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