Enzymes involved in the glycidaldehyde (2,3-epoxy-propanal) oxidation step in the kinetic resolution of racemic glycidol (2,3-epoxy-1-propanol) by Acetobacter pasteurianus U. Wandel* ,1 , S. Salgueiro Machado, J.A. Jongejan, J.A. Duine Department of Microbiology and Enzymology, Kluyver Laboratory for Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands Received 12 April 2000; accepted 27 September 2000 Abstract It is already known that kinetic resolution of racemic glycidol (2,3-epoxy-1-propanol) takes place when Acetobacter pasteurianus oxidizes the compound to glycidic acid (2,3-epoxy-propionic acid) with glycidaldehyde (2,3-epoxy-propanal) proposed to be the transient seen in this conversion. Since inhibition affects the feasibility of a process based on this conversion in a negative sense, and the chemical reactivity of glycidaldehyde predicts that it could be the cause for the phenomena observed, it is important to know which enzyme(s) oxidise(s) this compound. To study this, rac.- as well as ( R)-glycidaldehyde were prepared by chemical synthesis and analytical methods developed for their determination. It appears that purified quinohemoprotein alcohol dehydrogenase (QH-ADH type II), the enzyme responsible for the kinetic resolution of rac.-glycidol, also catalyses the oxidation of glycidaldehyde. In addition, a preparation exhibiting dye-linked aldehyde dehydrogenase activity for acetaldehyde, most probably originating from molybdohemoprotein aldehyde dehydroge- nase (ALDH), which has been described for other Acetic acid bacteria, oxidised glycidaldehyde as well with a preference for the ( R)-enantiomer, the selectivity quantified by an enantiomeric ratio (E) value of 7. From a comparison of the apparent kinetic parameter values of QH-ADH and ALDH, it is concluded that ALDH is mainly responsible for the removal of glycidaldehyde in conversions of glycidol catalysed by A. pasteurianus cells. It is shown that the transient observed in rac.-glycidol conversion by whole cells, is indeed ( R)-glycidaldehyde. Since both QH-ADH and ALDH are responsible for vinegar production from ethanol by Acetobacters, growth and induction conditions optimal for this process seem also suited to yield cells with high catalytic performance with respect to kinetic resolution of glycidol and prevention of formation of inhibitory concentrations glycidaldehyde. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Acetobacter pasteurianus; Alcohol dehydrogenase (ADH); Aldehyde dehydrogenase (ALDH); Glycidaldehyde oxidation; Kinetic resolution 1. Introduction Several members of the genus Acetobacter are used for the production of vinegar from ethanol. This process occurs in two steps, the oxidation of ethanol to acetaldehyde and of acetaldehyde to acetic acid. The main activity for these steps derives from dye-linked, NAD(P)-independent, membrane- integrated dehydrogenases located at the outer surface of the cytoplasmic membrane [1]. The oxidation of ethanol to acetaldehyde is catalysed by a quinohemeprotein alcohol dehydrogenase (QH-ADH type II) containing pyrroloquino- line quinone (PQQ) and haem c as cofactors. The purified enzyme oxidises a broad range of alcohols to their corre- sponding aldehydes [1– 4], exhibiting enantioselectivity to- ward several racemic alcohols [5,6], but also oxidises sev- eral aldehydes to their corresponding acids [1– 4]. Oxidation of acetaldehyde to acetic acid is also catalysed by molyb- dohemeprotein aldehyde dehydrogenase (ALDH), contain- ing a molybdopterin and heme as cofactors [12]. The en- zyme oxidises several aliphatic and aromatic aldehydes to their corresponding acids [7–11]. Homochiral glycidol (2,3-epoxy-1-propanol) and its de- rivatives [13] as well as glycidaldehyde (2,3-epoxy-pro- panal) [14] are important synthons for the production of enantiomerically pure pharmaceuticals like -blockers and antiviral substances and specialties like chiral lactones and ferro-electric liquid crystals. The demand for these valuable intermediates is expected to increase in the near future [15]. * Corresponding author. 1 Current address: Hercules B.V., Hercules European Research Center, P.O. Box 252, 3770 AG Barneveld, The Netherlands. www.elsevier.com/locate/enzmictec Enzyme and Microbial Technology 28 (2001) 233–239 0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0141-0229(00)00321-5