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题名: 苍白杆菌降解三氯卡班及其降解关键酶的基因克隆与表达
作者: 云 慧1
学位类别: 博士
答辩日期: 2017-06
授予单位: 中国科学院大学
授予地点: 北京
导师: 王爱杰
关键词: 三氯卡班,Ochrobactrum sp.,酰胺酶TccA,氯苯胺,生物降解 ; Triclocarban, Ochrobactrum sp., amidase TccA, chloroanilines,biodegradation
其他题名: Degradation of Triclocarban by Ochrobactrum sp.and the Gene Cloning and Expression of Key Enzyme
学位专业: 环境工程
中文摘要: 三氯卡班(Triclocarban,TCC)是一种广谱氯代芳香抗菌剂,大量的应用于 日常生活各类个人护理用品、消毒液以及备用品中,对革兰氏阴性、阳性菌和真 菌都有一定的抑制作用。由于TCC 水溶性极差,导致污水处理过程中活性污泥 吸附是其主要的去除途径,最后随剩余污泥排放到环境中。环境中的TCC 非常 稳定,在各种水体和底质环境中均被频繁检出,不仅会影响水生藻类的活性,还 对动物的生殖能力和内分泌造成影响,并且TCC 及其转化产物为潜在诱癌物, 同时也具有诱发微生物抗性基因产生的风险。我国是TCC 的使用大国,近些年 来TCC 在我国乃至全球开始得到广泛的关注。利用微生物进行TCC 的降解是一 种有效、经济和绿色的去除方法。因此,富集分离出高效的TCC 降解菌,丰富 TCC 的降解菌种资源,研究其降解分子机理,发掘其生物修复潜质,为强化生 物修复TCC 污染环境提供理论与技术基础。 本研究旨在于针对二级出水及环境介质中TCC 残留进行进一步的生物强化 处理,以期达到去除、脱毒无害化的效果,丰富TCC 降解菌的微生物多样性, 重点挖掘TCC 双酰胺键水解酶编码关键基因,为表征TCC 在生物修复过程中的 催化活性提供重要的分子标记;同时在分子和生化水平上揭示TCC 的微生物降 解途径和分子机理。 本研究从不同地点分离得到两株三氯卡班降解菌,两菌不能利用TCC 为唯 一碳源进行生长,水解TCC 中双酰胺键生成4-氯苯胺(4CA)和3,4-二氯苯胺 (DCA),两株菌经16S rRNA 基因鉴定均属于Ochrobactrum sp.,命名为菌株 TCC-1 和TCC-2,可在温度和pH 范围为20℃-40℃和5.2-9.0 下降解TCC,增大 接种量和提供共基质可以促进TCC 的降解和产物的积累,两株菌可以耐受较高 的TCC 浓度(>30mg/L)。 通过构建降解菌TCC-2 的基因组DNA 文库,获得了一个阳性克隆子U135, 能有效降解TCC 的双酰胺键生成4CA 和DCA,该基因编码的酰胺酶命名为TccA (GenBank 登录号KU753911),核酸序列大小为1425bp,编码474aa。与NCBI 数据库中Swiss-Prot 专业数据库中蛋白序列进行比对发现,TccA 与Rhodococcus sp. Oct1 中生化鉴定的邻硝基乙酰苯胺水解酶OctH 最高同源性38%,与来自 Streptomyces 推定酰胺酶同源性介于51%-53%,TccA 为一个全新的具有酰胺键 水解功能的酰胺酶。进一步比对分析发现,TccA 含有酰胺酶家族中高保守氨基 酸motif 区域GGSSGG,和该家族催化保守三分子(Lys98-Ser176-Ser200)。在 E. coli BL21 中高效表达了TccA,确定其分子量为50 kDa,为单体蛋白。 TccA 在温度低于70℃和pH 在5.0-10.0 之间时,酶的催化活性保持较好, 在偏碱性pH 条件下,酶的稳定性和催化活性都与最适反应条件差异不大。TccA 反应最适温度为35℃,最适pH 为8.0。1 mM 金属离子中仅Ag+对该酶有强烈抑 制作用,化学抑制剂1,10-邻菲啰啉、对甲基磺酰氟、吐温-80 和焦碳酸二乙酯可 抑制40%-60%酶催化活性。TccA 作用底物具有广谱性,不仅可以水解TCC 及其 脱卤同系物4,4’-二氯苯基脲(4,4’-dichlorocarbanilide, DCC)和双苯基脲 (carbanilide, NCC)的双酰胺键,还可以水解部分除草剂、杀虫剂和化学合成中 间体,包括除草剂敌稗,利谷隆,苯胺灵,氯苯胺灵,除虫剂除虫脲,化学合成 中间体4-氯苯基脲,4-溴苯基脲,1-(3,4-二溴苯基)脲,乙酰苯胺,医药品对 乙酰氨基酚和植物生长调节剂氯吡脲,其中TCC,DCC,NCC,除虫脲和氯吡 脲可以水解两个酰胺键。通过测定酶活力参数,最高值对应底物NCC,其次为 TCC,均大于DCC。该酶的表达不受底物的诱导,经RT-qPCR 确认TccA 为组 成型表达,这对于去除环境中低浓度底物残留具有重要意义。通过比较两株TCC 降解菌tccA 基因附近的区域发现,含有多个编码转座酶和整合酶的序列,推测 TCC 降解菌属Ochrobactrum 中该基因可能由基因水平转移进化而来。 基于TCC 大量存在的沉积物和生物质通常氧气不足,因此在微氧/厌氧条件 下以硝酸盐作为电子受体进行Ochrobactrum sp. TCC-1 的培养,同样可以水解 TCC,DCC,NCC 为氯苯胺和苯胺产物。微氧及碳氮比高的条件更利于降解菌 生物量积累,及促进底物水解效率。在厌氧剩余污泥体系中批次补充一定的电子 受体硝酸盐,发现可以支持菌株TCC-1 的定殖和底物TCC 水解,氯苯胺产物持 续积累,生物量也可以稳定在一定的水平,这些结果为基于生物刺激和生物强化 的TCC 污染环境生物修复奠定了理论基础。 另外,从污水处理厂剩余污泥中分离得到TCC 降解产物氯苯胺的高效降解 菌,该菌可以利用3,4-二氯苯胺作为唯一碳源、氮源和能源生长,该菌经16S rRNA 基因鉴定属于Diaphorobacter 菌属,命名为Diaphorobacter sp. LD72。不同的碳 氮源对该菌生长和底物降解的影响不同,丙氨酸和有机氮源对生长和降解的促进 明显,菌株LD72 也可以降解苯胺,间位和对位取代的一氯和二氯苯胺和溴代的 苯胺,4-氟苯胺,对不同浓度的底物适应性也较强,降解过程伴随当量摩尔的卤 离子释放,基于基因组测序分析推测双加氧酶参与卤代苯胺降解过程。当将氯苯 胺的降解菌LD72 与TCC 的降解菌TCC-2 共培养作用于TCC 的降解时,可以实 现TCC 的矿化,这为后期环境中TCC 的彻底去除奠定实验基础.
英文摘要: Triclocarban (TCC) is a synthetic broad-spectrum haloaromatic antimicrobial agent, which has been extensively produced and employed in various disinfectant, school supplies, and household and personal care products. TCC primarily inhibits Gram-positive bacteria and is also partly active against Gram negative bacteria as well as fungi. Because of its low water solubility, it is difficult to be treated sufficiently during the waste water treatment plant, which would be massively adsorbed on sewage sludge and ultimately discharged into environments. TCC is very stable, so it is frequently detected in various aquatic and terrestrial environments, like surface water, soil and sediments. Therefore, it is unavoidable to cause diverse negative influence to both human beings and ecological environments. It is reported that TCC is not only toxic to aquatic alga, but also has reproductive and endocrine toxicity to animals and humans.TCC and some of its transformation products are potential inducer of cancer. Meanwhile, after a long time of exposure, it is possible to lead to the cross-resistance and the development of other antibiotics resistance genes. As China is one of the largest consumers of TCC in the world, the related environmental problems caused by TCC would be very serious; so much attention has been paid in recent years. Biological treatment is generally considered as an efficient, economic and environment-friendly way for pollutants removal. Therefore, isolation of effective TCC-degrading bacteria, enriching the bacterial species resources, understanding the degradation mechanism in molecular level and disclosing its degradation potential are meaningful, which would lay basic foundations for future application and theoretical researches. The aim of this study is to realize the TCC removal or detoxification, which is from the secondary effluent and those discharged into environments, by isolated TCC-degrading bacteria. The degradation mechanism would be explained from the point of molecular view, which may be helpful to understand the TCC degradation pathway and potential application. Two TCC-degrading bacteria were isolated from different places, both of which could not grow with TCC but hydrolyze it into 4-chloroaniline (4CA) and 3,4-dichloroaniline (DCA). They were identified as Ochrobactrum sp. based on their 16S rRNA genes. They were similar with each other. They can degrade TCC at 20 oC to 40℃ and pH ranging from 5.2 to 9.0. TCC degradation and chloroanilines accumulation were enhanced when the inoculums were increased or when glucose was supplied. The reason was that obvious growth could occur with glucose and more biomass meant faster degradation. The TCC concentration of 30 mg/L showed no growth inhibition on the strain TCC-1 and the strain TCC-2. Based on the construction of genome DNA library, a positive clone, U135, which could degrade TCC was screened. The insert fragment was sequenced and analyzed for open read fragment (ORF). Among the 6 ORFs, a 1,425-bp fragment was blasted and had some similarity with biochemically identified amidase genes. It coded 474 amino acids and had a highest alignment of 38% with the biochemical identified p-nitroacetanilide amidases OctHD from Rhodococcus sp. Oct1, so it was identified as amidase gene tccA. When aligned with putative proteins from those species of Streptomyces, the similarity could be between 51%-53%, so TccA was considered as a novel amidase. Multiple alignment of the amino acid sequence of TccA with the biochemically characterized amidases revealed high conservation of the Gly/Ser-rich motif (GGSS[GS]G) and the catalytic triad (Ser-Ser-Lys) of the amidase signature enzyme family. The tccA fragment was cloned into the vector pET-29a(+) and transformed into E. coli BL21(DE3) for heterogenous expression. TccA was further purified by resin. The molecular weight of TccA was 50.73 kDa, and it was a monomer. When the temperature was lower than 70 oC and pH in the range of 5.0-10.0, the activity of TccA was well maintained. The optimal temperature and pH for function were 35 oC and 8.0, respectively. Most of tested metal ions had negligible influence on this enzyme except Ag+, which made it inactivity. Histidine modifier diethyl pyrocarbonate (DEPC), chelator 1, 10-phenanthroline, serine protease inhibitor phenylmethylsulfonyl fluoride and Tween-80 decreased TccA activity by about 40-60%. TccA could hydrolyze many substrates, including the TCC dehalogenated congeners 4,4’-dichlorocarbanilide (DCC) and carbanilide (NCC), insecticide diflubenzuron, herbicides linuron, propanil, propham, chlorpropham,chemical intermediates 4-chloracetanilide, acetanilide, 4-chlorophenylurea, 4-bromophenylurea, 1-(3,4-dichlorophenyl)urea, other chemicals forchlorfenuron and acetaminophen. The two amide bonds of TCC, DCC, NCC, diflubenzuron and forchlorfenuron could be hydrolyzed simultaneously by TccA. The specific enzyme values for TCC, NCC and DCC illustrated that NCC was the most affinity substrates for TccA, which was the biggest and followed by TCC and DCC. By RT-qPCR, tccA was confirmed to be transcribed constitutively without the induction of substrates, which was significant for the removal of low-concentration substrate residues. The amidase was also found in the genome of strain TCC-1, which had highly similar nearby sequences near the region of tccA, and some of them were related with integrase and transposase, suggesting the origin by horizontal gene transfer. In most cases where TCC is massively accumulated, like sediment and soil, oxygen is generally limited. So some tests were carried out under micro-aerobic and anaerobic conditions. With nitrate as electron acceptor, strain Ochrobactrum sp.TCC-1 could also hydrolyze the amide bonds of TCC, DCC and NCC and form chloroanilines and aniline. By comparison, more biomass could be accumulated with relative higher carbon and nitrogen ratio, which in turn enhanced the TCC removal and products formation. This was also the case for the micro-aerobic condition. The TCC hydrolysis in wastewater sewage sludge was also tested supplemented with nitrate and strain TCC-1. By amending nitrate intermittently, strain TCC-1 colonized and maintained the TCC-hydrolyzing activity, which results would lay a basic foundation for the potential bioremediation of TCC-contaminated anoxic sites with bioaugmentation and biostimulation. In addition, to realize the absolutely mineralization of TCC, strains for chloroanilines degradation were isolated from wastewater sewage sludge. The strain could use DCA as sole carbon, nitrogen source and energy source and was identified as Diaphorobacter sp. based on the 16S rRNA gene. With additional different carbon and/or nitrogen sources, strain Diaphorobacter sp.LD72 had different growth and substrates degradation ability, and alanine and organic nitrogen markedly spurred the growth and degradation. Aniline, 3-chloroaniline, 4-chloroaniline, 3-broroaniline, 4-broroaniline and 3,4-dibromoaniline and 4- furoaniline were all biodegraded by this strain, accompanied with stoichiometric amount of halide ions release and small amount of soluble metabolites accumulated in the medium. The elimination of these chloroanilines seemed to occur via cleavage by dioxygenase. When strain LD72 and strain TCC-2 were co-cultured in medium amended with TCC, TCC could be hydrolyzed and finally mineralized. This would lay a basic experimental foundation for the future TCC removal in environments.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38739
Appears in Collections:中科院环境生物技术重点实验室_学位论文

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作者单位: 1.中国科学院生态环境研究中心

Recommended Citation:
云 慧. 苍白杆菌降解三氯卡班及其降解关键酶的基因克隆与表达[D]. 北京. 中国科学院大学. 2017.
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