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

Alpha-galactosidase AgaB of Bacillus stearothermophilus was subjected to directed evolution in an effort to modify its regioselectivity. The wild-type enzyme displays a major 1,6 and minor 1,3 regioselectivity. We used random mutagenesis and staggered extension process (StEP) to obtain mutant enzymes displaying modified regioselectivity. We developed a screening procedure allowing first the elimination of AgaB mutants bearing the 1,6 regioselectivity and secondly the selection of those retaining a 1,3 regioselectivity. Our results show that, among the evolved enzymes that have lost most of their activity towards the 1,6 linkage both in hydrolysis and in synthesis, one (E901) has retained its 1,3 activity. However the transglycosylation level reached by this mutant is quite low versus that of the native enzyme. This work constitutes the first example of modification of glycosylhydrolase regioselectivity by directed evolution.