肠膜明串珠菌ATCC 12291蔗糖磷酸化酶的酶学性质及转糖苷分子改造

根据GenBank数据库公布的肠膜明串珠菌ATCC 12291中蔗糖磷酸化酶基因序列，构建重组表达质粒pQE-lmsp。在大肠杆菌Escherichia coli M15/pREP4中诱导表达，镍亲和层析纯化蛋白，进行酶学性质测定和重组酶转糖苷活性的研究。以蔗糖为底物对LMsp进行酶学性质分析，其最适温度和最适pH值分别为40 ℃和6.5；Km值和Vmax值分别为（34.16±1.219）mmol/L和（370.5±6.049）μmol/（mg·min）。当以葡萄糖-1-磷酸为供体时，对于大多数单糖及其糖醇类受体均具有转糖苷活性，尤其对L-阿拉伯糖具有75%的转化效率。然后通过对LMsp进行同源建模和氨基酸序列比对分析，进行分子改造，并比较酶学性质及转糖苷活性的变化。获得7 个单点和联合的突变体T180A、T219V、P236S、T180A-T219V、T180A-P236S、T219V-P236S、T180A-T219V-P236S，其中突变体T180A、P236S对L-山梨糖的转糖苷活性分别有13.7%和15%的提高。本研究通过对LMsp的研究，发现其具有良好的酸碱稳定性和对L-阿拉伯糖的高转糖苷活性，并且获得了对于L-山梨糖转糖苷活性提高的突变体，丰富了有关于蔗糖磷酸化酶的性质，并且对该酶的分子改造提供了经验依据。

HE Hehe,LIN Houmin,KOU Lidan,QIN Fenglan,WEI Yutuo,HUANG Ribo,DU Liqin

A recombinant expression plasmid named pQE-lmsp was constructed based on the sequence of the sucrose phosphorylase gene from Leuconostoc mesenteroides ATCC 12291 published in the GenBank database. The plasmid pQE-lmsp was expressed in Escherichia coli M15/pREP4. The recombinant protein LMsp was purified by NI-NTA affinity chromatography. The enzymatic properties, especially transglycoside activity of LMsp were studied. Using sucrose as substrate, the optimum temperature and pH were 40 ℃ and 6.5, respectively. The Km and Vmax values were (34.16 ± 1.219) mmol/L and (370.5 ± 6.049) μmol/(mg·min), respectively. When glucose-1-phosphate was used as donor, it had transglycoside activity on most monosaccharides, especially L-arabinose, which had 75% conversion efficiency. Then, molecular modification of LMsp was performed by homology modeling and amino acid sequence alignment analysis. A total of 7 single- and multiple-point mutants were obtained, including T180A, T219V, P236S, T180A-T219V, T180A-P236S, T219V-P236S, and T180A-T219V-P236S, and the transglycoside activities of T180A and P236S on L-sorbose were increased by 13.7% and 15%, respectively compared to that of LMsp. In this study, we found the sucrose phosphorylase LMsp had good pH stability and high transglycoside activity on L-arabinose. Further, the mutants with improved transglycoside activity on L-sorbose were obtained. This study enriches the properties of sucrose phosphorylase and provides a basis for molecular modification.