title: [Expression of hemolytic phospholipase C from Pseudomonas aeruginosa in Escherichia coli].
abstract: OBJECTIVE
  In this study, we constructed a recombinant Escherichia coli (E. coli) strain to produce hemolytic phospholipase C and optimized the fermentation conditions.
  
  
  METHODS
  We screened a high phospholipase C activity strain, Pseudomonas aeruginosa (P. aeruginosa) 41, through yolk borax plate method, and cloned the hemolytic phospholipase C gene (plcH) from it. The plcH was inserted into pET-28a (+) and then obtained the recombinant expression plasmid (pET28a-plcH). We selected the correct recombinant plasmid and transformed it into E. coli BL21 (DE3). Furthermore, we determined the PLC activity and hemolytic activity in positive transformants on yolk borax plate and columbia blood agar plate. Finally, we optimized the fermentation conditions.
  
  
  RESULTS
  We successfully constructed a recombinant E. coli strain (E. coli BL21 (DE3)/pET28a-plcH) that showed significant phospholipase C activity. Moreover, hemolytic phospholipase C of the recombinant strain showed strong hemolytic activity. The enzyme activity of phospholipase C was 722.9 +/- 0.47 U/mL with 5% of inoculation amount, 200 r/min for 4 hours at temperature of 37, induced by 0.9 mmol/L IPTG for 14 hours.
  
  
  CONCLUSIONS
  We constructed a recombinant E. coli strain with high hemolytic phospholipase C activity under optimized fermentation conditions. It is the first time in domestic to successfully clone and express phospholipase C gene from P. aeruginosa in E. coli. These research results are helpful to advance the industrialization and application of phospholipase C.
authors.affiliation: State Key Laboratory of Food Science and Technology, National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China. zhaojinxing18621@163.com
authors.forename: Jinxing
authors.initials: J
authors.lastname: Zhao
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhenghua
authors.initials: Z
authors.lastname: Gu
authors.forename: Zhongyang
authors.initials: Z
authors.lastname: Ding
authors.forename: Guiyang
authors.initials: G
authors.lastname: Shi
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial / Q000235:genetics; D053878:Hemolytic Agents / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins; D013696:Temperature; D013997:Time Factors; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes
chemical_list: D053878:Hemolytic Agents; D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C477519:sphingomyelin synthase, Pseudomonas aeruginosa