产碱杆菌DN25中降氰酶的分离纯化及生化特性

以一株可降氰的产碱杆菌DN25为酶来源,通过超滤、30 mg/mL硫酸鱼精蛋白沉淀、30%～70%硫酸铵盐析和Phenyl-Toyopearl 650M疏水层析等步骤,获得比活力为44 U/mg的纯化酶制剂.在确定酶浓度、反应时间等氰降解活力测定条件后开展酶学性质研究,试图为将来氰降解代谢机理的深入研究和菌株的基因工程改造提供理论基础.研究结果表明,此纯化酶催化氰化物水解的最适pH值为8.0,最适温度为30℃.该酶在pH 7.0～8.0区域稳定,而在pH>9时会很快失活;在30℃保存10 h,酶活力保持稳定,高于60℃,酶快速失活.加入甘氨酸稳定剂,在60℃下保存20 min酶活仍可保留19.6%.酶促反应动力学符合米氏双曲线方程,测得米氏常数Km为3.11 mmol/L,最大反应速率Vmax为0.23 mmolL-1min-1.

Purification and Biochemical Characteristics of Cyanide-degrading Enzyme from Alcaligenes sp.DN25

The cyanide-degrading enzyme from Alcaligenes sp.DN25 was purified through ultrafiltration,precipitation with 30 mg/mL protamine sulfate,30%~70% fractional ammonium sulphate precipitation and hydrophobic chromatography on Phenyl-Toyopearl 650M,and the pure enzyme with the specific activity of 44 U/mg was obtained.After the proper reaction conditions including enzyme concentration and reaction time were determined for the cyanide-degrading activity assay,the purified enzyme properties were then studied in order to provide a theoretical basis for the future researches on cyanide-degradation mechanism and genetic engineering of strain DN25.The results showed that the optimal pH and temperature were 8.0 and 30 ℃,respectively.Good stability of the enzyme was observed at pH 7.0~8.0 and its activity decreased quickly when pH reached up to 9.0.The activity of the purified enzyme could keep stable when preserved at 30 ℃ for 10 h while fast deactivation happened at 60 ℃.However,with glycine added,the enzyme activity still remained 19.6% after incubated at 60 ℃ for 20 min.The degradation of cyanide by the purified enzyme followed a typical Michaelis-Menten kinetics,with Km of 3.11 mmol/L and Vmax of 0.23 mmol L-1 min-1.