Cloning, Expression, and Characterization of P450 Monooxygenase CYP102H1 from Nocardia farcinica

Abstract

To isolate a new P450 monooxygenase belonging to the CYP102 family, CYP102H1 of Nocardia farcinica IFM 10152 (i.e., pnf11 580) was cloned, expressed, and partially characterized. CYP102H1 gene was amplified from pNF1 of N farcinica and cloned into expression vectors (i.e., pTrc99A, pET28a(+)). When Escherichia coli BL21(DE3) codon+ strain was transformed with pET28a-CYP102H1 and the culture was induced with 1.0 mM isopropyl-beta-D-thio-galactoside in a complex medium at 30 degrees C, CYP102H1 could be expressed in soluble form even though soluble form was not dominant. The enzyme showed typical features of heme proteins; a spectrum of reduced CO bound form showed typical maximum at 450 nm. When the biotransformation of linoleic acid was carried out in a reconstituted system consisting of CYP102H1 and redox partner proteins of Pseudomonas putida (i.e., CamAB), omega 1-hydroxylinoleic acid was detected with gas chromatography/mass spectrometry analysis, suggesting that CYP102H1 catalyzes oxygenation of linoleic acid at omega-1 position, which is typical in CYP102A subfamily members.