Optimization, purification, and characterization of hydroxylamine oxidoreductase from Acinetobacter sp. Y1

摘要

Hydroxylamine oxidoreductase (HAO) is a key enzyme involved in ammonium removal pathway. To further study the enzyme, HAO was purified from heterotrophic nitrifier Acinetobacter sp. Y1 and its property was investigated. Results of single-factor experiments showed that the optimal carbon source, nitrogen source, and C/N ratio were trisodium citrate, ammonium sulfate, and 14, respectively, with incubation time of 16 H. DEAE Sefinose(TM) FF anion-exchange chromatography was used to purify HAO, followed by Sefinose(TM) CL-6B gel filtration chromatography. SDS-PAGE revealed that a 47 kDa enzyme was purified successfully, with a purification fold of 7.32 and a recovery rate of 19.40%. The optimized enzyme activity of purified HAO was tested at pH 8.0 and 30 degrees C. The results showed that the activity was increased by 43.78% and 25.64% in the presence of 1 mM Fe2+ and Fe3+, respectively. HAO activity was increased with the increase of Na+ and K+, Mn2+, Zn2+, Cu2+, Ca2+, Ba2+ inhibited the HAO activity at three concentrations. In addition, HAO activity was activated by ethylenediaminetetraacetic acid at 0.4 mM, and a negative effect arose as the dose increased. The purified enzyme from Y1 is different from other reported HAOs. Further study should be conducted to investigate the enzyme.