Enzyme cascade converting cyclohexanol into ε-caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

Abstract

The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity VMAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml−1. For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a VMAX(app) of 491 mU ml−1 was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.