南极海洋石油烃低温降解菌Shewanella sp.NJ49加氧酶及其基因研究

【作者】 梁强；

【导师】 缪锦来；

【作者基本信息】 国家海洋局第一海洋研究所 ， 海洋生物学， 2011， 硕士

【摘要】 近年来,随着南极地区人类活动的增加,尤其是旅游和科研活动的增加,南极环境污染状况正日益加剧;石油烃污染是南极环境污染中最引人注目的问题,大量的石油制品以各种方式进入南极水域,对南极环境构成巨大的威胁。生物修复是消除海洋石油烃污染的重要途径,然而南极海洋系统独特的低温,高盐和强紫外辐射的环境条件,使得普通的常温微生物很难发挥其生物修复功能;本文以从南极海水海冰样品中筛选到的能降解石油烃的低温降解菌为研究对象,以期为海洋石油烃污染的低温生物修复技术应用奠定基础。本文以从南极海水样品中筛选到的能够高效降解烷烃和芳香烃的南极石油烃低温降解菌Shewanella sp. NJ49为材料菌株,对其烷烃降解特性、石油烃微生物降解的拉曼光谱实时监测、降解酶的定域、单加氧酶的分离纯化及理化性质和加氧酶的分子生物学表征等方面进行了研究,主要获得以下结果:南极石油烃低温降解菌Shewanella sp. NJ49可以在低温环境下高效降解正十六烷烃。将激光光镊拉曼光谱技术应用于烷烃微生物低温降解的研究,实时监测烷烃降解过程中降解菌单细胞、细胞群生长过程和降解前后培养基中产物的变化,初步推断南极菌NJ49的拉曼光谱峰值所代表的物质,其中位于783 cm-1、812 cm-1峰值处主要代表物质是磷脂类和油酸,1004 cm-1附近的峰主要代表物质是脂类,峰值1156 cm-1处代表的物质是葡聚糖类,1370 cm-1处是核苷酸类遗传物质,1435 cm-1和1516 cm-1处为蛋白质类,首次从细菌细胞水平获得对南极细菌低温降解正十六烷的新认识。对南极石油烃低温降解菌Shewanella sp. NJ49的降解酶定域研究表明,正烷烃的微生物降解通过氧化酶系酶促反应进行;南极菌NJ49降解正十六烷过程中起主要作用的是胞外酶和膜周酶(尤其以胞外酶为主),其中胞外酶和膜周酶通过正十六烷烃末端氧化完成其初步降解,并生成正十六醛、正十六醇等产物;胞内酶在正十六烷降解过程中所起作用较小,GC-MS图谱分析结果与上述结论一致。采用细胞静息技术获得胞外酶后,用超滤的方法收集分子量分别为>100000、30000-100000、10000-30000、5000-10000等4种不同分子量区段的酶,测定并比较酶活性,气相色谱分析结果可知分子量为3-10万区段的酶为降解正十六烷的关键酶;经硫酸铵盐析、酶的透析与超滤浓缩和葡聚糖凝胶SephardexG-75柱层析后,SDS-PAGE凝胶电泳结果显示得到了由α、β、γ三个亚基组成,分子量分别为56kD、45kD和36kD的单加氧酶。南极石油烃低温降解菌Shewanella sp. NJ49中存在能够降解石油烃的单加氧酶alkB,通过基因克隆和基因组步移技术得到长度为1176 bp的单加氧酶alkB全长序列;实时荧光定量PCR表达结果显示正十六烷浓度、温度、高盐和紫外辐射对南极菌NJ49降解正十六烷的能力均有不同程度的影响,对应温度梯度的最大相对表达量是15℃,单加氧酶alkB对应正十六烷浓度的最大相对表达量的是20 mg/L,对应UV-B紫外辐射时间梯度的最大相对表达量是6 h,对应高盐不同时间梯度的最大相对表达量是90‰NaCl 2 h。此外,通过基因克隆和基因组步移技术,从南极石油烃低温降解菌Shewanella sp. NJ49克隆到全长为924 bp的双加氧酶序列;实时荧光定量PCR表达结果显示萘浓度、温度和紫外辐射对南极菌NJ49降解萘的能力均有不同程度的影响,南极菌NJ49双加氧酶对应的最大相对表达量的萘浓度是50 mg/L,温度梯度是15℃,UV-B紫外辐射时间梯度中双加氧酶对应的最大相对表达量是4 h。更多还原

【Abstract】 In recent years, the Antarctic environmental pollution is aggravating with the increase of human activities, especially for the increase of Antarctic tourism and scientific research activities. Petroleum hydrocarbon pollution is one of the most remarkable problems for the Antarctic environment pollution. A lot of petroleum products entered into the Antarctic waters in a variety of ways and caused huge threat for the Antarctic environment. Bioremediation is an important way to eliminate offshore oil hydrocarbon pollution, however, the normal temperature microbe is hard to exert its biological repair function because of the unique environment conditions of Antarctic ocean systems such as high salt, low temperature and the strong ultraviolet radiation. In order to lay the foundation for the application of low-temperature bioremediation technology of marine oil hydrocarbon pollution, the low temperature bacteria that can degrade petroleum hydrocarbon and be separated from Antarctic sea ice samples as the research object.The psychrophilic bacterium NJ49 that can degrade alkane and aromatic hydrocarbons efficiently and be separated from Antarctic sea ice samples as materials strains with hexadecane used as sole carbon source. The alkane degradation characteristics of Antarctic psychrophilic degradation bacterium, Raman spectroscopy real-time monitoring of petroleum hydrocarbon microbial degradation, localization of degradation enzyme, purification and physical and chemical properties of hydroxylase and molecular characterization of oxygenase were researched. The results were as follows:Antarctic psychrophilic degradation bacterium Shewanella sp. NJ49 could degrade hexadecane efficiently at low-temperature environment. The variation of individual cells of degradation bacteria in the process of alkane degradation and the variation of products in the process of cells growth were monitored synchronously by Laser optical tweezers Raman spectroscopy. The representative materials of the Raman spectrum peak for Antarctic psychrophilic bacterium NJ49 were preliminary inferred. The phospholipids and oleic acid were mainly located in the peak of 783 cm- 1 and 812 cm^-1 and the lipid was located in the peak of 1004 cm^-1. The peak of 1156 cm^-1 was mainly represented the glucan. The genetic material of nucleotide was located in the peak of 1370 cm^-1 and the protein was located in the peak of 1435 cm^-1 and 1516 cm^-1. The new cognition of alkane degradation of Antarctic psychrophilic bacteria was obtained from the level of individual and colonial cells.The research of localization of degradation enzyme of Antarctic psychrophilic bacteria indicated that micro-biological degradation of alkane needed the enzymatic reaction of oxidase system. The extracellular enzyme played key role in the process of hexadecane degradation of Antarctic psychrophilic bacterium NJ49. It accomplished the preliminary degradation of hexadecane by the terminal oxidation and produced the products of n-hexadecanal and cetyl alcohol. The intracellular enzyme played little role in the process of hexadecane degradation. The analysis result of GC-MS was in accordance with this conclusion.The extracellular enzyme was obtained by the cell resting technology. Four enzymes of different molecular weight (greater than 100000, 30000^-100000, 10000-30000 and 5000^-10000, respectively) were collected by ultrafiltration and then determined and compared the enzymatic activity. Gas chromatography analysis indicated that the enzyme of molecular weight of 30000^-100000 was the key enzyme of hexadecane degradation. Polyacrylamide gel electrophoresis revealed purified hydroxylase was obtained finally. It was composed ofα,βandγsubunits but the contents were less.Monooxygenase can degrade oil hydrocarbon and exist in Antarctic psychrophilic bacterium NJ49. The gene full-length sequence of 1176 bp of monooxygenase was cloned by genome walking method. The expression results of real-time fluorescence quantitative PCR technology showed concentration of hexadecane, temperature, high salt and ultraviolet radiation influenced the ability of hexadecane degradation with varying degrees for Antarctic psychrophilic bacterium NJ49. The maximum relative express quantities of monooxygenase were 20 mg/L of hexadecane, 15 ?C of temperature gradient, 90‰NaCl 2 h of time gradient of high salt, 6 h of time gradient of ultraviolet radiation, respectively.In addition, The gene full-length sequences of 924 bp of dioxygenase was also cloned by genome walking method from Antarctic psychrophilic bacterium NJ49. The expression results of real-time fluorescence quantitative PCR technology revealed that concentration of naphthaline, temperature and ultraviolet radiation affected the ability of naphthaline degradation with varying degrees for Antarctic psychrophilic bacterium NJ49. The maximum relative express quantities of dioxygenase were 50 mg/L of naphthaline, 15 ?C of temperature gradient, 4 h of time gradient of ultraviolet radiation, respectively.更多还原