Marine Bacterium Bacillus Sp Research Articles

Purification and Characterization of a Novel Fibrinolytic Enzyme from Marine Bacterium Bacillus sp. S-3685 Isolated from the South China Sea.

A novel fibrinolytic enzyme, BSFE1, was isolated from the marine bacterium Bacillus sp. S-3685 (GenBank No.: KJ023685) found in the South China Sea. This enzyme, with a molecular weight of approximately 42 kDa and a specific activity of 736.4 U/mg, exhibited its highest activity at 37 °C in a phosphate buffer at pH 8.0. The fibrinolytic enzyme remained stable over a pH range of 7.5 to 10.0 and retained about 76% of its activity after being incubated at 37 °C for 2 h. The Km and Vmax values of the enzyme at 37 °C were determined to be 2.1 μM and 49.0 μmol min-1 mg-1, respectively. The fibrinolytic activity of BSFE1 was enhanced by Na+, Ba2+, K+, Co2+, Mn2+, Al3+, and Cu2+, while it was inhibited by Fe3+, Ca2+, Mg2+, Zn2+, and Fe2+. These findings indicate that the fibrinolytic enzyme isolated in this study exhibits a strong affinity for fibrin. Moreover, the enzyme we have purified demonstrates thrombolytic enzymatic activity. These characteristics make BSFE1 a promising candidate for thrombolytic therapy. In conclusion, the results obtained from this study suggest that our work holds potential in the development of agents for thrombolytic treatment.

Characterization and Application of a New β-Galactosidase Gal42 From Marine Bacterium Bacillus sp. BY02.

β-Galactosidase plays an important role in medicine and dairy industry. In this study, a new glycoside hydrolase family 42 (GH42) β-galactosidase-encoding gene, gal42, was cloned from a newly isolated marine bacterium Bacillus sp. BY02 and expressed in Escherichia coli. Structural characterization indicated that the encoding β-galactosidase, Gal42, is a homotrimer in solution, and homology modeling indicated that it retains the zinc binding sites of the Cys cluster. The reaction activity of Gal42 was significantly increased by Zn2+ (229.6%) and other divalent metal ions (Mn2+, Mg2+, and Co2+), while its activity was inhibited by EDTA (53.9%). Meanwhile, the thermo-stability of the Gal42 was also significantly enhanced by 5 and 10 mM of zinc ion supplement, which suggested that the “Cys-Zn” motif played important roles in both structural stability and catalytic function. Furthermore, Gal42 showed effective lactose hydrolysis activity, which makes the enzyme hydrolyze the lactose in milk effectively. These properties make Gal42 a potential candidate in food technology.

Purification, characterization and immunostimulatory activity of a novel exopolysaccharide from Bacillus sp. H5

Crude exopolysaccharides from extracellular polymeric substances produced by the marine bacterium Bacillus sp. H5 were fractionated using DEAE-Sepharose FF and Sephadex G-75 chromatography. The high molecular weight fraction (89.0 kD) from the neutral fraction was designated EPS5SH; it contained mannose, glucosamine, glucose, and galactose in a molar ratio of 1.00: 0.02: 0.07: 0.02. Infra-red, gas chromatography–mass spectrometry, electrospray ionisation-tandem mass spectrometry analysis and nuclear magnetic resonance revealed EPS5SH was a mannan with α-(1 → 4)-Manp, α-(1 → 2)-Manp, α-(1 → 4, 6)-Manp and β-terminal-Manp. Preliminary in vitro experiments revealed that EPS5SH significantly upregulated nitric oxide synthesis and release of pro-inflammatory factors in murine macrophage RAW264.7 cells. Western blot experiments verified the immunostimulatory effects of EPS5SH through the modulation of the NF-κB and MAPK signalling pathways. In conclusion, EPS5SH was a novel immunostimulatory mannan.

Cloning and Characterization of a Cold-adapted Chitosanase from Marine Bacterium Bacillus sp. BY01.

Chitosanase plays an important role in the production of chitooligosaccharides (CHOS), which possess various biological activities. Herein, a glycoside hydrolase (GH) family 46 chitosanase-encoding gene, csnB, was cloned from marine bacterium Bacillus sp. BY01 and heterologously expressed in Escherichia coli. The recombinant chitosanase, CsnB, was optimally active at 35 °C and pH 5.0. It was also revealed to be a cold-adapted enzyme, maintaining 39.5% and 40.4% of its maximum activity at 0 and 10 °C, respectively. Meanwhile, CsnB showed wide pH-stability within the range of pH 3.0 to 7.0. Then, an improved reaction condition was built to enhance its thermostability with a final glycerol volume concentration of 20%. Moreover, CsnB was determined to be an endo-type chitosanase, yielding chitosan disaccharides and trisaccharides as the main products. Overall, CsnB provides a new choice for enzymatic CHOS production.

Characterization of Antifungal Lipopeptide Biosurfactants Produced by Marine Bacterium Bacillus sp. CS30.

This study was initiated to screen for marine bacterial agents to biocontrol Magnaporthe grisea, a serious fungal pathogen of cereal crops. A bacterial strain, isolated from the cold seep in deep sea, exhibited strong growth inhibition against M. grisea, and the strain was identified and designated as Bacillus sp. CS30. The corresponding antifungal agents were purified by acidic precipitation, sequential methanol extraction, Sephadex LH-20 chromatography, and reversed phase high-performance liquid chromatography (RP-HPLC), and two antifungal peaks were obtained at the final purification step. After analysis by mass spectrometry (MS) and tandem MS, two purified antifungal agents were deduced to belong to the surfactin family, and designated as surfactin CS30-1 and surfactin CS30-2. Further investigation showed that although the antifungal activity of surfactin CS30-1 is higher than that of surfactin CS30-2, both of them induced the increased generation of reactive oxygen species (ROS) and caused serious damage to the cell wall and cytoplasm, thus leading to the cell death of M. grisea. Our results also show the differences of the antifungal activity and antifungal mechanism of the different surfactin homologs surfactin CS30-1 and surfactin CS30-2, and highlight them as potential promising agents to biocontrol plant diseases caused by M. grisea.

Biosynthesis of Pd and Au as nanoparticles by a marine bacterium Bacillus sp. GP and their enhanced catalytic performance using metal oxides for 4-nitrophenol reduction.

Recovery of noble metals using marine bacteria is becoming an attractive research area because the marine microbes can better adapt to unfavorable environment than terrestrial microorganisms. In this study, we first reported that a marine Bacillus sp. GP was capable of producing Pd and Au NPs in the presence of sodium lactate. Ultraviolet visible spectrometer (UV-vis), transmission electron microscopy (TEM), X-ray diffraction patterns (XRD), X-ray photoelectron spectroscopy (XPS) and fourier transform infrared spectroscopy (FTIR) analyses were employed to explain the process and mechanism of Pd(II)/Au(III) reduction through GP. Additionally, we also found that bio-Pd/Au NPs could be used as catalysts in chemical reduction of 4-nitrophenol (4-NP). Moreover, the catalytic activity of bio-Pd NPs could be enhanced by Fe3O4, Al2O3 and SiO2, which is beneficial for practical application. The k1 (k2) values of Fe3O4, Al2O3 and SiO2 supplemental systems were approximately 1.28-1.69 (1.15-1.69), 1.42-1.75 (1.53-1.91) and 1.07-1.73 (1.14-1.49) fold, respectively, compared to that of control systems.

Purification and Characterization of a Novel Alginate Lyase from the Marine Bacterium Bacillus sp. Alg07.

Alginate oligosaccharides with different bioactivities can be prepared through the specific degradation of alginate by alginate lyases. Therefore, alginate lyases that can be used to degrade alginate under mild conditions have recently attracted public attention. Although various types of alginate lyases have been discovered and characterized, few can be used in industrial production. In this study, AlgA, a novel alginate lyase with high specific activity, was purified from the marine bacterium Bacillus sp. Alg07. AlgA had a molecular weight of approximately 60 kDa, an optimal temperature of 40 °C, and an optimal pH of 7.5. The activity of AlgA was dependent on sodium chloride and could be considerably enhanced by Mg2+ or Ca2+. Under optimal conditions, the activity of AlgA reached up to 8306.7 U/mg, which is the highest activity recorded for alginate lyases. Moreover, the enzyme was stable over a broad pH range (5.0–10.0), and its activity negligibly changed after 24 h of incubation at 40 °C. AlgA exhibited high activity and affinity toward poly-β-d-mannuronate (polyM). These characteristics suggested that AlgA is an endolytic polyM-specific alginate lyase (EC 4.2.2.3). The products of alginate and polyM degradation by AlgA were purified and identified through fast protein liquid chromatography and electrospray ionization mass spectrometry, which revealed that AlgA mainly produced disaccharides, trisaccharides, and tetrasaccharide from alginate and disaccharides and trisaccharides from polyM. Therefore, the novel lysate AlgA has potential applications in the production of mannuronic oligosaccharides and poly-α-l-guluronate blocks from alginate.

Optimization, Production, and Partial Purification of Thermostable -Amylase Produced by Marine Bacterium Bacillus sp. NRC12017

α-Amylase biosynthesis was illustrated under stress circumstances of high temperature and high salinity in aerobically cultivated culture of a newly isolated moderately thermophilic bacterium of spore-forming Bacillus sp. NRC12017 in medium containing starch, peptone and yeast extract. Maximum yield (18.41 U/ml) was took place at pH 6.5 with 200 µl inoculum size at 45°C and an incubation time of 3 days. The ideal volume of the fermentation broth was found to be 15 ml in 100 ml Erlenmeyer flask, supplementation of starch at 2.5% and peptone plus yeast extract at 0.7% brought about the greatest production of α-amylase. Two fractions of α-amylase activities, designated FIII and FIV, were refined from culture filtrate utilizing ammonium sulfate (60 and 80%) and showed the main band at 30 KDa. Both fractions had the highest activity at 250 µl starch in a reaction mixture and pH 6.0-7.0. FIII and FIV showed that a temperature of 50 and 55°C and a reaction time of 20 and 30 min were the best available conditions for their activities, respectively, both fractions were stable up to 65°C and the activity was decreased drastically to 3.22 and 3.26 % when heated at 70°C. Concerning pH stability, a broad range of pH stability (5.0-11.0) was obtained by FIII and FIV.

Pumilacidin-Like Lipopeptides Derived from Marine Bacterium Bacillus sp. Strain 176 Suppress the Motility of Vibrio alginolyticus

Bacterial motility is a crucial factor during the invasion and colonization processes of pathogens, which makes it an attractive therapeutic drug target. Here, we isolated a marine bacterium (Vibrio alginolyticus strain 178) from a seamount in the tropical West Pacific that exhibits vigorous motility on agar plates and severe pathogenicity to zebrafish. We found that V. alginolyticus 178 motility was significantly suppressed by another marine bacterium, Bacillus sp. strain 176, isolated from the same niche. We isolated, purified, and characterized two different cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The two related CLPs have a pumilacidin-like structure and were both effective inhibitors of V. alginolyticus 178 motility. The CLPs differ by only one methylene group in their fatty acid chains. In addition to motility suppression, the CLPs also induced cell aggregation in the medium and reduced adherence of V. alginolyticus 178 to glass substrates. Notably, upon CLP treatment, the expression levels of two V. alginolyticus flagellar assembly genes (flgA and flgP) dropped dramatically. Moreover, the CLPs inhibited biofilm formation in several other strains of pathogenic bacteria without inducing cell death. This study indicates that CLPs from Bacillus sp. 176 show promise as antimicrobial lead compounds targeting bacterial motility and biofilm formation with a low potential for eliciting antibiotic resistance.IMPORTANCE Pathogenic bacteria often require motility to establish infections and subsequently spread within host organisms. Thus, motility is an attractive therapeutic target for the development of novel antibiotics. We found that cyclic lipopeptides (CLPs) produced by marine bacterium Bacillus sp. strain 176 dramatically suppress the motility of the pathogenic bacterium Vibrio alginolyticus strain 178, reduce biofilm formation, and promote cellular aggregation without inducing cell death. These findings suggest that CLPs hold great promise as potential drug candidates targeting bacterial motility and biofilm formation with a low overall potential for triggering antibiotic resistance.

The optimal of xylanase production conditions using empty fruit bunch raw biomass by marine bacterium Bacillus sp LBF-001

Several xylanases have been studied recently, but few xylanases are from marine microorganisms have been reported. Marine bacterium Bacillus sp. LBF-001 was isolated from Pari Island Kepulauan Seribu in Indonesia. The purposes of this study are to identify of 16S rDNA gene from marine bacterium LBF-001 and to optimize medium conditions including kind and concentration of biomass, nitrogen source, pH and temperature. With 16S rDNA gene analysis that LBF-001 isolate have 99% identity with Bacillus pumilus HT-Z4-B2 (KJ526885). Fermentation for producing xylanases was done by using several agricultural residues under solid-state fermentation (SSF). The optimum condition for xylanase production by isolate Bacillus sp. LBF-001 are 2.5% empty fruit bunch, 0.6% lactose broth, pH values was 6.5, temperature condition was 30oC, under submerged fermentation with shaking at 150 rpm for 48 hours fermentation and giving the xylanase activity 10.85 U/mL.

Photosynthetic inhibition and oxidative stress to the toxic Phaeocystis globosa caused by a diketopiperazine isolated from products of algicidal bacterium metabolism.

Algicidal bacteria have been turned out to be available for inhibiting Phaeocystis globosa which frequently caused harmful algal blooms and threatened to economic development and ecological balance. A marine bacterium Bacillus sp. Ts-12 exhibited significant algicidal activity against P. globosa by indirect attack. In present study, an algicidal compound was isolated by silica gel column, Sephadex G-15 column and HPLC, further identified as hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, cyclo-(Pro-Gly), by GC-MS and (1)H-NMR. Cyclo-(Pro-Gly) significantly increased the level of reactive oxygen species (ROS) within P. globosa cells, further activating the enzymatic and non-enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and ascorbic acid (AsA). The increase in methane dicarboxylic aldehyde (MDA) content showed that the surplus ROS induced lipid peroxidation on membrane system. Transmission electron microscope (TEM) and flow cytometry (FCM) analysis revealed that cyclo-(Pro-Gly) caused reduction of Chl-a content, destruction of cell membrane integrity, chloroplasts and nuclear structure. Real-time PCR assay showed that the transcriptions of photosynthesis related genes (psbA, psbD, rbcL) were significantly inhibited. This study indicated that cyclo-(Pro-Gly) from marine Bacillus sp. Ts-12 exerted photosynthetic inhibition and oxidative stress to P. globosa and eventually led to the algal cells lysis. This algicidal compound might be potential bio-agent for controlling P. globosa red tide.

Purification and characterization of cellulase from a marine Bacillus sp. H1666: A potential agent for single step saccharification of seaweed biomass

• Marine bacterium Bacillus sp. utilized without prior treated agricultural waste. • Cellulase production by Bacillus sp. in single step from wheat straw/rice bran. • Cellulase: molecular mass of 40 kDa, thermo-stable and active at wide pH 3–9. • High saccharification efficacy as tested on seaweed Ulva . In this study, 115 marine bacterial isolates were screened for cellulase enzymatic activity and enzyme with a molecular mass of 40 kDa was purified from culture supernatant of the marine bacterium Bacillus sp. H1666 using ion exchange and size exclusion chromatography method. Growth of bacterial strain H1666 with efficient cellulase enzyme production was observed on untreated wheat straw and rice bran. The biochemical properties of the extracted cellulase were studied and enzyme was found active over a range of pH 3–9. The optimum cellulase activity was observed at pH 7 and temperature 50 °C. The enzyme was also shown to be slightly thermo-stable with 40% residual activity at 60 °C for 4 h. The potential applicability of enzyme was tested on dried green seaweed ( Ulva lactuca ) and 450 mg/g increase in glucose yield was obtained after saccharification. MALDI TOF–TOF analysis of cellulase peptide fingerprint showed similarity to the sequence of the glycoside hydrolase family protein.

Biochemical characterization of a raw starch degrading α-amylase from the Indonesian marine bacterium Bacillus sp. ALSHL3

An Indonesian marine bacterial isolate, which belongs to genus of Bacillus sp. based on 16S rDNA analysis and was identified as Bacillus filicolonicus according to its morphology and physiology, produced a raw starch degrading α-amylase. The partially purified α-amylase using a maize starch affinity method exhibited an optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme retained 72% of its activity in the presence of 1.5 M NaCl. Scanning electron micrographs showed that the α-amylase was capable of degrading starch granules of rice and maize. This α-amylase from Bacillus sp. ALSHL3 was classified as a saccharifying enzyme since its major final degradation product was glucose, maltose, and maltotriose.

Synthesis of bacillamide 3 and its analogue

The bacillamide 1 is a new algaecide from the marine bacterium Bacillus sp. SY-1. Its analogues bacillamide 3 and alkaloid 4 were firstly synthesized effectively from d-alanine. The key step was a coupling reaction via the mixed anhydride. All structures were confirmed by 1H NMR and 13C NMR. The final compounds were confirmed by 1H NMR, 13C NMR and HRMS and the results are consistent with the reported natural products.

Three new cytotoxic cyclic acylpeptides from marine Bacillus sp.

Three new cytotoxic cyclopeptides belonging to iturin class have been isolated from marine bacterium Bacillus sp. These three compounds have the cytotoxic activities. Their structures were determined based on chemical analysis and all kinds of spectroscopic techniques.