Production of prebiotic oligosaccharides by glucansucrase from bacillus licheniformis th4-2

Abstract

The aim of the present study was to use glucansucrase from Bacillus licheniformis TH4-2 in the glucosyl transfer reaction for production of prebiotic oligosaccharide. Experimental steps started with optimization of cultivation condition for high glucansucrase production. The optimized condition was 42 hours of cultivation in the medium containing 5 % soluble starch from cassava supplemented with 0.4% ovalbumin at pH 6.5, 45o C. The enzyme was then purified to homogeneity by ammonium sulfate precipitation, DEAE-Cellulose and Sephadex G-100 column chromatography. The specific activity of the enzyme was 112 fold increased with 28% yields of total activity. The molecular mass of the purified enzyme was 64 kDa as measured by SDS-PAGE. The optimum pH and temperature were 6.0 and 45oC, respectively. The apparent Km and Vmax values for sucrose substrate were 38.14 mM and 0.042 mole/min, respectively. The enzyme was able to synthesize a variety of prebiotic oligosaccharides from sucrose donor using various saccharides as glucosyl acceptor, such as G2 (maltose) to G7 (maltoheptaose), lactose, melibiose, cellobiose, raffinose, palatinose and lactulose. Melibiose was found to be one of the efficient and interesting glucosyl acceptors. The apparent Km and Vmax values for melibiose acceptor were 148 mM and 0.0072 mole/min, respectively. From product analysis using TLC and HPLC, the optimum condition for oligosaccharide production obtained was 15% (w/v) melibiose (acceptor), 5% (w/v) sucrose (donor), glucansucrase concentration 5U/ml at pH 6.0, 45oC for 24 hours. After optimization, two product peaks from HPLC profile were obtained from glucosyl transfer to melibiose acceptor, main product (product A at Rt 8.3 min) and minor product (product B at Rt 10.3 min) with the yields of 17.2% and 3.3%,respectively. Reaction products were then prepared in larger scale, where similar percent yield of the two products was obtained. The main product was isolated from other sugars present in the reaction mixtures by Sephadex LH-20 column. The structured identification of the main product by MS and NMR revealed the trisaccharide structure of 504 daltons of melibiose linked by an -1,6 bond with one molecule of glucose. The main product can support significant growth of Lactobacillus acidophilus.