Microbial monooxygenase amperometric biosensor for monitoring of Baeyer-Villiger biotransformation

A whole-cell amperometric biosensor consisting of genetically engineered Escherichia coli immobilised in polyelectrolyte membrane onto a miniaturised oxygen electrode was developed and used for monitoring of biotransformation based on Baeyer-Villiger oxidation. Baeyer-Villiger oxidation is commonly performed using microorganisms overexpressing Baeyer-Villiger monooxygenase enabling the production of enantiopure lactones or esters used in pharmaceutical industry. The biorecognition element, genetically modified E. coli overexpressing either cyclopentanone monooxygenase or cyclohexanone monooxygenase was immobilised in the form of solid polyelectrolyte complex gel membrane made of cellulose sulphate, sodium alginate and poly(methylene-co-guanidine) and attached to the surface of miniaturised oxygen electrode. The time response of the biosensor was 30s, the linear range of the calibration curve (R(2)=0.9993) was 8-130 μM and the sensitivity was 1.8 nA μM(-1) (RSD=5.0%) for substrate of Baeyer-Villiger oxidation (±)-cis-bicyclo[3.2.0]hept-2-en-6-one as analyte. The biosensor sensitivity was assessed for two other commercially available substrates, 4-methylcyclohexanone and 3-methylcyclohexanone. No interferences from ampicillin, citric acid, acetic acid, ethanol, methanol, glucose and products of Baeyer-Villiger oxidation (1R, 5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one and (1S, 5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one were detected. After 1 week of storage at 4°C the biosensor sensitivity was without changes. The biosensor was employed for monitoring of Baeyer-Villiger biotransformation and the results were correlated with gas chromatography. Till now, this is the first described biosensor based on Baeyer-Villiger monooxygenase and the first reported application of biosensor for monitoring of biotransformation based on Baeyer-Villiger oxidation.