液化沙雷氏菌磷脂酶A1的克隆表达及乳糖自诱导发酵

Cloning, expression of phospholipase A1 from Serratia liquefaciens and auto-induction fermentation by lactose

摘要:

为了利用大肠杆菌高效生产重组磷脂酶，克隆了液化沙雷氏菌磷脂酶A1的编码基因pla，分别使用pET-28a(+) 和pET-20b(+) 载体，实现了磷脂酶A1在大肠杆菌BL21(DE3) 中的功能表达。重组菌利用载体pET-28a(+) 在原始信号肽的介导下胞外PLA1酶活达40.8 U/mL，占总酶活的91％。重组菌转接至优化后的发酵诱导培养基：蛋白胨10 g/L，酵母粉5 g/L，葡萄糖0.8 g/L，乳糖5 g/L，25 mmol/L Na2HPO4，25 mmol/L KH2PO4和1 mmol/L MgSO4；菌体生长6 h后，添加7.5 g/L的甘氨酸，37 ℃恒温发酵24 h，重组菌胞外PLA1酶活达到128.7 U/mL。

Abstract:

To produce recombinant phospholipase A1 (PLA1) by Escherichian coli, the pla gene encoding PLA1 was amplified from Serratia liquefaciens by PCR and cloned into two vectors pET20-b(+) and pET28-a(+). The two recombinant plasmids were then transformed into E. coli BL21 (DE3) individually to express PLA1. E. coli BL21(DE3)/pET28a-pla yielded extracellular PLA1 with an activity of 40.8 U/mL in batch cultivations of shaken flasks by auto-induction, which was accounted for 91% of total enzyme activity. On the basis of primal auto-induction medium, the optimized fermentation medium of PLA1 contained tryptone 10 g/L, yeast extract 5 g/L, glucose 0.8 g/L, lactose 5 g/L, Na2HPO4 25 mmol/L, KH2PO4 25 mmol/L and 1 mmol/L MgSO4 (final concentration). Glycine (7.5 g/L) was added 6 h after inoculated. After incubated at 37 °C for 24 h, extracellular enzyme activity reached 128.7 U/mL.