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  本文選題：枯草桿菌蛋白酶 + 熱穩(wěn)定性改造��； 參考：《北京工業(yè)大學(xué)》2014年碩士論文

【摘要】：對蛋白酶進(jìn)行熱穩(wěn)定性改造是近年來的研究熱點(diǎn)，具有十分重要的理論意義和實(shí)際價值。一方面，蛋白酶熱穩(wěn)定性改造工作可以幫助人們更加深入地探討蛋白質(zhì)的熱穩(wěn)定性機(jī)理，對于蛋白質(zhì)的結(jié)構(gòu)、功能等方面會有更系統(tǒng)、全面的認(rèn)識；另一方面，由于天然蛋白酶對溫度的耐受性低，嚴(yán)重制約了其在工業(yè)生產(chǎn)中的應(yīng)用，使其催化效率高、對底物的立體選擇性等優(yōu)點(diǎn)不能很好地得到發(fā)揮，因此迫切需要好的嗜熱性改造方法來指導(dǎo)蛋白酶熱穩(wěn)定性的改造，促進(jìn)和發(fā)展蛋白酶在實(shí)際生活中的應(yīng)用。 目前對于嗜熱改造效果的判定，主要是利用實(shí)驗(yàn)加溫測定的方法，該方法能夠很好地判斷蛋白酶的熱穩(wěn)定性是否得到提高，但該方法耗時耗力，因此需要找到更簡便、更快捷的方法去判定蛋白嗜熱性改造的效果，這對蛋白熱穩(wěn)定性改造工作具有十分重要的促進(jìn)作用。 本文以枯草桿菌蛋白酶（subtilisin BPN'）為研究對象，以探索評判蛋白酶嗜熱突變效果的方法為研究目的，選取了有可靠實(shí)驗(yàn)資料的6個突變點(diǎn)，運(yùn)用分子動力學(xué)模擬方法，在四種不同模擬條件下，對這6個突變體與1個野生型蛋白進(jìn)行了多種參量的對比分析，提取5個有效參數(shù)（ΔBOND、ΔELECT、ΔANGLE、ΔVDW、ΔDIHED），進(jìn)一步確定了前4個參數(shù)為特征有效參數(shù)，建立了枯草桿菌蛋白酶嗜熱性改造效果的評判方法；利用該方法對枯草桿菌蛋白酶的其他6個突變體以及色氨酸合成酶α亞基（Tryptophan synthase alpha-subunit）的嗜熱突變體效果進(jìn)行評判，評判結(jié)果與實(shí)驗(yàn)資料完全吻合，證明該方法可用于蛋白酶嗜熱性改造突變效果的評判；最后將該方法運(yùn)用到枯草桿菌蛋白酶的嗜熱性改造實(shí)際工作中，對5個隨機(jī)突變體進(jìn)行嗜熱改造效果的判定，判定結(jié)果證明這5個突變體為有效突變。 利用分子動力學(xué)模擬對嗜熱改造效果進(jìn)行評判的方法，可以較好地判別出嗜熱改造效果的好壞，，并且操作簡便、易于實(shí)現(xiàn)，對今后指導(dǎo)蛋白酶進(jìn)行有效突變、熱穩(wěn)定性改造有重要意義。
[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.
【學(xué)位授予單位】：北京工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R378;R3411

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本文編號：2059881