Structural study and immobilization of β-1,4-endoglucanase

黃牛瘤胃中存在厭氧性瘤胃真菌所產的內切聚葡萄糖酶，EglA，被分類於醣苷水解酶家族5。由於廣泛的受質專一性，因此在工業上具有應用潛力。為進一步瞭解酶與基質之間的相互關係，實驗運用了蛋白質結構學探討酶與受質之相互作用。在實驗中發現，因為EglA活性區被周圍的酶之N端六個組氨酸空間阻礙，酶晶體無法藉由浸泡基質溶液方式得到其交互作用的結構，而是要透過共結晶的方式得到酶與受質交互作用之晶體。將催化中心之谷氨酸154突變為丙胺酸，並且與受質纖維三糖共結晶，蛋白質結構分析，發現受質纖維三糖與酶活性區-3，-2與-1位置有廣泛的氫鍵的交互網絡。 為提高反應活性，酶固定化是一酵素工程研究方向。本研究將EglA固定於聚二甲基矽氧烷、紋理性矽晶片、矽晶片與銦錫氧化物，發現EglA在聚二甲基矽氧烷與矽晶片上有顯著固定狀況。藉由反應曲面法與中央合成設計實驗分析固定聚二甲基矽氧烷之EglA與固定於矽晶片之EglA其最適反應溫度與酸鹼度，結果顯示，固定在兩材料EglA其最適溫度與酸鹼度都比未固定化的EglA要高。固定於聚二甲基矽氧烷之EglA可重複使用六次，較固定於矽晶片之EglA可重複使用三次為佳。綜合研究結果，聚二甲基矽氧烷是用於固定化發展酶晶片或工業運用上較佳的材料。 此外，多種不同酵素固定化以達到一系列反應產物被認為有效促進受質催化。研究使用從硫磺礦硫化葉菌發現之內切聚葡萄糖酶SSO1354，使其與油體膜蛋白組成融合蛋白以超音波震盪方式組成人造油體。人造油體固定SSO1354 (AOB-SSO1354)、人造油體固定聚木糖酶CDBFV (AOB-CDBFV) 或人造油體固定SSO1354/CDBFV (AOB-SSOO1354/CDBFV) 以薄層層析方式分析產物特性，AOB-SSO1354分解羧甲基纖維素的產物為纖維二糖與纖維寡糖，分解燕麥木聚糖的產物為木二糖﹔而AOB-CDBFV分解燕麥木聚糖的產物為木二糖與低聚木糖。於兩種酶最適作用條件測定其分解天然基質稻稈能力，AOB-SSO1354/CDBFV反應時具協同作用。可應用於系列反應具有未來工業上的運用潛力。The endoglucanase EglA from rumen fungus Piromyces rhizinflata belongs to the GH5 family of glycoside hydrolase (GH) family 5. EglA showed promise in a wide range of industrial applications because of its broad substrate specificity. To understand the interaction between enzyme and substrate, the crystallization of EglA was interested. Because the active site was blocked by the N-terminal His tag of a neighbouring protein molecule in the crystal, enzyme–substrate complexes could not be obtained by soaking but were prepared by cocrystallization. The E154A mutant structure with a cellotriose bound to the -3, -2 and -1 subsites showed an extensive hydrogen-bonding network between the enzyme and the substrate. Since EglA has the promise industrial application, further improving the activity by immobilization is a considerable method. EglA was immobilized on different supporting materials including poly(dimethylsiloxane)(PDMS), Si wafer, textured Si wafer, and indium-tin-oxide-coated (ITO-coated) glass. The binding abilities of PDMS and Si wafer toward EglA were significantly higher than those of the other supporting materials. The optimized temperature and pH conditions for EglA immobilized on PDMS and on Si wafer were further determined by a response surface methodology (RSM) combined with a central composite design (CCD). The results indicated that the optimum pH and temperature values as well as the specific β-glucanase activity of EglA on PDMS were higher than those of free-form EglA. In addition, EglA immobilized on PDMS could be reused up to 6 times with detectable enzyme activity, while the enzyme activity of Eg1A on Si wafer was undetectable after 3 cycles of enzyme reaction. The results demonstrate that PDMS is an attractive supporting material for EglA immobilization and could be developed into an enzyme chip or enzyme tube for potential industrial applications. To investigate the digestion of natural complex substrate, rice straw, the thermostable endoglucanase SSO1354 from Sulfolobus solfataricus and thermostable xylanase CDBFV from Neocallimastix patriciarum were considered to be the good target in the enzyme immobilization by artificial oil bodies (AOBs). The formation of AOB-SSO1354, AOB-CDBFV and AOB-SSO1354/CDBFV were investigated by SDS-PAGE and confocal microscopy using antibody labeling. The products after AOB-SSO1354/CDBFV digestion were xylobiose, xylo-oligosaccharides, cellobiose and cello-oligosaccarides, which were demonstrated by thin layer chromatography (TLC). The synergic effect of SSO1354 and CDBFV was observed by hydrolysis of rice straw by AOB-SSO1354/CDBFV.Contents 謝誌 ii 中文摘要 iii Abstract iv Contents vi Table of figures x Table of tables xii Motivation 1 Chapter I. Research background 2 1. Plant cell wall biomass and fibrolytic enzymes 2 2. Application of fibrolytic enzymes 2 3. Classification of fibrolytic enzymes 3 4. The hydrolysis mechanism of glycosyl hydrolase family 5 (GH5) 4 5. Characteristic of EglA and SSO1354 endoglucanase 4 6. Natural enzyme immobilization - cellulosome 5 7. Enzymatic immobilization 6 (1) The interaction between enzymes and carriers 7 (2) Platforms for enzyme immobilization and co-localization 10 Chapter II. Research design and flow chart 16 1. Structural strategies 16 2. Physical stabilization 16 (1) Physical absorption 16 (2) Aritficial oil bodies 17 Chapter III. Substrate binding characteristic of the β-endoglucanase EglA from ruminal fungus Piromyces rhizinflata 19 1. Introduction 19 2. Materials and methods 21 (1) Chemicals and reagents 21 (2) Expression and purification of the recombinant EglA 21 (3) Point mutation by site-directed mutagenesis 22 (4) Crystallization 23 (5) Measurement of the enzymatic activity 23 3. Results 25 (1) Expression and purification of EglA 25 (2) Overall structure of EglA 25 (3) Enzyme-substrate interactions 26 (4) Protein-protein interactions 27 (5) Crystal packing interaction 27 4. Discussion 29 Chapter IV. Immobilization of Piromyces rhizinflata β-glucanase EglA on poly(dimethylsiloxane) and Si wafer and prediction of optimum reaction for enzyme activity 37 1. Introduction 37 2. Materials and Methods 39 (1) Supporting materials for enzyme immobilization 39 (2) Expression and purification of the recombinant EglA 39 (3) Immobilization of EglA on the supporting materials 39 (4) AFM observation of the supporting materials with or without EglA 40 (5) Optimum pH and temperature of the immobilized EglA 40 (6) β-Glucanase activity assay 41 (7) Reusability of the immobilized EglA 41 3. Results 42 (1) Immobilization of EglA on various supporting materials 42 (2) Surface morphology of PDMS and Si wafer with and without EglA 42 (3) Reaction conditions for optimal enzyme activity of immobilized EglA 42 (4) The reusability of immobilized EglA 44 4. Discussion 45 Chapter V. Co-immobilization of cellulase and xylanase on artificial oil body for efficient degradation of a complex cellulosic substrate 55 1. Introduction 55 2. Material and methods 57 (1) Bacterial culture and plasmid manipulation 57 (2) Cloning of xylanase and endoglucanas expression vectors 57 (3) Protein expression 58 (4) Immobilization of glycoside hydorlase on AOBs 59 (5) SDS-PAGE and Western blot 59 (6) Enzyme activity assay 60 (7) Confocal immunofluorescence microscopic analysis 60 (8) TLC analysis 61 (9) Reusability of AOB-SSO1354, AOB-CDBFV and AOB-SSO1354/CDBFV 61 3. Results 63 (1) Confocal immunofluorescence microscopic analysis of AOB-SSO1354, AOB-CDBFV and AOB-SSO1354/CDBFV 63 (2) Specific activities of AOB- SSO1354, AOB-CDBFV and AOB-SSO1354/CDBFV 63 (3) TLC of products analysis of AOB-SSO1354, AOB-CDBFV and AOB-SSO1354/CDBFV 64 (4) Reusability of AOB-SSO1354, AOB-CDBFV and AOB-SSO1354/CDBFV 64 (5) Enzyme activity on rice straw 64 4. Discussion 66 Conclusion 76 References 78 Appendix 8