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

Abstract

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

Keywords: Alcohol dehydrogenase; Cascade; Cyclohexanone monooxygenase; Enzyme fusion.

Fig. 1

Cascade reaction from cyclohexanol to ɛ-carpolactone, involving an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO)

Fig. 2

Blue native PAGE stained with Coomassie and zymography simultaneously. The HMW Native Marker ladder (GE Healthcare) values are given in kDa. The dark, purple stain results from the zymography, whereas the lighter, blue stain is caused by the Coomassie blue G-250 treatment