J. Microbiol. Biotechnol. (2015), 25(1), 89–97 http://dx.doi.org/10.4014/jmb.1409.09087 Research Article Review jmb

Characterization of AprE176, a Fibrinolytic from Bacillus subtilis HK176 Seon-Ju Jeong1, Kyeong Heo1, Ji Yeong Park1, Kang Wook Lee2, Jae-Yong Park3, Sang Hoon Joo4, and Jeong Hwan Kim1,2*

1Division of Applied Life Science (BK21 Plus), Graduate School, and 2Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea 3Department of Food Science and Nutrition, and 4College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea

Received: September 29, 2014 Revised: October 8, 2014 Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a Accepted: October 13, 2014 Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 unit/mg protein

First published online and the optimum pH and temperature were pH 8.0 and 40°C, respectively. Error-prone PCR October 15, 2014 was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after

*Corresponding author ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first Phone: +82-55-772-1904; using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest Fax: +82-55-772-1909; activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity E-mail: [email protected]

of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (kcat/Km) of M179 was not different from the wild-type enzyme owing to reduced affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45°C, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176th residue of M179, threonine, was located near the cation- compared with the wild type. This probably caused tight binding of M179 with Ca2+, which increased the pISSN 1017-7825, eISSN 1738-8872 thermostability of M179.

Copyright© 2015 by The Korean Society for Microbiology Keywords: Bacillus subtilis, fibrinolytic enzyme, error-prone PCR, thermostability and Biotechnology

Introduction Nattokinase secreted by Bacillus subtilis natto is the most well-known example [32]. Similar fibrinolytic are Cardiovascular diseases such as acute myocardial also secreted by Bacillus species and they have been infarction, ischemic heart disease, and high blood pressure purified. Their properties, including molecular weight, are the major causes of human death throughout the world. optimum pH and temperature, stability, and substrate Fibrin accumulation in the blood vessels is the main cause specificity, have been reported [1, 36]. Nattokinase is of the diseases. Thrombolytic agents such as tissue produced as a preproenzyme and processed into an active (t-PA), (UK), and enzyme with 275 amino acids. Nattokinase has a catalytic (SK) have been widely used for the treatment triad consisting of Asp32, His64, and Ser221 residues and two of thrombosis. However, these agents can cause serious calcium-binding sites that stabilize the three-dimensional side effects, such as bleeding, short half-lives, high cost, structure [25]. allergic reactions, and large therapeutic doses [15]. Recently, The activity and stability of fibrinolytic enzymes are fibrinolytic enzymes from Bacillus species have been actively important properties to be considered if the enzymes are studied because of their potential as fibrinolytic agents. intended to be used for the prevention and treatment of

January 2015 ⎪ Vol. 25⎪ No. 1 90 Jeong et al.

thrombosis. Engineering of a fibrinolytic enzyme has been size of lysis zone was compared with those caused by plasmin reported [7]. Error-prone PCR is a method widely used to (Sigma, St. Louis, MO, USA) with known concentrations. introduce various mutations into a specific gene [4]. The method does not require detailed information on the Construction of pET176 structure of a protein and accurate predictions of the amino The aprE176 gene was amplified by PCR using total DNA from B. subtilis HK176 as the template and the primer pair 51F (5’- acid substitutions at the proper sites [20]. The half-life of a AGGATCCCAAGAGAGCGATTGCGGCTGTGTAC-3’, BamHI site lipase from Bacillus sp. at 40°C was increased by 3-fold and underlined) and 51R (5’-AGAATTCTTCAGAGGGAGCCACCC the catalytic activity of the mutant increased by 2~5-fold GTCGATCA-3’, EcoRI site underlined) [16]. aprE176 without its over the wild type after error-prone PCR [14]. The signal sequence was amplified using the primer pair petF (5’- thermostability and pH stability of a xylanase from AGAGGATCCGATGGCAGGGAAATCA-3’, BamHI site underlined) Thermomyces lanuginosus DSM 5826 were improved by and petR (5’-AGACTCGAGCTGAGCTGCCGCCTG -3’, XhoI site error-prone PCR [31]. underlined). The PCR conditions were as follows: 94°C for 5 min, Bacillus sp. HK176 was isolated from cheonggukjang, a followed by 30 cycles of 94°C for 0.5 min, 60°C for 0.5 min, and Korean fermented soyfood, as a strain secreting highly 72°C for 1 min. Amplified aprE176 and the aprE176 without a active fibrinolytic enzymes (unpublished result). In this signal sequence were cloned into pET26b(+) (5.36 kb, Kmr), work, the structural gene of the major fibrinolytic enzyme, respectively. E. coli DH5α and E. coli BL21(DE3) competent cells aprE176, was cloned from HK176 and overexpressed in were prepared and transformed by electroporation as described previously [6]. Expression of aprE176 in E. coli was examined by E. coli. Error-prone PCR was done with aprE176 to get measuring the fibrinolytic activity of the transformant, and the mutants with increased fibrinolytic activities. One mutant, activity was expressed as plasmin unit/mg. M179, showed increased fibrinolytic activity and thermostability. The cloning of aprE176 and production of Purification of AprE176 M179 are described. The increased thermostability of M179 E. coli BL21(DE3) cells harboring recombinant pET26b(+) with and a possible cause for the improved thermostability are aprE176 or a mutant gene were cultivated in LB broth (500 ml) also discussed. containing kanamycin (30 µg/ml) at 37°C. When the OD600 value of the culture reached 0.8, IPTG (isopropyl β-D-1-thiogalactopyranoside) Materials and Methods was added (1 mM) to induce gene expression. After 15 h incubation at 20°C, cells were harvested by centrifugation and resuspended Bacterial Strains and Plasmids in 20 mM sodium phosphate buffer with 0.5 M NaCl and 10 mM E. coli DH5α, E. coli BL21(DE3), and B. subtilis HK176 were imidazole (pH 7.4). Cells were disrupted by sonication (Sonoplus grown in LB (Bacto tryptone 10 g, NaCl 10 g, yeast extract 5 g, per GM2070; Bandelin, Berlin, Germany) and the resulting cell extract liter) broth at 37°C with aeration. For cultivation of cells harboring was centrifuged at 12,000 ×g for 10 min at 4°C. The supernatant pHY300PLK (Takara Bio Inc., Shiga, Japan), tetracycline was and cell pellet were obtained. AprE176 and M179 (a mutant) were included in the culture medium at the concentration of 12 µg/ml. purified from the supernatant using a Ni-NTA column (GE For cultivation of cells harboring pET-26b(+) (Novagen, Madison, Healthcare, Uppsala, Sweden). The Bradford method [2] was used WI, USA) or pET176, kanamycin was included at the concentration to determine the protein concentration of the enzyme preparation, of 30 µg/ml. and bovine serum albumin was used as the standard.

Isolation of HK176 from Cheonggukjang Properties of AprE176 Commercial cheonggukjang products produced in several provinces Purified enzyme (1 µg) was incubated in buffers with different of Korea were purchased and used as sources of bacilli with pH values (pH 3-12) for 2 h at 37°C. Buffer of 50 mM concentration fibrinolytic activities. One gram of cheonggukjang was mixed with was used: citrate–NaOH (pH 3–5), sodium phosphate (pH 6–7), 9 ml of sterile 0.1% peptone water and homogenized using a Tris–HCl (pH 8–9), and glycine–NaOH (pH 10–12). After 2 h, the stomacher (Seward, London, UK), and the homogenate was serially remaining activity was measured by the fibrin plate method. The diluted using 0.1% peptone water. Aliquots (0.1 ml) of diluted optimum temperature for the fibrinolytic activity was determined samples were spreaded onto LB agar plates containing skim milk by measuring the activity after 30 min incubation (pH 8.0) at (2% (w/v)) and the plates were incubated for 12h at 37°C. temperatures ranging from 37°C to 60°C. The thermostability of Colonies with large lysis zones were selected and further the enzyme was evaluated by measuring the remaining activity of examined for the fibrinolytic activities by spotting on fibrin plates. enzyme, which was incubated at 45°C (pH 8.0) for different time The fibrinolytic activity was determined by the fibrin plate periods. The effects of metals and inhibitors on the activity were method [11]. Fibrin plates were incubated for 18 h at 37°C and the examined by incubating AprE176 in the presence of 5 mM metal

J. Microbiol. Biotechnol. Characterization of AprE176 91

ions or 1 mM inhibitors for 30 min at 40°C (pH 8.0) and measuring SWISS-MODEL Workspace [30], and Nat (PDB ID: the remaining activities. 4DWW) was used as the template structure. Predicted structures were compared using the PyMOL Molecular Graphics System Error-Prone PCR of aprE176 (ver. 1.5.0.4, Schrödinger, LLC., NY, USA) and exported as images. Error-prone PCR was performed using pET176, a pET26b(+) containing aprE176, as the template and according to the method Statistical Analysis of Cadwell and Joyce [5]. The reaction mixture (100 µl) consisted of All data are expressed as the mean ± SD values. The statistical

10 mM Tris–HCl (pH 8.3), 50 mM KCl, 7 mM MgCl2, 0.01% gelatin, analyses were performed using the SPSS program (SPSS, Inc., 0.2 mM dATP and dGTP, 1 mM dCTP and dTTP, pET176 (10 ng), Chicago, IL, USA). Duncan’s multiple range test was used to

0.5 mM MnCl2, and Taq polymerase (Takara, 5 units). petF and petR examine the difference among treatments. P values of 0.05 were were used as the primers (0.5 µM, each). The PCR was carried out considered significant, if not otherwise stated. for 30 cycles of 94°C for 1 min, 60°C for 0.5 min, and 72°C for 1 min. The PCR products were ligated with pET26b(+) after being digested Results and Discussion with BamHI and XhoI. The ligation mixture was introduced into E. coli BL21(DE3) by electroporation. Transformants, growing on LB Isolation and Identification of HK176 plates containing kanamycin (30 µg/ml) and skim milk (2% (w/v)), Cheonggukjang products produced in different provinces were screened for their proteolytic activities. After 48 h at 37°C, of Korea in 2012 were purchased for the screening of bacilli colonies with larger lysis zones than control were selected and with strong fibrinolytic activities. Colonies with strong inoculated into 5 ml of LB broth using sterile toothpicks. Plasmid activities were selected using skim-milk plates. DNA was prepared from each transformant for DNA sequencing. Selected isolates were further examined for their fibrinolytic Hydrolysis of Fibrinogen activities using fibrin plates. Among the isolates, HK176 Hydrolysis of fibrinogen was examined using purified AprE176 was the best in terms of the fibrinolytic activity. For the and M179 at 37°C. Fibrinogen (1 mg, bovine; MP Biochemicals, identification of HK176, the 700 bp recA gene [21] and 1,300 bp Illkirch, France) was dissolved in 1 ml of 20 mM Tris-HCl (pH 8.0) 16S rRNA gene were amplified and the sequences were and digestion started by adding enzyme (50 ng/ml). Aliquots determined. BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were taken at time points, mixed with 5× SDS sample buffer, and search for both genes indicated that HK176 was either a boiled for 5 min. SDS-PAGE was done using 12% acrylamide gel B. subtilis (99% identity) or a B. tequilensis (99% identity) and the gel was stained with Coomassie Brilliant Blue R-250. (results not shown). The RAPD-PCR profile of HK176 obtained by S30 primer matched well with that of B. subtilis Kinetics and Amidolytic Activity reference strains (results not shown) [21]. From these The amidolytic activity of AprE176 and M179 was assayed using results, HK176 was identified as a B. subtilis strain. the following substrates: N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma, S7388), N-Benzoyl-Phe-Val-Arg p-nitroanilide hydrochloride (Sigma, B7632), N-Benzoyl-Pro-Phe-Arg p-nitroanilide hydrochloride Cloning of aprE176 (Sigma, B2133), and N-(p-tosyl)-Gly-Pro-Lys 4-nitroanilide acetate aprE176 was amplified from genomic DNA of B. subtilis salt (Sigma, T6140). Fifty microliters of 10 mM substrate in 50 mM HK176 by PCR. The 1.5 kb fragment was cloned into an Tris-HCl (pH 8.0) was mixed with 10 µl of enzyme (1 µg) and E. coli-Bacillus shuttle vector, pHY300PLK, after digestion 440 µl of Tris-HCl (pH 8.0). After 10 min at 37°C, 500 µl of citrate- with EcoRI and BamHI, resulting in pHY176 (6.43 kb). NaOH (pH 3.0) was added and the mixture was put on ice Sequence analysis showed that the fragment was 1,491 bp immediately and centrifuged at 12,000 ×g for 5 min. The optical in length and contained the major fibrinolytic gene, density (OD ) of the supernatant was measured and the degree 410 aprE176. The sequence was deposited into GenBank under of hydrolysis was calculated from the absorbance value and molar the accession number of KJ572414. aprE176 encodes a extinction coefficient value of p-nitroanilide (8,800 M-1cm-1). protein of 382 amino acids (aa) consisting of signal sequence Kinetic parameters (V and K ) of AprE176 and M179 were max m (30 aa), propeptide (77 aa), and mature enzyme (275 aa). determined by measuring the release of p-nitroaniline from N- Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (0.01 to 0.9 mM), which This configuration was expected from comparison of the was dissolved in 100 mM phosphate buffer (pH 8.0) containing 4% amino acid sequence of AprE179 with those of similar fibrinolytic enzymes from bacilli [9, 29]. The size of mature (v/v) DMSO at 37°C [32]. The Vmax value was converted to the kcat value from the relationship, kcat = Vmax/(enzyme). and active AprE176 was estimated to be 27 kDa after processing. The ribosome-binding site (RBS) and putative Protein Structure Prediction promoter sequences (-35 and -10) were located upstream of The structures of AprE176 and M179 were predicted by using the ORF (Fig. 1) [11]. The nucleotide sequence of aprE176

January 2015 ⎪ Vol. 25⎪ No. 1 92 Jeong et al.

Fig. 1. Nucleotide sequence of aprE176. The deduced amino acid sequence is shown above the nucleotide sequence. Putative -35 and -10 promoter sequences are underlined. The RBS and transcription terminator are also underlined. The ends of the pre ( ▼ ) and pro sequences (▽) are marked. showed 99% similarity to those of fibrinolytic genes of proaprE176 without the signal sequence was amplified for B. amyloliquefaciens CH51 (EU414203), B. amyloliquefaciens overexpression in E. coli BL21(DE3). The 1.1 kb fragment MJ5-41 (JF739176), B. subtilis JZ06 (EU386607), and B. subtilis was cloned into pET26b(+), resulting in pET176. In pET176, D-2 (JQ730856). The translated amino acid sequence of the pelB signal sequence in the vector directed the secretion AprE176 was aligned with those of other homologous of AprE176 into the periplasm of E. coli, and aprE176 was proteins (Fig. 2). AprE176 showed higher similarity with transcribed by the T7 promoter. The T7 promoter system other fibrinolytic enzymes such as 99.7% with AprE3-17 was also used for the expression of other fibrinolytic genes from B. licheniformis CH3-17 (ACU32756) [13], 99.4% with in E. coli [8, 22]. AprE51 from B. amyloliquefaciens CH51 (ACA34903) [16], 98.9% with peptidase S8 from B. amyloliquefaciens LFB112 Purification of AprE176 (AHC41550), subtilisin DFE from B. amyloliquefaciens DC-4 After centrifugation of sonicated E. coli cells, the (AAZ66858) [29], and subtilisin DJ-4 from Bacillus sp. DJ-4 supernatant and cell pellet were obtained. Fibrinolytic (AAT45900) [17], 86.3% with nattokinase from B. subtilis activity was detected from both the supernatant and pellet, subsp. natto (ACJ11220) [26], and 85.6% with subtilisin E but higher activity was detected from the supernatant (data from B. subtilis 168 (CAA74536). The nucleotide sequence not shown). Thus, the supernatant was used for the of aprE176 was similar to those of B. amyloliquefaciens and purification of AprE176. A Ni-NTA column was used for B. subtilis strains and the deduced amino acid sequence the purification of AprE176, and bound AprE176 was showed the highest similarity to that of AprE3-17 from eluted as described by the supplier. A 27 kDa single band B. licheniformis. When the amino acid sequences of mature was observed when the eluate was analyzed by SDS-PAGE enzymes were compared, AprE176 differed from AprE3-17 (data not shown). The specific activity of the purified at a single amino acid, and differed from AprE51 at two AprE176 was 216.8 ± 5.4 plasmin unit/mg protein. For the amino acids (Fig. 2). M179 purification, the exact same procedures were used.

J. Microbiol. Biotechnol. Characterization of AprE176 93

Fig. 2. Alignment of amino acid sequence of AprE176 with homologous enzymes. AprE51 (B. amyloliquefaciens CH51, ACA34903), AprE3-17 (B. licheniformis CH3-17, ACU32756), peptidase S8 (B. amyloliquefaciens LFB112, YP_008949402), subtilisin DFE (B. amyloliquefaciens DC-4, AAZ66858), subtilisin DJ-4 (Bacillus sp. DJ-4, AAT45900), nattokinase (B. subtilis subsp. natto, ACJ11220), and subtilisin E (B. subtilis 168, CAA74536). Amino acids different from those of other proteins are marked in a box.

Fig. 3. Effects of pH and temperature on the activity of AprE176. (A) Residual fibrinolytic activities were measured after 2 h at different pH values. (B) Residual fibrinolytic activities were measured after 30 min in Tris-buffer (pH 8.0) at different temperatures.

Properties of AprE176 The activity decreased rapidly at 50°C and was completely AprE176 maintained more than 80% of its activity after inactivated at 55°C. The optimum pH and temperature of 2 h at pH between 7.0 and 10.0, but the activity decreased AprE176 are similar to those of nattokinase from B. subtilis rapidly at pH below 6.0 (Fig. 3A). The optimum pH was natto B-12 [35]. These activities are lower than those of an 8.0. The optimum temperature was 40°C at pH 8.0 (Fig. 3B). enzyme from B. amyloliquefaciens DC-4 (pH 9.0 and 48°C,

January 2015 ⎪ Vol. 25⎪ No. 1 94 Jeong et al.

Table 1. Effects of chemical reagents on the activity of AprE176. (M57, M111, M179) that showed higher fibrinolytic Metal ions Relative activity Inhibitors Relative activity activities than wild type were selected by measuring the (5 mM)a (%) (1 mM) (%) clear zone on the fibrin plate. In addition, a mutant (M11) None 100 PMSF 0.0 ± 0.0 showing significantly decreased activity was also selected K+ 98.5 ± 1.5 EDTA 4.5 ± 0.2 for comparison. Sequence analysis of four mutants showed Mg2+ 102.3 ± 0.8 EGTA 6.6 ± 0.6 that the amino acid substitutions were V84A/K213M, Ca2+ 117.1 ± 1.6 SDS 83.6 ± 0.7 K213N, G157S, and A176T for M11, M57, M111, and M179, Cu2+ 92.7 ± 1.4 Cantharidic acid 95.6 ± 1.5 respectively. The specific activities of purified M11, M57, M111, and M179 were 10.9 ± 1.6, 277.1 ± 15.7, 300.4 ± 21.2, Mn2+ 93.4 ± 2.2 Epstatin A 99.3 ± 0.7 and 479.8 ± 12.2 plasmin unit/mg protein, respectively. Zn2+ 56.8 ± 1.1 Bestatin 89.8 ± 1.4 Among them, M179 showed the highest fibrinolytic E-64 87.0 ± 1.4 activity, and this value was 2.2-fold higher than that of a The counterion for the tested metals was chloride. All values are the mean ± SD wild-type AprE176. M111 showed 1.4-fold higher activity (n =3). and M57 showed 1.3-fold higher activity. Changes in the 84th and 213th amino acids at the same time almost completely respectively) [28] and an enzyme from Bacillus sp. CK11-4 destroyed the activity (M11). (pH 10.5 and 70°C, respectively) [19]. The optimum pH of AprE176 (pH 8.0) was quite different from that of AprE3-17 Hydrolysis of Fibrinogen by AprE176 and M179 (pH 6.0) [13] and AprE51 (pH 6.0) [16]. The mature Fibrinogen is the precursor of fibrin, composed of three AprE176 differs from AprE3-17 and AprE51 at a single pairs of disulfide-bonded polypeptide chains (Aα, Bβ, and amino acid and two amino acids, respectively. The result γ). Both AprE176 and M179 quickly degraded the Aα chain indicates that even a single amino acid difference can of fibrinogen, which disappeared within 30 sec (Fig. 4). The cause significant difference in the enzyme properties. Aα chain was hydrolyzed first, followed by the Bβ chain. AprE176 was completely inactivated by 1 mM PMSF This indicated that AprE176 possesses strong α-fibrinogenase (phenylmethylsulfonyl fluoride), a well-known inhibitor of and moderate β-fibrinogenase activities. The degradation serine . EDTA (ethylenediaminetetraacetic acid) pattern was similar to those observed in some other and EGTA (ethylene glycol tetraacetic acid) also inhibited fibrinolytic enzymes such as AprE2 from B. subtilis CH3-5 the fibrinolytic activity of AprE176. The results showed [12], TPase from B. subtilis TP-6, and plasmin [18]. M179 that AprE176 is a and also a metalloprotease. degraded Aα and Bβ chains completely within 20 min, Unlike AprE176, AprE3-17 retained more than half of its indicating that M179 had improved β-fibrinogenase activity. initial activity after 30 min exposure to PMSF and EDTA Both enzymes failed to degrade the γ-chain in 20 min. [13], and AprE51 retained 83% activity after 1 h exposure to EDTA at 45°C [16]. The result again proves the uniqueness Amidolytic Activities and Kinetics of AprE176 and M179 of AprE176 from other similar enzymes. Ca2+ increased the The amidolytic activities of AprE176 and M179 were activity by 17%, but Cu2+, Mn2+, and Zn2+ reduced the determined using four different synthetic substrates. The activity by 7%, 7%, and 43%, respectively (Table 1). Ca2+ increased the activity of many fibrinolytic enzymes [10, 18]. Zn2+ inhibited several fibrinolytic enzymes, including AprE176. For example, 5 mM Zn2+ completely inactivated nattokinase from B. subtilis YJ1 [37] and decreased the activity of subtilisin DJ-4 from Bacillus sp. DJ-4 by 72.88% [17].

Error-Prone PCR of aprE176 Error-prone PCR was done with aprE176 for the purpose of obtaining mutant enzymes with improved fibrinolytic Fig. 4. Hydrolysis of fibrinogen by AprE176 (A) and M179 (B). activities. E. coli BL21(DE3) transformants (550 colonies) Lane M, size marker (DokDo-MARK, ELPIS-Biotech. Inc., Daejeon, were initially screened using skim-milk plates and further Korea.); 1, no enzyme treatment; 2, 30 sec; 3, 1 min; 4, 2 min; 5, 3 min; screened using fibrin plates. Subsequently, three clones 6, 4 min; 7, 5 min; 8, 10 min; 9, 20 min. A 12% acrylamide gel was used.

J. Microbiol. Biotechnol. Characterization of AprE176 95

Table 2. Kinetic parameters of AprE176 and M179. -1 -1 -1 Enzyme Km (mM) kcat (s ) kcat/Km (s mM ) AprE176 0.453 ± 0.036b 122.851 ± 7.863b 281.715a M179 0.537 ± 0.027a 151.429 ± 1.541a 282.961a All values are the mean ± SD (n = 3). Values in the same column with different superscripts are statistically different by Duncan’s multiple range test (p = 0.05). most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe- pNA, a preferred substrate for subtilisin and . AprE176 and M179 showed no activity for the other substrates, indicating that AprE176 is a subtilisin-like serine protease. The amidolytic activities of AprE176 and M179 were 28.63 ± 0.20 and 39.84 ± 0.04 mM/min/mg protein, respectively, when N-succinyl-Ala-Ala-Pro-Phe-pNA was used as the substrate. M179 showed 1.39-fold higher Fig. 5. Temperature stability of AprE176 and M179. Thermal stability was determined at 45°C in Tris-HCl buffer (pH 8.0) amidolytic activity than AprE176. The kinetic constants, Km for various times. AprE176, -●-; M179, - ■ -. and kcat, were determined for AprE176 and M179 by measuring the initial rates of hydrolysis of N-succinyl-Ala- Ala-Pro-Phe-pNA (Table 2). Although M179 showed 5 h at 45°C, M179 still retained 36% activity but AprE176 enhanced fibrinolytic activity and catalytic turnover (kcat), had only 11% activity. The thermostability of an alkaline it had a lower substrate affinity (Km) than AprE176. As a protease (BgAP) from Bacillus gibsonii was increased more result, the catalytic efficiency (kcat/Km) of M179 was not than 90-fold (half-life at 60°C) by directed evolution [24]. different from that of AprE176. The loop area of the BgAP surface was replaced by In the case of a keratinase from B. licheniformis BBE11-1, a substitutions to negatively charged amino acids, which single amino acid change (N122Y) caused the increase of contributed to the increase of the thermostability, probably the catalytic efficiency by 5.6-fold compared with wild- through increasing ionic and hydrogen bond interactions type keratinase. The 122th residue of mutant (N122Y) was in mutated BgAP. The half-life of a keratinase at 60°C from located 6.42 Å, 6.63 Å, and 2.45 Å from Asp32, His63, and B. licheniformis BBE11-1 was increased 8.6-fold by four Ser220, respectively. Some intramolecular interactions among amino acid substitutions (N122Y, N217S, A193P, N160C) the aromatic 122th residue, the (Asp32, His63, [23]. Increase in the thermostability was explained to be the Ser220), and substrate influenced to increase the catalytic result of increased interactions, such as hydrophobic efficiency [23], whereas other substitutions (N217S, A193P, interaction, cation-pi interaction, and hydrogen bond, in the and N160C), which were located far from the active site, mutant. Interactions such as hydrogen bonds, hydrophobic did not significantly change the Km or kcat [23]. For a serine interactions, and electrostatic interactions are well known alkaline protease from B. pumilus CBS, the catalytic efficiency to be the main factors for protein thermostability [33]. was increased 42-fold by mutations (L31I/T33S/N99Y) To explain the increased thermostability of M179 [10]. The substitutions occurred at residues surrounding compared with wild-type ArpE176, molecular modeling the catalytic residue, Asp32, in serine alkaline proteases. For was used. Three-dimensional models of AprE176 and M179 the above examples, changes occurred near the active site, were constructed using the known structure of nattokinase and this enhanced the catalytic efficiency. In the case of (4DWW). Nattokinase and AprE176 share 76% identity M179, the 176th residue was located far from the catalytic in amino acid sequences, and homology-based model site, and this might be the reason why the catalytic structures could be built using SWISS-MODEL Workspace efficiency did not increase. (http://swissmodel.expasy.org/workspace/). Based on the model structures, A176T mutation might be responsible for Thermostability of M179 the increased thermostability of M179. According to the M179 possessed improved thermal resistance compared model structure of AprE176, three amino acids (Gly169, with AprE176. Purified M179 retained 67% residual activity Tyr171, and Val174) are located within 3.0 Å radius from a after 150 min incubation at 45°C, whereas AprE176 had 50% calcium ion (Fig. 6A). For M179, the hydroxyl group of activity (Fig. 5). When the incubation time was extended to Thr176 is located closely (2.9 Å) to the calcium ion, whereas

January 2015 ⎪ Vol. 25⎪ No. 1 96 Jeong et al.

improved thermostability. Further improvements are necessary, and studies on the roles of important amino acids for the activity of AprE176 should also be continued.

Acknowledgments

This work was supported by a grant from Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) (High Value- Added Food Technology Development Program, 2012, 112066-03-SB010), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. S.-J. Jeong and J. Y. Park were supported by BK21 Plus Program, MOE, Republic of Korea.

References

1. Agrebi R, Haddar A, Hmidet N, Jellouli K, Manni L, Nasri Fig. 6. Predicted structures of calcium-binding pocket. (A) AprE176, (B) M179. The purple ball indicates a calcium ion. M. 2009. BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: purification, biochemical and molecular characterization. Process Biochem. 44: 1252-1259. Ala176 of AprE176 does not show any interaction with 2. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the calcium ions. Bacillus fibrinolytic enzymes (subtilisin E [34], principle of protein-dye binding. Anal. Biochem. 72: 248-254. Carlsberg [25], and BPN [3]) have two calcium-binding 3. Bryan P, Alexander P, Strausberg S, Schwarz F, Lan W, sites, Ca A and Ca B sites. The Ca A site is located near the Gilliland G, Gallagher DT. 1992. Energetics of folding N-terminus and has higher calcium-binding affinity, whereas subtilisin BPN’. Biochemistry 31: 4937-4945. Ca B site has weaker binding affinity. The occupation of the 4. Bryan PN. 2000. Protein engineering of subtilisin. Biochim. Ca B site is an important factor for the increase of thermal Biophys. Acta 1543: 203-222. resistance. Increased thermal stability by increased calcium 5. Cadwell RC, Joyce GF. 1994. Mutagenic PCR. Genome Res. 3: concentration was shown by Pantoliano et al. [27] at S136-S140. calcium concentrations ranging from 0.1 to 100 mM. It was 6. Dower WJ, Miller JF, Ragsdale CW. 1988. High efficiency also reported, in the X-ray crystal structure study using transformation of E. coli by high voltage electroporation. subtilisin, that the occupancy of the Ca B site was increased Nucleic Acids Res. 16: 6127-6145. as the calcium concentration was increased. 7. Estell DA, Graycar TP, Wells JA. 1985. Engineering an enzyme by site-directed mutagenesis to be resistant to From the model structure, it is suspected that Thr176 of chemical oxidation. J. Biol. Chem. 260: 6518-6521. M179 is more likely located in the Ca B site. The existence 8. Ghasemi Y, Dabbagh F, Ghasemmian A. 2012. Cloning of a of an extra amino acid with favorable interaction with fibrinolytic enzyme (subtilisin) gene from Bacillus subtilis in calcium ions could possibly increase the affinity with Escherichia coli. Mol. Biotechnol. 52: 1-7. calcium ions, resulting in increase of the thermostability of 9. Heo K, Cho KM, Lee CK, Kim GM, Shin JH, Kim JS, Kim the enzyme. Increased thermostability is desirable if JH. 2013. Characterization of a fibrinolytic enzyme secreted AprE176 and its derivatives are incorporated into foods by Bacillus amyloliquefaciens CB1 and its gene cloning. J. that are expected to be heat treated. Microbiol. Biotechnol. 23: 974-983. AprE176 possesses strong fibrinolytic activity, which can 10. Jaouadi B, Aghajari N, Haser R, Bejar S. 2010. Enhancement be utilized for the production of fermented foods or drugs of the thermostability and the catalytic efficiency of Bacillus preventing cardiovascular diseases caused by fibrin clots. pumilus CBS protease by site-directed mutagenesis. Biochimie Mature AprE176 shows different physicochemical properties, 92: 360-369. 11. Jeong S-J, Kwon G-H, Chun J, Kim JS, Park C-S, Kwon DY, such as pH optimum and inhibition by chemicals, from Kim JH. 2007. Cloning of fibrinolytic enzyme gene from enzymes with quite similar amino acid sequences. The Bacillus subtilis isolated from cheonggukjang and its expression property of AprE176 can be improved by directed evolution in protease-deficient Bacillus subtilis strain. J. Microbiol. methods, and M179, a derivative of AprE176, possesses Biotechnol. 17: 1018-1023.

J. Microbiol. Biotechnol. Characterization of AprE176 97

12. Jeong S-J, Cho KM, Lee CK, Kim GM, Shin JH, Kim JS, Kim U. 2012. Increasing activity and thermal resistance of Bacillus JH. 2014. Overexpression of aprE2, a fibrinolytic enzyme gibsonii alkaline protease (BgAP) by directed evolution. gene from Bacillus subtilis CH3-5, in Escherichia coli and the Biotechnol. Bioeng. 110: 711-720. properties of AprE2. J. Microbiol. Biotechnol. 24: 969-978. 25. McPhalenf CA, James MNG. 1988. Structural comparison of 13. Jo H-D, Kwon G-H, Park J-Y, Cha J, Song Y-S, Kim JH. two serine proteinase-protein inhibitor complexes: Eglin-C- 2011. Cloning and overexpression of aprE3-17 encoding the Subtilisin Carlsberg and CI-2-Subtilisin Novo. Biochemistry major fibrinolytic protease of Bacillus licheniformis CH 3-17. 27: 6582-6598. Biotechnol. Bioproc. Eng. 16: 352-359. 26. Nakamura T, Yamagata Y, Ichishima E. 1992. Nucleotide 14. Khurana J, Singh R, Kaur J. 2011. Engineering of Bacillus sequence of the subtilisin NAT gene, aprN, of Bacillus lipase by directed evolution for enhanced thermal stability: subtilis (natto). Biosci. Biotechnol. Biochem. 56: 1869-1871. effect of isoleucine to threonine mutation at protein surface. 27. Pantoliano MW, Whitlow M, Wood JF, Rollence ML, Finzel Mol. Biol. Rep. 38: 2919-2926. BC, Gilliland GL, et al. 1988. The engineering of binding 15.Killer M, Ladurner G, Kunz AB, Kraus J. 2010. Current affinity at metal ion binding sites for the stabilization of endovascular treatment of acute stroke and future aspects. proteins: subtilisin as a test case. Biochemistry 27: 8311-8317. Drug Discov. Today 15: 640-647. 28. Peng Y, Huang Q, Zhang R-H, Zhang Y-Z. 2003. 16. Kim GM, Lee AR, Lee KW, Park J-Y, Chun J, Cha J, et al. Purification and characterization of a fibrinolytic enzyme 2009. Characterization of a 27 kDa fibrinolytic enzyme from produced by Bacillus amyloliquefaciens DC-4 screened from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang. J. douchi, a traditional Chinese soybean food. Comp. Biochem. Microbiol. Biotechnol. 19: 997-1004. Phys. B 134: 45-52. 17. Kim S-H, Choi N-S. 2000. Purification and characterization 29. Peng Y, Yang XJ, Xiao L, Zhang YZ. 2004. Cloning and of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened expression of a fibrinolytic enzyme (subtilisin DFE) gene from doen-jang. Biosci. Biotechnol. Biochem. 64: 1722-1725. from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis. Res. 18. Kim SB, Lee DW, Cheigh CI, Choe EA, Lee SJ, Hong YH, et Microbiol. 155: 167-173. al. 2006. Purification and characterization of a fibrinolytic 30. Schwede T. 2003. SWISS-MODEL: an automated protein subtilisin-like protease of Bacillus subtilis TP-6 from an homology-modeling server. Nucleic Acids Res. 31: 3381-3385. Indonesian fermented soybean, tempeh. J. Ind. Microbiol. 31. Stephens DE, Singh S, Permaul K. 2009. Error-prone PCR of Biotechnol. 33: 436-444. a fungal xylanase for improvement of its alkaline and 19. Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, et al. 1996. thermal stability. FEMS Microbiol. Lett. 293: 42-47. Purification and characterization of a fibrinolytic enzyme 32. Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. produced from Bacillus sp. strain CK 11-4 screened from 1987. A novel fibrinolytic enzyme (nattokinase) in the chungkook-jang. Appl. Environ. Microbiol. 62: 2482–2488. vegetable cheese natto; a typical and popular soybean food 20. Kim YW, Choi JH, Kim JW, Park C, Kim JW, Cha H, et al. in the Japanese diet. Experientia 43: 1110-1111. 2003. Directed evolution of Thermus maltogenic amylase 33. Tina KG, Bhadra R, Srinivasan N. 2007. PIC: protein toward enhanced thermal resistance. Appl. Environ. Microbiol. interactions calculator. Nucleic Acids Res. 35: W473-W476. 69: 4866-4874. 34. Uehara R, Angkawidjaja C, Koga Y, Kanaya S. 2013. 21. Kwon GH, Lee HA, Park JY, Kim JS, Lim J, Park CS, et al. Formation of the high-affinity calcium binding site in pro- 2009. Development of a RAPD-PCR method for identification subtilisin E with the insertion sequence IS1 of Pro-Tk- of Bacillus species isolated from cheonggukjang. Int. J. Food subtilisin. Biochemistry 52: 9080-9088. Microbiol. 129: 282-287. 35. Wang C, Du M, Zheng D, Kong F, Zu G, Feng Y. 2009. 22. Lee S-Y, Yu S-N, Choi H-J, Kim K-Y, Kim S-H, Choi Y-L, et Purification and characterization of nattokinase from Bacillus al. 2013. Cloning and characterization of a thermostable and subtilis Natto B-12. J. Agric. Food Chem. 57: 9722-9729. alkaline fibrinolytic enzyme from a soil metagenome. Afr. J. 36. Yeo WS, Seo MJ, Kim MJ, Lee HH, Kang BW, Park JU, et al. Biotechnol. 12: 6389-6399. 2011. Biochemical analysis of a fibrinolytic enzyme purified 23. Liu B, Zhang J, Fang Z, Gu L, Liao X, Du G, Chen J. 2013. from Bacillus subtilis strain A1. J. Microbiol. 49: 376-380. Enhanced thermostability of keratinase by computational 37.Yin LJ, Lin HH, Jiang ST. 2010. Bioproperties of potent design and empirical mutation. J. Ind. Microbiol. Biotechnol. nattokinase from Bacillus subtilis YJ1. J. Agric. Food Chem. 58: 40: 697-704. 5737-5742. 24. Martinez R, Jakob F, Tu R, Siegert P, Maurer K-H, Schwaneberg

January 2015 ⎪ Vol. 25⎪ No. 1