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Structural study and immobilization of β-1,4-endoglucanase
endoglucanase,immobilization,EglA,SSO1354,poly(dimethylsiloxane),response surface methodology,artificial oil bodies,
|Publication Year :||2014|
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.
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