【作者】 苏靖；

【导师】 张智武；

【作者基本信息】 内蒙古农业大学 ， 食品科学， 2010， 硕士

【摘要】 微生物中的各种酶类具有巨大的潜在应用价值。它的开发和利用,对于提高人们生活质量有重要意义。对于这些蛋白酶的理化性质、生物活性和催化机理等方面的基础研究有助于发现它更广阔的应用前景。如今,酶类在食品发酵工业生产,生物制剂等方面已得到广泛的应用。本文借助结构生物学中晶体学的方法,通过获悉蛋白酶的空间结构来推测酶的功能,探讨催化机理。研究中以激烈火球菌(Pyrococcus furiosus)中潜在的参与脂肪酸合成酶类PFS(PF2046)和脆弱拟杆菌(Bacteroides.fragilis 9343)中的糖合成酶FKP为研究对象,将其基因构建至原核表达载体pMCSG7中,使用大肠杆菌E.coli BL21(DE3)大量表达出带有His标签的可溶性融合蛋白,经镍离子柱亲和层析,离子交换及分子筛层析纯化后,得到纯度达95%以上蛋白。使用商品化试剂盒进行晶体生长条件筛选,解析了PFS的三维结构,并对其功能进行初步研究。同时得到了FKP蛋白的微晶,经过结晶条件优化获得分辨率3.5 ?的晶体,为FKP蛋白的结构解析奠定基础。对这两个酶的性质和结构的认识,为深度利用这两种酶开发食品制剂或在食品及相关工业上的广泛应用提供详了理论切入点。更多还原

【Abstract】 Many microbiology enzymes have great potential in industrial application and have played very important roles in human life. Studies on the Physiochemical Characteristics、biological activity and catalytic mechanism of enzymes are important for their possible wider applications. Nowadays, enzymes have been commonly applied in food fermentation and biomedical regent development.In this thesis, two enzymes were selected to probe the crystal structures in order to provide clue to understand their function and catalytic mechanism, including a putative fatty acid synthetase PFS (PF2046) from Pyrococcus furio sus and a pyrophosphorylase FKP from Bacteroides.fragilis 9343. Both of these genes were cloned into the expression vector pMCSG7 and used E.coli BL21 (DE3) as a host for large-scale protein expression. The bacteria were broken and the soluble portion was purified by nickel affinity column, followed by ion-exchange and size-exclusion chromatography. The purity of the two enzymes was above 95%. Protein crystallization was performed by using commercial screening kits. PFS’s structure was solved through the B-factor sharpening method when protein crystal was big enough for data collection. FKP’s small crystal was diffracted to 3.5 ? and was very close to structure solving. Structure and biochemistry characterization of these two enzymes will provide people more information to develop new food additives or their extensive application in food and related industry.更多还原