β-1,4-內切聚葡萄糖酶之結構研究與其固定化

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

曾芝文(Chih-Wen Tseng)

指導教授 ： 劉嚞睿

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00456

摘要

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。

關鍵字

內切聚葡萄糖酶 ； 酶固定化 ； 反應曲面法 ； 聚二甲基矽氧烷 ； 人造油體

並列摘要

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.

並列關鍵字

endoglucanase ； immobilization ； EglA ； SSO1354 ； poly(dimethylsiloxane) ； response surface methodology ； artificial oil bodies

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