假单胞菌NyZ12中环己胺氧化酶基因的克

发布时间：2018-04-26 17:38

本文选题：假单胞菌NyZ12 + 环己胺　；参考：《武汉轻工大学》2016年硕士论文

【摘要】：环己胺(化学式C6H13N)又被称为氨基环己烷或六氢苯胺,是一种重要的精细化工中间体,在使用和生产的过程中,环己胺被释放到了环境中,实验已证实该化合物有致癌性。由于环己胺在工业生产上的广泛应用和对人体的明显危害性,有必要采取一定的措施对工业中产生的废弃胺进行降解;而微生物降解成本低,能耗少,是消除环己胺对环境污染和残留的有效途径之一。目前报道的以环己胺为碳氮源生长的纯培养物仅有两株,一株是Iwaki H.等人分离筛选到的革兰氏阳性短杆菌IH-35A,另一株是由中国科学院武汉病毒研究所周宁一研究组分离的假单胞菌NyZ12。假单胞菌NyZ12是一株可以降解环己胺的革兰氏阴性菌。本研究将筛选出的假单胞菌NyZ12进行了全基因组测序。通过比对分析,将可能编码环己胺氧化酶的基因进行克隆表达,之后运用各种实验方法确定了真正的编码基因,进一步诱导表达纯化该酶,检测了环己胺氧化酶的活力。主要研究内容和结论如下:1.首先研究了野生菌NyZ12的生长情况及降解环己胺的能力,实验证明该野生菌可在20h内降解10mmol/L的环己胺,确定了分光光度法检测环己胺浓度的的方法。而且通过实验确定了环己胺胺氧化酶是受底物诱导型表达的。2.将全基因组测序结果通过生物信息学分析比对,找到五个可能编码环己胺氧化酶的候选基因amo2631、amo4207、amo5539、amo0425、amo4637,分别克隆到载体pUC18上,并转化到大肠杆菌DH5?中。3.运用分光光度法检测pUC18系列的重组工程菌对底物环己胺的降解实验,结果证明只有DH5?[pUC18-2631]能够降解底物环己胺,因此初步判断出了这五个基因中编码环己胺氧化酶的一个关键基因是amo2631。4.通过荧光定量PCR研究了这五个基因在底物诱导之后的表达量,结果发现amo2631基因的表达量明显上调,这跟我们做的转录组分析结果是一致的。进一步证明了amo2631很有可能是编码环己胺氧化酶的基因。5.为了使目的基因表达形成可溶性的蛋白并便于纯化,将这五个基因加入His标签克隆到pVLT33载体上并转入DH5?中。用气相方法检测pVLT33系列的重组工程菌对底物环己胺的降解情况和产物生成情况,结果证明只有DH5?[pVLT33-2631]气相检测到了中间产物环己酮的生成,证明了amo2631为NyZ12编码环己胺氧化酶的关键基因。6.大量诱导表达pVLT33系列的工程菌,使其融合表达而不形成包涵体,用辣根过氧化物酶间接检测粗酶液降解环己胺的能力。实验证明DH5?[pVLT33-2631]粗酶浓度为914.97U/m L,证明了其具有催化底物环己胺的酶活性。7.诱导表达重组菌DH5?[pVLT33-2631],利用融合蛋白所带的组氨酸标签使用金属亲和层析柱纯化得到纯酶,测定环己胺氧化酶的比活力。
[Abstract]:Cyclohexylamine (C6H13N), also known as aminocyclohexane or hexahydroaniline, is an important fine chemical intermediate. In the process of use and production, cyclohexylamine is released into the environment. Due to the wide application of cyclohexylamine in industrial production and the obvious harm to human body, it is necessary to take some measures to degrade the waste amine produced in industry, while the microbial degradation cost is low and the energy consumption is low. It is one of the effective ways to eliminate environmental pollution and residue of cyclohexylamine. At present, there are only two pure cultures grown with cyclohexylamine as carbon and nitrogen source, one of which is Iwaki H. The other strain was Pseudomonas pseudomonas isolated from Zhouning Research Institute of Wuhan virus Research Institute of Chinese Academy of Sciences. Pseudomonas NyZ12 is a gram-negative bacterium that can degrade cyclohexylamine. In this study, the whole genome of Pseudomonas NyZ12 was sequenced. The genes that may encode cyclohexylamine oxidase were cloned and expressed by comparative analysis. After that, the true coding gene was determined by various experimental methods, and the activity of cyclohexylamine oxidase was detected by further induction, expression and purification of cyclohexylamine oxidase. The main contents and conclusions are as follows: 1. Firstly, the growth of wild strain NyZ12 and its ability to degrade cyclohexylamine were studied. It was proved that the wild strain could degrade cyclohexylamine of 10mmol/L within 20 hours, and the method of detecting the concentration of cyclohexylamine by spectrophotometry was determined. Furthermore, it was confirmed that cyclohexylamine oxidase was substrate-inducible. By bioinformatics analysis and comparison, five candidate genes amo2631, amo4207amo5539, amo0425amo4637 were cloned into the vector pUC18 and transformed into Escherichia coli DH 537. No. 3. The degradation of cyclohexylamine substrate by recombinant engineering bacteria of pUC18 series was detected by spectrophotometry. The results showed that only DH 5- [pUC18-2631] could degrade cyclohexylamine. One of the key genes encoding cyclohexylamine oxidase is amo2631.4. Fluorescence quantitative PCR was used to study the expression of these five genes after substrate induction. It was found that the expression of amo2631 gene was significantly up-regulated, which was consistent with the results of transcriptome analysis. It is further proved that amo2631 may be the gene encoding cyclohexylamine oxidase. In order to make the target gene express soluble protein and be easy to purify, the five genes were inserted into the pVLT33 vector and cloned into pVLT33 vector. In. The degradation of cyclohexylamine and the production of cyclohexylamine by recombinant engineering bacteria of pVLT33 series were detected by gas phase method. The results show that only DH 5- [pVLT33-2631] gas phase detects the formation of cyclohexanone, the intermediate product. It is proved that amo2631 is a key gene encoding cyclohexylamine oxidase in NyZ12. A large number of engineering bacteria expressing pVLT33 series were induced and expressed without inclusion bodies. Horseradish peroxidase was used to indirectly detect the ability of crude enzyme solution to degrade cyclohexylamine. The results showed that the concentration of DH 5- [pVLT33-2631] crude enzyme was 914.97U/m L, and that DH 5- [pVLT33-2631] had the enzyme activity of catalyzing cyclohexylamine. The recombinant strain DH 5- [pVLT33-2631] was induced and purified by metal affinity chromatography with histidine label carried by fusion protein to determine the specific activity of cyclohexylamine oxidase.
【学位授予单位】：武汉轻工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X172;Q78

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