【作者】 江慧芳；

【导师】 王雅琴；

【作者基本信息】 北京化工大学 ， 生物化工， 2007， 硕士

【摘要】 脂肪酶(Lipase，EC3．1．1．3)是一类催化脂肪酸甘油酯水解的酶类，它作为生物催化剂的重要组成部分有着广泛的用途。米根霉(Rhizopus oryzae)脂肪酶具有良好的1，3位置专一性，它能催化甘油三酯产生脂肪酸和单甘酯，单甘酯是一种重要的非离子型表面活性剂，已广泛地应用于食品、乳制品和化妆品等行业。因此，利用基因手段，构建产酶活力高的基因工程菌对生产单甘酯等具有重要的意义，是当前研究的热点，另外，脂肪酶活力至今仍然没有统一的测定方法，如何准确测定酶活力大小，对研究脂肪酶的特性及应用具有重要意义。本论文利用本实验室保存的含米根霉(Rhizopus oryzae 3．2686)脂肪酶基因ROL的重组质粒pGMT-ROL，设计两对特异性引物定向克隆甘油三酰酯脂肪酶基因ROL，分别与与载体pET-28a和pET-11c连接，构建成表达重组载体pET-28a-ROL和pET-11c-ROL。采用氯化钙法，将重组表达载体分别转入受体菌组BL21(DE3)和Origami DE3中，分别通过Kana抗性平板和Amp抗性平板筛选初步筛选获得阳性克隆。通过菌落PCR和提取重组质粒做筛选，筛选到的重组子，用IPTG做诱导基因表达，表达产物经SDS-PAGE电泳分析，可鉴定重组菌表达了重组蛋白，约35KD。另外，本文比较和改进了酸碱滴定法，对硝基苯酚比色法和铜皂法三种常用的脂肪酶活力测定方法。具体比较了其所耗时间，测出酶活大小的准确性，灵敏度。为对硝基苯酚比色法找到了有效的方法来终止反应，改进的对硝基苯酚比色法具有重复性好的特点。另外还对其中的铜皂法进行了改进，用甲苯代替了苯，取得了较好的结果，改进的铜皂法具有毒性小、快捷的优点。通过对几种测定方法的比较改进和优缺点的讨论，为以后测定脂肪酶活力提供选择依据。更多还原

【Abstract】 Lipase is a typical enzyme which catalyzes the hydrolysis of long chain triglycerides. They constitute the most important group of biocatalysts for biotechnological applications. Lipase from Rhizopus oryzae has 1,3 positional specificity, it can hydrolyze triacylglycerol producing fatty acid and monoglyceride (MG), MG is an important non-ionic surfactants, widely used as emulsifier in the food stuff and other industries. As a result, today people focus on the construction of gene recombinant strain with high enzyme activity by gene measuses.Besides,. There is still no uniform method to measure the activity of lipase exactly , while it is important in reasearching of characters and amplication of lipases.In This thesis , the gene which encoding lipase ROL from Rhizopus oryzae was amplified by PCR and cloned into vector pGM-T was saved in our lab.The Recombinant plasmid Is named pGMT-ROL, and I designed primer pair 4 and 4.2 to clone the lipase gene ROL from pGMT-ROL,separated connect with pET-28a and pET-11c. The recombinant plasmid are named pET-28a-ROL and pET-11c-ROL, and pET-28a-ROL was transferred into E.coli.BL21 and pET-11c-ROL was transferred into E.coli.Origami DE3. Expression Recombinant strains were obtained separated by Kana and Amp plates screening ,the Expression Recombinant strains were farther filtered by strain PCR, and The engineered strain was expressed the gene ROL by the induction of IPTG. The expression products of ROL gene was analysis by SDS-PAGE, the result indicated that ROL gene was functionally expressed in E.coliBL21 and Origami DE3. Recombinant lipase had a molecular mass of 35kD.On the other side,This thesis discusses three assays in common use,including the spectrophotometry assay with the synthetic substrate p-NP palmitate, the spectrophotometric assay using the formation of copper soaps and the alkaly titrition assay.We compare the consuming time of reaction, vericity and repeatity of methods.We have found an effective method to terminate the reaction for the spectrophotometry assay with the substrate p-NP palmitate,this modified method shows good repeatity and takes less time relatively.In addition,we also improved the copper soaps assay by using toluene insead of benzene,it has merits of less toxicity and good repeatity. Through discussing merits and demerits of these assays and improvement of them ,This thesis provides chosen basises of assays for lipase activity measure.更多还原