Enzyme and Microbial Technology 37 (2005) 718–722 Hydroxylation of androstenedione by resting Rhodococcus sp. cells in organic media B. Angelova a,b,∗ , P. Fernandes b,c , A. Cruz b , H.M. Pinheiro b , S. Mutafov a , J.M.S. Cabral b a Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology, Acad. G. Bonchev St., Block 26, 1113 Sofia, Bulgaria b Centro de Engenharia Biologica e Quimica, Instituto Superior T´ ecnico, Av. Rovisco Pais, 1049–001 Lisboa, Portugal c Universidade Lus´ ofona de Humanidades e Tecnologias, Av. Campo Grande 376, 1749–024 Lisboa, Portugal Received 1 July 2004; received in revised form 14 April 2005; accepted 28 April 2005 Abstract The use of organic solvent-based media for microbial 9-hydroxylation of androstenedione (AD) by resting Rhodococcus sp. cells was evaluated. Product yields higher than 90% were observed in a two-phase phthalate-buffer transformation medium (3:1, v/v) containing 1.0 g L -1 AD. The results suggest that in these conditions the reaction of  1 -dehydrogenation is considerably inhibited. The successful hydroxylation reaction with a product yield exceeding 60% was found to take place also in a phthalate medium under simultaneous forced aeration and maintenance of a minimum water content of about 3% (v/v). © 2005 Elsevier Inc. All rights reserved. Keywords: Rhodococcus; Steroids; Bis-(2-ethylhexyl) phthalate; Microbial transformation 1. Introduction The microbial transformation of steroid compounds added into the reaction medium as solutions in water immiscible or- ganic solvents proved to be a suitable approach for improving the process effectiveness by overcoming the low solubility of steroid substrates in water-based reaction media [1]. Organic- aqueous two-phase transformation media were successfully applied in the microbial side-chain cleavage of sitosterol [2–5] and dehydrogenation of 6-methyl-hydrocortisone-21- acetate [6] but not in the microbial steroid hydroxylation processes. The authors of the rare publications dealing with microbial steroid hydroxylation in the presence of water im- miscible organic solvents are prone to justify the loss of 11- hydroxylating activity of the Aspergillus ochraceus strain used with the inhibiting properties of the tested solvents [7,8]. There are still no reports on the applicability of water immis- cible organic solvents as media for carrying out microbial 9-steroid hydroxylation. ∗ Corresponding author. Tel.: +359 2 979 3154; fax: +359 2 87 00 109. E-mail address: blaga@microbio.bas.bg (B. Angelova). The microbial 9-hydroxylation of 4-androstene-3,17- dione (androstenedione, AD) into 9-hydroxy-4-androstene- 3,17-dione (9-hydroxy-androstenedione, 9-OH-AD) is a key process for manufacturing of corticosteroids [1]. It was shown to be effectively performed by growing [9] and rest- ing Rhodococcus sp. cells [10,12] according to the scheme on Fig. 1. It is important to note that both the 9-hydroxylating and the  1 -dehydrogenating activities of Rhodococcus sp. cells are inducible. Moreover, the process of their induction takes place in the C/N deficient transformation medium and proceeds consecutively, and the synthesis of the  1 -steroid dehydrogenase follows the synthesis of the 9-steroid hy- droxylase. Thus, the efficiency of the hydroxylating reaction could be increased to nearly stoichiometric values by block- ing the development of the undesired  1 -steroid dehydro- genating activity [12]. This paper reports the successful 9-hydroxylation of 4-androstene-3,17-dione carried out by resting Rhodococ- cus sp. cells in the presence of the water immiscible or- ganic solvent bis-(2-ethylhexyl) phthalate (phthalate). The paper summarises the experimental results obtained in both mono-phase phthalate medium and in two-phase media com- 0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.04.014