The identity of the acid/base catalyst of the Family 35 beta-galactosidases from Xanthomonas manihotis (BgaX) has been confirmed as Glu184 by kinetic analysis of mutants modified at that position. The Glu184Ala mutant of BgaX is shown to function as an efficient thioglycoligase, which synthesises thiogalactosides with linkages to the 3 and 4 positions of glucosides and galactosides in high (> 80%) yields. Kinetic analysis of the thioglycoligase reveals glycosyl donor K-m values of 1.5-21 mu M and glycosyl acceptor K. values from 180 to 500 mu M. This mutant should be a valuable catalyst for the synthesis of metabolically stable analogues of this important glycosidic linkage. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.