In situ encapsulation of laccase in microfibers by emulsion electrospinning: Preparation, characterization, and application Yunrong Dai, Junfeng Niu * , Jia Liu, Lifeng Yin, Jiangjie Xu State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, PR China article info Article history: Received 8 May 2010 Received in revised form 6 July 2010 Accepted 8 July 2010 Available online 13 July 2010 Keywords: Emulsion electrospinning Microfiber membranes Laccase Enzyme immobilization Biodegradation abstract Laccase from Trametes versicolor was successfully in situ encapsulated into the poly(D,L-lactide) (PDLLA)/ PEO–PPO–PEO (F108) electrospun microfibers by emulsion electrospinning. The porous morphology of electrospun microfibers was observed with scanning electron microscope, and the core–shell structure of microfibers and existence of laccase in microfibers were proved by laser confocal scanning microscopy micrograph. In this study, fibrous porosity and core–shell structure are advantageous to the activity and stability preservation of immobilized laccase. The activity of immobilized laccase could retain over 67% of that of the free enzyme. After 10 successive runs in the enzyme reactor, the immobilized laccase could also maintain 50% of its initial activity. Crystal violet dye was successfully degraded by the PDLLA/ F108-laccase electrospun microfiber membranes. It was observed that the immobilized laccase possessed a broadening pH range of catalysis activity compared to free laccase. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Laccase (p-diphenol: dioxygen oxidoreductases, EC 1.10.3.2) is a copper-containing oxidase, which is reported to be rather unspe- cific and has a wide range of substances. It is able to catalyze one- electron oxidation of various dyes, phenols, chlorinated phenols, aromatic substrates, and a series of other oxidizable compounds, with concomitant reduction of dioxygen to two molecules water (Monje et al., 2010; Arica et al., 2009). These characteristics of lac- case endow it with potential applications in various fields, such as textile, pulp and paper industry, biodegradation and biotransfor- mation of pollutant, etc. (Fillat et al., 2010; Arica et al., 2009). One of the main drawbacks of using free laccase in wastewater treatment is that it easily loses its activity in aqueous solutions, which limits its use. Immobilization of laccase has been a popular strategy for biotransformation applications of laccase, since it of- fers advantages over the use of free one, including increase in sta- bilities of enzymes, better control of the enzymatic reaction, and permission to repeated use (Qiu et al., 2009; Zhu et al., 2007). Among the various immobilization techniques such as adsorption (Córdova et al., 2009), crosslinking (Kim et al., 2005), encapsulation (Patel et al., 2006) and so on, the encapsulation method was con- sidered as a good choice for enzyme immobilization, as it may per- mit to have the enzyme molecules fully embedded in the supports and without the possibility of interacting with the external inter- face (Mateo et al., 2007). Emulsion electrospinning, as a novel and simple method of encapsulating enzymes in situ, has sparked increasing interest in recent years (Li et al., 2010; Xu et al., 2005). It can prepare the core–shell structured fibers by electrospinning either a water-in-oil (W/O) or an oil-in-water (O/W) emulsion, and the enzyme as the water phase of emulsion can be directly encapsu- lated into the core of electrospun fibers. Emulsion electrospin- ning had been successfully applied to the in situ encapsulation of lysozyme and BSA into the core of electrospun fibers, and the core–shell structure of fibers could protect the structural integrity and bioactivity of encapsulated protein (Li et al., 2010; Yang et al., 2008). Moreover, the microfiber membranes obtained from electrospinning possess many extraordinary prop- erties, including high surface-to-volume ratio, porous structure, which is favorable for the improvement of the mass-transfer rate of substrate to the active sites of enzymes; and superior mechan- ical properties, which make the microfiber membranes easy to be recovered from a reaction solution (Greiner and Wendorff, 2007; Li and Xia, 2004). Thus, electrospun microfiber membranes (EMFMs) themselves are viewed as the excellent supports for en- zymes immobilization (Wang et al., 2009), and emulsion elec- trospinning is also considered to be an efficient method for in situ immobilization of enzyme. However, in previous studies, the model enzymes were mainly lysozyme (Yang et al., 2008), a-chymotrypsin (Herricks et al., 2005) and so on, and the applications were mostly confined to tissue engineering and enzyme release. There are few reports about immobilized laccase by emulsion electrospinning for treatment of environmental pollutants so far. 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.07.027 * Corresponding author. Tel./fax: +86 10 5880 7612. E-mail address: junfengn@bnu.edu.cn (J. Niu). Bioresource Technology 101 (2010) 8942–8947 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech