【作者】 张莹；

【导师】 张会图；

【作者基本信息】 天津科技大学 ， 微生物与生化药学（理）， 2018， 硕士

【摘要】 脂肪酶在不同的体系中可以催化脂类物质水解和转酯等反应的发生,并被广泛应用于食品、洗涤及饲料等生产行业。来源于耶氏解脂酵母的脂肪酶LipY2除了具有催化活性高、生产工艺稳定等优点,还有强的耐酸性,因此能够适应人体消化系统内的酸性环境,适合应用到食品、医疗等行业当中。但野生型LipY2耐热性不强,致使其生产及运输的成本较高,使用范围也受到了一定的限制。因此,本研究首先利用分子动力学模拟研究了温度对LipY2结构的影响,解析其热不稳定的分子机制。然后进一步理性设计耐热的脂肪酶并利用系统的实验证明这些突变体的热稳定性,以期获得热稳定性高的LipY2变异体。本研究利用分子动力学模拟的方法在分析了不同温度下脂肪酶LipY2的结构变化情况,发现位于蛋白质表面的Ala111-His126和Gly207-Lys221两个区域受温度影响较大。进一步对该区域进行理论与折叠熵的计算,确定了五个耐热的突变体,分别命名为S115P、T117I、G207P、V213P和K215G。利用分子动力学模拟分析突变体在不同温度下的构象变化情况,结果表明这五个突变体的稳定性与野生型LipY2相比都有所提高。借助定点突变获取了突变体的目的基因,在毕赤酵母GS115中实现表达和蛋白纯化后,对各突变体的酶学性质进行系统研究。结果表明,位于Gly207-Lys221区域的三个突变体G207P、V213P和K215G的热稳定性有所提高,在50℃保温100 min,剩余酶活分别是野生型的1.25倍、1.6倍和1.26倍,且V213P突变体的最适温度由35℃提高至40℃,其它突变体的最适温度保持不变。处于Ala111-His126区域的两个突变体温度稳定性没有提高,但S115P突变体的比酶活较野生型LipY2相比,从1047.51 U/mg提高至1279.50 U/mg。这些研究结果进一步说明利用分子动力学模拟设计突变体的方法在改善酶学性质方面具有指导作用。更多还原

【Abstract】 Lipase can catalyze the hydrolysis of lipids and transesterification in different systems,and is widely used in food,washing and feed industries.The LipY2 derived from Yarrowia lipolytica has the advantages of high catalytic activity,stable production process,and strong acid resistance,so it can adapt to the acidic environment in the human digestive system and is suitable for applications in the food and medical industries.However,the heat-resistance of wild-type LipY2 is not strong,resulting in high production and transportation costs,and the scope of use is also limited.Therefore,this study first used molecular dynamics simulations to study the effect of temperature on the structure of LipY2,and to resolve the thermal instability of the molecular mechanism.Then further rational design of heat-resistant lipases and the use of systematic experiments to prove the thermal stability of these mutants,in order to obtain LipY2 variants with a high thermal stability.In this study,molecular dynamics simulation method was used to analyze the structural changes of LipY2 at different temperatures.It was found that the two regions Alalll-His126 and Gly207-Lys221 located on the surface of the protein were greatly influenced by temperature.Then calculate the theoretical and fold entropy of the region,and on this basis,determine five heat-resistant mutants,named S115P,T117I,G207P,V213P,and K215G.Then,molecular dynamics simulations were performed to analyze the conformational transitions of these mutants at different temperatures.The results showed that the stability of the five mutants was improved compared to wild-type LipY2.The target genes of the mutants were obtained by site-directed mutagenesis.After the expression and protein purification in Pichia pastoris GS115,the enzymatic properties of each mutant were systematically studied.The results showed that the thermostability of the three mutants G207P,V213P and K215G in the Gly207-Lys221 region was increased.After incubation at 50℃ for 100 min,the remaining enzyme activities were 1.25,1.6 and 1.26 times that of the wild type,respectively.The optimum temperature of V213P mutant was increased from 35℃ to 40℃.The temperature stability of the two mutants in the Ala111-Hisl26 region did not increase,but the specific enzyme activity of the S115P mutant increased from 1047.51 U/mg to 1279.50 U/mg compared to the wild-type LipY2.These findings further demonstrate that the use of molecular dynamics simulations to design mutants has a guiding role in improving enzymatic properties.更多还原