麦芽糖转糖基酶论文：麦芽糖转糖基酶在毕赤酵母中表达及活性分析.docVIP

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-α-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SalⅠ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-α-Glucanotransferase family, catalyzing intramolecular transglycosylation ofα-1,4-glucans to produce cyclicα-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-α-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 s