Purification and properties of a β-galactosidase with high galactosyl transfer activity from Cryptococcus laurentii OKN-4

Abstract

β-Galactosidase of Cryptococcus laurentii OKN-4 was solubilized from a cell wall preparation by Zymolyase-20T, and purified by column chromatographies on DEAE-Sephadex A-50, TSK gel Toyo Pearl HW-55S and TSK gel DEAE-5PW. The purified β-galactosidase was homogenous on polyacrylamide disc gel electrophoresis, and the molecular weight was estimated to be about 200,000 by gel filtration on Toyo Pearl HW-55S and about 100,000 by SDS-PAGE. The enzyme showed the optimum pH at 4.3, and was stable at pH's between 2.8 and 9.3. The optimum temperature of the enzyme was 60°C, and it was stable at temperatures below 57.5°C for 10 min incubation. The K m values of the enzyme were 18.2 and 11.4 mM, and those of V max 76.9 and 5.3 μmol/min/mg protein for O- nitrophenyl-β- d - galactoside and lactose, respectively. The enzyme was strongly inhibited by Hg 2+, Ag +, 2-mercaptoethanol, glucose, maltose and maltotriose. It produced 4'GL ( O-β- d- galactopyranosyl-(1→4)-O-β- d- galactopyranosyl-(1→4)- d- glucopyranose) in a yield of 28.2% from 2.5% lactose solution by galactosyl transfer reaction. The enzyme also produced other galactooligosaccharides, including di-, tri-, and tetrasaccharides. The galactosyl transfer reaction was also observed in a 1% lactose solution. The enzyme had several acceptors and gave transfer products from lactose, lactitol, xylose, arabinose, ribose, glucose and galactose.