Comparative study of new alpha-galactosidases in transglycosylation reactions

We have studied the potential of several newly cloned alpha-galactosidases to catalyze the regioselective synthesis of disaccharides using 4-nitrophenylgalactoside as a donor. The kinetics of the reactions were followed by in situ NMR spectroscopy. The following thermophilic enzymes have been tested: Aga 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-galactosidase from Thermus brockianus (strain IT360). Two other non-thermophilic alpha-galactosidases have also been evaluated: Aga 1 (Streptococcus mutans, strain Ingbritt) and Raf A (Escherichia coli, strain D1021). For all of the enzymes studied, high regioselectivity was observed leading to two (1 --> 6)-disaccharides: 4-nitrophenyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside and methyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside, which were obtained in 54% (Aga B) and 20% (Aga T) yields, respectively.