定向进化提高来源于Arthrobacter ramosus的MTHase的热稳定性

Improved the Thermostability of MTHase from Arthrobacter ramosus by Directed Evolution

麦芽寡糖基海藻糖水解酶(maltosyltrehalose hydrolase,MTHase)是以淀粉或麦芽糊精为底物制备海藻糖的关键酶之一。来源于Arthrobacter ramosus的MTHase,表达量好,比活高,但热稳定性差,限制了其工业化应用。采用定向进化技术,筛选得到L137M和A216T两个突变体,在60℃下,野生型和两个突变体的半衰期(t1/2)分别为15.5、20.5和23.3 min,L137M和A216T的t1/2分别比野生型提高1.3和1.5倍。进一步考察了L137M、A216T和野生型酶在60℃分别和同样来源于Arthrobacter ramosus的麦芽寡糖基海藻糖合成酶(maltosyltrehalose synthase,MTSase)复配制备海藻糖,在相同的加酶量下,野生型、L137M和A216T的海藻糖转化率分别为51.3%,52.6%和55.7%,两个突变体的转化率都比野生型提高,说明突变体提高的热稳定性有利于海藻糖的转化率。

Maltosyltrehalose hydrolase(MTHase)is one of the key enzymes for trehalose production using starch or maltodextrin as the substrate.MTHase from Arthrobacter ramosus has a good-level expression and high specific activity,but poor thermostability,which limits its industrial application.In this study,mutants of L137M and A216T were screened using the directed evolution.The half-life(t1/2,at 60℃)of wild-type,L137M and A216T were 15.5,20.5 and 23.3 min,respectively.The half-period t1/2 of L137M and A216T were 1.3-and 1.5-fold than that of the wild type,respectively.The performances of L137M and A216T and wild-type enzymes were further investigated by their trehalose yields from MTSase at 60℃.Under the same amount of enzyme,the trehalose conversion rates by wild-type,L137M and A216T were 51.3%,52.6%and 55.7%,respectively.The trehalose conversion rates by the two mutants were higher than that by the wild-type,indicating that the improved thermostability of the mutants is beneficial for industrial trehalose production.