蔗糖磷酸化酶在大肠杆菌中的表达及优化

Expression of sucrose phosphorylase in E.coli and optimization its induction temperature

从实验室分离得到的一株长双歧杆菌Bifidobacterium longum基因组中克隆得到蔗糖磷酸化酶基因,并对其进行了生物信息学分析。以p ET-22b(+)为载体构建蔗糖磷酸化酶基因的表达载体p ET-22b(+)/spase,在E.coli Rosetta(DE3)系统中实现了蔗糖磷酸化酶的异源重组表达,其胞内酶活达到2.0 U/mg。在此基础上分别更换p ET-22b(+)中pel B信号肽为Omp A、Mal E、Tor A和Suf I这4条信号肽,用于分泌表达蔗糖磷酸化酶,但结果显示这4条信号肽均未能实现蔗糖磷酸化酶的分泌,且对胞内酶活影响不大。随后考察诱导温度对蔗糖磷酸化酶产量的影响,结果发现诱导温度为25℃时酶活较为理想。这一结果为利用基因工程技术实现蔗糖磷酸化酶的异源表达打下了基础,为生产实践提供了一定的指导意义。

The spase gene from Bifidobacterium Iongum stored in our lab was cloned and analyzed. The spase gene was cloned to the plasmid pET-22b（＋）, resulted in the recombinant plasmid pET-22b（＋）/ spase. For further heterogenous expression of spase, the recombinant plasmid pET-22b（＋）/spase was transformed to E. coli Rosetta （DE3）. The enzyme activity of spase of the recombinant E. coli reached 2.0 U/mg intracellularly, but enzyme activity of spase was not detected in culture supernatant. To realize the secreted expression of spase, the spase gene was fused with these four signal peptides OmpA, MalE, TorA and Sufl, respectively. However, there was still no spase enzyme activity detected extracellularly. Then induction temperature for producing spase was optimized. The yield of spase was highest when the induction temperature was 25℃. The results present here have guiding significance for industrial production of spase from Bifidobacterium Iongum.