A novel α-galactosidase from the thermophilic probiotic <i>Bacillus coagulans</i> with remarkable protease-resistance and high hydrolytic activity

<div><p>A novel α-galactosidase of glycoside hydrolase family 36 was cloned from <i>Bacillus coagulans</i>, overexpressed in <i>Escherichia coli</i>, and characterized. The purified enzyme Aga-BC7050 was 85 kDa according to SDS-PAGE and 168 kDa according to gel filtration, indicating that its native structure is a dimer. With <i>p</i>-nitrophenyl-α-d- galactopyranoside (pNPGal) as the substrate, optimal temperature and pH were 55 °C and 6.0, respectively. At 60 °C for 30 min, it retained > 50% of its activity. It was stable at pH 5.0–10.0, and showed remarkable resistance to proteinase K, subtilisin A, α-chymotrypsin, and trypsin. Its activity was not inhibited by glucose, sucrose, xylose, or fructose, but was slightly inhibited at galactose concentrations up to 100 mM. Aga-BC7050 was highly active toward pNPGal, melibiose, raffinose, and stachyose. It completely hydrolyzed melibiose, raffinose, and stachyose in < 30 min. These characteristics suggest that Aga-BC7050 could be used in feed and food industries and sugar processing.</p></div

Determination of the molecular weight and oligomeric state of Aga-BC7050 by analytical gel filtration.

<p>(A) SDS-PAGE analysis of Aga-BC7050. Lanes: M, protein molecular markers; 1, crude lysate; 2, purified Aga-BC7050. (B) Elution profile from the analysis of purified Aga-BC7050. (C) Calibration curve using protein standards: albumin, 66.0 kDa; lactate dehydrogenase, 140.0 kDa; catalase, 232.0 kDa; ferritin, 440.0 kDa; thyroglobulin, 669.0 kDa.</p

Effects of different metal ions and specific chemical reagents on the activity of Aga-BC7050.

<p>Effects of different metal ions and specific chemical reagents on the activity of Aga-BC7050.</p

Thin-layer chromatography (TLC) analysis of oligosaccharide degradation by Aga-BC7050.

<p>Lane S shows the oligosaccharide standards. Control group (0 min) represents the substrate (melibiose, raffinose, stachyose) without the addition of α-galactosidase.</p

Effects of sugars on the activity of Aga-BC7050.

<p>Effects of sugars on the activity of Aga-BC7050.</p

Effect of temperature and pH on the enzymatic properties of purified Aga-BC7050.

<p>(A) Optimal temperature, (B) thermostability assay, (C) optimal pH, (D) pH stability assay. The effect of temperature was determined using different temperatures and McIlvaine buffer (pH 6.0). The pH profiles were measured at 55 °C with different buffers including McIlvaine (pH 4.0–8.0) and Britton-Robinson (pH 7.0–11.0). Error bars represent standard deviations.</p

Kinetic parameters of Aga-BC7050.

<p>Kinetic parameters of Aga-BC7050.</p

Hydrolysis of different substrates by Aga-BC7050.

<p>Hydrolysis of different substrates by Aga-BC7050.</p

Amino acid sequence alignment of Aga-BC7050 with other glycoside hydrolase (GH) family 36 α-galactosidases.

<p>The sequences are indicated with their genus initials, including <i>Enterococcus cecorum</i>, <i>Geobacillus stearothermophilus</i>, <i>Lactobacillus acidophilus</i>, <i>Bacillus megaterium</i>, <i>Bacillus halodurans</i>, <i>Aspergillus niger</i>, and <i>Rhizopus</i> sp. The gaps are indicated by hyphens. Similar or identical amino acid residues at the same position are highlighted. The asterisks show putative catalytic residues.</p