Gene cloning, expression, and reducing property enhancement of nitrous oxide reductase from Alcaligenes denitrificans strain TB.

Nitrous oxide (N2O) is a potent greenhouse gas and tends to accumulate as an intermediate in the process of bacteria denitrification. To achieve complete reduction of nitrogen oxide (NOx) in bacteria denitrification, the structural gene nosZ encoding nitrous oxide reductase (N2OR) was cloned from Alcaligenes denitrificans strain TB (GenBank JQ044686). The recombinant plasmid containing the nosZ gene was built, and the expression of nosZ gene in Escherichia coli was determined. Results show that the nosZ gene consisting of 1917 nucleotides achieves heterologous expression successfully by codon optimization strategy under optimal conditions (pre-induction inoculum OD600 of 0.67, final IPTG concentration of 0.5 mM, inducing time of 6 h, and inducing temperature of 28 °C). Determination result of gas chromatography confirms that N2O degradation efficiency of recombinant E. coli is strengthened by at least 1.92 times compared with that of original strain TB when treated with N2O as substrate. Moreover, N2OR activity in recombinant strain is 2.09 times higher than that in wild strain TB, which validates the aforementioned result and implies that the recombinant E. coli BL21 (DE3)-pET28b-nosZ is a potential candidate to control N2O accumulation and alleviate greenhouse effect. In addition, the N2OR structure and the possible N2O binding site in Alcaligenes sp. TB are predicted, which open an avenue for further research on the relationship between N2OR activity and its structure.