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.

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