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-  2018 

重组嗜热乳糖酶在毕赤酵母中的表达、纯化与活性分析

DOI: 10.3969/j.issn.1673-1689.2018.08.005

Keywords: 嗜热乳糖酶 激烈热球菌 毕赤酵母 分泌表达 纯化
thermophile lactase
,Pyrococcus furious,Pichia pastoris,secretory expression,purification

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Abstract:

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

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