微囊藻毒素降解菌CJ5厌氧降解MCLR过程的研究
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摘要
微囊藻毒素(Microcystins, MCs)是由水华蓝藻产生的一类环状七肽肝毒素,在水华水体环境中频繁出现,给生态系统及人类健康带来严重威胁。已有研究表明,微生物降解是水环境中MCs去除的主要途径之一,并且有望用于水环境中MCs污染的去除。已经从环境样品中分离并鉴定出40多株MCs降解菌,对MCs的降解过程及机理也有了一定的认识。但是,目前关于MCs微生物降解的研究大部分集中在好氧降解方面,且分离出的降解菌也大都为好氧降解菌,对缺氧和厌氧条件下MCs的降解过程了解很少。
最新的研究结果表明,在缺氧厌氧条件下MCs也可以被湖泊沉积物中的土著微生物迅速降解,说明该过程也是MCs在环境中归趋的一条重要途径。但是,目前关于MCs厌氧降解过程的认识还很匮乏。本实验室从滇池沉积物中分离纯化出一株MCs厌氧降解菌CJ5,其16S rDNA序列分析结果表明该菌为Acidaminobacter sp.,它可以在厌氧条件下快速降解MCs。为了进一步深入了解MCs的厌氧降解过程,本文以CJ5为研究对象,通过室内模拟实验,研究了初始接种量、温度、pH值、外加碳氮源及初始MCLR浓度等条件对CJ5菌厌氧降解MCLR的影响；通过提取CJ5菌的胞内酶和胞外酶并分别验证其MCLR降解活性,以确定CJ5菌中MCs降解酶的位置；根据文献报道的MCs降解基因m1rA的引物MF-MR,以CJ5菌基因组DNA作为模板进行PCR扩增,检测该菌是否含有m1rA基因,以推断其可能的降解途径。具体结果如下：
(1)CJ5菌厌氧降解MCLR过程受温度和pH值的影响较大,在温度为20℃-30℃条件下,MCLR能够被CJ5菌快速降解,12天内可将初始浓度约为4mg/L的MCLR完全降解,并且无迟滞期。而在低温(10℃、15℃)条件下MCLR降解缓慢,分别经过了6天和8天的迟滞期后MCLR才开始降解,第20天时分别降解68.3%和72.8%。在中性及碱性(pH=7.0-10.0)条件下MCLR可被CJ5菌迅速降解,12天内降解到检测限以下,而在酸性条件下MCLR降解缓慢,在pH为4.0和5.0时,需要近20天的迟滞期后MCLR才开始降解,第40天才能降解到检测限以下。外加碳源和氮源对CJ5厌氧降解MCLR无显著影响。
(2)酶活测试结果表明,CJ5菌中的MCs降解酶为胞内酶,这与已经报道的MCs好氧降解菌的降解酶类似。CJ5菌粗酶降解MCs的过程中生成了一种新的降解产物,该产物既不同于已报道的MCs好氧降解中间产物,也与CJ5菌降解MCLR的产物Adda不同。由于该产物在238nm处有特征吸收峰,说明该产物含有完整的Adda基团,这一结果说明CJ5菌的降解途径可能与目前已知的MCs好氧降解途径不同。由于EDTA, PMSF,盐酸苯甲脒,胃蛋白酶抑制剂和胰蛋白酶抑制剂均对CJ5菌粗酶降解MCLR无抑制作用,并且对该产物的产生和累积没有影响,说明该产物可能是CJ5厌氧降解MCs的起始反应的初始产物。
(3)以CJ5菌DNA为模板,利用优化的PCR反应条件扩增m1rA基因,结果均未扩增出目标条带,而阳性对照样品可以扩增出相应条带,说明CJ5菌可能不含m1rA基因。这一结果也进一步证明CJ5菌降解MCs的机理不同于已知的好氧降解途径。
Microcystins(MCs) is a group of heptapeptide hepatotoxin produced by cyanobacteria, which are widely distributed in eutrophic water. Its potent hepatotoxicity and tumor promotion activity have posed risks to ecosystem and human health. It has been reported that biodegradation is the main pathway that leads to the decrease of MCs in eutrophic water environment, and biodegradation process is considered as a potential method to get rid of MCs contamination from water bodies. To date, about40MC-degrading bacteria have been isolated and identified from different environmental samples, and the mechanism of MCs degradation by pure isolate also have been investigated to some extent. However, most of studies on MCs biodegradation have been restricted to under aerobic conditions and all MC-degrading bacteria isolated from environmental samples are aerobic bacteria. Little information is available about the MCs anoxic or anaerobic degradation and its mechanisms.
Recent studies confirmed that MCs can be degraded by the sediment samples under anoxic and anaerobic conditions, suggesting that anoxic or anaerobic degradation maybe an important pathway to remove MCs in natural environment. But until now no futher study on anaerobic degradation of MCs was reported. An anaerobic MCs-degrading baterium CJ5was isolated from the sediment of Lake Dianchi previously. It can degrade MCs effectively under anaerobic condition and was identified as Acidaminobacter sp. through the analysis of16S rDNA sequence. In present study, the anaerobic MCs degradation process by CJ5was further investigated. The effects of initial inoculum, temperature, pH value, extra carbon and nitrogen source and initial concentration of MCLR on the degradation of MCs were studied in the static experiments. The position of degradation enzyme which responsible for the MCLR degradation was investigated through enzymatic degradation experiments. PCR amplification was performed to testify whether strain CJ5contains mlrA gene. The main results are as followed:
(1) Temperature and pH value have more significant influence on anaerobic degradation of MCLR. MCLR can be rapidly degraded at20℃-30℃, while the degradation of MCLR was slow at low temperatures (10℃、15℃), with a6days or8days lag time before degradation, respectively. MCLR was easily degraded under neutral and alkaline (pH=7.0-10.0) conditions and was degraded completely within12days without obvious lag phase. However, about20days lag time were needed prior to the onset of microcystin degradation under acid conditions (pH=4.0and5.0), and. MCLR was completely degraded untill day40. In addition, extra carbon or nitrogen sources have no significant effect on the degradation ability of CJ5.
(2) The results of enzyme assay showed that the MCs-degradation enzymes belong to intracellular enzymes. A new kind of degradation product was detected during the degradation of MCLR by the intracellular enzyme of CJ5, which was different both from the final product Adda produced during the degradation of MCLR by CJ5strain and from the products produced during aerobic MCLR degradation. The fact that this product possess the characteristic UV absorption spectra (λmax=238nm) of Adda suggested that it contains the intact Adda group. The protease inhibitor experiments showed that this product might be the initial product poduced during anaerobic degradation of MCLR by CJ5. These results indicated that the enzymatic degradation pathway of MCLR by CJ5was different from that by other aerobic MC-degraders.
(3) No mlrA gene homologue was detected in CJ5, suggesting that CJ5might degrade MCs through a new pathway different from aerobic bacteria.
最新的研究结果表明,在缺氧厌氧条件下MCs也可以被湖泊沉积物中的土著微生物迅速降解,说明该过程也是MCs在环境中归趋的一条重要途径。但是,目前关于MCs厌氧降解过程的认识还很匮乏。本实验室从滇池沉积物中分离纯化出一株MCs厌氧降解菌CJ5,其16S rDNA序列分析结果表明该菌为Acidaminobacter sp.,它可以在厌氧条件下快速降解MCs。为了进一步深入了解MCs的厌氧降解过程,本文以CJ5为研究对象,通过室内模拟实验,研究了初始接种量、温度、pH值、外加碳氮源及初始MCLR浓度等条件对CJ5菌厌氧降解MCLR的影响；通过提取CJ5菌的胞内酶和胞外酶并分别验证其MCLR降解活性,以确定CJ5菌中MCs降解酶的位置；根据文献报道的MCs降解基因m1rA的引物MF-MR,以CJ5菌基因组DNA作为模板进行PCR扩增,检测该菌是否含有m1rA基因,以推断其可能的降解途径。具体结果如下：
(1)CJ5菌厌氧降解MCLR过程受温度和pH值的影响较大,在温度为20℃-30℃条件下,MCLR能够被CJ5菌快速降解,12天内可将初始浓度约为4mg/L的MCLR完全降解,并且无迟滞期。而在低温(10℃、15℃)条件下MCLR降解缓慢,分别经过了6天和8天的迟滞期后MCLR才开始降解,第20天时分别降解68.3%和72.8%。在中性及碱性(pH=7.0-10.0)条件下MCLR可被CJ5菌迅速降解,12天内降解到检测限以下,而在酸性条件下MCLR降解缓慢,在pH为4.0和5.0时,需要近20天的迟滞期后MCLR才开始降解,第40天才能降解到检测限以下。外加碳源和氮源对CJ5厌氧降解MCLR无显著影响。
(2)酶活测试结果表明,CJ5菌中的MCs降解酶为胞内酶,这与已经报道的MCs好氧降解菌的降解酶类似。CJ5菌粗酶降解MCs的过程中生成了一种新的降解产物,该产物既不同于已报道的MCs好氧降解中间产物,也与CJ5菌降解MCLR的产物Adda不同。由于该产物在238nm处有特征吸收峰,说明该产物含有完整的Adda基团,这一结果说明CJ5菌的降解途径可能与目前已知的MCs好氧降解途径不同。由于EDTA, PMSF,盐酸苯甲脒,胃蛋白酶抑制剂和胰蛋白酶抑制剂均对CJ5菌粗酶降解MCLR无抑制作用,并且对该产物的产生和累积没有影响,说明该产物可能是CJ5厌氧降解MCs的起始反应的初始产物。
(3)以CJ5菌DNA为模板,利用优化的PCR反应条件扩增m1rA基因,结果均未扩增出目标条带,而阳性对照样品可以扩增出相应条带,说明CJ5菌可能不含m1rA基因。这一结果也进一步证明CJ5菌降解MCs的机理不同于已知的好氧降解途径。
Microcystins(MCs) is a group of heptapeptide hepatotoxin produced by cyanobacteria, which are widely distributed in eutrophic water. Its potent hepatotoxicity and tumor promotion activity have posed risks to ecosystem and human health. It has been reported that biodegradation is the main pathway that leads to the decrease of MCs in eutrophic water environment, and biodegradation process is considered as a potential method to get rid of MCs contamination from water bodies. To date, about40MC-degrading bacteria have been isolated and identified from different environmental samples, and the mechanism of MCs degradation by pure isolate also have been investigated to some extent. However, most of studies on MCs biodegradation have been restricted to under aerobic conditions and all MC-degrading bacteria isolated from environmental samples are aerobic bacteria. Little information is available about the MCs anoxic or anaerobic degradation and its mechanisms.
Recent studies confirmed that MCs can be degraded by the sediment samples under anoxic and anaerobic conditions, suggesting that anoxic or anaerobic degradation maybe an important pathway to remove MCs in natural environment. But until now no futher study on anaerobic degradation of MCs was reported. An anaerobic MCs-degrading baterium CJ5was isolated from the sediment of Lake Dianchi previously. It can degrade MCs effectively under anaerobic condition and was identified as Acidaminobacter sp. through the analysis of16S rDNA sequence. In present study, the anaerobic MCs degradation process by CJ5was further investigated. The effects of initial inoculum, temperature, pH value, extra carbon and nitrogen source and initial concentration of MCLR on the degradation of MCs were studied in the static experiments. The position of degradation enzyme which responsible for the MCLR degradation was investigated through enzymatic degradation experiments. PCR amplification was performed to testify whether strain CJ5contains mlrA gene. The main results are as followed:
(1) Temperature and pH value have more significant influence on anaerobic degradation of MCLR. MCLR can be rapidly degraded at20℃-30℃, while the degradation of MCLR was slow at low temperatures (10℃、15℃), with a6days or8days lag time before degradation, respectively. MCLR was easily degraded under neutral and alkaline (pH=7.0-10.0) conditions and was degraded completely within12days without obvious lag phase. However, about20days lag time were needed prior to the onset of microcystin degradation under acid conditions (pH=4.0and5.0), and. MCLR was completely degraded untill day40. In addition, extra carbon or nitrogen sources have no significant effect on the degradation ability of CJ5.
(2) The results of enzyme assay showed that the MCs-degradation enzymes belong to intracellular enzymes. A new kind of degradation product was detected during the degradation of MCLR by the intracellular enzyme of CJ5, which was different both from the final product Adda produced during the degradation of MCLR by CJ5strain and from the products produced during aerobic MCLR degradation. The fact that this product possess the characteristic UV absorption spectra (λmax=238nm) of Adda suggested that it contains the intact Adda group. The protease inhibitor experiments showed that this product might be the initial product poduced during anaerobic degradation of MCLR by CJ5. These results indicated that the enzymatic degradation pathway of MCLR by CJ5was different from that by other aerobic MC-degraders.
(3) No mlrA gene homologue was detected in CJ5, suggesting that CJ5might degrade MCs through a new pathway different from aerobic bacteria.
引文
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