Robert Messner

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains.

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and β-octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.

Disruption of the Trichoderma reesei cbh2 gene coding for cellobiohydrolase II leads to a delay in the triggering of cellulase formation by cellulose

The role of the major conidial-bound cellulase - cellobiohydrolase I1 (CBH 11) - in the triggering of cellulase formation in the fungus Trichodkrma reesei was investigated by comparing the mutant strain QM 9414 with a recombinant strain unable to produce CBH 11. For this purpose, the cbh2 gene was isolated from a chromosomal gene bank of T. reesei, cloned into pGEM-7Zf( + ), and disrupted by insertion of the homologouspyr4 gene in its coding region to yield the plasmid vector pSB3. Transformation of the auxotrophic, pyr4-negative strain T. reesei TU-6 with pSB3 yielded 23 stable prototrophs, of which three were unable to produce CBH I1 - assessed by means of a monoclonal antibody - during growth on lactose or in the presence of sophorose. However, they formed cellobiohydrolase I (CBH I) at a rate comparable to strain QM 9414 under these conditions. Southern analysis of DNA of some CBH 11- and CBH 11+ transformants confirmed that pSB3 had integrated at the cbh2 locus in the CBH 11- strains. The latter displayed normal growth on glucose or maltose as carbon source. They showed retarded growth on cellulose as sole carbon source, however, and exhibited a lag in the time course of CBH I and EG I formation, although producing roughly the same final cellulase activities. It is concluded from these results that CBH I1 is not essential for induction of cellulase formation by cellulose, but that it contributes significantly to the formation of lower molecular mass inducers in the early phase of growth of the fungus on cellulose.

A monoclonal antibody against the alkaline extracellular β-glucosidase from Trichoderma reesei: reactivity with other Trichoderma β-glucosidases

Monoclonal antibodies were produced against the major extracellular β-glucosidase (76 kDa, isoelectric point 8.4) from Trichoderma reesei, which were able to detect as little as 10 ng of β-glucosidase on Western blots. They recognized a single protein of 76 kDa within the extracellular proteins secreted by T. reesei grown on either cellulose or lactose upon SDS-polyacrylamide gel electrophoresis (PAGE)/Western blotting, which usually was accompanied by one or two closely migrating antigenic proteins, thus indicating microheterogeneity. The antibodies did not react with β-glucosidase from commercial preparations of other Trichoderma sp., nor with those from Aspergillus niger or Penicillium funiculosum. The antibody did, however, recognize the partially purified β-glucosidase solubilized from T. reesei cell membranes and that solubilized from cell walls. It did not recognize the partially purified intracellular β-glucosidase. Separation of crude culture filtrates from T. reesei by FPLC always exhibited one major (IP 8.4) and two minor (IP 4.2 and 4.5) peaks of β-glucosidase activity, which, however, appeared similar in SDS-PAGE/Western blotting and immunostaining. Rechromatofocusing of the ‘acid’ peaks always produced peaks at IP 8.4. Culture filtrates from fermentations run at pH < 4 or from old cultures (older than 7 days) exhibited additional β-glucosidase peaks (IP 5.7–6), which, however, did not react with the antibody. Partial proteolysis of β-glucosidase by trypsin led to rapid appearance of antigenicity in a small peptide fragment of about 20 kDa, concomitantly with disappearance of the 76 kDa band. SDS-PAGE of the degraded β-glucosidase showed that newly formed proteins of 50 and 37 kDa had been formed. Upon chromatofocusing of partially proteolyzed β-glucosidase, two peaks of activity appeared at pH 5.7 and 6.1. From these results it is concluded that the multiplicity of β-glucosidase from T. reesei is due to anomalous chromatographic behaviour and partial proteolysis of a single enzyme species.

O-glycosylation of proteins by membrane fractions of Trichoderma reesei QM 9414

In order to investigate O-glycosylation of proteins in the fungus Trichoderma reesei QM 9414, a membrane preparation was isolated and used to study the glycosylation of endogenous proteins. Exogenously added GDP-[U-14C]mannose was used to mannosylate both endogenous lipid and protein. The kinetics of mannosylation together with pulse-chase experiments with cold GDP-mannose revealed that lipid was labelled before protein. The lipid was identified as mannosyl phosphoryl dolichol (Dol-P-Man) by TLC together with an authentic standard from yeast. Addition of tsushimycin, a specific inhibitor of Dol-P-Man synthesis, completely blocked transfer of mannose from GDP-[U-14C]mannose to endogenous lipid. The mannosyl units transferred to endogenous protein could be released by β-elimination, and were shown to consist mainly of tetra-, di- and monomannosyl chains. Mannosylation of endogenous proteins occurred at a lower rate with membranes isolated from glycerol-grown cells. This could be overcome by addition of cold GDP-mannose, suggesting a limitation of endogenous GDP-mannose and/or dolichol phosphate in glycerol-grown (i.e. catabolite-repressed) cells.

Evidence for a single, specific β-glucosidase in cell walls from Trichoderma reesei QM9414

Abstract Cell walls were isolated from mycelia of Trichoderma reesei QM9414 after growth on cellulose, lactose, cellobiose, and glycerol as a carbon source and used to investigate some properties of the wall-associated β-glucosidase. Thermal inactivation studies provided evidence that the β-xylosidase activity is not a side-specificity of β-glucosidase. This was further substantiated by the findings that the activity against para-nitrophenol-β- d -glucoside (PNPG) and cellobiose, but not against para-nitrophenyl-β- d -xyloside, was strongly inhibited by nojirimycin, a reputed inhibition of β-glucosidases. The following findings were obtained in favor of a single β-glucosidase being responsible for both PNPG and cellobiose cleavage: (i) activity of the cell-wall-bound enzyme against both substrates displays a constant ratio, irrespective of the conditions of growth; (ii) activity with one substrate is inhibited in a competitive manner by the other substrate; (iii) the effect of temperature inactivation is comparable on both activities. From these data we conclude that the cell-wall-bound β-glucosidase activity from T. reesei is due to a single, specific β-glucosidase, closely resembling the one found in the extracellular medium.

Presence, transcription and translation of cellobiohydrolase genes in several Trichoderma species

SummaryEight different species of Trichoderma (T. virgatum, T. longibrachiatum, T. harzianum, T. pseudokoningii, T. polysporum, T. koningii, T. todica, T. saturnisporum), and three strains of T. reesei [QM 6a (wildtype), QM 9123 and QM 9414 (derived mutants)] were found to contain single copies of the cellobiohydrolase genes cbh1 and cbh2 in their genome. This was demonstrated by hybridization of the respective chromosomal DNAs with the corresponding gene fragments of T. reesei QM 9414. According to the relative position of cbh1 and cbh2 in Southern blots, T. harzianum, T. virgatum and T. saturnisporum were clearly distinguishable as unique species. Despite the presence of both cbh genes, these species did not form detectable cellobiohydrolase (CBH) I or II, or exhibit any cellulase activity. All other taxa were identical with respect to the genomic position of cbh1, formed two groups with respect to the position of cbh2, and produced varying amounts of CBH I and II. In all cases CBH I and II production correlated with the relative amount of cbh1- and cbh2-mRNA found. This was particularly true for the three strains of T. reesei, which secreted different amounts of CBH I and II, their efficiency to transcribe cbh1 and cbh2 having been increased as a result of mutation for higher cellulase production.