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Cloning and Heterologous Expression in E. coli of Phospholipase C Gene from Bacillus cereus

LIU Fei-fei,ZHANG Liang,GU Zheng-hua,DING Chong-yang,SHI Gui-yang   

  1. 1. National Engineering Laboratory for Cereal Fermentation Technology, Wuxi 214122, China;
    2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Wuxi 214122, China
  • Online:2013-06-15 Published:2013-06-03

Abstract:

As phospholipase C (PLC) could hydrolyze egg yolk and produce a zone of opalescence surrounding colonies on
an egg yolk agar plate, one phospholipase C producing strain of Bacillus cereus 12 was isolated from 600 strains preserved
in our laoratory. Phosphatidylcholine-preferring phospholipase C (PC-PLC) gene pcplc1 was amplified from the isolated
strain. Recombinant plasmid pET28a(+)-pcplc1 was constructed and transformed into E. coli BL21(DE3). After subsequent
indution by isopropyl β-D-1-thiogalactopyranoside (IPTG), an approximately 33 kD protein was detected in the cell lysate
supernatant by SDS-PAGE. Meanwhile, the recombinant protein showed significant PLC activity on egg yolk agar plate.
Induction time, induction temperature and IPTG concentration were investigated and optimized induction conditions for
pcplc1 expression were obtained as follows: 4% of inoculum amount, 1.5 h of initial culture, 4 h of induced culture in the
presence of 0.2 mmol/L of IPTG, and 25 ℃ of culture temperature. Unde the optimized conditions, maximum PLC activity
in the supernatant was observed to be (30.24 ± 0.18) U/mL (crude enzyme activity) .

Key words: phospholipase C, cloning and expression, IPTG, optimization