Purified fish protease having high specific activity and method for producing the same

Abstract

The present invention relates to a method for producing fish proteases from fish viscera, preferably from cod (Gadus genus) viscera. The fish proteases produced according to the present invention have high specific enzymatic activity and are useful in food applications, biomedical applications, histology and tissue culture.

Description

Technical Field

[0001] Field of the Invention The present invention relates to a method for producing a fish protease from fish viscera, preferably the viscera of cod (Gadus genus). The fish protease produced according to the present invention has high specific enzyme activity and is useful for food applications, biomedical applications, histology and tissue culture.

[0002] Background The proteolytic activity of trypsin (EC 3.4.21.4) was first described in pancreatic secretions by W. Kuhne in 1876 ("Ueber das Trypsin (Enzym des Pankreas)", Verhandlungen des naturhistorisch-medicinischen Vereins zu Heidelberg, vol. 1, no. 3, pages 194-198).

[0003] This enzyme hydrolyzes C-terminal peptide bonds to lysine and arginine amino acid residues about 100 times faster than basic hydrolysis. Trypsin has been identified in all animals, including insects, fish, and mammals, since its first discovery. Trypsin from each source has slightly different activity, but the natural substrate for the enzyme is any peptide containing lysine or arginine.

[0004] Human trypsin hydrolyzes peptide bonds after arginine or lysine residues, and its activity is optimal at pH 7.5 - 8.5 and in the presence of calcium ions. Furthermore, human trypsin has an optimal operating temperature of about 37°C.

[0005] Fish trypsin, for example, trypsin isolated from Atlantic cod, has different optimal temperature ranges (since poikilothermic animals such as fish survive at low body temperatures). For example, cod trypsin includes trypsin I, which has a maximum temperature activity range of 4 - 65 °C and a maximum activity at 55 °C, and trypsin Y, which has an activity range included in 2 - 30 °C and a maximum activity at 21 °C (Gudmundsdottir A et al., 2005 Mar - Apr;7(2):77 - 88); Hindawi Publishing Corporation; BioMed Research International, Volume 2013, Article ID 749078). Another relevant difference between mammalian trypsin and fish trypsin is their thermal stability. For example, fish trypsin is completely inactivated by a pasteurization process, while mammalian trypsin is not inactivated. As a protein, trypsin exhibits various molecular weights depending on the source. For example, a molecular weight of 23.3 kDa has been reported for trypsin from bovine and porcine sources. On the other hand, fish trypsin I, trypsin X, and trypsin Y isolated from Atlantic cod have molecular weights of 23.9 kDa, 23.9 kDa, and 25.1 kDa, respectively (Bjarki Stefansson et al., Characterization of cold - adapted Atlantic cod (Gadus morhua) trypsin I - Kinetic parameters, autolysis and thermal stability; Comparative Biochemistry and Physiology, Part B; (2010) 186 - 194).The commercial uses of trypsin include, inter alia, the development of cell and tissue culture protocols (Soleimani M.; Nadri S. A, Nature protocols (2009), 4(1), 102-6), protein identification by peptide sequencing techniques (Schuchert-Shi et al., Analytical Biochemistry (2009), 387(2), 202-207) and their use in the medical field for modeling the degradation of articular cartilage in osteoarthritis (Wang S. et al., Connective tissue research (2010), 51(1), 36-47). In particular, fish trypsin (Atlantic cod trypsin I) has already proven its usefulness in various industrial applications, including the production of all-natural seafood flavors from lobster, shrimp, crab and other seafood (Bjarnason, J.B. et al., Psychrophilic proteinases from Atlantic cod ACS Symposium Series (1993), 516 (Biocatalyst Design for Stability and Specificity), 68-82). More recently, pollock trypsin has shown high efficacy in the degradation of native proteins and in vitro anti-pathogenic efficacy against HSV-1 and RSV, opening up new perspectives for new therapeutic uses of fish proteases (BioMed Research International Volume 2013, Article ID 749078, http: / / dx.doi.org / 10.1155 / 2013 / 749078). An important aspect of the use of proteases and marine organism trypsins is their stability, as they are subject to autolysis. For this reason, they should be stored at very low temperatures (-20 to -80 °C) to prevent degradation. Autolysis can be controlled by keeping these proteases at pH 3 or by using proteases modified by reductive methylation.Serine proteases (a class of proteases that also includes trypsin) show a recovery of activity when adjusted to return the pH to p8 (F.M. Pohl European J. Biochem. 7 (1968), 146 - 152; Aizawa, N.; Yokohama Medical Bulletin (1960), 11, 101 - 10), but significant losses of activity when working under very alkaline conditions (pH 10) have been described (B.K. Khangembam et al., International Aquatic Research, December 2012, 4:9). Commercially available marine proteases (trade name Accutase) extracted from the intestine of crustaceans and identified by CAS registration number 534583 - 22 - 7 are also temperature - sensitive: they are stable at 4 °C for 60 days, but when stored at 37 °C, 75% of their enzyme activity is rapidly (within 90 minutes) lost.

[0006] Standardization of these characteristics of purified trypsin and proteases, regarding stability and enzyme activity, is important from the perspective of their commercial use. This is because they can be verified for their use as production reagents (e.g., in food chemistry) as well as for use in histology and tissue culture. The pyloric caeca of the Atlantic cod, which function as digestive organs, are fishery by - products and can be utilized as an inexpensive starting material for the isolation of fish proteases including trypsin. This pyloric caeca contains a large amount of digestive enzymes, such as serine proteases (Asgeirsson B. et al., Eur J Biochem 180(1), 85 - 94).

[0007] The best-known members of these serine protease families from Pacific cod are trypsin, chymotrypsin, elastase, serine collagenase, and brachyurin (Halfon S, Craik CS (1998) "Family S1 of trypsin (clan SA)" In: Handbook of Proteolytic Enzymes, Barrett AJ, Rawlings ND, Woessner JF, eds. (San Diego, Calif.: Academic Press) pp 5-12). More specifically, three native trypsin isoenzymes called trypsin I, II, and III were isolated from the pyloric ceca of Pacific cod. Trypsin I is the most abundant, best-characterized form and also exhibits the highest catalytic efficiency. This efficiency is approximately 20-fold higher than that of its mesophilic bovine analog. However, the known methods for purifying proteases from fish viscera (Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology (1995), 110(4), 707-17; Journal of Agricultural and Food Chemistry, 39 (10), Pages 1738-42 (1991)) are very complex and may require multiple purification steps, especially when including multiple chromatographic purifications such as (NH4)2SO4 fractionation and hydrophobic interaction chromatography, affinity chromatography, or ion exchange chromatography. Based on the data reported in the literature, it should be noted that the overall yield of trypsin purification from the crude extract is low, and the resulting specific enzyme activity is in the range of a few units / mg.

[0008] Furthermore, the need to stabilize trypsin from autolysis during long purification processes, working in the presence of Ca 2+ and at low temperature, implies high costs for scale-up to industrial scale. Therefore, a simpler and more effective purification method is needed.

[0009] Description of the Invention The present invention relates to a method for purifying fish protease from fish viscera, comprising: a) extracting crude enzyme from fish viscera using calcium chloride buffer (pH 7), filtering, and ultrafiltrating; b) extracting the ultrafiltrate with an aqueous CaCl2 solution having a conductivity of 52 - 62 mS in a pH range of 7.8 - 8.2, and subsequently performing depth filtration; c) purifying the filtrate by hydrophobic interaction chromatography using an agarose-based matrix having a linear alkyl ligand or aryl ligand as the stationary phase, eluting with a buffer having a low salt content, and then eluting with an aqueous mixture of a water-miscible organic solvent and a polyol; d) dialyzing; e) optionally, lyophilizing.

[0010] The protease obtained by the method of the present invention has an average specific enzyme activity of 240 U / mg ± 40 U / mg trypsin, a chymotrypsin activity of 4 ± 2 U / mg, a collagenase activity of 0.04 ± 0.02 U / mg, and a protease activity of 65 ± 10 U / mg.

[0011] The method provides a total yield of solid fish protease of 0.06 - 0.11% by weight of the cod viscera used as the starting material.

[0012] The fish viscera is preferably cod viscera.

[0013] The extraction process of step a) is carried out at a temperature of 4 - 25°C.

[0014] The pH calcium chloride buffer preferably has a final concentration of 20 mM.

[0015] Ultrafiltration is preferably carried out using a membrane with a cut-off of 1 kDa. On the other hand, the agarose-based matrix of the stationary phase used in step c) exhibits an aryl ligand and a particle size distribution of 50 - 100 microns.

[0016] The chromatography elution with a buffer of low salt content in step c) is preferably carried out using an aqueous solution of 1.5 M sodium acetate, the water-miscible organic solvent is isopropanol, and the polyol is glycerol.

[0017] Step c) is preferably carried out at a temperature of 4 to 25 °C.

[0018] Detailed description: Definitions Cell lines tested for the detachment and dissociation of substratum-dependent cells from the surface PC12 cells (a cell line derived from a pheochromocytoma of the rat adrenal medulla) have specific receptors and are thought to respond to epidermal growth factor (EGF) (Huff and Guroff, 1979). The presence of such receptors may reflect a hitherto unrecognized role of EGF during neuronal development or, alternatively, may correlate with the neoplastic nature of PC12 cells. PC12 cells provide a model for studying the chemical disruption of processes related to neuronal differentiation, neurotransmitter synthesis, storage and release, ion channel function and regulation, and the interaction of compounds with membrane-bound receptors.

[0019] Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells that can be generated directly from adult cells. These cells can differentiate into hepatic lineages and provide an accurate model for liver diseases, drug screening, and drug toxicity testing (Curr Stem Cell Res Ther. 2015;10(3):208-15).

[0020] Neural progenitor cells (NPCs) are pluripotent stem cells that have the ability to differentiate into neurons and glial cells (oligodendrocytes and astrocytes). Thus, the successful proliferation of neural progenitor cells in vitro offers significant therapeutic potential for cell therapy applications.

[0021] Human osteosarcoma epithelial cells (U2OS) may meet the need for an in vivo metastasis model for osteosarcoma.

Brief Description of the Drawings

[0022]

Figure 1

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Figure 6

[0023] Enzyme tests used Trypsin activity. The enzyme activity was analyzed according to the method described in the United States Pharmacopeia 41st edition.

[0024] Chymotrypsin activity. The enzyme activity was analyzed according to the method described in the United States Pharmacopeia 41st edition.

[0025] Type I collagenase activity. The enzyme activity was analyzed according to the protocol described in the literature (Mandl, I. J. Clin. Invest. 32, 1323. 1953, Moore, S. et al., J. Biol. Chem. 176, 367. 1948).

[0026] Protease activity. The enzyme activity was analyzed according to the method described in the United States Pharmacopeia 41st edition.

[0027] The consistency of the claimed method was confirmed using different batches of cod viscera (captured at different times during the year) as starting materials. The resulting fish proteases were demonstrated to have limited variability between batches, with this variability not exceeding a range of ±15% with respect to the specific enzyme activity obtained.

[0028] The purified fish protease prepared according to the method of the present invention exhibits a specific enzyme profile. On the other hand, in fact, commercially available marine organism trypsin (for example, the marine organism protease identified by CAS registration number 534583-22-7) shows that about 26-49% of the total enzyme activity exhibits trypsin activity and about 47-67% exhibits type I collagenase activity, while the fish protease prepared according to the method of the present invention shows that about 89-66% of the total enzyme activity exhibits trypsin activity and only 0.011-0.015% exhibits collagenase activity.

[0029] Surprisingly, when the fish protease prepared according to the method of the present invention is utilized in histology for cell detachment, it is less harmful to cells and results in an improvement in survival rate compared to other marine organism proteases. Specifically, when the NPC and PC12 cell lines are treated with the fish protease obtained according to the present invention, the survival rate and the number of recovered cells are approximately 5-10% higher than those of other trypsins / proteases of mammalian and marine organism origin.

[0030] Detailed description: Method The frozen (-20 °C) cod viscera were thawed at 20-25 °C and then combined with 20 mM calcium chloride dihydrate extraction buffer at a relative ratio of 1 kg of fish viscera to 2 L of calcium chloride buffer. The resulting mixture was adjusted to a final pH of 7 by adding 50% w / v sodium hydroxide solution and stirring at 4 °C for 8-12 hours.

[0031] Large viscera chunks were separated by filtering through a net (1 mm cut-off).

[0032] The filter aid was added to the obtained mixture while stirring at 20 - 25°C, and then pressure filtration was performed.

[0033] Suitable filter aids are diatomaceous earth, perlite, and cellulose added in an amount containing 3 - 7% by weight relative to the volume of the mixture. Preferably, diatomaceous earth is used in an amount of 5% w / v relative to the mixture.

[0034] The filtered extract was concentrated by ultrafiltration (1 kDa cut-off) to 42% of the initial volume and stored at 4°C if not used immediately in the next purification process. The concentrated fish extract was warmed to 20 - 25°C, calcium chloride dihydrate was added to reach a final concentration of 1.0 - 1.8 w / v, preferably 1.4% w / v. Sodium acetate was added to reach a final concentration of 1.0 - 2.0 M, preferably 1.5 M, and the pH was adjusted to the range of 7.8 - 8.2, preferably 8.0, using a 5 M aqueous sodium hydroxide solution.

[0035] When the conductivity of the mixture is in the range of 54 - 62 mS, preferably 56 - 60 mS, the exact concentration of the salt can be obtained.

[0036] After stirring this mixture at 20 - 25°C for 1 hour, a filter aid was added and the resulting suspension was filtered. Suitable filter aids are diatomaceous earth, perlite, and cellulose added in an amount containing 1 - 4% by weight relative to the volume of the mixture. Preferably, diatomaceous earth is used in an amount of 2% w / v relative to the mixture.

[0037] Then, the resulting suspension was filtered by a filter press equipped with a suitable depth filter. The operating pressure used for this filtration should include 50 - 70 psi, preferably 60 psi, and the suitable depth filter to be used should have a cut-off included in 6 - 9 microns. The preferred depth filter is a cellulose filter sheet or a rigid media filter containing a porous metal, ceramic, or plastic media. Preferably, the XE-400 filter sheet (Carlson filtration) is used.

[0038] Subsequently, the obtained filtrate was purified by hydrophobic interaction chromatography (HIC). The ratio of the volume of the feed material to the volume of the stationary phase used is included in 4 to 8 volumes / volume, preferably 6.7. The preferred particle size of the stationary phase is included in 50 to 100 microns, preferably 75 microns. The stationary phase provides an agarose-based matrix having various immobilized ligands, for example, linear alkyl ligands or aryl ligands. The preferred ligand is an aryl ligand. Before use, the stationary phase is washed with at least 3 bed volumes of a 0.1 M aqueous sodium hydroxide solution and then with 3 bed volumes of distilled water. Subsequently, the column is adjusted with a solution prepared at a pH value included in 7.8 to 8.2, preferably 8.0, obtained by the addition of a 5 M sodium hydroxide solution, using an aqueous sodium acetate solution at a concentration included in 1.0 to 2.0 M, preferably 1.5 M, at 4 BV. This solution and the eluent are supplied at a flow rate of 1 / 10 ml / min and a pressure included in 10 to 20 psi, preferably 15 psi, with respect to the total volume of the stationary phase. After absorption, the bound solute is eluted by stepwise or gradient elution with a buffer having a low salt content and then with an aqueous mixture of a water-miscible organic solvent and a polyol. Suitable water-miscible organic solvents have a log P (hydrophobicity) included in -0.31 to +0.25 and include, for example, n-propanol, isopropanol, and ethanol, preferably isopropanol. Suitable polyols include glycerol, ethylene glycol, ethylene glycol, and sorbitol, preferably glycerol.

[0039] Dissolution is preferably carried out under the following conditions: 2 bed volumes of 1.5 M sodium acetate (pH 8), then 3 bed volumes of 10% v / v cellosolve solution and 5% v / v isopropanol solution (diluted with distilled water). The eluate was collected in about 1 bed volume of four fractions each. The elution fractions having trypsin activity were pooled together and concentrated to 1 / 10 of the starting volume by ultrafiltration (using an ultrafiltration membrane with a 1 kDa cut-off). The concentrated solution was then diluted 1 / 2.2 while stirring with 20 mM aqueous CaCl2 (pH 8), dialyzed back to the original volume, and then lyophilized to obtain purified fish protease. 33 - 60 g of purified fish protease having an average trypsin enzyme activity of 240 U / mg, chymotrypsin activity of 5 U / mg and collagenase activity of 0.04 U / mg was recovered from 52 Kg of cod viscera. Optionally, the method of the present invention can be stopped after the dialysis step (i.e., avoiding the last lyophilization step) to obtain fish protease in an aqueous solution useful for the preparation of an enzyme liquid formulation having a defined enzyme activity.

[0040] The consistency of the method was checked using different batches of cod viscera caught at different times of the year as starting materials. The obtained fish protease was confirmed to have limited variability between batches. This variability does not exceed a range of ±15% with respect to the obtained enzyme activity.

[0041] The present invention will be described in more detail in the following examples.

[0042] Example 1 Extraction of crude enzyme 52 kg of fish viscera was thawed overnight at room temperature and then combined with 103 L of 20 mM calcium chloride dihydrate extraction buffer at a relative ratio of 1 kg of fish viscera: 2 L of calcium chloride buffer. The resulting mixture (about 150 L) was adjusted to a final pH value of 7 by addition of 50% w / v aqueous sodium hydroxide solution and stirred overnight at 4°C.

[0043] The large visceral mass was separated by filtration through a net, leaving 130 L in the tank. To the resulting mixture, 6.5 kg of diatomaceous earth (5% w / v) was added while stirring at room temperature, and then it was pressure-filtered through 14 XE-400 filter sheets (7 cassettes). The filtered extract (100 L) was concentrated to 42 L using a 1×1 ultrafiltration spiral membrane (1 kDa cut-off). The membrane was washed and then concentrated with 100 L of 0.1 M aqueous sodium hydroxide solution, followed by reverse osmosis against water.

[0044] 2 L of the concentrated fish extract was taken out for lyophilization, and the remaining extract was stored at 4 °C until purification.

[0045] Example 2 Hydrophobic interaction chromatography (HIC) purification All purification steps were carried out at 20 - 25 °C. The concentrated fish extract was warmed to 20 - 25 °C, and 58.82 g of calcium chloride dihydrate was added. 4.93 kg of sodium acetate was added to obtain a concentration of 1.5 M. The pH was adjusted to 8 using 5 M aqueous sodium hydroxide solution.

[0046] The conductivity was checked to confirm that the correct concentration was reached (adjusted to 56 - 60 mS and was 57.4 mS). After stirring for 1 hour, 0.8 kg of diatomaceous earth (2% w / v) was added, and the resulting suspension was clarified by pressure-filtering through 4 XE-400 filter sheets (1 cassette). 40 L of feed material was obtained.

[0047] 6 L of Capto-phenl high sub column resin was conditioned with 20 L of 0.1 M aqueous sodium hydroxide solution, followed by 20 L of reverse osmosis water.

[0048] The column pressure was maintained at 15 psi and the flow rate was maintained at 417 mL / min. The column was equilibrated using 40 L of 1.5 M sodium acetate (pH 8). The eluate was also checked for conductivity (predicted value 62 mS).

[0049] The main feed was added to the column, washed with 15 L of equilibration buffer, and then eluted with 20 L of a solution of 10% v / v glycerol and 5% v / v isopropanol (diluted with reverse osmosis water). The eluate was collected into four fractions of 5 L each, and the fractions with trypsin activity were pooled (15 L was collected).

[0050] Example 3 Eluate concentration, dialysis, lyophilization Concentration and diafiltration were carried out using a crossflow ultrafiltration unit (Pall Filtron, USA) with a 1 kDa membrane. The selected eluate was concentrated to a final volume of 1.7 L. To this solution, 1 L of 20 mM CaCl2 aqueous solution (pH 8) was added with stirring.

[0051] Example 3 Eluate concentration, dialysis, lyophilization Concentration and diafiltration were carried out using a crossflow ultrafiltration unit (Pall Filtron, USA) with a 1 kDa membrane. The selected eluate was concentrated to a final volume of 1.7 L. To this solution, 1 L of 20 mM CaCl2 aqueous solution (pH 8) was added with stirring.

[0052] The resulting solution had a solids content of 4.3%. Further, 1 L of CaCl2 solution was added, and the resulting solution showed a solids content of 4.5%. The volume was reduced to 1.75 L by dialysis, and then the concentrated and diafiltered eluate was lyophilized to give 33 g of powder. The specific enzyme activity of this powder was 240 U / mg trypsin, 5 U / mg chymotrypsin, and 0.04 U / mg collagenase. The isolated enzyme showed multiple bands on SDS-PAGE. The major ones are indicated by arrows in Figure 1. The estimated molecular weight was 25,000.

[0053] Optionally, the production process of fish protease can be stopped after the dialysis step (i.e., the final lyophilization step is avoided) in order to obtain fish protease in an aqueous solution of the desired concentration.

[0054] These solutions can be used for the preparation of the final enzyme formulation in a phosphate buffered saline solution (PBS solution) containing potassium chloride, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, tetrasodium ethylenediaminetetraacetate and phenol red at a pH value of 7.2 to 7.6.

[0055] These enzyme formulations can contain potassium chloride and potassium dihydrogen phosphate at 0.1 - 0.3 g / L, NaCl at 7 - 9 g / L, disodium hydrogen phosphate at 1.0 - 1.3 g / L, phenol red at 2.4 mg / L and sodium ethylenediaminetetraacetate in the range of 0.3 - 0.6 mM, and include Dulbecco's phosphate buffered saline (Dulbecco, R et al. J. Exp. Med., 99, 167 - 182 (1954)).

[0056] Example 4 Analysis characteristics of fish trypsin purified according to the present invention The characteristics of the fish trypsin isolated according to the present invention are reported in the following table and figures.

[0057] Temperature range optimal for enzyme activity: Tables 1 and 2 show the enzyme activity of the prepared fish protease in the temperature range included between 5 and 70 °C. The main trypsin activity was tested.

[0058] [Table 1]

[0059] pH range optimal for enzyme activity: Tables 2 and 3 show the enzyme activity of the prepared fish trypsin in the pH range included between 3 and 12. The main trypsin activity was tested.

[0060] [Table 2]

[0061] Time-dependent stability data on enzyme activity at various pH values: Table 3 and Figure 4 show the enzyme activity of the prepared fish trypsin in the pH range of 3 - 10 at a temperature of 5°C for 3 hours. The same data in Table 3 are shown in graph form as relative enzyme activity (with the enzyme activity at time zero taken as 100%; Figure 4). The main trypsin activity was tested.

[0062]

Table 3

[0063] Cell culture: Biological “in vivo” tests Comparative in vitro biological tests in PC12 cells, human glioma, and human astrocytes were performed using fish proteases prepared according to the method of the present invention, a compound with registration number 534583 - 22 - 7, and mammalian trypsin having a trypsin activity of about 3000 U / ml in a 0.25 w / v% trypsin - 1 mM EDTA / 4Na solution (containing phenol red) (Wako, Japan 209 - 16941). The fish proteases prepared according to the present invention recovered more PC12 cells than trypsin and the compound with registration number 534583 - 22 - 7 (Table 4). Figure 5 shows optical microscope observations of PC12 cells before and after treatment with fish proteases prepared according to the claimed method, Accutase® and mammalian trypsin.

[0064]

Table 4

[0065] The survival rates obtained with fish proteases prepared according to the claimed method are comparable to the survival rates obtainable with the compound with CAS registration number 534583 - 22 - 7 and trypsin, depending on the cell type. The best results for these fish proteases were obtained with PC12 cells (Table 5).

[0066]

Table 5

[0067] To compare the fish protease obtained by the method of the present invention with mammalian trypsin having a trypsin activity of about 500 - 600 U / ml (0.05% trypsin / 0.53 mM EDTA 10X in HBSS 1X, sterile (Wisent Bioproducts, Quebec, Canada) and the compound with CAS registration number 534583 - 22 - 7, further comparative studies were conducted using the following cell lines: iPSC (induced pluripotent stem cells), NPC (neural progenitor cells), and U2OS (human osteosarcoma epithelial cells). In particular, attention was paid to the characteristics of cell attachment and viability as well as the final cell morphology.

[0068] For this purpose, 5,000 cells / well in a 12 - well plate were cultured for 5 days. When they reached 70% confluence, the experiment was started. Then, the medium was aspirated, rinsed with PBS, 0.5 mL of dissociating agent was added, and incubated at 37°C for 3 minutes until dissociation (monitored under a microscope until the optimal dissociation time was determined).

[0069] [Table 6]

[0070] DMEM (Dulbecco's Modified Eagle Medium) was added to stop the enzymatic reaction, and then the cells were recovered by centrifugation at 1200 rpm for 3 minutes. The cells were resuspended in the culture medium, and the cell count was determined by using a lunar automatic cell counter (Table 7). Treatment with the fish protease of the present invention for iPSC and NPC did not affect cell attachment and viability as observed with trypsin.

[0071] [Table 7]

[0072] The cells were reseeded onto 12-well plates containing the medium and observed under an optical microscope one week later (Figure 6). Based on this observation, it was confirmed that the cells treated with the fish protease of the present invention did not change their morphology and appeared normal.

[0073] The purified fish protease prepared according to the method of the present invention exhibits a specific enzyme profile characterized by trypsin activity of about 60 - 80% of the total enzyme activity. The collagenase activity can be substantially ignored (Table 8).

[0074]

Table 8

Claims

Claim 1 A method for purifying tar protease from the internal organs of tar, comprising: The protease has an average specific enzyme activity of 240 U / mg ± 40 U / mg of trypsin, a chymotrypsin activity of 4 ± 2 U / mg, a collagenase activity of 0.04 ± 0.02 U / mg, and a protease activity of 65 ± 10 U / mg. The average specific enzyme activity of the trypsin, the chymotrypsin activity, and the protease activity are determined by the method described in Article 41 of the United States Pharmacopeia. The collagenase activity is determined by the method described in Mandl, I. J. Clin. Invest. 32, 1323. 1953, or Moore, S. et al., J. Biol. Chem. 176, 367. 1948. a) Extracting crude enzyme from the internal organs of tar using a calcium chloride buffer (pH 7) at a concentration of 20 mM, filtering, and ultrafiltrating; b) Extracting the ultrafiltrate with an aqueous CaCl solution having a conductivity of 52 to 62 mS in a pH range of 7.8 to 8.2, and subsequently performing depth filtration; ２ ​ c) Using an agarose-based matrix with a linear alkyl ligand or an aryl ligand as the stationary phase, eluting with a 1.5 M aqueous sodium acetate solution, and then purifying the filtrate by hydrophobic interaction chromatography eluting with an aqueous mixture of isopropanol and glycerol; d) Dialyzing; A method comprising: Claim 2 The method according to claim 1, wherein step a) is carried out at a temperature of 4 to 25°C. Claim 3 The method according to claim 1, wherein the ultrafiltration in step a) is carried out using a membrane having a cut-off of 1 kDa. Claim 4 The method according to claim 1, wherein the agarose-based matrix of the stationary phase used in step c) exhibits an aryl ligand and a particle size distribution of 50 to 100 microns. Claim 5 The method according to claim 1, wherein step c) is carried out at a temperature of 4 to 25°C. Claim 6 The method according to claim 1, further comprising: e) lyophilizing. Claim 7 The method according to claim 1, wherein the tar protease is isolated directly into an aqueous solution from step d) at a desired final concentration. **Claim 8**: A protease having an estimated molecular weight of 25,000, an average specific enzymatic activity of trypsin of 240 U / mg ± 40 U / mg, a chymotrypsin activity of 4 ± 2 U / mg, a collagenase activity of 0.04 ± 0.02 U / mg, and a protease activity of 65 ± 10 U / mg, wherein the average specific enzymatic activity of the trypsin, the chymotrypsin activity, and the protease activity are determined by the method described in the 41st edition of the United States Pharmacopeia, and the collagenase activity is determined by the method described in Mandl, I. J. Clin. Invest. 32, 1323. 1953 or Moore, S. et al., J. Biol. Chem. 176, 367. 1948. Tharaprotease. **Claim 9** A formulation comprising the tharaprotease according to claim 8, 0.1 - 0.3 g / L potassium chloride and potassium dihydrogen phosphate, 7 - 9 g / L NaCl, 1.0 - 1.3 g / L disodium hydrogen phosphate, 2.4 mg / L phenol red, and sodium ethylenediaminetetraacetate at a final concentration in the range of 0.3 - 0.6 mM. **Claim 10** Use of the tharaprotease according to claim 8 for food, biomedical applications (excluding methods of treating humans), histology, and tissue culture. **Claim 11** Use of the tharaprotease according to claim 8 in histology and in the tissue culture of PC12 and NPC cell lines.

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