Mutant of bacterium Clostridium histolyticum, a process for the obtaining thereof, and its use in the production of clostripain-free collagenase
This invention relates to a novel mutant of bacterium Clostridium histolyticum, to a process for the obtaining thereof by means of a chemical mutagenic agent, and to its use in an improved process for the production of clostripain-free collagenase (clostridiopeptidase A). 
     It is well known that the enzyme collagenase is used not only for scientific purposes but also finds its place in human and veterinary therapy, in particular for the treatment of skin disorders. In combination with some non-specific proteases it accelerates the process of wound healing by removing necrotic tissue and consequently, by improving epithelization. Collagenase is also used in surgery in cases of tissue transplantation and for tissue disintegration. 
     In human medicine collagenase is used in the therapy of burns of different degrees, decubitus, skin ulcers, crusts, etc. Epithelization and recovery of the skin by means of collagenase, in addition to being more efficient and rapid, also prevents the formation of keloids and hvdertrophic growth as a result of formed collagen disintegration under the action of collagenase. Various species of microorganisms cultivated under defined conditions are known to synthesize collagenase. 
     In the course of collagenase purification process the accompanying enzyme clostripain causes great difficulties since its chemical and physical properties are very similar to those of collagenase. Medicinal preparations do not contain clostripain. 
     Maschman, E.O. (Biochem, Z., 297, 284, 1938) was the first to describe collagenase obtained by cultivation of bacterium Clostridium perfringens. Among all organisms which synthesize collagenase, Clostridium histolyticum was found to be the best producer. MacLennan, J.D., Piandi, 1. and Howes, E.I. (J. Clin. Inv., 32, 1317, 1953) have described the conditions for growth of Clostridium histolyticum. They investigated the composition of liquid medium which consisted mostly of proteose-peptone, inorganic salts and vitamin solution. Bacterium was cultivated at a temperature of 370° C. and at pH 7.2.  Berman, S. , Lowenthal, J P., Webster, M.E., Altieri, P.L. and Gochenour, E.B. (J. Bact., 82, 5829 1961) also performed investigations of the conditions for growth of Clostridium histolyticum with the aim of producing collagenase. They succeeded in cultivating Clostridium histolyticum in a nutrient medium containing no inorganic salts but only proteose-peptone, enzymatically hvdrolized proteins of casein and soy (tryptic soy broth) and vitamin solution. Such composition of nutrient medium also conditioned other values required for satisfactory bacterial growth and biosyrthesis of collagenase, such as pH value of 8.5 and temperature of 30° C. 
     Lettl, A. (J.Hyg.Epidemiol.Microbio.Immunol., 18, (4)47, 1974) succeeded in substituting proteose-peptone with other hydrolized proteins in the composition of nutrient medium used for cultivation of Clostridium, histolyticum. 
     Clostridium histolyticum is an anaerobic bacterium and for its cultivation in a liquid medium anaerobic conditions must be secured. Takahashi, S. and Seifert, S. (J.Appl.Bact., 35, 47, 1972) used in their investigations the reducing agents sodium thioglycolate and sodium bisulfite to obtain anaerobic conditions needed for bacterial growth. Optimal results, i.e. the highest collagenase yield was obtained when the mentioned reducing agents were used in a 1:1 ratio. 
     Chiulli, A.J. and Wegman E.H. (U.S. Pat. No. 3,705,083) described the production of collagenase by cultivation of a mutant of Clostridium histolyticum which showed improved properties relative to lower toxicity compared to parenteral toxicity and a possibility of inhibiting growth of other bacteria. Bacterium Clostridium histolyticum recovers in the medium the enzyme collagenase together with greater amounts of other active extracellular proteases (non-specific proteases, alpha-toxin, accompanying brown pigment), in particular clostripain (clostridiopeptidase B). Thus, it has been found that commercially available preparations contain traces of clostripain (Keil, B., Mol.Cell Biochem., 23, 87, 1979). Difficulties are encountered in the course of purification process and individual isolation from fermentation medium due to very, similar chemical and physical properties of these proteases. 
     Bacterium Clostridium histolyticum cultivated under known anaerobic submerged conditions in a medium containing conventional sources of carbon, nitrogen, mineral salts and vitamin solutions, at a temperature of 32°-370° C. and at pH range of 7.2-8.8, over a period of 10-24 hours secretes in the medium the enzyme collagenase (clostridiodeptase A), clostripain (clostridiopeptidase B) and other non-specific proteases. The problems encountered in such enzyme production consist in the presence of both enzymes, collagenase and clostripain in the filtrate. In the course of collagenase separation from clostripain, a large portion of collagenase is lost, resulting in reduced enzyme yield and consequently, increased costs per product unit. 
     We have now surprisingly found that the use of a novel mutant of bacterium Clostridium histolyticum in the production of collagenase solves all the aforesaid problems. 
     Hence, one object of the present invention is a novel mutant of bacterium 
     Clostridium histolyticum, designated Clostridium histolyticum K-26 clo 88, which was deposited: 
     i) at the Institute of Biochemical Engineering, Laboratory of Microbiology, Zagreb, Pierottijeva 6, on May 22, 1990, under the accession number 4043, 
     ii) and in conformance with the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure, pursuant to Rule 7.1, 
     at the National Collection of Agricultural and Industrial Microorganisms, Department of Microbiology and Biotechnology, University of Horticulture and Food Industry , Somloi ut 14-16, H-1118 Budapest, Hungary on June 4, 1991 , under the accession number NCAIM B (P) 001 145. 
     A further object of the present invention is a process of obtaining said novel mutant Clostridium histolyticum K-26 clo 88, by treating spores of Clostridium histolyticum K-26 (deposited at the Institute of Biochemical Engineering, Laboratory of Microbiology, Zagreb, Pierottijeva 6, on May 22, 1990, under the accession number 4042) with the mutagenic agent N-methyl-N&#39;-nitro-N-nitrosoguanidine for 45 and 90 minutes at about room temperature, whereupon the spores are separated by centrifugation, suspended in a physiological solution, inoculated into a solid medium, and incubated at 37° C. for about 3 days under amaerobic conditions. 
     The novel mutant Clostridium histolyticum K-26 clo 88 of the present invention differs from the parental strain Clostridium histolyticum K-26 in two essential characteristics. As, it can be seen from FIGS. 1 and 2 (enclosed) obtained by electron micrography, the mutant has lost its motility system which is a fundamental characteristic of the parenteral strain classified according to Bergey&#39;s Manual of Systematic Bacteriology (Sneath, P.H.A., Mair, N.S., Scharpe, F.E., Holt, J.G.; Eds:Williams and Wilking, Baltimore, London, Los Angeles, Sidney, Vol.2, 1170,  In distinction to the parenteral strain the second new characteristic of the novel mutant consists in its inability to biosynthesize the accompanying enzyme clostripain. This property was discovered by means of immunological precipitation reaction of anticlostripain and anticollagenase serum with mutant cultivation filtrates, as well as by method of analysis described in following text. 
     Each grown colony is tested for enzyme biosynthesis by both biochemical analysis and immuno-precipitation test. Presence or absence of clostripain is determined on the basis of appearance of a precipitation line between the filtrate of Clostridium histolytlicum K-26 culture and rabbit anticlostripain serum. 
     A further object of the present invention is a process for the production of clostripain-free collagenase, wherein said novel mutant Clostridium histolyticum K-26 clo 88 is cultivated in a nutrient medium containing proteose-peptone, enzymatically hydrolyzed proteins of casein and soy (tryptic soy broth), a vitamin solution, e.g. riboflavin, nicotinamide, pyridoxine, panthothenic acid, a reducing agent, e.g. Na-thioglycolate, and inorganic salts, if required, e.g. NaHPO 4 , mgSO 4 , FeSO 4 , for 10 to 24 hours, at a temperature ranging from 28° to 37° C. and a pH of 7.2 to 8.9, tinder anaerobic submersed conditions. During cultivation the enzyme collagenase recovers in liquid medium. The product is clostripain-free. Cultivation is followed by purification and isolation. The collagenase yield is increased by approx. 10 to 20% in comparison to usual methods for collagenase production. Activity of obtained collagenase was determined by applying the method after Gassman, V. and Nordwig, A. (Hoppe-Seyler&#39;s Z. Phys. Chem., 322, 267, 1960) with synthetic substrate. 
     Clostripain activity was determined using a modified method described in the catalogue of the firm Worthington, 1987, p.47. 
     Procedure for non-specific proteolytic activity determination was derived from the definition of casein activity unit in compliance with the catalogue of the firm Sigma, 1990, p.333. For incubation and precipitation were applied common conditions required for proteolytic activity determination. Determination of released amino groups was carried out, according to a modified colorimetric ninhydrin method after Rosen, H. (Arch. Biochem.Biochys. , 67, 10, 1957). 
     In order to maintain the activity of collagenase during possible freezing and lyophilization, liquid enzyme preparation is treated by adding carbohydrates, such as saccharose, maltose or the like, at concentrations ranging from 5×1O -3  M to 2.5×1O -2  M. For stabilization of activity of lyophilized collagenase preparation soluble proteins are added (enzymatically hydrolized casein (Fluka), Peptone I in powder form (Torlak), acid hydrolized casein (Serva), etc.) at concentrations ranging from 10 to 50 mg/mL of liquid enzyme preparation. 
    
    
     This invention is illustrated by the following examples: 
     EXAMPLE 1 
     The spores of Clostridium histolyticum K-26 are treated with 3 mg/mL of N-methyl-N&#39;-nitro-N-nitrosoguanidine (MNNG) dissolved in 10 ml- of TRIS buffer for 45 and 90 minutes. Mutagenic MNNG is removed by centrifugation and decantation of supernatant. The spores are suspended in a physiological solution, inoculated on agar medium in an adequate dilution and incubated under anaerobic conditions at 370° C. for 3 days. 
     The colonies grown in a solid medium are inoculated into liquid nutrient medium and after two days of growth at 370° C. the culture is pipetted into a well, 8 mm in diameter. In another adjacent well of the same size, placed at 2 cm distance, rabbit anticlostripain serum is pipetted. Gel diffusion of clostripain antibodies and clostripain leads to their linkage and formation of a precipitation line. In this way each mutant is controlled several times in addition to biochemical assay. Absence of precipitation line is an evidence that mutants are not producing clostripain. Obtained mutants are then lyophilized and stored for further use. 
     EXAMPLE 2 
     Lyophilized parenteral strain K-26 of the bacterium Clostridium histolyticum was used for inoculation of pre-fermentation nutrient medium containing the following: 
     
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proteose-peptone     50     g/L                                           
tryptic soy broth    15     g/L                                           
Na.sub.2 HPO.sub.4   9      g/L                                           
MgSO.sub.4 × 7H.sub.2 O                                             
                     0.08   g/L                                           
FeSO.sub.4 × 7H.sub.2 O                                             
                     0.014  g/L                                           
vitamin solution     5      mL/L                                          
vitamin solution:                                                         
pantothenic acid     200    mg/L                                          
nicotinamide         200    mg/L                                          
pyridoxine           200    mg/L                                          
thiamine HCl         200    mg/L                                          
riboflavin           20     mg/L                                          
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     Used medium had a natural pH value. Sterilization was performed at 1200° C. for 30 minutes. whilst vitamin solution was sterilized at 1150° C. for 15 minutes. Immediately prior to inoculation of medium with microorganism the solution of vitamins was added. The microorganism was. cultivated for a period of 20 hours at a temperature of 37° C. in a stationary way. 
     The second stage of bacterium cultivation was performed in a 2 Lit glass bioreactor (Multigene, New Brunswick, N.Y.) under the following conditions: 1500 mL of medium of the same composition as the pre-fermentation medium, but with addition of 10% of inoculum and with pH value of 7.4 and a temperature of 30° C. The fermentation was conducted for 20 hours, under occasional nitrogen bubbling conditions for the purposes of anaerobiosis and mild mixing at 50 rpm. 
     
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Enzyme yield:                                                             
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collagenase           1.32   U/mL                                         
clostripain           3.0    U/mL                                         
non-specific proteases                                                    
                      820    U/mL                                         
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     EXAMPLE 3 
     In this example the parenteral strain K-26 of Clostridium histolyticum was used for inoculation of pre-fermentation nutrient medium (as in Example 2). After 20 hours of cultivation 10% of this inoculum was used for inoculation of a nutrient medium of the following composition: 
     
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proteose-peptone     50     g/L                                           
tryptic soy broth    15     g/L                                           
vitamin solution     5      mL/L                                          
Na-thioglycolate     0.57   g/L                                           
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     Composition of vitamin solution was identical to that described in Example 2 with pH value 7.4. Medium was sterilized at 1200° C. for 30 min and cultivation was performed in a 2-Lit glass bioreactor (Multigene, New Brunswick, N.Y. ) with 1500 mT of described medium, at a temperature of 370° C. for 7 hours. 
     
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Enzyme yield:                                                             
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collagenase           1.32   U/mL                                         
clostripain           2.02   U/mL                                         
non-specific proteases                                                    
                      790    U/mL                                         
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     EXAMPLE 4 
     This example relates to the use of the lyophilized mutant of Clostridium histolyticum K-26 clo 88, obtained as described in Example 1, for inoculation of pre-fermentation medium of the composition and cultivation conditions as described in Example 2. Ten percent of inoculum was used for inoculation of liquid medium. The fermentation was conducted for 7 hours. 
     
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Enzyme yield:                                                             
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collagenase           1.2    U/mL                                         
clostripain           0.0    U/mL                                         
non-specific proteases                                                    
                      630    U/mL                                         
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     EXAMPLE 5 
     This example relates to the use of the lyophilized mutant of Clostridium histolyticum K-26 clo 88, obtained as described in Example 1, which was inoculated into a nutrient medium of the composition as described in Example 3. Cultivation of aforementioned bacterium was performed at 28° C. for 8-10 hours. 
     
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Enzyme yield:                                                             
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collagenase           2.72   U/mL                                         
clostripain           0.0    U/mL                                         
non-specific proteases                                                    
                      466    U/mL                                         
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     EXAMPLE 6 
     Lyophilized mutant of Clostridium histolyticum K-26 clo 88, obtained as described in Example 1, was used in this example as an inoculum for pre-fermentation medium as described in this Example 2. Cultivated microorganism was used as an inoculum for the second stage of fermentation as described in Example 3. The only difference was the pH value of 8.7 and a temperature of 30° C. Cultivation was interrupted after 10 hours. 
     
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Enzyme yield:                                                             
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collagenase           4.4    U/mL                                         
clostripain           0.0    U/mL                                         
non-specific proteases                                                    
                      376    U/mL                                         
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     EXAMPLE 7 
     This example shows the procedure of enzyme isolation from a sterile ultrafiltrate of fermentation medium of the mutant Clostridium histolyticum K-26 clo 88 which does not produce clostripain. Concentrated culture filtrate, obtained as described in Examples 4, 5 and 6, was treated with 0.03-0.05 M of calcium chloride and acetone at the concentrations of 18-33%. Fractional precipitation was achieved by adding ammonium sulphate first to 20-30% saturation and then to 70-80% saturation and finally, by a two-stage treatment with calcium phosphate gel, whilst treatment with acetone was omitted. Active lyophilized, clostripain-free collagenase preparation was obtained with utilization amounting to 75-85%. 
     
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Enzyme yield:                                                             
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collagenase           2.2    U/mg                                         
clostripain           0.0    U/mg                                         
non-specific proteases                                                    
                      45     U/mg                                         
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     EXAMPLE 8 
     This example shows the procedure of collagenase isolation from a sterile ultrafiltrate of fermentation medium of the mutant Clostridium histolyticum K-26 clo 88, obtained as described in Examples 4, 5 and 6 and treated as indicated in Example 7, but by avoiding treatment with ammonium sulphate to 30% saturation. A white active, lyophilized collagenase preparation, clostripain-free, was obtained with utilization ranging between 75 and 90%. 
     
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Enzyme yield:                                                             
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collagenase           2.5    U/mg                                         
clostripain           0.0    U/mg                                         
non-specific proteases                                                    
                      45     U/mg                                         
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     EXAMPLE 9 
     This example shows the procedure of collagenase isolation from samples obtained as described in Examples 4, 5 and 6, and purified in a way described in Examples 7 and 8, but by avoiding treatment with calcium phosphate gel. A white, lyophilized, clostripain-free collagenase preparation was obtained with utilization ranging between 75 and 90%. 
     
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Enzyme yield:                                                             
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collagenase           2.6    U/mg                                         
clostripain           0.0    U/mg                                         
non-specific proteases                                                    
                      44     U/mg                                         
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     EXAMPLE 10 
     This example illustrates a method for purification of liquid product obtained by cultivation and treatment as described in Examples 3 and 7. The purification procedure consists in the application of sample to the column containing Sephadex G-75. Application to the column and eluation of protein fractions was performed with buffer 0.01 11 TRIS HC1 at pH 7.2 and with addition of 0.01 M calcium acetate. Gel filtration procedure enabled identification of three protein fractions. Analysis confirmed collagenase activity in the first fraction, reduced collagenase activity in the second, whilst in the third fraction brown pigment was identified, in addition to activity of non-specific proteases, as follows: 
     
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I fraction                                                                
collagenase           34.2   U/mL                                         
clostripain           0.0    U/mL                                         
non-specific proteases                                                    
                      0.0    U/mL                                         
II fraction                                                               
collagenase           10.5   U/mL                                         
clostripain           0.0    U/mL                                         
non-specific proteases                                                    
                      0.0    U/mL                                         
III fraction                                                              
collagenase           0.0    U/mL                                         
clostripain           0.0    U/mL                                         
non-specific proteases                                                    
                      535    U/mL                                         
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     EXAMPLE 11 
     This example describes the desalination process of samples of purified collagenase fractions. Liquid ultrafiltrated fractions of pure enzyme obtained as described in Example 10 were taken and applied to the column containing Sephadex G-25. Sample application and eluation were performed using distilled water. One fraction of salt-free pure enzyme was obtained. Activity of pure enzyme was 38.2 U/mL. 
     EXAMPLE 12 
     This example refers to preservation of collagenase activity in liquid preparation during freezing and lyophilization. The following procedure was employed to a liquid preparation obtained as described in Examples 7-11 carbohydrates were added, such as for example saccharose, maltose and the like, in the concentrations ranging from 5×10 -3  M to 2.5×10 -2  M. 
     EXAMPLE 13 
     This example describes the ways by which it is possible to stabilize lyophilized collagenase preparation. The procedure consists in adding to liquid collagenase preparation, obtained as described in Examples 7-11, different soluble proteins such as enzymatically hydrolized casein (Fluka), Pepton 1 in powder form (Torlak), acid hydrolized casein (Serva) and others, in concentrations ranging from 10 to 50 mg/ml of liquid enzyme preparation. Thus, lyophilized collagenase-preparation shows its unchanged activity.