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

利用易错PCR技术结合高通量筛选技术对源于Sulfolobus acidocaldarius ATCC 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Km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。

A mutant D-6 of MTSase from Sulfolobus acidocaldarius ATCC 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 Km value 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.