Field of the invention

The invention is based on the new strain of bacterium Clostridium histolyticum, using it for production of crude collagenase with a high yield, collagenase prepared using this strain and application of the collagenase for isolation of Langerhans islets.

State of the art

Collagenase is a protease which is able to degrade the collagen protein (the basic component of intercellular mass, e.g. in connective tissue). Collagenases are commonly used in research, particularly for disintegration of intercellular mass to release cells. In this respect, clostridiopeptidase A isolated from Clostridium histolyticum proved to be useful (the name of the bacterium, i.e. histolyticum, reflects its ability to degrade tissues). Enzyme collagenase is not only used for research purposes; it has found its place in human and veterinary medicine, especially for treatment of skin diseases. In connection with certain non-specific proteases the collagenase removes necrotic tissue from wounds and thus accelerates healing and improves epithelialization. Collagenase is used in tissue transplantation and for tissue disintegration. It is also applied for treatment of (acid) bums of various degrees, decubiti, skin ulcers, scabs, etc. Not only the, epithelialization of skin is fast and effective after treatment with collagenase, the collagenase treatment also prevents formation of keloids (enlarged scars of tumour-like appearance) and hypertrophic growth as a result of formation of decomposed collagen.

Various strains of microorganisms, cultured under defined conditions, are known to synthesize collagenase. it was found, that among all organisms that are able to synthesize collagenase, Clostridium histolyticum is the best producer. Maclennan J.D., Mandl I. and Howes E.I.: J. Clin. Inv. 32, 1317 (1953) described conditions needed for growth of Clostridium histolyticum. They examined composition of the liquid environment which mainly consists of proteose peptone, inorganic salts and a vitamin solution. Bacteria are cultured at 37 °C and pH 7.2. Berman S., Lewenthal J. P., Webster M. E., Altieri P.L and Gochenour R.B.: J. Bact. 82, 582 (1961) also examined growth conditions of Clostridium histolyticum with the aim to produce collagenase. They were successful in culturing of Clostridium histolyticum in medium which did not contain any inorganic salts; it only contained proteose peptone, enzymatically hydrolysed proteins of casein and soya (soya culture medium) and a vitamin solution. Such a composition of culture medium also determined other parameters of conditions required for satisfactory growth and biosynthesis of collagenase, such as the value of pH 8.5 and a temperature 30 °C and higher.

Clostridium histolyticum is an anaerobic bacterium and anaerobic conditions must be ensured for culturing of this bacterium in liquid environment. Takahashi S. and Seifert S.: J. Appl. Bact. 35, 47 (1972) used reducing agents sodium thioglycolate and sodium bisulfite in order to achieve anaerobic conditions necessary for bacterial growth. The optimum results, i.e. the highest yield of collagenase, was achieved when the above-mentioned reducing agents were used in the ratio 1:1.

Up to the present, considerable number of procedures using various buffering systems (and mostly including precipitation for isolation of a functional protein) has been applied for collagenase extraction from different sources (both bacterial and other). However, these procedures are performed at physiological pH 7.4 - 7.6 which is not optimal for production of bacterial collagenase and at low temperature which prevents degradation of the enzyme. The essence is that commonly used physiological pH is not the most suitable for Clostridium. E.g. Yoshida, E. and H. Noda, 1965. Isolation and characterization of collagenase I and II from Clostridium histolyticum. Biochem. Biophys. Acta, 10593: 562-574. DOI: 10.1016/S0926- 6593(65)80239-9; or Sakamoto, S., P. Goldhaber and MJ. Glimcher, 1972. The further purification and characterization of mouse bone collagenase. Calcified Tissiue Res., 10: 142- 151. DOI: 10.1007/BF02012544; or Bond, M.D. and H.E. Van Wart, 1984. Characterization of the individual collagenases from Clostridium histolyticum. Biochemistry, 19: 3085-3091. DOI: 10.1021/bi00308 a036; or Matsushita, O., K. Yoshihara, S.I. Katayama, J. Minami and A. Okabe, 1994. Purification and characterization of a Clostridium perfringens 120-Kilodalton collagenase and nucleotide sequence of the corresponding gene. J. Bacteriol., 176: 149-156. PMCID: PMC205026. Buffers other than Tris-HCI are also used for purification of collagenase, e.g. sodium bicarbonate which also should maintain the stability of collagenase. In bicarbonate buffers a higher pH is used, which is in our case suitable for collagenase from Clostridium histolyticum; however, in such purification protocols the collagenase is isolated from snails. Indra, D., K. Ramalingam and M. Babu, 2005. Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica. Comparative Biochem. Phys., 142: 1-7. DOI: 10.1016/J.CBPC.2005.02.004.

The amounts of used ions appropriate for stability and effective action of enzymes degrading extracellular components are different in the particular approaches. Klimova, O.A., S.I. Borukhov, N.I. Solovyeva, T.O. Balaevskaya and A.Y. Strongin, 1990. The isolation and properties of collagenolytic proteases from crab hepatopancreas. Biochem. Biophys. Res. Commun., 166: 1411-1420. DOI: 10.1016/0006-291x(90)91024-M

By combining use of a specific production strain from a MB Pharma collection and suitable culturing conditions, sufficient production of the required enzymes occurs. A combination of collagenase produced by specific strain of Clostridium histolyticum and implementation of optimum conditions of collagenase purification is unique. At first we stabilize the enzyme through dialysis to a buffer of a higher pH (8) which is more natural for Clostridium histolyticum collagenase than physiological pH in the above-mentioned procedures. Only through gradual dialysis to buffers of a lower pH, fluent transition to physiological conditions more appropriate for clinical practice, is ensured. Crude collagenase enzymes produced up to now lack sufficient efficacy for Langerhans islets (LI) isolation. This can be caused by poor production of individual proteins or incorrect preparation process. For high yield of LI it is necessary to use highly efficient purified collagenase enzymes and mixtures of these enzymes. Combination of a new production strain CCM 8656 and circumscribed enzyme production procedures (see examples) enables production of enzymatic mixture, that is more efficient than the crude collagenases available and its efficacy is comparable to efficacy of purified collagenases mixture. The objective of this invention is to provide a specific Clostridium histolyticum strain using which the product of the collagenase enzyme is obtained and in its final form can be used for cleavage of connective structures of pancreas in order to receive vital Langerhans islets which can be used in a transplantation treatment of diabetes. Collagenase is a mixture of up to 12 proteolytic enzymes and other proteins received from a filtrate of Clostridium histolyticum cultures. Pancreas digestion is performed in an isolation chamber which was designed by Dr. Ricordi et al. and which is used in almost all isolation centres (Berkova Z., Zacharovova K., K z J., Jirak D., Girman P., Dovolilova E., Koblas T., Hajek M., Saudek F.: The impact of islet labeling with superparamagnetic nanoparticles for magnetic resonance imaging on islet vitality; 10th world congress of IPITA, Geneva, Switzerland, 4.-7.5.2005). It is a closed system in which a collagenase solution circulates, and while it is slightly agitated, the cells release gradually.

Summary of the invention

The substance of the solution is bacterium Clostridium histolyticum CCM 8656 producing collagenase. Collagenase produced by the strain has the following characteristics:

It is a mixture of two collagenases coll and col 2 produced by the strain of bacterium Clostridium histolyticum (CCM 8656) with molecular weight of 116 kDa and 126 kDa. The protein mixture contains collagenase and its natural degraded parts, which represent the most of the dry matter in the final product. On gel SDS PAGE both stripes must be apparent, and there is a possibility of occurrence of lower molecular weight fragments which still poses catalytic activity. Both types of collagenase consist of two peptide domains which are able on their own to decompose collagen. The rest of the protein consists of a binding domain which binds the whole enzyme to substrate, and even though it increases its activity via bonding to collagen it is not necessary for the activity. Smaller molecules of the catalytic domains thus complement activity of relatively large molecule of the complete enzymes thanks to better diffusion. Collagenase must have specific activity higher than 700 PZS/g. This unit (PZS/g) is defined as a number of micromols of substrate degraded by one gram of enzyme within one minute at a temperature of 25 °C. Clostripain activity may be also desirable; however, it is not a must. Clostripain may be the required admixture to the resulting collagenase mixture. Unit U/mg is such enzyme activity of clostripain which catalyses hydrolysis of 1 μιτιοΙ of substrate (BAEE) in 1 minute at a temperature of 25 K, pH 7.6 and presence of 2.5 mmol/l DTT. The optimum value of activity should range within 1.2-1.45 U/mg. It is therefore a naturally produced mixture of collagenolytic enzymes prepared by partial purification (see examples) and this mixture is suitable for isolation of LI.

Another subject of the invention is the use of "crude collagenase" in isolation of Langerhans islets.

Examples of carrying out the Invention

The strain of bacterium Clostridium histolyticum with working identification MB204 is deposited in the CCM - Czech Collection of Microorganisms, Kamenice 5, 625 00 Brno of 20/01/2016 under No. CCM 8656. The strain Clostridium histolyticum CCM 8656 which we have cultivated (via selection by long-term passaging) was obtained from a mixture of 3 strains that were obtained from a German collection of strains as strains DSM 627, 1126 and 2158.

Culturing is generally performed in tryptone medium, suitable for sufficient production of collagenase: (Tryptone 60 g/l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Na2HP04 3.4 g/l) pH 8.4, before inoculation, 100 μΙ of vitamin K of storage concentration 1% and 100 μΙ of L-cysteine of storage concentration 0.5 g/ml is added to the culturing medium. The culture is cultivated 24 hours at 37 °C ±1 °C without agitation. In the strain, using controlled evolution and long-term passaging (adaptive laboratory evolution) (Dragonits, 2013. Dragosits M, Mattanovich D. Adaptive laboratory evolution - principles and applications for biotechnology. Microbial Cell Factories. 2013;12:64. doi:10.1186/1475-2859-12-64.), one copy of the gene for collagenase was deleted. This selectively appropriate deletion was achieved using selective pressure and long-term culturing in laboratory. Even despite this deletion the strain is able to produce a large amount of both types of collagenases. It may be caused by a strong promotor which supplies production of genes from two points in genome. Into the culturing medium collagen was added as a substrate, which supported selectively production of enzyme which should degrade it. Thus natural selection of strain which is used for production of bacterial collagenase was achieved. Composition of the tryptone medium suitable for sufficient production of collagenase with collagen: (Tryptone 60 g/l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Na2HP04 3.4 g/l, collagen 50 g/l) pH 8.4, before inoculation, 100 μΙ of vitamin K of storage concentration 1% and 100 μΙ L-of cysteine of storage concentration 0.5 g/ml are added into the cultivation medium. The culture is cultivated 72 hours at 37 °C ±1 °C without agitation, ideally in anaerobic conditions. Genes encoding collagenase ColG and ColH were identified previously in C. histolyticum type strain JCM1403 (ATCC19401, DSM 2158) (Matsushita O, Jung C-M, Katayama S, Minami J, Takahashi Y, Okabe A. Gene Duplication and Multiplicity of Collagenases in Clostridium histolyticum. Journal of Bacteriology. 1999;181(3):923-933.

Yoshihara K, Matsushita O, Minami J, Okabe A. Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase. Journal of Bacteriology. 1994;176(21):6489-6496.) Production of 116 kDa collagenase and 98 kDa gelatinase was mentioned in Yoshirara et al. One of the collagenases produced by strain CCM 8656 differs in molecular weight (126 kDa) from the collagenase produced by the strain DSM 2158. Genes and their protein products are identical for the strains DSM 2158 a CCM 8656, but the comparison of crude collagense production between these two strains (Table 1) shows higher efficacy of collagenase produced by CCM 8656. The whole genome sequence of DSM 2158 is not available and its comparison to genome sequence of CCM 8656 is not possible. It is thus unclear what is the cause of the difference in collagenase efficacy. It is a markedly changed phenotypic manifestation though. In our experiments the same procedure of collagenase production was used for both strains CCM 8656 and DSM2158. The efficacy of collagenase produced by the strain DSM 2158 was always lower than 320 PZS/g and the yield of LI was poor (Table 1). The efficacy of collagenase produced by the strain CCM 8656 was always higher than 900 PZS/g and when this collagenase was used for LI isolation, the yield was higher than 1000 LI, the quality of LI was better and the time of isolation was shorter.

Production strain Average Count of Note

collagenase isolated

efficacy PZS/g Langerhans

islets (LI)

of efficacy collagenase

MB

201 and MB 202 are newly isolated strains and they are compared to collection strains and data from literature.

*Dendo M, Maeda H, Yamagata Y, Murayama K, Watanabe K, Imura T, Inagaki A, Igarashi Y, Katoh Y, Ebina M, Fujimori K, Igarashi K, Ohuchi N, Satomi S, Goto M. Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation.Transplantation. 2015 Jul;99(7):1349-55. doi: 10.1097/TP.0000000000000662.

**De Vos P, Smink AM, Paredes G, et al. Enzymes for Pancreatic Islet Isolation Impact Chemokine-Production and Polarization of Insulin-Producing β-Cells with Reduced Functional Survival of Immunoisolated Rat Islet-Allografts as a Consequence. Blondeau B, ed. PLoS ONE. 2016;ll(l):e0147992. doi:10.1371/journal.pone.0147992

Characteristics of strain Clostridium histolyticum CCM 8656: Clostridium histolyticum is an anaerobic gram-positive bacterium. Bacterial cells are mobile peritrichal straight rods with a size of 0.5-0.9 ^χ 1.3-9.2 μηι and they form in pairs or short chains. Cells are capable of sporulation; in anaerobic conditions their cell wall contains meso-DAP, glutamic acid and alanine. During cultivation on blood plaits colonies are large, reaching 0.5 to 2 mm in diameter, of circular to irregular shape, flat to slightly convex, of white-grey colour, glossy with granular surface mosaic. Similar colonies can be also cultivated in aerobic conditions but considerably less colonies grow which are much smaller. Therefore cultivation is thus suitable mainly in anaerobic conditions, when a large amount of the required collagenase is produced. Bacteria are able to grow at temperatures from 25 °C to 45 °C but the optimum growth temperature is 37 °C. The strain Clostridium histolyticum CCM 8656 is strongly proteolytic and produces considerable amounts of various proteolytic enzymes including collagenase, elastinase, neutral proteases and clostripain. The strains of this bacterium are sensitive to chloramphenicol, erythromycine and tetracycline. The strains are toxic but in long cultivation their precursor-proteases may degrade own toxins. Specific 16S RNA (GenBank accession number 16S rRNA of gene: M59094) shows that this strain is similar to the strains Clostridium limosum (97.2%) and Clostridium proteolyticum (96.1%).

Collagenase for isolation of Langerhans islets is received as a metabolite of the bacterium Clostridium histolyticum. This enzyme has a synergic effect in the process of degradation of collagen and other extracellular components. Collagenase for isolation of Langerhans islets is generally appropriate for isolation of cells from a number of animal tissues. The product is provided with basic information about enzymatic activity. The optimum concentration of enzyme and the specific conditions for using of degradation of various tissues has to be defined empirically. The enzyme is not of animal origin.

Collagenase for isolation of Langerhans islets is suitable for cell separations of e.g. tumour cells, separation of murine kidney cells, cells of lung tissue and various epithelial tissues. The enzyme can be also used for isolation of hepatocytes. Considering the selective collagenolytic activity which primarily does not damage cell membranes, the enzyme can be used as generally dispersing cell agent, also in the conditions of cell cultures. It is generally applicable that organs with a higher content of collagen can be incubated with collagenase for isolation of Langerhans islets for a longer time and at concentrations higher than when working with other proteolytic enzymes, without the cells losing their viability. Alternative applications of collagenase according to the invention are in preparation of various cell cultures, in research of medicinal products based on enzymatic activity of collagenase and in various transplantation programmes.

3 Flowchart of the Technological Procedure

Lyophilization Final testing

Dispatching

Example 1:

Opening and inoculating of a cab: Bacterial culture Clostridium histolyticum, strain No. CCM 8656 is stored in a freezing medium at -80 °C or in a form of lyophilizate in a glass vial at 4 °C. 100 μΙ of de-frost culture is inoculated to 100 ml of a liquid medium which is sterilized immediately before inoculation and then temperature-adjusted to a temperature 37°C. The culture medium, suitable for cultivation of anaerobic bacteria is thus deaerated. Composition of tryptone medium suitable for sufficient production of collagenase: (Tryptone 60 g l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Na ₂ HP0 ₄ 3.4 g/l) pH 7.8, before inoculation, 100 μΙ of vitamin K of storage concentration 1 % and 100 μΙ of L-cysteine of storage concentration 0.5 g/ml are added to the culturing medium. The culture is cultivated 24 hours at 37 °C ±1 °C without agitation. After cultivation purity of bacterial strains is verified through growth on solid media (anaerobic cultivation) and morphology of cells is assessed microscopically.

Preparation of large-scale cultivation:

Bacterial culture Clostridium histolyticum, strain No. 8656 cultivated in 100 ml of medium is collected from the bottom part of a bottle. 5 ml of culture is inoculated to 500 ml of a liquid medium which is sterilized immediately before inoculation and then temperature-adjusted to a temperature 37 °C. The culture medium, suitable for cultivation of anaerobic bacteria is thus deaerated. Composition of tryptone medium suitable for sufficient production of collagenase: (Tryptone 60 g/l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Na ₂ HP0 ₄ 3.4 g/l) pH 7.8, before inoculation, 100 μΙ of vitamin K of storage concentration 1 % and 100 μΙ of L- cysteine of storage concentration 0.5 g/ml are added to the culturing medium. The culture is cultivated 24 hours at 37 °C ±1 °C without agitation.

Cultivation in Fermentor:

19 litres of cultivation medium is prepared in a fermentor (Tryptone 60 g/l, peptone 1.5 g/l, NaCl 2.5 g/l, glucose 1.25 g/l, Ha ₂ HP0 ₄ 3.4 g/l) pH 7.8 before inoculation 20 ml of vitamin K (storage concentration 1 %) and 20 ml of L-cysteine (storage concentration 0.5 g/ml) are added. Fermentor is inoculated with 500 ml of bacterial culture from the previous step of cultivation. Mixed inoculum is sucked aseptically to the prepared cultivation medium. After inoculation sample 0 is taken. Culturing proceeds at +37 °C + 1°C. Culturing conditions are adjusted depending on bacterial growth. Since cultivation starts, pH is adjusted on a continuous basis with a 2M solution of NaOH to value of 7.8 ± 0.5. Culturing proceeds without stirring or aeration !

Cultivation is finished if 2 consecutive samplings do not show a significant increment and pH does not change. If at least pH changes, cultivation lasts for 40 - 48 hours.

Processing of enzyme:

When culturing is completed, 20 litres of the culture medium containing both bacteria and raw collagenase is drained into an appropriate vessel with 9.46 kg ammonium sulphate. The whole mixture is stirred so that ammonium sulphate is dissolved; pH is then adjusted to a value of 6.8 - 7.6. The mixture is left to sediment for 4 - 7 days at 4 °C. After 4 - 7 days supernatant is sucked off using a peristaltic pump while maximum volume of sediment is preserved without stirring it. Sediment containing precipitated collagenase is transferred to dialysing hoses (approx. 0.3 - 1 litre of sediment), the dialysing hose is placed in 10 I of buffer Tris CI (Tris 0.75 g/l, CaCI ₂ 0.484 g/l pH 10) and left to dialyse for 3 hours. Then the buffer is replaced with other 10 1 of TRIS HCI for 24 hours. Then the buffer is replaced with 10 1 of TRIS HCI of a different pH (Tris 0.36 g/l, CaCI ₂ 0.242 g/l pH 7.5 - 8.5) for 24 hours. Then it is again replaced with fresh Tris III. If dialysate appears to be to thick, replacement of the Tris buffer may be done two more times within further 24 hours.

When dialysis is completed, content of dialysing hoses is poured out to cuvettes and centrifugated (3500* g) for 45 minutes. Supernatant is poured into a sterile vessel and filtered through a 0.45μιη filter. The ultrafiltration in cartridges Millipore 50 kDa follows. Using this method leads to concentrating and reducing of the lysate volume. When collagenase is concentrated to the volume of 120 ml, filtration through a 0.45μιτι filter is performed. Then filling in vials and lyophilisation of the product follows.

Example 2:

Inoculation and culturing of bacterial strain Clostridium histolyticum CCM 8656 is the same as in Example 1. When culturing in the fermentor is completed, bacteria C. histolyticum is removed by centrifugation (7 000x g) and precipitation of collagenase with ammonium sulphate is performed only in supernatant. Precipitate containing sulphate with precipitated protein is left to sediment for 4 - 6 days at 4 °C. After dialysis against buffering solutions Tris HCI buffers (the same buffers as in Example 1), then ultrafiltration follows again in cartridges millipore with "cut off" 50 kDa. When protein is thickened, the sample is filled by 10 ml in vials and lyophilised.

Example 3:

Inoculation and culturing of bacterial strain Clostridium histolyticum CCM 8656 is the same as in Example 1. However, dialysis is followed by ultrafiltration through ultrafiltration cartridges, at first with "cut-off" 300 kDa for removal of ballast proteins of a large molecular weight. Thus volume is increased because flow-through is maintained and then the solution is ultrafiltrated in a cartridge "cut off" 50 kDa for removal of smaller proteins and degraded parts. In the course of concentrating, Tris-HCI buffer (Tris 0.36 g/l, CaCl ₂ 0.242 g/l pH 7.5 - 8.5) is continuously added. When protein is thickened, the sample is filled by 10 ml in vials and lyophilised.

Example 4:

Inoculation and culturing of bacterial strain Clostridium histolyticum CCM 8656 is the same as in Example 1. When culturing in the fermentor is completed, bacteria Clostridium histolyticum are removed by centrifugation (7 OOOx g). However, in this case precipitation with ammonium sulphate is not used; protein is directly thickened. Trough ultrafiltration cartridges at first with "cut off" 300 kDa ballast proteins of a large molecular weight are removed. Thus volume is increased because flow-through is maintained and then the solution is ultrafiltered in a cartridge with "cut off" 50 kDa for removal of smaller proteins and degraded parts, and volume is reduced. When protein is thickened, the sample is filled by 10 ml in vials and lyophilised.

Method:

Individual preparation methods for optimal production and purification of desired enzymes from the strain CCM 8656 are described in individual examples in this document. Mixture of enzymes, produced directly from bacterial culture, that leads to efficient LI isolation is also mentioned. A few methods of collagenase purification using precipitation with ammonium sulphate has been described. However, we use different concentrations for optimal removal of undesired ballast proteins and preserving of enzymatically active proteins (clostripain and neutral protease) to support characteristics of the final collagenase, for its use in isolation of Langerhans islets. The method uses only precipitation with ammonium sulphate, dialysis and an ultrafiltration system, which is a simple, easy-to-do method, compared to the costly and demanding purification method using chromatography. Moreover, large collagenase volumes can be processed using this method, compared to chromatographic methods. This method preserves other proteins which in a proper ratio support collagenase activity. Since bacterial culture is processed in the production, the intracellular proteins get to the final product. In other cases, only supernatant after culturing is processed, and here the supporting enzymes are lost. The method is also innovative in using two pH values, at first precipitate is dialysed against a buffer of a high pH. This process has been implemented in order to increase protein stability and achieve a higher yield. Dendo et al. (2015) demonstrates that the synergy of collagenase and clostripain positively affects the amount of LI obtained from pancreases of experimental animals. Combination of enzymes causes high yield >1000 LI, similarly as in case of our crude collagenase (Table 1). But the publication deals with highly purified recombinant proteins. The collagenase mentioned in the publication is not produced directly from C. histolyticum, it is a GMO product - a recombinant protein produced in E. coli. Adjusting to the desired concentration is performed separately for each enzyme. In our case the mixture of enzymes comes directly from specific strain C. histolyticum CCM 8656 and the quality of the mixture, showed by the amount of isolated LI, is comparable to recombinant collagenase. The advantage of natural collagenase production is the possibility of (legislatively) easier process of production and application in clinical and laboratory practice than in case of GMO products, recombinant proteins. More studies focus on the appropriate ratio of collagenases and other enzymes used for LI isolation. All of them agree on achieving the highest efficacy by mixing purified collagenases, neutral proteases, clostripain, etc. In our case the mixture of crude collagenase is similarly effective for LI isolation as the mixture of individually purified enzymes. Using crude collagenases always leads to lower yields of LI, as illustrated by Vos et al., 2016 (Table 1). At the beginning of discussion of this publication different contradictory studies discussing the role of neutral proteases are mentioned. The authors suggest that mixture of enzymes could be contaminated by undesirable enzymes. In our case the resulting product is an enzymatic mixture with parameters comparable to mixture of purified collagenases and other proteases.

Sequencing and PCR of the gene for collagenase:

Before preparing the DNA for sequencing it was necessary to verify which of the possible types of collagenase is produced by the strain used for production. Based on similarity and origin of strains, a gene for collagenase with the size of 3967 bp was selected. For the selected sequence a pair of primers for PCR (polymerase chain reaction) was suggested (KG Up: GGGATTATCTATGAAAAAAAA, KG Low: AATTATTTATTTACCCTTAACTCA) and PCR for confirmation of the gene in the bacterium genome was performed. The reaction confirmed presence of the just selected gene which enabled us to identify the protein product of the gene for other analyses of protein. Mainly the sequence of amino acids and size of protein (126 kDa) were important.

The collection of applicant MB PHARMA s.r.o. currently disposes of three strains of bacterium Clostridium histolyticum which produce not only bacterial collagenase but a significant amount of clostripain and neutral proteases. These enzymes may, but may not be required for production of the final product. In the case they are absent, they must be then added separately in order to isolate the Langerhans islets. Clostripain and neutral proteases are present in the mixture of proteins produced by C. histolyticum CCM 8656 using the methods mentioned above.

Working identification numbers of the three strains are: MB 201, MB 202 and MB 204 (CCM 8656), and for the purpose of the mass production the strain Clostridium histolyticum MB 204 (CCM 8656) was selected. Although the gene for collagenase in this production strain was confirmed using PCR, for better characterization it is better to determine the whole sequence of genome DNA. Thus, the genes for clostripain and potential neutral proteases can be found. Therefore, to ensure successful sequencing, DNA of very good quality and concentration must be prepared. A large volume of data must be processed for sequencing, and finished sequences of contigs must be then annotated. All three strains of C. histolyticum were sequenced, and annotating of their genomes showed that they code genes of two types of collagenase - collagenase proved using PCR (col 2) and the second type of collagenase of molecular weight 116 kDa (col 1). Strain M 201 contained a gene for col 1 in two copies an gene for col 2 in one copy only; on the contrary, strain MB 202 has gene col 1 in one copy and gene col 2 in two copies. The production strain MB 204 had both genes col 1 and col 2 in one copy only.

Genomes of all strains include a gene for clostripain. This enzyme can help collagenase to better disintegrate tissues during isolation of Langerhans islets. The main component of the collagenase final mixture should be collagenase, while clostripain should be a minor component. This was demonstrated.

All strains of Clostridium histolyticum dispose of gene for production of clostripain but alternatively, it would be more appropriate for the proper ratio of collagenase and clostripain to produce both enzymes separately and produce a mixture of the proper ratio just before the application. Clostripain has molecular weight 43.4 kDa so it would be possible to produce it relatively easily also as a recombinant protein e.g. in E. coli. Our strain CCM 8656 produces clostripain in an optimal amount to support the efficacy of collagenase in LI isolation.

It is a mixture of two collagenases coll and col2 produced by the bacterium Clostridium histolyticum CCM 8656 with molecular weight of 116 kDa and 121 kDa.

Coll, Mw: 116,3 kDa, pi: 5.82

Sequence:

MKRKCLSKRLMLAITMATIFTVNSTLPIYAAVDKNNATAAVQNESKRYTVSYLKTLNYYD LVDLLVKTEIEN

LPDLFQYSSDAKEFYGNKTRMSFIMDEIGRRAPQYTEIDHKGIPTLVEVVRAGFYLG FHNKELNEINKRSFK

ERVIPSILAIQKNPNFKLGTEVQDKIVSATGLLAGNETAPPEVVNNFTPILQDCIKN IDRYALDDL SKALFNV

LAAPTYDITEYLRATKEKPENTPWYGKIDGFINELKKLALYGKINDNNSWIIDNGIY HIAPLGKLHSNN IGIE

TLTEVMKVYPYLSMQHLQSADQIKRHYDSKDAEGNKIPLDKFKKEGKEKYCPKTYTF DDG VIIKAGARVE

EEKVKRLYWASKEVNSQFFRVYGIDKPLEEGNPDDILTMVIYNSPEEYKLNSVLYGY DTNNGGMYIEPEGT

FFTYEREAQESTYTLEELFRHEYTHYLQGRYAVPGQWGRTKLYDNDRLTWYEEGGAE LFAGSTRTSGILPR

KSIVSNIHNTTRNNRY LSDTVHSKYGASFEFYNYACMFMDYMYNKDMGILNKLNDLAKNNDVDGYDN

YIRDLSSNYALNDKYQDHMQERIDNYENLTVPFVADDYLVRHAYKNPNEIYSEISEV AKLKDAKSEVKKSQY FSTFTLRGSYTGGAS GKLEDQKAMNKFIDDSLKKLDTYSWSGYKTLTAYFTNY VDSSNRVTYDVVFHGY

LPNEGDSKNSLPYGKINGTY GTEKE IKFSSEGSFDPDGKIVSYEWDFGDGNKSNEENPEHSYDKVGTYT

VKLKVTDD GESSVSTTTAEIKDLSENKLPVIYMHVP SGALNQ VVFYGKGTYDPDGSIAGYQWDFGDG

SDFSSEQNPSHWTKKGEYTVTLRVMDSSGQMSEKTMKIKITDPVYPIGTEKEPNNSK ETASGP!VPGIPVS

GTIENTSDQDYFYFDVITPGEVKIDINKLGYGGATWVVYDENNNAVSYATDDGQNLS GKF ADKPGRYYI

HLYMFNGSYMPYRINIEGSVGR

Col 2, Mw: 126,2, pi: 5.62

Sequence:

MKKNILKILMDSYSKESKIQTVRRVTSVSLLAVYLTMNTSSLVLAKPIENTNDTSIKNVE KLRNAPNEENSKK

VEDSKNDKVEHV NIEEAKVEQVAPEVKS STLRSASIANTNSEKYDFEYLNGLSYTELTNLI NIKWNQIN

GLFNYSTGSQKFFGDKNRVQAIINALQESGRTYTANDM GIETFTEVLRAGFYLGYYNDGLSYLNDRNFQ

DKCIPAMIAIQKNPNFKLGTAVQDEVITSLGKLIGNASANAEVVNNCVPVLKQFREN LNQYAPDYVKGTA

VNELIKGIEFDFSGAAYEKDVKTMPWYG IDPFINELKALGLYGNITSATEWASDVGIYYLS FGLYSTNRN

DIVQSLEKAVDMYKYGKIAFVAMERITWDYDGIGSNGKKVDHDKFLDDAEKHYLPKT YTFDNGTFIIRAG

DKVSEEKIKRLYWASREVKSQFHRVVGNDKALEVGNADDVLTMKIFNSPEEYKFNTN INGVSTDNGGLYIE

PRGTFYTYERTPQQSIFSLEELFRHEYTHYLQARYLVDGLWGQGPFYEKNRLTWFDE GTAEFFAGSTRTSG

VLPRKSILGYLAKD VDHRYSLKKTLNSGYDDSDWMFYNYGFAVAHYLYEKDMPTFIKMNKAILNTDVKS

YDEIIKKLSDDANKNTEYQNHIQELADKYQGAGIPLVSDDYLKDHGYK ASEVYSEISKAASLTNTSVTAEKS

QYFNTFTLRGTYTGETSKGEF DWDEMS KLDGTLESLAKNSWSGY TLTAYFTNYRVTSDN VQYDVVF

HGVLTDNADISNNKAPIA VTGPSTGAVGRNIEFSGKDSKDEDG IVSYDWDFGDGATSRGKNSVHAYKK

AGTYNVTLKVTDDKGATATESFTIEIKNEDTTTPITKEMEPNDDIKEANGPIVEGVT V GDLNGSDDADTFY

FDVKEDGDVTIELPYSGSSNFTWLVYKEGDDQNHIASGIDKNNSKVGTFKSTKGRHY VFIYKHDSASNISYS

LNI GLGNEKL EKENNDSSDKATVIPNFNTTMQGSLLGDDSRDYYSFEVKEEGEVNIELDKKDEFGVTWT

LHPESNINDRITYGQVDGNKVSNKVKLRPG YYLLVYKYSGSGNYELRVN

In the protein mixture collagenase and its natural degraded parts represent the most of the dry matter in the final product. It may also contain clostripain or neutral proteases detectable using an appropriate method for measuring of activity of those admixtures. However, presence of both non-degraded collagenases and possibly their parts is essential. There must be two stripes visible on gel 12% SDS-PAGE, corresponding to the size 116 kDa and 126 kDa, and there is also a possibility of occurrence of stripe of lower molecular weight protein, which still has catalytic activity. If zymogram is used, lytic zones of proteins of lower molecular weight may be visible. Smaller molecules of the catalytic domains thus complement activity of relatively large molecules of the complete enzymes thanks to better diffusion. Collagenase must have specific activity higher than 700 PZS/g. This unit is defined as a number of micromols of substrate degraded by one gram of enzyme within one minute at a temperature of 25 °C. Clostripain activity may be also desirable; however, it is not a must. Clostripain may be the required admixture to the resulting collagenase mixture. Unit U/mg is such enzyme activity of clostripain which catalyses hydrolysis of 1 μπιοΙ of substrate (BAEE) in 1 minute at a temperature of 25 ^c, pH 7.6 and presence of 2.5 mmol/l DTT. The optimum value of activity should range within 1.2-1.45 U/mg.

Strain Clostridium histolyticum MB 204 stored in the collection as CCM 8656 had much higher yield of collagenase than other tested strains commonly used for production of collagenase. For assessment of the optimal collagenase production procedure we compared the C. histolyticum strains DSM 627, 1126, 2158 and strains MB 201, MB 202 and MB 204. The mixture of enzymes produced from the strain MB 204 was significantly more efficient in LI isolation compared to the other tested strains. The gene encoding collagenase in MB 204 has the same nucleotide sequence as the analogous gene in the strain 2158, but the strain MB 204 produces more efficient mixture of enzymes, thus a markedly different phenotypic manifestation is observed at equal cultivation conditions. Even though the genes encoding collagenase are identical in these two strains, the expression of the protein in the cell can be affected by various regulation mechanisms, in which the two strains differ. Although compared to commercially used bacteria this strain contains one copy of the gene for collagenase type I and one copy of the gene for collagenase type II; however, the yield of collagenase increased, most probably thanks to expression caused by a strong promotor. Moreover, the strain produces a minimal amount of toxic proteins, and during preparation undesirable proteins are degraded which is a big advantage for practical use in transplantation medicine. The new method of production in combination with the new strain leads to production of stable collagenase from Clostridium histolyticum. Cultivation of relatively small volume gives sufficient yields of both types of collagenase in the final product. Although partial degradation occurs, it is more likely beneficial in combination with sufficient amount of non- degraded part of the enzyme, as the degraded parts still retain their proteolytic activity.

Enzyme collagenase is used for digestion of pancreatic tissue, during which Langerhans islets (LI) are released and can be used for treatment of diabetes. Isolated LI using collagenase must meet strict criteria so they can be transplanted to diabetic patients. The most important monitored parameters of isolation are as follows: duration of digestion, digestion quality, quality of separation of cells from exocrine tissue and quality of cells after isolation and culturing. Quality of cells is evaluated using dying of viable and dead cells, using glucose- stimulated secretion of insulin with beta cells of LI (expressed as stimulation index) and using measuring of oxygen consumption. Using collagenase according to the invention resulted in achieving the digestion time of 15 min and average yield 1000 LI which retain sufficient vitality (more than 95 %). This collagenase is thus suitable for isolation of LI for transplantation purposes.

In cases of collagenase production described in literature (DeVos et al. 2018) the Sigma Aldrich crude collagenase from C. histolyticum was used, where the production process and C. histolyticum strain differs from ours. In case of Sigma Aldrich collagenase, the enzymatic mixture containing collagenase, clostripain and neutral proteases is produced without producing of individual components. However, the efficacy of Sigma Aldrich collagenase is lower than the efficacy of our collagenase. The yield of LI 256 (± 21) and 174 (± 23) is lower than in case of collagenase produced using our unique process and the strain CCM 8656, that allowed us to isolate 1147 (± 231) LI. This result is similar to result that was achieved using purified commercial collagenase (VitaCyte collagenase which does not contain clostripain and neutral proteases), when 905 ± 257 LI were isolated. Table 1 likewise shows the great difference between Sigma Aldrich and VicaCyte collagenases. The efficacy of our collagenase is thus comparable rather to efficacy of purified enzymes and the collagenase markedly differs from other crude enzymatic mixtures. This can be explained by using different production strain, different procedure and partial purification. Moreover, the authors of the publication say that individual batches of collagenase differ in efficacy. Due to our standardized procedure we achieved similar efficacy among different batches. Western blott (Figure 1) shows higher amount of nondegraded collagenase in our sample compared to Sigma Aldrich collagenase. So far yields of LI were poor when crude collagenase mixtures were used. For high yields of LI using of individually purified enzymes in mixtures was necessary, as described by Dendo et al. 2015. In our case the highly efficient crude collagenase is produced using cultivation and partial purification from the unique

C. hlstolyticum strain CCM 8656.

In case of collagenase produced according to our invention (Figure 1) it is clear, that most of the enzyme is not degraded. The western blott also shows that the SERVA collagenase is degraded and the Sigma Aldrich collagenase is partially degraded and the degradation is clearly higher than in case of collagenase produced according to our invention.