【作者】 王伟；

【导师】 刘万顺； 孙谧；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 低温过氧化氢酶(EC1.11.1.6)是好氧低温生物代谢必需的抗氧化酶。低温过氧化氢酶研究对于认识海洋微生物在低温下有氧代谢活动规律有重要意义。本研究从南极海水中筛选出高活性过氧化氢酶菌株,鉴定为芽孢杆菌属(Bacillus)细菌。详细研究了芽孢杆菌目(Bacillales)中产芽孢细菌尤其是芽孢杆菌属细菌的16S rDNA系统学,给出了新的分组结果。从海洋革兰氏阳性菌中分离纯化了过氧化氢酶并研究其性质,分析了低温酶和同源的中温酶差别不明显的现象。从南极表层海水中分离细菌并筛选高过氧化氢酶活性的菌株,获得的49株细菌用硫代硫酸钠滴定法初筛后,其过氧化氢酶活性都高于大肠杆菌的酶活。用紫外分光光度法复筛后有15株菌的过氧化氢酶活性高于藤黄微球菌的酶活。其中N2a菌株活性最高,超声破碎细胞后活性达1200 U/mg。49株细菌的形态与生理生化特征表明它们属于同种细菌。进一步测定N2a的脂肪酸含量与16S rDNA序列后将其鉴定为芽孢杆菌属的细菌。N2a是专性好氧,能运动,产芽孢的杆菌,主要脂肪酸为iso-C14:1,iso-C15:0,anteiso-C15:0和iso-C14:0。N2a与B. barbaricus、B. arsenicus,B. macauensis和B. gelatini的16S rDNA序列相似性分别为99.4%、97.9%、96.1%和95.3%。N2a的表型(4℃生长,7%NaCl生长)和脂肪酸含量(不饱和脂肪酸占40%以上)体现出低温海洋菌的特征,不同于和N2a的16S rDNA序列相近的菌株。所以N2a可能是Bacillus新种。好氧产芽孢的细菌的系统学长期以来研究较少,特别是Bacillus属及相近属的系统学存在问题。本文根据16S rDNA序列,用邻位连接法(NJ)、简约法(MP)、最小进化法(ME)、最大似然法(ML)和贝叶斯推断(BI)五种算法构建了181种芽孢杆菌属及相关属细菌的系统进化树。BI算法得到的进化树最理想,其次是ML。各种系统进化树都表明,Bacillus不是单系群,芽孢杆菌属及相关属细菌可以划分为9个组。芽孢杆菌属模式种B. subtilis属于组1。N2a菌株属于组7。组4,组6和组8的细菌在表型上有一致性,分别为嗜热菌、嗜盐菌和嗜碱菌,而其它各组内的细菌在表型上无一致性。组2,组4和组8是由芽孢杆菌属及相关属细菌组成的,说明目前芽孢菌的分类系统和16S rDNA系统进化树不完全一致。组9主要由芽孢杆菌科除Bacillus以外的属组成,其中的4种芽孢杆菌可能属于其它的属。还有4种芽孢杆菌位于9个组之外,应建立新属或划入其它属。从Bacillus sp. N2a菌体中分离纯化其过氧化氢酶(BNC),采用50 mmol/L pH7.5 Tris?Cl缓冲液。菌体超声波破碎并离心后加入硫酸铵粉末,收集40%至70%饱和度之间的沉淀,样品透析后使用?KTA FPLC进行离子交换和分子筛层析。DEAE-Sephadex A-25阴离子交换层析以NaCl梯度洗脱,在0.13 mol/L NaCl时收集活性峰。聚乙二醇20000浓缩样品后使用Sephacryl S200HR分子筛层析。得到的过氧化氢酶在变性和非变性条件下电泳均为单一条带,比活80000 U/mg。Sephacryl S200HR分子筛测定BNC分子量为230 kD,SDS-PAGE测得亚基分子量为56 kD,表明BNC是同源四聚体。Bio-Rad Rotofor等电聚焦电泳测得BNC的pI=4.2。BNC最适温度25℃,最适pH6-11,活性受叠氮钠、盐酸羟胺和巯基乙醇的抑制。这些性质表明BNC属于小亚基单功能过氧化氢酶。BNC的活化能13 kJ,表明它同其它小亚基单功能过氧化氢酶类似,属于非温度依赖性的酶。BNC在4℃和25℃的催化效率分别为3.6×106和4×106 M-1s-1。BNC低温下高催化效率保证代谢产生的过氧化氢被迅速清除。BNC的热稳定性低于来自中温芽孢杆菌的小亚基过氧化氢酶,可以认为BNC属于低温酶。BNC来自在自然环境中自由生活的细菌,可以作为低温小亚基单功能过氧化氢酶的代表。BNC和同源中温酶的活化能、催化效率及热稳定性差别不大,是低温酶的特例。更多还原

【Abstract】 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.更多还原