Elsevier

Analytical Biochemistry

Volume 357, Issue 2, 15 October 2006, Pages 257-261
Analytical Biochemistry

Kinetic analysis of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase using an amperometric biosensor

https://doi.org/10.1016/j.ab.2006.07.019Get rights and content

Abstract

An amperometric biosensor for the detection of cellobiose has been introduced to study the kinetics of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase. By use of a sensor in which pyrroloquinoline quinone-dependent glucose dehydrogenase was immobilized on the surface of electrode, direct and continuous observation of the hydrolysis can be achieved even in a thick cellulose suspension. The steady-state rate of the hydrolysis increased with increasing concentrations of the enzyme to approach a saturation value and was proportional to the amount of the substrate. The experimental results can be explained well by the rate equations derived from a three-step mechanism consisting of the adsorption of the free enzyme onto the surface of the substrate, the reaction of the adsorbed enzyme with the substrate, and the liberation of the product. The catalytic constant of the adsorbed enzyme was determined to be 0.044 ± 0.011 s−1.

Section snippets

Materials

Cellobiohydrolase I (CBHI) was purified from a commercial enzyme preparation from Trichoderma viride as follows. First, 5 g of Meicelase (Meiji Seika) was dissolved in 50 ml of 0.01 M (M = mol dm−3) acetate buffer (pH 5.0) and dialyzed with a dialysis membrane (Viskase, cutoff molecular weight of 12,000–14,000) for 20 h at 5 °C. The precipitate was removed by centrifugation, and the supernatant was applied to a 5-ml HiTrap Q HP column (Amersham Biosciences). The column was washed with the same buffer,

Results and discussion

Fig. 1 shows the current response of PQQ–GDH–BQ–CPE for the successive addition of 0.01 mM cellobiose in 0.1 M acetate buffer (pH 5.0) at 40 °C. On each addition of cellobiose, the current began to increase and reached a steady-state value within 1 min. Fig. 2 shows the plot of the catalytic steady-state current (Is) against [Cel]. The Is value was proportional to [Cel] up to 0.5 mM. From the slope of the regression line in Fig. 2, the sensitivity was determined to be 0.83 ± 0.06 μA mM−1. The limit of

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (18780077 to H.T.) and by a Grant-in-Aid for Scientific Research from the Fukui-ken University Science Foundation. The authors are grateful to Yasuteru Fujimoto for his assistance in the experiments.

References (20)

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