Screening, Identification And Characterization Of A Production Switch To Sugar-based ¦Â-galactosidase Glycosidase Huge Bacillus (bacillus Megaterium)

Oligosaccharides, which have great physiological functions, have been paid much attention in recent years. Oligosaccharides are classically defined as glycosides that contain2-10 sugar moieties. Galacto- oligosaccharides (GOS) are oligosaccharides formed by 1-4 mono- saccharides of galactose transferred to galacto-moieties of lactose. In now days, the commercial GOS are mainly synthesizied by the method of enzyme-catalysed synthesis.Many β-galactosidases (EC.3.2.1.23) have been isolated to produce GOS. Using lactose as substrate, P-galactosidase can catalyze the hydrolysis of lactose and the self-transgalactosylation of it. The proportion hydrolysis to transgalactosylation reaction, the concentrations and the structures of the galacto-oligosacchandes depend on the source of the enzyme. Among of the linkages of galacto-oligosacchandes, (1â†'6) glycosidic link is the major.One bacterium strain producing β-galactosidase with transgalactosyla- tion activity was screened from 97 strains of microorganisms stored in our laboratory using hydrolysis activity on o-nitrophenyl-β-D-galacto- pyranoside followed by transgalactosylation activity on lactose. Phenotypic analyses including morphology and physiology characteristics and 16S rDNA sequence analyses were carried out. The GenBank accession number of the 16S rDNA was DQ267829. By making a comprehensive view on all the results of taxonomy, the strain was identified as Bacillus megaterium 2-37-4-1. The growth condition for production of P-galactosidase of B. megaterium 2-37-4-1 was investigated. The optimal carbon source and nitrogen source were 1% lactose and 0.5 % peptone plus 0.5% yeast extract, respectively. High yield of the enzyme was obtained at 37℃ for 18h. Effects of pH, the concentration of lactose, reaction temperature and time on the enzyme-catalyzed transgalactosylation activity were studied and the reaction products were analyzed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). In 50mM phosphate buffer (pH 7.5) containing 40% lactose, reacted at 55℃ with shaking for 24h, the transgalactosylation product was composed of 25.68% galacto- oligosaccharides (GOS), 33.02% disaccharide including lactose and transgalctosylated disaccharide, 26.37% glucose and 14.92% galactose.The purification and properities of B. megaterium had been investigated. The p-galactosidase was purified using ammonium sulfate precipitation, DEAE-Sepharose,...