596 † To whom correspondence should be addressed. E-mail: bschun@pknu.ac.kr Korean J. Chem. Eng., 27(2), 596-601 (2010) DOI: 10.1007/s11814-010-0063-4 RAPID COMMUNICATION Transfer rate measurement of lysozyme by liquid-liquid extraction using reverse micelles with dense CO 2 Sun-Mi Jung*, Un-Mi Shin*, Md. Salim Uddin*, Sun-Young Park**, Hideki Kishimura***, Gordon Wilkinson****, and Byung-Soo Chun* ,† *Institute of Food Science, Faculty of Food Science & Biotechnology, Pukyong National University, Busan 608-737, Korea **School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5001, Australia ***Research Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan ****School of Chemical Engineering, University of Adelaide, Adelaide, SA 5005, Australia (Received 23 March 2009  accepted 24 May 2009) Abstract −Lysozyme was extracted from aqueous solution into i-octane using reverse micelles in the presence of pressurized CO 2 . A squat vessel with two independent stirrers was used to measure the mass transfer of the lysozyme across a planar interface. Mass transfer coefficient, k L of the lysozyme from the aqueous to the organic phase was meas- ured at selected ionic strengths, pH, sodium bis(2-ethylhexyl) sulfosuccinate (AOT) surfactant concentrations, tempera- tures and pressurized CO 2 . The mass transfer rate of lysozyme was higher in high temperature (318 K) and pressure (20 MPa). pH of 9 in aqueous phase showed highest mass transfer rate of lysozyme. The application of pressurized CO 2 markedly increased the mass transfer rate of lysozyme comparing to conventional non-pressurized system. Key words: Lysozyme, Reverse Micelle, Mass Transfer, Pressurized CO 2 , Sodium Bis(2-ethylhexyl) Sulfosuccinate (AOT) INTRODUCTION With recent developments in biotechnology, protein separation using reverse micelles is considered for commercial applications. The separation of proteins using reverse micelles is rather easy to scale up and can be operated continuously [1]. Organic solvents containing reverse micelles have great potential as novel media for bioseparation and biocatalysis [2,3]. In particular, protein extraction using reverse micelles has gained much attention because liquid- liquid extraction can be performed, which is especially attractive for use in large-scale, continuous processing [4,5]. In a reverse micellar extraction process, water soluble proteins are transferred from an aqueous phase to an organic one containing a surfactant, in the form of micelles. Mass transfer rates and other characteristics need quantitation to support design studies enabling commercial utilization of this technique. Some research works have been carried out on the interfacial transport of proteins across an aqueous-organic interface. Dekker et al. [6] investigated mass transfer rates in the extraction of α-amylase with a reverse micelle phase of the cationic surfactant trioctylmethylammonium chloride (TOMAC) in i -octane. Dungan et al. [7] studied the transport of α-chymot- rypsin and cytochrome C between a bulk aqueous and an AOT- i - octane reverse micelle phase. The mass transfer processes in lyso- zyme extraction by AOT- i -octane reverse micelles were also stud- ied by Lye et al. [8]. In this work lysozyme was selected as a model protein. Lysozyme is an industrially useful enzyme. Egg-white, which contains 3.5 % (w/w) lysozyme, is a convenient source [9] and the anionic surfac- tant, AOT, used in this process, is known to form spherical nanom- eter-sized molecular aggregates in a variety of non-polar solvents. The work has been done under an atmosphere of high-pressure CO 2 which, by dissolving in the i -octane, caused expansion of the organic phase [10]. Carbon dioxide as a supercritical fluid has many excel- lent properties such as non-toxicity, non-explosiveness, ready avail- ability and easy of removal from the extracted products [11]. In con- trast to previous work using CO 2 , a significant effect was observed on the rate of transport of protein into the reverse micellar system. The purpose of this study was to explore the recovery efficiency at different conditions by measuring the mass transfer rates of lyso- zyme from a bulk aqueous phase via reverse micelles to a bulk or- ganic phase which had been expanded by pressurized CO 2 . EXPERIMENTAL 1. Materials AOT (99%) was obtained from Aldrich Co. (USA), AR grade i -octane (99%) from Junsei Chemical Co. (Japan) and lysozyme (mucopeptide N-acetyl muramyl hydrolase, E. C. 3.2.1.17, molec- ular weight 14.3 kDa, pI 11.1) from hen egg-white obtained from Sigma Chemical Co. (USA). All other reagents were AR grade from Sigma. Aqueous solutions were prepared by dissolving each of lysozyme (0.2 g/L) and KCl (0.1 M to 0.4 M, used to regulate ionic strengths) in Milli-Q water. Organic solutions were prepared by dissolving the desired amount of AOT in i -octane. The pH of aqueous solutions was adjusted within the range of 3.8 to 11.3 by addition of 0.1 M HCl or 0.1 M NaOH. The purity of CO 2 was 99%. 2. Method The extraction of lysozyme was performed in a liquid-liquid flat vessel designed by Lewis [12] using the high-pressure apparatus shown in Fig. 1. Mass transfer rates were measured in a stirred cy-