Rodriguez-Gomez, Divanery; Hobley, Timothy John

    2013-11-01

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

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

    PubMed

    Zimoch-Korzycka, Anna; Bobak, Łukasz; Jarmoluk, Andrzej

    2016-01-01

    The aim of this study was to evaluate the impact of cellulase (C) on the biological activity of chitosan/hydroxypropyl methylcellulose (CH/HPMC) film-forming hydrosols. The hydrolytic activity of cellulase in two concentrations (0.05% and 0.1%) was verified by determination of the progress of polysaccharide hydrolysis, based on viscosity measurement and reducing sugar-ends assay. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging effect, the ferric reducing antioxidant power (FRAP), and microbial reduction of Pseudomonas fluorescens, Yersinia enterocolitica, Bacillus cereus, and Staphylococcus aureus were studied. During the first 3 h of reaction, relative reducing sugar concentration increased progressively, and viscosity decreased rapidly. With increasing amount of enzyme from 0.05% to 0.1%, the reducing sugar concentration increased, and the viscosity decreased significantly. The scavenging effect of film-forming solutions was improved from 7.6% at time 0 and without enzyme to 52.1% for 0.1% cellulase after 20 h of reaction. A significant effect of cellulase addition and reaction time on antioxidant power of the tested film-forming solutions was also reported. Film-forming hydrosols with cellulase exhibited a bacteriostatic effect on all tested bacteria, causing a total reduction. PMID:27608008

  5. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  6. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  7. Response to Comment on "Revealing nature's cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA".

    PubMed

    Brunecky, Roman; Alahuhta, Markus; Xu, Qi; Donohoe, Bryon S; Crowley, Michael F; Kataeva, Irina A; Yang, Sung-Jae; Resch, Michael G; Adams, Michael W W; Lunin, Vladimir V; Himmel, Michael E; Bomble, Yannick J

    2014-05-01

    Gusakov critiques our methodology for comparing the cellulolytic activity of the bacterial cellulase CelA with the fungal cellulase Cel7A. We address his concerns by clarifying some misconceptions, carefully referencing the literature, and justifying our approach to point out that the results from our study still stand. PMID:24812382

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

    PubMed

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

    2016-09-01

    Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development. PMID:27470141

  9. Recyclable Thermoresponsive Polymer-Cellulase Bioconjugates for Biomass Depolymerization

    PubMed Central

    Mackenzie, Katherine J.; Francis, Matthew B.

    2013-01-01

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

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

    SciTech Connect

    Wilson, D.B.

    1992-01-01

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

  11. Screening of cellulases for biofuel production: online monitoring of the enzymatic hydrolysis of insoluble cellulose using high-throughput scattered light detection.

    PubMed

    Jäger, Gernot; Wulfhorst, Helene; Zeithammel, Erik U; Elinidou, Efthimia; Spiess, Antje C; Büchs, Jochen

    2011-01-01

    A new prospective cellulase assay simultaneously combining high-throughput, online analysis and insoluble cellulosic substrates is described. The hydrolysis of three different insoluble cellulosic substrates, catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast), was monitored using the BioLector - allowing online monitoring of scattered light intensities in a continuously shaken microtiter plate. Cellulase activities could be quantitatively assayed using the BioLector. At low cellulase/cellulose ratios, the Michaelis-Menten parameters of the cellulase mixture were mainly affected by the crystallinity index of the cellulose. Here, the apparent maximum cellulase activities inversely correlated with the crystallinity index of the cellulose. At high cellulase/cellulose ratios the particle size of the cellulose, defining the external surface area accessible to the cellulases, was the key determining factor for cellulase activity. The developed technique was also successfully applied to evaluate the pH optimum of cellulases. Moreover, the non-hydrolytic deagglomeration of cellulose particles was investigated, for the first time, using high-throughput scattered light detection. In conclusion, this cellulase assay ideally links high-throughput, online analysis and realistic insoluble cellulosic substrates in one simple system. It will considerably simplify and accelerate fundamental research on cellulase screening.

  12. Hydrolysis of Chlorella by Cellulomonas sp. YJ5 cellulases and its biofunctional properties.

    PubMed

    Yin, Li-Jung; Jiang, Shann-Tzong; Pon, Shen-Hwei; Lin, Hsin-Hung

    2010-01-01

    Both 10% and 20% (w/w) Chlorella suspensions were hydrolyzed by 150 to 350 U/mL of cellulases from a 3-d cultivation of Cellulomonas sp. YJ5. Higher chlorophyll, reducing sugars and soluble proteins, and lower residual insoluble solid were observed on both samples after 30-min hydrolysis by various concentrations of cellulases at 50 °C. Decrease in insoluble solid, increases in soluble proteins, peptides and chlorophyll contents, and microscopic observation indicated obvious lysis of cell walls occurred during 60- to 180-min hydrolysis. Significant increases in soluble proteins, peptides, Fe(2+) chelating ability, trolox equivalent antioxidation capacity (TEAC), and reducing power was obtained after 3-h hydrolysis by 150 U/mL of cellulase. These data suggested that cellulolysis technology has high application potential in Chlorella industry.

  13. Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module

    PubMed Central

    Yao, Chaoxiang; Junaid, Muhammad; Lu, Zhenghui; Zhang, Houjin; Ma, Yanhe

    2015-01-01

    Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme. PMID:26562160

  14. Synthesizing a Cellulase like Chimeric Protein by Recombinant Molecular Biology Techniques

    PubMed Central

    Banerjee, Hirendra Nath; Krauss, Christopher; Smith, Valerie; Mahaffey, Kelly; Boston, Ava

    2016-01-01

    In order to meet the Renewable Fuels Standard demands for 30 billion gallons of biofuels by the end of 2020, new technologies for generation of cellulosic ethanol must be exploited. Breaking down cellulose by cellulase enzyme is very important for this purpose but this is not thermostable and degrades at higher temperatures in bioreactors. Towards creation of a more ecologically friendly method of rendering bioethanol from cellulosic waste, we attempted to produce recombinant higher temperature resistant cellulases for use in bioreactors. The project involved molecular cloning of genes for cellulose-degrading enzymes based on bacterial source, expressing the recombinant proteins in E. coli and optimizing enzymatic activity. We were able to generate in vitro bacterial expression systems to produce recombinant His-tag purified protein which showed cellulase like activity. PMID:27468362

  15. 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.

  16. Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module.

    PubMed

    Zhang, Huaidong; Zhang, Guimin; Yao, Chaoxiang; Junaid, Muhammad; Lu, Zhenghui; Zhang, Houjin; Ma, Yanhe

    2015-01-01

    Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme.

  17. Cellulase-assisted extraction and antioxidant activity of the polysaccharides from garlic.

    PubMed

    Pan, Saikun; Wu, Shengjun

    2014-10-13

    In the present study, the polysaccharides were prepared from garlic by using a cellulase-assisted extraction method and the antioxidant activity of garlic polysaccharides (GPs) was evaluated. To improve the yield of GPs, the influences of the several factors such as extraction time, temperature, pH, and cellulase amount on the extraction efficiency were studied. The optimal conditions for extraction of GPs were determined as follows: time, 80 min; temperature, 45 °C; pH, 5; cellulase amount, 8000 U/g. Under the optimised extraction conditions, the yield of GPS reached up to 35.34%. The GPs product exhibited strong antioxidant activity including hydroxyl radical scavenging activity, 2,2-diphenyl-β-picrylhydrazyl radical scavenging activity, and reducing power. The results suggest that the GPs could be used as potential antioxidants.

  18. Ethanol production from xylan-removed sugarcane bagasse using low loading of commercial cellulase.

    PubMed

    Li, Jingbo; Zhou, Pengfei; Liu, Hongmei; Wu, Kejing; Xiao, Wenjuan; Gong, Yingxue; Lin, Jianghai; Liu, Zehuan

    2014-07-01

    Xylan was always extracted as the feedstock for xylooligosaccharides production. The xylan-removed residue may contain high content of cellulose and thus had a possibility to be converted into ethanol. After soaked in 12% of NaOH at room temperature overnight, solubilization of cellulose, xylan, and lignin was 4.64%, 72.06%, and 81.87% respectively. The xylan-removed sugarcane bagasse (XRSB) was enzymatically hydrolyzed by using decreased cellulase loadings. The results showed that 7.5 FPU/g cellulose could obtain a cellulose conversion yield of 82%. Increasing the cellulase loading did not result in higher yield. Based on this, bioethanol production was performed using 7.5 FPU/g cellulose by employing fed-batch fermentation mode. The final ethanol concentration reached 40.59 g/L corresponding to 74.2% of the theoretical maximum. The high titer ethanol and low cellulase loading may reduce the overall cost.

  19. The addition of accessory enzymes enhances the hydrolytic performance of cellulase enzymes at high solid loadings.

    PubMed

    Hu, Jinguang; Chandra, Richard; Arantes, Valdeir; Gourlay, Keith; van Dyk, J Susan; Saddler, Jack N

    2015-06-01

    The pretreatment process used and the nature of the biomass feedstock will influence the role that accessory enzymes can play in synergistically interacting with cellulases to effectively deconstruct the substrate. The work reported here assessed the possible boosting effects of the xylanase and lytic polysaccharide monooxygenase (AA9, formerly known as GH61) on the hydrolytic potential of cellulase enzyme mixtures during hydrolysis of steam pretreated poplar and corn stover at high (10-20% w/v) substrate concentrations. A higher proportion of xylanase was required when the substrate had a relatively high xylan content and at high substrate concentrations. In contrast, a relatively small amount of AA9 (about 2 mg/g cellulose) was enough, regardless of the nature or concentration of the substrate. The overall protein loading required to achieve effective hydrolysis of high concentrations of pretreated biomass substrates could be substantially reduced by optimizing the ratio of enzymes in the "cellulase" mixture.

  20. Increasing yield of nanocrystalline cellulose preparation process by a cellulase pretreatment.

    PubMed

    Beltramino, Facundo; Roncero, M Blanca; Vidal, Teresa; Torres, Antonio L; Valls, Cristina

    2015-09-01

    In this work the introduction of a cellulase treatment prior to NCC isolation was assessed. NCC was produced using sulfuric acid at two different concentrations (62 and 64% wt.). The effect of pore size for filtration step was also assessed. The smaller acid dose leaded to yields up to 65-70% and average size up to 160 nm. It also produced crystals with reduced sulfur content (0.6-1%). Cellulase pretreatment influenced NCC characteristics, as it increased overall yield a 12%, increased average particle size around 35 nm and reduced NCC sulfur content up to a 0.8%. We found that different conditions of enzymatic treatments led to quantitative differences on their effects on NCC. Acetate buffer used for enzymatic treatments was found to counteract effects of acid. The evidence presented in this work suggested that pretreating fibers with this cellulase represents a very interesting option to partially replace chemicals on NCC isolation.

  1. Application of cellulase for the modification of corn stalk: leading to oil sorption.

    PubMed

    Peng, Dan; Lan, Zhoulin; Guo, Chuling; Yang, Chen; Dang, Zhi

    2013-06-01

    In this work, a new biotechnological procedure was developed using cellulase as a modifier to produce oil sorbent from corn stalk (CMCS). Cellulase treatment of raw corn stalk (RCS) with enzyme loading of 100 U/g at 45°C for 6h resulted in high oil sorption capacity. The sorption capacities of vegetable oil, diesel and crude oil by CMCS were 18.47, 16.15 and 27.23 g/g, respectively, which were found to be much higher than RCS. XRD, BET and SEM were applied to characterize RCS and CMCS. The effects of sorbent dose (0.1-0.5 g), initial oil amount (5-30 g), and the sorption kinetics were also studied. This work demonstrated that corn stalk modified by cellulase is an efficient and environment-friendly biosorbent for the removal of spilled oil. PMID:23618599

  2. Expression of cellulase genes in Rhodobacter capsulatus by use of plasmid expression vectors.

    PubMed Central

    Johnson, J A; Wong, W K; Beatty, J T

    1986-01-01

    Broad-host-range plasmid vectors were constructed for expression of heterologous genes in the photosynthetic bacterium Rhodobacter capsulatus. These plasmids utilize an RK2-derived replicon for maintenance and conjugative transfer and the R. capsulatus rxcA promoter to obtain transcription of genes within appropriately positioned DNA fragments. The expression vectors were used to obtain synthesis of endoglucanase and exoglucanase in R. capsulatus from cellulase genes present on exogenously derived DNA fragments. The cellulase genes were expressed either by use of their native translation initiation signals or by in-frame fusion with the rxcA B870 beta gene translation initiation signals to form a hybrid protein. The level of cellulase gene expression was found to be modulated in response to the extent of aeration of plasmid host cultures. Images PMID:3090019

  3. 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. PMID:27427671

  4. 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.

  5. Development of alkaline fuel cells.

    SciTech Connect

    Hibbs, Michael R.; Jenkins, Janelle E.; Alam, Todd Michael; Janarthanan, Rajeswari; Horan, James L.; Caire, Benjamin R.; Ziegler, Zachary C.; Herring, Andrew M.; Yang, Yuan; Zuo, Xiaobing; Robson, Michael H.; Artyushkova, Kateryna; Patterson, Wendy; Atanassov, Plamen Borissov

    2013-09-01

    This project focuses on the development and demonstration of anion exchange membrane (AEM) fuel cells for portable power applications. Novel polymeric anion exchange membranes and ionomers with high chemical stabilities were prepared characterized by researchers at Sandia National Laboratories. Durable, non-precious metal catalysts were prepared by Dr. Plamen Atanassovs research group at the University of New Mexico by utilizing an aerosol-based process to prepare templated nano-structures. Dr. Andy Herrings group at the Colorado School of Mines combined all of these materials to fabricate and test membrane electrode assemblies for single cell testing in a methanol-fueled alkaline system. The highest power density achieved in this study was 54 mW/cm2 which was 90% of the project target and the highest reported power density for a direct methanol alkaline fuel cell.

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

    PubMed Central

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

    1997-01-01

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

  7. Phytobezoar by aloe vera as long term complication after oesophagectomy resolved using cellulase

    PubMed Central

    Pinos, Nathalie; Moreno-Merino, Sergio; Congregado, Miguel

    2015-01-01

    Bezoars are uncommon diseases caused by the presence of indigestible mass of strange material in the gastrointestinal tract. Gold-standard treatment remains unclear and there are not clinical guidelines to follow. We present a very rare case of 53-year-old man suffering phytobezoar in a gastroplasty after oesophagectomy due to aloe vera ingestion as natural medicine. Finally it was solved with cellulase. Therefore, this is a scarcely complication after esophagectomy. Cellulase is a very good option to treat phytobezoar avoiding reintervention in this kind of patient. PMID:26094056

  8. 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.

  9. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2009-11-01

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

  11. 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.

  12. Method for the detection and differentiation of cellulase components in polyacrylamide gels

    SciTech Connect

    Bartley, T.D.; Murphy-Holland, K.; Eveleigh, D.E.

    1984-01-01

    Endoglucanase and exoglucanase components of cellulase can be detected and differentiated after polyacrylamide gel electrophoresis by performing activity stains. Endoglucanase activity was visualized in carboxymethyl cellulose agar replicas of gels by staining with Congo red. General ..beta..-1,4-glucanase activity was located by soaking the gel in a solution of NaBH/sub 4/-reduced cellulo-oligosaccharides, and detecting the formation of reducing sugars by reaction with triphenyl tetrazolium chloride. Endoglucanases are active in both assays, while exoglucanases can be distinguished by their activity in the cellulo-oligosaccharide assay only. This methodology has facilitated the purification and characterization of cellulase components from Trichoderma reesei and Microbispora bispora.

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

    PubMed

    Kanellis, A K; Kalaitzis, P

    1992-02-01

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

  14. Multigene families of Cellulomonas flavigena encoding endo-beta-1,4-glucanases (CM-cellulases).

    PubMed

    Akhtar, M W; Duffy, M; Dowds, B C; Sheehan, M C; McConnell, D J

    1988-12-30

    Multiple genes coding for endo-beta-1,4-glucanases (CM-cellulases) have been isolated from a newly discovered highly cellulolytic strain of Cellulomonas flavigena. Clones of C. flavigena DNA were isolated in Escherichia coli and screened for gene expression on CM-cellulose plates staining with congo red. Six clones produced CM-cellulase activity as detected in liquid assays, and on activity gels. They fell into three groups within which the sequences cross-hybridised. There were small differences in the pH and temperature optima of the enzymes encoded by representatives of the three groups of clones.

  15. 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

  16. Structural changes and enzymatic response of Napier grass (Pennisetum purpureum) stem induced by alkaline pretreatment.

    PubMed

    Phitsuwan, Paripok; Sakka, Kazuo; Ratanakhanokchai, Khanok

    2016-10-01

    Napier grass is a promising energy crop in the tropical region. Feasible alkaline pretreatment technologies, including NaOH, Ca(OH)2, NH3, and alkaline H2O2 (aH2O2), were used to delignify lignocellulose with the aim of improving glucose recovery from Napier grass stem cellulose via enzymatic saccharification. The influences of the pretreatments on structural alterations were examined using SEM, FTIR, XRD, and TGA, and the relationships between these changes and the enzymatic digestibility of cellulose were addressed. The extensive removal of lignin (84%) in NaOH-pretreated fibre agreed well with the high glucan conversion rate (94%) by enzymatic hydrolysis, while the conversion rates for fibre pretreated with Ca(OH)2, NH3, and aH2O2 approached 60%, 51%, and 42%, respectively. The substantial solubilisation of lignin created porosity, allowing increased cellulose accessibility to cellulases in NaOH-pretreated fibre. In contrast, high lignin content, lignin redeposition on the surface, and residual internal lignin and hemicellulose impeded enzymatic performance in Ca(OH)2-, NH3-, and aH2O2-pretreated fibres, respectively.

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

    PubMed

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

    2015-12-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. 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. PMID:26883662

  20. Enzymatic Saccharification of Lignocellulosic Residues by Cellulases Obtained from Solid State Fermentation Using Trichoderma viride

    PubMed Central

    Sartori, Tanara; Tibolla, Heloisa; Prigol, Elenizi; Colla, Luciane Maria; Costa, Jorge Alberto Vieira; Bertolin, Telma Elita

    2015-01-01

    The aim of this study was to verify the viability of lignocellulosic substrates to obtain renewable energy source, through characterization of the cellulolytic complex, which was obtained by solid state fermentation using Trichoderma viride. Enzymatic activity of the cellulosic complex was measured during saccharification of substrates filter paper, eucalyptus sawdust, and corncob, and compared with the activity of commercial cellulase. The characterization of the enzymes was performed by a 22 Full Factorial Design, where the pH and temperature were the variables of study. Enzymatic saccharification of different substrates appearedviable until 12 to be viable until 12 h; after this period the activity decreased for both enzymatic forms (cellulolytic complex and commercial cellulase). The enzymatic activity of the commercial cellulase was favored with the use of corncob as substrate, while the cellulolytic complex does not show any difference in its specificity by the substrates studied. The largest activities of both enzymes were obtained in the temperature and pH range between 40°C and 50°C and 4.8 and 5.2, respectively. The cellulolytic complex obtained appeared to be viable for the saccharification of lignocellulosic residues compared with the commercial cellulase. PMID:26137476

  1. 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.

  2. 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…

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

    PubMed

    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.

  4. Genome sequence and annotation of Trichoderma parareesei, the ancestor of the cellulase producer Trichoderma reesei

    DOE PAGES

    Yang, Dongqing; Pomraning, Kyle; Kopchinskiy, Alexey; Karimi, Aghcheh Razieh; Atanasova, Lea; Chenthamara, Komal; Baker, Scott E.; Zhang, Ruifu; Shen, Qirong; Freitag, Michael; et al

    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.

  5. Cellulase a key enzyme in fermentation: Annual report, 1985--1986

    SciTech Connect

    Eveleigh, D.E.; Macmillan, J.D.

    1986-05-04

    Many microbial cellulase systems are comprised of multiple components namely, endoglucanase (EG), cellobiohydrolase (CBH), and betaglucosidabe. These three types of enzyme of the cellulase complex act synergistically but their mechanism of interaction in the hydrolysis of crystalline cellulose is poorly understood. As monoclonal antibodies (McAb) can be used as both snesitive and specific protein probes, we proposed the preparation of McAb specific for cellulases components, which should allow direct analysis of these enzymes. We have been generating murine McAb that react specifically with cellobiohydrolases and endoglucanases. To date a McAb specific for a fungal (Trichoderma reesei) cellobiohydrolase I (CBH I) that does not show cross reactivity toward endoglucanases, has been obtained (Riskeet et al; 1986a). This McAb preparation has been used for purification of CBH I via affinity chromatography. Thus the purified McAb to CBH I was immobilized on CnBr-Sepharose-4b to yield an immunomatrix which was analogous studies are also being developed with the cellulase form Microbispora bispora. 5 refs.

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

    PubMed

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

    2015-12-01

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

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

    PubMed

    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

  8. 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

  9. 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. PMID:23544556

  10. 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.

  11. Novel cellulases from an extremophilic filamentous fungi Penicillium citrinum: production and characterization.

    PubMed

    Dutta, Tanmay; Sahoo, Rupam; Sengupta, Rajib; Ray, Sougata Sinha; Bhattacharjee, Arindam; Ghosh, Sanjay

    2008-04-01

    The enzymatic hydrolysis of cellulose has potential economical and environment-friendly applications. Therefore, discovery of new extremophilic cellulases is essential to meet the requirements of industry. Penicillium citrinum (MTCC 6489) that was previously isolated from soil in our laboratory, produced alkali tolerant and thermostable cellulases. Endoglucanase and filter paper activity hydrolase (FPAse) production of P. citrinum were studied using wheat bran substrate in solid state and submerged culture. Zymogram analysis of endoglucanase revealed the presence of two isoforms differing in molecular weight. One of them was 90 kDa and other one was 38 kDa. Partially purified endoglucanase showed two different peaks at pH 5.5 and 8.0, respectively, in its pH optima curve. But FPase showed only one peak (at pH 6.5) in its pH optima curve. Cellulase of P. citrinum is thermostable in nature. The present work reports for the first time, the alkali stable cellulase from alkali tolerant fungus Penicillium citrinum. Thermostable endoglucanase from P. citrinum may have potential effectiveness as additives to laundry detergents.

  12. Enzymatic Saccharification of Lignocellulosic Residues by Cellulases Obtained from Solid State Fermentation Using Trichoderma viride.

    PubMed

    Sartori, Tanara; Tibolla, Heloisa; Prigol, Elenizi; Colla, Luciane Maria; Costa, Jorge Alberto Vieira; Bertolin, Telma Elita

    2015-01-01

    The aim of this study was to verify the viability of lignocellulosic substrates to obtain renewable energy source, through characterization of the cellulolytic complex, which was obtained by solid state fermentation using Trichoderma viride. Enzymatic activity of the cellulosic complex was measured during saccharification of substrates filter paper, eucalyptus sawdust, and corncob, and compared with the activity of commercial cellulase. The characterization of the enzymes was performed by a 2(2) Full Factorial Design, where the pH and temperature were the variables of study. Enzymatic saccharification of different substrates appearedviable until 12 to be viable until 12 h; after this period the activity decreased for both enzymatic forms (cellulolytic complex and commercial cellulase). The enzymatic activity of the commercial cellulase was favored with the use of corncob as substrate, while the cellulolytic complex does not show any difference in its specificity by the substrates studied. The largest activities of both enzymes were obtained in the temperature and pH range between 40°C and 50°C and 4.8 and 5.2, respectively. The cellulolytic complex obtained appeared to be viable for the saccharification of lignocellulosic residues compared with the commercial cellulase. PMID:26137476

  13. Production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles

    SciTech Connect

    Webb, C.; Fukuda, H.; Atkinson, B.

    1986-01-01

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

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

    PubMed

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

    2014-01-01

    Laboratory bench scaling was done and an average of 1.85 fold increase by Response Surface Methodology (RSM) optimization was obtained. It was found that the predicted value (4.96 IU/ml) obtained by RSM is in close accordance with observed activity 5.14 IU/ml. Endoglucanases are mainly induced by CMC while Wheat bran (natural substrate) exoglucanase is more active when induced by avicel and cellulose. Addition of substrate beyond a level caused inhibition of cellulase production. The molecular weight of protein as determined by SDS-PAGE is very similar to molecular weight of cellulase of Trichoderma viride (T. viride) cellulase and Trichoderma reesei (T. reesei) endoglucanase. T. reesei β-glucosidase has high enzymatic activity on CMC substrate when compared with T. viride β-glucosidase. Secondary structure analysed by using Circular Dichroism confirmed that composition of celluase system is very similar to other analysed species. The cellulase was found to be active in pH range of 4.8-5.5; while temperature range varied from 50°C to 70°C. Although the enzymatic activity produced by mutants were lesser than the parent, but in one case mutants of Trichoderma reesei's BGL has shown higher activity on cellulose. PMID:25392792

  15. Hydrodynamic and kinetic study of cellulase production by Trichoderma reesei with pellet morphology.

    PubMed

    Yu, Liang; Chao, Yapeng; Wensel, Pierre; Chen, Shulin

    2012-07-01

    Numerical simulations and experimental validation were performed to understand the effects of hydrodynamics on pellet formation and cellulase production by filamentous T. reesei. The constructed model combined a steady-state multiple reference frame (MRF) approach describing mechanical mixing, oxygen mass transfer, and non-Newtonian flow field with a transient sliding mesh approach and kinetics of oxygen consumption, pellet formation, and enzyme production. The model was experimentally validated at various agitation speeds in a two-impeller Rushton turbine fermentor. Results from simulation and experimentation showed that higher agitation speeds led to increases in the pellet diameter and the proportion of pelletized (vs. filamentous) forms of the biomass. It also led to increase in dissolved oxygen mass transfer rate in shear-thinning fluid and cellulase productivity. The extent of these increases varied considerably among agitation speeds. Pellet formation and morphology were presumably affected within a viscosity-dependent shear-rate range. Cellulase activity and cell viability were shown to be sensitive to impeller shear. A maximum cellulase activity of 3.5 IU/mL was obtained at 400 rpm, representing a twofold increase over that at 100 rpm. PMID:22252572

  16. 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.

  17. 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

    2015-01-01

    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.

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

    PubMed

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

    2014-04-01

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

  19. 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

  20. 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

  1. Effect of pH on cellulase production and morphology of Trichoderma reesei and the application in cellulosic material hydrolysis.

    PubMed

    Li, Chen; Yang, Zhenhua; Zhang, Ronglin He Can; Zhang, Dongyuan; Chen, Shulin; Ma, Lijuan

    2013-12-01

    A low-cost of cellulase achieved through improving fermentation technology remains a key requirement for commercialization of cellulosic biofuels and biochemicals. pH plays a very important role in the process of cellulase synthesis by Trichoderma reesei. In this work, effects of pH on the production and production rates of three cellulase components (endoglucanase, exoglucanase, β-glucosidase) and mycelial morphology were studied. Production rates of the cellulase components were kept highest and the mycelial morphology was maintained at the optimal status by developing a phased pH control strategy in order to improve cellulase production. Cellulase production in terms of filter paper activity and β-glucosidase production in batch fermentation increased 17.6% and 22%. Saccharification efficiency of the enzyme obtained by pH control was evaluated by hydrolyzing pretreated corn cob. Saccharification yield increased significantly (up to 26.2%) compared with that without pH control. These results add new knowledge on approach for improving cellulase production.

  2. Effect of pH on cellulase production and morphology of Trichoderma reesei and the application in cellulosic material hydrolysis.

    PubMed

    Li, Chen; Yang, Zhenhua; Zhang, Ronglin He Can; Zhang, Dongyuan; Chen, Shulin; Ma, Lijuan

    2013-12-01

    A low-cost of cellulase achieved through improving fermentation technology remains a key requirement for commercialization of cellulosic biofuels and biochemicals. pH plays a very important role in the process of cellulase synthesis by Trichoderma reesei. In this work, effects of pH on the production and production rates of three cellulase components (endoglucanase, exoglucanase, β-glucosidase) and mycelial morphology were studied. Production rates of the cellulase components were kept highest and the mycelial morphology was maintained at the optimal status by developing a phased pH control strategy in order to improve cellulase production. Cellulase production in terms of filter paper activity and β-glucosidase production in batch fermentation increased 17.6% and 22%. Saccharification efficiency of the enzyme obtained by pH control was evaluated by hydrolyzing pretreated corn cob. Saccharification yield increased significantly (up to 26.2%) compared with that without pH control. These results add new knowledge on approach for improving cellulase production. PMID:24129314

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

    PubMed

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

    2013-09-10

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

  4. Alkaline fuel cell performance investigation

    NASA Technical Reports Server (NTRS)

    Martin, R. E.; Manzo, M. A.

    1988-01-01

    An exploratory experimental fuel cell test program was conducted to investigate the performance characteristics of alkaline laboratory research electrodes. The objective of this work was to establish the effect of temperature, pressure, and concentration upon performance and evaluate candidate cathode configurations having the potential for improved performance. The performance characterization tests provided data to empirically establish the effect of temperature, pressure, and concentration upon performance for cell temperatures up to 300 F and reactant pressures up to 200 psia. Evaluation of five gold alloy cathode catalysts revealed that three doped gold alloys had more that two times the surface areas of reference cathodes and therefore offered the best potential for improved performance.

  5. Alkaline fuel cell performance investigation

    NASA Technical Reports Server (NTRS)

    Martin, R. E.; Manzo, M. A.

    1988-01-01

    An exploratory experimental fuel cell test program was conducted to investigate the performance characteristics of alkaline laboratory research electrodes. The objective of this work was to establish the effect of temperature, pressure, and concentration upon performance and evaluate candidate cathode configurations having the potential for improved performance. The performance characterization tests provided data to empirically establish the effect of temperature, pressure, and concentration upon performance for cell temperatures up to 300 F and reactant pressures up to 200 psia. Evaluation of five gold alloy cathode catalysts revealed that three doped gold alloys had more than two times the surface areas of reference cathodes and therefore offered the best potential for improved performance.

  6. Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments.

    PubMed

    Kumar, Rajeev; Wyman, Charles E

    2009-06-01

    Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO(2)) pretreatments were measured at 4 degrees C. Langmuir adsorption parameters were then estimated by non-linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4 degrees C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO(2) pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly

  7. Alkaline Water and Longevity: A Murine Study.

    PubMed

    Magro, Massimiliano; Corain, Livio; Ferro, Silvia; Baratella, Davide; Bonaiuto, Emanuela; Terzo, Milo; Corraducci, Vittorino; Salmaso, Luigi; Vianello, Fabio

    2016-01-01

    The biological effect of alkaline water consumption is object of controversy. The present paper presents a 3-year survival study on a population of 150 mice, and the data were analyzed with accelerated failure time (AFT) model. Starting from the second year of life, nonparametric survival plots suggest that mice watered with alkaline water showed a better survival than control mice. Interestingly, statistical analysis revealed that alkaline water provides higher longevity in terms of "deceleration aging factor" as it increases the survival functions when compared with control group; namely, animals belonging to the population treated with alkaline water resulted in a longer lifespan. Histological examination of mice kidneys, intestine, heart, liver, and brain revealed that no significant differences emerged among the three groups indicating that no specific pathology resulted correlated with the consumption of alkaline water. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline water of long-lived mouse models.

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

    PubMed

    Chang, Chia-Jung; Lee, Cheng-Chung; Chan, Yueh-Te; Trudeau, Devin L; Wu, Mei-Huey; Tsai, Chih-Hsuan; Yu, Su-May; Ho, Tuan-Hua David; Wang, Andrew H-J; Hsiao, Chwan-Deng; Arnold, Frances H; Chao, Yu-Chan

    2016-01-01

    Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity. PMID:26986867

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

    PubMed

    Chang, Chia-Jung; Lee, Cheng-Chung; Chan, Yueh-Te; Trudeau, Devin L; Wu, Mei-Huey; Tsai, Chih-Hsuan; Yu, Su-May; Ho, Tuan-Hua David; Wang, Andrew H-J; Hsiao, Chwan-Deng; Arnold, Frances H; Chao, Yu-Chan

    2016-01-01

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

  10. Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production

    PubMed Central

    2013-01-01

    Background Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed. Results To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called “promoter-based gene assembly and simultaneous overexpression” (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol. Conclusion Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker, were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host

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

    PubMed Central

    Chan, Yueh-Te; Trudeau, Devin L.; Wu, Mei-Huey; Tsai, Chih-Hsuan; Yu, Su-May; Ho, Tuan-Hua David; Wang, Andrew H.-J.; Hsiao, Chwan-Deng; Arnold, Frances H.; Chao, Yu-Chan

    2016-01-01

    Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity. PMID:26986867

  12. Cellulase-Xylanase Synergy in Designer Cellulosomes for Enhanced Degradation of a Complex Cellulosic Substrate

    PubMed Central

    Moraïs, Sarah; Barak, Yoav; Caspi, Jonathan; Hadar, Yitzhak; Lamed, Raphael; Shoham, Yuval; Wilson, David B.; Bayer, Edward A.

    2010-01-01

    Designer cellulosomes are precision-engineered multienzyme complexes in which the molecular architecture and enzyme content are exquisitely controlled. This system was used to examine enzyme cooperation for improved synergy among Thermobifida fusca glycoside hydrolases. Two T. fusca cellulases, Cel48A exoglucanase and Cel5A endoglucanase, and two T. fusca xylanases, endoxylanases Xyn10B and Xyn11A, were selected as enzymatic components of a mixed cellulase/xylanase-containing designer cellulosome. The resultant mixed multienzyme complex was fabricated on a single scaffoldin subunit bearing all four enzymes. Conversion of T. fusca enzymes to the cellulosomal mode followed by their subsequent incorporation into a tetravalent cellulosome led to assemblies with enhanced activity (~2.4-fold) on wheat straw as a complex cellulosic substrate. The enhanced synergy was caused by the proximity of the enzymes on the complex compared to the free-enzyme systems. The hydrolytic properties of the tetravalent designer cellulosome were compared with the combined action of two separate divalent cellulase- and xylanase-containing cellulosomes. Significantly, the tetravalent designer cellulosome system exhibited an ~2-fold enhancement in enzymatic activity compared to the activity of the mixture of two distinct divalent scaffoldin-borne enzymes. These results provide additional evidence that close proximity between cellulases and xylanases is key to the observed concerted degradation of the complex cellulosic substrate in which the integrated enzymes complement each other by promoting access to the relevant polysaccharide components of the substrate. The data demonstrate that cooperation among xylanases and cellulases can be augmented by their integration into a single designer cellulosome. PMID:21157512

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

    SciTech Connect

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

    2011-02-17

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

  14. 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. PMID:27397800

  15. 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.

  16. Cellulase production by Trichoderma harzianum in static and mixed solid-state fermentation reactors under nonaseptic conditions

    SciTech Connect

    Deschamps, F.; Giuliano, C.; Asther, M.; Huet, M.C.; Roussos, S.

    1985-09-01

    Cellulase production from lignocellulosic materials was studied in solid-state cultivation by both static and mixed techniques under nonaseptic conditions. The effects of fermentation conditions, such as moisture content, pH, temperature, and aeration, on cellulase production by Trichoderma harzianum using a mixture of wheat straw (80%) and bran (20%) were investigated. With a moisture content of 74% and a pH of 5.8, 18 IU filter paper activity and 198 IU endoglucanase activity/g initial substrate content were obtained in 66 hours. The extension from static column cultivation to stirred tank reactor of 65 l capacity gave similar yields of cellulase.

  17. Thermal stability of Trichoderma reesei c30 cellulase and aspergillus niger; -glucosidase after ph and chemical modification

    SciTech Connect

    Woodward, J.; Whaley, K.S.; Zachry, G.S.; Wohlpart, D.L.

    1981-01-01

    Treatment of Trichoderma reesei C30 cellulase at pH 10.0 for 1 h at room temperature increased its pH and thermal stability. Chemical modification of the free epsilon-amino groups of cellulase at pH 10.0 resulted in no further increase in stability. Such chemical modification, however, decreased the thermal stability of the cellulose-cellulase complex. On the contrary, the chemical modification of Aspergillus niger glucosidase with glutaraldehyde at pH 8.0 increased the thermal stability of this enzyme.

  18. Alkaline detergent recycling via ultrafiltration

    SciTech Connect

    Steffani, C.; Meltzer, M.

    1995-06-01

    The metal finishing industry uses alkaline cleaners and detergents to remove oils and dirt from manufactured parts, often before they are painted or plated. The use of these cleaners has grown because environmental regulations are phasing out ozone depleting substances and placing restrictions on the use and disposal of many hazardous solvents. Lawrence Livermore National Laboratory is examining ultrafiltration as a cleaning approach that reclaims the cleaning solutions and minimizes wastes. The ultrafiltration membrane is made from sheets of polymerized organic film. The sheets are rolled onto a supporting frame and installed in a tube. Spent cleaning solution is pumped into a filter chamber and filtered through the membrane that captures oils and dirt and allows water and detergent to pass. The membrane is monitored and when pressure builds from oil and dirt, an automatic system cleans the surface to maintain solution flow and filtration quality. The results show that the ultrafiltration does not disturb the detergent concentration or alkalinity but removed almost all the oils and dirt leaving the solution in condition to be reused.

  19. Grace DAKASEP alkaline battery separator

    NASA Technical Reports Server (NTRS)

    Giovannoni, R. T.; Lundquist, J. T.; Choi, W. M.

    1987-01-01

    The Grace DAKASEP separator was originally developed as a wicking layer for nickel-zinc alkaline batteries. The DAKASEP is a filled non-woven separator which is flexible and heat sealable. Through modification of formulation and processing variables, products with a variety of properties can be produced. Variations of DAKASEP were tested in Ni-H2, Ni-Zn, Ni-Cd, and primary alkaline batteries with good results. The properties of DAKASEP which are optimized for Hg-Zn primary batteries are shown in tabular form. This separator has high tensile strength, 12 micron average pore size, relatively low porosity at 46-48 percent, and consequently moderately high resistivity. Versions were produced with greater than 70 percent porosity and resistivities in 33 wt percent KOH as low as 3 ohm cm. Performance data for Hg-Zn E-1 size cells containing DAKASEP with the properties shown in tabular form, are more reproducible than data obtained with a competitive polypropylene non-woven separator. In addition, utilization of active material is in general considerably improved.

  20. The design of alkaline fuel cells

    NASA Astrophysics Data System (ADS)

    Strasser, K.

    1990-01-01

    Alkaline fuel cells recently developed have yielded satisfactory operation even in the cases of their use of mobile and matrix-type electrolytes; the advantages of realistic operation have been demonstrated by a major West German manufacturer's 100 kW alkaline fuel cell apparatus, which was operated in the role of an air-independent propulsion system. Development has begun for a spacecraft alkaline fuel cell of the matrix-electrolyte configuration.

  1. Insights into the effect of dilute acid, hot water and alkaline pretreatment on cellulose accessible surface area and overall porosity of Populus

    SciTech Connect

    Meng, Xianzhi; Wells, Tyrone; Sun, Qining; Huang, Fang; Ragauskas, Arthur J.

    2015-06-19

    Pretreatment is known to render biomass more reactive to cellulase by altering the chemical compositions as well as physical structures of biomass. Simons stain technique along with mercury porosimetry were applied on the acid, neutral, and alkaline pretreated materials to measure the accessible surface area of cellulose and pore size distribution of Populus. Results indicated that acid pretreatment is much more effective than water and alkaline pretreatment in terms of cellulose accessibility increase. Further investigation suggests that lignin does not dictate cellulose accessibility to the extent that hemicellulose does, but it does restrict xylan accessibility which in turn controls the access of cellulase to cellulose. The most interesting finding is that severe acid pretreatment significantly decreases the average pore size, i.e., 90% average size decrease could be observed after 60 min dilute acid pretreatment at 160 °C; moreover, the nano-pore space formed between coated microfibrils is increased after pretreatment, especially for the acid pretreatment, suggesting this particular type of biomass porosity is probably the most fundamental barrier to effective enzymatic hydrolysis.

  2. Insights into the effect of dilute acid, hot water and alkaline pretreatment on cellulose accessible surface area and overall porosity of Populus

    DOE PAGES

    Meng, Xianzhi; Wells, Tyrone; Sun, Qining; Huang, Fang; Ragauskas, Arthur J.

    2015-06-19

    Pretreatment is known to render biomass more reactive to cellulase by altering the chemical compositions as well as physical structures of biomass. Simons stain technique along with mercury porosimetry were applied on the acid, neutral, and alkaline pretreated materials to measure the accessible surface area of cellulose and pore size distribution of Populus. Results indicated that acid pretreatment is much more effective than water and alkaline pretreatment in terms of cellulose accessibility increase. Further investigation suggests that lignin does not dictate cellulose accessibility to the extent that hemicellulose does, but it does restrict xylan accessibility which in turn controls themore » access of cellulase to cellulose. The most interesting finding is that severe acid pretreatment significantly decreases the average pore size, i.e., 90% average size decrease could be observed after 60 min dilute acid pretreatment at 160 °C; moreover, the nano-pore space formed between coated microfibrils is increased after pretreatment, especially for the acid pretreatment, suggesting this particular type of biomass porosity is probably the most fundamental barrier to effective enzymatic hydrolysis.« less

  3. Alkaline and alkaline earth metal phosphate halides and phosphors

    SciTech Connect

    Lyons, Robert Joseph; Setlur, Anant Achyut; Cleaver, Robert John

    2012-11-13

    Compounds, phosphor materials and apparatus related to nacaphite family of materials are presented. Potassium and rubidium based nacaphite family compounds and phosphors designed by doping divalent rare earth elements in the sites of alkaline earth metals in the nacaphite material families are descried. An apparatus comprising the phosphors based on the nacaphite family materials are presented herein. The compounds presented is of formula A.sub.2B.sub.1-yR.sub.yPO.sub.4X where the elements A, B, R, X and suffix y are defined such that A is potassium, rubidium, or a combination of potassium and rubidium and B is calcium, strontium, barium, or a combination of any of calcium, strontium and barium. X is fluorine, chlorine, or a combination of fluorine and chlorine, R is europium, samarium, ytterbium, or a combination of any of europium, samarium, and ytterbium, and y ranges from 0 to about 0.1.

  4. 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

  5. Decreased cellulase and xylanase production in the fungus Talaromyces cellulolyticus by disruption of tacA and tctA genes, encoding putative zinc finger transcriptional factors.

    PubMed

    Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko

    2015-03-01

    Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is one of the important strains for industrial cellulase production. An understanding of the control of cellulase gene expression in T. cellulolyticus is insufficient because only a few transcriptional factors related to cellulase gene expression have been identified. In the present study, we disrupted seven putative transcription regulator genes that showed similarity with cellulase or hemicellulase regulator genes in other filamentous fungi and investigated whether these genes are related to cellulase and xylanase production. Among the seven genes, five (tclA, tbgA, tlaA, tmcA, tclB2) had a smaller effect on cellulase and xylanase activities when culturing with cellulose. On the other hand, disruption of tacA and tctA, which are respectively homologues of ace1 (repressor of cellulase) and ctf1 (inducer of cutinase), led to a decrease in cellulase and hemicellulase production due to effects at both the enzymatic and transcriptional levels, indicating that tacA and tctA have positive roles in cellulase and xylanase production in T. cellulolyticus. These results suggest that cellulase and xylanase gene regulation in T. cellulolyticus differs from that in other filamentous fungi and imply that unknown transcriptional mechanisms function in T. cellulolyticus.

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

    PubMed

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

    2015-01-01

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

  7. 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.

  8. 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. PMID:27268435

  9. Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei.

    PubMed

    Geng, Alei; Zou, Gen; Yan, Xing; Wang, Qianfu; Zhang, Jun; Liu, Fanghua; Zhu, Baoli; Zhou, Zhihua

    2012-11-01

    A metagenomic fosmid library containing 1 × 10(5) clones was constructed from a biogas digester fed with pig ordure and rice straw. In total, 121 clones with activity of 4-methylumbelliferyl-cellobiosidase were screened from the metagenomic library. A novel GH5 cellulase gene exo2b was identified from a sequenced clone EXO02C10 and expressed in Escherichia coli BL21. The corresponding recombinant Exo2b protein showed high specific activity toward both carboxymethylcellulose (CMC; 260 U/mg protein) and β-D-glucan from barley (849 U/mg), with an optimal pH and temperature of 7.5 and 58 °C, respectively. Exo2b showed stable activity at a wide pH range from 5.5 to 9.0 and was highly thermostable at 60 °C in the presence of 60 mM cysteine. Residual activity was maintained at nearly 100% when Exo2b was incubated at 60 °C for 15 h. A thin-layer chromatography analysis of the hydrolysis products confirmed that Exo2b was an endo-β-1,4-glucanase and it could also produce oligosaccharide smaller than cellotetraose. The fragment encoding the Exo2b catalytic domain was then fused with the cbh1 gene from Trichoderma reesei, and the fused gene was successfully expressed in T. reesei Rut-C30. Compared to that of the parent strain, the filter paper activity and CMCase activity of the secreted proteins of a selected transformant A1 increased by 24% and 18%, respectively. Besides, the glucose concentration from the hydrolysis of pretreated corn stover by the A1 secreted proteins increased by 19.8%. The present study demonstrated the potential application of metagenome originated cellulase genes to modify cellulase producing fungi.

  10. Genetic approach to secretion and hyperproduction of cellulase by Trichoderma. Progress report, April 21, 1983-July 16, 1984

    SciTech Connect

    Montenecourt, B.S.; Sands, J.A.

    1984-07-16

    Research progress is reported in the delineation of the genetics of cellulase secretion in Trichoderma reesei through the construction of a series of hypersecretory mutants and temperature sensitive mutants and analysis of the genetic lesions through the parasexual cycle and protoplast fusion. The effects of secretion inhibitors, glycosylation inhibitors and membrane perturbing agents on synthesis and secretion of T. reesei cellulases were also studied. (ACR)

  11. Screening of optimal cellulases from symbiotic protists of termites through expression in the secretory pathway of Saccharomyces cerevisiae.

    PubMed

    Todaka, Nemuri; Nakamura, Risa; Moriya, Sigeharu; Ohkuma, Moriya; Kudo, Toshiaki; Takahashi, Haruo; Ishida, Nobuhiro

    2011-01-01

    For direct and efficient ethanol production from cellulosic materials, we screened optimal cellulases from symbiotic protists of termites through heterologous expression with Saccharomyces cerevisiae. 11 cellulases, belonging to glycoside hydrolase families 5, 7, and 45 endoglucanases (EGs), were confirmed to produce with S. cerevisiae for the first time. A recombinant yeast expressing SM2042B24 EG I was more efficient at degrading carboxylmethyl cellulose than was Trichoderma reesei EG I, a major EG with high cellulolytic activity.

  12. Alkaline pH sensor molecules.

    PubMed

    Murayama, Takashi; Maruyama, Ichiro N

    2015-11-01

    Animals can survive only within a narrow pH range. This requires continual monitoring of environmental and body-fluid pH. Although a variety of acidic pH sensor molecules have been reported, alkaline pH sensor function is not well understood. This Review describes neuronal alkaline pH sensors, grouped according to whether they monitor extracellular or intracellular alkaline pH. Extracellular sensors include the receptor-type guanylyl cyclase, the insulin receptor-related receptor, ligand-gated Cl- channels, connexin hemichannels, two-pore-domain K+ channels, and transient receptor potential (TRP) channels. Intracellular sensors include TRP channels and gap junction channels. Identification of molecular mechanisms underlying alkaline pH sensing is crucial for understanding how animals respond to environmental alkaline pH and how body-fluid pH is maintained within a narrow range.

  13. 13 C-metabolic flux analysis in heterologous cellulase production by Bacillus subtilis genome-reduced strain.

    PubMed

    Toya, Yoshihiro; Hirasawa, Takashi; Morimoto, Takuya; Masuda, Kenta; Kageyama, Yasushi; Ozaki, Katsuya; Ogasawara, Naotake; Shimizu, Hiroshi

    2014-06-10

    The great potential of Bacillus subtilis to produce biomaterials would be further enhanced by the development of strains with deletions of non-essential genomic regions. Here, using stationary (13)C-metabolic flux analysis ((13)C-MFA), we investigated the metabolism during cellulase production by the genome-reduced B. subtilis strain MGB874. We transformed MGB874 and wild-type strains with the heterologous cellulase gene, and cultured these on a synthetic medium containing glucose as carbon source. The addition of glutamate and the genome reduction enhanced cellulase production, which led us to use (13)C-MFA to assess the effects of glutamate addition and gene deletions on metabolism. We found that there was a significant increase in the flux in the pentose phosphate (PP) pathway, whereas the fluxes of reactions from acetyl-CoA to α-ketoglutarate were repressed in the presence of glutamate. We hypothesize that the increase in the PP pathway flux was caused by the decrease of citrate synthase flux through the accumulation of glycolytic intermediates. Excess NADPH produced by the PP pathway may affect the increase in cellulase production. Furthermore, the fluxes on glycolysis and the acetate formation of the cellulase-producing wild-type strain were significantly larger than that of the cellulase-producing MGB874 strain when the strains were cultured with glucose and glutamate.

  14. 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.

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

    SciTech Connect

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

    2011-06-13

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

  16. Reducing non-productive adsorption of cellulase and enhancing enzymatic hydrolysis of lignocelluloses by noncovalent modification of lignin with lignosulfonate.

    PubMed

    Lou, Hongming; Wang, Mengxia; Lai, Huanran; Lin, Xuliang; Zhou, Mingsong; Yang, Dongjie; Qiu, Xueqing

    2013-10-01

    Four fractions of one commercial sodium lignosulfonate (SXP) with different molecular weight (MW) and anionic polymers were studied to reduce non-productive adsorption of cellulase on bound lignin in a lignocellulosic substrate. SXP with higher MW had stronger blocking effect on non-productive adsorption of a commercial Trichoderma reesi cellulase cocktail (CTec2) on lignin measured by quartz crystal microgravimetry with dissipation monitoring. Linear anionic aromatic polymers have strong blocking effect, but they would also reduce CTec2 adsorption on cellulose to decrease the enzymatic activity. The copolymer of lignin and polyethylene glycol (AL-PEG1000) has strong enhancement in enzymatic hydrolysis of lignocelluloses, because it not only improves the cellulase activity to cellulose, but also blocks the non-productive cellulase adsorption on lignin. Apart from improving the cellulase activity to cellulose, the enhancements of enzymatic hydrolysis of lignocellulose by adding AL-PEG1000 and SXPs are the result of the decreased cellulase non-productive adsorption on lignin.

  17. Alkaline-resistance model of subtilisin ALP I, a novel alkaline subtilisin.

    PubMed

    Maeda, H; Mizutani, O; Yamagata, Y; Ichishima, E; Nakajima, T

    2001-05-01

    The alkaline-resistance mechanism of the alkaline-stable enzymes is not yet known. To clarify the mechanism of alkaline-resistance of alkaline subtilisin, structural changes of two typical subtilisins, subtilisin ALP I (ALP I) and subtilisin Sendai (Sendai), were studied by means of physicochemical methods. Subtilisin NAT (NAT), which exhibits no alkaline resistance, was examined as a control. ALP I gradually lost its activity, accompanied by protein degradation, but, on the contrary, Sendai was stable under alkaline conditions. CD spectral measurements at neutral and alkaline pH indicated no apparent differences between ALP I and Sendai. A significant difference was observed on measurement of fluorescence emission spectra of the tryptophan residues of ALP I that were exposed on the enzyme surface. The fluorescence intensity of ALP I was greatly reduced under alkaline conditions; moreover, the reduction was reversed when alkaline-treated ALP I was neutralized. The fluorescence spectrum of Sendai remained unchanged. The enzymatic and optical activities of NAT were lost at high pH, indicating a lack of functional and structural stability in an alkaline environment. Judging from these results, the alkaline resistance is closely related to the surface structure of the enzyme molecule.

  18. Characterization of Novel Cellulase-producing Bacteria Isolated From Rotting Wood Samples.

    PubMed

    Paudel, Yagya Prasad; Qin, Wensheng

    2015-11-01

    Seventeen bacterial isolates were screened for their cellulase activity by carboxymethyl cellulose (CMC) plate assay. The bacterial strain K1 showed the largest depolymerized region in CMC plate assay and was further studied for quantitative cellulase activity. On the basis of 16S rDNA sequence analysis, the strain K1 was found to be Bacillus sp. This strain produced the maximum CMCase at pH 6 and 50 °C in the presence of peptone (1%) as a source of nitrogen. The CMCase activity was stimulated by Ca(2+) (2 mM) by 20% over the control. The CMCase activity of this Bacillus sp. K1 was highly induced when lactose was used as a source of carbon during fermentation.

  19. Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum.

    PubMed

    Yuan, Shuo-Fu; Wu, Tzu-Hui; Lee, Hsiao-Lin; Hsieh, Han-Yu; Lin, Wen-Ling; Yang, Barbara; Chang, Chih-Kang; Li, Qian; Gao, Jian; Huang, Chun-Hsiang; Ho, Meng-Chiao; Guo, Rey-Ting; Liang, Po-Huang

    2015-02-27

    We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.

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

    PubMed

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

    2013-04-01

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

  1. Kinetic study on enzymatic hydrolysis of cellulose by cellulase from Trichoderma viride

    SciTech Connect

    Ohmine, K.; Ooshima, H.; Harano, Y.

    1983-08-01

    Pure cellulose (Avicel) was hydrolyzed batchwise at 50 degrees C and pH 4.8 by cellulase from Trichoderma viride (Meicelase CEP). Then the effects of the crystallinity of cellulose as well as the thermal deactivation and product (cellobiose and glucose) inhibition to cellulase on the hydrolysis rate were quantitatively investigated. While these factors had evidently retarded the enzymatic hydrolysis of cellulose to a significant extent, the hydrolysis rates observed could not be explained. For practical purposes, an empirical, simple rate expression was developed which included only one parameter: an overall rate retardation constant. This empirical rate expression held for the hydrolysis of at least two kinds of cellulosic materials: Avicel and tissue paper. (Refs. 10)

  2. Enzymatic hydrolysis of cellulose: evaluation of cellulase culture filtrates under use conditions

    SciTech Connect

    Mandels, M.; Medeiros, J.E.; Andreotti, R.E.; Bissett, F.H.

    1981-09-01

    Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 hours. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given time was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration. (Refs. 28).

  3. Biochemical Characterization and Structural Analysis of a Bifunctional Cellulase/Xylanase from Clostridium thermocellum*

    PubMed Central

    Yuan, Shuo-Fu; Wu, Tzu-Hui; Lee, Hsiao-Lin; Hsieh, Han-Yu; Lin, Wen-Ling; Yang, Barbara; Chang, Chih-Kang; Li, Qian; Gao, Jian; Huang, Chun-Hsiang; Ho, Meng-Chiao; Guo, Rey-Ting; Liang, Po-Huang

    2015-01-01

    We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities. PMID:25575592

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

    PubMed

    Qian, Zhi-Gang

    2014-01-30

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

  5. Screening for cellulases with industrial value and their use in biomass conversion.

    PubMed

    Jüergensen, Julia; Ilmberger, Nele; Streit, Wolfgang R

    2012-01-01

    Cellulose is an easily renewable and highly occurring resource. To take advantage of this great potential, there is a constant need of new cellulose degrading enzymes. In industrial applications enzymes have to function under extreme conditions like high temperature, very acidic or basic pH and different solvents. Cellulases have a huge area of application, for example the textile and food industry as well as the generation of bioethanol as an alternative energy source. They have the ability to yield a great energetic potential, but there is still a lack of economical technologies to conquer the stability of the cellulose structure. Via metagenomic research and well-directed screening, it is possible to detect new cellulases, which are active under tough industrial conditions.

  6. Screening of fungi isolated from environmental samples for xylanase and cellulase production.

    PubMed

    Ja'afaru, Mohammed Inuwa

    2013-01-01

    The aim of this work is to select filamentous fungal strains isolated from saw dust, soil, and decaying wood with the potential to produce xylanase and cellulase enzymes. A total of 110 fungi were isolated. Fifty-seven (57) of these fungi were isolated from soil samples, 32 from sawdust, and 19 from decaying wood. Trichoderma and Aspergillus had the highest relative occurrence of 42.6% and 40.8%, respectively. Trichoderma viride Fd18 showed the highest specific activity of 1.30 U mg(-1) protein for xylanase, while the highest cellulase activity of 1.23 U mg(-1) was shown by Trichoderma sp. F4. The isolated fungi demonstrated potential for synthesizing the hydrolytic enzymes. PMID:24416614

  7. Cellulase activity screening using pure carboxymethylcellulose: application to soluble cellulolytic samples and to plant tissue prints.

    PubMed

    Johnsen, Hanne R; Krause, Kirsten

    2014-01-01

    Reliable, rapid and inexpensive detection of cellulolytic enzymes that can be used for a wide variety of biological and environmental samples are currently in high demand. Here, a new cellulase detection protocol is described that circumvents problems observed with popular agar-based methods by exploiting the ability of carboxymethylcellulose (CMC) to form gel-like surfaces on its own. These pure CMC-layers are sensitive to cellulolytic degradation and stainable by Gram's iodine without showing unwelcome reactions with other enzymes. The staining intensity negatively correlates with the enzyme activity and can be used for quantification. Cellulase activities are not obstructed by high sugar contents (e.g., in plant material) which limit the applicability of other quantification methods, making our new method particularly attractive for screening of plant extracts. A useful variant of this new method is its applicability to plant tissue prints for spatial mapping of the cellulolytic activity in a zymogram-like fashion. PMID:24413752

  8. 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.

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

    PubMed

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

    2013-01-01

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

  10. 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.

  11. Diverse genes of cellulase homologues of glycosyl hydrolase family 45 from the symbiotic protists in the hindgut of the termite Reticulitermes speratus.

    PubMed

    Ohtoko, K; Ohkuma, M; Moriya, S; Inoue, T; Usami, R; Kudo, T

    2000-12-01

    Diverse genes encoding cellulase homologues belonging to glycosyl hydrolase family 45 were identified from the symbiotic protists in the hindgut of the termite Reticulitermes speratus through the use of consensus PCR and the screening of a cDNA library. Fifteen full-length cDNA clones were isolated and sequenced, which encoded polypeptides consisting of 218-221 amino acid residues showing up to 63% identity to known family 45 cellulases. The cellulase sequences of the termite symbiotic protists were phylogenetically monophyletic, showing more than 75% amino acid identity with each other. These enzymes consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. By whole-cell in situ hybridization using oligonucleotide probes specific for regions conserved in some of the sequences, the origin of the genes was identified as symbiotic hypermastigote protists. The presence of diverse cellulase homologues suggests that symbiotic protists of termites may be rich reservoirs of novel cellulase sequences.

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

    PubMed

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

    2014-11-01

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

  13. Cellulase production by Trichoderma longi, Aspergillus niger and Saccharomyces cerevisae cultured on waste materials from orange.

    PubMed

    Omojasola, P F; Jilani, O P

    2008-10-15

    The wastes materials from the sweet orange (Citrus sinensis) were used as substrate for the production of cellulase. The rind, the pericarp or albedo and the pulp were hydrolyzed by cellulolytic enzymes of Trichoderma longibrachiatum, Aspergillus niger and Saccharomyces cerevisiae after they were treated with alkali and steam. The amount of glucose released from the substrates following the secretion of cellulase by the three microorganisms was measured. The orange wastes released amounts of glucose ranging from 0.76-0.96 mg mL(-1) by Trichoderma longibrachiatum, 0.90-1.08 mg mL(-1) by A. niger and 0.60-0.76 mg mL(-1) by S. cerevisiae after five days of fermentation. The conditions of the fermentation were then varied to determine their effect on cellulase production. Fermentation parameters varied were time, pH, substrate concentration, temperature and inoculum size. After this, conditions that produced highest amounts of glucose were combined in an optimization experiment. Glucose production under optimized conditions were 0.94 mg mL(-1) by T. longibrachiatum, 0.83 mg mL(-1) by A. niger and 0.67 mg mL(-1) by S. cerevisae. The activity of the test organisms' cellulase against CMC on the orange wastes was also determined with T. longibrachiatum producing 3.86 mg mL(-1), A. niger 2.94 mg mL(-1) and S. cerevisiae 2.30 mg mL(-1) glucose amounts all from orange pulp. PMID:19137846

  14. Nano-structured cellulose nanocrystals-xyloglucan multilayered films for the detection of cellulase activity

    NASA Astrophysics Data System (ADS)

    Guyomard-Lack, A.; Cerclier, C.; Beury, N.; Jean, B.; Cousin, F.; Moreau, C.; Cathala, B.

    2012-11-01

    Multilayered cellulose nanocrystals-xyloglucan (CN-XG) films were assembled by spincoating at two CN concentrations inducing different structural colourations. Internal structure of both films was probed by neutron reflectometry (NR) revealing different thicknesses and architectures. The impact of film structure on degradation process by cellulases was revealed by a change in colours of the films, as CN and XG are both sensitive to cellulolytic enzymes.

  15. Cellulose hydrolysis by the cellulases produced by Talaromyces emersonii when grown on different inducing substrates

    SciTech Connect

    Moloney, A.P.; Considine, P.J.; Coughlan, M.P.

    1983-04-01

    Pulp obtained from the processing of sugar beet at a local factory is mixed with molasses and sold as cattle food. However, the value of the pulp would be increased considerably if its constituent cellulose and hemicellulose fractions could be converted to fermentable sugars. To this end we are investigating the enzymic hydrolysis of beet pulp using the cellulase system produced by the thermophilic fungus, Talaromyces emersonii. In this Communication, we report on the initial results of studies. (Refs. 21).

  16. Product inhibition of cellulases studied with 14C-labeled cellulose substrates

    PubMed Central

    2013-01-01

    Background As a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched. To be economically feasible, the hydrolysis of lignocellulose must be conducted at a high concentration of solids, which results in high concentrations of hydrolysis end-products, cellobiose and glucose, making the relief of product inhibition of cellulases a major challenge in the process. However, little quantitative information on the product inhibition of individual cellulases acting on cellulose substrates is available because it is experimentally difficult to assess the hydrolysis of the heterogeneous polymeric substrate in the high background of added products. Results The cellobiose and glucose inhibition of thermostable cellulases from Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum acting on uniformly 14C-labeled bacterial cellulose and its derivatives, 14C-bacterial microcrystalline cellulose and 14C-amorphous cellulose, was studied. Cellulases from Trichoderma reesei were used for comparison. The enzymes most sensitive to cellobiose inhibition were glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs), followed by family 6 CBHs and endoglucanases (EGs). The strength of glucose inhibition followed the same order. The product inhibition of all enzymes was relieved at higher temperatures. The inhibition strength measured for GH7 CBHs with low molecular-weight model substrates did not correlate with that measured with 14C-cellulose substrates. Conclusions GH7 CBHs are the primary targets for product inhibition of the synergistic hydrolysis of cellulose. The inhibition must be studied on cellulose substrates instead of on low molecular-weight model substrates when selecting enzymes for lignocellulose hydrolysis. The advantages of using higher temperatures are an increase in the catalytic efficiency of enzymes and the relief of

  17. Effect of colloidal materials on cellulase production by Trichoderma reesei Rut-C30

    SciTech Connect

    Duff, S.J.B.; Cooper, D.G.; Fuller, O.M.

    1985-04-01

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

  18. Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass

    PubMed Central

    2014-01-01

    Background Previous research focused on pretreatment of biomass, production of fermentable sugars and their consumption to produce ethanol. The main goal of the work was to economise the production process cost of fermentable sugars. Therefore, the objective of the present work was to investigate enzyme hydrolysis of microcrystalline cellulose and hemp hurds (natural cellulosic substrate) using free and immobilised enzymes. Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability. Results Up to 94% protein binding was achieved during immobilisation of cellulase on nanoparticles. Successful binding was confirmed using Fourier transform infrared spectroscopy (FTIR). The free and immobilised enzymes exhibited identical pH optima (pH 4.0) and differing temperature optima at 50°C and 60°C, respectively. The K M values obtained for the free and immobilised enzymes were 0.87 mg/mL and 2.6 mg/mL respectively. The immobilised enzyme retained 50% enzyme activity up to five cycles, with thermostability at 80°C superior to that of the free enzyme. Optimum hydrolysis of carboxymethyl cellulose (CMC) with free and immobilised enzymes was 88% and 81%, respectively. With pretreated hemp hurd biomass (HHB), the free and immobilised enzymes resulted in maximum hydrolysis in 48 h of 89% and 93%, respectively. Conclusion The current work demonstrated the advantages delivered by immobilised enzymes by minimising the consumption of cellulase during substrate hydrolysis and making the production process of fermentable sugars economical and feasible. The activity of cellulase improved as a result of the immobilisation, which provided a better stability at higher temperatures. The immobilised enzyme provided an advantage over the free enzyme through the reusability and longer

  19. Production of SCP and cellulase by Aspergillus terreus from bagasse substrate

    SciTech Connect

    Garg, S.K.; Neelakantan, S.

    1982-01-01

    The fermentation of 1.0% untreated bagasse under optimum cultural and nutritional conditions with Aspergillus terreus GN1 indicated that the maximum rate of protein and cellulase production could be obtained during three days of submerged fermentation. Even though 16.4% protein recovery, 0.55 units CMCase/mL, and 0.027 FPase units/mL were obtained on the seventh day, the rates of increase in protein recovery and cellulase production were slower than those obtained up to three days, which were 14.3% protein recovery, 0.45 units cMCase/mL, and 0.019 units FPase/mL. There was an initial lag in the utilization of cellulose up to two days due to the utilization of the water-soluble carbohydrate present in untreated bagasse. Cellulose utilization and water-soluble carbohydrate content during fermentation were correlated with protein recovery and enzyme production. The protein and cellulase production during three days fermentation with 1.0% untreated and treated bagasse were compared and the protein content of the total biomass was calculated into constituent protein contributed by the fungal mycelium and the undegraded bagasse. The total biomass recovered with untreated and treated bagasse was 1020 and 820 mg/g bagasse substrate, respectively, and contained 14.3 and 20.6% crude protein, respectively. The contribution of fungal biomass and undegraded bagasse was 309 and 711, and 373 and 447 mg/g untreated and treated bagasse substrates, respectively. In an 8-L-flask trial during three days of fermentation, the recovery of SCP and cellulase were 66 g and 32,400 units (Sigma) for treated bagasse and 82 g and 8200 units (Sigma) for untreated bagasse, respectively. (Refs. 18).

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

    SciTech Connect

    Not Available

    2012-07-01

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

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

    PubMed

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

    2010-12-01

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

  2. Effect of culture phasing and mannanase on production of cellulase and hemicellulase by mixed culture of Trichoderma reesei D 1-6 and Aspergillus wentii Pt 2804

    SciTech Connect

    Ghose, T.K.; Panda, T.; Bisaria, V.S.

    1985-09-01

    Significant increase in extracellular cellulase and hemicellulase activities was observed in the biosynthesis of cellulase enzyme in mixed culture fermentation of Trichoderma reesei D 1-6 and Aspergillus wentii Pt 2804 when the A. wentii inoculation was phased by 15 hours. The optimal conditions of fermentation by the mixed culture have been established. Presence of mannanase has been found to affect the release as well as activity of cellulase enzyme produced in mixed culture.

  3. Study of the cellulases produced by three mesophilic actinomycetes grown on bagasse as substrate

    SciTech Connect

    Van Zyl, W.H.

    1985-09-01

    The cellulases that strains of Streptomyces albogrisolus, S. nitrosporeus, and Micromonospora melanosporea produce when grown on untreated ballmilled bagasse were investigated. Optimum conditions for extracellular cellulase production and activity were determined to be growth at pH 6.7-7.4 and 25-35 degrees C for 4-5 days and assay at pH 5.0-6.0 and 45-55 degrees C, respectively. The endoglucanases were thermally stable at 50 degrees C, but the Avicelases had a half-life of approximately 24 hours at this temperature. Nearly half of the endoglucanases and almost all of the Avicelases were absorbed on ballmilled bagasse after 15 minutes incubation at 50 degrees C. The ..beta..-glucosidases were found to be mainly intracellular or cell wall bound. These mesophilic actinomycetes concomitantly produced xylanases and ..beta..-xylosidases with cellulases that, apart from cellobiose and glucose, also release xylose from bagasse. This feature may be advantageous in the commercial application of the enzymes of mesophilic actinomycetes for the saccharification of natural cellulosic substrates.

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

    DOE PAGES

    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. Adsorptionmore » 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.« less

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

    PubMed Central

    Niu, Hongxing; Shah, Nilay; Kontoravdi, Cleo

    2016-01-01

    Improved understanding of heterogeneous cellulose hydrolysis by cellulases is the basis for optimising enzymatic catalysis-based cellulosic biorefineries. A detailed mechanistic model is developed to describe the dynamic adsorption/desorption and synergistic chain-end scissions of cellulases (endoglucanase, exoglucanase, and β-glucosidase) upon amorphous cellulose. The model can predict evolutions of the chain lengths of insoluble cellulose polymers and production of soluble sugars during hydrolysis. Simultaneously, a modelling framework for uncertainty analysis is built based on a quasi-Monte-Carlo method and global sensitivity analysis, which can systematically identify key parameters, help refine the model and improve its identifiability. The model, initially comprising 27 parameters, is found to be over-parameterized with structural and practical identification problems under usual operating conditions (low enzyme loadings). The parameter estimation problem is therefore mathematically ill posed. The framework allows us, on the one hand, to identify a subset of 13 crucial parameters, of which more accurate confidence intervals are estimated using a given experimental dataset, and, on the other hand, to overcome the identification problems. The model’s predictive capability is checked against an independent set of experimental data. Finally, the optimal composition of cellulases cocktail is obtained by model-based optimisation both for enzymatic hydrolysis and for the process of simultaneous saccharification and fermentation. PMID:26865832

  6. 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.

  7. 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.

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

    PubMed

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

    2015-04-01

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

  9. 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. PMID:26341925

  10. 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

  11. Essential 170-kDa subunit for degradation of crystalline cellulose by Clostridium cellulovorans cellulase

    SciTech Connect

    Shoseyov, O.; Doi, R.H. )

    1990-03-01

    The cellulase complex from Clostridium cellulovorans has been purified and its subunit composition determined. The complex exhibits cellulase activity against crystalline cellulose as well as carboxymethylcellulase (CMCase) and cellobiohydrolase activities. Three major subunits are present with molecular masses of 170, 100, and 70 kDa. The 100-kDa subunit is the major CMCase, although at least four other, minor subunits show CMCase activity. The 170-kDa subunit has the highest affinity for cellulose, does not have detectable enzymatic activity, but is necessary for cellulase activity. Immunological studies indicate that the 170-kDa subunit is not required for binding of the catalytic subunits to cellulose and therefore does not function solely as an anchor protein. Thus this core subunit must have multiple functions. The authors propose a working hypothesis that the binding of the 170-kDa subunit converts the crystalline cellulose to a form that is capable of being hydrolyzed in a cooperative fashion by the associated catalytic subunits.

  12. 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

  13. Genetic approach to secretion and hyperproduction of cellulases by Trichoderma. Progress report, August 1985-April 1986

    SciTech Connect

    Montenecourt, B.S.; Sands, J.A.

    1986-01-01

    The multienzyme cellulase complex of the mesophilic fungus Trichoderma reesei has been studied. Our laboratory has focused on the production of high yielding mutants and the analysis of these mutants with respect to the events involved in secretion of the enzymes. The effect of glycosylation inhibitors, tunicamycin and 2-deoxyglucose and membrane perturbing agents (ethanol and phenylethanol) have been studied at both the biochemical and morphological levels. Antibodies conjugated to gold-protein A particles have localized cellobiohydrolase in the endoplasmic reticulum, Golgi and secretory vesicle regions, confirming that this general pathway, which has been firmly identified in yeast and mammalian cells, also exists in Trichoderma. Temperature sensitive secretion mutants have been isolated and one mutant, LU-ts 1, was shown to be ts for cellulase and not for protease, amylase and xylanase. At the non-permissive temperature heterogeneous protein bands are exhibited by this mutant which are inactive. This characteristic of ts for secretion is reversible. At the permissive temperature normal cellulase enzymes are secreted. 2 refs.

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

    PubMed

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

    2008-06-01

    In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant. PMID:18417637

  15. Aryl thioglycoside-based affinity purification of exo-acting cellulases.

    PubMed

    Piyachomkwan, K; Penner, M H

    1998-01-15

    The influence of ligand-coupling chemistry and mobile-phase composition on the interaction of exo-acting cellulases with an immobilized complementary ligand was investigated. p-Aminophenyl 1-thio-beta-D-cellobioside (APTC) was used as a representative affinity ligand to which exo-acting cellulases (cellobiohydrolases, CBHs) preferentially bind. A "crude" cellulase preparation from the fungus Trichoderma reesei served as an enzyme source. The adsorption properties of the two principal exo-acting CBHs in this preparation, CBH I and CBH II, are shown to be distinctly different under several scenarios. Their relative affinities, based on column elution behavior and partition equilibrium experiments, are shown to be highly dependent on the functional groups employed for ligand coupling, the extent of functional group hydrolysis, the composition of the mobile phase, and the inherent nature of the enzymes. The dependency on the chemistry of the supporting matrix was illustrated using agarose supports containing cyanate ester, N-hydroxy-succinimide, and epoxy functional groups. When compared under apparent optimal conditions, the affinity of CBH II for immobilized APTC was approximately 10-fold that of CBH I. However, selective adsorption of CBH I or CBH II can be achieved by adjusting experimental parameters. PMID:9451508

  16. Comparison of three ionic liquid-tolerant cellulases by molecular dynamics.

    PubMed

    Jaeger, Vance; Burney, Patrick; Pfaendtner, Jim

    2015-02-17

    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

  17. Establishment of a Simple Lactobacillus plantarum Cell Consortium for Cellulase-Xylanase Synergistic Interactions

    PubMed Central

    Moraïs, Sarah; Shterzer, Naama; Grinberg, Inna Rozman; Mathiesen, Geir; Eijsink, Vincent G. H.; Axelsson, Lars; Lamed, Raphael; Bayer, Edward A.

    2013-01-01

    Lactobacillus plantarum is an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including its ability to ferment both pentoses and hexoses, as well as its high acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent ability to deconstruct lignocellulosic substrates. As a first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding for a GH6 cellulase and a GH11 xylanase from a highly active cellulolytic bacterium into L. plantarum. For this purpose, we employed the recently developed pSIP vectors for efficient secretion of heterologous proteins. Both enzymes were secreted by L. plantarum at levels estimated at 0.33 nM and 3.3 nM, for the cellulase and xylanase, respectively, in culture at an optical density at 600 nm (OD600) of 1. Transformed cells demonstrated the ability to degrade individually either cellulose or xylan and wheat straw. When mixed together to form a two-strain cell-based consortium secreting both cellulase and xylanase, they exhibited synergistic activity in the overall release of soluble sugar from wheat straw. This result paves the way toward metabolic harnessing of L. plantarum for novel biorefining applications, such as production of ethanol and polylactic acid directly from plant biomass. PMID:23811500

  18. Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing.

    PubMed

    Le Crom, Stéphane; Schackwitz, Wendy; Pennacchio, Len; Magnuson, Jon K; Culley, David E; Collett, James R; Martin, Joel; Druzhinina, Irina S; Mathis, Hugues; Monot, Frédéric; Seiboth, Bernhard; Cherry, Barbara; Rey, Michael; Berka, Randy; Kubicek, Christian P; Baker, Scott E; Margeot, Antoine

    2009-09-22

    Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.

  19. Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing

    SciTech Connect

    Le Crom, Stphane; Schackwitz, Wendy; Pennacchiod, Len; Magnuson, Jon K.; Culley, David E.; Collett, James R.; Martin, Joel X.; Druzhinina, Irina S.; Mathis, Hugues; Monot, Frdric; Seiboth, Bernhard; Cherry, Barbara; Rey, Michael; Berka, Randy; Kubicek, Christian P.; Baker, Scott E.; Margeot, Antoine

    2009-09-22

    Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels, such as ethanol, and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions and 18 larger deletions leading to the loss of more than 100 kb of genomic DNA. From these events we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild type strain QM6a. Our analysis provides the first genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus, and suggests new areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.

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

    PubMed

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

    2016-01-01

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

  1. 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. PMID:22915066

  2. Essential 170-kDa subunit for degradation of crystalline cellulose by Clostridium cellulovorans cellulase.

    PubMed Central

    Shoseyov, O; Doi, R H

    1990-01-01

    The cellulase complex from Clostridium cellulovorans has been purified and its subunit composition determined. The complex exhibits cellulase activity against crystalline cellulose as well as carboxymethylcellulase (CMCase) and cellobiohydrolase activities. Three major subunits are present with molecular masses of 170, 100, and 70 kDa. The 100-kDa subunit is the major CMCase, although at least four other, minor subunits show CMCase activity. The 170-kDa subunit has the highest affinity for cellulose, does not have detectable enzymatic activity, but is necessary for cellulase activity. Immunological studies indicate that the 170-kDa subunit is not required for binding of the catalytic subunits to cellulose and therefore does not function solely as an anchor protein. Thus this core subunit must have multiple functions. We propose a working hypothesis that the binding of the 170-kDa subunit converts the crystalline cellulose to a form that is capable of being hydrolyzed in a cooperative fashion by the associated catalytic subunits. Images PMID:2107547

  3. 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

  4. 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

  5. Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with a cell surface-engineered yeast strain.

    PubMed

    Matano, Yuki; Hasunuma, Tomohisa; Kondo, Akihiko

    2013-03-01

    Enzymatic hydrolysis of cellulosic material is an essential step in the bioethanol production process. However, complete cellulose hydrolysis by cellulase is difficult due to the irreversible adsorption of cellulase onto cellulose. Thus, part of the cellulose remains in crystalline form after hydrolysis. In this study, after 96-h hydrolysis of Avicel crystalline cellulose, 47.1 % of the cellulase was adsorbed on the cellulose surface with 10.8 % crystalline cellulose remaining. In simultaneous saccharification and fermentation of 100 g/L Avicel with 1.0 filter paper unit/mL cellulase, a wild-type yeast strain produced 44.7 g/L ethanol after 96 h. The yield of ethanol was 79.7 % of the theoretical yield. On the other hand, a recombinant yeast strain displaying various cellulases, such as β-glucosidase, cellobiohydrolase, and endoglucanase, produced 48.9 g/L ethanol, which corresponds to 87.3 % of the theoretical yield. Higher ethanol production appears to be attributable to higher efficiency of cellulase displayed on the cell surface. These results suggest that cellulases displayed on the yeast cell surface improve hydrolysis of Avicel crystalline cellulose. Indeed, after the 96-h simultaneous saccharification and fermentation using the cellulase-displaying yeast, the amount of residual cellulose was 1.5 g/L, one quarter of the cellulose remaining using the wild-type strain, a result of the alleviation of irreversible adsorption of cellulases on the crystalline cellulose.

  6. Hydrolysis of insoluble cellulose to glucose catalyzed by cellulase-containing liposomes in an aqueous solution of 1-butyl-3-methylimidazolium chloride.

    PubMed

    Yoshimoto, Makoto; Tanimura, Kazuhiko; Tokunaga, Kazuki; Kamimura, Akio

    2013-01-01

    The liposome containing cellulase from Trichoderma viride was prepared under the condition that an appreciable amount of cellulase was incorporated in lipid membranes. The liposomal cellulase and free enzyme were examined in their hydrolytic activities to insoluble cellulose powder CC31 in the acetate buffer solution (pH 4.8) of 15 w/w% [Bmim][Cl] (1-butyl-3-methylimidazolium chloride). The mean diameter and size distribution of cellulase-containing liposome were practically unchanged under the above condition. The free cellulase was deactivated more rapidly than the liposomal cellulase in catalyzing the hydrolysis of 2.0 g/l CC31 at 45°C in the presence of [Bmim][Cl] for 48 h. The activities of liposomal and free cellulase to cellobiose as soluble substrate were less susceptible to [Bmim][Cl] than their cellulolytic activities to CC31, meaning that β-glucosidase is relatively stable among the three enzyme components of cellulase. The rate of glucose production could be appreciably improved by the pretreatment of CC31 with [Bmim][Cl] alone at 120°C for 30 min followed by the liposomal cellulase-catalyzed hydrolysis of the substrate at 45°C at the [Bmim][Cl] concentration of 15 w/w%. PMID:23813807

  7. Process for extracting technetium from alkaline solutions

    DOEpatents

    Moyer, Bruce A.; Sachleben, Richard A.; Bonnesen, Peter V.

    1995-01-01

    A process for extracting technetium values from an aqueous alkaline solution containing at least one alkali metal hydroxide and at least one alkali metal nitrate, the at least one alkali metal nitrate having a concentration of from about 0.1 to 6 molar. The solution is contacted with a solvent consisting of a crown ether in a diluent for a period of time sufficient to selectively extract the technetium values from the aqueous alkaline solution. The solvent containing the technetium values is separated from the aqueous alkaline solution and the technetium values are stripped from the solvent.

  8. Inorganic-organic separators for alkaline batteries

    NASA Technical Reports Server (NTRS)

    Sheibley, D. W. (Inventor)

    1978-01-01

    A flexible separator is reported for use between the electrodes of Ni-Cd and Ni-Zn batteries using alkaline electrolytes. The separator was made by coating a porous substrate with a battery separator composition. The coating material included a rubber-based resin copolymer, a plasticizer and inorganic and organic fillers which comprised 55% by volume or less of the coating as finally dried. One or more of the filler materials, whether organic or inorganic, is preferably active with the alkaline electrolyte to produce pores in the separator coating. The plasticizer was an organic material which is hydrolyzed by the alkaline electrolyte to improve conductivity of the separator coating.

  9. Alkaline sorbent injection for mercury control

    DOEpatents

    Madden, Deborah A.; Holmes, Michael J.

    2003-01-01

    A mercury removal system for removing mercury from combustion flue gases is provided in which alkaline sorbents at generally extremely low stoichiometric molar ratios of alkaline earth or an alkali metal to sulfur of less than 1.0 are injected into a power plant system at one or more locations to remove at least between about 40% and 60% of the mercury content from combustion flue gases. Small amounts of alkaline sorbents are injected into the flue gas stream at a relatively low rate. A particulate filter is used to remove mercury-containing particles downstream of each injection point used in the power plant system.

  10. Alkaline sorbent injection for mercury control

    DOEpatents

    Madden, Deborah A.; Holmes, Michael J.

    2002-01-01

    A mercury removal system for removing mercury from combustion flue gases is provided in which alkaline sorbents at generally extremely low stoichiometric molar ratios of alkaline earth or an alkali metal to sulfur of less than 1.0 are injected into a power plant system at one or more locations to remove at least between about 40% and 60% of the mercury content from combustion flue gases. Small amounts of alkaline sorbents are injected into the flue gas stream at a relatively low rate. A particulate filter is used to remove mercury-containing particles downstream of each injection point used in the power plant system.

  11. 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.

  12. Composite seal reduces alkaline battery leakage

    NASA Technical Reports Server (NTRS)

    Clatterbuck, C. H.; Plitt, K. F.

    1965-01-01

    Composite seal consisting of rubber or plastic washers and a metal washer reduces alkaline battery leakage. Adhesive is applied to each washer interface, and the washers are held together mechanically.

  13. Ratiometric electrochemical detection of alkaline phosphatase.

    PubMed

    Goggins, Sean; Naz, Christophe; Marsh, Barrie J; Frost, Christopher G

    2015-01-11

    A novel ferrocene-derived substrate for the ratiometric electrochemical detection of alkaline phosphatase (ALP) was designed and synthesised. It was demonstrated to be an excellent electrochemical substrate for the ALP-labelled enzyme-linked immunosorbent assay (ELISA).

  14. Evaluation of the alkaline electrolysis of zinc

    SciTech Connect

    Meisenhelder, J.H.; Brown, A.P.; Loutfy, R.O.; Yao, N.P.

    1981-05-01

    The alkaline leach and electrolysis process for zinc production is compared to the conventional acid-sulfate process in terms of both energy saving and technical merit. In addition, the potential for industrial application of the alkaline process is discussed on the basis of present market conditions, possible future zinc market scenarios, and the probability of increased secondary zinc recovery. In primary zinc production, the energy-saving potential for the alkaline process was estimated to be greater than 10%, even when significantly larger electrolysis current densities than those required for the sulfate process are used. The principal technical advantages of the alkaline process are that it can handle low-grade, high-iron-content or oxidized ores (like most of those found in the US) in a more cost- and energy-efficient manner than can the sulfate process. Additionally, in the electrowinning operation, the alkaline process should be technically superior because a dendritic or sponge deposit is formed that is amenable to automated collection without interruption of the electrolysis. Also, use of the higher current densities would result in significant capital cost reductions. Alkaline-based electrolytic recovery processes were considered for the recycling of zinc from smelter baghouse dusts and from the potential source of nickel/zinc electric-vehicle batteries. In all comparisons, an alkaline process was shown to be technically superior and, particularly for the baghouse dusts, energetically and economically superior to alternatively proposed recovery methods based on sulfate electrolysis. It is concluded that the alkaline zinc method is an important alternative technology to the conventional acid zinc process. (WHK)

  15. Toxicity of alkalinity to Hyalella azteca

    USGS Publications Warehouse

    Lasier, P.J.; Winger, P.V.; Reinert, R.E.

    1997-01-01

    Toxicity testing and chemical analyses of sediment pore water have been suggested for use in sediment quality assessments and sediment toxicity identification evaluations. However, caution should be exercised in interpreting pore-water chemistry and toxicity due to inherent chemical characteristics and confounding relationships. High concentrations of alkalinity, which are typical of sediment pore waters from many regions, have been shown to be toxic to test animals. A series of tests were conducted to assess the significance of elevated alkalinity concentrations to Hyalella azteca, an amphipod commonly used for sediment and pore-water toxicity testing. Toxicity tests with 14-d old and 7-d old animals were conducted in serial dilutions of sodium bicarbonate (NaHCO3) solutions producing alkalinities ranging between 250 to 2000 mg/L as CaCO3. A sodium chloride (NaCl) toxicity test was also conducted to verify that toxicity was due to bicarbonate and not sodium. Alkalinity was toxic at concentrations frequently encountered in sediment pore water. There was also a significant difference in the toxicity of alkalinity between 14-d old and 7-d old animals. The average 96-h LC50 for alkalinity was 1212 mg/L (as CaCO3) for 14-d old animals and 662 mg/L for the younger animals. Sodium was not toxic at levels present in the NaHCO3 toxicity tests. Alkalinity should be routinely measured in pore-water toxicity tests, and interpretation of toxicity should consider alkalinity concentration and test-organism tolerance.

  16. Technetium recovery from high alkaline solution

    DOEpatents

    Nash, Charles A.

    2016-07-12

    Disclosed are methods for recovering technetium from a highly alkaline solution. The highly alkaline solution can be a liquid waste solution from a nuclear waste processing system. Methods can include combining the solution with a reductant capable of reducing technetium at the high pH of the solution and adding to or forming in the solution an adsorbent capable of adsorbing the precipitated technetium at the high pH of the solution.

  17. Alkaline tolerant dextranase from streptomyces anulatus

    DOEpatents

    Decker, Stephen R.; Adney, William S.; Vinzant, Todd B.; Himmel, Michael E.

    2003-01-01

    A process for production of an alkaline tolerant dextranase enzyme comprises culturing a dextran-producing microorganism Streptomyces anulatus having accession no. ATCC PTA-3866 to produce an alkaline tolerant dextranase, Dex 1 wherein the protein in said enzyme is characterized by a MW of 63.3 kDa and Dex 2 wherein its protein is characterized by a MW of 81.8 kDa.

  18. Alkaline Water and Longevity: A Murine Study

    PubMed Central

    Magro, Massimiliano; Corain, Livio; Ferro, Silvia; Baratella, Davide; Bonaiuto, Emanuela; Terzo, Milo; Corraducci, Vittorino; Salmaso, Luigi; Vianello, Fabio

    2016-01-01

    The biological effect of alkaline water consumption is object of controversy. The present paper presents a 3-year survival study on a population of 150 mice, and the data were analyzed with accelerated failure time (AFT) model. Starting from the second year of life, nonparametric survival plots suggest that mice watered with alkaline water showed a better survival than control mice. Interestingly, statistical analysis revealed that alkaline water provides higher longevity in terms of “deceleration aging factor” as it increases the survival functions when compared with control group; namely, animals belonging to the population treated with alkaline water resulted in a longer lifespan. Histological examination of mice kidneys, intestine, heart, liver, and brain revealed that no significant differences emerged among the three groups indicating that no specific pathology resulted correlated with the consumption of alkaline water. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline water of long-lived mouse models. PMID:27340414

  19. Performed surfactant-optimized aqueous alkaline flood

    SciTech Connect

    Thigpen, D.R.; Lawson, J.B.; Nelson, R.C.

    1991-11-26

    This paper describes improvement in a process for recovering oil from an acidic oil reservoir by injecting an aqueous alkaline solution comprising water, sodium chloride, and alkaline material for reacting with the reservoir oil forming a petroleum acid soap to form an in-situ surfactant system. The improvement comprises: selecting a preformed cosurfactant which is soluble in both the aqueous solution and the reservoir oil and has a solubility ratio which is grater than the solubility ratio of the petroleum acid soap where the solubility ratio is the ratio of solubility in the aqueous alkaline solution to the solubility in the reservoir oil; combining with the alkaline solution an amount of the preformed cosurfactant which will result in the in-situ surfacant system having a salinity about equal to a salinity which results in minimal interfacial tension between the oil in the reservoir and the in-situ surfactant system at reservoir temperature, wherein the amount of the preformed cosurfactant is about 0.3 percent by weight in the aqueous alkaline solution; and injecting the cosurfactant-aqueous alkaline solution mixture into the reservoir to displace oil toward a fluid production location.

  20. 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.

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

    PubMed Central

    Rahman, Mohammad M.; Inoue, Akira; Ojima, Takao

    2014-01-01

    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, 66, 45, and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., 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 40°C, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55°C 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 β-1,4-glucanase (EC 3.2.1.4). A cDNA of 1013 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. PMID:25147784

  2. The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene Expression

    PubMed Central

    Atanasova, Lea; Fekete, Erzsébet; Paholcsek, Melinda; Sándor, Erzsébet; Aquino, Benigno; Druzhinina, Irina S.; Karaffa, Levente; Kubicek, Christian P.

    2014-01-01

    Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex. PMID:25386652

  3. An ionic liquid tolerant cellulase derived from chemically polluted microhabitats and its application in in situ saccharification of rice straw.

    PubMed

    Xu, Jiaxing; He, Bingfang; Wu, Bin; Wang, Bin; Wang, Chenghua; Hu, Lei

    2014-04-01

    A cellulase-producing fungus was isolated from chemically polluted microhabitats by [Amim][Cl] enrichment and identified as Aspergillus fumigatus. The maximum activity of the cellulase in 30% (v/v) ionic liquids (ILs) was detected in [Emim][DMP], [Amim][Cl] and [Emim][MA] as 127%, 111% and 109%, respectively, of its activity in buffer, suggesting its superior performance in high concentration ILs. Strikingly, although its initial activity varied in each IL, its half-life was longer in most ILs than in buffer, evidence of a high conformational stability of the enzyme that is essential for maintaining the remaining activity in relevant media. It noteworthy that 1-3M NaCl can activate the cellulase somewhat. More gratifyingly, a compatible IL-cellulase system based on the cellulase was developed, and its use significantly improved the saccharification rate of rice straw from 53% to 88% versus the control, demonstrating its potential for efficient transformation of lignocellulose to glucose in a single-step process.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-08-01

    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 soluble 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 biofilm formation in cellulosic biomass hydrolysis may promise an improved cellulosic biofuel process. PMID:26257364

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

    PubMed Central

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

    2016-01-01

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

  7. Evaluation of bacterial expansin EXLX1 as a cellulase synergist for the saccharification of lignocellulosic Agro-industrial wastes.

    PubMed

    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.

  8. Observing and modeling BMCC degradation by commercial cellulase cocktails with fluorescently labeled Trichoderma reseii Cel7A through confocal microscopy.

    PubMed

    Luterbacher, Jeremy S; Walker, Larry P; Moran-Mirabal, Jose M

    2013-01-01

    Understanding the depolymerization mechanisms of cellulosic substrates by cellulase cocktails is a critical step towards optimizing the production of monosaccharides from biomass. The Spezyme CP cellulase cocktail combined with the Novo 188 β-glucosidase blend was used to depolymerize bacterial microcrystalline cellulose (BMCC), which was immobilized on a glass surface. The enzyme mixture was supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A, which served as a reporter to track cellulase binding onto the physical structure of the cellulosic substrate. Both micro-scale imaging and bulk experiments were conducted. All reported experiments were conducted at 50 °C, the optimal temperature for maximum hydrolytic activity of the enzyme cocktail. BMCC structure was observed throughout degradation by labeling it with a fluorescent dye. This method allowed us to measure the binding of cellulases in situ and follow the temporal morphological changes of cellulose during its depolymerization by a commercial cellulase mixture. Three kinetic models were developed and fitted to fluorescence intensity data obtained through confocal microscopy: irreversible and reversible binding models, and an instantaneous binding model. The models were successfully used to predict the soluble sugar concentrations that were liberated from BMCC in bulk experiments. Comparing binding and kinetic parameters from models with different assumptions to previously reported constants in the literature led us to conclude that exposing new binding sites is an important rate-limiting step in the hydrolysis of crystalline cellulose.

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

    PubMed

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

    2010-03-01

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

  10. Enhanced enzymatic hydrolysis and ethanol production from cashew apple bagasse pretreated with alkaline hydrogen peroxide.

    PubMed

    da Costa, Jessyca Aline; Marques, José Edvan; Gonçalves, Luciana Rocha Barros; Rocha, Maria Valderez Ponte

    2015-03-01

    The effect of combinations and ratios between different enzymes has been investigated in order to assess the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen peroxide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic hydrolysis conducted with cellulase complex and β-glucosidase in a ratio of 0.61:0.39, enzyme loading of 30FPU/g(CAB-AHP) and 66CBU/g(CAB-AHP), respectively, using 4% cellulose from CAB-AHP, turned out to be the most effective conditions, with glucose and xylose yields of 511.68 mg/g(CAB-AHP) and 237.8 mg/g(CAB-AHP), respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an ethanol yield of 61.8kg/ton(CAB), corresponding to 15 g/L ethanol and productivity of 3.75 g/( Lh). The ethanol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80% yield and 74.2kg/ton(CAB).

  11. Enhanced enzymatic hydrolysis and ethanol production from cashew apple bagasse pretreated with alkaline hydrogen peroxide.

    PubMed

    da Costa, Jessyca Aline; Marques, José Edvan; Gonçalves, Luciana Rocha Barros; Rocha, Maria Valderez Ponte

    2015-03-01

    The effect of combinations and ratios between different enzymes has been investigated in order to assess the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen peroxide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic hydrolysis conducted with cellulase complex and β-glucosidase in a ratio of 0.61:0.39, enzyme loading of 30FPU/g(CAB-AHP) and 66CBU/g(CAB-AHP), respectively, using 4% cellulose from CAB-AHP, turned out to be the most effective conditions, with glucose and xylose yields of 511.68 mg/g(CAB-AHP) and 237.8 mg/g(CAB-AHP), respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an ethanol yield of 61.8kg/ton(CAB), corresponding to 15 g/L ethanol and productivity of 3.75 g/( Lh). The ethanol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80% yield and 74.2kg/ton(CAB). PMID:25545094

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Uptake of arsenic by alkaline soils near alkaline coal fly ash disposal facilities.

    PubMed

    Khodadoust, Amid P; Theis, Thomas L; Murarka, Ishwar P; Naithani, Pratibha; Babaeivelni, Kamel

    2013-12-01

    The attenuation of arsenic in groundwater near alkaline coal fly ash disposal facilities was evaluated by determining the uptake of arsenic from ash leachates by surrounding alkaline soils. Ten different alkaline soils near a retired coal fly ash impoundment were used in this study with pH ranging from 7.6 to 9.0, while representative coal fly ash samples from two different locations in the coal fly ash impoundment were used to produce two alkaline ash leachates with pH 7.4 and 8.2. The arsenic found in the ash leachates was present as arsenate [As(V)]. Adsorption isotherm experiments were carried out to determine the adsorption parameters required for predicting the uptake of arsenic from the ash leachates. For all soils and leachates, the adsorption of arsenic followed the Langmuir and Freundlich equations, indicative of the favorable adsorption of arsenic from leachates onto all soils. The uptake of arsenic was evaluated as a function of ash leachate characteristics and the soil components. The uptake of arsenic from alkaline ash leachates, which occurred mainly as calcium hydrogen arsenate, increased with increasing clay fraction of soil and with increasing soil organic matter of the alkaline soils. Appreciable uptake of arsenic from alkaline ash leachates with different pH and arsenic concentration was observed for the alkaline soils, thus attenuating the contamination of groundwater downstream of the retired coal fly ash impoundment.

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

    PubMed

    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.

  15. Improved production of reducing sugars from rice husk and rice straw using bacterial cellulase and xylanase activated with hydroxyapatite nanoparticles.

    PubMed

    Dutta, Nalok; Mukhopadhyay, Arka; Dasgupta, Anjan Kr; Chakrabarti, Krishanu

    2014-02-01

    Purified bacterial cellulase and xylanase were activated in the presence of calcium hydroxyapatite nanoparticles (NP) with concomitant increase in thermostability about 35% increment in production of d-xylose and reducing sugars from rice husk and rice straw was obtained at 80°C by the sequential treatment of xylanase and cellulase enzymes in the presence of NP compared to the untreated enzyme sets. Our findings suggested that if the rice husk and the rice straw samples were pre-treated with xylanase prior to treatment with cellulase, the percentage increase of reducing sugar per 100g of substrate (starting material) was enhanced by about 29% and 41%, respectively. These findings can be utilized for the extraction of reducing sugars from cellulose and xylan containing waste material. The purely enzymatic extraction procedure can be substituted for the harsh and bio-adverse chemical methods.

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

    PubMed

    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

  17. [Hydrolysis of cellulose by fungi. II. Production of cellulases by Trichoderma harzianum by fermentation in liquid media].

    PubMed

    Roussos, S; Raimbault, M

    1982-01-01

    Microcristalline cellulose (cellulose Avicel, Merck) supported growth of Trichoderma harzianum and induced production of cellulases in liquid cultures. After 50 h growth, the total cellulasic activities present in both the supernatant and the mycelium were 3,000 IU/l of carboxymethyl cellulose, 400 IU/l of filter paper activity, and 4 IU/l of cotton activity corresponding to 1.7 g/l of proteins. Cellulase production could be increased by a preliminary treatment of cellulose, and pH regulation during growth. The influence of inoculum concentration was studied and an optimum of 3 x 10(7) conidia/g dry weight of substrate was demonstrated. Using a synthetic culture medium, a soluble factor of germination was demonstrated which could be leached out by 3 successive washings of conidia.

  18. 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

  19. A constitutive expression system for cellulase secretion in Escherichia coli and its use in bioethanol production.

    PubMed

    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.

  20. Endogenous cellulase production in the leaf litter foraging mangrove crab Parasesarma erythodactyla.

    PubMed

    Bui, T H Hanh; Lee, Shing Yip

    2015-01-01

    The sesarmid crab Parasesarma erythodactyla consumes large amounts of mangrove leaf litter but its biochemical capacity for cellulose digestion is poorly known. We demonstrate the presence of endo-β-1,4-glucanase, β-glucosidase and total cellulase activities in the digestive juice of this crab. The highest total cellulase activity was observed at mildly acidic pH (5 to 6) and temperature between 30 and 50°C. A 1752bp cDNA containing an open reading frame of 1386bp encoding a putative endo-β-1,4-glucanase (EG) of 461 amino acids was identified in the crab's hepatopancreas using polymerase chain reaction (PCR), cloning and sequencing techniques. P. erythodactyla endo-β-1,4-glucanase (PeEG) contains a glycosyl hydrolase family 9 (GHF9) catalytic domain with all catalytically important residues conserved, and shows high sequence identity to GHF9 EGs reported from other arthropods. The endogenous origin of PeEG was confirmed by PCR amplification of a ~1.5kb DNA fragment, containing a phase 1 intron flanked by two exon sequences identical to the cDNA, from genomic DNA isolated from the crab's muscle tissue. PeEG encoding cDNA is the first endogenous EG sequence reported from the brachyuran crabs. Using degenerate primers, we also isolated 204bp cDNA fragments with sequences affiliated to EG from the hepatopancreas of eight other mangrove crabs of the Sesarmidae (Neosarmatium trispinosum and Sesarmoides borneensis), Macrophthalmidae (Ilyograpsus daviei, Australoplax tridentata, and Macrophthalmus setosus), Varunidae (Pseudohelice subquadrata), Heloeciidae (Heloecius cordiformis), and Ocypodidae (Uca perplexa) families, suggesting that endogenous cellulase production may be a common characteristic among the detritivorous mangrove crabs.

  1. Probing carbohydrate product expulsion from a processive cellulase with multiple absolute binding free energy methods.

    PubMed

    Bu, Lintao; Beckham, Gregg T; Shirts, Michael R; Nimlos, Mark R; Adney, William S; Himmel, Michael E; Crowley, Michael F

    2011-05-20

    Understanding the enzymatic mechanism that cellulases employ to degrade cellulose is critical to efforts to efficiently utilize plant biomass as a sustainable energy resource. A key component of cellulase action on cellulose is product inhibition from monosaccharide and disaccharides in the product site of cellulase tunnel. The absolute binding free energy of cellobiose and glucose to the product site of the catalytic tunnel of the Family 7 cellobiohydrolase (Cel7A) of Trichoderma reesei (Hypocrea jecorina) was calculated using two different approaches: steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations. For the SMD approach, three methods based on Jarzynski's equality were used to construct the potential of mean force from multiple pulling trajectories. The calculated binding free energies, -14.4 kcal/mol using SMD and -11.2 kcal/mol using FEP/MD, are in good qualitative agreement. Analysis of the SMD pulling trajectories suggests that several protein residues (Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381) play key roles in cellobiose and glucose binding to the catalytic tunnel. Five mutations (R251A, D259A, D262A, W376A, and Y381A) were made computationally to measure the changes in free energy during the product expulsion process. The absolute binding free energies of cellobiose to the catalytic tunnel of these five mutants are -13.1, -6.0, -11.5, -7.5, and -8.8 kcal/mol, respectively. The results demonstrated that all of the mutants tested can lower the binding free energy of cellobiose, which provides potential applications in engineering the enzyme to accelerate the product expulsion process and improve the efficiency of biomass conversion. PMID:21454590

  2. Probing Carbohydrate Product Expulsion from a Processive Cellulase with Multiple Absolute Binding Free Energy Methods*

    PubMed Central

    Bu, Lintao; Beckham, Gregg T.; Shirts, Michael R.; Nimlos, Mark R.; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.

    2011-01-01

    Understanding the enzymatic mechanism that cellulases employ to degrade cellulose is critical to efforts to efficiently utilize plant biomass as a sustainable energy resource. A key component of cellulase action on cellulose is product inhibition from monosaccharide and disaccharides in the product site of cellulase tunnel. The absolute binding free energy of cellobiose and glucose to the product site of the catalytic tunnel of the Family 7 cellobiohydrolase (Cel7A) of Trichoderma reesei (Hypocrea jecorina) was calculated using two different approaches: steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations. For the SMD approach, three methods based on Jarzynski's equality were used to construct the potential of mean force from multiple pulling trajectories. The calculated binding free energies, −14.4 kcal/mol using SMD and −11.2 kcal/mol using FEP/MD, are in good qualitative agreement. Analysis of the SMD pulling trajectories suggests that several protein residues (Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381) play key roles in cellobiose and glucose binding to the catalytic tunnel. Five mutations (R251A, D259A, D262A, W376A, and Y381A) were made computationally to measure the changes in free energy during the product expulsion process. The absolute binding free energies of cellobiose to the catalytic tunnel of these five mutants are −13.1, −6.0, −11.5, −7.5, and −8.8 kcal/mol, respectively. The results demonstrated that all of the mutants tested can lower the binding free energy of cellobiose, which provides potential applications in engineering the enzyme to accelerate the product expulsion process and improve the efficiency of biomass conversion. PMID:21454590

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

    DOE PAGES

    Wei, Hui; Wang, Wei; Alahuhta, Markus; Vander Wall, Todd; Baker, John O.; Taylor, Larry E.; Decker, Stephen R.; Himmel, Michael E.; Zhang, Min

    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

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

    SciTech Connect

    Wei, Hui; Wang, Wei; Alahuhta, Markus; Vander Wall, Todd; Baker, John O.; Taylor, Larry E.; Decker, Stephen R.; Himmel, Michael E.; Zhang, Min

    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 Trichoderma 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

  5. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of cellulose-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.

  6. Cellulase production in continuous and fed-batch culture by Trichoderma reesei MCG80

    SciTech Connect

    Allen, A.L.; Andreotti, R.E.

    1982-01-01

    Continuous culture of Natick's strain MCG80 of Trichoderma reesei at a dilution rate of 0.028 h/sup -1/ has yielded a cellulase titer of over 61 U/mL using 5% lactose as the sole carbon source. Enzyme productivity at this dilution rate is 168 IU/L/h. Repeated fed-batch cultures using this strain on lactose as the carbon source have titers of 10 IU/mL with productivities in excess of 100 IU/L/h. 5 figures, 1 table.

  7. Suggested improvements to the standard filter paper assay used to measure cellulase activity.

    PubMed

    Coward-Kelly, Guillermo; Aiello-Mazzari, Cateryna; Kim, Sehoon; Granda, Cesar; Holtzapple, Mark

    2003-06-20

    Two suggestions can be found in the literature to improve the reproducibility of the Mandels' filter paper assay: add supplemental cellobiase and increase the boiling time for color development. Here we provide data that strongly supports adding supplemental cellobiase. Adding supplemental cellobiase increased assay response by 56%. Cellulases from different sources have different cellobiase activities, which would cause significant variation in the assay response. There is no need for additional boiling time-5 minutes is sufficient. For maximum reproducibility, it is essential that the water bath vigorously boil so that temperature excursions are minimized.

  8. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1998-05-26

    Bacteria which produce large amounts of cellulose-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.

  9. Suggested improvements to the standard filter paper assay used to measure cellulase activity.

    PubMed

    Coward-Kelly, Guillermo; Aiello-Mazzari, Cateryna; Kim, Sehoon; Granda, Cesar; Holtzapple, Mark

    2003-06-20

    Two suggestions can be found in the literature to improve the reproducibility of the Mandels' filter paper assay: add supplemental cellobiase and increase the boiling time for color development. Here we provide data that strongly supports adding supplemental cellobiase. Adding supplemental cellobiase increased assay response by 56%. Cellulases from different sources have different cellobiase activities, which would cause significant variation in the assay response. There is no need for additional boiling time-5 minutes is sufficient. For maximum reproducibility, it is essential that the water bath vigorously boil so that temperature excursions are minimized. PMID:12673775

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

    PubMed

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

    2014-01-01

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

  11. Effect of chemical treatments on the degradability of cotton straw by rumen microorganisms and by fungal cellulase

    SciTech Connect

    Miron, J.; Ben-Ghedalia, D.

    1981-12-01

    Three different chemical treatments-sulfur dioxide, ozone, and sodium hydroxide-were applied on cotton straw, and the effect on cell-wall degradability was assessed by using rumen microorganisms and Trichoderma reesei cellulase. Sulfur dioxide (applied at 70 degrees Celcius for 72 hours) did not change the lignin content of cotton straw but reduced the concentration of hemicellulose by 48%. Ozone exerted a dual effect, both on lignin (a 40% reduction) and hemicellulose (a 54% decrease). The treatment with NaOH did not solubilize cell-wall components. The in vitro organic matter digestibility with rumen fluid of cotton straw was increased significantly by ozone and SO/sub 2/ treatments, by 120% and 50%, respectively, but not by NaOH. T. reesei cellulase was applied on the chemically pretreated cotton straw at a low level (6 filter paper U/g straw, organic matter). The highest level of reducing sugars (30.6 g/100 g organic matter) was obtained with the O/sub 3/-cellulase combination, which solubilized 64% of the cellulose and 88% of the hemicellulose. The SO/sub 2/- and the NaOH-pretreated cotton straw were hydrolyzed by T. reesei cellulase to the same extent. The rumen fluid digestibility of the enzymatically hydrolyzed straw was not increased over that obtained with the chemical pretreatments. However, the fermentability of the combined treatments was increased markedly. In the O/sub 3/-cellulase-treated cotton straw, 83% of the rumen fluid digestible material consisted of highly fermentable components. Although ozone proved to be the most potent pretreatment for enzymic saccharification, the absolute result was modest. The limited effect of the combined O/sub 3/-cellulase treatment was probably associated with the pretreatment limitations, but not with the enzyme level. A hypothesis has been suggested as to the location of lignin and hemicellulose in the cell-wall unit of cotton straw. (Refs. 18).

  12. 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.

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

    SciTech Connect

    Danna, K.J.

    1997-06-01

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

  14. 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.

  15. Photoelastic response of alkaline earth aluminosilicate glasses.

    PubMed

    Smedskjaer, Morten M; Saxton, Scott A; Ellison, Adam J; Mauro, John C

    2012-02-01

    Understanding the structural origins of the photoelastic response in oxide glasses is important for discovering new families of zero-stress optic glasses and for developing a predictive physical model. In this Letter, we have investigated the composition dependence of the stress optic coefficient C of 32 sodium aluminosilicate glasses with different types of alkaline earth oxides (MgO, CaO, SrO, and BaO). We find that most of the composition dependence of the stress optic response can be captured by a linear regression model and that the individual contributions from the alkaline earths to C depend on the alkaline earth-oxygen bond metallicity. High bond metallicity is required to allow bonds to be distorted along both the bonding direction and perpendicular to it. These findings are valuable for understanding the photoelastic response of oxide glasses.

  16. Alkaline Capacitors Based on Nitride Nanoparticles

    NASA Technical Reports Server (NTRS)

    Aldissi, Matt

    2003-01-01

    High-energy-density alkaline electrochemical capacitors based on electrodes made of transition-metal nitride nanoparticles are undergoing development. Transition- metal nitrides (in particular, Fe3N and TiN) offer a desirable combination of high electrical conductivity and electrochemical stability in aqueous alkaline electrolytes like KOH. The high energy densities of these capacitors are attributable mainly to their high capacitance densities, which, in turn, are attributable mainly to the large specific surface areas of the electrode nanoparticles. Capacitors of this type could be useful as energy-storage components in such diverse equipment as digital communication systems, implanted medical devices, computers, portable consumer electronic devices, and electric vehicles.

  17. Alkaline earth filled nickel skutterudite antimonide thermoelectrics

    DOEpatents

    Singh, David Joseph

    2013-07-16

    A thermoelectric material including a body centered cubic filled skutterudite having the formula A.sub.xFe.sub.yNi.sub.zSb.sub.12, where A is an alkaline earth element, x is no more than approximately 1.0, and the sum of y and z is approximately equal to 4.0. The alkaline earth element includes guest atoms selected from the group consisting of Be, Mb, Ca, Sr, Ba, Ra and combinations thereof. The filled skutterudite is shown to have properties suitable for a wide variety of thermoelectric applications.

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

    SciTech Connect

    Stipanovic, Arthur J

    2014-11-17

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

  19. Multicomponent cellulase production by Cellulomonas biazotea NCIM-2550 and its applications for cellulosic biohydrogen production.

    PubMed

    Saratale, Ganesh D; Saratale, Rijuta G; Lo, Yung-Chung; Chang, Jo-Shu

    2010-01-01

    Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM-2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl(2) as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H(2) energy was examined with seven H(2)-producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H(2) production performance with the hydrolysate of SCB and xylan. The cumulative H(2) production and H(2) yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H(2)/g reducing sugar (or 1.91 mmol H(2)/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H(2) production and H(2) yield of 87.02 mL/L and 5.03 mmol H(2)/g reducing sugar (or 4.01 mmol H(2)/g cellulose), respectively.

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

    PubMed

    Sciessere, L; Cunha-Santino, M B; Bianchini, I

    2011-07-01

    Due to the connection between enzymatic activity and degradation of different fractions of organic matter, enzyme assays can be used to estimate degradation rates of particulate and dissolved organic carbon in freshwater systems. The aim of this study was to quantify and model the enzymatic degradation involving the decomposition of macrophytes, describing temporal activity of cellulases (EC 3.2.1.4 and EC 3.2.1.91) and xylanase (EC 3.2.1.8) during in situ decomposition of three aquatic macrophytes (Salvinia sp., Eichhornia azurea and Cyperus giganteus) on the surface and water-sediment interface (w-s interface) of an oxbow lagoon (Óleo lagoon) within a natural Brazilian Savanna Reserve. Overall, the enzymatic degradation of aquatic macrophytes in Óleo lagoon occurred during the whole year and was initiated together with leaching. Xylanase production was ca. 5 times higher than cellulase values due to easy access to this compound by cellulolytic microorganisms. Enzymatic production and detritus mass decay were similar on the surface and w-s interface. Salvinia sp. was the most recalcitrant detritus, with low mass decay and enzymatic activity. E. azurea and C. giganteus decomposition rates and enzymatic production were high and similar. Due to the physicochemical homogeneity observed in the Óleo lagoon, the differences between the decay rates of each species are mostly related with detritus chemical quality. PMID:24031706

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

    PubMed Central

    Kilikian, B.V.; Afonso, L.C.; Souza, T.F.C.; Ferreira, R.G.; Pinheiro, I.R.

    2014-01-01

    Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77. PMID:24948946

  2. Expression of recombinant cellulase Cel5A from Trichoderma reesei in tobacco plants.

    PubMed

    Garvey, Megan; Klinger, Johannes; Klose, Holger; Fischer, Rainer; Commandeur, Ulrich

    2014-01-01

    Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems. PMID:24962636

  3. Engineered thermostable fungal cellulases exhibit efficient synergistic cellulose hydrolysis at elevated temperatures.

    PubMed

    Trudeau, Devin L; Lee, Toni M; Arnold, Frances H

    2014-12-01

    A major obstacle to using widely available and low-cost lignocellulosic feedstocks to produce renewable fuels and chemicals is the high cost and low efficiency of the enzyme mixtures used to hydrolyze cellulose to fermentable sugars. One possible solution entails engineering current cellulases to function efficiently at elevated temperatures in order to boost reaction rates and exploit several other advantages of a higher temperature process. Here, we describe the creation of the most stable reported fungal endoglucanase, a derivative of Hypocrea jecorina (anamorph Trichoderma reesei) Cel5A, by combining stabilizing mutations identified using consensus design, chimera studies, and structure-based computational methods. The engineered endoglucanase has an optimal temperature that is 17°C higher than wild type H. jecorina Cel5A, and hydrolyzes 1.5 times as much cellulose over 60 h at its optimum temperature compared to the wild type enzyme at its optimal temperature. This enzyme complements previously engineered highly active, thermostable variants of the fungal cellobiohydrolases Cel6A and Cel7A in a thermostable cellulase mixture that hydrolyzes cellulose synergistically at an optimum temperature of 70°C over 60 h.The thermostable mixture produces three times as much total sugar as the best mixture of the wild type enzymes operating at its optimum temperature of 60°C, clearly demonstrating the advantage of higher temperature cellulose hydrolysis.

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

    PubMed

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

    2015-01-01

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

  5. Detecting Cellulase Penetration Into Corn Stover Cell Walls by Immuno-Electron Microscopy

    SciTech Connect

    Donohoe, B. S.; Selig, M. J.; Viamajala, S.; Vinzant, T. B.; Adney, W. S.; Himmel, M. E.

    2009-06-15

    In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild-, moderate- and high-severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme- and polymer-specific antibodies. Low severity dilute-acid pretreatment (20 min at 100 C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120 C) allowed the enzymes to penetrate {approx}20% of the cell wall, and the high severity (20 min pretreatment at 150 C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute-acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high-resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes.

  6. Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates

    PubMed Central

    2013-01-01

    Introduction Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules. Method We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements. Results Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film. Conclusion Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film. PMID:23819686

  7. Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library.

    PubMed

    Nguyen, Nhung Hong; Maruset, Lalita; Uengwetwanit, Tanaporn; Mhuantong, Wuttichai; Harnpicharnchai, Piyanun; Champreda, Verawat; Tanapongpipat, Sutipa; Jirajaroenrat, Kanya; Rakshit, Sudip K; Eurwilaichitr, Lily; Pongpattanakitshote, Somchai

    2012-01-01

    Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.

  8. Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis

    PubMed Central

    Brás, Joana L. A.; Cartmell, Alan; Carvalho, Ana Luísa M.; Verzé, Genny; Bayer, Edward A.; Vazana, Yael; Correia, Márcia A. S.; Prates, José A. M.; Ratnaparkhe, Supriya; Boraston, Alisdair B.; Romão, Maria J.; Fontes, Carlos M. G. A.; Gilbert, Harry J.

    2011-01-01

    Clostridium thermocellum is a well-characterized cellulose-degrading microorganism. The genome sequence of C. thermocellum encodes a number of proteins that contain type I dockerin domains, which implies that they are components of the cellulose-degrading apparatus, but display no significant sequence similarity to known plant cell wall–degrading enzymes. Here, we report the biochemical properties and crystal structure of one of these proteins, designated CtCel124. The protein was shown to be an endo-acting cellulase that displays a single displacement mechanism and acts in synergy with Cel48S, the major cellulosomal exo-cellulase. The crystal structure of CtCel124 in complex with two cellotriose molecules, determined to 1.5 Å, displays a superhelical fold in which a constellation of α-helices encircle a central helix that houses the catalytic apparatus. The catalytic acid, Glu96, is located at the C-terminus of the central helix, but there is no candidate catalytic base. The substrate-binding cleft can be divided into two discrete topographical domains in which the bound cellotriose molecules display twisted and linear conformations, respectively, suggesting that the enzyme may target the interface between crystalline and disordered regions of cellulose. PMID:21393568

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

    PubMed Central

    Sciessere, L.; Cunha-Santino, M. B.; Bianchini, I.

    2011-01-01

    Due to the connection between enzymatic activity and degradation of different fractions of organic matter, enzyme assays can be used to estimate degradation rates of particulate and dissolved organic carbon in freshwater systems. The aim of this study was to quantify and model the enzymatic degradation involving the decomposition of macrophytes, describing temporal activity of cellulases (EC 3.2.1.4 and EC 3.2.1.91) and xylanase (EC 3.2.1.8) during in situ decomposition of three aquatic macrophytes (Salvinia sp., Eichhornia azurea and Cyperus giganteus) on the surface and water-sediment interface (w-s interface) of an oxbow lagoon (Óleo lagoon) within a natural Brazilian Savanna Reserve. Overall, the enzymatic degradation of aquatic macrophytes in Óleo lagoon occurred during the whole year and was initiated together with leaching. Xylanase production was ca. 5 times higher than cellulase values due to easy access to this compound by cellulolytic microorganisms. Enzymatic production and detritus mass decay were similar on the surface and w-s interface. Salvinia sp. was the most recalcitrant detritus, with low mass decay and enzymatic activity. E. azurea and C. giganteus decomposition rates and enzymatic production were high and similar. Due to the physicochemical homogeneity observed in the Óleo lagoon, the differences between the decay rates of each species are mostly related with detritus chemical quality. PMID:24031706

  10. Engineered thermostable fungal cellulases exhibit efficient synergistic cellulose hydrolysis at elevated temperatures.

    PubMed

    Trudeau, Devin L; Lee, Toni M; Arnold, Frances H

    2014-12-01

    A major obstacle to using widely available and low-cost lignocellulosic feedstocks to produce renewable fuels and chemicals is the high cost and low efficiency of the enzyme mixtures used to hydrolyze cellulose to fermentable sugars. One possible solution entails engineering current cellulases to function efficiently at elevated temperatures in order to boost reaction rates and exploit several other advantages of a higher temperature process. Here, we describe the creation of the most stable reported fungal endoglucanase, a derivative of Hypocrea jecorina (anamorph Trichoderma reesei) Cel5A, by combining stabilizing mutations identified using consensus design, chimera studies, and structure-based computational methods. The engineered endoglucanase has an optimal temperature that is 17°C higher than wild type H. jecorina Cel5A, and hydrolyzes 1.5 times as much cellulose over 60 h at its optimum temperature compared to the wild type enzyme at its optimal temperature. This enzyme complements previously engineered highly active, thermostable variants of the fungal cellobiohydrolases Cel6A and Cel7A in a thermostable cellulase mixture that hydrolyzes cellulose synergistically at an optimum temperature of 70°C over 60 h.The thermostable mixture produces three times as much total sugar as the best mixture of the wild type enzymes operating at its optimum temperature of 60°C, clearly demonstrating the advantage of higher temperature cellulose hydrolysis. PMID:24916885

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

    PubMed

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

    2015-01-01

    In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95%) of glycosyl hydrolase families including GH2 (24 genes), GH8 (27 genes), GH10 (19 genes), GH11 (14 genes) and GH36 (17 genes) families was retrieved, and certain sequences of GH2 (10.61%), GH8 (3.33%), and GH11 (18.42%) families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect. PMID:26197317

  12. Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library.

    PubMed

    Nguyen, Nhung Hong; Maruset, Lalita; Uengwetwanit, Tanaporn; Mhuantong, Wuttichai; Harnpicharnchai, Piyanun; Champreda, Verawat; Tanapongpipat, Sutipa; Jirajaroenrat, Kanya; Rakshit, Sudip K; Eurwilaichitr, Lily; Pongpattanakitshote, Somchai

    2012-01-01

    Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement. PMID:22790926

  13. Mathematical modeling and optimization of cellulase protein production using Trichoderma reesei RL-P37

    SciTech Connect

    Tholudur, A.; Ramirez, W.F.; McMillan, J.D.

    1999-07-01

    The enzyme cellulase, a multienzyme complex made up of several proteins, catalyzes the conversion of cellulose to glucose in an enzymatic hydrolysis-based biomass-to-ethanol process. Production of cellulase enzyme proteins in large quantities using the fungus Trichoderma reesei requires understanding the dynamics of growth and enzyme production. The method of neural network parameter function modeling, which combines the approximation capabilities of neural networks with fundamental process knowledge, is utilized to develop a mathematical model of this dynamic system. In addition, kinetic models are also developed. Laboratory data from bench-scale fermentations involving growth and protein production by T. reesei on lactose and xylose are used to estimate the parameters in these models. The relative performance of the various models and the results of optimizing these models on two different performance measures are presented. An approximately 33% lower root-mean-squared error (RMSE) in protein predictions and about 40% lower total RMSE is obtained with the neural network-based model, the RMSE in predicting optimal conditions for two performance indices, is about 67% and 40% lower, respectively, when compared with the kinetic models. Thus, both model predictions and optimization results from the neural network-based model are found to be closer to the experimental data than the kinetic models developed in this work. It is shown that the neural network parameter function modeling method can be useful as a macromodeling technique to rapidly develop dynamic models of a process.

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

    PubMed

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

    2013-11-01

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

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

    PubMed

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

    2016-05-20

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

  16. Effect of Cellulases and Xylanases on Refining Process and Kraft Pulp Properties

    PubMed Central

    Przybysz Buzała, Kamila; Kalinowska, Halina; Derkowska, Małgorzata

    2016-01-01

    Samples of bleached kraft pine cellulosic pulp, either treated with an enzyme preparation (a Thermomyces lanuginosus xylanase, an Aspergillus sp. cellulase, and a multienzyme preparation NS-22086 containing both these activities) or untreated, were refined in a laboratory PFI mill. The treatment with cellulases contained in the last two preparations significantly improved the pulp’s susceptibility to refining (the target freeness value of 30°SR was achieved in a significantly shorter time), increased water retention value (WRV) and fines contents while the weighted average fiber length was significantly reduced. These changes of pulp parameters caused deterioration of paper strength properties. The treatment with the xylanase, which partially hydrolyzed xylan, small amounts of which are associated with cellulose fibers, only slightly loosened the structure of fibers. These subtle changes positively affected the susceptibility of the pulp to refining (refining energy was significantly reduced) and improved the static strength properties of paper. Thus, the treatment of kraft pulps with xylanases may lead to substantial savings of refining energy without negative effects on paper characteristics. PMID:27557079

  17. 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.

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

    NASA Astrophysics Data System (ADS)

    Prior, Bernard A.; Day, Donal F.

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

  19. Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

    PubMed Central

    Garvey, Megan; Fischer, Rainer; Commandeur, Ulrich

    2014-01-01

    Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems. PMID:24962636

  20. Optimization of cellulase production by Enhydrobacter sp. ACCA2 and its application in biomass saccharification

    PubMed Central

    Premalatha, Nagaiah; Gopal, Nellaiappan O.; Jose, Polpass Arul; Anandham, Rangasamy; Kwon, Soon-Wo

    2015-01-01

    Cellulase finds use in saccharification of lignocellulosic agroresidues to fermentable sugars which can be used for production of commercially important metabolites. This study reports endoglucanase (CMCase) production by Enhydrobacter sp. ACCA2. The CMCase activity of the strain ACCA2 was successively improved by optimization of range of physical and nutritional parameter in a set of non-statistical and statistical experiments. Initial non-statistical selection of carbon source, incubation time, temperature and pH resulted in 1.07 fold increase of CMCase activity. In a subsequent statistical method, response surface methodology, optimization of medium components such as carboxymethylcellulose, peptone, NaCl, MgSO4, K2HPO4, and (NH4)2SO4 yielded further increase up to 2.39 fold CMCase activity. The cellulolytic potential was evaluated in biomass saccharification with different plant materials and the results revealed that the enzyme produced by strain may have significant commercial values for industrial saccharification process. Moreover, this is the first report of cellulase production by an Enhydrobacter spp. PMID:26500615