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PubMed1981

The action on cellulose and its derivatives of a purified 1,4-beta-glucanase from Trichoderma koningii.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.

Carboxymethylcellulose Sodium↗

PubMed1983

Cellobiohydrolase from Trichoderma reesei.

A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.

Cellulose 1,4-beta-Cellobiosidase↗

PubMed1984

The disulphide bridges in a cellobiohydrolase and an endoglucanase from Trichoderma reesei.

The positions of the disulphide bridges of the 1,4-beta-glucan cellobiohydrolase (CBH I) of the fungus Trichoderma reesei have been investigated. The results can be summarized as follows. (1) The enzyme contains 12 disulphide bridges and no free cysteine residues. (2) The location of six disulphide bridges have been determined experimentally. (3) The bonding patterns of the two disulphide bridges in the C-terminal region is suggested on the basis of internal homology. (4) The remaining four disulphide bridges are put into two groups, each containing four half-cystine residues where two are adjacent. (5) A repeating bonding pattern is observed along the peptide chain and a non-local disulphide bond with an unusually long separation distance links the N-terminal and the C-terminal region. (6) The disulphide-bonded CNBr peptides of a 1,4-beta-glucan glucanohydrolase (endoglucanase II) from T. reesei have been isolated and a disulphide bonding pattern is suggested on the basis of the sequence homology between the two enzymes.

Amino Acid Sequence↗

PubMed1985

A new appraisal of the endoglucanases of the fungus Trichoderma reesei.

The properties and enzymic activity of endoglucanases (EC 3.2.1.4) of the fungus Trichoderma reesei were studied by means of immunological methods and by using polyglycosidic substrates. Endoglucanases exist in the culture liquid as a series of immunologically related components. The most active endoglucanase component has an Mr of 43 000 and pI value of 4.0. The most abundant components have a value of pI about 5.0, an Mr of 56 000-67 000 and specific activity only one-fifth of that of the pI-4.0 component. During purification and storage the endoglucanases are spontaneously modified; the relative proportion of components having greater Mr values, more alkaline pI values and lower specific activities is increased. The hexose content of the endoglucanase components is 2-7%. Endoglucanases hydrolyse soluble beta-1,4 glycans. The enzymes described here differ from endoglucanase preparations described previously in not showing activity towards insoluble substrates. The role of endoglucanases in wood hydrolysis is consequently limited to the stage where wood constituents are already in soluble form.

Cellulase↗

PubMed1989

Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei.

Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose by the cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilum were determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme from both organisms attacks these substrates in a random manner. Turnover numbers are, however, low and do not increase appreciably as a function of the degree of polymerization of the substrates. The active-site topology of the CBH I from T. reesei was further probed by equilibrium binding experiments with cellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC), spatially restricted as deduced from the apparent independency of the thermodynamic parameters. It appears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, and subsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric group can be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnover numbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzyme increase as a function of chain length, indicative of an extended binding site (A-F). Its architecture allows for specific binding of beta-(1----4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophore in subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of some ligands common to both type of enzyme were compared: these substantiate the conclusions reached above.

Cellulose 1,4-beta-Cellobiosidase↗

PubMed1990

Studies of the cellulolytic system of the filamentous fungus Trichoderma reesei QM 9414. Substrate specificity and transfer activity of endoglucanase I.

Endoglucanase I from the filamentous fungus Trichoderma reesei catalyses hydrolysis and glycosyl-transfer reactions of cello-oligosaccharides. Initial bond-cleaving frequencies determined with 1-3H-labelled cello-oligosaccharides proved to be substrate-concentration-dependent. Using chromophoric glycosides and analysing the reaction products by h.p.l.c., kinetic data are obtained and, as typical for an endo-type depolymerase, apparent hydrolytic parameters (kcat., kcat./Km) increase steadily as a function of the number of glucose residues. At high substrate concentrations, and for both free cellodextrins and their aromatic glycosides, complex patterns (transfer reactions) are, however, evident. In contrast with the corresponding lactosides and 1-thiocellobiosides, and in conflict with the expected specificity, aromatic 1-O-beta-cellobiosides are apparently hydrolysed at both scissile bonds, yielding the glucoside as one of the main reaction products. Its formation rate is clearly non-hyperbolically related to the substrate concentration and, since the rate of D-glucose formation is substantially lower, strong indications for dismutation reactions (self-transfer) are again obtained. Evidence for transfer reactions catalysed by endoglucanase I further results from experiments using different acceptor and donor substrates. A main transfer product accumulating in a digest containing a chromophoric 1-thioxyloside was isolated and its structure elucidated by proton n.m.r. spectrometry (500 MHz). The beta 1-4 configuration of the newly formed bond was proved.

Cellulase↗

PubMed1991

Purification and characterization of an endoxylanase from Trichoderma koningii G-39.

Trichoderma koningii G-39 produced xylanases in submerged culture using oat spelt xylan or crystalline cellulose, Avicel, as the sole carbon source. A low-Mr xylanase was purified from the culture filtrate by ion-exchange chromatography on SP-Trisacryl-M and gel filtration on Fractogel TSK HW-50F. It was homogeneous on SDS/PAGE and isoelectric focusing. A typical procedure provided about 11-fold purification with 4.5% protein yield and 50% activity recovery. The purified enzyme has an Mr value of about 21,500 and a pI of 8.9. Its specific activity was 6100 units/mg of protein, with optimal activity towards 0.5% xylan at about pH 5.5 and 60 degrees C. The purified enzyme had no activity against CM-cellulose with a degree of substitution of 0.63. It also showed no beta-xylosidase activity. The Km and Vmax. values, as determined with the soluble fraction of oat spelt xylan as substrate, were 0.70 mg/ml and 1.85 x 10(6) mumol/min per mg of enzyme respectively. Hg2+ (1 mM) and SDS (10 mM) completely inhibited xylanase activity, whereas Ca2+ showed no significant effect on the enzyme activity at 1 mM, but gave 80% inhibition at 10 mM. The enzyme contained about 4.4% carbohydrate and showed an immunological relationship to a cellobiohydrolase from the same fungal strain.

Amino Acids↗

PubMed1992

Mechanisms of thermoinactivation of endoglucanase I from Trichoderma reesei QM 9414.

The mechanism of irreversible thermoinactivation of endoglucanase I from Trichoderma reesei has been determined at 70 degrees C at the pH of maximum enzyme activity. The time-course of thermoinactivation did not follow first-order kinetics and kinetic constants of the process were dependent on enzyme concentration, suggesting that aggregation was the main process leading to irreversible inactivation. The enzyme was extremely resistant to urea, which in fact seemed to stabilize it against temperature. Disulphide exchange, deamidation and hydrolysis of peptide bonds were also responsible for the loss of enzyme activity at 70 degrees C.

Ammonium Sulfate↗

PubMed1994

Cellulose hydrolysis by the cellulases from Trichoderma reesei: adsorptions of two cellobiohydrolases, two endocellulases and their core proteins on filter paper and their relation to hydrolysis.

Separate binding of several purified cellulolytic components of Trichoderma reesei on to filter paper was studied and concomitant hydrolysis rates evaluated. Enhancement of mass transfer from the bulk liquid to the solid substrate by agitation has two different effects on adsorption depending on the type of enzyme: (i) the fraction of cellobiohydrolase II (CBH II) and endoglucanase III (EG III) bound at equilibrium is increased, whereas (ii) the rate but not the extent of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) adsorption is affected. The adsorption of CBH I core, a component lacking the cellulose-binding domain (CBD), is, however, not significantly influenced by mass transfer. The CBH I interdomain peptide (present in CBH I core b) does not participate in adsorption but enhances stability. The adsorption of CBH I core proteins is a fully reversible process whereas that of the intact CBH I is not. Thus, the interaction of the CBD with filter paper apparently accounts for the mass-transfer-limited binding rate and also for the irreversible adsorption of intact CBH I. Adsorption isotherms at 50 degrees C indicate very similar relative association constants for the intact cellulases (0.24-0.30 l/g of cellulose), but drastically reduced values for CBH I core proteins (0.03 l/g of cellulose). The specific activities of adsorbed CBH I and of its core proteins are identical and a linear relationship between adsorption and rates of hydrolysis is found only for these enzymes. Thus, non-productive binding on to cellulose seems evident in the case of CBH II and EG III but not CBH I.

Adsorption↗

PubMed1989

Mechanism by which cellulose triggers cellobiohydrolase I gene expression in Trichoderma reesei.

The expression of cellobiohydrolase I mRNA from Trichoderma reesei, measured by Northern blot hybridization, is controlled by the nature of carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Cellobiohydrolase I mRNA was undetectable when antibodies to the major members of the cellulolytic system were present in the culture medium prior to the addition of cellulose. These antibodies had no repressive effect if sophorose was used as an inducer. The results strongly suggest that a low constitutive cellulolytic system catalyzes the formation of a soluble inducer from cellulose and that this inducer triggers the expression of the cellobiohydrolase I gene transcript, most probably at the transcription level.

Cellulose↗

PubMed1994

Characterization of ech-42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism.

A gene (ech-42; previously named ThEn-42) coding for one of the endochitinases produced by the biocontrol agent Trichoderma harzianum IMI206040 was cloned and characterized. Expression of the cDNA clone in Escherichia coli resulted in bacteria with chitinase activity. This chitinase has been shown to have lytic activity on Botrytis cinerea cell walls in vitro. The ech-42 gene was assigned to a double chromosomal band (chromosome V or VI) upon electrophoretic separation and Southern analysis of the chromosomes. Primer extension analysis indicated that transcription of the gene begins preferentially 109 bp upstream of the translation initiation codon. Expression of ech-42 was strongly enhanced during direct interaction of the mycoparasite with a phytopathogenic fungus when confronted in vitro and by growing it in minimal medium containing chitin as sole carbon source. Similarly, light-induced sporulation resulted in high levels of transcript, suggesting developmental regulation of the gene. The implications of these findings are discussed.

Amino Acid Sequence↗

PubMed1983

Microbial degradation of trifluralin by Aspergillus carneus, Fusarium oxysporum and Trichoderma viride.

3H-Trifluralin was synthesized by condensation of 3H-4-chloro-3,5-dinitro-alpha, alpha, alpha-trifluorotoluene with di-n-propylamine. After incubation of trifluralin with Aspergillus carneus, Fusarium oxysporum and Trichoderma viride for 10 days, a small percentage (less than 10%) of unchanged herbicide was recovered in the extractable fraction. This indicates a fairly rapid degradation of the herbicide by the fungal species. Other than trifluralin, the culture medium contained at least five labelled products: 2,6-dinitro-N-n-propyl-alpha, alpha, alpha-trifluoro-p-toluidine; 2,6-dinitro-alpha, alpha, alpha-trifluoro-p-toluidine; 2-amino-6-nitro-alpha, alpha, alpha-trifluoro-p-toluidine, 2,6-dinitro-4-trifluoromethyl phenol and a major polar product which constituted more than 50% of the total extractable transformation products. A pathway, which simulates that of aerobic degradation of the herbicide in soil, is suggested for the microbiological degradation of trifluralin.

Aspergillus↗

PubMed1991

Isolation, characterization, and primary structure of a base non-specific and adenylic acid preferential ribonuclease with higher specific activity from Trichoderma viride.

In order to elucidate the structure-function relationship of RNases belonging to the RNase T2 family (base non-specific and adenylic acid-preferential RNase), an RNase of this family was purified from Trichoderma viride (RNase Trv) to give three closely adjacent bands with RNase activity on slab-gel electrophoresis in a yield of 20%. The three RNases gave single band with the same mobility on slab-gel electrophoresis after endoglycosidase F digestion. The enzymatic properties including base specificity of RNase Trv were very similar to those of typical T2-family RNases such as RNase T2 from Aspergillus oryzae and RNase M from A. saitoi. The specific activity of RNase Trv towards yeast RNA was about 13-fold higher than that of RNase M. The complete primary structure of RNase Trv was determined by analyses of the peptides generated by digestion of reduced and carboxymethylated RNase Trv with Staphylococcus aureus V8 protease, lysylendopeptidase and alpha-chymotrypsin. The molecular weight of the protein moiety deduced from the sequence was 25,883. The locations of 10 half-cystine residues were almost superimposable upon those of other RNases of this family. The homologies between RNase Trv and RNase T2, RNase M, and RNase Rh (Rhizopus niveus) were 124, 132, and 92 residues, respectively. The sequences around three histidine residues, His52, His109, and His114, were highly conserved in these 4 RNases.

Adenosine Monophosphate↗

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↗

PubMed1975

Purification and properties of an exo-cellulase component of novel type from Trichoderma miride.

An enzyme extract from Cellulase-Onozuka, a commercial product of Trichoderma viride, was fractionated by Amberlite CG-50 column chromatography into three cellulase [EC 3.2.1.4] groups, peaks I to III. A noval enzyme, which has both beta-glucosidase [EC 3.2.1.21] and exo-carboxymethyl-cellulase (exo-CMCase) properties was obtained from peak III by extensive purification throuh consecutive column chromatography. The enzyme was homogeneous on ultracentrifugation, SDS-gel and cellulose acetate film electrophoreses and molecular sieve chromatography on Bio-Gel P-150. The molecular weight of this enzyme was estimated to be 53,000. The enzyme appeared to release cellobiose residues one by one from the nonreducing end of higher cellooligosaccharides and CM-cellulose (CMC), but to release glucosyl residues from reduced cellotriose and beta-cellobioside, resembling a beta-glucosidase in this respect. Furthermore, this exo-CMCase also attacked xylan exo-wise to produce xylobiose moleculaes one by one, but it scarcely attacked insoluble cellulose, except for a cellodextrin apparently rich in amorphous structure.

Animals↗

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↗

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↗

PubMed1978

Mutarotation of hydrolysis products by different types of exo-cellulases from Trichoderma viride.

Mutarotation of products from p-nitrophenyl beta-D-cellobioside and cellopentaitol by two different types of exo-cellulases from Trichoderma viride was investigated. It was found that an exo-cellulase of glucosidase type produced from the former substrate D-glucose which was mutarotated in a downward direction, while another exo-cellulase of Avicelase type produced from the latter substrate cellobiose which was mutarotated in an upward direction.

Animals↗