In order to establish a new route for epsilon-caprolactone production from the corresponding cyclohexanol with an internal cofactor recycling for NADPH, a recently redesigned thermostable polyol dehydrogenase (PDH) and the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus were combined. First, the expression of PDH could be improved 4.9-fold using E. calf C41 with co-expression of chaperones. Both enzymes were also successfully co-immobilized on glutaraldehyde-activated support (Relizyme (TM) HA403). Cyclohexanol could be converted to epsilon-caprolactone (epsilon-CL) with 83% conversion using the free enzymes and with 34% conversion using the co-immobilized catalysts. Additionally, a preparative scale biotransformation of epsilon-caprolactone starting from cyclohexanol was performed using the soluble enzymes. The epsilon-CL could be isolated by simple extraction and evaporation with a yield of 55% and a purity of >99%. (c) 2013 Elsevier Inc. All rights reserved.