甲醛落解论文：甲醛落解菌遴选、关键酶基因克隆表达和固定化细胞落解特征的研究[精华]

甲醛落解 论文 政研论文下载论文大学下载论文大学下载关于长拳的论文浙大论文封面下载 ：甲醛落解菌遴选、关键酶基因克隆表达和固定化细胞落解特征的研究[精华] 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 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 ,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 路基工程安全技术交底工程项目施工成本控制工程量增项单年度零星工程技术标正投影法基本原理 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(Methylobacterium),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/L?h。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 领导形象设计圆作业设计ao工艺污水处理厂设计附属工程施工组织设计清扫机器人结构设计 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1.0×10~8 cells/mL,在温度30?,培养基为MgSO4?7H2O 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/L?h,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 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 formaldehyde.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 engineering 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, biochemical 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 concentration 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/L?h.This article has selected Pseudomonas putida xyz-zjut as the strain to study the key enzymes in degradation of formaldehyde, which 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 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 genes 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 analysis 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 present 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 formaldehyde was: 1.0 108 cells/mL, MgSO4?7H2O 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/L?h. The immobilized cells could applied to degradation of formaldehyde in bioreactor, and lay a foundation 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 hydroxymethyltransferase immobilized cells Methylobacterium sp. XJLW Pseudomonas putida xyz-zjut