Heterologous Expression and Characterization of Thermostable Levansucrase (BsSacB) from Bacillus subtilis BB03

Abstract

Thermostable levansucrase encoding gene (sacB) with its promoter was isolated from Bacillus subtilis BB03. BsSacB is composed of 1419 bp, encoding 473 amino acid residues and with promoter regions TTGCAA (-35) and TAGAAT (-10). The amino acid sequence analysis of BsSacB showed conserved motifs of microbial fructosyltransferase belonging to Glycoside Hydrolase family 68 (GH68). Comparative analysis of the protein structure was carried out with 3D model of BsSacB constructed using PyMol software. BsSacB gene was expressed in Escherichia coli and the purified enzyme demonstrated thermo stability up to 50°C with a high hydrolytic activity at pH 6.0 and presence of Ca2+ promoted the activity by 15%. An attempt was made to further characterize the thermostable recombinant levansucrase from B.subtilis BB03 using crude sucrose rich substrates. Optimal levan production was seen with molasses (1.94±0.04 g/L) at 15 h as compared to cane juice (1.0±0.01 g/L) at 12 h. An increase in levan formation with gradual decrease in hydrolytic activity was distinctly evident in both the molasses and juice media.