Engineering a diaphorase via directed evolution for enzymatic biofuel cell application

Abstract

Abstract Background Diaphorase (DI) has received wide attention as the key anodic enzyme mediating the electron transfer and electric energy generation in enzymatic biofuel cells (EBFCs). Lowering the anodic pH may be a useful strategy for constructing high-performance in EBFCs. However, most DI suffered from the poor activity at low pHs. Therefore, it is necessary to modify the activity and its acidic tolerance to further improve the performance of the EBFC. Results This paper attempts to improve the enzyme activity of DI originated from Geobacillus stearothermophilus under acidic conditions through directed evolution. Three rounds of random mutagenesis by error-prone PCR of the GsDI gene followed by high-throughput screening allowed the identification of the mutant 3–8 (H37Q, S73T, F105L, S68T, G61S, D74V) exhibiting a 4- or 7-fold increase in the catalytic activity at pH 5.4 or 4.5 compared to that of the wild type. And the pH stability of mutant 3–8 was significantly better than that of wild type and showed a 1.3 times higher in the stability at pH 5.4. The EBFC anode equipped with 0.5 mg of mutant 3–8 achieved a maximum current of 40 μA at pH 5.4, much higher than that with the same loading of the wild type enzyme. Conclusion The GsDI has been improved in the specific activity and pH stability by directed evolution which leads to the improvement of the EBFC performance. Also, the enlarged catalytic channel of mutant and decreased B-factor may be beneficial for the activity and stability. These results suggest that this engineered DI will be a useful candidate for the construction of enhanced EBFCs.

Keywords

• Diaphorase
• Enzyme engineering
• Directed evolution
• Enzymatic biofuel cell
• Bioelectrocatalysis