Document Type : Research Article
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Abstract
Introduction: Hen egg white lysozyme (HEWL) is one of the proteins that has become increasingly important in many industrial aspects, including food and drug industries as well as high-technologies such as nanotechnology due to its specific properties. Its high thermal stability makes it a good natural food additive and sweetener. It has the antimicrobial activity against a wide variety of microorganisms such as Bacillus stearothermophilus, Micrococcus spp, Clostridium botulinum,Listeria monocytogenes as well as fungi. This activity remains inover a broad pH and ionic strength ranges. Thanks to its numerous advantages, it has been commonly used as an alternative to chemical antimicrobial agents in food industry such as sausage and dairy industries. HEWL has also high tendency to convert to protaneous well-ordered nanofibrils that is employed in nanotechnology. Furthermore, based on its well-known biochemical and biophysical features, HEWL is widely used as a model protein in the studies associated to the structure and function of proteins. Given theimportance increasing of lysozyme, development of an effective and simple as well as inexpensive method to purify this protein is required.
Material and methods: All salts and organic solvents were obtained from Merck (Darmstadt, Germany).Fresh eggs were purchased from a local market. After separating the white part from the yolk of eggs, it was diluted and filtered through linen. The filtered solution was then precipitated with 30 and 50% ammonium sulfate. The pellet of 50% ammonium sulfate dissolved in tris buffer (pH:7 with 2.5 M of urea) and was dialyzed for 15 hours.Thereafter, the sample was loaded on the cation exchange chromatography on a column of SP Sepharose Fast Flow. The washing buffers include buffer A (tris 30mM, pH:7 with urea 2M) and buffer B (tris 30 mM pH:7,ammonium sulfate 0.7 M and urea 2M) which were gradiently exchanged. The structural characters of the purified protein were analyzed by Far-UV CD (AVIV 215 spectropolarimeter, USA) and intrinsic fluorescence (usingVarian Cary Eclipse fluorescence spectrophotometer, Mulgrave, Australia) methods. To estimate the percentage of secondary structure in proteins the CD data was deconvoluted by CDNN software. In the next step, the enzymatic activity of the purified protein was tested based on Shugar’s method with some modifications and by using different strain.In this regard, the turbidity of cultured Micrococcus lysodeikticus was measured at 450 nm by T80+ Double Beam UV/Vis spectrophotometer (Leicestershire, England) after threating the cultures with purified protein and the enzymatic activity compared with the activity of an equal concentration of standard HEWL obtained from Sigma-Aldrich (USA).Bradford assay was carried out to determine the concentration of proteins
Results& Discussion: The subject of this study was to develop an effective, simple and inexpensive method topurify HEWL which has been able to scale up practically. In the first step, the filtered diluted white part of egg was precipitated withdifferent concentrations of ammonium sulfate. SDS-PAGE analysis indicated that the final pellet achievedby precipitation with 50% of ammonium sulfate had less impurity of the high molecular weight proteins. Salting out is an inexpensive and non-invasive purification method which can be used easily. Different proteins based on their specific chemical and physical properties precipitate at different concentrations of the salt. Ammonium sulfate is a common salt widelyusing for this purpose because of its inexpensive cost and high strength for salting out of different proteins. After ammonium sulfate precipitation step, the pellet was dissolved and consequently dialyzed in the presence of 2.5 M urea for 15 hours. After centrifugation, the sample solution was loaded on a cation exchange column chromatography. PI of HEWL is 11.35and the employ of a kind of cation exchange resin is appropriateto use for chromatography.After that, the column was washed with different proportions of buffers A and B. Each washed fraction of chromatography was collected and then analyzed with SDS-PAGE. The high purified protein (with more than 98% purity) was obtained in the fraction after washing with a solution of 4A+6B. Special activity of the final product was calculated which was considerably comparable to the activity of the standard enzyme. Furthermore, structural studies by circular dichroism (CD) spectroscopy indicated that the spectrum of the purified protein was very comparable with the standard HEWL. Deconvolution of CD data by CDNN software also showed that there were mostly same secondary structures in both proteins.Assessment of the intrinsic fluorescence intensity is also a valuable method to consider about integrity of tertiary structure of proteins based on exposing rate of hydrophobic residues especially tryptophan. The data extracted from CD and fluorescence spectroscopies confirmed that the purified HEWL had similar secondary and tertiary structure with the standard protein.
Conclusion: This study has developed an experimental method to purify HEWL which is simple, fast, and low cost with high efficiency.
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