Purification and Biochemical Characterization of a Novel Fibrinolytic

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Purification and biochemical characterization of a novel fibrinolytic enzyme from culture supernatant of Cordyceps militaris Xiaolan Liu, Narasimha kumar Kopparapu, Xi Shi, Yong ping Deng, Xi qun Zheng, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505717e • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Purification and biochemical characterization of a novel fibrinolytic

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enzyme from culture supernatant of Cordyceps m ilitaris

3

Xiaolan Liu1,

2

Narasimha-kumar Kopparapu1 Xi Shi1 Yongping Deng1 Xiqun Zheng*1

4

Jianping Wu*2

5

1

6

Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006,

7

China

8

2

9

Edmonton, AB, Canada

Heilongjiang Provincial Key University Laboratory of Processing Agricultural

Department of Agricultural, Food and Nutritional Science, University of Alberta,

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*Correspondence to:

11

(1) Prof. Dr. Xi-qun Zheng, College of Food and Bioengineering, Qiqihar University,

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Qiqihar, 161006, China (E-mail address: [email protected]).

13

(2) Prof. Dr. Jian-ping Wu, Department of Agriculture, Food and Nutritional Science,

14

University of Alberta, Edmonton, Alberta, Canada E-mail: [email protected]

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A Brief running title: Fibrinolytic enzyme from Cordyceps militaris

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*Corresponding author:

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Phone: +86-04522738168; Fax: +86-04522725454; Email:[email protected];

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ABSTRACT:

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A novel fibrinolytic enzyme from Cordyceps militaris was produced by submerged

25

culture fermentation, purified and biochemically characterized. The enzyme was

26

purified to homogeneity with an overall yield of 4.0% and a specific activity of 1682

27

U/mg. The molecular weight and pI of the enzyme were 32 kDa and 9.3±0.2,

28

respectively. The optimal pH and temperature of the enzyme were 7.4 and 37 °C,

29

respectively. The enzyme activity was inhibited by Fe2+, phenylmethane sulfonyl

30

fluoride (PMSF), aprotinin, and pepstatin, but not by N-tosyl-L-phenylalanine

31

chloromethyl ketone (TPCK) and ethylenediamine tetracetic acid (EDTA). Three

32

internal

33

EKNVGSTVNLLSYDGNK, and TDATSVLLDGYNVSAVNDLVAK were obtained.

34

The enzyme could hydrolyze fibrin(ogen) directly and cleave the α-chains more

35

efficiently than β- and γ- chains suggesting that it is a plasmin like protein. It

36

degraded thrombin, which indicated that it can act as an anticoagulant and prevent

37

thrombosis. Intravascular thrombosis is one of the major reasons of cardiovascular

38

diseases. Based on these results, the purified enzyme can be developed as a natural

39

agent for oral fibrinolytic therapy or prevention of thrombosis.

40

KEY WORDS: Cordyceps militaris, Mushroom, Fibrinolytic enzyme, Fermentation,

41

Purification

peptides

of

the

enzyme,

APQALTVAAVGATWAR,

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INTRODUCTION

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Cardiovascular diseases (CVDs), representing ~29% of all global death, are the

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leading cause of death.1 Intravascular thrombosis is one of the major factors causing

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CVDs.2-3 The formation of thrombus is a very complicated physiological process.

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Fibrin is the major protein component of blood clot/thrombus, which is formed from

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fibrinogen by the action of thrombin (EC 3.4.21.5). Fibrin can be degraded into fibrin

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degradation products (FDP) by plasmin (EC 3.4.21.7) which is formed from

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plasminogen by plasminogen activators. Therefore, under normal physiological

53

conditions, fibrin clot formation and fibrinolysis are well balanced, while in

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unbalanced state, the clots are not lysed which results in thrombosis.3

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Fibrinolytic agents are widely used for the prevention and treatment of

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thrombosis. Based on their mode of action, fibrinolytic agents can be divided into

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plasminogen activators (indirect type) which activate plasminogen into plasmin

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and plasmin like enzyme (direct type) which act directly on thrombus or fibrin clot.

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Currently, most fibrinolytic agents used clinically are plasminogen activators such as

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tissue-type plasminogen activator (t-PA), a urokinase-type plasminogen activator

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(u-PA), and the bacterial plasminogen activator, streptokinase. 3, 6-7 However, there are

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drawbacks associated with these agents such as excessive bleeding caused by

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proteolytic degradation of other blood protein factors, low specificity towards fibrin,

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and high cost.4 Therefore, there is a growing interest in exploring alternatives from

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natural sources which show less or no side effects.

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Over the past decade, fibrinolytic enzymes from various Asian traditional

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1, 8-11

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fermented foods have been reported.

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and also used in oriental traditional medicine; recently, the potential of mushroom as a

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source of bioactive compounds, including fibrinolytic enzymes, has been extensively

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studied.

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bodies, mycelia and culture broth of some of the edible and medicinal mushrooms.5, 7,

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14- 17

12-13

Mushrooms are usually consumed as food

Fibrinolytic enzymes have been purified and characterized from fruiting

These fibrinolytic enzymes can be used in many medicinal applications. 4, 11-12

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Cordyceps militaris is a traditional Chinese medicinal mushroom belonging to

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Clavicipitaceae and Ascomycotina.18 Extracts of Cordyceps militaris were reported to

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possess

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anti-inflammatory, and antioxidant activities. 18-19 Although fibrinolytic enzymes have

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been reported from fruiting bodies of Cordyceps militaris,

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enzymes from cultured supernatant are scarce. 18 Artificial culturing of fruiting bodies

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of mushrooms is performed using specific solid medium, however the growth time is

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too long to be economically attractive.13 Submerged culture has advantages over

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artificial culture in terms of shortened culture time and efficient production.21 The

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objectives of the present study are production of a novel fibrinolytic enzyme from

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Cordyceps

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characterization of the enzyme .

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MATERIALS AND METHODS

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Materials and chemicals. Sephadex G-25, CM-Sepharose FF, Phenyl-Sepharose HP,

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Superdex 75 16/60, preparative columns, and IEF calibration kit were purchased from

various

properties

militaris

by

such

submerged

as

antifungal,

fermentation

antibacterial,

3, 20

studies on fibrinolytic

culture,

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anticancer,

purification

and

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GE Life sciences (Pittsburgh, PA, USA). Bovine fibrinogen and thrombin were

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purchased from the Tianjin Blood Institute (Tianjin, China). Human fibrinogen was

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purchased from Sigma Chemical Co. (St. Louis, MO, USA). Soybean trypsin inhibitor

91

(SBTI),

92

phenylmethanesulfonyl fluoride (PMSF), aprotinin, pepstatin and low molecular

93

weight SDS-PAGE Protein standard kit (ranging from 14.4 kDa to 97.4 kDa) were

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purchased from Sangon (Shanghai, China).

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Strain and culture conditions. Cordyceps militaris strain was stored on PDA slants.

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The mycelia were transferred from the slants to plate medium. The mycelia were

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inoculated aseptically into 250 mL shake flasks containing 50 mL of fermentation

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media composed of 2% sucrose and 5% soybean cake powder. Fermentation was

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carried out for 5 days at 23 °C, 180 rpm. The fermentation broth was centrifuged

100

(10,000 rpm, 10 min, 4 °C) and the supernatant was considered as the crude enzyme

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extract.

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Enzyme assay and Protein determination. Fibrinolytic activity was determined by

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using the method of Astrup and Mullertz22 with slight modifications. Fibrin plates

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were prepared as follows: fibrinogen (0.4%, w/v) dissolved in 5 mL of 100 mM

105

barbital sodium-chlorhydric acid buffer, pH 7.8, was mixed with 5 mL of 0.5% (w/v)

106

agarose solution along with 1 mL of thrombin solution (200 U/mL) and poured into

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Petri plates; after standing for 1 h at room temperature for clot formation, the plate

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was used to measure fibrinolytic activity. In brief, 10 µL of the enzyme solution was

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carefully placed on the fibrin plate, incubated at 37 °C for 6 h and the diameter of the

N-α-tosyl-L-phenylalanine

chloromethyl

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ketone

(TPCK),

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lytic circle was measured. The diameter was directly proportional to the potency of

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the fibrinolytic activity and the unit of the enzyme activity was determined according

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to a calibration curve using urokinase as standard. The protein content was estimated

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by Lowry method,23 using bovine serum albumin as a standard. The carbohydrate

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content of the purified enzyme was quantified by anthrone colorimetric method using

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glucose as a standard 24.

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Purification of fibrinolytic enzyme. Crude sample was subjected to 0-20%

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saturation of (NH4)2SO4 precipitation. The supernatant obtained by centrifugation

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(10,000 rpm for 20 min at 4 °C) was buffer exchanged on a Sephadex G-25 column

119

(1.6×50 cm) using 0.02 mol/L sodium phosphate buffer, (pH 7.4). After buffer

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exchange, the enzyme solution was loaded onto a Phenyl-Sepharose HP column

121

(1.6×30 cm) which was earlier equilibrated with 0.02 mol/L sodium phosphate buffer,

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(pH 7.4) containing 30% saturated (NH4)2SO4. Bound proteins were eluted with a

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decreasing linear gradient of 30–0% (NH4)2SO4 in 0.02 mol/L sodium phosphate

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buffer, (pH 7.4). The active fractions were collected, buffer exchanged using 0.02

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mol/L sodium phosphate buffer, pH 6.0 and loaded onto a cation exchange column

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CM-Sepharose FF (2.6×20 cm) which was previously equilibrated with the same

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buffer. Fractions exhibiting the most potent fibrinolytic activity were pooled and

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further purified on a Superdex 75 gel filtration column (1.6×60 cm) using 0.02 mol/L

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sodium phosphate buffer containing 0.3 mol/L NaCl (pH 6.0). Finally, the purified

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active fractions were pooled, lyophilized and used for further characterization. All the

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purification steps were performed at 4 °C unless otherwise stated. 6

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Determination of molecular weight and isoelectric point. The molecular weight of

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the purified enzyme was determined by SDS-PAGE (under reducing denaturing

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conditions) as described by Laemmli,25 using a 12% polyacrylamide gel. Protein

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bands were visualized by staining the gel with Coomassie brilliant blue R-250. For

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calibration, an SDS-PAGE mid-range molecular weight standard kit (Sangon biotech,

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Shanghai, China) comprising of rabbit phosphorylase B (97.4 kDa), bovine serum

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albumin (66.2 kDa), ovalbumin (42.7 kDa), bovine carbonic anhydrase (31.0 kDa),

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and chicken egg white lysozyme (14.4 kDa) was used.

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The native molecular weight of the enzyme was determined by gel filtration

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chromatography using a Superdex 75 (1.6×60 cm) column in 20 mM phosphate buffer

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(pH 7.4) containing 300 mM NaCl. Gel filtration low molecular weight calibration kit

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comprising

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chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) was used. The void

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volume of the column was determined by using blue dextran 2000.

of

bovine

serum

albumin

(67

kDa),

ovalbumin

(43

kDa),

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The isoelectric point (pI) of the purified enzyme was determined by isoelectric

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focussing electrophoresis (IEF) using homogenous polyacrylamide gels (7.5%

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polyacrylamide with 3% cross linkage) containing carrier ampholytes (3–9) 26.

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Q-TOF2 tandem mass spectrometry sequencing. To determine the partial amino

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acid sequence by Q-TOF2 tandem mass spectrometry, the purified enzyme was

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subjected to SDS-PAGE. Protein band was excised from the gel and submitted for

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amino acid sequencing using high performance liquid chromatography-electrospray

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tandem mass spectrometry (HPLC-ESI-MS/MS) at National Center for Biomedical 7

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Analysis, Military Academy of Medical Sciences (Beijing, China). Mass spectral

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sequencing was performed using a Q-TOF II mass analyzer (Q-TOF2, Micromass

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Ltd., Manchester, UK). Peptide sequencing was performed using a palladium-coated

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borosilicate electrospray needle (Protana, Denmark). The mass spectrometer was used

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in positive ion mode with a source temperature of 80 °C, and a potential of 800 V was

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applied to the nanospray probe. MS/MS spectra were transformed using MaxEnt3

160

(MassLynx, Micromass), and amino acid sequences were interpreted manually using

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PepSeq (BioLynx, Micromass).

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Effects of environmental factors on enzyme activity. The optimal temperature of

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the purified enzyme was determined by measuring the activity at different

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temperatures (16–60 °C) in sodium barbital buffer (pH 7.4). The thermal stability of

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the enzyme was determined by incubating the enzyme at different temperatures

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(23–60 °C) for 0.5 h, in sodium barbital buffer (pH 7.4) and the residual activities

167

were assayed.

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To determine the optimal pH of the purified fibrinolytic enzyme, the enzyme

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activity was assayed at 37 °C in different buffers within the range of pH 6.8–8.2. pH

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stability of the purified enzyme was determined by incubating the enzyme in different

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buffers ranging from pH 3-12 for 1 h, and the residual activities were assayed by

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fibrin plate method. The buffers used were: citrate-phosphate (pH 3.0-7.0), barbital

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sodium-chlorhydric acid buffer (pH 8.0-9.0), Na2CO3-NaHCO3 (pH 9.5-10.5),

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Na2HPO4-NaOH buffer (pH 11.0-12.0).

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Effects of various factors such as 1) metal ions (Cu2+, Co2+, K+, Fe3+, Fe2+, Ca2+, 8

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Mg2+, Mn2+, Na+ and Zn2+) at a concentration of 5 mM at 37 °C for 18 h; 2) protease

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inhibitors such as pepstatin, PMSF (Phenylmethyl sulfonyl fluoride), aprotinin, TPCK

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(N-α-tosyl-L-phenyl alanine chloromethyl ketone), SBTI (Soybean trypsin inhibitor),

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Lys and metal chelator EDTA (Ethylenediamine tetraacetic acid) at concentrations of

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2.0 and 10 mM (2.5 and 10 mM in case of EDTA and Lys) at 37 °C for 10 min; 3)

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various protective agents and other reagents (5 mmol/L reduced glutathione; 5

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mmol/L oxidized glutathione, 20 mmol/L cysteine, 0.5% β- mercaptoethanol, 1%, w/v,

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bovine serum albumin, 1% gelatin, 1% peptone), organic substances (10%, v/v,

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acetone, 10% glycerol), and denaturants (urea and SDS) at 37 °C for 4 h, on the

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activity were determined as above.

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Simulated gastric and blood environment effect on enzyme activity. To prepare

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simulated gastric juice solution, 10% HCl solution (pH 3.0) was first autoclaved

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(121 °C, 15 min), cooled down to 37°C, and mixed with pepsin (1 g per 100 ml).

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Simulated blood solution (Clorox solution - commonly used for mammalian heart

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perfusion test) was prepared by dissolving 0.9 g NaCl, 0.042 g KCl, 0.024 g CaCl2,

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0.02 g NaHCO3, 0.2 g glucose into 100 mL de-ionized water, and stored at 4 ºC until

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further use.

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The activity of the enzyme in simulated gastric juice and simulated blood was

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tested in six different combinations.

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1. 100 µL water +50 µL purified enzyme;

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2. 50 µL of artificial gastric juice +50 µL of broth + 50 µL purified enzyme;

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3. 50 µL of artificial gastric juice +50 µL H2O + 50µL purified enzyme 9

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4. 50 µL artificial gastric juice +50 µL 10% maltose + 50 µL purified enzyme;

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5. 50 µL artificial gastric juice +25 µL broth +25 µL 10% maltose + 50 µL purified

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enzyme;

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6. 100 µL Clorox solution +50 µL purified enzyme.

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Simulated gastric and Clorox solutions were added to the purified enzyme

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separately and incubated at 37 ºC for 4 h. The effect of simulated gastric juice in

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presence of protein and carbohydrate was also evaluated. The residual activities were

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measured by standard fibrin plate method.

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Storage stability of purified enzyme. (1) Impact of freeze-thawing on storage of the

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purified enzyme. Purified fibrinolytic enzyme (0.151mg/mL) was stored at -20 °C and

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-70 °C and repeated freeze-thawing was carried out for 2, 4, 6, 8, 11, 13, 15, and 17

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times and the residual enzyme activity was determined.

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(2) Effect of stabilizers on storage stability of the purified enzyme. To appropriate

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volume of purified enzyme (0.066 mg/mL), an equal volume of different

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concentrations of glycerol and sucrose (5%, 10%, 15%, 20%) were added, mixed and

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stored at 4 °C refrigerator for one week. The residual activity was measured by

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standard assay.

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(3) Long-term storage stability of the purified enzyme. The purified enzyme (both in

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liquid (0.151mg/mL) and lyophilized forms) was stored at different temperatures

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(25 °C, 4 °C, -20 °C, -80 °C) for 1 day, 2 days, 1, 2, 3, 4, 7 weeks and 1 year. The

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residual activities were determined.

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Measurement of thrombin activity of the purified enzyme. Thrombin activity of 10

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the purified enzyme was measured as follows. 40 µL of different concentrations of

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fibrinogen (10, 20, 40, 80 and 100 mg/ml) was mixed with 10 µL of the enzyme

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(0.075mg/mL) and the clotting time was recorded. Bovine thrombin (500 U/mL) was

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used as a positive control.

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Activation of plasminogen (Plasminogen activator activity). Activation of

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plasminogen by the purified enzyme was analyzed by fibrin plate method. Fibrin

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plates were prepared as described earlier. Usually, commercially available fibrinogen

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contains traces of plasminogen, so the plasminogen-free plates were prepared by

228

using similar method, but were heated at 85 °C for 30 min to inactivate plasminogen.

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The presence of plasminogen activity was measured by difference in area of lytic

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zone in two different plates.

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In vitro dissolution of blood clots by the purified enzyme. To 0.4890 g of blood

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clot, 500 µL (0.075mg/mL) of enzyme solution was added and incubated at 37 ºC for

233

6 h. Samples at different time intervals, 10, 20, 40, 60, 90, 120, 180, 240, 300, and

234

360 min were centrifuged briefly at 3000 rpm for 30s, the supernatant was transferred

235

and the residual clot weight was recorded. The clot was re-dissolved in the

236

supernatant and the reaction was continued for further dissolution. Finally the rate of

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clot dissolution was calculated as follows:

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Clot dissolution rate = [(clot weight before dissolving – clot weight after dissolving) /

239

clot weight before dissolving] × 100%.

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Fibrin(ogen)olytic activity of the enzyme. Human blood fibrinogen degradation.

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Fibrinogenolytic activity was measured by a modified fibrinogenolytic assay 11

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Fibrinogen (23 µL of 2% human fibrinogen in 0.05 mol/L Tris-HCl buffer (pH 7.6))

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was mixed with the purified enzyme (23 µL of 0.075mg/mL) and incubated at 37 °C

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for different time intervals (1 min, 5 min, 15 min, 25 min, 1 h, 1.5 h, 2 h, 4 h and 24

245

h). After the indicated time intervals, aliquots were transferred onto ice and analyzed

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by SDS-PAGE to examine the cleavage patterns of the fibrinogen chains.

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Fibrin degradation. Fibrinolytic activity was measured by a modified fibrinolytic

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assay. To 23 µL of fibrinogen (2% human fibrinogen in 0.05 mol/L Tris-HCl buffer

249

(pH 7.6)), 10 µL of thrombin was added. After coagulation, 23 µL of purified enzyme

250

(0.075mg/mL) was added and incubated at 37 °C for different time intervals (3 min, 6

251

min, 15 min, 25 min, 1 h, 1.5 h, 2 h, 4 h and 24 h). After the indicated time intervals,

252

aliquots were transferred onto ice and analyzed by SDS-PAGE to examine the

253

cleavage pattern of the fibrin.

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Analysis of In vitro fibrinolytic and anticoagulant effect. To study the fibrinolytic

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and anticoagulant effect in vitro, purified enzyme (0.075 mg/mL), fibrinogen (Fbg 10

256

mg/mL), and thrombin (Tb, 0.5 U/µL) were mixed in three ways as follows:

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(1) Human fibrinogen + thrombin (clot formation time was recorded) + purified

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enzyme (clot dissolution time was recorded).

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(2) Human fibrinogen (37 °C for 5 min) + purified enzyme and Tb mixture;

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subsequently incubated in a 37 ºC (time required for clot formation and complete

261

dissolution was recorded).

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(3) Human fibrinogen + purified enzyme (incubated at 37 °C water bath for 5 min) +

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Tb 12

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The samples were incubated at 37 ºC and the time required for clot formation and

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dissolution were recorded. The degradation patterns of the samples were analyzed by

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SDS-PAGE. Fibrinogen was used as control.

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Proteolytic effect of purified enzyme on blood proteins. The effect of purified

268

enzyme on blood protein components was studied by incubating purified enzyme (23

269

µL) with equal volume of human serum albumin (HSA) (15 mg/mL), human

270

immunoglobulin G (IgG), and human thrombin at 37 ºC for 4 h. Samples were

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analyzed by electrophoresis.

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RESULTS

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Purification and characterization of fibrinolytic enzyme. A submerged culture

274

method was developed to prepare fibrinolytic enzyme. The fibrinolytic enzyme

275

extract was first subjected to 20% ammonium sulfate precipitation. The precipitated

276

materials were discarded and after buffer exchange, the supernatant was subjected to

277

hydrophobic interaction chromatography on phenyl sepharose FF column; the active

278

fractions were pooled and further purified by cation exchange chromatography,

279

followed by Superdex 75 gel filtration chromatography (Figure 1). An overall yield

280

of 4.0% was obtained with 41.3 fold increase in purity. The specific activity of the

281

purified enzyme was 1682 U/mg. A summary of purification steps is presented in

282

Table 1.

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The purified fibrinolytic enzyme appeared to be a single homogenous band on

284

SDS-PAGE (Figure 2a). The molecular weight of the purified enzyme was

285

determined under native and denatured conditions. Its molecular weight was 13

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determined to be 32 kDa by both gel filtration chromatography (data not shown) and

287

SDS-PAGE (Figure 2a), indicating that the enzyme is monomeric in nature. The

288

isoelectric point (pI) of the purified enzyme was determined to be 9.3±0.2 (Figure 2b).

289

The purified enzyme was found to be a glycoprotein as identified by Molish test

290

with a carbohydrate content of 0.98% (w/v).

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The partial amino acid sequences of the purified enzyme were determined by

292

Q-TOF2 mass spectrometer. Three internal peptides were obtained and their amino

293

acid

294

TDATSVLLDGYNVSAVNDLVAK. All the three peptides were analyzed using

295

NCBI-BLAST database for sequence similarity with earlier reported fibrinolytic

296

enzymes and the results are summarized in Table 2.

297

Effects of environmental factors on enzyme activity. The optimal pH of the

298

purified enzyme was 7.4 (Figure 3a), and exhibited stability at pH ranging from pH

299

6.0-11.0 (Figure 3b). The enzyme exhibited maximum activity at 37 °C (Figure 3c)

300

and showed thermo-stability in the temperature range of 23-60 °C, and was highly

301

stable up to 47 °C retaining >80% of its activity when incubated for 30 min (Figure

302

3d). Enzyme activity and thermo-stability decreased slowly above 50 °C.

sequences

are

APQALTVAAVGATWAR;

EKNVGSTVNLLSYDGNK;

303

Effect of various metal ions, protease inhibitors and other reagents on fibrinolytic

304

activity of the purified enzyme was also studied. The activity was strongly inhibited

305

by Fe3+ (46.7%) but was not affected by Fe2+ (94.5%), however, the activity was

306

enhanced by Ca2+ (131.9%) and Cu2+ (114.1%) at a concentration of 5 mM (Table 3).

307

The enzyme activity was strongly inhibited by serine protease inhibitors and aspartic 14

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protease inhibitor, but not by chymotrypsin inhibitor and metallo-protease inhibitor

309

(Table 4). These results indicated that metal ion is not present in the active site of the

310

enzyme and is not involved in enzyme activity.

311

The enzyme activity was inhibited by -SH group reactive agents, which indicated

312

that the sulfhydryl group of the enzyme was necessary for its activity. BSA (80.5%

313

activity) slightly inhibited the enzyme activity, while gelatin (120% activity) and

314

peptone (127% activity) enhanced the enzyme activity (Table 5).

315

Stability studies of the purified enzyme. The enzyme was found to be highly stable

316

even after repeated cycles of freeze-thawing. This property is very useful during

317

industrial production and clinical applications of the enzyme. The effect of stabilizers

318

such as glycerol and sucrose on storage stability of the purified enzyme was also

319

investigated. The enzyme was stable for one week at 4 °C in presence of 15% glycerol

320

without any loss of activity (Data not shown). Sucrose did not exhibit any protective

321

role at the same experimental conditions as glycerol.

322

Stability of the purified enzyme both in liquid and lyophilized forms was studied.

323

The enzyme was stable in liquid form up to one day at 25 °C and 4 °C while in

324

lyophilized form, the enzyme was stable after one month of storage at same

325

temperature (25 °C and 4 °C), retaining 78% and 83% of its initial activity,

326

respectively. Storage of both the forms at -20 °C and -80 °C did not result in loss of

327

any activity up to one month.

328

The stability of the enzyme was further analyzed after 14 months of storage. The

329

liquid form of enzyme was stable and retained 56% and 76% of its activity when 15

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330

stored at -20 °C and -80 °C for 14 months. Meanwhile, the lyophilized form of

331

enzyme was highly stable at 4 °C, -20 °C and -80 °C for the same period of time and

332

retained 62%, 83%, and 101% of its activity, respectively.

333

Simulated gastric and blood environment effect on enzyme. As shown in Figure 4a,

334

the enzyme lost its activity completely after 4 h of incubation in gastric juice (lane 3);

335

retained ~60% of its relative activity in the presence of protein broth (lane 2) or

336

protein broth and maltose (lane 5). Maltose did not show any protective effect (lane 4).

337

The enzyme also tolerated simulated blood environment (Clorox solution); 60% of its

338

relative activity was retained after 4 h of incubation. Therefore the enzyme should be

339

administered after a meal or intravenously; the loss of the enzyme activity after 4 h of

340

incubation implied its possible advantage over the current fibrinolytic agents

341

associated with bleeding due to excessive plasmin activity.

342

Determination of thrombin-like activity of purified enzyme. The coagulation time

343

required for clotting at different concentrations of human fibrinogen by bovine

344

thrombin is 3-4 min. Thrombin specifically hydrolyzes Aα and Bβ subunits of

345

fibrinogen and forms insoluble fibrin clot. The purified enzyme was unable to clot

346

fibrinogen even at the highest concentrations of 100 mg/mL indicating that the

347

enzyme did not have thrombin-like activity but acts directly on fibrin clots (data not

348

shown).

349

Activation of plasminogen (Plasminogen activator activity). Plasminogen activator

350

activity of the purified enzyme was examined. The purified enzyme did not act as

351

plasminogen activator since the area of diameter of the lytic circle was almost similar 16

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in both plasminogen and plasminogen-free fibrin plates (Figure 4b and 4c).

353

In vitro dissolution of blood clots by the purified enzyme. The in vitro effect of

354

purified enzyme on dissolution of blood clots and its dissolution rate was measured at

355

different time intervals (Table 6). The purified enzyme could dissolve the blood clots;

356

the dissolution substantially increased at prolonged incubation time and the clots

357

completed disappeared within 6 h (Table 6).

358

Fibrin(ogen)olytic

359

fibrin(ogen)olytic activity and the degradation patterns of fibrin(ogen) by the purified

360

enzyme were analyzed by SDS-PAGE (Figure 5a and 5b). In control, fibrinogen was

361

separated into Aα, Bβ and γ chains. When fibrinogen was incubated with the purified

362

enzyme, the Aα band disappeared first, followed by the Bβ and γ bands. This indicates

363

that the enzyme degraded the Aα chain of fibrinogen first, followed by the Bβ and γ

364

chains. The Aα chain was degraded within 5 min of incubation, while the Bβ and γ

365

chains were degraded 1 h after incubation (Figure 5a). This implies that the cleavage

366

sites of Aα and Bβ chains were different from thrombin. The low molecular weight

367

degradation products increased with incubation time.

activity

of

purified

enzyme.

The

enzyme

exhibited

368

The fibrin degradation pattern by the purified enzyme was also analyzed (Figure

369

5b). The fibrin α-chain was hydrolyzed within 3 min, followed by the γ-γ chain in 25

370

min of incubation. The β chain was hydrolyzed slowly and degraded completely in 1

371

h.

372

Analysis of in vitro fibrinolytic and anticoagulant effect. When fibrinogen and

373

thrombin were pre-mixed and then the enzyme was added, the time required for 17

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374

formation of clot was 9.75 s, and for dissolution it was 3 min 20s. Although the clot

375

was dissolved completely, the subunits of the fibrin were not degraded completely as

376

shown in SDS-PAGE (Figure 6a); the α-subunit was degraded completely, while β

377

and γ subunits were still visible.

378

When fibrinogen was first pre-activated, and then the purified enzyme and

379

thrombin were added, it took 1 min 41s for clot formation, which was much longer

380

than the 1st combination (9.75s). However, the newly formed fibrin clot was dissolved

381

by the enzyme within a short period of 2 min 15 s, which was significantly shorter

382

than 1st combination (3 min 20s). The difference in time required for clot formation

383

and dissolution could be due to the fact that both processes might have occurred

384

simultaneously in the system. Degree of degradation of each subunit of the fibrin

385

between two reactions was further confirmed by SDS-PAGE (Figure 6a, lane 2). As

386

shown in Fig. 6a, lane 3, human fibrinogen subunit was thoroughly hydrolyzed; as a

387

result no observable large protein bands could be seen on the gel.

388

Effect of purified enzyme on blood plasma proteins. The effect of purified enzyme

389

on different blood plasma proteins was studied. As shown in Figure 6b, human IgG

390

and serum albumin were partially hydrolyzed by the enzyme. It also degraded human

391

thrombin to some extent (Figure 6b).

392

DISCUSSION

393

In this study, a novel fibrinolytic enzyme from the mushroom, Cordyceps militaris

394

was produced by submerged culture fermentation. During fermentation, soybean

395

cake was used as a major component of the culture medium, which is different from 18

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that of other reports.29 The enzyme was purified to homogeneity by using

397

combination of various chromatographic steps. The molecular weight of the purified

398

enzyme was 32 kDa. The molecular weight of mushroom fibrinolyic enzymes are in

399

the range of 17 kDa to 100 kDa.28 Our value is similar to the enzyme purified from

400

Pleurotus ostreatus mycelia (32 kDa),17 but is smaller than that of fibrinolytic enzyme

401

from Flammulina velutipes (37 kDa), and Cordyceps militaris (52 kDa).7,

402

molecular weight of the purified enzyme is not similar to any of the reported

403

fibrinolytic enzymes from Cordyceps sp.3, 18, 20, 29 The isoelectric point (pI) of the

404

purified enzyme was determined to be 9.3±0.2, which is higher than the fibrinolytic

405

enzymes from Cordyceps militaris (8.1), and Rhizopus chinensis 12 (8.5)3, 26. The

406

partial amino acid sequences of the purified enzyme were determined by Q-TOF2

407

mass spectrometer. Peptides 1, 2 and 3 exhibited 94%, 93% and 73% similarity with

408

alkaline serine protease, Alp1 from Cordyceps militaris CM01 respectively

409

(Accession no. XP_006670629.1) (Table 2).

20

The

410

Mushroom fibrinolytic enzymes exhibited optimal activity in the range of pH

411

5.0-10.0.28 The optimal pH of the purified enzyme for the fibrinolytic activity is 7.4,

412

which is the physiological pH of humans. Its optimum pH is similar to fibrinolytic

413

enzymes from fruiting bodies of Cordyceps militaris and Tricholoma saponaceum,15,

414

20

415

commune enzymes.5, 7, 16 The enzyme exhibited maximal activity at 37 °C, which is

416

the same as the enzyme purified from fruiting bodies of Cordyceps militaris20, but

417

higher than the enzyme purified from Armillaria mellea, Flammulina velutipes, and

but higher than that of Flammulina velutipes, Armillaria mellea and Schizophyllum

19

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Cordyceps militaris.7, 16, 18 Mushroom fibrinolytic enzymes have optimal activity at

419

temperatures ranging from 20-60 °C.28

420

The enzyme activity was strongly inhibited by serine protease inhibitors and

421

aspartic protease inhibitor, but not by chymotrypsin inhibitor and metallo-protease

422

inhibitor. Our results are similar to the fibrinolytic enzyme from Cordyceps

423

militaris,20 but are in contrast to other fibrinolytic enzymes from fruiting bodies of

424

Korean Cordyceps militaris where the enzyme activity was strongly inhibited by

425

TPCK, EDTA and metal ions.3, 18 Most fibrinolytic enzymes purified from mushrooms

426

are inhibited by the metal chelator EDTA..5,

427

specificities, mushroom fibrinolytic enzymes are classified into two groups, viz serine

428

proteases (those inhibited by PMSF, SBTI, aprotinin) and metalloproteases (those

429

inhibited by EDTA). Therefore, the purified fibrinolytic enzyme from this study is a

430

serine protease. SDS and urea strongly inhibited the enzyme activity indicating that

431

these reagents might have denatured the enzyme.

7, 17, 30

Based on their inhibitory

432

Glycerol and sucrose are used as thermo-protective agents for storage of proteins

433

or enzymes. Cryo-protective effect of sucrose on stability of purified enzyme was

434

found to be weaker than glycerol. Therefore it is recommended to add 15% glycerol

435

for improving its storage stability. Results from stability studies indicate that the

436

liquid form of enzyme stored at 25 °C and 4 °C was stable only for a short time, and

437

the rate of loss of activity of freeze-dried powder was less than that of solution form.

438

The lyophilized powder of the enzyme can be stored for more than one year at -80 °C.

439

After one year of storage at -20 °C, the enzyme was more stable in freeze-dried 20

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powder retaining more than 80% of its activity as compared to the liquid enzyme

441

solution where it lost about half of its activity. Therefore, it is recommended that the

442

purified enzyme be lyophilized, and stored in a deep freezer for long term storage

443

(more than one year). For the first time, stability of a purified fibrinolytic enzyme

444

from mushrooms was studied and reported.

445

The enzyme retained 60% of its relative activity in the presence of an appropriate

446

protein solution (broth) which acts as a protective agent. It also tolerated simulated

447

blood environment (Clorox solution). Therefore the enzyme should be administered

448

after a meal or intravenously; the loss of the enzyme activity after 4 h of incubation

449

implied its possible advantage over the current fibrinolytic agents associated with

450

bleeding due to excessive plasmin activity. Fibrinolytic enzyme from fermented

451

shrimp paste was resistant to the digestive enzymes, pepsin and trypsin; and tolerated

452

in vitro physiological conditions of stomach and small intestine models for 1 h.6

453

Fibrinolytic enzymes degrade thrombus in two ways, either by degrading fibrin

454

directly, or by indirectly degrading fibrin through activation of plasminogen into

455

plasmin.5 The purified enzyme in this study did not act as plasminogen activator since

456

the area of diameter of the lytic circle was almost similar in both plasminogen and

457

plasminogen-free fibrin plates. In plasminogen-free plates, the lytic circles are not

458

condensed and appeared slightly larger than the plasminogen plates. It is possible that

459

pre-treatment of fibrin plates at high temperature might have changed the fibrin

460

structure which resulted in less condensed lytic zone. These preliminary results

461

indicate that Cordyceps militaris fibrinolytic enzyme is a plasmin like protein which 21

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462

can degrade fibrin directly, but not a plasminogen activator which can participate in

463

thrombolysis. These results are dissimilar with the earlier reported fibrinolytic

464

enzymes from Schizophyllum commune and Bacillus sp. which exhibited enhanced

465

activities in plasminogen rich plates.5, 9-10

466

The purified enzyme could dissolve the blood clots; the dissolution substantially

467

increased at prolonged incubation time and the clots completely disappeared within 6

468

h (Table 6). Similar result was reported for fibrinolytic enzyme purified from Bacillus

469

subtilis DC33.9 Our study showed for the first time the presence of thrombolytic

470

activity in mushroom fibrinolytic enzyme.

471

Degradation of fibrin(ogen) by purified enzyme was studied. Fibrinolytic

472

enzymes from Cordyceps militaris and Flammulina velutipes also exhibited similar

473

degradation patterns.7, 20 In addition, the enzyme can hydrolyze fibrin(ogen) chains

474

more quickly than the fibrinolytic enzyme from fruiting bodies of Korean Cordyceps

475

militaris (requires 4 h for complete degradation).3 However, some fibrinolytic

476

enzymes cannot hydrolyze all three chains of fibrin(ogen). It was reported that

477

fibrinolytic enzyme from Bacillus sp. could hydrolyze only Bβ and γ-chains of

478

fibrinogen.10 Fibrinolytic enzymes from the mushrooms Armillariella mellea and

479

Tricholoma saponaceum have been shown to hydrolyze Aα and Bβ chains of

480

fibrinogen with equal efficiency but γ-chain was resistant to hydrolysis.14-15 It is

481

noteworthy that the purified enzyme in this study can hydrolyze all three chains of

482

fibrinogen more efficiently and quickly.

483

During in vitro fibrinolytic and anticoagulant studies when human fibrinogen was 22

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484

pre-mixed with the purified enzyme, the enzyme degraded human fibrinogen. As a

485

result, fibrinogen was not activated by thrombin, or even if activated it could no

486

longer have the capacity to crosslink required for clot formation, which was further

487

observed by electrophoresis (Figure 6a). As shown in Figure 6a, lane 3, human

488

fibrinogen subunit was thoroughly hydrolyzed; as a result no observable large protein

489

bands could be seen on the gel. These results indicate that the purified enzyme could

490

act as an anticoagulant as well as a fibrinolytic agent. It could suppress the formation

491

of blood clots during coagulation process, and has the potential to develop into a

492

novel thrombolytic drug. The fibrinolytic enzyme from fermented shrimp paste also

493

exhibited similar kind of characteristics.6

494

Human IgG and serum albumin were partially hydrolyzed by the enzyme. IgG,

495

the main human immunoglobulin, constitutes about 70% of human plasma globulin,

496

which is the most important antibody in the primary immune response. Human serum

497

albumin is the main component in the formation of the osmotic pressure of plasma

498

gum, and plays an important role in maintaining normal body fluid distribution, blood

499

volume and blood pressure. Thrombin is the most important clotting factor in the

500

process of thrombosis. It also degraded human thrombin to some extent (Figure 6b).

501

These results indicate that during normal fibrin clot formation, thrombin activity can

502

also be inhibited and the enzyme can prevent thrombosis. Similar degradation was

503

observed for the fibrinolytic enzyme from Bacillus subtilis DC33.9 Therefore, the use

504

of this enzyme for fibrinolytic therapy might cause some mild undesirable effects

505

such as partial degradation of human IgG and serum albumin but can also prevent 23

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506

thrombosis by hydrolyzing thrombin. In contrast to the present results, the fibrinolytic

507

enzyme purified from fermented shrimp paste, fruiting bodies of mushrooms

508

Armillariella mellea and Tricholoma saponaceum did not show any proteolytic effect

509

on the blood proteins.6, 14-15

510

To conclude, a 32 kDa novel fibrinolytic enzyme with specific activity of 1682

511

U/mg was purified from Cordyceps militaris. The optimal condition for this enzyme

512

was at human physiological pH (7.4) and temperature (37 °C). As a plasmin like

513

protein, this enzyme could degrade fibrin directly, and cleaved α-chains more

514

efficiently than β- and γ- chains. It degraded thrombin, which indicates that it can act

515

as an anticoagulant and prevent thrombosis. Intravascular thrombosis is one of the

516

major reasons of cardiovascular diseases. Hence, the enzyme may be applied as a

517

potential therapeutic agent for treating thrombosis.

518

Abbreviations used: BSA, bovine serum albumin, SDS-PAGE, sodium dodecyl

519

sulfate polyacrylamide gel electrophoresis; EDTA , ethylenediamine tetracetic acid;

520

TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; SBTI, soybean trypsin

521

inhibitor; PMSF, phenylmethane sulfonyl fluoride.

522 523 524 525 526 527 24

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REFERENCES

529

(1) Mine, Y.; Wong, A. H.; Jiang, B. Fibrinolytic enzymes in Asian traditional

530

fermented foods. Food Res. Int. 2005, 38, 243–250.

531

(2) Peng, Y.; Yang, X.; Zhang, Y. Microbial fibrinolytic enzymes: an overview of

532

source, production, properties, and thrombolytic activity in vivo. Appl. Microbiol.

533

Biotechnol. 2005, 69 (2), 126-132.

534

(3) Choi, D. B.; Cha W. S.; Park, N.; Kim, H. W.; Lee, J. H.; Park, J. S.; Park, S. S.

535

Purification and characterization of a novel fibrinolytic enzyme from fruiting

536

bodies of Korean Cordyceps militaris. Bioresour. Technol. 2011, 102, 3279–3285.

537

(4) Wu, B.; Wu, L.; Chen, D.; Yang, Z.; Luo, M. Purification and characterization of

538

a novel fibrinolytic protease from Fusarium sp. CPCC480097. J. Ind. Microbiol.

539

Biotechnol. 2009, 36, 452-459.

540

(5) Park, I. S.; Park, J. U.; Seo, M. J.; Kim, M. J.; Lee, H. H.; Kim, S. R.; Kang, B.

541

W.; Choi, Y. H.; Joo, W. H.; Jeong, Y. K. Purification and biochemical

542

characterization of a 17 kDa fibrinolytic enzyme from Schizophyllum commune. J.

543

Microbiol. 2010, 48(6), 836-841.

544 545

(6) Wong, K. A. H.; Mine, Y. Novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning. J. Agri. Food Chem. 2004, 52, 980–986.

546

(7) Park, S. E.; Li, M. H.; Kim, J. S.; Sapkota, K.; Kim, J. E.; Choi, B. S.; Yoon, Y.

547

H.; Lee, J. C.; Lee, H. H.; Kim, C. S.; Kim, S. J. Purification and characterization

548

of a fibrinolytic protease from a culture supernatant of

549

mycelia. Biosci. Biotechnol. Biochem. 2007, 71(9), 2214-2222. 25

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Flammulina velutipes

Journal of Agricultural and Food Chemistry

550

(8) Sumi, H.; Hamada, H.; Tsushima, H.; Mihara, H.; Muraki, H. A novel fibrinolytic

551

enzyme (Nattokinase) in the vegetable cheese Natto, a typical and popular

552

soybean food in the Japanese diet. Experientia 1987, 43, 1110-1111.

553

(9) Wang, C. T.; Ji, B. P.; Li, B.; Nout, R.; Li, P. L.; Ji, H.; Chen, L. F. Purification

554

and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated

555

from Chinese traditional Douchi. J. Ind. Microbiol. Biotechnol. 2006, 33(9),

556

750–758.

557

(10) Hua, Y.; Jiang, B.; Mine, Y.; Mu, W. M. Purification and characterization of a

558

novel fibrinolytic enzyme from Bacillus sp. nov. sk006 isolated from an Asian

559

traditional fermented shrimp paste. J. Agric. Food Chem. 2008, 56, 1451–1457.

560

(11) Choi, N. S.; Song, J. J.; Chung, D. M.; Kim, Y. J.; Maeng, P. J.; Kim, S. H.

561

Purification and characterization of a novel thermo acid-stable fibrinolytic

562

enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented

563

Anchovy-joet. J. Ind. Microbiol. Biotechnol. 2009, 36, 417-426.

564

(12) Waseer, S. P. Medicinal mushrroms as a source of antitumor and

565

immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 2002, 60,

566

258-274.

567

(13) Lee, S. H.; Hwang, H. S.; Yun, J. W. Production of polysaccharides by submerged

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mycelial culture of entomopathogenic fungus Cordyceps takaomontana and their

569

apoptotic effects on human neuroblastoma cells. Korean J. Chem. Eng. 2009,

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26(4), 1075-1083.

571

(14) Kim, J. H.; Kim, Y. S. A fibrinolytic metalloprotease from the fruiting bodies of 26

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an edible mushroom, Armillariella mellea. Biosci. Biotechnol. Biochem. 1999, 63,

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2130–2136.

574

(15) Kim, J. H.; Kim, Y. S. Characterization of a metalloenzyme from a wild

575

mushroom, Tricholoma saponaceum. Biosci. Biotechnol. Biochem. 2001, 65(2),

576

356–362.

577

(16) Lee, S. Y.; Kim, J. S.; Kim, J. E.; Sapkota, K.; Shen, M. H.; Kim, S.; Chun, H. S.;

578

Yoo, J. C.; Choi, H. S.; Kim, M. K.; Kim, S. J. Purification and characterization

579

of fibrinolytic enzyme from cultured mycelia of Armillaria mellea. Protein

580

Expresssion and Purif. 2005, 43(1), 10–17.

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(17) Shen, M. H.; Kim, J. S.; Sapkota, K.; Park, S. E.; Choi, B. S.; Kim, S.; Lee, H. H.;

582

Kim, C. S.; Chun, H. S.; Ryoo, C. I.; Kim, S. J. Purification, characterization, and

583

cloning of fibrinolytic metalloprotease from Pleurotus ostreatus mycelia. J.

584

Microbiol. Biotechnol. 2007, 17(8), 1271–1283.

585

(18) Cui, L.; Dong, M. S.; Chen, X. H.; Jiang, M.; Lv, X.; Yan, G. A novel fibrinolytic

586

enzyme from Cordyceps militaris, a Chinese traditional medicinal mushroom.

587

World J. Microbiol. Biotechnol. 2008, 24, 483–489.

588 589

(19) Ng, T. B.; Wang, H. X. Pharmacological actions of Cordyceps, a prized folk medicine. J. Pharm. Pharmacol. 2005, 57, 1509-1519.

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(20) Kim, J. S.; Sapkota, K.; Park, S. E.; Choi, B. S.; Kim, S.; Nguyen, T. H.; Kim, C.

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S.; Choi, H. S.; Kim, M. K.; Chun, H. S.; Park, Y.; Kim, S. J. A fibrinolytic

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enzyme from the medicinal mushroom Cordyceps militaris. J. Microbiol. 2006,

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44, 622–631. 27

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594

(21) Pandee, P.; Kittikul, A. H.; Masahiro, O.; Dissara, Y. Production and properties of

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a fibrinolytic enzyme by Schizophyllum commune BL23. Songklankarin J. Sci.

596

Technol. 2008, 30(4), 447-453.

597 598 599 600 601 602 603 604

(22) Astrup, T.; Mullertz, S. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys. 1952, 40, 346–351. (23) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 264–275. (24) Yemm, E. W.; Willis, A. J. The estimation of carbohydrate in plant extracts by anthrone. Biochem. J. 1954, 57(3), 508-514. (25) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–686.

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(26) Liu, X. L.; Du, L. X.; Lu, F. P.; Zheng, X. Q.; Xiao, J. Purification and

606

characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12. Appl.

607

Microbiol. Biotechnol. 2005, 67, 209-214.

608

(27) Koh, Y. S.; Chung, K. H.; Kim, D. S. Biochemical characterization of a

609

thrombin-like enzyme and a fibrinolytic serine protease from snake (Agkistrodon

610

saxatilis) venom. Toxicon 2001, 39(4), 555-560.

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(28) Lu, C. L.; Chen, S. N.

612

Protein

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337-363.http://www.intechopen.com/books/protein-structure/fibrinolytic-enzyme

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s-from-medicinal-mushrooms.

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Structure,

Fibrinolytic enzymes from medicinal mushrooms. In Faraggi

E.,

Ed.;

Intech:,

2012;

pp.

(29) Li, H. P.; Hu, Z.; Yuan, J. L.; Fan, H. D.; Chen, W.; Wang, S. J.; Zheng, S. S.; 28

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Zheng, Z. L.; Zou, G. L. A novel extracellular protease with fibrinolytic activity

617

from the culture supernatant of Cordyceps sinensis: purification and

618

characterization. Phytotherapy Res. 2007, 21(12), 1234–1241.

619

(30) Lee, S. Y.; Kim, J. S.; Kim, J. E.; Sapkota, K.; Shen, M. H.; Kim, S.; Chun, H. S.;

620

Yoo, J. C.; Choi, H.S.; Kim, M. K.; Kim, S. J. Purification and characterization of

621

fibrinolytic enzyme from cultured mycelia of Armillaria mellea. Protein

622

Expresssion and Purif. 2005, 43(1), 10–17.

623

Note:

624

This work was supported by National Natural Science Foundation of P. R. China

625

(NSFC) (No. 31171744); Natural Science Foundation of Heilongjiang province (No.

626

ZD201305), P. R. China; and Program for Young Teachers Scientific Research at

627

Qiqihar University (2012k-Z08).

628 629 630 631 632 633 634 635 636 637 29

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638

FIGURE LEGENDS

639

Figure 1. Purification of fibrinolytic enzyme from Cordyceps militaris by using

640

Superdex 75 gel filtration chromatography column.

641

Figure 2. Analysis of the purified fibrinolytic enzyme. (a) SDS-PAGE under reducing

642

denaturing conditions. Lane 1: Molecular weight marker; lane 2: purified enzyme (b)

643

Iso-electric focusing (IEF). Lane 1: pI Marker; lane 2: purified enzyme

644

Figure 3. Effect of pH and temperature on fibrinolytic activity of the enzyme. (a)

645

Optimal pH of the purified enzyme was checked at 37 °C in 20 mM of different

646

buffers: citrate-phosphate (pH 3.0-7.0), barbital sodium-chlorhydric acid buffer (pH

647

8.0-9.0), Na2CO3-NaHCO3 (pH 9.5-10.5), Na2HPO4-NaOH buffer (pH 11.0-12.0). (b)

648

For pH stability, the enzyme was incubated at 37 °C for 1 h over range of pH buffers

649

and the residual activities were determined. (c) Optimal temperature of the purified

650

enzyme was determined at different temperatures (16–60 °C) in sodium barbital

651

buffer (pH 7.4). (d) For temperature stability, the enzyme was incubated at different

652

temperatures (23–60 °C) for 0.5 h, in sodium barbital buffer (pH 7.4), and the residual

653

activities were measured by standard fibrin plate method.

654

Figure 4. (a) Effect of simulated gastric and blood environments on purified enzyme.

655

The activity of the enzyme in simulated gastric juice and simulated blood was tested

656

in six different combinations. Lane 1, enzyme in water (control); lane 2, enzyme in

657

artificial gastric juice and protein broth; lane 3, enzyme in artificial gastric juice; lane

658

4, enzyme in artificial gastric juice and maltose; 5, enzyme in artificial gastric juice,

659

maltose and protein broth; lane 6, enzyme in Clorox solution. (b) Analysis of 30

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660

plasminogen activation by purified fibrinolytic enzyme on plasminogen-free fibrin

661

plate; and (c) Activation of plasminogen by purified fibrinolytic enzyme on

662

plasminogen-rich fibrin plate. 10 µL each of the purified enzyme was placed at four

663

spots (1-4) on each plate and incubated at 37 °C for 6 h. Activity was quantified by

664

measuring the area of lysis (1-4 circles) and compared between plasminogen-free and

665

rich plates.

666

Figure 5. Analysis of cleavage pattern of fibrin(ogen) by purified enzyme. (a)

667

SDS-PAGE analysis of reduced human fibrinogen after digestion by the purified

668

enzyme; lane C, control; lanes 1-9, degradation pattern of fibrinogen at different time

669

intervals of 1 min, 5 min, 15 min, 25 min, 1 h, 1.5 h, 2 h, 4 h and 24 h, respectively.(b)

670

SDS-PAGE analysis of reduced human fibrin after digestion by the purified enzyme;

671

lane C, control; lanes 1-9, degradation pattern of fibrinogen at different time intervals

672

of 3 min, 6 min, 15 min, 25 min, 1 h, 1.5 h, 2 h, 4 h and 24 h, respectively.

673

Figure 6. (a) In vitro fibrinogen degradation and anticoagulant effect of purified

674

enzyme. Lane C, Control; lane 1, Fbg and Tb were pre-mixed and the enzyme was

675

added after the formation of the fibrin clot (1st combination); lane 2, Fibrinogen was

676

first pre-activated, then purified enzyme and thrombin mixture were added (2nd

677

combination); lane 3, Human fibrinogen was pre-mixed with purified enzyme (3rd

678

combination). (b) Effect of purified enzyme on blood proteins. Lane 1, 3, 5 represents

679

positive controls of human serum albumin (HSA); human IgG; and human thrombin;

680

respectively; lanes 2, 4, 6 represent their respective degradation patterns by the

681

enzyme. 31

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Table 1. Summary of purification steps of fibrinolytic enzyme from Cordyceps militaris

Specific Volume

Protein

Activity

Recovery

Purification step

activity (mL)

(mg)

(U)

Fold

(%) (U/mg)

Culture supernatant

40

118.6

4822.9

100

40.7

1

20% (NH4)2SO4 precipitation

39

47.8

3547.3

73.6

74.3

1.8

Phenyl-Sepharose HP

78

4.0

2500.1

51.8

632.8

15.5

CM-Sepharose FF

68

1.4

1228.7

25.5

893.8

22

Superdex 75

16

0.1

193.0

4.0

1682

41.3

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Table 2. Comparison of internal peptide fragments of fibrinolytic enzyme from Cordyceps militaris with earlier reported proteases

Position of Source

Ident

first

a

Sequence

amino acid

Peptide 1

Positi

ity

ves

(%)

(%)

APQALTVAAVGATWAR

Cordyceps militaris

Accession No.

Present study

283

APQAITVAAVGATWAR

94

93

XP_006670629.1

283

APQAITVAAVNSEWSR

69

68

XP_008593638.1

260

APQAITVGAVDASW

71

71

ENH77499.1

CM01 Beauveria bassiana ARSEF 2860 Colletotrichum orbiculare

MAFF

240422 Peptide 2

Cordyceps militaris

EKNVGSTVNLLSYDGNK

Present study

381

NVGSTVNLLSYNGN

93

100

XP_006670629.1

381

NVGKTVNLLSYNGN

86

92

XP_008593638.1

CM01 Beauveria bassiana ARSEF 2860

Peptide 3

Cordyceps militaris

Present study

TDATSVLLDGYNVSAVNDLVAK

210

TADTSVILDGYN-WAVNDIVAK

73

72

XP_006670629.1

226

TSVILDGYN-WAVNDIVSK

74

73

CAL25580.1

223

TSVILDGYN-WAVNDIATK

68

68

XP_007602907.1

CM01 Trichoderma harzianum Colletotrichum fioriniae PJ7 a

Identical amino acid sequences are underlined.

33

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Table 3. Effect of various metal ions on fibrinolyitc activity of purified enzyme

Metal ion (5 mM)

Residual activity (%)

Control

100

Cu+2

114.1

Fe+2

94.5

Fe+3

46.7

Zn+2

101.7

Co+2

103.1

K

112.5

Na

91.2

Mg+2

110.3

Ca+2

131.9

Mn+2

104.3

34

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Table 4. Effect of protease inhibitors on fibrinolytic activity of purified enzyme

Concentration Inbihitor

Activity (%) (mmol/L)

Control

100 2.5

103.7

10

86.6

2.5

115.1

10

133.0

2

83.3

10

90.7

2

50.3

10

47.0

2

0

10

0

2

0

10

0

2

0

EDTA

Lys

TPCK

SBTI

PMSF

Aprotinine

Pepstatin

35

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Table 5. Effect of various reagents on fibrinolytic activity of purified enzyme

Residual activity Reagent

Concentration (%)

Control

-

100

Reduced glutathione

5 mmol/L

0

Oxidized glutathione

5 mmol/L

0

Cysteine

20 mmol/L

34.0

β- Mercaptoethanol

0.5%

0

Bovine serum albumin (BSA)

1%

80.5

Gelatin

1%

120.3

Peptone

1%

127.1

Acetone

10%

39.3

Glycerol

30%

93.3

SDS

0.1%

0

Urea

8M

0

36

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Table 6. Analysis of dissolution of blood clots by purified enzyme at different time intervals

Time (min)

Weight (g)

Dissolution rate (%)

0

0.4890

0

10

0.4597

6.0

20

0.4290

12.3

40

0.4079

16.6

60

0.3745

23.4

90

0.3347

31.6

120

0.1887

61.4

180

0.1647

66.3

240

0.1277

73.9

300

0.1001

79.5

360

0.0956

80.5

37

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Figure 1

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(b)

(a)

Purified enzyme

Purified enzyme

Figure 2

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(a)

(b)

(c)

(d)

Figure 3

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(a)

(b)

(c)

Figure 4

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(b)

Figure 5

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(a)

(b)

Figure 6

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