Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: Application for textile dye degradation

Abstract

Non-porous poly(glycidyl methacrylate/ethyleneglycol dimetacrylate) (poly(GMA/EGDMA)) beads were prepared by suspension polymerization. The enzyme (i.e. laccase) was covalently immobilized onto plain and spacer-arm attached poly(GMA/EGDMA) beads. The amount of immobilized enzyme on the plain and spacer-arm attached beads was determined as 5.6 and 4.9 mg/g, respectively. The maximum activity (V_max) and Michaelis constant (K_m) of laccase immobilized on the spacer-arm attached beads, were found to be 77.6 U/min and 0.47 mM, respectively. Finally, the immobilized laccase was operated in a batch system, and textile dye Reactive Red 120 was successfully decolorized in the enzyme reactor.

Introduction

Immobilization can be defined as the fixation of the biocatalysts (e.g. enzymes, microorganisms and organelles) to insoluble solid supports. The most important advantage of immobilization is that it makes continuous bioreactor operations possible and this is especially useful in the production of fine chemicals and bio-treatment of industrial and agricultural wastes (Mosbach et al., 1976, Yoshida et al., 1979, Arica and Bayramoglu, 2004, Arica et al., 1999, Arica et al., 2004). A large number and quantity of synthetic dyes are being increasingly used in the textile, paper, pharmaceutical, cosmetics and food industries. Over 7 × 10⁵ t of approximately 10,000 different dyes and pigments are produced annually worldwide, of which about 50,000 t are discharged into the environment (Lewis, 1999, Slokar and Majcen Le Marechal, 1998). Hence, removal of these textile dyes from textile industry effluent is a critical issue. The removal could be effectively achieved by adsorption and electrochemical degradations, however, these processes may generate hazardous by-products (Gianfreda et al., 2003, Ciullini et al., 2008, Soares et al., 2001). The applications of laccase and other oxidative enzymes on specialty wastewater treatment were increasingly reported (Gianfreda et al., 2003, Ko and Chen, 2008). Recently, enzymatic oxidative polymerization has been achieving much interest in the treatment of wastewaters containing toxic aromatic compounds (Tanaka et al., 2003; Bayramoglu and Arica, 2008).

Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) is a multi-copper oxidase produced by many plants and numerous fungi and it is able to oxidize a wide range of xenobiotic compounds such as synthetic dyes, chlorinated phenolics, and polycyclic aromatic hydrocarbons. Compared to other enzymes, laccase is reported to be rather unspecific and a wide range of substances are suitable for laccase for different application processes (Aktas and Tanyolac, 2003, Bayramoglu and Arica, 2008, Rittstieg et al., 2002, Ikehata et al., 2004) has been extensively reported.

In immobilized enzyme system, the most important criterion is the diffusion of the substrate that is the target molecules for enzymatic reaction, within the reaction medium. Diffusivity of the substrate must be carefully evaluated and the construction of the enzymatic reaction system should be carried out accordingly (Arica, 2000, Shao et al., 2007, Bayramoglu et al., 2004). Non-porous materials, to which enzymes are attached to the surfaces, are subject to minimum diffusion limitation while enzyme loading per unit mass of support is usually low. Whereas, porous materials can afford high enzyme loading, but suffer a much greater diffusional limitation to large molecular weight substrate (Yang et al., 2008, Bayramoglu et al., 2005, Arica and Bayramoglu, 2006). On the other hand, if an enzyme is coupled directly to the polymeric support, steric hindrance between the support surface and the immobilized enzyme will occur (Fernandez-Lorente et al., 2007). For this reason, enzyme molecules should be moved away from the support surface to some extent using a spacer-arm (Arica et al., 2004, Arica, 2000).

In this study, the activity behavior of the laccase immobilized on both plain poly(GMA/EGDMA) and poly(GMA/EGDMA)-DAH-GA beads were investigated. The modified beads with the immobilized enzyme were operated in a batch system for the degradation of a model textile reactive dye (i.e., Reactive Red 120).