CH619981A5 - Process for the preparation of a superoxide dismutase enzyme - Google Patents

Abstract

The enzyme is extracted from a culture of a marine bacterial strain, especially a photobacterium, by maintaining an aqueous dispersion of the microorganism at 4 DEG C, then for a few minutes at 50-60 DEG C while its pH is at 6.5-8, cooling to 4 DEG C, centrifugation, fractional precipitation of the enzyme using the supernatant fraction by addition of a neutral salt and renewed centrifugation. The enzyme thus obtained is useful as inhibitor of the oxidation of foodstuffs and other oxidisable materials.

Description

The present invention relates to a process for the preparation of a superoxide dismutase enzyme useful as an oxidation inhibitor, in particular as an oxidation inhibitor for food and other oxidizable compounds or media.

Superoxide dismutases have already been described, extracted from beef erythrocytes (Markovitz, "J. Biol. Chem." 234, p. 40,

1959) and "Escherichia coli (Keele and Fridovich," J. Biol. Chem. ", 245, p. 6176.1970).

Superoxide dismutases are enzymes capable of inducing the dismutation of superoxide ions, depending on the reaction:

2O2 ~ + 2H + -> Ü202 + O2

They therefore contribute to achieving a self-protection system for the articles or organisms in which they are found, because the Û2 ~ ions which are produced during the oxidation reactions under the effect of molecular oxygen are very active and attack, among others, proteins, by oxidation of tryptophane among other amino acids, and nucleic acids.

For the purposes of the present invention, the term oxidizable substance means any substance capable of being degraded by an oxidation involving the intervention of superoxide ions.

According to the invention, an effective superoxide dismutase is prepared from marine bacterial strains, such as, for example, strains of Photobacterium phosphoreum, Photobacterium leiognathi or Photobacterium sepia among others. Among these various strains, there may be mentioned the strains of Photobacterium phosphoreum No. ATCC 11040, Photobacterium leiognathi No. ATCC 25521, Photobacterium sepia No. ATCC 15 709.

All of these microorganisms are known. In particular, Photobacterium leiognathi has been described by Boisvert, Châtelain and Bassot ("Annals of the Institut Pasteur" (1967), 112, p. 520-524); Photobacterium sepia has been described by Nicholas and McElroy in "J. Gen. Microbiology ”(1964), 35, p. 401-410.

We have been able to determine the remarkable antioxidant effect provided by these superoxide dismutases on fungi, apples and potatoes, among other foods.

The method according to the invention consists in dispersing in water and maintaining a marine bacterial culture at approximately 4 ° C., then adjusting the pH of the medium to 6.5-8, and preferably approximately 7, to bring the mix at 50-60 ° C for a few minutes, cool it to around 4 "C and, at this temperature, centrifuge, fractionate precipitation of the enzyme using neutral salts on the supernatant, centrifuge at Preferably, a second fractional precipitation of the neutral salt enzyme is carried out on the supernatant fraction and centrifuged again.

As a variant, the process according to the invention can be completed by a purification step, advantageously consisting in dissolving in a phosphate buffer pH 7.8 of the precipitate collected in the last centrifugation and in dialysis of the solution thus obtained, against the same phosphate buffer pH 7.8.

According to another variant of the process according to the invention, the enzyme extracted from the marine bacterial culture is purified, after taking up the product of the last centrifugation with phosphate buffer pH 7.8, and dialysis against this same buffer, by chromatography. which is preferably a column chromatography and in particular a chromatography on three successive columns which are respectively: a first column of Sephadex G 200 gel, a column of Sephadex diethylaminoethyl (DEAE-Sephadex A-50) and a second column of gel of Sephadex G 200.

The bacterial culture serving as an enzyme source in the process according to the invention is, for example, a culture of strains of Photobacterium phosphoreum, Photobacterium leiognathi, or Photobacterium sepia.

To adjust the pH of the dispersion of bacteria in water to 6.5-8, 2N ammonia is used, for example, and potassium chloride, preferably 3M KCl, is then added.

The mixture thus constituted is then brought to a temperature of 50 to 60 ° C, for only a few minutes and preferably for 3 to 4 minutes. The mixture is then cooled to approximately 4 ° C., the temperature at which all the following stages of the process according to the invention are carried out.

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Each of the fractional precipitations constituting stages of this process is carried out, as indicated above, by means of neutral salts in aqueous solution, and preferably by means of ammonium sulphate (NH4) 2SO4.

In an advantageous embodiment of the method according to the invention, the first fractional precipitation is carried out by means of ammonium sulphate (NI-L ^ SC ^ added in an amount such that the mixture or enzymatic extract treated is thus brought at a final concentration of approximately 30 to 35% of saturation at 4 ° C. It should however be noted that it is possible to replace all or part of the ammonium sulphate by one or more other suitable salts and the total amount of said salts then an equivalent and appropriate amount so that it brings the mixture to a final concentration of approximately 30 to 35% of saturation at 4 ° C.

Alternatively, one can use an ultrafiltration system, such as for example a system implementing a porous device such as porous tubes, and in particular a system using a first porous tube retaining, by concentrating them, molecules having a molecular weight greater than 40,000, and a second porous tube letting through the desired enzyme molecules but retaining the remaining bacteria, thereby providing a sterile enzyme extract.

Likewise, the second fractional precipitation is advantageously carried out by means of ammonium sulphate (NH ^ SCU added in an amount such that the mixture or enzymatic extract treated is thus brought to a final concentration of 70 to 75% approximately of the saturation at 4 ° C.

The superoxide dismutases obtained from different marine bacterial strains by the process according to the invention are all superoxide dismutases comprising non-hematinic iron, while the enzymes exhibiting superoxide dismutase activity which have been described previously and which are erythrocupréine and the enzyme extracted from Escherichia coli contain divalent cations which are copper and zinc respectively for the first, and manganese for the second.

The search for the presence of a metal in the superoxide dismutases considered can be carried out by an analysis with an atomic absorption spectrograph.

It is also possible to carry out a colorimetric test which, in the present case, consists in staining an electrophoretic migration gel of an enzyme preparation by means of a specific dye of the ferrous iron Fe2 +, bathophenanthroline, possibly in the presence a reducing agent such as hydrazine. For this test, the gel is cut in half lengthwise and one of the two parts is placed in coomassie blue, and the other part in bathophenanthroline. The test is positive if a pink ring is obtained in the latter case, at the level of the protein band. However, as is known, such an appearance of a pink ring can be considered as an indication of the presence of ferrous iron, here in the protein superoxide dismutase.

Radioactive iron can also be used to label the protein and determine its stoichiometry for the divalent metal cation present.

The superoxide dismutase extracted from marine bacterial cultures by the process presently described has a molecular weight of approximately 40,000 ± 2,500 and a pHi or isoelectric point of approximately 4 to 7 and exhibits a maximum of enzymatic activity for a pH of 8.5 to 10 approximately, with an optimum at pH 9.5 approximately.

The active enzyme can be maintained over a long period of time by storing it in a 70-80% solution of neutral ammonium sulfate (NH4) 2 SO4 at 4 ° C.

The invention is described in more detail in the examples below.

Example 1:

Photobacterium leiognathi bacteria of strain No. ATCC 25521 were cultivated on a synthetic medium containing, in grams per liter:

NaCl

30

Na2HP04, 12H20

18.7

KH2PO4

2

MgS04, 7.20 AM

0.2

(NH4) 2HP04

0.5

Glucose

1.5

Glycerol

1.5

Trypticase

5

Yeast extract

5

This medium was brought to pH 7.2 with sodium hydroxide and sterilized for 1 h at 110 ° C.

A 1 1 preculture was used, grown overnight and distributed between 4 Erlenmeyer flasks containing 250 ml of medium each, to inoculate a fermenter of 121 of medium.

The culture was continued for 12 h at 20 ° C with strong aeration and thus 100 g of bacteria were obtained, expressed by weight of wet product.

135 g by wet weight of Photobacterium leiognathi bacteria, from several cultures, were dispersed in 650 ml of water and allowed to stand at 4 ° C overnight. 2N ammonia was added until the pH of the medium was brought to 7-8 and then 18 ml of 3M KCl was added.

The mixture was brought to 58 ° C and kept at this temperature for 4 min, after which it was cooled to 4 ° C and centrifuged for 10 min at the speed of 10,000 rpm. Still operating at 4 ° C., the supernatants were adjusted to 35% saturation with solid ammonium sulfate at pH 8; it was centrifuged and the supernatant was brought to 75% ammonium sulphate saturation and allowed to stand overnight at 4 ° C. The precipitated protein was harvested by centrifugation and stored in a 75% ammonium sulfate solution.

The activity of a solution of 9 mg of protein per milliliter (biuret) was 40 units / mg, while it was, for comparison, 0 unit / mg for catalase in solution of 9 mg of protein / ml.

After a further fractional precipitation using ammonium sulphate with a concentration gradient of 35 to 75% of saturation, a superoxide dismutase was obtained which, in solution at 14.8 mg protein / ml (biuret), exhibited an activity of 134 units / mg to 500 units / mg.

The protein precipitate was dissolved in pH 7.8 phosphate buffer and dialyzed for 48 h at 4 ° C. The enzyme superoxide dismutase was stored at -20 ° C.

To determine the activity of the latter, a tank was used placed in front of a photomultiplier and containing the reaction mixture below:

Luminol 10 ~ 3M 0.3 ml

Phosphate buffer 10 ~ 3M pH 7.8 0.3 ml

EDTA IO "3M 0.3 ml

Water 1.0 ml

Xanthine oxidase (lmg / ml) 0.050 ml

The reaction was started by injecting into the tank 1 ml of hypoxanthine 3 x 10 ~ 4 M.

A photon flux was then emitted, which> gave rise, under the action of the photomultiplier, to a current whose intensity was measured at the picoampmeter and the variations in this intensity were also recorded.

By introducing into the reaction mixture, before initiation of the reaction, 5 μl of a solution of superoxide dismutase prepared as indicated above, an inhibition of the emission of light was obtained and it was arbitrarily considered that 1 unit of the enzyme superoxide dismutase would be the amount of enzyme that causes a 50% inhibition of light emission.

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In UV spectroscopy, the superoxide dismutase extracted gave the classical spectrum of non-hematinic proteins, with the shoulder of tryptophan at 290 m | i.

To determine the molecular weight, two known techniques were used, one consisting in determining a sucrose centrifugation gradient and the other using a Sephadex G 200 gel.

To do this, we used the following markers, whose molecular weight was known (p.M.):

P.M.

Yeast ADH 150000

Bovine albumin 66000

Peroxidase 40200

It was concluded that a molecular weight of 40,000 ± 2,500.

To determine the subunits of the protein structure of the enzyme, a polyacrylamide gel electrophoresis was added with 10% SDS (sodium dodecylsulfate).

Only one type of subunit was observed, with a molecular weight of approximately 21,000. The molecular weight of the superoxide dismutase obtained can therefore be estimated at 21,000 x 2, or 42,000.

Still on the polyacrylamide gel, a red band was obtained in the presence of bathophenanthroline and hydrazine, precisely at the level of the superoxide dismutase band revealed by coomassie blue.

A colorimetric assay of a protein solution was carried out and a value was obtained for iron which, by estimating the molecular weight of the enzyme at 42000, gave approximately 2 iron atoms per mole.

Atomic absorption spectrometry of a 0.2 mg / ml protein solution confirmed this figure.

We can therefore reasonably estimate the number of iron atoms per mole of superoxide dismutase at 2.

Furthermore, the enzyme extracted according to this example has been found not to suffer any appreciable loss of activity after 5 min at the temperature of 70 ° C.

Electrofocusing of the superoxide dismutase made it possible to evaluate the pHi or isoelectric point of this enzyme at 4.4.

The enzyme exhibited maximum activity at a pH of around 9.5.

An identical enzyme with similar characteristics was obtained from a bacterial strain of Photobacterium leiognathi No. ATCC 25 587.

Example 2:

A culture of bacteria of Photobacterium sepia of strain No. ATCC 15709 was subjected to lysis by stirring in cold water, at the rate of 1 g of wet weight of bacteria per 4 ml of water. The mixture was then centrifuged at 16,000 rpm for 20 min at 4 ° C and then the lysate was cooled to 4 ° C and 3M KCl was added to the light yellow supernatant to a final concentration of 0.1 M .

The solution was heated for 3 to 4 min in a water bath brought to 60 ° C. It was then cooled to 4 ° C and clarified by centrifugation.

The supernatant was subjected to fractional precipitation by addition of solid ammonium sulfate. The active fraction precipitated between 45 and 75% saturation with ammonium sulfate and was separated by centrifugation; it was redissolved in the minimum volume of K2HPO4 5 x 10 "" 3M, pH 7.8, and dialyzed overnight against the same phosphate buffer. The product of this dialysis was then directed to a column of Sephadex G 100 or G 200 gel, equilibrated with 5 × 10 -3 M phosphate buffer, pH 7.8. The active fraction eluted was concentrated using a Diaflo PM-10 ultracentrifugation membrane, and then dialyzed overnight against 5x10-3 M K2HPO4, pH 7.8.

The enzyme superoxide dismutase was adsorbed on a column of DEAE-Sephadex A-50 buffered with K2HPO4

5 x 10-3 M, pH 7.8. The protein was eluted from the column with a linear gradient of K2HPO4, pH 7.8 (from 5 x 10-3 M to 3 x 10 _1 M). The superoxide dismutase was thus eluted at a phosphate concentration of 1.4 × 10 −1 M and it was then concentrated. A new filtration was carried out under the same conditions as the previous one on a column of DEAE-Sephadex A- 50. The enzyme was thus eluted at a phosphate concentration of 1.6 x 10-1 M and concentrated.

The protein thus extracted and purified gave a unique band to acrylamide gel electrophoresis (100 ng of protein for one gel).

Thus, 3 mg of pure superoxide dismutase was obtained from 20 g of frozen cells of Photobacterium sepia (with a superoxide dismutase activity of 250 to 5000 units / mg).

The active enzyme was maintained over a long period of time by storing in a 70-80% solution of (NH4) 2SO4 at 4 ° C.

The molecular weight of the purified enzyme was determined by ultracentrifugation at 45,000 rpm and for 16 h at 4 ° C. The sedimentation rate was measured by the method of Martin and Ames, with a gradient linear in sucrose from 5 to 20 (in weight / volume) and using Beckman Spinco model L2-65B equipment, with a SW 65 K rotor. A sedimentation coefficient of 3.2 was found for this dismutase, against a coefficient 4.82 and 7.4 respectively for alcohol dehydrogenases from horse liver and yeast. From this sedimentation constant, the molecular weight of the superoxide dismutase extracted was therefore calculated to be approximately 42,500.

It has also been established that the molecule of this protein contains 2 iron atoms.

Acrylamide gel electrophoresis gave a unique band for dismutase corresponding to a molecular weight of 20,000 to 20,500, thus showing that the protein molecule was composed of two identical subunits.

The superoxide disinnutase has also been shown to be very resistant to the proteolytic action of trypsin, since a treatment for 60 min of 150 (xg of this superoxide dismutase with 10 ng of trypsin at 20 ° C did not cause any change in the enzymatic activity, no more besides than in the electrophoretic mobility of the undissociated protein.

The enzyme obtained was very stable with respect to heat: no loss of enzymatic activity appeared after 30 min at 20 ° C, 30 ° C and even 40 ° C; after 15 min at 50 ° C, a 28% decrease in activity appeared; after 30 min at 50 ° C, the loss of activity was still only 50%, and it was only 10% and 50% after 3 min and 10 min respectively at 60 ° C.

Electrofocusing of the superoxide dismutase made it possible to evaluate the pHi or isoelectric point of this enzyme at 4.1.

It was determined by means of an ion solution Oi ~ prepared electrolytically, that the enzyme exhibited a maximum of activity for a pH of 8.5 to 10, with an optimum at pH 9.5.

Example 3:

We treated a strain N ° ATCC 11040 of Photobacterium phosphoreum bacteria which are marine bacteria and consequently have a high internal salt concentration.

The lysis of the bacteria was spontaneous in a solution of EDTA 10-3 M, pH 7.8, at 4 ° C.

Cellular debris was removed by centrifugation at 16,000 rpm for 20 min and at 0 ° C.

Preliminary studies had shown that the enzyme superoxide dismutase was stable at 50 ° C and we therefore eliminated the other proteins, themselves thermolabile, by carrying the lysate for 3 min at 50 ° C, after adding KCl until 0.1 molar, then the lysate was cooled to 4 ° C and the proteins were separated by standard centrifugation.

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Still at 4 ° C., a fractional precipitation was carried out using ammonium sulphate (NH4) 2SO4 added in an amount such that the mixture or enzymatic extract treated is brought to a concentration gradient from 0 to 30% approximately of the saturation at 4 ° C. The precipitated fraction was removed by centrifuging for 45 min at 16,000 rpm and at 0 ° C.

The amount of neutral ammonium sulfate required to bring it to 75% saturation at 4 ° C was added to the supernatant.

The precipitated fraction was combined by centrifugation similar to the previous one.

In order to purify the superoxide dismutase thus extracted, the collected precipitate was dissolved in a little 5 × 10 −3 M phosphate buffer, pH 7.8 and dialyzed against this same buffer for 48 h at 4 ° C., to obtain an extract (A).

The proteins still present were then separated according to the size of their molecules, using a Sephadex G 100 gel, the grain crosslinking of which is such that it excludes proteins having a molecular weight greater than 100,000. , which therefore come out very quickly outside the gel. Molecules with a lower molecular weight enter the gel and elute more or less quickly depending on their size.

To do this, the Sephadex resin was swollen for 3 h in water, it was degassed and filtered through Büchner to remove the water; it was placed in the filter pad and poured into a column 50 cm long by 3 cm inside diameter. The column was balanced by passing 21 of buffer.

The enzyme extract (A) from dialysis was taken up beforehand to concentrate it on a Diaflo millipore membrane (P.M. —10) under nitrogen pressure, to a volume of 5 ml. This concentrate was deposited on the column prepared as indicated above and was eluted by passing 500 ml of 5 x 10-3 M phosphate buffer, pH 7.8. The flow rate was 1 drop every 8 s and 2.5 ml fractions were collected; those with significant activity were pooled and concentrated on the Diaflo membrane. A concentrated extract (B) was thus obtained.

A new purification step was carried out by chromatography on an ion exchange resin based on DEAE Sephadex, resin on which the proteins are eluted according to their charge, by buffers of increasing ionic strength.

To prepare the column, we put it to swell in water,

then it was degassed and washed in the following solutions:

0.5 M NaOH

KH2PO4 0.5 M

taking care to rinse the resin with distilled water between each step, to a pH close to neutral. After filtration through Buchner, the resin was suspended in 0.1 M phosphate buffer pH 7.8 and placed in a column 30 cm long by 3 cm inside diameter. The column was balanced by passing 300 ml of 0.1 M buffer through it.

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The concentrated extract (B) was placed on the column thus prepared. Once this extract was completely adsorbed, it was eluted with 500 ml of phosphate buffer pH 7.8, composed of 250 ml of 0.1 M phosphate buffer to which 250 ml of 0.5 M phosphate buffer were gradually added. flow rate was 1 drop every 5 s and the volume of the collected fractions 2.5 ml. The active fractions were combined and precipitated with ammonium sulphate, then stored in this form at -18 to -20 ° C.

The purity of the enzyme superoxide dismutase could be checked by polyacrylamide gel electrophoresis.

A determination of the molecular weight of the enzyme by means of an elution study during filtration on Sephadex G 200 made it possible, using the relationship Log (PM) = f (elution volume) and known markers, to assign to the superoxide dismutase extracted a molecular weight of approximately 40,000.

This molecular weight was also determined by centrifugation in sucrose gradients: sucrose gradients of 5 to 20% were poured into 5 x 10 _ 3M phosphate buffer, pH 7.8, by gradually mixing 2.60 ml of the 20% solution to 225 ml of the 5% solution, in a suitable device.

Various protein markers of known molecular weight and superoxide dismutase have been deposited on these sucrose gradients. The equilibrium was achieved by centrifugation at 45,000 rpm, at 5 ° C and for 22 h. The tubes were then pierced through the bottom and the 10-drop fractions were collected, on which the enzymatic activities presented by the different markers used were measured. After plotting the line representing the variation in molecular weight as a function of the number of the elution fraction, the molecular weight of the superoxide dismutase extracted from Photobacterium phosphoreum was estimated at approximately 40,000, which confirms the previous result.

To determine the molecular weight of the protein subunits, the latter, as well as markers whose molecular weight of the subunits was known, were subjected to electrophoretic migration on polyacrylamide gel in the presence of sodium dodecylsulfate. The graphical resolution of the values found made it possible to assign the value 20,000 to the molecular weight of each subunit of the superoxide dismutase molecule.

The latter proved to be very stable still at 50 ° C. and to exhibit a maximum of enzymatic activity for a pH of approximately 9.5.

Electrofocusing of the superoxide dismutase led to evaluating the pHi or isoelectric point of this enzyme at 4.2.

A colorimetric test as described previously made it possible to detect the presence of ferrous iron in the protein, a pink ring appearing at the level of the protein band in an electrophoretic migration gel previously added with bathophenanthroline. The number of ferrous iron atoms per molecule has been estimated to be almost 2.

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Claims (12)

619981 1. Method for preparing a superoxide dismu-tase enzyme, characterized in that the superoxide dismu-tase enzyme is extracted from cultures of marine bacterial strains, said extraction comprising the succession of steps consisting in dispersing in water and maintain a marine bacterial culture at approximately 4 ° C., then bring the mixture to 50-60 ° C. for a few minutes while its pH is between 6.5 and 8, to cool said mixture to approximately 4 ° C. and, at this temperature, to centrifuge, to carry out on the supernatant fraction a fractional precipitation of the enzyme by means of a neutral salt and to centrifuge again. 2. Method according to claim 1, characterized in that, after the second centrifugation, a second fractional precipitation of the enzyme with neutral salts is carried out on the supernatant fraction and centrifuged a third time. 2 3. Method according to claim 1, characterized in that said marine strains are strains of Photobacterium leiognathi, Photobacterium phosphoreum or Photobacterium sepia. 4. Method according to claim 3, characterized in that said marine strains are chosen from a strain of Photobacterium leiognathi N ° ATCC 25521, a strain of Photobacterium phosphoreium N ° ATCC 11040 and a strain of Photobacterium sepia N ° ATCC 15709. 5. Method according to claim 1, characterized in that it comprises the dissolution, in phosphate buffer pH 7.8, of the precipitate collected in the last centrifugation and a dialysis of the solution thus obtained, against the same phosphate buffer pH 7 , 8. 6. Method according to claim 1, characterized in that the product of the last centrifugation is taken up with phosphate buffer pH 7.8 and chromatography of the solution obtained. 7. Method according to claim 1, characterized in that said mixture is brought to 50-60 ° C for 3 to 4 min. 8. Method according to claim 1, characterized in that said neutral salt is neutral ammonium sulfate (NH ^ SO-t. 9. Method according to claim 1, characterized in that the first fractional precipitation is carried out by means of (NH4) 2SC> 4 added in an amount such that the mixture or enzymatic extract treated is thus brought to a final concentration of 30 at 35% of saturation at 4 ° C. 10. Method according to claim 1, characterized in that the second fractional precipitation is carried out by means of (NH4) 2SC> 4 added in an amount such that the mixture or enzymatic extract treated is thus brought to a final concentration of 70 to 75% of saturation at 4 ° C. 11. Method according to claim 1, characterized in that the extract obtained is sterilized by ultrafiltration. 12. Method according to claim 11, characterized in that one operates by means of an ultrafiltration device comprising a first porous tube retaining, by concentrating them, molecules having a molecular weight greater than 40000 and a second porous tube letting through the desired enzyme molecules, but retaining the remaining bacteria, thereby providing a sterile enzyme extract.