Improvement of catalytic activity of lipase from Candida rugosa via sol–gel encapsulation in the presence of calix(aza)crown Arzu Uyanik, Nejdet Sen, Mustafa Yilmaz ⇑ Department of Chemistry, Selcuk University, Konya 42075, Turkey article info Article history: Received 21 October 2010 Received in revised form 23 December 2010 Accepted 27 December 2010 Available online 1 January 2011 Keywords: Candida rugosa lipase Enantioselective hydrolysis Sol–gel encapsulation Calix(aza)crowns Naproxen abstract Lipase from Candida rugosa (CRL) was encapsulated within a chemically inert sol–gel support in the pres- ence of calix(aza)crowns as the new additives. The catalytic activity of the encapsulated lipases was eval- uated both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yields of the calix(aza)- crown based encapsulated lipases were higher than that of the free lipase. Improved enantioselectivity was observed with the calix(aza)crown-based encapsulated lipases as compared to encapsulated free lipase. The reaction of Naproxen methyl ester resulted in 48.4% conversion for 24 h and 98% enantiomeric excess for the S-acid, corresponding to an E value of >300 (E = 166 for the encapsulated free enzyme). Moreover, the encapsulated lipases were still retained about 18% of their conversion ratios after the sixth reuse in the enantioselective reaction. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are widely used as versatile biocatalysts in industrial biotechnology and mod- ern organic chemistry. Depending on the nature of substrate and reaction conditions, lipases catalyze a wide range of enantio- and regio-selective reactions such as hydrolysis, esterifications, transe- sterifications, aminolysis and ammonolysis (Vaidya et al., 2008; Liljeblad et al., 2010). Many efforts have been made over the years with the aim of improving catalytic activity and operational stability of industrial enzymes through immobilization. Enzyme immobilization is also known to offer several advantages such as reusability, ease of prod- uct separation, greater control over catalysis and process. Lipases from several resources have been immobilized on different sup- ports either by covalent binding (Da Rós et al., 2010), physical adsorption (Tzialla et al., 2010), ionic interactions or by entrap- ment (Reetz et al., 2003; Yilmaz et al., 2011). Several factors includ- ing choice of support and selection of an immobilization strategy may affect the activity, recovery and reusability of enzymes in an immobilization process (Soares et al., 2001; Yang et al., 2009). It is also known that immobilization strategies may influence the catalytic and enantioselective properties of the enzyme (Palomo et al., 2002; Barbosa et al., 2010). Therefore, use of various immo- bilization strategies may provide immobilized lipases with differ- ent activity/selectivity characteristics (Reetz et al., 2003; Mateo et al., 2007). Among the available immobilization methods, entrapment of enzymes in an inorganic polymer matrix is a method that has received a considerable attention in recent years. This method which was pioneered by Avnir et al. (1994) is based on sol–gel processes. The application of the sol–gel material in the immobili- zation of lipases is well documented (Reetz, 1997). This approach has several advantages because mechanical entrapment of enzymes using sol–gel materials allows stabilization of the protein tertiary structure caused by the tight gel network. Moreover, good results were obtained in a number of studies in which enzyme entrapment concerns the use of inorganic matrices such as silica gel (Soares et al., 2006). Research in the area of sol–gel based enzyme immobilization has encompassed several aspects, such as the chemical composition of the sol–gel, catalyst type, water to precursors ratio, and drying and aging processes. The effect of additives on the activity of immobilized lipase preparation by sol–gel process has also been investigated in the literature. Many additives such as polyethyleneglycol, glycerol, polyvinylalcohol, ionic liquids, sugars, crown ethers, cyclodextrins, calixarenes etc. have already been tested (Reetz et al., 2003; Sahin et al., 2009; Yilmaz et al., 2010). The calixarenes are an important class of macrocycles widely used in supramolecular chemistry. They are basically cyclic oligo- mers built with phenol units through methylene bridges. They are recognized as molecular receptors and form a variety of com- plexes with metal ions, anions, and neutral molecules (Gutsche, 1998). They can be easily functionalized both at the phenolic OH 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.12.105 ⇑ Corresponding author. Tel.: +90 332 2233873; fax: +90 332 2412499. E-mail address: myilmaz42@yahoo.com (M. Yilmaz). Bioresource Technology 102 (2011) 4313–4318 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech