Thermoprecipitation of lysozyme from egg white using copolymers of N-isopropylacrylamide and acidic monomers

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Abstract

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33–35°C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 104–105 times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer–lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.

Introduction

Lysozyme is an industrially useful enzyme. Egg white, which contains 3.5% w/w lysozyme, is a convenient source for the recovery of lysozyme (Ruckenstein and Zeng, 1997). Lysozyme is a positively charged enzyme at physiological pH (pI=10) (Wilkinson and Dorrington, 1975) and thus exhibits strong electrostatic interactions with negatively charged carboxylate and sulfonate groups. Therefore, cation exchange resins are widely used in recovering lysozyme from egg white (Li-Chan et al., 1986, Shinano et al., 1993, Shuichi et al., 1994, Vachier et al., 1995). However, ion exchange columns suffer from the common problems such as column clogging, flow rate limitation, pressure drop etc. (Freifelder, 1982).

These problems are eliminated in affinity precipitation as the binding of target molecule with the affinity ligand and subsequent precipitation of the complex occurs in homogeneous solution (Senstad and Mattiasson, 1989, Morris et al., 1993, Gupta and Mattiasson, 1994). Sternberg and Hershberger (1974) reported the use of polyacrylic acid (PAA) for the formation of PAA-lysozyme polyelectrolyte complex. Chern et al. (1996) used pH sensitive submicron acrylic latex as well as Eudragit L100 for lysozyme precipitation. The recovery of lysozyme in these cases was 30 and 7%, respectively. The main reason for this low recovery is the acidic pH, used for precipitation of polymer–lysozyme complex. Since polymer–lysozyme interactions are pH and ionic strength dependent, there is substantial dissociation of the complex, when the pH is reduced. Thus, temperature shift is a better alternative over pH shift for lysozyme precipitation. However, to our knowledge, there are no reports on the use of thermoprecipitation for lysozyme recovery.

In this communication, we report up to 90% lysozyme recovery from egg white using copolymers of N-isopropylacrylamide (NIPAM) and various acidic monomers. These copolymers also exhibited higher reusability than that exhibited by the polymer containing N-acetylglucosamine (NAG), a natural ligand for lysozyme.

Section snippets

Materials

Acrylic acid, methacrylic acid and NIPAM were purchased from Aldrich Chemical Company Inc., Milwaukee, WI, USA. Lysozyme (3×crystallized, dialyzed and lyophilized, Lot No. 57H7045), Micrococcus lysodeikticus (Micrococcus luteus ATCC No. 4698, Lot No. 38H8619), NAG (Lot No. 98H0758) were purchased from Sigma Chemical Company, St. Louis, MO, USA. The manufacturer's definition of unit activity of lysozyme was the change in the absorbance (ΔA450) of 0.001 per s at pH 6.6, 25°C using a 1.6 ml

Results and discussion

The objective of this work was to demonstrate the use of thermoprecipitating polymers for the separation of lysozyme from egg white. We selected acidic monomers for co-polymerization with NIPAM as the resulting polymers exhibit excellent control over LCST (Galaev and Mattiasson, 1993b).

Conclusions

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of NIPAM with different acidic monomers. Polymers exhibited electrostatic binding with lysozyme and inhibited its bacteriolytic activity. Concentration of acidic groups required to attain I50 by the polymers was 104–105 times lower than that of corresponding free monomers due to the multiple binding interactions between polymer and lysozyme. Lysozyme activity was recovered ≥90% from egg white. Polymer exhibited

Acknowledgements

AAV would like to acknowledge the financial support rendered by the Council of Scientific and Industrial Research, New Delhi, India in the form of a Senior Research Fellowship.

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