毕赤酵母展示海藻糖合成酶及其性质研究

Displaying of Trehalose Synthase Through Pichia pastoris and its Enzymatic Properties

摘要:

利用毕赤酵母表面展示天蓝色链霉菌（Streptomyces coelicolor）海藻糖合成酶（Trehalose synthesase，TS，EC 5.4.99.16）并研究其酶学性质。人工合成酵母内源壁蛋白（GPI-modified wall proteins，GCW14）与天蓝色链霉菌海藻糖合成酶组合DNA（GCW14-TS），将其亚克隆到pPIC9k载体后转化到GS115酵母菌中，成功构建了pPIC9k -GCW14-TS载体，并将TS展示在毕赤酵母GS115表面，然后对其酶学性质进行研究。发现其在30~45 ℃温度范围都能发挥作用，最适pH为8.5，K+、Mg2+等金属离子对其有促进作用且重复使用5次后仍然可以保持75%以上的酶活性。该基因工程菌具有海藻糖合成酶固定化酶性质，可用于将麦芽糖转化为海藻糖的产业化生产。

Abstract:

The study aimed to display trehalose synthase（TS.EC 5.4.99.16）on the surface of Pichiia pastoris and investigate its enzymatic properties. Synthesized gene from Streptomyces coelicolor trehalose synthase and yeast cell wall protein GCW14 was cloned to pPIC9k carrier and transformed into yeast GS115 and thus the pPIC9k-GCW14-TS carrier was constructed to display TS. Then the properties of the recombinant trehalose synthase were determined. Results showed that the recombinant trehalose synthase possessed the optimal temperature 30~45 ℃ and pH 8.5，K+ and Mg2+ increased its enzymatic capacity and more than 75% of the activity was still remained after it was reused for 5 times. Therefore，this recombinant strain can be repeatedly use as a whole cell catalyst to convert maltose into trehalose.