A novel transglycosylating P-galactosidase was purified from Enterobacter agglomerans B1. It was a homodimer of similar to 248 kDa. The optimal pH and temperature for oNPGal hydrolysis were 7.5-8.0 and 37-40 degrees C, respectively. The Km values for oNPGal and lactose were 0.06 and 114 mM, 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, beta- or alpha-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. (c) 2007 Elsevier Inc. All rights reserved.