Culture medium for growing Hathewaya histolytica (or clostridium histolyticum) and for the production of one or more proteases

Abstract

The present invention relates to a liquid culture medium comprising yeast extract, glycine, arginine, glutamine, serine, threonine, magnesium ions, calcium ions, selenium, and water.The present invention further relates to a liquid feed composition for use as a feed in a fed-batch process, comprising at least yeast extract, threonine, serine, and water.The present invention relates to a kit of parts comprising a liquid culture medium and a liquid feed composition, the use of the composition for growing Hathewaya histolytica (Clostridium histolyticum) and obtaining one or more proteases from the culture supernatant, and a method related to the growth.

Description

[Technical field]

[0001] Introduction The present invention relates to a liquid culture medium comprising yeast extract, glycine, arginine, glutamine, serine, threonine, magnesium ions, calcium ions, selenium (e.g. in the form of sodium selenite), and water. The present invention further relates to a liquid feed composition for use as a feed in a fed-batch process comprising at least yeast extract, threonine, serine, and water. The present invention relates to a kit of parts comprising the liquid culture medium and the liquid feed composition, the use of the liquid culture medium and the liquid feed composition for growing Hathewaya histolytica (formerly Clostridium histolyticum) and obtaining one or more proteases, and methods related to the growth. [Background technology]

[0002] Review of the Prior Art Collagen is the main structural component of mammalian organisms, and accounts for a large proportion of the total protein content of the skin and other parts of the animal body. In humans, collagen is particularly important in the wound healing process and the natural aging process. Various skin injuries, such as bumps, surgery, infections, and accidents, are often characterized by irregular accumulations of fibrous tissue that is rich in collagen and has an increased proteoglycan content. In addition to replacing damaged or destroyed normal tissue, excessive and disfiguring deposition of new tissue can form during the healing process.

[0003] Numerous diseases and conditions are associated with excessive collagen deposition and the irregular accumulation of collagen-rich fibrous tissue. Such diseases and conditions are collectively referred to herein as "collagen-mediated diseases." Collagenase is an enzyme that has the specific ability to digest collagen. Collagenase has been used to treat a variety of collagen-mediated diseases, such as Peyronie's disease and Dupuytren's disease, and to remove necrotic tissue from wounds.

[0004] Additionally, collagenases and other proteases (such as neutral proteases and / or clostripain) are used in vitro for tissue dissociation and isolation of a variety of cells, such as pancreatic islet cells, hepatocytes, and tumor cells. These cells have found numerous applications in research and clinical practice. One common source of crude collagenase or protease mixtures is from bacterial fermentation processes, particularly from the fermentation of Hathewaya histolytica (formerly Clostridium histolyticum).

[0005] Hathewaya (Clostridium) is a genus of Gram-positive bacteria. Hathewaya (Clostridium) bacteria are anaerobic and are commonly found in soil, water, and the intestinal tract of humans and other animals. The species Hathewaya histolytica (Clostridium histolyticum) can produce collagenolytic enzymes as well as other enzymes with proteolytic activity, such as collagenase (EC 3.4.24.3), neutral proteases, and clostripain (EC 3.4.22.8). An important product of the growth process is bacterial collagenase, which possesses proteolytic activity against collagen. These enzymes have been classified by Bond et al. (see Non-Patent Document 1) as type I and II collagenases (hereinafter "collagenase I" and "collagenase II") according to their relative activity. Collagenase and other proteases are secreted into the culture medium and can be obtained from the culture supernatant.

[0006] In the biotechnology industry, many enzymes for use in pharmaceutical applications are produced in large-scale fermentation processes. For example, Hathewaya histolytica (Clostridium histolyticum) is grown to produce proteases, including, for example, collagenase, neutral protease, and / or clostripain. Usually, in such processes, the culture medium (nutrient medium, fermentation medium) contains animal-derived components, such as meat (tissue) peptones of bovine or porcine origin. Since adventitious agents (e.g., viruses) originating from the animal-derived growth medium may be present in the final product, it is desirable to develop culture media that are free of mammalian pathogenic agents. There are particular safety-related concerns regarding prions and BSE. In recent years, regulatory authorities have requested that these concerns be addressed. Therefore, the industry is turning towards culture media that are free of animal substances.

[0007] US Patent No. 5,999, 137 discloses a growth medium for Hathewaya histolytica (Clostridium histolyticum) that includes water, fish gelatin, and peptones from non-mammalian sources, with fish peptones excluded. The only examples of peptones from non-mammalian sources are plant peptones. This growth medium is plagued by the fact that fish gelatin and peptones from non-mammalian sources are complex components that contain a wide variety of compounds, most of which are unknown. Furthermore, these growth media are prepared from natural sources and are therefore subject to natural variations in their content.

[0008] Yeast extract and plant peptones have been considered in recent years as alternatives to culture medium components of animal origin. However, yeast extract alone cannot provide all the nutrients necessary for the satisfactory growth of certain bacteria. Patent document 2 discloses an animal product-free culture medium for bacteria of the genus Clostridium (now Hathewaya), in particular Clostridium histolyticum (now Hathewaya histolytica), which comprises water, a non-animal origin peptone or its derivatives, yeast extract, and the amino acids cysteine ​​and arginine. The non-animal origin peptone is derived from plants (plant peptone). Plant peptones are used in the fermentation of Clostridium histolyticum (now Hathewaya histolytica) to produce collagenase I and collagenase II according to patent document 3. Plant peptones have the disadvantage that they are complex and undefined mixtures, and that the production of raw materials is often subject to large batch variability, especially when weather conditions or planting areas change. Furthermore, the composition of plant peptones can vary considerably depending on the production process and source. Therefore, it is difficult or simply not possible to obtain a constant product quality or to find alternative sources. For these reasons, plant peptones are not desirable as ingredients for most industrial processes for bacterial growth. This is especially true for the growth of Hathewaya histolytica (Clostridium histolyticum) and the production of proteases. During growth, Hathewaya histolytica (Clostridium histolyticum) secretes collagenase I, collagenase II, clostripain, and neutral protease. The activity of all these enzymes in the supernatant is strongly influenced by the composition of the plant peptone used for growth and can vary by more than five-fold depending on the source or raw material of the plant peptone, the manufacturer, the production process, and depending on the batch. As outlined above, such product variability and reliance on a single source is less than optimal for industrial production processes, especially in the pharmaceutical industry. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] European Patent Application Publication No. 2133415 [Patent Document 2] European Patent Application Publication No. 2865748 [Patent Document 3] International Publication No. 2007 / 089851 [Non-patent literature]

[0010] [Non-Patent Document 1] Bond, MD, van Wart, HE; ​​Biochemistry, 23, 3077-91 (1984) Summary of the Invention

[0011] Problem to be solved by the invention The object of the present invention is to provide a liquid culture medium and / or liquid feed composition that (i) supports the growth of bacteria, (ii) stimulates the secretion of enzymes with proteolytic activity into the culture medium, and (iii) reduces product and process variability. It is particularly preferred that the medium stimulates high volumetric activity (enzyme activity per culture volume) in the culture supernatant. A further object of the present invention is to provide a new type of culture medium that allows the growth of bacteria without the addition of animal- and / or plant-derived components. The medium should furthermore contain as few components as possible to avoid any contamination with undesirable substances and to avoid excessively complex effects on the fermentation process. It is particularly preferred that such a culture medium allows the efficient growth of Hathewaya histolytica (Clostridium histolyticum) species and the production of one or more proteases, such as collagenase I, collagenase II, neutral protease, and / or clostripain. It is a further object of the present invention to provide a liquid culture supernatant of Hathewaya histolytica (Clostridium histolyticum) that can be used to isolate one or more proteases contained therein. It is also an object of the present invention to provide a process for the production of proteases with low degradation of proteases. In addition, the culture medium can provide the following benefits: a) higher batch reliability than culture media containing animal or plant peptones; b) all components can be obtained from multiple sources with suitable and consistent quality; c) the activity of collagenase in the supernatant of Hathewaya histolytica (Clostridium histolyticum) is 400 U / L or more (PZ activity). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In a first aspect, the present invention provides a method for producing a composition comprising the steps of: 2.5-100g / L yeast extract, 1.0-30g / L glycine, 1.0-35g / L arginine, 0.20-0.80g / L glutamine, Serine at 0.20-1.0 g / L, 0.3-3.0 g / L threonine, 0.4-12mmol / L magnesium ion (Mg 2+ ), e.g. 100-3000 mg / L magnesium sulfate heptahydrate (MgSO4×7H2O), 0.34 to 3.4 mmol / L calcium ion (Ca 2+ ), e.g. 50-500 mg / L calcium chloride dihydrate (CaCl2×2H2O), 0.00095-0.0152 mmol / L selenium, e.g., 0.25-4.00 mg / L sodium selenite pentahydrate (Na2SeO3×5H2O), and water The present invention relates to a liquid culture medium comprising, consisting essentially of, or consisting of:

[0013] As used herein, the term "consisting essentially of" is intended to mean that the composition (a) necessarily contains the recited ingredients, and (b) accepts unrecited ingredients that do not materially affect the basic and novel characteristics of the composition.

[0014] The liquid culture medium according to the invention is suitable as a culture medium for bacteria of the genus Hathewaya (Clostridium), in particular for Hathewaya histolytica (Clostridium histolyticum; e.g., Clostridium histolyticum G11 and / or ATCC® 21000®). It provides the nutrients required for sufficient growth of the bacteria and for the effective production of one or more proteases, such as collagenase I, collagenase II, neutral protease, and / or clostripain, optionally in combination with the feed composition according to the invention. In one embodiment, the fermentation of Hathewaya histolytica (Clostridium histolyticum) with the culture medium of the invention and optionally with the feed composition of the invention results in a collagenase activity in the supernatant of 400 PZ units / L or more, in another embodiment 600 PZ units / L or more, in another embodiment 1200 PZ units / L or more. According to Wuensch, one PZ unit catalyzes the hydrolysis of 1 μmol of 4-phenylazobenzyl-oxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine (PZ) per minute at 25° C. and pH 7.1 (Wuensch, E. & Heidrich, HG (1963) Hoppe-Seyler's Z. Physiol. Chem. 333, 149-51).

[0015] The use of a yeast extract concentration lower than 2.5 g / L is in principle possible, but may reduce bacterial growth and product yield. The use of a yeast extract concentration higher than 100 g / L is in principle possible, but may not provide significant benefits in terms of bacterial growth or protease secretion. In some embodiments, the concentration of yeast extract in the liquid culture medium is in the range of 2.5-80, 5.0-60, or 10-30 g / L. In another embodiment, the concentration of yeast extract in the liquid culture medium is in the range of 17-25 g / L. In some embodiments, the liquid culture medium comprises 20 g / L of yeast extract.

[0016] In principle, any yeast extract can be used. Yeast extract and methods for preparation are well known in the art. Contrary to animal or plant peptone, yeast extract is produced by a more controlled industrial process, including fermentation of yeast in industrial bioreactors. For this reason, batch reliability is higher than for plant or animal-derived components. Different batches of yeast extract produced by the same process provide similar performance in terms of bacterial growth and protease production. Furthermore, yeast extracts produced by different processes and / or different producers can substitute for each other and provide similar performance in terms of bacterial growth and protease production. In summary, yeast extract provides increased process robustness compared to animal and plant peptone.

[0017] In particular, any commercially available yeast extract can be used. Non-limiting examples are "BD Yeast Extract Technical" (Becton Dickinson and Company, Miami, FL, USA, product number: 288610), "BD Yeast Extract" (Becton Dickinson, product number: 212730), "BD Bacto Yeast Extract" (Becton Dickinson, product number: 212730). "BD Yeast Extract Technical" (Becton Dickinson, product number: 288610) provides high yields of collagenase and other proteases, and fermentation with this yeast extract is reproducible. Further suitable yeast extracts are, for example, "Difco Yeast Extract, low-dusting (LD), product number: 210933", yeast extracts produced by Ohly, Kerry HY-Yeast series, and Yeast Extract product number: A1552 from AppliChem.

[0018] Unless otherwise stated in the text, the yield and concentration of collagenase is measured by the PZ activity of the supernatant. The activity of neutral protease in the supernatant can be measured, for example, according to Lin, Y.-C. et al. (1969) J. Biol. Chem. 244, 789-93; or Kunitz, M. 1947. Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol. 30: 291-310; or as described in Example 1. The activity of clostripain in the supernatant can be measured, for example, according to Kezdy, FJ et al. Biochemistry, 1965, 4, 2302-2308; or as described in Example 2.

[0019] The liquid culture medium of the present invention comprises glycine or a pharma- ceutically acceptable salt thereof at a concentration of 1.0-30 g / L. The use of a concentration of glycine lower than 1.0 g / L is in principle possible, but may reduce bacterial growth and result in lower PZ activity in the supernatant. Concentrations of glycine higher than 30 g / L may have little benefit to cell growth or PZ activity in the supernatant. In one embodiment, the concentration of glycine in the liquid culture medium of the present invention is in the range of 5-8 g / L. In another embodiment, the concentration of glycine in the liquid culture medium is in the range of 5-7 g / L. In some embodiments, the liquid culture medium comprises 6.5 g / L of glycine.

[0020] The liquid culture medium of the present invention comprises arginine or a pharma- ceutically acceptable salt thereof at a concentration of 1.0-35 g / L. The use of arginine at a concentration lower than 1.0 g / L is possible in principle, but may reduce bacterial growth and result in lower PZ activity in the supernatant. Arginine at a concentration higher than 35 g / L may have little benefit on cell growth or PZ activity in the supernatant. In one embodiment, the concentration of arginine in the liquid culture medium of the present invention is in the range of 5-8 g / L. In another embodiment, the concentration of arginine in the liquid culture medium of the present invention is in the range of 6-7 g / L. In some embodiments, the liquid culture medium comprises 6.7 g / L of arginine.

[0021] The liquid culture medium of the present invention comprises glutamine or a pharma- ceutically acceptable salt thereof at a concentration of 0.20-0.80 g / L. Glutamine has a positive effect on bacterial growth and on PZ activity in the supernatant. The use of glutamine at a concentration lower than 0.20 g / L is in principle possible, but may reduce bacterial growth and result in lower PZ activity in the supernatant. Glutamine at a concentration higher than 0.80 g / L may have little benefit on cell growth or PZ activity in the supernatant. In one embodiment, the concentration of glutamine in the liquid culture medium of the present invention is in the range of 0.25-0.40 g / L. In another embodiment, the concentration of glutamine in the liquid culture medium of the present invention is in the range of 0.30-0.35 g / L. In some embodiments, the liquid culture medium comprises 0.32 g / L of glutamine.

[0022] The liquid culture medium of the present invention comprises serine or a pharma- ceutically acceptable salt thereof at a concentration of 0.20-1.0 g / L. Serine has a positive effect on bacterial growth but a negative effect on productivity. Productivity as defined herein is the ratio of PZ activity to bacterial growth. Bacterial growth can be determined by known methods, for example by measuring turbidity. In some embodiments, bacterial growth is determined by measuring optical density at 600 nm. The use of serine at a concentration lower than 0.20 g / L is possible in principle, but may result in lower bacterial growth. Serine concentrations higher than 1.0 g / L may reduce productivity. In one embodiment, the concentration of serine in the liquid culture medium of the present invention is in the range of 0.37-0.50 g / L. In another embodiment, the concentration of serine in the liquid culture medium of the present invention is in the range of 0.37-0.47 g / L. In some embodiments, the liquid culture medium comprises 0.42 g / L of serine.

[0023] The liquid culture medium of the present invention comprises threonine or a pharma- ceutically acceptable salt thereof at a concentration of 0.3-3.0 g / L. Lower concentrations of threonine within this range provide higher PZ activity in the supernatant than higher concentrations within this range. Use of a concentration of threonine lower than 0.3 g / L will result in lower bacterial growth. Concentrations of threonine higher than 3.0 g / L may reduce productivity. In one embodiment, the concentration of threonine is not higher than 3.0 g / L. In one embodiment, the concentration of threonine in the liquid culture medium of the present invention is in the range of 0.5-1.50 g / L. In another embodiment, the concentration of threonine in the liquid culture medium of the present invention is in the range of 0.8-1.2 g / L. In some embodiments, the liquid culture medium comprises 1 g / L of threonine.

[0024] The liquid culture medium according to the present invention contains 0.4 to 12 mmol / L of magnesium ions (Mg 2+), for example 100-3000 mg / L of magnesium sulfate heptahydrate (MgSO4×7H2O). In one embodiment, the liquid culture medium according to the present invention contains 1.2-6.9 mmol / L of magnesium ions (Mg 2+ In one embodiment, the concentration of magnesium sulfate heptahydrate in the liquid culture medium is in the range of 300 to 1700 mg / L. In another embodiment, the liquid culture medium according to the invention contains 2.8 to 5.3 mmol / L of magnesium ions (Mg 2+ In another embodiment, the magnesium sulfate heptahydrate concentration is in the range of 700-1300 mg / L. In some embodiments, the liquid culture medium comprises 995 mg / L of magnesium sulfate heptahydrate. The liquid culture medium according to the present invention comprises 0.34-3.4 mmol / L of calcium ions (Ca 2+ ), for example 50-500 mg / L calcium chloride dihydrate (CaCl2×2H2O). In one embodiment, the liquid culture medium according to the invention contains 0.68-2.0 mmol / L calcium ions (Ca 2+ In one embodiment, the concentration of calcium chloride dihydrate in the liquid culture medium is in the range of 100-300 mg / L. In another embodiment, the liquid culture medium according to the invention contains 1.0-2.0 mmol / L of calcium ions (Ca 2+). In another embodiment, the calcium chloride dihydrate concentration is in the range of 150-300 mg / L. In some embodiments, the liquid culture medium comprises 260 mg / L calcium chloride dihydrate. In one embodiment, the anions for magnesium and calcium ions may be selected from the group consisting of chloride, sulfate, phosphate, and acetate. In other embodiments, magnesium sulfate may be used for magnesium ions and / or calcium chloride may be used for calcium ions. The liquid culture medium according to the invention comprises 0.00095-0.0152 mmol / L selenium, for example 0.25-4.00 mg / L sodium selenite pentahydrate (Na2SeO3×5H2O). In one embodiment, the liquid culture medium according to the invention comprises 0.0027-0.0061 mmol / L selenium. In one embodiment, the concentration of sodium selenite pentahydrate in the liquid culture medium is in the range of 0.70-1.60 mg / L. In another embodiment, the liquid culture medium according to the invention comprises 0.0038-0.0053 mmol / L of selenium. In another embodiment, the sodium selenite pentahydrate concentration is in the range of 1.00-1.40 mg / L. In some embodiments, the liquid culture medium comprises 1.17 mg / L of sodium selenite pentahydrate. In some embodiments, sodium selenite (Na2SeO3) or its pentahydrate is used to provide selenium to the liquid culture medium of the invention, i.e., the medium comprises sodium selenite. Other selenium-containing compounds or compositions may be used. It is well within the knowledge of a person skilled in the art to select an appropriate form of selenium. It is well known that the selenium introduced into the medium may be in an appropriate form that has sufficient bioavailability. This makes the selenium contained in the medium available to the bacteria. Whether a compound or composition provides sufficient selenium to the medium may be easily determined by comparing the yield of the desired protease in the fermentation. This yield may be compared to the yield obtained by using sodium selenite. If necessary, the amount of a compound or composition other than sodium selenite or its pentahydrate used to provide selenium can be adjusted to obtain the same or similar results.

[0025] The liquid culture medium of the present invention described herein may contain water, such as purified water or water for injection. Purified water is water that has been mechanically filtered or treated to remove impurities and make it suitable for use, especially pharmaceutical use. Water for injection is even more purified and further specified, for example, in the European or US Pharmacopoeia. In one embodiment, the liquid culture medium contains water in an amount of at least 50, 60, or 70 weight percent, based on the total weight of the liquid culture medium. In some embodiments, the liquid culture medium contains water in an amount of at least 78 weight %, or at least 90 weight %, or at least 95 weight %. In some embodiments, water is the remainder of the liquid culture medium. In some embodiments, the liquid culture medium contains purified water or water for injection according to the European or US Pharmacopoeia, for example, in the amount specified above.

[0026] In some embodiments, the liquid culture medium is used for the fermentation of Hathewaya histolytica (Clostridium histolyticum) and the production of at least one protease selected from the group consisting of collagenase I, collagenase II, neutral protease, and clostripain.

[0027] In some embodiments, the liquid culture medium contains 10-30 g / L yeast extract, 5-8 g / L glycine, 5-8 g / L arginine, 0.25-0.40 g / L glutamine, 0.37-0.50 g / L serine, 0.50-1.50 g / L threonine, 1.2-6.9 mmol / L magnesium ions (Mg 2+ ), 0.68 to 2.0 mmol / L calcium ion (Ca 2+), 0.0027-0.0061 mmol / L selenium, and water. In some embodiments, the liquid culture medium comprises, consists essentially of, or consists of 10-30 g / L yeast extract, 5-8 g / L glycine, 5-8 g / L arginine, 0.25-0.40 g / L glutamine, 0.37-0.50 g / L serine, 0.50-1.50 g / L threonine, 300-1700 mg / L magnesium sulfate heptahydrate (MgSO4×7H2O), 100-300 mg / L calcium chloride dihydrate (CaCl2×2H2O), 0.70-1.60 mg / L sodium selenite pentahydrate (Na2SeO3×5H2O), and water. In some embodiments, the liquid culture medium contains 17-25 g / L yeast extract, 5-7 g / L glycine, 6-7 g / L arginine, 0.30-0.35 g / L glutamine, 0.37-0.47 g / L serine, 0.8-1.2 g / L threonine, 2.8-5.3 mmol / L magnesium ions (Mg 2+ ), 1.0-2.0mmol / L calcium ion (Ca 2+ ), 0.0038-0.0053 mmol / L selenium, and water. In some embodiments, the liquid culture medium comprises, consists essentially of, or consists of 17-25 g / L yeast extract, 5-7 g / L glycine, 6-7 g / L arginine, 0.30-0.35 g / L glutamine, 0.37-0.47 g / L serine, 0.8-1.2 g / L threonine, 700-1300 mg / L magnesium sulfate heptahydrate, 150-300 mg / L calcium chloride dihydrate, 1.00-1.40 mg / L sodium selenite pentahydrate, and water. In some embodiments, the liquid culture medium comprises, consists essentially of, or consists of 20 g / L yeast extract, 6.5 g / L glycine, 6.7 g / L arginine, 0.32 g / L glutamine, 0.42 g / L serine, 1 g / L threonine, 995 mg / L magnesium sulfate heptahydrate, 260 mg / L calcium chloride dihydrate, and 1.17 mg / L sodium selenite pentahydrate, and water (e.g., purified water).

[0028] Since yeast extract can be prepared in a reproducible manner, the liquid culture medium of the present invention provides a stable and reproducible culture medium with high batch stability, which provides high process robustness for the growth of Hathewaya histolytica (Clostridium histolyticum) and the production of proteases such as collagenase I, collagenase II, neutral protease, and clostripain. In some embodiments, the liquid culture medium does not contain any components derived from animals or plants. Such animal or plant-derived components may contain adventitious factors and / or these components may vary between manufacturers and even between different batches of the same manufacturer. Using a liquid culture medium without animal or plant-derived components for the growth of Hathewaya histolytica (Clostridium histolyticum) and the production of proteases provides more safety to the end consumer of the protease and improves the reliability of the liquid culture medium of the present invention in fermentation.

[0029] The term "animal derived" or "animal derived" or "animal-derived" as used herein shall mean any material of animal origin. The term "plant derived" or "plant derived" or "plant-derived" as used herein shall mean any material of plant origin. In some embodiments, the term "animal derived" or "animal derived" or "animal-derived" as used herein shall mean any material of animal origin and any material of non-animal origin that has been processed using one or more animal-derived materials (e.g., animal-derived enzymes). In some embodiments, the term "plant derived" or "plant derived" or "plant-derived" as used herein shall mean any material of plant origin and any material of non-plant origin that has been processed using one or more plant-derived materials (e.g., plant-derived enzymes). The term "component" or "ingredients" as used herein shall mean any material of the contents of a liquid culture medium or liquid feed composition.

[0030] In some embodiments, the liquid culture medium or liquid feed composition does not contain glucose. In some embodiments, the liquid culture medium or liquid feed composition does not contain a sugar selected from the group consisting of glucose, galactose, lactose, mannose, raffinose, fucose, sucrose, and arabinose. In some embodiments, the liquid culture medium or liquid feed composition does not contain any sugar.

[0031] In a further embodiment, the liquid culture medium includes an antifoaming agent. The antifoaming agent suppresses the generation of foam in the culture medium and during fermentation. Any commercially available antifoaming agent may be used. One suitable antifoaming agent is XIAMETER™ ACP-1500 (EU) antifoaming compound from Dow. The antifoaming agent may be added in an appropriate amount to suppress foaming to an acceptable level. Small-scale fermentation may be performed without the addition of an antifoaming agent. In large-scale fermentation, an antifoaming agent may be added.

[0032] In some embodiments, the liquid culture medium has a pH value in the range of 6.5 to 8.2. In some embodiments, the pH value of the liquid culture medium is in the range of 7.2 to 8.1 or 7.5 to 8.0. The pH value can be adjusted by any known method, for example by addition of a buffer with an appropriate buffering capacity or by addition of an acid or a base. In some embodiments, for example in pre-cultures and fermentations with a supernatant volume of about 0.5 liters or less, a buffer can be added. For example, 3-(N-morpholino)propanesulfonic acid buffer (MOPS buffer) can be used. In some embodiments, for example in fed-batch processes and / or fermentation processes where the supernatant volume is greater than about 1 liter, the pH value can be continuously measured and an acid or base (or both over the time course of the growth process) can be added to keep the pH value in the desired range. For example, phosphoric acid and / or sodium hydroxide can be used. In some embodiments, for example in fermentation processes where the supernatant volume is about 0.5 to about 1 L, one of two methods can be used.

[0033] In further embodiments, the liquid culture medium is sterilized. In some embodiments, the composition does not contain any self-replicating organisms. Sterilization can be achieved by standard methods known to those skilled in the art, for example, by heat treatment, such as, for example, autoclaving. In some embodiments, the sterilized liquid culture medium contains an inoculum of Hathewaya histolytica (Clostridium histolyticum). In some embodiments, the liquid culture medium does not contain any self-replicating organisms, but does contain Hathewaya histolytica (Clostridium histolyticum). In some embodiments, such compositions are prepared by adding an inoculum of Hathewaya histolytica (Clostridium histolyticum) to the sterilized liquid culture medium of the present invention.

[0034] In a second aspect, the present invention provides a method for producing a composition comprising the steps of: at least 20 g / L yeast extract, Threonine at least 3 g / L; at least 1.5 g / L serine, and water The present invention relates to a liquid feed composition, for use as a feed in a fed-batch process, for example for the growth of Hathewaya histolytica (Clostridium histolyticum), comprising, consisting essentially of, or consisting of:

[0035] As used herein, the term "fed-batch culture" is intended to mean an operating technique in biotechnology processes in which one or more nutrients are fed (fed) to a vessel, e.g., a bioreactor, during growth and the product remains in the vessel until the end of the run. For further details on fed-batch processes, see, e.g., Tsuneo Yamane and Shoichi Shimizu (Fed-batch Techniques in Microbial Processes; Advances in Biochem Eng. / Biotechnol 1984,30:147-194).

[0036] The liquid feed composition and the liquid culture medium are of different compositions. In some embodiments, the liquid feed composition comprises at least 30, 40, or 50 g / L yeast extract. In some embodiments, the liquid feed composition comprises at least 4, 5, 6, 7, 8, 9, or 10 g / L threonine. In some embodiments, the liquid feed composition comprises at least 2.0, 2.5, 3.0, 4.5, 5.0 g / L serine. In some embodiments, the liquid feed composition comprises, consists essentially of, or consists of 50-200 g / L yeast extract, 3-60 g / L threonine, and 1.5-40 g / L serine, and water. In some embodiments, the liquid feed composition comprises, consists essentially of, or consists of 70-130 g / L yeast extract, 3-10 g / L threonine, and 1.5-10 g / L serine, and water. In some embodiments, the liquid feed composition comprises, consists essentially of, or consists of 100 g / L yeast extract, 5 g / L threonine and 2.5 g / L serine, and water. The liquid feed composition of the present invention described herein comprises water, such as purified water or water for injection, in an amount of at least 50, 60, or 65 weight percent based on the total weight of the liquid feed composition. In some embodiments, the liquid feed composition comprises water, such as purified water or water for injection, in an amount of at least 70 weight%, or at least 80 weight%, or at least 95 weight%. In some embodiments, water is the remainder of the liquid feed composition.

[0037] In some embodiments, the liquid feed composition has a pH value in the range of 6.5 to 8.2.

[0038] In further embodiments, the liquid feed composition is sterilized. In some embodiments, the composition does not contain any self-replicating organisms. Sterilization can be achieved by standard methods known to those skilled in the art, for example by heat treatment, such as by autoclaving.

[0039] In a third aspect, the present invention relates to a kit of parts comprising a liquid culture medium of the present invention and a liquid feed composition of the present invention, which combination is useful for the fermentation of, for example, Hathewaya histolytica (Clostridium histolyticum; e.g., Clostridium histolyticum G11 and / or ATCC™ 21000™), e.g., in a fed-batch process, and for the production of one or more proteases.

[0040] A fourth aspect of the invention is a liquid culture supernatant of Hathewaya histolytica (Clostridium histolyticum, e.g. Clostridium histolyticum G11 and / or ATCC™ 21000™), comprising the liquid culture medium of the invention and / or the liquid feed composition of the invention, and one or more proteases. The one or more proteases may be selected from the group consisting of collagenase I, collagenase II, neutral protease, and clostripain. In some embodiments, the supernatant of a Hathewaya histolytica (Clostridium histolyticum) liquid culture prepared using the liquid culture medium and / or liquid feed composition of the present invention has a collagenase activity of at least 400 PZ units / L (Wuensch units), or at least 500 PZ units / L, or at least 600 PZ units / L, or at least 700 PZ units / L, or at least 800 PZ units / L, or at least 900 PZ units / L, or at least 1000 PZ units / L, or at least 11000 PZ units / L, or at least 1200 PZ units / L.

[0041] An embodiment of this aspect of the present invention is a culture supernatant of Hathewaya histolytica (Clostridium histolyticum) containing one or more proteases obtainable by the method of: (a) providing an inoculum of Hathewaya histolytica (Clostridium histolyticum) in a sterilized liquid culture medium according to the present invention; (b) growing the bacteria, whereby the bacteria secrete one or more proteases into the liquid phase; (c) separating solids, such as cellular material and other particulate matter, from the liquid phase; thereby obtaining a culture supernatant from Hathewaya histolytica (Clostridium histolyticum) containing one or more proteases. In some embodiments, the supernatant is obtained from a liquid fed-batch culture. Thus, in some embodiments, the supernatant can be obtained by a method comprising: (a) providing an inoculum of Hathewaya histolytica (Clostridium histolyticum) in a sterilized liquid culture medium according to the present invention; (b) growing the bacteria by fed-batch operation; (c) adding a liquid feed composition according to the present invention; and (d) separating solids, such as cellular material and other particulate matter, from the liquid phase; thereby obtaining a culture supernatant from Clostridium histolyticum containing one or more proteases. In some embodiments, the one or more proteases can be selected from the group including collagenase I, collagenase II, neutral protease, and clostripain.

[0042] In a fifth aspect, the present invention relates to the use of a liquid culture medium according to the invention for growing Hathewaya histolytica (Clostridium histolyticum; e.g. Clostridium histolyticum G11 and / or ATCC™ 21000™) and obtaining from the culture supernatant at least one protease having collagenase activity, such as collagenase I and / or collagenase II and / or neutral protease and / or clostripain. In a sixth aspect, the present invention relates to the use of the liquid feed composition of the present invention for growing Hathewaya histolytica (Clostridium histolyticum; e.g. Clostridium histolyticum G11 and / or ATCC™ 21000™) and obtaining at least one protease having collagenase activity from the culture supernatant, such as collagenase I and / or collagenase II and / or neutral protease and / or clostripain.

[0043] In a seventh aspect, the present invention provides a method for producing a composition comprising the steps of: - growing Hathewaya histolytica (Clostridium histolyticum; e.g., Clostridium histolyticum G11 and / or ATCC™ 21000™) in a liquid culture medium of the invention and obtaining at least one protease from the culture supernatant; The present invention relates to a method comprising the steps of:

[0044] In some embodiments, collagenase I, collagenase II, neutral protease, and / or clostripain are obtained using the methods described above. In some embodiments of the methods of the present invention, the pH value of the culture is controlled so that it is in the range of 6.5 to 8.2. In some embodiments, the pH value of the culture is controlled so that it is in the range of 7.2 to 8.1 or 7.5 to 8.

[0045] In some embodiments of the methods of the invention, growth is preferably carried out in a fed-batch operation, preferably by using a sterilized liquid feed composition described herein as a feed. In some embodiments of such fed-batch processes, the pH value of the culture is controlled so that it is in the range of 7.2 to 7.8.

[0046] In some embodiments, the method according to the present invention comprises: (a) providing an inoculum of Hathewaya histolytica (Clostridium histolyticum bacteria; e.g., Clostridium histolyticum G11 and / or ATCC™ 21000™) in a sterilized liquid culture medium according to the present invention; (b) growing the bacteria and optionally adding a liquid feed composition according to the invention, whereby the bacteria secrete one or more proteases into the liquid phase; (c) separating solids, e.g., cellular material and other particulate matter, from the liquid phase, thereby obtaining a supernatant; (d) obtaining one or more proteases from the supernatant; thereby producing one or more proteases from Hathewaya histolytica (Clostridium histolyticum). The process includes:

[0047] The culture during the fermentation process can be monitored continuously or at one or more time points during growth, for example by determining the turbidity and / or by determining the protease activity in samples of the culture supernatant, which can be taken at one or more time points during growth. In some embodiments, the addition of the liquid feed composition is started when the bacterial growth rate reaches the exponential phase. In some embodiments, the addition of the liquid feed composition is started when the bacterial growth rate reaches about 40%, 50%, 60%, or 70% of the maximum turbidity. The bacteria can be grown for 15 to 19 hours. Since the enzyme ratio changes over time, the growth time can be adapted according to the target enzyme or enzymes. Suitable methods for monitoring the growth and bacterial growth phase are disclosed, for example, in US Pat. No. 5,999,366. The protease or proteases can be obtained from the culture supernatant by any known method, for example in crude form or purified. Thus, in some embodiments, step (d) can include one or more purification steps, such as, for example, filtration and / or column chromatography. WO 2020 / 164721 describes several exemplary procedures for obtaining one or more proteases from the culture supernatant of Hathewaya histolytica (Clostridium histolyticum). EXAMPLES

[0048] Analysis method: The determination of Wuensch activity in the supernatant was performed according to Wuensch, E., Heidrich, HG; Hoppe-Seyler's Zeit. Physiol. Chem., 333, 149-51 (1963). One PZ unit activity according to Wuensch is the activity that catalyzes the hydrolysis of 1 μmol of 4-phenyl-azobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine per minute at 25° C. and pH 7.1. All values ​​of Wuensch units in this specification are given in PZ units / liter (U / L), i.e., Wuensch units per liter of culture medium, unless otherwise stated.

[0049] Clostripain and neutral protease activities were determined as described in Examples 1 and 2.

[0050] The turbidity was measured offline on a Mettler Toledo UV-VIS spectrophotometer UV5Nano at 600 nm (OD600). The turbidity is measured against a blank sample (medium without inoculum). If necessary, the solution must be diluted so that the measured value is in the measuring range of OD600 up to 0.6.

[0051] material: In all experiments, Clostridium histolyticum G11 and / or ATCC™ 21000™ were used. ATCC™ 21000™ is available from ATCC (American Type Culture Collection). As yeast extract, "BD Yeast Extract" (Becton Dickinson, product no.: 288610) was used. In the experiments described below, all cultures were inoculated with 2 volume percent of a preculture. One vial of lyophilized Hathewaya histolytica (Clostridium histolyticum G11 or ATCC™ 21000™) mixed with 1 mL of liquid culture medium (the vial contains a minimum of 10 6 The vial was determined to contain 35 × 10 CFU; analysis revealed that the vial 6 Precultures were prepared by inoculation of liquid culture medium with the contents of a vial (containing 100 CFU / vial) and anaerobic incubation at 37° C. in shake flasks.

[0052] As bioreactor a Multifors bioreactor (Infors GmbH, Einsbach, Germany) was used. Further materials, reagents and equipment are described in the Examples below.

[0053] Example 1: Activity assay of neutral proteases using a continuous photometric assay with the substrate N-(3-[furyl]acryloyl)-glycine leucine amide (FAGLA) Principle: Neutral protease from Hathewaya histolytica (or Clostridium histolyticum) catalyzes the hydrolysis of the peptide bond in the substrate FAGLA between the amino acids glycine and leucine. In the photometric assay, substrate conversion was continuously measured by the decreasing absorbance at 345 nm. Hence, the decrease in absorbance or the resulting negative slope is a direct measure of substrate conversion and hence of enzyme activity.

[0054] Unit definition: 1 FAGLA unit (1 U) is defined as the hydrolysis of 1 μmol of N-[3-(2-furyl)acryloyl]-glycine-L-leucineamide per minute.

[0055] Equipment: Temperature controlled UV-VIS spectrophotometer (e.g., Cary 50, 60, or 100, Agilent), circular shaker (e.g., Scientific Industries, Vortex Genie2), UV cuvette semi-micro (e.g., Brand, part number 759150), semi-micro quartz cuvette (e.g., Hellma), PD-10 desalting column (e.g., GE Healthcare, part number 17-0851-01).

[0056] Reagents: Purified water, European Pharmacopoeia (Ph.Eur.), 2-morpholinoethanesulfonic acid monohydrate, abbreviation MES (e.g. Merck, item no. 7 1.060126), calcium chloride dihydrate (e.g. Merck, item no. 1.02382), dimethylsulfoxide for spectroscopy, abbreviation DMSO (e.g. Merck, item no. 1.02950.), Triton® X-100 (e.g. Sigma-Aldrich, item 23,472-9), 2-propanol (e.g. Merck, item 1.01040), 1 mol / l sodium hydroxide solution (Merck, item 1.09137), FA-Gly-Leu-NH2 (e.g. Bachem, item 4003615), current reference substance neutral protease NB (REF00236), tris(hydroxymethyl)aminomethane, abbreviation Tris (e.g. Merck, item 1.08382.), 1 mol / l hydrochloric acid (e.g. Merck, item 1.09057).

[0057] Reagent solutions: a) MES buffer pH 6.5: 10.67 g (0.05 mol) of 2-morpholinoethanesulfonic acid monohydrate and 0.735 g (0.005 mol) of calcium chloride dihydrate were dissolved in approximately 800 ml of purified water. Then 10 ml of 2-propanol (1% (v / v)) was added to the buffer mixture. The pH was adjusted to 6.5 at room temperature with 1 mol / l sodium hydroxide solution and filled up to 1000 ml with purified water. 0.1 ml of Triton X-100 was added to the buffer by slowly pipetting a highly viscous Triton X-100 solution (0.01% (v / v)) while stirring by using a 100 μl piston stroke pipette. The surfactant was added to the buffer mixture and finally the tip was placed into the buffer. The solution was stirred until homogeneity was reached. The resulting buffer solution has a concentration of 0.05 mol / l 2-morpholinoethanesulfonic acid and 0.005 mol / l calcium chloride.

[0058] b) FAGLA substrate solution (concentration = 2.5 mmol / l): First, a 0.1 M FAGLA solution was prepared (36.9 mg of FAGLA was dissolved in 1.2 ml of dimethyl sulfoxide). Then, 1 ml of the 0.1 M FAGLA solution was mixed with 39 ml of MES buffer pH 6.5 described in a).

[0059] c) Reference substance: Approximately 12-20 mg of reference substance Neutral Protease NB (Nordmark Biochemicals, Uetersen, Germany) were weighed out and adjusted to a concentration of 10 mg / ml with MES buffer pH 6.5 as described in a). This solution was stored on ice. Further dilutions (within the reasonable working range described below) were prepared from this solution with MES buffer pH 6.5. Approximately 2.5-3.6 mg / ml of reference substance was used. The absorbance change should be within the reasonable working range. Analyte solutions were prepared at room temperature immediately before the determination with MES buffer pH 6.5 and measured immediately. The activity of the reference substance was measured in duplicate for at least one dilution within the reasonable working range.

[0060] d) Tris buffer pH 9.5: 0.606 g (0.005 mol) tris(hydroxymethyl)aminomethane and 0.37 g (0.0025 mol) calcium chloride dihydrate were dissolved in approximately 400 ml purified water. The pH was adjusted to 9.5 at room temperature with 1 mol / l hydrochloric acid and filled up to 500 ml with purified water. The resulting buffer had a concentration of 0.01 mol / l tris(hydroxymethyl)aminomethane and 0.005 mol / l calcium chloride.

[0061] Sample preparation: At least one duplicate measurement was performed. Depending on the activity, samples were diluted to a reasonable working range (see below). Samples from the concentrate of cell-free culture supernatant were rebuffered on a PD-10 column equilibrated with Tris buffer pH 9.5 before measurement.

[0062] execution: The tests were carried out in a temperature-controlled UV-VIS spectrophotometer at +30° C. and a wavelength of 345 nm. The change in absorbance at 345 nm was determined over a time interval of 5 minutes.

[0063] The FAGLA substrate solution was pre-warmed in a water bath at +30° C. for approximately 30 minutes before the actual measurement. Alternatively, a semi-microcuvette containing 0.8 ml substrate solution can be pre-heated directly in the UV-VIS photometer for at least 10 minutes before the measurement. Before starting the series of measurements, the photometer was calibrated against MES buffer pH 6.5 without substrate solution. For the measurement, 0.8 ml pre-heated substrate solution was placed in the cuvette. Then, 0.2 ml sample solution was added and immediately pre-mixed by pipetting up (approximately 3 piston strokes) and the whole reaction mixture was mixed with a disposable stirring spatula. The measurement was then started immediately. The final volume in the test was 1 ml. At least one duplicate measurement was performed for each sample. A specific substrate concentration of 2 mmol / l was below the saturation value, i.e. the specific activity measured depends on the substrate concentration. Therefore, the substrate concentration in the assay must be strictly adhered to and only activities determined at the same substrate concentration are comparable.

[0064] Reasonable working area: A prerequisite for an evaluable activity measurement is an absorbance change per minute (ΔA 345 nm / min) in a reasonable working range of -0.01 to -0.035, and the direction of the negative slope must be linear. If the value (ΔA 345 nm / min) is too high, a stronger dilution must be used, if the value is too low, a lower dilution should be used.

[0065] evaluation: The current reference material was evaluated. First, target values ​​and acceptance criteria were defined. The activity of the reference material and the samples was calculated from the average values ​​of the duplicate measurements. To calculate the negative absorbance change (ΔA / min) of the measurement results, the values ​​from 0 to 5 min were evaluated. Acceptance criteria for the evaluation: ΔA 345 nm / min of all measurements must be within the linear working range. The maximum deviation may be ≦8% of the mean value. In case of large deviations, the measurements were repeated.

[0066] The activity of samples with unknown concentrations was calculated using the following formula:

[0067]

number

[0068] The activity of a sample of known weight was calculated using the following formula:

[0069]

number

[0070] ΔA = Absorbance change per minute at 345 nm VF = sample dilution factor V=1ml (total volume) v = 0.2 ml (sample volume) ε=-0.317mM -1 cm -1 FAGLA extinction coefficient m = sample weight in mg

[0071] Example 2: Determination of clostripain activity in culture supernatants from Clostridium histolyticum principle: The enzyme clostripain catalyzes the hydrolysis of benzoyl-L-arginine ethyl ester (BAEE) in the presence of calcium and the reducing agent dithiothreitol (DTT), which is measured photometrically at 255 nm (Kezdy FJ, Lorand L, Miller KD.Titration of active centers in thrombin solutions.Standardization of the enzyme.Biochemistry 1965;4:2302-2308). If the reaction is pseudo-zero order, i.e., if the reaction is constant, the increase in absorbance per unit time is a direct measure of the enzyme concentration. One unit is the amount of enzyme activity that catalyzes the hydrolysis of 1 μmol BAEE per minute at 25° C. and pH 7.8 in the presence of 2.5 mM DTT.

[0072] Equipment: Spectrophotometer Cary 50 (Varian) with temperature controlled cuvette block vortex, rotator, quartz cuvettes, d=10 mm.

[0073] Reagents: Purified water, European Pharmacopoeia and USP, Dithiothreitol (DTT) (Serva, Part No. 20710), Na-benzoyl-L-arginine-ethyl ester HCl (BAEE) (Serva, Part No. 14600), Sodium dihydrogen phosphate monohydrate (Merck, Part No. 6346), 1N sodium hydroxide solution (Merck, Part No. 09137), Calcium acetate hydrate (Kraft, Part No. 15208)

[0074] Reagent solutions: 7.5 mM DTT solution: 57.8 mg DTT was dissolved in 50 ml purified water. The solution was prepared freshly every day. 1.5 mM BAEE solution: 51.8 mg BAEE was dissolved in 100 ml purified water. The solution was prepared freshly every day. 75 mM sodium phosphate buffer, pH 7.8: 10.35 g sodium dihydrogen phosphate was dissolved in approximately 800 ml purified water, and the pH was precisely adjusted to pH 7.8 at room temperature using sodium hydroxide solution. The solution was then filled up to 1000 ml with purified water.

[0075] Enzyme solvents: Solution A (without activation): 1.0 mM Ca(OAc)2: 40 mg calcium acetate hydrate was dissolved in 250 ml purified water. Solution B (with activation): 1.0 mM Ca(OAc)2 with 5 mM DTT: 38.4 mg DTT was dissolved in 50 ml of solution A. Solution C (with activation): 1.0 mM Ca(OAc)2 with 2.5 mM DTT: 19.2 mg DTT was dissolved in 50 ml of solution A. Solutions were prepared fresh every day.

[0076] Reference material: For each analysis, collagenase NB1 (Nordmark Biochemicals, Uetersen, Germany) was also analyzed as a reference material for identity.

[0077] Sample preparation: Protein concentrations in cell-free culture supernatants were determined by spectrophotometry by measuring absorbance at 280 nm. Three samples of cell-free culture supernatants were taken and each sample was diluted with solution B to a concentration of 20 mg of sample per ml of solution B.

[0078] Each of these diluted samples was divided as follows:

[0079]

number

[0080] Sample X and Sample Y are incubated at +4°C for 40 min to 4 h, then 100 μl of Sample X is further diluted with 900 μl of Solution C, and 200 μl of Sample Y is further diluted with 200 μl of Solution A to obtain Samples X and Y ready for measurement ("Sample Xm" and "Sample Ym").

[0081] execution: Activated sample Xm and non-activated sample Ym were measured in parallel. In addition, a zero measurement was performed. The following amounts of reagents (pre-heated to 25° C.) were pipetted into a quartz cuvette:

[0082] [Table 1]

[0083] Samples Xm and Ym, respectively, were added last, the contents of the cuvettes were mixed and the change in absorbance at 255 nm was measured immediately after mixing for 5 min at 25° C. The slope ΔA 255 nm / min was evaluated in the linear region of the curve. The measured slope of the absorbance at 255 nm per minute of the sample must be linear over the entire 5 min period, otherwise dilutions must be adapted.

[0084] Activity calculation: The activity was calculated using the following formula:

[0085]

number

[0086] ΔA 255nm : Gradient per minute at 255 nm V G : Total volume used in the test (V G =3.1ml) V P : Volume of sample used in the test (V P =0.1ml) 0.81: The molar absorption difference of BAEE relative to benzoyl-L-arginine at 255 nm, i.e., 810M -1 cm -1 Dilution factor: All dilution factors starting from the cell-free culture supernatant sample to the measurement

[0087] Example 3: Preparation of liquid culture medium 20g / L yeast extract, 6.5g / L glycine, 6.7g / L arginine, 0.32g / L glutamine, 0.42g / L serine, 1.00g / L threonine, 995mg / L magnesium sulfate heptahydrate (MgSO4×7H2O), 260mg / L calcium chloride dihydrate (CaCl2×2H2O), and 1.17mg / L sodium selenite pentahydrate (Na2SeO3×5H2O), as well as 10mg silicone-based antifoam agent (XIAMETER™ ACP-1500 (EU) antifoam compound) were added to a 1-liter flask pre-filled with about 600ml purified water, and then the flask was filled with purified water under stirring up to a total volume of 1 liter of liquid culture medium. Finally, the liquid culture medium was sterilized in an autoclave. After sterilization, the pH was adjusted to 7.5 by the addition of 10 M sodium hydroxide solution in water.

[0088] Example 4: Preparation of liquid culture medium for pre-culture 20 g of yeast extract, 6.5 g of glycine, 6.7 g of arginine, 0.32 g of glutamine, 0.42 g of serine, 1.00 g of threonine, 995 mg of magnesium sulfate heptahydrate (MgSO4 x 7H2O), 260 mg of calcium chloride dihydrate (CaCl2 x 2H2O), 1.17 mg of sodium selenite pentahydrate (Na2SeO3 x 5H2O), and 41.86 g of 3-(N-morpholino)propanesulfonic acid buffer, as well as 10 mg of a silicone-based antifoam agent (XIAMETER™ ACP-1500 (EU) antifoam compound) were added to a 1-liter flask pre-filled with approximately 600 ml of purified water. The flasks were then filled with purified water under stirring up to a total volume of 1 liter of liquid culture medium and the pH was adjusted to 7.9 by addition of 10 M sodium hydroxide solution in water. Finally, the liquid culture medium was sterilized in an autoclave.

[0089] Example 5: Preparation of a liquid feed composition 100 g of yeast extract, 5 g of threonine, and 2.5 g of serine were added to a 1-liter flask pre-filled with about 600 ml of purified water, and then the flask was filled with purified water under stirring up to a total volume of 1 liter of liquid culture medium. Finally, the liquid culture medium was sterilized in an autoclave.

[0090] Example 6: First pre-culture A 100 mL Erlenmeyer flask was filled with 100 mL of medium prepared according to Example 4. Approximately 1 mL of medium was removed from the flask and 1 vial of lyophilized Clostridium histolyticum G11 (approximately 35×10 6 The contents of ATCC™ 21000™ or ATCC™ 21000™ were mixed with the medium. The medium was inoculated with this mixture. The cultures thus prepared were grown for 24 hours (± 1 hour) without shaking. Growth was carried out in anaerobic pots (Anaerocult, manufacturer Merck) with the exclusion of oxygen in an incubator or in an anaerobic bench with an anaerobic gas atmosphere (85% nitrogen, 10% carbon dioxide, and 5% hydrogen). The ambient temperature was kept at 37°C.

[0091] Example 7: Second pre-culture For fermentation in the 1 L bioreactor, a 100 mL Erlenmeyer flask was filled with 100 mL medium prepared according to Example 4. The medium was inoculated with 5 mL of the first preculture (corresponding to 5% of the second preculture medium volume). The culture was grown for 12 hours (±0.5 hours) without shaking. Growth was performed under anaerobic conditions as disclosed in Example 6 and at an ambient temperature of 37° C. For fermentation in the 30 L bioreactor, a second preculture was prepared in a 1000 mL flask filled with 1000 mL medium prepared according to Example 4. The medium was inoculated with 50 mL of the first preculture (corresponding to 5% of the second preculture medium volume). The culture was grown for 12 hours (±0.5 hours) without shaking. Growth was performed under anaerobic conditions as disclosed in Example 6 and at an ambient temperature of 37° C.

[0092] Example 8: Main culture in a bioreactor For fermentation in the 1 L bioreactor, the starting volume of the medium (0.6 L) was inoculated with 12 mL of the second preculture (corresponding to 2% of the medium volume). For fermentation in the 30 L bioreactor, the starting volume of the medium (27 L) was inoculated with 540 mL of the second preculture (corresponding to 2% of the medium volume).

[0093] The fermentation parameters for each system and each scale are shown in Table 1 below:

[0094] Table 1: Fermentation parameters in two different bioreactors [Table 2]

[0095] The liquid culture medium was introduced into the bioreactor and sterilized (Multifors in autoclave; Techfors reactor itself). The bioreactor was then flushed with nitrogen and stirred for at least 3 hours before inoculation. A 2 volume percent inoculum was used. The corresponding amount of the second preculture of Example 7 was taken from the second preculture using a sterile syringe and the main culture was inoculated through the inoculation port of the bioreactor. For the 30 L scale, the preculture was poured into the bioreactor through a hose (the container with the preculture was placed on a balance to determine the exact amount of inoculum). The cultivation time was 15-19 hours, mainly 17 hours, and the addition of the liquid feed composition prepared according to Example 5 was started after 9 hours of fermentation.

[0096] The following table shows the amount of protease determined in the supernatant with Clostridium histolyticum G11:

[0097] [Table 3]

[0098] It can clearly be seen that successful scale-up for this process can be achieved without significant loss of activity for either collagenase, clostripain, or neutral protease.

[0099] The following table shows the amount of protease determined in the supernatant using Clostridium histolyticum ATCC™ 21000™:

[0100] [Table 4]

[0101] It can clearly be seen that large scale production of the enzyme can be successfully achieved using different strains of Clostridium histolyticum.

Claims

1. 2.5 to 100 g / L of yeast extract; 1.0 to 30 g / L of glycine; 1.0 to 35 g / L of arginine; 0.20 to 0.80 g / L of glutamine; 0.20 to 1.0 g / L of serine; 0.3 to 3.0 g / L of threonine; 0.4 to 12 mmol / L of magnesium ions (Mg ２＋ )and, 0.34 to 3.4 mmol / L of calcium ions (Ca ２＋ )and, 0.00095 to 0.0152 mmol / L of selenium; Water and A liquid culture medium comprising:

2. 2.5 to 100 g / L of yeast extract; 1.0 to 30 g / L of glycine; 1.0 to 35 g / L of arginine; 0.20 to 0.80 g / L of glutamine; 0.20 to 1.0 g / L of serine; 0.3 to 3.0 g / L of threonine; 100 to 3000 mg / L of magnesium sulfate heptahydrate; 50 to 500 mg / L of calcium chloride dihydrate; 0.25 to 4.00 mg / L of sodium selenite pentahydrate; Water and 2. The liquid culture medium of claim 1, comprising:

3. 10 to 30 g / L of yeast extract; 5-8 g / L of glycine; 5 to 8 g / L of arginine; 0.25 to 0.40 g / L glutamine; 0.37 to 0.50 g / L of serine; 0.50 to 1.50 g / L of threonine; 300 to 1700 mg / L of magnesium sulfate heptahydrate; 100 to 300 mg / L of calcium chloride dihydrate; 0.70 to 1.60 mg / L of sodium selenite pentahydrate; Water and 3. The liquid culture medium of claim 2, comprising:

4. 17-25 g / L of yeast extract; 5-7 g / L glycine; 6-7 g / L arginine; 0.30 to 0.35 g / L glutamine; 0.37 to 0.47 g / L of serine; 0.8 to 1.2 g / L of threonine; 700 to 1300 mg / L of magnesium sulfate heptahydrate; 150 to 300 mg / L of calcium chloride dihydrate; 1.00 to 1.40 mg / L of sodium selenite pentahydrate; Water and 4. The liquid culture medium of claim 3, comprising:

5. 10. The liquid culture medium of claim 1, which does not contain any components of animal or plant origin.

6. 10. The liquid culture medium of claim 1, having a pH value in the range of 6.5 to 8.

2.

7. 10. The sterilized liquid culture medium of claim 1.

8. 10. The liquid culture medium of claim 1, additionally comprising an inoculum of Hathewaya histolytica (Clostridium histolyticum).

9. at least 20 g / L of yeast extract; at least 3 g / L of threonine; at least 1.5 g / L serine; Water and 1. A liquid feed composition for use as a feed in a fed-batch process for the growth of Hathewaya histolytica (Clostridium histolyticum), comprising:

10. 10. The liquid feed composition according to claim 9, having a pH value in the range of 6.5 to 8.

2.

11. A sterilized liquid feed composition according to any one of claims 9 or 10.

12. 10. A kit of parts comprising the liquid culture medium of claim 1 and the liquid feed composition of claim 9.

13. 10. Use of the liquid culture medium according to claim 1 and / or the liquid feed composition according to claim 9 for growing Hathewaya histolytica (Clostridium histolyticum) and obtaining at least one protease from the culture supernatant.

14. 10. A method comprising the steps of growing Hathewaya histolytica (Clostridium histolyticum) in the liquid culture medium of claim 1 and obtaining at least one protease from the culture supernatant.

15. 15. The method of claim 14, wherein the propagation is carried out in a fed-batch operation by use of a liquid feed composition according to claim 9.