Optimization of the expression of levansucrase L17 in recombinant E. coli

Abstract

Levansucrases synthesize levans and fructooligosaccharides, which are of interest in the food and pharmaceutical industry. Leuconostoc mesenteroides Lm 17 produces levansucrase of about 120 kDa. The encoding gene from this strain was cloned and expressed in Escherichia coli BL21(DE3). The cloned gene encodes a 1022 amino acids long levansucrase with 96% identity to levansucrase LevS from L. mesenteroides NRRL B-512F strain. The induction and expression of the levansucrase gene were performed at temperatures between 15 °C and 37 °C, and concentration of the inducer isopropyl-β-D-thiogalactopyranoside from 0.1 to 2.0 (mmol/L)-1. We report for the first time recombinant expression of a levansucrase gene at a low temperature of induction, after cell biomass accumulation at 37 °C. The highest enzyme activity of 1.90 (U/mg)-1 was measured in TB medium at 18 °C temperature of induction, and concentration of the inducer from 0.1 to 1.0 (mmol/L)-1. The in situ analysis of the purified enzyme showed an active band of about 120 kDa, similar to the one produced by the native strain. The purified enzyme has a temperature optimum at 35 °C, pH optimum at 5.5, and Km = 64 (mmol/L)-1 of sucrose.

Keywords

• Expression optimization
• E. coli
• Leuconostoc mesenteroides
• levansucrase
• low temperature