| A Rhodococcus YL-1, which had high degrading capacity for nitriles was isolated and screened out from soil and wastewater polluted by nitriles. It can grow well with acrylonitrile and utilize it as carbon source, nitrogen source and energy, then biogegrade and catalyze it into acrylamide. After identified with morphology and physiological characteristics, it was assumed to belong to the Rhodococcus.The fermentation process of strain YL-1 was studied. It can produce nitrile hydratase during fermentation, biogegrade and catalyze acrylonitrile into acrylamide.The growth of strain and the activity expression of nitrile hydratase were parallelled. pH can influence the nitrile hydratase producing remarkably.So it is possibilly to increase the fermentation level and the biomass through the controlling of pH.YL-1 had the highest degrading capacity when φ (acrylonitrile) is 0. 2. The optimal temperature and pH were 30℃ and 7. 0, and the best volume of medium was 25 mL per 250 mL. After being cultured in broth for 30h under above conditions, its degradation rate of nitrile was 99. 4%, and for 40h , the biomass of the strain cell can reach to 10 g/L, the activity of nitrile hydratase can reach to 54 units per mg of dry cells.Strain YL-1 had a certain degrade capability of other nitriles such as formonitrile, acetonitrile, propionitrile, methylene cyanide,butyronitrile and butanedinitrile. The result showed that strain YL-1 had a large scale degrade capability of nitriles, and can be used to handle with environmental pollution and waste water treatment.Of strain YL-1, the acrylamide producing activity, from acrylonitrile, was found predominantly in the cells and not in the culture filtrate, when fresh culture broth was centrifuged. This suggested that the enzyme was located in the bacterial cell. And because the full cell can degrade acrylonitrile , it is suggested that the cell had no inhabit of the permeability of acrylonitrile and acrylamide.A bacterial strain having higher nitrle hydratase activity and acrylonitrile concentration tolerance than that of YL-1, numbered Rhodococcus sp YL-2, was developed by repeated subculturing of Rhodococcus sp. YL-1 in the broth containing acrylonitrile with slightly increasing acrylonitrile concentration. The specific nitrile hydratase activity of YL-2 increased up to 79. 8 units per mg of dry cells, 1. 7 times higher than that of YL-1.The nitrle hydratase producing conditions were studied and optimized. The results showed that the factors influencing nitrle hydratase activity mostly were the concentration of glucose, revulsant urea, Co2+ and pH. The optimum conditions were below: initial concentration of glucose 20 g/L, the adding amoumt of revulsant urea 0.06 g/L, the adding amoumt of Co2+ 0. 3 g/L, culture temperature 30℃ and pH 7.0. After being cultured 40 hours under the optimal conditions, the nitrle hydratase activity of YL-2 can reach to 134. 5 units per mg of dry cells, 1. 7 times higher than that before optimization. And for 34h,the degradation rate of nitrile was 99.5%.Reaserch of enzymatic properties showed that the optimal temperature and pH of nitrile hydratase were 30℃ and 7.0.It was stable under temperature 50°C and between pH 6.0 to 9.0.The activity of nitrilehydratase can' t be inhabited by Co2+,Fe2+,Zn2+, Mn2+, Mg2+ and Al3+, and obviously inhabited by Ag+, Ba2+, Hg2+, Pb2+, EDTA and SDS. Tween 80 and 60 can inhabit it also, but the concentration change of Tween 80 affected it remarkably than that of Tween 60.Strain YL-2 had the resistance to ampicillin,gentamycin and low concentration of erythromycin, and has no resistance to vancomycin and ciprofloxacin. The plasmid was isolated from the cell of YL-2. The experiment of plasmid elimination confirmed that the degradation gene of acrylonitrile was exist in plasmid. But the strain whose plasmid was eliminated still had the esistance to ampicillin, gentamycin and low concentration of erythromycin. The results showed that the drug resistant gene was not exist in plasmid. |
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