GB1573684A

GB1573684A - Process for the production of lysozyme

Info

Publication number: GB1573684A
Application number: GB27854/77A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1977-07-04
Filing date: 1977-07-04
Publication date: 1980-08-28
Also published as: FI60576B; FI60576C; FR2396764B1; FR2396764A1; NL7807231A; DE2828944A1; DE2828944C2; BE868714A; FI782151A7

Description

(54) PROCESS FOR THE PRODUCTION OF LYSOZYME (71) We, LORENZO FERRARI, of 8 Via Biella, 20143 Milan, Italy, and CARLO TRINCHERA, of 44 Via Tito Vignoli, 20146 Milan, Italy, both Citizens of Italy, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention is concerned with a new and improved process for the production of lysozyme.

Before its introduction into medical therapy some 20 years ago, lysozyme (EC 3.2.1.17), which is an enzyme possessing an antibacterial and immunising activity which was isolated by Fleming in 1922, was only used in bacteriological laboratories in limited amounts, for example, for studying the structures and components of bacterial cell walls.

Due to the favourable clinical results in combating inflammation and infections and, subsequently, as a result of its use in conjunction with foodstuffs as a preservative and mould preventive, the demand for large quantities of a pure lysozyme has increased and numerous industrial processes for the production of lysozyme have been patented.

Lysozyme is an enzyme which can be better defined as N-acetyl-muramido-glycanohydrolase. It has a basic character and a molecular weight of about 15,000. It is fairly stable in an acid medium and less stable in an alkaline medium. It is particularly active against grampositive bacteria, from which it frees substances which can be specifically determined with amino-sugar reagents.

Its primary structure as a basic polypeptide, formed from 129 amino-acid radicals, has been particularly studied by Jolles (Exp. Ann. Bioch. Med., 27th Series, page 1, pub.

Masson, Paris. 1966).

In our British Patent Specification No. 1,110,466 we have described and claimed a process for the production of lysozyme from egg-white, wherein egg-white is contacted with a weakly acidic ion exchange resin at a pH of 6 - 7 at a temperature below ambient temperature, separating the resin, eluting contaminating proteins from the resin with a salt solution having a concentration of not more than 0.2M and a pH of not more than 7, then eluting the lysozyme with an aqueous solution of a salt and precipitating the lysozyme from the eluate by increasing the salt concentration therein.

The present invention is concerned with a process for the preparation of lysozyme, preferably from egg-white but also from other starting materials containing lysozyme, be they of vegetable or animal origin.

By means of the process according to the present invention, which is based on the extraction of lysozyme from egg-white or from other lysozyme-containing material by weak cationic resins under special experimental conditions, various objects are achieved which are more advantageous than those of the methods at present known: the egg-white used is not contaminated by extraneous substances and is not modified with regard to its natural alkaline pH of about 9; furthermore, since the extraction time is of limited duration, the egg-white does not undergo any deterioration of its physical characteristics. for example, its binding and rising properties, so that it can be used for food purposes in the same way as the original egg-white and, consequently, its commercial value also remains practically unchanged; the simplified mode of operation and the short working times result in reduced production costs; finally, in the various operational steps, all those substances, the presence of which in the effluent water could result in serious contamination problems, are eliminated. Preferably, the process according to the present invention only makes use of sodium chloride and sodium hydroxide, the solutions of which are re-used in successive production cycles. The inevitable but small losses of sodium chloride (originating partly from the neutralisation of sodium hydroxide by hydrochloric acid) can be discharged as effluent after simple dilution to the limits permitted by anti-pollution regulations.

Thus, according to the present invention, there is provided a process for the production of lysozyme, wherein a weakly acidic ion exchange resin containing carboxylic acid groups and preferably a carboxylic acid group-containing methacrylic resin (for example "Amberlite" CG50 or "Amberlite" IRC 50) ("Amberlite" is a Registered Trade Mark) is adjusted to a pH higher than 7.1 and preferably of from 8 to 9.5, for example with a 30% by weight aqueous solution of sodium hydroxide, whereafter, when the desired pH is obtained, the resin is washed with water and then contacted with a lysozyme-containing material, preferably egg-white.

Other alkalis which may be used include aqueous solutions of potassium hydroxide or ammonium hydroxide, the concentrations of which are not critical.

The use of an equilibrated resin with an alkaline pH provides two advantages, in addition to those previously mentioned, which have hitherto not been achieved in other industrial processes: a) in the first place, the pH of the mixture of the lysozyme-containing material, such as egg-white, and resin of about 9, i.e. about the normal pH of egg-white, reduces the possibility of contamination by bacteria or fungi during the processing operation.

Furthermore, the alkalising solution used in the process increases the rate of adsorption of the lysozyme by the resin so that the treatment time in this phase is substantially reduced, with substantial saving in tile costs of production. Thus, the process may be carried out at a near-ambient temperature (10 to 15 C.), which is economically advantageous, and the lysozyme-containing material especially egg-white, which is not substantially modified in its properties and bacterial content, can be used, after the removal of its lysozyme content, for other purposes.

b) another advantage. when the adsorption of the lysozyme is carried out within a pH range of 8 to 9, is that, after the period of contact, when the lysozyme-containing material, for example egg-white. has been removed and the resin has been washed with water to free it from the last traces of lysozyme-containing material, it is not necessary to wash the resin with a saline solution to remove any protein substances, because of the high degree of selectivity of the adsorption.

The lysozyme may be eluted with an aqueous solution of an inorganic salt, for example, of sodium nitrate, potassium nitrate, sodium chloride, potassium chloride, sodium sulphate, potassium sulphate or ammonium sulphate, or of organic salts, for example, sodium acetate, ethanolamine hydrochloride or pyridine hydrochloride.

Very good results have been obtained with 6 to 10% by weight aqueous solutions of ammonium sulphate and with 1.5 to 3% by weight aqueous solutions of sodium chloride.

The lysozyme may be separated from the eluate according to the following processes: a. precipitation by, addition of a salt to the eluted solutions; b. ultrafiltration of the eluate, followed by precipitation by the addition of a salt to the concentrate; c. concentration by ultrafiltration. followed by spray drying or lyophilisation of the solution.

Concentration by ultrafiltration may be carried out according to the following scheme:

(1% by wt. (lysozyme elution of 100 litres (3% by wt.

(sodium chloride sodium chloride ULTRAFILTRATION (2.5% by wt. (lysozyme 40 litres 3% by wt.

sodium chloride dilution with 30 litres water 1.45% by wt.

lysozyme 70 litres 1.7% litres1.7% by wt.

sodium chloride ULTRAFILTRATION (6.65% by wt. lysozyme 15 litres 1.7% by wt.

(sodium chloride +sodium chloride LYSOZYME The lysozyme obtained by the process according to the present invention can be used therapeutically and in foodstuffs without further treatment or, if desired, after salification with a non-toxic and physiologically acceptable inorganic or organic acid.

The following Examples are given for the purpose of illustrating the present invention: Example 1 11 litres of methacrylic resin of the carboxylic acid group-containing type, in which the ;arboxvl groups are in the free form ("Amberlite" IRC 50), are suspended in about 60 litres of water, A 20% by weight aqueous solution of sodium hydroxide is slowly added, with stirring, until the pH of the suspension is 8. The liquid is decanted off and the resin is repeatedly washed by decantation with demineralised water. After removing the water, the resin is contacted with 66 kg. of egg-white at a temperature of 10 C. until the content of lysozyme in the egg-white is about 300 y/ml. The resin is then washed with water and subsequently eluted with an aqueous solution of 3% by weight sodium chloride, about 25 litres of eluate being obtained containing 240 to 260 g. of lysozyme. The solution is concentrated by ultrafiltration (DDS membrane type 600) to a volume of 10 litres; after dilution with 7.5 litres of water, it is then concentrated again to a volume of about 3.8 litres and filtered. The pH is adjusted to 3.5 and about 85 g. of sodium chloride are added; after cooling, centrifuging and drying, 240 to 250 g. of lysozyme are obtained with a titre of 94 to 96cm .

Example 2 The process described in Example 1 is repeated except that the resin is equilibrated to pH 9. After elution, the pH of the eluate is adjusted to 9.5 and sodium chloride is added until the concentration thereof is 5% by weight. After cooling, 240 to 245 g. of lysozyme base separate out: it has a titre of > 94%.

Example 3 The process described in Example 1 is repeated. The concentrated solution obtained after ultrafiltration is adjusted to pH 3.3 with hydrochloric acid and spray dried. The lysozyme hydrochloride thus obtained has a titre of < 94%.

Example 4 The process described in Example 1 is repeated. After the lysozyme has been adsorbed and the resin has been washed with water, the lysozyme is eluted with an 8% by weight aqueous solution of ammonium sulphate. The concentration of the ammonium sulphate is then increased to 40% by weight to precipitate the lysozyme. In order to obtain lysozyme hydrochloride and to eliminate any traces of impurities, the precipitate is dissolved in filtered water, the pH is adjusted to 3.3 with hydrochloric acid and the lysozyme is precipitated by the addition of an aqueous solution of sodium chloride.

Example 5 1110 ml. of methacrylic resin of the carboxylic group-containing type ("Amberlite" IRC 50) in the acid form are placed in a 6-litre flask and a 30% by weight aqueous solution of sodium hydroxide slowly added dropwise, while stirring, until the suspension has a pH of 8.

Stirring is then terminated, the liquid is decanted off and the resin is filtered through a Buchner funnel and washed several times with demineralised water; the resin is then contacted with 6.6 kg. of egg-white at about ambient temperature (about 15"C.) until the lysozyme content of the egg-white is 100 to 200 y/ml. The resin is then washed with water, whereafter the lysozyme is eluted with a 3% by weight aqueous solution of sodium chloride.

The pH of the eluate is adjusted to about 9.5 to 9.8 and the pure basic lysozyme is precipitated by increasing the concentration of the sodium chloride to 5C/o.

Example 6 The procedure described in Example 5 is repeated except that the resin is adjusted to a pH of 9 with an aqueous sodium hydroxide solution. After elution of the resin with 2.5% by weight aqueous solution of sodium chloride, there are obtained 1900 to 2100 ml. of eluate containing 1.4 to 1.5% by weight of lysozyme. The solution is then subjected to ultrafiltration (DDS membrane type 600) until the volume is reduced to 800 ml., whereafter the solution is diluted with 600 ml. of water and a second ultrafiltration concentration is carried out until the volume is 300 ml. The solution. which may have become turbid during the concentration, is filtered, the pH is adjusted to 3.5 with hydrochloric acid and 6 g.

sodium chloride are added. Cooling results in the precipitation of 24.5 to 25.5 g. lysozyme hydrochloride with a titre of 95 to 97to.

Example 7 On completion of the elution. the resin used in Example 6 is washed with demineralised water until chlorides can no longer be detected. The resin is then contacted with 6.6 kg. of egg-white. followed by the procedure described in Example 5. The pH of the eluate is adjusted to 3.5 with hydrochloric acid and sodium chloride is added up to a concentration thereof of 5% by weight. After standing for some time in a refrigerator. 24 g. of lysozyme hydrochloride separate out.

WHAT WE CLAIM IS: 1. A process for the production of lysozyme, wherein a weakly acidic ion exchange resin containing carboxylic acid groups is adjusted to a pH higher than 7.1. washed with water and contacted with a lysozyme-containing material, whereafter adsorbed lysozyme is eluted from the resin.

2. A process according to claim 1, wherein the resin is adjusted to a pH of from 8 to 9.5.

3. A process according to claim 1 or 2. wherein the resin used is a carboxylic acid group-containing methacrylic resin.

4. A process according to any of the preceding claims. wherein the pH of the resin is adjusted with an aqueous solution of sodium hydroxide. potassium hydroxide or ammonium hydroxide.

5. A process according to any of the preceding claims. wherein the lysozyme is eluted from the resin with an aqueous solution of an inorganic salt or organic salt.

6. A process according to claim 5. wherein elution is carried out with a 6 to 10% by weight aqueous solution of ammonium sulphate or with a 1.5 to 3% by weight aqueous solution of sodium chloride.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. 9. After elution, the pH of the eluate is adjusted to 9.5 and sodium chloride is added until the concentration thereof is 5% by weight. After cooling, 240 to 245 g. of lysozyme base separate out: it has a titre of > 94%. Example 3 The process described in Example 1 is repeated. The concentrated solution obtained after ultrafiltration is adjusted to pH 3.3 with hydrochloric acid and spray dried. The lysozyme hydrochloride thus obtained has a titre of < 94%. Example 4 The process described in Example 1 is repeated. After the lysozyme has been adsorbed and the resin has been washed with water, the lysozyme is eluted with an 8% by weight aqueous solution of ammonium sulphate. The concentration of the ammonium sulphate is then increased to 40% by weight to precipitate the lysozyme. In order to obtain lysozyme hydrochloride and to eliminate any traces of impurities, the precipitate is dissolved in filtered water, the pH is adjusted to 3.3 with hydrochloric acid and the lysozyme is precipitated by the addition of an aqueous solution of sodium chloride. Example 5 1110 ml. of methacrylic resin of the carboxylic group-containing type ("Amberlite" IRC 50) in the acid form are placed in a 6-litre flask and a 30% by weight aqueous solution of sodium hydroxide slowly added dropwise, while stirring, until the suspension has a pH of 8. Stirring is then terminated, the liquid is decanted off and the resin is filtered through a Buchner funnel and washed several times with demineralised water; the resin is then contacted with 6.6 kg. of egg-white at about ambient temperature (about 15"C.) until the lysozyme content of the egg-white is 100 to 200 y/ml. The resin is then washed with water, whereafter the lysozyme is eluted with a 3% by weight aqueous solution of sodium chloride. The pH of the eluate is adjusted to about 9.5 to 9.8 and the pure basic lysozyme is precipitated by increasing the concentration of the sodium chloride to 5C/o. Example 6 The procedure described in Example 5 is repeated except that the resin is adjusted to a pH of 9 with an aqueous sodium hydroxide solution. After elution of the resin with 2.5% by weight aqueous solution of sodium chloride, there are obtained 1900 to 2100 ml. of eluate containing 1.4 to 1.5% by weight of lysozyme. The solution is then subjected to ultrafiltration (DDS membrane type 600) until the volume is reduced to 800 ml., whereafter the solution is diluted with 600 ml. of water and a second ultrafiltration concentration is carried out until the volume is 300 ml. The solution. which may have become turbid during the concentration, is filtered, the pH is adjusted to 3.5 with hydrochloric acid and 6 g. sodium chloride are added. Cooling results in the precipitation of 24.5 to 25.5 g. lysozyme hydrochloride with a titre of 95 to 97to. Example 7 On completion of the elution. the resin used in Example 6 is washed with demineralised water until chlorides can no longer be detected. The resin is then contacted with 6.6 kg. of egg-white. followed by the procedure described in Example 5. The pH of the eluate is adjusted to 3.5 with hydrochloric acid and sodium chloride is added up to a concentration thereof of 5% by weight. After standing for some time in a refrigerator. 24 g. of lysozyme hydrochloride separate out. WHAT WE CLAIM IS:

1. A process for the production of lysozyme, wherein a weakly acidic ion exchange resin containing carboxylic acid groups is adjusted to a pH higher than 7.1. washed with water and contacted with a lysozyme-containing material, whereafter adsorbed lysozyme is eluted from the resin.

7. A process according to any of the preceding claims, wherein the lysozyme is

separated from the eluate either (i) by precipitating by the addition of a salt or (ii) by ultrafiltration of the eluate, followed by precipitation by the addition of a salt to the concentrate or (iii) by concentration by ultrafiltration, followed by spray drying or lyophilisation of the solution.

8. A process according to any of the preceding claims, wherein the lysozyme obtained is salified with a non-toxic and physiologically acceptable inorganic or organic acid.

9. A process according to claim 1 for the production of lysozyme, substantially as hereinbefore described and exemplified.

10. Lysozyme, whenever obtained by the process according to any of claims 1 to 9.