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.
Highlights
Levans are prebiotic agents with potential health benefits, selectively support intestinal health and act as low calorie sweeteners (Byun et al, 2014; Kang et al, 2009)
The present study report the isolation of BsSacB from B.subtilis with its own promoter inducible in presence of sucrose that can effectively be expressed in E.coli
Microbacterium laevaniformans was used for levan production (10.4 g/L in 48 h) using date syrup as source of sucrose and the results suggest that increase in fermentation time caused a decrease in levan yield (Moosavi-Nasab et al, 2010)
Summary
Levans are prebiotic agents with potential health benefits, selectively support intestinal health and act as low calorie sweeteners (Byun et al, 2014; Kang et al, 2009). They are used as blending component in cosmetics to provide cell-proliferating and skin moisturizing effects (Abdel-Fattah et al, 2012). Levans are synthesized by bacterial levansucrases which belong to GH68 family They synthesize fructan oligosaccharides and levan by transferring fructosyl group of non-activated sucrose into fructan polymers with β (2→6) linkage (Seibel et al, 2006). Poor availability and low stability of characterized microbial enzymes generates the need for investigation on levansucrases with improved physico-chemical properties (Maiorano et al, 2008)
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