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Sample records for alkaline cellulase egl-237

  1. Alkaline cellulases from alkaliphilic Bacillus: enzymatic properties, genetics, and application to detergents.

    PubMed

    Ito, S

    1997-05-01

    We have isolated a number of alkaliphilic Bacillus that produce alkaline exoenzymes and found a possible use for alkaline cellulase (carboxymethylcellulase) as an additive for improving the cleaning effect of detergents. Enzymatic properties of some candidate cellulases fulfilled the essential requirements for enzymes to be used practically in laundry detergents. Here I describe the properties and possible catalytic mechanism of the hydrolytic reaction and the gene for the industrial alkaline cellulase produced by one of the isolates, Bacillus sp. KSM-635.

  2. Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme.

    PubMed

    Shirai, T; Ishida, H; Noda, J; Yamane, T; Ozaki, K; Hakamada, Y; Ito, S

    2001-07-27

    The crystal structure of the catalytic domain of alkaline cellulase K was determined at 1.9 A resolution. Because of the most alkaliphilic nature and it's highest activity at pH 9.5, it is used commercially in laundry detergents. An analysis of the structural bases of the alkaliphilic character of the enzyme suggested a mechanism similar to that previously proposed for alkaline proteases, that is, an increase in the number of Arg, His, and Gln residues, and a decrease in Asp and Lys residues. Some ion pairs were formed by the gained Arg residues, which is similar to what has been found in the alkaline proteases. Lys-Asp ion pairs are disfavored and partly replaced with Arg-Asp ion pairs. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high pH range.

  3. Production of alkaline cellulase by fungi isolated from an undisturbed rain forest of peru.

    PubMed

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

    2012-01-01

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

  4. Treatment of cotton with an alkaline Bacillus spp cellulase: activity towards crystalline cellulose.

    PubMed

    Caparrós, Cristina; López, Carmen; Torrell, Marc; Lant, Neil; Smets, Johan; Cavaco-Paulo, Artur

    2012-02-01

    We analysed the influence of several enzymatic treatment processes using an alkaline cellulase enzyme from Bacillus spp. on the sorption properties of cotton fabrics. Although cellulases are commonly applied in detergent formulations due to their anti-redeposition and depilling benefits, determining the mechanism of action of alkaline cellulases on cotton fibres requires a deeper understanding of the morphology and structure of cotton fibres in terms of fibre cleaning. The accessibility of cellulose fibres was studied by evaluating the iodine sorption value and by fluorescent-labelled enzyme microscopy; the surface morphology of fabrics was analysed by scanning microscopy. The action of enzyme hydrolysis over short time periods can produce fibrillation on cotton fibre surface without any release of cellulosic material. The results indicate that several short consecutive treatments were more effective in increasing the fibre accessibility than one long treatment. In addition, no detectable hydrolytic activity, in terms of reducing sugar production, was found.

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

    PubMed

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

    2016-12-01

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

  6. 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

  7. High external pH enables more efficient secretion of alkaline α-amylase AmyK38 by Bacillus subtilis

    PubMed Central

    2012-01-01

    Background Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system. Results Compared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l-1) of AmyK38 was obtained using strain MGB874. Conclusions We demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38. PMID:22681752

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

    PubMed

    Gomaa, Eman Zakaria

    2013-01-01

    The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg(2+), Fe2+ and Ag(+) showed a stimulatory effect on protease activity and ions of Fe(2+), Mg(2+), Ca(2+), Cu(2+) and Ag(+) caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

  9. Production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching.

    PubMed

    Thomas, Leya; Sindhu, Raveendran; Binod, Parameswaran; Pandey, Ashok

    2015-06-01

    Here, we described the production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching. Various process parameters affecting xylanase production by B. pumilus were optimized by adopting a Plackett-Burman design (PBD) as well as Response surface methodology (RSM). These statistical methods aid in improving the enzyme yield by analysing the individual crucial components of the medium. Maximum production was obtained with 4% yeast extract, 0.08% magnesium sulphate, 30 h of inoculum age, incubation temperature of 33.5 degrees C and pH 9.0. Under optimized conditions, the xylanase activity was 372 IU/ml. Media engineering improved a 5-fold increase in the enzyme production. Scanning electron microscopy (SEM) showed significant changes on the surface of xylanase treated pulps as a result of xylan hydrolysis. Increased roughness of paper carton fibres was apparent in scanning electron micrograph due to opening of the micro fibrils present on the surface by xylanase action. The untreated pulp did not show any such change. These results demonstrated that the B. pumilus MTCC 5015 xylanase was effective in bio-bleaching of paper carton.

  10. Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases

    PubMed Central

    2014-01-01

    Background Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). Results Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. Conclusion The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw. PMID:24766728

  11. 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.

  12. 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.

  13. 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.

  14. Thermostable Cellulases: Why & How?

    SciTech Connect

    Kumar, Manoj

    2010-04-19

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

  15. Engineering Cellulases for Biorefinery

    SciTech Connect

    Kumar, Manoj

    2010-06-27

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

  16. Thermostable Cellulases: Why & How?

    SciTech Connect

    Kumar, Manoj

    2010-03-24

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

  17. Cellulases and biofuels.

    PubMed

    Wilson, David B

    2009-06-01

    There is a major international effort to develop renewable alternatives to fossil fuels. One approach is to produce a liquid fuel by enzymatically hydrolyzing carbohydrate polymers in biomass to sugars and fermenting them to ethanol. Cellulose is the main polymer in biomass and cellulases can hydrolyze it to cellobiose, which can be converted to glucose by beta-glucosidase. Extensive research is being carried out to try to obtain cellulases with higher activity on pretreated biomass substrates by screening and sequencing new organisms, engineering cellulases with improved properties and by identifying proteins that can stimulate cellulases. Despite extensive research on cellulases there are major gaps in our understanding of how they hydrolyze crystalline cellulose, act synergistically, and the role of carbohydrate binding modules.

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

    PubMed

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

    2016-07-01

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

  19. Chimeric enzymes with improved cellulase activities

    DOEpatents

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

    2015-03-31

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

  20. 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

  1. Visualizing cellulase activity.

    PubMed

    Bubner, Patricia; Plank, Harald; Nidetzky, Bernd

    2013-06-01

    Commercial exploitation of lignocellulose for biotechnological production of fuels and commodity chemicals requires efficient-usually enzymatic-saccharification of the highly recalcitrant insoluble substrate. A key characteristic of cellulose conversion is that the actual hydrolysis of the polysaccharide chains is intrinsically entangled with physical disruption of substrate morphology and structure. This "substrate deconstruction" by cellulase activity is a slow, yet markedly dynamic process that occurs at different length scales from and above the nanometer range. Little is currently known about the role of progressive substrate deconstruction on hydrolysis efficiency. Application of advanced visualization techniques to the characterization of enzymatic degradation of different celluloses has provided important new insights, at the requisite nano-scale resolution and down to the level of single enzyme molecules, into cellulase activity on the cellulose surface. Using true in situ imaging, dynamic features of enzyme action and substrate deconstruction were portrayed at different morphological levels of the cellulose, thus providing new suggestions and interpretations of rate-determining factors. Here, we review the milestones achieved through visualization, the methods which significantly promoted the field, compare suitable (model) substrates, and identify limiting factors, challenges and future tasks.

  2. 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

  3. Bioprospecting thermophiles for cellulase production: a review.

    PubMed

    Acharya, Somen; Chaudhary, Anita

    2012-07-01

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

  4. 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.

  5. 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.

  6. Exo-endo cellulase fusion protein

    SciTech Connect

    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

    Kumar, Manoj

    2011-04-26

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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. Structure and function of a novel cellulase 5 from sugarcane soil metagenome.

    PubMed

    Alvarez, Thabata M; Paiva, Joice H; 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.

  17. 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.

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

    PubMed

    Lobanok, A G; Pavlovskaia, Zh I

    1977-01-01

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

  19. 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

  20. 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).

  1. Thermal tolerant cellulase from Acidothermus cellulolyticus

    DOEpatents

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

    2006-06-13

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

  2. 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.

  3. Hydrolysis of lignocelluloses by penicillium funiculosum cellulase

    SciTech Connect

    Mishra, C.; Rao, M.; Seeta, R.; Srinivasan, M.C.; Deshpande, V.

    1984-04-01

    Enzymatic hydrolysis of cellulose is a promising method for the conversion of waste cellulose to glucose. During the past few years, the development of this technology has proceeded rapidly, with significant advances made in enzyme production, pretreatment, and hydrolysis. A variety of fungi are reported to produce cellulases but among these Trichoderma reesei and its mutants are powerful producers of cellulases. However, the search for new and possibly better sources of cellulase is continued due to the low levels of beta-glucosidase of T. reesei. Penicillium funiculosum produces a complete cellulase having endo-beta-1,4-glucanase (15-20 U/mL), exo-beta-1,4-glucanase (1.5-2.0 U/mL), and high beta-glucosidase (8-10 U/mL). The saccharification of alkali-treated cotton and bagasse by P. funiculosum enzyme was 70 and 63%, respectively. It was possible to obtain glucose concentration as high as 30% using 50% bagasse. It is of interest that the percent saccharification of cellulosic substrates with the Penicillium enzyme is comparable to that of T. reesei cellulase when the same amount of filter paper activity is used, although the endo-glucanase activity of the latter is two to three times higher. This communication reports the studies on saccharification of lignocelluloses by P. funiculosum cellulase and certain studies on the kinetic aspects. (Refs. 15).

  4. Outlook for cellulase improvement: screening and selection strategies.

    PubMed

    Percival Zhang, Y-H; Himmel, Michael E; Mielenz, Jonathan R

    2006-01-01

    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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Inhibition of cellulase by fermentation products

    SciTech Connect

    Takagi, M.

    1984-12-01

    Cellulosic materials are important resources for the production of fuels and chemicals. One of the processes designed to utilize cellulosic materials is enzymatic hydrolysis followed by fermentation to final products. It was shown that simultaneous saccharification and fermentation (SSF) is effective in the production of ethanol from cellulose. The acceleration of the rate of cellulose degradation in SSF was caused by less inhibition of cellulase by ethanol than by glucose and/or cellobiose. In order to apply the SSF method effectively to the production of substances derived from glucose by fermentation, several conditions should be satisfied. One of them is the coincidence of conditions of enzymatic hydrolysis and fermentation of hydrolyzate, such as pH and temperature. The second condition is that the inhibition of cellulase by the final product is less than that by glucose and/or cellobiose. There has been a comprehensive review and some reports on inhibition of cellulase by saccharides such as glucose, cellobiose, xylose, lactose, maltose, metals, and some chemicals such as dyes, detergents, halogenated compounds, and phenolic compounds. In this communication, the inhibition of cellulase by several substances derived by fermentation of glucose is reported. 7 references.

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

    PubMed

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

    2016-08-01

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

  13. 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.

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

    PubMed

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

    2015-12-01

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

  15. 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.

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

    PubMed

    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.

  17. 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

  18. 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.

  19. 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.

  20. The adsorptive and hydrolytic performance of cellulase on cationised cotton.

    PubMed

    Hao, Longyun; Wang, Rui; Liu, Jingquan; Liu, Rongzhan

    2012-06-05

    In this research, the cotton fabrics were cationised by a cationic agent to change their surface electric properties. The cationised cotton fabrics were then bio-polished by cellulase to explore the influence of cationisation on the adsorptive and hydrolytic performance of cellulase. The experimental results from cellulase adsorption reveal the cationisation of cotton can obviously improve the binding efficiency of cellulase protein mainly by the improved electrostatic attraction between oppositely charged cellulase and cationised cotton. The adsorption parameters calculated can further prove this improvement trend. Through measuring the concentration of reducing sugars released and weight loss of cotton during the bio-polishing, it is found the hydrolytic activity toward cotton is partially damaged by the cationisation. The reason can be attributed to the movement restriction and dysfunction of cellulase protein by the cationisation.

  1. 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.

  2. Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates.

    PubMed

    Tu, Maobing; Chandra, Richard P; Saddler, Jack N

    2007-01-01

    The recycling of cellulase enzymes is one potential strategy for reducing the cost of the enzymatic hydrolysis step during the bioconversion of lignocellulosics to ethanol. To determine the influence of lignin on the post-hydrolysis distribution of cellulase enzymes between the liquid and solid phases, the hydrolysis of Avicel was compared to an organosolv-pretreated Douglas fir substrate with a lignin content of 3.0%. After a 12 h hydrolysis reaction on Avicel, 90% of the added cellulases (including beta-glucosidases) remained "free" in the liquid phase compared to only 65% in the case of the hydrolysis of the organosolv-pretreated Douglas fir substrate. The readsorption of free cellulases by supplementing the hydrolysis reaction with fresh substrate was explored as a potential means of recovering the free cellulases that remain in the liquid phase after hydrolysis. The Langmuir adsorption isotherm was used to develop a model predicting that 82% of the free cellulases could be recovered via readsorption onto fresh substrates during the hydrolysis of an ethanol-pretreated mixed softwood substrate with a lignin content of 6%. Recoverable free cellulase values of 85% and 88% based on cellulase activity and protein content, respectively, were obtained after experimental verification of the model. The readsorption of free cellulases onto fresh lignocellulosic substrates was shown to be an effective method for free enzyme recovery.

  3. 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.

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

    PubMed

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

    2014-01-03

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

  5. 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.

  6. 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.

  7. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  8. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  9. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  10. 21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Cellulase enzyme preparation derived from....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase enzyme.... reesei). The enzyme, cellulase, catalyzes the endohydrolysis of 1,4-beta-glycosidic linkages in...

  11. 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.

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

    SciTech Connect

    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.

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

    DOEpatents

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

    2015-04-07

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

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

    SciTech Connect

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

    2016-12-20

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

  15. 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.

  16. 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.

  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. 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.

  19. Immobilization of Cellulase on Magnetic Nanocarriers

    PubMed Central

    Roth, Hans‐Christian; Schwaminger, Sebastian P.; Peng, Fei

    2016-01-01

    Abstract The constant increase in the number of sustainable products on the global markets demands new biotechnological processing strategies such as the purification and recovery of biocatalysts. Superparamagnetic iron oxide nanoparticles exhibit excellent recovery properties as carrier materials in enzyme catalysis. We present the simple and fast electrostatic assembly of cellulase (CEL) and low‐priced silica‐coated magnetic nanoparticles, which demonstrates stable enzyme bonding and excellent colloidal stability. The high CEL loading (0.43 g g−1), without leaching of biocatalyst and high recovery yields (75 %), could be sustained over ten magnetic recycling steps. The highlight of this study is the preservation of a high enzymatic activity and, therefore, the outstandingly high lifecycle stability. PMID:27957407

  20. Cellulase immobilization on magnetic nanoparticles encapsulated in polymer nanospheres.

    PubMed

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

    2017-04-01

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

  1. 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.

  2. Enhanced production and application of acidothermophilic Streptomyces cellulase.

    PubMed

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

    2016-01-01

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

  3. 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

  4. Factorial optimization of a six-cellulase mixture

    SciTech Connect

    Kim, E.; Irwin, D.C.; Walker, L.P.; Wilson, D.B.

    1998-06-05

    A factorial experimental design approach was used to optimize mixtures of six cellulases (five Thermomonospora fusca cellulases and plus/minus Trichoderma reesei CBHI along with {beta}-glucosidase) so as to maximize the glucose produced from filter paper. Optimized mixture A and mixture B produced glucose at 25 and 8.3 {micro}mol glucose/{micro}mol enzyme/min, respectively, which are 8 and 1.5 times higher than the sum of the activity of the individual cellulases. In both mixtures, the glucose yield depended on the ratio and the cellulases used. Most enzymes showed synergistic interactions that increased the glucose yield. The yield of glucose with the optimum mixtures depended on the total enzyme concentration.

  5. Cellulase hyperproducers constructed from polyploids of Lentinus edodes.

    PubMed

    Toyama, H; Toyama, N

    2000-01-01

    A mycelial mat of Lentinus edodes was treated with 0.01% (w/v) colchicine solution for 240 h at 26 degrees C and autopolyploidization occurred. The mycelia were treated subsequently with the haploidizing reagent, benomyl, and fanshaped sectors were produced from colonies. Among such sectors, cellulase hyperproducers could be selected. The cellulase productivity of the hyperproducer, L1, did not decrease through five generations.

  6. Stimulation of Cellulases by Small Phenolic Compounds in Pretreated Stover.

    PubMed

    Zhao, Junying; Chen, Hongzhang

    2014-03-27

    The effect of small phenolic compounds in pretreated stover on celluase activity is crucial but has not yet been fully elucidated. This work investigated the effects of both phenolic acid and phenolic aldehyde on cellulase activity. The model substances of small phenolic compounds identified in steam exploded corn stover were used to examine their individual effects on cellulase activity. It was found that phenolic aldehyde significantly inhibited cellulase activity at 0.05-8 g/L. However, phenolic acids might have a concentration-dependent effect on cellulase activity: significant inhibition at 0.05 g/L and slight stimulation at 2-4 g/L. Small phenolic compounds mixture might also have a concentration-dependent effect on cellulase activity: significant stimulation at 2-8 g/L and slight inhibition at 0.05-1 g/L. The small phenolic compounds in pretreated stover were proven to be able to significantly stimulate enzymatic hydrolysis of stover. On the basis of these results, it is proposed that the concentration-dependent effects of small phenolic compounds on cellulase activity should be considered while removing them after pretreatment.

  7. 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.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

  10. 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.

  11. 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.

  12. Cellulase immobilization onto the reversibly soluble methacrylate copolymer for denim washing.

    PubMed

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

    2013-06-20

    Cellulase treatment of denim fabrics is an environmentally friendly way for producing desired worn look. In this work, the enzymatic treatments of the denim fabrics, i.e., bio-washing, using native cellulase and cellulase immobilized with reversibly soluble copolymer (Eudragit S-100), immobilized-cellulase, have been investigated. According to the analyses of the lightness (CIE L value), color strength (K/S value) and color variations, at a cellulase concentration level of 6% o.w.f., the denim fabrics treated with the immobilized cellulase showed decoloration and color effect close to the native cellulase. However, the immobilized cellulase treatment of the denim fabrics showed lower weight loss and considerably higher tensile strength than those treated with the native cellulase. Both the native and immobilized cellulases improved the crystalline indice and the apparent crystallite size of the fiber sample compared with the control ones. The amorphous portion of the cellulose suffered more hydrolysis by the native cellulase than the immobilized cellulase. Scanning electron microscope pictures (SEM) and digital pictures further indicated that the immobilized cellulase can efficiently remove indigo dyestuffs on the surfaces of the denim fabrics without the problem of excessive damage to the fibers.

  13. 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.

  14. 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.

  15. Behavior of lignin-binding cellulase in the presence of fresh cellulosic substrate.

    PubMed

    Nonaka, Hiroshi; Kobayashi, Ai; Funaoka, Masamitsu

    2013-05-01

    A model lignin-binding cellulase was prepared from Trichoderma reesei cellulase and lignocresol, which was synthesized from softwood or hardwood lignin. Filter paper was incubated with the lignocresol-cellulase complex, and it was observed that only a limited amount of cellulase migrated to the filter paper. The cellulase adsorption isotherms for the lignocresols and filter paper were fitted to a Langmuir absorption model, and the determined Langmuir constants were as follows: softwood lignocresol>hardwood lignocresol>filter paper. The calculations demonstrated that lignin-binding cellulase can potentially be recovered by the addition of a sufficient quantity of cellulosic substrate. As a result, the lignocresol-binding cellulase is highly stable and lignocresol can potentially be used for immobilizing cellulase in the active state.

  16. 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.

  17. 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.

  18. Feasibility Assessment for the Use of Cellulase in Biomass Conversion for Human Application

    DTIC Science & Technology

    2007-11-02

    Cellulase In Biomass Conversion For Human Application Contract No: DAAD19-03-C-0113; Proposal No: 45611-LS-000 Michael Ward, Lynn Tierney...fungal cellulases to release glucose from cellulose in the human gut has been evaluated. Cellulase stability in simulated gastric and intestinal...conversion to soluble sugars by different cellulases under simulated gastric and intestinal conditions was also investigated. Up to 30% conversion of

  19. 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.

  20. 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.

  1. Hydrolysis of cellulose by purified cellulase components: Synergistic effects

    SciTech Connect

    Woodward, J.; Lee, N.E.

    1987-01-01

    The hydrolysis of cellulose by purified cellulase components is reported. The adsorption of purified cellobiohydrolases (CBH I and II) and endoglucanases (EG I and II) from Trichoderma reesei strain L27 to microcrystalline cellulose (Avicel) has been studied. Scatchard analysis of the adsorption data gave the maximum amount of each component that bound to Avicel at saturation. Hydrolysis of Avicel was thus carried out by saturating and non-saturating (50% saturation) concentrations of cellulase components alone and in combination with each other, and it was found that the greatest amount of synergism between them was observed when Avicel was incubated with non-saturating concentrations of enzyme. Synergism was observed between CBH I and CBH II, as well as between EG I and CBH I; however, inhibition of hydrolysis occurred using a combination of EG I and EG II. Synergism between cellulase components may be significant during cellulose hydrolysis only when non-saturating enzyme concentrations are used. 4 refs., 3 figs.

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

    PubMed

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

    2013-08-01

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

  3. 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...

  4. 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...

  5. 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...

  6. 21 CFR 173.120 - Carbohydrase and cellulase derived from Aspergillus niger.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  7. 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...

  8. 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.

  9. 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.

  10. 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).

  11. Enhancing Cellulase Commercial Performance for the Lignocellulosic Biomass Industry

    SciTech Connect

    Jarnigan, Alisha

    2016-06-07

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

  12. 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.

  13. Hybrid cellulase aggregate with a silica core for hydrolysis of cellulose and biomass.

    PubMed

    Sutarlie, Laura; Yang, Kun-Lin

    2013-12-01

    Cellulase is an important enzyme for hydrolyzing cellulose to form glucose. To recycle cellulase after the reaction, cellulase is often immobilized on solid supports but its activity is also compromised. In this study, we show a new hybrid cellulase aggregate with a silica core, which is prepared by physical adsorption of cross-linked cellulase on a highly porous solid support silica gel. The hybrid cellulase aggregate exhibits highest activity at pH 4.8 and 51°C, similar to the optimum condition of free cellulase. This hybrid cellulase aggregate can produce 3.4 g/L of glucose within 2 h, which is two times higher than glucose produced by using cross-linked cellulase aggregate alone (without silica core). Another advantage of the hybrid cellulase aggregate is that it can settle down naturally after the hydrolysis of cellulose, thanks to the presence of the silica core. To show its practical applications, we also study the hydrolysis of palm oil fiber by using the hybrid cellulase aggregate. Up to 5.0 g/L of glucose can be produced within 24h, and this process can be repeated five times with only 19% decrease in activity.

  14. 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.

  15. 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.

  16. 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

  17. 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)

  18. 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.

  19. 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.

  20. Chapter Three -- Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels

    SciTech Connect

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

    2015-10-24

    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.

  1. CBH1 homologs and varian CBH1 cellulase

    DOEpatents

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

    2014-07-01

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

  2. 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.

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

    PubMed

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

    2017-04-01

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

  4. 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.

  5. The productive cellulase binding capacity of cellulosic substrates.

    PubMed

    Karuna, Nardrapee; Jeoh, Tina

    2017-03-01

    Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases. Productive cellulase binding to cellulose results in hydrolysis and can be quantified by measuring hydrolysis rates. In this study, we measured the productive Trichoderma reesei Cel7A (TrCel7A) binding capacity of five cellulosic substrates from different sources and processing histories. Swollen filter paper and bacterial cellulose had higher productive binding capacities of ∼6 µmol/g while filter paper, microcrystalline cellulose, and algal cellulose had lower productive binding capacities of ∼3 µmol/g. Swelling and regenerating filter paper using phosphoric acid increased the initial accessibility of the reducing ends to TrCel7A from 4 to 6 µmol/g. Moreover, this increase in initial productive binding capacity accounted in large part for the difference in the overall digestibility between filter paper and swollen filter paper. We further demonstrated that an understanding of how the productive binding capacity declines over the course of the hydrolysis reaction has the potential to predict overall saccharification time courses. Biotechnol. Bioeng. 2017;114: 533-542. © 2016 Wiley Periodicals, Inc.

  6. 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.

  7. 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.

  8. Alkaline flooding injection strategy

    SciTech Connect

    French, T.R.; Josephson, C.B.

    1992-03-01

    The objective of this project is to improved alkali-surfactant flooding methods, and this includes determining the proper design of injection strategy. Several different injection strategies have been used or suggested for recovering heavy oils with surfactant-enhanced alkaline flooding methods. Oil recovery was compared for four different injection strategies: (1) surfactant followed by polymer, (2) surfactant followed by alkaline polymer, (3) alkaline surfactant followed by polymer, and (4) alkali, surfactant, and polymer mixed in a single formulation. The effect of alkaline preflush was also studied under two different conditions. All of the oil recovery experiments were conducted under optimal conditions with a viscous, non-acidic oil from Hepler (KS) oil field. The coreflood experiments were conducted with Berea sandstone cores since field core was not available in sufficient quantity for coreflood tests. The Tucker sand of Hepler field is a Class I fluvial dominated deltaic reservoir, as classified by the Department of Energy, which has been selected as the site of a DOE-sponsored field pilot test.

  9. Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation.

    PubMed

    Vinzant, T B; Adney, W S; Decker, S R; Baker, J O; Kinter, M T; Sherman, N E; Fox, J W; Himmel, M E

    2001-01-01

    Polysaccharide degrading enzymes from commercial T. reesei broth have been subjected to "fingerprint" analysis by high-resolution 2-D gel electrophoresis. Forty-five spots from 11 x 25 cm Pharmacia gels have been analyzed by LC-MS/MS and the resulting peptide sequences were compared to existing databases. Understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. These studies suggest follow-on work comparing induced and noninduced T. reesei cells at the proteome level, which may elucidate substrate-specific gene regulation and response.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Improvement of halophilic cellulase production from locally isolated fungal strain

    PubMed Central

    Gunny, Ahmad Anas Nagoor; Arbain, Dachyar; Jamal, Parveen; Gumba, Rizo Edwin

    2014-01-01

    Halophilic cellulases from the newly isolated fungus, Aspergillus terreus UniMAP AA-6 were found to be useful for in situ saccharification of ionic liquids treated lignocelluloses. Efforts have been taken to improve the enzyme production through statistical optimization approach namely Plackett–Burman design and the Face Centered Central Composite Design (FCCCD). Plackett–Burman experimental design was used to screen the medium components and process conditions. It was found that carboxymethylcellulose (CMC), FeSO4·7H2O, NaCl, MgSO4·7H2O, peptone, agitation speed and inoculum size significantly influence the production of halophilic cellulase. On the other hand, KH2PO4, KOH, yeast extract and temperature had a negative effect on enzyme production. Further optimization through FCCCD revealed that the optimization approach improved halophilic cellulase production from 0.029 U/ml to 0.0625 U/ml, which was approximately 2.2-times greater than before optimization. PMID:26150755

  16. Biomining active cellulases from a mining bioremediation system.

    PubMed

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

    2013-09-20

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

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

    PubMed

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

    2017-02-01

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

  18. Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers

    PubMed Central

    Maki, Miranda L; Broere, Michael; Leung, Kam Tin; Qin, Wensheng

    2011-01-01

    The wide variety of bacteria in the environment permits screening for more efficient cellulases to help overcome current challenges in biofuel production. This study focuses on the isolation of efficient cellulase producing bacteria found in organic fertilizers and paper mill sludges which can be considered for use in large scale biorefining. Pure isolate cultures were screened for cellulase activity. Six isolates: S1, S2, S3, S4, E2, and E4, produced halos greater in diameter than the positive control (Cellulomonas xylanilytica), suggesting high cellulase activities. A portion of the 16S rDNA genes of cellulase positive isolates were amplified and sequenced, then BLASTed to determine likely genera. Phylogenetic analysis revealed genera belonging to two major Phyla of Gram positive bacteria: Firmicutes and Actinobacteria. All isolates were tested for the visible degradation of filter paper; only isolates E2 and E4 (Paenibacillus species) were observed to completely break down filter paper within 72 and 96 h incubation, respectively, under limited oxygen condition. Thus E2 and E4 were selected for the FP assay for quantification of total cellulase activities. It was shown that 1% (w/v) CMC could induce total cellulase activities of 1652.2±61.5 and 1456.5±30.7 μM of glucose equivalents for E2 and E4, respectively. CMC could induce cellulase activities 8 and 5.6X greater than FP, therefore CMC represented a good inducing substrate for cellulase production. The genus Paenibacillus are known to contain some excellent cellulase producing strains, E2 and E4 displayed superior cellulase activities and represent excellent candidates for further cellulase analysis and characterization. PMID:21969070

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Advanced alkaline water electrolysis

    NASA Astrophysics Data System (ADS)

    Wakabayashi, N.; Torikai, E.; Kawami, Y.; Takenaka, H.

    Results are presented of experimental studies of possible separators and electrodes for use in advanced, high-temperature, high-pressure alkaline water electrolyzers. Material evaluations in alkaline water electrolyzers at temperatures from 100 to 120 C have shown a new type polytetrafluoroethylene membrane impregnated with potassium titanate to be the most promising when the separator is prepared by the hydrothermal treatment of a porous PFTE membrane impregnated with hydrated titanium oxide. Measurements of cell voltages in 30% KOH at current densities from 5 to 100 A/sq dm at temperatures up to 120 C with nickel electrodes of various structures have shown the foamed nickel electrode, with an average pore size of 1-1.5 mm, to have the best performance. When the foamed nickel is coated by fine powdered nickel, carbonyl nickel or Raney nickel to increase electrode surface areas, even lower cell voltages were found, indicating better performance.

  4. 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).

  5. 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

  6. 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.

  7. 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.

  8. 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.

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

    PubMed

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

    2017-01-02

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

  10. Influence of rice straw polyphenols on cellulase production by Trichoderma reesei.

    PubMed

    Zheng, Wei; Zheng, Qin; Xue, Yiyun; Hu, Jiajun; Gao, Min-Tian

    2017-02-12

    In this study, we found that during cellulase production by Trichoderma reesei large amounts of polyphenols were released from rice straw when the latter was used as the carbon source. We identified and quantified the phenolic compounds in rice straw and investigated the effects of the phenolic compounds on cellulase production by T. reesei. The phenolic compounds of rice straw mainly consisted of phenolic acids and tannins. Coumaric acid (CA) and ferulic acid (FA) were the predominant phenolic acids, which inhibited cellulase production by T. reesei. When the concentrations of CA and FA in the broth increased to 0.06 g/L, cellulase activity decreased by 23% compared with that in the control culture. Even though the rice straw had a lower tannin than phenolic acid content, the tannins had a greater inhibitory effect than the phenolic acids on cellulase production by T. reesei. Tannin concentrations greater than 0.3 g/L completely inhibited cellulase production. Thus, phenolic compounds, especially tannins are the major inhibitors of cellulase production by T. reesei. Therefore, we studied the effects of pretreatments on the release of phenolic compounds. Ball milling played an important role in the release of FA and CA, and hot water extraction was highly efficient in removing tannins. By combining ball milling with extraction by water, the 2-fold higher cellulase activity than in the control culture was obtained.

  11. Evidence for the presence of a cellulase gene in the last common ancestor of bilaterian animals.

    PubMed Central

    Lo, Nathan; Watanabe, Hirofumi; Sugimura, Masahiro

    2003-01-01

    Until recently, the textbook view of cellulose hydrolysis in animals was that gut-resident symbiotic organisms such as bacteria or unicellular eukaryotes are responsible for the cellulases produced. This view has been challenged by the characterization and sequencing of endogenous cellulase genes from some invertebrate animals, including plant-parasitic nematodes, arthropods and a mollusc. Most of these genes are completely unrelated in terms of sequence, and their evolutionary origins remain unclear. In the case of plant-parasitic nematodes, it has been suggested that their ancestor obtained a cellulase gene via horizontal gene transfer from a prokaryote, and similar suggestions have been made about a cellulase gene recently discovered in a sea squirt. To improve understanding about the evolution of animal cellulases, we searched for all known types of these enzymes in GenBank, and performed phylogenetic comparisons. Low phylogenetic resolution was found among most of the sequences examined, however, positional identity in the introns of cellulase genes from a termite, a sea squirt and an abalone provided compelling evidence that a similar gene was present in the last common ancestor of protostomes and deuterostomes. In a different enzyme family, cellulases from beetles and plant-parasitic nematodes were found to cluster together. This result questions the idea of lateral gene transfer into the ancestors of the latter, although statistical tests did not allow this possibility to be ruled out. Overall, our results suggest that at least one family of endogenous cellulases may be more widespread in animals than previously thought. PMID:12952640

  12. 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.

  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.

  14. Useful halophilic, thermostable and ionic liquids tolerant cellulases

    SciTech Connect

    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.

  15. C1-Cx revisited: intramolecular synergism in a cellulase.

    PubMed Central

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

    1994-01-01

    Endoglucanase A (CenA) from the bacterium Cellulomonas fimi is composed of a catalytic domain and a nonhydrolytic cellulose-binding domain that can function independently. The individual domains interact synergistically in the disruption and hydrolysis of cellulose fibers. This intramolecular synergism is distinct from the well-known intermolecular synergism between individual cellulases. The catalytic domain corresponds to the hydrolytic Cx system and the cellulose-binding domain corresponds to the nonhydrolytic C1 system postulated by Reese et al. [Reese, E. T., Sui, R. G. H. & Levinson, H. S. (1950) J. Bacteriol. 59, 485-497] to be required for the hydrolysis of cellulose. PMID:7972069

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. Enzymatic deinking of secondary fibers: cellulases/hemicellulases versus laccase-mediator system.

    PubMed

    Ibarra, David; Concepción Monte, M; Blanco, Angeles; Martínez, Angel T; Martínez, María J

    2012-01-01

    The use of enzymes has been suggested as an environmentally friendly alternative to complement conventional chemical deinking in the recycling of recovered paper. This study compares the use of cellulases/hemicellulases versus the laccase-mediator system for deinking printed fibers from newspapers and magazines. For this purpose, two commercial enzyme preparations with endoglucanase and endoxylanase activities (Viscozyme Wheat from Aspergillus oryzae and Ultraflo L from Humicola insolens, Novozymes) and a commercial laccase (NS51002 from Trametes villosa, Novozymes), the latter in the presence of synthetic or natural (lignin-related) mediators, were evaluated. The enzymatic treatments were studied at the laboratory scale using a standard chemical deinking sequence consisting of a pulping stage; an alkaline stage using NaOH, sodium silicate and fatty acid soap; and a bleaching stage using hydrogen peroxide. The handsheets were then prepared and their brightness, residual ink concentration, and strength properties were measured. Among the different enzymatic treatments assayed, both carbohydrate hydrolases were found to deink the secondary fibers more efficiently. Brightness increased up to 3-4% ISO on newspaper fibers, being Ultraflo 20% more efficient in the ink removal. Up to 2.5% ISO brightness increase was obtained when magazine fibers were used, being Viscozyme 9% more efficient in the ink removal. Regarding the laccase-mediator system, alone or in combination with carbohydrate hydrolases, it was ineffective in deinking both newspaper and magazine fibers, resulting in pulps with worse brightness and residual ink concentration values. However, pulp deinking by the laccase-mediator system was displayed when secondary fibers from printed cardboard were used, obtaining up to 3% ISO brightness increase and lower residual ink concentrations.

  10. 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.

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

    PubMed

    Han, Wei; He, Ming

    2010-05-01

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

  12. Adsorption of cellulase on cellulose: effect of physicochemical properties of cellulose on adsorption and rate of hydrolysis

    SciTech Connect

    Lee, S.B.; Shin, H.S.; Ryu, D.D.Y.

    1982-11-01

    In the cellulase-cellulose reaction system, the adsorption of cellulase on the solid cellulose substrate was found to be one of the important parameters that govern the enzymatic hydrolysis rate of cellulose. The adsorption of cellulase usually parallels the rate of hydrolysis of cellulose. The affinity for cellulase varies depending on the structural properties of cellulose. Adsorption parameters such as the half-saturation constant, the maximum adsorption constant, and the distribution coefficient for both the cellulase and cellulose have been experimentally determined for several substrates. These adsorption parameters vary with the source of cellulose and the pretreatment methods and are correlated with the crystallinity and the specific surface area of cellulose substrates. The changing pattern of adsorption profile of cellulase during the hydrolysis reaction has also been elucidated. For practical utilization of cellulosic materials, the cellulose structural properties and their effects on cellulase adsorption, and the rate of hydrolysis must be taken into consideration. (Refs. 24).

  13. Optimization of Cellulase Production by Halobacillus sp. QLS 31 Isolated from Lake Qarun, Egypt.

    PubMed

    Korany, Ahmed H; Ali, Amal E; Essam, Tamer M; Megahed, Salwa A

    2017-02-24

    A halophilic cellulase-producing bacterium was isolated from a sediment sample collected from Lake Qarun (Fayoum Province, Egypt). Molecular identification based on 16S rDNA amplification and sequencing revealed 99% homology with Halobacillus sp. and hence was designated as Halobacillus sp. QLS 31. Medium composition and culture conditions were optimized for enhancing the production of cellulase enzyme using the Plackett-Burman statistical design. Ten variables were evaluated for their influence on cellulase production. Carboxymethyl cellulose (CMC), zinc sulfate (ZnSO4), and inoculum size were found to exert a significant effect on cellulase productivity by Halobacillus sp. QLS 31. The maximum specific activity of cellulase enzyme was 48.08 U/mg. Following the predicted conditions, a 7.5-fold increase in cellulase specific activity (175.47 U/mg) was achieved compared to the basal medium (23.19 U/mg) under the following optimized conditions: temperature (30 °C), fermentation time (2 days ), pH value (9), CMC concentration (1%), inoculum size (1%), yeast extract concentration (0.1%), ammonium sulfate ((NH3)2SO4) concentration (0.1%), sodium chloride (NaCl) concentration (20%), and metal inducers: ZnSO4 (0.1%) and Ca/Mg ratio (0.01%). Thus, the results of this study provide an important basis for more efficient, cheap industrial cellulase production from halophilic Halobacillus sp. QLS 31.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Cellulases without carbohydrate-binding modules in high consistency ethanol production process

    PubMed Central

    2014-01-01

    Background Enzymes still comprise a major part of ethanol production costs from lignocellulose raw materials. Irreversible binding of enzymes to the residual substrate prevents their reuse and no efficient methods for recycling of enzymes have so far been presented. Cellulases without a carbohydrate-binding module (CBM) have been found to act efficiently at high substrate consistencies and to remain non-bound after the hydrolysis. Results High hydrolysis yields could be obtained with thermostable enzymes of Thermoascus aurantiacus containing only two main cellulases: cellobiohydrolase I (CBH I), Cel7A and endoglucanase II (EG II), Cel5A. The yields were decreased by only about 10% when using these cellulases without CBM. A major part of enzymes lacking CBM was non-bound during the most active stage of hydrolysis and in spite of this, produced high sugar yields. Complementation of the two cellulases lacking CBM with CBH II (CtCel6A) improved the hydrolysis. Cellulases without CBM were more sensitive during exposure to high ethanol concentration than the enzymes containing CBM. Enzymes lacking CBM could be efficiently reused leading to a sugar yield of 90% of that with fresh enzymes. The applicability of cellulases without CBM was confirmed under industrial ethanol production conditions at high (25% dry matter (DM)) consistency. Conclusions The results clearly show that cellulases without CBM can be successfully used in the hydrolysis of lignocellulose at high consistency, and that this approach could provide new means for better recyclability of enzymes. This paper provides new insight into the efficient action of CBM-lacking cellulases. The relationship of binding and action of cellulases without CBM at high DM consistency should, however, be studied in more detail. PMID:24559384

  19. Comparative performance of precommercial cellulases hydrolyzing pretreated corn stover

    PubMed Central

    2011-01-01

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

  20. 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.

  1. 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.

  2. 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.

  3. 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).

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  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. 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.

  12. 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)...

  13. 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.

  14. 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.

  15. Water hyacinth as carbon source for the production of cellulase by Trichoderma reesei.

    PubMed

    Deshpande, Pradnya; Nair, Sajitha; Khedkar, Shubhangi

    2009-09-01

    Water hyacinth (Eichhornia crassipes), an aquatic weed common to the subtropic/tropical regions, was utilized as an inexpensive lignocellulosic substrate for production of cellulase by Trichoderma reesei. The effects of process parameters like substrate pretreatment, substrate concentration, initial medium pH, mode of inoculation, and incubation temperature on cellulase production were investigated. Under optimal conditions, a maximal cellulase activity of 0.22 +/- 0.04 IU/ml (approximately 73.3 IU/g cellulose) was recorded at the end of 15-day incubation period. Specific activity of the enzyme was 6.25 IU/mg protein. Hydrolysis of 1% substrate (water hyacinth) using crude enzyme dosage of 1.2 IU/g water hyacinth showed 28.7% saccharification in 1 h. The observations in present study indicate that saccharification of cellulose from water hyacinth was significantly higher by laboratory-produced cellulase than the commercial blend.

  16. 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.

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

    PubMed

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

    2010-05-01

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

  18. 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).

  19. The effect of additional autopolyploidization in a slow growing cellulase hyperproducer of Trichoderma.

    PubMed

    Toyama, H; Toyama, N

    2001-01-01

    M14-2 is a cellulase hyperproducer derived from Trichderma reesei QM 6a, but with a growth rate lower than that of the original strain. When M14-2 was autopolyploidized followed by haploidization and selection, the strain with both a higher cellulase productivity per mycelia and a higher growth rate could be obtained as M14-2B. This strain seemed to be constructed using gene sources amplified by additional autopolyploidization.

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

    PubMed

    Liang, Wenjuan; Cao, Xuejun

    2012-07-01

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

  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. 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.

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

    PubMed

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

    2017-02-01

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

  4. 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.

  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.

  6. 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.

  7. 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.

  8. Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30.

    PubMed

    Zhang, Qin; Lo, Chi-Ming; Ju, Lu-Kwang

    2007-03-01

    Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (beta-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.

  9. Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome

    PubMed Central

    Garg, Roma; Srivastava, Ritika; Brahma, Vijaya; Verma, Lata; Karthikeyan, Subramanian; Sahni, Girish

    2016-01-01

    Cellulase catalyzes the hydrolysis of β-1,4-linkages of cellulose to produce industrially relevant monomeric subunits. Cellulases find their applications in pulp and paper, laundry, food and feed, textile, brewing industry and in biofuel production. These industries always have great demand for cellulases that can work efficiently even in harsh conditions such as high salt, heat, and acidic environments. While, cellulases with high thermal and acidic stability are already in use, existence of a high halotolerant cellulase is still elusive. Here, we report a novel cellulase Cel5R, obtained from soil metagenome that shows high halotolerance and thermal stability. The biochemical and functional characterization of Cel5R revealed its endoglucanase activity and high halostability. In addition, the crystal structure of Cel5R determined at 2.2 Å resolution reveals a large number of acidic residues on the surface of the protein that contribute to the halophilic nature of this enzyme. Moreover, we demonstrate that the four free and non-conserved cysteine residues (C65, C90, C231 and C273) contributes to the thermal stability of Cel5R by alanine scanning experiments. Thus, the newly identified endoglucanase Cel5R is a promising candidate for various industrial applications. PMID:28008971

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

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

    PubMed Central

    Saliu, Bolanle Kudirat; Sani, Alhassan

    2012-01-01

    Corn cob is a major component of agricultural and domestic waste in many parts of the world. It is composed mainly of cellulose which can be converted to energy in form of bioethanol as an efficient and effective means of waste management. Production of cellulolytic enzymes were induced in the fungi Aspergillus niger and Penicillium decumbens by growing them in mineral salt medium containing alkali pre-treated and untreated corn cobs. The cellulases were characterized and partially purified. Alkali pre-treated corn cobs were hydrolysed with the partially purified cellulases and the product of hydrolysis was fermented using the yeast saccharomyces cerevisae to ethanol. Cellulases of A. niger produced higher endoglucanase and exoglucanase activity (0.1698 IU ml-1 and 0.0461 FPU ml-1) compared to that produced by P. decumbens (0.1111 IU ml-1 and 0.153 FPU ml-1). Alkali pre-treated corn cob hydrolysed by cellulases of A. niger yielded 7.63 mg ml-1 sugar which produced 2.67 % (v/v) ethanol on fermentation. Ethanol yield of the hydrolysates of corn cob by cellulases of P. decumbens was much lower at 0.56 % (v/v). Alkali pre-treated corn cob, hydrolysed with cellulases of A. niger is established as suitable feedstock for bioethanol production. PMID:27418920

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

    PubMed

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

    2017-02-10

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

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

    PubMed

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

    2017-01-23

    Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surface display system. This work provides key insights in engineering the cellulase cocktail in a consolidated bioprocessing yeast strain. Biotechnol. Bioeng. 2017;9999: 1-7. © 2017 Wiley Periodicals, Inc.

  14. 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

  15. EXTRACELLULAR ENZYME SYSTEM UTILIZED BY THE ROT FUNGUS STEREUM SANGUINOLENTUM FOR THE BREAKDOWN OF CELLULOSE: I. STUDIES ON THE ENZYME PRODUCTION. II. PURIFICATION OF THE CELLULASE. III. CHARACTERIZATION OF TWO PURIFIED CELLULASE FRACTIONS,

    DTIC Science & Technology

    focusing method. Also, the separation of two cellulase peaks from culture solutions of the rot fungus Stereum sanguinolentum has been demonstrated...homogeneous cellulase peak upon rechromatography on a DEAE-Sephadex A-50 column as well as on column electrophoresis. The results strongly indicate that S...sanguinolentum only excretes one cellulase enzyme in a culture solution with powdered cellulose as the carbon source. (Author)

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. Relationships between the addition rates of cellulase or glucose and silage fermentation at different temperatures.

    PubMed

    Zhang, Jian-Guo; Kawamoto, Hidenori; Cai, Yi-Min

    2010-06-01

    The influence of the application rates of cellulase preparation and glucose on silage fermentation at different temperatures was studied with the straw of naked barley (Hordeum vulgare L. emand Lam) and guineagrass (Panicum maximum Jacq.). Addition rate of cellulase and glucose, temperature and their interaction had significant effects on pH value, lactic acid content, butyric acid content and propionic acid content of naked barley straw silage and significant effects on all the parameters of guineagrass silage (P < 0.01). Temperature and interaction had significant effect on acetic acid content (P < 0.05) and no significant effect on NH(3)-N content of naked barley straw silage (P > 0.05). Under all the temperatures, the pH values of barley straw and guineagrass silages were reduced by cellulase and glucose addition even at the lowest rate (P < 0.05), compared with their corresponding control. Lactic acid contents of silages were the highest within the same temperature and same additive when glucose and cellulase were added at the highest rates, whereas the effect of cellulase and glucose addition on butyric acid production varied with their application rates and silage storage temperature. The addition rate of restricting butyric acid fermentation was lower at 20 degrees C than that at 30 degrees C, and it was the lowest at 40 degrees C where cellulase and glucose addition restricted butyric acid fermentation even at 0.1 g/kg and 10 g/kg, respectively, when compared to the control. While the addition rate was lower than the above level, cellulase and glucose addition also promoted butyric acid fermentation.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. A Kinetic Model for the Enzymatic Action of Cellulase

    PubMed Central

    Ting, Christina L.; Makarov, Dmitrii E.; Wang, Zhen-Gang

    2009-01-01

    We develop a mechanochemical model for the dynamics of cellulase, a two-domain enzyme connected by a peptide linker, as it extracts and hydrolyzes a cellulose polymer from a crystalline substrate. We consider two random walkers, representing the catalytic domain (CD) and the carbohydrate binding module (CBM), whose rates for stepping are biased by the coupling through the linker and the energy required to lift the cellulose polymer from the crystalline surface. Our results show that the linker length and stiffness play a critical role in the cooperative action of the CD and CBM domains and that, for a given linker length, the steady state hydrolysis shows a maximum at some intermediate linker stiffness. The maximum hydrolysis rate corresponds to a transition of the linker from a compressed to an extended conformation, where the system exhibits maximum fluctuation, as measured by the variance of the separation distance between the two domains and the dispersion around the mean hydrolysis speed. In the range of experimentally known values of the parameters of our model, improving the intrinsic hydrolytic activity of the CD leads to a proportional increase in the overall hydrolysis rate. PMID:19292431

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

    PubMed

    Subramaniyan, S; Prema, P

    2000-02-01

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

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

    DOE PAGES

    Bali, Garima; Meng, Xianzhi; Deneff, Jacob I.; ...

    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. 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.

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

    PubMed

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

    2017-01-01

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

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

    PubMed

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

    2017-03-01

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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Zinc electrode in alkaline electrolyte

    SciTech Connect

    McBreen, J.

    1995-12-31

    The zinc electrode in alkaline electrolyte is unusual in that supersaturated zincate solutions can form during discharge and spongy or mossy zinc deposits can form on charge at low overvoltages. The effect of additives on regular pasted ZnO electrodes and calcium zincate electrodes is discussed. The paper also reports on in situ x-ray absorption (XAS) results on mossy zinc deposits.

  3. (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.

  4. Interrelationships between cellulase activity and cellulose particle morphology

    DOE PAGES

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

    2016-06-11

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

  5. Interrelationships between cellulase activity and cellulose particle morphology

    SciTech Connect

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

    2016-06-11

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

  6. 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.

  7. 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.

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

    PubMed

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

    2016-08-01

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

  9. 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

  10. 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.

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

    PubMed

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

    2017-01-01

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

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

    PubMed

    Shida, Yosuke; Furukawa, Takanori; Ogasawara, Wataru

    2016-09-01

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

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

    PubMed

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

    2014-02-01

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

  14. 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.

  15. The accessible cellulose surface influences cellulase synergism during the hydrolysis of lignocellulosic substrates.

    PubMed

    Hu, Jinguang; Gourlay, Keith; Arantes, Valdeir; Van Dyk, J S; Pribowo, Amadeus; Saddler, Jack N

    2015-03-01

    Effective enzymatic hydrolysis of insoluble cellulose requires the synergistic action of a suite of cellulase components. Most previous studies have only assessed cellulase synergism on model cellulosic substrates. When the actions of individual and combinations of cellulases (Cel7A, Cel6A, Cel7B, Cel5A) were assessed on various pretreated lignocellulosic substrates, Cel7A was shown to be the major contributor to overall cellulose hydrolysis, with the other enzymes synergistically enhancing its hydrolytic efficiency, at least partially, by facilitating Cel7A desorption (assessed by a double-sandwich enzyme-linked immunosorbent assay). When the influences of various substrate physicochemical characteristics on the effectiveness of enzyme synergism were assessed, a strong relationship was observed between cellulose accessibility (as determined by the cellulose binding module technique) and the degree of synergism, with greater synergy observed on the more disorganized/accessible cellulose surface.

  16. Recycling cellulase from enzymatic hydrolyzate of acid treated wheat straw by electroultrafiltration.

    PubMed

    Chen, Guoqiang; Song, Weijie; Qi, Benkun; Lu, Jianren; Wan, Yinhua

    2013-09-01

    This work explores the feasibility of recycling cellulase by electroultrafiltration (EUF), an ultrafiltration process enhanced by an electric field, to reduce the cost of enzymatic transformation of cellulose. The effect of electric field under different operating conditions (buffer concentration, acid treated wheat straw concentration, current and temperature) on flux during EUF was examined. The results showed that EUF was effective to reduce concentration polarization (CP) and enhance filtration flux in recycling cellulase. The flux improvement by the electric field could be strengthened at low buffer concentration (5 mM) and relatively low temperature (room temperature) and high current (150 mA). The flux for 2% (substrate concentration, w/v) lignocellulosic hydrolyzate increased by a factor of 4.4 at 836 V/m and room temperature, compared to that without electric field. This work shows that under appropriate operating conditions EUF can efficiently recycle cellulase from lignocellulosic hydrolyzate and thus substantially reduce hydrolysis cost.

  17. 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.

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

    PubMed Central

    Adeoyo, Olusegun Richard

    2011-01-01

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

  19. 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.

  20. 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.

  1. 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.

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

    SciTech Connect

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

    2015-05-30

    This study took advantage of resorufin cellobioside as a fluorescent substrate to determine the distribution of cellulase activity in cellulosic biomass fermentation systems. Cellulolytic biofilms were found to express nearly four orders greater cellulase activity compared to planktonic cultures of Clostridium thermocellum and Caldicellulosiruptor obsidiansis, which can be primarily attributed to the high cell concentration and surface attachment. The formation of biofilms results in cellulases being secreted close to their substrates, which appears to be an energetically favorable stategy for insoluble substrate utilization. For the same reason, cellulases should be closely associated with the surfaces of suspended cell in 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 biofilmformation in cellulosic biomass hydrolysismay promise an improved cellulosic biofuel process.

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

    PubMed Central

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

    2016-01-01

    Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces (S.) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies, and Streptomyces flavofuscus), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus, and Streptomyces ambofaciens. Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers (S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and xylanase

  4. 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.

  5. 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.

  6. Extracting xylooligosaccharides in wheat bran by screening and cellulase assisted enzymatic hydrolysis.

    PubMed

    Zhao, Xiaoqing; Dong, Cao

    2016-11-01

    Functional xylooligosaccharides (XOS) from wheat bran were carried out by screening and cellulase assisted enzymatic hydrolysis. First, wheat bran was crushed and screened, getting the optimal experimental particle size of raw materials (>125μm), and 72.43% of starch. Wheat bran was then repeated colloid milled and hydrolysed with cellulase, releasing about 45% of xylan (higher than the untreated wheat bran (about 29%)). Finally, membrane treatment (20kDa and 1kDa cutoff) was chosen to eliminate impurities and enriched XOS. In the designed process, 51mg purified XOS was obtained from 1g dry wheat bran powder.

  7. 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.

  8. Effects of Cellulase Supplementation on Nutrient Digestibility, Energy Utilization and Methane Emission by Boer Crossbred Goats

    PubMed Central

    Wang, Lizhi; Xue, Bai

    2016-01-01

    This study examined the effect of supplementing exogenous cellulase on nutrient and energy utilization. Twelve desexed Boer crossbred goats were used in a replicated 3×3 Latin square design with 23-d periods. Dietary treatments were basal diet (control, no cellulase), basal diet plus 2 g unitary cellulase/kg of total mixed ration dry matter (DM), and basal diet plus 2 g compound cellulase/kg of total mixed ration DM. Three stages of feeding trials were used corresponding to the three treatments, each comprised 23 d, with the first 14 d as the preliminary period and the following 9 d as formal trial period for metabolism trial. Total collection of feces and urine were conducted from the 4th d of the formal trial, and gas exchange measures were determined in indirect respiratory chambers in the last 3 d of the formal trial. Results showed that cellulase addition had no effect (p>0.05) on nutrient digestibility. Dietary supplementation of cellulase did not affect (p>0.05) N intake and retention in goats. Gross energy (GE) intake, fecal energy and urinary energy excretion, heat production were not affected (p>0.05) by the cellulase supplementation. Total methane emission (g/d), CH4 emission as a proportion of live weight or feed intake (DM, organic matter [OM], digestible DM or digestible OM), or CH4 energy output (CH4-E) as a proportion of energy intake (GE, digestible energy, or metabolizable energy), were similar (p>0.05) among treatments. There was a significant (p<0.001) relationship between CH4 and live weight (y = 0.645x+0.2, R2 = 0.54), CH4 and DM intake (y = 16.7x+1.4, R2 = 0.51), CH4 and OM intake (y = 18.8x+1.3, R2 = 0.51) and CH4-E and GE intake. Results from this study revealed that dietary supplementation of cellulase may have no effect on nutrient digestibility, nitrogen retention, energy metabolism, and methane emission in goat. PMID:26732445

  9. The secondary alkaline zinc electrode

    NASA Astrophysics Data System (ADS)

    McLarnon, Frank R.; Cairns, Elton J.

    1991-02-01

    The worldwide studies conducted between 1975 and 1990 with the aim of improving cell lifetimes of secondary alkaline zinc electrodes are overviewed. Attention is given the design features and characteristics of various secondary alkaline zinc cells, including four types of zinc/nickel oxide cell designs (vented static-electrolyte, sealed static-electrolyte, vibrating-electrode, and flowing-electrolyte); two types of zinc/air cells (mechanically rechargeable consolidated-electrode and mechanically rechargeable particulate-electrode); zinc/silver oxide battery; zinc/manganese dioxide cell; and zinc/ferric cyanide battery. Particular consideration is given to recent research in the fields of cell thermodynamics, zinc electrodeposition, zinc electrodissolution, zinc corrosion, electrolyte properties, mathematical and phenomenological models, osmotic pumping, nonuniform current distribution, and cell cycle-life perforamnce.

  10. 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.

  11. 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

  12. 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.

  13. 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…

  14. 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...

  15. 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.

  16. 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.

  17. Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis.

    PubMed

    de Castro, Aline Machado; de Albuquerque de Carvalho, Marcelle Lins; Leite, Selma Gomes Ferreira; Pereira, Nei

    2010-02-01

    The objective of this work is to investigate the utilization of two abundant agricultural residues in Brazil for the production and application of cellulolytic enzymes. Different materials obtained after pretreatment of sugarcane bagasse, as well as pure synthetic substrates, were considered for cellulase production by Penicillium funiculosum. The best results for FPase (354 U L(-1)) and beta-glucosidase (1,835 U L(-1)) production were observed when sugarcane bagasse partially delignified cellulignin (PDC) was used. The crude extract obtained from PDC fermentation was then partially characterized. Optimal temperatures for cellulase action ranged from 52 to 58 degrees C and pH values of around 4.9 contributed to maximum enzyme activity. At 37 degrees C, the cellulases were highly stable, losing less than 15% of their initial activity after 23 h of incubation. There was no detection of proteases in the P. funiculosum extract, but other hydrolases, such as endoxylanases, were identified (147 U L(-1)). Finally, when compared to commercial preparations, the cellulolytic complex from P. funiculosum showed more well-balanced amounts of beta-glucosidase, endo- and exoglucanase, resulting in the desired performance in the presence of a lignocellulosic material. Cellulases from this filamentous fungus had a higher glucose production rate (470 mg L(-1) h(-1)) when incubated with corn cob than with Celluclast, GC 220 and Spezyme (312, 454 and 400 mg L(-1) h(-1), respectively).

  18. 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

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

    DOE PAGES

    Yang, Dongqing; Pomraning, Kyle; Kopchinskiy, Alexey; ...

    2015-08-13

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

  20. 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.

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

    PubMed

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

    2017-02-07

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

  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.

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

    PubMed

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

    2015-01-09

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

  4. 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

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

    PubMed

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

    2009-05-01

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

  6. Alkaline hydrogen peroxide pretreatment of cashew apple bagasse for ethanol production: study of parameters.

    PubMed

    Correia, Jessyca Aline da Costa; Júnior, José Edvan Marques; Gonçalves, Luciana Rocha B; Rocha, Maria Valderez Ponte

    2013-07-01

    The alkaline hydrogen peroxide (AHP) pretreatment of cashew apple bagasse (CAB) was evaluated based on the conversion of the resultant cellulose into glucose. The effects of the concentration of hydrogen peroxide at pH 11.5, the biomass loading and the pretreatment duration performed at 35°C and 250 rpm were evaluated after the subsequent enzymatic saccharification of the pretreated biomass using a commercial cellulase enzyme. The CAB used in this study contained 20.56 ± 2.19% cellulose, 10.17 ± 0.89% hemicellulose and 35.26 ± 0.90% lignin. The pretreatment resulted in a reduced lignin content in the residual solids. Increasing the H2O2 concentration (0-4.3% v/v) resulted in a higher rate of enzymatic hydrolysis. Lower biomass loadings gave higher glucose yields. In addition, no measurable furfural and hydroxymethyl furfural were produced in the liquid fraction during the pretreatment. The results show that alkaline hydrogen peroxide is effective for the pretreatment of CAB.

  7. Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification. [Trichoderma reesei

    SciTech Connect

    Gould, J.M.

    1984-01-01

    Approximately one-half of the lignin and most of the hemicellulose present in agricultural residues such as wheat straw and corn stover are solubilized when the residue is treated at 25/sup 0/C in an alkaline solution of hydrogen peroxide. The delignification reaction is most efficient when the ratio of hydrogen peroxide to substrate is at least 0.25 (w/w) and the pH is 11.5. The supernatant fraction from a given pretreatment, after addition of makeup peroxide and readjustment of the pH, can be recycled to treat at least six additional batches of substrate, resulting in a substantial concentration of hemicellulose and soluble lignin degradation products. Hydrolysis of the insoluble fraction with Trichoderma reesei cellulase after alkline peroxide treatment yields glucose with almost 100% efficiency, based upon the cellulose content of the residue before treatment. These data indicate that alkaline peroxide pretreatment is a simple and efficient method for enhancing the enzymatic digestibility of lignocellulosic crop residues to levels approaching the theoretical maximum.

  8. Environmentally safe treatment of black liquor with Comamonas sp. B-9 under high-alkaline conditions.

    PubMed

    Zheng, Yu; Chai, Liyuan; Yang, Zhihui; Chen, Yuehui; Shi, Yan; Wang, Yangyang

    2014-02-01

    The strain Comamonas sp. B-9 was isolated from steeping fluid of erosive bamboo slips derived from Kingdom Wu during the Three-Kingdoms Dynasty of ancient China (A.D. 220-280). It could be used to treat black liquor (BL) with high-alkaline pH and with an initial chemical oxygen demand (COD) of 18,000-25,000 mg L(-1) , without the addition of other carbon and nitrogen sources. The results revealed that Comamonas sp. B-9 was capable of reducing the COD, color, and lignin content of BL by up to 56.8, 35.3, and 43.5%, respectively. High levels of laccase, manganese peroxidase, cellulase, and xylanase enzymatic activities were also observed, and these enzymes could play an important role in the biotreatment of BL. Further, GC-MS analysis showed that most of the compounds detected in BL after biotreatment with Comamonas sp. B-9 were diminished, while 4-methyl benzaldehyde, 3,4,5-trihydroxybenzoic acid ethyl ester, and 4-hydroxy-3,5-dimethoxy benzaldehyde were produced as metabolites. The presented results indicate that Comamonas sp. B-9 has potential application for the treatment of wastewaters from pulp and paper processing with high COD load under high-alkaline conditions.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  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.

  15. Diclofenac salts. III. Alkaline and earth alkaline salts.

    PubMed

    Fini, Adamo; Fazio, Giuseppe; Rosetti, Francesca; Angeles Holgado, M; Iruín, Ana; Alvarez-Fuentes, Josefa

    2005-11-01

    Diclofenac salts containing the alkaline and two earth alkaline cations have been prepared and characterized by scanning electron microscopy (SEM) and EDAX spectroscopy; and by thermal and thermogravimetric analysis (TGA): all of them crystallize as hydrate when precipitated from water. The salts dehydrate at room temperature and more easily on heating, but recovery the hydration, when placed in a humid environment. X-ray diffraction spectra suggest that on dehydration new peaks appear on diffractograms and the lattice of the salts partially looses crystallinity. This phenomenon is readily visible in the case of the calcium and magnesium salts, whose thermograms display a crystallization exotherm, before melting or decomposing at temperatures near or above 200 degrees C; these last salts appear to form solvates, when prepared from methanol. The thermogram of each salt shows a complex endotherm of dehydration about 100 degrees C; the calcium salt displays two endotherms, well separated at about 120 and 160 degrees C, which disappear after prolonged heating. Decomposition exotherms, before or soon after the melting, appear below 300 degrees C. The ammonium salt is thermally unstable and, when heated to start dehydration, dissociates and leaves acidic diclofenac.

  16. 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.

  17. Thermal stability of Trichoderma reesei C30 cellulase and Aspergillus niger. beta. -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 ..beta..-glucosidase with glutaraldehyde at pH 8.0 increased the thermal stability of this enzyme.

  18. 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.

  19. 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.

  20. 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.

  1. 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; ...

    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

  2. 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

  3. Alkaline and alkaline earth metal phosphate halides and phosphors

    DOEpatents

    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. 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.

  5. 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.

  6. 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.

  7. Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

    PubMed Central

    Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

    2012-01-01

    Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the

  8. A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection

    PubMed Central

    2014-01-01

    Background Most of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales’ procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development. Results We report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 μM. In contrast to the other commonly used methods, the Schales’ procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin. Conclusion The ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional

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

    PubMed Central

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

    2012-01-01

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

  11. 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)

  12. Closed type alkaline storage battery

    SciTech Connect

    Hayama, H.

    1980-06-10

    The alkaline storage battery employs a metallic hat shaped terminal closure which has a piercing needle as well as a puncturable metallic diaphragm positioned below the piercing needle. The needle is fixed by caulking at its peripheral edge portion to a edge of the closure. A comparatively thick and hard metal plate is placed on the inner surface of the diaphragm and is applied to an open portion of a tubular metallic container which has a battery element. A peripheral edge portion of the closure, the diaphragm and the metallic plate are clamped in airtight relationship through a packing between the caulked end portion and an inner annular step portion of the metallic container of the battery. A lead wire extends from one polarity electrode of the battery element and is connected to a central portion of the metallic plate.

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

    PubMed

    Zhang, Fei; Bai, Fengwu; Zhao, Xinqing

    2016-10-01

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

  14. 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.

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

    PubMed

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

    2016-01-01

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

  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. 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.

  18. 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.

  19. 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.

  20. Cellulase finishing of woven, cotton fabrics in jet and winch machines.

    PubMed

    Cortez, J M; Ellis, J; Bishop, D P

    2001-08-23

    Some authors have reported that as the applied agitation rate increases, the apparent activity of the endoglucanases from Trichoderma reesei towards cotton cellulose increases more markedly than does the apparent activity of the cellobiohydrolases. This suggests that the quality of cellulase finishing effects on cellulosic textiles may be machine-type dependent. The present work using total crude, endoglucanase-rich and cellobiohydrolase-rich cellulases from T. reesei confirmed that the final properties of woven, cotton fabrics treated under realistic processing conditions in a jet machine, were measurably and perceivably different from those of the same fabrics, treated using the same processing conditions of temperature, time, pH, enzyme concentration and fabric to liquor ratio, but in a winch machine. The results are interpreted in terms of the effects of agitation rate on the adsorption-desorption behaviour of the T. reesei endoglucanases and cellobiohydrolases.

  1. Cellulase production through solid-state tray fermentation, and its use for bioethanol from sorghum stover.

    PubMed

    Idris, Ayman Salih Omer; Pandey, Ashok; Rao, S S; Sukumaran, Rajeev K

    2017-03-20

    The production of cellulase by Trichoderma reesei RUT C-30 under solid-state fermentation (SSF) on wheat bran and cellulose was optimized employing a two stage statistical design of experiments. Optimization of process parameters resulted in a 3.2-fold increase in CMCase production to 959.53IU/gDS. The process was evaluated at pilot scale in tray fermenters and yielded 457IU/gDS using the lab conditions and indicating possibility for further improvement. The cellulase could effectively hydrolyze alkali pretreated sorghum stover and addition of Aspergillus niger β-glucosidase improved the hydrolytic efficiency 174%, indicating the potential to use this blend for effective saccharification of sorghum stover biomass. The enzymatic hydrolysate of sorghum stover was fermented to ethanol with ∼80% efficiency.

  2. Occupational asthma caused by cellulase and lipase in the detergent industry.

    PubMed

    Brant, A; Hole, A; Cannon, J; Helm, J; Swales, C; Welch, J; Taylor, A Newman; Cullinan, P

    2004-09-01

    Three employees from two different detergent companies were investigated for occupational asthma, using skin prick tests, serum specific IgE, and specific bronchial challenge. Two were challenged with lipase and one with cellulase. All three cases had immunological evidence of sensitisation to the detergent enzymes with which they worked. Bronchial challenge in each provoked a reproducible dual asthmatic response, which reproduced their work related symptoms. These are the first reported cases of occupational asthma attributable to cellulase and lipase in the detergent industry. Four of the most common enzymes used in this industry have now been reported to cause occupational asthma; continued vigilance and caution are needed when working with these or other enzymes.

  3. 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.

  4. 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.

  5. 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.

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

    PubMed

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

    2017-01-01

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

  7. 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).

  8. 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.

  9. Evolution of alkaline phosphatases in primates.

    PubMed Central

    Goldstein, D J; Rogers, C; Harris, H

    1982-01-01

    Alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] in placenta, intestine, liver, kidney, bone, and lung from a variety of primate species has been characterized by quantitative inhibition, thermostability, and immunological studies. Characteristic human placental-type alkaline phosphatase occurs in placentas of great apes (chimpanzee and orangutan) but not in placentas of other primates, including gibbon. It is also present in trace amounts in human lung but not in lung or other tissues of various Old and New World monkeys. However, a distinctive alkaline phosphatase resembling it occurs in substantial amounts in lungs from Old World monkeys but not New World monkeys. It appears that duplication of alkaline phosphatase genes and mutations of genetic elements controlling their tissue expression have occurred relatively recently in mammalian evolution. Images PMID:6950431

  10. 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.

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

    PubMed

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

    2011-01-01

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

  12. 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.

  13. 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

  14. 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).

  15. 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

  16. 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.

  17. Adsorption of Clostridium thermocellum cellulases onto pretreated mixed hardwood, avicel, and lignin

    SciTech Connect

    Bernardez, T.D.; Lyford, K.; Hogsett, D.A.; Lynd, L.R. . Thayer School of Engineering)

    1993-09-20

    Adsorption of Avicel-hydrolyzing activity was examined with respect to: mixed hardwood flour pretreated with 1% sulfuric acid for 9 s at 220C (PTW220), lignin prepared from PTW220 by either acid or enzymatic hydrolysis, and Avicel. Experiments were conducted at 60C for all materials, and also at 25C for PTW220. Based on transient adsorption results and reaction rates, times were selected at which to characterize adsorption at 60C as follows: PTW220, 1 min; lignin, 30 min; and Avicel, 45 min. Similar results were obtained for adsorption of cellulase activity to PTW220 at 25 and 60C, and for lignin prepared by enzymatic and acid hydrolysis. For all materials, adsorption was described well by a Langmuir equation, although the reversibility of adsorption was not investigated. Langmuir affinity constants (L/g) were: PTW220, 109; lignin, 17.9; Avicel, 4.3; cellulose from PTW220, [ge]187. Langmuir capacity constants were 760 for PTW220 and 42 for Avicel; the cellulase binding capacity of lignin appeared to be very high under the conditions examined, and could not be determined. At low and moderate cellulase loadings at least, the majority of cellulase activity adsorbed to PTVV220 is bound to the cellulosic component. The results indicate that PTW220, and its cellulose component in particular, differ radically from Avicel with respect to adsorption. Avicel-hydrolyzing activity and CMC-hydrolyzing activities were found to bind to Avicel with a constant ratio of essentially one, consistent with adsorption of a multi-activity complex.

  18. 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.

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

    NASA Astrophysics Data System (ADS)

    Donaldson, Lloyd; Vaidya, Alankar

    2017-03-01

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

  20. Statistical optimization of process conditions for cellulase production by liquid state bioconversion of domestic wastewater sludge.

    PubMed

    Alam, Md Zahangir; Muyibi, Suleyman A; Wahid, Rosmaziah

    2008-07-01

    A two-level fractional factorial design (FFD) was used to determine the effects of six factors, i.e. substrate (domestic wastewater sludge - DWS) and co-substrate concentration (wheat flour - WF), temperature, initial pH, inoculum size and agitation rate on the production of cellulase enzyme by Trichoderma harzianum in liquid state bioconversion. On statistical analysis of the results from the experimental studies, optimum process conditions were found to be temperature 32.5 degrees C, substrate concentration (DWS) 0.75% (w/w), co-substrate (WF) concentration 2% (w/w), initial pH 5, inoculum size 2% (v/w) and agitation 175 rpm. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) of 0.975. Cellulase activity reached 10.2 FPU/ml at day 3 during the fermentation process which indicated about 1.5-fold increase in production compared to the cellulase activity obtained from the results of design of experiment (6.9 FPU/ml). Biodegradation of DWS was also evaluated to verify the efficiency of the bioconversion process as a waste management method.

  1. 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

  2. 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.

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

    PubMed Central

    Donaldson, Lloyd; Vaidya, Alankar

    2017-01-01

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

  4. Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator.

    PubMed

    Zhang, Xiaoyue; Li, Yonghao; Zhao, Xinqing; Bai, Fengwu

    2017-01-01

    The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved.

  5. 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.

  6. 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.

  7. Use of fungi for the bioconversion of rice straw into cellulase enzyme.

    PubMed

    Khan, Munir H; Ali, S; Fakhru'l-Razi, A; Alam, Z

    2007-05-01

    Cellulase production was carried out by solid state bioconversion (SSB) method using rice straw, a lignocellulosic material and agricultural waste, as the substrate of three Trichoderma spp. and Phanerochaete chrysosporium in lab-scale experiments. The results were compared to select the best fungi among them for the production of cellulase. Phanerochaete chrysosporium was found to be the best among these species of fungi, which produced the highest cellulase enzyme of 1.43 IU/mL of filter paper activity (FPase) and 2.40 IU/mL of carboxymethylcellulose activity (CMCase). The "glucosamine" and "reducing sugar" parameters were observed to evaluate the growth and substrate utilization in the experiments. In the case of Phanerochaete Chrysosporium, the highest glucosamine concentration was 1.60 g/L and a high concentration of the release of reducing sugar was measured as 2.58 g/L obtained on the 4th day of fermentation. The pH values were also recorded. The range of the pH was about 5.15 to 5.56 in the case of Phanerochaete Chrysosporium.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. Degradation of Distillery Lees (Shochu kasu) by Cellulase-Producing Thraustochytrids.

    PubMed

    Taoka, Yousuke; Nagano, Naoki; Kai, Hiroyuki; Hayashi, Masahiro

    2017-01-01

    Single cell oils produced by oleaginous microorganisms have attracted increasing interests as a petroleum alternative energy. Marine eukaryotes, thraustochytrids were heterotrophic, and can grow rapidly and accumulate large amount of lipids containing functional fatty acids, such as docosahexaenoic acid (DHA) in their cells body. In this investigation, thraustochytrids isolated from marine environment were cultured in the medium containing an industrial waste and an unused resource, distillery lees (Shochu kasu) to produce biofuel or functional fatty acids by microorganisms. Sixty-nine thraustochytrids and Schizochytrium aggregatum ATCC 28209 were screened for cellulase production, and the activities were detected using sodium carboxymethyl cellulose (CMC) as a substrate. Based on the screening test, strain TM02Bc identified to Schizochytrium sp. was selected for the Shochu kasu degradation test and compared with S. aggregatum ATCC 28209 previously known as a cellulase-producing thraustochytrid. Strains TM02Bc and ATCC 28209 were cultured in artificial seawater containing Shochu kasu for 15 days. The two strains could degrade Schochu kasu, especially that from sweet potato Shochu (Imo Shochu). Cellulase (CMCase) and protease activities were detected in culture supernatant of both strains, and the ratio of polyunsaturated fatty acids (PUFAs) significantly increased as a result of incubation of Shochu kasu with two strains. This preliminary study indicated that strain TM02Bc was a potent candidate for Shochu kasu treatment and fatty acid production.

  13. 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

  14. Analysis of casein biopolymers adsorption to lignocellulosic biomass as a potential cellulase stabilizer.

    PubMed

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

    2012-01-01

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

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

    PubMed

    Kim, Seonghun; Kim, Chul Ho

    2012-01-01

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

  16. 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

  17. 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.

  18. 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.

  19. 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%.

  20. Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

    PubMed

    Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

    2016-11-14

    This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of

  1. 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.

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

    PubMed

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

    2017-03-01

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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

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

    PubMed

    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.

  8. 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

  9. High-coverage gene expression profiling analysis of the cellulase-producing fungus Acremonium cellulolyticus cultured using different carbon sources.

    PubMed

    Hideno, Akihiro; Inoue, Hiroyuki; Fujii, Tatsuya; Yano, Shinichi; Tsukahara, Kenichiro; Murakami, Katsuji; Yunokawa, Harunobu; Sawayama, Shigeki

    2013-06-01

    The gene expression of a cellulase-producing fungus, Acremonium cellulolyticus, was investigated after culturing with three different carbon sources: glycerol, lactose, and Solka-Floc powdered cellulose (SF). High-coverage gene expression profiling (HiCEP) analysis, a method requiring no prior sequence knowledge, was used to screen genes upregulated at the early stage of cellulase production. SF was used as a strong inducer of cellulase production, lactose was used as an inducer of the expression of cellulase genes at the early stage of the culture, and glycerol was used as a negative control. Approximately 15,000 transcript-derived fragments (TDFs) were detected in each sample prepared from the culture grown for 16 h. Based on the expression profiles of the cultured cells, 36 fragments upregulated in both the SF and lactose cultures were selected and sequenced. The deduced gene products of 31 TDFs were likely related to biomass degradation, sugar metabolism, transcriptional regulation, protein modification and metabolism, cell wall recycling, fatty acid and polyketide biosynthesis, and other functions. Quantitative real-time reverse-transcriptase polymerase chain reaction analysis verified that almost all of the transcripts obtained by HiCEP analysis were upregulated in the SF and lactose cultures grown for 18 h. Some of the TDFs in the SF culture were further upregulated over the course of 72 h. The gene products from these TDFs would provide insight into improving the cellulase productivity of A. cellulolyticus.

  10. 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

    2016-08-10

    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.

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

    PubMed Central

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

    2013-01-01

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

  12. 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.

  13. Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid.

    PubMed

    Santos, Victor T O; Esteves, Paula J; Milagres, Adriane M F; Carvalho, Walter

    2011-08-01

    This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H(2)SO(4) with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, β-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2(3) central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19-4.81%, w/w) and loadings of enzymes (1.9-38.1 FPU/g bagasse) and Tween 20 (0.0-0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.

  14. 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

  15. In vitro solubility of calcium, iron, and zinc in rice bran treated with phytase, cellulase, and protease.

    PubMed

    Wang, Yaqin; Cheng, Yongqiang; Ou, Keqin; Lin, Li; Liang, Jianfen

    2008-12-24

    Absorption of minerals is inhibited by phytic acid, fiber, and protein because of the chelates formed. Response surface method (RSM) was used in this study to evaluate the effect of application of commercial phytase, protease, and cellulase in rice bran on the in vitro solubility of calcium (IVCa), iron (IVFe), and zinc (IVZn). It is shown that IVCa and IVZn were significantly improved by the application of phytase and cellulase, and the models of two second-order polynomials are recommended for prediction, with coefficients at R(2) = 0.86 and R(2) = 0.88, respectively. Interaction between protease and cellulase also significantly affected IVCa and IVZn. Cellulase had more efficiency than phytase on IVCa. Differing in its effect on Ca and Zn, phytase had a significant linear correlation with IVFe, and none of the other processing parameters exerted a significant effect. The largest increment for IVFe, IVCa, and IVZn occurred in the treatment with applications of phytase at 2.5 U g(-1) and protease and cellulase at 0.2% and 1%, respectively, which were 3.3, 3.6, and 4.3 times, respectively, that of the untreated material.

  16. Halostable cellulase with organic solvent tolerance from Haloarcula sp. LLSG7 and its application in bioethanol fermentation using agricultural wastes.

    PubMed

    Li, Xin; Yu, Hui-Ying

    2013-12-01

    A haloarchaeal strain LLSG7 with cellulolytic activity was isolated from the saline soil of Yuncheng Salt Lake, China. Biochemical and physiological characterization along with 16S rRNA gene sequence analysis placed the isolate in the genus Haloarcula. Cellulase production was strongly influenced by the salinity of the culture medium with the maximum obtained in the presence of 25 % NaCl. Substrate specificity tests showed that the crude cellulase was a multicomponent enzyme system, and zymogram analysis revealed that five different endoglucanases were secreted by strain LLSG7. Optimal cellulase activity was at 50 °C, pH 8.0, and 20 % NaCl. In addition, it was highly active and stable over broad ranges of temperature (40-80 °C), pH (7.0-11.0), and NaCl concentration (17.5-30 %). The cellulase displayed remarkable stability in the presence of non-polar organic solvents with log P ow ≥ 1.97. The crude cellulase secreted by strain LLSG7 was further applied to hydrolyze alkali-pretreated rice straw and the enzymatic hydrolysate was used as the substrate for bioethanol fermentation by Saccharomyces cerevisiae. The yield of ethanol was 0.177 g per gram of pretreated rice straw, suggesting that it might be potentially useful for bioethanol production.

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

    DOE PAGES

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

    2015-05-30

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

  18. 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

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

    PubMed

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

    2014-01-01

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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. [Alkaline phosphatase in Amoeba proteus].

    PubMed

    Sopina, V A

    2005-01-01

    In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.

  6. 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...

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

    PubMed

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

    2014-05-10

    A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39-0.40s(-1)) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.

  8. Study of protein adsorption on indigo particles confirms the existence of enzyme--indigo interaction sites in cellulase molecules.

    PubMed

    Gusakov, A V; Sinitsyn, A P; Markov, A V; Sinitsyna, O A; Ankudimova, N V; Berlin, A G

    2001-04-27

    Adsorption of several crude and purified cellulases (from Trichoderma reesei, Penicillium verruculosum and Chrysosporium lucknowense) on indigo particles and Avicel cellulose was studied. Much higher amounts of protein were bound to indigo than to cellulose under similar conditions. For different purified enzymes, the quantity of bound protein per mg of adsorbent (indigo or cellulose) varied in the range of 57-111 and 0-62 microg x mg(-1), respectively. However, in general, the enzyme adsorption on indigo was less specific than the adsorption on cellulose. Three endoglucanases, having the highest indigo-binding ability, demonstrated the best washing performance in the process of enzymatic denim treatment. These data confirmed our previous findings that certain cellulases, which have indigo-binding sites (clusters of closely located aromatic and other non-polar residues) on the surface of their molecules, may remove indigo from the denim fabric better than cellulases with lower content of hydrophobic residues exposed to solvent.

  9. 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

  10. 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.

  11. Acetone-butanol-ethanol production from corn stover pretreated by alkaline twin-screw extrusion pretreatment.

    PubMed

    Zhang, Yuedong; Hou, Tongang; Li, Bin; Liu, Chao; Mu, Xindong; Wang, Haisong

    2014-05-01

    In this study, the alkaline twin-screw extrusion pretreated corn stover was subjected to enzymatic hydrolysis after washing. The impact of solid loading and enzyme dose on enzymatic hydrolysis was investigated. It was found that 68.2 g/L of total fermentable sugar could be obtained after enzymatic hydrolysis with the solid loading of 10 %, while the highest sugar recovery of 91.07 % was achieved when the solid loading was 2 % with the cellulase dose of 24 FPU/g substrate. Subsequently, the hydrolyzate was fermented by Clostridium acetobutylicum ATCC 824. The acetone-butanol-ethanol (ABE) production of the hydrolyzate was compared with the glucose, xylose and simulated hydrolyzate medium which have the same reducing sugar concentration. It was shown that 7.1 g/L butanol and 11.2 g/L ABE could be produced after 72 h fermentation for the hydrolyzate obtained from enzymatic hydrolysis with 6 % solid loading. This is comparable to the glucose and simulated hydrozate medium, and the overall ABE yield could reach 0.112 g/g raw corn stover.

  12. 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).

  13. 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.

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

    DOE PAGES

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

    2014-10-16

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

  15. 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

  16. 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.

  17. 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.

  18. 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.

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

    PubMed Central

    2012-01-01

    Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment. PMID:23062472

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

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

    PubMed

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

    2014-07-01

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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Adsorption of cellulase Aspergillus niger on a commercial activated carbon: kinetics and equilibrium studies.

    PubMed

    Daoud, Fatima Boukraa-Oulad; Kaddour, Samia; Sadoun, Tahar

    2010-01-01

    The adsorption kinetics of cellulase Aspergillus niger on a commercial activated carbon has been performed using a batch-adsorption technique. The effect of various experimental parameters such as initial enzyme concentration, contact time and temperature were investigated. The pseudo-first-order and pseudo-second-order kinetic models were used to describe the kinetic data which shows that the adsorption of the enzyme followed the pseudo-second-order rate expression and the rate constants were evaluated. The Langmuir and Freundlich adsorption isotherm models were applied to describe the equilibrium isotherms, and the isotherm constants were determined. It was found that Langmuir model was more suitable for our data. The activation energy of adsorption was also evaluated for the adsorption of enzyme onto activated carbon. It was found 11.37 kJ mol(-1). Thermodynamic parameters Delta G(0), Delta H(0) and DeltaS(0) were calculated, indicating that this process can be spontaneous and endothermic. The adsorption enthalpy and entropy were found 11.12 kJ mol(-1) and 0.084 kJ mol(-1)K(-1), respectively. At 30 degrees C and at pH 4.8, 1g activated carbon adsorbed about 1565 mg of cellulase, with a retention of 70% of the native enzyme activity up to five cycles of repeated batch enzyme reactions.

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

    PubMed

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

    2017-03-28

    Producing fuels, chemicals, and materials from renewable resources to meet societal demands remains an important step in the transition to a sustainable, clean energy economy. The use of cellulolytic enzymes for the production of nanocellulose enables the coproduction of sugars for biofuels production in a format that is largely compatible with the process design employed by modern lignocellulosic (second generation) biorefineries. However, yields of enzymatically produced nanocellulose are typically much lower than those achieved by mineral acid production methods. In this study, we compare the capacity for coproduction of nanocellulose and fermentable sugars using two vastly different cellulase systems: the classical "free enzyme" system of the saprophytic fungus, Trichoderma reesei (T. reesei) and the complexed, multifunctional enzymes produced by the hot springs resident, Caldicellulosiruptor bescii (C. bescii). We demonstrate by comparative digestions that the C. bescii system outperforms the fungal enzyme system in terms of total cellulose conversion, sugar production, and nanocellulose production. In addition, we show by multimodal imaging and dynamic light scattering that the nanocellulose produced by the C. bescii cellulase system is substantially more uniform than that produced by the T. reesei system. These disparities in the yields and characteristics of the nanocellulose produced by these disparate systems can be attributed to the dramatic differences in the mechanisms of action of the dominant enzymes in each system.

  8. Temperature sensitivity of cellulase adsorption on lignin and its impact on enzymatic hydrolysis of lignocellulosic biomass.

    PubMed

    Zheng, Yingfu; Zhang, Songping; Miao, Shida; Su, Zhiguo; Wang, Ping

    2013-07-10

    Unproductive enzyme adsorption is an important factor in addition to steric hindrance of lignin that limits the enzymatic hydrolysis of lignocellulosic biomass. While both are important factors, enzymatic hydrolysis of pretreated biomass is most likely conducted in the presence of certain amount of lignin residues that may not necessarily present accessibility hindrance, but can competitively absorb the enzyme. This paper presents a study with purified lignin samples to elucidate the role of unproductive enzyme adsorption. It appeared that lignin adsorbed cellulase quickly at 4 °C with adsorption equilibrium reached within 1h, similar to that observed for crystalline cellulose. Increasing temperature to 50 °C (typical hydrolytic reaction condition) facilitated the rate of cellulase adsorption on cellulose with a peak of adsorption reached at 0.25 h; however, adsorption on lignin was surprisingly slower and took over 12h to reach equilibrium, which was accompanied with a 10-fold increase in adsorption capacity. Despite the high adsorption capacity of lignin (which is comparable to that of cellulose) at 50 °C, the presence of added lignin imposed only minimal impact on the enzyme apparent activity, most likely due to the slow adsorption kinetics of lignin.

  9. Utilization of anaerobically treated distillery spent wash for production of cellulases under solid-state fermentation.

    PubMed

    Acharya, Bhavik K; Mohana, Sarayu; Jog, Rahul; Divecha, Jyoti; Madamwar, Datta

    2010-10-01

    Pollution caused by distillery spent wash on one hand has stimulated the need to develop new technologies to treat the waste and on the other, forced us to reevaluate the efficient utilization of its nutritive potential for production of various high value compounds. In this study, anaerobically treated distillery spent wash was used for the production of cellulases by Aspergillus ellipticus under solid-state fermentation using wheat straw as a substrate. The interactions between distillery effluent concentration, initial pH, moisture content and inoculum size were investigated and modeled using response surface methodology (RSM) involving Box-Behnken design (BBD). Under optimized conditions, filter paper activity, beta-glucosidase and endo-beta-1,4-glucanase activities were found to be 13.38, 26.68 and 130.92 U/g of substrate respectively. Characterization of endo-beta-1,4-glucanase and beta-glucosidase was done after partial purification by ammonium sulfate fractionation followed by desalting. The partially purified endo-beta-1,4-glucanase and beta-glucosidase showed maximum activity at 60 degrees C. Saccharification studies performed with different lignocellulosic substrates showed that wheat bran was most susceptible to enzymatic hydrolysis. The study suggests that anaerobically treated distillery spent wash can be used as a viable nutrient source for cellulase production under solid-state fermentation by A. ellipticus.

  10. Molecular cloning of glycoside hydrolase family 45 cellulase genes from brackish water clam Corbicula japonica.

    PubMed

    Sakamoto, Kentaro; Toyohara, Haruhiko

    2009-04-01

    We previously reported endogenous Glycoside Hydrolase Family (GHF) 9 beta-1,4-glucanase gene, CjCel9A, from common Japanese freshwater clam Corbicula japonica. Here we identified another endogenous beta-1,4-glucanase genes which belong to GHF45 (CjCel45A, CjCel45B). Both genes encode ORF of 627 bp corresponding to 208 amino acids. CjCel45A and CjCel45B are different in 5' and 3'-untranslated regions and six nucleotides in the ORF. CjCEL45 has only one GHF45 catalytic domain without any carbohydrate binding modules as is the case with other molluskan GHF45 enzymes. Phylogenetic analysis and genomic structure of CjCel45 gene implies that this gene is likely to be acquired from fungi by common ancestor of mollusks. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCel45A is likely to be expressed in the secretory cells in the digestive gland, suggesting that this cellulase is produced in the same site as CjCEL9A. CjCEL45A was successfully expressed in E. coli cells and zymographic analysis of the recombinant CjCEL45A showed that CjCEL45A is a functional beta-1,4-glucanase. The finding of multiple cellulase genes in C. japonica strongly supports our hypothesis that this species function as a cellulose decomposer in estuarine environments.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

    2015-07-21

    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.

  16. The isolation and functional identification on producing cellulase of Pseudomonas mendocina.

    PubMed

    Zhang, Jianfeng; Hou, Hongyan; Chen, Guang; Wang, Shusheng; Zhang, Jiejing

    2016-09-02

    The straw can be degraded efficiently into humus by powerful enzymes from microorganisms, resulting in the accelerated circulation of N,P,K and other effective elements in ecological system. We isolated a strain through screening the straw degradation strains from natural humic straw in the low temperature area in northeast of china, which can produce cellulase efficiently. The strain was identified as Pseudomonas mendocina by using morphological, physiological, biochemical test, and molecular biological test, with the functional clarification on producing cellulase for Pseudomonas mendocina for the first time. The enzyme force constant Km and the maximum reaction rate (Vmax) of the strain were 0.3261 g/L and 0.1525 mg/(min.L) through the enzyme activity detection, and the molecular weight of the enzyme produced by the strain were 42.4 kD and 20.4 kD based on SDS-PAGE. The effects of various ecological factors such as temperature, pH and nematodes on the enzyme produced by the strain in the micro ecosystem in plant roots were evaluated. The result showed that the optimum temperature was 28°C, and the best pH was 7.4∼7.8, the impact heavy metal was Pb(2+) and the enzyme activity and biomass of Pseudomonas mendocina increased the movement and predation of nematodes.

  17. 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

  18. Use of cellulases and recombinant cellulose binding domains for refining TCF kraft pulp.

    PubMed

    Cadena, Edith M; Chriac, A Iulia; Pastor, F I Javier; Diaz, Pilar; Vidal, Teresa; Torres, Antonio L

    2010-01-01

    The modular endoglucanase Cel9B from Paenibacillus barcinonensis is a highly efficient biocatalyst, which expedites pulp refining and reduces the associated energy costs as a result. In this work, we set out to identify the specific structural domain or domains responsible for the action of this enzyme on cellulose fibre surfaces with a view to facilitating the development of new cellulases for optimum biorefining. Using the recombinant enzymes GH9-CBD3c, Fn3-CBD3b, and CBD3b, which are truncated forms of Cel9B, allowed us to assess the individual effects of the catalytic, cellulose binding, and fibronectin-like domains of the enzyme on the refining of TCF kraft pulp from Eucalyptus globulus. Based on the physico-mechanical properties obtained, the truncated form containing the catalytic domain (GH9-CBD3c) has a strong effect on fibre morphology. Comparing its effect with that of the whole cellulase (Cel9B) revealed that the truncated enzyme contributes to increasing paper strength through improved tensile strength and burst strength and also that the truncated form is more effective than the whole enzyme in improving tear resistance. Therefore, the catalytic domain of Cel9B has biorefining action on pulp. Although cellulose binding domains (CBDs) are less efficient toward pulp refining, evidence obtained in this work suggests that CBD3b alters fibre surfaces and influences paper properties as a result.

  19. Structural and biochemical studies of GH family 12 cellulases: improved thermal stability, and ligand complexes.

    PubMed

    Sandgren, Mats; Ståhlberg, Jerry; Mitchinson, Colin

    2005-11-01

    In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.

  20. 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.

  1. 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.

  2. Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis.

    PubMed

    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

    2011-03-29

    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.

  3. 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

  4. Adsorption characteristics of cellulase and β-glucosidase on Avicel, pretreated sugarcane bagasse, and lignin.

    PubMed

    Machado, Daniele Longo; Moreira Neto, João; da Cruz Pradella, José Geraldo; Bonomi, Antonio; Rabelo, Sarita Cândida; da Costa, Aline Carvalho

    2015-01-01

    Although adsorption is an essential step in the enzymatic hydrolysis of lignocellulosic materials, literature reports controversial results in relation to the adsorption of the cellulolitic enzymes on different biomasses/pretreatments, which makes difficult the description of this phenomenon in hydrolysis mathematical models. In this work, the adsorption of these enzymes on Avicel and sugarcane bagasse pretreated by the hydrothermal bagasse (HB) and organosolv bagasse (OB) methods was evaluated. The results have shown no significant adsorption of β-glucosidase on Avicel or HB. Increasing solids concentration from 5% (w/v) to 10% (w/v) had no impact on the adsorption of cellulase on the different biomasses if stirring rates were high enough (>100 rpm for Avicel and >150 rpm for HB and OB). Adsorption equilibrium time was low for Avicel (10 Min) when compared with the lignocellulosic materials (120 Min). Adsorption isotherms determined at 4 and 50 °C have shown that for Avicel there was a decrease in the maximum adsorption capacity (Emax) with the temperature increase, whereas for HB increasing temperature increased Emax . Also, Emax increased with the content of lignin in the material. Adsorption studies of cellulase on lignin left after enzymatic digestion of HB show lower but significant adsorption capacity (Emax = 11.92 ± 0.76 mg/g).

  5. High activity cellulase production by recombinant Trichoderma reesei ZU-02 with the enhanced cellobiohydrolase production.

    PubMed

    Fang, Hao; Xia, Liming

    2013-09-01

    The cbh1 strong promoter was employed to over-express the cbh2 gene for enhancing cellobiohydrolase (CBH) production in Trichoderma reesei because CBH II component has higher specific activity than CBH I and is an important component in cellulase. The recombinant plasmid pCAMBIA1300-hph-PsCT containing strong expression cassette was constructed and transformed into T. reesei via optimized Agrobacterium-mediated transformation, producing 324 positive T. reesei transformants for the two steps of screening. Ten fast-growing T. reesei transformants were selected, amongst which C10 was found to have the highest filter paper activity 28.92±2.45 IU/mL, 4.3-fold higher than that of ZU-02, 6.71±0.79 IU/mL. C10 also has the highest cellobiohydrolase activity 122.44±7.42 U/mL, 5.4 times higher than that of ZU-02, 22.49±2.27 U/mL. The cellulase from C10 performed better (93.06±2.83%) than the one from ZU-02 in enzymatic hydrolysis because the exo-exo-synergism played a role.

  6. 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.

  7. The Relation Between Promoter Chromatin Status, Xyr1 and Cellulase Ex-pression in Trichoderma reesei.

    PubMed

    Mello-de-Sousa, Thiago M; Rassinger, Alice; Derntl, Christian; Poças-Fonseca, Marcio J; Mach, Robert L; Mach-Aigner, Astrid R

    2016-04-01

    The ascomycete Trichoderma reesei is used for the production of plant cell wall-degrading enzymes in industrial scale. The interplay of the transactivator Xyr1 and the repressor Cre1 mainly regulates the expression of these enzymes. During induc-ing conditions, such as in the presence of sophorose, the transcription of the two major cellulase-encoding genes, cbh1 and cbh2, is activated as well as the expression of xyr1. In the presence of D-glucose carbon catabolite repression mediated by Cre1 takes place and the expression of Xyr1 and the plant cell wall-degrading enzymes is down-regulated. In this study we compare the chromatin status of xyr1, cbh1, and cbh2 promoters in the wild-type strain and the Cre1-deficient strain Rut-C30. Chromatin rearrangement occurs in the xyr1 promoter during induction on sophorose. Chromatin opening and protein-DNA interactions in the xyr1 promoter were detected especially in a region located 0.9 kb upstream the translation start co-don, which bears several putative Cre1-binding sites and a CCAAT-box. Moreover, the xyr1 promoter is overall more acces-sible in a cre1-truncated background, no matter which carbon source is present. This makes the xyr1 regulatory sequence a good target for promoter engineering aiming at the enhancement of cellulase production.

  8. 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.

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

    PubMed

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

    2016-09-01

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

  10. 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

  11. Functional characterization of two M42 aminopeptidases erroneously annotated as cellulases.

    PubMed

    Dutoit, Raphaël; Brandt, Nathalie; Legrain, Christianne; Bauvois, Cédric

    2012-01-01

    Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome.

  12. Synergistic properties of cellulases from Clostridium cellulovorans in the presence of cellobiose.

    PubMed

    Yamamoto, Kosuke; Tamaru, Yutaka

    2016-03-01

    An anaerobic mesophile, Clostridium cellulovorans, produces a multienzyme complex called the cellulosome and actively degrades polysaccharides in the plant cell wall. C. cellulovorans also changes cellulosomal subunits to form highly active combinations dependent on the carbon substrate. A previous study reported on the synergistic effects of exoglucanase S (ExgS) and endoglucanase H (EngH) that are classified into the glycosyl hydrolase (GH) families 48, and 9, respectively. In this study, we investigated synergistic effects of ExgS and EngK, a GH9 cellulase different from EngH. In addition, since EngK was known to produce cellobiose as its main product, the inhibition on cellulase activity of EngK with cellobiose was examined. As a result, the effect of cellobiose inhibition on EngK coexistent with ExgS was found to be much lower than that with EngH. Thus, although EngH and EngK are in the same GH9 family, enzymatic activity in the presence of cellobiose was significantly different.

  13. 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.

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

    PubMed Central

    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

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

    PubMed

    Behera, Sudhanshu S; Ray, Ramesh C

    2016-05-01

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

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

    PubMed

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

    2011-11-01

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

  17. Intermediate range order in alkaline borate glasses

    NASA Astrophysics Data System (ADS)

    Crupi, C.; Carini, G.; Ruello, G.; D'Angelo, G.

    2016-03-01

    We describe the neutron diffraction patterns of a series of alkaline borate glasses at different metal oxide content. Strong differences are observed in the intermediate range order as a function of the specific alkaline ion and of its concentration. On these results, we propose that the first sharp diffraction peak arises from correlations of atoms of voids and show that the compositional variation of this peak intensity in alkaline borate glasses is due to changes in the distribution of void sizes within the three-dimensional network. We argue that our interpretation in terms of interstitial (empty and/or filled) voids, having different sizes, provides a general explanation for all anomalous behaviours revealed for the first sharp diffraction peak.

  18. Laser direct write of planar alkaline microbatteries

    NASA Astrophysics Data System (ADS)

    Arnold, C. B.; Kim, H.; Piqué, A.

    We are developing a laser engineering approach to fabricate and optimize alkaline microbatteries in planar geometries. The laser direct-write technique enables multicapability for adding, removing and processing material and provides the ability to pattern complicated structures needed for fabricating complete microbattery assemblies. In this paper, we demonstrate the production of planar zinc-silver oxide alkaline cells under ambient conditions. The microbattery cells exhibit 1.55-V open-circuit potentials, as expected for the battery chemistry, and show a flat discharge behavior under constant-current loads. High capacities of over 450 μAhcm-2 are obtained for 5-mm2 microbatteries.

  19. 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.

  20. 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.

  1. Effects of dietary cellulase and xylanase addition on digestion, rumen fermentation and methane emission in growing goats.

    PubMed

    Lu, Qi; Jiao, Jinzhen; Tang, Shaoxun; He, Zhixiong; Zhou, Chuanshe; Han, Xuefeng; Wang, Min; Kang, Jinhe; Odongo, N E; Tan, Zhiliang

    2015-01-01

    The objective of this study was to evaluate the effectiveness of supplementation of cellulase and xylanase to diets of growing goats to improve nutrient digestibility, utilisation of energy and mitigation of enteric methane emissions. The experiment was conducted in a 5 × 5 Latin square design using five goats with permanent rumen fistulae and five treatments consisted of two levels of cellulase crossed over with two levels of xylanase plus unsupplemented Control. The cellulase (243 U/g) derived from Neocallimastix patriciarum was added at 0.8 and 1.6 g/kg dry matter intake (DMI) and the xylanase (31,457 U/ml) derived from Aspergillus oryzae was fed at 1.4 and 2.2 ml/kg DMI. There were no differences in apparent digestibility of organic matter, neutral detergent fibre, acid detergent fibre and rumen fermentation parameters (i.e. ammonia-nitrogen [N], volatile fatty acids) among all treatments. Dietary cellulase and xylanase addition did not influence energy and N utilisation. But compared to xylanase addition at the higher dose, at the low xylanase dose the retained N, the availability of retained N and digested N were increased (p < 0.01). Moreover, enzyme addition did not affect the enteric methane emission and community diversity of ruminal methanogens. The present results indicated that previous in vitro findings were not confirmed in ruminant trials.

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

    PubMed

    Liu, Gang; Zhang, Jian; Bao, Jie

    2016-01-01

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

  3. Purification and characterization of cellulase from North Pacific krill (Euphausia pacifica). Analysis of cleavage specificity of the enzyme.

    PubMed

    Tsuji, Akihiko; Sato, Shiori; Kondo, Ayumi; Tominaga, Keiko; Yuasa, Keizo

    2012-01-01

    Krill are filter feeders that consume algae, plankton and detritus, indicating that krill possess an adequate cellulose digesting system. However, less is known about the enzymatic properties of crustacean cellulases compared to termite cellulases. In the present study, 48 kDa-cellulase was highly purified from krill (Euphausia pacifica) in an effort to determine the cleavage specificity of the enzyme. The most notable characteristic of the enzyme was its high activity against both lichenan and carboxymethyl cellulose. The enzyme hydrolyzed internal β-1,4 glycosidic bonds within lichenan as well as carboxymethyl cellulose to release oligosaccharides and glucose. The effects of pH and temperature on the activity and stability of both enzyme activities were almost identical. Cello-oligosaccharides with a degree of polymerization of 4-6 were hydrolyzed by the enzyme, and the same endo-products, cellotriose, cellobiose and glucose, were produced from these oligosaccharides. Neither cellotriose nor cellobiose was hydrolyzed by the enzyme. The enzyme digested filter paper and sea lettuce to produce cellobiose, cellotriose and glucose as major products. Although amino acid sequence homology of the enzyme with termite cellulases and the presence of oligosaccharides in the enzyme suggested that the enzyme is produced by krill itself, further analysis is necessary.

  4. Production of cellulases from Aspergillus niger NS-2 in solid state fermentation on agricultural and kitchen waste residues.

    PubMed

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

    2012-07-01

    Various agricultural and kitchen waste residues were assessed for their ability to support the production of a complete cellulase system by Aspergillus niger NS-2 in solid state fermentation. Untreated as well as acid and base-pretreated substrates including corn cobs, carrot peelings, composite, grass, leaves, orange peelings, pineapple peelings, potato peelings, rice husk, sugarcane bagasse, saw dust, wheat bran, wheat straw, simply moistened with water, were found to be well suited for the organism's growth, producing good amounts of cellulases after 96 h without the supplementation of additional nutritional sources. Yields of cellulases were higher in alkali treated substrates as compared to acid treated and untreated substrates except in wheat bran. Of all the substrates tested, wheat bran appeared to be the best suited substrate producing appreciable yields of CMCase, FPase and β-glucosidase at the levels of 310, 17 and 33 U/g dry substrate respectively. An evaluation of various environmental parameters demonstrated that appreciable levels of cellulases could be produced over a wide range of temperatures (20-50 °C) and pH levels (3.0-8.0) with a 1:1.5 to 1:1.75 substrate to moisture ratio.

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

    PubMed Central

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

    2014-01-01

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

  6. The Alkaline Diet: Is There Evidence That an Alkaline pH Diet Benefits Health?

    PubMed Central

    Schwalfenberg, Gerry K.

    2012-01-01

    This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy. Many books written in the lay literature on the alkaline diet were also reviewed and evaluated in light of the published medical literature. There may be some value in considering an alkaline diet in reducing morbidity and mortality from chronic diseases and further studies are warranted in this area of medicine. PMID:22013455

  7. The alkaline diet: is there evidence that an alkaline pH diet benefits health?

    PubMed

    Schwalfenberg, Gerry K

    2012-01-01

    This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy. Many books written in the lay literature on the alkaline diet were also reviewed and evaluated in light of the published medical literature. There may be some value in considering an alkaline diet in reducing morbidity and mortality from chronic diseases and further studies are warranted in this area of medicine.

  8. Negative Electrode For An Alkaline Cell

    DOEpatents

    Coco, Isabelle; Cocciantelli, Jean-Michel; Villenave, Jean-Jacques

    1998-07-14

    The present invention concerns a negative electrode for an alkaline cell, comprising a current collector supporting a paste containing an electrochemically active material and a binder, characterized in that said binder is a polymer containing hydrophilic and hydrophobic groups, said polymer being selected from an acrylic homopolymer, copolymer and terpolymer, an unsaturated organic acid copolymer and an unsaturated acid anhydride copolymer.

  9. Alkaline electrochemical cells and method of making

    NASA Technical Reports Server (NTRS)

    Hoyt, H. E.; Pfluger, H. L. (Inventor)

    1970-01-01

    Equilibrated cellulose ether membranes of increased electrolytic conductivity for use as separators in concentrated alkaline electrochemical cells are investigated. The method of making such membranes by equilibration to the degree desired in an aqueous alkali solution mantained at a temperature below about 10 C is described.

  10. Kinetics of the alkaline hydrolysis of nitrocellulose.

    PubMed

    Christodoulatos, C; Su, T L; Koutsospyros, A

    2001-01-01

    Cellulose nitrate (nitrocellulose) is an explosive solid substance used in large quantities in various formulations of rocket and gun propellants. Safe destruction of nitrocellulose can be achieved by alkaline hydrolysis, which converts it to biodegradable products that can then be treated by conventional biological processes. The kinetics of the alkaline hydrolysis of munitions-grade nitrocellulose in sodium hydroxide solutions were investigated in completely mixed batch reactors. Experiments were conducted using solutions of alkaline strength ranging from 0.1 to 15% by mass and temperatures in the range of 30 to 90 degrees C. Regression analysis of the kinetic data revealed that alkaline hydrolysis of nitrocellulose is of the order 1.0 and 1.5 with respect to nitrocellulose and hydroxide concentration, respectively. The activation energy of the hydrolysis reaction was found to be 100.9 kJ/mol with a preexponential Arrhenius constant of 4.73 x 10(13). Nitrite and nitrate, in a 3:1 ratio, were the primary nitrogen species present in the posthydrolysis solution. The kinetic information is pertinent to the development and optimization of nitrocellulose chemical-biological treatment systems.

  11. MERCURIC CHLORIDE CAPTURE BY ALKALINE SORBENTS

    EPA Science Inventory

    The paper gives results of bench-scale mechanistic studies of mercury/sorbent reactions that showed that mercuric chloride (HgC12) is readily adsorbed by alkaline sorbents, which may offers a less expensive alternative to the use of activated carbons. A laboratory-scale, fixed-b...

  12. ISSUES WITH ALKALINE TREATMENT OF SLUDGE

    EPA Science Inventory

    This presentation begins with a discussion of the use of lime and other alkaline materials from the very earliest times to the present for killing bacteria, viruses and parasites and for controlling odors in wastewaters and sludge. It answers the question "How did EPA arrive at i...

  13. Cellulase variants

    DOEpatents

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

    2015-07-14

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

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

    PubMed

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

    2017-01-27

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2015-07-10

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

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

    PubMed

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

    2014-09-01

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

  18. Alkaline earth metal catalysts for asymmetric reactions.

    PubMed

    Kobayashi, Shū; Yamashita, Yasuhiro

    2011-01-18

    The group 2 alkaline earth metals calcium (Ca), strontium (Sr), and barium (Ba) are among the most common elements on Earth, abundant in both the sea and the Earth's crust. Although they are familiar in our daily lives, their application to organic synthesis has, so far, been limited. Some particularly useful properties of these elements include (i) low electronegativity, (ii) a stable oxidation state of +2, meaning that they can potentially form two covalent bonds with anions, and (iii) the ability to occupy a variety of coordination sites due to their large ionic radius. Furthermore, the alkaline earth metals, found between the group 1 and group 3 elements, show mild but significant Lewis acidity, which can be harnessed to control coordinative molecules via a Lewis acid-base interaction. Taken together, these characteristics make the metals Ca, Sr, and Ba very promising components of highly functionalized acid-base catalysts. In this Account, we describe the development of chiral alkaline earth metal catalysts for asymmetric carbon-carbon bond-forming reactions. Recently prepared chiral alkaline earth metal complexes have shown high diastereo- and enantioselectivities in fundamental and important chemical transformations. We chose chiral bisoxazoline (Box) derivatives bearing a methylene tether as a ligand for chiral modification. These molecules are very useful because they can covalently coordinate to alkaline earth metals in a bidentate fashion through deprotonation of the tether portion. It was found that chiral calcium-Box complexes could successfully promote catalytic asymmetric 1,4-addition and [3 + 2] cycloaddition reactions with high diastereo- and enantioselectivities. Both the calcium-Box complexes and chiral strontium-bis-sulfonamide and chiral barium-BINOLate complexes could catalyze asymmetric 1,4-addition reactions with high enantioselectivities. Furthermore, we designed a calcium-neutral coordinative ligand complex as a new type of chiral alkaline

  19. Informational suppression to investigate structural functional and evolutionary aspects of the Erwinia chrysanthemi cellulase EGZ.

    PubMed

    Bortoli-German, I; Haiech, J; Chippaux, M; Barras, F

    1995-02-10

    The cellulase EGZ produced by the plant pathogen Erwinia chrysanthemi belongs to family 5 of the beta-glycohydrolases (also referred to as cellulase family A), which contains over 40 members from Gram-negative and Gram-positive bacteria and fungi. Amber mutations were introduced into 16 codons of the celZ gene encoding EGZ. Targeted residues included: (1) two Glu, two His and one Arg residue, strictly conserved throughout family 5; (2) one Arg and one His residue conserved in sub-family 5-2; and (3) one His and six Arg residues not conserved at all. Each amber allele was introduced into 13 Escherichia coli strains each carrying a different suppressor tRNA that inserts an amino acid at the mutated position. In vivo stability of the mutated forms of EGZ and their cellulase activity were analysed as well as suppression efficiency. For some positions of particular interest, missense mutations were introduced into the celZ gene either to confirm the effect of the suppressor-mediated amino acid substitution or to broaden the spectrum of mutations available. The substitution patterns of the two Glu positions were interpretable in the light of the stereospecificity of the reaction catalysed by EGZ: Glu133 and Glu220 are proposed to act as a proton donor and as a nucleophile, respectively, forming the glycosyl-enzyme intermediate. Substitution at His-occupied positions, including two non-conserved positions, yielded proteins affected in their catalytic activity but not their in vivo stability. In particular, evidence was obtained for His at position 98 to be involved in interactions with the substrate. The view that Arg residues are important in stabilizing proteins was supported by the identification of three Arg residues, whose substitution yielded thermosensitive forms of EGZ. In addition, Pro substitutions of any of the six Arg residues altered protein stability in vivo but the substitutions scored almost neutral for activity. Five positions, predicted to be within

  20. Cellulase production from a new strain Acremonium strictum isolated from the Brazilian Biome using different substrates.

    PubMed

    Goldbeck, Rosana; Ramos, Mayla M; Pereira, Gonçalo A G; Maugeri-Filho, Francisco

    2013-01-01

    The objective of the present study was to evaluate the production of cellulolytic enzymes by Acremonium strictum isolated from Brazilian Biome using different substrates. Fermentations were initially carried out using commercial substrates, including microcrystalline cellulose (AVICEL® and SERVACEL®) and carboxymethylcellulose (CMC). This was followed by fermentations performed using lignocellulosic biomass: sugarcane bagasse pretreated at different intensities. The fermentations were carried out in shakers at 150 rpm, 30 °C for 240 h. Four enzyme activities were determined: CMCase, FPase, cellobiase and β-glucosidase. Among the substrates used, results showed that the sugarcane bagasse submitted to mild pretreatment was that which induced the microorganism to produce greater cellulolytic activities. This substrate was employed in the optimization study of cellulase production by A. strictum.

  1. Ethanol production from oil palm trunks treated with aqueous ammonia and cellulase.

    PubMed

    Jung, Young Hoon; Kim, In Jung; Kim, Jae Jin; Oh, Kyeong Keun; Han, Jong-In; Choi, In-Geol; Kim, Kyoung Heon

    2011-08-01

    Oil palm trunks are a possible lignocellulosic source for ethanol production. Low enzymatic digestibility of this type of material (11.9% of the theoretical glucose yield) makes pretreatment necessary. An enzymatic digestibility of 95.4% with insoluble solids recovery of 49.8% was achieved after soaking shredded oil palm trunks in ammonia under optimum conditions (80°C, 1:12 solid-to-liquid ratio, 8h and 7% (w/w) ammonia solution). Treatment with 60 FPU of commercial cellulase (Accellerase 1000) per gram of glucan and fermentation with Saccharomyces cerevisiae D(5)A resulted in an ethanol concentration of 13.3g/L and an ethanol yield of 78.3% (based on the theoretical maximum) after 96 h. These results indicate that oil palm trunks are a biomass feedstock that can be used for bioethanol production.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

    Chylenski, Piotr; Felby, Claus; Østergaard Haven, Mai; Gama, Miguel; Selig, Michael J

    2012-08-01

    Comparative studies between commercial Trichoderma reesei cellulase preparations show that, depending on the preparation and loading, total protein precipitation can be as high as 30 % under standard hydrolysis conditions used for lignocellulosic materials. ATR-IR and SDS-PAGE data verify precipitates are protein-based and contain key cell wall hydrolyzing enzymes. Precipitation increased considerably with incubation temperature; roughly 50-150 % increase from 40 to 50 °C and 800 % greater at 60 °C. All of the reported protein losses translated into significant, and often drastic, losses in activity on related 4-nitrophenyl substrates. In addition, supplementation with the non-ionic surfactant PEG 6,000 decreased precipitation up to 80 % in 24 h precipitation levels. Protein precipitation is potentially substantial during enzymatic hydrolysis of lignocelluloses and should be accounted for during lignocellulose conversion process design, particularly when enzyme recycling is considered.

  4. Metabolic engineering of inducer formation for cellulase and hemicellulase gene expression in Trichoderma reesei.

    PubMed

    Seiboth, Bernhard; Herold, Silvia; Kubicek, Christian P

    2012-01-01

    The filamentous fungus T. reeseiis today a paradigm for the commercial scale production of different plant cell wall degrading enzymes mainly cellulases and hemicellulases. Its enzymes have a long history of safe use in industry and well established applications are found within the pulp, paper, food, feed or textile processing industries. However, when these enzymes are to be used for the saccharification of cellulosic plant biomass to simple sugars which can be further converted to biofuels or other biorefinery products, and thus compete with chemicals produced from fossil sources, additional efforts are needed to reduce costs and maximize yield and efficiency of the produced enzyme mixtures. One approach to this end is the use of genetic engineering to manipulate the biochemical and regulatory pathways that operate during enzyme production and control enzyme yield. This review aims at a description of the state of art in this area.

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

    PubMed

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

    2016-06-10

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

  6. Fractionation of cellulase and fermentation inhibitors from steam pretreated mixed hardwood.

    PubMed

    Kim, Youngmi; Kreke, Thomas; Hendrickson, Rick; Parenti, Josh; Ladisch, Michael R

    2013-05-01

    The purpose of liquid hot water and steam pretreatment of wood is to fractionate hemicelluloses, partially solubilize lignin, and enhance enzyme hydrolysis of cellulose. The pretreatment also solubilizes sugar oligomers, lignin-derived phenolic compounds, acetic acid, and furan derivatives that inhibit cellulase enzymes and/or impede fermentation of hydrolysates by yeasts. This work extends knowledge of the relative contribution of identified inhibitors, and the effect of temperature on their release when pretreated materials are washed and filtered with hot water. Dramatic yield improvements occur when polymeric or activated carbon adsorbs and removes inhibitors. By desorbing, recovering, and characterizing adsorbed molecules we found phenolic compounds were strong inhibitors of enzyme hydrolysis and fermentation of concentrated filtrates by Saccharomyces cerevisiae wine yeast NRRL Y-1536 or xylose fermenting yeast 424A (LNH-ST). These data show that separation of inhibitors from pretreatment liquid will be important in achieving maximal enzyme activity and efficient fermentations.

  7. Effect of repeated applications of buprofezin and acephate on soil cellulases, amylase, and invertase.

    PubMed

    Raju, M Naga; Venkateswarlu, K

    2014-10-01

    The impact of repeated applications of buprofezin and acephate, at concentrations ranging from 0.25 to 1.0 kg ha(-1), on activities of cellulases, amylase, and invertase in unamended and nitrogen, phosphorous, and potassium (NPK) fertilizer-amended soil planted with cotton was studied. The nontarget effect of selected insecticides, when applied once, twice, or thrice on soil enzyme activities, was dose-dependent; the activities decreased with increasing concentrations of insecticides. However, there was a rapid decline in activities of enzymes after three repeated applications of insecticides in unamended or NPK-amended soil. Our data clearly suggest that insecticides must be applied judiciously in pest management in order to protect the enzymes largely implicated in soil fertility.

  8. Cellulase production from spent sulfite liquor and paper-mill waste fiber

    SciTech Connect

    Qu Yinbo; Zhao Xin; Gao Peiji; Wang Zunong

    1991-12-31

    Since a high proportion of the overall cost of the conversion of cellulosics to useful products is the expense of cellulose production (1), it is desirable to develop new processes for producing large amounts of cellulase inexpensively. So far, most of the research work on cellulose production has been carried out using milled cellulose powder and inorganic salts as substrates, which significantly increases the cost of enzyme production. In order to reduce the cost of raw materials, we tried to develop from industrial wastes a new medium for the production of cellulose. In this report, we describe a simple method by which an all-waste medium, which was composed of spent ammonium sulfite liquor and cellulosic waste of a paper mill, and a catabolite derepression mutant of Penicillium decumbens were used to produce the enzyme efficiently.

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

    PubMed Central

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

    2013-01-01

    The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v) sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v) soy bran; 0.1% (w/v) wheat bran; and a solution of salts. The highest filter paper activity (FPA) (1.95  ±  0.04 IU·mL−1) was obtained on the seventh day in the medium containing 0.5% (w/v) sorbitol and 0.5% (w/v) cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day (9.99 ± 0.75 IU·mL−1) in the medium containing 0.75% (w/v) sorbitol and 0.75% (w/v) cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v) sorbitol and 0.25% (w/v) cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems. PMID:24058733

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

    PubMed

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

    2010-05-24

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

  11. Process optimization and production kinetics for cellulase production by Trichoderma viride VKF3.

    PubMed

    Nathan, Vinod Kumar; Esther Rani, Mary; Rathinasamy, Gunaseeli; Dhiraviam, Kannan Narayanan; Jayavel, Sridhar

    2014-01-01

    Microbial cellulases are the enzymes widely studied due to their enormous applications in biochemical industry. Among 12 fungal isolates isolated from mangrove plant debris and soil sample collected from Valanthakad Mangroves, Kerala, India, 3 of them were found to exhibit cellulolytic activity. Among them, the most potent isolate which exhibited maximum cellulolytic activity was identified as Trichoderma viride VKF3 [Gene bank accession number- JX683684.1] based on colony morphology, microscopic observation and molecular centeracterization using D1/D2 region amplification. The isolate T. viride VKF3 was found to be non-phytopathogenic against the selected plants. Neighbour joining tree depicted its least divergence rate from the root taxon HM466686.1. T. viride VKF3 was grown under dynamic carbon, nitrogen sources, pH and temperature of the medium to draw out the optimum conditions for cellulase production. Protein stability kinetics and biomass production was also studied upto 11(th) day of incubation. It was evident from the study, that dextrose and beef extract could be used as major carbon and nitrogen sources in submerged fermentation at pH 9.0 and incubation temperature of 25°C to get maximum CMCase yield. Optimum enzyme recovery period was identified between 5(th) to 9(th) days of incubation beyond which the enzyme activity was reduced. By comparing two fermentation methods, submerged fermentation was found to be the best for maximum enzyme production. But utilization of substrates like sugarcane bagasse and cassava starch waste in the SSF offers a better scope in biodegradation of solid waste contributing to solid waste management.

  12. Cellulase system of a free-living, mesophilic clostridium (strain C7).

    PubMed Central

    Cavedon, K; Leschine, S B; Canale-Parola, E

    1990-01-01

    The enzymatic activity responsible for crystalline cellulose degradation (Avicelase activity) by a mesophilic clostridium (strain C7) was present in culture supernatant fluid but was not detected in significant amounts in association with whole cells or in disrupted cells. Cells of the mesophilic clostridium lacked cellulosome clusters on their surface and did not adhere to cellulose fibers. The extracellular cellulase system of the mesophilic clostridium was fractionated by Sephracryl S-300 gel filtration, and the fractions were assayed for Avicelase and carboxymethylcellulase activities. The Avicelase activity coincided with an A280 peak that eluted in the 700,000-Mr region. Nondenaturing polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 700,000-Mr fractions showed that Avicelase was present as a multiprotein aggregate that lost the ability to hydrolyze crystalline cellulose when partially dissociated by sodium dodecyl sulfate treatment. Proteins resulting from the partial dissociation of the aggregate retained carboxymethylcellulase activity. An Avicelase-deficient mutant of strain C7 (strain LS), which was not capable of degrading crystalline cellulose, lacked the Avicelase-active 700,000-Mr peak. The results indicated that an extracellular 700,000-Mr multiprotein complex, consisting of at least 15 proteins, is utilized by the mesophilic clostridium for the hydrolysis of crystalline cellulose. At least six different endo-1,4-beta-glucanases may be part of the cellulase system of strain C7. Sephacryl S-300 column fractions, corresponding to an A280 peak in the 130,000-Mr region, contained carboxymethylcellulase-active proteins that may serve as precursors for the assembly of the Avicelase-active complex by the mesophilic clostridium. Images PMID:2376559

  13. Aspergillus niger β-Glucosidase Has a Cellulase-like Tadpole Molecular Shape

    PubMed Central

    Lima, Marisa A.; Oliveira-Neto, Mario; Kadowaki, Marco Antonio S.; Rosseto, Flavio R.; Prates, Erica T.; Squina, Fabio M.; Leme, Adriana F. P.; Skaf, Munir S.; Polikarpov, Igor

    2013-01-01

    Aspergillus niger is known to secrete large amounts of β-glucosidases, which have a variety of biotechnological and industrial applications. Here, we purified an A. niger β-glucosidase (AnBgl1) and conducted its biochemical and biophysical analyses. Purified enzyme with an apparent molecular mass of 116 kDa forms monomers in solution as judged by native gel electrophoresis and has a pI value of 4.55, as found for most of the fungi of β-glucosidases. Surprisingly, the small angle x-ray experiments reveal that AnBgl1 has a tadpole-like structure, with the N-terminal catalytic domain and C-terminal fibronectin III-like domain (FnIII) connected by the long linker peptide (∼100 amino acid residues) in an extended conformation. This molecular organization resembles the one adopted by other cellulases (such as cellobiohydrolases, for example) that frequently contain a catalytic domain linked to the cellulose-binding module that mediates their binding to insoluble and polymeric cellulose. The reasons why AnBgl1, which acts on the small soluble substrates, has a tadpole molecular shape are not entirely clear. However, our enzyme pulldown assays with different polymeric substrates suggest that AnBgl1 has little or no capacity to bind to and to adsorb cellulose, xylan, and starch, but it has high affinity to lignin. Molecular dynamics simulations suggested that clusters of residues located in the C-terminal FnIII domain interact strongly with lignin fragments. The simulations showed that numerous arginine residues scattered throughout the FnIII surface play an important role in the interaction with lignin by means of cation-π stacking with the lignin aromatic rings. These results indicate that the C-terminal FnIII domain could be operational for immobilization of the enzyme on the cell wall and for the prevention of unproductive binding of cellulase to the biomass lignin. PMID:24064212

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

    PubMed Central

    2010-01-01

    Although measurements of crystallinity index (CI) have a long history, it has been found that CI varies significantly depending on the choice of measurement method. In this study, four different techniques incorporating X-ray diffraction and solid-state 13C nuclear magnetic resonance (NMR) were compared using eight different cellulose preparations. We found that the simplest method, which is also the most widely used, and which involves measurement of just two heights in the X-ray diffractogram, produced significantly higher crystallinity values than did the other methods. Data in the literature for the cellulose preparation used (Avicel PH-101) support this observation. We believe that the alternative X-ray diffraction (XRD) and NMR methods presented here, which consider the contributions from amorphous and crystalline cellulose to the entire XRD and NMR spectra, provide a more accurate measure of the crystallinity of cellulose. Although celluloses having a high amorphous content are usually more easily digested by enzymes, it is unclear, based on studies published in the literature, whether CI actually provides a clear indication of the digestibility of a cellulose sample. Cellulose accessibility should be affected by crystallinity, but is also likely to be affected by several other parameters, such as lignin/hemicellulose contents and distribution, porosity, and particle size. Given the methodological dependency of cellulose CI values and the complex nature of cellulase interactions with amorphous and crystalline celluloses, we caution against trying to correlate relatively small changes in CI with changes in cellulose digestibility. In addition, the prediction of cellulase performance based on low levels of cellulose conversion may not include sufficient digestion of the crystalline component to be meaningful. PMID:20497524

  15. Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

    PubMed Central

    Su, Xiaoyun; Mackie, Roderick I.

    2012-01-01

    Thermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses of Caldicellulosiruptor bescii Cel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins of C. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1 ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparent kcat of the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in the Km led to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide. PMID:22247178

  16. Adenosine 3', 5'-cyclic monophosphate levels in Thermomonospora curvata during cellulase biosynthesis

    SciTech Connect

    Fennington, G.; Neubauer, D.; Stutzenberger, F.

    1983-01-01

    The enzymatic degradation of cellulose requires the synergistic activity of at least three enzymes: exo-beta-1,4-glucanase (EC3.2.1.91), endo-beta-1,4-glucanase (EC3.2.1.4), and beta-glucosidase (EC3.2.1.21). Despite extensive studies on a variety of cellulolytic bacteria and fungi, the mechanism(s) regulating the biosynthesis of this inducible catabolic enzyme complex remains unknown. The intracellular concentrations of cyclic nucleotides such as adenosine 3',5'-cyclic monophosphate (cAMP) have been shown to play a major role in mediating catabolite repression of enzyme biosynthesis. The cAMP acts through a cAMP receptor protein (termed CRP or CAP) which is a dimer having two identical subunits each capable of binding one molecule of cAMP. The N-terminal domain of the CRP binds the cAMP while the C-terminal domain binds to DNA at the promotor region of a cAMP-dependent operon and stimulates transcription by promoting the formation of a preinitiation complex between RNA polymerase and the DNA. Intracellular cAMP levels in E. coli (the prototype organism for such studies) are influenced by the type and availability of carbon source used for growth. High intracellular cAMP levels should lead to higher concentrations of cAMP-CRP complexes which should increase the transcription rates for cAMP-dependent operons (such as the lac operon of beta-galactosidase) and indeed the differential rate of beta-galactosidase biosynthesis correlates to intracellular cAMP levels. In the case of cellulase, catabolite repression by glucose or other readily metabolizable compounds closely controls production in an apparently similar manner and therefore a correlation may exist between enzyme biosynthesis and intracellular cAMP levels. This communication describes the fluctuation in cAMP levels during cellulase induction and repression in the thermophilic actinomycete, Thermomonospora curvata.

  17. The sulphate-reduction alkalinity pump tested

    NASA Astrophysics Data System (ADS)

    Meister, Patrick; Petrishcheva, Elena

    2016-04-01

    Carbonate precipitation has been suggested to be induced by alkalinity increase during sulphate reduction under anoxic conditions. This mechanism may explain the formation of carbonate deposits in shallow marine environments, either within a redox stratified sediment inhabited by phototrophic microbial mats or in shallow water within the photic zone where sulphidic water is upwelling onto the shelf. The alkalinity pump may work as long as the sulphide is not reoxidized to sulphate, a process that would acidify the surrounding. The alkalinity effect of sulphate reduction was recently tested by Aloisi (2008) for microbial mats using a model approach. He found that sulphate reduction does not significantly increase or even decrease carbonate saturation and is unlikely to have played a significant role through Earth history. The model considers many environmental factors, including the effect of carbonate precipitation itself on the carbonate equilbrium and on the alkalinity. We used a modified version of Aloisi's (2008) model to simulate the saturation states of aragonite, calcite and dolomite without the effects of carbonate precipitation. This is necessary to evaluate the effect of microbial metabolisms exclusively on carbonate saturation, since carbonate precipitation is only the consequence, but not the cause of oversaturation. First results show that the saturation state is increased in the zone of phototrophic CO2 uptake. In contrast, the saturation state is strongly decreased in the zone where dissolved oxygen overlaps with dissolved sulphide. Aerobic sulphide oxidation consumes most of the HS- and dissipates most of the alkalinity produced in the sulphate reduction zone below. Hence, our results are consistent with the findings of Aloisi (2008), and they even more clearly show that sulphate reduction does not induce carbonate precipitation nor contributes to carbonate precipitation in combination with phototrophic CO2 uptake. The alkalinity effect of sulphate

  18. [Leucocyte alkaline phosphatase in normal and pathological pregnancy (author's transl)].

    PubMed

    Stark, K H; Zaki, I; Sobolewski, K

    1981-01-01

    The activities of leucocyte alkaline phosphatase were determined in 511 patients with normal and pathological pregnancy. Mean values were compared and the enzyme followed up, and the conclusion was drawn that leucocyte alkaline phosphatase was no safe indicator of foetal condition. No direct relationship were found to exist between leucocyte alkaline phosphatase, total oestrogens, HSAP, HLAP, HPL, and oxytocinase.

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

    PubMed

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

    2017-02-01

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

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

    PubMed

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

    2016-12-01

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

  1. Surfactant-enhanced alkaline flooding field project

    SciTech Connect

    French, T.R.

    1991-10-01

    The Tucker sand of Helper (KS) field is a candidate for surfactant-enhanced alkaline flooding. The geology of the Helper site is typical of many DOE Class I reservoirs. The Tucker sand of Helper field was deposited in a fluvial dominated deltaic environment. Helper oil can be mobilized with either chemical system 2 or chemical system 3, as described in this report. Oil fields in the Gulf Coast region are also good candidates for surfactant-enhanced alkaline flooding. The results from laboratory tests conducted in Berea sandstone cores with oil brine from Helper (KS) field are encouraging. The crude oil is viscous and non-acidic and, yet, was mobilized by the chemical formulations described in this report. Significant amounts of the oil were mobilized under simulated reservoir conditions. The results in Berea sandstone cores were encouraging and should be verified by tests with field core. Consumption of alkali, measured with field core, was very low. Surfactant loss appeared to be acceptable. Despite the good potential for mobilization of Helper oil, certain reservoir characteristics such as low permeability, compartmentalization, and shallow depth place constraints on applications of any chemical system in the Tucker sand. These constraints are typical of many DOE Class I reservoirs. Although Hepler field is not a perfect reservoir in which to apply surfactant- enhanced alkaline flooding, Hepler oil is particularly amenable to mobilization by surfactant-enhanced alkaline systems. A field test is recommended, dependent upon final evaluation of well logs and cores from the proposed pilot area. 14 refs., 21 figs., 10 tabs.

  2. The alkaline earth intercalates of molybdenum disulfide

    NASA Technical Reports Server (NTRS)

    Somoano, R. B.; Hadek, V.; Rembaum, A.; Samson, S.; Woollam, J. A.

    1975-01-01

    Molybdenum disulfide has been intercalated with calcium and strontium by means of the liquid ammonia technique. Chemical, X-ray, and superconductivity data are presented. The X-ray data reveal a lowering of crystal symmetry and increase of complexity of the structure upon intercalation with the alkaline earth metals. The Ca and Sr intercalates start to superconduct at 4 and 5.6 K, respectively, and show considerable anisotropy regarding the critical magnetic field.

  3. Alkaline earth cation extraction from acid solution

    DOEpatents

    Dietz, Mark; Horwitz, E. Philip

    2003-01-01

    An extractant medium for extracting alkaline earth cations from an aqueous acidic sample solution is described as are a method and apparatus for using the same. The separation medium is free of diluent, free-flowing and particulate, and comprises a Crown ether that is a 4,4'(5')[C.sub.4 -C.sub.8 -alkylcyclohexano]18-Crown-6 dispersed on an inert substrate material.

  4. Oxidation catalysts on alkaline earth supports

    DOEpatents

    Mohajeri, Nahid

    2017-03-21

    An oxidation catalyst includes a support including particles of an alkaline earth salt, and first particles including a palladium compound on the support. The oxidation catalyst can also include precious metal group (PMG) metal particles in addition to the first particles intermixed together on the support. A gas permeable polymer that provides a continuous phase can completely encapsulate the particles and the support. The oxidation catalyst may be used as a gas sensor, where the first particles are chemochromic particles.

  5. Inhibition of Alkaline Phosphatase by Several Diuretics

    DTIC Science & Technology

    1980-01-01

    August 20th, 1979) . . Summary , . Acetazolamide, furosemide, ethacrynic acid and chlorothiazide, diuretics of considerable structural diversity, inhibit...Ki is calculated to be 8.4, 7.0, 2.8 and 0.1 mmol/l for acetazolamide, furosemide, ethacrynic acid and chlorothiazide, respectively. Chlorothiazide...is a much more potent inhibitor of alkaline phos- phatase than the other three diuretics. The combination of ethacrynic acid and cysteine, itself an

  6. Alkaline flooding for enhanced oil recovery

    SciTech Connect

    Gittler, W.E.

    1983-09-01

    There are over 12 active projects of varying size using one of 3 major types of alkaline agents. These include sodium silicate, caustic soda, and soda ash. Among the largest pilots currently is the THUMS project in the Wilmington field, California. Plans called for the injection of a 4% weight concentration of sodium orthosilicate over a 60% PV. Through the first 3 yr, over 27 million bbl of chemicals have been injected. Gulf Oil is operating several alkaline floods, one of which is located off shore in the Quarantine Bay field, Louisiana. In this pilot, sodium hydroxide in a weight concentration of 5 to 12% is being injected. Belco Petroleum Corp. has reported that their pilot operating in the Isenhour Unit in Wyoming is using a .5% weight concentration of soda ash in conjunction with a polymer. Other uses for alkaline agents in chemical flooding include the use of silicate as a preflush or sacrificial agent in micellar/polymer and surfactant recovery systems. In addition, caustic has been tested in the surface-mixed caustic emulsion process while orthosilicate has been tested in a recovery method known as mobility-controlled caustic floods.

  7. Alkaline phosphatase of Physarum polycephalum is insoluble.

    PubMed

    Furuhashi, Kiyoshi

    2008-02-01

    The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg(2+)-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.

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

    PubMed

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

    2014-11-01

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

  9. Mesozoic mafic alkaline magmatism of southern Scandinavia

    NASA Astrophysics Data System (ADS)

    Tappe, Sebastian

    2004-11-01

    More than 100 volcanic necks in central Scania (southern Sweden) are the product of Jurassic continental rift-related mafic alkaline magmatism at the southwest margin of the Baltic Shield. They are mainly basanites, with rarer melanephelinites. Both rock groups display overlapping primitive Mg-numbers, Cr and Ni contents, steep chondrite-normalized rare earth element patterns (LaN /YbN = 17 27) and an overall enrichment in incompatible elements. However, the melanephelinites are more alkaline and have stronger high field strength element enrichment than the basanites. The existence of distinct primary magmas is also indicated by heterogeneity in highly incompatible element ratios (e.g. Zr/Nb, La/Nb). Trace element modelling indicates that the magmas were generated by comparably low degrees of melting of a heterogeneous mantle source. Such a source can best be explained by a metasomatic overprint of the mantle lithosphere by percolating evolved melts. The former existence of such alkaline trace element-enriched melts can be demonstrated by inversion of the trace element content of green-core clinopyroxenes and anorthoclase which occur as xenocrysts in the melanephelinites and are interpreted as being derived from crystallization of evolved mantle melts. Jurassic magmatic activity in Scania was coeval with the generation of nephelinites in the nearby Egersund Basin (Norwegian North Sea). Both Scanian and North Sea alkaline magmas share similar trace element characteristics. Mantle enrichment processes at the southwest margin of the Baltic Shield and the North Sea Basin generated trace element signatures similar to those of ocean island basalts (e.g. low Zr/Nb and La/Nb) but there are no indications of plume activity during the Mesozoic in this area. On the contrary, the short duration of rifting, absence of extensive lithospheric thinning, and low magma volumes argue against a Mesozoic mantle plume. It seems likely that the metasomatic imprint resulted from the

  10. Alkaline and ultrasound assisted alkaline pretreatment for intensification of delignification process from sustainable raw-material.

    PubMed

    Subhedar, Preeti B; Gogate, Parag R

    2014-01-01

    Alkaline and ultrasound-assisted alkaline pretreatment under mild operating conditions have been investigated for intensification of delignification. The effect of NaOH concentration, biomass loading, temperature, ultrasonic power and duty cycle on the delignification has been studied. Most favorable conditions for only alkaline pretreatment were alkali concentration of 1.75 N, solid loading of 0.8% (w/v), temperature of 353 K and pretreatment time of 6 h and under these conditions, 40.2% delignification was obtained. In case of ultrasound-assisted alkaline approach, most favorable conditions obtained were alkali concentration of 1N, paper loading of 0.5% (w/v), sonication power of 100 W, duty cycle of 80% and pretreatment time of 70 min and the delignification obtained in ultrasound-assisted alkaline approach under these conditions was 80%. The material samples were characterized by FTIR, SEM, XRD and TGA technique. The lignin was recovered from solution by precipitation method and was characterized by FTIR, GPC and TGA technique.

  11. Alkaline solution/binder ratio as a determining factor in the alkaline activation of aluminosilicates

    SciTech Connect

    Ruiz-Santaquiteria, C.; Fernandez-Jimenez, A.; Palomo, A.

    2012-09-15

    This study investigates the effect of the alkaline solution/binder (S/B) ratio on the composition and nanostructure of the reaction products generated in the alkaline activation of aluminosilicates. The experiments used two mixtures of fly ash and dehydroxylated white clay and for each of these, varying proportions of the solution components. The alkali activator was an 8 M NaOH solution (with and without sodium silicate) used at three S/B ratios: 0.50, 0.75 and 1.25. The {sup 29}Si, {sup 27}Al MAS NMR and XRD characterisation of the reaction products reveal that for ratios nearest the value delivering suitable paste workability, the reaction-product composition and structure depend primarily on the nature and composition of the starting materials and the alkaline activator used. However, when an excess alkaline activator is present in the system, the reaction products tend to exhibit SiO{sub 2}/Al{sub 2}O{sub 3} ratios of approximately 1, irrespective of the composition of the starting binder or the alkaline activator.

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

    PubMed

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

    2013-11-01

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

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

    PubMed

    Nonaka, Hiroshi; Kobayashi, Ai; Funaoka, Masamitsu

    2013-07-01

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

  14. Enhancing Cellulase Production in Thermophilic Fungus Myceliophthora thermophila ATCC42464 by RNA Interference of cre1 Gene Expression.

    PubMed

    Yang, Fan; Gong, Yanfen; Liu, Gang; Zhao, Shengming; Wang, Juan

    2015-07-01

    The role of CRE1 in a thermophilic fungus, Myceliophthora thermophila ATCC42464, was studied using RNA interference. In the cre1-silenced strain C88, the filter paper hydrolyzing activity and β-1,4-endoglucanase activity were 3.76-, and 1.31-fold higher, respectively, than those in the parental strain when the strains were cultured in inducing medium for 6 days. The activities of β-1,4-exoglucanase and cellobiase were 2.64-, and 5.59-fold higher, respectively, than those in the parental strain when the strains were cultured for 5 days. Quantitative reverse-transcription polymerase chain reaction showed that the gene expression of egl3, cbh1, and cbh2 was significantly increased in transformant C88 compared with the wild-type strain. Therefore, our findings suggest the feasibility of improving cellulase production by modifying the regulator expression, and an attractive approach to increasing the total cellulase productivity in thermophilic fungi.

  15. “Newton’s cradle” proton relay with amide–imidic acid tautomerization in inverting cellulase visualized by neutron crystallography

    PubMed Central

    Nakamura, Akihiko; Ishida, Takuya; Kusaka, Katsuhiro; Yamada, Taro; Fushinobu, Shinya; Tanaka, Ichiro; Kaneko, Satoshi; Ohta, Kazunori; Tanaka, Hiroaki; Inaka, Koji; Higuchi, Yoshiki; Niimura, Nobuo; Samejima, Masahiro; Igarashi, Kiyohiko

    2015-01-01

    Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the “Newton’s cradle”–like proton relay pathway of the catalytic cycle. Amide–imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions. PMID:26601228

  16. Optimization of synergism of a recombinant auxiliary activity 9 from Chaetomium globosum with cellulase in cellulose hydrolysis.

    PubMed

    Kim, In Jung; Nam, Ki Hyun; Yun, Eun Ju; Kim, Sooah; Youn, Hak Jin; Lee, Hee Jin; Choi, In-Geol; Kim, Kyoung Heon

    2015-10-01

    Auxiliary activity family 9 (AA9, formerly known as glycoside hydrolase family 61 or polysaccharide monooxygenase) is a group of fungal proteins that were recently found to have a significant synergism with cellulase in cellulose hydrolysis via the oxidative cleavage of glycosidic bonds of cellulose chains. In this study, we report the active expression of a recombinant fungal AA9 from Chaetomium globosum (CgAA9) in a bacterial host, Escherichia coli, and the optimization of its synergistic activity in cellulose hydrolysis by using cellulase. The recombinant CgAA9 (0.9 mg/g cellulose) exhibited 1.7-fold synergism in the hydrolysis of Avicel when incubated with 0.9 filter paper units of Celluclast 1.5 L/g cellulose. The first study of the active expression of AA9 using a bacterial host and its synergistic optimization could be useful for the industrial application of AA9 for the saccharification of lignocellulose.

  17. "Newton's cradle" proton relay with amide-imidic acid tautomerization in inverting cellulase visualized by neutron crystallography.

    PubMed

    Nakamura, Akihiko; Ishida, Takuya; Kusaka, Katsuhiro; Yamada, Taro; Fushinobu, Shinya; Tanaka, Ichiro; Kaneko, Satoshi; Ohta, Kazunori; Tanaka, Hiroaki; Inaka, Koji; Higuchi, Yoshiki; Niimura, Nobuo; Samejima, Masahiro; Igarashi, Kiyohiko

    2015-08-01

    Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the "Newton's cradle"-like proton relay pathway of the catalytic cycle. Amide-imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

  18. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein.

    PubMed

    Haarmeyer, Carolyn N; Smith, Matthew D; Chundawat, Shishir P S; Sammond, Deanne; Whitehead, Timothy A

    2017-04-01

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

  19. Cellulase Ss (CelS) is synonymous with the major cellobiohydrolase (subunit S8) from the cellulosome of Clostridium thermocellum.

    PubMed

    Morag, E; Bayer, E A; Hazlewood, G P; Gilbert, H J; Lamed, R

    1993-11-01

    The controversy regarding the identity of a major cellulosomal component type from two different strains of Clostridium thermocellum has been resolved. The principal cellobiohydrolase, subunit S8, from the cellulosome of strain YS has been demonstrated to be synonymous with cellulase component Ss (CelS) from the cellulosome of ATCC strain 27405. This component is not related to any other cellulosomal subunit or cloned endoglucanase in this organism.

  20. Improved production of reducing sugars from rice straw using crude cellulase activated with Fe₃O₄/alginate nanocomposite.

    PubMed

    Srivastava, Neha; Singh, Jay; Ramteke, Pramod W; Mishra, P K; Srivastava, Manish

    2015-05-01

    Effect of Fe3O4 nanoparticles (NPs) and Fe3O4/Alginate nanocomposites (NCs) have been investigated on production and thermostability of crude cellulase enzyme system obtained by newly isolated thermotolerant Aspergillus fumigatus AA001. Fe3O4 NPs and Fe3O4/Alginate NCs have been synthesized by co-precipitation method and characterized through various techniques. In presence of Fe3O4 NPs and Fe3O4/Alginate NCs, filter paper activity of crude cellulase was increased about 35% and 40%, respectively in 72 h as compared to control. Fe3O4/Alginate NCs treated crude enzyme was thermally stable up to 8h at 70°C and retained 56% of its relative activity whereas; control samples could retain only 19%. Further, the hydrolysis of 1.0% alkali treated rice straw using Fe3O4/Alginate NCs treated cellulase gave much higher sugar productivity than control at optimal condition. These findings may be utilized in the area of biofuels and biowaste management.