Identification of fibrinogen-induced nattokinase WRL101 from Bacillus subtilis WRL101 isolated from Doenjang

1 Department of Food Science and Technology, Catholic University of Daegu, Korea. 2 Advanced Radiation Technology Research Institute, KAERI, Jeonbuk Branch Institute, Jeongeup, Korea. 3 Department of Pharmaceutical Engineering, Konyang University, Nonsan 320-711, Korea. 4 Department of Biochemistry and Health Science, Changwon National University, Changwon, Korea. 5 Institute Bioindustry Research Center, KRIBB, Jeonbuk, Korea. 6 Woori Life Science Co., Ltd., Cheonan-si, Chungnam, Korea.


INTRODUCTION
Accumulation of fibrin in the blood vessels usually results in thrombosis, leading to myocardial infarction and other cardiovascular diseases (Kim et al., 1996).Fibrin is formed from fibrinogen by activated thrombin (EC 3.4.21.5) and lysed by plasmin (EC 3.4.21.7), which is generated from plasminogen by tissue type plasminogen activator (tPA).The major thrombolytic agents are classified into two types.One is plasminogen activators, such as urokinase, tPA (tissue type plasminogen activator), and streptokinase, which activate plasminogen to plasmin.The other type is plasmin-like proteins, such as fibrolase from snake venom and lumbrokinase (Mihara et al., 1991) from earthworm, which can directly degrade the fibrin.The former three agents are currently being used as thrombolytic agents; however, they are expensive and have undesirable side effects such as gastrointestinal bleeding and allergic reactions.Therefore, the search for alternative, safer thrombolytic agents from various sources is ongoing.Many bacterial fibrinolytic enzymes were discovered from fermented foods, such as natto (Sumi et al., 1987(Sumi et al., , 1990;;Fujita et al., 1993;Chang et al., 2000;Kwon et al., 2011;Wang et al., 2011), shiokara (Sumi et al., 1995) in Japanese food, Chungkook-Jang (Kim et al., 1996), Doenjang (Kim and Choi, 2000) and Jeot-Gal (Kim et al., 1997;Choi et al., 2009b) in Korean food, douchi (Peng et al., 2003) in Chinese food and Tempeh (Kim et al., 2006) in Indonesian food.The fibrinolytic enzymes were successively obtained from different microorganisms, the most important among which is the genus Bacillus.In particular, nattokinase produced by Bacillus natto screened from natto was reported that the enzyme not only hydrolyzed thrombi in vivo, but also converted plasminogen to plasmin (Sumi et al., 1990).Oral administration of the enzyme showed that it could enhance fibrinolytic activity in plasma and the production of tPA, and its fibrinolytic activity was retained in the blood for more than 3 h.These results suggest that NK may be a potent natural agent for oral thrombolytic therapy.Subsequently, many fibrinolytic enzymes were identified in different traditional fermented foods, such as CK of Bacillus sp.strain CK 11-4 from Chungkook-Jang (Kim et al., 1996) and subtilisin DJ-4 of Bacillus sp.DJ-4 from Doenjang (Kim and Choi, 2000) in Korea, a fibrinolytic enzyme of B. subtilis IMR-NK1 from Taiwanese soil (Chang et al., 2000), subtilisin DFE of B. amyloliquefaciens DC-4 from douchi in China (Peng et al., 2003) and a subtilisin-like fibrinolytic protease of B. subtilis TP-6 from Tempeh in Indonesia (Kim et al., 2006).
So far, many researchers have focused their efforts on isolating and screening of microorganisms for fibrinolytic enzyme production with higher activity and on purifying and characterizing newly found enzymes.To achieve high product yields, it is a prerequisite to select a proper medium.Little information, however, is available on the optimized media for fibrinolytic enzyme production.Here, we applied fibrinogen (or fibrin) as substrate for production of fibrinolytic enzyme (nattokinase WRL101) from B. subtilis WRL101 isolated from Doenjang, a popular soybean fermented food in Korea.With a fibrinogen-contained TSB medium, FIN-WRL101 was easily purified and its nucleotide and amino acid sequences were determined.
Also, the amino acid sequence of FIN-WRL101 and its enzymatic properties with those of other subtilisins (BPN', Carlsberg, and DJ-4) were compared.

Enzyme assay
Quantitative analysis of fibrinolytic activity was conducted by the standard fibrin plate method (Asrup and Müllertz, 1952).Fibrinogen [5 ml of 0.6% (w/v)] solution in a 50 mM sodium phosphate buffer (pH 7.4) was mixed with the same volume of 2% (w/v) agarose solution and 0.1 ml of thrombin (10 NIH units/ml) in a Petri dish.The solution was left for 1 h at room temperature to form a fibrin clot layer.Caseinolytic activity was assayed by using the casein plate   -11 are 6, 12, 18, 24, 36, 48, 72, 96, 120, 144, and 168 h cultivation, respectively.Asterisked bands (*) are subtilisin determined by Nterminal amino acid sequencing analyzer.
method.Casein [5 ml of 0.6% (w/v)] solution in a 50 mM sodium phosphate buffer (pH 7.4) was mixed with the same volume of 2% (w/v) agarose solution in a Petri dish.20 µl (0.1 μg) of sample solution were applied to a fibrin plate and incubated at 37°C for 12 h.The same volume of plasmin solution (1 NIH unit/ml) was also incubated on a fibrin plate as a positive control for fibrinolytic protease activity.

Enzyme purification
All purification steps were carried out at 4°C.The buffers used were as follows: buffer A, 50 mM Tris-HCl buffer (pH 7.4) and buffer B, 50 mM Tris-HCl buffer (pH 7.4) containing 0.1 M NaCl.
To purify the fibrinolytic enzyme, B. subtilis WRL101 was cultured at 37°C in TSB containing bovine fibrinogen (1.0%, w/v) in 2-L Erlenmeyer flasks with shaking (150 rpm) for 2 days.The fibrinolytic enzyme in the 500 ml culture supernatant was concentrated by ultrafiltration with PM-10 membrane (Amincon, Inc., Beverly, MA, USA).The concentrated sample was dialyzed against 20 volumes of Buffer A for one day with three buffer changes.The dialyzed suspension was loaded onto a CM-cellulose column (2.0 × 10 cm) equilibrated with Buffer A. Proteins were eluted with a 100 ml of linear gradient of 0 to 0.5 M NaCl in Buffer A. Fractions showing fibrinolytic activity were pooled and then dialyzed against Buffer B. The dialyzed enzyme was concentrated by lyophilization, and further purified by TSK gel filtration column (2.0 × 110 cm) (Toyopearl HW-55F, TOSOH, Kyoto, Japan) using Buffer B.

Amidolytic assay
The amidolytic activity was colorimetrically estimated with a Beckman DU-70 spectrophotometer by using various chromogenic substrates (Choi et al., 2009b).Assays were carried out in 50 mM glycine-NaOH buffer (pH 10.0), 0.2 ml of 0.5 mM substrate, and purified enzyme (0.3 μg/0.2 ml).The mixture was incubated at 37°C for 5 min, and the reaction was stopped by adding 0.1 ml of 50% acetic acid.Activity was determined from the change in absorbance at 405 nm due to the formation of p-nitroaniline.One unit was defined as the amount of enzyme releasing 1 μmol of substrate per minute.

SDS-PAGE and determination of N-terminal amino acid sequence of purified enzyme
SDS-PAGE was performed by the Laemmli (1970) method.Protein samples were diluted 5 times with SDS sample buffer comprised of 0.5 M Tris-HCl (pH 6.8), 10% (w/v) SDS, 20% (v/v) glycerol and 0.03% (v/v) bromophenol blue.After SDS-PAGE, the purified enzyme on the gel was transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting (Matsudaira, 1987) and stained with Coomassie blue.The stained material was excised and used for direct N-terminal sequencing by the automated Edman degradation method using a gas-phase protein sequencer model Procise 491 (ABI, Foster City, CA, USA).

PCR cloning of FIN-WRL101 gene
Using the amino acid sequence of FIN-WRL101, we searched the nucleotide sequence database of National Center for Biotechnology Information for a subtilisin homologue and found the nattokinase clone (Accession No.: AF368283.1).Chromosomal DNA from B. subtilis WRL101 was prepared by the method of Rochelle et al. (1992), and used as the template for PCR.FIN-WRL101 gene was amplified by PCR using NdeI-linked sense primer (5'-GGAATTCCATATGAGAAGCAAAAAATTGTGGATCA-3') and BamHI-linked antisense primer (5'-CGCGGATTCTTATTGTGCAGCTGCTTGT-3').PCR amplification was performed under the following conditions: 30 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 1.5 min.The PCR-amplified 1143 bp DNA fragment was extracted from agarose gel and then ligated into pGEM-T Easy vector (Promega) to generate pT-FIN-WRL101 plasmid.The FIN-WRL101 gene and its deduced amino acid sequences were determined (SolGent Co.).

Identification of a fibrinolytic enzyme-producing bacterium
In the present study, the bacterial strain (WRL101) prod-ucing a fibrinolytic enzyme, fibrinogen induced nattokinase WRL101 (FIN-WRL101), was isolated from Doenjang, a traditional Korean soybean fermented food.
Phylogenetic analysis of WRL101, based on the levels of similarity of the 16S rRNA sequences (deposited in GenBank under Accession No. JN400257), indicate that the strain belonged to the genus Bacillus and is closely related to the type strain of Bacillus subtilis strain JSU-2 (100%), and the strain was then named B. subtilis WRL101.

Effect of fibrin or fibrinogen on fibrinolytic enzyme production
Based on the observation, fibrin decreased FIN-WRL101 production.After 72 h cultivation in the fibrin-contained medium, the fibrinolytic activity was completely inhibited (Figure 2).Plus, no FIN-WRL101 band was detected at this time on the SDS gel.On the other hand, both the fibrinolytic activity and FIN-WRL101 band cultured in the fibrinogen-contained medium were increased.Using this medium, we can easily isolate FIN-WRL101 as follows:

Effect of pH and temperature on activity and stability
The effect of pH on the activity of FIN-WRL101 was determined in buffers of various pH values, and results show that FIN-WRL101 was active over a wide range of pH values from 5.0 to 12.0 and was most active at pH 11.0 (Figure 4A).The enzyme was very stable in the range of pH 7.0 to 10.0 at 37°C for 60 min, but became unstable out of this range.The effect of temperature on the fibrinolytic activity of the enzyme was examined at pH 11.0 (Figure 4B).The temperature showing the maximal enzyme activity was 47°C, which was comparable to those of nattokinase (Fujita et al., 1993), subtilisin DJ-4 (Kim and Choi, 2000) and subtilisin D5 (Choi et al., 2009a).

Effect of inhibitors and metal ions on fibrinolytic activity
The effects of various inhibitors and metal ions on the fibrinolytic activity of FIN-WRL101 are summarized in Table 2. FIN-WRL101 was inhibited by 1 mM PMSF, but EDTA, EGTA, Leupeptin and Aprotinin did not inhibit the fibrinolytic activity, indicating that FIN-WRL101 is a serine protease.In addition, the enzyme activity was inhibited by 5 mM of Cd 2+ , Cu 2+ and Zn

Comparison of the specific activity of FIN-WRL101 with other proteases
The specific activity (F/C, the ratio of fibrinolytic activity to caseinolytic activity) of FIN-WRL101 with other proteases was determined by measuring fibrinolytic and caseinolytic activities and calculating the F/C ratios.As shown in Table 3, the specific activity of FIN-WRL101 was 1.88 and 2.79 times higher than those of subtilisin BPN' and Carlsberg, respectively.But, it was 1.42 and 1.26 times lower than that of subtilisin D4 (Kim and Choi, 2000) and D5 (Choi et al., 2009a), respectively.

Amidolytic activity using synthetic substrates
The amidolytic activity of the FIS-WRL101 was investigated with several synthetic substrates.FIN-WRL101 only hydrolyzed Meo-Suc-Arg-Pro-Tyr-pNA (S-2586), a synthetic chromogenic substrate for chymotrypsin, and did not show activity on other tested synthetic substrates (Table 4).NK from Bacillus natto (Fujita et al., 1993) and subtilisin D5 from B. amyloliquefaciens DJ-5 (Choi et al., 2009a) also showed high activity for this substrate of chymotrypsin.

N-Terminal amino acid sequence of FIS-WRL101
The N-terminal amino acid sequence of FIN-WRL101 was analyzed by the automated Edman degradation  method after SDS-PAGE and electroblotting.The sequence of the first 13 residues was found to be AQSVPYGISQIKA, which is identical to that of subtilisin NAT (formerly designated Nattokinase from B. subtilis natto) (Fujita et al., 1993), subtilisin E (from B. subtilis sp.) (Wong et al., 1984) and subtilisin D5 (from B. amyloliquefaciens DJ-5) (Choi et al., 2009a) (Table 5).Amino acids A-Q (positions 1 and 2) and I-K-A (positions 11, 12 and 13) are the almost conserved amino acid residues of the N-terminal sequence of these subtilisins from Bacillus spp.Together, the results for synthetic substrate specificity, effect of inhibitors, and the Nterminal amino acid sequences, indicate that FIN-WRL101 is a subtilisin-like serine-type fibrinolytic enzyme that occurs as a monomer.

Fibrinogenolytic activity of FIN-WRL101
FIN-WRL101 showed high fibrinogenolytic activity, degrading predominantly the Aα-chain of human fibrinogen within 10 min.By comparison, FIN-WRL101 degraded the B-chain slowly and did not cleave the chains (Figure 5), indicating that it is an -fibrinogenase and different from subtilisin BPN' and FS33 (Wang et al., 2006).
In general, fibrin(ogen)olytic enzymes belong to as two classes, the ()-fibrinogenases (known as zincmetalloproteinases) and the -fibrinogenases (known as thermostable serine proteinases) (Siigur et al., 1996;Lee et al., 1999).On the basis of these results, FIN-WRL101 is a serine-type alkaline chymotrypsin-like ()fibrinogenase.Furthermore, our results demonstrate that FIN-WRL101 is highly specific for the A-chain of human fibrinogen.Hence, this study highlights the potential for FIN-WRL101 as an effective thrombolytic agent.Investigations to further characterize FIN-WRL101 are underway.
determined.The nucleotide sequence revealed only one open reading frame (ORF), composed of 1143 base pairs and 381 amino acid residues, which proved to be identical to that of subtilisin NAT (Nakamura et al., 1992), J (Jang et al., 1992) and amylosacchariticus (Kurihara et al., 1972), but differed from that of subtilisin BPN' (Kaneko et al., 1989) (1146 base pairs and 382 amino acids).The amino acid sequence of FIN-WRL101 was compared with the published sequences of the other subtilisins (Figure 6), and was found to show 98.7% identity with subtilisin NAT, 98.2% with subtilisin J, 98.4% with amylosacchariticus, 86.1% with subtilisin BPN' and 65.6% with subtilisin Carlsberg.FIN-WRL101 was found to be identical to subtilisin NAT, except for five amino acid substitutions (boxed); one in the signal peptide region and four in the mature subtilisin region (Ala-107, underlined).The catalytic triad (Asp-32, His-64 and Ser-221, using the numbering of mature FIN-WRL101) and the amino acid sequences near these amino acid residues were well conserved (Figure 6).Further works should be done concerning the performance of FIN-WRL101 enzyme in vivo.

Figure 1 .
Figure 1.Fibrinogenolysis or fibrinolysis shown by Bacillus subtilis WRL101.Fibrinogen (A and B) or fibrin (C and D) (1.0 %) contained TSB media.A and C show control media (before seeding of B. subtilis WRL101).B and D show the degradation of fibrinogen (B) or fibrin (D) by B. subtilis WRL101.

Figure 4 .
Figure 4. Effects of pH (A) and temperature (B) on the activity of FIN-WRL101.The enzyme solutions were incubated with buffer of various pHs, at 4 o C for 24 h.For the effect of temperature, the enzyme solutions were incubated at the indicated temperature for 30 min.

Table 2 .
Effects of metal ions and inhibitors on the activity of FIN-WRL101.

Table 3 .
Comparison of FIN-WRL101 with other proteases for activity.

Table 5 .
N-terminal amino acid sequence and molecular mass of FIN-WRL101, compared with other fibrinolytic enzymes in literature data.