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

• Bioengineering • Previous Articles     Next Articles

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

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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