Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester Elif Yilmaz, Keziban Can, Mehmet Sezgin, Mustafa Yilmaz ⇑ Department of Chemistry, Faculty of Science, Selcuk University, Konya 42075, Turkey article info Article history: Received 2 June 2010 Received in revised form 22 August 2010 Accepted 23 August 2010 Available online 26 August 2010 Keywords: Candida rugosa lipase Glass beads Immobilization Enantioselective hydrolysis S-Naproxen abstract Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol–gel encapsulation procedure and improved considerably by fluo- ride-catalyzed hydrolysis of mixtures of RSi(OCH 3 ) 3 and Si(OCH 3 ) 4 . The catalytic properties of the immo- bilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher con- version and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Candida rugosa lipase (CRL) is an important industrial lipase and due to its wide substrate specificity it is successfully utilized in a variety of hydrolysis and esterification reactions. The synthesis of several pharmaceuticals could be made possible due to its high ste- reoselectivity and regioselectivity (Takac and Bakkal, 2007). The versatility of lipase-catalyzed reactions has made it a unique industrial biocatalyst in drug synthesis, food and flavor making, and recently in the cosmetics and perfumery industries (Hung et al., 2003). A lot of effort is still being devoted to the search for new support materials and novel techniques. The type of support as well as the method of immobilization influences the activity and operational stability of immobilized lipases. By an appropriate choice of the immobilization process, operational costs of indus- trial processes involving lipases can be significantly reduced. The extent of stabilization depends upon the enzyme structure, the immobilization method, and the type of support (Mateo et al., 2007; Ozmen et al., 2009a). Lipases have been immobilized on var- ious supports by physical adsorption, covalent binding, ionic inter- actions or by entrapment (Yilmaz et al., 2009; Mateo et al., 2002, 2003; Guisan, 2006). A well-established sol–gel processing tech- nique consists in hydrolyzing adequate precursors in aqueous solu- tions to produce soluble hydroxylated monomers, followed by polymerization and phase separation to produce a hydrated metal or semi-metal oxide hydrogel (Brinker and Scherer, 1990). Re- moval of water from the wet gel, which is usually accompanied by changes in the structure of the pores and of the gel’s network, results in a porous xerogel. The most widely used precursors are al- kyl-alkoxysilanes. These precursors were used already in the mid- 1980s to prepare organically modified silicates (Ormosils) for the successful encapsulation of antibodies and enzymes (Avnir et al., 1994). Reetz et al. (2003) shown that hydrophobic sol gels may im- prove the activity of immobilized lipases (due to the hyperactiva- tion of these enzymes in the presence of these surfaces). They used the 18-crown-6, Celite or Tween 80 as additive on lipase immobilization process and reported that the sol–gel lipase immo- bilizates were excellent catalysts in the kinetic resolution of chiral alcohols and amines, recycling without any substantial loss in enantioselectivity. A large number of researchers have made studies with glass beads as enzyme carriers, which are inexpensive and renewable materials (Bhushan et al., 2008; Gomez et al., 2006). a-Amylase was covalently immobilized onto phthaloyl chlo- ride-containing amino group functionalized glass beads. The immobilized a-amylase exhibited better thermostability than the free one (Kahraman et al., 2007). The glutaraldehyde technique is very versatile and may be used in very different fashions (Betancor et al., 2006; Migneault, 2004). However, in terms of stabilization, the treatment with glutaraldehyde of proteins previously adsorbed in supports bearing primary amino groups offers in many cases very good results, because permit the crosslink between glutaral- dehyde molecules bound to the enzyme and glutaraldehyde mole- cules bound to the support. However, it implies the chemical modification of the whole enzyme surface (López-Gallego et al., 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.08.083 ⇑ Corresponding author. Tel.: +90 332 2233873; fax: +90 332 2412499. E-mail address: myilmaz42@yahoo.com (M. Yilmaz). Bioresource Technology 102 (2011) 499–506 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech