Shewanella mangrovi sp. nov., an acetaldehyde-degrading bacterium isolated from mangrove sediment.

A taxonomic study was carried out on strain YQH10T, which was isolated from mangrove sediment collected from Zhangzhou, China during the screening of acetaldehyde-degrading bacteria. Cells of strain YQH10T were Gram-stain-negative rods and pale brown-pigmented. Growth was observed at salinities from 0 to 11% and at temperatures from 4 to 42 °C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YQH10T is affiliated to the genus Shewanella, showing the highest similarity with Shewanella haliotis DW01T (95.7%) and other species of the genus Shewanella (91.4-95.6 %). The principal fatty acids were iso-C15 : 0 and C17 : 1ω8c. The major respiratory quinone was Q-8. The polar lipids comprised phosphatidylethanolamine and phosphatidylglycerol. The genomic DNA had a G+C content of 48.3 mol%. Strain YQH10T can completely degrade 0.02% (w/v) acetaldehyde on 2216E at 28 °C within 48 h. Based on these phenotypic and genotypic data, strain YQH10T represents a novel species of the genus Shewanella, for which the name Shewanella mangrovi sp. nov. is proposed. The type strain is YQH10T ( = MCCC 1A00830T = JCM 30121T).

Rhizobium marinum sp. nov., a malachite-green-tolerant bacterium isolated from seawater.

A motile, Gram-stain-negative, non-pigmented bacterial strain, designated MGL06T, was isolated from seawater of the South China Sea on selection medium containing 0.1 % (w/v) malachite green. Strain MGL06T showed highest 16S rRNA gene sequence similarity to Rhizobium vignae CCBAU 05176T (97.2 %), and shared 93.2-96.9 % with the type strains of other recognized Rhizobium species. Phylogenetic analyses based on 16S rRNA and housekeeping gene sequences showed that strain MGL06T belonged to the genus Rhizobium. Mean levels of DNA-DNA relatedness between strain MGL06T and R. vignae CCBAU 05176T, Rhizobium huautlense S02T and Rhizobium alkalisoli CCBAU 01393T were 20 ± 3, 18 ± 2 and 14 ± 3 %, respectively, indicating that strain MGL06T was distinct from them genetically. Strain MGL06T did not form nodules on three different legumes, and the nodD and nifH genes were also not detected by PCR or based on the draft genome sequence. Strain MGL06T contained Q-10 as the predominant ubiquinone. The major fatty acid was C18 : 1ω7c/C18 : 1ω6c with minor amounts of C19 : 0 cyclo ω8c, C16 : 0 and C18 : 1ω7c 11-methyl. Polar lipids of strain MGL06T included unknown glycolipids, phosphatidylcholine, aminolipid, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, an unknown polar lipid and aminophospholipid. Based on its phenotypic and genotypic data, strain MGL06T represents a novel species of the genus Rhizobium, for which the name Rhizobium marinum sp. nov. is proposed. The type strain is MGL06T ( = MCCC 1A00836T = JCM 30155T).

The first characterization of a Ca2+-dependent carbohydrate-binding module of ß-1,3-xylanase from Flammeovirga pacifica.

A novel carbohydrate binding module (CBM) was identified in a ß-1,3-xylanase from Flammeovirga pacifica, which showed only 25.0% sequence identity with the reported CBMs with the coverage of 36.4%. To verify its function, a truncated ß-1,3-xylanase (Xy13088-T) and a carbohydrate binding module (CBM3088) were expressed and purified. The thermostability and catalytic efficiency of the Xy13088-T declined significantly when compared with the full-length one, with the decreasing of the half-life and catalytic efficiency (Kcat/Km) by 90%. Interestingly, the CBM3088 showed the binding ability to ß-1,3-xylan only when Ca2+ existed, which was different from the reported CBMs of ß-1,3-xylanases. The maximum amount of CBM3088 binding to ß-1,3-xylan was 9.65 µmol/g of ß-1,3-xylan. The residues probably involved in the binding to the ß-1,3-xylan and Ca2+ were addressed by bioinformatics analysis.

π-π stacking interaction is a key factor for the stability of GH11 xylanases at low pH.

Acidic xylanases possess the unique features necessary for the tolerance of acidic environments, which may have great potentials for industrial purposes. However, factors controlling the pH-dependent stability of xylanases are only partially known. Here we proposed a residue interaction networks based method to analyze the differences of residue interactions between 6 pairs of experimentally verified acidic and neutral xylanases. They had very close numbers of aromatic amino acids, however extremely significant more (p < 0.001) π-π stacking interactions existed in acidic xylanases, which has not been reported before. Whereas the interactions between Tyrosine-Phenylalanine (Tyr-Phe) and Phenylalanine-Phenylalanine (Phe-Phe) were the main contributors. An equation quantitatively described the relationship between the optimal pH and the number of π-π stacking interactions was proposed. The predicted optimal pHs for three xylanases was 4.13, 6.7 and 6.1, while the experimental values of the optimum pHs were 4.6, 6.5 and 6.5, with an absolute error of 0.47, 0.2 and 0.4 pH unit, respectively. By counting the aromatic residue pairs forming π-π stacking in the 3D structure of an acidic (PDB ID: 1BK1, with an optimal pH of 2) and a neutral (PDB ID:1XXN, with an optimal pH of 6.5) xylanase, we found significant differences existed in the positions ranging from 145 to 166 in forming π-π stacking. Two phenylalanines at position 149 and 157 in the acidic xylanase, which involved in 7 π-π stacking interactions, played an important role in the stability of xylanase at low pH environment, which was further proved by a mutation experiment. A mutated xylanase with Phe149 → Ala149 and Phe157 → Ala157 was expressed and purified, resulting the optimal pH shifted from 2 to 4.5. The interaction networks based method paved a new way in underlying and engineering the acid-stability of xylanase, as well as the characteristics of other enzymes.

Molecular cloning and expression of a cold-adapted lipase gene from an Antarctic deep sea psychrotrophic bacterium Pseudomonas sp. 7323.

A psychrotrophic bacterium producing a cold-adapted lipase was isolated from the deep-sea sediment of Prydz Bay, Antarctic and identified as a Pseudomonas strain. Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp. 7323 (lipA) revealed that LipA is composed of 617 amino acid residues with a calculated molecular weight of 64,466 Da. LipA has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine. The lipase purified from the Escherichia coli transformant (rLipA) by metal-chelating chromatography exhibited the same electrophoretic mobility as did the wild-type lipase (wLipA) purified from strain 7323, and both enzymes were quite similar in physicochemical properties. The optimal temperature and pH value for the lipases activity were 30 degrees C and 9.0, respectively. They were unstable at temperatures above 25 degrees C and only retained half of their highest activity after incubation at 60 degrees C for 5 min. These results indicated that the enzymes were typical alkaline cold-adapted enzymes. Both enzymes were particularly activated by Ca2+. Additionally, the enzymes hydrolyzed p-nitrophenyl caprate and tributyrin at the highest velocity among the other p-nitrophenyl esters and triglycerides.

Purification and characterization of a cold-adapted alpha-amylase produced by Nocardiopsis sp. 7326 isolated from Prydz Bay, Antarctic.

An actinomycete strain 7326 producing cold-adapted alpha-amylase was isolated from the deep sea sediment of Prydz Bay, Antarctic. It was identified as Nocardiopsis based on morphology, 16S rRNA gene sequence analysis, and physiological and biochemical characteristics. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram activity staining of purified amylase showed a single band equal to a molecular mass of about 55 kDa. The optimal activity temperature of Nocardiopsis sp. 7326 amylase was 35 degrees C, and the activity decreased dramatically at temperatures above 45 degrees C. The enzyme was stable between pH 5 and 10, and exhibited a maximal activity at pH 8.0. Ca(2+), Mn(2+), Mg(2+), Cu(2+), and Co(2+) stimulated the activity of the enzyme significantly, and Rb(2+), Hg(2+), and EDTA inhibited the activity. The hydrolysates of soluble starch by the enzyme were mainly glucose, maltose, and maltotriose. This is the first report on the isolation and characterization of cold-adapted amylase from Nocardiopsis sp.

Characterization of a novel psychrophilic and halophilic ß-1, 3-xylanase from deep-sea bacterium, Flammeovirga pacifica strain WPAGA1.

ß-1, 3-Xylanase is one of the most important hydrolytic enzymes to prepare oligosaccharides as functional foods in seaweed industry. However, less than five ß-1, 3-xylanases have been experimentally expressed and characterized; moreover, none of them is psychrophilic and salt tolerant. Here, we mined a novel ß-1, 3-xylanase (Xyl512) from the genome of the deep-sea bacterium Flammeovirga pacifica strain WPAGA1 and biochemically characterized it in detail. The Xyl512 did not contain any carbohydrate-binding module; the catalytic domain of it belonged to the glycoside hydrolase family 26. The optimum temperature and pH of the purified ß-1, 3-xylanase was 20 °C and pH 7.0 in the condition of no NaCl. However, they shifted to 30 °C and 7.5 with 1.5 mol/L NaCl, respectively. In this condition (1.5 mol/L NaCl), the overall activity was 2-fold as high as that without NaCl. Based on the residue interactions and the electrostatic surfaces, we addressed the possible mechanism of its adaption to low temperature and relative high NaCl concentration. The Xyl512 showed significantly reduced numbers of hydrogen bonds leading to a more flexible structure, which is likely to be responsible for its cold adaptation. While the negatively charged surface may contribute to its salt tolerance. The ß-1, 3-xylanase we identified here was the first reported psychrophilic and halophilic one with functionally characterized. It could make new contributions to exploring and studying the ß-1, 3-xylanase for further associated investigations.

Expression and Characterization of a Novel Thermostable and pH-Stable ß-Agarase from Deep-Sea Bacterium Flammeovirga Sp. OC4.

A novel gene (aga4436), encoding a potential agarase of 456 amino acids, was identified in the genome of deep-sea bacterium Flammeovirga sp. OC4. Aga4436 belongs to the glycoside hydrolase 16 ß-agarase family. Aga4436 was expressed in Escherichia coli as a fusion protein and purified. Recombinant Aga4436 showed an optimum agarase activity at 50-55 °C and pH 6.5, with a wide active range of temperatures (30-80 °C) and pHs (5.0-10.0). Notably, Aga4436 retained more than 90%, 80%, and 35% of its maximum activity after incubation at 30 °C, 40 °C, and 50 °C for 144 h, respectively, which exhibited an excellent thermostability in medium-high temperatures. Besides, Aga4436 displayed a remarkable tolerance to acid and alkaline environments, as it retained more than 70% of its maximum activity at a wide range of pHs from 3.0 to 10.0 after incubation in tested pHs for 60 min. These desirable properties of Aga4436 could make Aga4436 attractive in the food and nutraceutical industries.