Purification, Characterization, Crystallization and Structure Modeling of a Commercial β-Glucosidase from Aspergillus niger

• Main Authors: Wen-Shiue Chang, 張文學
• Other Authors: Ping-Lin Ong
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/92237413712598296510

碩士 === 國立嘉義大學 === 生化科技學系研究所 === 99 === β-Glucosidase from Novozyme 188, a commercial product, was purified by 80% saturation ammonium sulfate precipitation, ferric sulfate decolorization, CM-SepharoseTM ion exchanger chromatography, DEAE-SepharoseTM ion exchanger chromatography and native polyacrylamide gel electrophoresis with gel slicing. The β-glucosidases was purified 18.1-fold with a recovery of 42.5% of the initial activity and a specific activity of 6.6 U/mg. The purified β-glucosidase was found to be a N-linked glycoprotein containing 19.2% (w/w) carbohydrate with phenol-sulfuric acid method. The optimal catalytic activity was observed at pH 4.0 and 60°C. It was also stable in the temperature range of 30 ~ 60 °C and pH range of 4.0 ~ 9.0. β-glucosidases show protease-resistant properties. It’s activity was decreased 23%, 29% and 83% by 5mM CuSO4, 5mM glucose and 5mM gluconolactone, respectively. It’s has nearly no activity in 10 ~ 40% organic solvents. Kinetic parameters of the β-glucosidases catalyze p-nitrophenyl- β-D-glucopyranoside were measured as Km = 0.9 mM and Kcat = 411.5 sec-1. The Ki of glucose and gluconolactone inhibitor for β-glucosidases are 4.97 mM and 24.48 μM, respectively. β-glucosidase with polysaccharide binding properties which is confirmed by competitive affinity chromatography with carboxymethyl cellulose（CMC）. The identity of β-glucosidases was confirmed as GI 145254958 and classified as family 3 by LC/MS/MS. The crystallization conditions of β-glucosidase were screened through 1008 conditions, and further modified to be around 19 ~ 22% polyethylene glycol 4000 and 0.1 M acetate buffer (pH5.0). The crystal is not good enough to be diffracted by x-ray, so it’s necessary to refine the crystallization conditions more.