Lysozyme from chicken egg white

Egg white-derived lysozyme has been used to extract genomic DNA from bacterial cells. It is used as an external standard for MALDI-TOF (Matrix Assisted Laser Desorption Ionization - Time of Flight) mass spectrometry

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One unit will produce a ΔA 450 of 0.001 / min at 25 ° C. at 25 ° C. using a suspension of Micrococcus licicus as a substrate in 6 mL reaction mixture (1 cm path length).

Lysozyme hydrolyzes peptidoglycan between β- (1 → 4) and N-acetyl-D-glucosamine residues between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in chitin . )button. Gram positive cells are very sensitive to this hydrolysis, as their cell walls have a high proportion of peptidoglycan. Gram negative bacteria are less susceptible to influence due to the low proportion of outer membrane and peptidoglycan. However, these cells can be hydrolyzed in the presence of EDTA which chelates the metal ions in the external bacterial membrane.

This enzyme is active in a wide pH range (6.0 to 0). At pH 2, maximum activity is observed over a broad range of ionic strength (0.02 - 0.100 M) than pH 2 (0.01 - 0.06 M).

Lysozyme is a catalytic enzyme that digests bacterial cell walls and is present in large amounts in egg white. Egg white, also known as protein, is a cytoplasm of non-fertilized egg cells and consists of about 40 different proteins dissolved in water (Lee-Huang et al., 1999). The main proteins in proteins are ovalbumin, ovotransferrin, ovomucoid, globulin, lysozyme, ovomucin, avidin and the like (Lee-Huang et al., 1999). Lysozyme accounts for about 5% of the total protein weight in proteins (Lee-Huang et al., 1999) and in this laboratory we use egg white solution as the source of lysozyme. The main objective of this experiment was to purify the lysozyme using size exclusion chromatography and to detect its structural properties and its concentration in the protein.

In this experiment, lysozyme was purified from egg white using CM Sepharose based ion exchange chromatography. The protein consists of a complex mixture of proteins such as ovalbumin, lysozyme, albumin, ovotransferrin, flavoprotein, G 2 globulin and G 3 globulin. Among them, lysozyme is a low molecular weight protein (molecular weight 14.3 kDa), which is a commercially important enzyme used as an effective antimicrobial agent, food additive and antiulcer agent and precursor of infectious drug is there. (Yilmaz, Bayramoglu, and Arca 2005). This experiment was designed to demonstrate the practical application of CM Sepahalose resin in the isolation of lysozyme from whole albumen. Here, lysozyme is chosen as the basic protein and the pH is maintained at 2 with Tris buffer and sodium chloride.

Purification of lysozyme from egg white using ion exchange column chromatography and confirmation of purity by biochemical and molecular techniques is the sole purpose of this experiment. Lysozyme was mixed with cationic CM agarose resin as positively charged protein. The displacer solution containing sodium chloride and Tris buffer is then injected continuously. The Na + ions present on the sodium chloride have a higher dynamic affinity for the CM separation binding sites. Because of this higher affinity, Na + competes with lysozyme previously supplied for adsorption sites and causes lysozyme elution. FIG. 1 shows that in chromatographic analysis of 16 fractions only fractions 7, 8, 9 and 10 showed potential lysozyme activity. Since the binding properties of lysozyme and its contaminating proteins may have similarities, it is necessary to measure the specific lysozyme activity using micrococcus suspensions.