earticle

영어

There is continuing interest in self-sufficient P450 engineering for the biotransformation of aromatic compounds like flavonoid, chalcone and lignin monomers with the object of increasing antioxidant effect. However there is no efficient screening system to analyze large number of mutant library against that substrate. In this study, we developed the high throughput screening system using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) to screen the P450 BM3 mutant library towards daidzein. The hydroxylation activity of the P450 BM3 F87A mutants saturated at Y51 has been shown to occur to varying degrees with small alterations in the structure of substrates. Reaction and extraction of mutant library were carried out in 96 deep-well plates. Reaction sample containing more than one substrate were spotted on the 384 MALDI target plate with a matrix and surfactant. To reduce
matrix ion interference, small molecule analytes have been investigated with respect to their suitability for use in analysis by surfactant applied MALDI-TOFMS. Hydroxylated metabolites were automatically detected within 2 hours. In this way developed method for the detection of the hydroxyated flavonoid and lignin monomers can be applied to screen the large number of mutant library against the different substrates at a same time, which is less time consuming and more accurate than the conventional screening methods.