高产β-葡萄糖苷酶工程菌株的构建及其在2,6-二甲氧基对苯醌发酵制备中的应用

摘要

构建高产β-葡萄糖苷酶的工程菌株并将其应用到2,6-二甲氧基对苯醌的发酵制备中.通过聚合酶链式反应从酿酒酵母CS1401的总DNA中扩增得到β-葡萄糖苷酶基因,并与载体pPICZαA连接后导入毕赤酵母X33中进行甲醇诱导表达;对表达成功的重组菌株进行β-葡萄糖苷酶活力测定;同时将酿酒酵母CS1401和重组菌株接种到小麦胚芽培养基中进行发酵并测定2,6-二甲氧基对苯醌的发酵产量.结果表明,酿酒酵母CS1401的β-葡萄糖苷酶基因在毕赤酵母X33工程菌株中得到高效表达,酶活力达到4.5 U/mL,为酿酒酵母CS1401的5倍;2,6-二甲氧基对苯醌的摇瓶发酵产量达到935.7 μg/g,比酿酒酵母CS1401提高了0.4倍.因此,高产β-葡萄糖苷酶的重组工程菌株在2,6-二甲氧基对苯醌的发酵制备中具有一定的应用前景.%An engineered strain producing high β-glucosidase activity was constructed and applied in the production of 2,6-dimethoxybenzoquinone.The β-glucosidase gene was amplified from the genomic DNA of Saccharomyces cerevisiae CS1401 using PCR.The amplified gene was then ligated to pPICZαA vector and transformed to Pichia pastoris X33 for expression under the induction of methanol.The activity of the recombinant enzyme was measured,and S.cerevisiae CS1401 and the recombinant strain were separately cultured in wheat germ medium for the production of 2,6-dimethoxybenzoquinone.The results indicated that the β-glucosidase gene of S.cerevisiae CS1401 was efficiently expressed in the engineered strain ofP.pastoris X33,giving a β-glucosidase activity of up to 4.5 U/mL,which was 5 times higher than that of S.cerevisiae CS1401.Besides,the yield of 2,6-dimethoxybenzoquinone produced by the recombinant strain in shaking flasks reached 935.7 tg/g,which was 40％ higher than that of S.cerevisiae CS1401.Therefore,this engineered strain has a promising prospect of application in the microbial production of 2,6-dimethoxybenzoquinone.