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At least 19 recordsLinked to original sources
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↗
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↗
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↗
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↗
PubMed1992

Biochemistry and genetics of actinomycete cellulases.

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.

Actinomycetales↗
PubMed1975

Regulation and in vitro translation of messenger ribonucleic acid for cellulase from auxin-treated pea epicotyls.

Polysomal RNA was isolated from pea epicotyls treated with (2,4-dichlorophenoxy)acetic acid, and fractionated on oligo(dT)-CELLULOSE TO YIELD POLY(A)-containing RNA. This RNA fraction was translated in a wheat embryo cell-free system and found to have more than 90% of the messenger activity in total polysomal RNA. Immunoprecipitation of the translation product by monospecific antibodies to pea cellulases (beta-1,4-glucan 4-glucanohydrolase, EC 3.2.1.4) indicated that cellulase was synthesized in this system. The immunoprecipitate co-migrated with the buffer-soluble cellulase component in sodium dodecyl sulfate-gel electrophoresis. Buffer-insoluble cellulase was not detected in the in vitro translation products. Fractionation of mRNA from membrane-bound and free polysomes and their subsequent translation indicated preferential synthesis of buffer-soluble cellulase on membrane-bound polysomes. With the above techniques for assaying buffer-soluble cellulase mRNA, a 10-fold increase in the level of this messenger per tissue segment was observed within 48 hours following (2,4-dichlorophenoxy)acetic acid treatment. There was no evidence for pre-existing untranslated message for cellulase in control tissues. Since there was no delay in the appearance of mRNA for cellulase, compared to a 24-hour lag in the increase of cellulase activity, it is suggested that translational as well as transcriptional controls are exerted on the biosynthesis of cellulase in vivo. Analysis of the rates of peptide chain initiation and elongation, both in vivo and in vitro, indicated that peptide chain elongation may be rate-limiting during the lag phase of cellulase development.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1975

Cellulases from Sporocytophaga myxococcoides. Purification and Properties.

Two extracellular cellulases active on carboxymethylcellulose have been isolated from the culture supernatant of Sporocytophaga myxococcoides by a series of gel-filtration and ion-exchange chromatography steps. Cellulase II, being present in highest amount, had a molecular weight determined by gel electrophoresis of 52000, pI 4.75 and a relatively broad pH optimum (5.5--7.5). Cellulase I had a molecular weight of 46000. pI was 7.5 and the pH optimum 6.5--7.5. Both cellulases had a very low carbohydrate content, possibly present as impurities. They had fairly similar amino acid compositions. The specific acitivity of cellulase I was about 6 times higher than that of cellulase II. Both cellulases acted as endoglucanases. A cell-associated cellulase, present in amounts corresponding to about 10% of total activity, was partly purified. It showed similarities with cellulase II.

Amino Acids↗
PubMed1985

Multiplicity in cellulases of Schizophyllum commune. Derivation partly from heterogeneity in transcription and glycosylation.

The white-rot fungus, Schizophyllum commune, secretes a member of each of three classes of cellulases: a beta-glucosidase, an exoglucanase, and an endoglucanase. Antibodies were developed to members of each of these three enzyme classes. Secretion of these cellulases is induced when a mycelium is transferred from a glucose to cellulose medium. The maximum level of cellulase transcripts, as indicated by the ability to direct biosynthesis of these cellulases in the rabbit reticulocyte cell-free translation system, occurred when the rate of secretion was maximum. This implied that initial regulation, at least, of cellulase biosynthesis occurs at the transcriptional level. There were two distinct mRNA-directed products for each of the cellulases, with sizes estimated to be, for the beta-glucosidase, 95700 and 93800, for the exoglucanase, 59300 and 58200, and for the carboxymethylcellulase, 40600 and 39400. The secreted cellulases are largely glycosylated, as indicated by their binding to concanavalin A and their incorporation of D-[3H]mannose. The labelled protein was fractionated on concanavalin-A-agarose; about 70% of the label was bound. A small amount of each of the cellulases appeared in the unbound fraction; the remainder appeared in fractions eluted with 10 mM methyl glucoside or with 100 mM methyl glucoside plus 500 mM methyl mannoside. These results indicated each of the cellulases had an additional heterogeneity in glycosylation, with the most heavily glycosylated and highest molecular weight form eluting last from the concanavalin-A-agarose. Although tunicamycin (5 micrograms/ml) blocked glycosylation, there was still some secretion but at a reduced rate which was more pronounced for the beta-glucosidase than for the carboxymethylcellulase activity. The size of the tunicamycin-secreted product in each case was, within experimental error, equivalent to that of the mRNA-directed one.

Agaricales↗
PubMed2026

Trichoderma reesei Nsd3 transcription factor: pleiotropic roles in development, stress response, secondary metabolism, and cellulase production.

Trichoderma reesei is known for its ability to secrete high amounts of cellulases, enzymes of fundamental importance in generating products from lignocellulosic biomass. Diverse signaling pathways and transcription factors (TFs) control the cellulolytic repertoire in T. reesei to ensure correct adaptation to the environment. Here, we analyzed RNA-Seq data and identified a new potential regulator of cellulase production in T. reesei: a novel TF named Nsd3, a homolog of NsdC from Aspergilli. Deletion of nsd3 reduced vegetative growth and conidiation on solid medium. Phenotypic characterization of the Δnsd3 strain showed that it is more sensitive to osmotic stress, but more resistant to cell wall and oxidative stresses. Our results showed that Nsd3 is a repressor of cellulase expression by directly regulating key genes in the cellulolytic pathway, an unreported role for this TF in fungi. Loss of nsd3 leads to a faster and more robust induction of cellulolytic genes, and higher cellulase and hemicellulase activities. Transcriptional profiling by RNA-Seq, chromatin accessibility profiling by ATAC-Seq, and protein-DNA interaction assays showed that sugar transporters are important targets of Nsd3 during cellulase expression regulation. Combined with microscopy and gene expression analyses, the ATAC-Seq data also highlighted Nsd3 as a central regulator of cell wall remodeling and organization. Furthermore, the transcriptomics also showed that Nsd3 regulates genes involved in secondary metabolism. These results showed that Nsd3 regulates several physiological processes and provide novel insights into the regulatory system of cellulases in T. reesei that can be used in the design of high-performance strains for biorefinery.IMPORTANCETrichoderma reesei is a key player in the production of hydrolytic enzymes for the degradation of lignocellulose biomass, and transcription factors are important targets for genetic engineering to construct cellulase-hyperproducing strains. Here, we identified the transcription factor Nsd3 and characterized its role as a regulator of cellulase production in T. reesei. We applied two powerful genomics methods (transcriptome sequencing and chromatin accessibility sequencing) to unravel the global role of Nsd3 and its regulatory mechanism. Nsd3 participates in various biological processes in T. reesei, including cell wall remodeling, calcium metabolism, and secondary metabolism, in addition to regulating the expression of sugar transporters. Protein-DNA interaction assays demonstrate that Nsd3 acts through important genes to regulate cellulase expression, including ace4, crt1, stp1, and cel1b. Our study provides mechanistic insights about how Nsd3 regulates diverse physiological processes in T. reesei. This work also applied ATAC-Seq for the first time to study chromatin accessibility in T. reesei.

ATAC-Seq↗
PubMed1990

Isolation and characterization of a cellulase gene family member expressed during avocado fruit ripening.

We present in this paper the structural analysis of two members of a small cellulase gene family, designated cel1 and cel2, from avocado. These genes were isolated by screening a lambda EMBL3 genomic library with a ripening-induced cellulase cDNA. Restriction endonuclease and Southern blot analyses showed that the cel1 gene is highly homologous to the cellulase cDNA and thus represents a ripening-related cellulase gene. The other cellulase gene, cel2, is closely related to cel1, but is divergent at its 5' end. The nucleotide sequence of a 5 kb region encompassing the cel1 gene was determined. Four previously characterized cellulase cDNAs from ripe fruit are identical to the eight exons of the cel1 gene. RNase protection and primer extension analyses were used to define the transcription start site of cel1 and to quantitate cel1 transcripts in ripening fruit. The cel1 mRNA was present at a low level in unripe fruit and increased 37-fold during ripening. Partial DNA sequence analysis of cel2 and comparison to the cel1 sequence revealed a high degree of similarity both at the DNA and deduced amino acid sequence levels. No characterized cellulase cDNAs derived from ripe fruit represent cel2 transcripts. These data suggest that the cel1 gene is responsible for a major portion, if not all, of the cellulase transcripts in ripe fruit. The DNA sequence of 1.4 kb of 5' flanking DNA of the cel1 gene was compared to the upstream sequence of other ethylene-regulated genes. Several interesting upstream sequence motifs were identified and are discussed.

Amino Acid Sequence↗
PubMed1977

Chemical modification of cellulase from Aspergillus niger.

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.

Aspergillus niger↗