Industrial attributes of ß-glucanase produced by Bacillus sp. CSB55 and its potential application as bio-industrial catalyst.

The ß-glucanase produced from Bacillus sp. CSB55 not only depicts the potent industrial characteristics but also relates as bio-industrial catalyst supporting the spontaneous formation of the products, high hydrolytic efficiency, and feasibility of the enzymatic reaction. A homogeneous ß-glucanase (GluB55) was purified via various purification processes resulting in 11.69% yield and 14.24-fold purity. Biochemical characterization of the purified enzyme revealed the molecular mass of approximately 40 kDa, which was verified by zymography. The optimum activity of GluB55 was determined at pH 7.2 and 55 °C. GluB55 could highly hydrolyze carboxymethylcellulose and was stable over a wide range of pH, retaining more than 70% residual activity at pH 5.8-11.0 and carried 100% thermostability as high as 60 °C. In addition, it showed 68% residual activity at 70 °C. The N-terminal amino acid sequence of GluB55 was Ala-Asn-Pro-Glu-Leu-Val-Asn-X-Gln-Ala-X-X-Ala-X-Gln-Gly. The enzyme activity was stimulated by Co2+ (158.6%), Zn2+ (211.1%), Mn2+ (264.4%), and Ba2+ (211.4%). Enzyme kinetics showed Km and Vmax values of 0.022 mg mL-1 and 994.56 ± 3.72 U mg-1, respectively. Q10 was calculated to be 1.12. ∆H, ∆G, and ∆S were low revealing that the formation of the transition phase and conversion to the product is very well organized. The lower the free energy change (∆G), the more feasible is the reaction.

Prospects for Bio-Industrial Application of an Extremely Alkaline Mannanase From Bacillus subtilis subsp. inaquosorum CSB31.

Mannan-degrading enzymes have been growing interest in bio-industrial applications, such as the pulp and paper, food, and pharmaceutical industries. In this study, an extremely alkaline mannanase (MnB31) is produced by Bacillus subtilis subsp. inaquosorum CSB31. MnB31 is purified to 17.92-fold with a 21.51% yield and specific activity of 1,796.13 U mg-1 by anion-exchange and gel filtration column chromatography. The biochemical characterization of MnB31 is performed, and the results are as follows: molecular weight of ≈47 kDa with an optimum temperature of 60 °C and pH of 12.5. The enzyme is strongly activated by Co2+ , Mn2+ , Na+ , and K+ , and inhibited by Zn2+ , Ni2+ , and Mg2+ . Halo-tolerance (10% NaCl), urea stability (3 M), and protease resistance are also observed. The kinetic parameters of MnB31 are found to be Km of 0.043 mg ml-1 , and Vmax of 1,046 ± 3.605 U mg-1 , respectively. In addition, the thermodynamical parameters are investigated; the activation energy (Ea ) is found to be 31.36 kJ mol-1 with a Kcat value of 156.9 × 104 s-1 , ΔH (28.59 kJ mol-1 ), ΔG (42.38 kJ mol-1 ), ΔS (-41.39 J mol-1 K-1 ), Q10 (1.40), ΔGE-S (-8.697 kJ mol-1 ), and ΔGE-T (-48.22 kJ mol-1 ). These results suggest that MnB31 has potential bio-industrial application, due to its greater hydrolytic efficiency and feasibility of enzymatic reaction.

Antimicrobial peptide from Bacillus subtilis CSB138: characterization, killing kinetics, and synergistic potency.

We studied the prospect of synergy between the antimicrobial peptide p138c and non-peptide antibiotics for increasing the potency and bacterial killing kinetics of these agents. The production of p138c was maximized in the late exponential growth phase of Bacillus subtilis CSB138. Purification of p138c resulted in a total of 4800 arbitrary units (AU) with 19.15-fold and 3.2% recovery. Peptide p138c was thermo-tolerant up to 50 °C and stable at pH 5.8 to 11. The biochemical nature of p138c was determined by a bioassay, similar to tricine-SDS-PAGE, indicating inhibition at 3 kDa. The amino acid sequence of p138c was Gly-Leu-Glu-Glu-Thr-Val-Tyr-Ile-Tyr-Gly-Ala-Asn-Met-X-Ser. Potency and killing kinetics against vancomycin-resistant Staphylococcus aureus improved considerably when p138c was synergized with oxacillin, ampicillin, and penicillin G. The minimal inhibitory concentration (MIC) of p138c showed a 4-, 8-, and 16-fold improvement when p138c was combined with oxacillin, ampicillin, and penicillin G, respectively. The fractional inhibitory concentration index for the combination of p138c and oxacillin, ampicillin, and penicillin G was 0.3125, 0.25, and 0.09, respectively. Synergy with non-peptide antibiotics resulted in enhanced killing kinetics of p138c. Hence, the synergy between antimicrobial peptide and non-peptide antibiotics may enhance the potency and bacterial killing kinetics, providing more potent and rapidly acting agents for therapeutic use. [Int Microbiol 20(1):43-53 (2017)].

Antimicrobial peptide from Bacillus subtilis CSB138: characterization, killing kinetics, and synergistic potency

We studied the prospect of synergy between the antimicrobial peptide p138c and non-peptide antibiotics for increasing the potency and bacterial killing kinetics of these agents. The production of p138c was maximized in the late exponential growth phase of Bacillus subtilis CSB138. Purification of p138c resulted in a total of 4800 arbitrary units (AU) with 19.15-fold and 3.2% recovery. Peptide p138c was thermo-tolerant up to 50 °C and stable at pH 5.8 to 11. The biochemical nature of p138c was determined by a bioassay, similar to tricine-SDS-PAGE, indicating inhibition at 3 kDa. The amino acid sequence of p138c was Gly-Leu-Glu-Glu-Thr-Val-Tyr-Ile-Tyr-Gly-Ala-Asn-Met-X-Ser. Potency and killing kinetics against vancomycin-resistant Staphylococcus aureus improved considerably when p138c was synergized with oxacillin, ampicillin, and penicillin G. The minimal inhibitory concentration (MIC) of p138c showed a 4-, 8-, and 16-fold improvement when p138c was combined with oxacillin, ampicillin, and penicillin G, respectively. The fractional inhibitory concentration index for the combination of p138c and oxacillin, ampicillin, and penicillin G was 0.3125, 0.25, and 0.09, respectively. Synergy with non-peptide antibiotics resulted in enhanced killing kinetics of p138c. Hence, the synergy between antimicrobial peptide and non-peptide antibiotics may enhance the potency and bacterial killing kinetics, providing more potent and rapidly acting agents for therapeutic use (AU)

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Antimicrobial peptide isolated from Bacillus amyloliquefaciens K14 revitalizes its use in combinatorial drug therapy.

The present study was performed to evaluate the antibacterial activities of an antimicrobial peptide (CSpK14) and the synergies thereof with ß-lactams against vancomycin-resistant Staphylococcus aureus (VRSA) and Enterococci (VRE). Our strain was isolated from fermented food (kimchi), which is 99.79 % homologous with Bacillus amyloliquefaciens subsp. plantarum FZB42(T). CSpK14 was purified to homogeneity by diammonium sulfate precipitation, concentration, dialysis, and followed by two-stage chromatographic separation, i.e., Sepharose Cl-6B and Sephadex G-25 chromatography, and had a molar mass of ~4.6 kDa via Tricine SDS-PAGE and in situ examination. It was stable at pH 6.0-11.5 and temperature up to 80 °C. In addition, it was also stable with various metal ions, solvents, and proteases. The N-terminal amino acid sequence was H-Y-D-P-G-D-D-S-G-N-T-G and did not show any significant homology with reported peptides. However, it shows some degrees of identity with alpha-2-macroglobulin and ligand-gated channel protein from different microorganisms. CSpK14 significantly reduced the minimum inhibitory concentrations (MICs) of ß-lactams and had no effect on non-ß-lactams against VRSA and VRE. MICs of CSpK14/oxacillin and CSpK14/ampicillin were reduced by 8- to 64-fold and 2- to 16-fold, respectively. The time killing assay between CSpK14/oxacillin (2.29-2.37 Δlog10CFU/mL at 24 h) and CSpK14/ampicillin (2.30-2.38 Δlog10CFU/mL at 24 h) being >2-fold and fractional inhibitory concentration index ˂0.5 revealed synergy. Furthermore, the biofilms formed by VRSA and VRE were reduced completely. CSpK14 was simple to purify, had low molecular mass, was stable over a wide pH range or tested chemicals, had broad inhibitory spectrum, and possessed potent synergistic antimicrobial-antibiofilm properties. CSpK14 synergistically enhanced the efficacy of ß-lactams and is therefore suitable for combination therapy.

A multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its compatibility as an industrial biocatalyst.

Bacillus sp. CSB39, isolated from popular traditional Korean food (Kimchi), produced a low molecular weight, thermostable mannanase (MnCSB39); 571.14U/mL using locust bean gum galactomannan as a major substrate. It was purified to homogeneity using a simple and effective two-step purification strategy, Sepharose CL-6B and DEAE Sepharose Fast Flow, which resulted in 25.47% yield and 19.32-fold purity. The surfactant-, NaCl-, urea-, and protease-tolerant monomeric protein had a mass of ∼30kDa as analyzed by SDS-PAGE and galactomannan zymography. MnCSB39 was found to have optimal activity at pH 7.5 and temperature of 70°C. The enzyme showed ˃55% activity at 5.0-15% (w/v) NaCl, and ˃93% of the initial activity after incubation at 37°C for 60min. Trypsin and proteinase K had no effect on MnCBS39. The enzyme showed ˃80% activity in up to 3M urea. The N-terminal amino acid sequence, ALKGDGX, did not show identity with reported mannanases, which suggests the novelty of our enzyme. Activation energy for galactomannan hydrolysis was 26.85kJmol(-1) with a Kcat of 142.58×10(4)s(-1). MnCSB39 had Km and Vmax values of 0.082mg/mL and 1099±1.0Umg(-1), respectively. Thermodynamic parameters such as ΔH, ΔG, ΔS, Q10, ΔGE-S, and ΔGE-T supported the spontaneous formation of products and the high hydrolytic efficiency and feasibility of the enzymatic reaction, which strengthen its novelty. MnCSB39 activity was affected by metal ions, modulators, chelators, and detergents. Mannobiose was the principal end-product of hydrolysis. Bacillus subtilis CSB39 produced a maximum of 1524.44U mannanase from solid state fermentation of 1g wheat bran. MnCSB39 was simple to purify, was active at a wide pH and temperature range, multi-stress tolerant and catalyzes a thermodynamically possible reaction, characteristics that suggests its suitability for application as an industrial biocatalyst.