Appl Microbiol Biotechnol (2007) 74:331338 DOI 10.

1007/s00253-006-0621-1

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Purification and characterization of fibrinolytic alkaline protease from Fusarium sp. BLB
Mitsuhiro Ueda & Toshihiro Kubo & Kazutaka Miyatake & Takumi Nakamura

Received: 6 June 2006 / Revised: 3 August 2006 / Accepted: 8 August 2006 / Published online: 13 January 2007 # Springer-Verlag 2007

Abstract Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH4)2SO4 and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was observed at pH 9.5 and 50C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50C. The enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. The Nterminal amino acid sequence of the enzyme showed a similarity to those of proteases from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus. Keywords Fibrinolytic activity . Alkaline serine protease . Fusarium sp

Introduction Blood clots (fibrin) are formed from fibrinogen by thrombin (EC 3.4.21.5) and are lysed by plasmin (EC 3.4.21.7), which is activated from plasminogen by tissue plasminogen activator (tPA). Although fibrin clot formation and fibrinolysis are maintained in balance by the biological system, thromboses, such as myocardial infraction, occur when clots are not lysed as a result of a disorder in the balance. Intravenous administration of urokinase and streptokinase was widely used for thrombosis therapy but these enzymes have a low specificity to fibrin. tPA was developed for the treatment of thrombosis because of its efficacy and strong affinity to fibrin (Sherry 1987). However, these enzymes are expensive, and patients may suffer from undesirable side effects such as gastrointestinal bleeding, allergic reaction, and resistance to repercussion (Blann et al. 2002; Bode et al. 1996; Turpie et al. 2002). Therefore, the search for thrombolytic agents from other sources is needed. Recently, potent fibrinolytic enzymes were discovered in fermented food products, such as Japanese natto (Sumi et al. 1987; Fujita et al. 1993), Korean Chang kook-Jang soy sauce (Noh et al. 1999; Kim et al. 1996), skipjack Shiokara (Sumi et al. 1995; Nakajima et al. 1993a,b), fermented shrimp paste (Wong and Mine 2004), and some marine creatures (Sumi et al. 1992). In particular, oral ingestion of natto or its enzyme can effectively enhance the release of an endogenous plasminogen activator in both animal models and human subjects (Sumi et al. 1987; Omura et al. 2005). In this study, to find a unique protease and to apply it in fibrinolysis, we conducted a screening of the fungi to produce the fibrinolytic enzyme. The fungus strain BLB isolated from a hibiscus leaves was named Fusarium sp. BLB, which had exhibited the highest activity toward

M. Ueda (*) : K. Miyatake Laboratory of Biocycle Engineering, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan e-mail: mueda@biochem.osakafu-u.ac.jp T. Kubo : T. Nakamura SODX Corporation, 1-3-14, Techno stage, Izumi, Osaka 594-1144, Japan

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fibrin. We isolated, purified, and characterized fibrinolytic alkaline protease from Fusarium sp. BLB.

Materials and methods Isolation of fibrinolytic enzyme-producing fungi A plant leaf sample was suspended in sterilized water and the water was spread on potato dextrose agar medium at pH 7.0 and incubated for 4872 h at 28C. The fungi were grown on the medium and we isolated the many fungi. The isolated fungi were cultured in 2% soy bean powder, 2% glucose, 0.5% polypepton, 0.2% yeast extract, 0.1% KH2PO4, and 0.1% MgSO4 at pH 7.0 for 4872 h at 28C. The culture supernatants were used with the crude fibrinolytic enzyme. The fibrinolytic enzymes were measured by fibrin plate assay as described in the following enzyme assay. The highest fibrinolytic enzyme activity was produced by strain BLB after growth for 72 h at 28C in a liquid medium containing 4% soy bean powder, 3% glucose, 0.2% yeast extract, 0.1% KH2PO4, and 0.1% MgSO4 at pH 7.0. Taxonomical studies

transferred into a jar fermenter containing 5 l of liquid medium (4% soy bean powder, 3% glucose, 0.2% yeast extract, 0.1% KH2PO4, and 0.1% MgSO4 at pH 7.0) and grown for 72 h at 28C to produce the fibrinolytic enzyme. The culture broth was centrifuged for 30 min at 12,000g and the supernatant was used for the subsequent purification of the enzyme. Enzyme assay Fibrin plate assay Fibrinolytic activity was determined as follows using a slightly modified method of that developed by Astrup and Mullertz (1952). A total of 10 ml of 0.4% human fibrinogen (Sigma) in 0.1 M phosphate buffer at pH 7.4 was poured into a 10-cm petri dish and then clotted by the addition of 0.2 ml of human thrombin (100 U/ml NIH; Sigma) in the same buffer. The clot was allowed to stand for 1 h at room temperature. After this, 30 l of the sample solution was carefully placed on a plate. The plate was incubated for 18 h at 37C. After measuring the dimensions of the clear zone, the number of units was determined according to standard curve using plasmin (Sigma). Protease assay

The morphological and cultural characteristics of the isolated fungi were analyzed according to the methods of Kornerup and Wanscher (1978). An internal transcriptional spacer (ITS)-5.8S rDNA fragment was amplified by PCR with ITS5 and ITS4 primers (White et al. 1990). 28S rDNA fragment was amplified by PCR with NL1 and NL4 primers (ODonnell 1993). The amplification was performed using a Thermal Cycler TP 2000 (Takara). The DNA sequence of the amplified fragment was analyzed using a DNA sequencer (ABI PRISM 377 DNA Sequencer, ABI). The sequences produced were assembled using AutoAssembler software (ABI) and edited for accuracy. Searches for identity were performed against the GenBank database by using Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology web site (http://www.ncbi.nih.gov/BLAST/). Sequences were added manually to an existing alignment sequence from fungal strain BLB. Neighbor-joining analysis was performed using the software package PAUP version 4.0b9 (Swofford 2002). Enzyme production A fibrinolytic enzyme-producing fungus was grown in 50 ml of liquid medium in a Sakaguchi flask (500 ml) containing 2% soy bean powder, 2% glucose, 0.5% polypepton, 0.2% yeast extract, 0.1% KH2PO4, and 0.1% MgSO4 at pH 7.0. Fifty milliliters of the seed culture was

Protease activity was assayed at 37C for 10 min by the casein FolinCiocalteau method (Oda and Murao 1974); 0.5 ml of the enzyme solution was added to 1.5 ml of 1.14% Hammarsten casein solution in a 0.1-M glycineNaOH buffer with a pH of 9.5. After incubation for 10 min, the reaction was stopped by the addition of 2.0 ml of 0.44 M trichloroacetic acid, followed by centrifugation at 15,000g for 10 min. The supernatant (0.5 ml) was mixed with 2.5 ml of 0.44 M sodium carbonate and 0.5 ml of FolinCiocalteu reagent. The optical density of the color developed at 37C for 20 min and was measured at 660 nm. One unit of enzyme activity was defined as the enzyme quantity that liberates 1 g of tyrosine per ml of the reaction mixture per min. Purification of fibrinolytic alkaline protease Solid ammonium sulfate was added to 3,580 ml of the cell-free culture broth to make a 70% saturated final concentration. The resulting precipitate was collected by centrifugation. The pellet was dissolved in a small volume of 20 mM sodium acetate buffer with a pH of 5.0 and the solution was dialyzed against the same buffer overnight at 4C. The dialyzate was put on a CM-Toyopearl 650M (Tosoh) column (4.5 28 cm) previously equilibrated with 20 mM sodium acetate buffer at pH 5.0. The column was washed with a 20-mM sodium acetate buffer at pH 5.0. The

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effect of temperature on proteinase activity was studied at 3060C. Effect of pH and temperature on enzyme stability Analysis of the effect of pH level on enzyme stability was carried out by incubating the enzyme for 1 h at 37C in the following buffers: glycineHCl, sodium acetate, phosphate, and glycineNaOH buffer. All buffers were 0.1 M. After incubation, the activity remaining was determined using the casein FolinCiocalteau method. For measuring its thermal stability, the purified protease was incubated in 50 mM sodium acetate buffer at pH 5.0 for 10 min at 1080C. After incubation, the activity remaining was determined using the casein FolinCiocalteau method. Hydrolysis of chromogenic substrate The hydrolytic activity of the protease was also studied using the following chromogenic substrate (all purchased from Sigma): D-Ile-Pro-Arg-p-nitroanilide dihydrochloride, D-Val-Leu-Lys-p-nitroanilide dihydrochloride, N--benzoyl-L -Arg-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Leu-pnitroanilide, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and N-glutaryl-L-Phe p-nitroanilide. Each reaction mixture contained 2.5 mM of substrate in 0.1 M glycineNaOH buffer at pH 9.5 in 4% DMSO. Effect of protease inhibitors The inhibition of the protease was determined by measuring its activity at a pH of 9.5 in the presence of protease inhibitors. Enzyme samples were separately incubated at 37C for 10 min with each of the following inhibitors: soy bean trypsin inhibitor (SBTI, Wako Pure Chemical Industries), Streptomyces subtilisin inhibitor (SSI, Daiwa Kasei), -aminocapronic acid (-ACA, Wako Pure Chemical IndusTable 1 Purification of fibrinolytic alkaline protease from Fusarium sp. BLB Step Total activitya (U) 239,000 80,700 20,100 16,700 Total protein (mg) 17,200 336 36.3 25.1 Specific activity (U/mg protein) 13.9 240 554 665 Yield (%) Fold

Fig. 1 Column chromatography of fibrinolytic alkaline protease on Superdex 75 HR 10/30. The protease was put on Superdex 75 HR 10/ 30. See the text for details. Straight line: protein (absorbance at 280 nm), filled circled: protease activity (U/ml), and open circle: fibrinolytic activity (10 IU/ml)

protease was eluted with a linear 0- to 0.5-M NaCl gradient in a 20-mM sodium acetate buffer at pH 5.0. Protease fractions were collected and the enzyme was precipitated by adding solid ammonium sulfate to 80% saturation. After centrifugation, the pellet was dissolved in a small volume of 20 mM of sodium acetate buffer at pH 5.0. The solution was applied onto a Superdex 75 HR 10/30 column (Amersham Bioscience) equilibrated with 20 mM of sodium acetate buffer at pH 5.0 containing 0.2 M NaCl and eluted with the same buffer. Active fractions were pooled and used as the purified enzyme solution (Fig. 1). Effect of pH and temperature on activity The activities of the purified enzyme were measured by using the synthetic substrates at 37C in glycineHCl (pH 4.56.0), phosphate (pH 6.07.5), TrisHCl (pH 7.59.0), and glycineNaOH (pH 9.011.5). All buffers were 0.1 M. The
Fig. 2 SDS-PAGE of the purified enzyme. A 12.5% polyacrylamide gel was used for analysis. Markers: phosphorylase B (97.4 kDa), bovine serum albumin (66.3 kDa), aldose (42.4 kDa), carbonic anhydrase (30.0 kDa), and trypsin inhibitor (20.1 kDa)

Culture filtrate 70% (NH4)2 SO4 ppt. CM-Toyopearl 650M Superdex 75 HR10/30
a

1.00 33.8 8.4 7.0

1 17.3 39.9 47.9

Protease activity was assayed by FolinCiocalteau method. The substrate used for assay was casein.

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tries), phenylmethylsulfonyl fluoride (PMSF, Wako Pure Chemical Industries), L-1-chloro-3-[-4-tosylamido]-4-phenyl-2-butanone (TPCK, Wako Pure Chemical Industries), diisopropylfluorophosphates (DFP, Wako Pure Chemical Industries), 2,2-bipyridyl, chymostatin, phenantrolin, ethylenediaminetetraacetic acid (EDTA, Wako Pure Chemical Industries), E-64 (Wako Pure Chemical Industries), and Streptomyces pepsin inhibitor (S-PI, Wako Pure Chemical Industries). Residual activity was then determined.

Protein determination Protein determination was performed using the methods of Bradford (1976) with bovine serum albumin (Wako Pure Chemical Industries) as a standard. For column chromatography, the protein concentration was determined by measuring the absorbance at 280 nm.

Fig. 3 Effect of pH on activity (a) and stability (b), and effect of temperature on activity (c), and stability (d). a The activities of purified enzymes were measured by using the D-Ile-Pro-Arg-p-nitroanilide dihydrochloride at 37C with sodium acetate (open circle), phosphate (filled circle), TrisHCl (filled square), and glycineNaOH (open square). All buffers were 0.1 M. b The effect of pH on enzyme stability was elucidated by incubating the enzyme for 1 h at 37C (filled square) or 24 h at 4C (open circle) in the following buffers: glycine

HCl, sodium acetate, phosphate, TrisHCl, and glycineNaOH. Remaining activity was measured using D-Ile-Pro-Arg-p-nitroanilide dihydrochloride at pH 9.5. c The effect of temperature on the protease activity was studied at 3060C. d For measuring its thermal stability, the purified protease was incubated using a 50-mM sodium acetate buffer at pH 5.0 for 10 min at 1080C. Remaining activity was measured using D-Ile-Pro-Arg-p-nitroanilide dihydrochloride at 37C

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Electrophoresis of protein Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done according to the methods of Laemmli (1970) using the molecular mass marker Daiichi II (Daiichi Pure Chemical) as a standard. Protein bands were detected by staining with Coomassie Brilliant Blue R-250. Determination of N-terminal amino acid sequence

rDNA and 28S rDNA. Macroconidia of this strain was observed to have a crescent shape at the base of the aerial hyphae. Microconidia also formed a spindle shape. Sequences of ITS-5.8S rDNA and 28S r DNA from isolated strain BLB indicated 99.8 and 99.6% homology with those of Fusarium sp. NRRL 22354, respectively (data not shown). From these results, it was shown that strain BLB belongs to the genus Fusarium. Purification of fibrinolytic alkaline protease

Proteins were separated using SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was washed extensively with water, stained with 0.25% Coomassie Brilliant Blue R-250, 5% aqueous methanol, and 7.5% acetic acid for 5 min, and destained with 90% aqueous methanol for 10 min. A portion of the membrane containing the desired protein band was cut out and the Nterminal amino acid sequence was determined by way of an automated protein sequencer (PPSQ-23, Shimazu).

Nucleotide sequence accession number The nucleotide sequences of ITS-5.8S rDNA and 28S rDNA from Fusarium sp. BLB will be available in the DDBJ/ EMBL/GenBank nucleotide sequence database under accession numbers: AB267087 and AB267088, respectively.

A fungal strain BLB isolated from the leaves of hibiscus has a high fibrinolytic enzyme activity (1,500 IU/ml culture broth). The protease was purified from Fusarium sp. BLB as described above. The purified protein appeared as a single band as determined by SDS-PAGE (Fig. 2). The yield and purity of the protease at each purification step are summarized in Table 1. The enzyme was purified by about 47.8-fold with a recovery of 7.0%. The molecular mass of the enzyme was estimated to be 27 kDa from SDS-PAGE. The protease activity fractions also have fibrinolytic activity (Fig. 1). Effect of pH and temperature on the protease activity The optimum pH of the purified protease was 9.5 (Fig. 3a). It was stable with a pH ranging from 2.5 to 11.5 for 20 h at 4C and from 3 to 9 for 1 h at 37C (Fig. 3b). The optimum temperature of the enzyme was 50C and stability was below 50C (Fig. 3c,d). Aminoterminal amino acid sequence analysis The N-terminal amino acid sequence of the enzyme was determined to be IVVGTTAASGGDFPIIVSIYYQGRAR. The protein showed the homology with proteases of Bos taurus bovine (McGrath et al. 2006), Streptomyces griseus (Nakajima et al. 1993b), Fusarium oxysporum (Rypniewski et al. 1993), Katsuwo pelamis digestive tract (Nakajima et al. 1993a), and Lumbricus rubellus (Nakajima et al. 1993b) (Fig. 4).

Results Identification of fibrinolytic alkaline protease-producing strain To investigate the taxonomic position of this strain BLB, we checked the morphological characterization of the strain and analyzed the nucleotide sequences of the ITS-5.8S

Table 2 Hydrolysis of chromogenic substrate by Fusarium sp. BLB fibrinolytic alkaline protease Substrates dihydrochloride dihydrochloride N--benzoyl-L-Arg-p-nitroanilide N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide N-glutaryl-L-Phe-p-nitroanilide
D-Val-Leu-Lys-p-nitroanilide D-Ile-Pro-Arg-p-nitroanilide

Relative activity (%) 100 21 2.8 0 0 0

Fig. 4 N-terminal amino acid sequence of the fibrinolytic proteases that show homology with Fusarium sp. BLB enzyme. Identical corresponding amino acid residues are underlined. 1) This paper, 2) Rypniewski et al. 1993, 3) Kato et al. 2005, 4) Mikes et al. 1996, 5) Nakajima et al. 1993a, and 611) Nakajima et al. 1993b

336 Table 3 Inhibition of fibrinolytic alkaline protease activity by various specific inhibitors Inhibitorsb SBTI SSI -ACA PMSF TPCK DFP 2,2-Bipyridyl Chymostatin Phenantrolin EDTA E-64 S-PI Concentration 0.01 mg/ml 0.001 mg/ml 1 mM 1 mM 0.1 mM 0.1 mM 1 mM 0.01 mg/ml 1 mM 1 mM 0.1 mM 0.1 mM Inhibition (%) 0 0 0 41 3 79 3 0 0 0 3 0

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zyme indicated only a slightly activity toward N--benzoylL-Arg-p-nitroanilide. No hydrolysis of N-glutaryl-L-Phe-pnitroanilide was observed. Effect of protease inhibitors on the enzyme activity The effects of various inhibitors on enzyme activity for N--benzoyl-L-Arg-p-nitroanilide were investigated as shown in Table 3. Protease activity was strongly inhibited by both DFP and PMSF serine protease inhibitors, whereas other inhibitors did not show any inhibitory activity.

Discussion We report the purification and characterization of a unique fibrinolytic alkaline protease from Fusarium sp. BLB. The enzyme was purified by CM-Toyopearl 650M and Superdex 75HR column chromatography. The chromatographic behavior of Fusarium protease resembles that of other Fusarium proteases (Pekkarinen and Jones 2002; Pekkarinen et al. 2002). The molecular weight of the purified protease was estimated at 27,000, which was similar in size to those from Fusarium sp. (Rypniewski et al. 1993; Pekkarinen and Jones 2002; Pekkarinen et al. 2002), L. rubellus (Nakajima et al. 1993b; Cho et al. 2004), and Aspergillus fumigatus (Larcher et al. 1992) (Table 4). It was stable over a broad pH range from 2.5 to 11.5. This enzyme was pH-resistant, which

Reaction conditions describes in Materials and methods SBTI Soy bean trypsin inhibitor, SSIStreptomyces subtilisin inhibitor, -ACA -aminocapronic acid, PMSF phenylmethylsulfonyl fluoride, TPCKL-1-chloro-3-[-4-tosylamido]-4-phenyl-2-butanone, DFP diisopropylfluorophosphate, S-PIStreptomyces pepsin inhibitor, and EDTA disodium ethylenediaminetetraacetate

Hydrolysis of chromogenic substrate When the hydrolytic activities of the protease were measured at pH 9.5 with various synthetic substrates, the results listed in Table 2 were obtained. The enzyme hydrolyzed D-Ile-Pro-Arg-p-nitroanilide dihydrochloride and D-Val-Leu-Lys-p-nitroanilide dihydrochloride. The enTable 4 Comparison of characterization of fibrinolytic proteases Source M.W. Opt. pH 9.5 7 9 911 412 911 7.4 7 10.5 37

pH stability 2.511.5 ND ND 111 ND 111 712 79 710.5 ND

Opt. temp. Heat stability Inhibitor (C) (C) 50 ND 3742 ND 50 ND ND 40 70 ND 50 ND 40 60 ND 60 ND 40 50 50 DFP, PMSF ND PMSF, chymostatin DFP, SBTI, aprotinin PMSF, SBTL TPCK, leupeptin DFP, SBTI, aprotinin DFP, negvon EDTA, 2,2bipyridine, phenanthroline PMSF EDTA, Cu2+

Fusarium sp. BLBa Fusariumsemitectumb Aspergillus fumigatusc Lumbricus rubellus F-I, II, IIId Lumbricus rubellus F16e Katsuwo pelamis digestive tractf Bacillus subtilis (natto)g Bacillus sp. KA 38 (Joet-Gal)h

27,000 ND 33,000 23,01329,667 24,60033,000 38,000 20,000 41,000

28,200 Bacillus sp. CK-11-4 (Chungkook-Jang)i Fibrinolytic enzyme in fermented shrimp pastej 18,000 ND Not determined a This paper b Buckley and Jeffries (1981) c Larcher et al. (1992) d Nakajima et al. (1993b) e Cho et al. (2004) f Nakajima et al. (1993a) g Fujita et al. (1993) h Kim et al. (1997) i Kim et al. (1996) j Wong and Mine (2004)

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is evident of being more stable than those of Bacillus subtilis (natto) (Fujita et al. 1993) and Bacillus sp. KA38 (Kim et al. 1997). The Fusarium protease had high activity toward DIle-Pro-Arg-p-nitroanilide dihydrochloride, which resembles the fibrinolytic enzyme (F-I-1 and FI-2) found in earthworms (Nakajima et al. 1993b). The substrate specificity of the Fusarium enzyme was similar to that of the fibrinolytic enzyme found in earthworms, but not similar to the enzyme from B. subtilis (natto) and A. fumigatus. It was established that the Fusarium enzyme acted on arginine and lysine residue attached to the carboxyl side of the splitting points of the chromogenic substrate. To clarify the type of Fusarium protease, the effect of various protease inhibitors toward enzyme activity was examined. The Fusarium protease activity was inhibited by PMSF and DFP, but not by other inhibitors, e.g., SBTI, SSI, and chymostatin. This strong inhibition caused by PMSF and DFP may be an indication that the Fusarium enzyme is a unique serine protease, and belongs to neither the chymotrypsin, trypsin, or subtilisin families (Tables 3 and 4). A comparison of the N-terminal amino acid sequence of the Fusarium protease with those of fibrinolytic proteases revealed the identity of the proteases from F. oxysporum (Rypniewski et al. 1993), S. griseus (Kato et al. 2005), B. taurus bovine (Mikes et al. 1996), K. pelamis digestive tract (Nakajima et al. 1993a), and L. rubellus (Nakajima et al. 1993b). The N-terminal amino acid sequence was highly homologous with that of the trypsinlike enzyme of F. oxysporum (Rypniewski et al. 1993). However, the characteristics of the enzyme are different from those of F. oxysporum trypsin. A potent fibrinolytic enzyme (nattokinase, NK) was previously isolated from the traditional Japanese fermented food, natto, by Sumi et al. (1987). This enzyme is an extracellular serine protease produced from B. subtilis (natto). Sumi et al. (1990) further demonstrated that oral ingestion of natto or NK capsules enhanced fibrinolysis in the canine plasma of an experimental thrombosic model. To confirm the physiological functions of the enzyme, the next step will be to examine the intestinal absorption of the enzyme in vitro by using human intestinal epithelial cells and to measure the fibrinolytic activity of the enzyme in the blood and the organs using animal model systems. The calculated fibrinolytic activity of the extract obtained from 1 g of wet natto corresponded to about 1,600 IU of urokinase (Sumi et al. 1987). The enzyme activity of Fusarium sp. BLB has 1,500 IU urokinase/ml of culture broth. It was thought that Fusarium has almost the same enzyme productivity as that of Bacillus natto. The fibrinolytic enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy like other potent fibrinolytic enzymes, such as NK and earthworm enzymes. The Fusarium protease was able to maintain the activity for long periods of time in vivo because of its high pH resistance. The

Fusarium protease shows a significant potential for food fortification and nutraceutical applications, such that their use could effectively prevent cardiovascular diseases. The detailed protein and gene structure of the Fusarium protease are currently under investigation.

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