| ชื่อโครงการวิจัย/ชื่อเรื่อง | Protein Engineering of a Glycoside Hydrolase Family 3 β-Glucosidase for Hydrolysis of Cellobiose and Xylobiose |
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| ชื่อนักวิจัย/ชื่อผู้แต่ง | ศิริพรรณ อาทรธุระสุข |
| คำสำคัญ | beta-glucosidase;Aspergillus niger;cellobiose;xylobiose;transglucosylation |
| หน่วยงาน | ภาควิชาชีวเคมี คณะวิทยาศาสตร์ มหาวิทยาลัยเกษตรศาสตร์ |
| ปีที่เผยแพร่ | 2560 |
| คำอธิบาย | Glycoside hydrolase family 3 β-glucosidase from Aspergillus niger ASKU28 (or AnBG) shows high application potential in biomass conversion and cellulosic ethanol industry by its potent cellobiose hydrolyzing activity. However, it has some undesirable properties such as its narrow substrate specificity and tranglucosylation activity which can reduce the yield of fermentable sugar obtained from actual cellulolysis process if performed under inappropriate condition. Previous studies revealed that 4 amino acids, namely W49, D73, W262 and Y492, locating in the binding pocket of AnBG are likely to be members of the key residues that determine substrate specificity and ratio of hydrolytic and transglucosidic activities of the enzyme. Therefore, the mutant enzymes with amino acid substitutions at these 4 positions have been constructed and their kinetic properties toward cellobiose, xylobiose, para-nitrophenyl-β-D-glucopyranoside (pNP-Glc) and para-nitrophenyl-β-D-xylopyranoside (pNP-Xyl), their transglucosylation activities and their effects in hydrolysis of cellulose and xylan have been investigated. At the subsite +1, substitution of W49 with F or Y slightly compromised the hydrolyzing efficiency toward pNP-Glc, pNP-Xyl and cellobiose. The W49F mutant slightly improved xylobiose hydrolyzing efficiency by reducing the Km value. The W49Y mutant also decreased the transglucosylation activity of AnBG toward pNP-Glc but not cellobiose. Both the W49F and W49Y muatants pertained the capability of AnBG in assisting hydrolysis of cellulose and xylan better than the commercial cellobiase. Substitution of Y492 with F or Y, gave different effects. The Y492F mutation exhibited reduced transglucosylation activities, and slightly improved hydrolyzing efficiency toward xylobiose substrates. The Y492F mutant also showed capability in assisting hydrolysis of cellulose and xylan better than the commercial cellobiase. The Y492W mutation decreased the hydrolyzing efficiency toward both glucoside and xyloside substrates, and did not significantly decrease transglucosylation activity of the enzyme. At the subsite -1, substitution of D73 with E resulted in a great loss of enzyme activity toward both xyloside and glucoside substrates, suggesting that D73 is a crucial residue that plays a role in enzyme catalysis, even though it is neither the acid/base catalyst nor the nucleophile of the enzyme. Substitution of W262, with Y did not help improving the hydrolysis of any substrates in this study. The W262Y mutant showed higher transglucosylation activity toward both pNP-Glc and cellobiose which would be useful for oligosaccharide synthesis application. |
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Protein Engineering of a Glycoside Hydrolase Family 3 β-Glucosidase for Hydrolysis of Cellobiose and Xylobiose is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 Thailand License.
