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

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

Effects of sugars on the activity of Aga-BC7050.

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

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

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

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

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

Development and evaluation of novel recombinant adenovirus-based vaccine candidates for infectious bronchitis virus and <i>Mycoplasma gallisepticum</i> in chickens

<p>Avian infectious bronchitis caused by the infectious bronchitis virus (IBV), and mycoplasmosis caused by <i>Mycoplasma gallisepticum</i> (MG) are two major respiratory diseases in chickens that have resulted in severe economic losses in the poultry industry. We constructed a recombinant adenovirus that simultaneously expresses the S1 spike glycoprotein of IBV and the TM-1 protein of MG (pBH-S1-TM-1-EGFP). For comparison, we constructed two recombinant adenoviruses (pBH-S1-EGFP and pBH-TM-1-EGFP) that express either the S1 spike glycoprotein or the TM-1 protein alone. The protective efficacy of these three vaccine constructs against challenge with IBV and/or MG was evaluated in specific pathogen free chickens. Groups of seven-day-old specific pathogen free chicks were immunized twice, two weeks apart, via the oculonasal route with the pBH-S1-TM-1-EGFP, pBH-S1-EGFP, or pBH-TM-1-EGFP vaccine candidates or the commercial attenuated infectious bronchitis vaccine strain H52 and MG vaccine strain F-36 (positive controls), and challenged with virulent IBV or MG two weeks later. Interestingly, by days 7 and 14 after the booster immunization, pBH-S1-TM-1-EGFP-induced antibody titre was significantly higher (<i>P </i>< 0.01) compared to attenuated commercial IBV vaccine; however, there was no significant difference between the pBH-S1-TM-1-EGFP and attenuated commercial MG vaccine groups (<i>P </i>> 0.05). The clinical signs, the gross, and histopathological lesions scores of the adenovirus vaccine constructs were not significantly different from that of the attenuated commercial IBV or MG vaccines (positive controls) (<i>P </i>> 0.05). These results demonstrate the potential of the bivalent pBH-S1-TM-1-EGFP adenovirus construct as a combination vaccine against IB and mycoplasmosis.</p