一株产胆固醇氧化酶的不动杆菌的筛选鉴定及特性研究

Screening of an Acinetobacter Strain Producing Cholesterol Oxidase and Study on Its Characteristic and Fermentation Conditions

目的:筛选鉴定产胆固醇氧化酶的菌株并对其酶的性质及发酵条件进行初步的研究。方法:利用唯一碳源的胆固醇平板筛选,酶活测定比较得酶活力最高的菌株;生理生化试验结合16S rDNA序列分析鉴定其种属,单因素及正交实验优化培养基及发酵条件。结果:所得菌株H4与产不动杆菌(Acinetobacter)有最近的亲缘关系,其胆固醇氧化酶作用的最适温度和pH分别为37℃和8.0,金属离子Mg2+、Zn2+、Fe2+对该酶具有一定激活作用,菌株产酶的最适培养基为(g/L):胆固醇1.5,蔗糖5,蛋白胨7,硝酸铵3,吐温1.0,pH7.5;最适培养条件为33℃,15mL培养基/100mL三角瓶,摇床培养(200r/min)48h,优化后发酵液酶活达135.8U/L。结论:获得了1株产胆固醇氧化酶的菌株H4,并初步鉴定为不动杆菌(Acinetobacter)。

Objective：Isolated and identified cholesterol oxidase producing strain and studied on its properties.Method：Isolated a strain with the highest cholesterol oxidase（COD） activity using the cholesterol selective medium and the photometry test;identified the strain with the physiological and biochemical experiments and analysis of the 16S rDNA gene sequence;used fermentation tests and orthogonal experiments to optimize cultivation condition.Result：Bacterial strain H4 was selected with the highest COD activity and showed strong homology with Acinetobacter;the optimum temperature and pH of the enzyme was 37℃ and 8.0;Mg2 ＋,Zn2 ＋,Fe2 ＋ can increase its activity;the optimum medium for the Acinetobacter strain was（g/L）：cholesterol 1.5,sucrose 5,peptone 7,NH4NO3 3,Tween80 1mL,pH7.5,the fermentation conditions best to COD activity were initial 33℃ culturing temperature for 48 h and 15mL liquid medium in 100mL flask,on this condition,the COD activity could reach 135.8U/L.Conclusion：A cholesterol oxidase producing strain（H4） was isolated and identified as Acinetobacter.