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PubMed1992

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.

Cations, Divalent↗

PubMed1990

Biological control of Penicillium digitatum by Trichoderma viride on postharvest citrus fruits.

In previous studies it was shown that Trichoderma viride, isolated from Spanish citrus packing houses, showed antagonistic activity against Penicillium digitatum in in vitro laboratory tests. In the present in vivo studies Navelina oranges, protected with aqueous suspension of T. viride (2.5 x 10(6) to 2.5 x 10(9) spores per ml), showed an increase in resistance toward P. digitatum. Oranges, inoculated with P. digitatum, did not produce lesions after 5 days when T. viride was applied 48 h or 72 h before inoculation.

Citrus↗

PubMed1990

Pilot scale production of a heterologous Trichoderma reesei cellulase by Saccharomyces cerevisiae.

Cellobiohydrolase II of Trichoderma reesei was produced in laboratory and pilot scale using a transformant strain of Saccharomyces cerevisiae harbouring a multicopy expression plasmid. Different strategies were compared for concentration and partial purification of the enzyme produced in a 200 1 pilot cultivation. After efficient separation of biomass and sub-cellular particulate matter, a combination of ultrafiltration and adsorbent treatment for removal of protein impurities was used to provide a concentrate for chromatographic purification. Effective purification of the CBH II protein was obtained by passing the concentrate through a column of DEAE Sepharose, on which almost all the yeast proteins were adsorbed. The purified enzyme reacted with antibodies prepared against T. reesei CBH II and catalyzed partial solubilization of crystalline cellulose to soluble sugars.

Biotechnology↗

PubMed1991

Pilot scale production of a Trichoderma reesei endo-beta-glucanase by brewer's yeast.

Endo-beta-glucanase I (EGI) of Trichoderma reesei was produced in laboratory and pilot scale using recombinant strains of "bottom-fermenting" Saccharomyces cerevisiae. The gene eg/1 was integrated in the chromosome or an expression cassette was inserted on a multicopy plasmid. Expression levels were compared in a laboratory scale bioreactor. The best EGI-producing strain was cultivated in pilot scale. Adsorbent treatment was used to remove endogenous yeast proteins and other impurities from the culture filtrate during concentration. Effective pilot scale one-step purification of the EGI protein was obtained using DEAE-Sepharose, on which EGI was weakly bound. The purified enzyme reacted with antibodies prepared against T. reesei EGI and catalyzed the hydrolysis of both insoluble and soluble substrates.

Fermentation↗

PubMed1991

The 1,4-beta-D-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei.

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.

Amino Acid Sequence↗

PubMed1993

Expression and characterisation of chymosin pH optima mutants produced in Trichoderma reesei.

The production of chymosin mutants designed to have altered pH optima using the cellulolytic filamentous fungus Trichoderma reesei is described. The strong promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has been used for the expression and secretion of active calf chymosin. Structural analysis of the hydrogen bonding network around the two active site aspartates 32 and 215 in chymosin have suggested that residues Thr 218 and Asp 303 may influence the rate and pH optima for catalysis. The chymosin mutants Thr218Ala and the double mutant Thr218Ala/Asp303Ala have been made by site-directed mutagenesis and expressed in T. reesei. Enzyme kinetics of the active enzyme T218A indicate a pH optimum of 4.2 compared to 3.8 for native chymosin B using a synthetic octa-peptide substrate, confirming the previous analysis undertaken in E. coli. The double mutant T218A/D303A exhibits a similar optimum of 4.4 to that reported for the D303A, indicating that the combination of these changes is not additive. The application of protein engineering in the rational design of specific modifications to tailor the properties of enzymes offers a new approach to the development of industrial processes.

Amino Acid Sequence↗

PubMed1994

On the safety of Trichoderma reesei.

Trichoderma reesei has a long history of safe use in industrial-scale enzyme production. Applications of cellulases and xylanases produced by this fungus are found in food, animal feed, pharmaceutical, textile and pulp and paper industries. T. reesei is non-pathogenic for man and it has been shown not to produce fungal toxins or antibiotics under conditions used for enzyme production. During recent years genetic engineering techniques have also been used to improve the industrial production strains of T. reesei and, in addition, considerable experience of safe use of recombinant T. reesei strains in industrial scale has accumulated. Thus, T. reesei can be generally considered not only a safe production organism of its natural enzymes but also a safe host for other harmless gene products.

Allergens↗

PubMed1995

Safety evaluation of beta-glucanase derived from Trichoderma reesei: summary of toxicological data.

Barlican, a beta-glucanase enzyme obtained from Trichoderma reesei, was produced by a fermentation process and subjected to a series of toxicological tests to document its safety for use as a feed additive. The enzyme product was examined for general oral toxicity, inhalation toxicity, irritation to eye and skin, skin sensitization and mutagenic potential. An extensive literature search on the production organism was also conducted. Furthermore, safety for target species was assessed in a 28-day oral toxicity study with broilers. A strong skin-sensitizing potential of the beta-glucanase enzyme was detected, but no other evidence of oral or inhalation toxicity, mutagenic potential, eye or skin irritancy was found. Feeding of the beta-glucanase enzyme at dietary levels up to 10,000 ppm in the 90-day subchronic toxicity study in rats did not induce noticeable signs of toxicity. In addition, no adverse effects were observed when broiler chicks were fed dietary concentrations of the beta-glucanase enzyme up to eight times the daily recommended dose. It is therefore concluded that this beta-glucanase preparation is safe for use in feed of the intended target species. However, some occupational health precautions should be taken to avoid skin contact and inhalation, as is the case for almost all enzyme proteins.

Administration, Oral↗

PubMed1988

Hydrolysis of cellulose by a mixture of Trichoderma reesei cellobiohydrolase and Aspergillus niger endoglucanase.

Two endoglucanase-containing fractions were separated from Aspergillus niger cellulase by gel filtration and fast protein liquid chromatofocusing (FPLC). They possessed no ability to bind to or hydrolyze insoluble microcrystalline cellulose (Avicel) but were active toward soluble carboxymethylcellulose. No synergism was observed between Trichoderma reesei cellobiohydrolase I and either endoglucanase from A. niger. These findings may indicate that the role of the endoglucanase component of cellulase in insoluble microcrystalline cellulose hydrolysis is dependent upon its ability to be adsorbed upon the substrate.

Aspergillus niger↗

PubMed1990

Kinetic mechanism of beta-glucosidase from Trichoderma reesei QM 9414.

beta-Glucosidase is a key enzyme in the hydrolysis of cellulose to D-glucose. beta-Glucosidase was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as carbon source. The enzyme hydrolyzed cellobiose and aryl beta-glucosides. The double-reciprocal plots of initial velocity vs. substrate concentration showed substrate inhibition with cellobiose and salicin. However, when p-nitrophenyl beta-D-glucopyranoside was the substrate no inhibition was observed. The corresponding kinetic parameters were: K = 1.09 +/- 0.2 mM and V = 2.09 +/- 0.52 mumol.min-1.mg-1 for salicin; K = 1.22 +/- 0.3 mM and V = 1.14 +/- 0.21 mumol.min-1.mg-1 for cellobiose; K = 0.19 +/- 0.02 mM and V = 29.67 +/- 3.25 mumol.min-1.mg-1 for p-nitrophenyl beta-D-glucopyranoside. Studies of inhibition by products and by alternative product supported an Ordered Uni Bi mechanism for the reaction catalyzed by beta-glucosidase on p-nitrophenyl beta-D-glucopyranoside as substrate. Alternative substrates as salicin and cellobiose, a substrate analog such as maltose and a product analog such as fructose were competitive inhibitors in the p-nitrophenyl beta-D-glucopyranoside hydrolysis.

Benzyl Alcohols↗

PubMed1991

Transglycosylation activity of cellobiohydrolase I from Trichoderma longibrachiatum on synthetic and natural substrates.

Using 4-methylumbelliferyl (MUF) beta-D-cellobioside as a substrate, the ability of cellobiohydrolase I from Trichoderma longibrachiatum to catalyze transglycosylation has been demonstrated. At substrate concentrations greater than 2 mM, the formation of MUF-tetrasaccharide was detected using HPLC. In the course of enzymatic reaction, a concentration of the transglycosylation product passed through a maximum, since at later stages of the reaction the product was further hydrolyzed. At MUF-beta-D-cellobioside concentrations of 2-10 mM, the maximum weight content of MUF-tetrasaccharide amounted to 1-4% of the total content of saccharides. In the reaction system, containing 2.5 mM MUF-beta-D-cellobioside and 10 mM MUF-beta-D-glucoside, MUF-trisaccharide was formed as the main transglycosylation product. In hydrolysis of natural substrates (cellulose and cellotriose) in the presence of MUF-beta-D-glucoside a formation of MUF-trisaccharide was also observed.

Cellobiose↗

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↗

PubMed1995

Family A cellulases: two essential tryptophan residues in endoglucanase III from Trichoderma reesei.

Three tryptophan residues are readily oxidised by N-bromosuccinimide in endoglucanase III from Trichoderma reesei. Evidence was obtained that the residue first modified is situated in the cellulose-binding domain and the second in the enzyme's catalytic site. The latter influences the binding and hydrolysis of soluble substrates. The modification of a third residue does not further affect the catalytic properties. The present results complement published data concerning other identified catalytic residues, and help to clarify the active site structure of family A cellulases.

Amino Acid Sequence↗

PubMed1992

Glucoamylase P gene of Hormoconis resinae: molecular cloning, sequencing and introduction into Trichoderma reesei.

The glucoamylase P gene of the fungus Hormoconis resinae has been cloned and sequenced from a genomic library. The gene consists of a 2153-bp protein coding region including three introns. The usual number of introns in cloned fungal glucoamylase genes has been four and in some cases five. Two of the glucoamylase P gene introns contain a sequence resembling the consensus sequence found near the 3' splice site in the introns of the fungus Trichoderma reesei cellobiohydrolase 1 (cbh1) gene. The H. resinae glucoamylase P gene, under its own promoter, was introduced into T. reesei, but no expression could be detected.

Amino Acid Sequence↗

PubMed1992

Light-activated adenyl cyclase from Trichoderma viride.

The effect of light on adenyl cyclase (E.C. 4.6.1.1) and 3':5'-cyclic-AMP-phosphodiesterase (E.C. 3.1.4.17) activity of Trichoderma viride was investigated. Adenyl cyclase proved to be a membrane-associated enzyme, requiring Mn2+ and was activated by light. In contrast, 3':5'-cyclic-AMP-phosphodiesterase showed no light-stimulated activity. The activity of 3':5'-cyclic-AMP-phosphodiesterase was present mainly in the cytosol and was stimulated by Mg2+.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Intraspecific hybridisation of Trichoderma pseudokoningii by anastomosis and by protoplast fusion.

Double auxotrophic and morphological mutants of Trichoderma pseudokoningii Rifai were fused by anastomosis and by protoplast fusion. The recovery of recombinants from heterokaryons on different selective media and from heterokaryotic colonies indicated the occurrence of parasexual events. Prototrophic colonies growing on minimal medium produced binucleate spores, green in colour, revealing a non-autonomous system for conidial pigmentation. Recombinants were obtained from these dikaryotic colonies suggesting the occurrence of a highly unstable diploid phase.

Cellulase↗

PubMed1992

Isolation and properties of a low molecular mass endoglucanase from Trichoderma reesei.

Optimal culture conditions for obtaining low molecular mass endoglucanase (EG) from culture fluids of Trichoderma reesei were determined. The purification of this unglycosylated EG in a two-step procedure is described. In contrast to most cellulases, this EG did not bind to ConA-affinity columns. The unglycosylated fraction of the culture fluid proteins was further purified by preparative isoelectric focusing. Conditions relevant to an enzyme assay for this EG were determined (pH optimum 5.8, temperature optimum 52 degrees C).

Cellulase↗

PubMed1986

Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene.

The filamentous fungus Trichoderma reesei produces several endoglucanases (EG) and cellobiohydrolases (CBH) which are involved in cellulose hydrolysis in a complex synergistic manner. We have cloned and sequenced the gene and the full-length cDNA coding for the major endoglucanase EG-I, and compared this to the cbh1 gene sequence to clarify the relationship between the EG and CBH classes of cellulases. The deduced 437-amino acids (aa) long EG-I protein with a 22-aa long signal peptide is 45% identical in aa sequence with CBH-I. The best conserved region is found at the C terminus and shows about 70% homology. The data suggest that the two enzymes have arisen from a common ancestor by gene duplication. Despite this, the intron positions have not been conserved in these genes which both contain two short introns. The deduced EG-I sequence contains six putative N-glycosylation sites, and a putative O-glycosylated region is found near the C terminus, closely resembling a similar region at the C terminus of CBH-I. Comparison of the aa sequences suggests that the evolutionary divergence of EG-I from CBH-I has involved four separate 10-20 aa "deletions" from the ancestral protein.

Amino Acid Sequence↗