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PubMedDate not supplied

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

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

Cellulase↗
PubMedDate not supplied

Shear deactivation of cellulase, exoglucanase, endoglucanase, and beta-glucosidase in a mechanically agitated reactor.

Shear deactivation of cellulase and its major component enzymes, viz., exoglucanase (exo-1,4-beta-D-glucan-4-cellobiohydrolase), endoglucanase (endo-1,4-beta-D-glucanhydrolase), and 1,4-beta-glucosidase, was carried out by exposing cellulase to shear in a mechanically agitated reactor in the presence as well as in the absence of the substrate cellulose. Cellulase was found to undergo deactivation when subjected to shear, and the extent of deactivation increased with increasing speed of agitation. Among the three major component enzymes of cellulase, exoglucanase showed rapid deactivation and contributed the most to cellulase deactivation. The presence of a substrate did not affect the deactivation of cellulase.

Cellulase↗
PubMed1979

The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and beta-glucosidases.

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000-48000 (C1), 30000-35000 (C2), 15000-18000 (C3), 10000-11000 (C4) and 4800-5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2-C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2-C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight beta-glucosidase (component B1, mol.wt. 350000-380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000-47000) predominated in older filtrates. 9. Intermediate molecular species of beta-glucosidase (B2, mol.wt. 170000-180000; B3, mol.wt. 83000-87000) were also found. 10. Cellulases C2-C5 acted in synergism with C1, particularly in the presence of beta-glucosidase.

Cellobiose↗
PubMed1996

Fine substrate specificities of four exo-type cellulases produced by Aspergillus niger, Trichoderma reesei, and Irpex lacteus on (1-->3), (1-->4)-beta-D-glucans and xyloglucan.

To investigate the fine substrate specificities of four highly purified exo-type cellulases (Exo-A from Aspergillus niger, CBHI and CBHII from Trichoderma reesei, and Ex-1 from Irpex lacteus), water-soluble substrates such as barley glucan, xyloglucan from tamarind (Tamarindus indica L.), and their oligosaccharides were employed. Four exo-type cellulases immediately hydrolyzed 3-O-beta-D-cellotriosylglucose to produce cellobiose and laminaribiose. In contrast, CBHII showed no hydrolytic activity towards 3(2)-O-beta-D-cello-biosylcellobiose, which was hydrolyzed to cellobiose by the other exo-type cellulases. These cellulases hydrolyzed the internal linkages of barley glucan and lichenan in an endo-type fashion to produce cellobiose and mix-linked oligosaccharides as main products. The DP-lowering activities of the four exo-type cellulases on barley glucan were in the order of Ex-1, CBHII, Exo-A, and CBHI. Based on gel permeation chromatography analysis of the hydrolysates, Ex-1 seemed to attack the internal cellobiosyl unit adjacent to beta-1,3-glucosidic linkages in barley glucan molecule more frequently than did the other cellulases. Xyloglucan was hydrolyzed only by CBHI and CBHII, and produced hepta-, octa-, and nona-saccharides. In addition, a xyloglucan tetradecasaccharide (XG14) was split only to heptasaccharide (XG7) by CBHI and CBHII.

Aspergillus niger↗
PubMed1990

Cloning and sequencing of the endo-cellulase cDNA from Robillarda sp. Y-20.

An endo-cellulase cDNA has been screened from a Robillarda sp. Y-20 cDNA expression library using polyclonal antibodies (immunoscreening). Western blot analysis showed that recombinant CMCase I fused to beta-galactosidase with the molecular weight of approximately 150,000 was expressed in Escherichia coli Y1090. Sequence analysis of the cloned cDNA revealed that it consisted of 1,136 nucleotides. By comparison of the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of the purified protein (residues 1 to 18, determined by protein sequencing), the cDNA was found to lack 44 nucleotides at its 5' end corresponding to residues 1 to 15. Therefore, the mature cellulase (CMCase I) was deduced to be composed of 375 amino acid residues and the molecular weight of its protein was calculated to be 41,004. Yaguchi et al. reported that the N-terminal amino acid sequence of an endo-beta-1,4-glucanase (endo-cellulase) from Schizophyllum commune was homologous with the active site of various hen egg-white type lysozymes, and the homology offered evidence for a lysozyme-type mechanism in enzymatic hydrolysis of cellulase [Biochem. Biophys. Res. Commun. (1983) 116, 408-411]. Pentillä et al. also pointed out that some amino acid homology was found between endo-glucanase I from Trichoderma reesei and the lysozyme of the phage T4 [Gene (1986) 45, 253-263]. From the result of sequence alignment of the endo-cellulase from Robillarda sp. Y-20 and four kinds of lysozymes, there was a possibility that the endo-cellulase from Robillarda sp. Y-20 also hydrolyzes carboxymethyl cellulose by a lysozyme-type mechanism.

Amino Acid Sequence↗
PubMed1976

Partial proteolysis of some cellulase components from Trichoderma viride and the substrate specificity of the modified products.

An endo-cellulase component [EC 3.2.1.4] or random type, F II, was obtained from "Cellulase Onozuka," a commercial product from Trichoderma viride, and was subjected to partial proteolysiats with a protease preparation of the same fungal origin. The resulting modified cellulase was fractioned by two steps of column chromatography, and the resulting patterns, together with the substrate specificity expressed in terms of the randomness of CMC hydrolysis and the immunological properties against anti-F II-rabbit se-um, were examined. The chromatographic patterns were very similar to those of cellulase subfractions without proteolytic treatment. Moreover, the immunological response of the modified cellulases from F II was mostly positive and their randomness of CMC hydrolysis was generally lower, compared with subfractions of F II which were not subjected to proteolysis. The subfractions of Peak III, which were obtained from F II by proteolysis, showed mostly negative immunological response and higher randomness of CMC hydrolysis compared with subfractions of Peak III which were not subjected to proteolysis. Thus, some limited proteolysis of cellulase components may, at least in part, be responsible for its multiplicity in vivo.

Amino Acids↗
PubMed1980

Purification and properties of a lower-molecular-weight endo-cellulase from Irpex lacteus (Polyporus tulipiferae).

A new endo-cellulase component of carboxymethyl cellulase (CMCase) type (En-1) was obtained by gel filtration and column chromatography from Driselase, a commerical enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The enzyme behaved as a single protein on polyacrylamide disc electrophoresis in the presence of sodium dodecyl sulfate (SDS). Its molecular weight was estimated to be 15,500 and it contained only 0.73% carbohydrate as glucose. The pattern of its amino acid composition is similar to those of other cellulases in respect of high contents of acidic amino acids, glycine, serine, and threonine. The cellulase was most active at pH 4.0 and was very stable in the pH range of 3.0 to 6.0, but was completely inactivated by heating at 70 degrees C for 10 min. A series of cellooligosacharides, including cellobiose, was formed by this enzyme from sodium carboxymethyl cellulose (CMC) as well as from water-insoluble celluloses. In the hydrolysis of CMC, the increase in the fluidity of the substrate was relatively large as compared with the simultaneous increase in reducing power. From this result and the pattern of hydrolysis products, En-1 was elucidated to be an endo-cellulase, and it showed the highest randomness among the cellulase components obtained so far from Irpex lacteus.

Amino Acids↗
PubMed1986

Molecular cloning and nucleotide sequence of the alkaline cellulase gene from the alkalophilic Bacillus sp. strain 1139.

The cellulase gene from the alkalophilic Bacillus sp. strain 1139 was cloned in Escherichia coli using pBR322. Plasmid pFK1 was isolated from transformants producing cellulase, and the cloned cellulase gene was found to be in a 4 X 6 kb HindIII fragment. The cellulase gene was subcloned in a functional state on a 2 X 9 kb DNA fragment and its nucleotide sequence was determined. The coding sequence showed an open reading frame encoding 800 amino acids. The pFK1-encoded cellulase had the same enzymic properties as the extracellular cellulase produced by the alkalophilic Bacillus sp. strain 1139, but its Mr was slightly higher.

Amino Acid Sequence↗
PubMed2001

Salicylic acid and ethylene pathways are differentially activated in melon cotyledons by active or heat-denatured cellulase from Trichoderma longibrachiatum.

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

Adaptation, Physiological↗
PubMed1991

Allergy from cellulase and xylanase enzymes.

Modern biotechnical methods have enabled production of many new types of potentially allergenic proteins. Enzymes have long been known to be respiratory allergens, but relatively few cases of skin allergy have been reported. Here we describe four patients who developed occupational allergic respiratory symptoms, three with bronchial asthma and one with allergic rhinitis, caused by cellulase and/or xylanase enzymes. Each patient also had urticarial symptoms after skin contact with these enzymes. In addition, one of the patients had allergic contact dermatitis from cellulase, and one from xylanase. Allergic contact dermatitis was verified by positive patch tests with the enzymes, and the immediate allergy was revealed by skin prick tests, specific IgE determinations (RAST) and RAST-inhibition tests. All patients had positive RASTs to both cellulase and xylanase. In the RAST inhibition test 20 microliters of cellulase brought about a 94% inhibition, indicating the specificity of the RAST. Xylanase (20 microliters, 5% w/v) gave an 92% inhibition of cellulase RAST, indicating cross-reactivity between cellulase and xylanase. Three patients have been able to continue at their previous places of work, but at different worksites. One of the patients requires continuous medication for asthma and had to change her job because of persistent symptoms.

Adult↗
PubMed1992

The bgl1 gene encoding extracellular beta-glucosidase from Trichoderma reesei is required for rapid induction of the cellulase complex.

We have used a targeted gene deletion event to remove the coding region for the bgl1 gene encoding an extracellular beta-glucosidase from the genome of the cellulolytic fungus Trichoderma reesei. The bgl1 null mutants were used to investigate the role of beta-glucosidase in the hydrolysis of cellulose and induction of the other cellulolytic enzyme components. In the absence of extracellular beta-glucosidase, growth of bgl1 null strains on several carbon sources was the same as that of the parent (as measured by mycelial dry weight). However, levels of extracellular protein and total endoglucanase production were seen to lag relative to those levels observed in the control strain. The mRNA levels of the CBHI, CBHII, EGI, and EGII cellulase genes (cbh1, cbh2, egl1 and egl3) showed a corresponding lag in induction, suggesting that the absence of extracellular beta-glucosidase has an effect on the co-ordinate regulation of the other cellulase genes at the level of transcription. The addition of a potent inducer of the cellulase complex (sophorose) resulted in normal rates of cellulase gene mRNA production and extracellular protein release. This indicates that the absence of beta-glucosidase is not affecting some intrinsic cellular ability to produce mRNA or secrete protein. These data suggest that a functional beta-glucosidase is at least partially responsible for the efficient induction of the depolymerase enzymes of the cellulase complex. The observation that the cellulase complex is induced, albeit after a lag, suggests that other enzymes are present that can substitute for the function of beta-glucosidase during induction.

Cellulase↗
PubMed1977

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.

Cellulase↗
PubMed1997

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

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

Cellulase↗
PubMed2005

Role of the bga1-encoded extracellular {beta}-galactosidase of Hypocrea jecorina in cellulase induction by lactose.

Lactose is the only soluble and economically feasible carbon source for the production of cellulases or heterologous proteins regulated by cellulase expression signals by Hypocrea jecorina (Trichoderma reesei). We investigated the role of the major beta-galactosidase of H. jecorina in lactose metabolism and cellulase induction. A genomic copy of the bga1 gene was cloned, and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence. This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular beta-galactosidase during growth on lactose. Its transcript was abundant during growth on l-arabinose and l-arabinitol but was much less common when the organism was grown on lactose, d-galactose, galactitol, d-xylose, and xylitol. Deltabga1 strains grow more slowly and accumulate less biomass on lactose, but the cellobiohydrolase I and II gene expression and the final cellulase yields were comparable to those of the parental strain. Overexpression of bga1 under the control of the pyruvate kinase promoter reduced the lag phase, increased growth on lactose, and limited transcription of cellobiohydrolases. We detected an additional extracellular beta-galactosidase activity that was not encoded by bga1 but no intracellular beta-galactosidase activity. In conclusion, cellulase production on lactose occurs when beta-galactosidase activity levels are low but decreases as the beta-galactosidase activities increase. The data indicate that bga1-encoded beta-galactosidase activity is a critical factor for cellulase production on lactose.

Cellulase↗
PubMed1978

Cellulase and beta-glucosidase production by a basidiomycete species.

The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.

Basidiomycota↗
PubMed2004

Presence and properties of cellulase and hemicellulase enzymes of the gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes.

Digestive juice from the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes exhibited total cellulase activity and activities of two cellulase enzymes; endo-beta-1,4-glucanase and beta-1,4-glucosidase. These enzymes hydrolysed native cellulose to glucose. The digestive juice of both species also contained laminarinase, licheninase and xylanase, which hydrolysed laminarin, lichenin and xylan, respectively, to component sugars. The pH optima of beta-1,4-glucosidase, endo-beta-1,4-glucanase and total cellulase from G. natalis were 4-5.5, 5.5 and 5.5-7, respectively. In the digestive juice from D. hirtipes, the corresponding values were 4-7, 5.5-7 and 4-9, respectively. The pH of the digestive juice was 6.69+/-0.03 for G. natalis and 6.03+/-0.04 for D. hirtipes and it is likely that the cellulases operate near maximally in vivo. In G. natalis, total cellulase activity and endo-beta-1,4-glucanase activity were higher than in D. hirtipes, and the former species can thus hydrolyse cellulose more rapidly. beta-1,4-glucosidase from G. natalis was inhibited less by glucono-d-lactone (K(i)=11.12 mmol l(-1)) than was the beta-1,4-glucosidase from D. hirtipes (K(i)=4.53 mmol l(-1)). The greater resistance to inhibition by the beta-1,4-glucosidase from G. natalis may contribute to the efficiency of the cellulase system in vivo by counteracting the effects of product inhibition and possibly dietary tannins. The activity of beta-1,4-glucosidase in the digestive juice of D. hirtipes was higher than that of G. natalis.

Analysis of Variance↗
PubMed2002

Synergism in binary mixtures of Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A on BMCC and Avicel.

In an earlier binding study conducted in our laboratory using Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A (formally Thermomonosporafusca E3, E4, and E5), it was observed that binding capacities for these three cellulases were 18-30 times higher on BMCC than on Avicel. These results stimulated an interest in how the difference in accessibility between the two cellulosic substrates would affect synergism observed with cellulase mixtures. To explore the impact of substrate accessibility on the extent of conversion and synergism, three binary T. fusca cellulase mixtures were tested over a range of cellulase ratios and total molar cellulase concentrations on Avicel and BMCC. Higher extents of conversion were observed for BMCC due to the higher enzyme to substrate ratio resulting from the higher binding. The processive endoglucanase, Cel9A, had four times the extent of conversion of the endocellulase Cel5A, while the exocellulase Cel6B had three times the extent of conversion of Cel5A. Approximately 500 nmol/g of the Cel9A+Cel6B mixture was needed to obtain 80% conversion, while the Cel6B+Cel5A and Cel9A+Cel5A mixtures required 1500 and 1250 nmol/g, respectively, to obtain 80% conversion. Thus, it appears that the more accessible structure of BMCC, as reflected by its binding capacity, results in relative higher processive activity.

Actinomycetales↗
PubMed2003

Automated filter paper assay for determination of cellulase activity.

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

Automation↗