Characterization of Trichoderma reesei cellobiohydrolase CeI7A secreted from Pichia pastoris using two different promoters

Citation
H. Boer et al., Characterization of Trichoderma reesei cellobiohydrolase CeI7A secreted from Pichia pastoris using two different promoters, BIOTECH BIO, 69(5), 2000, pp. 486-494
Citations number
31
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biotecnology & Applied Microbiology",Microbiology
Journal title
BIOTECHNOLOGY AND BIOENGINEERING
ISSN journal
0006-3592 → ACNP
Volume
69
Issue
5
Year of publication
2000
Pages
486 - 494
Database
ISI
SICI code
0006-3592(20000905)69:5<486:COTRCC>2.0.ZU;2-M
Abstract
Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotr ophic yeast Pichia pastoris was tested both under the P. pastoris alcohol o xidase (AOX1) pro meter and the glyceraldehyde-3-phosphate dehydrogenase (G AP) promoter in a fermenter. Production of Ce17A with the AOX1 promoter gav e a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N-glycosylation sites as compared to the native T. reesei protein, but les s extensive than Cel7A expressed in Saccharomyces cerevisiae. The k(cat) an d K-m values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH o ptimum also closely resembles that of purified T. reesei Cel7A. Furthermore , the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the o ther hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. Th e constitutive GAP promoter expression system of P. pastoris is nevertheles s well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site-direc ted and random mutants with, for instance, an altered optimum pH is possibl e, in contrast to the homologous T. reesei expression system. (C) 2000 John Wiley & Sons, Inc.