【作者】 吴吟妮；

【导师】 黄建忠；

【作者基本信息】 福建师范大学 ， 生化与分子生物学， 2005， 硕士

【摘要】 脂肪酶是一类广泛 应用于洗涤剂、医药、食品和皮革等方面的重要工业用酶,它普遍存在丁动植物各种组织及许多微生物中。脂肪酶是最早被研究的酶类之一,人们对它的研究己有上百年的历史。 我国脂肪酶的研究只有40多年历史,20世纪60年代末才有报道。1967年,中国科学院微生物研究所筛选到一株解枝假丝酵母(candida,lipolytica)AS2.1203。20世纪70年代末我国开始碱性脂肪酶的研究,施巧琴教授于1981年在国内成功筛选了碱性脂肪酶生产菌(Penicillium expansum),并且于1984年首次在江苏南通投入生产。进入90年代,我国科技工作者紧跟国际研究热点,脂肪酶的研究进入快速发展时期,这些都促进和加快了我国脂肪酶领域的研究工作。 扩展青霉FSl884碱性脂酶是多代筛选后得到的更优良的产酶菌株,本文对该碱性脂肪酶进行了表征、酶学特性、稳定性及结晶条件的研究,为研究该酶的催化特性及应用等方面提供了依据: 该碱性脂肪酶N-端氨基酸序列测定的结果是:ATADAAAFPDLHRAAKLSs A,等电点为5.44,用质谱分析得到该酶的肽质纹图谱,在图谱中该酶为双电荷峰,分子量为261380.0922Da,并且不含糖基。赖氨酸氨基,组氨酸咪唑基及二硫键是它的活性必需基团,而巯基是维持酶的特定结构的重要基团,该结构与碱性脂肪酶活力有一定关系。 该酶的最适作用pH为9.4,最适作用温度为36℃,在pH7～ll与20℃以下相对稳定:二价离子,特别是Ca2~+对该酶活力有促进作用;以橄榄油为底物时的K_m为0.035g/mL,对硝基苯棕榈酸为底物时的K_m为60.6lμg/mL。羧酸钠,单糖,双糖,多元醇对该碱性脂肪酶都能起到一定的稳定效果,尤其是单糖,对该碱性脂肪酶的热稳定性具有显著作用。 用吐温-80使在乳化液配制的SDS凝胶中电泳后的脂肪酶复性,在胶上出现透明圈,该方法有利于快速检测混合物中是否含有脂肪酶而且能直接判断出所含脂肪酶的大约分子量。 利用结晶试剂盒对该碱性脂酶进行结晶研究,对有结品析出的结晶条件细化后重新培养晶体,得到品形比较规则的晶体。更多还原

【Abstract】 Penicillium expansion PF898 is a mutant strain obtained by many generation mutagenesis, can produce an alkaline lipase at a high level. Both for broadening the industrial applicability and for furthering understanding of the relationship between structure and function, we have applied protein engineering to improve the characterise of the Penicillum expansum lipase （PEL）.Mutants of D92P、 G98A、 N148T、 E113V、 P163G、 P163L、 P163H、 P163W、 P163F、 P163R of PEL gene were obtained by in vitro site-directed mutagenesis using overlap extension PCR. The recombinant plasmid PEL-pPIC3.5K containing mutant genes were cloned into Escherichia coli TG1 strain and expressed in Pichia pastoris GSl 15 strain. The comparison results of the muatants with wild-type PEL showed that:（1） The optimum temperature for PEL-D92P-GS was higher by 5℃ than that of the wild type PEL-GS, while the thermostability of the mutant was similar to the wild type, the substitution of Pro for Asp at position 92 might introduce a pyrrolidine ring at a loop, and an increase of the frequency of proline occurrence at loop could make the protein structure more rigid, thus enhanced the optimum temperature of PEL.（2） Tribulyrin was hydrolysed by PEL-G98A-GS, but nearly no activity was detected towards olive oil. It showed that the 98^th Gly was essential for the lipase activity towards olive oil.（3） The catalytic activity of PEL-N148T-GS was 109.03 U mg^-1 at 40℃, which was 115% that of the wild type at the same conditions. The optimum temperature of mutant was the same to the wild type while it is sensitive to temperature.（4） The optimum temperature of the mutant PEL-E113V-GS （45℃） was higher by 5℃ than that of the wild type PEL-GS, while the thermostability of the mutant was some decreased to the wild type.（5）SDS-PAGE detection showed that the expression product PEL-P163G-GS、 PEL-P163L-GS、 PEL-P163H-GS、 PEL-P163W-GS、 PEL-P163F-GS、 PEL-P163R-GS were different from PEL-GS, and their yield decreased dramatically.更多还原