![]() Β-Galactosidases (E.C.3.2.1.23) are enzymes that catalyse the hydrolytic cleavage of galactose residues from the non-reducing end of β-galactosides. BgaC has transglycosylation activity producing prebiotic oligosaccharides.BgaC possesses sought-after properties for biotechnology, e.g.Bifidobacterium adolescentis BgaC β-galactosidase was selected from human faecal metagenome. ![]() adolescentis BgaC make it an ideal candidate for dairy industry applications and prebiotic manufacture. In addition, BgaC possessed transglycosylation activity to produce galactooligosaccharides (GOS) from lactose, as determined by TLC and HPLC analysis. It exhibited low product inhibition by galactose with a K i of 116 mM and high tolerance for glucose (66% activity retained in presence of 700 mM glucose). BgaC had a V max of 107 μmol/min/mg and a K m of 2.5 mM for ONPG and a V max of 22 μmol/min/mg and a K m of 3.7 mM for lactose. Kinetic parameters were determined using ortho-nitrophenyl-β- d-galactopyranoside (ONPG) and lactose substrates. It required a divalent metal ion co-factor maximum activity was detected with Mg 2+, while Cu 2+ and Mn 2+ were inhibitory. The enzyme had optimal activity at pH 7.0 and 37 ☌, with a wide range of pH (4–10) and temperature (0–40 ☌) stability. This gene was cloned with a hexahistidine tag, expressed in Escherichia coli and His-tagged-BgaC was purified using Ni 2+-NTA affinity chromatography and size filtration. The β-galactosidase gene found in the majority of the clones was BAD_1582 from Bifidobacterium adolescentis, subsequently named bgaC. ![]() Out of the 16,000 clones screened, 30 β-galactosidase-positive clones were identified. A human faecal metagenomic fosmid library was constructed and functionally screened to identify novel β-galactosidases. Members of the human gut microbiota use glycoside hydrolase (GH) enzymes, such as β-galactosidases, to forage on host mucin glycans and dietary fibres.
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