醣苷水解酵素家族的反應機制與重要催化殘基之鑑定

Abstract

摘要 一 本研究旨在探討Steptomyces matensis DIC-108菌株之β-1,3-葡聚五醣生產水解醣苷酵素(Laminaripentaose-producing β-1,3-glycanase, LPHase )屬於醣苷水解酵素第六十四家族的水解酵素(EC 3.2.1.39),藉以單取代反轉機制催化水解β-1,3醣苷鍵結，並專一性的生產有五個葡萄醣所構成的Laminaripentaose寡糖產物。 本實驗以設計24條人造設計的寡核苷酸引子，利用PCR技術，合成完整的LPHase人造基因，並重組建構於Prseta表現載體上，後轉殖進入大腸桿菌，BL21(DE3)系統中，以IPTG誘導過量表現，並以離子交換樹脂純化野生種與突變後的LPHase，純度均達90％以上。利用1H-NMR觀測LPHase酵素催化水解受質時醣立體選擇性作用，為一似SN2之單取代反轉構型機制。 為了瞭解LPHase之重要催化胺基酸殘基，因此探討所有同家族來原菌的胺基酸序列進行多重序列比對分析，發現五個高保留度的胺基酸，分別為D143、E154、D170、D376及D377，並對這些胺基酸進行突變，在這些突變株當中E154Q、E154G、D174N和D174G皆使酵素失去催化活性，接著對兩突變株E154G和D174G加入親核性陰離子試劑，確認Glu154為一般催化酸，而Asp170為一般催化鹼可以輔助酵素進行SN2反轉催化反應，本研究為第一個透過動力學檢測對醣苷水解酵素家族第六十四號之β-1,3-葡聚醣苷水解酵素，做完整的重要催化殘基位置鑑定 摘要 二 我們的目標是把內切型的幾丁聚醣酶轉換成外切型的幾丁聚醣酶，並且利用此酵素生產幾丁二醣，本實驗選擇來自於Bacillus circulans MH-K1(Csn)菌株的內切型幾丁二醣酶，根據蛋白質結構模擬，於Csn酵素中設計兩胜肽PCLGG及SRTCKP分別插入於Csn的可捲曲式胺基酸(loop) D115/T116和T222/G224之殘基後，使其在活性中心分開的位置形成雙硫鍵，避免受質反應時發生top-down反應 變異型(Chim)及野生型(WT)的幾丁聚醣酶都可以在大腸桿菌中被大量表現，且純化後均質度大於90％，酵素催化後的產物藉由LC-MS做分析，其結果發現WT-Csn催化後的產物具有混合的幾丁二醣、幾丁三醣、幾丁四醣，而Chim-Csn主要產生幾丁二醣。 另外，藉由聯結LC-MS的半定量分析以及黏度偵測，進行催化研究分析，發現隨著時間的變化，Chim-Csn催化甲殼素釋放幾丁二醣，溶液具有較低的黏滯還原速率。 綜合以上結果，藉由插入可撓曲式胺基酸，內切型的幾丁聚醣酶會轉換變成外切型的幾丁聚醣酶，不論是在於自然界或是人工設計的酵素中，這都是第一個外切型的幾丁聚醣酶，且只會產生單一的幾丁二醣產物

摘要 三 在自然界中有許多寡醣，特是別是含有蔗糖的寡醣，對於生物機制更是扮演重要的角色。例如:生長調控因子、訊號傳送的接受者(receptor)、細胞間的作用、抗基因反應(antigenic-response)。除此之外，母乳寡醣(HMO)中含有許多重要的蔗糖基寡醣，藉由與細菌表面的凝集素(lectin)反應，防止細菌與細胞中的醣類反應可以保護嬰兒不受細菌的感染，若調控蔗糖酵素的表現系統改變，將會使人體生理受到很大的影響。 為了瞭解此分解代謝酶的重要性，本研究將來自於Flavobacterium johnsoniae UW101菌株的岩藻糖苷酶(α-L-fucosidase)基因，轉殖到pET22b(+)的載體中，並成功於菌株E. coli BL21(DE3)pLys大量表現。 重組的野生種及突變酵素，經過管柱純化後，其純度達到95％以上。根據GH-29酵素家族胺基酸序列比對，選出六個殘基為Glu和Asp的胺基酸進行突變分析，作為探討酵素重要胺基酸殘基之依據。結果顯示突變株E56A、E261G和E291G嚴重喪失酵素催化活性，其K_cat值為1/9.8、1/8.33和1/22而D218G完全沒有展現酵素催化活性。本研究也更進一步使用疊氮化鈉，對D218G進行研究，發現Asp218為主要的親核性試劑。

PART I: Characterization and Identification of Essential Residues of the Glycoside Hydrolase Family 64 laminaripentaose-producing-β-1, 3-glucanase

ABSTRACT I

Laminaripentaose-producing β-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of β-1,3-glucan to release laminaripentaose as the predominant product. For study this novel enzyme, the gene of LPHase was reconstructed with PCR and overexpressed in E. coli. The recombinant wild-type enzyme and various mutants were further purified to >90 % homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with 1H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residues -- Asp143, Glu154, Asp170, Asp376 and Asp377, were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu154 functions as the general acid whereas Asp170 serves as the general base in a catalytic turnover. This work is the first report that provides a direct information for the identification of the essential residues of GH-64 through kinetic examination.

PART II: Structural Simulation and Protein Engineering for Converting an Endo-chitosanase to an Exo-chitosanase

ABSTRACT II The aim of this study was to produce chito-oligosaccharide with specific degrees of polymerisation. Our strategy was to convert an endo-chitosanase into an exo-type chitosanase and use it for chitobiose preparation. An endo-chitosanase from Bacillus circulans MH-K1 (Csn) was chosen as the candidate for protein engineering. Based on protein structure simulation, two peptides with five amino acids (PCLGG) and six amino acids (SRTCKP) were designed and inserted after the positions D115/T116 and T222/G224 of Csn, respectively. The inserted fragments are expected to form two loops at each band of the bilobate of Csn and further to form a disulphide bond across the clefts of the active site to prevent the ‘top-down’ action of the substrate binding. The chimeric chitosanase (Chim-Csn) and wild-type chitosanase (WT-Csn) were both successfully overexpressed in Escherichia coli and purified to >90% homogeneity. The enzymatic products were analysed by LC-MS. The results showed that a mixture of chitobiose, chitotriose and chitotetraose are the products of WT-Csn catalysis, whereas Chim-Csn produces chitobiose as the dominant product. Also, by combining semi-quantitative analysis of LC-MS and viscosity measurement, the time-course catalytic study showed that Chim-Csn released chitobiose from chitosan with a lower reduction rate of viscosity. All the results suggested that, by inserting two surface loops, the endo-type of Bacillus chitosanase is converted into an exo-type chitosanase. No matter whether in nature or artificiality, this is the first exo-type chitosanase that produces chitobiose as the sole end product.

PART III: Expression, Purification and Characterization of □-L-fucosidase from Flavobacterium johnsoniae UW101 ABSTRACT III Among the great variety of oligosaccharides, those containing fucose have an important role in nature. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions. For study this important catabolic enzyme, the codon optimized gene of □-L-fucosidase from Flavobacterium johnsoniae UW101 was cloned into pET22b (+) plasmid with His6 at C-terminus. Protein was successfully overexpressed in E. coli. BL21(DE3)pLys. The recombinant wild-type enzyme and various mutants were further purified to >95% homogeneity by Ni-column. Based on careful sequence alignment of GH-29 enzymes, six residues of glutamate and aspartate were selected for mutagenic study to determine the essential residues. Among the mutants, E56A, E261G and E291G lost catalytic activity significantly; their kcat values are 1/9.8, 1/8.33, 1/22, respectively, of that of the wild-type enzyme, whereas D218G was completely inactive. Further investigation with chemical rescue using sodium azide on D218G confirmed that Asp218 function as nucleophile.