Metagenomic identification, purification and characterisation of the Bifidobacterium adolescentis BgaC β-galactosidase

Daniel Mehabie Mulualem, Christy Agbavwe, Lesley A. Ogilvie, Brian V. Jones, Michelle Kilcoyne, Conor O’Byrne, Aoife Boyd

Research output: Contribution to journalArticlepeer-review

Abstract

Members of the human gut microbiota use glycoside hydrolase (GH) enzymes, such as β-galactosidases, to forage on host mucin glycans and dietary fibres. A human faecal metagenomic fosmid library was constructed and functionally screened to identify novel β-galactosidases. Out of the 16,000 clones screened, 30 β-galactosidase-positive clones were identified. The β-galactosidase gene found in the majority of the clones was BAD_1582 from Bifidobacterium adolescentis, subsequently named bgaC. This gene was cloned with a hexahistidine tag, expressed in Escherichia coli and His-tagged-BgaC was purified using Ni2+-NTA affinity chromatography and size filtration. The enzyme had optimal activity at pH 7.0 and 37 °C, with a wide range of pH (4–10) and temperature (0–40 °C) stability. It required a divalent metal ion co-factor; maximum activity was detected with Mg2+, while Cu2+ and Mn2+ were inhibitory. Kinetic parameters were determined using ortho-nitrophenyl-β-d-galactopyranoside (ONPG) and lactose substrates. BgaC had a Vmax of 107 μmol/min/mg and a Km of 2.5 mM for ONPG and a Vmax of 22 μmol/min/mg and a Km of 3.7 mM for lactose. It exhibited low product inhibition by galactose with a Ki of 116 mM and high tolerance for glucose (66% activity retained in presence of 700 mM glucose). In addition, BgaC possessed transglycosylation activity to produce galactooligosaccharides (GOS) from lactose, as determined by TLC and HPLC analysis. The enzymatic characteristics of B. adolescentis BgaC make it an ideal candidate for dairy industry applications and prebiotic manufacture. Key points • Bifidobacterium adolescentis BgaC β-galactosidase was selected from human faecal metagenome. • BgaC possesses sought-after properties for biotechnology, e.g. low product inhibition. • BgaC has transglycosylation activity producing prebiotic oligosaccharides. [Figure not available: see fulltext.]

Original languageEnglish
Pages (from-to)1063-1078
Number of pages16
JournalApplied Microbiology and Biotechnology
Volume105
Issue number3
Early online date11 Jan 2021
DOIs
Publication statusPublished - 28 Feb 2021

Keywords

  • Bifidobacterium
  • Enzyme screening
  • Metagenomic library
  • Transglycosylation
  • β-Galactosidase

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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