NADH再生系统的共建及石油降解酶制剂的制备

发布时间：2018-02-24 04:33

本文关键词： 石油烃固定化酶博伊丁假丝酵母菌酶修复 FDH　出处：《山东师范大学》2015年硕士论文　论文类型：学位论文

【摘要】：在石油的开采、存储、精炼以及之后的使用过程中不可避免的会产生一些泄漏和排放，从而对环境造成很大的破坏，导致土壤和地下水的严重污染，此外，这些污染对生物也同样具有很大的毒性。利用自然界中的微生物的代谢过程对石油烃的降解是目前来说最为有效的修复方式。而生物降解的本质是生物体内降解酶对石油烃分子的酶促降解，将微生物的细胞进行细胞破碎，从中提取石油烃降解酶，直接应用于石油烃的降解，可以避开石油烃分子从胞外到胞内的运输过程，提高石油烃的降解效率。本文通过表达来自博伊丁假丝酵母菌中的甲酸脱氢酶（NADH再生系统）并与石油降解酶进行共固定，构建石油降解及NADH再生的偶联系统，利用甲酸/甲酸脱氢酶辅酶再生系统可以为石油烃降解酶连续提供还原态NADH，实现石油酶降解及NADH再生的偶联，降低石油烃降解酶的使用成本；研究该系统的构建及石油降解特性，主要研究内容及结论如下：（1）首先提取了博伊丁假丝酵母菌（C.boidinii）的基因组，PCR扩增了NAD+依赖性的甲酸脱氢酶的基因（fdh），并对博伊丁假丝酵母菌的fdh基因进行了序列分析；（2）将fdh基因连接到载体pET28a(+)中，构建了质粒pET28a(+)-fdh，转化大肠杆菌BL21(DE3)，构建完成表达fdh的大肠杆菌菌株E.coli E.coliBL21（pET28a(+)-fdh）。（3）培养E.coli BL21（pET28a(+)-fdh），转接2h之后进行诱导，添加终浓度为1mmol/L的IPTG进行诱导，离心收集菌体、破碎，测定酶活性，结果显示当破碎功率为300W、破碎\间隔时间为2s\2s、工作总时间10min时，每克菌体得到的酶活性为1.19U；（4）将石油降解酶与FDH进行共固定，在每克硅藻土固定2mg石油降解酶的情况下，当甲酸脱氢酶的添加量为0.06U·g-1时，固定化酶的催化活性最高；（5）将石油降解酶与FDH进行共固定。当石油降解酶2mg、甲酸脱氢酶0.06U、甲酸钠0.167mnol时，在30°C条件下对100mg石油进行降解，10h时降解率达到最高，为83.3%。与前期不添加甲酸脱氢酶的石油降解酶相比，添加NADH再生系统后的石油降解酶对石油烃的降解效率明显提高，，经过进一步的验证后，有望应用于石油污染的工程修复中。
[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 掳C at 30 掳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 掳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.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X74;X172

【参考文献】