大肠杆菌高效表达屎肠球菌纤维素结合域谷氨酸脱羧酶的条件优化

Optimization of conditions for efficient expression of recombinant cellulose-binding domain-glutamate decarboxylase of Enterococcus faecium in Escherichia coli

为了规避大肠杆菌( Escherichia coli )自身基因组表达的天然GAD的干扰,以再生无定形纤维素(regenerated amorphous cellulose,RAC)特异性吸附纤维素结合结构域谷氨酸脱羧酶(cellulose-binding domain-glutamate decarboxylase,CBD-GAD)制备的RAC-CBD-GAD固定化酶作为评价指标,采用单因素和田口试验设计法对 E. coli GDMCC60445高效表达CBD-GAD的条件进行了优化。结果表明, E. coli GDMCC60445表达CBD-GAD的适宜培养基为改良LB (Luria-Bertani)培养基,其组成为胰蛋白胨8 g/L、酵母膏 6 g/L、NaCl 10 g/L 、pH 5.5;适宜的培养条件为温度37 ℃、摇床转速120 r/min、培养时间24 h。在该适宜条件下,RAC-CBD-GAD活力为(419.79±10.37) U/g,与田口法预测值一致,较优化前提高了(30.28±3.22)%。

In order to avoid the interference of natural GAD expressed by the own genome of Escherichia coli , the recombinant cellulose-binding domain glutamate decarboxylase (CBD-GAD) was firstly immobilized by regenerated amorphous cellulose (RAC), and then the RAC-CBD-GAD immobilized enzyme was isolated and used as an evaluation index to optimize the culture conditions for highly efficient expression of CBD-GAD in E. coli GDMCC60445 by one-factor-at-a-time and Taguchi methods. The results indicated that the optimal medium for E. coli GDMCC60445 to express CBD-GAD was modified Luria-Bertani (LB) medium, which was consisted of 8 g/L tryptone, 6 g/L yeast extract, 10 g/L sodium chloride, and pH 5.5. The suitable culture conditions were 37 ℃, 120 r/min and 24 h. Under the optimal conditions, the activity of RAC-CBD-GAD prepared from E. coli GDMCC60445 was (419.29±10.37) U/g, which was consistent with the predicted value of Taguchi and improved by (30.28± 3.22 )% as compared to the initial one.