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.
Biotechnology Progress – Wiley
Published: Jan 1, 1999
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