Cloning and Expression of Trichoderma reesei Cellobiohydrolase I in Pichia pastoris

Shubhada Godbole, Stephen R. Decker, Rafael A. Nieves, William S. Adney, Todd B. Vinzant, John O. Baker, Steven R. Thomas, Michael E. Himmel

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46 Scopus Citations

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.

Original languageAmerican English
Pages (from-to)828-833
Number of pages6
JournalBiotechnology Progress
Volume15
Issue number5
DOIs
StatePublished - 1999

NREL Publication Number

  • NREL/JA-580-27873

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