【作者】 李安章；

【导师】 徐洵； 邵宗泽；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 柴油食烷菌Alcanivorax dieselolei B-5T是本课题组分离鉴定的一株重要的石油降解菌,具有潜在的环境意义和生物学意义。实验发现柴油食烷菌B-5能够利用多种卤代烷烃为唯一的碳源和能量来源进行生长。1.柴油食烷菌B-5中的HLDs基因克隆表达与酶学特性分析本文中表达、纯化了柴油食烷菌B-5中的两个推定的HLDs,DadA和DadB;检测了这两个酶的底物范围和酶活特性；测定了DadB的稳态动力学常数、最适温度和最适pH;使用动态光散射技术分析了DadB在不同pH的溶液中的稳定性；通过同源建模研究了DadB酶学特征的结构学基础。DadA在已鉴定的HLDs中具有最严格的底物选择性,其底物范围明显小于其他已鉴定的HLDs,代表了一个新的酶活特异性亚家族。DadB表达纯化简单高效,具有宽广的底物范围和很高的催化活性,偏爱小分子(C2和C3)底物、溴代底物和氯代烯烃底物,其对不同程度的氯代、溴代、碘代的C：和C3链长的底物的整体酶活是目前已知的HLDs中最高的。DadB对6个卤代底物的稳态动力学常数基本与其他HLDs处于相近的数量级,对3-氯-2-甲基丙烯、4-溴丁腈等多个底物具有较高的转化数Kcat。另外,4-溴丁腈脱溴对DadB具有明显的底物自抑制效果。DadB酶活稳定性好。DadB在pH5.0到pH10.0范围内都有明显的活性,最高活性出现在pH8.0处,且在pH8.0-9.0范围内变动不大。DadB在20℃到55℃温度范围内都有酶活,最适温度为50℃。缓冲液pH低于7.5时,DadB易聚集形成不同聚合程度的聚合物。当缓冲液pH≥7.5时,DadB在溶液中以单体形式存在,能保持长时间的结构稳定。对DadA和DadB进行同源建模,发现二者采用典型的α,β-水解酶折叠方式,由核心结构域和帽子结构域两部分组成。DadA、DadB的催化五联体、活性位点的拓扑结构与已解析结构的HLDs基本一致。但是DadB具有更大的活性位点进出通道的开口,同时在阻隔主进出通道和狭槽通道的壁垒处采用Ile/Trp的组合,这可能是DadB偏爱小分子底物的重要原因。2. HLDs的系统进化分析本文对总共37株食烷菌基因组进行了分析,发现其中33株食烷菌中含有一共51条推定的HLDs序列。对食烷菌属中的HLDs序列进行系统发育分析,发现食烷菌属在不同的时间点通过至少4次相对独立的进化事件从其他种属中得到了不同的HLDs进化分支,随后这些HLDs可能还通过基因水平转移在食烷菌属内不同菌株之间进行了传递。本文还对目前几乎所有的HLDs进行了整体的系统发育研究。使用已鉴定的17条HLDs序列进行搜索,运用生物信息学技术从NCBI的nr数据库中一共找出421条推定的HLDs序列,加入本课题组获得的53条HLDs序列后进行系统发育分析,发现DadA及另外19条全部来自于食烷菌中的HLDs代表了HLDs家族的一个新的进化分支,这与DadA具有独特的酶活特性的现象相一致。本文对HLDs家族的天然功能及其对微生物和环境的重要作用进行了初步分析,提出海洋环境可能是HLDs序列的一大遗传资源库,并对HLDs的传播和进化起了重要作用。总之,本文第一次证明了食烷菌属存在具有脱卤活性的HLDs,分析了这些HLDs对食烷菌利用卤代烷烃作为唯一碳源进行生长的意义,鉴定得到了具有新奇的底物选择性和序列特异性的DadA和具有宽广的底物范围且对难降解的小分子底物具有目前已鉴定的HLDs中最高酶活的DadB。这些研究拓宽了HLDs遗传资源,为HLDs的实践应用提供了选择目标,为通过蛋白工程改造获得更有催化效率或更好的底物选择性的HLDs的研究提供了材料,并且证明了石油降解菌A. dieselolei B-5还具有卤代污染物降解方面的应用潜力。目前发现推定的HLDs在大约120个属中存在,本文以食烷菌属作为优良的模型系统,第一次对某一个种属内的HLDs的来源、进化、传递、扩散等事件进行了分析,对揭示HLDs的天然功能、探讨HLDs在如此之多的不同种属中分布的可能原因、研究HLDs对物种代谢及生存的作用和意义提供了重要的线索和思路。更多还原

【Abstract】 Alcanivorax dieselolei B-5T is an important oil degradation bacteria strain isolated and identified by our group and has potential meanings in environmental improvement and biological study. Carbon source experiments showed A. dieselolei B-5can use a variety of halogenated alkanes as sole carbon and energy source for growth.1. Cloning, expression and enzymatic analysis of HLDs in A. dieselolei B-5.We expressed and purified the two putitave HLDs in B-5, DadA and DadB; detected their enzyme activities and substrate specificity, measured the steady-state kinetic constants and the optimum temperature and pH of DadB, checked the stability of DadB in different buffers with different pH by the dynamic light scattering(DLS) methods, discussed the structural basis of DadB’s enzymology characteristics by homology modeling.DadA has the most stringent substrate selectivity in the identified HLDs, its substrate range significantly smaller than other HLDs identified, representing a new subfamily of specificity. Expression and purification of DadB are simple and efficient. DadB has broad substrates range and high activity profile, preferring small substrates (C2and C3), brominated substrates and chloroalkene substrates. As far as known, DadB has the highest systemic activity towards chlorinated, brominated, iodinated C2and C3substrates in all HLDs identified. Steady-state kinetic constants of DadB with6substrates are basiclly in the similar magnitude, while DadB has higher Kcat values with several substrates such as3-chloro-2-methylpropene and4-bromobutanenitrile. Moreover,4-bromobutanenitrile showed distinct substrate self-inhibition phenomenon.DadB has excellent stability of the enzyme acitivity. DadB keeps obvious activity between pH5.0to pH10.0and the peak occurred at pH8.0. DadB has activity from20℃to55℃with the optimum temperature at50℃. DadB aggregates very fast to form large polymers with different size in the buffer has a pH lower than7.5. When the buffer’s pH≥7.5, DadB exists in the form of monomer in solution and keep stable in a long time.Two structural models were obtained and they have typical α/β-hydrolase structural fold, consisting of the core domain and the cap domain. Topological structures of catalytic pentad amino acids in DadA and DadB are very consistent with the HLDs already had the three dimensional structures. But DadB has an open active cavity with greater access tunnel, also takes Ile/Trp couple as the residue barrier to separate the main tunnel and the slot tunnel. This may be an important cause of the preference of DadB for small substrates.2. Phylogenetic analysis of HLDsWe analyzed genomes of37Alcanivorax and found out51HLDs genes in33Alcanivorax. This paper analyzed the phylogeny of putative HLDs from Alcanivorax, proposed that Alcanivorax bacteria have obtained different evolutionary clades of HLDs from the other species in at least4independent evolutionary events occured at different time and subsequently these HLDs spread between different strains of Alcanivorax maybe through horizontal gene transfer.This paper also did the systematic phylogenitic analysis with almost all of HLDs can found now.Through bioinformatics technique,421putative HLDs sequences were found out from non-redundant protein sequences (nr) database of NCBI using17HLDs sequences identified as queries. Phylogenetic analysis with these obtained421HLDs together with53putative HLDs sequenced by our group shows that DadA and other19putative HLDs all from Alcanivorax represents a new evolutionary branch of HLDs. This maybe the reason why DadA has unique enzyme properties of activity. This paper preliminarily analysed the the natural functions and the important role of HLDs in the microorganism and in the environment, proposed that marine environment may be a large genetic resource pool of HLDs sequences and may play an important role in the propagation and evolution process of HLDs.In summary, this study proved for the first time there are HLDs with identified dehalogenation activity existing in Alcanivorax, analyzed the role of these HLDs for Alcanivorax to take halogenated compounds as sole carbon source. This paper obtained and identified two HLDs, DadA with novel substrate selectivity and sequence specificity, DadB with the most wide range of substrates and the highest activity in the knowen HLDs identified untill now to small substrates with serious recalcitrance. This paper expanded the genetic resources of HLDs, offered alternative targets for the practical applications of HLDs, provided research materials for improving catalytic efficiency and substrate selectivity of HLDs by protein engineering technique, and also proved oil degrading bacteria A. dieselolei B-5also has potential application in degradation of halogenated pollutants.Putative HLDs sequences has already been found in almost120genuses. Taking Alcanivorax as a good model, this paper for the first time analyzed the origin, evolution, transferring, spreading in a perticular genus, provided important clues and ideas for discussing the natural functions of HLDs and the possible reason of its spread in so many different genuses and for researching the roles and meanings for metabolism and survival.更多还原