偶氮染料高效降解基因工程菌构建与特性研究
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摘要
偶氮染料的生物降解已得到较为深入的研究，分离得到多种具有偶氮染料脱色活性的微生物，并有数种偶氮还原酶基因经大肠杆菌表达获得高纯度、高效率的偶氮还原酶，但是酶制剂的应用受到各种条件的限制，成本较高。因此，考虑提高微生物本身的偶氮染料脱色活性，以便于工程应用。
本文的目的是构建一株具有较高偶氮染料脱色活性的基因工程菌，并考察其对偶氮染料的脱色特性。该工程菌是以沼泽红假单胞菌／大肠杆菌穿梭质粒为载体，将外源基因(来自球形红细菌Rhodobacter sphaeroides AS1.1737的偶氮还原酶基因)导入受体菌(沼泽红假单胞菌Rhodopseudomonas palustris AS1.2352)，使受体菌表达外源偶氮还原酶，从而提高该工程菌的偶氮染料脱色能力。为达到以上目标，首先，在工程菌构建之前，通过考察外源偶氮还原酶基因的供体菌(球形红细菌)和受体菌(沼泽红假单胞菌)的偶氮染料脱色活性，以及受体菌细胞中质粒DNA的种数及其复制等相关情况，验证该构建途径的可行性；进而构建穿梭质粒及工程菌；最后，以原始受体菌为对照，考察工程菌的生长及脱色特性。
对球形红细菌Rhodobacter sphaeroides AS1.1737的菌体及其胞内粗酶的偶氮染料脱色活性进行考察。菌体在光照缺氧条件可将多种偶氮染料脱色，部分染料24小时脱色率可达90％以上。染料不能作为该菌生长的唯一碳源。35-40℃，中性偏碱环境(pH7-8)具有较高脱色率。超声破碎菌体，获得胞内粗酶，经Red Sepharose CL-6B凝胶纯化，粗酶中偶氮还原酶的含量有一定提高。以甲基橙作为模型染料时，50℃、pH 8.0为其粗酶酶促反应的最佳条件，并且该酶催化的脱色反应符合双底物的乒乓机理。甲基橙、NADH表观米氏常数分别为0.45 mM和1.25 mM。
前期研究发现，沼泽红假单胞菌Rhodopseudomonas palustris AS1.2352具有偶氮染料脱色能力，其胞内粗酶亦具有偶氮还原酶活性，催化过程符合乒乓机理。而纺织印染助剂中包括多种表面活性剂，对生物具有一定的毒性，因此，考察表面活性剂对该菌脱色能力的影响具有实际意义。实验发现，该菌对两种阴离子表面活性剂(LAS，Sodiumlinear alkylbenzene sulphonate；SDS，Sodium dodecyl sulfate)有较强的耐受能力，并且能够在好氧条件下将其降解；LAS对菌体生长的抑制作用明显大于SDS；0.1％ SDS存在时染料脱色受到一定的抑制，较高温度(30℃以上)时，抑制情况随温度提高迅速加重。
为考察沼泽红假单胞菌Rhodopseudomonas palustris AS1.2352中存在的质粒DNA的情况，根据该菌的特点和常规提取方法应用中遇到的问题，在碱裂解法基础上，对其
Biodegradation of azo dyes has been studied for years, and many microorganism degrading azo dyes were screened. Some azoreductase genes were cloned and expressed in E. coli efficiently, while enzyme application in waste water treatment is limited by its high cost. Then microorganism itself is asked to be more efficient to promote practical treatment.
The purpose of this dissertation is to construct a genetically engineered bacterium with higher azoreductase activity. In order to get this goal, the azoreductase gene from the donor (Rhodobacter sphaeroides AS 1.1737) is inserted in a Rhodopseudomonas palustris/E. coli shuttle vector, which is transformed into the acceptor (Rhodopseudomonas palustris AS 1.2352). And the feasibility of this process is proved by investigating the azo dye decolorization by donor and acceptor strains, and the knowledge on plasmids in acceptor cells.
The decolorization of azo dyes by cells and crude enzyme of Rhodobacter sphaeroides AS1.173 7 was investigated, and several kinds of azo dyes can be decolorized over 90% in 24 h. While azo dyes can not be the sole carbon source for growth of this strain. Besides, the optimal conditions of decolorization are 35 - 40℃ and pH 7-8. The crude enzyme extracted from this strain has azoreductase activity, but can not be well purified by Red Sepharose CL-6B. Making methyl orange as model dye, the optimal decolorization conditions are 50℃ and pH 8.0, and the catalysis corresponded to ping-pong mechanism, where the Michaelis-Menten constants for Methyl Red and NADH are 0.45 mM and 1.25 mM, respectively.
The azoreductase activities of the cells and intracellular crude enzyme of Rhodopseudomonas palustris AS1.2352 were reported, and LAS (Sodium linear alkylbenzene sulphonate) and SDS (Sodium dodecyl sulfate) were added in cultures to simulate practical dyeing wastewater. It is found that this strain can endure surfactant and degrade them aerobically, and inhibitions of LAS on growth and decolorization are higher than SDS. Besides, the optimal temperature of decolorization with SDS (30℃) is much lower than that without SDS, which shows that SDS made the decolorization of this strain sensitive to higher temperatures.
The plasmid extraction of Rhodopseudomonas palustris was optimized, so that the native plasmids of this strain could be obtained with higher quantity and quality for further operations. And four kinds of plasmids were found in this strain, one of which was identified
本文的目的是构建一株具有较高偶氮染料脱色活性的基因工程菌，并考察其对偶氮染料的脱色特性。该工程菌是以沼泽红假单胞菌／大肠杆菌穿梭质粒为载体，将外源基因(来自球形红细菌Rhodobacter sphaeroides AS1.1737的偶氮还原酶基因)导入受体菌(沼泽红假单胞菌Rhodopseudomonas palustris AS1.2352)，使受体菌表达外源偶氮还原酶，从而提高该工程菌的偶氮染料脱色能力。为达到以上目标，首先，在工程菌构建之前，通过考察外源偶氮还原酶基因的供体菌(球形红细菌)和受体菌(沼泽红假单胞菌)的偶氮染料脱色活性，以及受体菌细胞中质粒DNA的种数及其复制等相关情况，验证该构建途径的可行性；进而构建穿梭质粒及工程菌；最后，以原始受体菌为对照，考察工程菌的生长及脱色特性。
对球形红细菌Rhodobacter sphaeroides AS1.1737的菌体及其胞内粗酶的偶氮染料脱色活性进行考察。菌体在光照缺氧条件可将多种偶氮染料脱色，部分染料24小时脱色率可达90％以上。染料不能作为该菌生长的唯一碳源。35-40℃，中性偏碱环境(pH7-8)具有较高脱色率。超声破碎菌体，获得胞内粗酶，经Red Sepharose CL-6B凝胶纯化，粗酶中偶氮还原酶的含量有一定提高。以甲基橙作为模型染料时，50℃、pH 8.0为其粗酶酶促反应的最佳条件，并且该酶催化的脱色反应符合双底物的乒乓机理。甲基橙、NADH表观米氏常数分别为0.45 mM和1.25 mM。
前期研究发现，沼泽红假单胞菌Rhodopseudomonas palustris AS1.2352具有偶氮染料脱色能力，其胞内粗酶亦具有偶氮还原酶活性，催化过程符合乒乓机理。而纺织印染助剂中包括多种表面活性剂，对生物具有一定的毒性，因此，考察表面活性剂对该菌脱色能力的影响具有实际意义。实验发现，该菌对两种阴离子表面活性剂(LAS，Sodiumlinear alkylbenzene sulphonate；SDS，Sodium dodecyl sulfate)有较强的耐受能力，并且能够在好氧条件下将其降解；LAS对菌体生长的抑制作用明显大于SDS；0.1％ SDS存在时染料脱色受到一定的抑制，较高温度(30℃以上)时，抑制情况随温度提高迅速加重。
为考察沼泽红假单胞菌Rhodopseudomonas palustris AS1.2352中存在的质粒DNA的情况，根据该菌的特点和常规提取方法应用中遇到的问题，在碱裂解法基础上，对其
Biodegradation of azo dyes has been studied for years, and many microorganism degrading azo dyes were screened. Some azoreductase genes were cloned and expressed in E. coli efficiently, while enzyme application in waste water treatment is limited by its high cost. Then microorganism itself is asked to be more efficient to promote practical treatment.
The purpose of this dissertation is to construct a genetically engineered bacterium with higher azoreductase activity. In order to get this goal, the azoreductase gene from the donor (Rhodobacter sphaeroides AS 1.1737) is inserted in a Rhodopseudomonas palustris/E. coli shuttle vector, which is transformed into the acceptor (Rhodopseudomonas palustris AS 1.2352). And the feasibility of this process is proved by investigating the azo dye decolorization by donor and acceptor strains, and the knowledge on plasmids in acceptor cells.
The decolorization of azo dyes by cells and crude enzyme of Rhodobacter sphaeroides AS1.173 7 was investigated, and several kinds of azo dyes can be decolorized over 90% in 24 h. While azo dyes can not be the sole carbon source for growth of this strain. Besides, the optimal conditions of decolorization are 35 - 40℃ and pH 7-8. The crude enzyme extracted from this strain has azoreductase activity, but can not be well purified by Red Sepharose CL-6B. Making methyl orange as model dye, the optimal decolorization conditions are 50℃ and pH 8.0, and the catalysis corresponded to ping-pong mechanism, where the Michaelis-Menten constants for Methyl Red and NADH are 0.45 mM and 1.25 mM, respectively.
The azoreductase activities of the cells and intracellular crude enzyme of Rhodopseudomonas palustris AS1.2352 were reported, and LAS (Sodium linear alkylbenzene sulphonate) and SDS (Sodium dodecyl sulfate) were added in cultures to simulate practical dyeing wastewater. It is found that this strain can endure surfactant and degrade them aerobically, and inhibitions of LAS on growth and decolorization are higher than SDS. Besides, the optimal temperature of decolorization with SDS (30℃) is much lower than that without SDS, which shows that SDS made the decolorization of this strain sensitive to higher temperatures.
The plasmid extraction of Rhodopseudomonas palustris was optimized, so that the native plasmids of this strain could be obtained with higher quantity and quality for further operations. And four kinds of plasmids were found in this strain, one of which was identified
引文
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