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.
Cloning, Molecular ; Culture Media ; Escherichia coli ; genetics ; growth & development ; metabolism ; Fermentation ; Lactose ; pharmacology ; Phospholipases A1 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Serratia liquefaciens ; enzymology

Vertebral osteomyelitis or paraspinal abscess caused by Serratia species is rare and has been reported only in certain groups of people, such as intravenous drug users, immunocompromised hosts, and patients who have undergone invasive procedures. However, we report here that Serratia was recovered from a patient with vertebral osteomyelitis and epidural abscess without predisposing factors. The organism was initially identified as Serratia liquefaciens by a Vitek II-based assessment. It was finally identified as Serratia grimesii by 16S rRNA gene sequence analysis. We present the first case of vertebral osteomyelitis caused by S. grimesii in a patient without risk factors and who was managed successfully.
Abscess ; Drug Users ; Epidural Abscess ; Genes, rRNA ; Humans ; Immunocompromised Host ; Osteomyelitis ; Risk Factors ; Sequence Analysis ; Serratia ; Serratia liquefaciens ; Spine ; Spondylitis