Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/104—Aminoacyltransferases (2.3.2)
  • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y203/00—Acyltransferases (2.3)
  • C12Y203/02—Aminoacyltransferases (2.3.2)
  • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Definitions

• the present invention relates to an activated Factor XIII product with improved properties, a composition comprising the 5 activated Factor XIII, and a process for producing activated Factor XIII.
• Factor XIII also known as plasma transglutaminase is one of the components of the blood coagulation system, and circulates
• Activated Factor XIII catalyses the crosslinking of fibrin polymers by introducing covalent bonds between non-covalent fibrin polymers. More specifically, activated Factor XIII catalyses the formation of
• Factor XIII circulates as a tetrameric complex consisting of two a subunits (Mr of about 83 kD) containing the catalytic site of the enzyme and two b subunits (Mr of about 80 kD) (S.I. Chung et al., J. Biol. Chem. 249. 1974, pp. 940-950).
• the b subunits are cleaved off.
• a 4 kD fragment is cleaved off the N-terminal end of each of the a subunits (Schwartz et al., J. Biol. Chem. 248. 1973, pp. 1395-1407).
• the potential catalytic site is located in the a chain with cysteine at the active centre.
• Factor XIII Due to its function in the coagulation process, Factor XIII has been used for treating patients with postoperative wound healing disorders (Mishima et al., Chirur ⁇ . 55. 1984, pp. 803- 808) and scleroderma (Delbarre et al., Lancet 2., 1981, p.204) . Furthermore, Factor XIII has been used as a component of tissue 10 adhesives (US 4,414,976; US 4,453,939; US 4,377,572; US 4,362,567; US 4,298,598) and has been suggested for use in antifibrinolytic therapy for the prevention of postoperative bleeding and in the treatment of subarachnoid haemorrhage, ulcerative colitis and general wound healing.
• transglutaminase has been added to minced meat and fish paste (cf. for instance JP 2-255060 to Ajinomoto, JP 2-
• Transglutaminase has been added to gelatin to make highly polymerised gelatin products (cf. JP 2-86743 to Ajinomoto) .
• the present invention relates to activated stable Factor XIII.
• stable refers to the storage stability of the activated Factor XIII and is intended to indicate that the activated Factor XIII preparation retains at least 60% of its initial activity after about 3 months.
• the present invention relates to a composition
• a composition comprising activated stable Factor XIII in freeze- dried form or in the form of a frozen liquid concentrate.
• the invention relates to a method of producing a processed meat product with improved water-binding and consistency properties, the method comprising mixing the composition of the invention with a meat material and incubating the mixture for a period of time sufficient to let the activated Factor XIII react with proteins present in the meat material.
• this method may be used in the preparation of restructured meat products, e.g. processed ham, containing finely diced meat, or emulsified meat products such as sausages or chopped beef or pork, optionally together with soy protein.
• the Factor XIII composition may be added to the meat material before, during or after dicing or blending. After incubation, the mixture may be put into appropriate containers such as sausage casings or tins and boiled.
• the invention also relates to a method of producing a fish paste product with improved consistency properties, the method comprising mixing composition of the invention with a fish meat material and incubating the mixture for a period of time sufficient to let the activated Factor XIII react with proteins present in the fish meat material.
• the Factor XIII composition of the invention may be used for the production of sausage casings by crosslinking of collagen, for making gelatin gels, in cheese- making for improving the yield of cheese by crosslinking soluble whey proteins, in baking for strengthening gluten, and in the food industry for making edible protein films for wrapping meat or fish products.
• the Factor XIII composition may be used generally for the crosslinking of proteins, e.g. for immobilisation, precipitation or modification of the properties of a protein (such as changes in pi, hydrophilicity, hydrophobicity, etc.).
• activated Factor XIII of the invention for medical purposes.
• medical applications include, but are not limited to postoperative wound healing, inclusion in tissue adhesives, antifibrinolytic therapy, treatment of ulcerative colitis, cf. EP 268 772 and the references cited therein.
• the present invention relates to a process for producing activated Factor XIII, the process comprising contacting Factor XIII precursor with an immobilised proteolytic enzyme, and collecting the activated Factor XIII in a buffer solution containing one or more stabilisers, or contacting Factor XIII precursor in a buffer solution containing one or more stabilisers with an immobilised proteolytic enzyme.
• the term "Factor XIII precursor” is intended to indicate the zymogen form of Factor XIII, i.e. the a 2 dimer (2 x 83 kD) , also known as placental Factor XIII, or the a 2 b 2 tetramer, also known as plasma Factor XIII.
• the invention relates to a process of producing activated Factor XIII, the process comprising contacting Factor XIII precursor with a proteolytic enzyme in a buffer solution containing one or more stabilisers, followed by addition, after a suitable interval, of a protease inhibitor.
• the Factor XIII product of the invention may conveniently be provided in the form of a Factor XIII a 2 dimer (i.e. placental Factor XIII) .
• the Factor XIII product may be in the form of an a'a dimer or a'a' dimer.
• the a'a dimer form is one in which a 4 kD fragment has been cleaved off the N-terminal end of one of the a monomers
• the a'a' dimer form is one in which a 4 kD fragment has been cleaved off the N-terminal end of both a monomers.
• the Factor XIII product of the invention is advantageously a recombinant protein since this is a more reliable and economical source of Factor XIII than plasma.
• the preparation of recombinant Factor XIII in yeast is described in, for instance, EP 268 772 to ZymoGenetics as well as P.D. Bishop et al., Biochemistry 29. 1990, pp. 1861-1869, the contents of which are incorporated herein by reference.
• Factor XIII precursor as defined above may be activated with an immobilised proteolytic enzyme.
• suitable enzymes are thrombin, trypsin or a trypsin-like enzyme (e.g. a protease obtainable from a species of Fusarium. cf. WO 89/06270) .
• the proteolytic enzyme may suitably be immobilised by one of the procedures described in K. Mosbach (ed.), "Immobilized Enzymes” in Methods in Enzymology 44. Academic Press, New York, 1976, including covalent coupling to insoluble organic or inorganic supports, entrapment in gels and adsorption to ion exchange resins or other adsorbent materials.
• Suitable support materials for the immobilised enzyme are, for instance, plastics (e.g. polypropylene, polystyrene, polyvinylchloride, polyurethane, latex, nylon, teflon, dacron, polyvinylacetate, polyvinylalcohol or any suitable copolymer thereof) , polysaccharides (e.g. agarose or dextran) , ion exchange resins (both cation and anion exchange resins) , silicon polymers (e.g. siloxane) or silicates (e.g. glass) .
• plastics e.g. polypropylene, polystyrene, polyvinylchloride, polyurethane, latex, nylon, teflon, dacron, polyvinylacetate, polyvinylalcohol or any suitable copolymer thereof
• polysaccharides e.g. agarose or dextran
• ion exchange resins both cation and
• the Factor XIII precursor may be contacted with a proteolytic enzyme after which a protease inhibitor is added.
• the protease inhibitor may suitably be a trypsin inhibitor such as aprotinin or soybean trypsin inhibitor.
• the buffer solution into which the activated Factor XIII is collected is preferably a glycine, alanine or borate buffer.
• the stabiliser or stabilisers present in the buffer solution as well as in the final Factor XIII composition may be a chelating agent, for instance EDTA, EGTA or citrate.
• EDTA may be present in a concentration of 2-15 mM, preferably 3-12 mM, more preferably- 5-10 mM.
• Another stabiliser which may be present in the buffer solution and Factor XIII composition is a reducing agent or another substance capable of preventing oxidation of the active -SH at Cys314 of Factor XIII, e.g. a cysteine or sulfite, or an antioxidant such as ascorbic acid or glutathion.
• a suitable reducing agent is dithiothreitol (DTT) , which may be present in a concentration of 1-10 mM, preferably 2-7 mM, more preferably .2.5-5 mM.
• DTT dithiothreitol
• a further stabiliser which may be present in the buffer solution and Factor XIII composition is a sugar.
• suitable sugars are lactose, glucose, sucrose, maltose or trehalose.
• the sugar may be present in an amount of 0.5-5%, preferably 1-2%, by weight.
• a still further stabiliser which may be present in the buffer solution and Factor XIII composition is casein.
• calcium ions should be present.
• a currently preferred stabilising solution comprises 2% lactose, 2% casein, 10 mM EDTA, and 5 mM DTT in 10 mM glycine buffer, pH 8.0.
• the present composition is in freeze-dried form as this generally results in improved stability.
• rFXIII recombinant Factor XIII
• the homogenized cells were diluted approximately
• rFXIII was isolated by crystallization with ll%(w/v) Na-formiate at 25°C in 5 hours. The crystals were 10 harvested by centrifugation (4000 g in 30 minutes) and freezedried. In this way, two preparations of rFXIII were made: M57 with an initial activity of 285 g/kg, and F435 with an initial activity of 580 g/kg.
• composition of feed Unless otherwise specified, glycine buffer 1510 mM pH 8.0 was used as the solvent. The concentration of rFXIII in the feed was 0.5, 5, 10 and 15 mg/ml. The mixture of glycine buffer and rFXIII was stirred for 1.5 - 2 hours at room temperature to ensure complete dissolution of rFXIII. pH was adjusted several times with NaOH. The solution was clarified by 20 centrifugation (14400 x g for 10 minutes) followed by filtration through a 0.45 ⁇ m membrane filter.
• the %-yield calculated in the tables shown below is based on the assayed content of the feed (activated with thrombin) .
• the assay for Factor XIII activity was carried out by a fluorometric activity assay (referred to in P.D. Bishop et al., supra) .
• the whole content of the vials was dissolved in 100 ml TANEP buffer (0.1 M Tris-acetate, 0.15 M NaCl, 1 mM EDTA, 0.1% PEG 6000) pH 7,5 in order to avoid erroneous results due to inhomogenieties in the freeze-dried preparations. Samples were further diluted in TANEP buffer pH 7,5 prior to being assayed. The results shown are the average of two determinations.
• Casein is Hammersten casein (Merck Art. 2242) , D-
• A. Activation Feed composition 4.2 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. 10 mM DTT in TANEP buffer pH 7.5 was added to the effluent. The results of rFXIII activation appear from Table 1 below (cf. Fig. 1) .
• Feed composition 8,81 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%) , EDTA (10 mM) and DTT (10 mM) were added to the effluent. The results are shown in Table 2 below (cf. Fig. 2) . Table 2
• Feed composition 2,6 g F435 in 200 ml 10 mM glycine buffer were applied on a Sepharose column on which a trypsin-like protease derived from Fusarium (prepared as described in WO 89/06270) had been immobilised by adding 200 mg of the active enzyme mixed with coupling buffer to 4 g of CNBr-activated Sepharose 4B (available from Pharmacia, Sweden) at pH 7.5. An HR5/10 column was packed to a bed height of 43 mm (bed volume 0,84 ml). Trehalose (1%), EDTA (10 mM) and DTT (10 mM) were added to the effluent. The results are shown in Table 3 below (cf. Fig. 3) . 10
• Feed composition 6.99 g F435 in200 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%) , 10 mM EDTA and 10 mM DTT were added to the effluent. The results are shown in Table 4 below (cf. Fig. 204) .
• Feed composition 10.04 g M57 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above.
• EDTA 10 E5 or 20 mM (E10)
• lactose 2 LI
• 4 % L2
• casein 0 or 2 % Cl
• the activation yield is 60%, but when casein is added a yield of 75% is obtained.
• the freeze-dried preparations containing casein have the highest overall yield regardless of the level of lactose and EDTA.
• Feed composition 10.07 g M57 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Lactose (2%) and EDTA (10 mM) and varying amounts of casein (0, 1, 2 and 4%) were added to the effluent. Samples were assayed immediately, and some of them were frozen or freeze-dried, while others were kept in the refrigerator. The results are shown in Table 6 below.
• Feed composition 10.76 g M57 in 300 ml 10 mM glycine buffer pH 108.0 was applied on a trypsin-Sepharose column as described above. Casein (Hammarsten) 2% (C) , EDTA 10 mM (E) , DTT 5 mM (D) and 2% lactose (L) or sucrose (S) were added to the effluent as indicated in Table 7 below. Samples were assayed immediately. Some samples were freeze-dried, others were kept in the 15 refrigerator.
• Casein (Hammarsten) 2% (C) , EDTA 10 mM (E) , DTT 5 mM (D) and 2% lactose (L) or sucrose (S) were added to the effluent as indicated in Table 7 below. Samples were assayed immediately. Some samples were freeze-dried, others were kept in the 15 refrigerator.
• the activation yield is high, 80 - 90%.
• the freeze- dried preparations containing casein, EDTA, DTT and lactose/sucrose give a high over-all yield of about 60%. They show no sign of loss of activity over two months. The same composition results in the most stable samples when kept cold.
• Feed composition 2.51 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Casein 2% (C) , lactose 2% (L) , EDTA 10 mM (E) and DTT 0 - 20 mM were added to the effluent. Samples were assayed immediately. Some samples were freeze-dried, others were kept in the refrigerator. The results are shown in Table 8 below.
• Feed composition 4.82 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%), EDTA (10 mM) , DTT (10 mM) and casein (0-2%) were added to the effluent from the column. Samples were assayed immediately. Some samples were frozen and freeze-dried, others were kept in the refrigerator. The results are shown in Table 9 below.
• Example 3 (used as a control) was prepared, and the two samples were placed in a water bath for 2 hours at 30°C followed by cooling at 5°C for 24 hours. After the sausage skins had been removed, the control sample was still liquid, while the enzymatically treated sample had solidified into a sausage which was stable at room temperature for several days until it was microbially degraded.
• Example 3
• the ingredients were mixed and blended.
• the pH of the resulting mince was 5.51.
• the pH was adjusted to 7.0 with NaOH.
• the mince was divided into four portions as follows: 101. Control.
• the amount of Factor XIII in the mince is calculated from a percentage of protein in beef of 20%.
• the samples were tinned and heat treated in a water bath at 2530°C for 2.5 hours followed by treatment at 80°C for 1 hour.
• the tins were cooled in a water bath at about 20"C and subsequently in a refrigerator over night.
• the gel strength of the boiled mince was measured by means of an Instron dynamometer (available from Instron, FRG) , using the method described by P.-G. Klettner, Fleischwirtsch. 69 (2), 1989, pp. 225-226. The results of these experiments is shown in Figs. 5-7.
• the ordinate indicates the force required to compress a 22 mm slice of the mince to a thickness of 2 mm.
• the graph showing the gel strength figures for mince treated with 0.5% activated Factor XIII (Fig. 5) shows a distinct peak indicating breaking of the gel (as it should) .
• the graph for mince treated with inactive Factor XIII at the same dosage level shows a less distinct peak (Fig. 6) , while the graph for mince treated with 0.05% inactive Factor XIII shows no peak at all (Fig. 7) .
• the PVC skin was removed, and the gel strength af the sausages was measured on samples cut to a height of 25 mm by means of a Bloom gelometer available from Griffin and George Ltd. , Great Britain.
• the strength of the samples was 234 g and 142 g, respectively, corresponding to a gel strength improvement in the enzymatically treated gel of 65%.

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