アイテム詳細

要旨

Directed evolution is a very useful tool for targeted improvement of enzyme properties ^[1-7]. In the present dissertation it was used to improve the enantioselectivity of the cyclohexanone monooxygenases (CHMO) from Acinetobacter cal. NCIMB 9871 towards two different types of substrates. The CHMO is a 61 kDa NADPH dependent monomeric flavoprotein ^{[8, 9]} and catalyzes the introduction of an oxygen atom from molecular oxygen into a substrate ^[10]. Due to the ambivalent character of the peroxyflavin, the CHMO can catalyze Baeyer-Villiger reactions of electron-deficient substrates, such as cyclic ketones and the oxidation of electron-rich substrates, such as sulfides.
The CHMO-wt converts 4-hydroxycyclohexanone, which was used as the model substrate, leading to an ee of 9 % in slight favor of (R)-lactone ^[11]. In the course of the evolutionary project mutants were created which convert the 4-hydroxycyclohexanone to the (R)-lactone with ee of 90% and to the (S)-lactone with ee of 83%.
p-Methylbenzyl methyl sulfide was used as model substrate for the second substrate type and is converted by the CHMO-wt with an ee of 5% in slight favor of (R)-p-methylbenzyl methyl sulfoxide ^[12]. In the course of the same evolutionary project mutants were created converting the p-methylbenzyl methyl sulfide to (R)-or the (S)-sulfoxide with an ee > 99 %.
Sequencing and saturation studies identify amino acid position 432 as significant for the enantioselective conversion of both substrates.