Structural study and immobilization of β-1,4-endoglucanase
Date Issued
2014
Date
2014
Author(s)
Tseng, Chih-Wen
Abstract
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.
Subjects
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
Type
thesis
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