TR-04104; No of Pages 7

Thrombosis Research xxx (2010) xxxxxx

Contents lists available at ScienceDirect

Thrombosis Research
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s

Regular Article

Purication and characterization of a novel anticoagulant and brinolytic enzyme

produced by endophytic bacterium Paenibacillus polymyxa EJS-3
Fengxia Lu, Zhaoxin Lu , Xiaomei Bie, Zhengying Yao, Yufeng Wang, Yaping Lu, Yao Guo
College of Food Science and Technology, Nanjing Agricultural University, Weigang, Nanjing, 210095, P.R. China

a r t i c l e i n f o a b s t r a c t

Article history: Introduction: Endophytes may become a new source of thrombolytic agents for thrombosis treatment.
Received 28 March 2010 Materials and Methods: A novel brinolytic enzyme from Paenibacillus polymyxa EJS-3 (PPFE-I) was puried
Received in revised form 20 July 2010 with ammonium sulfate precipitation, hydrophobic chromatography, ion exchange and gel ltration
Accepted 4 August 2010 chromatography. The characterization of the enzyme was investigated by means of brinolysis plate,
Available online xxxx
hydrolysis of brinogen and anticoagulant effect in vitro.
Results: The brinolytic enzyme is puried to homogeneity with a purication of 14.5 fold and a recovery of
Keywords:
Paenibacillus polymyxa
3.3%. The enzyme was shown to have a molecular mass of 63.3 kDa by matrix assisted laser desorption
Fibrinolytic enzyme ionization time-of-ight mass spectrometry (MALDI-TOF-MS). The optimum temperature and pH value were
Fibrinolytic activity 37 C and 7.5, respectively. Results from the brinolysis pattern showed that the enzyme rapidly hydrolyzed
Endophytes the A-chain of brinogen, followed by the B-chains. It also hydrolyzed the -chains, but more slowly. It
was activated by metal ions such as Zn2+, Mg2+, and Fe2+, but inhibited by Ca2+ and Cu2+. Furthermore,
PPFE-I activity was inhibited strongly by PMSF, and it was found to exhibit a higher specicity for the
synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA for chymotrypsin, indicating that the enzyme is a
chymotrypsin-like serine protease. Additionly, PPFE-I showed a signicant anticoagulant effect in vitro.
Conclusion: The brinolytic enzyme PPFE-I from endophytic bacterium Paenibacillu polymyxa EJS-3 exhibits a
profound brinolytic activity.
2010 Elsevier Ltd. All rights reserved.

1. Introduction have some advantages in large quantity of production and oral

Thromboembolism is a medical complication with high morbidity
and mortality. Thrombolytic therapy is the best way to achieve
recanalization in these disesases nowadays. Thrombolytic agents have
been extensively used in the therapeutic treatment of thrombosis [1].
According to their action mechanisms, thrombolytic agents are of two
types. One is plasminogen activators, e.g. tissue-type plasminogen
activator (t-PA) [2] and urokinase [3], which activates plasminogen
into active plasmin to degrade brin. The other type is plasmin-like
proteins, e.g. nattokinase [4], which directly degrade brin. Despite
their widespread use, all these agents have hemorrhagic side effects,
short half-life in the body, and are also relatively expensive. It is
indispensable to search for novel brinolytic enzymes from various
sources since a variety of cardiovascular diseases and drawbacks in
the typical thrombolytic agents are being increasingly reported.
Many organisms are important sources of thrombolytic agents.
Effective thrombolytic agents have been identied and characterized
from animals [57], plants [8] and microorganisms [912]. Microorgan-
isms are important sources of thrombolytic agents [13], because they
administration for thrombotic diseases such as the acute myocardial
and cerebral infarctions [14]. So, various brinolytic enzymes were
successively discovered from different microorganisms, such as bacteria,
actinomyces, fungi, and algae. Of them, only few reports are from
endophytes origin.
Endophytes are a special group of microorganisms, which have been
demonstrated to be excellent producers of bioactive and structurally
novel metabolites [15]. A lot of novel bioactive products such as
antibiotics, anticancer and antidiabetic agents have been isolated from
endophytes [16]. Few studies, however, have been conducted on
brinolytic enzymes biosynthesized by endophytes [1720]. Prior to
our report, brinolytic enzymes had not yet been identied in the culture
supernatant of Paenibacillus polymyxa EJS-3, which isolated from tissues
of Stemona japonica (Blume) Miq, a Chinese traditional medicine. We,
therefore, describe the purication and characterization of the brino-
lytic enzyme from Paenibacillus polymyxa EJS-3 in this paper.
2. Materials and Methods
2.1. Chemicals

Corresponding author. Tel.: + 86 25 84396583; fax: +86 25 84396431. Hiprep phenyl FF, RESOURCEMQ and Sephacryl S-300HR were
E-mail address: fmb@njau.edu.cn (Z. Lu). purchased from GE Healthcare UK Ltd. (Little Chalfont, Buckinghamshire,

0049-3848/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.thromres.2010.08.003

Please cite this article as: Lu F, et al, Purication and characterization of a novel anticoagulant and brinolytic enzyme produced by
endophytic bacterium Paenibacillus polymyxa EJS-3, Thromb Res (2010), doi:10.1016/j.thromres.2010.08.003
e2 F. Lu et al. / Thrombosis Research xxx (2010) xxxxxx
England). Bovine brinogen, human brinogen, and bovine serum
albumin (BSA)were purchased from Sigma (USA). Thrombin and
urokinase were purchased from Chinese Medicine Testing Institute.
Phenylmethylsulfonyl uoride (PMSF), ethylenediaminetetraacetic acid
(EDTA), pepstatin A, Para-nitroanilide(pNA), H-D-Val-Leu-Lys-Pna,
Pyro-Glu-Gly-Arg-pNA, and N-Succinyl-Ala-Ala-Pro-Phe-pNA were
obtained from Sigma (USA). Acrylamide (Acr) and N, N-methy-lene-
bis-acrylamide (Bis) were from Bio-Rad (Hercules, CA). All other
reagents were analytical grade.
2.2. Bacterial strain and culture condition
Paenibacillus polymyxa EJS-3 producing brinolytic enzyme was
isolated from tissues of Stemona japonica (Blume) Miq, a Chinese
traditional medicine. The strain was grown at 33 C in a shaking water
bath in medium containing 1% tryptone, 0.5% beef extract, 0.5% NaCl,
pH 7.2. After 24 h cultivation, the seed culture broth 4% (v/v) was
transferred into fermentation medium (10% potato,0.5% tryptone,
0.5% yeast extract, 0.25% K2HPO4, 0.1% KH2PO4, 0.02 % MgSO4, 0.02%
CaCl2, pH7.2) and fermented at 33 C, 150 rpm for 72 h. Culture
supernatant of the strain was obtained by centrifugation (10,000 g,
15 min).
2.3. Purication of brinolytic enzyme
The brinolytic enzyme was puried by chromatographic proce-
dures including hydrophobic chromatography, anion exchange and
gel ltration chromatography. All purication steps were performed
at 4 C.
2.3.1. Hiprep phenyl FF column chromatography
The culture supernatant was treated using a two-step salting-out
procedure at 4 C with ammonium sulfate, rst at 30% saturation, and
then at 70% saturation. The formed precipitate was collected by
centrifugation at 10,000 g for 20 min. The crude enzyme precipitate
was dissolved in 20 mM Tris-HCl buffer (pH7.4) with 1 M ammonium
sulfate and applied to a Hiprep phenyl FF column (1.6 10 cm)
equilibrated with 20 mM Tris-HCl buffer (pH7.4) containing 1 M
ammonium sulfate,and then eluted with a linear gradient of 1-0 M
ammonium sulfate in the same buffer at a ow rate of 3 ml/min. The
active fractions were identied (section 2.4) , pooled and dialyzed
against 20 mM Tris-HCl buffer (pH 7.4) overnight at 4 C. The dialyzed
enzyme was concentrated by lyophilization.
2.3.2. RESOURCEM Q column chromatography
The lyophilized enzyme dissolved in 20 mM Tris-HCl buffer (pH
8.5) was loaded on a RESOURCEM Q column (1 6 cm) previously
equilibrated with the same buffer. Elution was performed at a ow
rate of 2 ml/min with a linear gradient of 0-1.5 M NaCl in the same
buffer. The active fractions were collected and concentrated by
lyophilization.
2.3.3. Sephacryl S-300 HR column chromatography
The lyophilized enzyme was dissolved in 20 mM Tris-HCl buffer
(pH 7.4), subjected to a Sephacryl S-300HR column (1.6 60 cm)
equilibrated with 20 mM Tris-HCl buffer (pH 7.4), and then carried
out at a ow rate of 0.5 ml/min with the same buffer. The active
fraction were pooled, concentrated and analyzed for purity by SDS-
PAGE.
2.4. Enzyme assay on brin plate
Enzyme activity was determined by the brin plate methods [21]
with minor modications. In brief, 15 ml of 0.8 mg/ml bovine
brinogen solution (in 0.1 M sodium phosphate buffer, pH 7.4) was
mixed with 1 ml of thrombin solution (7.5 U/ml) and 20 ml of 0.8%
Please cite this article as: Lu F, et al, Purication and characterization of a novel anticoagulant and brinolytic enzyme produced by
endophytic bacterium Paenibacillus polymyxa EJS-3, Thromb Res (2010), doi:10.1016/j.thromres.2010.08.003
agarose solution in 90 mm Petri dish. 10 l of the enzyme sample
solution was placed in each lter piece (5 mm diameter) and
incubated at 37 C for 18 h. After measuring the diameter of clear
zone, the units (IU) of the enzyme activities were determined
according to the standard curve using urokinase as a standard
brinolytic enzyme.
2.5. Protein concentration
Protein concentration was determined by the method of Bradford
[22] using bovine serum albumin as standard, measuring the
absorbance at 595 nm. After column chromatography, the protein
concentration was estimated by measuring the absorbance at 280 nm.
2.6. SDS-PAGE and SDS-brin zymography
SDS-PAGE was performed at room temperature by the method of
Laemmli [23], using 10% polyacrylamide gel and stained with
Coomassie Brilliant Blue R250.
Fibrin zymography was performed by Kim and Choi's Method [24
26]. A separating gel solution (12%, w/v) was prepared in the presence
of 0.12% bovine brinogen (w/v) and 150 l of thrombin (7.5 U/ml).
The enzyme samples (45 l) were diluted in a zymogram sample
buffer (5), which consisted of 0.5 M Tris-HCl (pH 6.8), 10% SDS, 20%
glycerol and 0.03% bromophenol blue. Then, diluted enzyme samples
were electrophoresed into the brin-copolymerized gel at a 14 mA
constant current in the cold room (at 4 C). After electrophoresis, the
gel was soaked in 2.5% Triton X-100 containing Tris (50 mM) buffer
(pH 7.4) for 30 min at room temperature. After washing the gel in
distillation water for 30 min, the gel was incubated in 30 mM Tris
buffer (pH 7.4) containing 200 mM NaCl, 10 mM CaCl2, and 0.02%
NaN3 at 37 C for 16 h. The gel was stained with Coomassie blue for
2 h and then destained.
2.7. Determination of molecular weight
The molecular weight of the enzyme was determined by MALDI-
TOF-MS. The dried mixtures of protein samples were dissolved in 10 l
acetonitrile (ACN)/H2O (v/v, 30:70) containing 0.1% v/v triuoroacetic
acid (TFA) for 1 h. Sample preparation was carried out according to the
dried droplet method [27], The matrix solution was prepared by
dissolving Sinapic acid (SA) in ACN/H2O (30:70 v/v) with 0.1% v/v TFA at
a concentration of 8 mg/ml. The extracted HMW-GS solution (total
60 ml) was mixed with SA solution at the ratio of 1:10 (v/v) and 2 ml of
this protein-SA mixture was deposited on to a 96-sample MALDI probe
tip, and dried at room temperature. MALDI-TOF mass spectrometric
experiments were carried out on a TOF mass spectrometer (Bruker
Daltonics, Bremen, Germany) equipped with smart-beam laser. The
instrument was used with the following parameters: laser intensity,
mass range 20-500 kDa, acceleration voltage 25 kV, grid voltage 92%,
guide wire 0.3%, and delay time 450 ns. The Bin size was set at 20 ns and
input bandwidth at 100 Hz. Spectra were obtained in positive linear ion
mode and were averaged from 50 laser shots to improve the S/N level.
All the samples were automatically accumulated in a random pattern
over the sample spot to provide the nal spectrum. The protein standard
was used as external standard for mass assignment.
2.8. Effect of temperature on brinolytic activity and stability
The optimal temperature for the puried enzyme was determined
by measuring residual activity after incubation at different tempera-
tures (25-60 C) for 18 h. The thermal stability of the enzyme was
evaluated by incubating the enzyme in various temperatures for 30-
150 min at pH 7.4, and then the residual activities were measured. The
remaining activity was assessed using the brin plate method.
 F. Lu et al. / Thrombosis Research xxx (2010) xxxxxx e3

2.9. Effect of pH on brinolytic activity and stability 3. Results

The optimal pH for the puried enzyme was determined to be
within a pH range of 4.0-11.0 at 37C. The pH stability of the
enzyme was examined by incubating the enzyme for 24 h at 37C
with different buffers, and the residual activities were determined.
The remaining activity was assessed using the brin plate method.
The following buffer systems were used: 100 mM sodium acetate
buffer; pH 4.0-5.0; 100 mM phosphate buffer; pH 6-7.5; 100 mM
Tris-HCl buffer; pH 8.0-9.0 and 100 mM glycineNaOH buffer pH
10.011.0.
2.10. Effect of metal ions and protease inhibitors on brinolytic activity
The effect of metal ions on the activity of the enzyme was
investigated using MgSO4, ZnSO4, MnSO4, FeSO4, KCl, CuSO4, and
CaCl2. The puried enzyme was pre-incubated with various metal ions
at a concentration of 5 mM for 6 h at 37 C and the residual activities
were determined. The activity of the enzyme in the absence of metal
ions was taken as 100%.
The effects of protease inhibitors were also studied using PMSF,
EDTA, DTT, and pepstatin A. The enzyme was pre-incubated with
these protease inhibitors for 30 min at 37 C and the residual activities
were determined. The activity of the enzyme assayed in the absence of
inhibitors was taken as 100%.
2.11. Amidolytic activity
3.1. Purication of brinolytic enzyme and molecular weight
The brinolytic enzyme from Paenibacillus polymyxa EJS-3 was
puried using a combination of chromatographic steps listed in
Table 1. Protein concentration and brinolytic activity were used as
indices of purication. After the ammonium sulfate precipitation, a
Hiprep phenyl FF column, RESOURCEM Q column and Sephacryl S-
300HR column were used to purify the enzyme to homogeneity. The
Hiprep phenyl FF column gave several brinolytic enzyme peaks. The
major fraction with brinolytic activity was collected and applied
onto the RESOURCEM Q column, which yielded one major peak
showing brinolytic activity in the washed fraction. The active
fraction was further puried by the Sephacryl S-300HR column
chromatography, and three peaks appeared, of which the second peak
had brinolytic activity (data not shown), and showed a single band
of approximately 63 kDa by SDS-PAGE (Lane 1 of Fig. 1) and brin-
zymography (Lane 2 of Fig. 1). This active protein was named as PPFE-
I. The enzyme was puried 14.5-fold, with a nal yield of 3.3% by these
purication steps. The specic activity of the nal enzyme preparation
was estimated to be 2,096 IU/mg protein. As shown in Lane 3 of Fig. 1,
the culture supernatant of Paenibacillus polymyxa EJS-3 contained
three extracellular proteases that exhibited some degree of brino-
lytic activity on brin-zymography.
The molecular mass of PPFE-I was estimated to be 63.3 kDa by
MALDI-TOF-MS (Fig. 2). This value is similar to the value estimated
by SDS-PAGE (Fig. 1) indicates that the enzyme is a monomeric
protein.

Amidolytic activity was measured spectrophotometrically by

using chromogenic substrates as follows [10]. The reaction mixture
(1 ml) contained 20 l of 0.05 mg/ml enzyme solution, 5 10-4 M
substrate, and 0.1 M sodium phosphate buffer (pH7.4). After
incubation for 5 min at 37 C with a spectrophotometer equipped
with a cuvette temperature controller, the amount of -nitroaniline
that was liberated was determined from the A405. One unit of
amidolytic activity (AU) was expressed as micromoles of substrate
hydrolyzed per minute per milliliter by the enzyme.
Km and Kcat for PPFE-I (1 g ) were determined with N-succinyl-
Ala-Ala-Pro-Phe-pNA as the substrate ( 0.1, 0.2, 0.4, 0.6, and 0.8 mM).
The reactions were performed at 0.1 M sodium phosphate bufferer
(pH 7.4) and 37 C for 3 min.
3.2. Effect of temperature on brinolytic activity and stability of PPFE-I
The effect of temperature on the brinolytic activity of PPFE-I was
examined at pH 7.4 (Fig. 3). The temperature showing the maximal
enzyme activity was 37 C (Fig. 3a). The relative activities at 30 and
40 C were about 84% and 86 %, respectively. The thermal stability
proles showed that the PPFE-I was stable at 30-40 C, and showed
25% and 0% residual activity after a 150 min incubation at 50 and
60 C, respectively (Fig. 3b).
3.3. Effect of pH on brinolytic enzyme activity and stability of PPFE-I

The effect of pH on the brinolytic activity of PPFE-I was

2.12. Fibrinogenolytic activity analysis
Fibrinogenolytic activity was measured by a modied brinogen-
olytic assay [28]. The brinogen solution (50 l of 2.5% human
brinogen in 50 mmol/L Tris-HCl buffer, pH7.4) was mixed with the
puried enzyme solution (10 l of 0.4 mg/ml) and incubated at 37 C
for 15 min, 30 min, 1 h, 2 h, 4 h and 8 h, respectively. After the
indicated time intervals, aliquots were transferred to ice and
separated by SDS-PAGE to examine the cleavage pattern of the
brinogen chains.
determined using buffers at various pHs. The optimum pH for the
brinolytic activity was found to be 7.5, and the relative activity was
about the same in the range of pH 7.0-8.0 (Fig. 4a). The pH stability of
the enzyme was investigated in the range of pH 4-11 by measuring
the residual activity after incubation at each pH for 24 h. The enzyme
was very stable in the range of pH 6.08.0 at 37 C for 24 h, but below
pH 6.0 or above pH 8.0, the enzyme stability abruptly decreased
(Fig. 4b).

Table 1
2.13. Evaluation of the anticoagulant effect of PPFE-I in vitro Purication of brinolytic enzyme from Paenibacillus polymyxa EJS-3.

Step Total Total brinolytic Specic Yield Purication

The anticoagulant effect of PPFE-I was observed by adding of 1 ml Protein activity activity (%)
fresh human whole blood to 1 ml 150 IU/ml of PPFE-I solution (0.9% (mg) (IU) (IU/mg)
NaCl, pH 7.4) in a glass test tube. The resulting solution was blended Culture supernatant 692 100220 144.8 100 1
and then incubated at 37C for 6 h. The fresh human whole blood was 30%-70% (NH4)2SO4 205.8 50819.4 246.9 50.7 1.7
obtained from healthy young male. As a positive or negative control, Hiprep phenyl FF 32.5 20964 645.1 20.9 4.5
200 IU/ml urokinase( 0.9% NaCl, pH 7.4) and normal saline were used RESOURCEM Q 9.1 10887.5 1196.4 10.9 8.3
Sephacryl S-300HR 1.6 3353.9 2096.2 3.3 14.5
instead of PPFE-I, respectively.

Please cite this article as: Lu F, et al, Purication and characterization of a novel anticoagulant and brinolytic enzyme produced by
endophytic bacterium Paenibacillus polymyxa EJS-3, Thromb Res (2010), doi:10.1016/j.thromres.2010.08.003
e4 F. Lu et al. / Thrombosis Research xxx (2010) xxxxxx

a
118kDa 100

80

Relative activity(%)
63kDa
49kDa 60

40

20

0
25 30 37 40 45 50 55 60
Temperature(C)

b
Fig. 1. SDS-PAGE of puried brinolytic enzyme. M: Protein markers; Lane 1: Puried 100
brinolytic enzyme on SDS-PAGE; Lane 2: Puried brinolytic enzyme on Fibrin

Relative activity(%)
zymography; Lane 3: Culture supernatant of Paenibacillus polymyxa. EJS-3 on Fibrin
80
zymography.

60

3.4. Effect of metal ions and protease inhibitors on brinolytic activity of 40

PPFE-I
20
The effects of metal ions and various protease inhibitors on the
brinolytic activity of PPFE-I is summarized in Table 2. The effects of
0
various metal ions on the brinolytic activity of PPFE-I were -10 10 30 50 70 90 110 130 150
investigated from the residual activities assay after incubation of the Time(min)
enzyme with different metal ions for 6 h at 37 C. The enzyme
30C 37C 40C 50C 60C
activities were found to be enhanced by the addition of Zn2+, Mg2+
and Fe2+, but to be inhibited by the addition of Ca2+ and Cu2+ ions, Fig. 3. Effect of temperature on the brinolytic activity (a) and stability (b) of PPFE-I.
and to be not affected by monovalent K+ ion. To classify the puried
enzyme, the brinolytic activity of PPFE-I was also examined in the
presence of various protease inhibitors. As shown in Table 2, the
activity of PPFE-I was completely inhibited by the serine protease
inhibitor PMSF, and was also affected by the metalloprotease inhibitor a
EDTA with. 24% of its original activity being lost. However, other 100
Relative activity(%)

inhibitors, DTT had not signicant effect on the activity of the enzyme.
80
These results suggested that PPFE-I is a serine protease.
60

40

20

0
4 5 6 7 8 9 10 11 12
pH

b
100
Relative activity(%)

80

60

40

20

0
4 5 6 7 8 9 10 11
pH

Fig. 2. MALDI-TOF mass spectrum of PPFE-I. Fig 4. Effect of pH on the brinolytic activity (a) and stability (b) of PPFE-I.

Please cite this article as: Lu F, et al, Purication and characterization of a novel anticoagulant and brinolytic enzyme produced by
endophytic bacterium Paenibacillus polymyxa EJS-3, Thromb Res (2010), doi:10.1016/j.thromres.2010.08.003
 F. Lu et al. / Thrombosis Research xxx (2010) xxxxxx e5

Table 2
Effect of metal ions and protease inhibitors on the brinolytic activity of PPFE-I.

Chemicals Concentration (mmol/L) Relative activity (%)

Control - 100
Ca2+ 5 80.1 0.7
Zn2+ 5 109.3 0.6
Cu2+ 5 66.7 2.7
Mg2+ 5 111.4 0.4
Mn2+ 5 104.6 0.8
Fe2+ 5 110.5 0.2
K+ 5 103.7 0.3
EDTA 1 76.3 2.7
PMSF 1 0
DTT 1 95.0 0.4
Pepstatin A 1 77.2 2.5

Fig. 5. Analysis of the pattern of brinogenolysis by PPFE-I. Lane 1:Fibrinogen control

without PPFE-I after 0 min incubation; Lanes 2-7:brinogenolytic products by PPFE-I
3.5. Amidolytic activity after 0.25, 0.5, 1, 2, 4, 8 h incubation.

The amidolytic activity of PPFE-I was investigated with several
synthetic substrates (Table 3). The enzyme exhibited the highest
degree of specicity for the substrate N-succinyl-Ala-Ala-Pro-Phe-
pNA (for subtilisin or chymotrypsin) and lesser effects for the
substrate H-D-Val-Leu-Lys-pNA (for plasmin). According to the
effects of inhibitors on PPFE-I and its activities with Synthetic
substrates, PPFE-I was considered to be as a chymotrypsin-like serine
protease. The Km and Kcat of PPFE-I for hydrolysis of N-succinyl-Ala-
Ala-Pro-Phe-pNA were calculated to be 0.20 mM and 38.6 S-1,
respectively.
3.6. Fibrinogenolytic activity analysis
have been reported from endophytic fungi [17,18]. But no work on
brinolytic enzymes from endophytic bacterium was observed.
We have found three extracellular proteases (118, 63, and 49 kDa)
secreted by Paenibacillus polymyxa EJS-3. One of the three proteases
(63 kDa) was puried to electrophoretic homogeneity by combina-
tion of chromatographic steps. As shown in Fig. 1, the molecular mass
of PPFE-I was estimated to be 63.3 kDa by MALDI-TOF-MS. This value
is similar to the value assessed by SDSPAGE and brin-zymography.
a

The brinogenolytic activity and the degradation patterns of

brinogen for the enzyme were investigated by SDS-PAGE. As shown
in Fig. 5, the enzyme rapidly hydrolyzed A-chains of brinogen at
rst, followed by B-chains. It also hydrolyzed the -chains, but more
slowly. However, all chains were completely hydrolyzed after 4 h
incubation with PPFE-I, as well as brinogen fragments of 10-40 kDa
were formed.

3.7. Evaluation of the anticoagulant effect of PPFE-I in vitro

No blood clots were observed in the test tube of PPFE-I incubated

with the human whole blood after 6 h (Fig. 6-a3, b3). In the test tube
of normal saline, clotting had occurred (Fig. 6-a1, b1). As a positive
control, the blood clots were partially formed in the test tube of UK
after 6 h (Fig. 6-a2, b2). The result indicated that the enzyme
exhibited an efcient anticoagulant effect in vitro.

4. Discussion 1 2 3
In this work, we described purication and characterization of a b
brinolytic enzyme, designated as PPFE-I, from Paenibacillus polymyxa
EJS-3, which isolated from tissues of Stemona japonica (Blume) Miq
and differed from other brinolytic enzyme producing strains
previously reported. Until recently, only few brinolytic enzymes

Table 3
Amidolytic activity of PPFE-I with different substrates.

Synthetic substrate Characteristics Concentration Amidolytic 1 2 3

(mM) activity
(AU)

H-D-Val-Leu-Lys-pNA For plasmin 0.5 42.2

Pyro-Glu-Gly-Arg-pNA For urokinase 0.5 2.6
N-Succinyl-Ala-Ala-Pro- For subtilisin or 0.5 141.8 Fig. 6. Effect on the anticoagulant of PPFE-I solution. a. Effect on the anticoagulant of
Phe-pNA chymotrypsin PPFE-I in vitro;b. Formation of blood clots after incubation with PPEF-I.1. 0.9%(w/v) NaCl
(as a negative control); 2. 200 IU/ml UK (as a positive control); 3. 150 IU/ml PPFE-I.

Please cite this article as: Lu F, et al, Purication and characterization of a novel anticoagulant and brinolytic enzyme produced by
endophytic bacterium Paenibacillus polymyxa EJS-3, Thromb Res (2010), doi:10.1016/j.thromres.2010.08.003
e6 F. Lu et al. / Thrombosis Research xxx (2010) xxxxxx
Generally, the molecular weights of brinolytic proteases from
microorganisms range between 27.7 and 44 kDa [13]. The molecular
mass of PPFE-I was higher than those of brinolytic enzymes from
Cordyceps militaris (52.0 kDa) [29], Bacillus sp. KDO-13 (45.0 kDa)
[30], Bacillus subtilis KCK-7 (44.0 kDa) [31], Bacillus sp. KA38
(41.0 kDa) [32] and Bacillus subtilis natto (27.7 kDa) [4,9], indicated
PPFE-I was a novel brinolytic enzyme.
On the basis of catalytic mechanisms, microbial brinolytic
enzymes are classied into three types: serine protease, e.g. NK[4],
subtilisin DFE[13], and CK[10]; metalloprotease, e.g. AMMP [33] and
mixture of both serine and metalloprotease, e.g. Fu-P[19] and FP84
[34]. To clarify the type of PPFE-I, the brinolytic activity of PPFE-I was
also examined and shown in Table 2. The activity of PPFE-I was
notably inhibited by PMSF, and was also affected by EDTA. These
results suggested that PPFE-I was a serine protease. This feature was
similar as observed in the brinolytic enzymes from Pseudomonas sp.
TKU015.[35] and Bacillus subtilis DC33[36].
The substrate specicity of PPFE-I had the highest specicity for N-
Succ-Ala-Ala-Pro-Phe-pNA; the studies achieved almost the same
level of NK, subtilisin DFE, Fu-P, and subtilisin IMR-NK1 [37].
According to the effects of inhibitors on PPFE-I and its activities
with synthetic substrates, PPFE-I was considered to be as a
chymotrypsin-like serine protease.
To investigate the induced brinolysis mechanism of PPFE-I, the
brinolytic activity of PPFE-I on plasminogen-free and plasminogen-
rich brin plates was examined [12,20]. PPFE-I formed a similar-size
clear lysis zone on both plate types, indicating that the enzyme was
not a plasminogen activators (data not shown). PPFE-I also exhibited
brinogenolytic activity by rapidly hydrolyzing the brinogen the A-
chain, and then the B- and the -chains. The brinogenolysis pattern
of PPFE-I is similar to those of AMMP and Fu-P, but was different from
the brinolytic enzymes of subtilisin FS33 [36] and AJ [38].
Furthermore, we also found that PPFE-I showed an efcient
anticoagulant effect. These results indicated that PPFE-I to be a
direct-acting brinolytic and brinogenolytic agent as it acted via
direct cleavage of brinogen and not by plasminogen activators such
as SK, UK, and tPA.
The overall ndings of PPFE-I in relation to the molecular weight,
effect of inhibitors and metal ions, substrate specicity, brino(geno)
lytic activity, and effect of anticoagulant indicate that PPFE-I is a novel
anticoagulant and brinolytic enzyme different from other known.
In conclusion, the brinolytic enzyme PPFE-I, obtained from
endophytic bacterium Paenibacillu polymyxa EJS-3, exhibits a pro-
found brinolytic activity Therefore, endophytes may become a new
source for thrombolytic agents to treat thrombosis. Further studies
on the physiological function of PPFE-I in vivo lysis of thrombi are
required, and the cloning, sequencing, and expression of the PPFE-I
gene from chromosomal DNA of Paenibacillus polymyxa EJS-3 are
proceeding.
Acknowledgements
This work was supported by National High Technology Research
and Development Program of China (No.2008AA10Z309), High-Tech
Development Program of Jiangsu Province (No.BG2007337) and
Natural Science Fund of Jiangsu Province (No.BK2007160).
References
[1] Ouriel K. Current status of thrombolysis for peripheral arterial occlusive disease.
Ann Vasc Surg 2002;16:797840.
[2] Collen D, Lijnen HR. Tissue-type plasminogen activator: a historical perspective
and personal account. J Thromb Haemost 2004;2:5416.
[3] Duffy MJ. Urokinase plasminogen activator and its inhibitor, PAI-1, as prognostic
markers in breast cancer: from pilot to level 1 evidence studies. Clin Chem
2002;48:11947.
Please cite this article as: Lu F, et al, Purication and characterization of a novel anticoagulant and brinolytic enzyme produced by
endophytic bacterium Paenibacillus polymyxa EJS-3, Thromb Res (2010), doi:10.1016/j.thromres.2010.08.003
[4] Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. A novel brinolytic enzyme
(nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in
the Japanese diet. Experientia 1987;43:11101.
[5] Mihara H, Sumi H, Yoneta T, Mizumoto H, Ikeda R, Seiki M. A novel brinolytic
enzyme extracted from the earthworm, Lumbricus rubellus. Jpn J Physiol 1991;41:
46172.
[6] Zhang Y, Wisner A, Xiong YL, Bon C. A novel plasminogen activator from snake
venom. J Biol Chem 1995;270:1024655.
[7] Ahn MY, Hahn BS, Ryu KS, Kim JW, Kim I, Kim YS. Purication and characterization
of a serine protease with brinolytic activity from the dung beetles, Catharsius
molossus. Thromb Res 2003;112:33947.
[8] Maurer HR. Bromelain: biochemistry, pharmacology and medical use. Cell Mol Life
Sci 2001;58:123445.
[9] Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S. Purication and
characterization of a strong brinolytic enzyme (nattokinase) in vegetable cheese
natto, a popular soybean fermented food in Japan. Biochem Biophys Res Commun
1993;197:13407.
[10] Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, et al. Purication and characterization
of a brinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from
Chungkook-Jang. Appl Environ Microbiol 1996;62:24828.
[11] Chitte RR, Dey S. Potent brinolytic enzyme from a thermophilic Streptomyces
megasporus strain SD5. Lett Appl Microbiol 2000;31:40510.
[12] Peng Y, Huang Q, Zhang RH, Zhang YZ. Purication and characterization of a
brinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from
douchi, a traditional Chinese soybean food. Comp Biochem Physiol Biochem Mol
Biol 2003;134:4552.
[13] Peng Y, Yang X, Zhang Y. Microbial brinolytic enzymes: an overview of source,
production, properties, and thrombolytic activity in vivo. Appl Microbiol
Biotechnol 2005;69:12632.
[14] Ko J, Yan J, Zhu L, Qi Y. Subtilisin QK, a brinolytic enzyme, inhibits the exogenous
nitrite and hydrogen peroxide induced protein nitration, in vitro and in vivo. J
Biochem Mol Biol 2005;38:57783.
[15] Strobel GA. Endophytes as sources of bioactive products. Microbiol Infect 2003;5:
53544.
[16] Guo B, Wang Y, Sun X, Tang K. Bioactive natural products from endophytes: A
review. Appl Biochem Microbiol 2008;44:13642.
[17] Li Y, Shuang JL, Yuan WW, Huang WY, Tan RX. Verticase: a Fibrinolytic Enzyme
Produced by Verticillium sp. Tj33, an Endophyte of Trachelospermum jasminoides. J
Integrat Plant Biol 2007;49:154854.
[18] Mitsuhiro U, Toshihiro K, Kazutaka M, Takumi N. Purication and characterization
of brinolytic alkaline protease from Fusarium sp. BLB. Appl Microbiol Biotechnol
2007;74:3318.
[19] Wu B, Wu LC, Chen DJ, Yang ZJ, Luo MY. Purication and characterization of a
novel brinolytic protease from Fusarium sp. CPCC 480097. J Ind Microbiol
Biotechnol 2009;36:4519.
[20] Lu FX, Sun LJ, Lu ZX, Bie XM, Fang YW, Liu S. Isolation and Identication of an
endophytic Strain EJS-3 producing novel brinolytic enzymes. Curr Microbiol
2007;54:4359.
[21] Astrup T, Mullertz S. The brin plate method for estimating brinolytic activity.
Arch Biochem Biophys 1952;40:34651.
[22] Bradford MM. A rapid and sensitive method for the quantitation of micro-
gramquantities of protein utilizing the principle of protein-dry binding. Anal
Biochem 1976;72:24854.
[23] Laemmli UK. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 1970;227:6805.
[24] Choi NS, Kim SH. Application of brin zymography for determining the optimum
culture time for protease activity. Biotechnol Tech 1999;13:899901.
[25] Choi NS, Kim SH. Two brin zymography methods for analysis of plasminogen
activators on gels. Anal Biochem 2000;281:2368.
[26] Kim SH. Choi NS, Lee WY. Fibrin zymography: A direct analysis of brinolytic
enzymes on gels. Anal Biochem 1998;263:1156.
[27] Kussmann M, Nordhoff E, Rahbek-Nielsen H, Haebel S, Rossel-Larsen M, Jakobsen
L, et al. Matrix-assissted laser desorption/ionization mass spectrometry sample
preparation techiques designed for various peptide and protein. Analytes J Mass
Spectrom 1997;32:593601.
[28] Koh YS, Chung KH, Kim DS. Biochemical characterization of a thrombin-like
enzyme from snake. Toxcion 2001;39:55560.
[29] Kim JS, Kumar S, Park SE, Choi BS, Kim S, Nguyen TH, et al. A brinolytic enzyme
from the medicinal mushroom Cordyceps militaris. J Microbiol 2006;44:62231.
[30] Lee SK, Bae DH, Kwon TJ, Lee SB, Lee HH, Park JH. Purication and characterization
of a brinolytic enzyme from Bacillus sp. KDO-13 isolated from soybean paste. J
Microbiol Biotechnol 2001;11:84552.
[31] Paik HD, Lee SK, Heo S, Kim SY, Lee H, Kwon TJ. Purication and characterization of
the brinolytic enzyme produced by Bacillus subtilis KCK-7 from Chungkookjang. J
Microbiol Biotechnol 2004;14:82935.
[32] Kim HK, Kim GT, Kim DK, Choi WA, Park SH, Jeong YK, et al. Purication and
characterization of a novel brinolytic enzyme from Bacillus sp. KA38 originated
from fermented sh. J Ferment Bioeng 1997;84:30712.
[33] Lee SY, Kim JS, Kim JE, Sapkota K, Shen MH, Kim S, et al. Purication and
characterization of brinolytic enzyme from cultured mycelia of Armillaria mellea.
Protein Expr Purif 2005;43:107.
[34] Simkhada JR, Mander P, Cho SS, Yoo JC. A novel brinolytic protease from
Streptomyces sp. CS684. Process Biochem 2010;45:8893.
[35] Wang SL, Chen HJ, Liang TW, Lin YD. A novel nattokinase produced by
Pseudomonas sp. TKU015 using shrimp shells as substrate. Process Biochem
2009;44:706.
 F. Lu et al. / Thrombosis Research xxx (2010) xxxxxx e7

[36] Wang CT, Ji BP, Li B, Rob N, Li PL, Ji H, et al. Purication and characterization of a
brinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional
Douchi. J Ind Microbiol Biotechnol 2006;33:7508.
[37] Chang CT, Fan MH, Kuo FC, Sung HY. Potent brinolytic enzyme from a mutant of
Bacillus subtilis IMR-NK1. J Agric Food Chem 2000;48:32106.
[38] Choi NS, Song JJ, Chung DM, Kim YJ, Maeng PJ, Kim SH. Purication and
characterization of a novel thermoacid-stable brinolytic enzyme from Staphy-
lococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet. J Ind
Microbiol Biotechnol 2009;36:41726.

Please cite this article as: Lu F, et al, Purication and characterization of a novel anticoagulant and brinolytic enzyme produced by
endophytic bacterium Paenibacillus polymyxa EJS-3, Thromb Res (2010), doi:10.1016/j.thromres.2010.08.003