α-1,6-葡聚糖酶基因的克隆及在毕赤酵母中的表达

摘要

The α-1,6-dextranase gene was amplified from a synthetic α-1, 6-dextranase gene derived from Penicillium minioluteum HI-4 and was inserted into plasmid pPICZαA. The recombinant plasmid named pPICZαA-Dex was identified by PCR and enzyme digestion,and was then electrotransformed into Pichia pastoris X33. The recombinant Pichia pastoris X33 was induced to express the dextranase using methanol. The enzyme activity reached 29. 2 U/mL after 120 h of induction. The size of the recombinant α-1 ,6-dextranase is approximately 67 ku analyzed by SDS-PAGE,which is consistent with the theoretical molecular weight.%根据朱黄青霉(Penicillium minioluteum HI-4)α-1,6-葡聚糖酶的基因序列,人工合成α-1,6-葡聚糖酶基因并将其插入毕赤酵母(Pichia pastoris)表达载体pPICZαA,PCR和双酶切验证结果均表明成功构建了重组质粒pPICZαA-Dex.重组质粒经SacⅠ线性化后整合到毕赤酵母X33基因组中进行表达.摇瓶发酵表明,重组毕赤酵母经甲醇诱导120 h产酶活力达到最高值29.2 U/ mL,SDS-PAGE结果显示在67 ku附近有明显的条带,与α-1,6-葡聚糖酶理论分子质量相符合.