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PubMed1975

Enzymatic studies on a cellulase system of Trichoderma viride. II. Purification and Properties of two cellulases.

Two cellulase [EC 3.2.1.4] components derived from Meicelase, a commercial crude cellulase preparation from Trichoderma viride, were purified by consecutive column chromatography, and were designated as cellulase II-A and cellulase II-B. Cellulases II-A and II-B were each homogeneous on polyacrylamide gel electrophoresis. The molecular weights of cellulases II-A and II-B were 30,000 and 43,000, respectively, on the basis of Sephadex G-100 gel filtration. Both enzymes contained 12-14% carbohydrates (as glucose). Some properties of the purified cellulases were investigated. The optimum pH and temperature for cellulases II-A and II-B were pH 4.5-5.0 and 60 degrees, and pH 4.5-5.0 and 50 degrees, respectively. Both enzymes were stable over the range of pH 5.0-7.0 at 4 degrees for 24 hr. Cellulases II-A and II-B retained 27 and 41% of the original CM-cellulose-saccharifying activities, respectively, after heating at 100 degrees for 10 min. Both enzymes were completely inhibited by some metal ions such as 1 mM Hg-2+, and partially by 1 mM Ag-+ and Cu-2+. However, Mg-2+, Fe-2+, and several other metal ions showed no inhibition at this concentration. The hydrolysis of CM-cellulose by cellulase II-A was more random than that by cellulase II-B.

Animals↗
PubMed1975

Enzymatic studies on a cellulase system of Trichoderma viride. III. Transglycosylation properties of two cellulase components of random type.

Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of rho-nitrophenyl beta-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from rho-nitrophenyl beta-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.

Animals↗
PubMed2002

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

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

Adsorption↗
PubMed1976

Xylanase activity of an endo-cellulase of carboxymethyl-cellulase type from Irpex lacteus (Polyporus tulipiferae).

An endo-cellulase [EC 3.2.1.4.] of carboxymethyl-cellulase type (F-1) which was fractionated from culture filtrate of Irpex lacetus and purified to electrophoretic and ultracentrifugal homogeneity, was found to show xylanase [EC 3.2.1.8.] activity. The activity was not removed from any of the intermediate fractions during the purification of the initial F-I peak, and the radio of xylanase to cellulase activity remained almost unchanged through the purification processes. The xylanase activity of F-I showed not only the same optiomal pH, heat stability, and pH stability as its cellulase activity, but also the same mobility as the cellulase activity upon cellulose acetate film and starch zone electrophoreses. The overall rates of hydrolysis of mixtures of variouis concentrations of CM-cellulose and xylan by F-1 coincided well with those calculated from the Michaelis-Menten treatment of two substances competing for the same active site of the enzyme. These results indicate that the xylanase activity of F-1 is intrinsic to the cellulase itself.

Basidiomycota↗
PubMed1976

Enzymatic studies on a cellulase system of Trichoderma viride. IV. Purification and properties of a less-random type cellulase.

A cellulase [EC 3.2.1.4] component was purified from a crude cellulase preparation of Trichoderma viride (Meicelase) by consecutive column chromatography procedures, and was designated as cellulase III. The enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme was estimated to be about 45,000 by gel filtration. The optimum pH and temperature of the enzyme were pH 4.5-5.0 and 50 degrees, respectively. The enzyme was stable over the range of pH 4.5-7.5 at 4 degrees for 24 hr, and retained 40% of the original carboxymethylcellulose-saccharifying activity after heating at 100 degrees for 10 min. The enzyme was completely inactivated by 1 mM Hg2+, and partially by 1 mM Ag+ and Cu2+. The enzyme was characterized as a less-random type cellulase on the basis of its action on carboxymethylcellulose. The enzyme split cellohexaose, retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products. The Km values of cellulase III for cellooligosaccharides decreased in parallel with increase of the chain length of the substrates, while Vmax values showed a tendency to increase. The enzyme produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase III preferentially attacked the aglycone linkage of p-nitrophenyl beta-D-cellobioside. The enzyme was found to catalyze the rapid synthesis of cellotetraose from cellobiose (condensation action).

Carboxymethylcellulose Sodium↗
PubMed1997

Role of four major cellulases in triggering of cellulase gene expression by cellulose in Trichoderma reesei.

The relative contributions of four major cellulases of Trichoderma reesei (1,4-beta-D-glucan cellobiohydrolase I [CBH I], CBH II, endo-1,4-beta-D-glucanase I [EG I], and EG II) to the generation of the cellulase inducer from cellulose were studied with isogenic strains in which the corresponding genes (cbh1, cbh2, egl1, and egl2) had been deleted by insertion of the Aspergillus nidulans amdS marker gene. During growth on lactose (a soluble carbon source provoking cellulase gene expression), these strains showed no significant alterations in their ability to express the respective other cellulase genes, with the exception of the strain containing delta cbh1, which exhibited an increased steady-state level of cbh2 mRNA. On crystalline cellulose as the only carbon source, however, significant differences were apparent: strains in which cbh2 and egl2, respectively, had been deleted showed no expression of the other cellulase genes, whereas strains carrying the cbh1 or egl1 deletion showed these transcripts. The delta cbh1-containing strain also showed enhanced cbh2 mRNA levels under these conditions. A strain in which both cbh1 and cbh2 had been deleted, however, was unable to initiate growth on cellulose. Addition of 2 mM sophorose, a putative inducer of cellulase gene expression, to such cultures induced the transcription of egl1 and egl2 and restored the ability to grow on cellulose. We conclude that CBH II and EG II are of major importance for the efficient formation of the inducer from cellulose in T. reesei and that removal of both cellobiohydrolases renders T. reesei unable to attack crystalline cellulose.

Cellulase↗
PubMed2002

Lactose metabolism and cellulase production in Hypocrea jecorina: the gal7 gene, encoding galactose-1-phosphate uridylyltransferase, is essential for growth on galactose but not for cellulase induction.

Lactose is at present the only soluble carbon source which can be used economically for the production by Hypocrea jecorina (= Trichoderma reesei) of cellulases or heterologous proteins under the control of cellulase expression signals. However, the mechanism by which lactose triggers the formation of cellulases is unknown. To enhance our understanding of lactose metabolism and its relationship to cellulase formation, we have cloned and characterized the gal7 gene (for galactose-1-phosphate uridylyltransferase) of H. jecorina. The gene encodes a polypeptide of 43.8 kDa, the sequence of which exhibits a moderate level of identity (about 50%) to that of the Gal7 proteins of Saccharomyces cerevisiae and Kluyveromyces lactis, and contains an active-site signature typical for galactose-1-phosphate uridylyltransferase family 1. H. jecorina gal7 is not clustered with other genes of galactose metabolism. A single 1.7-kb transcript is synthesized constitutively during the rapid growth phase and accumulated to twice this level during incubation in the presence of D-galactose and L-arabinose and the corresponding polyols (dulcitol, arabitol). A gal7 deletion mutant, constructed by replacing the gal7 reading frame by the H. jecorina pyr4 gene, was unable to grow on D-galactose between pH 4.5 and 7.5, thus proving that in H. jecorina gal7 is essential for metabolism of D-galactose, whereas the growth rate of the mutant on lactose was only reduced by about 50%. The rate of formation of cellobiohydrolase Cel7A and the abundance of the corresponding (cbh1) transcript during growth on lactose was only slightly lower in the absence of gal7, but a significant delay in decay of the cbh1 transcript was noted during later stages of growth. The results suggest that H. jecorina uses only the Leloir pathway for metabolism of D-galactose and lactose. Furthermore, we conclude that metabolism of lactose past the galactose-1-phosphate step is not essential for cellulase formation.

Amino Acid Sequence↗
PubMed2002

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.

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.

Adsorption↗
PubMed2000

Cellulase allergy and challenge tests with cellulase using immunologic assessment.

OBJECTIVES: This study attempted to develop and evaluate a challenge test for diagnosing allergic asthma and rhinitis due to cellulase. METHODS: Challenge tests in a chamber were performed on 11 persons sensitized to cellulase. Four different enzyme-lactose mixtures, starting from a 0.03% mixture, were used. The enzyme dust was generated from a dry enzyme preparation mixed with lactose powder, using pressurized air. The cellulase concentration in the air was measured with an immunochemical method. RESULTS: Nasal, pharyngeal, or bronchial symptoms could be elicited at cellulase air concentrations of 1 to 1300 microg/m3. A dose-response relationship was observed for symptoms in repeated challenge tests with increasing concentrations of cellulase. For 2 persons skin symptoms could also be reproduced. CONCLUSION: The challenge method proved to be a practical means with which to simulate conditions at the worksite and elicit the specific respiratory symptoms of the patients.

Adult↗
PubMed2003

Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans.

Clostridium cellulovorans produces a cellulase complex (cellulosome) as well as noncellulosomal cellulases. In this study, we determined a factor that affected the solubility of the cellulosomal cellulase EngB and the noncellulosomal EngD when they were expressed in Escherichia coli. The catalytic domains of EngB and EngD formed inclusion bodies when expressed in E. coli. On the other hand, both catalytic domains containing the C-terminal cellulose-binding domain (CBD) of EngD were expressed in soluble form. Fusion with the CBD of EngD also helped increased the solubility of cellulosomal cellulase EngL upon expression in E. coli. These results indicate that the CBD of EngD plays an important role in the soluble expression of the catalytic domains of EngB, EngL, and EngD. The possible mechanisms of solubilization by fusion of the catalytic domain with the CBD from EngD are discussed.

Amino Acid Sequence↗
PubMed1995

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity.

The celV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 bp and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of CelV1 was highly similar to that of CelV of another E. c. subsp. carotovora strain SCR1193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. CelV1 mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.

Amino Acid Sequence↗
PubMed1994

The use of conserved cellulase family-specific sequences to clone cellulase homologue cDNAs from Fusarium oxysporum.

Five cDNAs from the cellulolytic fungi Fusarium oxysporum that code for five distinct cellulase homologues have been cloned and sequenced. The cloning strategy exploited the hydrophobic cluster analysis-based cellulase family classification of Henrissat and Bairoch [Biochem. J. 293 (1993) 781-788] to design degenerate oligodeoxyribonucleotides (oligos) that encoded amino-acid sequences conserved in an intra-family, but not inter-family, manner among cellulases from different species. Polymerase chain reaction (PCR) experiments using F. oxysporum genomic DNA primed with these 'family-specific' oligos were used to rapidly generate PCR fragments which were in turn used to probe cDNA libraries. Two distinct cDNAs coding for cellulase C-family homologues and one cDNA each coding for homologues to the B, F and K families, were isolated in this manner. This approach is an example of the power of multiple sequence analysis to generate cross-species, homology-based probes to rapidly clone homologues in a species of interest.

Amino Acid Sequence↗
PubMed1991

Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase.

The amino acid sequences of cellulase from Bacillus subtilis (BSC) and that from an alkalophilic Bacillus sp. N-4 (NK1) show significant homology in most parts except for the C-terminal portions. Despite the high homology, the pH activity profiles of the two enzymes are quite different; BSC has its optimum pH at 6-6.5, whereas NK1 is active over a broad pH range from 6 to 10.5. In order to identify the structural features which determine such pH activity profiles, chimeric cellulases between BSC and NK1 were constructed using four restriction sites commonly present within the homologous coding sequences, and were produced in Escherichia coli. The chimeric cellulases showed various chromatographic behaviors, reflecting the origins of their C-terminal regions. The pH activity profiles of the chimeric enzymes in the alkaline range could be classified into either the BSC or NK1 type mainly depending on the origins of the fifth C-terminal regions. In the acidic range, the profile was determined only by the origin of the fourth enzyme region from the N terminus. Comparison of the kinetic parameters between pH 5 and 6 using p-nitrophenyl cellobioside as a substrate indicated that the fourth region is responsible for the pH-dependent change of the kcat value. Only a limited number of amino acids in the fourth region may affect on deprotonation of catalytic residues of the cellulases and modulate the catalytic activity in the acidic pH values.

Amino Acid Sequence↗
PubMed1996

The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase.

Bean leaf abscission (organ separation) correlates with the de novo accumulation of a pI9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5'-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between -1 and -200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5' to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar -210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the -210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.

Base Sequence↗
PubMed1993

Trichoderma reesei has no true exo-cellulase: all intact and truncated cellulases produce new reducing end groups on cellulose.

Adsorption to and formation of insoluble reducing end groups on cellulose was studied for intact enzymes and catalytic domains, 'cores', of the four major cellulases from Trichoderma reesei, CBH I, CBH II, EG I and EG III. Individual enzymes were incubated with NaBH4-reduced, phosphoric acid swollen Avicel (regenerated cellulose) or with filter paper. Adsorption onto regenerated cellulose was rapid (equilibration reached within 2 min), but was slow onto filter paper (not completed after 24 h). On both substrates, less was bound of the core domains than of the intact enzymes. After reaching a maximum in adsorption, all the core domains except CBH I core were released again. In general, the desorption of the core enzymes was much faster than the rate of substrate conversion. All enzymes produced new reducing end groups on both substrates, and thus none of them is a true exo-cellulase. However, both the rate of formation and the amount was considerably higher for the EG enzymes than for the CBH's, which may justify the classification of cellulases into two groups, although the difference is quantitative rather than qualitative. EG III was the most endo-active of the enzymes, and CBH I the least.

Adsorption↗
PubMed2000

Cellulase production of Trichoderma reesei Rut C 30 using steam-pretreated spruce. Hydrolytic potential of cellulases on different substrates.

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C 30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hemicellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and Iogen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C 30. However, the conversion of cellulose using these enzymes was higher than that obtained with Iogen or Celluclast cellulases using steam-pretreated spruce as substrate.

Cellobiose↗
PubMed2003

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

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

Base Sequence↗
PubMed1989

The mechanism of fungal cellulase action. Synergism between enzyme components of Penicillium pinophilum cellulase in solubilizing hydrogen bond-ordered cellulose.

Studies on reconstituted mixtures of extensively purified cellobiohydrolases I and II and the five major endoglucanases of the fungus Penicillium pinophilum have provided some new information on the mechanism by which crystalline cellulose in the form of the cotton fibre is rendered soluble. It was observed that there was little or no synergistic activity either between purified cellobiohydrolases I and II, or, contrary to previous findings, between the individual cellobiohydrolases and the endoglucanases. Cotton fibre was degraded to a significant degree only when three enzymes were present in the reconstituted enzyme mixture: these were cellobiohydrolases I and II and some specific endoglucanases. The optimum ratio of the cellobiohydrolases was 1:1. Only a trace of endoglucanase activity was required to make the mixture of cellobiohydrolases I and II effective. The addition of cellobiohydrolases I and II individually to endoglucanases from other cellulolytic fungi resulted in little synergistic activity; however, a mixture of endoglucanases and both cellobiohydrolases was effective. It is suggested that current concepts of the mechanism of cellulase action may be the result of incompletely resolved complexes between cellobiohydrolase and endoglucanase activities. It was found that such complexes in filtrates of P. pinophilium or Trichoderma reesei were easily resolved using affinity chromatography on a column of p-aminobenzyl-1-thio-beta-D-cellobioside.

Cellulase↗