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.Graphical
Highlights
Introduction β-Galactosidases (E.C.3.2.1.23) are enzymes that catalyse the hydrolytic cleavage of galactose residues from the non
The forward sequence of clone 9 had similarity with Bifidobacterium bifidum (83% identity) while the reverse sequence had a match with Olsenella uli (73% identity)
After 72-h incubation, there was little remaining lactose and the hydrolysis reaction was clearly favoured over transglycosylation as Glc and Gal were the most intense reaction products (Fig. 5c). These results indicate a balance of reactions with transglycosylation and hydrolysis both occurring simultaneously at all time points, but the balance favouring different reaction products shifting over time
Summary
Introduction βGalactosidases (E.C.3.2.1.23) are enzymes that catalyse the hydrolytic cleavage of galactose residues from the non-Bath, UK reducing end of β-galactosides. β-Galactosidases belong to the six glycoside hydrolase families of GH 1, GH 2, GH 35, GH 42, GH 59, and GH 147 within the Carbohydrate-Active enZymes (CAZy) database (http://www.cazy.org/) (Lombard et al 2014). Galactosidases (E.C.3.2.1.23) are enzymes that catalyse the hydrolytic cleavage of galactose residues from the non-. There has been a growing interest in identification and characterisation of β-galactosidases, primarily for application in dairy industries due to their hydrolysis of lactose into glucose and galactose (Adam et al 2004). The demand for lactose-free dairy products has increased due to the growing. Some βgalactosidases can carry out transglycosylation, whereby they transfer galactose residues to lactose acceptors to synthesise galactooligosaccharides (GOS) with various linkages and degrees of polymerisation (Reuter et al 1999; Torres et al 2010). The biotechnological applications of β-galactosidases encompass production of lactosefree dairy products and production of GOS with prebiotic properties
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