Expression and characterization of catechol 1,2-dioxygenase from Oceanimonas marisflavi 102-Na3.

The gene of catechol 1, 2-dioxygenase was identified and cloned from the genome of Oceanimonas marisflavi 102-Na3. The protein was expressed in Escherichia coli BL21 (DE3) and purified to homogeneity of a dimer with molecular mass of 69.2 kDa. The enzyme was highly stable in pH 6.0-9.5 and below 45 °C and exhibited the maximum activity at pH 8.0 and 30 °C. Being the first characterized intradiol dioxygenase from marine bacteria Oceanimonas sp., the enzyme showed catalytic activity for catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and pyrogallol. For catechol, Km and Vmax were 11.2 µM and 13.4 U/mg of protein, respectively. The enzyme also showed resistance to most of the metal ions, surfactants and organic solvents, being a promising biocatalyst for biodegradation of aromatic compounds in complex environments.

High Water Density at Non-Ice-Binding Surfaces Contributes to the Hyperactivity of Antifreeze Proteins.

Antifreeze proteins (AFPs) can bind to ice nuclei thereby inhibiting their growth and their hydration shell is believed to play a fundamental role. Here, we use molecular dynamics simulations to characterize the hydration shell of four moderately-active and four hyperactive AFPs. The local water density around the ice-binding-surface (IBS) is found to be lower than that around the non-ice-binding surface (NIBS) and this difference correlates with the higher hydrophobicity of the former. While the water-density increase (with respect to bulk) around the IBS is similar between moderately-active and hyperactive AFPs, it differs around the NIBS, being higher for the hyperactive AFPs. We hypothesize that while the lower water density at the IBS can pave the way to protein binding to ice nuclei, irrespective of the antifreeze activity, the higher density at the NIBS of the hyperactive AFPs contribute to their enhanced ability in inhibiting ice growth around the bound AFPs.

Discovery of α-Glucosidase Inhibitors from Marine Microorganisms: Optimization of Culture Conditions and Medium Composition.

Various studies showed that the suppression of α-glucosidase activity can impede the glucose absorption in our body, and therefore, it can be used to treat type 2 diabetes. Hence, the compounds with anti-α-glucosidase have gained considerable attention because of their potential application in diabetes treatment. In previous literature studies, these anti-α-glucosidase compounds were extracted from plants and fungus. Less studies are being conducted to identify the anti-α-glucosidase compounds in the microbial community. In this study, 23 marine bacterial strains were screened for their potential to suppress the α-glucosidase activity. The highest inhibitory activity was exhibited by isolated L06 which was identified as Oceanimonas smirnovii EBL6. The cultivation conditions, such as temperature and pH, were optimized to increase the production of α-glucosidase inhibitors by Oceanimonas smirnovii EBL6 strain. The result findings showed that the highest yield of α-glucosidase inhibitors can be obtained at the culture time of 120 h, fermentation temperature of 30 °C, and pH 4.6. Under these conditions, the inhibitory activity of α-glucosidase can reach 81%. The IC50 of n-butanol extract was 13.89 µg/ml, while standard acarbose was 31.16 µg/ml. Overall, these findings suggest that Oceanimonas smirnovii produces α-glucosidase inhibitors and could been applied in the biochemical and medicinal fields in the future.

Potential Anticoagulant Activity of Trypsin Inhibitor Purified from an Isolated Marine Bacterium Oceanimonas Sp. BPMS22 and its Kinetics.

Protease inhibitors control major biological protease activities to maintain physiological homeostasis. Marine bacteria isolated from oligotrophic conditions could be taxonomically distinct, metabolically unique, and offers a wide variety of biochemicals. In the present investigation, marine sediments were screened for the potential bacteria that can produce trypsin inhibitors. A moderate halotolerant novel marine bacterial strain of Oceanimonas sp. BPMS22 was isolated, identified, and characterized. The effect of various process parameters like salt concentration, temperature, and pH was studied on the growth of the bacteria and production of trypsin inhibitor. Further, the trypsin inhibitor was purified to near homogeneity using anion exchange, size exclusion, and affinity chromatography. The purified trypsin inhibitor was found to competitively inhibit trypsin activity with an inhibition coefficient, Ki, of 3.44 ± 0.13 µM and second-order association rate constant, kass, of 1.08 × 103 M-1 S-1. The proteinaceous trypsin inhibitor had a molecular weight of approximately 30 kDa. The purified trypsin inhibitor showed anticoagulant activity on the human blood samples.

Oceanimonas smirnovii sp. nov., a novel organism isolated from the Black Sea.

A slightly creamy, melanogenic, gram-negative, aerobic bacterium was isolated from seawater sample collected in the Karadag Natural Reserve of the Eastern Crimea, the Black Sea. The novel organism was chemoorganotrophic, had no obligate requirement in NaCl, tolerated to 12% NaCl, grew between 10 and 45 degrees C, was slightly alkaliphilic, and was not able to degrade starch, gelatin, agar, and Tween 80. 16S rRNA gene sequence-based analyses of the new organism revealed that Oceanimonas doudoroffii ATCC 27123T, Oceanimonas baumanii ATCC 700832T, and Oceanisphaera litoralis DSM 15406T were the closest relatives (similarity around 97%-96%). The G + C content of the DNA of the strain 31-13T was 55.5mol%. Phosphatidylethanolamine (49.0%), phosphatidylglycerol (41.8%), and diphosphatidylglycerol (9.2%) were the predominant phospholipids. The major fatty acids were 16:0 (24.1%), 16:1omega7 (40.3%), and 18:1omega7 (29.2%). On the basis of the significant differences demonstrated in the phenotypic and chemotaxonomic characteristics, it is suggested that the bacterium be classified as a novel species; the name Oceanimonas smirnovii sp. nov. is proposed. The type strain is 31-13T (UCM B-11076T = LMG 22147T = ATCC BAA-899T).

Structure of the O-specific polysaccharide from a marine bacterium Oceanisphaeralitoralis KMM 3654(T) containing ManNAcA.

The O-specific polysaccharide was isolated from the lipopolysaccharide of a marine bacterium Oceanisphaeralitoralis KMM 3654(T) and studied by chemical methods along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-specific polysaccharide of O. litoralis containing D-glucose and two residues of 2-acetamido-2-deoxy-D-mannuronic acid was established: →4)-α-D-Glcp-(1→4)-ß-D-ManpNAcA-(1→4)-ß-D-ManpNAcA-(1→.

Combined solvent and water activity stresses on turgor regulation and membrane adaptation in Oceanimonas baumannii ATCC 700832.

Oceanimonas baumannii ATCC 700832 is a Gram negative marine bacterium capable of utilising phenol as asole carbon source. The ability of the bacterium to tolerate low water activity when utilising either succinate or phenol as a substrate in minimal medium was studied. The membrane lipid and protein composition showed two discreet adaptive phases as salinity increased. Firstly, when NaCl concentration was increased from 0.15% (w/v), the minimum at which growth was observed, to 1% NaCl (w/v), the ratio of zwitterionic to anionic phospholipids in the membrane increased significantly. At the same time the ratio of saturated to unsaturated fatty acids and the total membrane protein decreased significantly. The second phase was observed when salinity was increased from 1% to 7% NaCl (w/v) as the ratio of zwitterionic to anionic phospholipids decreased and membrane protein increased. However, the ratio of saturated to unsaturated fatty acids was unaffected. Salinity also affected the tolerance of cultures to elevated levels of phenol. Cultures grown in 0.15% NaCl (w/v) could tolerate 12 mM phenol, whereas in the presence of 1% NaCl (w/v) cultures continued to grow in up to 20 mM phenol and in 7% NaCl (w/v) cultures 8 mM phenol could be tolerated. Changes to the composition of the membrane phospholipids and fatty acids were also observed when phenol concentrations were at the maximum that could be tolerated. Under such conditions the ratio of zwitterionic to anionic phospholipids decreased twofold compared to cultures utilising 4 mM phenol as the substrate, in all salinities except in 7% NaCl (w/v) cultures, where there was no significant effect. The ratio of saturated to unsaturated fatty acids increased significantly in all salinities compared to cultures grown with 4 mM phenol.

Identification of indole-3-acetic acid producing freshwater wetland rhizosphere bacteria associated with Juncus effusus L.

Production of indole-3-acetic acid (IAA), a key physiological feature of culturable, O2-tolerant bacteria associated with the freshwater macrophyte Juncus effusus L., was examined over a period of 2 years. Up to 74% of rhizobacteria identified and tested produced IAA. The number of indoleacetic acid producers decreased in winter. IAA was produced even when L-tryptophan, a precursor of IAA, was not added to the medium. Most of the IAA-producing strains were dominated by strains that were not identifiable to species level on the basis of API testing. Based on 16S rRNA gene sequencing and fatty acid analysis, it was found that IAA-producing rhizosphere bacteria associated with the freshwater wetland plant Juncus effusus L. are representatives of several families, including the Enterobacteriaceae, Pseudomonadaceae, Aeromonadaceae, Burkholderiaceae, and Bacillaceae. This study identifies numerous potentially important bacterial physiological groups of freshwater wetlands. Additionally, the study provides a baseline for monitoring and assessing the mutualistic relationships of wetland plants with rhizosphere bacteria in freshwater wetlands.