Study On Gene Cloning, Expression And Characterization Of β-D-galactosidase In Marinomonas Sp. BSi20414

A bactreial strain BSi20414 was isolated from Arctic sea ice showing high P-D-galactosidase activity. Based on the physiological and biochemical property and the phylogenetic analysis of the 16S ribosomal DNA sequence of this strain, it can be classed in the genus Marinomonas. The growth condition for production ofβ-D-galactosidase of BSi20414 was investigated in detail.The purification and properities ofβ-D-galactosidase produced by the strain BSi20414 had been further studied. The purifedβ-D-galactosidase was obtained through ammonium sulfate precioitation and ion exchange chromatography. The molecular weight is about 76 kDa. The optimum temperature ofβ-D-galactosidase is 60℃, the optimum pH is 6.0. Theβ-D-galactosidase is thermostable, pH stability of the P-galactosidase is ranged in 5.0-8.0. The km and Vmax ofβ-D-galactosidase towards ONPG are 13.465 mmol/L and 1.072 umol/L-min. This enzyme has marked feature of specific glycosidic bond. HPLC analysis showed that it can hydrolyze theβ-1-3 glycosidic bond, but can not hydrolyze theβ-1-4 andβ-1-6 glycosidic bond.We cloned the P-D-galactosidase gene from the strain BSi20414 and named galm gene. The galm gene was ligated to pET-22b (+) and PGEX-4T-1 respectively, expressed in E.coli BL21(DE3).