Abstract
Objectives
To find an efficient and cheap system for NAD+ regeneration
Results
A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD+ regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD+ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C
Conclusion
The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD+ regeneration.
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Supplementary information
Supplementary Fig. 1—Lineweaver-Burk plot of the effect of NADH concentration on NADH dehydrogenase activity. Enzyme kinetic parameters were obtained by measuring the rate of A340 value decrease at various concentrations of NADH ranging from 12.5 μM to 400 μM at 45 °C in Tris/HCl buffer (pH 7.5, 50 mM).
Supplementary Fig. 2—Lineweaver-Burk plot of the effect of potassium ferricyanide concentration on NADH dehydrogenase activity. Enzyme kinetic parameters were obtained by measuring the rate of A340 value decrease at various concentrations of potassium ferricyanide ranging from 6.25 μM to 200 μM at 30 °C in citrate/phosphate buffer (pH 5.6, 50 mM).
Supplementary Fig. 3—Residual activity of NADH dehydrogenase and laccase following incubation at 30 °C in citrate/phosphate buffer (pH 5.6, 50 mM).
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Jizhong Wang and Chengli Yang have contributed equally to this work.
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Wang, J., Yang, C., Chen, X. et al. A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD+ regeneration. Biotechnol Lett 38, 1315–1320 (2016). https://doi.org/10.1007/s10529-016-2106-3
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DOI: https://doi.org/10.1007/s10529-016-2106-3