Expression and characterization of a novel cytochrome P450 enzyme from Variovorax paradoxus S110 / 生物工程学报

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.

Construction of sugar-based microbial fuel cells by dissimilatory metal reduction bacteria / 生物工程学报

Dissimilatory Metal Reduction Bacteria play an important role in the anaerobic environment. This kind of bacteria gains energy by coupling the oxidation of organic acid or sugars to the reduction of metal oxides. The graphite electrode rode can also be used as the final electron acceptor due to its similarity to solid metal oxides. Based on this biological mechanism, Dissimilatory Metal Reduction Bacteria Rhodoferaxferrireducens was used to construct a suit of microbial fuel cells with sugars as fuel, and the process and mechanism of electricity generation was studied. Rhodoferax ferrireducens was inoculated into the anode chamber in which a graphite electrode served as the final electron acceptor and glucose as the sole electron donor. It was showed that current density was up to 158mA/m2 with the resistance of 510omega at the normal temperature (platform voltage was around 0.46V, the effectual electrode surface was 57cm2). Following 20days' growth a large amount of bacteria cells attached to the electrode surface had been observed through the SEM images. The plandtonic cell protein concentration was 140mg/L and the attached biomass of electrode surface was 1180mg/m2 determined by the Bradford method, which indicated quite a few bacteria attached to the electrode. By analyzing the voltage value measured by the data acquisition system, it was proved that microbial electricity generation attributed mainly to the electrochemically and biologically active cells attached to the electrode, while the planktonic cells had no ability to catalyze electricity generation and almost had not electrochemically and biologically active. Furthermore, this kind of microbial fuel cells exhibited a good electrochemical cycle property and proved to be efficient in biomass utilization and energy restore since other sugars like fructose, sucrose, even xylose, could be oxidized and finally decomposed. Vast waste biomass in the form of carbohydrates is discarded in the environment. Not only is contamination of the environment caused by the discarded biomass, but also abundant energy stored in the biomass is drained away in vain. The sugar-based microbial fuel cells constructed by Rhodoferax ferrireducens could effectively transform the energy stored in sugars into electricity. Meanwhile, the microbial fuel cells presented in this paper, which could work cleanly at normal temperature with a good cycle property, showed a promising future application in this field.