Title of Invention

PROCESS FOR SEPARATION AND PURIFICATION OF LYSOZYME, CONALBUMIN, AND OTHER PROTEINS FROM EGG WHITE

Abstract

The process is disclosed for the separation and purification for lysozyme (mucopeptide-N-acetyl-muromoylhydrolase), Conalbumin (ovotransferrin), and other proteins such as avidin and ovalbumin on a single chromatographic column or adsorbent resin, from egg white extract in high purity. The invented process of extracting and purifying these proteins from egg white includes preparation of egg white extract by diluting egg white with acidic buffer, incubation for 2 to 24 hours followed by flirtation. Adsorption on a high capacity ion exchanger or hydrophobic matrix is carried out in chromatographic packed bed, expanded bed, fiuidized bed or stirred tank. The adsorption is affected by contacting the egg white extract with a porous adsorption resin having a pore size of 10" to 10"6 meters, a grain diameter of at least 5 x 10"6 m, and a specific surface area of at least 200 m2/g so that the resin adsorbs the proteins. The bound lysozyme, conalbumin, and avidin are eluted sequentially or simultaneously by linear or step gradient of salt and/or pH. Unabsorbed fraction contains major protein as ovalbumin that is captured by using second column having anion exchange matrix and eluted by elevated ionic strength. Ovalbumin can be captured first on anion exchanger and unabsorbed fraction is processed to adsorb lysozyme, conalbumin, and avidin on cation exchanger, which are then eluted by salt gradient. The invented process can be operated at high flow rates up to 3000 cm/hr without compromising the adsorption efficiency of adsorbents. Recovery of eluted proteins is more than 85% depending upon raw material used for processing. Recovered lysozyme, conalbumin have more than 98% purity whereas purity of other proteins is in between 85% and 99% (on the basis of SDS page).

Full Text

FORM 2 THE PATENTS ACT, 1970 (39 of 1970) & The Patent Rules, 2003 COMPLETE SPECIFICATION (See Section 10 and rule 13) 1. Process for Separation and Purification of Lysozyme, Conalbumin, and other Proteins from Egg White Lali Arvind Mallinath. Indian National Chemical Engineering Division University Institute of Chemical Technology, Nathalal Parikh Marg, Matunga (East), Mumbai-400 019 Maharashtra India The following complete specification particularly describes the invention and the manner in which it is to be performed 23-5-2008 Field of Invention The present invention relates to a process for chromatographic separation and purification of all, few or one of the group of lysozyme, conalbumin, ovalbumin, and avidin form egg white, and/or related raw materials, by using adsorption and elution. Lysozyme, conalbumin, ovalbumin, and avidin obtained by present invention have various biochemical activities and may be used in pharmaceuticals, foods, drinks, cosmetics, and diagnostics. Background of invention (Prior art) The ever growing number of bacterial strains and viral diseases which are resistant to antibiotics have made it necessary to introduce new kinds of drugs for treatment of human beings as well as animals. Among different therapeutic enzymes Lysozyme or muramidase (E. C. 3.2.1.17) has the tremendous potential for various physiological and biological treatments. It has been observed that Lysozyme has various therapeutic properties such as antiviral, antibacterial, anti-inflammatory and antihistaminic effects. The antibacterial effect of Lysozyme appears to be based on the hydrolysis of the beta -1-4-glycoside bond between n-acetylomuraminic acid and n-acetyloglucosamine, both contained in the bacterial wall. Lysozyme can also be used in the cancer treatment, food-packaging film to provide the antiseptic effect and as food preservative. Avidin, Ovalbumin and Conalbumin are equally much important in diagnostics, various pharmaceutical applications, food industries etc. Although Lysozyme is found in many natural sources such as milk, egg- white, human tears etc. Egg white is the rich source of the lysozyme, conalbumin, ovalbumin, and avidin. It is economical to use egg white as a source for the purification. US-4966851 discloses the process for the isolation of Lysozyme and avidin from egg white. This isolation was done by contacting egg white with weakly acidic cation exchanger resin to which only lysozyme and avidin get adsorbed. Lysozyme is then eluted by three to four washes of low ionic strength buffer and avidin then eluted by high ionic strength buffer. This process doesn't claim about purification of Conalbumin and Ovalbumin. 2 US-4705755 discloses the batch apparatus for collecting the lysozyme from egg white. In this detailed description of apparatus has been given for the efficient contact of resin and egg white extract. This doesn't disclose the conalbumin, Ovalbumin and avidin recovery by such process. US-4518695 discloses the batch process, which claims to isolate lysozyme using weakly acidic cation exchange resin like Amberlite (Rohm and Hass), Duolite (Diamond Shamrock), Dowex (Dow Chemical Co.), Lebatit CNP (Bayer Co), and Diaion WK (Mitsubishi Kasei Kogyo K. K. Japan). Elution is done at pH 6.5 to 8.0 with sodium chloride or ammonium secondary phosphate. This process includes the addition of alkali to elute to maintain the neutral pH. Also this process doesn't claim about the purification of avidin, conalbumin and ovalbumin. US-4552845 discloses the affinity-based isolation of lysozyme at natural pH of egg white. This reference also deals with the preparation and composition of suitable affinity resins for the reversible, preferential binding of lysozyme in egg white. US-3940317 discloses isolation of lysozyme from mammalian and vegetables sources by adsorption on chitin and eluting the adsorbed lysozyme using aqueous acid solution. US-4104125 discloses method for isolation of human lysozyme using cation exchanger such as CM -cellulose, CM-Sephadex (carboxymethyl crosslinked dextran), Phospho-cellulose (P-cellulose) from Pharmacia Co. Sweden. Using Tris-HCl containing NaCl at basic pH eluted adsorbed lysozyme. US-3515643 discloses process for purification of lysozyme, which consists of contacting the egg white with weakly acidic ion exchanger resin at pH of 6.0-7.0. Preferred ion exchangers are Amberlite CG-150, and Amberlite IRC-50 (Rohm and Hass). US-3419471 discloses method for preparation of albumin free lysozyme using ion exchange filtration method. Suitable anion exchange cellulose used includes GE-cellulose, TEAE-Cellulose, DEAE-cellulose, and AE-cellulose. DEAE-Sephadex A-50. The pH is maintained between 6.0-10.5, preferably between 6.5-10.0 to allow adsorption of albumins to the resin and to allow albumin free lysozyme in extract. Muzzarelli et. al., (Biotech. Bioeng., 1978) discloses the preferential reversible adsorption of lysozyme on chitosan. Albumin fraction gets damaged due to use of 3 sulphuric acid prior to adsorption on chitosan. Jensen and Kleppe disclose an affinity between lysozyme and chitin but shows that lysozyme destroys the chitin by hydrolysis. Cherkasov et. al., (1967) discloses discloses method for separating lysozyme from hen egg white by diluting hem egg white extract, adjusting pH to 5.5 and contacting the treated solution with chitin powder at pH between 5.0-5.5. Lysozyme complexes with chitin, which is broken by, dilute acid such as 0.1 M acetic acid. Weaver et al., (J. food. Sci. 1977) discloses that deaminated chitin has a high specificity and capacity for lysozyme. This has got good stability for isolating lysozyme and allows fast flow rates. None of the method in the above mentioned references provides sequential separation of lysozyme, conalbumin, avidin and ovalbumin from single feed stock of chicken egg white. Thus there is need to develop the efficient separation and purification protocol for recovery of four proteins lysozyme, conalbumin, avidin and ovalbumin from same egg white. The process of present invention provides cost effective separation and purification of lysozyme, conalbumin, avidin and ovalbumin in high purity and recovery using low cost "high performance expanded bed/fluidized bed/packed bed chromatography" from egg white and related raw materials. The process of present invention employs low cost as synthetic polymer based matrices like SEPABEADS® (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy) and CELBEADS (Indigenous, Indian patent application No. 356/MUM/2003) for efficient separation and purification at an industrial level. According to the present invention it has become possible to recover high purity proteins such as lysozyme, conalbumin, avidin and ovalbumin into more active form which is cheap, specific, more efficient, easily performed, giving therapeutic proteins with easy compatibility with semi-continuous, and continuous system. The references discussed herein are provided solely for their disclosure prior to filing date of present application. Nothing herein to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention 4 Statement of the Invention This is an improved process for obtaining highly pure multiple proteins i.e. lysozyme, conalbumin, ovalbumin and avidin from egg white extract in single step, said process comprising of i) selective capture of the lysozyme, conalbumin, avidin and ovalbumin on an pre-equilibrated adsorbent from egg white extract ii) washing the adsorbents with suitable washing solution, iii) selective sequential elution of one or more of said adsorbed proteins from the said adsorbent using elution mobile phase, in high purity, and in an unchanged chemical form, and iv) Cleaning-In-Place of the adsorbent using regenerants for its reuse in next cycle after equilibration wherein (a) the equilibration was carried out at pH value of 3 to 7.5 and at ionic strength of 5 to 100 mM, (b) selective capture of said proteins on said adsorbent was carried out at pH value of 4.5 to 7.5 and at ionic strength of 5 to 100 mM, (c) washing of the said adsorbent was carried out equilibration solution, (d) selective sequential elution from said adsorbent was carried out with step gradient to obtain each protein in highly pure form. Selective elution of ovalbumin was carried out at pH value of 4 to 5 and ionic strength of 100 to 250 mM, followed by selective sequential elution of conalbumin at pH value of 5 to 7.5 and ionic strength of 50 to 150 mM, followed by next selective sequential elution of lysozyme at pH value of 6 to 11.5 and at ionic strength of 100 to 500 mM and last selective sequential elution of avidin at pH value of 8 to 11.5 and at ionic strength of 500 to 1000 mM, and (e) cleaning in place of adsorbent for reuse was carried out with 0.5 to 1.0 M sodium hydroxide In this process the lysozyme, conalbumin, and avidin are obtained in concentrated form and having yield of more than 85% and purity of more than 98% and the purified lysozyme has high specific activity, i.e. more than 3 x 105 Units/mg. This process is carried out in packed bed, expanded bed, fluidized bed column chromatography. Summary of Invention An objective of the present invention is to provide efficient one column separation and purification process for lysozyme, conaibumin, avidin and ovalbumin from chicken egg white extract (CHWE) on the basis of their molecular weight and isoelectric points. Present invention has achieved this objective by providing a process, operated on a single adsorbent column, for separation and purification of egg white lysozyme, conaibumin, avidin and ovalbumin in high purity, activity and high recovery with a simple and commercially viable process from egg white. The process has now been found to isolate highly active lysozyme, conaibumin, and avidin using "column chromatography" consisting: adsorption stage wherein said egg white extract is contacted with specific kind of strong or weak cation exchanger in an expanded bed or packed bed mode to thereby lysozyme, conaibumin, and avidin is adsorbed, whereas ovalbumin was passed unabsorbed under specified conditions. Ovalbumin is made to adsorb on anion exchangers filled in second column, then the conaibumin, lysozyme and avidin was eluted sequentially or simultaneously from first column by varying ionic strength and pH of eluting solution. Ovalbumin containing fraction was then collected from said second column using salt gradient. Lysozyme and conaibumin was eluted in high purity and in more than 10 fold concentration in elute compared to load with aqueous salt solution having pH greater than 4.0 from the said first column. Thus present invention relates to the purification of conaibumin, lysozyme ovalbumin, and avidin where said proteins were purified using "Column Chromatography." The present process describes isolation of conaibumin, lysozyme ovalbumin, and avidin from egg white extract. Inventors have found that, it is important to utilize synthetic matrix based weak or strong ion exchangers and mixed mode interacting resins. These types of matrices have high mechano-osmotic stability and negligible volume variation when used in high and low pH environment. Synthetic matrix based cation exchange resins used are SEPABEADS® (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy), the useful functionalzed matrix for protein purification. Indigenously developed rigid, gigaporous, cellulose based cation exchange resins, CELBEADS (Indian patent application No. 356/MUM/2003) are also suitable for adsorption of lysozyme, 6 conalbumin, ovalbumin and avidin in a packed bed or expanded bed mode showing high degree of stability even when used at high ionic strength and flow rate. Brief description herein accompanying the figures as follows Figure 1: Schematic view of preferred embodiment of the process according to the invention when egg white extract is processed and ovalbumin is recovered in flow through of cation exchange column. 1: column I (expanded bed/ (packed bed), 1A: Column I adaptor, 2: Peristaltic pump, 3:UV-spectrophotometer, 4: Chart recorder, SI: Equilibration Buffer, S2: Egg White Extract, 3: Washing Buffer, S4: Elution 1 Buffer, S5: Elution 2 Buffer, S6: Elution 3 buffer, S7: Regenerant Solution, S8: D. M. Water, S9: Purified conalbumin fraction, S10: purified lysozyme fraction, Sll: purified avidin fraction, S12: flow through of column containing ovalbumin (which is further purified by second column of anion exchanger). Tanks SI to S8 were connected in such way that there is no cross mixing of mobile phases which will create bad performance of column. Direction of cycle is from SI to S8. Arrow in the figure indicates flow direction. Figure 2: Show elution profile at 280 nm (solid line) of loading, washing, elution and step-gradient (dotted line) when conalbumin, lysozyme and avidin eluted from first column. Here conditions used are as: Column: Borosilicate Glass 15 mm i. d., Adsorbent: SEPABEADS(R) EB-CM, Bed height: 300 mm (settled), Feed: 1.0 liter of chicken egg white extract (CHWE), Flow velocity: 300 cm/hr at equilibration, feed application, and wash 100 cm/hr at elution and CIP, Elution: Step Gradient, Peak 1: conalbumin elution, Peak 2: lysozyme elution, Peak3: Avidin elution. 1: Loading, 2: Washing, 3:Elutionl, 4: Eltuon 2, 5: Elution 3 Figure 3: Show reduced SDS-PAGE of purified conalbumin, and lysozyme from chicken egg white extract (CHWE). Conalbumin and lysozyme show single band at 77kDa and 14.3 kDa respectively with respect to commercial lysozyme and molecular weight marker. Lanel: Egg white extract, lane 2: Unbound fraction obtained from cation exchange column, lane 3: Wash fraction, lane 4: Conalbumin peak fraction, lane 5: commercial lysozyme sample, lane 6: Lysozyme peak fraction, lane 7: Molecular Weight Marker Detail Description of the Present Invention The present invention has specific utility for large-scale production of high purity lysozyme, conalbumin, avidin and ovalbumin from egg white. In the present invented process the starting material used is egg white extract (EWE) obtained from chicken eggs. Polyacrylate based porous SEPABEADS® (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy), and rigid, gigaporous cellulose based CELBEADS (Indian patent application No. 356/MUM/2003) are used without adherence of particulate matter for efficient purification of egg white proteins. In the preferred embodiment of the present invention, the egg whites extract (EWE) is an extract obtained by breaking eggs, more preferably of chicken egg, separating the egg white and diluting it with buffered aqueous solution or aqueous solution containing salts or acid which acts as extraction media for lysozyme, conalbumin, and avidin. In a preferred embodiment of the present invention, change in one or more parameters selected from a group consisting of pH, ionic strength, salt content, hydrophobicity, charge, temperature, and pressure was done. This change may be obtained by using an extraction medium selected form the group consisting of inorganic or organic salts, acids or bases. In the preferred embodiment of present invention, the extraction media contains inorganic salts of food grade such as but not limited to sodium chloride, potassium chloride, calcium chloride, and sodium sulphate; organic salts such as sodium acetate, sodium citrate, sodium lactate, and acids such as but not limited to hydrochloric acid, phosphoric acid, sulphuric acid, acetic acid, citric acid and/or lactic acid. In the present context, the term "extraction media" relates to the aqueous solution used for dilution of egg white or equilibration of resins or washing solution for resins or elution mobile phase used to elute said proteins from the resins Molecular weight of lysozyme, conalbumin, avidin and ovalbumin are 14.3kDa, 77.7kDa, 68kDa, and 45kDa respectively whereas isoelectric points are 10.7, 6.0, 10.0, and 4.5 respectively. These differences in molecular weight and isoelectric point are exploited to separate said proteins from chicken egg white extract (CHWE). In the preferred embodiment, precipitation is carried out to prepare egg white extract (EWE) using low cost acids such as hydrochloric acid, phosphoric acid and sulphuric acid. However, food grade organic acids or salts of organic acids such as acetic acid, citric acid, and lactic acid may be particularly preferred. The egg white extract (EWE) is prepared by breaking the eggs and separating egg white which is then diluted 2 to 6 fold with buffer having concentration in the range of 5 to 500mM and pH of 2 to 5. After diluting the extract it was incubated for 4 to 24 hours to precipitate other proteins. The temperature used for incubation is in the range of 2 to 60 degree C. The egg white extract is then filtered through cheesecloth or muslin cloth or Whatman filter paper No. 1. In the adsorption step of the process, according to the claimed invention, any known porous resin having pore size: 10"8 to 10"6 m, grain size: at least 5"6 m, specific surface area: at least 200 m2 /g may be selected. The rigid, strong cation exchangers employed are, SEPABEADS® EB-SP, SEPABEADS® FP-SP (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy), CELBEADS-SP, (Indigenous), and weak cation exchangers are SEPABEADS® EB-CM, SEPABEADS® FP-CM (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy), CELBEADS-CM (Indigenous). The anion exchangers employed are selected from group of SEPABEADS® EB-QA (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy), CELBEADS-DEAE (indigenous), and STREAMLINE-DEAE (Amersham Biosciences, Sweden). The adsorption resins used in the adsorption steps can be identical or different; if different resins are employed, the difference may reside for example in the pore size, grain size, surface area, and the polarity of the surface or solubility index. The further conditions of the adsorption steps which are generally performed at room temperature, e.g. the optimum pH-value and the optimum contact time with the resin, may also vary depending on the quality of the egg white extract and on its actual composition. In the preferred embodiment of the present invention, cation or anion exchanger is first equilibrated with salt or buffered aqueous solution to a pH range of pH 3.0-7.5, such as pH 3.5- 6.7, e.g. pH 4.0-6.6, such as pH 4.5-6.5, e.g. pH 5.0-6.4, such as pH 5.0-6.0 and having ionic strength of 5.0 to 500mM. The salt/buffer can be selected from but not limited to sodium chloride, potassium chloride, phosphate buffer, acetate buffer, macclave buffer, citrate buffer, Tris-HCl buffer or borate buffer. Alternatively dilute acid solution can be used for which acid can be selected from but not limited to mineral acids hydrochloric acid, phosphoric acid, sulphuric acid or organic acids such as but not limited to acetic acid, citric acid, maleic acid, or malonic acid. Equilibration can be done in packed bed or in expanded bed. The filtrate recovered is used for loading in to the pre-equilibrated column at a higher flow rates. The filtrate used for loading has pH in the range of or may be adjusted to pH 3.0-7.5, such as pH 3.5- 6.7, e.g. pH 4.0-6.6, such as pH 4.5-6.5, e.g. pH 5.0-6.4, such as pH 5.0-6.0 and having ionic strength of 5.0 to 500mM. The unbound fraction (flow through fraction) of the said column was collected containing major protein as ovalbumin having purity in the range of 70 to 98%. In the preferred embodiment of the present invention, the optimal adsorption or other type of fixation of lysozyme, conalbumin, and avidin to the matrix takes place at the pH range of pH 2.0 to 9.0, e.g. in the range of pH 4.0 to 8.0, such as in the range of pH 5.0 to 7.0. Washing of the matrix after loading is an optimal step and in a preferred embodiment of the present invention, the optimal washing procedure is based on aqueous washing solution that does not interfere with the association of lysozyme, conalbumin, and avidin. The washing solution is typically selected from group consisting of tap water, de-mineralized water, filtered tap water, dilute salt solutions, diluted buffer solutions or any combination thereof. In the preferred embodiment of the present invention, the washing solution comprises a dilute salt solution selected form group consisting of sodium acetate, sodium chloride, or any other low cost, food grade mineral or organic salt. The concentration of the dilute salt solution is typically in the range of 0.02 to 0.2M. Lysozyme, conalbumin, and avidin bound to the resin are then sequentially desorbed as purified fractions using aqueous solution containing single salt or salt/s in aqueous buffered solution. Lysozyme, conalbumin, and avidin are eluted sequentially or simultaneously by salt solution in stepwise or linear gradient 0 to 1.5M. Salts used in the 10 elution phase may be same or different and having either same or different ionic strength at every step to elute proteins separately. In the present embodiment lysozyme and conalbumin elute and separate at ionic strength in the range of 0.02 to 0.8 M. Using different buffers, or same buffer having higher ionic strength, the eluted fractions can obtain the bound avidin, in expanded or packed bed mode. Elution of adsorbed conalbumin from the resin used in the adsorption technique is obtained by increasing the pH and ionic strength. In the preferred embodiment of the present invention, The pH of elution of conalbumin is in the range of pH 3.0-8.5, such as pH 3.5- 8.0, e.g. pH 5.0-7.6, such as pH 5.5-7.5, e.g. pH 5.5-7.4, such as pH 6.0-6.7, and ionic strength 10-500mM, e.g. 20-400mM, such as 25-250mM, e.g. 25-200mM, such as 50-150mM adjusted by any low cost, food grade salt or acid mentioned above. Elution of adsorbed lysozyme from the resin used in the adsorption technique is obtained by increasing the pH and ionic strength. In the preferred embodiment of the present invention, The pH of elution of lysozyme is in the range of pH 6.0-12.0, such as pH 6.5-11.8, e.g. pH 6.5.0-11.6, such as pH 6.8-11.5, e.g. pH 7.0-11.4, such as pH 7.5-10.7, and ionic strength 10-600mM, e.g. 20-400mM, such as 25-250mM, e.g. 25-200mM, such as 50-150mM adjusted by any low cost, food grade salt or acid mentioned above. Elution of adsorbed avidin from the resin used in the adsorption technique is obtained by increasing the pH and ionic strength. In the preferred embodiment of the present invention, the pH of elution of avidin is in the range of pH 5.0-12.5, such as pH 6.0- 12.0, e.g. pH 6.5-11.6, such as pH 7.0-11.5, e.g. pH 7.5-11.4, such as pH 8.0-10.7, and ionic strength 10-lOOOmM, e.g. 50-800mM, such as 75-750mM, e.g. 100-500mM, such as 200-500mM adjusted by any low cost, food grade salt or acid mentioned above. The ovalbumin obtained from the column as first product can be further purified by decreasing the pH and increasing ionic strength of obtained ovalbumin from the said cation exchange column, and loading on to a second anion exchange resin column. In the preferred embodiment of the present invention, the pH of elution of ovalbumin from the said second anion exchange column, is in the range of pH 3.0-8.5, such as pH 3.5- 8.0, e.g. pH 5.0-7.6, such as pH 5.5-7.5, e.g. pH 5.5-7.4, such as pH 6.0-6.7, and ionic strength 10-1000mM, e.g. 25-800mM, such as 50-750mM, e.g. 100-500mM, such as 200-500mM adjusted by any low cost, food grade salt or acid mentioned above. In the preferred embodiment of present invention ovalbumin is kept unabsorbed on said cation exchanger column by adjusting ionic strength and pH of egg white, while lysozyme, conalbumin, and avidin are made to adsorb. Thereafter, lysozyme, conalbumin and avidin are sequentially eluted as purified fractions, whereas the flow through of column containing ovalbumin is diverted to second column having anion exchanger as CELBEADS-DEAE (indigenous) or SEPABEADS EB-QA (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy), STREAMLINE-DEAE (Amersham Biosciences, Sweden) where the ovalbumin binds to the column, which was then washed with water or buffer and eluted with increasing salt concentration. The recovered ovalbumin has more than 85% purity. Whereas, alternatively, ovalbumin can be captured first on an anion exchanger such as CELBEADS-DEAE and lysozyme, conalbumin, and avidin, which remain unabsorbed under binding conditions used for ovalbumin, then further purified by use of cation exchanger. The adsorbed ovalbumin, lysozyme, conalbumin, and avidin can be eluted in same manner as described earlier. In the present context, the term "elution" relates to a liquid used to release adsorbed or fixed proteins from the resins used in n adsorption technique. In the preferred embodiment of the present invention, the amount of isolated proteins (lysozyme, conalbumin, ovalbumin, and avidin) relative to amount present in the egg white or egg white extract (EWE) is at least 80%, at least, 90%, or at least 100%. The recovered lysozyme, conalbumin and avidin have purity more than 90% or specifically in between 92 to 99% or more specifically 96 to 99% or exactly 98 to 99%. All proteins separated by said process show as single product bands in SDS-PAGE indicating their efficient separation by said process. Cleaning in place (CIP) of resins is carried out to reuse the resins by applying 0.5 to 1.0M NaOH followed by D. M. water wash till pH falls to neutral. Separating and purifying lysozyme conalbumin, avidin, and ovalbumin in accordance with present invention is cost effective and eliminates need of conventional methods. Specifically, invented process enables recovery (85% or higher), purity (90% or higher), and high activity of lysozyme. Quality control tests 1. Lysozyme activity was determined by terbidometric assay where one enzyme unit (U) is defined as the amount of enzyme that causes lysis of Micrococcus Lysodeikticus NCIM-2437 (National Chemical Laboratory, NCL, Pune, India) and gives a change on OD450 of 0.001 absorbance units per minute. (At pH 7.0 Phosphate buffer, and temperature 250Q 1. Purity was determined by applying 50 u.1 of protein sample on a UNO-Q. (2 ml) column with a full-scale sensitivity of 0.01 using Biologic Duoflow chromatography system from BioRad, USA. The elution program was a linear salt (0-1.0M NaCl) gradient in 25Mm Tris to 25mMTris-HCl, pH 8.1, at 4 ml/min. lysozyme, conalbumin, ovalbumin, and avidin elutes as separate peaks whose area under peak was determined and used to access purity. 2. Total protein content was determined by using Bradford reagent from Bio-Rad, USA. Simplification of the entire process using integration of operation, high purity of the product, yield and recovery makes it possible to reduce the overall processing cost and to utilize the proteins in food products, diagnostics, and pharmaceuticals. Thus process of present invention can be easily applied to industrial level. The following is the example carried out to demonstrate the present invention. However, the present invention should not be construed as limited to the description of these examples. Examplel: 1000 ml of chicken white extract (CHWE) was prepared by beating 7 eggs, collecting egg white fraction, and diluting it up to 1000 ml with chilled 25mM acetate buffer having pH 4.5. The extract was then incubated for 6 hours at 8 degree Celsius temperature followed by filtration using Whatman No. 1 filter paper. The filtrate recovered is used for separation and purification of lysozyme, conalbumin, avidin and ovalbumin. A 15 mm inner diameter and 600mm long borosilicate glass column was filled with 50 ml of the weak cation exchanger SEPABEADS EB-CM (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy). The settled bed height was 300mm. Column was then equilibrated with solution containing 25mM acetate buffer having pH 4.5. To this pre-equilibrated column 1000 ml of egg white extract is loaded containing 36.4x106 Units of Lysozyme, 744 mg of conalbumin, 60 mg of avidin and 2580 mg of ovalbumin at velocity of 300 cm/hr in an packed bed mode. The resin column was washed with 25 mM sodium acetate solution in a packed bed mode at linear velocity of 350 cm/hr to remove almost all unbound materials. Unbound fraction is collected separately and found to contain major protein as ovalbumin in 85% purity. Conalbumin and lysozyme was then eluted with 50mM solution of phosphate buffer of pH 6.8 and 100 mM potassium carbonate of pH 11.3 respectively. Finally avidin was eluted with 0.5 M carbonate buffer of pH 10.7. Recovered lysozyme has 340315 Units/mg of specific activity containing 73.6xl06 Units with fold purity of 103. 739.2 mg of conalbumin, 52 mg of avidin and 2475 mg of ovalbumin was recovered from the column. Yield of isolated lysozyme, conalbumin, avidin and ovalbumin are 100%, 99.3%, 86.7%, and 85%) respectively. Purity of eluted lysozyme, conalbumin, and avidin is 99.1%> (by SDS-PAGE method). Example2: The CHWE was prepared in the same manner as in example land applied to the column containing 50 ml of the strong cation exchanger SEPABEADS® EB-SP (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy). To this pre-equilibrated column 1000 ml of egg white extract is loaded containing 36.4x106 Units of Lysozyme, 744 mg of conalbumin, 60 mg of avidin and 2580 mg of ovalbumin at velocity of 300 cm/hr in an packed bed mode. The resin column was washed with 25 mM sodium acetate solution in a packed bed mode at linear velocity of 350 cm/hr to remove almost all unbound materials. Unbound fraction is collected separately and found to contain major protein as ovalbumin in more than 90%o purity. Conalbumin and lysozyme was then eluted with 80mM solution of phosphate buffer of pH 6.8 and 150 mM potassium carbonate of pH 11.3 respectively. Finally avidin was eluted with 0.7 M carbonate buffer of pH 10.7. Recovered lysozyme has 300745 Units/mg of specific activity containing total 65.2xl06 Units with fold purity of 98.8. 730.5 mg of conalbumin, 62 mg of avidin and 2242 mg of ovalbumin was recovered from the column. Purity of eluted lysozyme, conalbumin, and avidin is 98.2% (by SDS-PAGE method). Example 3: The 4 grams of ovalbumin recovered from unbound fractions in purity of around 85% was loaded on the 100 ml of SEPABEADS® EB-QA ((Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy) in up flow mode at 150 cm/hr linear flow velocity. The adsorbed resin were washed with two column volumes of 50mM sodium acetate and eluted with 0.2M of sodium dihydrogen phosphate pH 4.6. 3.8 grams of ovalbumin was recovered with 95.3% purity (by HPLC method). Example 4: The process was carried out in the same manner as that of example 1 on 2.5 cm diameter column with 30 cm bed height and containing SEPABEADS® EB-CM (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy). 5 liters of egg white extract was loaded followed by washing and elution. All said proteins recovered in more than 90% yield and 98% purity except 87.3% for ovalbumin which is recovered as unbound fraction. I claim, 1) An improved process for obtaining highly pure multiple proteins i.e. lysozyme, conalbumin, ovalbumin and avidin from egg white extract in single step, said process comprising of i) selective capture of the lysozyme, conalbumin, avidin and ovalbumin on an pre-equilibrated adsorbent from egg white extract if) washing die adsorbents with suitable washing solution, iii) selective sequential elution of one or more of said adsorbed proteins from the said adsorbent using elution mobile phase, in high purity, and in an unchanged chemical form, and iv) Cleaning-In-Place of the adsorbent using regenerants for its reuse in next cycle after equilibration wherein characterized that selective capture of said proteins on said adsorbent was carried out at pH value of 4.5 to 7.5 and at ionic strength of 5 to 100 mM, and selective sequential elution of multiple proteins from said adsorbent was carried out at pH value of 4 to 5 and ionic strength of 100 to 250 mM for ovalbumin, followed by selective sequential elution of conalbumin at pH value of 5 to 7.5 and ionic strength of 50 to 150 mM, followed by next selective sequential elution of lysozyme at pH value of 6 to 11.5 and at ionic strength of 100 to 500 mM and last selective sequential elution of avidin at pH value of 8 to 11.5 and at ionic strength of 500 to 1000 mM. 2) A process of claim 1 wherein the process comprises rigid porous polymeric synthetic ion exchange adsorbent, 3) A process claim 1 €B$,2 wherein the adsorbent employed are selected from group of, SEPABEADS® EB-SP, SEPABEADS® FP-SP SEPABEADS® EB-CM, SEPABEADS® FP-CM, SEPABEADS® EB-QA (Resindion S.r.l, Mitsubishi Chemical Corp. Binasco, Italy), CELBEADS-SP, CELBEADS-CM CELBEADS-DEAE (indigenous), and STREAMLINE-DEAE (Amersham Biosciences, Uppsala, Sweden). 16 3) A process of claim 1 wherein the adsorbent is SEPABEADS EB-CM to which lysozyme, conalbumin, and avidin adsorb selectively on the said adsorbent and unbound solution recovered contains ovalbumin. 4) A process of claim 1 wherein the extract, equilibration solution, washing solution, elution mobile phase and regenerants contains inorganic or organic salts, acids or bases from one or more of following (i) inorganic salts of food grade such as but not limited to sodium chloride, potassium chloride, calcium chloride, and sodium sulphate, (ii) organic salts such as sodium acetate, sodium citrate, sodium lactate, (iii) acids such as but not limited to hydrochloric acid, phosphoric acid, sulphuric acid, acetic acid, citric acid and/or lactic acid, and (iv) any suitable combination of one or more thereof chosen to achieve required selectivity. Further the mobile phase used for equilibration, washing, elution and regeneration comprises one or more of those mentioned above in this claim and applied to the adsorbent in one or more of a method comprising a change of pH and/or ionic strength in 'step gradient' or any suitable 'continuous gradient' elution, or any combinations thereof, 5) A process of claim 1 wherein (i) the equilibration was carried out at pH value of 3 to 7.5 and at ionic strength of 5 to 100 mM, (ii) selective capture of said proteins on said adsorbent was carried out at pH value of 4.5 to 7.5 and at ionic strength of 5 to 100 mM, (iii) washing of the said adsorbent was carried out equilibration solution, (iv) selective sequential elution from said adsorbent was carried out with step gradient to obtain each protein in highly pure form. Selective elution of ovalbumin was carried out at pH value of 4 to 5 and ionic strength of 100 to 250 mM, followed by selective sequential elution of conalbumin at pH value of 5 to 7.5 and ionic strength of 50 to 150 mM, followed by next selective sequential elution of lysozyme at pH value of 6 to 11.5 and at ionic strength of 100 to 500 mM and last selective sequential elution of avidin at pH value of 8 to 11.5 and at ionic strength of 500 to 1000 mM, and (v) cleaning in place of adsorbent for reuse was carried out with 0.5 to 1.0 M sodium hydroxide. 6) A process of claim 1 wherein the lysozyme, conalbumin, and avidin are obtained in concentrated form and having yield of more than 85% and purity of more than 98%. 7) A process of claim 1 wherein the purified lysozyme has high specific activity, i.e. more than 3 x 105 Units/mg • 8) A process of claim 1 wherein the process is carried out in packed bed, expanded bed, fluidized bed column chromatography. LALI ARVIND MALLINATH (Signature of Applicant) 18

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