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