重组嗜热乳糖酶在毕赤酵母中的表达、纯化与活性分析

Expression,Purification and Activity Assay of Recombinant Thermophile Lactase From Pichia pastoris

为获得耐高温的重组乳糖酶,PCR扩增激烈热球菌(Pyrococcus furious)乳糖酶基因、构建毕赤酵母分泌型表达载体pPICZαA/Lac并转化毕赤酵母X-33菌株。重组酵母转化子经甲醇诱导,重组嗜热乳糖酶实现了分泌表达并经蛋白印迹验证。纯化前上清液中乳糖酶总活力高达125 U/mL,经镍亲合纯化得重组酶,SDS-PAGE显示其纯度在95%以上,比活力1 800 U/mg,该酶最适温度在105℃左右且热稳定性好,具有很强的水解乳糖能力。

In order to obtain the recombinant thermostable lactase,a DNA fragment containing the mature lactase gene was amplified from Pyrococcus furious by PCR and cloned into pPICZaA,generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33.Recombinant yeast transformants with high-level recombinant lactase production was identified by Western blotting,secreting as much as 125 U/mL induction by methanol.The recombinant thermostability lactase was purified by Ni+-NTA affinity chromatography and SDS-PAGE results shown that the purity was over 95%.The specific activity was about 1 800 U/mg and the optimal temperature was about 105℃.It was also showed that the purified lactase from P.pastoris has a highly thermostability and strong ability to hydrolysis lactose.