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Thrombosis Research xxx (2010) xxxxxx

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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


administration for thrombotic diseases such as the acute myocardial
Thromboembolism is a medical complication with high morbidity and cerebral infarctions [14]. So, various brinolytic enzymes were
and mortality. Thrombolytic therapy is the best way to achieve successively discovered from different microorganisms, such as bacteria,
recanalization in these disesases nowadays. Thrombolytic agents have actinomyces, fungi, and algae. Of them, only few reports are from
been extensively used in the therapeutic treatment of thrombosis [1]. endophytes origin.
According to their action mechanisms, thrombolytic agents are of two Endophytes are a special group of microorganisms, which have been
types. One is plasminogen activators, e.g. tissue-type plasminogen demonstrated to be excellent producers of bioactive and structurally
activator (t-PA) [2] and urokinase [3], which activates plasminogen novel metabolites [15]. A lot of novel bioactive products such as
into active plasmin to degrade brin. The other type is plasmin-like antibiotics, anticancer and antidiabetic agents have been isolated from
proteins, e.g. nattokinase [4], which directly degrade brin. Despite endophytes [16]. Few studies, however, have been conducted on
their widespread use, all these agents have hemorrhagic side effects, brinolytic enzymes biosynthesized by endophytes [1720]. Prior to
short half-life in the body, and are also relatively expensive. It is our report, brinolytic enzymes had not yet been identied in the culture
indispensable to search for novel brinolytic enzymes from various supernatant of Paenibacillus polymyxa EJS-3, which isolated from tissues
sources since a variety of cardiovascular diseases and drawbacks in of Stemona japonica (Blume) Miq, a Chinese traditional medicine. We,
the typical thrombolytic agents are being increasingly reported. therefore, describe the purication and characterization of the brino-
Many organisms are important sources of thrombolytic agents. lytic enzyme from Paenibacillus polymyxa EJS-3 in this paper.
Effective thrombolytic agents have been identied and characterized
from animals [57], plants [8] and microorganisms [912]. Microorgan- 2. Materials and Methods
isms are important sources of thrombolytic agents [13], because they
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 agarose solution in 90 mm Petri dish. 10 l of the enzyme sample
albumin (BSA)were purchased from Sigma (USA). Thrombin and solution was placed in each lter piece (5 mm diameter) and
urokinase were purchased from Chinese Medicine Testing Institute. incubated at 37 C for 18 h. After measuring the diameter of clear
Phenylmethylsulfonyl uoride (PMSF), ethylenediaminetetraacetic acid zone, the units (IU) of the enzyme activities were determined
(EDTA), pepstatin A, Para-nitroanilide(pNA), H-D-Val-Leu-Lys-Pna, according to the standard curve using urokinase as a standard
Pyro-Glu-Gly-Arg-pNA, and N-Succinyl-Ala-Ala-Pro-Phe-pNA were brinolytic enzyme.
obtained from Sigma (USA). Acrylamide (Acr) and N, N-methy-lene-
bis-acrylamide (Bis) were from Bio-Rad (Hercules, CA). All other 2.5. Protein concentration
reagents were analytical grade.
Protein concentration was determined by the method of Bradford
2.2. Bacterial strain and culture condition [22] using bovine serum albumin as standard, measuring the
absorbance at 595 nm. After column chromatography, the protein
Paenibacillus polymyxa EJS-3 producing brinolytic enzyme was concentration was estimated by measuring the absorbance at 280 nm.
isolated from tissues of Stemona japonica (Blume) Miq, a Chinese
traditional medicine. The strain was grown at 33 C in a shaking water
2.6. SDS-PAGE and SDS-brin zymography
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
SDS-PAGE was performed at room temperature by the method of
transferred into fermentation medium (10% potato,0.5% tryptone,
Laemmli [23], using 10% polyacrylamide gel and stained with
0.5% yeast extract, 0.25% K2HPO4, 0.1% KH2PO4, 0.02 % MgSO4, 0.02%
Coomassie Brilliant Blue R250.
CaCl2, pH7.2) and fermented at 33 C, 150 rpm for 72 h. Culture
Fibrin zymography was performed by Kim and Choi's Method [24
supernatant of the strain was obtained by centrifugation (10,000 g,
26]. A separating gel solution (12%, w/v) was prepared in the presence
15 min).
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
2.3. Purication of brinolytic enzyme
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
The brinolytic enzyme was puried by chromatographic proce-
were electrophoresed into the brin-copolymerized gel at a 14 mA
dures including hydrophobic chromatography, anion exchange and
constant current in the cold room (at 4 C). After electrophoresis, the
gel ltration chromatography. All purication steps were performed
gel was soaked in 2.5% Triton X-100 containing Tris (50 mM) buffer
at 4 C.
(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
2.3.1. Hiprep phenyl FF column chromatography
buffer (pH 7.4) containing 200 mM NaCl, 10 mM CaCl2, and 0.02%
The culture supernatant was treated using a two-step salting-out
NaN3 at 37 C for 16 h. The gel was stained with Coomassie blue for
procedure at 4 C with ammonium sulfate, rst at 30% saturation, and
2 h and then destained.
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 2.7. Determination of molecular weight
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 The molecular weight of the enzyme was determined by MALDI-
ammonium sulfate,and then eluted with a linear gradient of 1-0 M TOF-MS. The dried mixtures of protein samples were dissolved in 10 l
ammonium sulfate in the same buffer at a ow rate of 3 ml/min. The acetonitrile (ACN)/H2O (v/v, 30:70) containing 0.1% v/v triuoroacetic
active fractions were identied (section 2.4) , pooled and dialyzed acid (TFA) for 1 h. Sample preparation was carried out according to the
against 20 mM Tris-HCl buffer (pH 7.4) overnight at 4 C. The dialyzed dried droplet method [27], The matrix solution was prepared by
enzyme was concentrated by lyophilization. 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
2.3.2. RESOURCEM Q column chromatography 60 ml) was mixed with SA solution at the ratio of 1:10 (v/v) and 2 ml of
The lyophilized enzyme dissolved in 20 mM Tris-HCl buffer (pH this protein-SA mixture was deposited on to a 96-sample MALDI probe
8.5) was loaded on a RESOURCEM Q column (1 6 cm) previously tip, and dried at room temperature. MALDI-TOF mass spectrometric
equilibrated with the same buffer. Elution was performed at a ow experiments were carried out on a TOF mass spectrometer (Bruker
rate of 2 ml/min with a linear gradient of 0-1.5 M NaCl in the same Daltonics, Bremen, Germany) equipped with smart-beam laser. The
buffer. The active fractions were collected and concentrated by instrument was used with the following parameters: laser intensity,
lyophilization. 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
2.3.3. Sephacryl S-300 HR column chromatography input bandwidth at 100 Hz. Spectra were obtained in positive linear ion
The lyophilized enzyme was dissolved in 20 mM Tris-HCl buffer mode and were averaged from 50 laser shots to improve the S/N level.
(pH 7.4), subjected to a Sephacryl S-300HR column (1.6 60 cm) All the samples were automatically accumulated in a random pattern
equilibrated with 20 mM Tris-HCl buffer (pH 7.4), and then carried over the sample spot to provide the nal spectrum. The protein standard
out at a ow rate of 0.5 ml/min with the same buffer. The active was used as external standard for mass assignment.
fraction were pooled, concentrated and analyzed for purity by SDS-
PAGE. 2.8. Effect of temperature on brinolytic activity and stability

2.4. Enzyme assay on brin plate The optimal temperature for the puried enzyme was determined
by measuring residual activity after incubation at different tempera-
Enzyme activity was determined by the brin plate methods [21] tures (25-60 C) for 18 h. The thermal stability of the enzyme was
with minor modications. In brief, 15 ml of 0.8 mg/ml bovine evaluated by incubating the enzyme in various temperatures for 30-
brinogen solution (in 0.1 M sodium phosphate buffer, pH 7.4) was 150 min at pH 7.4, and then the residual activities were measured. The
mixed with 1 ml of thrombin solution (7.5 U/ml) and 20 ml of 0.8% remaining activity was assessed using the brin plate method.

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 e3

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

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

Amidolytic activity was measured spectrophotometrically by


using chromogenic substrates as follows [10]. The reaction mixture 3.2. Effect of temperature on brinolytic activity and stability of PPFE-I
(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 The effect of temperature on the brinolytic activity of PPFE-I was
incubation for 5 min at 37 C with a spectrophotometer equipped examined at pH 7.4 (Fig. 3). The temperature showing the maximal
with a cuvette temperature controller, the amount of -nitroaniline enzyme activity was 37 C (Fig. 3a). The relative activities at 30 and
that was liberated was determined from the A405. One unit of 40 C were about 84% and 86 %, respectively. The thermal stability
amidolytic activity (AU) was expressed as micromoles of substrate proles showed that the PPFE-I was stable at 30-40 C, and showed
hydrolyzed per minute per milliliter by the enzyme. 25% and 0% residual activity after a 150 min incubation at 50 and
Km and Kcat for PPFE-I (1 g ) were determined with N-succinyl- 60 C, respectively (Fig. 3b).
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. 3.3. 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 determined using buffers at various pHs. The optimum pH for the
brinolytic activity was found to be 7.5, and the relative activity was
Fibrinogenolytic activity was measured by a modied brinogen- about the same in the range of pH 7.0-8.0 (Fig. 4a). The pH stability of
olytic assay [28]. The brinogen solution (50 l of 2.5% human the enzyme was investigated in the range of pH 4-11 by measuring
brinogen in 50 mmol/L Tris-HCl buffer, pH7.4) was mixed with the the residual activity after incubation at each pH for 24 h. The enzyme
puried enzyme solution (10 l of 0.4 mg/ml) and incubated at 37 C was very stable in the range of pH 6.08.0 at 37 C for 24 h, but below
for 15 min, 30 min, 1 h, 2 h, 4 h and 8 h, respectively. After the pH 6.0 or above pH 8.0, the enzyme stability abruptly decreased
indicated time intervals, aliquots were transferred to ice and (Fig. 4b).
separated by SDS-PAGE to examine the cleavage pattern of the
brinogen chains.

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

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

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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
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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