本文選題：假單胞菌NyZ12 + 環(huán)己胺��； 參考：《武漢輕工大學(xué)》2016年碩士論文

【摘要】：環(huán)己胺(化學(xué)式C6H13N)又被稱為氨基環(huán)己烷或六氫苯胺,是一種重要的精細(xì)化工中間體,在使用和生產(chǎn)的過程中,環(huán)己胺被釋放到了環(huán)境中,實(shí)驗(yàn)已證實(shí)該化合物有致癌性。由于環(huán)己胺在工業(yè)生產(chǎn)上的廣泛應(yīng)用和對人體的明顯危害性,有必要采取一定的措施對工業(yè)中產(chǎn)生的廢棄胺進(jìn)行降解;而微生物降解成本低,能耗少,是消除環(huán)己胺對環(huán)境污染和殘留的有效途徑之一。目前報(bào)道的以環(huán)己胺為碳氮源生長的純培養(yǎng)物僅有兩株,一株是Iwaki H.等人分離篩選到的革蘭氏陽性短桿菌IH-35A,另一株是由中國科學(xué)院武漢病毒研究所周寧一研究組分離的假單胞菌NyZ12。假單胞菌NyZ12是一株可以降解環(huán)己胺的革蘭氏陰性菌。本研究將篩選出的假單胞菌NyZ12進(jìn)行了全基因組測序。通過比對分析,將可能編碼環(huán)己胺氧化酶的基因進(jìn)行克隆表達(dá),之后運(yùn)用各種實(shí)驗(yàn)方法確定了真正的編碼基因,進(jìn)一步誘導(dǎo)表達(dá)純化該酶,檢測了環(huán)己胺氧化酶的活力。主要研究內(nèi)容和結(jié)論如下:1.首先研究了野生菌NyZ12的生長情況及降解環(huán)己胺的能力,實(shí)驗(yàn)證明該野生菌可在20h內(nèi)降解10mmol/L的環(huán)己胺,確定了分光光度法檢測環(huán)己胺濃度的的方法。而且通過實(shí)驗(yàn)確定了環(huán)己胺胺氧化酶是受底物誘導(dǎo)型表達(dá)的。2.將全基因組測序結(jié)果通過生物信息學(xué)分析比對,找到五個(gè)可能編碼環(huán)己胺氧化酶的候選基因amo2631、amo4207、amo5539、amo0425、amo4637,分別克隆到載體pUC18上,并轉(zhuǎn)化到大腸桿菌DH5?中。3.運(yùn)用分光光度法檢測pUC18系列的重組工程菌對底物環(huán)己胺的降解實(shí)驗(yàn),結(jié)果證明只有DH5?[pUC18-2631]能夠降解底物環(huán)己胺,因此初步判斷出了這五個(gè)基因中編碼環(huán)己胺氧化酶的一個(gè)關(guān)鍵基因是amo2631。4.通過熒光定量PCR研究了這五個(gè)基因在底物誘導(dǎo)之后的表達(dá)量,結(jié)果發(fā)現(xiàn)amo2631基因的表達(dá)量明顯上調(diào),這跟我們做的轉(zhuǎn)錄組分析結(jié)果是一致的。進(jìn)一步證明了amo2631很有可能是編碼環(huán)己胺氧化酶的基因。5.為了使目的基因表達(dá)形成可溶性的蛋白并便于純化,將這五個(gè)基因加入His標(biāo)簽克隆到pVLT33載體上并轉(zhuǎn)入DH5?中。用氣相方法檢測pVLT33系列的重組工程菌對底物環(huán)己胺的降解情況和產(chǎn)物生成情況,結(jié)果證明只有DH5?[pVLT33-2631]氣相檢測到了中間產(chǎn)物環(huán)己酮的生成,證明了amo2631為NyZ12編碼環(huán)己胺氧化酶的關(guān)鍵基因。6.大量誘導(dǎo)表達(dá)pVLT33系列的工程菌,使其融合表達(dá)而不形成包涵體,用辣根過氧化物酶間接檢測粗酶液降解環(huán)己胺的能力。實(shí)驗(yàn)證明DH5?[pVLT33-2631]粗酶濃度為914.97U/m L,證明了其具有催化底物環(huán)己胺的酶活性。7.誘導(dǎo)表達(dá)重組菌DH5?[pVLT33-2631],利用融合蛋白所帶的組氨酸標(biāo)簽使用金屬親和層析柱純化得到純酶,測定環(huán)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.
【學(xué)位授予單位】：武漢輕工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：X172;Q78

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