Semi-interpenetrating polymer networks (semi-IPNs) for entrapment of laccase and their use in Acid Orange 52 decolorization Ozgur Yamak a , N. Ayca Kalkan a , Serpil Aksoy a, *, Haydar Altinok b , Nesrin Hasirci c a Department of Chemistry, Faculty of Arts and Sciences, Gazi University, Teknikokullar, 06500 Ankara, Turkey b Department of Chemistry, Faculty of Arts and Sciences, Kırıkkale University, 71450 Kırıkkale, Turkey c Department of Chemistry, Faculty of Arts and Sciences, Middle East Technical University, 06351 Ankara, Turkey 1. Introduction Immobilization of enzymes on a solid support is a widely used technique because of their continuous or repeated use in industrial applications mainly in biotechnology, biomedicine and food technology [1,2]. Immobilization increases the stability and durability, and provides rapid separation of the enzyme from the reaction media leading economical operations. Although various polymeric supports can be used for immobilization of several enzymes [3–7], biological polymers have certain advan- tages such as low cost, nontoxic and biocompatible property, easy interactions with enzymes through their functional groups. Hydrogels of natural polymers such as gelatine, alginate, chitosan, xanthan, and agarose were used conveniently in both wet and dried states, although, these supports suffer from low mechanical strength and ease of microbial degradation [8,9]. Mechanical strength of the corresponding hydrogels can be enhanced by preparing interpenetrating polymer networks (IPNs) or semi- interpenetrating polymer networks (semi-IPNs) [10]. IPNs have been defined as combination of two polymers, each in network form, at least one which has been synthesized and/or cross-linked in the presence of the other. Semi-IPNs can be prepared by dissolving a preformed linear polymer in hydrophilic monomer and cross-linking agent mixture which is subsequently polymer- ized. In this way, a synthetic network is formed around primary polymer chain which is modifying the behavior of the hydrogel. Laccase (E.C.1.10.3.2 benzenediol:oxygen oxidoreductase) is a multi-copper protein capable of oxidizing various aromatic substrates by reducing molecular oxygen to water. This enzyme occurs in many species of fungi, bacteria and higher plants [11,12]. The substrate range is wide and includes several polyphenols, aromatic amines, and aminophenols [13]. Because of its low substrate specificity and its ability to oxidize many pollutants, laccase is a potentially attractive catalyst for bioremediation of environmental pollutants (especially organic substances such as chlorophenols or aromatic hydrocarbons of petrochemical indus- try; toxins of olive oil mill wastewater; etc.) [14–18]; delignifica- tion in paper industry [19]; and dye decolorization in textile industry [20]. Decolorization of waste dyestuffs released from various industries is an important problem since most dyes are quite resistant to degradation and their presence in aqueous environment may create crucial health hazard. Since most of them are highly stable to chemicals or light, their removal from the wastewater is not an easy process while even low concentrations Process Biochemistry 44 (2009) 440–445 ARTICLE INFO Article history: Received 25 August 2008 Received in revised form 14 November 2008 Accepted 10 December 2008 Keywords: Semi-interpenetrating polymer networks Enzyme immobilization Laccase Entrapment Decolorization Acid Orange 52 ABSTRACT Laccase enzyme (L) from Trametes versicolor was entrapped in three hydrogel structures namely poly(acrylamide-N-isopropylacrylamide), P(AAm-NIPA), and semi-interpenetrating networks of poly(- acrylamide)/alginate, P(AAm)/Alg, and poly(acrylamide-N-isopropylacrylamide)/alginate, P(AAm- NIPA)/Alg. The optimum temperatures for free and all immobilized systems were found to be 40 8C. For free and immobilized laccase systems of P(AAm-NIPA)-L, P(AAm)/Alg-L and P(AAm-NIPA)/Alg-L, K m values were found to be 6.7  10 3 , 8.8  10 2 , 5.5  10 2 and 1.8  10 2 mM; V max values were calculated as 1.8  10 3 , 2.5  10 2 , 1.5  10 2 and 6.1  10 3 mM min 1 , respectively. For free and the same immobilized systems, the enzymes retained 42%, 91%, 79% and 86% of their initial activities at the end of 56 days of storage. After using the mentioned immobilized systems repeatedly 10 times, they retained 77%, 71% and 84% of their original activities, respectively. For free and the same immobilized systems, decolorization of Acid Orange 52 (AO52) in 6 h were found to be 63%, 50%, 48% and 66%, respectively. Addition of 2,2 0 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), ABTS, into the assay medium increased these values up to 73%, 73%, 74% and 75%, respectively. ß 2008 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +90 312 2021103; fax: +90 312 2122279. E-mail address: seraksoy@gazi.edu.tr (S. Aksoy). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.12.008