title: [Overexpression, purification and characterization of phospholipase C from Acinetobacter calcoaceticus].
abstract: OBJECTIVE
  In this study, we constructed two recombinant Escherichia coli strains to produce phospholipase C (PLC) from Acinetobacter calcoaceticus. The recombinant enzymes were purified to homogeneity and characterized. [Methods] We cloned the PLC encoding gene plc1, plc2 from genome DNA of A. calcoaceticus ATCC17902. The amplified fragments were inserted into pET28a(+ to obtain expression plasmids. E. coli BL21 (DE3) harboring the above plasmids were cultivated and induced with isopropyl-beta-D-thiogalactopyranoside to express PLCs. The recombinant PLCs were purified by affinity chromatography and their catalytic properties were characterized.
  
  
  RESULTS
  Two PLCs from A. calcoaceticus were cloned and functional expressed in E. coli. The recombinant enzymes have activities of 31,160 +/- 418 U/mg for PLC1 and 13640 +/- 354 U/mg for PLC2, when using p-nitrophenyl phosphorycholine as substrate. The purified PLC1 and PLC2 exhibited optimum temperature at 65 degrees C and 50 degrees C, respectively. Their optimal pH were 8 and 7.5, respectively. PLC2 was stable under 40 degrees C and pH at 8, whereas the residual activity of PLC1 was less than 25% in the same condition. Mg2+ and Ca2+ stimulated two enzymes activity, whereas Zn2. stimulated PLC1 and inhibited PLC2. PLC1 and PLC2 hydrolyzed phosphatidylinositol.
  
  
  CONCLUSIONS
  It is the first time to express and characterize the PLC gene from A. calcoaceticus ATCC17902. These research results provide reference for the study of food-safety microbiological PLC.
authors: Yanhong
authors: Y
authors: Wang
authors: Liang
authors: L
authors: Zhang
authors: Zhenghua
authors: Z
authors: Gu
authors: Zhongyang
authors: Z
authors: Ding
authors: Guiyang
authors: G
authors: Shi
mesh_terms: D016954:Acinetobacter calcoaceticus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D007700:Kinetics; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010738:Type C Phospholipases