PURIFIED FISH PROTEASES WITH HIGH SPECIFIC ACTIVITIES AND ITS PROCESS OF PRODUCTION

Abstract

The invention concerns a process for the preparation of fish proteases from fish viscera, preferably from cod (Gadus genus) viscera. The fish proteases produced according to the invention have high specific enzymatic activity and are useful for food uses, for biomedical applications, in histology and tissue culture.

Claims

1. A process of purification of fish proteases from fish viscera, said proteases having an average specific enzymatic activity of Trypsin of 240 U/mg±40 U/mg, Chymotrypsin activity of 4±2 U/mg, Collagenase activity of 0.04±0.02 U/mg and a Protease activity of 65±10 U/mg, said method comprising: a) extracting crude enzyme from fish viscera with a calcium chloride buffer at pH 7, filtrating and ultrafiltrating to obtain a ultrafiltrate; b) extracting the ultrafiltrate with an aqueous solution of CaCl2 having conductivity from 52 to 62 mS in a pH range of 7.8÷8.2 followed by depth filtration to obtain a filtrate; c) purifying by hydrophobic interaction chromatography the filtrate obtained in step b) using as stationary phases an agarose base matrix with straight chain alkyl ligands or aryl ligands and eluting by elution with buffers of low salt content and then with an aqueous mixture of water miscible organic solvents and a polyol; d) dialyzing; e) Optional freeze-drying.

2. A process according to claim 1 wherein the fish viscera are cod viscera.

3. A process according to the claim 1 wherein step a) is carried out at a temperature between 4 and 25° C.

4. A process according to claim 1 wherein the calcium ions of step a) are derived from calcium chloride at a final concentration of 20 mM.

5. A process according to claim 1 wherein the ultrafiltrating step a) is carried out using a membrane with a 1 kDalton cut off.

6. A process according to claim 1 wherein the agarose base matrix of the stationary phase used in step c) presents aryl ligands and a particle size distribution between 50 and 100 microns.

7. A process according to claim 1 wherein the chromatographic elution with buffers of low salt content in step b) is done using a 1.5 M aqueous sodium acetate solution.

8. A process according to claim 1 wherein the water miscible organic solvent utilized in the chromatographic elution in step c) is isopropanol.

9. A process according to claim 1 wherein the polyol utilized as component of the chromatographic elution in step c) is glycerol.

10. A process according to the claim 1 wherein step c) is carried out at a temperature between 4 and 25° C.

11. A process according to claim 1 wherein step e) is not carried out and the fish proteases are isolated in an aqueous solution directly from the step d) at the desired final concentration.

12. Fish proteases with an average specific of Trypsin activity of 240±40 U/mg, Chymotrypsin activity of 4±2 U/mg, Collagenase activity of 0.04±0.02 U/mg and a Protease activity of 65±10 U/mg obtained by the process of claim 1.

13. Formulations comprising the fish proteases of claim 12, 0.1-0.3 g/L of potassium chloride and potassium dihydrogen phosphate, 7-9 g/L of NaCl, 1.0-1.3 g/L of disodium monohydrogen phosphate, 2.4 mg/L of phenol red and sodium ethylenediaminetetraacetate at a final concentration ranging between 0.3 and 0.6 mM.

14. (canceled)

15. Method of treating tissue cultures of PC12 and neuronal progenitor (NPC) cell lines with the fish proteases according to claim 12, said method comprising; treating tissue cultures of PC12 and NPC with said fish proteases; recovering said cells and replating same.

Description

DETAILED DESCRIPTION: PROCESS

[0036] Frozen (−20° C.) cod viscera were thawed at 20-25° C., then combined with 20 mM calcium chloride dihydrate extraction buffer in a relative ratio of 1 kg of fish viscera for 2 L calcium chloride buffer. The resulting mixture was adjusted to a final pH 7 by addition of sodium hydroxide 50% w/v water solution of and stirred for 8-12 hours at 4° C.

[0037] Large viscera chunks were separated by filtration with a net (1 mm cut-off).

[0038] A filter aid was added to the obtained mixture, under stirring at 20-25° C., which was then filter pressed.

[0039] Suitable filter aids are diatomaceous earth, perlite, cellulose added in a amounts comprised between 3 and 7% by weight with respect to the volume of the mixture. Preferably diatomaceous earth is used in an amount of 5% w/v with respect to the mixture.

[0040] The filtered extract was concentrated by ultrafiltration (1 kDa cut off) to 42% of the initial volume and stored at 4° C. if not immediately used in the next purification process. The concentrated fish extract was warmed to 20-25° C. and calcium chloride dihydrate was added to reach a final concentration comprised between 1.0 and 1.8 w/v, preferably 1.4% w/v. Sodium acetate was added to reach a final concentration between 1.0 and 2.0 M, preferably 1.5 M, and the pH adjusted in a range between 7.8 and 8.2, preferably 8.0, using 5 M sodium hydroxide water solution.

[0041] The correct concentration of the salts is obtained, when the conductivity of the mixture is comprised in a range between 54 and 62 mS, preferably between 56 and 60 mS.

[0042] After stirring the mixture for one hour at 20-25° C., a filter aid was added and the obtained suspension filtered. Suitable filter aids are diatomaceous earth, perlite, cellulose added in an amount comprised between 1 and 4% by with weight respect to the volume of the mixture. Preferably diatomaceous earth is used in an amount of 2% w/v with respect to the mixture.

[0043] The obtained suspension is then filtered using a filter press equipped with suitable depth filters. The operative pressure used for this filtration is comprised between 50 and 70 psi, preferably 60 psi, and the suitable depth filters employed should have a cut off comprised between 6 and 9 microns. Preferred depth filters are cellulose filters sheets or rigid media filter contains porous metal, ceramic or plastic media. Preferably XE-400 filter sheets are used (Carlson filtration).

[0044] Then the obtained filtrate was purified by hydrophobic interaction chromatography (HIC): the ratio of the volume of the feed material to the volume of the used stationary phase is comprised between 4 and 8 volume/volume, preferably 6,7. The preferred particle size of the stationary phase is comprised between 50 and 100 microns, preferably 75 microns. The stationary phases present an agarose base matrix with different immobilized ligands, such as straight chain alkyl ligands or aryl ligands. Preferred ligands are aryl ligands. The stationary phase prior use is washed with at least 3 bed volumes of 0.1 M sodium hydroxide aqueous solution and then with 3 bed volumes of distilled water. Then the column was conditioned with 4 BV with a solution prepared using a sodium acetate aqueous solution at a concentration comprised between 1.0 and 2.0 M, preferably 1.5 M, at a pH value comprised between 7.8 and 8.2, preferably 8.0, obtained by addition of a 5 M sodium hydroxide solution. The solution and the eluates are fed at 1/10 ml/minute flow rate with respect to the total volume of the stationary phase and at a pressure comprised between 10 and 20 psi, preferably 15 psi. After the absorption, the bound solutes are eluted by stepwise or gradient elution with buffers with low salt content and then with an aqueous mixture of water miscible organic solvents and a polyol. Suitable water miscible organic solvents have a log P (hydrophobicity) comprised between −0.31 and +0.25, such as n-propanol, isopropanol and ethanol, preferably isopropanol. Suitable polyols are glycerol, ethylene glycol, ethylene glycol and sorbitol, preferably glycerol.

[0045] The elution is preferably effected under the following conditions: 2 bed volumes of 1.5 M sodium acetate at pH 8, then elution with 3 bed volumes of a solution of 10% v/v glycerol and 5% v/v isopropanol (diluted with distilled water). Eluates were collected in 4 fractions of about 1 bed volume each. The eluted fractions with trypsin activity were pooled together, concentrated to 1/10 of the starting volume by ultrafiltration (using an ultrafiltration membrane with a cut off of 1 kDa). Then the concentrated solution was diluted under stirring to 1/2.2 with a 20 mM CaCl.sub.2 aqueous solution at pH 8, dialyzed to restore the original volume then freeze dried to afford a purified fish protease. 33-60 g of purified fish proteases with an average Trypsin enzymatic activity of 240 U/mg, Chymotrypsin activity of 5 U/mg and a collagenase activity of 0.04 U/mg were recovered from 52 Kg of cod viscera. Optionally, the process of the invention can be stopped after the dialysis step (i.e. avoiding the final freeze drying step) to obtain fish proteases in aqueous solution useful for the preparation of enzymatic liquid formulations having a defined enzymatic activity.

[0046] The consistency of the process was checked using different batches of cod viscera caught at different times of the year as starting material: the obtained fish proteases were confirmed to have a limited variability from batch to batch; this variability does not exceed the range of ±15% with respect to the obtained enzymatic activities.

[0047] The invention is illustrated in more detail in the following examples.

Example 1

[0048] Extraction of Crude Enzyme

[0049] 52 kg of fish viscera was thawed overnight at room temperature, then combined with 103 L of a 20 mM calcium chloride dihydrate extraction buffer in a relative ratio of 1 kg of fish viscera:2 L calcium chloride buffer. The resulting mixture (about 150 L) was adjusted to a final pH 7 value by addition of a sodium hydroxide water solution 50% w/v and stirred overnight at 4° C.

[0050] Large viscera chunks were separated by filtration with a net, leaving 130 L in the tank. 6.5 kg of diatomaceous earth (5% w/v) was added to the obtained mixture under stirring at room temperature, then filter pressed 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 cleaned prior to concentrating with 100 L 0.1M sodium hydroxide water solution followed by reverse osmosis against water.

[0051] 2 L of concentrated fish extract was removed for freeze drying, and the rest of the extract was stored at 4° C. until purification.

Example 2

[0052] Hydrophobic Interaction Chromatography (HIC) Purification

[0053] All purification steps were performed at 20-25° C. The concentrated fish extract was warmed to 20-25° C., and 58.82 g calcium chloride dihydrate was added. 4.93 kg of sodium acetate was added to obtain a 1.5 M concentration and the pH was adjusted to 8 using 5 M sodium hydroxide water solution.

[0054] The conductivity was checked to ensure that the correct concentration had been reached (adjusted to 56-60 mS; was 57.4 mS). After stirring for one hour, 0.8 kg of diatomaceous earth was added (2% w/v) and the obtained suspension was clarified by filter pressing through 4 XE-400 filter sheets (1 cassette). 40 L of feed material was obtained.

[0055] 6 L of Capto-phenyl high sub column resin was conditioned with 20 L of 0.1M sodium hydroxide aqueous solution followed by 20 L reverse osmosis water.

[0056] Column pressure was maintained at 15 psi and a flow rate of 417 mL/minute. 40 L of 1.5 M sodium acetate at pH 8 was used to equilibrate the column. The eluates were also checked for conductivity (expected value 62 mS).

[0057] Primed feed was added to the column, washed with 15 L of equilibration buffer, then eluted with 20 L of a solution of 10% v/v glycerol and 5% v/v isopropanol (diluted with reverse osmosis water). Eluate was collected in 4 fractions, 5 L each, and the fractions with trypsin activity were pooled (15 L collected).

Example 3

[0058] Eluate Concentration, Dialysis, Freeze Drying

[0059] Concentrations and diafiltrations were performed using a cross tangential ultrafiltration unit with a 1 kDa membrane (Pall Filtron, USA). The selected eluates were concentrated to a final volume of 1.7 L. This solution was added with 1 L of 20 mM CaCl.sub.2 water solution at pH 8 under stirring.

[0060] The resulting solution had 4.3% solids. Additional 1 L of CaCl.sub.2 solution was added and the resulting solution showed 4.5% solids. The volume was reduced to 1.75 L by dialysis, then the concentrated and diafiltered eluate was freeze dried to afford 33 g of powder. The specific enzymatic activity of this powder was: Trypsin 240 U/mg, Chymotrypsin 5 U/mg and Collagenase 0.04 U/mg. The isolated enzyme exhibited several bands on SDS-PAGE: the main one, indicated with an arrow in FIG. 1, with an estimated molecular weight of 25,000.

[0061] Optionally the production process of fish proteases can be stopped after the dialysis step (i.e. avoiding the final freeze-drying step) to obtain fish proteases in aqueous solution at the desired concentration.

[0062] These solutions can be used for the preparation of final enzymatic formulations in phosphate-buffered saline solutions (PBS solutions) containing potassium chloride, potassium dihydrogen phosphate, sodium chloride, disodium monohydrogen phosphate, tetrasodium ethylenediaminetetraacetate and phenol red at a pH value comprise between 7.2 and 7.6.

[0063] These enzymatic formulations may contain 0.1-0.3 g/L of potassium chloride and potassium dihydrogen phosphate, 7-9 g/L of NaCl, 1.0-1.3 g/L of disodium monohydrogen phosphate, 2.4 mg/L of phenol red and sodium ethylenediaminetetraacetate at a final concentration ranging between 0.3 and 0.6 mM and include the Dulbecco's phosphate-buffered saline solution (Dulbecco, R et al. J. Exp. Med., 99, 167-182 (1954)).

Example 4

[0064] Analytical characteristics of fish trypsin purified according to the invention

[0065] The characteristics of the fish trypsin isolated according to the invention are reported in the following Tables and in Figures.

[0066] Optimum temperature range for enzymatic activity: Table 1 and FIG. 2 show the enzymatic activity of the prepared fish protease in the temperature range comprised between 5 and 70° C. Tested the main trypsin activity.

TABLE-US-00001 TABLE 1 Temp. (deg. C.) Trypsin Activity (U/mg) 5 40 10 56 15 81 20 115 30 225 40 390 50 559 60 73 70 33

[0067] Optimum pH range for enzymatic activity: Table 2 and FIG. 3 show the enzymatic activity of the prepared fish trypsin in the pH range comprised between 3 and 12. The main trypsin activity has been tested.

TABLE-US-00002 TABLE 2 pH Trypsin Activity (U/mg) 3 0 4 3 5 15 6 83 7 147 8 173 9 160 10 0 11 1 12 2

[0068] Stability data for the enzymatic activity over time at different pH values: Table 3 and FIG. 4 show the enzymatic activity of the prepared fish trypsin in the pH range of 3-10 in 3 hours at the temperature of 5° C. The same data of the Table 3 are presented in graphic form as relative enzymatic activity (100% of enzymatic activity at time zero; FIG. 4). The main trypsin activity has been tested.

TABLE-US-00003 TABLE 3 trypsin activity (U/mg) time (minutes) pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 0 168 205 205 169 136 161 161 168 30 13 171 128 156 140 155 155 133 60 13 157 107 157 157 151 147 128 90 20 157 100 153 144 155 145 116 120 14 148 96 148 137 151 135 107 150 17 171 95 147 135 149 143 110 180 19 165 93 141 167 151 140 110

[0069] Cell culture: biological “in vivo” tests

[0070] Comparative in vitro biological tests in PC12 cells, Human Glioma and Human Astrocyte were carried out using fish proteases prepared according to the process of the invention, compound with registry number 534583-22-7 and mammalian trypsin having approximately 3000 U/ml trypsin activity 0.25 w/v % Trypsin-1 mM EDTA/4Na Solution with Phenol Red (Wako, Japan 209-16941). Fish proteases prepared according to the invention recovered more PC12 cells than trypsin and the compound with registry number 534583-22-7 (Table 4). FIG. 5 shows the optical microscope observation of PC12 cell before and after treatment with Fish proteases prepared according to the claimed process, Accutase® and mammalian trypsin.

TABLE-US-00004 TABLE 4 Survival rate (%) of PC12 cells after treatment with treatment with Fish proteases prepared according to the claimed process, compound with registry number 534583-22-7 and Mammalian Trypsin. PC12 cells Number of recovered cells Ratio to Survival rate Enzyme [×10{circumflex over ( )}5 cells/well] Trypsin [%] Fish proteases 1.325 1.1 98.8 Compound with registry 0.783 0.64 93.3 number 534583-22-7 Mammalian Trypsin 1.22 1 97.3

[0071] The survival rate obtained with fish proteases prepared according to the claimed process is comparable to that obtainable with the compound with CAS registry number 534583-22-7 and to trypsin, depending on cell type; the best results with these fish proteases were obtained on PC12 cells (Table 5).

TABLE-US-00005 TABLE 5 Survival rate [%] PC12 cells Human Glioma Human Astrocytes Fish proteases 98.8 78.0 90.0 Accutase ® 93.3 83.4 91.3 Mammalian Trypsin 97.3 98.1 97.0

[0072] Further comparatives studies were carried out using the following cell lines: iPSC (induced pluripotent stem cells), NPC (neuronal progenitor cells) and U2OS (human bone osteosarcoma epithelial cells) in order to compare fish proteases obtained by process of the invention in comparison with mammalian trypsin having approximately 500-600 U/ml trypsin activity (Trypsin 0.05%/EDTA 0.53 mM 10× IN HBSS 1×, STERILE (Wisent Bioproducts, Quebec, Canada) and with the compound with CAS registry number 534583-22-7, with particular attention to the characteristics of cell attachment and viability and the final cell morphology.

[0073] For this purpose, 5,000 cells/well in 12 well plate were cultured for 5 days, starting the experiment when the it reached 70% confluency; then the medium was aspirated, rinsed with PBS and 0.5 mL of dissociation reagent was added and incubated at 37° C. for 3 minutes until dissociation (dissociation was monitored under the microscope until the optimal time was determined).

TABLE-US-00006 TABLE 6 Optimized incubation time of different enzymes for efficient dissociation on different cell lines Incubation time Incubation time Incubation time with with with compound registry number Cell lines Fish proteases Trypsin 534583-22-7 iPSC 3 minutes 3 minutes 3 minutes NPC 3 minutes 3 minutes 3 minutes U2OS 14 minutes  3 minutes 3 minutes

[0074] DMEM (Dulbecco's Modified Eagle Medium) was added to stop the enzyme reaction, then the cells were recovered by centrifugation at 1200 rpm for 3 min. The cells were re-suspended the in-culture medium; the cells number was determined by using lunar automated cell counter (Table 7). Treatment with fish proteases of the invention on iPSCs and NPCs did not affect cell attachment and viability as observed with trypsin.

TABLE-US-00007 TABLE 7 Cell numbers and viability of different cell lines after the enzymatic treatments Compound registry Cell Fish Mammalian number lines test proteases Trypsin 534583-22-7 iPSC Cell number  0.5 × 10.sup.−5 1.1 × 10.sup.−5 2.95 × 10.sup.−5 Viability 89.63%  81.4% 92.06%  NPC Cell number 0.52 × 10.sup.−5 0.49 × 10.sup.−5  0.39 × 10.sup.−5 Viability 89.9% 86.9% 58.1% U2OS Cell number 3.52 × 10.sup.−5 2.1 × 10.sup.−5 4.55 × 10.sup.−5 Viability 95.1%  100% 96.8%

[0075] The cells were re-plated onto 12 well containing the medium and observed by optical microscopy after one week (FIG. 6). On the basis of this observations we confirmed that the cells treated with fish proteases of the invention did not change morphology and appeared healthy.

[0076] The purified fish proteases prepared according to the process of the invention present a peculiar enzymatic profile characterized by a trypsin activity of about 60-80% of the total enzymatic activity, collagenase activity being substantially negligible (Table 8).

TABLE-US-00008 TABLE 8 % of enzymatic activities of fish proteases obtained according to the process of the invention versus other commercially available proteases. Fish proteases solution Compound with registry Accutase TM XL after diafiltration and number 534583-22-7 AccuMax TM powder freeze drying (% of enzymatic activity) (% of enzymatic activity) (% of enzymatic activity) (% of enzymatic activity) Trypsin activity 29 49 26 78.0 Chymotrypsin activity 0 0 0 1.6 Collagenase type I activity 59 47 67 0.01 Protease activity 12 5 7 21.0