【作者】 李昂；

【导师】 周集体；

【作者基本信息】 大连理工大学 ， 环境工程， 2009， 博士

【摘要】 本论文旨在考察1株新分离的Dyella属菌株LA-4对联苯的降解特性并对其代谢基因进行分子遗传学研究。对菌株LA-4进行生理生化、Biolog分析及16S rDNA分子鉴定,考察其生长及降解联苯的特性,推测出菌株LA-4降解联苯的途径;将该菌株作为生物强化制剂进行生物强化降解联苯的研究,采用PCR-DGGE分子指纹技术对强化体系进行微生物群落动态解析;利用染色体步移法扩增得到完整的bph基因簇,并对其中的bphA1A2、bphA3、bphA4、bphB、bphC和mfphA基因进行克隆和表达。菌株LA-4是一株新的联苯降解菌,结合生理生化鉴定、菌株的形态观察、Biolog鉴定及16S rDNA序列分析,菌株LA-4归属于变形细菌Y亚类,Dyella属ginsengisoli菌种。菌株LA-4已作为专利菌种保存于中国普通微生物菌种保藏中心,注册编号为:CGMCC No.2723。菌株LA-4与Dyella ginsengisoli Gsoil 3046T（=DSM 18387T）的16SrDNA序列相似性高达98.22%,其16S rDNA序列登录GenBank,注册号为:EF191354。菌株LA-4对卡那霉素和链霉素具有抗性,对其它所试抗生素均无抗性。菌株LA-4的最适生长与降解条件为30℃,pH=6.0,摇床转速为150 r/min。加入的表面活性剂Tween-80浓度小于40 mg/L时会促进菌株LA-4对联苯的降解;菌株在生长降解过程中可以耐受3%的盐度;菌株具有抗金属离子Zn²⁺的能力。菌株LA-4的粗酶具有2,3-二羟基联苯1,2-双加氧酶活性,且为组成酶。联苯在菌株LA-4的作用下,有黄色中间产物产生,苯环发生了间位断裂反应。LC-MS分析中检测到主要降解中间产物为2-羟基-6-氧-6-苯基己二烯酸（HOPDA）和苯甲酸。将菌株LA-4的游离态细胞投加到联苯处理系统中进行生物强化降解研究,结果表明投加菌株LA-4的游离态细胞可以缩短处理系统的启动时间,投加不同接种量对强化效果有显著影响,投配比为3.96%的系统显示出最强的处理效果,在进水联苯浓度为500-1000 mg/L时,处理效果稳定。利用PCR-DGGE对强化体系进行微生物群落动态解析,结果表明活性污泥体系和投配比为1.98%的系统随着体系的不断运行,微生物的多样性逐渐降低;当进水联苯浓度逐渐升高时,投配比为1.98%的系统中,强化菌逐渐消失。而在投配比为3.96%的系统中可证明体系中始终有强化菌LA-4的存活。利用染色体步移的方法从菌株LA-4的基因组DNA中扩增得到完整的bph基因簇,全长为12,186 bp,全序列登录GenBank,序列号为EU258607。对整个基因簇进行解析,结果表明基因排列顺序为bphA1A2-ORF1-bphA3A4BCXOX1-ORF2-bphX2X3D,其中12个基因出现在已报道的bph基因簇中,ORF2基因首次在bph基因簇中发现,位于6phX1和bphX2基因之间。根据基因排列特点,说明其属于LB400型bph基因簇。ORF2基因与菌株Pseudomonas putida UCC22（BAB62053）中编码2-羟基粘康酸半醛水解酶（2-hydroxymuconic semialdehyde hydrolase）的基因相似性最高,确定其为编码芳环间位断裂产物水解酶的基因,命名为mfphA。菌株LA-4中的联苯降解酶与其他已知的相应酶的氨基酸序列相似性较低,最高相似性均在63-89%之间。将bphA1A2、bphA3、bphA4、bphB和bphC基因在大肠杆菌BL21（DE3）中进行表达。菌株LA-4联苯双加氧酶(BPDO_LA-4)基因的表达产物能够转化联苯/多氯联苯等物质,以联苯为底物时,酶的比活性最高,为134.9 nmol NADH/min（mg protein）。BPDO_LA-4对底物的选择性按以下顺序:联苯＞4-氯联苯＞2,5-二氯联苯＞2,2′-二氯联苯＞2,5,2′-三氯联苯＞3,3′-二氯联苯＞2,5,3′-三氯联苯＞4,4′-二氯联苯＞2,5,4′-三氯联苯。实验中未检测到联苯双加氧酶转化2,2′,5,5′-四氯联苯的活性。实验表明BPDO_LA-4还具有转化二苯并呋喃和黄烷酮的活性。通过GC-MS和紫外可见光谱分析确定了菌株LA-4的联苯双加氧酶在转化联苯及多氯联苯时仅存在2,3-位双加氧活性,而未发现其具有3,4-位加氧活性。通过对BphC LA-4的氨基酸序列分析,说明BphC LA-4为双域的Ⅰ类Fe（Ⅱ）依赖型外二醇双加氧酶。利用A|¨KTA蛋白质层析仪对bphC基因在大肠杆菌中的表达产物进行纯化。纯化后的BphC LA-4可以催化邻苯二酚类物质的间位开环反应,以2,3-二羟基联苯为底物时,酶的比活最高,为118.3 U/mg,酶反应的最适pH为8.0,最佳温度为40℃。BphC_LA-4对催化底物的选择性按以下顺序:2,3-二羟基联苯＞3-甲基邻苯二酚＞邻苯二酚＞4-氯邻苯二酚＞4-甲基邻苯二酚。根据MfphA的氨基酸序列分析和三级结构模拟的结果,说明mfphA基因所编码的酶属于α/β水解酶家族,可以在单环芳香化合物降解过程中水解芳环间位开环产物。将mfphA基因在大肠杆菌BL21（DE3）中进行表达,并利用A|¨KTA蛋白质层析仪对表达产物进行了纯化。纯化后的MfphA可以水解单环芳香化合物间位断裂产物,以2-羟基-6-氧-2,4-己二烯酸（HODA）为底物时酶的比活性最高,为2.0 U/mg;酶反应的最适pH为7.0,最佳温度为70℃,可以长期于低温下保存。MfphA对催化底物的选择性按以下顺序:6-methyl-HODA＞HODA＞5-methyl-HODA＞6-phenyl-HODA＞5-chloro-HODA。更多还原

【Abstract】 The purpose of this dissertation is to investigate the characteristics and molecular genetics research on catabolic genes of a newly isolated bacterium Dyella with the ability to degrade biphenyl.The physio- biochemical characterization,Biolog analysis and 16S rDNA molecular identification were performed.The characteristics of strain LA-4 on growth and biphenyl degradation were also investigated and the proposed metabolic pathway of biphenyl degradation by strain LA-4 was presumed.Meanwhile,bioaugmentation of biphenyl degradation with strain LA-4 as augmented inoculums was studied,and the PCR-DGGE fingerprints analysis was used to reveal the structural changes of microbial community. Finally,a complete bph gene cluster was amplified from strain LA-4 by genome walking method.The bphA1A2,bphA3,bphA4,bphB,bphC and mfphA genes were cloned and heterologously expressed in Escherichia coli.Strain LA-4 is a novel biphenyl-degrading bacterium.It was identified as genus Dyella ginsengisoli belonging to Gammaproteobacteria according to the morphological and physio-biochemical characteristics,Biolog analysis and 16S rDNA sequence analysis.Strain LA-4 has been deposited as a patent bacterium in China General Microorganism Culture Center with the accession number CGMCC 2723.The 16S rDNA sequence of strain LA-4 showed the highest similarity with that of Dyella ginsengisoli Gsoil 3046^T（=DSM 18387^T） （98.22%identity）.The 16S rDNA sequence of strain LA-4 has been deposited in the GenBank database under accession number EF191354.Strain LA-4 was resistant to kanamycin and streptomycin,and sensitive to other antibiotics.The optimal conditions for both growth and degradation were as follows: temperature 30℃,pH6.0 and shaking speed 150 r/min.The surfactant,Tween 80,could accelerate the biodegradation process when the concentration was less than 40 mg/L.And strain LA-4 was able to grow on and degrade biphenyl in the medium containing 3%NaCl.It could tolerate to Zn²⁺ in the biphenyl degradation.The crude cell extracts of strain LA-4 exhibited the 2,3-dihydroxybiphenyl dioxygenase activity,and it was proved that the enzyme was a constitutive extradiol dioxygenase.A yellow intermediate was observed during the biphenyl degradation by strain LA-4,and it was suggested that meta-cleavage occured.The major metabolites formed from biphenyl,such as 2-hydroxy-6-oxo-6- phenylhexa-2,4-dienoic acid（HOPDA） and benzoic acid,were identified by LC-MS. The suspended cells of strain LA-4 were inoculated into biphenyl treatment system,and the results showed that the cells of strain LA-4 can shorten the start-up time of the system.Different inoculums had the significant impacts on bioaugmentation,and the results demonstrated that the system with 3.96%inoculums had the strongest removal efficiency,which had the relatively stable function when the biphenyl concentration of influent was 500-1000 mg/L.The PCR-DGGE was used to reveal the structural changes of microbial community in the treatment system.The results showed that the microbial diversity was decreased in the activated sludge system and 1.98%inoculums system.As the biphenyl concentration was increasing,strain LA-4 was disappeared in 1.98%inoculums system.However,the introduced strain LA-4 was persistent in the augmented systems inoculated 3.96%strain.A complete 12,186 bp bph gene cluster was amplified by genome walking method,and the sequence of this cluster has been deposited in the GenBank database under accession number EU258607.Based on the sequence analysis,the gene organization of bph gene cluster from strain LA-4 was shown as bphA1A2-ORF1-bphA3A4BCXOX1-ORF2-bphX2X3D.12 ORFs in 13 ORFs obtained in this study showed homology with the previously characterized bph genes.Insterestingly,a novel ORF（ORF2） was firstly cloned from bph gene cluster, which was located between bphX1 and bphX2.The bph gene cluster from strain LA-4 belonged to LB400-type gene cluster.Meanwhile,it was determined that the ORF2 gene encoded a meta-fission hydrolase designated as MfphA,because the amino acid sequence of ORF2 exhibited 75%similarity with the sequence of 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas putida UCC22（BAB62053）.The sequence of all ORFs exhibited lower similarity（about 63-89%identities） with previously characterized bph genes.The bphA1A2,bphA3,bphA4,bphB,and bphC genes were cloned and heterologously expressed in Escherichia coli BL21（DE3）.The recombinant biphenyl dioxygenase(BPDO_LA-4) could biotransform biphenyl and various polychlorinated biphenyls（PCBs）.BPDO_LA-4 showed the best activity to transform biphenyl with the specific activity 134.9 nmol NADH/min（mg protein）.It was able to transform the biphenyl/PCBs in the following order: biphenyl＞4-CB＞2,5-CB＞2,2’-CB＞2,5,2’-CB＞3,3’-CB＞2,5,3’-CB＞4,4’-CB＞2,5,4’-CB.But the BPDO_LA-4 had no activity on 2,2’,5,5’- tetrachlorobiphenyl.Meanwhile, BPDO_LA-4 could also transform dibenzofuran and flavanone.It was detected that BPDO_LA-4 could introduce two hydroxyl into the biphenyl or PCB congeners at ortho and meta carbons, and 3,4-dihydroxylation was not observed by GC-MS and UV-Vis.According to the amino acid sequences analysis of BphC_LA-4,it was a two-domain typeⅠFe（Ⅱ）-dependent extradiol dioxygenase.The expression product of BphC_LA-4 in E. coli BL21 was purified using an（?）KTA explorer.The specific activity was 118.3 U/mg for 2,3-dihydroxybiphenyl,which was the best substrate for BphC_LA-4.The optimal pH for BphC_LA-4 was 8.0,and the enzyme showed its maximal activity at 40℃.According to the specificity constant(K_cat/K_m),BphC LA-4 was able to cleave dihydroxylated substrates in the following order of specificity:2,3-dihydroxybiphenyl＞3-methylcatechol＞catechol＞4-chlorocatechol＞4-methylcatechol.According to the amino acid sequences analysis and the three-dimensional structure of MfphA,it was indicated that MfphA encoded by the novel gene located in bph gene cluster belonged to theα/βhydrolase family,which could hydrolyze the meta-fission products（MFPs） during the biodegradation process of monocyclic compounds.The mfphA gene was heterologously expressed in E.coli,and the expression product was purified using an AKTA explorer.The purified MfphA could transform MFPs,and the highest specific activity was 2.0 U/mg for HODA among these substrates.MfphA showed the maximum activity at pH7.0,and the optimal temperature was 70℃.Meanwhile,MfphA was more stable for storage at low temperature.It was determined that MfphA was able to hydrolyze the substrates in the following specificity order:6-methyl-HODA＞HODA＞5-methyl-HODA＞6-phenyl-HODA＞5-chloro-HODA.更多还原