苯甲酸钠降解菌的筛选、龙胆酸1，2-双加氧酶基因的克隆与异源表达

	苯甲酸钠降解菌的筛选、龙胆酸1，2-双加氧酶基因的克隆与异源表达
	孙向楠 1,2
学位类型	硕士
导师	胡晓珂
	2015-05-25
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	海洋生物学
关键词	群落结构芽孢杆菌同源建模龙胆酸1 异源表达 2-双加氧酶
摘要	应用分子生态学方法，研究在不同芳香族化合物胁迫下，海洋沉积物中细菌群落结构的演替规律。在实验室建立驯化系统, 向海洋沉积物中分别添加芘和苯甲酸钠，采用Illumina MiSeq高通量测序技术分析实验组和对照组细菌群落16S rRNA基因V3-V4可变区数量的组成及变化，研究沉积物中添加芘、苯甲酸钠后，微生物群落多样性和群落结构的变化。实验结果表明：添加芘、苯甲酸钠极大地降低了海洋沉积物中菌群丰度和多样性；在添加苯甲酸钠实验组，变形杆菌门中海杆菌属(Marinobacter)，假海源菌属(Pseudidiomarina)的细菌得到显著富集；在添加芘实验组中拟杆菌门中的Balneola属，碳酸噬胞菌属(Aequorivita)得到极大富集。初步分析了海洋沉积物环境中不同多环芳烃胁迫下，细菌群落结构的演替特征，该实验结果为海洋环境中针对不同芳香烃的微生物修复提供理论依据。从海底沉积物的样品中，筛选出一株能够以苯甲酸钠为唯一碳源的芽孢杆菌，该海洋细菌能够通过龙胆酸途径降解苯甲酸钠。针对该途径中的关键酶龙胆酸1，2-双加氧酶的基因，运用Touch down PCR和TAIL PCR成功扩增出该基因的完整阅读框。该阅读框包含1143bp，编码379氨基酸残基，预测相对分子量为42.9 kDa。BLASTx比对显示这段基因编码的氨基酸序列与已发现或预测的其他龙胆酸1，2-双加氧酶相似性均低于59%。这段基因编码的氨基酸序列与Geobacillus kaustophilus GBlys中的龙胆酸1，2-双加氧酶具有52%的相似性，与Bacillus halodurans C-125中龙胆酸1，2-双加氧酶有50%相似性，与Paenibacillus sp. NyZ101中龙胆酸1，2-双加氧酶相似性为49%，与Escherichia coli O157:H7 str. Sakai中龙胆酸1，2-双加氧酶相似性为36%。通过分子建模模拟该蛋白亚基的分子结构，分析蛋白亚基与底物分子的对接结果，推测His₁₁₄，His₁₁₆，His₁₅₅与亚铁离子构成催化核心，Trp₉₁，Asp₇₂, Asp₁₈₉, Arg₇₄可能在催化过程中起了一定的作用。将该基因连入表达载体成功实现了酶蛋白的异源表达。研究表明该蛋白酶在30℃，pH 8.0条件下具有最大的酶活性，二价铁离子能够显著地提高酶活性。该蛋白酶不能催化与龙胆酸结构类似的一系列底物，具有严格的底物专一性。该实验结果为研究该酶的催化机制奠定了基础。
其他摘要	Microcosm experiments were set up in order to study the dynamic changes of the bacterial communities in marine sediments supplemented with different model PAH compounds pyrene and sodium benzoate. High-throughput sequencing technology Illumina Miseq targeted the 16S rRNA gene V3-V4 variable region was used to analysis the shift in the composition of the bacterial communities due to the addition of pyrene and sodium benzoate. Either pyrene or sodium benzoate could dramatically influence the bacterial community structures in marine sediments. Lower diversity and abundance were observed compared with the control group. Marinobacter and Pseudidiomarina became dominant community in the in experimental group treated with sodium benzoate. Balneola and Aequorivita were greatly enriched in the experimental group with the addition of pyrene. Our study will help to reveal the functional bacteria capable of degrading pyrene and sodium benzoate respectively. This result may also provide a theoretical basis for the bioremediation of PAH compounds in marine environment. Bacillus sp. LKG-A is a gram-positive benzoate degrader bacterium isolated from marine sediment, which degrades benzoate via the gentisate pathway to central metabolites. The godA gene, encoding the gentisate1, 2-dioxygenase responsible for catalysing the ring fission of gentisate with the formation of maleylpyruvate was cloned by touch-down PCR and thermal asymmetric interlaced PCR amplification. The open reading frame consists of 1143bp encoding a protein with 379 amino acids.The deduced amino acid sequence of godA with a theoretical Mr of 42.9 kDa displays no more than 59% sequence identity with those previous pubulished gentisate1, 2-dioxygenase. The sequence of the gdoA gene product shared similarity with few known polypeptides, including gentisate 1, 2-dioxygenase from Paenibacillus sp. NyZ101 (49%) or gentisate 1, 2-dioxygenase from Escherichia coli O157: H7 str. Sakai (36%), gentisate 1, 2-dioxygenase from Bacillus halodurans C-125 share 50% identity. Homology modeling were employed to construct the three-dimensional structure of the gentisate 1, 2-dioxygenase protein from Bacillus sp. LKG-A. Analyzing three-dimensional structure of the protein subunits by homology modeling and docking result, we speculated His₁₁₄, His₁₁₆, His₁₅₅ and ferrous ions were considered to constitute one catalytic core, and Trp₉₁, Asp₇₂, Asp₁₈₉, Arg₇₄may play a certain role in the catalytic process. The probable biochemical mechanism of the involvement of the protein in aromatic hydrocarbon oxidation has also been investigated. It was cloned into pET22b vector then transformed into E. coli BL21 (DE3) expressed by the induction with 1.0 mM of IPTG. The resulting product was purified to homogeneity and partially characterized. Studies showed that the dioxygenase had the largest activity at 30℃and pH 8.0. Ferrous ions could significantly improve its catalytic activity. The dioxygenase could not catalyze similar structure with gentisate, which was different from gentisate 1, 2-dioxygenase discovered earlier .The discoveries maight deepen our understanding of the gentisate metabolic pathways.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/7919
专题	中国科学院烟台海岸带研究所知识产出_学位论文
作者单位	1.中国科学院烟台海岸带研究所 2.中国科学院大学
第一作者单位	中国科学院烟台海岸带研究所
推荐引用方式 GB/T 7714	孙向楠. 苯甲酸钠降解菌的筛选、龙胆酸1，2-双加氧酶基因的克隆与异源表达[D]. 北京. 中国科学院研究生院,2015.

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