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

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.

A GH42 β‐galactosidase from Bifidobacterium thermophilum: Biochemical characterization and synthesis of galactosyl glycerol by reverse hydrolysis

“…As shown in Figure 4A, when p NPG was used as the substrate, the activity of BtGal42 was about 297.2% of that on o NPG, indicating that BtGal42 had a noticeable preference for the cleavage of β‐D‐anomeric linkages. This phenomenon was also observed for β‐galactosidases from B. adolescentis , 29 B. velezensis SW5, 41 and B. coagulans NL01 10 . No activity was detected when p NPαG, p NPGul, p NPLac, and p NPCel were used as substrates, indicating that BtGal42 acted exclusively on terminal β‐linked Gal residues.…”

“…The activities of β‐galactosidase from B. velezensis SW5, 41 B. longum CCRC 15708, 46 and P. barengoltzii 30 were also inhibited by Ca 2+ . BtGal42 was completely inhibited by Cu 2+ , Zn 2+ , and Fe 3+ , as the most reported bacterial β‐galactosidases 27,29,30,41 . On the other hand, it was found that Mg 2+ enhanced the activity of BtGal42 by within 15%.…”

“…The thermostability of BtGal42 was assayed by incubating BtGal42 at different temperature from 30 to 70°C for 2 h. The results showed that more than 80% of its original enzymatic activity was remained after incubation at 50°C for 2 h, while the enzyme showed a gradual inactivation at temperatures above 60°C (Figure 3D). The thermal stability of BtGal42 was lower than that of some thermostable β‐galactosidases, such as T. terrestris β‐galactosidase TtbGal1, 5 B. coagulans β‐galactosidase BcBga, 10 and P. torridus β‐galactosidase 39 ; however, BtGal42 exhibited a higher thermostability compared with β‐galactosidases from other Bifidobacterium species, including B. breve , 35 B. bifidum , 32,34 and B. adolescentis 29 …”

“…The amount of glucose released upon hydrolysis was determined using a glucose oxidase‐peroxidase assay kit (Biosino, Beijing, China) according to the manufacturer's protocol. One unit of enzymatic activity was defined as the amount of enzyme required to release 1 μmol glucose per minute 29 …”

“…One unit of enzymatic activity was defined as the amount of enzyme required to release 1 μmol glucose per minute. 29 Additionally, the enzymatic hydrolysis of lactose was evaluated using 50 g/L lactose and 5 U/g BtGal42 and carried out for 4 h. Self-condensation reaction was performed using 400 g/L lactose as the substrate serving as both glycosyl donor and acceptor for 24 h. The hydrolysis and transglycosylation products were analyzed by the thinlayer chromatography (TLC). 28 The mixture of butanol, acetic acid, and water at a ratio of 5:3:2 (v/v/v) was used as the developing solvent system.…”