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PubMed2003

Separation and quantification of cellulases and hemicellulases by capillary electrophoresis.

Cellulases and hemicellulases are two classes of enzymes produced by filamentous fungi and secreted into the cultivation medium. Both classes of enzymes consist of a subset of classes of which the fungi produce several enzymes with varying molecular mass and pI but similar enzymatic activities. Current methods are limited in their ability to quantify all of these enzymes when all are present simultaneously in a mixture. Five different cellulases (two cellobiohydrolases and three endoglucanases) and one hemicellulase (endoxylanase) were separated using capillary electrophoresis (CE) in a fused silica capillary at pH values close to neutral. The improvement of the separation of these six proteins by the addition of alpha,omega-diaminoalkanes with chain lengths from three to seven carbon units was investigated. Dynamically coating the capillary with 1,3-diaminopropane resulted in separation of the six enzymes and the reproducibility of the migration times was between 0.6 and 1.9%. Two cases-quantitative determination of the enzyme concentrations in cultivation samples and investigation of adsorption of the enzymes onto cellulose-demonstrated the advantages and perspectives of CE analysis of these broad groups of enzymes.

Adsorption↗
PubMed2003

Molecular cloning, transcriptional, and expression analysis of the first cellulase gene (cbh2), encoding cellobiohydrolase II, from the moderately thermophilic fungus Talaromyces emersonii and structure prediction of the gene product.

A gene (cbh2) encoding cellobiohydrolase II was isolated from the fungus Talaromyces emersonii by rapid amplification of cDNA ends techniques and the equivalent genomic sequence was subsequently cloned. This represents the first report of a key component of the cellulase regulon from this organism. DNA sequencing revealed that cbh2 has an open reading frame of 1377 bp, which encodes a putative polypeptide of 459 amino acids, and is interrupted by seven introns. The deduced amino acid sequence revealed that cbh2 has a modular structure with a predicted molecular mass of 47 kDa and consisting of a fungal type carbohydrate binding module separated from a catalytic domain by a proline/serine/threonine rich linker region. The deduced protein is homologous to fungal cellobiohydrolases in Family 6A of the glycosyl hydrolases. Profiles of cbh2 expression in T. emersonii investigated by Northern blot analysis revealed that expression is regulated at the transcriptional level. Expression of the T. emersonii cbh2 gene is induced by cellulose, xylan, xylose, and gentiobiose and clearly repressed by glucose. Putative regulatory element consensus sequences have been identified in the upstream regulatory sequence of the cbh2 gene including the catabolite repressor element and the activator of cellulase expression (Ace) binding sites. High sequence identity (67%) between the catalytic domain of Cel 6A from Trichoderma reesei and the T. emersonii cbh2 gene product allowed structure prediction for the 3D model of the T. emersonii catalytic domain to be a variant of the classical TIM alpha/beta fold.

Amino Acid Sequence↗
PubMed2001

The hydrolysis of barley beta-glucan by the cellulase EC 3.2.1.4 under dilute conditions is identical to that of barley solubilase.

Barley beta-glucan solubilase is an enzyme that degrades barley beta-glucan in extracts obtained from barley flour. The solubilase preferentially attacks the longer blocks of beta-(1-->4) linkages, i.e., those containing at least nine glucosyl residues. There is strong evidence to suggest that the solubilase derives from fungi associated with the husk of the grain. It was found that cellulase (EC 3.2.1.4) from Trichoderma sp. shows similar activity under dilute conditions. Since fungi associated with the husk of the grain are known to produce these types of cellulases, there is no need, based on current evidence, to propose the existence of a unique enzyme, i.e., solubilase, for the solubilising behaviour of enzymes in the barley grain.

Carbohydrate Conformation↗
PubMed1979

Analysis of cellulase proteins by high-performance liquid chromatography.

A new procedure using high-performance liquid chromatography for the rapid separation of cellulase proteins is described. The cellulase components of Trichoderma reesei are fractionated on a DEAE anion-exchange column using a phosphate buffer at pH 6.2. Activities of the individual components obtained from T. reesei QM6a, a wild strain, and several mutant strains have been determined. Each system examined contained beta-glucosidase, at least two exo-beta-1,4 glucanases and five endo-beta-1,4 glucanases with the endo-beta-1,4 glucanases accounting for 20--36% and the exo-beta-1,4 glucanases for 64--80% of the soluble protein.

Cellulase↗
PubMed2002

Comparative study on the fractionation of cellulases on some hydrophobic interaction chromatography adsorbents.

In this work, a comparative study for the fractionation of Trichoderma reesei cellulases on five different hydrophobic interaction chromatography adsorbents (Butyl-Sepharose 4 FF, Phenyl-Sepharose 6 FF, Octyl-Sepharose 4 FF, Epoxy-Sepharose CL-6B and Polypropylene glycol-Sepharose CL-6B) is shown. The influence of the mobile phase composition on the chromatographic behaviour of T. reesei cellulases complex was evaluated using different concentrations of ammonium sulphate in the eluent buffer. A selective separation of beta-glucosidase with two-fold increase in specific activity and good recoveries of cellobiase activity were obtained with Butyl-Sepharose 4 FF and Phenyl-Sepharose 6 FF using 7% (w/v) ammonium sulphate in the eluent buffer. A beta-glucosidase fractionation was also obtained with Epoxy-Sepharose CL-6B, using 13% (w/v) of the salt in the mobile phase.

Cellulase↗
PubMed2001

Guidelines to come to minimized tensile strength loss upon cellulase application.

Application of cellulase technology in the textile production process often results in a certain loss of tensile strength along with the desired performance. In this paper guidelines are given how to come to minimization or even prevention of tensile strength loss. Part of the considerations is based on the hypothesis given in the accompanying paper (Lenting and Warmoeskerken, 2001, J. Biotechnol.) concerning the mechanism of interaction between cellulase action and applied shear force. Recommendations given concern the enzyme choice, process parameters and enzyme targeting.

Cellulase↗
PubMed2001

Application of microassays for investigation of cellulase abrasive activity and backstaining.

Model microassays were used for testing the denim-washing performance and indigo backstaining for Trichoderma reesei and Chrysosporium lucknowense commercial cellulase preparations on a 'test-tube scale'. C. lucknowense preparation demonstrated a higher potential in the denim biostoning process. The performance of four purified cellulases (two endoglucanases and two cellobiohydrolases) from C. lucknowense on cotton textiles was assayed, and the key enzyme (endoglucanase with a molecular weight of 25 kDa) responsible for high abrasion effects on denim fabrics was found.

Cellulase↗
PubMed2001

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

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.

Cellulase↗
PubMed1997

Enzymatic properties of cellulases from Humicola insolens.

We present the analysis of the activities towards soluble and insoluble substrates of seven cellulases cloned from the saprophytic fungus Humicola insolens. The activity on the soluble polymer substrate carboxymethylcellulose (CMC) was used to determine the pH activity profiles of the five endoglucanases (EG), whereas cellotriose and reduced cellohexaose were used to determine the pH activity profiles of cellobiohydrolase I (CBH) and CBH II. All the EGs show optimal activity between pH 7 and 8.5, while CBH I and CBH II peak around pH 5.5 and 9, respectively. The catalytic activities of five of these cellulases were investigated under neutral and alkaline conditions using reduced cellohexaose as a substrate in a cellobiose oxidase coupled assay. EG I and CBH I both belong to family (7) according to a recent classification of glycosyl hydrolases. They both have activity against cellotriose. Therefore, they were studied using a coupled assay involving glucose oxidase. The activity on insoluble substrate (phosphoric-acid swollen cellulose) was assessed by the formation of reducing groups. The presence of a cellulose binding domain (CBD) lowers the apparent KM. This can be explained by the dispersing action of CBD. However, the CBD also reduces the apparent k(cat) probably by slowing down the mobility. EG I, EG II and EG III show similar activity towards CMC and amorphous cellulose, while EG V, EG VI, CBH I and CBH II have the highest catalytic rate on amorphous cellulose. In summary, Humicola insolens possesses a battery of cellulose-degrading enzymes which cooperate in the efficient hydrolysis of cellulose.

Cellulase↗
PubMed2002

Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study.

Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies. We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins. Cellulase C has a TIM barrel fold with an additional helical subdomain. We designed and produced a number of mutants with the aim to increase its thermostability. Five mutants were designed to create new electrostatic interactions. They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme. Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains. In another mutant, the small helical subdomain was deleted. This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function. A double mutant was designed to introduce a new disulfide bridge into the enzyme. This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme. Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior. We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein. Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects.

Calorimetry, Differential Scanning↗
PubMed2003

Biodegradation of wastepaper by cellulase from Trichoderma viride.

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

Biodegradation, Environmental↗
PubMed2004

Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass.

To investigate the production of cellulases and hemicellulases from Aspergillus niger KK2, solid state fermentation (SSF) was performed by using different ratios of rice straw and wheat bran. When A. niger KK2 was grown on rice straw alone as a solid support in SSF, the maximum FPase activity was 19.5 IU g(-1) in 4 days. Also, CMCase (129 IU g(-1)), beta-glucosidase (100 IU g(-1)), xylanase (5070 IU g(-1)) and beta-xylosidase (193 IU g(-1)) activities were concurrently obtained after 5-6 days of fermentation. The higher enzyme activities produced by A. niger KK2 is a significant advantage from the viewpoint of practical saccharification reaction. Cellulases and hemicellulases produced by A. niger KK2 might be applied to pulp and paper industry, feed industry and chemical industry.

Aspergillus niger↗
PubMed1995

Crystal structure of the catalytic domain of a bacterial cellulase belonging to family 5.

BACKGROUND: Cellulases are glycosyl hydrolases--enzymes that hydrolyze glycosidic bonds. They have been widely studied using biochemical and microbiological techniques and have attracted industrial interest because of their potential in biomass conversion and in the paper and textile industries. Glycosyl hydrolases have lately been assigned to specific families on the basis of similarities in their amino acid sequences. The cellulase endoglucanase A produced by Clostridium cellulolyticum (CelCCA) belongs to family 5. RESULTS: We have determined the crystal structure of the catalytic domain of CelCCA at a resolution of 2.4 A and refined it to 1.6 A. The structure was solved by the multiple isomorphous replacement method. The overall structural fold, (alpha/beta)8, belongs to the TIM barrel motif superfamily. The catalytic centre is located at the C-terminal ends of the beta strands; the aromatic residues, forming the substrate-binding site, are arranged along a long cleft on the surface of the globular enzyme. CONCLUSIONS: Strictly conserved residues within family 5 are described with respect to their catalytic function. The proton donor, Glu170, and the nucleophile, Glu307, are localized on beta strands IV and VII, respectively, and are separated by 5.5 A, as expected for enzymes which retain the configuration of the substrate's anomeric carbon. Structure determination of the catalytic domain of CelCCA allows a comparison with related enzymes belonging to glycosyl hydrolase families 2, 10 and 17, which also display an (alpha/beta)8 fold.

Binding Sites↗
PubMed2006

[Effect of straw apoplast protein on cellulase activity].

We studied the alteration of the maize straw apoplast proteins in the process of preservation, and analyzed the effects of apoplast proteins on Penicillum expansum cellulase activities. The results show that: the extractable apoplast proteins are gradually decreased during the preservation of maize straw. Meanwhile, their synergistic effects on P. expensum cellulose are also attenuated. The apoplast proteins extracted from fresh maize straw possess endogenous EG activities, which is unstable and completely vanished after 6 months preservation. The apoplast proteins from the preserved straw exhibit significant synergistic effect on FPA, cotton lyase and beta-glucosidase. The maximal synergistic values are 95.32%, 102.06% and 96.6%, respectively. But interestingly, they inhibit the CMCase activity (max. 49.52%). Apoplast proteins show distinctive synergy with betaG and EG, but have no effect on CBH activity. After eliminating the effect of endogenous EG, the apoplast proteins from fresh maize straw have enhanced synergistic or inhibiting effects on FPA, Cotton lyase, betaG and CMCase than those extracted from the preserved straw. Based on our observation, the apoplast proteins play important roles in regulating the cellulase activities. The detailed analysis of the related mechanisms will greatly benefit the studies of the natural biomaterials hydrolysis.

Cellulase↗
PubMedDate not supplied

Do enzymatic hydrolyzability and Simons' stain reflect the changes in the accessibility of lignocellulosic substrates to cellulase enzymes?

In an attempt to elucidate the impact of substrate accessibility to cellulases on the susceptibility of lignocellulosic substrates to enzymatic hydrolysis, a hydrogen peroxide treated, Douglas fir kraft pulp was dried using several methods with varying levels of intensity. Oven-drying at 50 and 100 degrees C, air-drying, and freeze-drying methods were employed to remove the interfibrillar water from the pulp samples. Subsequently, the never-dried and variably dried pulps were hydrolyzed using a commercial cellulase preparation supplemented with additional beta-glucosidase. Drying reduced the susceptibility of the substrates to enzymatic hydrolysis, which can be attributed to the hornifying effect that drying has on fibers. This effect was more pronounced for the fibers that were oven-dried at 100 degrees C (23% reduction) and 50 degrees C (15% reduction), and there was a good correlation between the Simons's stain results and the enzymatic digestibility of the dried pulps. These observations indicated that drying significantly reduced the population of larger pores and that the partial closure of larger pores created a large number of smaller pores that were not accessible to the displacement dye molecules (orange dye). The inaccessibility of the cellulose to the enzymes, due to the collapse or closure of the large pores, appears to be the primary reason for the lower susceptibility of the dried pulps to enzymatic hydrolysis.

Algorithms↗
PubMedDate not supplied

Effect of cellulase mole fraction and cellulose recalcitrance on synergism in cellulose hydrolysis and binding.

Elucidating the molecular mechanisms that govern synergism is important for the rational engineering of cellulase mixtures. Our goal was to observe how varying the loading molar ratio of cellulases in a binary mixture and the recalcitrance of the cellulose to enzymatic degradation influenced the degree of synergistic effect (DSE) and degree of synergistic binding (DSB). The effect of cellulose recalcitrance was studied using a bacterial microcrystalline cellulose (BMCC), which was exhaustively hydrolyzed by a catalytic domain of Cel5A, an endocellulase. The remaining prehydrolyzed BMCC (PHBMCC) was used to represent a recalcitrant form of cellulose. DSE was observed to be sensitive to loading molar ratio. However, on the more recalcitrant cellulose, synergism decreased. Furthermore, the results from this study reveal that when an exocellulase (Cel6B) is mixed with either an endocellulase (Cel5A) or a processive endocellulase (Cel9A) and reacted with BMCC, synergism is observed in both hydrolysis and binding. This study also revealed that when a "classical" endocellulase (Cel5A) and a processive endocellulase (Cel9A) are mixed and reacted with BMCC, only limited synergism is observed in reducing sugar production; however, binding is clearly increased by the presence of the Cel5A.

Bacteria↗
PubMed2006

Cellulase and protease preparations can extract pectins from various plant byproducts.

The use of protease and cellulase preparations to extract pectins from plant byproducts (chicory, cauliflower) was investigated. Different enzymatic preparations were characterized by their activities toward proteins, cellulose, and pectins. These preparations were then tested regarding pectin extraction, and extraction conditions (nature and concentration of enzyme, incubation time) were optimized. Enzymatic and acidic extractions were compared and also combined in sequential extractions. This study shows that it is possible to extract pectins by using cellulases and proteases. Enzymes can extract pectins with a higher yield ( approximately 35%) than acid (approximately 28%) but enzyme-extracted pectins have a smaller molar mass (300,000 g/mol) than acid-extracted pectins (500,000 g/ mol). Different hypotheses are tested and discussed to explain this mass difference.

Brassica↗
PubMed2026

Genomic and bioreactor evaluation of newly isolated cellulase-producing bacteria.

Cellulose is a renewable resource with broad biotechnological potential, and its hydrolysis by cellulases underpins applications such as biofuel production and biowaste valorization. This study combined isolation, screening, molecular identification, enzyme assays, genome sequencing, and bioreactor evaluation to characterize novel cellulase-producing bacteria. Preliminary screening identified and selected bacterial isolates by hydrolysis halo formation, with strains showing halos ≥ 20 mm selected for further quantitative assays. Streptomyces olivaceus C1_7A, Bacillus thuringiensis FBB7BB, and Bacillus licheniformis strains AT081C and AT082C showed the highest endoglucanase activities (from 0.11 to 0.26 U mL-1). Further testing on Arundo donax and Avicel led to the selection of B. thuringiensis FBB7BB and S. olivaceus C1_7A as the most promising strains for biotechnological application. Whole genome sequencing confirmed the presence of genes involved in polysaccharide degradation. Finally, lab-scale fermentation trials using 1-2% CMC showed that B. thuringiensis FBB7BB reached a maximum endo-1,4-β-glucanase activity of 0.30 ± 0.04 U mL-1 after 8 h at 37 °C, whereas S. olivaceus C1_7A achieved 0.36 ± 0.03 U mL-1 after 216 h at 30 °C.

Cellulase↗