Construction of recombinant Pichia strain GS115-Ch-Glu expressing ß-glucosidase and cyanide hydratase for cyanogenic glycosides detoxification

Abstract

A recombinant Pichia strain GS115-Ch-Glu expressing both ß-glucosidase and cyanide hydratase was constructed to remove cyanogenic glycosides in edible plants. The & ß-glucosidase could hydrolyze cyanogenic glycosides into cyanide and its gene (Glu) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). A cyanide hydratase could catalyze cyanide to formamide and its gene (Ch) was obtained by C (PCR). Both Glu and Ch genes were integrated into the genome of Pichia strain GS115 by homologous recombination. The engineering strain GS115-Ch-Glu was confirmed by multiple analytical methods, and was inoculated to induce  expression of the recombinant Glu and Ch genes. Sodium dodecyl sulfate  polyacrylamide gel electrophoresis (SDS-PAGE) showed that the fused  proteins of interest had specifically been expressed as 31 and 52 KDa, which corresponded to the predicted molecular weights of the two enzymes. The enzyme preparation containing cyanide hydratase and & ß-glucosidase produced by strain GS115-Ch-Glu could completely decompose cyanogenic glycosides in flaxseed power into cyanide, and further catalyze the  hydrolysis of over 80% cyanide.

Key words: Cyanogenic glycosides, recombinant strain, cyanide hydratase, ß-glucosidase, detoxification.