一株产胶原蛋白酶细菌的鉴定及产酶条件优化

Identification of a Collagenase-producing Bacterium and Optimization of Enzyme Producing Conditions

采用形态学特征、生理生化特征结合16SrDNA对一株产胞外胶原蛋白酶的菌株AL-13进行鉴定。获得安全、高效、高产量的生产胶原蛋白酶工艺。以胶原蛋白酶活性为指标,采用单因素实验优化温度、pH、接种量等产酶培养条件后,利用单因素结合响应曲面法优化产酶培养基。结果表明,AL-13鉴定为侧孢短芽孢杆菌(Brevibacillus laterosporus),最适产酶培养条件为:培养时间48h、培养温度25℃、初始pH8.0、接种量6%(v/v),最适产酶培养基为:葡萄糖8.14g/L、牛肉膏11.63g/L、氯化钙0.17g/L、磷酸氢二钾2.08g/L、氯化钠9.48g/L,在该产酶条件下胶原蛋白酶活力预测值为154.89U/mL,验证结果显示酶活为(153.06±3.73)U/mL,此结果与预测值接近,相对误差为0.04%。本实验成功优化了一株产胶原蛋白酶细菌的产酶条件,产酶条件优化后的酶活(153.06U/mL)较优化前(16.14U/mL)提高了9.5倍。

A strain AL-13 producing extracellular collagenase was identified by morphological,physiological and biochemical characteristics and 16S rDNA sequencing.In order to obtain a safe,efficient and high-yield method for collagenase processing, with the activity of collagenase as an index,single factor experiments was used to optimize the culture conditions of temperature,pH and inoculation quantity for collagenase producing.Response surface methodology combined with single factors were used to optimize the enzyme - producing medium.The results showed that,AL - 13 was identified as Brevibacillus laterosporus.The optimal enzyme production conditions were:Culture time 48 h,culture temperature 25 ℃,initial pH8.0, inoculum 6%( v /v).Optimal enzyme production medium were:Glucose 8.14 g /L,beef extract 11.63 g /L,CaCl2 0.17 g /L, K2HPO4 2.08 g /L,NaCl 9.48 g /L.Under the conditions of enzyme production,the predicted value of collagenase activity was 154.89 U/mL.And the obtained reliability enzyme activity was( 153.06 ± 3.73) U/mL under the optimized processing.The result was close to the predicted value,and the relative error was 0.04%.The enzyme production conditions of the collagenase - producing bacteria were successfully optimized,and the enzyme activity( 153.06 U/mL) optimized for enzyme production was increased by 9.5 times compared with that before optimization( 16.14 U/mL).