Process Biochemistry 46 (2011) 1688–1692 Contents lists available at ScienceDirect Process Biochemistry jo u rn al hom epa ge: www .elsevier.com/locate/procbio Short communication A novel nanoprotein particle synthesis: Nanolipase Rıdvan Say a,∗ , Rüstem Kec¸ ili a , Özlem Bic¸ en a , Filiz Yılmaz S¸ is¸ man a , Deniz Hür a , Adil Denizli b , Arzu Ersöz a a Department of Chemistry, Anadolu University, Eskis¸ ehir, Turkey b Department of Chemistry, Hacettepe University, Ankara, Turkey a r t i c l e i n f o Article history: Received 4 January 2011 Received in revised form 22 April 2011 Accepted 26 April 2011 Keywords: Nanolipase Photosensitive microemulsion polymerization Photosensitive cross-linking Proteinous polymeric particles a b s t r a c t Lipases (triacylglycerol acylhydrolases; E.C. 3.1.1.3) constitute a group of enzymes defined as car- boxylesterases that catalyse the hydrolysis (and synthesis) of long-chain acylglycerols at the lipid–water interface. In this study, a novel method has been developed to prepare nanoprotein particles carrying lipase using a photosensitive microemulsion polymerization process. The nanostructured lipases with photosensitive features have been characterized by transmission electron microscopy (TEM) and Zeta Sizer. The average particle size of nanolipases was found to be about 100 nm. Lipase nanoparticles were used in hydrolysis of paranitrophenyl palmitate (p-NPP) and the results were compared with free lipase. The parameters like pH, temperature and hydrolytic activity that affect p-NPP hydrolysis were investi- gated by using lipase nanoparticles and compared to free lipase. Lastly, reusability of lipase nanoparticles was investigated and according to our results, this novel lipase nanoparticles showed admirable potential in reusable catalyst. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Lipases (glycerol ester hydrolase, E.C. 3.1.1.3) have been widely used for enzymatic conversion in the various biotechnological applications in dairy industry, manufacturing of specialty chemi- cals, organic synthesis, and preparation of enantiomerically pure pharmaceuticals as they combine a broad range of substrate speci- ficity with a high regio- and enantio-selectivity for many reactions [1–3]. In most cases, especially in industrial applications, enzymes are preferably used in their immobilized states owing to many advantages, such as improved activity and stability of the immobi- lized enzyme, ready reutilization of the catalyst and possibility of continuous process operation. To date, numerous efforts have been towards the development of enzyme immobilization techniques, e.g. covalent attachment [4,5], physical adsorption [6] and encap- sulation [7–15]. Adsorption techniques are easy to perform, but the interactions between the enzyme and the support is relatively weak. Such biocatalysts are usually not stable enough for a long- term utilization. Covalent attachment normally leads to improved enzyme stability. However, partial deactivation is often obtained due to the conformational restrictions by the covalent bonding of enzyme moieties to the support [16]. ∗ Corresponding author at: Anadolu Universitesi, Fen Fakültesi, Kimya Bölümü, Yunus Emre Kampüsü, 26470 Eskis¸ ehir, Turkey. Tel.: +90 222 3350580x4823; fax: +90 222 3204910. E-mail address: rsay@anadolu.edu.tr (R. Say). Chemical conjugation of enzymes with synthetic polymers enables their colloidal stability or thermal stability to be enhanced, and their enzyme activity to be controlled [17–20]. However, chemical conjugation has the difficulty that the synthesis is not always easy, and that conjugation sometimes induces a decrease in the enzyme activity [20]. Bioconjugation is a useful technique for modifying the function of enzymes for pharmaceutical and biotechnological applications. The biconjugation approach entails the advantages of easy preparation and versatility for various pro- teins [21–23]. In this study, nanostructured lipase was prepared by the photo- sensitive cross-linking microemulsion polymerization technique. This technique is a new technique which allows the synthesis of nanoparticles with narrow size distribution [24]. Microemulsion polymerization is a complex heterogeneous process where trans- port of monomers, free radicals and other species (such as chain transfer agent, co-surfactant and inhibitors) between the aqueous and organic phases, takes place [25]. The polymerization rate is controlled by monomer partitioning between the phases, parti- cle nucleation, adsorption and desorption of radicals. The particle stability is affected by the amount and type of surfactant and the pH of the dispersing medium [26]. Although microemulsion polymerization is a complex heterogeneous process, but in this study, photosensitive cross-linking technique become more easy and efficient by using the particles which were synthesized accord- ing to ANADOLUCA (AmiNoAcid (monomer) Decorated and Light Underpinning Conjugation Approach) method. The proteinous nanoparticles prepared by photosensitive cross-linking were 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.04.011