Estimation of cellobiohydrolase I activity by numerical differentiation of dynamic ultraviolet spectroscopy.
1,4-beta-D-glucan cellobiohydrolase I (CBH I), p-nitrophenyl beta-D-cellobioside, p-nitrophenol and cellobiose show distinct ultraviolet spectra, allowing the design of an assay to track the dynamic process of p-nitrophenyl beta-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 nm (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 beta-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.
Wu
Bin
B
The State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
Zhao
Yue
Y
Gao
Pei-Ji
PJ
D000465:Algorithms; D019540:Area Under Curve; D020134:Catalytic Domain; D002475:Cellobiose / Q000737:chemistry; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008962:Models, Theoretical; D009596:Nitrophenols / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D014242:Trichoderma / Q000201:enzymology*; D001617:beta-Glucosidase / Q000378:metabolism
D009596:Nitrophenols; D002475:Cellobiose; D001617:beta-Glucosidase; D043366:Cellulose 1,4-beta-Cellobiosidase; C024836:4-nitrophenol