characterization of apre176, a fibrinolytic enzyme from ... · nattokinase secreted by bacillus...

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January 2015 Vol. 25 No. 1 J. Microbiol. Biotechnol. (2015), 25(1), 89–97 http://dx.doi.org/10.4014/jmb.1409.09087 Research Article jmb Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176 Seon-Ju Jeong 1 , Kyeong Heo 1 , Ji Yeong Park 1 , Kang Wook Lee 2 , Jae-Yong Park 3 , Sang Hoon Joo 4 , and Jeong Hwan Kim 1,2 * 1 Division of Applied Life Science (BK21 Plus), Graduate School, and 2 Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea 3 Department of Food Science and Nutrition, and 4 College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea Introduction Cardiovascular diseases such as acute myocardial infarction, ischemic heart disease, and high blood pressure are the major causes of human death throughout the world. Fibrin accumulation in the blood vessels is the main cause of the diseases. Thrombolytic agents such as tissue plasminogen activator (t-PA), urokinase (UK), and streptokinase (SK) have been widely used for the treatment of thrombosis. However, these agents can cause serious side effects, such as bleeding, short half-lives, high cost, allergic reactions, and large therapeutic doses [15]. Recently, fibrinolytic enzymes from Bacillus species have been actively studied because of their potential as fibrinolytic agents. Nattokinase secreted by Bacillus subtilis natto is the most well-known example [32]. Similar fibrinolytic enzymes are also secreted by Bacillus species and they have been purified. Their properties, including molecular weight, optimum pH and temperature, stability, and substrate specificity, have been reported [1, 36]. Nattokinase is produced as a preproenzyme and processed into an active enzyme with 275 amino acids. Nattokinase has a catalytic triad consisting of Asp 32 , His 64 , and Ser 221 residues and two calcium-binding sites that stabilize the three-dimensional structure [25]. The activity and stability of fibrinolytic enzymes are important properties to be considered if the enzymes are intended to be used for the prevention and treatment of Received: September 29, 2014 Revised: October 8, 2014 Accepted: October 13, 2014 First published online October 15, 2014 *Corresponding author Phone: +82-55-772-1904; Fax: +82-55-772-1909; E-mail: [email protected] pISSN 1017-7825, eISSN 1738-8872 Copyright © 2015 by The Korean Society for Microbiology and Biotechnology Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a 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 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40°C, respectively. Error-prone PCR was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (k cat /K m ) of M179 was not different from the wild-type enzyme owing to reduced substrate 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 176 th residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca 2+ , which increased the thermostability of M179. Keywords: Bacillus subtilis, fibrinolytic enzyme, error-prone PCR, thermostability

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  • January 2015⎪Vol. 25⎪No. 1

    J. Microbiol. Biotechnol. (2015), 25(1), 89–97http://dx.doi.org/10.4014/jmb.1409.09087 Research Article jmbReviewCharacterization of AprE176, a Fibrinolytic Enzyme 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 Korea3Department of Food Science and Nutrition, and 4College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea

    Introduction

    Cardiovascular diseases such as acute myocardialinfarction, ischemic heart disease, and high blood pressureare the major causes of human death throughout the world.Fibrin accumulation in the blood vessels is the main causeof the diseases. Thrombolytic agents such as tissueplasminogen activator (t-PA), urokinase (UK), andstreptokinase (SK) have been widely used for the treatmentof thrombosis. However, these agents can cause seriousside effects, such as bleeding, short half-lives, high cost,allergic reactions, and large therapeutic doses [15]. Recently,fibrinolytic enzymes from Bacillus species have been activelystudied because of their potential as fibrinolytic agents.

    Nattokinase secreted by Bacillus subtilis natto is the mostwell-known example [32]. Similar fibrinolytic enzymes arealso secreted by Bacillus species and they have beenpurified. Their properties, including molecular weight,optimum pH and temperature, stability, and substratespecificity, have been reported [1, 36]. Nattokinase isproduced as a preproenzyme and processed into an activeenzyme with 275 amino acids. Nattokinase has a catalytictriad consisting of Asp32, His64, and Ser221 residues and twocalcium-binding sites that stabilize the three-dimensionalstructure [25].

    The activity and stability of fibrinolytic enzymes areimportant properties to be considered if the enzymes areintended to be used for the prevention and treatment of

    Received: September 29, 2014

    Revised: October 8, 2014

    Accepted: October 13, 2014

    First published online

    October 15, 2014

    *Corresponding author

    Phone: +82-55-772-1904;

    Fax: +82-55-772-1909;

    E-mail: [email protected]

    pISSN 1017-7825, eISSN 1738-8872

    Copyright© 2015 by

    The Korean Society for Microbiology

    and Biotechnology

    Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a

    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 plasmin unit/mg protein

    and the optimum pH and temperature were pH 8.0 and 40°C, respectively. Error-prone PCR

    was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after

    ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first

    using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest

    activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity

    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 substrate 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-binding site compared with the

    wild type. This probably caused tight binding of M179 with Ca2+, which increased the

    thermostability of M179.

    Keywords: Bacillus subtilis, fibrinolytic enzyme, error-prone PCR, thermostability

  • 90 Jeong et al.

    J. Microbiol. Biotechnol.

    thrombosis. Engineering of a fibrinolytic enzyme has beenreported [7]. Error-prone PCR is a method widely used tointroduce various mutations into a specific gene [4]. Themethod does not require detailed information on thestructure of a protein and accurate predictions of the aminoacid substitutions at the proper sites [20]. The half-life of alipase from Bacillus sp. at 40°C was increased by 3-fold andthe catalytic activity of the mutant increased by 2~5-foldover the wild type after error-prone PCR [14]. Thethermostability and pH stability of a xylanase fromThermomyces lanuginosus DSM 5826 were improved byerror-prone PCR [31].

    Bacillus sp. HK176 was isolated from cheonggukjang, aKorean fermented soyfood, as a strain secreting highlyactive fibrinolytic enzymes (unpublished result). In thiswork, the structural gene of the major fibrinolytic enzyme,aprE176, was cloned from HK176 and overexpressed inE. coli. Error-prone PCR was done with aprE176 to getmutants with increased fibrinolytic activities. One mutant,M179, showed increased fibrinolytic activity andthermostability. The cloning of aprE176 and production ofM179 are described. The increased thermostability of M179and a possible cause for the improved thermostability arealso discussed.

    Materials and Methods

    Bacterial Strains and Plasmids

    E. coli DH5α, E. coli BL21(DE3), and B. subtilis HK176 were

    grown in LB (Bacto tryptone 10 g, NaCl 10 g, yeast extract 5 g, per

    liter) broth at 37°C with aeration. For cultivation of cells harboring

    pHY300PLK (Takara Bio Inc., Shiga, Japan), tetracycline was

    included in the culture medium at the concentration of 12 µg/ml.

    For cultivation of cells harboring pET-26b(+) (Novagen, Madison,

    WI, USA) or pET176, kanamycin was included at the concentration

    of 30 µg/ml.

    Isolation of HK176 from Cheonggukjang

    Commercial cheonggukjang products produced in several provinces

    of Korea were purchased and used as sources of bacilli with

    fibrinolytic activities. One gram of cheonggukjang was mixed with

    9 ml of sterile 0.1% peptone water and homogenized using a

    stomacher (Seward, London, UK), and the homogenate was serially

    diluted using 0.1% peptone water. Aliquots (0.1 ml) of diluted

    samples were spreaded onto LB agar plates containing skim milk

    (2% (w/v)) and the plates were incubated for 12 h at 37°C.

    Colonies with large lysis zones were selected and further

    examined for the fibrinolytic activities by spotting on fibrin plates.

    The fibrinolytic activity was determined by the fibrin plate

    method [11]. Fibrin plates were incubated for 18 h at 37°C and the

    size of lysis zone was compared with those caused by plasmin

    (Sigma, St. Louis, MO, USA) with known concentrations.

    Construction of pET176

    The aprE176 gene was amplified by PCR using total DNA from

    B. subtilis HK176 as the template and the primer pair 51F (5’-

    AGGATCCCAAGAGAGCGATTGCGGCTGTGTAC-3’, BamHI site

    underlined) and 51R (5’-AGAATTCTTCAGAGGGAGCCACCC

    GTCGATCA-3’, EcoRI site underlined) [16]. aprE176 without its

    signal sequence was amplified using the primer pair petF (5’-

    AGAGGATCCGATGGCAGGGAAATCA-3’, BamHI site underlined)

    and petR (5’-AGACTCGAGCTGAGCTGCCGCCTG -3’, XhoI site

    underlined). The PCR conditions were as follows: 94°C for 5 min,

    followed by 30 cycles of 94°C for 0.5 min, 60°C for 0.5 min, and

    72°C for 1 min. Amplified aprE176 and the aprE176 without a

    signal sequence were cloned into pET26b(+) (5.36 kb, Kmr),

    respectively. E. coli DH5α and E. coli BL21(DE3) competent cells

    were prepared and transformed by electroporation as described

    previously [6]. Expression of aprE176 in E. coli was examined by

    measuring the fibrinolytic activity of the transformant, and the

    activity was expressed as plasmin unit/mg.

    Purification of AprE176

    E. coli BL21(DE3) cells harboring recombinant pET26b(+) with

    aprE176 or a mutant gene were cultivated in LB broth (500 ml)

    containing kanamycin (30 µg/ml) at 37°C. When the OD600 value of

    the culture reached 0.8, IPTG (isopropyl β-D-1-thiogalactopyranoside)

    was added (1 mM) to induce gene expression. After 15 h incubation

    at 20°C, cells were harvested by centrifugation and resuspended

    in 20 mM sodium phosphate buffer with 0.5 M NaCl and 10 mM

    imidazole (pH 7.4). Cells were disrupted by sonication (Sonoplus

    GM2070; Bandelin, Berlin, Germany) and the resulting cell extract

    was centrifuged at 12,000 ×g for 10 min at 4°C. The supernatant

    and cell pellet were obtained. AprE176 and M179 (a mutant) were

    purified from the supernatant using a Ni-NTA column (GE

    Healthcare, Uppsala, Sweden). The Bradford method [2] was used

    to determine the protein concentration of the enzyme preparation,

    and bovine serum albumin was used as the standard.

    Properties of AprE176

    Purified enzyme (1 µg) was incubated in buffers with different

    pH values (pH 3-12) for 2 h at 37°C. Buffer of 50 mM concentration

    was used: citrate–NaOH (pH 3–5), sodium phosphate (pH 6–7),

    Tris–HCl (pH 8–9), and glycine–NaOH (pH 10–12). After 2 h, the

    remaining activity was measured by the fibrin plate method. The

    optimum temperature for the fibrinolytic activity was determined

    by measuring the activity after 30 min incubation (pH 8.0) at

    temperatures ranging from 37°C to 60°C. The thermostability of

    the enzyme was evaluated by measuring the remaining activity of

    enzyme, which was incubated at 45°C (pH 8.0) for different time

    periods. The effects of metals and inhibitors on the activity were

    examined by incubating AprE176 in the presence of 5 mM metal

  • Characterization of AprE176 91

    January 2015⎪Vol. 25⎪No. 1

    ions or 1 mM inhibitors for 30 min at 40°C (pH 8.0) and measuring

    the remaining activities.

    Error-Prone PCR of aprE176

    Error-prone PCR was performed using pET176, a pET26b(+)

    containing aprE176, as the template and according to the method

    of Cadwell and Joyce [5]. The reaction mixture (100 µl) consisted of

    10 mM Tris–HCl (pH 8.3), 50 mM KCl, 7 mM MgCl2, 0.01% gelatin,

    0.2 mM dATP and dGTP, 1 mM dCTP and dTTP, pET176 (10 ng),

    0.5 mM MnCl2, and Taq polymerase (Takara, 5 units). petF and petR

    were used as the primers (0.5 µM, each). The PCR was carried out

    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

    with BamHI and XhoI. The ligation mixture was introduced into

    E. coli BL21(DE3) by electroporation. Transformants, growing on LB

    plates containing kanamycin (30 µg/ml) and skim milk (2% (w/v)),

    were screened for their proteolytic activities. After 48 h at 37°C,

    colonies with larger lysis zones than control were selected and

    inoculated into 5 ml of LB broth using sterile toothpicks. Plasmid

    DNA was prepared from each transformant for DNA sequencing.

    Hydrolysis of Fibrinogen

    Hydrolysis of fibrinogen was examined using purified AprE176

    and M179 at 37°C. Fibrinogen (1 mg, bovine; MP Biochemicals,

    Illkirch, France) was dissolved in 1 ml of 20 mM Tris-HCl (pH 8.0)

    and digestion started by adding enzyme (50 ng/ml). Aliquots

    were taken at time points, mixed with 5× SDS sample buffer, and

    boiled for 5 min. SDS-PAGE was done using 12% acrylamide gel

    and the gel was stained with Coomassie Brilliant Blue R-250.

    Kinetics and Amidolytic Activity

    The amidolytic activity of AprE176 and M179 was assayed using

    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

    (Sigma, B2133), and N-(p-tosyl)-Gly-Pro-Lys 4-nitroanilide acetate

    salt (Sigma, T6140). Fifty microliters of 10 mM substrate in 50 mM

    Tris-HCl (pH 8.0) was mixed with 10 µl of enzyme (1 µg) and

    440 µl of Tris-HCl (pH 8.0). After 10 min at 37°C, 500 µl of citrate-

    NaOH (pH 3.0) was added and the mixture was put on ice

    immediately and centrifuged at 12,000 ×g for 5 min. The optical

    density (OD410) of the supernatant was measured and the degree

    of hydrolysis was calculated from the absorbance value and molar

    extinction coefficient value of p-nitroanilide (8,800 M-1cm-1).

    Kinetic parameters (Vmax and Km) of AprE176 and M179 were

    determined by measuring the release of p-nitroaniline from N-

    Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (0.01 to 0.9 mM), which

    was dissolved in 100 mM phosphate buffer (pH 8.0) containing 4%

    (v/v) DMSO at 37°C [32]. The Vmax value was converted to the kcatvalue from the relationship, kcat = Vmax/(enzyme).

    Protein Structure Prediction

    The structures of AprE176 and M179 were predicted by using

    SWISS-MODEL Workspace [30], and Subtilisin Nat (PDB ID:

    4DWW) was used as the template structure. Predicted structures

    were compared using the PyMOL Molecular Graphics System

    (ver. 1.5.0.4, Schrödinger, LLC., NY, USA) and exported as images.

    Statistical Analysis

    All data are expressed as the mean ± SD values. The statistical

    analyses were performed using the SPSS program (SPSS, Inc.,

    Chicago, IL, USA). Duncan’s multiple range test was used to

    examine the difference among treatments. P values of 0.05 were

    considered significant, if not otherwise stated.

    Results and Discussion

    Isolation and Identification of HK176

    Cheonggukjang products produced in different provincesof Korea in 2012 were purchased for the screening of bacilliwith strong fibrinolytic activities. Colonies with strongprotease activities were selected using skim-milk plates.Selected isolates were further examined for their fibrinolyticactivities using fibrin plates. Among the isolates, HK176was the best in terms of the fibrinolytic activity. For theidentification of HK176, the 700 bp recA gene [21] and 1,300 bp16S rRNA gene were amplified and the sequences weredetermined. BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi)search for both genes indicated that HK176 was either aB. subtilis (99% identity) or a B. tequilensis (99% identity)(results not shown). The RAPD-PCR profile of HK176obtained by S30 primer matched well with that of B. subtilisreference strains (results not shown) [21]. From theseresults, HK176 was identified as a B. subtilis strain.

    Cloning of aprE176

    aprE176 was amplified from genomic DNA of B. subtilisHK176 by PCR. The 1.5 kb fragment was cloned into anE. coli-Bacillus shuttle vector, pHY300PLK, after digestionwith EcoRI and BamHI, resulting in pHY176 (6.43 kb).Sequence analysis showed that the fragment was 1,491 bpin length and contained the major fibrinolytic gene,aprE176. The sequence was deposited into GenBank underthe accession number of KJ572414. aprE176 encodes aprotein of 382 amino acids (aa) consisting of signal sequence(30 aa), propeptide (77 aa), and mature enzyme (275 aa).This configuration was expected from comparison of theamino acid sequence of AprE179 with those of similarfibrinolytic enzymes from bacilli [9, 29]. The size of matureand active AprE176 was estimated to be 27 kDa afterprocessing. The ribosome-binding site (RBS) and putativepromoter sequences (-35 and -10) were located upstream ofthe ORF (Fig. 1) [11]. The nucleotide sequence of aprE176

  • 92 Jeong et al.

    J. Microbiol. Biotechnol.

    showed 99% similarity to those of fibrinolytic genes ofB. amyloliquefaciens CH51 (EU414203), B. amyloliquefaciensMJ5-41 (JF739176), B. subtilis JZ06 (EU386607), and B. subtilisD-2 (JQ730856). The translated amino acid sequence ofAprE176 was aligned with those of other homologousproteins (Fig. 2). AprE176 showed higher similarity withother fibrinolytic enzymes such as 99.7% with AprE3-17from B. licheniformis CH3-17 (ACU32756) [13], 99.4% withAprE51 from B. amyloliquefaciens CH51 (ACA34903) [16],98.9% with peptidase S8 from B. amyloliquefaciens LFB112(AHC41550), subtilisin DFE from B. amyloliquefaciens DC-4(AAZ66858) [29], and subtilisin DJ-4 from Bacillus sp. DJ-4(AAT45900) [17], 86.3% with nattokinase from B. subtilissubsp. natto (ACJ11220) [26], and 85.6% with subtilisin Efrom B. subtilis 168 (CAA74536). The nucleotide sequenceof aprE176 was similar to those of B. amyloliquefaciens andB. subtilis strains and the deduced amino acid sequenceshowed the highest similarity to that of AprE3-17 fromB. licheniformis. When the amino acid sequences of matureenzymes were compared, AprE176 differed from AprE3-17at a single amino acid, and differed from AprE51 at twoamino acids (Fig. 2).

    proaprE176 without the signal sequence was amplified foroverexpression in E. coli BL21(DE3). The 1.1 kb fragmentwas cloned into pET26b(+), resulting in pET176. In pET176,the pelB signal sequence in the vector directed the secretionof AprE176 into the periplasm of E. coli, and aprE176 wastranscribed by the T7 promoter. The T7 promoter systemwas also used for the expression of other fibrinolytic genesin E. coli [8, 22].

    Purification of AprE176

    After centrifugation of sonicated E. coli cells, thesupernatant and cell pellet were obtained. Fibrinolyticactivity was detected from both the supernatant and pellet,but higher activity was detected from the supernatant (datanot shown). Thus, the supernatant was used for thepurification of AprE176. A Ni-NTA column was used forthe purification of AprE176, and bound AprE176 waseluted as described by the supplier. A 27 kDa single bandwas observed when the eluate was analyzed by SDS-PAGE(data not shown). The specific activity of the purifiedAprE176 was 216.8 ± 5.4 plasmin unit/mg protein. For theM179 purification, the exact same procedures were used.

    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.

  • Characterization of AprE176 93

    January 2015⎪Vol. 25⎪No. 1

    Properties of AprE176

    AprE176 maintained more than 80% of its activity after2 h at pH between 7.0 and 10.0, but the activity decreasedrapidly at pH below 6.0 (Fig. 3A). The optimum pH was8.0. The optimum temperature was 40°C at pH 8.0 (Fig. 3B).

    The activity decreased rapidly at 50°C and was completelyinactivated at 55°C. The optimum pH and temperature ofAprE176 are similar to those of nattokinase from B. subtilisnatto B-12 [35]. These activities are lower than those of anenzyme from B. amyloliquefaciens DC-4 (pH 9.0 and 48°C,

    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.

  • 94 Jeong et al.

    J. Microbiol. Biotechnol.

    respectively) [28] and an enzyme from Bacillus sp. CK11-4(pH 10.5 and 70°C, respectively) [19]. The optimum pH ofAprE176 (pH 8.0) was quite different from that of AprE3-17(pH 6.0) [13] and AprE51 (pH 6.0) [16]. The matureAprE176 differs from AprE3-17 and AprE51 at a singleamino acid and two amino acids, respectively. The resultindicates that even a single amino acid difference cancause significant difference in the enzyme properties.AprE176 was completely inactivated by 1 mM PMSF(phenylmethylsulfonyl fluoride), a well-known inhibitor ofserine proteases. EDTA (ethylenediaminetetraacetic acid)and EGTA (ethylene glycol tetraacetic acid) also inhibitedthe fibrinolytic activity of AprE176. The results showedthat AprE176 is a serine protease and also a metalloprotease.Unlike AprE176, AprE3-17 retained more than half of itsinitial activity after 30 min exposure to PMSF and EDTA[13], and AprE51 retained 83% activity after 1 h exposure toEDTA at 45°C [16]. The result again proves the uniquenessof AprE176 from other similar enzymes. Ca2+ increased theactivity by 17%, but Cu2+, Mn2+, and Zn2+ reduced theactivity by 7%, 7%, and 43%, respectively (Table 1). Ca2+

    increased the activity of many fibrinolytic enzymes [10, 18].Zn2+ inhibited several fibrinolytic enzymes, includingAprE176. For example, 5 mM Zn2+ completely inactivatednattokinase from B. subtilis YJ1 [37] and decreased the activityof 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 purposeof obtaining mutant enzymes with improved fibrinolyticactivities. E. coli BL21(DE3) transformants (550 colonies)were initially screened using skim-milk plates and furtherscreened using fibrin plates. Subsequently, three clones

    (M57, M111, M179) that showed higher fibrinolyticactivities than wild type were selected by measuring theclear zone on the fibrin plate. In addition, a mutant (M11)showing significantly decreased activity was also selectedfor comparison. Sequence analysis of four mutants showedthat the amino acid substitutions were V84A/K213M,K213N, G157S, and A176T for M11, M57, M111, and M179,respectively. The specific activities of purified M11, M57,M111, and M179 were 10.9 ± 1.6, 277.1 ± 15.7, 300.4 ± 21.2,and 479.8 ± 12.2 plasmin unit/mg protein, respectively.Among them, M179 showed the highest fibrinolyticactivity, and this value was 2.2-fold higher than that ofwild-type AprE176. M111 showed 1.4-fold higher activityand M57 showed 1.3-fold higher activity. Changes in the84th and 213th amino acids at the same time almost completelydestroyed the activity (M11).

    Hydrolysis of Fibrinogen by AprE176 and M179

    Fibrinogen is the precursor of fibrin, composed of threepairs of disulfide-bonded polypeptide chains (Aα, Bβ, andγ). Both AprE176 and M179 quickly degraded the Aα chainof fibrinogen, which disappeared within 30 sec (Fig. 4). TheAα chain was hydrolyzed first, followed by the Bβ chain.This indicated that AprE176 possesses strong α-fibrinogenaseand moderate β-fibrinogenase activities. The degradationpattern was similar to those observed in some otherfibrinolytic enzymes such as AprE2 from B. subtilis CH3-5[12], TPase from B. subtilis TP-6, and plasmin [18]. M179degraded Aα and Bβ chains completely within 20 min,indicating that M179 had improved β-fibrinogenase activity.Both enzymes failed to degrade the γ-chain in 20 min.

    Amidolytic Activities and Kinetics of AprE176 and M179

    The amidolytic activities of AprE176 and M179 weredetermined using four different synthetic substrates. The

    Table 1. Effects of chemical reagents on the activity of AprE176.

    Metal ions

    (5 mM)aRelative activity

    (%)

    Inhibitors

    (1 mM)

    Relative activity

    (%)

    None 100 PMSF 0.0 ± 0.0

    K+ 98.5 ± 1.5 EDTA 4.5 ± 0.2

    Mg2+ 102.3 ± 0.8 EGTA 6.6 ± 0.6

    Ca2+ 117.1 ± 1.6 SDS 83.6 ± 0.7

    Cu2+ 92.7 ± 1.4 Cantharidic acid 95.6 ± 1.5

    Mn2+ 93.4 ± 2.2 Epstatin A 99.3 ± 0.7

    Zn2+ 56.8 ± 1.1 Bestatin 89.8 ± 1.4

    E-64 87.0 ± 1.4

    aThe counterion for the tested metals was chloride. All values are the mean ± SD

    (n = 3).

    Fig. 4. Hydrolysis of fibrinogen by AprE176 (A) and M179 (B).Lane M, size marker (DokDo-MARK, ELPIS-Biotech. Inc., Daejeon,

    Korea.); 1, no enzyme treatment; 2, 30 sec; 3, 1 min; 4, 2 min; 5, 3 min;

    6, 4 min; 7, 5 min; 8, 10 min; 9, 20 min. A 12% acrylamide gel was used.

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    January 2015⎪Vol. 25⎪No. 1

    most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA, a preferred substrate for subtilisin and chymotrypsin.AprE176 and M179 showed no activity for the othersubstrates, indicating that AprE176 is a subtilisin-like serineprotease. The amidolytic activities of AprE176 and M179were 28.63 ± 0.20 and 39.84 ± 0.04 mM/min/mg protein,respectively, when N-succinyl-Ala-Ala-Pro-Phe-pNA wasused as the substrate. M179 showed 1.39-fold higheramidolytic activity than AprE176. The kinetic constants, Kmand kcat, were determined for AprE176 and M179 bymeasuring the initial rates of hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-pNA (Table 2). Although M179 showedenhanced fibrinolytic activity and catalytic turnover (kcat),it had a lower substrate affinity (Km) than AprE176. As aresult, the catalytic efficiency (kcat/Km) of M179 was notdifferent from that of AprE176.

    In the case of a keratinase from B. licheniformis BBE11-1, asingle amino acid change (N122Y) caused the increase ofthe catalytic efficiency by 5.6-fold compared with wild-type keratinase. The 122th residue of mutant (N122Y) waslocated 6.42 Å, 6.63 Å, and 2.45 Å from Asp32, His63, andSer220, respectively. Some intramolecular interactions amongthe aromatic 122th residue, the active site (Asp32, His63,Ser220), and substrate influenced to increase the catalyticefficiency [23], whereas other substitutions (N217S, A193P,and N160C), which were located far from the active site,did not significantly change the Km or kcat [23]. For a serinealkaline protease from B. pumilus CBS, the catalytic efficiencywas increased 42-fold by mutations (L31I/T33S/N99Y)[10]. The substitutions occurred at residues surroundingthe catalytic residue, Asp32, in serine alkaline proteases. Forthe above examples, changes occurred near the active site,and this enhanced the catalytic efficiency. In the case ofM179, the 176th residue was located far from the catalyticsite, and this might be the reason why the catalyticefficiency did not increase.

    Thermostability of M179

    M179 possessed improved thermal resistance comparedwith AprE176. Purified M179 retained 67% residual activityafter 150 min incubation at 45°C, whereas AprE176 had 50%activity (Fig. 5). When the incubation time was extended to

    5 h at 45°C, M179 still retained 36% activity but AprE176had only 11% activity. The thermostability of an alkalineprotease (BgAP) from Bacillus gibsonii was increased morethan 90-fold (half-life at 60°C) by directed evolution [24].The loop area of the BgAP surface was replaced bysubstitutions to negatively charged amino acids, whichcontributed to the increase of the thermostability, probablythrough increasing ionic and hydrogen bond interactionsin mutated BgAP. The half-life of a keratinase at 60°C fromB. licheniformis BBE11-1 was increased 8.6-fold by fouramino acid substitutions (N122Y, N217S, A193P, N160C)[23]. Increase in the thermostability was explained to be theresult of increased interactions, such as hydrophobicinteraction, cation-pi interaction, and hydrogen bond, in themutant. Interactions such as hydrogen bonds, hydrophobicinteractions, and electrostatic interactions are well knownto be the main factors for protein thermostability [33].

    To explain the increased thermostability of M179compared with wild-type ArpE176, molecular modelingwas used. Three-dimensional models of AprE176 and M179were constructed using the known structure of nattokinase(4DWW). Nattokinase and AprE176 share 76% identityin amino acid sequences, and homology-based modelstructures could be built using SWISS-MODEL Workspace(http://swissmodel.expasy.org/workspace/). Based on themodel structures, A176T mutation might be responsible forthe increased thermostability of M179. According to themodel structure of AprE176, three amino acids (Gly169,Tyr171, and Val174) are located within 3.0 Å radius from acalcium ion (Fig. 6A). For M179, the hydroxyl group ofThr176 is located closely (2.9 Å) to the calcium ion, whereas

    Table 2. Kinetic parameters of AprE176 and M179.

    Enzyme Km (mM) kcat (s-1) kcat/Km (s

    -1mM-1)

    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).

    Fig. 5. Temperature stability of AprE176 and M179. Thermal stability was determined at 45°C in Tris-HCl buffer (pH 8.0)

    for various times. AprE176, -●-; M179, -■ -.

  • 96 Jeong et al.

    J. Microbiol. Biotechnol.

    Ala176 of AprE176 does not show any interaction withcalcium ions. Bacillus fibrinolytic enzymes (subtilisin E [34],Carlsberg [25], and BPN [3]) have two calcium-bindingsites, Ca A and Ca B sites. The Ca A site is located near theN-terminus and has higher calcium-binding affinity, whereasCa B site has weaker binding affinity. The occupation of theCa B site is an important factor for the increase of thermalresistance. Increased thermal stability by increased calciumconcentration was shown by Pantoliano et al. [27] atcalcium concentrations ranging from 0.1 to 100 mM. It wasalso reported, in the X-ray crystal structure study usingsubtilisin, that the occupancy of the Ca B site was increasedas the calcium concentration was increased.

    From the model structure, it is suspected that Thr176 ofM179 is more likely located in the Ca B site. The existenceof an extra amino acid with favorable interaction withcalcium ions could possibly increase the affinity withcalcium ions, resulting in increase of the thermostability ofthe enzyme. Increased thermostability is desirable ifAprE176 and its derivatives are incorporated into foodsthat are expected to be heat treated.

    AprE176 possesses strong fibrinolytic activity, which canbe utilized for the production of fermented foods or drugspreventing cardiovascular diseases caused by fibrin clots.Mature AprE176 shows different physicochemical properties,such as pH optimum and inhibition by chemicals, fromenzymes with quite similar amino acid sequences. Theproperty of AprE176 can be improved by directed evolutionmethods, and M179, a derivative of AprE176, possesses

    improved thermostability. Further improvements arenecessary, and studies on the roles of important aminoacids for the activity of AprE176 should also be continued.

    Acknowledgments

    This work was supported by a grant from Korea Instituteof 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 RuralAffairs, Republic of Korea. S.-J. Jeong and J. Y. Park weresupported by BK21 Plus Program, MOE, Republic of Korea.

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