Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382

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 res...

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Autores principales: Bultema, Jelle B., Kuipers, Bas J.H., Dijkhuizen, Lubbert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2014
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4250540/
https://www.ncbi.nlm.nih.gov/pubmed/25473598
http://dx.doi.org/10.1016/j.fob.2014.11.002
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author Bultema, Jelle B.
Kuipers, Bas J.H.
Dijkhuizen, Lubbert
author_facet Bultema, Jelle B.
Kuipers, Bas J.H.
Dijkhuizen, Lubbert
author_sort Bultema, Jelle B.
collection PubMed
description 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.
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spelling pubmed-42505402014-12-03 Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382 Bultema, Jelle B. Kuipers, Bas J.H. Dijkhuizen, Lubbert FEBS Open Bio Article 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. Elsevier 2014-11-12 /pmc/articles/PMC4250540/ /pubmed/25473598 http://dx.doi.org/10.1016/j.fob.2014.11.002 Text en © 2014 The Authors
spellingShingle Article
Bultema, Jelle B.
Kuipers, Bas J.H.
Dijkhuizen, Lubbert
Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382
title Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382
title_full Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382
title_fullStr Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382
title_full_unstemmed Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382
title_short Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382
title_sort biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of bacillus circulans atcc 31382
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4250540/
https://www.ncbi.nlm.nih.gov/pubmed/25473598
http://dx.doi.org/10.1016/j.fob.2014.11.002
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