Biochemical and structural characterization of levansucrase from bacillus licheniformis RN-01

Abstract

Levansucrase (Ls) is a fructosyltransferase belonging to family 68 of glycosyltransferase using sucrose as a sole substrate to synthesize levan, a fructose polymer. Bacillus licheniformis RN-01, isolated from soil in Thailand was found to produce high Ls activity. In sucrose containing medium, Ls was detected mainly in culture medium and membrane-bound fraction. To characterize the enzyme, ls of B. licheniformis RN-01 was cloned. The nucleotide sequence of 1793 bp revealed a single open reading frame of 1446 bp and the putative endogenous promoter at 5' of the structural gene. The encoded amino acid sequence contained 482 amino acids with N-terminal signal peptide of 29 residues. The ls was expressed under two expression systems, the endogenous promoter and T7 promoter. The best condition to produce Ls from the strain RN-01 (LsRN) was the expression under putative promoter by E. coli Top-10 in 3xLB medium for 36 h. LsRN was purified by DEAE-and Butyl Toyopearl-650M. The optimal condition for catalysis was at 50oC in sodium citrate pH 6.0. The Km, kcat, and kcat / Km values were 9.12 mM, 86.0 s-1, and 9.43 mM-1s-1 for hydrolysis reaction, and 6.94 mM, 32.3 s-1 and 4.65 mM-1s-1 for transfructosylation reaction. The polymer synthesized by LsRN was identified as a levan by 13C-NMR. The Mw of levan was determined by HPLC, the reaction temperature and the addition of NaCl affected the size of levan. By rational mutagenesis, N251 was identified at +2 subsite. Mutation at this residue abolished the synthesis of levan polymer. By error prone PCR, the surface residue Y246 was involved in control of product size distribution. Mutation at this residue shifted the main product from levan polymer to oligomer. For applicable use, levan nanoparticles (NPs) were enzymatically synthesized and their ability to encapsulate O-acetyl-α-tocopherol was demonstrated.