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PubMed1993

Rapid release of protoplasts from Eremothecium ashbyii in comparison with Trichoderma reesei and Penicillium chrysogenum using novozyme and funcelase.

Protoplast release in Eremothecium ashbyii, Trichoderma reesei, and Penicillium chrysogenum was achieved using commercially available enzymes, Novozyme 234 and Funcelase. A rapid release of protoplasts was observed in E. ashbyii, yielding nearly 4.0 x 10(7) protoplasts ml-1 in 10-35 min. The regeneration frequency of protoplasts from T. reesei, P. chrysogenum, and E. ashbyii using Funcelase was 51.77, 28.32, and 7.64%, respectively, and was higher in comparison with Novozyme-derived protoplasts.

Biotechnology↗

PubMed1993

Comparison of Ellman's reagent with N-(1-pyrenyl)maleimide for the determination of free sulfhydryl groups in reduced cellobiohydrolase I from Trichoderma reesei.

The enzyme cellobiohydrolase I (CBH I) from Trichoderma reesei was treated with 5 mM dithiothreitol at different pH values in order to reduce some or all of its 12 disulfide bridges. A discrepancy was found in the number of free sulfhydryl (SH) groups generated upon the reduction of CBH I when they were measured using N-(1-pyrenyl)maleimide (PM) or Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid). For example, the number of SH mol generated/mol CBH I at pH 8.5 was determined to be 16 and < 1 when measured using PM or Ellman's reagent, respectively. The low value obtained with Ellman's reagent may be due to the electrostatic repulsion between the carboxylic acid groups in CBH I and those in Ellman's reagent. The fluorimetric assay used for determining SH molecules in reduced CBH I, based on their reaction with PM, is described.

Cellulose 1,4-beta-Cellobiosidase↗

PubMed1990

The conserved terminal region of Trichoderma reesei cellulases forms a strong antigenic epitope for polyclonal antibodies.

The specificity of polyclonal antibodies (Pab) raised against Trichoderma reesei cellulases has been studied. cDNAs lacking regions coding for certain functional domains were produced by preparing series of 3'-end deletions from the cDNAs for two cellobiohydrolases, CBH I and CBH II, and an endoglucanase, EG I. The proteins coded by the full length cDNAs and the truncated proteins coded by the deleted cDNAs were expressed in yeast Saccharomyces cerevisiae, under the control of the ADC1 promoter. Each polyclonal antiserum showed cross-reactivity with other cellulases. Pabs for CBH I and CBH II both recognized EG I. Pab for EG I strongly recognized both CBH I and CBH II. By analyzing the truncated proteins, we found that these antibodies were almost entirely directed against the conserved tail of the cellulase enzymes.

Antibodies, Fungal↗

PubMed1993

Cloning of two isozymes of Trichoderma koningii glyceraldehyde-3-phosphate dehydrogenase with different sensitivity to koningic acid.

Koningic acid inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by binding to the SH group in the active center. The fungus Trichoderma koningii, the producer of koningic acid, contains two GAPDH isozymes (GAPDHs I and II). GAPDH I is inhibited 50% by 1.1.10(-3) M koningic acid, while GAPDH II is inhibited 50% at 6.8 x 10(-6) M. cDNAs of the two isozymes were cloned from T. koningii and their nucleotide sequences were determined. The sequence of coding region and codon usage in both clones were compared with each other and with those of the gene for Aspergillus nidulans GAPDH (enzyme activity is inhibited 50% by 2.7 x 10(-7) M koningic acid). Results indicated that GAPDH II is more closely related to A. nidulans GAPDH than GAPDH I. All essential amino acid residues, except 174 and 181, which are implicated in catalysis and binding of NAD and substrates, were conserved among A. nidulans GAPDH and GAPDHs I and II. Residues 174 and 181 are threonine in both A. nidulans GAPDH and GAPDH II, but alanine and serine, respectively, in GAPDH I. The side-chain of alanine-174 in GAPDH I can not replace threonine-174 functionally as threonine-174 side-chain forms a hydrogen bond with the catalytically essential histidine-176.

Amino Acid Sequence↗

PubMed1990

Comparison of the hydrolytic activity and fluorescence of native, guanidine hydrochloride-treated and renatured cellobiohydrolase I from Trichoderma reesei.

Guanidine hydrochloride (GdnHCl) is an effective agent for the elution of cellulase protein from unhydrolyzed cellulosic residues, but once eluted the enzyme is inactive. The studies described in this paper examine the effect of GdnHCl on the hydrolytic activity and tryptophan fluorescence of cellobiohydrolase I (CBH I) from Trichoderma reesei. CBH I was found to be completely inactivated by 0.25 M GdnHCl, but higher concentrations of GdnHCl were required to partially unfold this enzyme, as determined from the measurement of a decrease in its tryptophan fluorescence. Binding of CBH I to microcrystalline cellulose was prevented by 4 M GdnHCl, suggesting that a conformational change of CBH I resulted in the loss of substrate binding. Removal of the denaturant from CBH I by dialysis or gel filtration allowed the kinetics of the reactivation of CBH I, after 4 M GdnHCl treatment, to be studied. The fluorescence and specific hydrolytic activity of native and renatured CBH I were comparable. It is concluded, therefore, that GdnHCl may be used to elute cellulase components, such as CBH I, adsorbed on undigested cellulosic substrates since this component can easily be renatured and subsequently reused.

Cellulose↗

PubMed1991

A re-appraisal of multiplicity of endoglucanase I from Trichoderma reesei using monoclonal antibodies and plasma desorption mass spectrometry.

An endo beta-1,4-glucanase (EC 3.2.1.4, 1.4-(1,3;1,4)-beta-D-glucan 4 glucanhydrolase) was purified to apparent homogeneity from culture filtrates of Trichoderma reesei QM 9414. Identity of the protein with endoglucanase I (EG I) was examined by subjecting CNBr fragments of the protein to analysis by plasma desorption mass spectrometry. Seven non-glycosylated fragments, mapped on the eg1 gene sequence, could be identified, hence proving at least 39.4% identity of the amino acid sequence. No sign for microheterogeneity was observed. Purified EG I was used to prepare monoclonal antibodies. 17 stable clones were obtained, of which one--Mab EG 3--was used to analyze several commercial T. reesei cellulase preparations as well as culture filtrates from T. pseudokoningii and T. longibrachiatum for the presence of EG I. Most of them contained immunoreactive material migrating as a prominent 50-55 kDa band on SDS-PAGE, resembling EG I, but in some instances additional lower molecular weight bands were also observed. Cultivation of T. reesei at low pH led to an increase of these lower molecular weight bands. EG I was rather stable against proteolysis by papain in vitro, but after prolonged treatment, immunopositive products of 50 and 45 kDa were produced at the expense of the 55 kDa band. Our monoclonal antibodies failed to react with a low-molecular-weight endoglucanase, which was previously shown to be detectable with polyclonal antiserum against EG I. However, all monoclonals reacted with a 118 kDa protein which is most probably a dimer of EG I. These results are discussed with respect to the occurrence of multiple forms of EG I in T. reesei cellulase preparations.

Antibodies, Monoclonal↗

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↗