| Literature DB >> 10514252 |
S Godbole1, S R Decker, R A Nieves, W S Adney, T B Vinzant, J O Baker, S R Thomas, M E Himmel.
Abstract
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.Entities:
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Year: 1999 PMID： 10514252 DOI： 10.1021/bp9901116
Source DB: PubMed Journal: Biotechnol Prog ISSN： 1520-6033