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Chemistry and Industry of Forest Products ›› 2014, Vol. 34 ›› Issue (6): 135-140.doi: 10.3969/j.issn.0253-2417.2014.06.022

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Expression Purification and Directed Evolution of Cytochrome P450 from Bacillus megaterium

LI Xun, WANG Ting, WANG Liang-liang, WANG Fei   

  1. College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
  • Received:2014-03-17 Online:2014-12-25 Published:2015-08-18

Abstract: In order to express the cytochrome P450 enzyme (CYPs) from Bacillus megaterium ALA2 effectively, the CYPs genes (cyp) were cloned into pET20b (T7 promoter) and pTrc99A (Trc promoter) to yield pET20b-cyp and pTrc99A-cyp. Then they were transferred into the Escherichia coli BL21 (DE3) and Top10. The pTrc99A-cyp produced more recombinant CYPs than pET20b-cyp. The Ni-NTA purification of CYPs was optimized. The recombinant CYPs were eluted by 60 mmol/L imidazole at pH 8.1. The enzyme activity of purified recombinant CYPs was 2 095 U/mg and recovery rate reached 31%, and the recovery ratio was 2.32. The mutation positions of the threonine (T) and phenylalanine (F) were measured as 269 loci and 394 loci by reasonable experimental design. The activities of mutant enzyme T269S and T269R were increased by 32.0% and 29.0% respectively with palmitic acid as substrate, and 11.6% and 9.1% with oleic acid as substrate.

Key words: Bacillus megaterium, cytochrome P450, directed evolution, palmitic acid, oleic acid

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