食品科学 ›› 2019, Vol. 40 ›› Issue (20): 122-129.doi: 10.7506/spkx1002-6630-20181024-279

• 生物工程 • 上一篇    下一篇

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

何贺贺,林厚民,寇力丹,覃凤兰,韦宇拓,黄日波,杜丽琴   

  1. (亚热带农业生物资源保护与利用国家重点实验室,广西大学生命科学与技术学院,广西 南宁 530005)
  • 出版日期:2019-10-25 发布日期:2019-10-25
  • 基金资助:
    国家自然科学基金地区科学基金项目(21566003);广西自然科学基金项目(2018GXNSFAA138103)

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

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

  1. (State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning 530005, China)
  • Online:2019-10-25 Published:2019-10-25

摘要: 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。

关键词: 肠膜明串珠菌, 蔗糖磷酸化酶, 酶学性质, 转糖苷功能, 分子改造

Abstract: 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.

Key words: Leuconostoc mesenteroides, sucrose phosphorylase, enzymatic properties, transglycoside, molecular modification

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