基于麦芽糖诱导型启动子在枯草芽胞杆菌中异源表达麦芽四糖淀粉酶及其酶学性质的研究  

Heterologous Expression and Characterization of Maltotetraose Amylase in Bacillus subtilis Based on Maltose⁃Inducible Promoter

作　　者：丛贵龙 李明玉 姜梦彤 国丹 刘羽欣 王从纲 李宪臻[1] CONG Gui-long;LI Ming-yu;JIANG Meng-tong;GUO Dan;LIU Yu-xin;WANG Cong-gang;LI Xian-zhen(Coll.of Bio-Engin.,Dalian Eng.Uni.,Dalian 116000)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116000

出　　处：《微生物学杂志》2024年第1期23-31,共9页Journal of Microbiology

基　　金：辽宁省自然科学基金项目(20180550668)。

摘　　要：麦芽四糖淀粉酶(Maltotetraose amylase,Mta)可以从淀粉的非还原末端特异性依次切割第4个α-1,4糖苷键形成麦芽四糖,目前在食品、医疗保健和造纸等领域具有重要应用。构建安全、高效的表达系统强化麦芽四糖淀粉酶的重组表达,进而降低以其为核心酶的麦芽四糖生物转化过程的生产成本具有迫切的现实需求。本研究将源自Pseudomonas saccharophila(DSM 654)的麦芽四糖淀粉酶基因mta在枯草芽胞杆菌Bacillus subtilis中重组表达,利用麦芽糖诱导型启动子实现其安全高效表达,之后对重组酶进行分离纯化和酶学性质表征。结果显示,将携带麦芽糖诱导型启动子Pglv的表达载体转入B.subtilis WB800N中,成功构建工程菌后进行诱导表达,并且利用金属离子螯合层析技术成功获得了Mta纯酶。酶学性质研究结果显示其最适反应温度为55℃,最适反应pH为7.5。动力学常数Km为(1.26±0.17)g/L、kcat/Km为(2275.07±32.83)L/s·g,纯酶在4℃储存14 d后保留40%的酶活力。本研究成功构建出安全、高效表达重组麦芽四糖淀粉酶的表达系统,为其规模化制备和应用提供了新策略。Maltotetraose amylase can cut successively the fourthα⁃1,4 glycosidic bond from the non⁃reducing end of starch to produce maltotetraose,which is widely used to date in the food,healthcare,and paper industries.However,the urgent and realistic need to reduce production cost is to establish safe,high efficient expression system to strength⁃en the recombinant expression of maltotetraose amylase.In this study,a maltotetraose amylase gene mta from Pseudo⁃monas saccharophila(DSM 654)was expressed in Bacillus subtilis using a maltose⁃inducible promoter to realize its safe and high efficient expression,followed by isolation purification and feature characterization of the recombinant en⁃zyme.The results showed that recombinant plasmid carrying the maltose⁃inducible promoter Pglv with expression carrier was transformed into B.subtilis WB800N for the expression.After sucessful construction of the engineered strain′s ex⁃pression was induced and successfully obtained pure enzyme Mta adopting metal ion chelate chromatography.The re⁃sult of enzymological study showed that its optimized reaction temperature was at 55℃and pH at 7.5.And its kinetic constant Km was at(1.26±0.17)g/L,and its kcat/Km was at(2275.07±32.83)L/(s·g).The purified enzyme could preserve 40%of its original activity after 14 days of storage at 4℃.To summarized,in this study a safe and high efficient expression system of maltotetraose amylase was successfully constructed and provided a new strategy for its large⁃scale preparation and application.

关 键 词：麦芽四糖 麦芽四糖淀粉酶 枯草芽胞杆菌 异源表达 启动子 酶学性质 

分 类 号：Q936[生物学—微生物学]