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The photo-induced conidiation of Trichoderma viride is suppressed by ethidium bromide, acriflavin, lomofungin and 8-quinolinol at concentrations which do not inhibit the colony growth of this deuteromycete.
Cellulase produced by Trichoderma viride acted on carboxymethyl cellulose with a Km of 4.9 g substrate per litre, showing a pH optimum at 4.5 and a temperature optimum at 55 degrees C. Ag+, Hg2+, Zn2+, Cu2+ and N3- were inhibitory..
Endoglucanases II, III and IV (EC 3.2.1.4) from Trichoderma viride are highly active in degrading CM-cellulose or phosphoric acid swollen cellulose, and only slightly active on Avicel. The specific activities of the endoglucanases increase with the length of the cellooligosaccharide substrates. By rate and product analyses using high pressure liquid chromatography the mode of action of Endoglucanase III was differentiated from that of Endoglucanases II and IV. Endoglucanase III has a low affinity for cellobiose, reacts rapidly with cellotriose, and gradually increases in reactivity with cellooligosaccharides as degree of polymerization increases from four to six. In addition to cleaving internal glycosidic bonds of polymeric substrates, it preferentially cleaves cellobiosyl units from the non-reducing end of oligosaccharides. The cellobiosyl units are often, under initial reaction conditions, transferred to the substrate-acceptor. Endoglucanases II and IV show a preference for internal glycosidic bonds of cellooligosaccharides. The soluble products from the initial action of Endoglucanases II and IV on swollen cellulose are glucose, cellobiose, and cellotriose, which are slowly converted to glucose and some cellobiose.
Four electrophoretically distinct endo-1,4-beta-D-glucanases (EC 3.2.1.4) from Trichoderma viride have been identified and named as isozymes, Endoglucanases I, II, III and IV, according to their electrophoretic mobilities on polyacrylamide gels. Endoglucanases II, III and IV, the homogeneity of each of which was established by discontinuous gel electrophoresis and ultracentrifugation, had specific activities on CM-cellulose of 1010, 60 and 250 specific fluidity units/mg protein, respectively. These enzymes have similar pH optima (pH 4.0-4.5) and are labile at pH values greater than 8.0. The endoglucanases are high in acidic and hydroxylated amino acids and glycine, but low in basic amino acids. Values of 12.0, 10.3 and 13.1 have been determined for the epsilon 1%280 of purified Endoglucanases II, III and IV, respectively. Sedimentation equilibrium analysis has established the molecular weights of Endoglucanases II, III and IV to be 37 200, 52 000 and 49 500, respectively. The three endoglucanases contain mannose, galactose, glucose and glucosamine. Mannose is the principal neutral sugar in each enzyme. Endoglucanase II is distinguished by its low carbohydrate content, 4.5% (w/w), compared to Endoglucanases III and IV which contain 15.0% and 15.2% carbohydrate, respectively.
A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.
A glycoprotein enzyme, 1,4-beta-D-glucan cellobiohycrolase (EC 3.2.1.91) form C, was purified to electrophoretic homogeneity by a procedure which permitted isolation of gram quantities from a commercial Trichoderma viride culture filtrate preparation. Purified cellobiohydrolase C has an E1%/280 nm = 14.2 and degrades both microcrystalline and phosphoric acid-swollen cellulose to cellobiose. The cellobiohydrolase C contains 26.4, 4.8, 2.4 and 3.4 mol of mannose, glucose, galactose and glucosamine, respectively, per mol of enzyme (molecular weight, 48 400). Methylation analysis of cellobiohydrolase glycopeptides indicates an average carbohydrate chain length of two residues. Alkaline borohydride treatment of cellobiohydrolase C released neutral carbohydrate which is bound through an average of 16.7 O-glycosidic linkages to serine and threonine per molecule of enzyme. Glucosamine was not released from the protein by alkaline treatment. Analysis of alkaline borohydride-released carbohydrate by high pressure liquid chromatography demonstrated that an average enzyme molecule contains 8.8 mono-, 1.8 di-, 4.6 tri-, 1.2 tetra-, and 0.4 pentasaccharide chains. The linkages between the neutral monosaccharides are (1 leads to 6) as shown by gas chromatography - mass spectrometry of partially methylated residues. The (1 leads to 6) linkage is consistent with the stability of the linkages to alkaline conditions and the destruction of all neutral carbohydrate by periodate. Action of alpha-mannosidase indicates that some oligosaccharide chains contain alpha-mannose as the terminal residue.
Four electrophoretically distinct 1,4-beta-D-glucan cellobiohydrolase enzymes (exo-cellobiohydrolase, EC 3.2.1.91) from Trichoderma viride have been purified to homogeneity. Three enzymes (A, B, and C) were from a commercial T. viride preparation whereas the other (D) was from T. viride QM 9123 grown on cellulose in submerged culture. The enzymes were similar with respect to ultraviolet light absorption, amino acid and amino sugar composition, heat stability, molecular weight, specific activity, and carboxyterminal residues, indicating very nearly identical polypeptide portions. The enzymes also exhibited immunological cross-reactivity. The enzymes differed most in the content and composition of covalently bound neutral carbohydrate.
Cellobiohydrolase I from Trichoderma reesei catalyzes the hydrolysis of methyl beta-D-cellotrioside (Km = 48 microM, kcat = 0.7 min-1) with release of the beta-cellobiose (retention of configuration). The same enzyme catalyzes the trans-hydration of cellobial (Km = 116 microM, kcat = 1.16 min-1) and lactal (Km = 135 microM, kcat = 1.35 min-1), presumably with glycosyl oxo-carbonium ion mediation. Protonation of the double bond is from the direction opposite that assumed for methyl beta-cellotrioside, but products formed from these prochiral substrates are again of beta configuration. Cellobiohydrolase II from the same microorganism hydrolyzes methyl beta-D-cellotetraoside (Km = 4 microM, kcat = 112 min-1) with inversion of configuration to produce alpha-cellobiose. The other reaction product, methyl beta-cellobioside, is in turn partly hydrolysed by cellobiohydrolase II to form methyl beta-D-glucoside and D-glucose, presumably the alpha-anomer. Reaction with cellobial is too slow to permit unequivocal determination of product configuration, but clear evidence is obtained that protonation occurs from the si-direction, again opposite that assumed for protonating glycosidic substrates. These results add substantially to the growing evidence that individual glycosidases create the anomeric configuration of their reaction products by means that are independent of substrate configuration.
The p-nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HCl) that did not affect the tryptophan fluorescence of this enzyme. The Km of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combined reversibly with 1 molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.
Site directed mutagenesis has been performed to test hypotheses concerning the putative active sites of Trichoderma reesei cellobiohydrolase I and endoglucanase I. It is shown that mutagenesis of the residue E126, previously proposed to be the proton donor in CBHI, did not totally inactivate the enzyme while mutagenesis of the residue E127 in the homologous enzyme EGI resulted in complete loss of activity. These results are compared with those obtained in similar studies of other glucanases and the effects on enzymatic activity of hyperglycosylation of the yeast produced cellulases are discussed.
The function of the domains of Trichoderma reesei endoglucanase I (EGI) has been studied. Truncated EGI proteins were expressed from the 3'-end deleted cDNAs in the yeast Saccharomyces cerevisiae under the control of the ADC1 expression cassette. EGI protein was detected by monoclonal antibody EI-2 and EGI activity as cleared zones around growing colonies on agar plates containing hydroxyethylcellulose (HEC) covalently stained with Ostazin brilliant red (OBR). The results showed that the The-Ser-rich hinge region and the conserved 'tail' are not necessary for the efficient synthesis and secretion of EGI in yeast, but the intact core region is necessary for the enzymatic activity.
Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.
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.
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.
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.
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.
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.
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.