Purification And Kinetic Parameter Analysis Of β-galactosidase From Arthrobacter Sp.

β-galactosidase (EC3.2.1.23) is the bio-enzyme without toxicity and side effects. It can catalyze lactose to form glucose and galactose, or transfer the galactose to the receptor saccharides to produce gal-actooligosaccharides.β-galactosidase produced by Arthrobacter sp. has both hydrolysis and transgalactosyl activites. The purpose of this article is to purify and characterize this enzyme. The culture bacterial cells were gathered by centrifugation and disrupted by ultrasonication. The crude enzyme solution was precipitated by ammonium sulphate. Further enzyme purification was carried out by Phenyl-Sepharose, DEAE-Sepharose and p-aminobenzyl-1-thio-β-D-galacto pyranoside (PABTG) affinity chromatography. The purifiedβ-galactosidase was illustrated as a single band in SDS-PAGE with a purification fold of 31.4 and an apparent molecular weight of 30kD.When O-nitrophenyl-a-D-glucopyranoside (ONPG) was used as substrate, the maximum activity of theβ-galactosidase was determined at pH 6.6 and 37℃. Above 80% of the maximum activity of the enzyme was detected at pH 6.0-8.0. The ions of Mg2+,Mn2+,Co2+ K+, Cu2+ and Ag+ can strongly stimulate the enzyme activity. Metal ions can also increase the thermostability of the enzyme. No loss of the activity was found in the presence of 1 mmol/L of Mn2+ for 1 h at 45℃. The Km for ONPG is 4.64 mmol/L and the maximum reaction rate is 0.919μmol/min.When lactose was used as substrate, the maximum activity of the p-galactosidase was determined at 30℃and pH 6.5-7.0. The optimum lactose concentration was 3%.The transglycosylation ofβ-galactosidase from Arthrobacter sp. was also performed through the product analysis by HPLC. The results showed that the optimum pH and temperature for transglycosylation was 6.0 and 20℃respectively.