海藻糖是一种无色无味,性质稳定的天然双糖,具有保湿、抗辐射和保护生物分子结构的重要作用,其主要通过双酶法催化生产,但转化率较低。该研究成功构建麦芽寡糖基海藻糖合酶(maltooligosyltrehalose synthase, MTSase)、麦芽寡糖基海藻糖水解酶(maltooligosyltrehalose trehalohydrolase, MTHase)以及4-α葡糖基转移酶(4-α-glucanotransferase, 4αGT)的大肠杆菌表达系统,从而构建三酶法催化体系,并通过系统优化实现了海藻糖转化率的显著提升。首先,将麦芽寡糖基合酶MTSase、麦芽寡糖基水解酶MTHase以及4-α葡糖基转移酶4αGT整合至pBAD载体中,并在大肠杆菌TR07实现可溶性表达。为进一步提升表达水平,利用生物信息学工具对序列进行优化,获得表达MTSase的重组菌株TR07-1*和表达4αGT的重组菌株TR07-3*,酶活力相较于序列优化前分别提高了87%和52.8%。同时,将6种蛋白促融标签(谷胱甘肽巯基转移酶、麦芽糖结合蛋白、大肠杆菌转录延长的抗终止因子蛋白、蛋白质二硫键异构酶、小泛素样修饰蛋白和硫氧还蛋白)融合于酶的N端,并结合发酵诱导温度、诱导剂浓度和诱导时OD600值的优化,使MTSase和MTHase的酶活力相较初始条件分别提高了31.9%和15.2%。最终,将优化后的重组大肠杆菌应用到海藻糖的转化中,使转化率由78.65%提升至83.33%。该研究对海藻糖工业化生产具有借鉴意义。
Trehalose, a colorless, odorless, and chemically stable natural disaccharide, plays a vital role in moisture retention, radiation resistance, and biomolecular structure protection.It is typically produced through a dual-enzyme catalytic process;however, the conversion rate remains low.This study successfully constructed an Escherichia coli expression system for maltooligosyltrehalose synthase (MTSase), maltooligosyltrehalose trehalohydrolase (MTHase), and 4-α-glucanotransferase (4αGT), thereby establishing a three-enzyme catalytic system.Through systematic optimization, a significant improvement in the trehalose conversion rate was achieved.Initially, MTSase, MTHase, and 4αGT were integrated into the pBAD vector and solubly expressed in E.coli TR07.To further enhance the expression levels, bioinformatics tools were utilized for sequence optimization, resulting in recombinant strains TR07-1* (expressing MTSase) and TR07-3* (expressing 4αGT), which demonstrated 87% and 52.8% increased in enzyme activity, respectively, compared to the unoptimized strains.Additionally, six protein fusion tags (glutathione S- transferase,maltose binding protein,N-utilization substance A,protein disulfide isomerase,small ubiquitin-related modifier,thioredoxins) were fused to the N-terminus of the enzymes, and fermentation parameters, including induction temperature, inducer concentration, and OD600 at induction, were optimized.These modifications further improved the enzyme activities of MTSase and MTHase by 31.9% and 15.2%, respectively.Ultimately, the optimized recombinant E.coli system increased the trehalose conversion rate from 78.65% to 83.33%.This study provides a valuable reference for the industrial-scale production of trehalose.
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