Estimation of Cellobiohydrolase I Activity by Numerical Differentiation of Dynamic Ultraviolet Spectroscopy  

作　　者：Bin WU Yue ZHAO Pei-Ji GAO 

机构地区：[1]The State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China

出　　处：《Acta Biochimica et Biophysica Sinica》2006年第6期372-378,共7页生物化学与生物物理学报（英文版）

基　　金：This study was supported the grants from the National Natural Science Foundation of China(No.30370013);Natlonal Basic Research Program of China(No.2003CB716006 and No.2004CB719702)

摘　　要：1,4-β-D-glucan cellobiohydrolase Ⅰ （CBH Ⅰ）, p-nitrophenyl β-D-cellobioside, p-nitrophenol and cellobiose show distinct ultraviolet spectra, allowing the design of an assay to track the dynamic process of p-nitrophenyl β-D-cellobioside hydrolysis by CBH Ⅰ. Based on the linear relationship between p-nitrophenol formation in the hydrolysate and its first derivative absorption curve of AUC340-400 m （area under the curve）, a new sensitive assay for the determination of CBH Ⅰ activity was developed. The dynamic parameters of catalysis reaction, such as Vm and kcat, can all be derived from this result. The influence of β-glucosidase and endoglucanase in crude enzyme sample on the assay was discussed in detail. This approach is useful for accurate determination of the activity of CBHs.1,4-β-D-glucan cellobiohydrolase Ⅰ （CBH Ⅰ）, p-nitrophenyl β-D-cellobioside, p-nitrophenol and cellobiose show distinct ultraviolet spectra, allowing the design of an assay to track the dynamic process of p-nitrophenyl β-D-cellobioside hydrolysis by CBH Ⅰ. Based on the linear relationship between p-nitrophenol formation in the hydrolysate and its first derivative absorption curve of AUC340-400 m （area under the curve）, a new sensitive assay for the determination of CBH Ⅰ activity was developed. The dynamic parameters of catalysis reaction, such as Vm and kcat, can all be derived from this result. The influence of β-glucosidase and endoglucanase in crude enzyme sample on the assay was discussed in detail. This approach is useful for accurate determination of the activity of CBHs.

关 键 词：CELLOBIOHYDROLASE dynamic spectrum area under the curve （AUC） 

分 类 号：Q4[生物学—生理学]