ARTICLE Enantioselectivity of Candida rugosa Lipases (Lip1, Lip3, and Lip4) Towards 2-Bromo Phenylacetic Acid Octyl Esters Controlled by a Single Amino Acid Rungtiwa Piamtongkam, 1,2,3,4 Sophie Duquesne, 1,2,3 Florence Bordes, 1,2,3 Sophie Barbe, 1,2,3 Isabelle Andre ´, 1,2,3 Alain Marty, 1,2,3 Warawut Chulalaksananukul 5,6 1 Universite ´ de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; telephone: 33-5-61559439; fax: 33-5-61559400; e-mail: alain.marty@insa-toulouse.fr 2 CNRS, UMR5504, Toulouse, France 3 INRA, UMR792, Inge ´nierie des Syste `mes Biologiques et des Proce ´de ´s, Toulouse, France 4 Program in Biotechnology, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok, Thailand 5 Faculty of Science, Department of Botany, Chulalongkorn University, 254 Phyathai Road, Patumwan, Bangkok 10330, Thailand; telephone: 66-2821-5482; fax: 66-2821-5482; e-mail: warawut.c@chula.ac.th 6 Biofuels by Biocatalysts Research Unit, Faculty of Science, Department of Botany, Chulalongkorn University, Patumwan, Bangkok, Thailand Received 5 January 2011; revision received 25 February 2011; accepted 28 February 2011 Published online 9 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.23124 ABSTRACT: Enantiomer discrimination by enzymes is a very accurate mechanism, which often involves few amino acids located at the active site. Lipase isoforms from Candida rugosa are very good enzymatic models to study this phe- nomenon as they display high sequence homology (>80%) and their enantioselectivity is often pointed out. In the present work, we investigated three lipases from C. rugosa (Lip1, Lip3, and Lip4, respectively) towards the resolution of 2-bromo-arylacetic acid esters, an important class of che- mical intermediates in the pharmaceutical industry. All exhibited a high enantioselectivity, with Lip4 preferring the R-enantiomer (E-value ¼ 15), while Lip1 and Lip3 showed an S-enantioselectivity >200. A combination of sequence and structure analysis of the three C. rugosa lipases suggested that position 296 could play a role in S- or R- enantiomer preference of C. rugosa lipases. This led to the construction by site-directed mutagenesis of Lip1 and Lip4 variants in which position 296 was, respectively, exchanged by a Gly, Ala, Leu, or Phe amino acid. Screening of these variants for their enantioselectivity toward 2-bromo phenyl acetic acid octyl esters revealed that steric hindrance of the amino acid residue introduced at position 296 controls both the enantiopreference and the enantioselectivity value of the lipase: bulkier is the amino acid at position 296, larger is the selectivity towards the S-enantiomer. To investigate further these observations at an atomic level, we carried out a preliminary modeling study of the tetrahedral intermediates formed by Lip1 and Lip4 with the (R, S)-2-bromo-pheny- lacetic acid octyl ester enantiomers that provides some insight regarding the determinants responsible for lipase enantiodiscrimination. Biotechnol. Bioeng. 2011;108: 1749–1756. ß 2011 Wiley Periodicals, Inc. KEYWORDS: lipase; enantioselectivity; rational engineer- ing; Yarrowia lipolytica; Candida rugosa Introduction The use of racemic mixtures in pharmaceutical drugs is strongly regulated as one enantiomer can be the active molecule whereas the other enantiomer might be toxic (Smith, 2009). Thorough investigations are thus requested on the toxicological effects of each enantiomer before they can be accepted in drug composition. The commer- cialization of a single pure enantiomer is therefore often privileged. Nevertheless, classical methods used to separate Correspondence to: Alain Marty and Warawut Chulalaksananukul Contract grant sponsor: The Royal Golden Jubilee PhD Program Contract grant sponsor: Thailand Research Fund Contract grant number: PHD/0235/2547 Contract grant sponsor: French Embassy ß 2011 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 108, No. 8, August, 2011 1749