枯草杆菌蛋白酶嗜热性改造效果评判方法研究

发布时间：2018-06-24 03:50

本文选题：枯草杆菌蛋白酶 + 热稳定性改造　；参考：《北京工业大学》2014年硕士论文

【摘要】：对蛋白酶进行热稳定性改造是近年来的研究热点，具有十分重要的理论意义和实际价值。一方面，蛋白酶热稳定性改造工作可以帮助人们更加深入地探讨蛋白质的热稳定性机理，对于蛋白质的结构、功能等方面会有更系统、全面的认识；另一方面，由于天然蛋白酶对温度的耐受性低，严重制约了其在工业生产中的应用，使其催化效率高、对底物的立体选择性等优点不能很好地得到发挥，因此迫切需要好的嗜热性改造方法来指导蛋白酶热稳定性的改造，促进和发展蛋白酶在实际生活中的应用。目前对于嗜热改造效果的判定，主要是利用实验加温测定的方法，该方法能够很好地判断蛋白酶的热稳定性是否得到提高，但该方法耗时耗力，因此需要找到更简便、更快捷的方法去判定蛋白嗜热性改造的效果，这对蛋白热稳定性改造工作具有十分重要的促进作用。本文以枯草杆菌蛋白酶（subtilisin BPN'）为研究对象，以探索评判蛋白酶嗜热突变效果的方法为研究目的，选取了有可靠实验资料的6个突变点，运用分子动力学模拟方法，在四种不同模拟条件下，对这6个突变体与1个野生型蛋白进行了多种参量的对比分析，提取5个有效参数（ΔBOND、ΔELECT、ΔANGLE、ΔVDW、ΔDIHED），进一步确定了前4个参数为特征有效参数，建立了枯草杆菌蛋白酶嗜热性改造效果的评判方法；利用该方法对枯草杆菌蛋白酶的其他6个突变体以及色氨酸合成酶α亚基（Tryptophan synthase alpha-subunit）的嗜热突变体效果进行评判，评判结果与实验资料完全吻合，证明该方法可用于蛋白酶嗜热性改造突变效果的评判；最后将该方法运用到枯草杆菌蛋白酶的嗜热性改造实际工作中，对5个随机突变体进行嗜热改造效果的判定，判定结果证明这5个突变体为有效突变。利用分子动力学模拟对嗜热改造效果进行评判的方法，可以较好地判别出嗜热改造效果的好坏，，并且操作简便、易于实现，对今后指导蛋白酶进行有效突变、热稳定性改造有重要意义。
[Abstract]:The thermal stability modification of protease is a hot topic in recent years, which has very important theoretical and practical value. On the one hand, protease thermal stability modification can help people to explore the mechanism of protein thermal stability more deeply, and have a more systematic and comprehensive understanding of the structure and function of protein; on the other hand, Because of the low tolerance of natural protease to temperature, its application in industrial production is seriously restricted, and its catalytic efficiency is high, and its stereoselectivity to substrate can not be well developed. Therefore, a good thermophilic modification method is urgently needed to guide the transformation of protease thermal stability, and to promote and develop the application of protease in real life. At present, the determination of the effect of thermophilic transformation is mainly determined by the method of experimental heating measurement, which can well judge whether the thermal stability of protease has been improved or not, but the method is time-consuming and labor-consuming, so it needs to be found more conveniently. A more rapid method to determine the effect of protein thermophilic transformation, which has a very important role in improving the thermal stability of protein. In this paper, taking subtilisin BPN'as the research object and exploring the method to evaluate the effect of protease thermophilic mutation, six mutation sites with reliable experimental data were selected and the molecular dynamics simulation method was used. Under four different simulation conditions, the six mutants and one wild-type protein were compared and analyzed. Five effective parameters (螖 BOND, 螖 ELECT, 螖 Angle, 螖 VDW, 螖 DIHED) were extracted, and the first four parameters were determined as characteristic effective parameters. A method for evaluating the effect of thermophilic modification of Bacillus subtilis protease was established, and the effect of the other 6 mutants of Bacillus subtilis protease and the thermophilic mutants of tryptophan synthase alpha-subunit were evaluated. It is proved that this method can be used to evaluate the mutation effect of protease thermophilic modification. Finally, the method is applied to the practical work of thermophilic modification of Bacillus subtilis protease. The effect of thermophilic transformation on five random mutants was determined and the results showed that the five mutants were effective mutations. The method of evaluating the effect of thermophilic transformation by molecular dynamics simulation can be used to judge the effect of thermophilic transformation, and it is easy to operate and realize. Thermal stability modification is of great significance.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R378;R3411

【参考文献】