Current Biotechnology Send Orders for Reprints to reprints@benthamscience.net Current Biotechnology, 2019, 8, 3-14 3 RESEARCH ARTICLE Immobilization of Purified Fungal Laccase on Cost Effective Green Coconut Fiber and Study of its Physical and Kinetic Characteristics in Both Free and Immobilized Form Priyanka Ghosh 1 and Uma Ghosh 1,* 1 Department of Food Technology & Biochemical Engineering, Jadavpur University, Kolkata-700032, West Bengal, India A R T I C L E H I S T O R Y Received: January 03, 2019 Revised: January 27, 2019 Accepted: January 28, 2019 DOI: 10.2174/2211550108666190201151816 Abstract: Background: Laccases are important enzymes that have numerous applications in different biotechnological sectors. Objective: The aim was to purify laccase from Aspergillus flavus PUF5, successfully immobilize it on coconut fiber and characterize different physical and kinetic properties under both free and immobilize conditions. Methods: Laccase from A. flavus PUF5 was purified using ammonium sulfate precipitation, followed by DEAE column chromatography and gel filtration using Sephadex G100. The molecular weight was determined through SDS-PAGE (12%). It was immobilized on pretreated coconut fiber through cross- linking by glutaraldehyde (4% v/v). Physical and kinetic parameters like optimum temperature, pH, thermostability, the effect of additives, activation energy, K m and V max for free and immobilized lac- case were also analyzed. Recycling stability of the immobilized laccase was further determined. Results: The extracellular laccase (65 kDa) was purified up to homogeneity and was immobilized on acid-pretreated coconut fiber by 4% (v/v) glutaraldehyde solution at 30°C, pH 5.0. Activation energy (E a ) of free and immobilized laccase for oxidation of guaiacol was found to be 24.69 and 32.76 kJ mol -1 respectively. Immobilized laccase showed higher melting temperature (T m ) of (82.5°C) than free en- zyme (73°C). Km and Vmax for free and immobilized laccase were found to be 0.67 mM, 0.70 mM and 280 U/mg, 336 U/mg respectively when guaiacol was used as substrate. Additionally, in immobi- lized condition laccase retained ˃80% of its initial activity after use till six repeated cycles. Conclusion: The purified laccase enzyme and the cheap immobilization seem to be a prospective proc- ess for different biotechnological and industrial applications. Keywords: Laccase, Aspergillus flavus PUF5, purification, coconut fiber, glutaraldehyde, immobilization, characterization. 1. INTRODUCTION Laccases (EC 1.10.3.2) are multicopper oxidases that are able to catalyze the oxidation of a broad range of aromatic organic and inorganic compounds, which include di and polyphenols, substituted phenols, aromatic amines, diamines etc. Molecular oxygen acts as the electron acceptor in these oxidation reactions and gets reduced to water [1]. The sub- strates lose a single electron and form an unstable free radi- cal which may undergo further laccase based oxidation or *Address correspondence to this author at the Department of Food Technol- ogy & Biochemical Engineering, Jadavpur University, Kolkata-700032, West Bengal, India; Tel: 91-33-2414-6663; Fax: 91-33-2414-6822; E-mail: ughoshftbe@yahoo.co.in non-enzymatic reactions like hydration, disproportionation, and polymerization. The presence of laccases has been widely observed in plants, insects, bacteria and fungi which are actively engaged in pathogenesis, pigmentation, melanin production and spore coat resistance, degradation of lignin, etc. But among them, fungal laccases mainly from Ascomycetes, Basidiomycetes, and Deuteromycetes are studied most extensively. Fungal laccases are reported to catalyze the polymerization, de- polymerization, methylation or demethylation reactions in different phenolic compounds [2]. Therefore, fungal laccases have been widely implemented in industrial and biotechno- logical sectors, such as lignocellulose delignification, bleach- ing of paper and pulps, dye decolorization, detoxification of 2211-551X/19 $58.00+.00 © 2019 Bentham Science Publishers