嗜热放线菌海藻糖合成酶在地衣芽孢杆菌中的异源表达

Heterologous Expression of Trehalose Synthase from a Thermophilic Actinomyces in Bacillus licheniformis

摘要:

海藻糖合成酶可将麦芽糖异构化为海藻糖，是海藻糖酶法生物转化的中心酶制剂。为了实现海藻糖合成酶在食品安全型表达宿主中高效生产，进而应用于食品级海藻糖的酶法合成，构建了革兰氏阳性菌重组表达质粒并转化入地衣芽孢杆菌。重组质粒携带了来自于嗜热放线菌Thermomonospora curvata海藻糖合成酶的编码基因，在地衣芽孢杆菌木糖异构酶启动子及其阻遏蛋白的介导下表达，胞内海藻糖合成酶发酵活力为12.1 U/mL。考察了不同培养条件对重组菌生长和产酶的影响，结果显示质量分数4%的麦芽糊精和质量分数0.4%的豆饼粉分别为产酶适宜的碳源和氮源；菌体培养10 h后加入终质量浓度为1 g/dL的诱导剂，于37 ℃诱导12 h后重组菌产酶最高达到23.7 U/mL。

Abstract:

Trehalose synthase could transform maltose to trehalose via isomerization, which is a crucial enzyme in the process of trehalose biotransformation with enzymatic method. Recombinant expression plasmid was constructed in order to effectively produce food-grade trehalose synthase in food safety host. The plasmid harbored a trehalose synthase encoding gene from Thermomonosporacurvata, which was expressed under the control of a xylose operon from Bacillus megaterium. The enzyme activity yield of intracellular trehalose synthase reached to 12.1 U/mL after fermentation when expressed by xylose isomerase promoter and its repressor protein in Bacillus licheniformis. Furthermore, the effect of different cultural conditions on the enzyme production was investigated and the optimal condition was determined with 4% maltodextrin, 0.4% soybean powder, and 1% inducer added after 10 h incubation. The maximal yield was 23.7 U/mL after 12 h induction at 37 ℃.The study would contribute to the industrial application of trehalose synthase from both theoretical and experimental aspects.