Design and evaluation of new ligands for lysozyme recovery by affinity thermoprecipitation

Abstract

Ligands containing acetamido group and a spacer were conjugated with an acrylic monomer and copolymerized with N-isopropylacrylamide (NIPAM) to yield a thermoprecipitating polymer. The ability of the ligand to bind to lysozyme, which is the first step in the separation of lysozyme, is quantified in terms of I₅₀, the ligand concentration required to achieve 50% of the maximum attainable inhibition of lysozyme. The copolymers containing acetamido groups inhibit lysozyme far more efficiently than the corresponding polymers containing N-acetylglucosamine, the natural inhibitor for lysozyme. The amount and activity of lysozyme recovered from the aqueous solution as well as lysozyme–ovalbumin mixture increased with the length and the hydrophilicity of the spacer. These polymers also exhibited better recyclability.

Introduction

With the advent of biotechnology, newer separation processes based on membranes, two phase systems and chromatography have evolved. The efficiency of many of these processes has been improved by the incorporation of affinity ligands, which has led to a wide range of affinity based separation processes. Affinity membrane filtration (Ruckenstein & Zeng, 1997) and affinity chromatography (Hirano, Kaneko, & Kitagawa, 1991; Junowicz & Charm, 1975; Liapis et al., 1989; Mayes, Moore, Eisenthal, & Hubble, 1990; Safarik & Safarikova, 1993; Yamasaki, Fukumura, Ito, & Imoto, 1985) provide high selectivity but are beset with several practical difficulties. In affinity membrane filtration, the fractionation is carried out on the basis of molecular size. Selection of the membrane, which offers precise molecular weight cut off, is often difficult. At high pressures, the denaturation of enzyme, and the fouling of the membrane lead to poor product quality and productivity (Ehsani, Parkkinen, & Nystrom, 1997). The affinity chromatography method suffers from slow binding, low capacity and flow rate limitations due to pressure drop, etc. (Chern, Lee, Chen, & Yeh, 1996a). Affinity precipitation overcomes many of the above problems. It offers simple separation procedure, ease of scale up, amenability to continuous operation and recycling of the affinity ligand (Chern et al., 1996a). Affinity precipitation is now being looked upon as an alternative to fluidized bed adsorption (Eggert, Baltes, Garret-Flaudy, & Freitag, 1998).

Egg white, which contains about 3.5% lysozyme, is routinely used for the recovery of lysozyme on industrial scale (Godfrey & West, 1996). So far, different techniques for lysozyme separation have been developed which range from conventional salt precipitation (Alderton & Fevold, 1946) to modern ultrafiltration (Bozzano & Glatz, 1991; Ehsani et al., 1997; Iritani, Mukai, & Murase, 1997). Tyagi, Kumar, Sardar, Kumar, and Gupta (1996) used chitosan as an affinity macroligand for selective precipitation of lysozyme. But lysozyme also cleaves chitosan. Moreover, ligands containing glucose, e.g. N-acetylglucosamine (NAG), chitosan, chitin are susceptible to microbial attack, hydrolytic degradation and thus exhibit poor stability (Hirano et al., 1991). Chitin and chitosan can undergo transglycosylation and mutarotation, which complicate the kinetic interpretation of inhibition data (Davies, Neuberger, & Wilson, 1969; Neuberger & Wilson, 1967). Therefore, it is desirable to replace glucose moiety by stable synthetic ligands in the affinity based lysozyme separation. The first step in the recovery of lysozyme by a ligand bound to the thermoprecipitating polymer is the binding between lysozyme and the ligand. Galaev and Mattiasson (1993a) showed that for effective recovery an optimal binding between the substrate and the ligand was necessary. Apart from binding, steric effects also contribute to the recovery. An effective measure of both contributions is provided by the inhibition concentration I₅₀, which represents the ligand concentration required to achieve 50% of the maximum attainable inhibition of lysozyme. Indeed, Blake et al. (1967) reported that NAG inhibited lysozyme but glucosamine (devoid of N-acetyl group) did not. Thus, N-acetyl groups are crucial for inhibiting lysozyme. On the contrary, the type of sugar did not influence binding significantly (Rupley, 1967). It, therefore, appears that synthetic ligands containing N-acetyl groups could be used for lysozyme separation. The methodology employed in the present work for affinity thermoprecipitation of lysozyme is shown in Fig. 1.

In the present communication, we report the lysozyme inhibition efficiency of a series of N-acetylated ligands formed by acetylation of amino acids of increasing chain length. The inhibition efficiency is enhanced when the acetamido ligand is incorporated into a thermoprecipitating polymer. The protein and activity of lysozyme recovered from aqueous solution as well as from lysozyme–ovalbumin mixture exceeds the corresponding values obtained for NAG. The polymers based on synthetic ligands can be recycled more effectively than the polymers based on NAG.