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Abstract: A novel transglycosylating β-galactosidase was purified from Enterobacter agglomerans B1. It was a homodimer of ∼248kDa. The optimal pH and temperature for oNPGal hydrolysis were 7.5–8.0 and 37–40°C, respectively. The K m values for oNPGal and lactose were 0.06 and 114mM, respectively. The enzyme produced galacto-oligosaccharides in a 38% yield at the lactose concentration of 12.5% (w/v). When using oNPGal as donor, the enzyme was able to catalyze glycosyl transfer to a series of acceptors, including hexose, pentose, β- or α-disaccharides, hexahydroxy alcohol, cyclitol, and aromatic glycosides. This suggested the enzyme to be a potential synthetic tool for preparing galactose-containing chemicals. The gene encoding this enzyme was cloned by degenerate PCR and TAIL-PCR. It revealed an ORF of 3090 nucleotides encoding a 1029 amino-acid protein, which had been expressed in Escherichia coli. Transferase activities in both recombinant and natural enzymes were similar. [Copyright &y& Elsevier]