Purification and characterization of a recombinant β-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96

Abstract.

A β-galactosidase isoenzyme, β-GalI, from Bifidobacterium infantis HL96, was expressed in Escherichia coli and purified to homogeneity. The molecular mass of the β-GalI subunit was estimated to be 115 kDa by SDS-PAGE. The enzyme appeared to be a tetramer, with a molecular weight of about 470 kDa by native PAGE. The optimum temperature and pH for o-nitrophenyl-β-D-galactopyranoside (ONPG) and lactose were 60°C, pH 7.5, and 50°C, pH 7.5, respectively. The enzyme was stable over a pH range of 5.0–8.5, and remained active for more than 80 min at pH 7.0, 50°C. The enzyme activity was significantly increased by reducing agents. Maximum activity required the presence of both Na⁺ and K⁺, at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, divalent metal cations, and Cr³⁺, and to a lesser extent by EDTA and urea. The hydrolytic activity using lactose as a substrate was significantly inhibited by galactose. The K _m and V _max values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. β-GalI possesses strong transgalactosylation activity. The production rate of galactooligosaccharides from 20% lactose at 30 and 60°C was 120 mg/ml, and this rate increased to 190 mg/ml when 30% lactose was used.