HRP920480A2 - Clostridium histolyticum bacterium mutant, its use for aring collagenase enzymes without clostripaine - Google Patents

Description

The field of technology

C12N : 9/50 15/00 A 61 K

The invention relates to a new mutant of the bacterium Clostridium histolyticum, to its production by means of a chemical mutagenic agent, and to its use in a process for facilitated and increased production of the enzyme collagenase (clostridipeptidase A).

It is known that the enzyme collagenase, apart from its use for scientific purposes, is essential in human and veterinary therapy, especially in the treatment of skin. The activity of the enzyme consists in the fact that, in combination with some non-specific proteases, it accelerates the process of wound healing by removing necrotic tissue, thereby promoting tissue epithelization. Collagenase enzyme is also used in surgery during tissue transplantation and for tissue disintegration.

The collagenase enzyme is used in medicine for the treatment of various degrees of burns, decubitus, skin ulcers, scabs, etc. Epithelization and healing of the skin using the collagenase enzyme is more efficient and faster and prevents the formation of keloid and hypertrophic growths. This is the result of the breakdown of the collagen produced under the action of the collagenase enzyme. It is known that many types of microorganisms grown under certain conditions synthesize the collagenase enzyme.

When purifying the enzyme collagenase, the accompanying enzyme clostripain, which is very similar to collagenase in terms of its chemical and physical properties, presents great difficulties. Medicinal preparations do not contain clostripain. According to the invention "Procedure for obtaining purified collagenase enzyme from the bacterium Clostridium histolyticum K-26 by inhibition of clostripain"; by V. Derkos-Sojak, S. Gamulin, I. Udovicic, M. Holjevac, S. Cižmek and V. Delić (P-1294/90), its removal was achieved by inhibition with the specific inhibitor N-ethylmaleimide (NEM), however , with this procedure there is a loss in the utilization of the collagenase yield, so the discovery of a mutant that does not synthesize clostripain made it possible to obtain a better yield per unit of product, and also a higher utilization of the enzyme.

Maschman, E.O. (Biochem, Z.297, 284, 1938) was the first to describe the collagenase enzyme obtained by growing the bacteria Clostridium perfrigens. Of the many collagenase-synthesizing microorganisms, Clostridium histolyticum has been shown to be the best collagenase enzyme producer. MacLennan, J.D., Mandl, I. and Howes, E.L. (J.Clin.Inv.32,1317,1953) described the growth conditions of the bacterium Clostridium histolyticum. They investigated the composition of the liquid nutrient medium, in which the main part is proteose-peptone, inorganic salts and a vitamin solution. The bacterium was grown at a temperature of 37°C and a pH of 7.2.

Berman, S., Loventhal, J.P., Webster, M.E., Altieri, P.L. and Gochenour, R.B., (J. Bact. 82,582,1961) also investigated the growth conditions of Clostridium histolyticum in order to obtain collagenase. They managed to grow the bacterium Clostridium histolyticum in a nutrient medium without inorganic salts, and its composition is proteose-peptone, enzymatically decomposed casein and soy proteins (tryptic soy broth) and vitamin solutions. This composition of the nutrient medium conditioned other values for good bacterial growth and collagenase biosynthesis, such as pH 8.5 and a temperature of 30°C.

Lettl, A. (J.Hyg.Epidemiol.Microbiol.Immunol., 18,(4)47, 1974) managed to replace proteose-peptone, in the composition of the nutrient medium for the growth of the bacterium Clostridium histolyticum, with other degraded proteins.

Clostridium histolyticum is an anaerobic bacterium, and when growing in a liquid nutrient medium, anaerobic conditions must be ensured.

Takahashi S. and Seifter, S. (J. Appl.Bact. 35, 47, 1972) used in their research the reducing agents sodium thioglycolate and sodium bisulfite, to achieve anaerobic conditions for bacterial growth. The best results, i.e. the highest yield of the collagenase enzyme, were obtained when the mentioned reducing agents were in a 1:1 ratio.

Chiulli, A.J. and Wegman, E.H. (U.S. Patent 3,705,083) describes the production of a collagenase enzyme by growing a Clostridium histolyticum mutant, which showed improved properties in terms of lower toxicity than the parent and the ability to inhibit the growth of other bacteria.

The bacterium Clostridium histolyticum secretes the collagenase enzyme into the nutrient medium in a mixture with larger amounts of other active extracellular proteases (non-specific proteases, α-toxin, accompanying brown pigment), especially clostripain (clostridiopeptidase B, E.C.3.4.22.8.). Commercial preparations of collagenase were thus found to contain traces of clostripain (Keil, B., Mol. Cell. Biochem. 23, 87, 1979). Due to the very similar chemical and physical properties of these proteases, there are difficulties during the purification process and individual isolation from the fermentation medium.

The bacterium Clostridium histolyticum grown in the hitherto known manner submerged, anaerobically in a nutrient medium containing conventional sources of carbon, nitrogen, mineral salts and vitamin solutions, at a temperature of 32 to 37°C at a pH of 7.2 to 8.8 in a time of For 10 to 24 hours, it secretes the enzyme collagenase (clostridiopeptidase A), clostripain (clostridiopeptidase B) and other non-specific proteases into the substrate. The problem with obtaining enzymes in this way is that the filtrate contains both enzymes, collagenase and clostripain. When separating collagenase from clostripain, a large part of the collagenase is lost, which reduces its yield, which also means an increase in the price per product unit.

Our invention consists in treating the spores of Clostridium histolyticum K-26 with the chemical mutagenic agent N-methyl-N'-nitro-N-nitrosoguanidine (Delić, V., Hopwood D.A. and Friend, E.J., Mutation Res., 9 , 1 7, 1970) prepared a new mutant strain of the bacterium Clostridium histolyticum, deposited at the Department of Biochemical Engineering, Laboratory for Microbiology, Pierottieva 6, Zagreb. The obtained mutant, which is the first subject of this invention, differs from the parent strain in two essential properties. From the image (attached) obtained by electron micrography, it is evident that the mutant has lost its motility system. This property is the basic characteristic of the parent strain systematized according to Bergey's Manual of Systematic Bacteriology (Eds. P.H.A. sneth. N.S. Mait. M. Escharpe, J.G. Holt Wilking Baltimore, London, Los Angeles, Sidney, vol. 2. 1170,198).

Another new property of the mutant, in contrast to the parent strain, is the inability to biosynthesize the accompanying enzyme clostripain. This property of it was revealed by the immunological reaction of the precipitation of anti-clostrypain and anti-collagenase serum with filtrates from the cultivation of the mutant, and also by the analytical method, defined in the following text.

The procedure for obtaining a new mutant Clostridium histolyticum K 26 Klō 88 from the strain Clostridium histolyticum K-26, deposited in the Department of Biochemical Engineering, Laboratory for Microbiology, Zagreb, Pierottiev 6* is carried out by processing the spores of the strain Clostridium histolyticum K-26 with the chemical mutagenic agent N-methyl-N'-nitro-N-nitrosogranidine (MNNG). The time of action of MNNG on spores was 45 and 90 min. at room temperature. After that, the spores are separated by centrifugation, which are suspended in a physiological solution and inoculated with a suitable dilution onto a solid nutrient medium and incubated at 37°C under anaerobic conditions for 3 days. ;*Numbers under which the mentioned microorganisms were deposited:

1. Clostridium histolyticum K-26 4042

2. Clostridium histolyticum K-26 Klō 88 4043

Each grown colony is tested for enzyme biosynthesis by biochemical analysis, as well as using an immunoprecipitation test. Based on the appearance of a precipitation line between Clostridium histolyticum K-26 culture filtrate and rabbit anticlostipain serum, the presence or absence of clostripain can be concluded.

The invention relates to the use of a new mutant obtained from the bacterium Clostridium histolyticum K-26, in the process of collagenase enzyme production by submerged and anaerobic cultivation on nutrient media containing proteose-peptone, enzymatically degraded casein and soy proteins (tryptic soy broth), vitamin solution (e.g. riboflavin, nicotinamide, pyridoxine, pantothenic acid), reducing agent (e.g. Na-thioglycolate), and, if necessary, inorganic salts, e.g. NaHPO4, MgSO4, FeSO4. The cultivation temperature ranges from 28°C to 37°C, pH values from 7.2 to 8.9 and time from 10 to 24 hours. During cultivation, collagenase enzyme is secreted into the liquid nutrient medium. The product does not contain clostripain. The cultivation is followed by purification and isolation, so the utilization of the collagenase enzyme is increased, for example from 10 to 20%, in comparison with the usual methods of obtaining collagenase.

The method of Gassman, V. and Nordwig, A. (Hoppe-Esyler's Z. Phys. Chim. 322, 262, 1960) with a synthetic substrate was used to determine the activity of the collagenase enzyme obtained.

To determine clostripain activity, a modified method was used according to the catalog of the company "Worthington" from 1987, p.47.

The procedure for determining non-specific proteolytic activity was derived from the definition of a unit of caseinase activity according to the catalog of the company "Sigma" from 1990, p. 333. For incubation and precipitation, the usual conditions for determining proteolytic activity were applied, and the determination of released amino groups was performed using a modified colorimetric ninhydrin method according to Rosen. H. (Arch. Biochem. biophys. 67, 10, 1957).

In order to preserve the activity of the collagenase enzyme during possible freezing and lyophilization, the liquid enzyme preparation is processed by adding carbohydrates, such as sucrose, maltose and similar, in concentrations from 5x10-3 M to 2.5x10-2 M, and in order to stabilize the activity of the freeze-dried collagenase enzyme preparation, soluble proteins are added /enzymatically decomposed casein (Fluka), Peptone 1 in powder (Torlak), acid-degraded casein (Serva) and others/ in concentrations of 10 to 50 mg per milliliter of liquid enzyme preparation.

The invention is illustrated by the following examples.

Example 1

Clostridium histolyticum K-26 spores are treated with 3 mg/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) dissolved in 10 ml of TRIS buffer for 45 and 90 minutes. The mutagen MNNG is removed by centrifugation and decantation of the supernatant. The spores are suspended in a physiological solution and inoculated in suitable dilutions on a solid agar medium and incubated in anaerobic conditions at 37°C for 3 days.

Colonies grown on a solid nutrient medium are inoculated onto a liquid nutrient medium and cultured after two days of growth at 37 C. They are dripped into a pool of 0 8 mm, while rabbit anticlostripain serum is dripped into an adjacent pool of the same size, 2 cm away. Diffusion of clostripain antibodies and clostripain enzyme in the gel leads to their joining and the formation of a pre-epithelial line. Each mutant is controlled in this way several times with additional biochemical analysis. The absence of a precipitation line is evidence that the mutants do not produce clostripain. The obtained mutants are lyophilized and stored for further use.

Example 2

In this example, the lyophilized parental strain Clostridium histolyticum K-26 was used, with which the pre-fermenter medium of this composition is inoculated:

[image]

vitamin solution:

[image]

The substrate has a natural pH, sterilization takes 30 min. at 121°C, while sterilization of the vitamin solution takes 15 min. at 115°C. Immediately before the inoculation of the nutrient medium with the microorganism, a vitamin solution is added. Cultivation of the bacteria on this medium lasts 20 hours and in a stationary manner at a temperature of 37 C.

The second stage of bacterial cultivation was carried out in glass bioreactors with a volume of 2 L (Multigene, New Brunswick, N.Y.), under the following conditions: 1500 ml of nutrient medium with the same composition as the pre-fermenter with the addition of 10% inoculum, pH value 7.4, temperature 37°C , cultivation time 20 hours, with occasional blowing with nitrogen due to the establishment of anaerobiosis with mixing at 50 rpm.

Enzyme yield:

[image]

Example 3

In this example, the parental strain Clostridium histolyticum K-26 was used, with which the pre-fermenter nutrient medium was inoculated (as in example 2). After 20 hours of growing with 10% of that inoculum, the uninoculated nutrient medium with the following composition:

[image]

The composition of the vitamin solution is the same as described in example 2, the pH value is natural 7.4, the sterilization of the substrate is 30 min. at 120ºC, and cultivation was carried out in a 2 L glass bioreactor (Multigen New Brunswick, N.Y.) with 1500 ml of the described medium, cultivation time was 7 hours, at a temperature of 37°C.

Enzyme yield:

[image]

Example 4

In this example, the lyophilized mutant Clostridium histolyticum K-26 klº 88, obtained according to example 1, was used for inoculation of the pre-fermenter medium with the composition and growth conditions as described in example 2.

10% of the inoculum was used to inoculate the liquid nutrient medium. The cultivation time was 7 hours.

Enzyme yield:

[image]

Example 5

In this example, a lyophilized mutant of Clostridium histolyticum K-26 klō88, obtained according to example 1, was used, which was inoculated into a nutrient medium of the composition as described in example 3. Bacterial cultivation was carried out at a temperature of 28°C and a duration of 8-10 hours.

Enzyme yield:

[image]

Example 6

Lyophilized mutant Clostridium histolyticum K-26 klō 88; obtained according to example 1, was used in this example as an inoculum for the pre-fermentation medium as described in example 2. The cultured microorganism was now used as an inoculum for the second stage of fermentation as described in example 3, with the difference that the pH value was 8, 7, and the temperature is 30°C. Cultivation was stopped after 10 hours.

Enzyme yield:

[image]

Example 7

This example shows the method of isolating the enzyme from the sterile ultrafiltrate of the fermentation medium of the mutant Clostridium histolyticum k-26 Klō 88, which does not produce clostripain, by treating the concentrated culture filtrate obtained in examples 4, 5, 6 with 0.03-0.05 M calcium chloride, acetone from 18 to 33% by fractional precipitation with ammonium sulfate, first with the addition of 20-30% saturation, then from 70 to 80% saturation, and finally by two-stage treatment with calcium phosphate gel, indicated by omitting the treatment with acetone. An active lyophilized preparation of collagenase is obtained, which does not contain clostripain, with a utilization of 75 to 85%.

Enzyme yield:

[image]

Example 8

The example shows the method of isolating the collagenase enzyme from the sterile ultrafiltrate of the fermentation medium of the mutant Clostridium histolyticum K-25 klō 88, obtained as in example 4, 5, 6 and processed according to example 7, by omitting the treatment with ammonium sulfate up to 30% saturation. A white active lyophilized preparation of collagenase is obtained, which does not contain clostripain, with a yield of 75 to 90%.

Enzyme yield:

[image]

Example 9

The example shows the method of isolating collagenase enzyme from samples obtained as in example 4, 5 and 6 and purified as in example 7 and 8, by omitting the treatment with calcium phosphate gel, a white lyophilized preparation of collagenase is obtained, which does not contain clostripain. with a utilization of 75 to 90%.

Enzyme yield:

[image]

Example 10

This example illustrates a method of purifying a liquid preparation obtained by cultivation and processing as described in examples 3 and 7. The purification procedure consists in applying the sample to a Sephadex G-75 gel filtration column. Application of the sample and elution of protein fractions is carried out with buffer 0.01 M TRISx HCl at pH 7.2 with the addition of 0.01 M calcium acetate. Three protein fractions were identified by gel filtration. The analysis proved collagenase activity in the first fraction, in the second function a smaller collagenase activity was proved, and in the third the activity of non-specific proteases and brown pigment was proved as follows:

[image]

[image]

[image]

Example 11

This example describes the desalting of samples of purified collagenase enzyme fractions. The liquid ultrafiltered fractions of the pure enzyme obtained as in example 10 are taken and applied to a column with a Sephadex G 25 gel column. Application and elution of the sample is carried out with distilled water. One fraction of pure enzyme freed from salt is obtained. The activity of the pure enzyme was 38.2 I/ml.

Example 12

This example refers to the preservation of collagenase enzyme activity in a liquid preparation during freezing and lyophilization. The procedure is carried out by adding carbohydrates such as sucrose, maltose and the like to the liquid preparation obtained as described in examples 7 to 11, in concentrations from 5x10-3 M to 2.5x10-3 M.

Example 13.

This example describes methods of stabilizing a lyophilized collagenase enzyme preparation. The procedure is carried out by adding soluble proteins, such as enzymatically degraded casein (Fluka), Peptone 1 in powder (Torlak), acid-degraded casein (Serva) and others, to the liquid preparation of collagenase, obtained according to examples 7 and 11. concentrations from 10 to 50 mg per milliliter of liquid enzyme preparation. Thus, the lyophilized preparation of collagenase shows unchanged enzymatic activity.

Claims (3)

1. Mutant Clostridium hlstolytlcum K-26 klō 88, deposited at the Institute for Biochemical Engineering, Laboratory for Microbiology, Pierottieva 6, Zagreb, under number 4043. 2. Procedure for obtaining collagenase using the Clostridium histolyticum K-26 klō 88 mutant, indicated by the fact that spores of the Clostridium histolyticum K-26 strain are treated with N-methyl-N'-nitro-N-nitrosoguanidine for 45 and 90 minutes at room temperature temperature. After that, the spores are separated by centrifugation and suspended in a physiological solution and inoculated with a suitable dilution onto a solid nutrient medium and incubated at 37°C under anaerobic conditions for 3 days. Each grown colony is tested for enzyme biosynthesis by biochemical analysis and immunodiffusion test. 3. Method for obtaining collagenase without clostripain using the mutant Clostridium histolyticum K-26 klō 88 characterized by the fact that the mutant Clostridium histolyticum K-26 klō 88 is grown in a liquid nutrient medium containing proteose-peptone, enzymatically degraded casein and soy proteins and a solution of vitamins, the reducing substance Na-thioglycolate and nitrogen, in a period of 10 to 24 hours, at a temperature of 28 to 37°C, pH values of 7.2 to 8.9 in submerged and anaerobic conditions, after which a combination of precipitation agents such as From calcium chloride, acetone, ammonium sulfate and calcium phosphate gel, a purified preparation of collagenase is obtained, and the resulting product is treated with the addition of carbohydrates in concentrations from 5x10-3 M to 2.5x10-2 in order to preserve its activity during freezing and lyophilization of the liquid enzyme preparation. M, and in order to stabilize the activity of the lyophilized collagenase enzyme preparation, soluble proteins are added in concentrations of 10 to 50 mg per milliliter of liquid enzyme im preparation.