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

Characterization of Recombinant Sucrose Phosphorylase from Leuconostoc mesenteroides ATCC 12291 and Its Molecular Modification for Improved Transglucoside Activity

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.

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 pQElmsp 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 multiplepoint mutants were obtained, including T180A, T219V, P236S, T180A-T219V, T180A-P236S, T219V-P236S, and T180AT219V- 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.