Identification And Characterization Of A Catalase-Producing Halomonas Piezotolerans Bacterium NBT06E8~T Isolated From Hadal

Catalase(EC1.11.1.6)can effectively catalyze the decomposition of hydrogen peroxide and has broad potential applications in the field of textile,paper,pulping and food industries.Oxidative stress is hazardous.The basal antioxidant defence systems are essential for living microorganisms.These mechanisms between low temperature-high hydrostatic pressure and antioxidant are still unclear today,and the relationship has been only studied in Shewanella piezotolerans WP3.The result of WP3 displayed both pressure and oxygen tolerance were relatively low,so we need more relevant strains for more comprehensive studies.Therefore,new high pressure tolerance and antioxidant model strains for experimental study of antioxidant mechanisms is the prerequisite.Strain Halomonas piezotolerans NBT06E8^Tused in this study was obtained during an investigation of microbial-derived cold-adapted catalase to screen for H₂O₂-resistant strains.Sediment samples were collected at a depth of 8900 m from the New Britain Trench(153.8°E,6.4°S)in January 2017,and stored at 4°C until use.Bacteria were isolated mainly by plate screening using different concentrations of H₂O₂,and the concentration of H₂O₂was gradually increased during the screening process until the highest H₂O₂-tolerant strain was found and named NBT06E8^T.Phylogenetical trees based on 16S r RNA gene sequences were reconstructed using Neighbor-joining and maximum-parsimony.The most similar species were obtained based on the results of sequencing comparison,and 16S r RNA sequence similarity<97%was used as the cut-off threshold.The results of DNA-DNA(DNA–DNA hybridization values),ANI(average nucleotide identity values)and AAI(average amino acid identity)for the isolate were compared with those of the model strains,and statistical analysis showed that the isolate was a new species of Halomonas,based on these results and morphological,physiological and biochemical characteristics.The obtained peroxidase was then purified by gel-passed column and overnight dialysis to obtain the purified peroxidase,and the enzyme physiology of the purified peroxidase was studied,and the maximum enzyme activity and optimum enzyme activity temperature of the enzyme were obtained by measuring the physiological indicators of the enzyme activity,providing a basis for the industrial application of peroxidase from deep-sea microorganisms.The experiment on NBT06E8^Tstrain tolerant to a high concentration of H₂O₂was conducted.Most of previously cultured species in the genus Halomonas were isolated from salt or soda lakes around the world.Some species of Halomonas were isolated from non-aquatic habitats including soil,cheese,industrial brine,salty leaves and so on.A common feature of the genus Halomonas is salt tolerance,many Halomonas strains are able to grow at salinity up to 32.5%.In our experiment,we screened the new Halomonas strain NBT06E8^Twith resistant to high concentrations of H₂O_2.The physiological and biochemical indicators of the NBT06E8^Tstrain showed its cells are aerobic,motile,Gram-stain-negative,oxidase-and catalase-positive,rod-shaped,0.8–1?m wide and 1.5–1.8?m long.Colonies are yellow-coloured,circular,smooth and 1–2mm diameter after 2 days of incubation at 30°C on medium.Growth occurs at4–45°C(optimum,30°C),p H 5–11(optimum,p H 8–9)and 0.5–21%(w/v)Na Cl(optimum,3–7%,w/v).The optimum growth pressure of strain NBT06E8^Tis 0.1–30MPa with tolerance up to 60 MPa.It could tolerate 15 m M H₂O₂.The minimal inhibitory concentrations of Mn²⁺,Co²⁺,Cu²⁺and Zn²⁺for strain NBT06E8^Twere 300,3.5,35 and 80 m M,respectively.Results of 16S r RNA gene sequence analysis showed that strain NBT06E8^Tis closely related to Halomonas aquamarina DSM 30161^T(99.5%),Halomonas meridiana DSM 5425^T(99.43%)and Halomonas axialensis Althf1^T(99.35%).The digital DNA–DNA hybridization values between strain NBT06E8^Tand the three related type strains,H.aquamarina,H.meridiana and H.axialensis,were 30.5±2.4%,30.7±2.5%and 31.5±2.5%,respectively.The average nucleotide identity values between strain NBT06E8^Tand the three related type strains were 86.26,86.26 and 83.63%,respectively.The major fatty acids were summed feature 8(C18:1?7c and/or C18:1?6c)and C16:0.The predominant respiratory quinone detected was ubiquinone-9(Q-9).Based on its phenotypic and phylogenetic characteristics,we conclude that strain NBT06E8^Trepresents a novel species of the genus Halomonas.The Gen Bank accession numbers for the 16S r RNA gene and genome sequences of strain NBT06E8^Tare MN435603 and VWRT00000000,respectively.Catalase experiment:This purified catalase(Kat E)was obtained by heterologous expression in Escherichia coli and Ni-NTA agarose gel filtration chromatography.The characteristics of Kat E showed that the optimal substrate concentration,the optimal activity,the optimal temperature and the optimal p H were 60 m M,20°C,and p H7,respectively.The activity of Kat E was stable in the range of p H 5-7,and the optimal salinity was between 2-4%.The purified Kat E activity can reach 47448.4 U/mg,the K_mwas 54.03 mmol/L and the V_maxwas 71584 U/mg.In summary,the results of 16S RNA sequencing and whole-genome phylogenetic analysis show that NBT06E8^Tis a new species of Halomonas.The characteristics of the catalase indicate that the optimal temperature of the strain is 20?.New findings on oxidative stress mechanisms of this strain will broaden our knowledge of stress adaptation mechanisms of deep-sea bacteria and potentiate the biotechnological application of Halomonas species in the future.