Isolation And Identification Of Antarctic Marine Bacteria N2a With High Catalase Activity, Purification And Characterization Of A Psychrophilic Catalase And Phylogenetic Relationships Between Bacillus Species

Catalase (EC1.11.1.6) is a kind of high-active house-keeping protein among all organisms surviving in oxic environments, which scavenges H2O2 produced by cellular metabolism of O2 to prevent H2O2 from oxidizing lipids, proteins and nucleic acids. Studies on psychrophilic catalases are of significance to understand the metabolism of aerobic psychrophiles. Cold-adaptive catalases also have potential applications in dairy and water treatment in paper, food, textile and semiconductor industries. Bacteria with high catalase activities from Antarctic seawater have never been studied directly. Thus, an investigation of Antarctic marine bacteria containing high-activity hydroperoxidases is conducted by isolation and characterisation of the bacteria. The isolates are identified as Bacillus. The phylogeny of the new Bacillus species and the new genera of Bacillaceae have not been thoroughly studied. Therefore phylogenetic relationships between Bacillus species and related genera by reconstructing 16S rDNA phylogenetic trees using several algorithms are established. To better understand psychrophilic catalases, a novel cold adapted catalase (BNC) from the Bacillus sp. N2a is purified and characterized, and its activation energy, thermostability and kcat / Km are compared with several psychrophilic and mesophilic homologous. This catalase is reported as the first characterized psychrophilic catalase from Gram-positive bacteria.Forty-nine colonies are isolated from Antarctic surface seawater sample inoculated on Marine agar plates. After subcultured and identified by several morphological, biochemical and cultural methods, the Antarctic seawater isolates are all reciprocally similar. Cells are straight, Gram-variable, strictly aerobic and motile rods, 2.5–5 m long and 0.5–1 m wide. They form oval endospores subterminally. Cells grow on marine agar at pH 6–11 (optimally at pH 7.5–8) and tolerated up to 6% NaCl (w/v). Colonies are whitish, translucent, flat, smooth, and circular with regular margins and approximately 1 mm in diameter within 24 h when grown on marine agar at 20°C. Their cell extracts show high catalase activity of 1200±295 U/mg. High activity of the catalase at low temperatures is in accordance with the environmental conditions under which the microorganisms live. Identification of the Antarctic seawater isolates by several morphological, biochemical and cultural methods show that they belonged to the same species, among which the strain N2a is chosen as representative. This strain is determined to be a member of Bacillus according to its 16S rRNA gene and fatty acids profile. However, culturing properties (growth at 4°C and in 7% NaCl) and relatively high unsaturated fatty acids (>40%) of the strain N2a are different from those of the 16S rRNA gene phylogenetic relatives, exhibiting the characteristics of psychrotolerant marine bacteria. Thus these results support the hypothesis that the Antarctic isolates are novel species of the genus Bacillus.Neighbor-joining, maximum-parsimony, minimum-evolution, maximum-likelihood and Bayesian trees constructed based on 16S rRNA gene sequences from Genbank of 181 type strains of Bacillus species and related taxa manifest 9 phylogenetic groups, and the Bayesian tree is confirmed the best evolutionary tree, although the phylogeny of five species is uncertain. The phylogenetic analysis shows that Bacillus is not a monophyletic group. B. subtilis is in Group 1. Bacillus sp. N2a is in Group 7. Group 4, 6 and 8 respectively consist of thermophiles, halophilic or halotolerant bacilli and alkaliphilic bacilli, while the species in other groups differ much in the phenotype. Group 2, 4 and 8 consisting of Bacillus species and related genera demonstrate that the current taxonomic system does not agree well with the 16S rRNA gene evolutionary tree. The position of Caryophanaceae and Planococcaceae in Group 2 suggests that they might be transferred into Bacillaceae, and the heterogeneity of Group 2 implies that some Bacillus species in it might belong to several new genera. The close relationship between B. thermantarcticus and Geobacillus in Group 4 seem that B. thermantarcticus should be accommodated in Geobacillus. Group 9 is mainly comprised of the genera (excluding Bacillus) of Bacillaceae, so some Bacillus species in Group 9: B. salarius, B. qingdaonensis and B. thermcloacae might not belong to Bacillus. Four Bacillus species, B. schlegelii, B. tusciae, B. edaphicus and B. mucilaginosus are clearly placed outside the 9 groups. It is proposed to respectively elevate B. schlegelii and B. tusciae to the rank of genus, and B. edaphicus and B. mucilaginosus should be transferred into Paenibacillus.Catalase (BNC) from Bacillus sp. N2a is produced by the free-living bacterium from a cold habitat, rendering it more representative of cold-adapted catalases than any other reported psychrophilic catalases. After sonication of the harvested N2a strain cells, for the purification of BNC, a three-step protocol consisting of (NH4)2SO4 precipitation, anion exchange and gel filtration is developed. All purification steps are done at 4°C and chromatography was done by a Fast Protein Liquid Chromatography system (?KTA FPLC). The catalase is purified approximately 67-fold with a yield of about 26%. It shows a single protein band on SDS-PAGE and native PAGE. The purified catalase activity is about 80000 U/mg. The isoelectric point of the catalase is determined to be 4.2. BNC has a molecular mass of about 230 kD and is composed of four identical subunits of 56 kD. The catalase show optimal activity at 25°C and at pH range of 6-11. The enzyme could be inhibited by azide, hydroxylamine and mercaptoethanol. These characteristics suggest that BNC is a small-subunit monofunctional catalase. The activation energy of BNC is 13 kJ/mol and the apparent kcat/Km are 3.6×106 M-1 s-1 and 4×106 M-1 s-1 at 4°C and 25°C, respectively. High catalytic efficiency of BNC at low temperatures enables this bacterium to scavenge H2O2 efficiently. BNC can be categorized in psychrophilic enzymes according to its relatively low optimal temperature (25°C) and low thermostability. BNC exhibited activation energy, catalytic efficiency and thermostability comparable to some mesophilic homologues. Such similarity of enzymatic characteristics to mesophilic homologues, though uncommon among the cold-adapted enzymes in general, has also been observed in other psychrophilic small-subunit monofunctional catalases. The distinctive activation energy, thermostability and kcat / Km of small-subunit monofunctional catalases blur the boundary between psychrophilic and mesophilic catalases. The highly efficient mesophilic catalases with low activation energy have naturally adapted to the cold environment. In reverse, BNC exhibited cold-adapted characteristics not typical of psychrophilic enzymes.