麦芽糖转糖基酶论文：麦芽糖转糖基酶在毕赤酵母中的表达及活性分析.doc

麦芽糖转糖基酶论文：麦芽糖转糖基酶在毕赤酵母中的表达及活性分析【中文摘要】麦芽糖转糖基酶是一种4-葡聚糖转移酶,能催化直链淀粉环化生成大环糊精。大环糊精具有筒状疏水内腔和亲水外表结构,可以广泛应用于食品、医药、化工等行业。麦芽糖转糖基酶在生物(包括微生物)体内含量很低,不能满足制备大环糊精和科研的需要。本文从E.coli BL21(DE3)plysS中扩增得到麦芽糖转糖基酶基因(MalQ),构建了表达载体pPIC9K-MalQ,SalI线性化,电击转化入毕赤酵母(Pichia pastoris) GS115感受态细胞中,成功构建了一株基因工程菌pPIC9K-MalQ/GS115。利用0.8%甲醇诱导表达,表达上清进行转糖基活性分析,TLC结果显示表达上清液具有糖基转移酶作用,证实麦芽糖转糖基酶在毕赤酵母中成功表达。表达产生的麦芽糖转糖基酶和直链淀粉反应,制备得到一系列大环糊精的混合物。本文主要结论如下：(1)PCR扩增得到的MalQ基因,构建了表达载体pPIC9K-MalQ。对其进行酶切和测序分析。测序结果经BLAST比对,与GenBank中报道的E. coli BL21(DE3)MalQ基因的同源性为100%。(2)表达载体用Sal线性化,电击转化入毕赤酵母GS115感受态细胞中,构建了基因工程菌pPIC9K-MalQ/GS115。0.8%甲醇进行诱导表达。表达上清液与麦芽糖溶液反应,TLC结果显示：上清液具有糖基转移酶活性。SDS-PAGE电泳显示,上清液在78.5 kDa位置处有明显条带,与麦芽糖转糖基酶的理论分子量相符。葡萄糖氧化酶法测得表达上清液酶活118 U。证实麦芽糖转糖基酶在毕赤酵母中成功表达。确定了该酶的最适反应温度为30,最适反应pH为6.0。(3)基因工程菌pPIC9K-MalQ/GS115的表达上清液与直链淀粉反应,葡萄糖淀粉酶初步纯化,TLC结果显示：有聚合度大于9的一系列环状糊精生成。证实麦芽糖转糖基酶能催化淀粉生成大环糊精。【英文摘要】Amylomaltase is a member of 4-Glucanotransferase family, catalyzing intramolecular transglycosylation of-1,4-glucans to produce cyclic-1,4 glucans (cycloamyloses). Cyclodextrin with the structure of a hydrophobic inner cavity and a hydrophilic out surface that looks like a column. These properties are currently used in numerous applications in the food, pharmaceutical and chemical industries. But its low content in nature resources cannot meet the need of lib research or prodution of cycloamyloses.Gene MalQ was cloned from E.coli BL21(DE3) plysS, constructed expression plasmid pPIC9K-MalQ. The recombinant plasmid bearing MalQ gene was lincarized with Sal I and transformed into Pichia pastoris strain GS115. The positive clones were induced with methanol to express amylomaltase. The crude enzyme was demonstrated having 4-glucanotransferase activity with TLC. The amylomaltase also produced cycloamyloses with amylase. The main results are described as follows:(1) The gene MalQ was amplified from E.coli BL21(DE3) plysS by PCR, then constructed expression plasmid pPIC9K-MalQ. MalQ sequence was analyzed by BLAST and it displayed high similarity(100%) to gene MalQ of E.coli BL21(DE3) plysS reported in the GenBank.(2) The recombinant plasmid bearing MalQ gene was lincarized with Sal I and transformed into Pichia pastoris strain GS115, constructed the recombinant strain pPIC9K-MalQ/GS115. The positive clones were induced with 0.8% methanol to express amylomaltase. TLC showed the the crude enzyme had 4-glucanotransferase activity. SDS-PAGE showed a clear 78.5 kDa band. The enzyme activity which is 118 U. The optimal temperature and pH was 30,6.0, respectively.(3) The crude enzyme was used to treat amylase.The resulting mixture solution was treated by glucoamylase. TLC showed that there were existance of large cycloamylose. These all above indicated that the crude enzyme could catalyzed cyclization of amylose to produce cycloamylose.【关键词】麦芽糖转糖基酶 大环糊精 毕赤酵母 基因重组【英文关键词】Amylomaltase cycloamylose Pichia pastoris gene recombination【目录】麦芽糖转糖基酶在毕赤酵母中的表达及活性分析摘要3-4Abstract4第一章 绪论8-191.1 麦芽糖转糖基酶的研究进展8-91.1.1 麦芽糖转糖基酶的理化性质研究8-91.1.2 麦芽糖转糖基酶的应用研究91.2 环状糊精的研究进展9-121.2.1 环状糊精的理化性质研究9-101.2.2 制备大环糊精的酶10-111.2.3 大环糊精在工业中的的应用11-121.3 毕赤酵母表达系统介绍12-171.3.1 毕赤酵母的生物学性质131.3.2 外源蛋白在毕赤酵母中的表达策略13-161.3.3 影响外源蛋白表达的关键因素16-171.3.4 毕赤酵母表达系统尚存在的问题171.4 研究的背景和意义17-181.5 研究内容18-19第二章 pPIC9K-MalQ重组表达载体的构建19-312.1 材料20-212.1.1 菌株及质粒202.1.2 主要实验试剂与酶202.1.3 主要实验溶液及培养基的配置20-212.1.4 仪器设备212.2 方法21-272.2.1 E.coli BL21(DE3)基因组的提取21-222.2.2 E.coli DH5感受态细胞的制备22-232.2.3 pPIC9K质粒DNA的提取23-242.2.4 引物设计24-252.2.5 麦芽糖转糖基酶基因的PCR扩增252.2.6 PCR产物及质粒的双酶切25-262.2.7 双酶切产物的回收连接262.2.8 连接产物的转化26-272.2.9 阳性克隆的筛选272.2.10 阳性克隆的测序分析272.3 结果与分析27-302.3.1 麦芽糖转糖基酶的PCR扩增27-282.3.2 pPIC9K质粒DNA的提取28-292.3.3 阳性克隆子的酶切验证及测序分析29-302.4 本章小结30-31第三章 麦芽糖转糖基酶在毕赤酵母中的分泌表达31-433.1 材料31-333.1.1 菌种与质粒313.1.2 主要试剂313.1.3 主要溶液及培养基的配置31-333.2 方法33-373.2.1 毕赤酵母GS115菌株的培养33-343.2.2 毕赤酵母GS115感受态细胞的制备343.2.3 重组质粒pPIC9K-MalQ的线性化343.2.4 电击转化入GS115感受态细胞中34-353.2.5 毕赤酵母GS115重组子表型的鉴定及高拷贝菌株的筛选353.2.6 PCR验证毕赤酵母的整合353.2.7 麦芽糖转糖基酶的诱导表达35-363.2.8 表达产物的转糖基活性分析363.2.9 表达产物酶活的测定363.2.10 表达产物最适反应温度的探究363.2.11 表达产物对温度稳定性分析36-373.2.12 表达产物最适反应pH值的探究373.2.13 表达产物对pH稳定性分析373.3 结果与分析37-423.3.1 PCR验证毕赤酵母GS115的电击转化37-383.3.2 麦芽糖转糖基酶的诱导表达383.3.3 表达产物的活性分析及酶活测定38-393.3.4 表达产物最适反应温度的确定及对温度的稳定性分析39-413.3.5 表达产物最适反应pH的确定及对pH的稳定性分析41-423.4 本章小结42-43第四章 表达产物的应用研究43-484.1 材料444.1.1 菌株444.1.