Identification by saturation mutagenesis of a single residue involved in the a-galactosidase agab regioselectivity

Abstract

a-Galactosidase AgaB from Bacillus stearothermophilus displays a major a(1~6) and a minor a(1~3) regioselectivity in hydrolysis and transglycosylation. Its corresponding gene, agaB, was subjected to saturation mutagenesis at codon 442 in order to change its regioselectivity. The mutant genes were cloned and expressed in Escherichia coli using pBTac2 as vector. The regioselective activity of the mutants was determined using thin layer chromatography and nuclear magnetic resonance spectroscopy. A single point mutation, G442R, resulted in a mutant displaying an a(1~2) regioselectivity. Other amino acid substitutions at this site also gave mutants with altered regioselectivity and transglycosylation profiles. This is the first demonstration that single point mutations can lead to a strong modification of the regioselectivity of a glycosyl hydrolase. The kinetic parameters of the enzyme variants were determined and a preliminary investigation of possible substrates for condensation reactions conducted.