Modulation of the regioselectivity of a Bacillus alpha-galactosidase by directed evolution

alpha -galactosidase AgaB of Bacillus stearothermophilus was subjected to d irected evolution in an effort to modify its regioselectivity. The wild-typ e enzyme displays a major 1,6 and minor 1,3 regioselectivity. We used rando m mutagenesis and staggered extension process (StEP) to obtain mutant enzym es displaying modified regioselectivity. We developed a screening procedure allowing first the elimination of AgaB mutants bearing the 1,6 regioselect ivity and secondly the selection of those retaining a 1,3 regioselectivity. Our results show that, among the evolved enzymes that have lost most of th eir activity towards the 1,6 linkage both in hydrolysis and in synthesis, o ne (E901) has retained its 1,3 activity. However the transglycosylation lev el reached by this mutant is quite low versus that of the native enzyme. Th is work constitutes the first example of modification of glycosylhydrolase regioselectivity by directed evolution.