提升枯草桿菌脂肪酶的催化活性與改變其受質特異性

Abstract

Bacillus subtilis脂肪酶(B. subtilis lipase, BSL)已經被應用在食品、洗滌劑、皮革和廢油處理等方面。成熟具活性的BSL (mature BSL, mBSL)蛋白分子是由181個胺基酸組成，是目前已知分子量最小的脂肪酶，在pH 10具有最佳活性，屬於嗜鹼性脂肪酶，但最適作用溫度為35℃，耐熱性較差，在受質特異性方面，則對含短、中碳鏈脂肪酸受質有較高的催化活性。另一種嗜高溫古生菌Archaeoglobus fulgidus脂肪酶(AFL)具有嗜熱與嗜鹼的特性，最適作用條件分別為，pH值為10.0、溫度為90℃。BSL與AFL的X-ray結晶立體結構已經被解出，AFL的 C-端區域具獨特的功能，對於長碳鏈受質的結合及穩定酵素結構扮演重要角色，而mBSL與AFL的N-端區域具有相似結構。為了提升mBSL的耐熱特性，並改變其受質特異性，本研究藉由overlap PCR將mBSL與AFL的C-端區域基因融合，以獲得融合酵素mBSL-cAFL，同時亦配合電腦模擬針對融合酵素進行蛋白質工程。重組融合酵素於E. coli中表達和純化，經過性質分析後，發現突變株 mBSL-cAFL(181+239)在高溫下(50℃)對於短碳鏈脂肪酸受質的水解活性，較mBSL提升約57%，顯示AFL C-端區域具有提升mBSL熱穩定性的作用；而修改mBSL與AFL的C-端區域間的連結區結構[mBSL-cAFL(175+245) and mBSL-cAFL(175+linker+245)]，以及針對脂肪酸結合位所作的修飾[mBSL-cAFL(175+245)/F373A]，並無法提升對中、長碳鏈脂肪酸受質的水解活性；針對催化三元體中的Asp133所製作的突變株mBSL-cAFL(175+245)/D133A和mBSL-cAFL(175+245)/D133E，顯示融合酵素在高溫下可能是以具有催化二元體的形式來進行催化作用。為了擴展BSL的工業用途，需要進行更多的蛋白質工程，以便進一步提升其熱穩定性、與改變其受質特異性成為適合催化長碳鏈脂肪酸受質的脂肪酶。

關鍵字：枯草桿菌脂肪酶、Archaeoglobus fulgidus 脂肪酶

B. subtilis lipase (BSL) has been used in the fields of food, laundry, leather, and waste oil processing, etc. Mature form of BSL (mBSL), as the smallest lipase, is composed of 181 amino acid residues. It is an alkalophilic lipase with an optimal pH of 10. However, it is not thermostable with an optimal temperature of 35℃, and only shows higher activity on glycerol ester substrates with short to medium chain-length fatty acids. Lipase from Archaeoglobus fulgidus (AFL) has been proved to be a hyperthermophilic and alkalophilic enzyme with a optimal pH and temperature of 10.0 and 90℃, respectively. The three-dimensional structures of BSL and AFL have been resolved by X-ray crystallography. The unique C-terminal domain of AFL plays an important role in long-chain substrate binding and conferring protein stability. The structure of the mBSL exhibits high similarity to the N-terminal domain of AFL. To improve the thermostability and alter the substrate specificity of mBSL, in the present study we constructed fusion genes by overlap PCR to obtain fusion enzymes which are composed of the mBSL and the C-terminal domain of AFL. The fusion enzmes were also engineered in according to the information of computer modeling. The recombinant fusion enzymes were expressed and purified in E. coli. After characteristic analysis, the mBSL-cAFL(181+239) mutant increased 57% of activity toward short-chain fatty acid ester under higher temperature (50℃) when compared to mBSL. It revealed that the intramolecular interaction of the C-domain of AFL with the BSL may improve the thermostability of BSL. Changes of the linker structure between the mBSL and the C-terminal domain of AFL [mBSL-cAFL(175+245) and mBSL-cAFL(175+linker+245)] and modification of the substrate binding site [mBSL-cAFL(175+245)/F373A] did not improve the activities toward medium and long-chain fatty acid esters. Site-directed mutagenesis of the Asp133 of the catalytic triad [mBSL-cAFL(175+245)/D133A and mBSL-cAFL(175+245)/D133E] revealed that the fusion enzyme may act as a diad-containing lipase under higher temperature. To expand the industrial applications of BSL, more protein engineerings need to be done to improve its thermostability and alter the substrate specificity of BSL into preferrence for the substrates containing long-chain fatty acids.

Key word：Bacillus subtilis Lipase、Archaeoglobus fulgidus Lipase