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Characterizations and Fibrinolytic Activity of Serine Protease from Bacil-lus subtilis C10

Nguyen T.A. Thu1,2, Nguyen T.M. Khue1, Nguyen D. Huy3, Nguyen Q.D. Tien1, Do T.B. Thuy4 and Nguyen H. Loc1*

1Institute of Bioactive Compounds and Department of Biology, University of Sciences, Hue University, Hue, Vietnam; 2University of Medicine and Pharmacy, Hue University, Hue, Vietnam; 3Institute of Biotechnology, Hue University, Vietnam; 4University of Agriculture and Forestry, Hue University, Hue, Vietnam

Abstract: Background: Fibrinolytic enzymes, such as Nattokinases from Bacillus species are known to degrade the fibrin blood clots. They belong to serine protease group having commercial applica-tions, such as therapeutic agents and functional food formulation.

Objective: The present study reports some characteristics and fibrinolytic activity of serine protease from B. subtilis C10 strain that was isolated from shrimp shell.

Methods: Extracellular enzyme from B. subtilis C10 culture was harvested and partially purified by ammonium sulphate precipitation. Fibrinolytic activity of the enzyme was determined by zymography and measured by spectrophotometry with fibrinogen and thrombin used as substrates. The optimal temperature and pH for fibrinolytic activity were studied in the range of 31-43ºC and 5-10, respective-ly. The thermal and pH stability of enzyme was studied by incubating enzyme for 30 min in the same range of temperature and pH as above. The effect of some metal ions and reagents on fibrinolytic ac-tivity of enzyme was evaluated by concentrations of 5 mM and 5%, respectively.

Results: Zymogram analysis indicated the presence of four fibrinolytic enzymes with molecular weights of approximately 69, 67, 39 and 36 kDa. The optimal temperature and pH for enzyme activity were 37°C and 9, respectively. The thermal and pH stability ranged from 35-39°C and 8-10, respec-tively. Fibrinolytic activity reached a maximum value of about 400 U/mg protein after 16 h of C10 strain culture. Enzyme has been drastically inhibited by PMSF and SDS, and partially inhibited by EDTA, while Triton X-100 has significantly increased enzyme activity. Effects of ions such as Mg2+, Ca2+ and Mn2+ on enzyme were negligible, except Cu2+ and Zn2+ have strongly decreased its activity.

Conclusion: Results from the present study suggested that enzyme obtained from B. subtilis C10 could be serine protease that has a high fibrinolytic activity up to about 400 U/mg protein at the most appro-priate temperature and pH of 37ºC and 9. This activity can be improved up to 142% by incubating en-zyme with 5% Triton X-100 for 30 min.

A R T I C L E H I S T O R Y

Received: June 17, 2019 Revised: August 26, 2019 Accepted: September 11, 2019 DOI: 10.2174/1389201020666191002145415

Keywords: Bacillus subtilis, fibrinolytic activity, nattokinase, serine protease.

1. INTRODUCTION

Serine proteases, as a subclass of protease, are proteolytic enzymes with a serine residue (Ser) on the active site. These enzymes are grouped into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like [1]. Fibrinolytic proteases (e.g. tPA) belonging to serine proteases usually operate at neutral and alkaline pH, with an optimum between pH 8 and 10 [2]. Fibrinolytic proteases (also known as nattokinase) have an ability to degrade fibrin

*Address correspondence to this author at the Institute of Bioactive Com-pounds and Department of Biology, University of Sciences, Hue University, Hue, Vietnam; E-mail: nhloc@hueuni.edu.vn

which is normally formed from fibrinogen by the action of thrombin [3]. In human, the tPA catalyzes the conversion of plasminogen to plasmin for slicing fibrin to avoid thrombosis in blood vessels [4]. According to Di Cera [5], a typical ge-nome contains 2-4% of genes encoding for proteolytic en-zymes. Among these, serine proteases were considered as the most abundant and functionally diverse group.

On the basis of the catalytic mechanism, fibrinolytic pro-teases from microorganism are classified into three types, serine protease (e.g. nattokinase from Bacillus), metallo pro-tease (e.g. Armillaria mellea metallo protease), and mixture of both types of proteases above (e.g. protease from Strepto-myces) [6]. Some fibrinolytic proteases produced by Bacillus

2 Current Pharmaceutical Biotechnology, 2020, Vol. 21, No. 0 Thu et al.

species have also been reported. For example, plasmin-like serine protease and subtilisin family serine protease from B. subtilis QK02 [7], subtilisin FS33 from B. subtilis DC33 [8], subtilisin-like protease from B. subtilis TP-6 [9], serine pro-tease from B. amyloliquefaciens An6 [10], subtilisin like serine protease from B. subtilis B2805 [11], serine/metallo protease from B. subtilis BR21 [12] and B. subtilis HK176 [13] etc.

In the present work, partial purified proteases (known as serine protease due to their fibrinolytic activity and optimal pH in the range of 8-10) from B. subtilis C10 strain that iso-lated from shrimp shell has been characterized.

2. MATERIALS AND METHODS

2.1. Bacterial Isolation and Identification

B. subtilis C10 strain was isolated from shell waste of brackish water shrimp in Thua Thien Hue, Vietnam. Shell waste was collected from shrimp processing, then it was washed with tap water, dried and ground to powder. One gram of powder was suspended in 5 mL of sterile saline so-lution (0.5% KH2PO4, 0.5% MgSO4.7H2O and 0.5% NaCl, pH 7) and incubated at 37ºC for 48 hours. Pasteurized (15 min 80°C) dilutions (in 0.9% NaCl) of the culture were spread onto skim milk-LB agar plate (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar and 1% skim milk) and incubated again as described above. A clear zone of hydroly-sis surrounding the colony confirmed the protease produc-tion. Molecular identification of protease-producing isolates was conducted by 16S rRNA gene sequence analysis. 16S rRNA gene was amplified from genome by PCR with uni-versal primers. The amplicon was sequenced and compared through a BLAST search in database of Genbank (NCBI).

2.2. Bacterial Culture and Enzyme Production

B. subtilis C10 was grown in medium containing 1% peptone, 0.5% sodium chloride and 0.3% beef extract (pH 7) for 36 h at 37ºC on a shaker (MRC, LM-570) with a speed of 200 rpm [14]. The Td is calculated using the following for-mula [15]:

Where: t is duration of culture, a is final cell biomass, and b is initial cell biomass.

The serine protease production was carried out under the same condition using medium with 0.6% peptone, 0.5% so-dium chloride, 0.18% beef extract, and 1% skim milk (Sig-ma-Aldrich) as a substrate (pH 7). The supernatant from the culture was harvested after 16 h by centrifugation at 14,000 rpm/4ºC for 5 min on Mikro 22R machine (Hettich Co.) and used as a crude enzyme. For partial purification, crude en-zyme was precipitated by ammonium sulfate (70% satura-tion) at 4ºC for 2 h and then centrifuged at 15,000 rpm/4ºC for 10 min. The pellet was resuspended and dialyzed for fur-ther use.

2.3. Enzymatic Activity Assay

The proteolytic activity was determined by loading en-zyme into pre-punched hole on the assay plate containing 1.5% agar and 2% skim milk. The plate was incubated at 4ºC for 8 h for diffusing enzyme, and then at 37ºC for 5 h for proteolysis. Finally, the plate was stained with 0.1% amido black solution for detection of substrate hydrolysis. Protease activity assay using casein (Sigma-Aldrich) as a substrate was performed according to Anson’s method [16] with slight modifications. Reaction mixture consists of 1 mL of enzyme and 2 mL of 2% (w/v) casein (dissolved in 50 mM Tris-HCl, pH 7) was incubated at 50°C for 15 min. The hydrolytic re-action was then stopped with 5 mL of 5% (w/v) TCA and kept at RT for 20 min to precipitate the residue substrate. Tyrosine that released from casein was measured spectro-photometrically at a wavelength of 750 nm on Evolution 60S machine (Thermo Scientific). One unit of protease activity is defined as the amount of enzyme required to liberate 1 µmol of tyrosine per 1 mL per min under the standard assay condi-tions.

Nattokinase activity was determined as described by Deepak et al. [17]. The reaction mixture containing 140 µL of 50 mM Tris-HCl (pH 7) and 40 µL of 0.72% fibrinogen (Sigma-Aldrich) was incubated at 37ºC for 10 min, followed by the addition of 0.2 U thrombin (Sigma-Aldrich) and 10 µL of enzyme, and continue to keep at 37ºC for 30 min. The reaction was then terminated with 200 µL of 0.2 M TCA, the supernatant was recovered by centrifugation at 15,000 rpm/ 4ºC for 5 min, and the nattokinase activity was measured at a wavelength of 275 nm on UV-Vis spectrophotometer (Evo-lution 60S, Thermo Scientific). One unit of nattokinase ac-tivity is defined as the amount of enzyme required to in-crease the absorbance by 0.01 at 275 nm after 1 h incubation. The nattokinase specific activity is expressed as units of en-zyme per milligram of total proteins. Total protein concentration was determined by Brad-ford’s method [18] with bovine serum albumin (Sigma-Aldrich) as the standard. The absorbance was measured at a wavelength of 595 nm against the blank.

2.4. Zymography

Nattokinase zymogram was performed as described by Choi et al. [19] with slight modifications. The polyacryla-mide gel solution (12%, w/v) was prepared in the presence of fibrinogen (0.12%, w/v) and 100 µL of thrombin (10 NIH U/mL). Enzyme was dissolved in sample buffer (0.1 µg/µL), that consisted of 0.5 M Tris-HCl (pH 6.8), 10% SDS, 20% glycerol, and 0.03% bromophenol blue. SDS-PAGE was run at 4ºC on omniPAGE Mini Vertical System (Cleaver Scien-tific Ltd.) and the gel was then incubated in solution contain-ing 50 mM Tris-HCl (pH 7.4) and 2.5% Triton X-100 for 30 min at RT to remove SDS. In the next step, the gel was washed with ddH2O for 30 min to remove Triton X-100 and then incubated in reaction buffer [30 mM Tris-HCl (pH 7.4) and NaN3] at 37ºC for 12 h. Finally, the gel was stained with Coomassie Blue R-250 for 1 h to reveal the zones where fibrin was digested, and visualized as clear zones (unstained zones) of the zymogram gel.

batTd loglog

2log−

×=

Characterizations and Fibrinolytic Activity of Serine Protease Current Pharmaceutical Biotechnology, 2020, Vol. 21, No. 0 3

2.5. Characterization of Serine Protease

The optimal temperature and pH for fibrinolytic activity were investigated in a range of 31 to 43°C and 5 to 10, re-spectively. Buffers for optimal pH determination were 20 mM citrate buffer (pH 5-6), 20 mM phosphate buffer (pH 7-8), and 20 mM glycine-NaOH buffer (pH 9-10). The temper-ature and pH stability of enzyme were determined by incu-bating enzyme for 30 min at same temperatures and pHs of the optimal investigations without substrate, enzyme solution was then immediately cooled to 4°C. The relative activity of the enzyme was calculated by comparing with enzyme incu-bated at 37°C and pH of 7 (control). Effect of some metal ions and reagents on fibrinolytic activity were tested by incubating enzyme at an appropriate temperature and pH for 30 min with 5 mM metal ion (Mg2+, Cu2+, Ca2+, Zn2+, and Mn2+) or 5% reagents (PMSF, SDS, Tri-ton X-100, and EDTA). The relative activity of the enzyme was calculated by comparing with enzyme incubated under similar condition without metal ions or reagents (control).

2.6. Statistical Analysis

Each bacterial culture and biochemical experiment was repeated thrice and the averages were compared by one-way ANOVA (Duncan’s test at a level of 0.05) using SPSS pro-gram.

3. RESULTS AND DISCUSSION

3.1. Growth of B. subtilis C10

Bacillus subtilis C10 exhibited a simple pattern for the growth curve and shown the changes in the size of a bacterial population over time during culture. B. subtilis C10 cells reached a maximum biomass of approximately 1.1×1010 cells/mL after 16 h of culture, corresponding to Td of bio-mass is about 3.6 h. During the period of 16-20 h, the cells seem to enter the stationary stage, so the differences in cell density are non-significant (p>0.05). After 20 h, the cells entered into a dead phase. Total extracellular protein content had a peak of approximately 0.35 mg/mL at 16 h (Fig. 1).

Posada-Uribe et al. [20] obtained a maximum biomass of 8.78×109 CFU/mL from B. subtilis EA-CB0575 after 96 h of culture at 30°C with a shaking speed of 150 rpm. Mean-while, the study of Sahoo et al. [21] showed that B. subtilis MTCC.736 achieved the highest number of live active cells after 10 h of culture, at the late log phase or the early station-ary phase, which corresponds to about 106 cells/mL. The growth of B. subtilis MTCC.121 has been also recorded that the lag phase exists for first 4 h followed by a log phase for 12 h and stationary phase exists for 20 h when bacterial cells were cultured at 30ºC with a shaking speed of 150 rpm [22]. According to Hakim et al. [23], the growth of B. subtilis AKAL7 has peaked with cell density of about 1011 CFU/mL after 24 h of culture in basal medium at 37ºC and 120 rpm.

3.2. Enzyme Activity

Proteolytic action of extracellular enzyme from B. sub-tilis C10 was determined by hydrolytic plate with skim milk as a substrate. Data from (Fig. 2A) showed that D-d was about 3.8 cm with D as the diameter of the clear zone and d as the diameter of pre-punched hole for loading enzyme. Protease activity had peaked about 75 U/mg protein after 16 h of culture (Fig. 2B). A study of Chantawannakula et al. [24] showed that the crude enzyme of B. subtilis 38 strain exhibited the high pro-teolytic action on skim milk agar plate with a clear zone of 480 mm2 (~ 24.72 mm diameter). Gelatin was also used as a substrate in B. subtilis culture, Pant et al. [25] found the maximum proteolytic zone (diameter of 22 mm) on agar plate after overnight incubation. Protease productivity reached the highest level with a total activity of up to 475.56 U/mL after 36 h of culture at 45ºC and pH 10. B. subtilis I-2 strain has produced a maximum protease amount of 797.28 U/mL (calculated against 1 µg tyrosine was released) in me-dium where soybean meal was used as nitrogen source at 37ºC with a shaking speed of 180 rpm [26].

Zymography was carried out for the detection of fibrino-lytic activity of serine proteases produced by B. subtilis C10 strain. Zymogram showed the presence of four clear zones with the molecular weight of approximately 36, 39, 67 and

Fig. (1). Time course of growth and total extracellular protein of B. subtilis C10. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

4 Current Pharmaceutical Biotechnology, 2020, Vol. 21, No. 0 Thu et al.

69 kDa against a background of the gel stained with bromo-phenol blue. Among them, the 39 kDa zone displayed the higher fibrinolytic activity (Fig. 3A). A gene coding 39 kD serine protease with fibrinolytic activity has been also isolat-ed from B. subtilis C10 and deposited in NCBI with acces-sion number as KU341112 (data not shown). Fibrinolytic activity also reached the highest value of about 400 U/mg protein after 16 h of culture (Fig. 3B).

Bajaj et al. [26] found three iso-forms in fibrin zymogra-phy of B. subtilis I-2 with molecular weights of approximate-ly 42, 48 and 60 kDa. However, Yogesh and Halami [12] found six prominent clear zones in the fibrin zymogram gel that related to protease from B. subtilis BR21 strain. Mean-while in some other B. subtilis strains, there is only one clear zone with molecular weight of about 27 kDa [27], 28 kDa [28], 35 kDa [29] or 45 kDa [30]. In general, fibrinolytic enzymes are very different in molecular weight and amount in B. subtilis strains.

Fig. (3). A) SDS-PAGE (lane 1) and fibrin zymography (lane 2) of serine protease from B. subtilis C10. M: protein weight marker. B) Fibrinolytic activity of serine protease from B. subtilis C10. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

3.3. Characteristics of Enzyme

The results from (Fig. 4) showed that the enzyme from B. subtilis C10 exhibited the highest fibrinolytic activity at an optimal temperature of 37ºC and pH of 7 (approx. 100%); however, considerable activities were observed at 35-39ºC (relative activity was approx. 80%). Thermostability investi-gation indicated that enzyme activity was quite stable at 31-39ºC (relative activity of about 90%) for 30 min but it de-creased drastically at 41ºC or above. Proteases have main-tained more than 95% of the fibrinolytic activity at pH range of 8-10 with the optimum at pH 9 and 37ºC (relative activity was more than 115%); enzymes also exhibited poor activity in the acidic pH 5-6 (50-55%). pH stability analysis showed that proteases also possessed remarkable stability at pH 8-10 (90-98%). In the acidic pH (5-6), the enzyme retained more 60% activity (Fig. 5). Alekseeva et al. [11] showed subtilisin-like serine prote-ase from B. subtilis B2805 had a maximum activity in pH range from 7.5 to 10 with the optimum was found at pH 8.

Fig. (2). Proteolytic activity of total extracellular protease from B. subtilis C10. A) agar plate with skim milk as substrate, B) total protease activity. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

Characterizations and Fibrinolytic Activity of Serine Protease Current Pharmaceutical Biotechnology, 2020, Vol. 21, No. 0 5

Fig. (4). Optimal temperature and thermostability of serine protease from B. subtilis C10. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

Fig. (5). Optimal pH and pH stability of serine protease from B. subtilis C10. (A higher resolution/colour version of this figure is available in the electronic copy of the article). However, it only retained about 50 and 60% at pH 6 and 11, respectively. The enzyme remained stable within 30 min at 40°C. At 45°C the remaining activity of enzyme was about 40%, and the enzyme was inactivated completely at 60°C. According to Bajaj et al. [26], fibrinolytic protease from B. subtilis I-2 showed maximum activity at optimal temperature of 50ºC and its thermostability was remained at 30-50ºC for 60 min, the enzyme activity decreased drastically at 60ºC or above. Enzyme displayed maximum activity in the pH range of 7-9 with optimum at pH 8 and it possessed remarkable stability at pH 7-10. In the acidic pH (5-6), enzymes has low activity and poor thermostability. Although the optimal tem-perature and thermostability are different from fibrinolytic protease of B. subtilis C10, they all belong to alkaline prote-ase group that acts within a pH range of 8-10.

Matthews [31] indicated that metal ions can modulate protein-protein, protein-substrate, and other intermolecular

interactions. In this study, Cu2+ and Zn2+ ions significantly reduced the fibrinolytic activity of serine protease from B. subtilis C10 (relative activity was about 32 and 37%, respec-tively). The reason may be that these ions have inhibited central site of enzyme and also conformational changes in the enzyme molecule, the most probably in lysine-binding sites [32]. Other divalent ions have negligible influence on enzyme activity. For Mg2+ ion, enzyme activity slightly in-creased by about 107%; while for Ca2+ and Mn2+ ions, en-zyme activity remained around 87%.

As the known PMSF is an irreversible inhibitor of the serine protease [33] and SDS is a denaturing agent [34], so they usually cause a significant inhibition of the enzyme activity. In the present work, PMSF and SDS also displayed a stronger inhibition of protease than EDTA, relative activity of the enzyme is only about 7 and 16%, respectively. Mean-

6 Current Pharmaceutical Biotechnology, 2020, Vol. 21, No. 0 Thu et al.

while, Triton X-100 increased enzyme activity by more than 142% (Table 1).

Table 1. Effect of metal ions and reagents on fibrinolytic ac-

tivity of serine protease from B. subtilis C10.

Metal Ions and Reagents Relative Activity (%)

Control 100

Ca2+ 87.42 ± 4.01

Cu2+ 32.01 ± 2.78

Zn2+ 37.33 ± 2.59

Mn2+ 86.59 ± 5.77

Mg2+ 107.23 ± 7.52

PMSF 7.45 ± 0.81

SDS 16.44 ± 1.01

EDTA 70.51 ± 4.55

Triton X-100 142.48 ± 8.19

A report showed that EDTA and all the metal ions tested (Cu2+, Mn2+, Ca2+, Co2+, Zn2+ and Mg2+) at 2 mM caused significant activity decrease of fibrinolytic protease from B. subtilis I-2, except Fe2+ which only reduced slightly the en-zyme activity [26]. While fibrinolytic activity of Nattokinase from B. amyloliquefaciens DC-4 was inhibited by Zn2+, Cu2+, EDTA, Mn2+ and Fe2+. Ca2+ and Mg2+ did not activate en-zyme, but Na+ and K+ slightly increased its activity. Triton X-100 resulted in a complete loss of fibrinolytic activity of enzyme [28]. In general, our results obtained above suggested that the enzyme obtained from B. subtilis C10 could be a serine pro-tease. B. subtilis C10 could be of potential use for cost-effective production of fibrinolytic protease that could be useful for medical applications. Further enhancement of pro-tease production from B. subtilis C10 could be attempted by using statistical optimization approaches and genetic engi-neering tools.

LIST OF ABBREVIATION

ANOVA = Analysis of Variance CFU = Colony Forming Unit EDTA = Ethylenediaminetetraacetic Acid LB = Luria-Bertani NIH = National Institute of Health PAGE = Polyacrylamide Gel Electrophoresis PMSF = Phenylmethylsulfonyl Fluoride RT = Room Temperature SDS = Sodium Dodecyl Sulfate TCA = Trichloroacetic Acid

Td = Doubling Time tPA = Tissue Plasminogen Activator

ETHICS APPROVAL AND CONSENT TO PARTICI-PATE

The study was only performed on bacteria Bacillus sub-tilis without any impact on humans or animals.

HUMAN AND ANIMAL RIGHTS

The study was not carried out on human and animal.

CONSENT FOR PUBLICATION

The study has received approval from Hue University, Vietnam for publication and all authors have consented to submit this manuscript to the Current Pharmaceutical Bio-technology.

AVAILABILITY OF DATA AND MATERIALS

The article does not contain data or materials from any sources.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

This work was supported by the University of Sciences, Hue University, Vietnam. ORCID ID - Nguyen T.A. Thu: no ORCID ID - Nguyen T.M. Khue: no ORCID ID - Nguyen D. Huy: 0000-0002-9146-1122. ORCID ID - Nguyen Q.D. Tien: 0000-0002-7330-3139. ORCID ID - Nguyen H. Loc: 0000-0002-6387-0359. ORCID ID - Do. T.B. Thuy: No.

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