《甲醛降解论文：甲醛降解菌筛选、关键酶基因克隆表达和固定化细胞降解特性的研究.doc》由会员分享，可在线阅读，更多相关《甲醛降解论文：甲醛降解菌筛选、关键酶基因克隆表达和固定化细胞降解特性的研究.doc（9页珍藏版）》请在三一文库上搜索。
1、 甲醛降解论文：甲醛降解菌筛选、关键酶基因克隆表达和固定化细胞降解特性的研究【中文摘要】甲醛作为一种原生毒素,对生物体有很强的毒害作用,是室内空气污染的主要污染物之一。如何有效的控制空气中的甲醛污染,已引起人们的高度关注。通过生物法降解甲醛将成为去除甲醛的有利手段。本文旨在筛选甲醛降解菌,对甲醛代谢的关键酶进行克隆分析,并对固定化细胞的降解条件进行研究,为构建高耐受高效率降解甲醛的工程菌和生物法去除室内甲醛污染的应用奠定基础。利用以甲醛为唯一碳源的基本培养基,分离得到了一株甲醛降解新菌株,经形态学观察、生理生化特性鉴定及16S rDNA的同源性分析,初步鉴定该菌株属于甲基杆菌属(Methylo
2、bacterium),命名为Methylobacterium sp. XJLW。该菌株在初始驯化阶段,甲醛耐受浓度为0.1g/L,且在液体培养基中生长缓慢。实验室已经分离得到的一株甲醛降解菌株Pseudomonas putida xyz-zjut,该菌株的甲醛最高耐受浓度为6g/L,降解效率高达0.114g/Lh。经比较,选取实验室已经分离的高活性高耐受甲醛菌株Pseudomonas putida xyz-zjut作为甲醛关键酶基因的研究对象。根据NCBI中公布的Pseudomonas putida基因序列,设计了两对特异性引物,克隆了甲醛同化作用关键酶丝氨酸羟甲基酶(SHMT)和甲醛异化作用
3、关键酶甲醛脱氢酶(FDH)的基因。丝氨酸羟甲基酶基因大小为1254 bp,能编码417个氨基酸,甲醛脱氢酶基因全长1206 bp,编码401个氨基酸,与已报道的Pseudomonas putida F1及Pseudomonas putida KT2440具有较高的同源性。将甲醛脱氢酶基因连接至原核表达质粒pET-28b中,通过PCR,酶切鉴定及SDS-PAGE表明fdh基因成功克隆至载体pET-28b上,构建好的pET-28b-fdh在宿主E. coli BL21(DE3)成功获得了表达, SDS-PAGE中的目标蛋白质大小为43 kDa,与预期一致。但在检测工程菌降解甲醛的情况时发现甲醛降解
4、效率没有明显的提高,推测原因可能是甲醛脱氢酶虽然表达了,但是大部分蛋白在宿主中是以包涵体形式存在,使得蛋白不具有甲醛脱氢酶活性。以海藻酸钙为包埋载体固定Pseudomonas putida xyz-zjut降解甲醛取得较好的效果。固定化细胞降解甲醛的海藻酸钙颗粒中的最适细胞密度为1.0108 cells/mL,在温度30,培养基为MgSO47H2O 0.2 g/L,(NH4)2SO4 2.4 g/L,微量元素母液0.1 mL,pH值9的条件下,甲醛的降解效率高达0.107 g/Lh,高于国内外同类研究水平。该固定化细胞颗粒可应用于降解甲醛的反应器中,为生物法降解甲醛奠定基础。【英文摘要】As
5、a native toxin, formaldehyde has strong toxic effect on the organism and is one of the main pollutants of indoor air pollution. How to control formaldehyde pollution in the air has caused great concern, and degradation of formaldehyde by microorganism will be an effective way for the removal of form
6、aldehyde.The aim of the research was to isolate a strain which can metabolize formaldehyde, then investigate the key enzymes in degradation of formaldehyde and the characteristics of immobilized cells by gel beads. The research provided a basic study for constructing a high tolerance and efficient e
7、ngineering bacteria for formaldehyde degradation and the application for biological treatment technology about the indoor formaldehyde pollution.One bacterial strain which was capable to use formaldehyde as a sole carbon source was isolated from soil. Based on morphological, physiological, biochemic
8、al properties and 16S rDNA homology analysis, the strain was characterized as Methylobacterium and named as Methylobacterium sp. XJLW. The concentration of formaldehyde-tolerant by the strain was 0.1g/L in the initial acclimation period, and the strain grown slowly in liquid medium. The concentratio
9、n of formaldehyde-tolerant of Pseudomonas putida xyz-zjut which was as isolated by our laboratory up to 6 g/L, and the degradation rate of formaldehyde was up to 0.114 g/Lh.This article has selected Pseudomonas putida xyz-zjut as the strain to study the key enzymes in degradation of formaldehyde, wh
10、ich was isolated by our laboratory and also proved to effectively degrade formaldehyde. Two pairs of specific primers was designed to amplify serine hydroxymethyltransferase （SHMT） and formaldehyde dehydrogenase （FDH） genes from Pseudomonas putida xyz-zjut, according to the genome of the strain from
11、 GenBank. The result showed serine hydroxymethyltransferase gene obtained from Pseudomonas putida xyz-zjut was 1254 bp in size and encoded 417 amino acids, and formaldehyde dehydrogenase gene was 1206 bp in size and encoded 401 amino acds. The DNA fragments had high homology with the key enzyme gene
12、s form Pseudomonas putida F1 and Pseudomonas putida KT2440. The engineering strain E. coli BL21（DE3）with recombinant vector pET-28b-fdh was obtained by introducing formaldehyde dehydrogenase gene into the expression vector pET-28b. The results from PCR, restriction enzyme digestion and SDS-PAGE anal
13、ysis revealed that the recombinant expression vector pET-28b-fdh had been constructed and expressed in the host of E. coli BL21（DE3） successfully. Unfortunately, the efficiency of formaldehyde degradation of engineering strain was improved insignificantly. It was speculated that the target protein p
14、resent in the inclusion body in the host, and does not have the activity of formaldehyde dehydrogenase.Using alginate as immobilized supporter, the immobilized cells of Pseudomonas putida xyz-zjut could degrade formaldehyde effectively. The optimum conditions that the immobilized cells metabolize fo
15、rmaldehyde was: 1.0 108 cells/mL, MgSO47H2O 0.2 g/L,（NH4）2SO4 2.4g/L,trace elements 0.1 mL/L, temperature 30, adjust pH to 9. Under these conditions, the effect of formaldehyde degradation was 0.107 g/Lh. The immobilized cells could applied to degradation of formaldehyde in bioreactor, and lay a fou
16、ndation for the further research of application of biological formaldehyde degradation.【关键词】甲醛降解 甲醛脱氢酶 丝氨酸羟甲基转移酶 固定化细胞 Methylobacterium sp. XJLW Pseudomonas putida xyz-zjut【备注】索购全文在线加好友：1.3.9.9.3.8848 同时提供论文写作一对一指导和论文发表委托服务【英文关键词】formaldehyde degradation formaldehyde dehydrogenase serine hydroxymeth
17、yltransferase immobilized cells Methylobacterium sp. XJLW Pseudomonas putida xyz-zjut【目录】甲醛降解菌筛选、关键酶基因克隆表达和固定化细胞降解特性的研究摘要3-5ABSTRACT5-7第一章 文献综述12-291.1 室内甲醛污染的来源12-141.2 甲醛的污染治理方法14-171.2.1 通风换气式法141.2.2 控制温湿度法141.2.3 物理吸附法14-151.2.4 化学法15-161.2.4.1 化学反应法151.2.4.2 臭氧氧化法151.2.4.3 空气负离子技术15-161.2.4.4
18、光催化法161.2.4.5 常温催化氧化法161.2.5 生物法16-171.2.5.1 植物吸收法16-171.2.5.2 微生物降解法171.3 甲醛在微生物中的代谢途径17-221.3.1 甲醛代谢的同化途径18-201.3.1.1 丝氨酸循环18-191.3.1.2 核酮糖单磷酸途径（RuMP）途径19-201.3.2 甲醛代谢的异化途径20-221.3.2.1 谷胱甘肽（GSH）依赖型异化途径20-211.3.2.2 叶酸-依赖型异化途径21-221.3.2.3 甲醛环化氧化途径221.4 国内外研究现状22-231.5 本文研究的意义及内容23-24参考文献24-29第二章 甲醛降
19、解新菌株的分离纯化及鉴定29-442.1 引言29-302.2 材料30-322.2.1 甲醛降解菌样品的采集302.2.2 培养基及试剂30-312.2.2.1 培养基30-312.2.2.2 试剂312.2.3 药品与试剂31-322.2.4 仪器322.3 方法32-372.3.1 分光光度法测定甲醛浓度32-332.3.2 样品甲醛浓度的测定332.3.3 菌株的筛选及分离纯化332.3.4 菌株的鉴定33-372.3.4.1 革兰氏染色33-342.3.4.2 透射电镜观察342.3.4.3 生理生化特征34-352.3.4.4 分子生物学鉴定35-372.4 结果与分析37-412
20、.4.1 菌株的筛选及分离纯化372.4.2 菌株的鉴定37-412.4.2.1 革兰氏染色37-382.4.2.2 透射电镜观察382.4.2.3 生理生化特征38-392.4.2.4 分子生物学鉴定39-412.5 小结41参考文献41-44第三章 Pseudomonas putida xyz-zjut 甲醛降解关键酶基因的克隆和表达44-663.1 引言44-453.2 材料45-483.2.1 菌株和质粒453.2.2 主要试剂45-463.2.3 实验仪器46-473.2.4 PCR 引物473.2.5 主要培养基及试剂47-483.3 方法48-533.3.1 菌株xyz 菌基因组
21、DNA 的提取48-493.3.2 PCR 扩增丝氨酸羟甲基转移酶（SHMT）和甲醛脱氢酶（FDH）的基因493.3.3 凝胶回收纯化DNA49-503.3.4 连接反应503.3.5 大肠杆菌感受态细胞的制备50-513.3.6 转化大肠杆菌感受态细胞513.3.7 试剂盒快速小量提取质粒DNA51-523.3.8 酶切反应523.3.9 蛋白电泳检测重组蛋白的表达52-533.4 结果与分析53-643.4.1 丝氨酸羟甲基转移酶的克隆53-573.4.2 甲醛脱氢酶的克隆与表达57-643.4.2.1 甲醛脱氢酶基因片段的获得57-623.4.2.2 pET28b-fdh 重组质粒的构建
22、62-633.4.2.3 重组蛋白在E. coli BL21 中的蛋白表达分析63-643.5 小结64-65参考文献65-66第四章 固定化Pseudomonas putida xyz-zjut 降解甲醛的研究66-794.1 引言66-674.2 材料67-694.2.1 实验菌种674.2.2 培养基及试剂674.2.3 药品与试剂67-684.2.4 仪器68-694.3 方法69-714.3.1 样品甲醛浓度的测定694.3.2 菌种的预培养及活化694.3.3 固定化颗粒的制备694.3.4 菌体固定化后的甲醛降解效果69-704.3.5 不同培养基对固定化颗粒降解甲醛的影响704
23、.3.5.1 蛋白胨水培养基与生理盐水对固定化颗粒降解甲醛的效果比较704.3.5.2 无机盐培养基与生理盐水对固定化颗粒降解甲醛的效果比较704.3.6 固定化菌株在不同菌浓下的甲醛降解效果70-714.3.7 固定化菌株在不同温度下的甲醛降解效果714.3.8 固定化菌株在不同 pH 的甲醛降解效果714.4 结果与分析71-774.4.1 菌体固定化后对甲醛的降解效果71-724.4.2 不同培养基对固定化细胞降解甲醛的影响72-744.4.2.1 蛋白胨水培养基与生理盐水对固定化颗粒降解甲醛的效果比较72-734.4.2.2 无机盐培养基与生理盐水对固定化颗粒降解甲醛的效果比较73-744.4.3 固定化菌株在不同菌浓下的甲醛降解效果74-754.4.4 固定化菌株在不同温度下的甲醛降解效果75-764.4.5 固定化菌株在不同pH 下的甲醛降解效果76-774.5 小结77参考文献77-79第五章 实验总结与展望79-81致谢81-82攻读学位期间发表的学术论文和专利目录82