Biotechnology Letters 23: 1633–1639, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 1633 Immobilization of the epoxide hydrolase from Aspergillus niger S. Karboune, L. Amourache, H. Nellaiah, C. Morisseau & J. Baratti ∗ Universit´ e de la M´ editerran´ ee, Biocatalyse et Chimie Fine, CNRS UMR 6111, Facult´ e des Sciences de Luminy, case 901, 13288 Marseille Cedex 9, France ∗ Author for correspondence (Fax: +33 491 829 362; E-mail: baratti@luminy.univ-mrs.fr) Received 12 July 2001; Revisions requested 20 July 2001; Revisions received 27 July 2001; Accepted 27 July 2001 Key words: Aspergillus niger, diffusional limitations, epoxide hydrolase, immobilization Abstract Three methods for the immobilization of the epoxide hydrolase from the fungus Aspergillus niger were tested. The highest immobilization yield (90%) and retention of activity (65%) were obtained by adsorption onto DEAE- cellulose compared to adsorption onto hydrophobic porous polypropylene and covalent linkage using Eupergit resin. The enzymatic properties of the immobilized enzyme were similar to those of the free enzyme with respect to the effect of temperature and pH on both activity and stability as well as the effect of solvent (DMF) on activity. The kinetic parameters were affected leading to lower K M(app) and higher Vm (app) . Introduction Epoxide hydrolases catalyse the hydration of the oxi- rane ring of epoxides into the corresponding diols (Archelas & Furstoss 1998, Omiecinski et al. 2000). When one of the carbon atom the oxirane ring is substituted for instance by a phenyl radical, the cor- responding epoxide is chiral (styrene oxide). Usually, epoxide hydrolases recognize the two enantiomers and thus can be used to resolve racemic mixtures of chemi- cally synthesized epoxides (Archelas & Furstoss 1999, Orru & Faber 1999). Chiral epoxides and their corre- sponding vicinal diols can be easily prepared by this method with high enantiomeric purity (Archelas & Furstoss 1997). These compounds are important chiral building blocks for the organic synthesis of fine chem- icals (Archelas & Furstoss 2001, Genzel et al. 2001, Manoj et al. 2001). The epoxide hydrolase from the fungus Aspergillus niger has been studied in some detail in our laboratory and by others at the molecular level (Arand et al. 1999, Morisseau et al. 1999, Zou et al. 2000). This fungal enzyme shows high activity and good enantioselectiv- ity when used with mono-substituted epoxides such as the para-nitrostyrene oxide (pNSO). The enzyme is highly enantioselective for the hydrolysis of pNSO with enantiomeric ratio of around 40 (Moussou et al. 1998, Pedragosa-Moreau et al. 1993). Reactors for the enzymatic resolution of pNSO were run at 330 mM (around 13 g l -1 ) and produced pure (R)-pNSO with 50% yield and 99% enantiomeric exces (ee) (Morisseau et al. 1997, Nellaiah et al. 1996). However, the enzyme was not very stable in the conditions used for these reactors with a half-life around 28 h. Thus, the enzyme could not be re-used af- ter utilization and reactors should be operated without loss of the enzyme preparation (Manoj et al. 2001). Thus, we aim at the evaluation of immobiliza- tion for enzyme re-utilization and continuous reactor operation. Materials and methods Enzyme preparation Aspergillus niger was from the Museum d’Histoire Naturelle (Paris, France). It was cultivated and the enzyme was partly purified according to a previously published procedure (Morisseau et al. 1997). The lyophilized preparation was stored at 4 ◦ C. The pow- der was dissolved in 10 mM Tris/HCl buffer at pH 7