本文選題：華根霉脂肪酶 + 理性設(shè)計　； 參考：《江南大學(xué)》2014年碩士論文

【摘要】：脂肪酶是繼蛋白酶和淀粉酶之后的第三大酶制劑，而根霉脂肪酶作為微生物脂肪酶的一種重要來源，因其高效的催化效率等特點，已有多種根霉脂肪酶制成商品化酶制劑，在食品、醫(yī)藥、化工及環(huán)保等多個行業(yè)表現(xiàn)出廣闊的應(yīng)用前景。華根霉脂肪酶是本實驗室具有自主知識產(chǎn)權(quán)的脂肪酶，前期在酶的穩(wěn)定性和外源表達(dá)量等方面已經(jīng)取得較好的研究成果，擴(kuò)大了其工業(yè)應(yīng)用范圍。 為了獲得耐熱性更高的華根霉脂肪酶，，同時研究其蛋白結(jié)構(gòu)與功能的關(guān)系，本文一方面通過理性設(shè)計的思路，運用定點突變方法提高華根霉脂肪酶的耐熱性；另一方面在畢赤酵母表達(dá)系統(tǒng)中研究了華根霉脂肪酶前肽對脂肪酶折疊和分泌的影響，主要研究內(nèi)容包括： （1）獲得了耐熱性提高的突變酶。通過同源建模和序列比較，運用重疊延伸PCR方法對華根霉脂肪酶基因proRCL進(jìn)行定點突變，構(gòu)建了突變脂肪酶D190V。畢赤酵母表達(dá)的突變酶D190V與野生型酶r27RCL相比，D190V的最適溫度比r27RCL高5℃，65℃下的半衰期提高了一倍，Km值下降了23.3%。通過結(jié)構(gòu)分析，定點突變D190V提高該酶穩(wěn)定性的主要原因可能在于提高了突變位點所在的α螺旋的穩(wěn)定性以及增強(qiáng)了穩(wěn)定蛋白質(zhì)結(jié)構(gòu)的氫鍵作用力。 （2）考察了脂肪酶前肽對脂肪酶體外折疊的輔助作用，比較了全長前肽的脂肪酶proRCLCNQ、攜帶截短前肽的脂肪酶r27RCLC和不含前肽的成熟肽脂肪酶mRCLC在28℃、4種GSH和GSSG濃度下的體外復(fù)性差異。不添加GSH和GSSG時，變性的脂肪酶可以自發(fā)折疊成活性的酶蛋白，而前肽有助于該過程的進(jìn)行；當(dāng)GSH/GSSG濃度比從0提高到2:1，復(fù)性6h的數(shù)據(jù)顯示，proRCLCNQ、r27RCLC和mRCLC的復(fù)性率分別由13.6%、23.7%、8.7%提高至19.7%、74.5%、40.9%。復(fù)性率提高幅度由高到低順序為：r27RCLC mRCLC proRCLCNQ，說明在不同氧化還原劑濃度下，r27RCLC上的截短前肽可以促進(jìn)脂肪酶的自發(fā)折疊，而在較高還原劑濃度下proRCLCNQ上的全長前肽未能促進(jìn)酶的折疊。前肽N端的1-67氨基酸對脂肪酶的折疊不是必需的，而68-94位氨基酸則對酶的折疊有明顯的促進(jìn)作用。 （3）考察了前肽序列對脂肪酶表達(dá)分泌的影響，比較了不同截短前肽序列的proRCLC基因在畢赤酵母中表達(dá)分泌差異。華根霉脂肪酶前肽N端的1-16位氨基酸對酶蛋白的表達(dá)分泌不是必需的，截去后反而提高了目標(biāo)蛋白的表達(dá)和分泌。截去1-67位氨基酸，脂肪酶不能表達(dá)。截去1-35或者1-47位氨基酸的結(jié)果相似，不僅影響細(xì)胞的生長、降低目標(biāo)蛋白的表達(dá)量和胞外的分泌，還造成胞外的雜蛋白量明顯增多。
[Abstract]:Lipase is the third major enzyme preparation after protease and amylase. As an important source of microbial lipase, Rhizopus lipase has been made into commercial enzyme preparation due to its high catalytic efficiency. In food, medicine, chemical and environmental protection and other industries show broad application prospects. Rhizopus chinensis lipase is a lipase with independent intellectual property rights in our laboratory. Good research results have been obtained in the field of enzyme stability and exogenous expression in the early stage, which has expanded its industrial application scope. In order to obtain Rhizopus chinensis lipase with higher heat resistance and to study the relationship between its protein structure and function, on the one hand, the heat resistance of Rhizopus chinensis lipase was improved by site-directed mutagenesis through rational design. On the other hand, the effects of lipase prepeptide of Rhizopus chinensis on lipase folding and secretion were studied in Pichia pastoris. A mutant enzyme with improved heat resistance was obtained. By homologous modeling and sequence comparison, the lipase gene proRCL of Rhizopus chinensis was mutated by overlapping extended PCR method, and the mutant lipase D190V was constructed. The optimum temperature of the mutant D190V expressed by Pichia pastoris was more than that of the wild type enzyme r27RCL. The optimum temperature of D190V was 5 鈩

本文編號：1802629