本文關(guān)鍵詞： 石油烴 固定化酶 博伊丁假絲酵母菌 酶修復(fù) FDH　出處：《山東師范大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：在石油的開采、存儲、精煉以及之后的使用過程中不可避免的會產(chǎn)生一些泄漏和排放，從而對環(huán)境造成很大的破壞，導(dǎo)致土壤和地下水的嚴(yán)重污染，此外，這些污染對生物也同樣具有很大的毒性。利用自然界中的微生物的代謝過程對石油烴的降解是目前來說最為有效的修復(fù)方式。而生物降解的本質(zhì)是生物體內(nèi)降解酶對石油烴分子的酶促降解，將微生物的細(xì)胞進(jìn)行細(xì)胞破碎，從中提取石油烴降解酶，直接應(yīng)用于石油烴的降解，可以避開石油烴分子從胞外到胞內(nèi)的運(yùn)輸過程，提高石油烴的降解效率。 本文通過表達(dá)來自博伊丁假絲酵母菌中的甲酸脫氫酶（NADH再生系統(tǒng)）并與石油降解酶進(jìn)行共固定，構(gòu)建石油降解及NADH再生的偶聯(lián)系統(tǒng)，利用甲酸/甲酸脫氫酶輔酶再生系統(tǒng)可以為石油烴降解酶連續(xù)提供還原態(tài)NADH，實現(xiàn)石油酶降解及NADH再生的偶聯(lián)，降低石油烴降解酶的使用成本；研究該系統(tǒng)的構(gòu)建及石油降解特性，主要研究內(nèi)容及結(jié)論如下： （1）首先提取了博伊丁假絲酵母菌（C.boidinii）的基因組，PCR擴(kuò)增了NAD+依賴性的甲酸脫氫酶的基因（fdh），并對博伊丁假絲酵母菌的fdh基因進(jìn)行了序列分析； （2）將fdh基因連接到載體pET28a(+)中，構(gòu)建了質(zhì)粒pET28a(+)-fdh，轉(zhuǎn)化大腸桿菌BL21(DE3)，構(gòu)建完成表達(dá)fdh的大腸桿菌菌株E.coli E.coliBL21（pET28a(+)-fdh）。 （3）培養(yǎng)E.coli BL21（pET28a(+)-fdh），轉(zhuǎn)接2h之后進(jìn)行誘導(dǎo)，添加終濃度為1mmol/L的IPTG進(jìn)行誘導(dǎo)，離心收集菌體、破碎，測定酶活性，結(jié)果顯示當(dāng)破碎功率為300W、破碎\間隔時間為2s\2s、工作總時間10min時，每克菌體得到的酶活性為1.19U； （4）將石油降解酶與FDH進(jìn)行共固定，在每克硅藻土固定2mg石油降解酶的情況下，當(dāng)甲酸脫氫酶的添加量為0.06U·g-1時，固定化酶的催化活性最高； （5）將石油降解酶與FDH進(jìn)行共固定。當(dāng)石油降解酶2mg、甲酸脫氫酶0.06U、甲酸鈉0.167mnol時，在30°C條件下對100mg石油進(jìn)行降解，10h時降解率達(dá)到最高，為83.3%。 與前期不添加甲酸脫氫酶的石油降解酶相比，添加NADH再生系統(tǒng)后的石油降解酶對石油烴的降解效率明顯提高，，經(jīng)過進(jìn)一步的驗證后，有望應(yīng)用于石油污染的工程修復(fù)中。
[Abstract]:In the exploitation, storage, refining and subsequent use of oil, there will inevitably be some leakage and discharge, which will cause great damage to the environment, leading to serious soil and groundwater pollution, The biodegradation of petroleum hydrocarbons is the most effective way of remediation, and the essence of biodegradation is biodegradation in vivo. Enzymatic degradation of petroleum hydrocarbon molecules by enzyme, When the microorganism cells were broken and the petroleum hydrocarbon degrading enzymes were extracted from them, they could be directly applied to the degradation of petroleum hydrocarbons, which could avoid the transport process of petroleum hydrocarbon molecules from the extracellular to the intracellular, and improve the degradation efficiency of petroleum hydrocarbons. In this paper, the coupling system of petroleum degradation and NADH regeneration was constructed by expression of formate dehydrogenase nadh regeneration system from Candida Boitin and co-immobilized with petroleum degrading enzyme. By using formic acid / formic acid dehydrogenase coenzyme regeneration system, the reduced NADHs can be continuously provided for petroleum hydrocarbon degrading enzymes, the coupling of petroleum enzyme degradation and NADH regeneration can be realized, and the use cost of petroleum hydrocarbon degrading enzymes can be reduced. The main contents and conclusions of this system are as follows:. (1) the genomic DNA of C. boidinii was extracted from C. boidinii to amplify the NAD dependent formic dehydrogenase gene, and the fdh gene of C. boidinii was sequenced. (2) the fdh gene was ligated into the vector pET28a (), and the plasmid pET28a( -fdh) was constructed, which was transformed into E. coli BL21 (DE3), and the E. coli strain E.coli BL21pET28a (pET28a) was constructed. E. coli BL21 (pET28a) was transferred for 2 hours, then induced by IPTG with final concentration of 1 mmol / L. The bacteria were collected by centrifugation, and the enzyme activity was measured. The results showed that when the power of breakage was 300W, the time of crushing\ interval was 2s\ 2s, and the total working time was 10min, the results showed that, when the breaking power was 300W, the breaking time was 2s / 2s, and the total working time was 10min. The enzyme activity per gram of bacteria was 1.19 U; When the amount of formate dehydrogenase was 0.06 U 路g ~ (-1), the catalytic activity of the immobilized enzyme was the highest when the amount of formate dehydrogenase was 0.06 U 路g ~ (-1). The biodegradation rate of 100mg oil reached the highest at 30 擄C at 30 擄C for 10 h, when the oil degrading enzyme was 2 mg, formate dehydrogenase was 0.06 U and sodium formate was 0.167 mnol, the degradation rate reached the highest at 30 擄C, and the degradation rate was 83.3%. Compared with the petroleum degradation enzyme without formate dehydrogenase in the early stage, the degradation efficiency of petroleum hydrocarbon by adding NADH regeneration system is obviously improved. After further verification, it is expected to be applied to the engineering remediation of oil pollution.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X74;X172

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 任磊,黃廷林;黃土高原的石油類物質(zhì)坡面徑流污染研究[J];中國給水排水;2000年11期

2 梁生康,王修林,汪衛(wèi)東,李希明;高效石油降解菌的篩選及其在油田廢水深度處理中的應(yīng)用[J];化工環(huán)保;2004年01期

3 羊明;徐巖;穆曉清;肖榮;;一種新的高立體選擇性羰基還原酶的性質(zhì)及分離[J];化工進(jìn)展;2006年09期

4 何良菊,魏德洲,張維慶;土壤微生物處理石油污染的研究[J];環(huán)境科學(xué)進(jìn)展;1999年03期

5 丁明宇,黃健,李永祺;海洋微生物降解石油的研究[J];環(huán)境科學(xué)學(xué)報;2001年01期

6 王洪祚,劉世勇;酶和細(xì)胞的固定化[J];化學(xué)通報;1997年02期

7 張

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