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A NHase high-producing strain TQD-58 from Rhodococcus ruber CGMCC3090 was closed and bred by both parents inactivated protoplast fusion technique. The two parental strains L08-03 and LUV30-06 were derived from a NHase-producing strain R. ruber CGMCC3090 by UV irradiation, UV and LiCl mutating respectively. The protoplasts of L08-03 and LUV30-06 were fully inactivated by heating at 55°C for 7 min and by UV irradiation (30W UV light and vertical distance 30cm) for 90 sec respectively. The highest fusion frequency (7.01×10-2) between L08-03 and LUV30-06 was obtained by fusion (90s) at the conditions of adding Polyethylene glycol (PEG, 40%). One hyper yielding fusant TQD-58 was screened and its NHase activity with 3-cyanopyridine as substrate was up to 4296.8 U/mL, which was increased by 20.79% and 10.71% compared with that of parental strains L08-03 and LUV30-06, respectively. The genetic stability of TQD-58 was confirmed by successive subculture for 15 times.