Biosynthesis and Structural Characterization of Levan by a Recombinant Levansucrase from Bacillus subtilis ZW019

Abstract

PurposeThe yield of levan extracted from microbial fermentation broth is low, so in vitro catalytic synthesis of levan by levansucrase is expected to be one of the industrial production approaches of levan.MethodsThe recombinant plasmid pET-28a-AcmA-Z constructed in a previous study was used to produce levansucrase. The effects of temperature, pH, and metal ions on the levan formation activity of the levansucrase were investigated. The polymer was analyzed by means of HPIC, FTIR, and NMR techniques.ResultsThe recombinant levansucrase could be easily purified in one step and the purified enzyme had a single band clearly visible in SDS-PAGE. The conditions for enzymatic reactions were optimal at pH 5.2 and 40 ℃, and the activity of enzymes was stimulated by K+ and Ca2+. The yield of levan biosynthesis from 10% (w/v) sucrose with 6.45 U/g sucrose of levansucrase was 30.6 g/L. The molecular weight of the levan was approximately 1.56 × 106 Da, as measured by GPC. HPIC analysis showed that the monosaccharide composition of the levan was fructose and glucose. The results of FTIR and NMR analysis indicated that the polymer produced by the recombinant levansucrase was β-(2,6) levan.ConclusionsThe results of this study provide a basis for the large-scale production of levan by enzymatic methods.Graphical Abstract