粪产碱杆菌D-氨基酰化酶的分离纯化及发酵条件优化

Isolation,Purification and Fermentation Conditions Optimization of Alcaligenes Faecalis D-Aminoacylase

采用热激法将含粪产碱杆菌D-氨基酰化酶基因载体的质粒导入大肠杆菌,筛选重组子,对该工程菌产生的D-氨基酰化酶用Ni 2+柱进行分离纯化;通过单因素实验优化D-氨基酰化酶的发酵条件。结果表明,纯化后的D-氨基酰化酶比活力为456.71U·mg-1,纯化回收率为50%,纯化倍数为12.4倍;最佳发酵条件为:37℃通气培养至菌体浓度OD600值为0.6时,加入0.5mmol·L-1诱导剂IPTG诱导5h,在此条件下,D-氨基酰化酶酶活力为90.9U·mL-1。

Heat shock method was applied to transform recombinant plasmid containing Alcaligenes faecalis D-aminoacylase gene into Escherichia coli,and then the recombinants were screened.D-Aminoacylase from the engineering bacteria was isolated and purified by Ni-NTA affinity chromatography.The fermentation conditions of D-aminoacylase were optimized by a single factor experiment.The results showed that D-aminoacylase was purified up to 12.4times with a recovery rate of 50%,and its specific activity was 456.71U·mg-1.The optimum fermentation conditions were as follows：after aerobic cultured at 37 ℃to OD600 value of 0.6,induced5 hwith addition of 0.5mmol·L-1 IPTG.Under above conditions,D-aminoacylase activity was 90.9U·mL-1.