Lysozyme is a kind of natural lyase, which can specifically hydrolyze the peptidoglycan structure of the cell wall of many microorganisms, especially Gram-positive bacteria. Its antibacterial protection mechanism is significant. It has been a long development process since Nicolle first isolated the dissolving factor from
Bacillus subtilis, and the World Health Organization (WHO) and many countries and regions determined that lysozyme can be used as a non-toxic and safe additive. Nowadays, lysozyme is widely used in medicine [
1], food, scientific research, and other fields [
2,
3,
4], especially in a variety of industries. Therefore, the production and purification of lysozyme has become very important. Lysozyme is an alkaline protein with stable chemical properties. It can maintain its original structure and activity under a wide temperature and pH range. It has a high isoelectric point and is mostly positively charged. It is a high molecular weight compound with a variety of dissociable multivalent amphoteric electrolytes. It has different amounts of positive or negative charges at different pH values. In acidic and near neutral environments, there are many positive charges. Most other proteins are acidic proteins. When they coexist with other proteins, the interaction between molecules is easy to combine into a certain macromolecule, so it is difficult to separate lysozyme directly.
Lysozyme, because of its non-specificity, can be used as a cellular immune protein in various organisms, such as birds, mammals, and bacteria. Among them, the content inegg white is particularly rich. The traditional process usually adopts the combination of ultrafiltration and chromatography to remove the force of enzyme molecules and other proteins, so as to achieve the purpose of separating lysozyme. The common way to obtain lysozyme is to use egg white as raw material. However, the process of extracting lysozyme from egg white is limited by the source of egg white. Because egg white has only about 3.5% lysozyme, extraction has high production cost, it is a complicated operation, and it has very limited output and low profit, which is not conducive to amplification, and it is difficult to realize large-scale industrial production. Therefore, it is necessary to produce substitutes and reduce production costs to solve the problem of reducing enzyme production. Microbial fermentation with genetically engineered bacteria is an effective way to solve the above problems. However, it is an important challenge to separate lysozyme from the fermentation broth, because the metabolism of microorganisms involves multiple integrated processes, so that in addition to lysozyme, there are many complex components in the fermentation broth: water, residual substrates, by-products, and macromolecules (such as protein and polysaccharide). The first step in the treatment of fermentation broth is to remove various insoluble impurities such as microbial cells and residual substrates, which can be solved by centrifugal filtration. Then, it is necessary to remove the impurity protein and other macromolecular substances in the clear liquid as much as possible. Ultrafiltration can be used for further treatment. Ultrafiltration membrane technology can push water and small molecular substances through the membrane and release them into the permeate according to the molecular weight of the target product and the pressure difference between the two sides of the membrane; lysozyme is intercepted during this process. It is an excellent fermentation liquid purification technology. Important aspects include obtaining clearer permeate; less energy demand; recyclable material; simple and efficient operation; improved purity; and assuredlysozyme activity [
5,
6]. However, the viscous substances in the feed liquid are easy to adsorb, block the membrane pores, and form a filter cake layer, resulting in concentration polarization on the surface of the ultrafiltration membrane, increasing the resistance and affecting the transmittance, rejection, and membrane flux. Therefore, adjusting the ionic strength in the feed liquid, weakening the force between molecules, selecting the appropriate ultrafiltration membrane pressure, and improving the membrane flux are of great significance to improving the purity of the feed liquid. Lysozyme cannot be completely separated from other impurities in the feed solution by using biofilm, and it needs further refining and purification. Ion exchange technology can complete the refining and purification process, select the appropriate resin as the filler, and use the difference of binding force between lysozyme and exchange groups in the resin to complete the purification of lysozyme in the process of adsorption, binding, and elution. There are many factors affecting the adsorption–desorption process, such as resin type, time, eluent concentration and dosage, etc.,and optimizing the operating conditions of ion exchange is particularly important for the commercial production of lysozyme [
7]. The whole process is easy to operate, has low cost, has no need to add any chemical reagent, and is safe and efficient. In particular, ultrafiltration technology has mild conditions; does not cause changes in temperature and pH; can prevent denaturation, inactivation, and autolysis of lysozyme molecules; ensures the activity of lysozyme to the greatest extent; and provides good contact conditions for ion exchange. The macroporous resin has many and large pores, large surface area, many active centers, fast diffusion speed, short distance, high efficiency, short processing time, easy adsorption and exchange, strong pollution resistance, and a stable structure, and is renewable and recyclable [
8]. The purpose of this study is to combine ultrafiltration membrane and ion exchange technology, improve the extraction process, seek the best process parameters, ensure the enzyme activity to the greatest extent, extract lysozyme step by step from the fermentation broth, purify and refine lysozyme, and lay a foundation for microbial fermentation to produce lysozyme and realize industrial production.