Radical-based photoinactivation of fatty acid photodecarboxylases.
Anal Biochem
; 600: 113749, 2020 07 01.
Article
em En
| MEDLINE
| ID: mdl-32348726
ABSTRACT
Fatty acid photodecarboxylases (FAP) are a recently discovered family of FAD-containing, light-activated enzymes, which convert fatty acids to n-alkanes/alkenes with potential applications in the manufacture of fine and speciality chemicals and fuels. Poor catalytic stability of FAPs is however a major limitation. Here, we describe a methodology to purify catalytically stable and homogeneous samples of recombinant Chlorella variabilis NC64A FAP (CvFAP) from Escherichia coli. We demonstrate however that blue light-exposure, which is required for photodecarboxylase activity, also leads to irreversible inactivation of the enzyme, especially in the absence of palmitate substrate. Photoinactivation is attributed to formation of protein based organic radicals, which were observed by EPR spectroscopy. To suppress photoinactivation, we prepared stable and catalytically active FAP in the dark. The steady-state kinetic parameters of CvFAP (kcat 0.31 ± 0.06 s-1 and KM 98.8 ± 53.3 µM) for conversion of palmitic acid to pentadecane were determined using gas chromatography. Methods described here should now enable studies of the catalytic mechanism and exploitation of FAPs in biotechnology.
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MEDLINE
Assunto principal:
Carboxiliases
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Ácidos Graxos
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article