FI60576C - FOERFARANDE FOER FRAMSTAELLNING AV LYSOZYM - Google Patents

Description

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Ma m 11 utlAggningsskrift cU5 / 6 C (45) Patent granted 27 12 1982. ligSÄfj Patent meddelat '^ (51) Kv.lk.3 / lnt.ci.3 C 12 N 9/56 3 UOM IM FINLAND (21) Pitunttlh »k« mu * - Patuntansekning 782131 (22) Hakemlipilvi - Ansäkningidag 0 ^ · 07 · 7 8 * '(23) Alkupttvl — GUtlghetsdag 0 ^ .07.78 (41) Interpreters Swept - Bllvit offentilj 05.01.79

Patent and Registration Office .... .......

_. . . . (44) Date of publication of the contract. - "

Patent- och registerstyrelsen Antökan utlagd och utl.akrlft «n publkerad 30.10.8l (32) (33) (31) Pyydetty etuoikeus— Baglrd priority 0U .07.77

United Kingdom-Storbritannien (GB) 2785 ^ / 77 Proven-Styrkt (71) (7?) Lorenzo Ferrari, 8, Via Biella, 201 ^ 3 Milan,

Carlo Trinchera, UU, Via Tito Vignoli, 201 ^ * 6 Milan,

Italia-Italien (IT) (7 ^) Oy Borenius & Co Ab (5U) Method for the preparation of lysozyme - Förfarande för fram-ställning av lysozym

The invention relates to a new and improved process for the preparation of lysozyme and its salts formed with non-toxic and physiologically tolerable acids from egg white, the weakly acidic ion exchange resin being contacted with egg white, after which the lysozyme is eluted from the resin with an aqueous salt solution and lysed.

Before lysozyme was first used in pharmacotherapy about twenty years ago, Fleming isolated in 1922 lysozyme (EC 3.2.1.17), an antibacterial and immunosuppressive enzyme previously used to a limited extent in bacteriological laboratories, e.g., bacterial cell wall structures and bacterial cell wall structures. Due to the advantageous clinical results obtained in the treatment of inflammation and infections, and later due to the use of lysozyme in foods as a preservative and anti-mold agent, the demand for pure lysozyme has greatly increased and numerous industrial methods for producing lysozyme have been patented.

Lysozyme is an enzyme that can be better defined as N-acetyl-2,60576 muramido-glycan hydrolase. It is alkaline in nature and has a molecular weight of about 15,000. It is reasonably stable in an acidic medium but less stable in a basic medium. It is very effective against gram-positive bacteria, from which it releases substances that can be determined using amino-sugar reagents.

Jolles (Exp. Ann. Bioch. Med., 27th Series, page 1, published by Masson,

Paris, 1966) has specifically studied its basic structure as a basic polypeptide composed of 129 amino acid residues.

Applicant's GB Patent Publication No. 1,110,466 describes a process for preparing lysozyme from egg white, wherein the egg white is contacted with a slightly acidic ion exchange resin at pH 6-7 and below room temperature, after which the resin is separated, is not more than 0.2 M and the pH is not more than 7, after which the lysozyme is eluted with an aqueous salt solution and precipitated from the eluate by increasing the salt concentration of the solution.

The invention provides a new, better process for the preparation of lysozyme, preferably from egg white, but also from other lysozyme-containing starting materials which may be of plant or animal origin.

Thanks to the method according to the invention, which is based on the extraction of lysozyme from egg white or other lysozyme-containing material by means of weakly cationic resins under special experimental conditions, advantages are obtained compared to currently known methods: The egg white used is not contaminated with foreign substances and its natural alkaline pH is about 9 Whereas the processing time is short so that the physical properties of the egg white, such as its binding and lifting properties, are not impaired so that it can be used in food in the same way as the original egg white, so that its commercial value remains virtually unchanged; 60576 finally, at the various stages of treatment, all substances whose presence in sewage could cause serious pollution problems are eliminated. In the practice of the process according to the invention, only sodium chloride and sodium hydroxide are used, the solutions of which can be reused in successive production cycles. The inevitable but small losses of sodium chloride (due in part to the neutralization of sodium hydroxide with hydrochloric acid) can be safely eliminated.

According to the invention, there is thus provided a process for the preparation of lysozyme characterized in that a methacrylic resin containing carboxylic acid groups is used as the ion exchange resin, the pH of which is first adjusted to a value greater than 7.1 and preferably between 8 and 9.5, the resin is washed with water and contact with egg white at a temperature of 10 to 15 ° C, after which the adsorbed lysozyme is eluted from the resin with an aqueous solution of an inorganic salt, preferably either a 6 to 10% by weight aqueous solution of ammonium sulfate or a 1.5 to 3% by weight aqueous solution of sodium chloride, and that the lysozyme then separated by precipitation with said inorganic salt from the eluate, possibly concentrated by ultrafiltration, or by ultrafiltration of the concentrated eluate, and spraying! by gradual drying, and that the resulting lysozyme is, if desired, converted into a salt with a non-toxic and physiologically tolerable acid.

The pH of the resin can be adjusted, for example, with 30% aqueous sodium hydroxide solution or with aqueous solutions of potassium hydroxide or ammonium hydroxide, the concentrations of which are not critical.

The use of a balanced resin with a pH on the basic side provides two advantages in addition to those mentioned above, which have not been achieved so far in other industrial processes:

In particular, the pH of the egg white and resin mixture is about 9, i.e. approximately the normal pH of the egg white, which reduces the risk of bacterial or fungal contamination during egg white processing. In addition, the alkaline solution used in the process of the invention increases the rate of lysozyme adsorption to the resin so that the processing time of this step is considerably shortened, which correspondingly reduces the manufacturing cost. Thus, the method can be carried out at approximately room temperature (10 to 15 ° C), which is an economic advantage, and the egg white, the properties and bacterial content of which have not been substantially altered, can be used for other purposes after its lysozyme content has been removed, (b)

Another advantage of lysozyme adsorption at pH 8-9 is that after the contact period, after the egg white has been removed and the resin has been washed with water to remove the last residues of the egg white, the resin does not need to be washed with saline to remove protein because the selectivity of adsorption is very high.

Lysozyme can be eluted with an aqueous solution of an inorganic salt, for example, sodium nitrate, potassium nitrate, sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, or ammonium sulfate can be used, but elution can also be performed with organic salts, e.g.

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

The lysozyme can be separated from the eluate by the following methods: a. The enzyme is precipitated by adding salt to the eluted solutions, b. The eluate is ultrafiltered, followed by the addition of salt to the concentrate to precipitate the enzyme, c. concentrated by ultrafiltration, followed by spray-drying or lyophilization of the solution.

Concentration by ultrafiltration can be performed according to the following formula; 5 60576 Elution of 100 liters (1% lysozyme! (3% sodium chloride ultrafiltration! \ K 40 liters (2.5% lysozyme (3% sodium chloride dilution with 30 liters of water)

V

7 0 liters (1, 4-5% lysozyme (1.7% sodium chloride

V

ultrafiltration 15 liters (6.65% lysozyme! (1.7% sodium chloride i + sodium chloride ^ lysozyme

The lysozyme obtained by the process of the invention can be used without further treatment in medicine and foodstuffs, but the lysozyme can also be converted into a salt by means of a non-toxic and physiologically tolerable inorganic or organic acid, if desired.

The following examples illustrate the invention.

Example 1

Suspend in about 60 liters of water 11 liters of methacrylic resin of the carboxylic acid group type, in which the carboxyl groups are free ("Amberlite IRC 50"). A 20% aqueous solution of sodium hydroxide is added slowly with stirring until the pH of the suspension is 8. The liquid is removed by decantation and the resin is washed repeatedly by decantation using demineralized water. After the water has been removed, the resin is contacted with egg white (66 kg) at about 10 ° C until the lysozyme content of the egg white is about 300 γ / ml. The resin is then washed with water and then eluted with 3% aqueous sodium chloride to give about 25 liters of eluate containing 240-260 g of lysozyme. The solution is concentrated by ultrafiltration (DDS membrane, type 600) to a volume of 10 liters. Dilute with 7.5 liters of water, then concentrate again to a volume of about 3.8 liters and filter. The pH is adjusted to 3.5 by adding hydrochloric acid, followed by about 85 g of sodium chloride. Cool, centrifuge and dry to give 240-250 g of lysozyme with a purity of 94-96%.

Example 2

The procedure of Example 1 is repeated except that the pH of the resin is equilibrated to 9. After elution, the pH of the eluate is adjusted to 9.5 and sodium chloride is added until the concentration is 5%. After cooling the reaction mixture, 240-245 g of lysozyme are separated, purity> 94%.

Example 3

The procedure of Example 1 is repeated. The pH of the concentrated solution obtained after ultrafiltration is adjusted to 3.3 with hydrochloric acid, followed by spray-drying. The lysozyme hydrochloride thus obtained is> 94% pure.

Example 4

The procedure of Example 1 is repeated. After the lysozyme is adsorbed and the resin is washed with water, the lysozyme is eluted with 8% aqueous ammonium sulfate. The ammonium sulfate concentration is then added to 40% to precipitate lysozyme. To obtain lysotime hydrochloride and to remove all impurities, the precipitate is dissolved in filtered water, the pH is adjusted to 3.3 with hydrochloric acid and the lysozyme is precipitated by adding aqueous sodium chloride solution.

Example 5

To a 6 liter flask add 1110 ml of acidic methacrylic resin of the carboxyl group type ("Amberlite IRC 50") and slowly add dropwise, with stirring, 30% aqueous sodium hydroxide solution, 7 60576 until the pH of the suspension = 8. Stirring is then stopped, the liquid is removed by decantation and decanted. the resin is filtered through a Buchner funnel and washed several times with demineralized water.

The resin is then contacted with the egg white (6.6 kg) at about room temperature (about 15 ° C) until the lysozyme concentration of the egg white is 100-200 γ / ml. The resin is then washed with water and the lysozyme is eluted with 3% aqueous sodium chloride solution. The pH of the eluate is adjusted to about 9.5 to 9.8, and pure basic lysozyme is precipitated by increasing the sodium chloride concentration to 5%.

Example 6

The procedure of Example 5 is repeated except that the pH of the resin is adjusted to 9 with aqueous sodium hydroxide solution. The resin is eluted with 2.5% aqueous sodium chloride to give 1900-2100 ml of eluate with 1.4-1.5% lysozyme. The solution is then ultrafiltered (DDS membrane, type 600) until the volume is reduced to 800 ml, at which time the solution is diluted with 600 ml of water and concentrated by ultrafiltration a second time until the volume is 300 ml. During concentration, the cloudy solution is filtered, the pH is adjusted to 3.5 with hydrochloric acid, and 6 g of sodium chloride are added. Upon cooling, 24.5-25.5 g of lysozyme hydrochloride precipitates, purity 95-97%.

Example 7

At the end of the elution, the resin used in Example 6 is washed with demineralized water until no more chlorides can be detected. The resin is then contacted with egg white (6.6 kg), after which the treatment described in Example 5 is repeated. The pH of the eluate is adjusted to 3.5 with hydrochloric acid, and sodium chloride is added until its concentration is 5%. The reaction mixture is allowed to stand in the refrigerator for some time to give 24 g of lysozyme hydrochloride.

Claims (1)

A method for producing a lysozyme or a salt thereof with a non-toxic and physiologically tolerable oxygen from an egg white, the weakly acidic ion exchange resin being contacted with an egg white resin, the lysozyme being eluted from the resin with an aqueous salt solution, and the lysozyme eluted from the resin. The pH is first adjusted to a value greater than 7.1 and preferably between 8 and 9.5, the resin is washed with water and contacted with egg white at a temperature of 10-15 ° C, after which the adsorbed lysozyme is eluted from the resin with an aqueous solution of an inorganic salt, preferably either an aqueous solution of ammonium sulphate 6 to 10% by weight or an aqueous solution of sodium chloride 1,5 to 3% by weight, and that the lysozyme is then separated from the eluate by precipitation with said inorganic salt, a is optionally concentrated by ultrafiltration, or by ultrafiltration of the concentrated eluate and spray-drying or freeze-drying, and that the lysozyme obtained is, if desired, converted into a salt by means of a non-toxic and physiologically tolerable acid. For use in the preparation of lysozymes or in the presence of phytologically compatible oye as a physiologically compatible syringe, the colors and the color of the lyso-resin rings in contact with the lysozyme are separated from the resin by the resin of the resin. ur eluate, kännetecknat av att jonbytar-resart utgörs av ett methacrylharts, som innehäller carboxylsyragrupper, att hartsets pH först regulleras account högre än 7,1, företrädesvis account mellan 8 ooh 9,5, det tvättas med vatten och bringa i kontakt med contact at a temperature of 10 to 15 ° C, the adsorbent lysozyme is eluted from the resin with the addition of water to an organic site, with the addition of 6 to 10% by weight of ammonium sulphate or with 1.5 to 3% by weight Vatenlösning av natriumklorid och att att lysozymet därefter avskiljs genom utfällning frän eluatet medelst nämnda oorgannsait, vilket eluat eventuellt concentrate genom ultrafiltrering ell Medels The ultrafiltration of the concentrated eluate and the spraying or lyophilization, including lysozymes, are self-contained, and are suitable for use as a gift or physiologically compatible solution.