Large accumulation of nitrate in soil has resulted in "salt stress" and soil secondary salinization. Bacillus megaterium NCT-2 which was isolated from secondary salinization soil showed high capability of nitrate reduction. The genes encoding assimilatory nitrate and nitrite reductase from NCT-2 were cloned and over-expressed in Escherichia coli. The optimum co-expression condition was obtained with E. coli BL21 (DE3) and 0.1mM IPTG for 10h when expression was carried out at 20°C and 120rpm in Luria-Bertani (LB) medium. The molecular mass of nitrate reductase was 87.3kDa and 80.5kDa for electron transfer and catalytic subunit, respectively. The large and small subunit of nitrite reductase was 88kDa and 11.7kDa, respectively. The purified recombinant enzymes showed broad activity range of temperature and pH. The maximum activities were obtained at 35°C and 30°C, pH 6.2 and 6.5, which was similar to the condition of greenhouse soils. Maximum stimulation of the enzymes occurred with addition of Fe3+, while Cu2+ caused the maximum inhibition. The optimum electron donor was MV+Na2S2O4+EDTA and MV+Na2S2O4, respectively. Kinetic parameters of Km and Vmax were determined to be 670μM and 58U/mg for nitrate reductase, and 3100μM and 5.2U/mg for nitrite reductase. Results of quantitative real-time PCR showed that the maximum expression levels of nitrate and nitrite reductase were obtained at 50mM nitrate for 8h and 12h, respectively. These results provided information on novel assimilatory nitrate and nitrite reductase and their properties presumably revealed adaption of B. megaterium NCT-2 to secondary salinization condition. This study also shed light on the role played by the nitrate assimilatory pathway in B. megaterium NCT-2.
Keywords: Bacillus megaterium NCT-2; Characterization; Nitrate reductase; Nitrite reductase; Over-expression; Secondary salinization.
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