Coupled reactions by coupled enzymes: alcohol to lactone cascade with alcohol dehydrogenase–cyclohexanone monooxygenase fusions

• DOI: 10.1007/s00253-017-8501-4
• Publication Date: 2017-09-15T06:53:54Z
• Authors: Friso S. Aalbers; Marco W. Fraaije
• Keywords: Cyclohexanol; Phosphofructokinase 2

The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). ADH can use NADP+ to oxidize cyclohexanol form and NADPH. Both products are then used by CHMO produce ε-caprolactone. In this study, these two redox-complementary were fused, create self-sufficient bifunctional enzyme that convert alcohols esters or lactones. Three different genes fused gene coding thermostable CHMO, in both orientations (ADH-CHMO CHMO-ADH). All six fusion could be produced purified. For three ADHs, we found clear difference between orientations: one showed expected activity, low no activity. activity each correlated its oligomerization state. fusions retained stability was hardly affected. TbADH-TmCHMO selected perform reaction, producing ε-caprolactone from cyclohexanol. By circumventing substrate product inhibition, > 99% conversion 200 mM achieved 24 h, 13,000 turnovers per molecule.