Use of isolated cyclohexanone monooxygenase from recombinant Escherichia coli as a biocatalyst for Baeyer-Villiger and sulfide oxidations

F Zambianchi, P Pasta, G Carrea, S Colonna, N Gaggero, John Woodley

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The performance, in Baeyer-Villiger and heteroatom oxidations, of a partially purified preparation of cyclohexanone monooxygenase obtained from an Escherichia coli strain in which the gene of the enzyme was cloned and overexpressed was investigated. As model reactions, the oxidations of racemic bicyclo[3.2.0]hept-2-en-6-one into two regioisomeric lactones and of methyl phenyl sulphicle into the corresponding (R)-sulphoxide were used. Enzyme stability and reuse, substrate and product inhibition, product removal, and cofactor recycling were evaluated. Of the various NADPH regeneration systems teste, 2-propanol/alcohol dehydrogenase from Thermoanerobium brockii appeared the most suitable because of the low cost of the second substrate and the high regeneration rate. Concerning enzyme stability, kosmotropic salts were the only additives able to improve it (e.g., half-life from 1 day in diluted buffer to 1 week in I M sodium sulphate) but only under storage conditions. Instead, significant stabilization under working conditions was obtained by immobilization on Eupergit C (half-life approximately 2.5 days), a procedure that made it possible to reuse the catalyst up to 16 times with complete substrate (5 g . L(-1)) conversion at each cycle. Reuse of free enzyme was also achieved in a membrane reactor but with lower efficiency. Water-organic solvent biphasic systems, which would overcome substrate inhibition and remove from the aqueous phase, where reaction takes place, the formed product, were unsuccessful because of their destabilizing effect on cyclohexanone monooxygenase. More satisfactory was continuous substrate feeding, which shortened reaction times and, very importantly, yielded in the case of bicyclo[3.2.0]hept-2-en-6-one (10 g . L(-1)) both lactone products with high optical purity (enantiomeric excess greater than or equal to96%), which was not the case when all of the substrate was added in a single batch. (C) 2002 Wiley Periodicals, Inc.
Original languageEnglish
JournalBiotechnology and Bioengineering (Print)
Issue number5
Pages (from-to)489-496
Publication statusPublished - 2002
Externally publishedYes


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