[Cloning, expression of phospholipase A1 from Serratia liquefaciens and auto-induction fermentation by lactose].
To produce recombinant phospholipase A(1) (PLA(1)) by Escherichian coli, the pla gene encoding PLA(1) 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 PLA(1). E. coli BL21(DE3)/pET28a-pla yielded extracellular PLA(1) 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 PLA(1) 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 degrees C for 24 h, extracellular enzyme activity reached 128.7 U/mL.
Key Laboratory of Industrial Biotechnology of Ministry of Education, National Engineering Laboratory for Cereal Fermentation Technology, State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Jinlei
J
Yan
Liang
L
Zhang
Zhenghua
Z
Gu
Zhongyang
Z
Ding
Guiyang
G
Shi
D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005285:Fermentation; D007785:Lactose / Q000494:pharmacology*; D054466:Phospholipases A1 / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D044090:Serratia liquefaciens / Q000201:enzymology*
D003470:Culture Media; D011994:Recombinant Proteins; D054466:Phospholipases A1; D007785:Lactose