title: Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382.
abstract: The Bacillus circulans ATCC 31382 β-galactosidase (BgaD) is a retaining-type glycosidase of glycoside hydrolase family 2 (GH2). Its commercial enzyme preparation, Biolacta N5, is used for commercial-scale production of galacto-oligosaccharides (GOS). The BgaD active site and catalytic amino acid residues have not been studied. Using bioinformatic routines we identified two putative catalytic glutamates and two highly conserved active site histidines. The site-directed mutants E447N, E532Q, and H345F, H379F had lost (almost) all catalytic activity. This confirmed their essential role in catalysis, as general acid/base catalyst (E447) and nucleophile (E532), and as transition state stabilizers (H345, H379), respectively.
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Jelle B
authors.initials: JB
authors.lastname: Bultema
authors.affiliation: FrieslandCampina Research, Stationsplein 4, 3818 LE Amersfoort, The Netherlands.
authors.forename: Bas J H
authors.initials: BJ
authors.lastname: Kuipers
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Lubbert
authors.initials: L
authors.lastname: Dijkhuizen
keywords: Bacillus circulans; Catalytic residue; DNA, deoxyribonucleic acid; GH2, glycoside hydrolase family 2; GOS, galacto-oligosaccharides; Galacto-oligosaccharides; Mutagenesis; PCR, polymerase chain reaction; SD, standard deviation; Tm, melting temperatures; Transglycosylation activity; o-NPG, ortho-nitrophenyl-β-galactoside; β-Galactosidase