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- Title
Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382.
- Authors
Bultema, Jelle B.; Kuipers, Bas J.H.; Dijkhuizen, Lubbert
- 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.
- Subjects
GALACTOSIDASES; BACILLUS circulans; OLIGOSACCHARIDES; AMINO acid residues; BIOINFORMATICS; HISTIDINE
- Publication
FEBS Open Bio, 2014, Vol 4, p1015
- ISSN
2211-5463
- Publication type
Article
- DOI
10.1016/j.fob.2014.11.002