Expression, Characterization, and Engineering Arabinofuranosidase-xylanase for Degrading Hemicelluloses for Biofuel Production

植物細胞細胞壁是由許多纖維素與半纖維素透過不同的醣酯鍵交錯形成，故分解其細胞壁需由許多不同酵素催化一連串的反應來達成。其中，聚木醣 (xylan)占半纖維素組成的最大宗，以β-1,4-鍵結的木醣長鏈結構為主幹，又依取代基的不同而形成多種異質聚木醣(heteroxylan)，常見的取代基包括α-1,2-或α-1,3-阿拉伯呋喃醣基(α-1,2- and/or α-1,3- arabinofuranosyl residues)與乙醯基-1,4-甲基-D-葡萄醣醛酸基(acetyl,4-O-methyl-D-glucuronosyl residues)。異質聚木醣分解的過程先可透過阿拉伯呋喃醣酶(α-L-arabinofuranosidase)的作用，將側支上的阿拉伯呋喃醣去除。先前的文獻報告指出，一種來自梭狀芽孢桿菌屬(Clostridium thermocellum)的阿拉伯呋喃醣酶(EC 3.2.1.55)，命名為CtAraf51A，其催化中心的七個胺基酸：第27號麩胺酸(Glu27)、第72號天門冬胺酸(Asn72)、第172號天門冬胺酸(Asn172)、第178號色胺酸(Trp178)、第244號酪胺酸(Tyr244)、第292號麩胺酸(Glu292) (親核基nucleophile)以及第352號麩醯胺酸(Gln352)與此酵素的催化及受質的結合息息相關。觀察此蛋白質的立體結構後，發現第178號色胺酸與第244號酪胺酸位於催化中心頂端，因此提出第178號色胺酸與第244號酪胺酸可以決定受質專一性的假設。 藉由單定點突變法將第178號色胺酸與第244號酪胺酸分別轉變為丙胺酸 (Alanine)，結果發現此二類單點突變都使阿拉伯呋喃醣酶活性下降許多(第178號色胺酸轉變為丙胺酸使活性下降四十倍，第244號酪胺酸轉變為丙胺酸使活性下降七十倍)。再進一步以雙定點突變法將上述二胺基酸轉變為丙胺酸，會使得雙突變的阿拉伯呋喃醣酶完全喪失活性。又另外發現原本的阿拉伯呋喃醣酶同時具有些許木醣苷酶(β-xylosidase)活性，但所有突變的酵素皆已不再具有水解雙木醣(xylobiose)之活性。 催化聚木醣分解同時也需要聚木醣酶(xylanase)的作用，其隨意的切斷沒有取代基或側鏈的主幹而釋放出寡木醣或雙木醣，再由木醣苷酶將雙木醣水解成木醣(xylose)。為了能夠更有效地催化聚木醣分解，接著建構一N端為阿拉伯呋喃醣酶、C端為聚木醣酶的融合酵素，此融合酵素同時具有聚木醣酶、木醣苷酶與阿拉伯呋喃醣酶之活性。分別以人工合成的間-硝基苯酚-α-阿拉伯呋喃醣 (p-nitrophenyl-α-L-arabinofuranoside)與樺木聚木醣(beechwood xylan)作為受質，觀察發現單一酵素與融合酵素的催化最適溫度與酸鹼質並無明顯差異。阿拉伯呋喃醣酶與融合酵素作用最適溫度與酸鹼質皆為65 oC，pH 6.5，同樣地，聚木醣酶與融合酵素催化最適溫度與酸鹼質亦為65 oC，pH 6.5。進一步做酵素動力學測定，阿拉伯呋喃醣酶的Km與kcat分別為294±43Due to a composite structure of polysaccharides, containing celluloses and hemicelluloses, depolymerization of the plant cell wall requires diversely sequential enzyme actions. Xylan, the major component of hemicelluloses, consists of β-1,4-linked xylopyranose units usually including different substituent groups, such as α-1,2- and/or α-1,3- arabinofuranosyl and acetyl, 4-O-methyl-D-glucuronosyl residues. An important enzyme in heteroxylan digestion is α-L-arabinofuranosidase (α-L-AFase), which releases L-arabinofuranosyl residues from side chains. n previous study, the crystal structure of an α-L-AFase (EC 3.2.1.55), Araf51A from Clostridium thermocellum, called “CtAraf51A” has been solved [1]. The co-crystal structures of the complexes reveal seven critical residues responsible for catalysis and substrate binding, which are Glu27, Asn72, Asn172, Trp178, Tyr244, Glu292 (nucleophile), and Gln352. The X-ray crystal structure of CtAraf51A shows Trp178 and Tyr244 are located at the head of the active center, which may be responsible for substrate specificity of CtAraf51A. In order to confirm this hypothesis, the single mutant, W178A and Y244A, and W178A/Y244A double mutant were constructed in this study. W178A mutant and Y244A mutant showed much lower α-L- AFase activity than the wild-type CtAraf51A (W178A with a 40-fold and Y244A with 70-fold lower kcat, respectively). Furthermore, double mutant W178A/Y244A had abolished activity of α-L-AFase. Interestingly, wild-type CtAraf51A could accommodate xylopyranosidic substrates, but all of the mutants could not hydrolyze the pyranosidic synthetic substrates. egradation of xylan also needs a key component, xylanase which cleaves internal glycosidic bonds by random hydrolysis of xylan backbone, resulting in xylo-oligosaccharides and xylobiose. In order to degrade xylan more efficiently, a hybrid enzyme with CtAraf51A in the N-terminal and truncated form of Xyn10Z (tXyn10Z) from C. thermocellum (Araf-tXyn) in the C-terminal, containing xylanase, β-xylosidase, and α-L-AFase activities was further constructed. The individual and hybrid enzymes shared similar pH and temperature profiles when assays were performed with 4-nitrophenyl-α-L-arabinofuranoside (4NPA) and beechwood xylan. This hybrid enzyme had the optimum for α-L-AFase activity at pH 6.5 and 65 oC and the highest xylanase activity at pH 6.5 and 65 oC similar to two individual enzymes, CtAraf51A and tXyn10Z. Moreover, the Km and kcat values of the individual CtAraf51A were determined to be 294±43中文摘要.....1BSTRACT.....3BBREVIATIONS.....61)INTRODUCTION.1 The cellulase system of Clostrdium thermocellum.....7.2 Hemicellulose.....8.3 Classification of xylanse.....9 .4 Classification of Arabinan-hydrolyzing enzymes.....10.5 Dual-function α-arabinofuranosidase/β-xylosidase enzymes.....11.6 Natural and artificial fusion enzymes with dual activities.....12.7 Application of different hemicellulases.....13.8 Specific aim of this study.....142)MATERIALS AND METHODS.1 Materials.....15.2 Bacterial strain and growth condition.....15.3 Plasmid construction.....15.4 Preparation of Mutant CtAraf51A Enzyme.....16.5 Computer modeling of the substrates binding with wild-type and mutant CtAraf51As.....17.6 Recombinant Fusion Gene Construction.....18.7 Expression and Purification of Enzymes.....19.8 Substrate specificity.....20.9 pH and Temperature Optima Determination.....20.10 Thermostability Tests.....21.10 Measurements of the Km and kcat Values.....22.11 Enzyme Activities against Natural Substrates Using End Products Determination.....233)RESULTS.1 Expression and Purification of Mutant CtAfaf51A.....24.2 Kinetic parameters of Wild-type and Mutant CtAraf51As.....25.3 Comparison of 4NPA-CtAraf51A complex with 4NPX-CtAraf51A complex.....26.4 Docking of CtAraf51A with 4NPA.....26.5 Construction and Expression of the Individual and Hybrid enzymes.....28.6 pH Optimum of the Individual and Hybrid Enzymes.....29.7 Temperature Optimum of the Individual and Hybrid Enzymes.....29.8 Kinetic studies of the individual and hybrid enzymes.....30.9 Activities toward Natural Substrates.....314)DISCUSSION.....32EFERENCE.....37 ABLE.....44 IGURE.....4