重組嗜熱嗜鹼性 Archaeoglobus fulgidus 脂肪酶之結構與功能分析

Structure and Function of a Recombinant Thermoalkalophilic Lipase from Archaeoglobus fulgidus

劉曉蓉(Hsiao-Jung Liu)

指導教授 ： 李冠群

國立臺灣師範大學/理學院/生命科學研究所/碩士(2009年)

摘要

從嗜高溫古生菌Archaeoglobus fulgidus 基因體所選殖的新型脂肪酶(lipase) 基因AFL 已經成功地在大腸桿菌中被表達，並且以X-ray 結晶繞射的方法解出立體結構。Archaeoglobus fulgidus lipase(AFL) 為嗜熱性與嗜鹼性的脂肪酶，由474 個胺基酸組成，包含18個胺基酸的訊息胜肽以及456 個胺基酸的mature lipase (mAFL)，其結構上分為N-端以及C-端，活性中心位於N-端，C-端則含有受質脂肪酸官能基結合部位。本研究主要藉由蛋白質工程和生化分析，配合蛋白質立體結構的觀察，探討AFL 之結構與功能的關係，並找出對脂肪酶活性有影響的胺基酸。在AFL 的立體結構中，發現在兩個功能區域交界之處有許多的ion pairs，其中一對(K184、D370 及E372)以基因定位突變的方式破壞正負電荷引力之後，發現突變種K184A 、D370N 與E372Q 之活性顯著下降，分析K184A 後發現，最適反應溫度由野生種的70~90℃ 改變為僅侷限於90℃，顯示ion pair在兩個區域的交互作用扮演重要的角色。在脂肪酶與受質的結合部位中，推測對受質結合有影響的三個胺基酸：A32，S332 與E339 ，由基因定位突變後，突變種A32W 的受質專一性和野生種的長碳鏈相比，改變為中碳鏈之酯類，並且對中短鏈長的脂肪酸活性比野生種XIII高；而突變種S332W 與E339W 的活性皆低於野生種。在進行X-ray 結晶時觀察到鈣或鎂離子結合於C- 端區域的 D405、D409與D431 上，經由酵素動力學實驗及熱穩定性實驗發現，鈣離子的結合在高溫反應下，有助於AFL 與受質的親和力，並在90℃時穩定酵素結構，鎂離子則沒有幫助，證明鈣離子對於脂肪酶在高溫下有提升穩定性的影響。以三酸甘油脂為受質，分析AFL 的活性後發現有介面活化作用產生，亦即當三酸甘油質達到不溶於水的濃度時，AFL 的活性急遽上升，因此證明了AFL 確實為一脂肪酶而非酯酶，其結構上的口蓋（lid）可能會隨著油水介面的產生而開啟。由鈣離子結合部位的突變種分析，以酵素動力學和熱穩定性的實驗證明D405、D409 與D431 為鈣離子結合部位。以上結果闡述了此嗜熱、嗜鹼的脂肪酶特性以及催化機制和結構上的關係，並提供未來改造AFL 的線索，以因應在生物技術上的應用。

關鍵字

Archaeoglobus fulgidus ； 脂肪酶 ； 嗜熱嗜鹼性 ； 鈣離子 ； 熱穩定性

並列摘要

A novel lipase gene (AFL) from Archaeoglobus fulgidus has been previously cloned and functionally expressed in E. coli. AFL is a thermoalkalophilic lipase. Its three-dimensional structure has been already resolved by X-ray crystography. AFL is composed of 474 amino acid residues with a N-terminal signal peptide (18 amini acid residues) and mature lipase gene (456 amino acid). The N-terminal of AFL contains the catalytic triad and the substrate binding site (tunnel) is located at the C-erminal domain. According to the informations of three-dimensional structure, protein engineering and biochemical characters assay were performed to investigate the structure-function relationship of AFL. In the N- and C-terminal domain interface, K184, D370 and E372 form a electrostatic interaction network to stabilize these two domains. The activities of the mutants K184A, D370N and E372Q dromatically decreased. The optimal temperatur of mutant K184A became narrowly at 90℃ as compared with the broad range one (70-90℃) of wild-type AFL. A32, S332 and E339 located in the substrate binding tunnel were predicted to be involved in the substrate specificity of AFL. Through site-directed mutagenesis and activity assay the substrate specificity of the mutant A32W changed to favor the hydrolysis of middle chain-length esters as compared with the long-chain specificity of wild-type AFL, while the activities of S332W and E339W were lower than wild-type AFL. In the C-terminal domain, there is a putative divalent cation binding site composed of D405, D409 and D431. From kinetics assay, thermostabilty assay and thermo-dynamic assay, the binding of calcium benefited the substrate affinity of AFL at high temperature, and also enhanced the thermostability at the 90℃. On the other hand, magnesium did not affect the substrate affinity and stability of AFL. Through interfacial activation assay using triglycerides as substrate, AFL is proved to be a true lipase rather than a carboxylesterase. A drastic increase in lipase activity occurred when the solubility limit of tricaprylin was exceeded. This means that the apparent rate of hydrolysis correlate with the degree of micellar formation. Consistent with observations on other lipases, the lid conformation may change from closed to open form in the presence of lipid interface. Site-directed mutagenesis assays were performed to identify D405, D409 and D431 which form the cation binding site. These results revealed the structural basis of the thermoalkalophilic characteristics and the catalytic mechanism of AFL, and provides important clues for the engineering of AFL in biotechnological applications.

並列關鍵字

Archaeoglobus fulgidus ； lipase ； thermoalkalophilic ； calcium ； thermostability

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