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首页> 外文期刊>African Journal of Biotechnology >Construction of recombinant Pichia strain GS115-Ch-Glu expressing -glucosidase and cyanide hydratase for cyanogenic glycosides detoxification
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Construction of recombinant Pichia strain GS115-Ch-Glu expressing -glucosidase and cyanide hydratase for cyanogenic glycosides detoxification

机译：表达-葡糖苷酶和氰化物水合酶的重组毕赤酵母菌株GS115-Ch-Glu的生氰苷解毒

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摘要

A recombinant?Pichia?strain GS115-Ch-Glu expressing both β-glucosidase and cyanide hydratase was constructed to remove cyanogenic glycosides in edible plants. The β-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 ofPichia?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 β-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.
机译：构建了既表达β-葡糖苷酶又表达氰化物水合酶的重组毕赤酵母菌株GS115-Ch-Glu，以去除食用植物中的氰化物。 β-葡萄糖苷酶可将氰基糖苷水解为氰化物，并通过逆转录聚合酶链反应（RT-PCR）扩增其基因（Glu）。氰化物水合酶可催化氰化物生成甲酰胺，并通过ΔC（PCR）获得其基因（Ch）。通过同源重组将Glu和Ch基因整合到毕赤酵母GS115的基因组中。通过多种分析方法确认了工程菌株GS115-Ch-Glu，并对其进行了诱导以诱导重组“ Glu”和“ Ch”基因的表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）显示融合感兴趣的蛋白质已明确表达为31和52 KDa，与两种酶的预测分子量相对应。 GS115-Ch-Glu菌株生产的含有氰化物水合酶和β-葡萄糖苷酶的酶制剂可以将亚麻籽粉中的氰化物完全分解为氰化物，并进一步催化80％以上的氰化物水解。

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