運用基因工程嵌入伴護蛋白功能區塊以建構新型的角蛋白酶

Engineering novel keratinases through an intramolecular chaperone swapping approach

10.6342/NTU.2014.01573

黃婷資

角蛋白酶 ； 類枯草桿菌蛋白 ； Aqualysin ； 內分子伴護蛋白酶 ； 功能區置換 ； keratinase ； subtilisin-like protease ； aqualysin ； intramolecular chaperone ； domain swapping

臺灣大學生化科學研究所學位論文

2014年

碩士

吳世雄

英文

角蛋白酶是一種蛋白質水解酵素，他能降解結構非常堅硬且一般酵素無法分解的的角蛋白。近年來，這類角蛋白酶已漸漸受到大眾的重視，無論是在環境保護或是生物技術應用方面，都具有很高的應用價值。然而，雖然目前已有很多角蛋白酶被發現，但其複雜的水解機制仍然存留許多不解。 先前，我們成功地從台灣本土嗜熱菌分離出一個新的角蛋白酶，從結構比對結果，我們發現一個與之結構極為相似的蛋白質，其命名為aqualysin，此蛋白酶來自另一株更耐高溫的嗜熱菌Thermus aquaticus，卻從未有相關文獻指出其具有角蛋白酶的活性；因此，我們利用偶氮角蛋白 (azokeratin) 當作水解受質，證實aqualysin確實具有高活性的角蛋白水解功能，並測得其活性最佳條件。此外，藉由分析具有不同功能性片段的aqualysin，我們發現此蛋白酶必須在有N端內分子伴護蛋白區塊 (intramolecular chaperone, NAQ) 的存在下，才能將蛋白適當摺疊成具有活性的構型。為了進一步證明NAQ是否具有角蛋白水解功能的記憶，並引導蛋白酶摺疊成具有角蛋白酶活性的構型，我們將NAQ置換到來自台灣本土嗜熱菌，其不具角蛋白酶活性的類枯草桿菌蛋白SPR2216之中。幸運地，SPR2216在嵌入NAQ之後，成功的獲得了原本並不具備的角蛋白酶活性；而這也證明了NAQ 對於幫助蛋白酶區塊獲得角蛋白水解功能記憶的過程中，扮演了非常重要的角色。因此，運用此基因工程的方法，將能提供有效的資訊來建構出新型且具有不同特性的角蛋白酶。至於NAQ是如何幫助蛋白酶摺疊，以及更細節的分子機制，還需要更進一步的研究探討。

Keratinases are proteolytic enzymes responsible for the degradation of highly recalcitrant keratin substrates. They have gradually captured the attention in the realms of environmental and biotechnological application. Although these enzymes have widely been discovered, the complex mechanism of keratinolysis has not yet been well-understood. Previously, we successfully identified a novel keratinase from Meiothermus taiwanensis. Based on the structural similarity, we found a subtilisin-like protease from Thermus aquaticus named aqualysin, which has not yet been reported as a keratinase. Based on azokeratin assays, we confirmed that aqualysin is a highly efficient keratinase. Furthermore, the results from the truncate studies suggested that propeptide of aqualysin (NAQ), an intramolecular chaperone, was an indispensable domain for the keratinolytic activity. To verify whether NAQ possessed the protein memory for keratinolysis and guided protease to fold as a keratinase, we swapped NAQ to a subtilisin-like protease, SPR2216 from Meiothermus taiwanensis, which possessed no keratinolytic activity. Fortunately, we successfully obtained NAQ-fused SPR2216 which possessed gain-of-function for keratinolysis. Importantly, we demonstrated the significant role of NAQ for protease domains to acquire the memory of “keratinase-fold”. These findings would be very useful for generating new keratinases with additional characteristics. However, more studies should be made to further elucidate the detailed molecular mechanisms of how NAQ helped protease domain fold.

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