Regulatory Controls in Relation to Overproduction of Fungal Cellulases

Montenecourt, Bland S.; Nhlapo, S. D.; Trimiño-Vazquez, H.; Cuskey, S M; Schamhart, D. H. J.; Eveleigh, D. E.

doi:10.1007/978-1-4684-3980-9_4

Cited by 21 publications

(9 citation statements)

References 25 publications

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“…The presence of lactose in the growth medium significantly increased the synthesis of the epb and cbh1 transcripts in T. reesei ( Fig. 2A and B; compare lanes 1, 2, and 3), as expected (13,22).…”

Section: Resultssupporting

confidence: 84%

Expression and Secretion of Barley Cysteine Endopeptidase B and Cellobiohydrolase I in Trichoderma reesei

Nykanen¹,

Saarelainen²,

Raudaskoski³

et al. 1997

Appl Environ Microbiol

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Localization of expression and secretion of a heterologous barley cysteine endopeptidase (EPB) and the homologous main cellobiohydrolase I (CBHI) in a Trichoderma reesei transformant expressing both proteins were studied. The transformant was grown on solid medium with Avicel cellulose and lactose to induce the cbh1 promoter for the synthesis of the native CBHI and the recombinant barley protein linked to a cbh1 expression cassette. Differences in localization of expression between the two proteins were clearly indicated by in situ hybridization, indirect immunofluorescence, and immunoelectron microscopy. In young hyphae, native-size recombinant epb mRNA was localized to apical compartments. In older cultures, it was also seen in subapical compartments but not in hyphae from the colony center. The recombinant EPB had a higher molecular weight than the native barley protein, probably due to glycosylation and differential processing in the fungal host. As was found with its transcripts, recombinant EPB was localized in apical and subapical compartments of hyphae. The cbh1 mRNA and CBHI were both localized to all hyphae of a colony, which suggests that the endogenous CBHI was also secreted from these. In immunoelectron microscopy, the endoplasmic reticulum and spherical vesicles assumed to contribute to secretion were labeled by both CBHI and EPB antibodies while only CBHI was localized in elongated vesicles close to the plasma membrane and in hyphal walls. The results indicate that in addition to young apical cells, more mature hyphae in a colony may secrete proteins.

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Section: Resultssupporting

confidence: 84%

Expression and Secretion of Barley Cysteine Endopeptidase B and Cellobiohydrolase I in Trichoderma reesei

Nykanen¹,

Saarelainen²,

Raudaskoski³

et al. 1997

Appl Environ Microbiol

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“…The induction of endoglucanase by cellulose, an insoluble molecule which cannot enter the cell, is held to be mediated by its soluble catabolite sophorose (8,22,26,34). When T. reesei cultures pregrown in glucose or glycerol were transferred to a medium containing sophorose, however, it was interesting to see that the number of endoglucanase forms secreted was considerably lower than on the cellulose-grown culture (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Cellulolytic enzyme synthesis in Trichoderma reesei is subject to control by both induction and carbon catabolite repression (22,26). The mechanism by which an insoluble extracellular substance like cellulose can induce its own catabolism has been the subject of controversial explanations.…”

mentioning

confidence: 99%

“…We have recently presented evidenice that low levels of a conidial endoglucanase and a plasma membrane-boundglucosidase are involved in cellulase induction in T. reesei QM9414 (15). The latter enzyme has been shown to form sophorose-the putative in vivo inducer (8,15,20,21,22,26,34,35)-in vitro (8). The mechanism of carbon catabolite repression has not been dealt-with; it has only been shown that it is not restricted to the presence of glucose but can be observed with several other sugars or glucose metabolites (25).…”

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confidence: 99%

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Differential regulation of synthesis of multiple forms of specific endoglucanases by Trichoderma reesei QM9414

1988

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A method consisting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent detection of endoglucanases by blotting with a polyclonal antibody against endoglucanase I was used to investigate the effect of induction and carbon catabolite derepression on the synthesis of multiple forms of endoglucanase I by Trichoderma reesei. Five forms appeared upon growth on cellulose, whereas four and only two appeared upon growth on lactose (carbon catabolite derepression) and induction by sophorose in a resting cell system, respectively. All endoglucanases detected resembled endoglucanase I in their specificity, since they exhibited no activity toward xylan or paranitrophenyl-,-D-lactobioside. A small (25-kiodalton) endoglucanase only appeared during growth on cellulose. None of the multiple forms arose by postsecretional modification. The results indicate that sophorose may not be the only compound mediating cellulose induction of the specific endoglucanases in T. reesei.

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“…No carboxymethylcellulase could be detected (cf. Montenecourt et al, 1981), but P-glucosidase was present. A separation of intracellular pglucosidase by chromatofocusing revealed two peaks of activity, occurring at each extreme of the fractionation range (Fig.…”

Section: Identijication Of Early Multiple Formsmentioning

confidence: 96%

Carboxymethylcellulase and -Glucosidase Secretion by Protoplasts of Trichoderma reesei

et al. 1985

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Novozym 234, a commercially available enzyme from Trichoderma harzianum, has been used to prepare protoplasts from Trichoderma reesei QM 9414. Optimal conditions were: 20 h old mycelium, 0.9 M-KCl as the osmotic stabilizer, 0.1 % (w/v) Novozym 234 and 18 h incubation at 28 "C. To prevent damage to the protoplasts during isolation the incubation mixtures were agitated by vibration, so avoiding shaking or stirring. More than 95% of the protoplasts were viable and were also metabolically intact as shown by their ability to take up [14C]leucine and incorporate it into cellular protein. Freshly prepared protoplasts could be induced by sophorose to produce and secrete carboxymethylcellulase and P-glucosidase. Optimal conditions were : 0.9 M-KCl as osmotic stabilizer, phosphate buffer pH 6.0, 7 mM-sophorose and lo7 protoplasts ml-' . Separation of the secreted proteins by fast protein liquid chromatography revealed at least seven peaks of carboxymethylcellulase activity and P-glucosidase activity, indicating that some multiplicity of the forms of these enzymes is already present at the secretion stage.

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Regulatory Controls in Relation to Overproduction of Fungal Cellulases

Cited by 21 publications

References 25 publications

Expression and Secretion of Barley Cysteine Endopeptidase B and Cellobiohydrolase I in Trichoderma reesei

Expression and Secretion of Barley Cysteine Endopeptidase B and Cellobiohydrolase I in Trichoderma reesei

Differential regulation of synthesis of multiple forms of specific endoglucanases by Trichoderma reesei QM9414

Carboxymethylcellulase and -Glucosidase Secretion by Protoplasts of Trichoderma reesei

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