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

被引：28

作者：

Bultema, Jelle B. ^{[1
]}

Kuipers, Bas J. H. ^{[2
]}

Dijkhuizen, Lubbert ^{[1
]}

机构：

[1] Univ Groningen, Groningen Biomol Sci & Biotechnol Inst GBB, NL-9747 AG Groningen, Netherlands

[2] FrieslandCampina Res, NL-3818 LE Amersfoort, Netherlands

来源：

FEBS OPEN BIO | 2014年 / 4卷

关键词：

beta-Galactosidase; Galacto-oligosaccharides; Bacillus circulans; Catalytic residue; Mutagenesis; Transglycosylation activity; ESCHERICHIA-COLI; METAL-BINDING; GLU-416; STATE; MG2+;

D O I：

10.1016/j.fob.2014.11.002

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The Bacillus circulans ATCC 31382 beta-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. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

引用

页码：1015 / 1020

页数：6

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