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Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp.strain CK 11-4


APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1996, p. 2482–2488 0099-2240/96/$04.00 0 Copyright 1996, American Society for Microbiology

Vol. 62, No. 7

Puri?cation and Characterization of a Fibrinolytic Enzyme Produced from Bacillus sp. strain CK 11-4 Screened from Chungkook-Jang
WONKEUK KIM,1* KEEHYUN CHOI,1 YONGTAEK KIM,1 HYUNGHWAN PARK,1 JANGYOON CHOI,1 YOONSOO LEE,1 HOONIL OH,2 IKBOO KWON,1 AND SHINYOUNG LEE3 Department of Biotechnology, Institute of R & D, Lotte Group, Yangpyung-Dong, Youngdeungpo-Gu, Seoul,1 Department of Fermentation Engineering, Kangweon National University, Hyojah-Dong, Chuncheon-Shi, Kangwon-Do,3 and Department of Food Science & Technology, Sejong University, Kwangjin-Gu, Seoul,2 (South) Korea
Received 18 December 1995/Accepted 19 April 1996

Bacillus sp. strain CK 11-4, which produces a strongly ?brinolytic enzyme, was screened from ChungkookJang, a traditional Korean fermented-soybean sauce. The ?brinolytic enzyme (CK) was puri?ed from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong ?brinolytic activity. The optimum temperature and pH were 70 C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ?rst 14 amino acids of the N-terminal sequence of CK are Ala-Gln-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of ?brinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys- NA). A similar result was also obtained from ?brin plate analysis. Bacillus spp. produce a variety of extracellular and intracellular proteases. An alkaline protease (subtilisin), a neutral metalloprotease, and an esterase are secreted into media, whereas at least two intracellular serine proteases are produced within Bacillus spp. (8, 16, 24, 25, 27, 30). In particular, the production of subtilisin protease has been exploited commercially for use in laundry detergents and for other applications (6, 10). The usage of protease for thrombolytic therapy by oral administration has been assessed (26, 33, 37). Blood clots (?brin) are formed from ?brinogen 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 ?brin clot formation and ?brinolysis are maintained in balance by the biological system, thromboses, such as myocardial infarction, occur when clots are not lysed as a result of a disorder of the balance (39). Intravenous administration of urokinase and streptokinase has been widely used for thrombosis therapy but these enzymes have a low speci?city to ?brin and are expensive. tPA has been developed for the treatment of thrombosis because of its ef?cacy and stronger af?nity to ?brin (27). Oral administration of the ?brinolytic enzyme nattokinase (NK) (32), revealed to be the same as subtilisin NAT (19) and which is produced from Bacillus NAT in the traditional Japanese fermented food, Natto, has been reported to enhance ?brinolytic activity in plasma and the production of tPA (31). A ?brinolytic enzyme produced from Bacillus subtilis has also been reported (5), but it did not show the same level of plasminogen activator activity as does NK.
* Corresponding author. Mailing address: Department of Biotechnology, Lotte Group Institute of R & D, 23, 4-Ga, Yangpyung-Dong, Youngdeungpo-Gu, Seoul, (South) Korea. Phone: (02) 670-6533. Fax: (02) 634-6184. Electronic mail address: bio00@bora.dacom.co.kr. 2482

Bacillus sp. strain CK 11-4, which produces a strongly ?brinolytic enzyme, was screened in our laboratory from Chungkook-Jang, a traditional Korean fermented-soybean sauce, to develop the enzyme for use as a thrombolytic agent. In this paper, we report the puri?cation and some characteristics of the ?brinolytic enzyme produced from Bacillus sp. strain CK 11-4.
MATERIALS AND METHODS Strain. Bacillus sp. strain CK 11-4 producing ?brinolytic enzyme (CK) was isolated from Chungkook-Jangs collected from various regions in Korea. Enzyme production. Bacillus sp. strain CK 11-4 was grown on basal medium containing 0.3% beef extract, 0.5% peptone, 1% soytone, and 1% milk casein. The pH was adjusted to 7.0 with 1 M HCl or 1 M NaOH. For the seed culture, one colony per plate was inoculated into 5 ml of basal medium and incubated at 37 C in a shaking water bath for 16 h. The seed culture broth (1 ml) was transferred to 1 liter of basal medium in a jar fermenter and fermented at 40 C, at an air ?ow rate of 1 vol/vol/min, for 16 h. Crude enzyme preparation. Cells were separated from the 1-liter culture broth by centrifugation (10,000 g, 15 min), and the supernatant ?uid was added to 3 volumes of acetone. The mixture of supernatant and acetone was allowed to stand at 4 C for 1 day. After centrifugation (10,000 g, 15 min) of the mixture, the resultant precipitate was lyophilized and used for the following experiments. Enzyme assay. Fibrinolytic activity was determined by both the modi?ed plasminogen-free ?brin plate method and the plasminogen-rich ?brin plate method (1) by using 1 U of plasmin (Sigma, St. Louis, Mo.) per ml as a standard ?brinolytic protease. The ?brinogen (plasminogen-free) solution [2.5 ml of 1.2% (wt/vol) human ?brinogen (Sigma) in 0.1 M sodium phosphate buffer, pH 7.4] was mixed with 0.1 ml of thrombin solution (100 NIH U/ml; Sigma) and 7.4 ml

TABLE 1. Puri?cation steps of CK from Bacillus sp. strain CK 11-4
Step Vol (ml) Total protein (mg) Total activity (U) Sp act (U/mg) Fold puri?cation Yield (%)

Culture broth Acetone precipitation CM-cellulose Toyo-pearl HW 55

500 10 10 1

48 45.4 14.2 6.1

920 915 902 874

19.2 20.2 63.5 143.3

1 1.1 3.3 7.5

100 99.5 98 95

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a ?nal inhibitor concentration of 0.1 or 1.0 mM. Enzyme samples were separately incubated at 37 C for 10 min with each of the following inhibitors (3): phenylmethylsulfonyl ?uoride (a serine protease inhibitor), EDTA, ε-aminocaproic acid, E64 [an effective cysteine protease inhibitor produced by Aspergillus japonicus TPR-64, L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-buthan], pepstatin A (a transition state analog that is a potent inhibitor of cadepsin D, pepsin, rennin, and many microbial aspartic proteases), and 2,4-dinitrophenol. Residual activity was then determined. Determination of the N-terminal amino acid sequence of the enzyme. After SDS-PAGE, puri?ed enzyme on polyacrylamide gel was transferred to a polyvinylidene di?uoride membrane by electroblotting (17) and stained with Ponceau S solution containing 5% acetic acid. The stained portion was excised and used for N-terminal sequencing directly. Restriction enzyme mapping of CK gene. PCR was performed with chromosomal DNA of Bacillus sp. strain CK 11-4 by using primer 1, 5 -ATGATGAGG AAAAAGAGTTTTTGGC-3 , and primer 2, 5 -CATCCGACCATAATGGAA CGGATTC-3 . The resultant PCR product was isolated and inserted in pGEM7Zf( ) vector (Promega) at an SmaI site and transformed to Escherichia coli JM109. Plasmid containing PCR product was obtained from transformant culture and analyzed with several restriction enzymes.

RESULTS Puri?cation of ?brinolytic enzyme from Bacillus sp. strain CK 11-4. Strain CK 11-4 is an aerobic, spore-forming, grampositive, motile, rod-shaped, and catalase- and oxidase-producing microorganism. It is clear that the bacterium should belong to the genus Bacillus. Temperature and pH ranges for growth were 28 to 55 C and pH 6 to 10, respectively (41). CK was puri?ed from the culture supernatant of Bacillus sp. strain CK 11-4 by the procedure described in Materials and Methods. Its puri?cation procedure is summarized in Table 1. CK was easily and quickly puri?ed by consecutive chromatog-

FIG. 1. Effect of pH on ?brinolytic activity and stability of CK from Bacillus sp. strain CK 11-4 at 30 C. F, activity; E, stability.

of 1% (wt/vol) agarose solution in a petri dish (100 by 15 mm). In the case of plasminogen-rich ?brin plate, 2.0 ml of ?brinogen (1.5% [wt/vol]) and 0.5 ml of plasminogen (10 U/ml) were used. After the dishes were allowed to stand for 30 min at room temperature to form ?brin clots, ?ve holes were made on a ?brin plate by suction by using a capillary glass tube (1-mm-diameter). Two microliters of sample solution was dropped into each hole and incubated at 37 C for 8 h. After measuring the dimension of the clear zone, the number of units was determined according to standard curve by using plasmin. Caseinolytic activity was assayed by the following procedure. A mixture (1 ml) containing 0.7 ml of 0.1 M sodium phosphate buffer (pH 7.5), 0.1 ml of 2% -casein, and 0.1 ml of enzyme solution was incubated for 5 min at each temperature, mixed with 0.1 ml of 1.5 M trichloroacetic acid, allowed to stand at 4 C for 30 min, and then centrifuged at room temperature. The A275 for the supernatant was measured and converted to the amount of tyrosine equivalent. One unit of caseinolytic activity (CU) was de?ned as the amount of enzyme releasing 1 mol of tyrosine equivalent per min. Amidolytic activity was measured spectrophotometrically by using chromogenic substrates as follows. The reaction mixture (1 ml) contained 20 l of enzyme solution, 5 10 4 M substrate, and 0.1 M sodium phosphate buffer (pH 7.4). After continuous measurement for 5 min at 37 C with a spectrophotometer equipped with a cuvette temperature controller, the amount of -nitroaniline that was liberated was determined from the A405. One unit of amidolytic activity (AU) was expressed as micromoles of substrate hydrolyzed per minute per milliliter by the enzyme. Each value is the mean of three determinations. Enzyme puri?cation. Crude enzyme was dialyzed against 5 liters of 10 mM glycine-NaOH buffer (pH 10.0, four buffer changes for 12 h each). The dialysate was adjusted to pH 6.0 with 0.2 M HCl, mixed for 2 h with 500 ml of carboxymethyl cellulose equilibrate with buffer A (10 mM sodium phosphate buffer, pH 6.0), and ?ltered through ?lter paper (Whatman No. 1). The cellulose was washed three times with 500 ml of buffer A, packed into a glass column (5 by 60 cm) and eluted by a 0 1 N NaCl gradient at a rate of 2.0 ml/min at 4 C. The active fractions (220 ml) were added to 660 ml of acetone and allowed to stand at 4 C for 18 h. The precipitates were collected by centrifugation and then lyophilized. For further puri?cation, gel ?ltration with Toyo-pearl HW 55 gel equilibrated with 10 mM glycine-NaOH buffer (pH 10.0) was performed. The active fractions were precipitated with acetone and then lyophilized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done according to Laemmli (15) by using a 10 to 20% gradient polyacrylamide gel and a 4% stacking gel at 4 C. Effect of pH on the ?brinolytic activity of CK. Because ?brin is insoluble, 800 l of ?brinogen solution (2%) was added to 100 l of thrombin solution (100 NIH U/ml). The mixture was incubated at 37 C for 1 h and then centrifuged. The precipitate was washed twice with 1 ml of each buffer and vortexed for dispersion of ?brin formed in the reaction. Puri?ed enzyme was dissolved either in 0.1 M sodium acetate buffer (pH 4.0 to 6.0), sodium phosphate buffer (pH 6.5 to 8.0), or glycine-NaOH buffer (pH 8.5 to 12.0). The enzyme solution (100 l) was then added to the dispersed ?brin solution. Other reaction conditions were the same as described earlier. Effect of protease inhibitor. Puri?ed and lyophilized enzyme was dissolved in 10 mM glycine-NaOH buffer (pH 10.0) and mixed with each salt solution to give

FIG. 2. Effect of temperature on ?brinolytic activity (a) and stability (b) of CK at pH 10.0. (b) F, 40 C; s, 50 C; ?, 60 C; ?, 70 C; }, 80 C.

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KIM ET AL. TABLE 2. Effect of protease inhibitor on CK activitya

APPL. ENVIRON. MICROBIOL.

Inhibitorb 0.1 mM

Concn 1.0 mM

DNP ε-ACA EDTA PMSF E64 Pepstatin A
a b

92.6 97.8 97.1 41.4 96.7 92.4

90.4 96.1 78.6 0 92.6 38.2

Reaction conditions described in Materials and Methods. DNP, 2,4-dinitrophenol; ε-ACA, ε-aminocaproic acid; PMSF, phenylmethylsulfonyl ?uoride.

raphy with carboxymethyl cellulose and Toyo-pearl HW 55. The ?nal speci?c activity of the puri?ed enzyme increased more than 7.5-fold, and its protein content was about 12.7% on the basis of content in the culture supernatant (Table 1). The puri?ed sample of CK migrated as a single protein band (Fig. 8), and its apparent molecular weight was estimated to be approximately 28,200 by SDS-PAGE. From the results of gel chromatography, activity staining (renature SDS-PAGE; data not shown), and denature SDS-PAGE, CK was considered to be a monomeric protein. Effect of pH on ?brinolytic activity and stability of CK. The optimum pH for the ?brinolytic activity of CK was around 10 to 12, and the enzyme activity decreased rapidly at levels below pH 6.0. As shown in Fig. 1, the enzyme was very stable in the range of pH 7 to 10.5 at 30 C for 20 h. Above pH 11.0, enzyme stability abruptly decreased. Although CK showed high activity around pH 10 to 12 in the presence of substrate, its stability was decreased during incubation in the absence of substrate at a pH above 11.0. Effect of temperature on ?brinolytic activity and stability of CK. The effect of temperature on the ?brinolytic activity of CK was examined at pH 10.0 (Fig. 2a). The temperature showing the maximal enzyme activity was 70 C. After a 60-min incubation, CK was very stable at 40 C, and showed 58.7, 48.6, 32.8, and 31.2% residual activity at 50, 60, 70, and 80 C, respectively (Fig. 2b). Effect of inhibitors on CK activity. When the enzyme (600 U/ml) was incubated at room temperature for 10 min in 10 mM glycine-NaOH buffer (pH 10.0) with 1 mM phenylmethylsulfonyl ?uoride, enzyme activity was completely inhibited. Enzyme activity was partially inhibited by EDTA, ε-aminocaproic acid, E64, and pepstatin A, but 2,4-dinitrophenol did not signi?cantly inhibit CK (Table 2). In view of the effects of pH, temperature, and inhibitors, CK can apparently be classi?ed as a thermophilic alkaline serine protease. Comparison of CK with other proteases for ?brinolytic ac-

tivity. The ?brinolytic activity of CK was compared with that of other proteases that were already known. After the activity units of each enzyme were converted to caseinolytic activity for uni?cation of the enzyme unit, ?brinolytic activities were measured and the ratios of ?brinolytic activity to caseinolytic activity were calculated. As shown in Table 3, ?brinolytic activity of CK was 2.6-, 3.2-, and 7.9-fold higher than that of the fungal protease of Aspergillus oryzae, trypsin from bovine pancreas, and subtilisin Carlsberg, respectively. Comparison of CK with other proteases on amidolytic activity on several synthetic substrates. Amidolytic activity of CK was compared with that of NK, subtilisin BPN , and subtilisin Carlsberg on several chromogenic substrates (Table 4). CK, subtilisin BPN , and subtilisin Carlsberg showed no activity on the synthetic substrate for trypsin (Bz-DL-Arg- NA), but NK showed some activity. CK, NK, and subtilisin BPN showed no activity when the synthetic substrate for urokinase (pyro-GluGly-Arg- NA) was used, but subtilisin Carlsberg showed some activity. CK had a similar level of amidolytic activity to that of subtilisin BPN , and a higher speci?city for the synthetic substrate for plasmin (H-D-Leu-Lys- NA) than did NK. N-terminal amino acid sequence of CK. After SDS-PAGE, puri?ed enzyme on polyacrylamide gel was transferred to a polyvinylidene di?uoride membrane by electroblotting and stained with Ponceau S. The stained portion was cut out and used for N-terminal sequencing. The N-terminal amino acid sequence of the ?rst 14 residues of CK was analyzed by a protein sequencer. The sequence was Ala-Gln-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp, which is identical to that of subtilisin Carlsberg but different from that of NK, as shown in Fig. 3. Although the N-terminal sequence of the ?rst 14 amino acids of CK and subtilisin Carlsberg are identical, the enzymes showed different substrate speci?city. Comparison of restriction map of the CK gene with that of subtilisin Carlsberg and its isoforms. For the differentiation of CK and subtilisin Carlsberg, PCR was performed ?ve times to eliminate Taq DNA polymerase error. Each PCR product was inserted into pGEM-7Zf( ) vector and transformed to E. coli JM109. Each plasmid containing the PCR product of the CK gene was digested with several restriction enzymes, and the same results were obtained from all of the restriction maps. A comparison with the restriction enzyme maps of subtilisin Carlsberg and its isoforms, shown in Fig. 4, indicates that the restriction enzyme sites of the CK gene are different from that of Bacillus licheniformis NCIB 6816 (subtilisin Carlsberg) (9) at the PstI, AccI, and HindII sites. Differences are also evident with B. licheniformis 11594 (20) at the NcoI and AccI sites, with B. licheniformis 14353 (21) at the PstI site, and with B. licheniformis 15413 (22) at the HindII, EcoRI and NdeI sites. On the restriction enzyme map, CK was most similar to that of B.

TABLE 3. Comparison of CK with other proteases for ?brinolytic activitya
Protease Caseinolytic activity (U) Fibrinolytic activity (U) Fibrinolytic/caseinolytic activity (%)

Protease from Bacillus sp. strain CK 11-4 Protease from B. licheniformis (type VIII) Protease from A. oryzae (type XIII) Trypsin from bovine pancreas (type I) Protease (K) from Tritirachium album (type XXVIII) Protease from Streptomyces griseus (type XXI) Subtilisin BPN (type XXVII) Subtilisin Carlsberg (type VIII)
a

352 423 127 334 426 395 438 325

257 41 35 75 129 80 142 30

73.0 (100) 9.7 (13.3) 27.6 (37.8) 22.5 (30.8) 15.3 (20.9) 20.3 (27.8) 32.4 (44.4) 9.2 (12.6)

Reaction conditions described in Materials and Methods.

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TABLE 4. Comparison of speci?city of CK with nattokinase, subtilisin BPN , and subtilisin Carlsberg with chromogenic synthetic substrates
Enzyme activitya Substrate CK ( mol/min/ml) Nattokinaseb (nmol/min/ml) Subtilisin BPN ( mol/min/ml) Subtilisin Carlsberg ( mol/min/ml)

H-D-Val-Leu-Lys- NAc Bz-DL-Arg- NAd H-D-Phe-Pip-Arg- NAe H-D-Val-Leu-Arg- NA pyro-Glu-Gly-Arg- NAf
a b c

424.3 (100) 0 (0) 21.7 (5.1) 16.9 (4.0) 0 (0)

68.5 (100) 18.0 (26.3) 14.0 (20.4) 13.5 (19.7) 0 (0)

119.7 (100) 0 (0) 6.8 (5.7) 3.4 (2.6) 0 (0)

462.5 (100) 0 (0) 50.3 (10.9) 25.9 (5.6) 19.6 (4.2)

Values in parentheses are percentages calculated on the basis of enzyme activity to H-D-Val-Leu-Lys- NA. Reference 27. Synthetic substrate for plasmin. d Synthetic substrate for trypsin. e Synthetic substrate for thrombin. f Synthetic substrate for urokinase.

licheniformis 14353, but the ?rst amino acid of the N-terminal amino acid sequence of CK is alanine rather than glycine, as it is for the B. licheniformis 14353 protease. These results demonstrate that CK is not identical to subtilisin Carlsberg or its isoforms. Effect of plasminogen addition on amidolytic activity of CK. When CK was added to the mixture of substrate solution (H-D-Val-Leu-Lys- NA) and plasminogen, amidolytic activity increased about twofold at the initial stage of the reaction compared with when CK alone was added to the substrate solution (Fig. 5). A similar result was obtained from ?brin plate analysis (Fig. 6, Table 5). In plasminogen-free ?brin plate, the ?brinolytic activity of a mixture of CK and plasminogen showed a 1.6-fold increase above the level of ?brinolytic activity with CK alone. In plasminogen-rich ?brin plate, the ?brinolytic activity of CK was 1.5-fold higher than the activity of CK in plasminogen-free ?brin plate. When the activity was spectrophotometrically measured on the range of 0.000 0.035 with a small amount of CK (0.1 U) added to the mixture of plasminogen and substrate, a stair-like reaction curve, with repeated and rather random increasing reaction-rate and decreasing reaction-rate stages, was obtained (Fig. 7). This phenomenon apparently appeared in the initial state of the reaction, and disappeared by degrees during further incubation time until the reaction rate was similar to that of the control. The mixture of CK and plasminogen was analyzed by SDSPAGE after incubation for 2 min (Fig. 8). Plasminogen was degraded by CK, and the bands of degraded products showed approximate molecular weights of 25,600, 34,600, and 37,600 in

comparison with a CK band of 28,200. In the products from plasminogen, a product with a molecular weight of about 28,200 was newly produced when that was compared with the addition amount of CK. DISCUSSION This article describes the puri?cation and characterization of CK produced from Bacillus sp. strain CK 11-4 for assessment of its application as a thrombosis agent. As mentioned above, intravenous administration of urokinase and streptokinase has been widely used for thrombosis therapy. Fibrinolytic therapy by oral drug administration has been recently investigated in animal models in which enteric-coated urokinase capsules were given to normal and experimental dogs with saphenous vein thrombosis (34). Sumi et al. reported that intravenous administration did not show any clear thrombolytic effect, but oral administration enhanced ?brinolytic activity. In another study, Sumi et al. (31) reported that when NK was given to human subjects by oral administration, ?brinolytic activity and the amounts of tPA and ?brin degradation product in plasma increased about twofold. On the basis of these reports, strains producing ?brinolytic enzyme were isolated from Chungkook-Jangs, a traditional Korean soybean-fermented food, obtained from various regions of Korea. Among them, strain CK 11-4 showed the strongest ?brinolytic activity and was identi?ed as a Bacillus spp. CK was puri?ed from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic-hydrophilic and strong ?brinolytic activity. The N-terminal sequences of the ?rst 14 amino acids of CK

FIG. 3. Comparison of N-terminal amino acid sequence of CK with those of other proteases. CK, extracellular protease from Bacillus sp. strain CK 11-4 (this study); Carls, subtilisin Carlsberg (extracellular protease from B. licheniformis [24]); BPN , subtilisin BPN (extracellular protease from B. amyloliquefaciens [34]); NAT, subtilisin NAT [NK, extracellular protease from B. subtilis (natto) (16)]; Mesen, extracellular protease from Bacillus mesentericus (32); Amylosacc, extracellular protease from Bacillus amylosacchariticus (38); SubE, subtilisin E (extracellular protease from B. subtilis [31]).

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FIG. 4. Comparison of restriction enzyme map of CK with that of subtilisin Carlsberg and its isoforms. Square box shows sequence of ?rst 14 amino acids. H3, HindIII; Sc, ScaI; ER, EcoRI; Sa, SalI; Ns, NsiI; Nd, NdeI; Ps, PstI; Ac, AccI; H2, HindII; and Nc, NcoI. Bar, 100 kb.

and subtilisin Carlsberg are identical. But because the enzymes showed different substrate speci?city and because the level of CK activity to ?brin was about eight times higher than that of subtilisin Carlsberg, these enzymes are not considered to be identical, in spite of the high homology of their amino acid sequences. For the differentiation of CK and subtilisin Carlsberg, a restriction enzyme map of the CK gene was compared with that of subtilisin Carlsberg and its isoforms. The restriction

enzyme sites of the CK gene were different from those of B. licheniformis NCIB6816 (subtilisin Carlsberg) at the PstI and AccI sites, as mentioned above. Full sequencing of the CK gene is proceeding, and a partial sequence (about 400 bp) has been obtained. This sequence is different from that of any other protease, although a high homology is evident (data not shown).

FIG. 5. Amidolytic activity of CK and a mixture of CK and plasminogen with chromogenic substrate (H-D-Val-Leu-Lys- NA). F, CK; E, CK and plasminogen (0.5 U).

FIG. 6. Fibrinolytic activity of CK, plasminogen, and a mixture of CK and plasminogen in a plasminogen-free ?brin agarose plate. C, 0.1 U of plasmin as control; 1, 0.33 U of CK; 2, mixture of CK (0.33 U) and plasminogen (0.5 U); 3, 0.5 U of plasminogen. The resultant data are shown in Table 5.

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TABLE 5. Fibrinolytic activity of CK, plasminogen, and CK-plasminogen mixture in ?brin agarose platea
Fibrin agarose plate Sample Fibrinolytic activity (U) Fold

Plasminogen-free Plasminogen-richc
a b c

CK CK CK

plasminogenb

0.331 0.536 0.494

1 1.6 1.5

See also Fig. 6. A total of 0.5 U of plasminogen was used. Plasminogen-rich ?brin plate contained 5 U (0.5 U/ml) of plasminogen per plate.

It is interesting that when CK was added to a mixture of plasminogen and substrate, ?brinolytic activity was increased relative to the control level, although plasminogen did not show activity. Was CK activated by digest products produced from plasminogen as stimulators? In that case, because CK reacted with two substrates, chromogenic substrate and plasminogen, the activity must be less than that for one chromogenic substrate reaction. When albumin or casein was added to the reaction mixture, the chromogenic activity decreased (data not shown). As shown in Fig. 7, at the reaction rate-increasing stage (arrow a), active product seemed to be produced from plasminogen by CK, but it was then degraded or inactivated (arrow b). For the fractionation, puri?cation, and N-terminal amino acid sequencing of active product(s) in the reaction mixture of CK and plasminogen, zymogram electrophoresis (13) was used. But except for an active band of CK, no other active band was found. When gel ?ltration chromatography with high performance liquid chromatography and capillary electrophoresis was used, an active band was not found either. So, as the stability of the active product was low and the quantity produced was small, the active product is likely to be dif?cult to detect. To our knowledge, this phenomenon has not been reported before.

FIG. 8. PAGE gel of puri?ed CK, plasminogen, and the products after reaction of CK and plasminogen. Lane 1, CK (2 g); lanes 2 and 6, molecular weight markers; lane 3, CK (1 g); lane 4, mixture of plasminogen (20 g) and CK (1 g) after 1-min incubation; lane 5, plasminogen (20 g); lane 7, CK (3 g).

There are several studies that have reported on the intestinal absorption of serum albumin (40), lipase (23), 131I-elastase (11), and Serratia protease (18). On the basis of perfusion experiments in dogs and rats, Kitaguchi et al. (12), Hijikata et al. (7) and Klocking et al. (14) have reported that several serine ¨ proteases release plasminogen activators. Bernik and Oller (2) observed activation of a plasminogen proactivator of the human kidney by trypsin treatment. In view of these reports, it can be suggested that CK can be given orally for use as a thrombolytic agent. Bacillus spp. have been recognized as being safe for humans (4). Future studies will test CK in vivo, and the cloning, sequencing, and expression of the CK gene from chromosomal DNA of Bacillus sp. strain CK 11-4 is proceeding.
REFERENCES 1. Astrup, T., and S. Mullertz. 1952. The ?brin plate method for estimating ¨ ?brinolytic activity. Arch. Biochem. Biophys. 40:346–351. 2. Bernik, M. B., and E. P. Oller. 1977. Regulation of ?brinolysis through activation and inhibitor of activator of plasminogen proactivator (preurokinase). Thromb. Haemostasis 38:136. 3. Beynon, R. J., and J. S. Bond (ed.). 1989. Proteolytic enzyme; a practical approach. IRL Press, Oxford. 4. de Boer, A. S., and B. Diderichsen. 1991. On the safety of Bacillus subtilis and B. amyloliquefaciens: a review. Appl. Microbiol. Biotechnol. 36:1–4. 5. Fayek, K. I., and S. T. El-Sayed. 1980. Fibrinolytic activity of an enzyme produced by Bacillus subtilis. Z. Ernaehrwiss. 19:21–23. 6. Godfrey, T., and J. R. Reichelt (ed.). 1983. Industrial enzymology. Nature Press, New York. 7. Hijikata, A., M. Hirata, and H. Kitaguchi. Effect of proteases on plasminogen activator release from isolated perfused dog leg. Thromb. Res. 20:521– 531. 8. Hiroshi, S., and H. Kadota. 1976. Intracellular proteases of Bacillus subtilis. Agric. Biol. Chem. 40:1047–1049. 9. Jacobs, M., M. Eliasson, M. Uhlen, and J.-I. Flock. 1985. Cloning, sequenc? ing, and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res. 13:8913–8926. 10. Kame, M., H. Koda, A. Kato, and T. Koma. 1973. Detergency and mechanism of soil removal in detergent-enzyme system. JAOCS 11:464–469. 11. Katayama, K., and T. Fujita. 1972. Studies on biotransformation of elastase. II. Intestinal absorption of 131I-labeled elastase in vivo. Biochim. Biophys. Acta 288:181–189. 12. Kitaguchi, H., A. Hijikata, and M. Hirata. 1979. Effect of thrombin on plasminogen activator release from isolated perfused dog leg. Thromb. Res. 16:407–420. 13. Kleiner, D. E., and W. G. Stetler-Stevenson. 1994. Quantitative zymography: detection of picogram quantities of gelatinase. Anal. Biochem. 218:325–329.

FIG. 7. Amidolytic activity of CK and a mixture of CK and plasminogen. 0.1 U of CK and 1.0 U of plasminogen were used in a narrow spectrometric range. E, CK; F, CK plus plasminogen; (a), reaction rate-increasing stage in which plasminogen was seemingly activated by CK; (b), reaction rate-decreasing stage in which activated plasminogen was seemingly degraded by CK.

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14. Klocking, H. P., C. Jablonowsky, and F. Markwardt. 1981. Studies on the ¨ release of plasminogen activator from the isolated rat lung by serine protease. Thromb. Res. 23:375–379. 15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685. 16. Mantsala, P., and H. Zalkin. 1980. Extracellular and membrane-bound pro¨ ¨¨ teases from Bacillus subtilis. J. Bacteriol. 141:493–501. 17. Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene di?uoride membrane. J. Biol. Chem. 262: 10035–10038. 18. Miyata, K., S. Hirai, T. Yashiki, and K. Tomoda. 1980. Intestinal absorption of Serratia protease. J. Appl. Biochem. 2:111–116. 19. Nakamura, T., Y. Yamagata, and E. Ichishima. 1992. Nucleotide sequence of the subtilisin NAT gene, arpN, of Bacillus subtilis (natto). Biosci. Biotechnol. Biochem. 56:1869–1871. 20. Pan, F. M., S. T. Chen, and S. H. Chiou. 1995. Sequence characterization of the precursor of one mutant subtilisin from Bacillus licheniformis (strain 11594). NCIB accession no. X91260. Unpublished data. 21. Pan, F. M., S. T. Chen, and S. H. Chiou. 1995. Sequence characterization of the precursor of one mutant subtilisin from Bacillus licheniformis (strain 14353). NCIB accession no. X91261. Unpublished data. 22. Pan, F. M., S. T. Chen, and S. H. Chiou. 1995. Sequence characterization of the precursor of one mutant subtilisin from Bacillus licheniformis (strain 15413). NCIB accession no. X91262. Unpublished data. 23. Papp, M., S. Feher, G. Folly, and E. J. Horvath. 1977. Absorption of pancreatic lipase from the duodenum into lymphatics. Experientia 33:1191– 1192. 24. Prestidge, L., V. Gage, and J. Spizizen. 1971. Protease activities during the course of sporulation in Bacillus subtilis. J. Bacteriol. 107:815–823. 25. Price, C. W., M. A. Gitt, and R. H. Doi. 1983. Isolation and physical mapping of the gene encoding the major factor of Bacillus subtilis RNA polymerase. Proc. Natl. Acad. Sci. USA 80:4074–4078. 26. Sasaki, K., S. Moriyama, Y. Tanaka, H. Sumi, N. Toki, and K. C. Robbins. 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. 27. Sherry, S. 1987. Recombinant tissue plasminogen activator(rt-PA): is it the thrombolytic agent of choice for an evolving acute myocardial infarction? Am. J. Cardiol. 59:984–989. 28. Smith, E. L., R. J. DeLange, W. H. Evans, M. Landon, and F. S. Markland. 1968. Subtilisin Carlsberg V. The complete sequence: comparison with subtilisin BPN ; evolutionary relationships. J. Biol. Chem. 243:2184–2191. 29. Srivastava, O. P., and A. I. Aronson. 1981. Isolation and characterization of

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