Cloning and expression of Trichoderma reesei cellobiohydrolase I in Pichia pastoris

被引：45

作者：

Godbole, S ^{[1
]}

Decker, SR ^{[1
]}

Nieves, RA ^{[1
]}

Adney, WS ^{[1
]}

Vinzant, TB ^{[1
]}

Baker, JO ^{[1
]}

Thomas, SR ^{[1
]}

Himmel, ME ^{[1
]}

机构：

[1] Natl Renewable Energy Lab, Biotechnol Ctr Fuels & Chem, Golden, CO 80401 USA

来源：

BIOTECHNOLOGY PROGRESS | 1999年 / 15卷 / 05期

关键词：

D O I：

10.1021/bp9901116

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Pichia pastoris was transformed with the Trichoderma reesei cbh1 gene, and the recombinant enzyme was purified and analyzed kinetically and by circular dichroism. The P. pastoris rCBH I was recognized by MoAb raised to T. reesei CBH I but was found in multiple molecular weight species on SDS-PAGE gels. Carbohydrate content determination and SDS-PAGE western analysis indicated that the recombinant protein was hyperglycosylated, although a species very similar in molecular weight to the T. reesei enzyme could be isolated chromatographically. The P. pastoris rCBH I also demonstrated activity toward soluble and insoluble substrates (i.e., pNPL and Sigmacell), although at a level significantly lower than the wild-type enzyme. More seriously, the yeast-expressed enzyme showed non-wild-type secondary structure by circular dichroism. We conclude that P. pastoris may not serve as an adequate host for the site-directed mutagenesis of T. reesei CBH I.

引用

页码：828 / 833

页数：6