ORIGINAL PAPER Baeyer–Villiger Oxidation of Cyclohexanone in Aqueous Medium with In Situ Generation of Peracid Catalyzed by Perhydrolase CLEA Georgina Cha ´vez • Jo-Anne Rasmussen • Michiel Janssen • Gashaw Mamo • Rajni Hatti-Kaul • Roger A. Sheldon Ó Springer Science+Business Media New York 2013 Abstract A perhydrolase, immobilized as a cross linked enzyme aggregate (CLEA), was employed to catalyze the in situ formation of peracetic acid (PAA) from ethylene glycol diacetate (EGDA) and hydrogen peroxide. The produced PAA was used for the Baeyer–Villiger oxidation of cyclohexanone, which afforded caprolactone in 63 % yield. The effect of type and amount of acyl donor, solvent, pH, temperature and ratio of cyclohexanone to hydrogen peroxide on the production of caprolactone was studied. The highest caprolactone yield was obtained with 100 mM EGDA as the acyl donor at pH 6 and room temperature using a ratio of cyclohexanone to hydrogen peroxide ratio of 1:4. Interestingly, the perhydrolase CLEA exhib- ited the highest activity in aqueous medium in contrast to the well studied lipase B from Candida antarctica. The perhydrolase CLEA proved to be a very efficient catalyst; the K m and V max values were 118 mM and 56.3 lmol min -1 , respectively. Keywords Perhydrolase Á CLEA Á Baeyer–Villiger Á Oxidation Á Epoxidation Á Aqueous medium Á Caprolactone 1 Introduction In the past decade, environmental concern has increased dramatically and this has led to formulation of strict leg- islation with regard to safety of industrial processes and waste disposal. The recent United Nations Conference on Sustainable Development, in Rı ´o de Janeiro, Brazil [1] pointed out that the management of chemicals is crucial and extra effort must be made to enhance technologies for the production of environmentally benign chemicals. Diminishing the use of chemicals is a key point in achieving this goal. Considering that the solvent contri- bution to the total waste produced in a process can be around 80 % in some cases [2], the use of organic solvents should be diminished or avoided to make a process greener. In this sense, the use of water is an environmentally friendly alternative to some traditional solvents used in chemical processes [3], with the added advantages of its low impact on health issues, and being an economical and readily available resource. The use of enzymes is another way to contribute to the greenness of an organic synthesis as enzymes generally work under mild conditions, i.e. physiological pH, mild temperature and atmospheric pressure. Safety issues can also be solved by the use of enzymes for the in situ generation of reagents avoiding the direct handling of dangerous substances. As an example, the current industrial production of e-caprolactone is based on the oxidation of cyclohexanone with peracetic acid (PAA) at 50 °C and atmospheric pressure [4]. However, the risks associated with the transport, storage and handling of large amounts of concentrated PAA solutions make in situ generation an interesting option. Moreover, indus- trial grade PAA is much more expensive than H 2 O 2 and contains up to 45 % acetic acid to stabilize it, which needs to be neutralized and generates waste. In short, in situ G. Cha ´vez (&) Á G. Mamo Á R. Hatti-Kaul Department of Biotechnology, Lund University, P.O. Box 124, 221 00 Lund, Sweden e-mail: georgina.chavez@biotek.lu.se G. Cha ´vez Á J.-A. Rasmussen Á M. Janssen Á R. A. Sheldon (&) CLEA Technologies BV, Delftechpark 34, 2628 XH Delft, The Netherlands e-mail: r.sheldon@clea.nl G. Cha ´vez Instituto de Investigaciones Fa ´rmaco Bioquı ´micas, Universidad Mayor de San Andre ´s, Casilla Postal, 3239 La Paz, Bolivia 123 Top Catal DOI 10.1007/s11244-013-0190-3