Comparative study of new alpha-galactosidases in transglycosylation reactions

We have studied the potential of several newly cloned a-galactosidases to c atalyze the regioselective synthesis of disaccharides using 4-nitrophenylga lactoside as a donor. The kinetics of the reactions were followed by in sit u NMR spectroscopy. The following thermophilic enzymes have been tested: Ag a A and an isoenzyme Aga B obtained from the strain KVE39 and Aga 285 from the strain IT285 of Bacillus stearothermophilus; Aga T is an alpha-galactos idase from Thermus brockianus (strain IT360). Two other non-thermophilic oc -galactosidases have also been evaluated: Aga I (Streptococcus mutans, stra in Ingbritt) and Raf A (Escherichia coli, strain D1021). For all of the enz ymes studied, high regioselectivity was observed leading to two (1 --> 6)-d isaccharides: 4-nitrophenyl alpha-D-galactopyranosyl-( 1 --> 6)-alpha-D-gal actopyranoside and methyl alpha-D-galactopyranosyl-(1 --> 6)-alpha-D-galact opyranoside, which were obtained in 54% (Aga B) and 20% (Aga T) yields, res pectively. (C) 2000 Elsevier Science Ltd. All rights reserved.