Abstract: kerD,a keratinase gene derived from Stenotrophomonas maltophilia BBE11-1,was optimized by Pichia codon,and then the recombinant vector pPIC9k-kerD was constructed;the recombinant vector was integrated into the Pichia SMD1168 genome,and Mut⁺ recons were screened;recons resisting G418 were induced by methanol,and the best recombinant strain with the highest enzyme production was screened. The recombinant enzyme was purified and detected by SDS-PAGE,while part of the enzymatic properties was investigated. We found that the optimal reaction pH of recombinant enzyme was 10,and the optimal reaction temperature was 60℃. In order to further increase the yield of recombinant enzyme,multi-carbon source feeding strategy,using methanol mixed with sorbitol and mannitol as carbon source,was used to optimize the formation of producing keratinase by the recombinant strain. The results showed that when mixing ratio of methanol to mannitol was 20∶0.5,fermenting 168 h,production of recombinant keratinase reached 2 048 U/mL,which was improved 87.2% from single methanol carbon.

Key words: keratinase, Pichia pastoris, enzymatic properties, double carbon source, optimization of fermentation