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Overexpression of aprE2, a Fibrinolytic Enzyme Gene from Bacillus subtilis CH3-5, in Escherichia coli and the Properties of AprE2
Overexpression of aprE2, a Fibrinolytic Enzyme Gene from Bacillus subtilis CH3-5, in Escherichia coli and the Properties of AprE2
Journal of Microbiology and Biotechnology. 2014. Jul, 24(7): 969-978
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : January 16, 2014
  • Accepted : April 11, 2014
  • Published : July 28, 2014
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About the Authors
Seon-Ju Jeong
Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea
Kye Man Cho
Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea
Chang Kwon Lee
Mong-Go Foods Co., Ltd., Changwon 641-465, Republic of Korea
Gyoung Min Kim
Namhae Garlic Research Institute, Namhae, Gyeongnam 668-812, Republic of Korea
Jung-Hye Shin
Namhae Garlic Research Institute, Namhae, Gyeongnam 668-812, Republic of Korea
Jong Sang Kim
School of Applied Biosciences and Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea
Jeong Hwan Kim
Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea
jeonghkm@gnu.ac.kr

Abstract
The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at 20℃, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at 37℃. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of 1,069.4 ± 42.4 U/mg protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and 40℃, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded Aα and Bβ chains of fibrinogen quickly, but not the γ-chain. AprE2 exhibited the highest specificity for N -succinyl-Ala-Ala-Pro-Phe-pNA. The K m and k cat /K m of AprE2 was 0.56 mM and 3.10 × 10 4 S -1 M -1 , respectively.
Keywords
Introduction
Thrombotic disease is one kind of lethal complication caused by the accumulation of thrombi in the blood vessels [24 , 28] . Thrombolytic therapies have been extensively investigated as means of medical treatments. Thrombolytic agents are important components of normal homeostatic response since they can dissolve fibrin clots and maintain normal blood flow at vascular injury sites [1] . The major thrombolytic agents used for the lysis of fibrin clots are urokinase, streptokinase, and tissue plasminogen activator. Although these agents have been used widely, they suffer from some shortcomings such as short half lives, high costs, and risk of hemorrhagic complications [33] .
During the past few decades, research has been focused on looking for more safe and economical alternatives to replace current thrombolytic agents [25] . Among animal, plant, and microbial sources, promising fibrinolytic enzymes have been isolated from bacilli isolated from Asian fermented foods such as cheonggukjang , deonjang , douchi , jeotgal , meju , natto , and tempeh [13 , 16 - 18 , 20 , 28 , 32 , 37] . Fermented soy foods are especially rich sources for bacilli, and some bacilli secrete proteases with strong fibrinolytic activities [29] . Nattokinase, secreted by some strains of B. subtilis , is the most well-known enzyme and commercially utilized as a health-promoting ingredient [34 , 36] . Nattokinase not only hydrolyzes fibrin clot directly but also activates plasmin by cleaving the plasminogen activator inhibitor [7 , 35] . Cheonggukjang , a traditional Korean fermented soy food, is similar to natto . Both are fermented soybean foods, and fermentation is carried out by Bacillus species. Bacillus species with strong fibrinolytic activities have been isolated from cheonggukjang , and fibrinolytic enzymes and their genes have been characterized [15 , 21 , 22 , 40] . B. subtilis CH3-5 with strong fibrinolytic activity was isolated from cheonggukjang , and the main fibrinolytic gene was cloned and expressed in a protease-deficient B. subtilis host [12] . AprE2 is a plasmin-like protease that directly degrades fibrin clot and has antiplatelet and antithrombotic activities [11 , 30] . Detailed enzymatic properties of AprE2, however, have not been studied so far. Neither was it produced in E. coli . In this work, aprE2 was overexpressed in E. coli using an expression vector, pET26b(+). Active enzyme was produced after IPTG induction either by continued growth at low temperature (20℃) or by renaturation of inactive enzyme produced during growth at 37℃. The AprE2 was purified by affinity chromatography. The properties and kinetic parameters of the purified AprE2 were examined.
Materials and Methods
- Bacterial Strains and Plasmids
E. coli BL21 (DE3) and B. subtilis CH3-5 were grown in LB (Bacto tryptone 10 g/l, NaCl 10 g/l, yeast extract 5 g/l) broth at 37℃ with aeration. For the cultivation of E. coli cells harboring plasmid pET26b(+) (5.36 kb, Km R ; Novagen, Merck KGaA, Darmstadt, Germany) or pET3-5, kanamycin (Sigma, St. Louis, Mo, USA) was included in the medium at the concentration of 30 μg/ml.
- Construction of plasmid pET3-5
The aprE2 gene (DQ997812) from B. subtilis CH3-5 was amplified using the following primer pairs: pET3-5F, 5’-AGA GGATCC GAT GGCCGGAAAAAGC-3’ ( Bam HI site underlined) and pET3-5R, 5’-AGA CTCGAG TTGTGCAGCTGCTTG-3’ ( Xho I site underlined). The PCR conditions were as follows: 94℃, 5 min, followed by 30 cycles consisting of 94℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 1 min. The amplified 1.1 kb PCR fragment was inserted into pET26b(+) after being digested with Bam HI and Xho I, resulting in pET3-5. E. coli BL21(DE3) competent cells were prepared and transformed by electroporation as described previously [6] .
- Expression of aprE2
E. coli BL21(DE3) cells harboring pET3-5 were grown in LB broth with 30 μg/ml kanamycin at 37℃. IPTG was added at a concentration of 1 mM when the OD 600 of the culture reached 0.8. Incubation was continued for 20 h at different temperatures (15℃, 20℃, 25℃, 30℃, and 37℃). At each time point (2, 4, 10, and 20 h), the culture was centrifuged and cell pellets were resuspended in PBS (phosphate-buffered saline; 2.7 M NaCl, 86 mM sodium phosphate dibasic, 54 mM potassium chloride, 28 mM potassium phosphate monobasic, pH 7.4). Cells were lysed by sonication using a Sonoplus cell disruptor (Bandelin, Berlin, Germany). Whole cell lysates were used to measure the fibrinolytic activities by the fibrin plate method, and fibrinolytic activity was expressed as U (plasmin unit)/mg protein, as described previously [12] . Whole cell lysates were centrifuged at 12,000 ×g for 10 min at 4℃ and the supernatant was the soluble fraction. The pellet (insoluble fraction) was resuspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4) and analyzed by SDS-PAGE.
- In Vitro Renaturation of AprE2
E. coli BL21 (DE3) harboring pET3-5 was cultivated in LB broth with 30 μg/ml kanamycin at 37℃ for 10 h after IPTG induction. Cells were harvested by centrifugation at 12,000 × g for 10 min at 4℃, resuspended in binding buffer, and disrupted by sonication using Sonoplus (Bandelin, Berlin, Germany) (5 cycles of 1 min each, with cooling for 2 min between cycles). Cell extracts were centrifuged at 12,000 × g for 20 min at 4℃ and the obtained pellet was used as the insoluble fraction. The insoluble fraction was dissolved in 6 M guanidine-HCl, stood for 4 h at room temperature, and then dialyzed against an excess volume of buffer for 12 h at 4℃. Three different buffers were used: Tris-HCl (2, 1, 0.5, 0.1, and 0.05 M, pH 7.0), sodium acetate (2, 1, 0.5, 0.1, and 0.05 M, pH 7.0), and PBS. Buffers contained reducing agents (5 mM cysteine and 1 mM cystine). Dialysates were centrifuged for 20 min at 12,000 × g at 4℃ to remove precipitates. Buffer change of the dialyzates was done using binding buffer and Amicon filters (MWCO 12,000; Millipore, Billerica, MA, USA). Then purification of renatured AprE2 was done by using a Ni-NTA column (GE healthcare, Uppsala, Sweden) as described below.
- Purification of AprE2
E. coli BL21 (DE3) harboring pET3-5 was cultivated in 500 ml of LB broth with 30 μg/ml kanamycin. When the OD 600 of culture reached 0.8, IPTG was added at 1 mM concentration. Growth was continued for 10 h at 37℃ or 20 h at 20℃. Cells grown for 20 h at 20℃ were collected by centrifugation at 12,000 × g for 10 min at 4℃, resuspended in binding buffer, and disrupted by sonication as described above. After centrifugation, supernatant (soluble fraction) was obtained and AprE2 in the soluble fraction was purified using a Ni-NTA column. Cells grown for 10 h at 37℃ after IPTG induction were treated as described above, by in vitro renaturation of AprE2. Renatured and buffer-changed AprE2 was purified using a Ni-NTA column like AprE2 in the soluble fraction. Bound AprE2 was eluted from the column by stepwise elution with sodium phosphate buffer (20 mM, pH 7.4) containing imidazole and 0.5 M NaCl. The starting imidazole concentration was 20 mM and the final concentration was 500 mM. One microliter of each fraction (1 ml) was spotted onto a fibrin plate and the plate was incubated for 1 h at 37℃. Active fractions were pooled and dialyzed against 20 mM sodium phosphate buffer (pH 7.4) for 24 h. After dialysis, AprE2 was concentrated by using an Amicon filter (MWCO 12,000; Millipore). Protein concentration was measured by the Bradford method using bovine serum albumin (BSA) as a standard.
- SDS-PAGE and Fibrin Zymography
SDS-PAGE and zymography were done for protein samples from different purification stages. For SDS-PAGE, samples were loaded onto a 12% or 15% acrylamide gel after being boiled for 5 min. For the fibrin zymography, samples were loaded without boiling. The acrylamide gel with fibrin was prepared as described previously [12] . As a size standard, Dokdo-MARK (broad range, Elpis Biotech. Inc., Deajeon, Korea) was used.
- MALDI-TOF Mass Spectrometric Analysis of Fibrinolytic Enzyme
In-gel trypsin digestion of a protein spot and MALDI-TOF-MS spectrometric analysis were carried out as described by Lee et al . [23] . A protein band was excised from a gel stained by Coomassie brilliant blue R-250. Mass spectrometric analysis was performed using a MALDI-TOF-MS Voyager Biospectrometry Workstation (PE Biosystems, Norwalk, CT, USA). The spectra were analyzed with data explorer software (PE Biosystems) and searched against the taxonomy of all entries in the nonredundant NCBI database using the Mascot peptide mass fingerprinting program ( http://www.matrixscience.com ).
- Properties of AprE2
AprE2 in the soluble fraction from culture induced for 20 h at 20℃ was purified on a N i-NTA column and u sed for the tests. The optimum pH for the stability of AprE2 was determined between pH 3 and 11 using citrate-NaOH (50 mM, pH 3-5), sodium phosphate (50 mM, pH 6-7), and Tris-HCl (50 mM, pH 7- 11). One microgram of AprE2 was incubated in each buffer for 2 h at 37℃ and then the remaining activities were measured by the fibrin plate method [12 , 16] . To find the optimum temperature, 1 μg of enzyme was incubated at pH 7 for 30 min at different temperatures (37-60℃) and then the remaining activities were measured. AprE2 was exposed to 5 mM metal ions or 1 mM inhibitors for 30 min at 40℃ and pH 7. Then the remaining activities were measured.
- Hydrolysis of Fibrinogen
Hydrolysis of fibrinogen (bovine; MP Biochemicals, Illkirch, France) by AprE2 was examined. One milligram of fibrinogen was dissolved in 1 ml of 20 mM sodium phosphate buffer (pH 7.0) containing AprE2 (50 ng). Incubation was continued at 37℃ and aliquots were taken out at time intervals and mixed with 5× SDS sample buffer. After being boiled for 5 min, samples were analyzed by SDS-PAGE using a 12% acrylamide gel.
- Kinetics and Amidolytic Activity Measurements
The amidolytic activity of AprE2 was examined by using the following substrates: N -Succinyl-Ala-Ala-Pro-Phe- p -nitroanilide (S7388, Sigma), N -Benzoyl-Phe-Val-Arg- p -nitroanilide hydrochloride (B7632, Sigma), N -Benzoyl-Pro-Phe-Arg- p -nitroanilide hydrochloride (B2133, Sigma), and N -( p -tosyl)-Gly-Pro-Lys-4-nitroanilide acetate salt (T6140, Sigma). First, 50 μl of 10 mM substrate in sodium phosphate buffer (50 mM, pH 7.0) was mixed with 10 μl of AprE2 (1 μg) and 440 μl of sodium phosphate buffer. After 10 min incubation at 40℃, 500 μl of citrate-NaOH buffer (pH 3.0) was added and the mixture was put on ice immediately and centrifuged at 12,000 × g for 5 min. The OD 410 nm of the supernatant was read. The degree of hydrolysis was calculated from the absorbance values and molar extinction coefficient value of p -nitroanilide (8,800 M -1 cm -1 ). Kinetic parameters of AprE2 were determined at 37℃ by measuring the release of p -nitroaniline from the chromogenic substrate, N -Succinyl-Ala-Ala-Pro-Phe- p -nitroanilide, in sodium phosphate buffer (100 mM, pH 7.0) containing 4% (v/v) DMSO [32] . V max and K m were determined from the initial rate measurements at different substrate concentrations ranging from 0.01 to 0.9 mM. The V max value was converted to k cat from the relationship k cat = V max /[enzyme].
Results and Discussion
- Expression of aprE2 in E. coli BL21(DE3)
In pET3-5, the aprE2 gene with its prosequence was inserted just downstream of the pelB signal sequence. aprE2 also had an extra six His codons at the 3’ end ( Fig. 1 ). When soluble and insoluble fractions of culture after IPTG induction were analyzed by SDS-PAGE, a thick 40 kDa band was observed from the insoluble fractions of culture grown at 30℃ and 37℃ ( Figs. 2 A and 2 B, arrow “a”). Although the band intensities were quite high, the fractions did not show any fibrinolytic activities ( Fig. 2 F). MALDI-TOF mass analysis was done for the 40 kDa band, and amino acid sequences of five peptides matched with those of AprE2. Thus, the 40 kDa band was the fusion protein consisting of the signal peptide of PelB and pro-AprE2. Unprocessed AprE2 consists of signal peptide (29 amino acids), prosequence (77 aa), and mature enzyme (275 aa), and the mature, active AprE2 has an apparent size of 29 kDa as determined by SDS-PAGE [12] . The calculated size of pro-AprE2 (352 aa) was 36,120.28 Da and the size of the PelB signal peptide was 3,288.98 Da ( Fig. 1 ). The sum is 39,409.26 Da and this value is quite close to the 40 kDa band (a in Fig. 2 ).
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A schematic presentation of pET3-5. (A) Structure of aprE2 in pET3-5: T7 promoter (PT7), signal sequence (pelB leader), prosequence of aprE2 (pro), mature aprE2 gene (aprE2), 6× histidine tag (His-tag). (B) Nucleotide sequence at the 5’ region of aprE2. The pelB leader sequence and BamHI site are the underlined, and aprE2 prosequence is indicated by a grey box.
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AprE2 production in E. coli BL21(DE3) at different growth temperature after IPTG induction. After IPTG induction, culture continued to grow at 37°C (A), 30°C (B), 25°C (C), 20°C (D), and 15°C (E), respectively. M, DokDo-MARK (broad range; Elpis Biotech. Inc., Daejeon, Korea.); 0, soluble fraction from uninduced cells; lanes 1-4, soluble fraction from cells grown for 2 h after induction (1), 4 h (2), 10 h (3), 20 h (4); lanes 5-8, insoluble fraction from cells grown for 2 h after induction (5), 4 h (6), 10 h (7), 20 h (8). Arrows, a and b, indicate PelB-proAprE2 and mature AprE2, respectively. (F) Fibrinolytic activities of whole cell lysates. (G) MALDI-TOF mass spectrometric analysis of band a. Matched peptides are underlined and they covered 30% of AprE2.
A processed and active 29 kDa band ( Figs. 2 C, 2 D, and 2 E, arrow “b”) was observed from soluble and insoluble fractions of cells grown at 25℃, 20℃, and 15℃ after IPTG induction. Although the band intensities were low, fibrinolytic activities were detected from total cell proteins ( Fig. 2 F). Culture cultivated for 20 h at 20℃ had the highest fibrinolytic activity.
It was suspected that some AprE2 retained the active form during prolonged cultivation at lower temperatures. A reduced rate of AprE2 synthesis at lower temperatures might help AprE2 to fold correctly as observed for subtilisin E [10] . The presence of the 77 aa prosequence might help AprE2 fold correctly. Previous study showed that the prosequence (77 aa) of subtilisin E helped the formation of enzymatically active subtilisin from prepro-subtilisin overproduced in E. coli [10] . The prosequence was essential for the maturation of prepro-subtilisin into active subtilisin, and the presequence could be replaced by other presequences such as that of E. coli OmpA. The critical role of the prosequence for maturation was confirmed by producing propeptide (77 aa) and examining its effect. When artificially produced propeptide was provided, denatured subtilisin was able to refold and became active enzyme [26] . These results and other studies confirm that prosequences of some enzymes play an important role for the conversion of inactive enzyme into active, mature form.
AprE2 can be overproduced in E. coli by cultivating at 37℃ after IPTG induction. In this case, inactive AprE2 is produced, not properly processed, and forming inclusion bodies. It is well known that inactive proteins are often accumulated inside E. coli cells in quantity when heterologous genes are overexpressed [2 , 41] . Overproduced proteins often fail to maintain the active 3-D conformation in E. coli . Rather, they accumulate as a bulk of inactive, denatured proteins known as inclusion bodies. Since the yields of active AprE2 from culture grown at lower temperatures are not great, methods to improve the yield should be developed.
Zhang et al . [42] tried overproduction of subtilisin DFE, a fibrinolytic protein secreted by Bacillus amyloliquefaciens DC-4, in E. coli . Subtilisin DFE was produced as a fusion protein with thioredoxin, which was expected to help the correct folding of denatured proteins and thus enhance the solubility of fusion proteins. Trx-pro-subtilisin DFE was produced efficiently by 6 h cultivation at 30℃ after IPTG induction [42] . SDS-PAGE analysis showed that Trx-prosubtilisin DFE was present both in soluble and insoluble fractions, but only the soluble fraction showed fibrinolytic activity. Active subtilisin DFE was restored by dialyzing the inactive inclusion bodies against 200 mM sodium potassium phosphate buffer [42] . Han et al . [8] tried to produce a fibrinolytic enzyme from Bacillus sp. zlw -2 in E. coli . A gene encoding prepro-enzyme was cloned into pET28a(+), resulting in pET28a-NK1. The fibrinolytic activity of E. coli BL21(DE3) harboring pET28a-NK1 reached the highest value at 30℃ but decreased at higher temperatures. More studies are required to find out efficient and economical methods for the conversion of inactive inclusion bodies into active and soluble forms.
- Renaturation and Purification of AprE2
The insoluble fraction of E. coli cells (10 h cultivation at 37℃ after IPTG induction) was obtained by centrifugation and dissolved in 6 M guanidine HCl. Dialysis was done using three different buffers. After 12 h dialysis, fibrinolytic activities were detected from samples dialyzed against 2 M Tris-HCl, 2 M sodium acetate, 1 M sodium acetate, and 0.5 M sodium acetate. Activities were not detected from samples dialyzed against Tris-HCl (1, 0.5, 0.1, and 0.05 M), sodium acetate (0.1 and 0.05 M), and PBS buffers. The results are similar to those observed in subtilisin DFE [42] . Subtilisn DFE regained activity in the presence of 0.2 M sodium potassium phosphate buffer, but failed in 0.01 M sodium potassium phosphate buffer and 0.02 M Tris-HCl (pH 8.0). It was explained that subtilisin DFE regained activity in high salt buffers because the pro-peptide is highly charged [42] .
Tris-HCl (2 M) and sodium acetate (0.5 M) buffers were compared for the efficiency of renaturation of AprE2 in the insoluble fraction from cells grown for 10 h at 37℃. Although the insoluble fraction had no fibrinolytic activity, this fraction dialyzed against 2 M Tris-HCl or 0.5 M sodium acetate buffer regained activity and the specific activity was 110 ± 0.4 or 109 ± 1.7 U/mg protein, respectively ( Table 1 ). Both buffers were effective similarly for the renaturation of inactive AprE2 during dialysis. Dialyzates were filtered with Amicon units (MWCO 12,000) for the purpose of concentration and buffer change. Binding buffer was used for buffer change and the specific activity increased over 1.5-fold ( Table 1 ).
Purification of AprE2 fromE. coliBL21(DE3) containing pET3-5.
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aInsoluble fraction dialyzed against 2 M Tris-HCl (pH 7). bInsoluble fraction dialyzed against 0.5 M sodium acetate (pH 7). cDialyzate was concentrated and buffer-changed by using Amicon filter and binding buffer.
Purification of AprE2 was done from the soluble fraction of cells grown for 20 h at 20℃ and also from the renatured insoluble fraction of cells grown for 10 h at 37℃. Ni-NTA columns were used and purification was successful. SDS-PAGE analysis showed that the purified protein was the same, 29 kDa in size ( Figs. 3 A and 3 B, lanes 2, 6, and 9) from both fractions. Purified AprE2 showed fibrinolytic activities on a fibrin gel ( Fig. 3 C). Samples with fibrinolytic activities showed halos on the top of the fibrin gel, and the so-called binding mode [15] was not observed in lane 3 of Fig. 3 C (insoluble fraction). The size of the halo is proportional to the strength of fibrinolytic activity of a sample (personal observation). Purified AprE2 (lanes 6 and 9) showed bigger halos than less-purified samples. Purified AprE2 from the soluble fraction showed a 20.96-fold increase in specific activity (1,069.4 ± 42.4 U/mg protein) ( Table 1 ). The specific activity increased to 380 ± 2.2 U/mg protein for dialyzate against 2M Tris-HCl, and 359 ± 2 U/mg protein for dialyzate against 0.5 M sodium acetate. Total fibrinolytic units of the soluble fraction was 57.7 U, and those of the insoluble fraction dialyzed against 2 M Tris-HCl and 0.5 M sodium acetate were 532 U and 689 U, respectively. AprE2 from soluble fraction had a higher specific activity but the total unit was much less than that of the insoluble fraction. Purified AprE2 from insoluble fractions dialyzed against 2 M Tris-HCl or 0.5 M sodium acetate had lower specific activities, but total units were 10-fold higher than that from the soluble fraction. This was caused by the reduced rate of AprE2 synthesis at low temperature, 20℃ in the case of the soluble fraction. Thus, the insoluble fraction may be a better source for AprE2 if AprE2 is needed in a large amount, although denaturation and renaturation steps are required. The efficiency of renaturation of inactive AprE2 was not high and needs to be improved. Studies on the factors affecting the efficiency of renaturation should be done. Then, large-scale production and purification of active AprE2 should be tried to establish more efficient procedures.
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Purified AprE2 from soluble and insoluble fractions. (A) AprE2 from soluble fraction. M, DokDo-MARK; 1, soluble fraction; 2, AprE2 after Ni-NTA column. (B) AprE2 from insoluble fraction. M, DokDo-MARK; 3, denatured insoluble fraction; 4, dialyzate against 2 M Tris-HCl (pH 7); 5, dialyzate after buffer changed (2 M Tris-HCl to binding buffer); 6, AprE2 after Ni-NTA column; 7, dialyzate against 0.5 M sodium acetate (pH 7); 8, dialyzate after buffer changed (0.5 M sodium acetate to binding buffer); 9, AprE2 after Ni-NTA column. (C) Fibrin zymogram of samples from (B). Each lane is the same as (B).
- Effects of pH and Temperature on the Fibrinolytic Activity of AprE2
AprE2 was most stable at pH 7.0 ( Fig. 4 A). AprE2 maintained higher activity at pH 7-10 (100%, 88%, 94%, and 94% of the activity remained at pH 7, 8, 9, and 10, respectively). No activity was detected at pH 5 and below. The optimum pH of AprE2 is similar with those of many microbial fibrinolytic enzymes, such as pH 7 of a major fibrinolytic enzyme from B. amyloliquefaciens [16] , pH 7 of B. subtilis A1 metalloprotease [14] , and pH 7.5 of B. subtilis Jin7 nattokinase [3] . The pH stability of AprE2 is similar to that of B. subtilis KCK-7 [27] . The effect of temperature on AprE2 was examined at pH 7 and the optimum temperature was 40℃ ( Fig. 4 B). After 30 min at 45℃, 90% activity remained. If the temperature was increased further to 55℃ and above, no activity remained after 30 min incubation. The results showed that AprE2 has moderate heat stability. It is expected that AprE2 is completely destroyed if a product containing AprE2 is heated at a temperature higher than 55℃. The optimum temperature of AprE2 is similar with those of B. subtilis natto B-12 [36] and Bacillus sp. KA38 [17] but lower than those of other Bacillus strains, such as B. subtilis KCK-7 (60℃) [27] and Bacillus sp. strain CK11-4 (70℃) [21] .
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pH and temperature stabilities of AprE2. (A) pH stability. -●-, citrate-NaOH buffer; -○-, sodium phosphate buffer; -▼-, Tris-HCl buffer. (B) Temperature stability.
- Effects of Metals and Inhibitors
The effects of metals and inhibitors on the activity of AprE2 were examined ( Table 2 ). Mg 2+ enhanced the fibrinolytic activity by 15%, and K + , Fe 2+ , and Zn 2+ slightly enhanced it. The activity was inhibited by Cu 2+ (3.5% inhibition) and Co 2+ (11.97% inhibition). The fibrinolytic activity was completely inhibited by PMSF but not affected by SDS, EDTA, and EGTA. The results showed that AprE2 is a serine protease but not a metal protease. Almost all fibrinolytic enzymes are inhibited by PMSF, but the effects of metal chelating agents such as EDTA and metal ions are variable depending upon the specific enzyme. For example, subtilisin DJ-4 from Bacillus sp. DJ-4 was inhibited by 1 mM EDTA (12% inhibition), and severely inhibited by Cb 2+ , Cu 2+ , Ni 2+ , and Zn 2+ [19] . Nattokinase from Pseudomonas sp. TKU015 w as activated by 5 mM EDTA and Fe 2+ but not affected by many metal ions, including Mg 2+ , Ca 2+ , Cu 2+ , Ba 2+ , Mn 2+ , and Zn 2+ [38] .
Effects of metal ions and inhibitors on the activity of AprE2.
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aThe counterion for the tested metals was chloride. All values are themean ± SD (n = 3).
- Hydrolysis of Fibrinogen
AprE2 completely degraded Aα and Bβ chains of fibrinogen in 1 h at 37℃, whereas the γ chain was not degraded even after 24 h ( Fig. 5 ). The Aα chain was the most sensitive and hydrolyzed almost immediately. This pattern was similar to that obtained with plasmin and TPase from Bacillus subtilis TP-6 [18] . TPase completely degraded the Aα and Bβ chains, whereas the γ chain was not completely digested after 6 h. The pattern is different from that of a fibrinolytic protease FP84 from Streptomyces sp. CS684 where FP84 hydrolyzed Bβ chains but did not cleave Aα and γ chains [31] .
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Fibrinogen hydrolysis by AprE2. M, size marker (DokDo-MARK); 1, control (no enzyme treatment); 2, 5 min; 3, 10 min; 4, 30 min; 5, 1 h; 6, 3 h; 7, 6 h; 8, 12 h; 9, 24 h. A 12% acrylamide gel was used. Fibrinogen bovine (MP Biomedicals, Illkirch, France); alpha chain (66 kDa), beta chain (54 kDa), and gamma chain (48 kDa).
- Amidolytic Activity of AprE2 and Kinetics
The amidolytic activity of AprE2 was determined by using four different synthetic substrates. The most sensitive substrate was N -succinyl-Ala-Ala-Pro-Phe- p NA, which is the substrate for subtilisin and chymotrypsin ( Table 3 ). Nattokinase from B. subtilis natto also showed the highest activity for N -succinyl-Ala-Ala-Pro-Phe- p NA [7] . The kinetic constants, K m and k cat , were determined from the initial rates for the hydrolysis of N -succinyl-Ala-Ala-Pro-Phe- p NA. The apparent K m and k cat of AprE2 were 0.559 ± 0.032 mM and 17.344 ± 0.990 S -1 , respectively. k cat /K m was 3.1 × 10 4 S -1 M -1 and this value seemed similar to those of some fibrinolytic enzymes reported ( Table 4 ). Specifically, the catalytic efficiency of AprE2 is similar to that of NK from B. natto (3.4 × 10 4 S -1 M -1 ) but lower than that of subtilisin FS33 or TPase.
Amydolytic activity of AprE2.
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apNA: p-nitroanilide. All values are the mean ± SD (n = 3).
Catalytic efficiencies of fibrinolytic enzymes from bacilli.
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Catalytic efficiencies of fibrinolytic enzymes from bacilli.
So far, a nattokinase gene from B. subtilis natto was overexpressed in E. coli by a T7 promoter system [39] , and a gene encoding subtilisin DFE, cloned from B. amyloliquefaciens DFE isolated from Chinese douchi , was also overexpressed in E. coli by the T7 system [42] . However, no attempts have been done for the overexpression of a fibrinolytic gene from Bacillus species isolated from Korean fermented soy foods such as cheonggukjang . As far as the authors know, this is the first report on the overexpression of a fibrinolytic gene of a Bacillus strain from cheonggukjang and the renaturation of an overproduced inactive enzyme. Cheonggukjang and other Korean fermented soy foods such as doenjang are rich sources for bacilli secreting fibrinolytic enzymes, which could be utilized as therapeutic agents preventing and/or curing vascular diseases caused by fibrin accumulations inside blood vessels. In this respect, studies on the mass production of fibrinolytic enzymes are important. Moreover, methods to renature inactive inclusion bodies need to be improved. The methods described in this work, especially renaturation of inactive AprE2, are useful as the first step for overproduction of fibrinolytic enzymes for various applications. Unlike Bacillus hosts, AprE2 is accumulated inside E. coli cells as inclusion bodies, which can be easily recovered by centrifugation. If the yield and purity of active AprE2 can be improved through future studies, the development of functional foods and drugs is possible.
Acknowledgements
This work was supported by a grant f rom IPET (High Value-added Food Technology Development Program, 2012, 112066-3), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. S.-J. Jeong was supported by BK21 Plus from MOE, Republic of Korea.
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