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Purification and biochemical characterization of a fibrinolytic enzyme


? 2001 Kluwer Academic Publishers. Printed in the Netherlands.

World Journal of Microbiology & Biotechnology 17: 89±92, 2001.

89

Puri?cation and biochemical characterization of a ?brinolytic enzyme from Bacillus subtilis BK-17
Yong Kee Jeong1,*, Jeong Uck Park2, Hyun Baek1, Sung Hoon Park3, In Soo Kong4, Dong Wan Kim5 and Woo Hong Joo6 1 Department of Microbiology, Dong-Eui University, Pusan 614-714, Korea 2 Department of Microbiology, College of Medicine, Gyeongsang National University, Chinju 660-280, Korea 3 Department of Chemical Engineering, Pusan National University, Pusan 609-735, Korea 4 Department of Biotechnology and Bioengineering, Pukyong National University, Pusan 608-737, Korea 5 Department of Microbiology, Changwon National University, Changwon 641-773, Korea 6 Department of Biology, Changwon National University, Changwon 641-773, Korea *Author for correspondence: Department of Biology, Dong-Eui University, Gayadong, Chingu, Pusan 614-714, Korea. Tel.: 82-51-890-1534, Fax: 82-51-894-0840, E-mail: ykjeong@hyomin.dongeui.ac.kr
Received 19 July 2000; accepted 17 January 2001

Keywords: Amidolytic activity, Bacillus subtilis BK-17, fibrinolytic enzyme, homologous proteins, N-terminal amino acid sequencing, protease inhibitor, thrombolytic agent

Summary A ?brinolytic enzyme from Bacillus subtilis BK-17 has been puri?ed to homogeneity by gel-?ltration and ionexchange chromatography. Compared to the crude enzyme extract, the speci?c activity of the enzyme increased 929fold with a recovery of 29%. The subunit molecular mass of the puri?ed enzyme was estimated to be 31 kDa by SDS±PAGE. The N-terminal amino acid sequence of the puri?ed ?brinolytic enzyme was: A-Q-S-V-P-Y-G-V-S-QI-K-A-P-A-A-H-N. The sequence was highly homologous to the ?brinolytic enzymes nattokinase, subtilisin J and subtilisin E from Bacillus spp. However, there was a substitution of three amino acid residues in the N-terminal sequence. The amidolytic activity of the puri?ed enzyme for several substrates was assessed. In comparison with nattokinase and CK (?brinolytic enzyme from a Bacillus spp.), which showed strong ?brinolytic activity, the amidolytic activity of the enzyme for the synthetic substrate, kallikrein (H-D-Val-Leu-Arg-pNA, S-2266) increased 2.4- and 11.8-fold, respectively. Introduction Fibrin is the main protein component of the blood clot, and it is normally formed from ?brinogen by the action of thrombin (EC 3.4.21.5). Accumulation of ?brin in the blood vessels usually increases thrombosis, leading to myocardial infarction and other cardiovascular diseases. The blood clot ?brin is lysed by plasmin, which is activated from plasminogen by tissue plasminogen activator (tPA) (Kim et al. 1997). The typical thrombolytic agents for therapeutic purposes include urokinase (Wun & Voet 1982; Sasaki et al. 1985) and a tissue-type plasminogen activator (tPA) (Pennica et al. 1983). These are plasmin activators and convert plasminogen to plasmin, which degrades ?brin. These activators are of human origin and generally safe, but are expensive. Some ?brinolytic enzymes produced by microbes have also been reported. Among them, streptokinase produced by Streptococcus haemolyticus (Medved et al. 1966) and staphylokinase produced by Staphylococcus aureus (Lijnen et al. 1991; Arai et al. 1995) have been studied most extensively. They are also plasminogen activators, but not in common use because of their side e?ects. Bacillus spp. produce a variety of extracellular and intracellular proteases including nattokinase (Nakamura et al. 1992), an enzyme from B. amylosaccharolyticus (Yoshimoto et al. 1988), Subtilisin J (Jang et al. 1992) and subtilisin E (Jain et al. 1998; Yang et al. 2000). The ?brinolytic enzymes from Bacillus sp. have attracted interest as thrombolytic agents because of their e?ciency in the ?brinolytic process including plasmin activation. They also have an industrial application in detergent production. The subtilisins from Bacillus sp. have been identi?ed as having speci?c protease activity (Vasantha et al. 1984; Wong et al. 1984; Nakamura et al. 1992). Kim et al. (1996) identi?ed a ?brinolytic enzyme from Bacillus sp. strain CK 11-4, which was isolated from a traditional Korean fermented-soybean sauce. The novel ?brinolytic enzyme was also puri?ed and characterized from Bacillus sp. KA 38, which originated from fermented ?sh (Kim et al. 1997).

90 In this paper, a ?brinolytic enzyme from B. subtilis BK-17, which was isolated from a decaying rice plant, was puri?ed and biochemically characterized as a potential thrombolytic agent. Materials and Methods Bacterial strain and culture condition Bacillus subtilis strain BK-17 producing a ?brinolytic enzyme was isolated from a decaying rice plant in Korea. The strain was aerobically cultured at 37 °C overnight using Luria±Bertani medium consisting of 1% Bacto-tryptone (Sigma, USA), 1% sodium chloride, and 0.5% Bacto-yeast extract (Sigma, USA). Enzyme puri?cation and electrophoresis All puri?cation steps were carried out at room temperature except for centrifugation, which was conducted at 4 °C. Protein concentration was measured by the Bradford method at each puri?cation step. The crude enzymes were saturated up to 75% by using ethanol and followed by centrifugation at 15,000 ? g for 30 min at 4 °C. The precipitate was then dialysed against 5 l of 20 mM Tris-HCl (pH 7.5) at 4 °C for 12 h. The enzyme solution was loaded on to a DEAE-Sephadex A-50 column pre-equilibrated with 20 mM Tris-HCl (pH 7.5). The eluates were passed through a Sephadex G-75 column using Tris-HCl bu?er and lyophilized. SDS± PAGE was done according to the method of Laemmli (1970) by using a 10±15% gradient polyacrylamide gel and 4% stacking gel at 4 °C. Assay of enzyme activity Fibrinolytic activity was determined by both the plasminogen-free ?brin plate method and the plasminogen-rich ?brin plate method (Astrup & Mullertz 1952). Pla? sminogen-free ?brin plate was made up of the ?brinogen solution [2.5 ml of 1.2% human ?brinogen (Sigma, USA) in 0.1 M sodium phosphate bu?er, pH 7.4], 10 U of thrombin solution (Sigma, USA) and 1% agarose. Plasminogen-rich ?brin plate was made up 2 ml of 1.5% ?brinogen and 5 U plasminogen. The sterilized paper disc (5 mm in diameter) was overlayered on the ?brin plate. To observe the ?brinolytic activity of the enzymes, 100 ll of the puri?ed protein solution was carefully dropped on to the disc and incubated at 37 °C for 18 h. The activity of a ?brinolytic enzyme was determined by

Y.K. Jeong et al. measuring the dimension of the clear zone on the ?brin plate and plotting a standard curve made by varying the quantity of plasmin. Amidolytic activity was measured spectrophotometrically by using chromogenic substrates. The reaction mixture (1 ml) contained 20 ll of enzyme solution, 5 ? 10)4 M chromogenic substrate, and 0.1 M sodium phosphate bu?er (pH 7.4). After incubation for 5 min at 37 °C, the amount of p-nitroaniline that was liberated was determined from the spectrophotometric absorption at 405 nm. One unit of amidolytic activity was expressed as nmol of substrate hydrolysed per min per ml by the enzyme. N-terminal amino acid sequencing The puri?ed ?brinolytic enzyme on SDS±PAGE gel was electroblotted on to a polyvinylidene di?uoride membrane (Bio-Rad, USA) and stained with Coomassie Brilliant Blue R-250. The stained protein portion was excised and the amino acids of the N-terminal sequence were determined by the automated Edman method using a gas-phase protein sequencer (Model 476A, Applied Biosystems, USA). E?ect of metal ions and protease inhibitors The e?ect of metal ions on the ?brinolytic activity was investigated by using CaCl2, CoCl2, CuSO4, MgCl2, MnSO4, FeCl3, and ZnCl2. The e?ect of the protease inhibitors were studied by using diisopropyl phosphoro?uoridate (DFP), tosyllysine chloromethyl-?uoride (TLCK), phenylmethane sulphonyl ?uoride (PMSF), eaminocaproic acid (e-ACA), t-4-aminomethyl-cyclohexane carboxylic acid (t-AMCHA), p-tosyl-L -arginine methylester hydrochloride (TAME), ethylene diamine tetraacetic acid (EDTA), aprotin, soybean trypsin inhibitor (SBTI), chymotrypsin, leupeptin, antipain, amido-4-glutaminobutane t-epoxysuccinyl-L -leucyl (t-ESLB). The concentrations of the metal ions and protease inhibitors in the reaction were 1 and 0.1 mM, respectively. Results Puri?cation of the ?brinolytic enzyme from B. subtilis BK-17 The ?brinolytic enzyme was puri?ed to electrophoretic homogeneity by the steps listed in Table 1. After the

Table 1. Puri?cation steps of the ?brinolytic protease from B. subtilis BK-17. Step Culture broth Ethanol precipitation DEAE Sephadex A-50 Sephadex G-75 Total protein (mg) 5099.0 477.5 30.5 1.6 Total activity (U) 345,000 300,400 289,000 9,980 Speci?c activity (U/mg) 68 630 9,475 63,165 Yield (%) 100 87 83 29 Puri?cation (Fold) 1 9 139 929

A ?brinolytic enzyme from B. subtilis ammonium sulphate precipitation, subsequent DEAESephadex A-50 chromatography and Sephadex G-75 gel ?ltration were used to purify the enzyme to homogeneity. The DEAE-Sephadex A-50 anion exchange column yielded two major peaks; one showing high ?brinolytic activity, the other showing no activity. The eluted proteins were subjected to SDS±PAGE, showing two bands (Figure 1, Lane b). The speci?c activity of the ?brinolytic enzyme increased 139-fold with a 83% yield on the basis content in the culture supernatant. After the elution of the ?brinolytic enzyme by Sephadex G-75 gel ?ltration, the speci?c activity of the protein increased to 932-fold with a 29% yield. The apparent subunit molecular weight of the puri?ed ?brinolytic enzyme was estimated to be approximately 31 kDa (Figure 1, Lane c), which was distinguished from the subunit molecular weight of other ?brinolytic enzymes derived from Bacillus spp. (Yoshimoto et al. 1988; Jang et al. 1992; Nakamura et al. 1992; Jain et al. 1998). N-terminal amino acid sequencing As shown in Figure 2, the ?rst 18 amino acid residues of N-terminal sequence of the puri?ed ?brinolytic enzyme were A-Q-S-V-P-Y-G-V-S-Q-I-K-A-P-A-H-A-N-A-AH-N. The sequence of the 31 kDa enzyme was used to search for homology of the N-terminal amino acid sequence with that of other known ?brinolytic enzymes (Altschul et al. 1990). The amino sequence of the enzyme showed approximately 83% homology with the N-terminal sequence of nattokinase, the enzyme from B. amylosaccharolyticus, subtilisin J, and subtilisin

91

Figure 2. Comparison of BK with other proteases for N-terminal amino acid sequence. The consensus sequences of the puri?ed 31 kDa ?brinolytic enzyme, nattokinase precursor, amylosacchariticus precursor, subtilisin J, and subtilisin E from Bacillus spp. are indicated by the box.

E from Bacillus spp. respectively, carrying a substitution of 3 amino acid residues in the N-terminal sequence. The eighth amino acid residue, isoleucine in the sequence of the above enzymes was substituted by valine. The ?brinolytic enzyme also carried alanine and asparagine instead of leucine and serine in the sixteenth and eighteenth amino acid residues of the sequence, respectively. However, no other ?brinolytic enzyme was homologous to the 31 kDa protein with regard to N-terminal amino acid sequence. E?ect of metal ions and protease inhibitors The e?ect of metal ions such as Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+ and Zn2+ on the ?brinolytic activity was carefully examined. None of the ions activated the ?brinolytic activity of the 31 kDa enzyme. Mg2+, Mn2+, and Co2+ ions did not have any signi?cant e?ect on the enzyme activity. However, Ca2+, Cu2+ and Fe2+ ions inhibited the ?brinolytic enzyme 29, 25 and 29%, respectively. Of note, Zn2+ inhibited the enzyme up to 39%. The in?uence of the protease inhibitors including TLCK, e-ACA, p-tosyl-L -arginine methyl ester hydrochloride, chymostatin, leucopeptin and antipain on the activity was also observed. The activity of the puri?ed ?brinolytic enzyme was signi?cantly decreased by chymostatin. However, the other inhibitors did not have a notable effect on the protease activity. Discussion As shown in Table 2, the amidolytic activity of the puri?ed ?brinolytic enzyme on several chromogenic substrates was compared with that of nattokinase, which showed strong ?brinolytic activity for the use of oral thrombosis therapy (Sumi et al. 1983; Toki et al. 1985), and CK which was one of the ?brinolytic enzymes recently isolated from Bacillus sp. (Kim et al. 1996). As shown in Table 2, the activity of the 31 kDa enzymes on the synthetic substrate for plasmin (H-D-VL-pNA) was increased 1.3-fold (Table 2). However, the enzyme did not show any amidolytic activity on the synthetic substrate for urokinase (pyro-Glu-Gly-

Figure 1. Polyacrylamide gel electrophoresis of the puri?ed ?brinolytic enzyme. Lane a, protein molecular mass markers; lane b, eluted proteins through DEAE-anion exchange column; lane c, puri?ed ?brinolytic enzyme from DEAE-anion exchange columm and Sephadex G-75 gel ?ltration. The arrow indicates the puri?ed ?brinolytic enzyme from B. subtilis BK-17.

92
Table 2. Amidolytic activity of the 31 kDa ?brinolytic enzyme, nattokinase and CK for the synthetic substrates. Substrate Enzyme activity 31 kDa enzyme Nattokinase CK (nM/min/ml) (nM/min/ml) (lM/min/ml) H-D-Val-Leu-Lys-pNAa H-D-Phe-Pip-Arg-pNAb H-D-Val-Leu-Arg-pNAc H-D-He-Pro-Arg-pNAd 90 10 42 45 (100) (11) (47) (50) 69 (100) 14 (20.4) 14 (19.7) ND 424 (100) 21 (5.1) 17 (4.0) ND

Y.K. Jeong et al.
Astrup, T. & Mullertz, S. 1952 The ?brin plate method for estimating ? ?brinolytic activity. Archives of Biochemistry and Biophysics 40, 346±351. Jain, S.C., Shinde, U., Li, Y., Inouye, M. & Berman, H.M. 1998 The crystal structure of an autoprocessed Ser221Cys-subtilisin. Epropeptide complex at 2.0-A resolution. Journal of Molecular Biology 284, 137±144. Jang, J.S., Kang, D.O., Chun, M.J. & Byun, S.M. 1992 Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis. Biochemical and Biophysical Research Communications 184, 277±282. Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Oh, H., Kwon, I. & Lee, S. 1996 Puri?cation and characterization of a ?brinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Applied and Environmental Microbiology 62, 2482±2488. Kim, H.K., Kim, G.T., Kim, D.K., Choi, W.A., Park, S.H., Jeong, Y.K. & Kong, I.S. 1997 Puri?cation and characterization of a novel ?brinolytic enzyme from Bacillus sp. KA38 originated from fermented ?sh. Journal of Fermentation and Bioengineering 84, 307±312. Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680±685. Lijnen, H.R., van Hoef, B., de Cock, F., Okada, K., Ueshima, S., Matsuo, O. & Collen, D. 1991 On the mechanism of ?brin-speci?c plasminogen activation by staphylokinase. Journal of Biological Chemistry 239, 11,826±11,832. Medved, L.V., Solovjov, D.A. & Ingham, K.C. 1966 Domain structure, stability and interactions in streptokinase. European Journal of Biochemistry 239, 333±339. Nakamura, T., Yamagata, Y. & Ichishima, E. 1992 Nucleotide sequence of the subtilisin NAT gene, aprN of Bacillus subtilis (natto). Bioscience, Biotechnology and Biochemistry 56, 1869±1871. Pennica, D., Holmes, W.E., Kohr, W.J., Harkins, R.N., Vehar, G.A., Ward, C.A., Bennett, W.F., Yelverton, E., Seeburg, P.H., Heyneker, H.L., Goeddel, D.V. & Collen, D. 1983 Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 301, 214±221. Sasaki, K., Moriyama, S., Tanaka, Y., Sumi, H., Toki, N. & Robbins, K.C. 1985 The transport of 125I-labeled human high molecular weight urokinase across the intestinal tract in a dog model with stimulation of synthesis and/or release of plasminogen activators. Blood 66, 69±75. Sumi, H., Maruyama, M., Yoneta, T. & Mihara, H. 1983 Activation of plasma ?brinolysis after intrarectal administration of high molecular weight urokinase and its derivative. Acta Haematologica 70, 289±295. Toki, N., Sumi, H., Sasaki, K., Boreisha, I. & Robbins, K.C. 1985 Transport of urokinase across the intestinal tract of normal human subjects with stimulation of synthesis and/or release of urokinasetype proteins. Journal of Clinical Investigation 75, 1212±1222. Vasantha, N., Thompson, L.D., Rhodes, C., Banner, C., Nagle, J. & Filpula, D. 1984 Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. Journal of Bacteriology 159, 811±819. Wong, S.L., Price, C.W., Goldfarb, D.S. & Doi, R.H. 1984 The subtilisin E gene of Bacillus subtilis is transcribed from a sigma-37 promoter in vivo. Proceedings of the National Academy Sciences of the USA 81, 1184±1188. Wun, D. & Voet, J.G. 1982 Isolation and characterization of urokinase from human plasma. Journal of Biological Chemistry 257, 3276± 3283. Yang, Y., Jiang, L.L., Yang, S., Zhu, L., Yujie, W. & Zhenjie, L. 2000 A mutant subtilisin E with enhanced thermostability. World Journal of Microbiology and Biotechnology 16, 249±251. Yoshimoto, T., Oyama, H., Honda, T., Tone, H., Takeshita, T., Kamiyama, T. & Tsuru, D. 1988 Cloning and expression of subtilisin amylosacchariticus gene. Journal of Biochemistry 103, 1060±1065.

The values in parentheses are percentages calculated on the basis of enzyme activity to H-D-Val-Leu-Lys-pNA. Each value is the mean of three determinations. ND indicates `not determined'. a Synthetic substrate for plasmin. b Synthetic substrate for thrombin. c Synthetic substrate for kallikrein. d Synthetic substrate for serine protease.

Arg-pNA, S-2444). The puri?ed ?brinolytic enzyme showed the lowest amidolytic activity on the synthetic substrate for thrombin (H-D-Phe-Pip-Arg-pNA, S2238). Of note, the puri?ed ?brinolytic enzyme from B. subtilis BK-17 showed the highest amidolytic activity on the synthetic substrate for kallikrein (H-D-Val-LeuArg-pNA, S-2266). In comparison with nattokinase and CK, the activity of the 31 kDa enzyme for kallikrein increased 2.4- and 11.8-fold, respectively. The ?brinolytic enzyme formed a clear lysis zone on the plasminogen-rich and plasminogen-free ?brin plates. This result may re?ect that the ?brinolytic enzyme was able to degrade ?brin clots in two ways; one, by forming plasmin from plasminogen (i.e. plasminogen activator type), the other, without plasmin (direct ?brinolysis type). The ?rst 18 amino acid residues of N-terminal sequence of the 31 kDa protein carried signi?cant homology with the sequences of other ?brinolytic enzymes including nattokinase, the enzyme from B. amylosaccharolyticus, subtilisin J, and subtilisin E from Bacillus sp. except for a substitution of three amino acid residues (Figure 2). This ?nding may suggest that the 31 kDa ?brinolytic enzyme could have a horizontal evolution relationship with the other ?brinolytic enzymes. Acknowledgment This work was supported by a Grant-in-Aid from the Ministry of Agriculture, South Korea (1996).

References
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. 1990 Basic local alignment search tool. Journal of Molecular Biology 215, 403±410. Arai, K., Mimuro, J., Madoiwa, S., Matsuda, M., Sako, T. & Sakata, Y. 1995 E?ect of staphylokinase concentration of plasminogen activation. Biochimica et Biophysica Acta 1245, 69±75.


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