易错PCR技术提高源于/Sulfolobus acidocaldarius麦芽寡糖基海藻糖合成酶活性

Improving the Activity of MTSase from Sulfolobus acidocaldarius by Error-Prone PCR

DOI：10.3969/j.issn.1673-1689.2020.03.003

中文关键词: 麦芽寡糖基海藻糖合成酶 嗜酸热硫化叶菌 易错PCR

英文关键词: maltooligosyl trehalose synthase,Sulfolobus acidocaldarius,error-prone PCR

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中文摘要:

利用易错PCR技术结合高通量筛选技术对源于/Sulfolobus acidocaldariusATCC 33909 的MTSase进行定向进化，获得一个酶活提高的突变体酶D-6。基因分析表明，突变体酶发生了2个位点突变：G432D/G586D，其中突变位点G586D位于Aβ8与AEα14的loop环上。将野生型MTSase和突变体酶D-6进行蛋白质纯化后，测定其酶学性质，结果表明：突变体酶D-6的比活力为野生型MTSase的1.22倍；野生型的/K_m值为4.74 mmol/L，而突变体D-6为2.77 mmol/L，表明其对底物亲和性较野生型有所提高。

英文摘要:

A mutant D-6 of MTSase from/Sulfolobus acidocaldariusATCC 33909 was obtained through directed evolution using error-prone PCR and high-throughput screening. Gene analysis showed that there were two mutations G432D/G586D with the G586D on the loop of Aβ8 and AEα14. The enzymatic properties of wild-type MTSase and mutant D-6 was determined. The results showed that the specific activity of mutant D-6 was 1.22 times that of wild-type MTSase. The/K_mvalue of the mutant D-6 was 2.77 mmol/L while that of wild-type was 4.74 mmol/L. Thus, the substrate affinity of mutant increased compared to the wild-type.

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