Identification of Marinobacter psychrotrophilus sp. nov., a Novel Psychrotrophic Protease-producing Bacterium Isolated from Antarctic Ocean and Characterization of the Alkaline Cold-active Protease

摘要从南极海域沉积物中筛选到一产蛋白酶的菌株R2，细胞呈短杆状，单个排列，无鞭毛，大小为10~15μm×2~3μm，好氧，革兰氏染色为阴性；生长温度范围在5~30ºC(最适生长温度为10~15ºC)，生长pH范围在6~12之间(最适生长pH值为7)，生长盐度范围在0~20%之间(最适生长盐度为3%)；能够利用多种单一碳源作为能量来源；具有氨苄青霉素、卡那霉素、链霉素抗性；基因组DNA的G+C含量为56.3±0.2mol%。基于16SrDNA序列的系统发育分析表明该菌株属于海杆菌属(Marinobacter)，与M.hydrocarbonoclasticus和M.aquaeo...ABSTRACTA strain R2, which produces a protease, was isolated from the Antarctic Oceansediment samples. The cells of the strain R2 were neutrophilic, aerobic,Gram-negative, separate motile rods, with a size of 10~15 μm × 2~3 μm. Growthwas observed between 5 and 30ºC(optimum temperature: 10~15ºC), between pH 6 and 12(optimum: pH 7), and between NaCl concentrations of 0 and 20% (w/v)(optimu...学位：理学硕士院系专业：生命科学学院生物学系_生物化学与分子生物学学号：20022607

SMALL-SCALE DNA EXTRACTION FROM DEEP SEA SEDIMENT AND APPLICATION

采用化学裂解和酶解相结合的方法 ,进行了深海沉积物中微量DNA的提取 ,并采用DNA吸附树脂进行纯化。结果表明 ,该方法能有效地去除沉积物中的腐殖酸等抑制剂 ,每克湿重沉积物样品可得到DNA约 1 6 μg,回收率可达 95 % ,所得到的DNA分子片段均在 2 3kb左右 ,纯化后的DNA可直接应用于各种分子生物学操作。利用细菌 1 6SrDNA通用引物对所提取的深海沉积物DNA进行了PCR RFLP及系统发育分析 ,各主要细菌类群均能检出 ,证实该方法可以应用于深海等极端环境中微生物的多样性调查、系统发育分析以及特殊功能基因的筛选 ,同时还可应用于环境样品中生物量的半定量估计In recent years, several protocols based on the extraction of DNA from environmental samples have been developed for the microbial diversity study in nature. We report an efficient and nonselective protocol to extract the small-scale DNA from deep sea sediment with minimal shearing. The direct lysis technique involving SDS and lysozyme treatments was used because it could yield more DNA with less bias from sediment. The addition of PVPP (polyvinylpolypyrrolidone) and CTAB (Cetyl-trimethyl-ammonium Bromide) could remove the humic acid and other inhibitors from sediment efficiently. About 16 microgramme pure DNA with the size of 23kb, which could be used directly for molecular operation, was recovered from one gram deep sea sediment after purification by resin. The DNA recovery ratio was about 95%. PCR-RFLP (Restriction Fragment Length Polymorphism) analysis was done by universal bacterial 16S rDNA primers using the purified total DNA as template. The analysis of 16S rDNA sequences indicated that the majority of bacteria taxa in the sample could be detected. The method could be applied in analyzing the diversity and phylogenesis of natural microbial communities in extreme environment with low biomass; in screening for gene with special function; and in semi-quantifying the biomass of environmental sample.国家重点基础研究专项经费资助项目,G2 0 0 0 0 785 0 0号;; 中国大洋协会资助项目 ,DY1 0 5 4 2 4

Cold-active Protease from Antarctic Bacterium Marinobacter sp. Strain R2:Fermentation Condition and Enzyme Properties

从南极海域沉积物中筛选到一产蛋白酶的菌株R2,经细菌学形态特征鉴定及16SrDNA序列分析鉴定,该菌株属于海杆菌属(Marinobacter).生长特性研究表明该菌株属于兼性嗜冷菌,其最适生长温度为10～15℃.R2菌株能利用多种碳源产酶,最适产酶温度为20℃.粗酶液经硫酸铵盐析、DEAEcellulose 52柱层析进行初步分离纯化后进行酶学性质的研究.该菌株所分泌蛋白酶的最适作用温度为20℃,最适pH值为9～10,对热敏感,是典型的低温碱性蛋白酶.Ca2+、Mn2+、Cu2+对该蛋白酶有较为明显的激活作用,而Cd2+、Co2+则能抑制酶活.高浓度的变性剂、PMSF、AEBSF和EDTA都能抑制该蛋白酶,表明该酶属于丝氨酸蛋白酶,且金属离子对酶活性中心构象有关键性的影响.A strain R2,which produces protease,was isolated from the Antarctic Ocean sediment samples.Results of morphology and 16S rDNA sequence analysis showed that R2 belonged to Marinobacter.The Marinobacter sp.strain R2 was facultative psychrophile because it grew best at 10～15℃.The strain could produce protease with many kinds of single carbonaceous substance,and produced maximum protease activity at 20℃.The primary purification was performed by ammonium sulfate fractionation and column chromatography with DEAE cellulose-52.The optimum condition for the protease activity was 20℃ and pH 9～10,which suggested the enzyme was a typical alkaline cold-active protease.The protease activity was stimulated by Ca~(2+)、Mn~(2+)、Cu~(2+),and was inhibited by Cd~(2+)、Co~(2+).This enzyme appeared to be a serine protease,on the basis of its sensitivity to PMSF and AEBSF.From the result that EDTA showed obvious inhibiting effect on the enzyme,it can be suggested that metal ions played an important role in the conservation of enzyme conformation for the catalytic activity.国家海洋局青年基金(98310);; 国家海洋局项目(科01303)资

Screening and molecular identification of cold-active chitinase-producing bacteria from Antarctica

从南极普里兹湾深海底泥样品中分离得到一批嗜冷细菌,对这些嗜冷细菌进行了产几丁质酶菌株的筛选,对其中一株具有较高产酶活性的菌株AC167所分泌的几丁质酶进行了性质分析,并通过16S rDNA序列分析进行了菌株的分子鉴定。该菌株最适生长温度为10℃,最高生长温度为25℃,是典型的嗜冷菌,经16S rDNA序列比较及系统发育分析鉴定为假单胞菌属(Pseudomonas)。该菌株只在低于25℃的条件下分泌几丁质酶,酶的最适反应温度为30°C,属于低温酶。An amount of psychrophilic bacteria was isolated from deep sea sediment collected from Prydz Bay,Antarctica.The strain AC167,which showed high cold-active chitinase activity,was screened from these bacteria.It was identified as a Pseudomonas species by 16S rDNA sequence comparison.The optimal and highest temperatures for the growth of Pseudomonas strain AC167 were 10℃ and 25℃ respectively,indicating that it was a typical psychrophilic bacterium.The chitinase produced by Pseudomonas strain AC167 belonged to cold-active enzyme because it was most active at 30℃ and was secreted only at temperatures under 25℃.国家自然科学青年基金资助项目(4406029);; 国家海洋局青年基金资助项目(98310