The gene (bglT) encoding Tca beta-glycosidase (Thermus caldophilus GK24 beta-glycosidase) was overexpressed under the control of the trp promoter on a high-copy-number plasmid, pTRPES, in Escherichia coli W3110. The purified Tca beta-glycosidase enzyme was used in a galactosyl-transfer reaction to synthesize galacto-oligosaccharides from lactose. The optimum temperature and pH for the enzyme to synthesize galacto-oligosaccharides from 30% (w/v) lactose were 80 degrees C and 6.0, respectively. The major product of the reaction was a trisaccharide. The thermostable Tca beta-glycosidase produced galacto-oligosaccharides efficiently during the hydrolysis of lactose.