Method for producing a microbial collagenase enzyme

Abstract

The present invention relates to a method for producing collagenase enzymes by means of microorganisms in high purity and yield, in order to obtain collagens suitable for use in technical fields such as food, cosmetics and skin care, medicine, pharmaceuticals, leather processing and textile products, as well as sports supplements. By means of the method disclosed in the invention, it is further possible to obtain environmentally sustainable collagenase enzymes that are stable for industrial use without incurring high costs and time loss.

Description

[0001] METHOD FOR PRODUCING A MICROBIAL COLLAGENASE ENZYME

[0002] TECHNICAL FIELD

[0003] The present invention relates to a method for producing collagenase enzymes by means of microorganisms in high purity and yield, in order to obtain collagens suitable for use in technical fields such as food, cosmetics and skin care, medicine, pharmaceuticals, leather processing and textile products, as well as sports supplements. By means of the method disclosed in the invention, it is further possible to obtain environmentally sustainable collagenase enzymes that are stable for industrial use without incurring high costs and time loss.

[0004] PRIOR ART

[0005] Collagenase enzyme is a metalloproteinase capable of degrading the collagen molecule. This enzyme specifically cleaves the triple helical structure of collagen into smaller peptides. Naturally found in microorganisms and in the digestive systems of certain animals, collagenase may also be obtained for industrial production through methods such as microbial fermentation and genetic modification. Due to its ability to cleave collagen at specific sites, the collagenase enzyme has a wide range of applications in various industrial, medical, and cosmetic fields.

[0006] One of the principal areas of use of collagenase is medical applications. In particular, in wound healing, tissue engineering, and surgical repair processes, collagenase contributes to the reorganization and softening of collagen-based tissues. In medical treatments, especially in diseases such as Dupuytren’s contracture that result from collagen accumulation, collagenase-based drugs target collagen degradation. Furthermore, during wound debridement, collagenase is employed to effectively remove necrotic tissues, thereby enabling faster wound healing. In the cosmetics industry, collagenase plays a critical role in the production of hydrolyzed collagen used in skin care products to enhance skin elasticity and reduce wrinkles. Hydrolyzed collagens processed with collagenase are incorporated into skin masks, serums, and anti-aging products, increasing skin flexibility and providing a younger appearance. The small peptides produced through collagenase treatment penetrate the skin barrier more effectively, providing efficient moisturization and skin regeneration. In the food industry, collagenase enzyme is used in meat tenderization processes. By breaking down the collagen content of meat tissues, collagenase contributes to making the meat softer and more flavorful. Additionally, collagenase used inthe production of hydrolyzed collagen is employed in the manufacture of protein-rich foods. Such products are included in protein-enriched food items such as sports nutrition and health supplements.

[0007] One of the conventional methods for producing collagenase enzyme is through animal-derived sources. In this method, collagenase enzyme is isolated from the internal organs of fish or other animals to achieve production. However, this process is highly costly and labor-intensive. Moreover, the enzyme obtained from animal sources is generally found in mixture with other proteins, which leads to a high risk of impurities. Extensive purification steps are required, which not only complicates the production process but also increases costs and reduces production efficiency.

[0008] Another method for producing collagenase enzyme is natural fermentation processes. In this method, collagenase is obtained from microorganisms through fermentation. However, natural fermentation processes are dependent on specific microorganisms, which limits production diversity and makes it difficult to achieve optimal results in terms of efficiency. In addition, natural fermentation is sensitive to environmental factors such as temperature, pH, and nutrients. Since the control of these conditions is challenging, the risks of impurity and the formation of undesirable by-products are high, which adversely affects enzyme efficiency.

[0009] In the state of art, collagenase enzyme may also be obtained from microorganisms of the Clostridium species. Although it is possible to obtain collagenase enzyme from Clostridium bacteria through natural fermentation, these bacteria also produce different proteins and enzymes in their natural environments, making it difficult to obtain collagenase in high purity. This necessitates additional purification processes to achieve the desired collagenase enzyme purity, thereby rendering the process more complex and costly. Fermentations based on Clostridium may also lead to the formation of undesirable proteolytic activities in the by-products, which may disrupt the structural integrity of collagen molecules and reduce product quality.

[0010] Another disadvantage of conventional fermentation methods for collagenase enzyme is that they are time-consuming and costly. Conventional fermentation processes take longer to complete, which decreases production efficiency while requiring higher labor and energy input. Due to the risks of by-products and impurities, the produced enzyme requires additional purification and quality control processes. This increases production costs and reduces commercial competitiveness.As a result, all of the aforementioned problems have made it necessary in the relevant technical field to provide an innovation for obtaining collagenase enzyme that offers high efficiency and purity, while ensuring low costs and environmentally sustainable production.

[0011] BRIEF DESCRIPTION OF THE INVENTION

[0012] The present invention relates to a method for obtaining collagenase enzyme, in order to eliminate the above-mentioned disadvantages and to provide new advantages to the relevant technical field.

[0013] An object of the invention is to provide a method that enables the obtaining of collagenase in high purity by reducing undesirable by-products. Thus, it becomes possible to obtain collagenase enzyme in an impurity-free and effective form.

[0014] Another object of the invention is to provide a method for producing collagenase with higher efficiency and requiring less energy and labor compared to conventional methods. Thus, the production of collagenase enzyme can be achieved at lower costs and through environmentally friendly processes.

[0015] A further object of the invention is to provide a method for producing collagenase enzyme, wherein autocatalytic activity is prevented through genetic modifications performed in microorganisms and culture medium optimizations for microbial production. Thus, the stability and long-term effectiveness of the collagenase enzyme can be ensured.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] In this detailed description, the subject matter of the invention is a method for obtaining microbial collagenase enzyme, which is explained with examples provided solely for a better understanding of the subject and without creating any limiting effect.

[0018] In the present invention, the term “microbial collagenase enzyme” refers to the form of collagenase enzyme produced by means of microorganisms. In this context, genetic modification processes are carried out on microorganisms within the scope of the invention. Genetic information enabling the microorganisms to produce collagenase enzyme with the desired properties is introduced. This process involves the insertion of specific genes into the genetic structure of the microorganisms in order to increase collagenase enzyme production and enhance its purity.One of the objectives of the invention is to obtain collagenase enzyme in high purity. The way to achieve this is by carrying out suitable genetic modifications in order to prevent the microorganisms from producing other proteases, undesirable proteins, or other compounds.

[0019] In the present invention, the production of microbial collagenase enzyme is achieved through the use of collagenase production cassettes designed by the inventors, which contain genetic structural units. The collagenase production cassette subject to the invention comprises at least one promoter, at least one terminator, at least one collagenase production gene, at least one selection marker, and at least one homology region for integration into the genome of the organism.

[0020] Subsequently, the production cassette designed in the invention is transferred into the target microorganisms in question, and within these organisms, collagenase enzyme production is carried out in a controlled manner.

[0021] As mentioned, the collagenase production cassette in question comprises at least one promoter. A promoter, as is known, is the DNA region that initiates the transcription of the collagenase production gene (target gene). It regulates gene expression by enabling the binding of RNA polymerase. If preferred, either a constitutive promoter or an inducible promoter may be used.

[0022] The collagenase production cassette comprises at least one terminator. The terminator ensures the proper termination of transcription by providing a termination signal. It enables the synthesis of mRNA at the desired lengths and ensures post-transcriptional stability.

[0023] The collagenase production cassette comprises at least one selection marker. The said selection marker comprises the pyrG gene. This selection marker makes it possible to reduce environmental and regulatory concerns.

[0024] The collagenase production cassette comprises at least one homology region containing DNA sequences designed to enable integration into the genome of the target organism in which production will be carried out. These regions are DNA segments that exhibit a high degree of similarity with a specific locus in the target genome. In a preferred embodiment, the said homology region is obtained from the target organism to which the cassette will be transferred.The inventors incorporate into the collagenase production cassette a collagenase production gene by providing its modification. This production gene is responsible for encoding the collagenase enzyme having the targeted SEQ 2 sequence listing. Although the organism from which it is isolated is not critical within the scope of the present invention, what is essential is obtaining a gene having a DNA sequence that enables the production of a collagenase enzyme with the desired properties.

[0025] In the most preferred embodiment, the collagenase production cassette comprises, as the production gene, a DNA sequence having the SEQ 1 sequence listing.

[0026] ATGTTGTCGGAACCAGTTAGTCAAGTGACCGAGCAGCATGCCCACTCTGCTCATACTC ACGGCGTGGAGTTCAACCGGGTTGAATACCAGCCTACTGCCACCCTGCCTATCCAGC CCAGCAAGGCTACCCGCGTTCAGTCGTTGGAGTCCCTCGATGAATCCTCTACCGCCT GCGACCTGGAGGCTCTTGTCACTGAAAGCTCGAACCAGCTGATCTCTGAGATCCTTA GCCAGGGTGCCACCTGTGTCAATCAGCTTTTCTCTGCTGAGAGCCGCATCCAGGAAT CGGTGTTCTCCTCTGATCACATGTACAACATCGCCAAGCACACCACTACCCTTGCTAA GGGCTATACCGGCGGTGGATCTGATGAGTTGGAAACTCTCTTCCTGTACCTTCGTGC CGGTTACTATGCTGAGTTCTATAACGACAATATCTCTTTCATCGAGTGGGTCACTCCT GCCGTGAAGGAAAGCGTTGATGCCTTCGTCAACACCGCTTCGTTCTACGAGAACTCC GACCGGCATGGTAAAGTGCTCTCTGAAGTTATCATCACTATGGATAGCGCCGGATTG CAGCACGCTTATCTCCCCCAGGTCACCCAGTGGCTGACTCGCTGGAACGACCAGTAC GCCCAGCATTGGTATATGCGTAACGCTGTTAATGGCGTCTTCACCATCCTTTTCGGCG GTCAATGGAACGAGCAGTTCGTGCAGATCATCGGCAATCAGACTGATCTGGCCAAGG CTCTTGGTGACTTCGCTCTCCGTGCCTCCTCCATTGGAGCTGAGGATGAGTTCATGG CCGCTAACGCTGGACGGGAATTGGGCCGCCTCACCAAGTACACTGGTAATGCCTCCT CTGTCGTGAAGTCGCAGCTCTCCCGTATCTTCGAGCAGTACGAAATGTATGGTCGGG GAGACGCTGTTTGGCTGGCCGCTGCCGATACTGCCTCGTACTATGCTGACTGCTCCG AGTTCGGCATCTGTAACTTCGAGACTGAACTTAAGGGTTTGGTTCTCTCTCAGACTTAT ACCTGCAGCCCAACCATCCGTATCTTGTCCCAGAATATGACTCAGGAACAGCACGCT GCCGCTTGTTCGAAGATGGGTTACGAGGAAGGATATTTCCATCAGTCCCTCGAAACC GGCGAACAGCCGGTCAAGGATGACCACAACACTCAGTTGCAAGTGAATATCTTCGAT AGCTCGACCGACTACGGCAAGTATGCCGGTCCTATCTTCGACATCTCTACCGACAAC GGAGGCATGTACTTGGAGGGAGATCCAAGCCAGCCGGGCAACATCCCCAATTTCATC GCCTACGAGGCCTCCTACGCCAACGCTGACCATTTCGTTTGGAACCTGGAGCATGAA TACGTCCACTATCTGGATGGACGCTTCGACCTTTACGGTGGATTCTCTCACCCTACCG AGAAGATCGTTTGGTGGAGCGAGGGCATCGCCGAATATGTCGCTCAGGAGAACGATA ATCAGGCCGCTTTGGAAACCATCCTCGACGGTTCTACTTACACCCTCAGCGAAATCTT

[0027]

[0028] CGAAACTACCTATGATGGATTCGATGTGGACCGCATCTACCGTTGGGGCTATCTTGCC GTTCGTTTCATGTTCGAGAACCATAAGGATGACGTCAATCAGATGTTGGTGGAAACCC GGCAGGGTAACTGGATCAATTACAAGGCCACTATCACCCAGTGGGCTAACCTCTATC AGTCGGAGTTCGAACAGTGGCAGCAGACTCTGGTGTCCAACGGAGCCCCCAATGCT GTTATCACCGCTAATTCGAAGGGCAAGGTCGGAGAGTCCATCACCTTCTCCTCTGAAA ACTCGACTGATCCAAATGGCAAGATCGTGTCCGTTCTCTGGGATTTCGGCGACGGTT CCACTTCTACCCAGACTAAGCCGACTCATCAGTACGGCTCGGAGGGTGAATATAGCG TCTCGCTGTCCGTGACCGACTCCGAGGGTCTTACCGCCACTGCTACCCACACTGTTG TCATCAGCGCCTTGGGCGGTAACGATACCCTCCCACAGGACTGCGCTGTTCAGTCTA AGGTCAGCGGAGGCCGCTTGACTGCCGGAGAACCTGTCTGTCTCGCTAATCAGCAGA CCATCTGGCTGTCCGTGCCCGCCGTTAACGAGTCCTCCAATCTGGCTATCACTACCG GAAACGGCACTGGTAATCTGAAGCTGGAGTATTCTAACAGCGGTTGGCCGGATGACA CCAATCTCCACGGATGGTCTGACAACATCGGAAATGGCGAGTGCATCACTCTGTCGA ACCAGTCCAATTACTGGGGCTATGTCAAGGTGAGCGGTGATTTTGAGAACGCAGCGA TTGTGGTTGATTTTGACGCCCAGAAGTGTCGGCAGTGA

[0029]

[0030] Table 1. Sequence listing of the DNA sequence enabling the production of the collagenase enzyme.

[0031] By means of the coding of the collagenase production gene having the sequence listing given in Table 1 and other similar sequence listings, a collagenase enzyme comprising the amino acid sequence having the SEQ 2 sequence listing is obtained.

[0032] MLSEPVSQVTEQHAHSAHTHGVEFNRVEYQPTATLPIQPSKATRVQSLESLDESSTACDL EALVTESSNQLISEILSQGATCVNQLFSAESRIQESVFSSDHMYNIAKHTTTLAKGYTGGG SDELETLFLYLRAGYYAEFYNDNISFIEWVTPAVKESVDAFVNTASFYENSDRHGKVLSEVI ITMDSAGLQHAYLPQVTQWLTRWNDQYAQHWYMRNAVNGVFTILFGGQWNEQFVQIIG NQTDLAKALGDFALRASSIGAEDEFMAANAGRELGRLTKYTGNASSVVKSQLSRIFEQYE MYGRGDAVWLAAADTASYYADCSEFGICNFETELKGLVLSQTYTCSPTIRILSQNMTQEQ HAAACSKMGYEEGYFHQSLETGEQPVKDDHNTQLQVNIFDSSTDYGKYAGPIFDISTDNG GMYLEGDPSQPGNIPNFIAYEASYANADHFVWNLEHEYVHYLDGRFDLYGGFSHPTEKIV WWSEGIAEYVAQENDNQAALETILDGSTYTLSEIFETTYDGFDVDRIYRWGYLAVRFMFE NHKDDVNQMLVETRQGNWINYKATITQWANLYQSEFEQWQQTLVSNGAPNAVITANSKG KVGESITFSSENSTDPNGKIVSVLWDFGDGSTSTQTKPTHQYGSEGEYSVSLSVTDSEGL TATATHTVVISALGGNDTLPQDCAVQSKVSGGRLTAGEPVCLANQQTIWLSVPAVNESSN LAITTGNGTGNLKLEYSNSGWPDDTNLHGWSDNIGNGECITLSNQSNYWGYVKVSGDFE NAAIVVDFDAQKCRQ

[0033]

[0034] Table 2. Sequence listing of the collagenase enzyme subject to the invention.

[0035] As mentioned, the collagenase enzyme having the SEQ 2 sequence listing as the intended final product is obtained from collagenase production cassettes comprising a collagenase production gene such as SEQ 1 used in the production process. These DNA fragments may be genetically modified and optimized to provide different technical advantages.

[0036] The collagenase production cassettes contain the genetic instructions necessary for the efficient and accurate synthesis of the target collagenase enzyme. As mentioned previously, the collagenase production cassette may comprise, as the production gene, a DNA sequence having a sequence listing different from the DNA sequence having the SEQ 1 sequence listing.

[0037] The collagenase production cassette subject to the invention is transferred into target organisms by means of at least one vector, in order to enable the obtaining of the collagenase enzyme having the SEQ 2 sequence listing. In the present invention, the target organism is preferably selected from at least one of the group consisting of Aspergillus oryzae, Aspergillus niger, Pichia pastoris, Trichoderma reesei, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio splendidus, Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, and Vibrio harveyi.

[0038] In the most preferred embodiment, Aspergillus oryzae is used as the microorganism. This microorganism is a species of fungus. The reason for using this fungal species is that it has a long history of safe use in the food industry and is recognized as “Generally Recognized as Safe” (GRAS) by the United States Food and Drug Administration (FDA). This safety profile makes it an ideal choice particularly for biotechnological production. In addition, Aspergillus oryzae is highly efficient in terms of enzyme production capacity; it can produce large amounts of enzymes and readily secrete them into the culture medium. This feature increases purity in the fermentation medium and facilitates the extraction process. It is also easy to genetically modify and to insert target genes, thereby allowing optimization for the production of specific enzymes such as collagenase. Moreover, Aspergillus oryzae can be fed with low-cost nutrient sources and is resistant to various environmental conditions, which reduces production costs and increases economic efficiency. Owing to these characteristics, Aspergillus oryzae stands out as a reliable, efficient, and sustainable microorganism for biotechnological enzyme production.In the present invention, DNA molecules such as bacterial plasmids or viral DNA are generally used as vectors for transferring the collagenase gene into microorganisms such as Aspergillus oryzae. Plasmids, being small circular DNA molecules obtained from bacteria, can be easily manipulated and are preferred due to their high gene-carrying capacity. Viral DNA, on the other hand, is effective particularly in difficult gene transfers because of its natural ability to deliver genetic material into cells. In addition, fungal plasmids specifically developed for fungal cells or artificial chromosome vectors such as Yeast Artificial Chromosomes (YACs), which have the capability of carrying large gene fragments, can be used for delivering genetic material into microorganisms such as Aspergillus oryzae.

[0039] The gene encoding the collagenase enzyme (SEQ 1 or similar sequence listings) described above is inserted into a carrier DNA fragment provided by a vector, after which transfer into the target microorganism is carried out. The recombinant DNA containing the genetic information is transferred into the microorganism, such as Aspergillus oryzae or strains of Vibrio, by means of the vector. The transfer of genetic material into microorganisms by the vector can be carried out using methods known in the art. Examples of such methods include at least one of electroporation, chemical transformation, biolistics, or microinjection. In this way, Aspergillus oryzae becomes a modified microorganism carrying this new genetic information.

[0040] The microorganism subject to the invention begins to produce collagenase enzyme in accordance with the newly acquired genetic information. This process is carried out in a fermentation medium optimized as disclosed in the invention. Another innovative aspect of the invention is the configuration of the said fermentation medium.

[0041] In the invention, the fermentation medium is carried out under aseptic conditions, and contamination is prevented to ensure genetic stability.

[0042] In the fermentation medium, a culture medium optimization is carried out. This culture medium provides the essential raw materials for the growth of the microorganisms that will perform the fermentation processes and for enzyme production. These raw materials supply carbon, nitrogen, and various mineral sources.

[0043] The culture medium subject to the invention comprises at least one carbon source. It may contain at least one of glucose, maltose, lactose, or dextrin group as the carbon source. These carbon sources provide energy production and growth for the microorganisms.In the most preferred embodiment, dextrin is included in the culture medium as the carbon source. Dextrin is more rapidly and easily absorbable for enzyme production compared to more complex carbohydrates. In addition, dextrin provides a stable carbon source and balances microbial growth.

[0044] The culture medium subject to the invention comprises at least one of potassium chloride, magnesium chloride, sodium chloride, or calcium chloride. These compounds support the growth of microorganisms and optimize the enzyme production process. Potassium chloride contributes to osmotic balance and enzyme activation, while magnesium chloride supports the effective use of ATP and functions as a cofactor for enzymes essential for protein synthesis. Sodium chloride regulates osmotic pressure and aids in cell stability, whereas calcium chloride increases cell wall stability. The presence of these compounds in the culture medium is necessary to enhance the efficiency of the fermentation process and to provide a stable environment for the production of the target collagenase enzyme.

[0045] The culture medium subject to the invention comprises at least one nitrogen source. The said nitrogen source provides the nitrogen required for protein and nucleic acid synthesis and plays a role in maintaining the pH balance of the medium. In a preferred embodiment, the culture medium comprises ammonia as the nitrogen source.

[0046] The culture medium subject to the invention comprises at least one phosphorus source. In the present invention, phosphoric acid is added as the phosphorus source.

[0047] In a preferred embodiment of the fermentation medium, air passed through filters in the range of 0.1 to 0.4 microns is supplied. This filtered air is used to prevent contamination in the fermentation medium.

[0048] In the fermentation medium, at least one target microorganism containing collagenase production cassettes comprising the production gene with the SEQ 2 sequence listing is included, in an amount ranging from 5% to 15% by weight, to enable the production of the collagenase enzyme.

[0049] The pH value of the fermentation medium subject to the invention is in the range of 3.5 to 7.5. This pH range is determined both to provide the optimal growth conditions for the target microorganism and to increase the production efficiency of the collagenase enzyme having the SEQ 2 sequence listing. Low pH prevents contamination, while neutral orslightly acidic pH supports the metabolic activities of the microorganism. In addition, this range is critical for maintaining enzyme stability and activity during fermentation.

[0050] The temperature of the fermentation medium subject to the invention is in the range of 28 to 35 °C. This temperature range is determined for the optimal growth of the genetically modified target microorganisms and for the effective production of the collagenase enzyme having the SEQ 2 sequence listing. This range ensures that the metabolic activities of the target microorganism occur at the highest level, while at the same time maintaining enzyme stability and increasing efficiency. Controlled temperature conditions are of critical importance for the fermentation process to yield consistent and reproducible results.

[0051] The fermentation period subject to the invention is in the range of 72 hours to 140 hours.

[0052] The dissolved oxygen content of the fermentation medium subject to the invention is in the range of 3 to 17 mg / mL. Dissolved oxygen is required for energy production in cellular respiration; at low levels it causes oxygen deficiency, whereas at high levels it creates oxidative stress for microorganisms. Accordingly, the dissolved oxygen content characterized in the invention is within the ranges at which such technical drawbacks do not occur and the preferred microorganisms are able to effectively maintain their activities.

[0053] In accordance with the determined parameters, fermentation processes are carried out to produce the collagenase enzyme. The target microorganisms grow and develop in a sterilized culture medium containing optimized nutrients under aseptic conditions. This culture medium provides the carbon, nitrogen, and various minerals required by the target microorganisms. When the enzyme production level reaches its maximum point, the process is allowed to continue for a period and then terminated to obtain the product. This product is a liquid mixture containing microbial collagenase enzyme and is collected for subsequent stages of filtration and purification. The fermentation process is terminated when the transition to dry mass occurs and the increase in protein level ceases.

[0054] Accordingly, the obtained product is filtered by means of at least one of a vacuum rotary drum filter or a press filter method. Through this filtration process, the microorganisms are separated from the liquid. The microbial residues are collected as solids, and only the collagenase enzyme remains in the liquid phase.

[0055] After this process, the separated liquid product is passed through ceramic or polymer membrane filters having porosity values in the range of 0.1 to 0.4 microns. This process allows the collagenase enzymes to pass through the filter while preventing microorganismsfrom remaining in the liquid. Thus, a collagenase enzyme solution purified from impurities, primarily the production microorganism, is obtained.

[0056] In a preferred embodiment, the enzyme solution is further subjected to ultrafiltration processes. The purpose of applying ultrafiltration is to process the collagenase enzyme solution in a cross-flow ultrafiltration unit so as to reach a specific concentration. As a result of these processes, the collagenase enzyme solution becomes more concentrated and attains the desired purity levels.

[0057] As a result of the preferred filtration processes, the liquid containing collagenase enzymes with specific purity values and concentration intensity is subjected to drying processes. The said drying processes are carried out at a temperature in the range of 90 to 150 °C. During the drying process, the product is dried with the aim of preserving its activity and maintaining the required moisture content.

[0058] In a preferred embodiment, the drying process is carried out by means of a spray-drying method applied to the solution containing collagenase enzyme. Upon completion of the drying processes, the collagenase enzyme obtained in purified form is subjected to quality control tests. These tests are related to whether the product complies with the relevant regulations and standards. The scope of protection of the invention is independent of what these regulations and tests are. Subsequently, suitable packaging processes are carried out for delivery to the user.

[0059] The purity value of the collagenase enzyme subject to the invention is in the range of 50% to 99%.

[0060] The production efficiency of the collagenase enzyme subject to the invention is in the range of 1 to 100 g / L.

[0061] The production method subject to the present invention is essentially carried out through two optimizations. The first of these is the genetically modified DNA sequence, which enables microorganisms to produce collagenase enzymes. The other optimization is the creation of a culture medium that allows these microorganisms to perform fermentation processes with high efficiency. With the said components and the corresponding parameters, the fermentation conditions have been optimized.With all of these technical teachings, it becomes possible to obtain a purer, more effective, and economical collagenase enzyme suitable for industrial applications. Thus, it is possible to obtain collagenase enzyme usable in various technical fields such as food, cosmetics, medicine, and pharmaceuticals.

[0062] By using the collagenase production cassette subject to the invention, the collagenase enzyme obtained through the method for producing microbial collagenase enzymes in the said target microorganisms is an innovative microbial enzyme distinguished by its high collagenase activity. The said collagenase enzyme has a collagenase activity of 400 CDU / mg, a gelatinase activity of 600 LGU / mg, and a caseinase activity of 4.0 caseinase / mg.

[0063] Accordingly, the collagenase activity (Collagenase Degrading Unit - CDU) subject to the invention is defined as the amount of enzyme that releases one micromole of L-leucine from collagen within 5 hours under conditions of 37 °C and pH 7.4. This value determines the capacity of the enzyme to degrade the collagen substrate and is an important criterion in terms of collagenase specificity.

[0064] In the present invention, the gelatinase activity (Livzym Gelatinase Unit - LGU) is defined as the amount of enzyme that releases one micromole of L-leucine from gelatin within one hour under conditions of 45 °C and pH 7.4. Gelatinase activity represents the ability of the enzyme to degrade the gelatin substrate.

[0065] In the present invention, the caseinase activity is defined as the amount of enzyme that releases one micromole of L-leucine from a casein substrate within 5 hours under conditions of 37 °C and pH 7.4. Caseinase activity is a parameter used to measure the presence of non-specific proteases.

[0066] The high CDU value of the collagenase enzyme obtained by the method subject to the invention emphasizes its specific affinity for the collagen substrate, while the low caseinase activity demonstrates that the effect of non-specific proteases is minimal.

[0067] Figure 1 illustrates the relative activity of the collagenase enzyme subject to the invention in relation to pH levels. According to the information provided in Figure 1, the activity of the enzyme begins to increase around pH 5 and reaches its maximum level at pH 8. After pH 8, the relative activity of the collagenase enzyme is observed to decrease up to pH 10.Figure 2 presents the SDS-PAGE image of the collagenase enzyme subject to the invention together with other proteins. In Figure 2, the band of approximately ~88 kDa indicated by the white arrow represents the collagenase enzyme of the invention. Other byproduct proteins produced are also visible on the gel. For reference, a commercial collagenase enzyme and a commercial protein ladder (Prestained protein marker, #S5331, Genaxxon Bioscience, Germany) were included in the gel image for molecular weight comparison. The molecular weight comparison serves as an important reference for confirming that the produced collagenase is of the correct size and for evaluating its purity level.

[0068] The scope of protection of the invention is defined in the claims provided herewith and shall by no means be limited to the examples described in this detailed description. It is evident that a person skilled in the art may, in light of the foregoing, develop similar embodiments without departing from the main concept of the invention.

Claims

CLAIMS1. A method for producing collagenase enzymes by means of microorganisms in order to obtain collagens suitable for use in technical fields such as food, cosmetics and skin care, medicine, pharmaceuticals, leather processing and textile products, and sports supplements, characterized by comprising the following process steps:- obtaining a collagenase production cassette comprising at least one promoter, at least one terminator, at least one collagenase production gene enabling the production of collagenase enzyme having the SEQ 2 sequence listing, at least one selection region, and at least one homology region, - transferring the obtained production cassette into at least one target microorganism by means of at least one vector, wherein said target microorganism is at least one selected from the group consisting of Aspergillus oryzae, Aspergillus niger, Pichia pastoris, Trichoderma reesei, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio splendidus, Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, and Vibrio harveyi,introducing the obtained microorganisms into a fermentation medium comprising a culture medium and carrying out fermentation processes,wherein the pH value of the said fermentation medium is in the range of 3.5 to 7.5,- subjecting the liquid product obtained from the fermentation processes to filtration processes to obtain a filtrate product,- applying drying processes to the obtained filtrate product.

2. A method according to claim 1, characterized in that at least one bacterium belonging to the vibrio strain family is used in obtaining the said collagenase production gene.

3. A method according to claim 1 or claim 2, characterized in that the said collagenase production gene has the SEQ 1 sequence listing.

4. A method according to any of the preceding claims, characterized in that the target microorganism is the fungal species Aspergillus oryzae.

5. A method according to any of the preceding claims, characterized in that bacterial plasmids or viral DNA are used as the vector.

6. A method according to any of the preceding claims, characterized in that bacterial plasmids or viral DNA are used as the vector.- A method according to any of the preceding claims, characterized in that the said culture medium comprises:- dextrin as a carbon source,- at least one selected from the group consisting of potassium chloride, sodium chloride, and calcium chloride as a mineral source,- phosphoric acid as a phosphorus source,- ammonia as a nitrogen source.

7. A method according to any of the preceding claims, characterized in that air passed through filters having a value in the range of 0.1 to 0.4 microns is supplied to the fermentation medium in order to prevent contamination in the fermentation environment.

8. A method according to any of the preceding claims, characterized in that at least one target microorganism is added to the fermentation medium in an amount ranging from 5% to 15% by weight.

9. A method according to any of the preceding claims, characterized in that the temperature of the fermentation medium is in the range of 28 to 35 °C.

10. A method according to any of the preceding claims, characterized in that the dissolved oxygen content of the fermentation medium is in the range of 3 to 17 mg / mL.

11. A method according to any of the preceding claims, characterized in that the said filtration process is carried out by passing through ceramic or polymer membranes having a porosity in the range of 0.1 to 0.4 microns.

12. A method according to claim 10, characterized in that an ultrafiltration process is carried out following the said filtration process.

13. A method according to any of the preceding claims, characterized in that the said drying process is carried out at a temperature in the range of 90 to 150 °C.

14. Collagenase enzyme having the SEQ 2 sequence listing, obtained by a method according to any of the preceding claims.

15. A collagenase enzyme according to claim 13, characterized in that its optimum operating pH range is from 6 to 10.

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