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Fibrinolytic Enzymes from
Medicinal Mushrooms
Chung-Lun Lu and Shiu-Nan Chen
College of Life Science, National Taiwan University,
Taiwan
1. Introduction
Mushrooms, a special group of macrofungi, are not plants and thus do not use
photosynthesis for nutrient acquisition. Mushrooms are fleshy, have the spore-bearing
fruiting body of fungi, and typically grow above ground on soil, or other food sources. Most
mushrooms are Basidiomycota or Agaricomycetes. The fruiting body of mushrooms is an
important food and is used in many cuisines worldwide. Additionally, several species have
been consumed extensively as a crude drug or folk tonics; in East Asia, these mushrooms
are considered as medicinal mushrooms. Some medicinal mushrooms can be cultured and
are an abundant source of natural proteins and polysaccharides. These mushrooms have
garnered considerable attention for clinical research and for modern scientific and medicinal
researches investigating their biological functions (Kino et al., 1989).
1.1 Functional mushrooms
In recent decades, medicinal mushrooms have been used to improve human health and
strengthen the immune system, which have become significant issues in medical and
pharmacological researchers (Guillamon et al., 2010; Kwok et al., 2005). The activities that
have been identified include immunomodulatory and hypocholesterolemic actions, and
antitumor, anti-inflammatory, anti-allergic, anticoagulation, and antithrombin activity as
well as fibrino(geno)lysis stimulation (Lu et al., 2010b; Wang et al., 1995). Based on the
medicinal potentials of edible mushrooms, scientists and businesses have devoted
considerable effort to increase the quality of cultivated mushroom. It’s estimated that
approximately 14,000 species of mushrooms exist (Miles & Chang, 2004); as edible and
medicinal mushrooms account for only a small proportion. More than 10 million metric tons
of edible and medicinal mushrooms are cultivated annually worldwide. Products from
cultivated mushrooms have been used extensively as food additives, health foods, and for
medicinal purposes to treat cancers and circulatory disorders (Kim et al., 2003; Mao et al.,
2005; Park et al., 2001). Improvements in mushroom cultivation technology have brought
countless benefits to commercial production and laboratory applications of mushroom.
1.2 Bioactive compounds in mushrooms
Many bioactive compounds, such as cordycepin, polysaccharides, polysaccharide-peptide
complex, ergosterol, mannitol, peptides and protein/protease, in various mushrooms have

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338

Protein Structure

chemotherapeutic or medicinal activity (Das et al., 2010). The sources of these bioactive
compounds include fruiting body, mycelia, cultivation broth, submerged cultivation
mycelia and fermentation derivatives (Cheung, 1996; Fiore & Kakkar, 2003; Kwok et al.,
2005; Sugimoto et al., 2007; Wang et al., 1995; Wong et al., 2011; Wu et al., 2010, Yamamoto
et al., 2005; Yoon et al., 2003). This chapter focuses on the fibrinolytic enzymes derived from
medicinal mushrooms. The function and characterization of these fibrinolytic enzymes are
described in detail. Technologies for purification and characterization of fibrinolytic enzyme
from Schizophyllum commune are discussed.

2. Haemostasis and antithrombotic studies
Cardiovascular diseases are the leading cause of death worldwide. A common cause of
cardiovascular diseases is abnormal fibrin accumulation in the blood vessels or a fibrin clot
adhering to the unbroken vessel walls of the endoepithelium. An abnormal clot formation is
called a “thrombus”. Thrombosis can stop blood circulation in vessels (arteries or veins),
and may cause a hypoxiation syndrome such as acute myocardial infarction, high blood
pressure, ischemic heart, and stroke (Mine et al., 2005). In response to the high mortality
rates associated with thrombosis, antithrombotic studies and clinical therapies are
progressing rapidly.
2.1 Mechanisms of coagulation
The balance of circulation blood in a liquid or clotted state is called haemostasis, which
includes two complementary mechanisms: blood coagulation and fibrinolysis.
Coagulation limits blood loss from a damaged vessel via clot formation. After the
rehabilitation of the vessel’s endoepithelium, fibrinolysis system processes dissolve clots
and recover circulating blood (Takada et al., 1994). When a blood vessel is damaged by
external force (e.g. a cut or scrape), this damage induces the platelet activation and
aggregation of clotting plasma proteins. Platelets adhere to the subendothelium and
simultaneously activate a coagulation cascade that induces fibrin production (Heemskerk
et al., 2002). This coagulation cascade is the outcome of multiple interdependent
interactions among plasma proteins (tissue factors), platelets, prothrombin, thrombin,
fibrinogen, and fibrin. Via the intrinsic and extrinsic pathways, soluble fibrinogen is
converted into insoluble fibrin. Fibrins construct a mesh structure over the platelet plug,
sealing the injury site (Gentry, 2004; Norris, 2003; Wolberg, 2007).
2.2 Fibrinolysis
Under normal conditions, fibrin clots formed in blood vessels should be disassembled
rapidly and removed by the fibrinolysis system effectively. The key enzyme in the
fibrinolysis system is plasmin, a serine protease, which is activated from proenzyme
plasminogen via a tissue-type plasminogen activator (tPA) or a urinary plasminogen
activator (u-PA) trigger. The fibrinolysis system is regulated by a number of orchestrated
interactions between fibrin, specific inhibitors, and plasminogen; plasminogen activators are
indeed necessary that generate clot degradation (Medved & Nieuwenhuizen, 2003). Collen
(1999) demonstrated that a fibrin formation can trigger activation of fibrinolytic system and
generate of active plasmin; the latter substance may then degrade fibrin into soluble fibrin
degradation products (FDPs), followed by clot disintegration (Collen, 1999).

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Many snake venoms. 2005).. streptokinase. C-type lectins. metalloproteinases. These enzymes may have great potential for antithrombotic therapy.3 Clinical thrombolytic agents Pharmacologic dissolution of an established thrombus is now an accepted therapeutic approach for thrombotic occlusive disease. which have a molecular weight of 25-32 kDa. 2001). a safer thrombolytic agent is need for treating thrombolytic processes. Sumi et al. which consist of a multitude of biologically active proteins and peptides. lumbrokinase (LK). which can degrade fibrin and inhibit fibrin clot formation (Chen et al. Widespread systemic activation of fibrinolysis leads to potentially life-threatening side effects.Fibrinolytic Enzymes from Medicinal Mushrooms 339 2.. 1987). and a fibrino(geno)lytic function which can digest fibrin and fibrinogen. To overcome these risks. Recent studies have shown that LK enzymes can dissolve blood fibrin clots and inhibit platelets activation and aggregation. and tissues (Clemetson et al. In 1991. 1991. Moreover. and anisoylated plasminogen streptokinase-activator complex is effective in restoring blood flow in occluded arteries and veins (Liu et al.intechopen. 1991. Studies of LK demonstrated that these enzymes. In 2008. However. Overview of fibrinolytic enzymes Fibrinolytic enzymes have been found in natural sources.1 Fibrinolytic enzymes from animals Earthworms have been used for their antithrombotic effect in East Asia traditional fork medicine for a thousand years. found in the earthworm’s body cavity and digestive organs. However. Their specific characteristics. perform PA and plasmin activities.. alter the balance between coagulation and anticoagulation. also exist in snake venoms. 1993. Notably. blood cells. such as rapid degradation.. a thrombin-like function. applications of earthworms have been investigated intensively. 1991).. some fibrinolytic enzymes exhibit activity similar to that of a PA. disintegrins and phospholipases... 2002). (Mihara et al. 2007). Their activity resembles that of plasmin. such as fibrinolysis. facilitating their development as antithrombotic drugs (Fan et al. Each may act selectively on different blood coagulation factors. including recombinant tPA (r-tPA). fibrinogenolysis and the proteolytic effect.plasminogen activators (PA).. Intravenous infusion of commercial thrombolytic agents . Venom proteases may involve activation or inactivation of each factor related to coagulation and fibrinolysis. was first extracted from the Lumbricus rubellus by Mihara et al. Intestinal absorption experiments for LK enzymes have also showed that these heterologous proteolytic enzymes have potent properties. resulting in wide-ranging therapeutic applications. which stimulates fibrinogen forward clotting processes. absorption and efficacy of earthworm fibrinolytic enzyme d (EFE-d) was enhanced when delivered in water-in-oil (w/o) microemulsions to rats (Cheng et al. rokinase. 3. these agents are expensive and have a number of drawbacks. a proteolytic enzyme. In this century.com . their precise physiological and biochemical mechanism remains unclear.. Further study has showed that venom www. 2008). These biological molecules have been classified as serine proteases. uncontrollable acceleration of fibrinolysis and haemorrhage. u-PA. are lethal to humans by adversely altering haemostasis. such that LK enzymes can be administered to treat stroke patients as well those with cardiovascular diseases (Nakajima et al. Mihara et al.. Tang et al. 3.

were demonstrated to be effective in thrombolytic therapy. Moise & Kashyap.or -chain of fibrinogen. 1996. streptomyces. including those of Agkistrodon acutus. A. is fermented by the microorganism Bacillus subtilis natto. which were found with fibrin and plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251) digestion activity (Sumi et al. a metalloproteinase inhibitor. meat. A. A. 1995). Sumi et al. 1994). 2005). Natto a popular soybean food in Japan. fungi. several venom fibrin(ogen)olytic enzymes and genetically recombined venom fibrin(ogen)olytic enzymes have been examined using animal models and a promising result was obtained (Gasmi et al. Tempeh). 1987. have been investigated extensively worldwide.g. Korean Chungkook-Jang soy sauce. and Crotalus atrox. piscivorus piscivorus. A.. the Bacillus genus contains microbial species that usually are used for fermentation during fermented-foods production. The first commercial fibrinolytic enzyme. 1987). and algae (Peng et al. Fermentation is carried out by edible bacteria or fungi (Kim et al. with molecular masses of approximately 25 and 60 kDa.. a serine proteinase inhibitor.. (2005) presented an overview of microbial fibrinolytic enzymes. Sugimoto et al. Sumi et al.. Swenson and Markland (2005) characterized venom fibrino(geno)lytic metalloproteinases and serine proteinases. respectively (Swenson & Markland. For example. Toombs. 1996. respectively. Kimchi). The recombinant fibrinolytic enzyme produced by recombinant technology is now mass-produced. actinomyces.. Additionally.. www.com . Nattokinase (NK). 2005). 2008. Fibrino(geno)lytic enzymes have been isolated in snake venom. In 1997. Marsh & Fyffe. was purified and characterized from Natto.5 kDa protein corrected to native streptokinase has a peptide sequence that was successfully used to treat Thrombus (Avilan et al. and dairy products. Two well-known plasminogen activators. rhodostoma. The -chain and -chain fibrinogenases can be defined as venom enzymes degrading preferentially (although not exclusively) either the .intechopen. dochi. salt-fermented fish. Studies of fermented foods showed that fibrinolytic enzymes may be purified from such sources as Japanese Natto. these enzymes are currently examined under examination in preclinical and clinical experiments for their thrombolytic efficacy and haemostatic safety. and Indonesia soy products (e. the streptokinase gene was cloned and its expression in the non-pathogenic Escherichia coli was characterized. A.. and bacilli). g.. 1997. Microbial fibrinolytic enzymes are derivatives from bacteria (e.. Two sub-classes of proteinases that have distinct sensibility to enzymatic inhibitors. fermented shrimp paste. The most significant characteristic of the amino acid composition of an enzyme is very high levels of Asx and Glx residues (Hahn et al.2 Fibrinolytic enzymes from microbial Peng et al. Most fermented foods are derived from raw materials such as beans. 2004). The recombinant 47. Based on the high fibrinolytic activity of the fibrinolytic enzyme process in venom. and phenylmethanesulfonylfluoride (PMSF). 2001). fish. halys brevicaudus. such as EDTA.340 Protein Structure contains two groups of fibrino(geno)lytic enzyme. streptokinase and staphylokinase from Streptococcus hemolyticus and Streptococcus aureus. 3. 1995. the recombinant forms of streptokinase have low antigenicity and high fibrin-selective activity in human circulatory system (Collen & Lijnen. Nattokinases. Recently..g. grains. 1997). piscivorus conami. 2007. Wong & Mine. vegetables. The mechanism of action of venom fibrin(ogen)olytic metalloproteinases and serine proteinases differs. the side effect of the recombinant fibrinolytic enzyme is reduced. contortrix. fermented vegetables (e. and they target different amino acid sequences in fibrin(ogen).. Unlike native streptokinase.

a novel NK protein (NKCP) with both antithrombotic and fibrinolytic effects was discovered. 1995. Kim et al. Sumi et al. non-polluted substances for growing mediums are becoming rare. from supernatant of B. fruiting body. The products of mushroom cultivation include hyphae. NK was characterized as a substilin-like serine protease. and similar organic items. (1990) further demonstrated that Natto or NK capsules administered orally enhance fibrinolysis in canine plasma. rm-subDJ-4 does not. technology for mushroom submerged path culture has been established in the laboratory and at an industrial level. ground. Moreover. 4. Previously. 2001.0–4.. In 2005. 1982).0) properties than native subtilisin DJ-4. Mushrooms (fungi) may generate a new generation www.. Notably. the fruiting body was cultured in growing mediums comprising woodchips. as the fibrinolytic enzyme was purified from common edible sources. straw. 1999. 1990). base on its high sensitivity to protein substrate Suc-Ala-Ala-Pro-Phe-pNA for substilin (Fujita et al.. Since air. Two fibrinolytic enzymes. Spores may create new individuals when in an environment suitable for growth. Many fibrinolytic enzymes were discovered from these products recently. however.g. Table 1 lists fibrinolytic enzymes derived from mushrooms. The rf-subDJ-4 had higher heat. Tricholoma saponaceum. 2000). a traditional Korean fermented soybean paste (Kim & Choi. and -chains of fibrinogen. now desired mushroom cultures can be successfully created. NK has great potential as a drug candidate for treating and preventing thrombus in animal models (Fujita et al. Sumi et al. subtilis QK02 culture broth were purified and characterized in 2004. In the last decade. 2006). Mushroom fibrinolytic enzymes The fruiting body of mushrooms can produce and disperse a large number of spores within a short period. The mushroom fruiting body can be collected from nature and cultured via a sterilized growth medium made of organic substances for saprophytic utilization.intechopen. coffee grounds. cultivation of mushroom fruiting body is space occupied and labor-intensive.and acid-resisting (pH 3. Kim & Kim. This was the first study of fibrinolytic enzyme from mushrooms and their application as therapeutic agents. logs. and derivatives that dissolve in broth.com . sawdust. trees) and ground water can be pollute. after seeding spores or the mycelium of fungi on artificial solid or liquid culture medium under controlled temperature and moisture. 4. -. plants (e.1 Fibrin(ogen)olytic enzyme of mushroom The proteolytic complex from fungus Flammulina velutipes was studied by gel chromatography and the activities of the enzyme complex were compared with those of Aspergillus terricola and Streptomyces griseus proteinases (Morozova et al. Thereafter. oral administration is likely safe. Fibrinolytic recombinant full-subtilisin DJ-4 (rf-subDJ-4) and mature-subtilisin DJ-4 (rmsubDJ-4) were expressed by a pET29 vector system (Choi et al. and Cordyceps militaris (Choi & Shin. Armillaria mellea.. Moreover.. 2004). 2004). 1998. culture broth.Fibrinolytic Enzymes from Medicinal Mushrooms 341 In 1993. QK-1 and QK-2. QK-1 is a plasmin-like serine protease and QK-2 is a subtilisin family serine protease (Ko et al.. Dosedependent prolongations of both prothrombin time (PT) and active partial thromboplastin time (APTT) were observed in rats administered NKCP intraduodenally (Omura et al.. However. 2005).. A strong fibrinolytic subtilisin doenjang (DJ)4 was purified from doenjang. mycelium. 1993). rfsubDJ-4 has the same abilities as the hydrolyzed -.. fibrin(ogen)olytic proteases were discovered in the fruiting of Pleurotus ostreatus.

. Choi and Sa (2000) isolated a metalloprotease with fibrinolytic activity from cultured mycelium of Ganoderma lucidum (Choi & Sa.10-phenanthroline) and metal ions. Fibrinolytic enzymes derived from mushrooms when a fresh environment and nutrients are provided. The mycelium may grow stably without pollutants. and the carbon source. Park et al.. 2008) FFP1 FFP2 CSP FP no name 32 kDa 42 kDa 31 kDa 27 kDa 21.. 2005. Broth for submerged cultivation is rich in mushroom derivatives. Nutrients and differences between growth mediums and submerged broth contribute to the particular metabolite of mushrooms. Changing culture medium parameters... 1997. Over the last 50 years. 2007) (Kim et al. 1991).5 kDa 18. Archer & Peberdy. were identified (Kim et al. 1998) (Kim & Kim. 2007) * N means undetermined Table 1..5 kDa (Sugimoto et al.. 2007) (Ueda et al. 2008.. Mushrooms grown in medium or submerged broth may differ morphologically. FFP1 and FFP2).intechopen. protease secretion was increased significantly during submerged cultivation. metalloprotease from F. including metalloprotease from A. BLB FP N N (Li et al. markedly alter the extracellular performance of fungi mycelium (Archer et al. Fukushima et al.e. 2010a) 28. Fibrinolytic metalloproteases are sensitive to metalloprotease inhibitors (e.com . 2001) (Kim et al. velutipes FVP-1. 2005) (Park et al.342 Protein Structure Mushroom From fruiting body Flammulina velutipes Pleurotus ostreatus Armillaria mellea Tricholoma saponaceum Cordyceps militaris From mycelium Ganoderma lucidum Armillaria mellea Flammulina velutipes Perenniporia fraxinea From culture broth Fomitella fraxinea Protease Fibrinogen Native Degradation Mol. 1995.. 2007) (Lu et al. mellea (AMMP). (1991) reported that when soy sauce oil was the carbon source for Asperyillus oryzae. Wt. Several extracellular fibrinolytic enzymes have been purified from submerged broth of Fomitella fraxinea (i. 2006) Cordyceps sinensis Fusarium sp..32 kDa (Lee et al.. 2000) (Lee et al. 1999) (Kim & Kim. protein content. Bobowicz-Lassociska & Grajek...g.1 kDa 52 kDa (Choi & Shin. 1982) 24 kDa 18. www. 2006) metalloprotease N AMMP FVP-1 metalloprotease 100 kDa 21 kDa 37 kDa 42 kDa (Choi & Sa. EDTA and 1... 1995. Reference Two protease N* (no name) Metalloprotease Metalloprotease TSMEP1 no name N (Morozova et al. 2008). Lee et al.. Kadimaliev et al.. 2000). salt concentrations. such as osmotic pressure. BLB Schizophyllum commune From recombinant source Fusarium sp. and metalloprotease from Perenniporia fraxinea. 2007). specific protease production was stimulated selectively by the oils (Fukushima et al. fibrin(ogen)olytic proteases from mushroom mycelium. mushroom derivatives with biological activities for humans have been investigated extensively. Furthermore. In the following decade..

e. which including fibrin. mella fruiting body and mycelia. T. lucidum.e... we expect that the high production efficiency of mushroom fibrin(ogen)olytic enzymes can be achieved by well-designed cultivation processes.. and identified the N-terminal amino acid (Ueda et al.e. militaris had high sequence identify with subtilisin PR1J (GeneBank. Previous studies have showed that most fibrinolytic serine protease from traditional fermented foods belong to subtilisin of Bacillus origin such as nattokinase. and substrate specificity. have markedly different N-terminal sequences. P.e. 2007. BLB (i. The purification stratagem for these extracellular fibrinolytic enzymes differs from those of fruiting bodies or mycelium. except for metalloprotease derived from A. These fibrin(ogen)olytic enzymes.. F.2 Characteristics of medicinal mushroom fibrin(ogen)olytic enzymes Fibrin(ogen)olytic enzymes including those from P..e. 2007). The Nterminal sequences of most fibrinolytic enzymes have been determined. commune (Lee et al. (2007) first purified a fibrinolytic protease from Fusarium sp. C.. CSP) were found to have a broad substrate specificity for synthetic substrates. 2006).. optimal pH.Fibrinolytic Enzymes from Medicinal Mushrooms 343 Cordyceps sinensis (i. BLB. 2006. Via study of N-terminal sequence alignment. commune. Fusarium sp. the optimal temperature is 20-60°C. Serine fibrinolytic enzymes from mushrooms of F. Nutrition and the physical environment may be the dominant factor for growth of fungi and the rate at which extracellular proteases are produced in a submerged culture system.. 2007).com . have been identified. 2005). sinensis. BLB may demonstrate that manufacturing mushroom fibrinolytic enzymes may be possible in the future.. fibrinogen. 2007).. Ueda et al. This development of FP from Fusarium sp. 2007). BLB (FP).e. Aspergillus oryzae. Extracellular secreted protease of fungi is in some cases the fungi metabolite that may reflect cultivation conditions. ostreatus. fibrinolytic protease from Fusarium sp. BLB. Fusarium sp. CSP) and S. 2006). and C. www. inhibited by metalloprotease inhibitors) according to protease inhibitor specificity. inhibitors. 4. fraxinea also has 20% identify with Nattokinase and subtilisin E (Lee et al. The optimal pH for these mushroom fibrin(ogen)olytic enzymes is 5-10... FFP1 from F. The hydrolytic activity of FP toward synthetic peptide subtracts is higher than that of proteases from Bacillus subtilis natto. F. inhibited by serine protease inhibitors) and metalloprotease (i. Ueda et al.intechopen. fraxinea (i. 2004). mellea. Li et al. A. FP). and S. C. (2007) cloned an FP gene encoding a novel protease derived from Fusarium sp. Table 2 lists their biochemical properties. Sugimoto et al. CAC95048) (Kim et al. sinensis (i. a chymotrypsin-like serine protease from mushroom C.. 2010a. FFP1).. The protease activity of mushroom serine fibrinolytic enzymes can be irreversibly inhibited by PMSF but no other protease inhibitors. including molecular weight. thermal stability. velutipes. subtilisin DFE and subtilisin QK-1 (Peng et al. Previous studies have demonstrated that the fibrinolytic enzymes may be available in the fruiting bodies of some mushrooms but not in the cultured mycelium (Kim et al. 2008). casein. militaris. In laboratory works. Fungi derivatives in submerged broth are biological molecules considered important for human health (Papagianni. Streptomyces griseus and commercial plasmin (Sugimoto et al. substrates for subtilisin and substrates for plasmin. BLB had higher fibrin degradation and plasminogen activation than Nattokinase (Sugimoto et al. fraxinea. An overview of microbial fibrinolytic enzymes (Table 2) showed that mushroom fibrinolytic enzymes may be classified as serine protease (i. G. Lu et al.. saponaceum.. Base on the critical influence that produce mushroom extracellular secreted protease in submerged culture system.. fraxinea.

0. 1998) Zn. Cu. to 50°C metalloprotease) Armillaria mellea pH 7... AMMP) Fomitella fraxinea pH 5.. Co Cu. 2001) Ca. Co Yes 1. 35~40°C Mn. 45~55°C (18.5 kDa. aprotinin PMSF Yes DFP.5. 2007) (Sugimoto et al. 37°C militaris (52 kDa ) Fomitella fraxinea pH 10.Fe Hg.3 kDa. TSMEP1) Armillaria mellea pH 6. Mg fraxinea (42 kDa.. FP) Fusarium sp.4. 40°C (32 kDa. FVP-1) Perenniporia pH 6.10phenanthr oline. FFP1) Cordyceps sinensis pH 7.0~7. 33°C (21 kDa. Co YES Yes N N Yes N N Mn. 2006) Mushroom Protease Optimal pH and temperature Serine protease typeCordyceps pH 7.10phenanthr oline. BLB N (28. CSP) Fusarium sp.10Yes phenanthr oline.. 2006) (Lee et al. 40°C (42 kDa. 2008) Hg. Review of medicinal mushroom fibrin(ogen)olytic enzymes www. Ni Yes (Lee et al. 2005) (Lee et al. FP) Metalloprotease type-Flammulina N velutipes (metalloprotease) Pleurotus pH 7. PMASF PMSF.1 kDa. Yes Fe3+ 1. metalloprotease) Ganoderma lucidum (100 kDa. Cu Yes Yes (Kim & Kim. Mg. Mg Cu. Fe2+. FFP2) Flammulina pH 6. 2007) N N N EDTA N (Morozova et al.. Mg Cu. metalloprotease) Schizophyllum pH 5.... Table 2.Zn Yes EDTA Yes (Kim et al. BLB pH 9. EDTA 1. EDTA EDTA.com Yes (Park et al. 2000) pH 7. metalloprotease) PMSF. metalloprotease) Tricholoma saponaceum (18. 1982) Zn. Co N Yes N (Choi & Shin.5~8. Co. up Zn. 2010) *N means undetermined. Yes EGTA Cu. EDTA EDTA Yes Zn. 1999) pH 7. up ostreatus (24 kDa. 2007) (Ueda et al.5.5 kDa. Cu. 40°C (31 kD. PMSF Yes Cu.intechopen. Yes Hg N N N N N N Yes (Kim et al. Co. 30~40°C Zn. 2006) (Li et al. Mg Hg Yes Yes (Kim & Kim.344 Protein Structure Ion induced Ion inhibit Fibrinogen Protein degrade inhibitor NTerminal sequence Reference Ca.10phenanthr oline.5~9. 2007) . Yes Co EDTA Yes (Lu et al.Mg velutipes (37 kDa.10phenanthr oline 1. EDTA 1. 45°C Mg commune (21. 50°C (27 kDa.Fe. 20~30°C Mn.. Co to 60°C N Yes (assume) N (Choi & Sa.

Surface of plate agar was pure colour and without contamination. Additionally... T. 2010b. malt extract. The presence of Zn2+ was detected in metalloprotease from P. lucidum. P. 2007). was transferred to 50ml YM broth for 5 days at 25°C. 2006. saponaceum. dextrose. the manufacturing capacity of SK from haemolytic streptococci is limited with a high price tag. or mycelium. 2010). a piece of plate agar covered with grown mycelia. S. was like white flannelette. For mass production. Lu et al.. commune was cultured in YM agar plate (contains peptone. Kim & Kim. Peng et al. Lu et al. commune is a good biological resource for scientists whom study the functional substance. G. The mycelia grew and covered plate surface. Park et al. In our study. 1981). ostreatus and G. mellea (AMMP). commune. Mg2+-dependent protease activity was found in metalloproteases from F. A. Lu & Chen. (2005) illustrated that microbial fibrinolytic enzymes. especially those from food-grade microorganisms.com . 2010. S. Zn2+-dependent protease activity exists in metalloprotease derived from F. P.2% agar) at 25°C for one week. 1982. The production. 2008. Morozova et al. The fast growing and easy cultivation raise the universal uses for S. Lee et al.. As a seed culture. mellea.10phenanthroline predominantly .. have potential to be developed as functional food additives and drugs to prevent or cure thrombotic disease (Peng et al. Production. Therefore. Proteases were purified in the fruiting body. A. 1998. commune Schizophyllum commune derivatives have been suspected with antithrombotic effect for human beings by scientists (Okamura-Matsui et al. fraxinea and S. Kim et al. lucidum by mass spectrometry. both of these metalloproteases have Zn2+-dependent protease activity. purification and characterization of mushroom fibrinolytic enzymes Streptokinase (SK) from streptococci is a widely used therapeutic agent for acute myocardial infarction.. fibrin(ogen)olytic enzymes from non-toxic mushrooms has gradually become the centre of attention for investigators in thrombolytic therapy. ostreatus. Pharmacological uses of SK take risks of causing potential myocardium and liver damages due to the residual bacteriohemolysin in the manufacturing process (Zhang et al. or culture supernatant. with an undefined ion structure..1 Submerged cultivation of S. F. The activity of mushroom fibrinolytic metalloproteases can be inhibited by EDTA and 1. Lee et al. commune (Choi & Shin... P. 5. commune is a ubiquitous white rot and widespread fungus in existence.. 2005).. fraxinea (FFP2). 2010a. 2001). 5. the seed culture then transferred to 15L YM broth for additional www. velutipes. velutipes (FVP-1). This medicinal mushroom has been an additive in traditional folk medicine and the subject of genetic analysis.Fibrinolytic Enzymes from Medicinal Mushrooms 345 Mushroom fibrinolytic metalloproteases were discovered in F. fraxinea and S. The metal ions dependent activity is not only a character of mushroom fibrin(ogen)olytic enzymes but also can be considered as a critical point for proteases under the clinical application for thrombolytic therapy (Lu & Chen. 2010). mellea and T. purification and characterization of an fibrin(ogen)olytic protease from S. commune in science researches and medicinal applications. However. yet the detail mechanism is unclear. commune were discussed in this section (Lu et al. saponaceum (TSMEP1). yeast extract and 0.intechopen. 2001. 1999). Base on the high protease and extracellular biological substance production ratio in submerged cultivation (Desrochers et al.. Mushroom of S. A. 1999. 2005. fraxinea.

Eluted fractions were analyzed for protease activity to azocasein and recorded (Fig.com . One is the pre-treatment of resource. Eluted fractions were analyzed for protease activity to azocasein (see in Fig. The 2nd dialysis substrate was loaded on Mono QTM 5/50 GL column at flow rate of 1 ml/min. 2). In step 3. 1A). Mono QTM 5/50 GL column was preequilibrated with 20mM Tris buffer (pH 8. 8.45μm membrane and fractionized by cross-flow filtration. Figure 1A illustrated the result of elution processes with column buffer without (NH4)2SO4 in a stepwise manner of 60 min interval at flow rate of 1 ml/min.000×g. to collect the <100-kDa fraction. 30min at 4°C. commune Mycelia in submerged culture broth were removed by centrifugation at 4°C. The protein concentration of each eluted fraction was detected with A280 in real time by ÄKTA purifier 10 (GE Healthcare). The growth rate increases with time so that the logarithm of the amount of fungus mycelium increases with time. 5. Colonies of fungi in submerged cultivation grow exponentially. including the crude protein extraction from fruiting body. 1B). In step 2. Protease purification was then performed by fast performance liquid chromatography. The 3rd dialysis substrate was loaded with same buffer at flow rate of 1. The ceramic cross-flow filtration is done before protein precipitation. Figure 2 showed the purification purity by SDS-PAGE (Fig. or mycelium. protein precipitated and dialyzed against 20mM Tris (pH 8. an ion exchange column. The concentrated fraction (molecular weight 3 -100 kDa) was precipitated by saturated ammonium sulfate at 4°C for 8 hour followed by centrifugation at 10. that will obstruct the protein purification. that is different from yeast cells grow at a constant rate.intechopen. which www. by linear gradient of 20-fold column volume to 40% 1M NaCl. which was further concentrated by 3-kDa ceramic column filtration. a size exclusion column. The active fractions were pooled.2 Purification of fibrinolytic enzyme from S. The protein pellet was dissolved in ddH2O and followed by dialysis with FPLC column buffer containing 50mM sodium phosphate (pH 8.0) as 3rd dialysis substrate for next step of chromatography. for 30min.0) as 2nd dialysis substrate for next step of chromatography. Due to the extracellular polysaccharide with huge molecular weight present in broth.0) and 1M (NH4)2SO4 as 1st dialysis substrate. Measuring activity to azocasein is a general selected detection method to identify target protease fractions. Elution was carried out with the same buffer but containing 1M NaCl.0). a hydrophobic interaction column. or culture supernatant. The purified protease then can be applied for more. Superdex 75 10/300 GL column was preequilibrated with 50mM sodium phosphate buffer. protein precipitated and dialyzed against 50mM sodium phosphate buffer (pH 8. the purified fraction containing target protease is available. Equilibrated 1st dialysis substrate was loaded on column at flow rate of 1 ml/min. Another is the chromatography stratagem. The supernatant was filtered by 0. Phenyl SepharoseTM High Performance beaded packing column was preequilibrated with column buffer.2 ml/min. 1C). After examination of protease activity to azocasein (see in Fig.200 ×g. Combination of liquid chromatography and protease activity screening. Fibrinolytic enzyme purification from mushroom may divide into two processes.346 Protein Structure 7 days with shaking platform and air exchangeable cover. the active fractions were pooled. In step 1.

Fibrinolytic Enzymes from Medicinal Mushrooms 347 including the combination of ion exchange.intechopen. hydrophobic interaction and size exclusion chromatography. www. Techniques with selectivity are highly independent of protease resources and the enzyme properties.com .

www. Fig. (C) Eluted fractions of Superdex 75 10/300 GL column. 2. commune fibrinolytic enzyme (A) Eluted fractions of Phenyl SepharoseTM High Performance column. 1. Chromatography purification of S. (B) Eluted fractions of Mono Q column.com .intechopen. SDS-PAGE result of chromatographic purification M: protein markers. Lane 4: Protein with highest protease activity eluted from Superdex 75 10/300 GL column. Lane 1: 1st dialysis substrate.348 Protein Structure Fig. Lane 3: 3rd dialysis substrate. Lane 2: 2nd dialysis substrate.

3 Characterization of fibrinolytic enzyme from S. and the protease activity to azocasein then examined. (B) Temperature effects. 3.Fibrinolytic Enzymes from Medicinal Mushrooms 349 5. commune To study the effects of pH and temperature to fibrinolytic enzyme from S. Optimal protease activity reveal at pH 5. commune (A) pH effects. www. The effects of pH and temperature on protease activity of fibrinolytic enzyme from S. commune. purified enzyme was incubated in various pH and temperature respectively. the data was illustrated in Fig. Fig.0 and 45°C.com .intechopen. 3A and 3B.

CuSO4.com . Protease activity of mixed solution was then examined by azocasein assay. Pb(NO3)2. pepstatin A (aspartyl peptidases inhibitor). aprotinin (serine protease) and phosphoramidon (endopeptidase inhibitor) respectively.350 Protein Structure Three replicates of fibrinolytic enzyme were mixed with HgCl2. EDTA (metalloprotease inhibitor). CaCl2. and inhibits by EDTA. benzamidine hydrochloride hydrate (trypsin. The result indicated that the protease shows Mg2+-dependent activity. Figure 4 demonstrated the relative protease activity to the control treatment (100%=1) (Fig. 4. Fig. commune (A) Divalent cations effects. CoCl2. www. C4H6O4Zn·2H2O. The effects of divalent cations and protein inhibitors on protease activity of fibrinolytic enzyme from S. (B) Protein inhibitors effects. MgCl2. ddH2O (control) and protease inhibitors of PMSF (serine protease inhibitor). trypsin-like protease inhibitor). 4A & 4B).intechopen.

fibrinolytic enzymes from microorganisms show low homology except the fibrinolytic enzyme from substilisin group. (2) X means amino acid undetermined. and incubated for 24 hours. fibrinogenolytic assay. Purified protease were loaded on a fibrin plate. Fibrinolytic enzymes from medicinal mushroom are novel proteases with fibrin and fibrinogen degradation activity. which synthesized by the method described by Astrup and Mullertz (Astrup & Mullertz. Table 3. the excess stimulation of plasmin that may cause haemorrhage within patients. A piece of clot was placed on Petri dish. 1952). In order to identify the clinical functions and risks of medicinal mushroom fibrinolytic enzymes. Fibrin plate was made at room temperature in a petri dish containing 1.351 Fibrinolytic Enzymes from Medicinal Mushrooms N-terminal sequence of protease is another character for protein identification. Pleurotus ostreatu. substilisin DFE (Peng et al.. and 10U thrombin. The result showed that the purified S. 1992) . 2003). but the side effect.2μg and 0. 5). commune A A X2) A A Q T T X L S N-terminal amino acid sequence S V P Y G I Y N G C S S F V G C S A Y N G X T X Y V G X S P Y N G X S S S S (1) GFMEP. POMEP. Antithrombotic effect of mushroom fibrinolytic enzymes Current clinical thrombolytic agents are used to convert plasminogen to active enzyme.2% human fibrinogen. 6. AMMEP and TSMEP are metalloendopeptidases from Grifola frondosa. it demonstrated the distinctive feature of the fibrinolytic enzyme from S. Comparison of N-terminal amino acid sequence of mushroom fibrinoytic enzyme 6. and the dynamic tracking of blood clot formation. 1984). Rat blood was withdrawn without anticoagulant and stayed for clot formation in a tube. followed by dropping 0.5% agarose.5μg fibrinolytic enzyme on clot disks and incubated in 20°C for 8 hours to observe the clot degradation www. Armillariella mellea and Tricholoma saponaceum.1 Fibrinolytic activity assay and clot degradation test Fibrinolytic activity was determined by artificial fibrin plate assay. Plasmin from human plasma (3U/mg protein) was applied as a positive control. such as substilisin NAT (Nakamura et al.. commune to other mushroom. acting on the surface of thrombus that avoids excessive induction of systemic fibrinolytic system. Newly thrombolytic agents now are developed for fibrin-specific property.intechopen. which degrades fibrin and process antithrombotic effect. 0.com . It’s an effective way to decompose harmful thrombus in circulation system. plasmin. substilisin E (Wong et al. commune fibrinolytic enzyme display stronger fibrinolytic activity than commercial plasmin (Fig.. which directly break the clot and interfere the clotting system. However. mushroom fibrinolytic enzymes were examined by various antithrombotic examinations. such as fibrinolytic assay. plasminogen activation assay. Table 3 illustrated the results of Nterminal sequence comparison. Enzyme1) NK & substilisin DFE GFMEP POMEP AMMEP TSMEP Fibrinolytic enzyme from S.

5μg fibrinolytic enzyme digested the clot disks. Phosphate buffer was dropped on clot disk as the control treatment and another clot disk as the blank without any treatment.1. respectively. but opposite results were observed in control and blank.0μg human plasmin (zones D) showed the equal digestion effects to 0. 0.intechopen. and H: 0. 5.352 Protein Structure effects. G.0μg of human plasmin.com . Clot degradation assay of fibrinolytic enzyme from S. commune 0.5μg fibrinolytic enzyme from S.2. Fibrinolytic activity of the fibrinolytic enzyme from S.2μg and 0. F.6). Figure 6 demonstrated that S. and D: 0. commune.25. commune Fibrin digestion zones A. commune (zone G). Blank: blank blood clot disk. Control: clot disk treated with PBS. 0. www. respectively. B.5.5μg fibrinolytic enzyme. Treatment of 1. Fig. C. and 1μg of fibrinolytic enzyme S. commune fibrinolytic enzyme digests the blood clot dominantly (Fig. 0.5μg: blood clot disks treated with 0. and 1. 6. Treatment of 0.2μg and 0.5. Fig. 0. Fibrin digestion zones E.2μg and 0.8.

As the results in Fig.and -chains. and shows three bands on the SDS-PAGE. and it was less efficient in digesting the -chain (Fig.2 Fibrinogenolytic activity assay A fibrinogenolytic degradation assay was performed using a 1% fibrinogen solution mixed with the protease from S.intechopen.5μg purified enzyme with plasminogen (0. resulted in the digestion of S2251 and an increase in absorbance.Fibrinolytic Enzymes from Medicinal Mushrooms 353 6. In this assay. commune failed to digest the S2251 directly and without plasminogen activation activity. the presence of urokinase. www. In this assay. The fibrinogenolytic activity assay was carried out by the incubation of fibrinolytic enzyme with fibrinogen. and -chains of fibrinogen. fibrinolytic enzyme from S. Most of the chain was digested after 22 hours of incubation. Black arrows: Fibrinogen degradation products. Degradation of the -chain occurred after 6 hours of incubation. The purified fibrinolytic enzyme from S. Fibrinogen is composed of peptides -. the fibrinolytic enzyme completely digested all of peptide chains in 30 hours.5 hour after the reaction. commune was mixed with plasminogen. commune at 37°C. the reaction mixture was removed and SDS-PAGE electrophoresis was performed. At different time intervals. . 6. . plasmin substrate S2251and without fibrin. human plasmin and urokinase were used as positive control for S2251 digestion and plasminogen activation. Lane 1~7: Time course sampling in reaction. 7). 8. 7. and means : . which is known to activate plasminogen. Fig. Fibrinogenolytic assay of the fibrinolytic enzyme from S. Fibrinolytic enzyme from S. M: Protein marker.1UN) and a 10mM plasmin-specific substrate S2251 at 37°C.3 Plasminogen activation assay Plassminogen activation assay was performed by incubating 12. commune displayed a higher activity in digesting the -chain and -chain.com . Substrate S2251 can be cleaved by plasmin and generated measurable p-nitroaniline (p-NA). commune . Significant degradation of the fibrinogen -chain and -chain occurred within 0. Afterward.

commune fibrinolytic enzyme applied in rat citrated blood. Plasminogen activation assay of fibrinolytic enzyme from S. Four TEG parameters.intechopen. since clotting initiation completed normally. Urokinase was used as positive treatment to activate plasminogen and resulted in fibrinolysis after reaching of MA (Fig.0001 UN. commune is not a serine protease for S2251 and does not activate plasminogen in vitro. commune ○ : 1 μg purified enzyme and plasminogen at 0. 8.1UN plasminogen. In the presence of fibrin.354 Protein Structure Fig. ● : 20 μg urokinase. G. commune In previous study. The result of TEG reaction time (R) indicated that the protease did not affect the clotting aggregation by the platelets. Fibrinolysis after MA was determined by measuring the loss of clot strength. : plasminogen at 0. Fibrinolytic enzyme from S. The anticoagulant activity was performed by inhibition of thrombin and hydrolyzation of fibrin and fibrinogen (Choi & Sa. fibrinolytic enzyme from S. 9). Fibrinolytic enzyme from S.com . □ : 1 μg purified protease. * : 1 μg human plasmin (≧3 units/mg protein). commune was mixed with CaCl2 and citrated rat blood to initiate recalcification. In our research. lucidum fibrinolytic enzyme was examined in human plasma by activated partial thromboplastin time (APTT) and thrombin time (TT). and the maximum vertical amplitude of the developed clot (MA) were measured. and the coagulation processes were monitored by thromboelastrography (TEG®) analysis. 6. The protease-digested zone in the plasminogen-rich fibrin plate was then compared to that of the plasminogen-free fibrin plate. 2000). In order to test the plasminogen activation while fibrin present.4 Coagulation effects of protease from S. Fibrinolytic www. commune was applied on a plasminogen-rich fibrin plate containing 0. regardless of whether fibrin exists or not. including the reaction time for clot initiation (R). digest representation were equal on the plasminogen-rich and plasminogen-free plates (data not shown).0001 UN. the time to reach a 20-mm level of clot formation (K). the slope angle from R to K ( value). S.0001 UN. ■ : 1 μg urokinase and plasminogen at 0.

TEG parameters Reaction time (R) Coagulation time (K) Velocity of clot ( ) Maximum amplitude (MA) Range of values 2.. C. citrated blood treated by S. Thromboelastography tracings of citrated blood added fibrinolytic enzyme from S.intechopen.51b 59. commune and human urokinase Curve A: Human urokinase 0.14 81. Additionally.3.22 165.14 90.2 μg 1. a regulatory manipulation of S. 2002).6 μg 1. (n=8). clotting velocity depresses and significant decrease of clotting strength were occurred (Table 4).2-4. Magnesium therapy in coronary heart disease has recently been proposed and documented in clinical trials (Shechter et al.5 μg 100a 92. commune 1. Effects of various concentrations of fibrinolytic enzyme from S.6. Thus. commune to citrated blood by thromboelastography analysis In an experiment of coexist with Mg2+ ion. D.5.21 100 96. Curve B. magnesium ion stimulate activity of fibrinolytic enzyme from S. 0.com .17b 100 99. 1999. Fig. Clotting time prolongation. 0.355 Fibrinolytic Enzymes from Medicinal Mushrooms enzyme of S.07 86.8-24. P<0.05 a Table 4.96 127.43 88.2-81.76 92. commune fibrinolytic enzyme activity by magnesium supplementation can be expected in the future. 1. E and F: Purified protease from S. commune suppressed blood coagulation without excessive fibrinolysis. commune was proved in study. 0 (control)μg added respectively. The cost reducing effect by magnesium supplementation is also an innovative ideal for the currently used fibrin-specific antithrombotic agents.74b 100 94.2. 9.8-3. www.44 98. Whiss & Andersson.7 min 0.3 min 50. 10).3 μg 0.1° 75. Coagulation suppression effect here we predicate may resulted from fibrinolysis and platelet-medicated clot retraction.67 97. The supplementation of Mg2+ ion stimulated the depression of blood clot amplitude (Fig.69 101.5μg added.00 73.5 mm Amount of fibrinolytic enzyme in TEG 0 μg 0. b Statistically significant compared to control samples. commune fibrinolytic enzyme and Mg2+ ion.07b TEG Parameters with the addition of protease are presented as the relative index obtained by normalized to the values in control sample (0μg) which are set to 100%.

Alphabetical lowercase means significant variation between other treatments.5mM).5mM MgCl2. C. So far. militaris. fibrin(ogen)olytic identification and characterization. T. BLB and S. such as P. crude protein extraction. 0. Purification and characterization of fibrinolytic enzyme from S. saponaceum. A. ostreatus.6μg&Mg: citrated blood mixed with 0.6 / 1.6 / 1.2μg fibrinolytic enzyme. Mg: citrated blood mixed with MgCl2 (2. 7. Fusarium sp. F. Clinical application of several plasminogen activators results in activation of circulating plasminogen. While scientists and clinicians are in the search for a better. Most of them have been universal dietetic additives and traditional fork medicine for a long time. pharmacologic intervention of an established thrombus has become an ideal therapeutic approach for thrombotic occlusive disease. Fibrin(ogen)olytic enzymes were discovered from mushrooms.com . G. protein chromatography.6μg fibrinolytic enzyme and 2. however. commune were illustrated in this chapter. many mushroom fibrinolytic enzymes were investigated which perform superior fibrinolytic activity than known thrombolytic agents and inhibit thrombus www. risk accompanied with this treatment option may include a life-threatening haemorrhage caused by the systemic activation of fibrinolytic mechanism. C. mellea. F. Blood clot amplitude analysis of fibrinolytic enzyme coexist to Mg2+ Control: citrated blood mixed with PBS.05. safer therapeutic agent with fewer side effects. Conclusion In recent decades. commune. a difference was considered statistically significant at p < 0.intechopen. velutipes.356 Protein Structure Fig. The low antigenicity and allergic affections of these mushroom fibrinolytic enzymes when human taken orally may be expected definitely.2μg: citrated blood mixed with 0. 0. fraxinea. fraxinea. which successfully digest abnormal blood clot occluded in arteries or vein. sinensis. the methods for purification and characterization of medicinal mushroom fibrinolytic enzyme were carried out by mushroom cultivation. TEG values were expressed relative to the control sample (at 100%). and might be a representative model for purification of medicinal mushroom fibrinolytic enzyme. lucidum. the discovery of the fibrinolytic enzymes from medicinal mushroom may shed the light for the modern thrombolytic therapies. In previous studies. P. 10.

9. D. such as platelets. prothrombin. commune as the model. thrombin. T. M. (1952). Heterologous protein secretion by Aspergillus niger growing in submerged culture as dispersed or aggregated mycelia. Most notably.. the researches and therapeutic development are never finished to search a more effective therapy for such coronary patients. F. D. Avilan. pp. mushroom fibrinolytic enzyme suppressed blood coagulation without excessive fibrinolysis by prolonging clotting time and decreasing clotting velocity. Although the therapeutic results are still ambivalent.. Applied Microbiology Biotechnology. C. Although the description in this chapter showed the investigation of mushroom fibrinolytic enzyme is currently at stage of in vitro and ex-vivo experiment. As results have shown. B. Brazilian Journal of Medical Biological Research.12.Fibrinolytic Enzymes from Medicinal Mushrooms 357 formation. J.4. but rather reduced the likelihood of thrombus formation without increasing the risk of haemorrhage. Vol.2. The fibrin plate method for estimating fibrinolytic activity. expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli. 346-351. Vol. M. Taiwan) for critical reading of manuscript. Mushroom fibrinolytic enzyme did not offset haemostasis. the initiation of clotting system and plasminogen activation occurrences were maintained at a normal level. these enzymes revealed significant metal ion dependent activity. Vol. No. These data indicated that the safer antithrombotic effect of mushroom fibrinolytic enzyme and the board application may use as thrombolytic agents clinically. Han Yin-Yi (Trauma Department. Feei Sun (Council of Agriculture. which monitored the clotting and fibrinolysis action in blood. The molecular biology of secreted enzyme production by fungi. J. 1427-1430. References Archer. However. J. Metal ion therapy in coronary heart disease has recently been proposed. Acknowledgement The authors would like to thank Dr. National Health Research Institutes. No. B. 8. No. fibrinogen. & Puig. As fibrinolytic effect is not the only solution for thrombus occurrence and decomposition. plasminogen. 44. A. Bastidas. Consequently.30. D. 157-160. & Peberdy.com . pp. A. (1995). J. Cloning. We also thank Dr. (1997). www..40. pp. Review of characters from medicinal mushroom fibrinolytic enzymes. Critical Reviews Biotechnology.. Vol. (1997). and fibrin. No. pp. Yarzabal. Kurt M. 273-306. Mackenzie. L. S. Fibrinolytic enzymes interfere in antithrombotic interaction may occur amongst plasma proteins. Cruz. plasmin. & Mullertz. Lin (Division of Medical Engineering Research.intechopen. Taiwan Agricultural Chemicals and Toxic Substance Research Institute) and Dr. the effectiveness of medicinal mushroom fibrinolytic enzyme in antithrombotic assays indicated the possible clinical application of mushroom fibrinolytic enzyme in the future. National Taiwan University Hospital) for equipment and technical assistance in TEG® analysis. the therapeutic effect of mushroom fibrinolytic enzyme were further examined in citrated blood on thromboelastography.17. & Ridout. Archives Biochemistry Biophysics.. the result is a significant decrease in clot strength. The inhibition or the stimulation of enzyme activity by metal ion may provide a pharmaceutical conservation and a critical control manipulation in clinical uses. Astrup. Jurgensen.1-2. Archer. using S.

286-292. No.84. Vol. Y. H. Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat.7. P. Desrochers. Masuda.197. Vol. Biochemical and Biophysical Research Communications. 1416-1418. 71-76. Vol. C.3. Choi. Zhang.3. M. No. (1996). No. Q. T. Vol. (1991). M. Characterization of water-in-oil microemulsion for oral delivery of earthworm fibrinolytic enzyme. Vol. S. 277-287. 1340-1347.3.. www. pp.. P. Collen.com . F. 2887-2892. K. Medicinal uses of the mushroom Cordyceps militaris: current state and prospects. Hong. (2008). H. Nutrition Research. 683-694. & Kim. Li. Applied and Environmental Microbiology. M. K. H. R. T. D. K..62. Vol.intechopen. V. B. (1993). pp. M. DJ-4..28. H. & Lijnen. Vol. M. pp. Chen. 1387-1391. A. No. Q. Y.1. No. Hong. (1981). Toxicon.6. Zhou. FEMS Microbiolgy Letters Vol. Fibrinolytic and antithrombotic protease from Ganoderma lucidum. K. Cheung. a popular soybean fermented food in Japan. Vol. Acta Biotechnologica. R.2. pp.. & Nishimuro. and fibrin(ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. Fitoterapia. 259-270.16. S & Sa. H. Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide. S. Li. Journal of Control Release. Vol. Fiore. Wu. expression. Kariya. & Sakakibara.. Q.18. Vol. Chen. Changes in protein secretion of Aspergillus niger caused by the reduction of the water activity by potassium chloride. (2007). & He..10.. Cloning.. J. Fan. pp. R.358 Protein Structure Bobowicz-Lassociska. H. N. pp. pp. 1721-1725. No. 82. & Grajek. F.. (1999). W. Choi. S. J. pp.2.-S. L. Y. Biochimica et Biophysica Acta. (2010). (1994). 222-228. Vol. Vol. Chang. (2001). pp... Snake venom proteins affecting platelets and their applications to anti-thrombotic research. Biophysical Research Communications. L. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto.29.1. K. S. D.13. S. (1998). & Clemetson. (2003).3. Fujii. M.. Fujita. A. Vol. L. H. Current Pharmaceutical Design. No. M. Liu.3. K. & Nishimuro. The plasminogen (fibrinolytic) system. (2000). Q.. G. Collen. No. No.. & Shin. (2004). X. D. Immunological properties of the fibrinolytic enzyme (fibrolase) from southern copperhead (Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography. X. M. Vol. 680-686. A. Thrombosis and Haemostasis. Biological and Pharmaceutical Bulletin. Guan. pp.129. & Kakkar..1.8.. pp. S. Cheng. Jr. No. S. pp.92.. a fibrin-specific plasminogen activator with therapeutic potential? Blood.41.10. Staphylokinase. Asada. Ito. 545-552.81. No... Sakurai. Fan. The hypocholesterolemic effect of two edible mushrooms: Auricularia auricula (tree-ear) and Tremella fuciformis (white jelly-leaf) in hypercholesterolemic rats. K. K. No. No. J.236. Jae Maeng. No.1526.. Y. S. H.311. pp. & Markland. W. M. Some features of intestinal absorption of intact fibrinolytic enzyme III-1 from Lumbricus rubellus. High Production of beta-Glucosidase in Schizophyllum commune: Isolation of the Enzyme and Effect of the Culture Filtrate on Cellulose Hydrolysis. No. 41-48. Mycologia. Bioscience Biotechnology Biochemistry. Wang.15. M. (1995). Jurasek. Hou. C. & Paice. 961-968. pp. Nomura. Das.. Purification and characterization of cysteine protease from Pleurotus ostreatus. V. M. C.. (1995). Ito. Y. & Zhang. R. No. Vol.. Choi.. Clemetson. 325-331. Lu.. pp. Fujita.

428-435. 186-193. Vol. Production and characterization of exopolysaccharides from an enthomopathogenic fungus Cordyceps militaris NG3. No. D. No. Thrombosis Haemostasis. E. (2003). S. pp. & Kim. & Tsunoo. Y. pp.-H. & Dukhovskis. the fibrinolytic enzymes from eastern cottonmouth moccasin venom (Agkistrodon piscivorus piscivorus). Watanabe. C. S. J. 233-242. Kim..-E. 582-588. 2130-2136. (1989). P. Nguyen. Kim. pp. A. & Kim. S.-S.. Bioscience. Lee.. Rostagno. A. A. Isolation and characterization of a new immunomodulatory protein.. from Ganoderma lucidium.. J..44. 33. A. Nadezhina. A.-S. A. B. V. B.. Shimizu. H. Chabchoub.. 2482-2488.7.. I. H. Villares. Y. Ko. K. S. J. No.34.. O.44.. Kim. P. S.1. and Biochemistry. & Kim. Biotechnology.. & Dellagi. K.3..... A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom. Vol. Toxicon... 990-998. H. Vol. V. Sapkota. S. and Biochemistry. D'arrigo. S. (1995). Y. (1997).com . & Kim. strain CK 11-4 screened from Chungkook-Jang. Chang. A....112. Lee. Part 8. and Biochemistry. (1999). K.5. Xu. K. K. M. Kim.86.. W. No. S. Fukase. Vol. H. M. I. Garcia-Lafuente. S. H.63. W. Vol.19. Karoui. Applied and Environmental Microbiology. Hwang. Yamaoka.. J. Vol.. Vol.-K. Choi. Gentry. Platelet activation and blood coagulation. Purification and characterization of fibrinolytic metalloprotease from Perenniporia fraxinea mycelia. (1996). pp. Vol. 472-478. & Motai. Bioscience. J. Kim. Biotechnology Progress.6. S. 238-251.264.2. pp. J. Kim. Gasmi.. 1722-1725. M. Kim.-S. H... & Kim. A. Vol. Kim. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. Veterinary Journal. Choi.. H. J. Y. J. The Journal of Biological Chemistry. M.5. Choi.88. Y. S.. Tanaka. H. Bevers.. (2001). A.2. (2000). Itoh. W.62. pp.. 622-631. Park. Elayeb... Tricholoma saponaceum. S.. K. Biotechnology. T. W. A. 929-941. W. Vol.. J.. Characterization of a metalloenzyme from a wild mushroom. Y. Lavrova.. T. No.-E. S.. Stimulation of protease production by Aspergillus-Oryzae with oils in continuous culture. Kwon. No. Oh. & Alfredo Martinez. V.. Sapkota. ling zhi-8 (LZ-8).81. Bioscience. Kino. (2010).. Comparative aspects of blood coagulation.65..168. S.2.. N. Edible mushrooms: Role in the prevention of cardiovascular diseases. I. H. M. S. Armillariella mellea. Guillamon. H. 356-362.7. Guermazi.12. K. & Lee. Kim. Vol.intechopen. Purification and characterization of piscivorase I and II.. S. A fibrinolytic enzyme from the medicinal mushroom Cordyceps militaris. Prikladnaia Biokhimiia Mikrobiologiia.. Further characterization and thrombolytic activity in a rat model of a fibrinogenase from Vipera lebetina venom. (2002). P. Applied Microbiology and Biotechnology. Choi. Kim. J. S.. Parshin. H. www. No. Biotechnology.. J. Kim.-J. Kim. Increased secretion of lignolytic enzymes by the Lentinus tigrinus fungus after addition of butanol and toluene in submerged cultivation. & Lindhout. Revin. (2004). A. 715-723. strain DJ-4 screened from Doen-Jang. Park.Fibrinolytic Enzymes from Medicinal Mushrooms 359 Fukushima. M. B. C. & Choi..-S. Y. Y. (1991). Kim. S. (2008). Park. S. Kim.7. H. N. S. No. Mycological Research. No. Atykian. T. M.. E. S. J. W. Kim... K.. Chun. Heemskerk. H. H. (2006). pp.. Choi. S. Vol.. pp. 586-590. Yamashita. Journal of Microbiology. Vol. pp. pp. (2008). pp. Vol. No. Park.3. Thrombosis Research. Y. Choi. Fitoterapia. Kim. Kadimaliev. Hahn. C. M. K. C. Park. pp. E.. No. J.8. No. pp.64. No. pp. A. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. & Yun. No.. Kim. Lozano.

117-122.. Vol. G.-K.. (2003). N. Kim. P. Wang. & Chen. Process Biochemistry. Lumbricus rubellus. S. double-blind. (1991). J. T. N. & Zhong. Z. H. Zheng.-S. Alfimeprase for the treatment of acute peripheral arterial occlusion. pp.-E. G. Baik. C. (2005). J.21. Kim. C.43. W. Vol. 782-784. Zhonghua Xin Xue Guan Bing Za Zhi. pp.. Yoo.89. Magnesium enhanced fibrinolytic activity of protease from Schizophyllum commune... J. C. Comparative Biochemistry and Physiology.18. The American Journal of Chinese Medicine. W.72. The Japanese Journal of Physiology. H. & Maruyama. C. Kwok. & Jiang. S.360 Protein Structure Ko. 423-426. Kim.intechopen. ISBN 0-8493-1043-1.. S. 10-17. Expert Opinion on Biological Therapy. Vol. L. A. Yoneta.-J.. Mushrooms: Cultivation. S. No.. N. Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea. and Environmental Impact. Lu. Nutritional Value. Chun. Sapkota. M..1. (2010a). S. & Gao. H. N. Marsh. Thrombosis Haemostasis. 1667-1672. J. L. L. (1996).. Vol.. pp. C. & Zou. Ikeda.55. (2004).9-10. H. L. Z. H. Lee. & Man. pp. No.. pp. Seiki. Taiwania. No. 264-271. (2005). & Chang.. Bollettino della Societă Italiana Biologia Sperimentale. K.3. No.-C. No. Y. N. R. [Study on early fibrinolytic therapy to avoid acute myocardial infarction].Journal of Food and Drug Analysis.2. Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02. S. A.-B. M. pp. F. Xu.. Chauvatcharin. W. Vol. pp.. Wang.2. Y. Xu. Purification and characterization of a novel fibrinolytic protease from Schizophyllum commune. No. Food Research International. (2010b). Analysis of the component constitution and substrate specificity of a fibrinolytic www. & Kim.. C. Anesthesia and Analgesia. 1234-1241. 683-689. C. (2006). Journal of Microbiology and Biotechnology. N.8. L. J. (1982). Medicinal Effect. Choi. B. Lu. Lu.12.9. Chen. Mao.-S. H.6. G.. 409-419. & Nieuwenhuizen.5. 461-472.. Vol. Ng. Mine. Eksriwong. Optimization of carbon source and carbon/nitrogen ratio for cordycepin production by submerged cultivation of medicinal mushroom Cordyceps militaris. Yuan. table of contents. Miles. Vol. pp. Sumi. D. Liu. E.1.. A novel fibrinolytic enzyme extracted from the earthworm. N. Protein Expression and Purification. M. Fomitella fraxinea..-S. L. 65-74.-S. Fibrinolytic enzymes in Asian traditional fermented foods. Florida: CRC Press. pp. J.. (2005).5. Lee. M. N. Purification and characterization of two novel fibrinolytic proteases from mushroom. Zheng. P. W. T. Denisova. Kuang. S. 243-250.. Yan.. Mizumoto. & Qi. Zhang. No. C Toxicology Pharmacology. P.. & Kashyap. M.41.. Mihara.38. S.. Falina. No. 1223-1231. (2007). (2005).101. Y. & Chen. V. G. pp. No. H. pp. Protease purified from Schizophyllum commune culture broth digests fibrins without activating plasminogen. L. (2005).38.3. S. A. H. J. P. No. pp. A novel extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis: purification and characterization. V.. pp. J. (2004).2. Y. S. 263-278. J. & Park. Vol. S.2. L. Kim. Medved. R. (2008). S. L.. Morozova. T.. Barkova.137.. No. 69-76. (2010). Lam. Hu. Vol. Vol. Kwan Wong. Vol.40. R.-S. Y. & Psurtseva. W. Phytotherapy Research. Vol. placebo-controlled study of the platelet and global hemostatic effects of Ganoderma lucidum (Ling-Zhi) in healthy volunteers.33. P. Shen.-Y.. P..-J. S. Zhu. Moise. V.-H... J. No. No. Vol. No. Li.-T. R.com . Vol.16. randomized. Yao. & Chen. C..3. M. & Fyffe. S. Boca Raton... H.. No. Practical applications of snake venom toxins in haemostasis.. Molecular mechanisms of initiation of fibrinolysis by fibrin. Fan. H. Vol. Li. L. N. pp. K. A prospective. Chen.. J. pp. X.

pp. H. Biotechnology Advances. J.22. Li. S. K.. H. M.. Sugimoto. Park. M. Merz. A. (1990). Vol.3. and Biochemistry.. (2004). (2004). J.. Vol. pp. Vol. Characteristics of a cheese-like food produced by fermentation of the mushroom Schizophyllum commune. Biotechnology. Vol. Shechter... T. Vol. C. Yoon. Fungal morphology and metabolite production in submerged mycelial processes.. Lee.Fibrinolytic Enzymes from Medicinal Mushrooms 361 preparation from the fungus Flammulina velutipes. Bioscience. X.. N. Fusarium sp BLB. (2003). J.155. No.. Vol. www. H. Dwyer. No. Vol. Bioscience. T. pp. pp. Morimiya. Hitosugi. Yamagata. H. T. Peng. Clinical Obstetrics & Gynaecology. J. (2001). 167-173.. & Kim.99. Microbial fibrinolytic enzymes: an overview of source.... E. Meisel.intechopen. H. Y. & Yun. Park. Molloy.10. Kim. Oral magnesium supplementation inhibits platelet-dependent thrombosis in patients with coronary artery disease. Fukuda. W. Peng. 11811185. No..1. Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis. H. Takeno.3.9. Papagianni. E. T. M. (2005). Y. and thrombolytic activity in vivo. Lumbricus rubellus. and Biochemistry.17. Vol. Biokhimiia. Xiao..84. Sapkota.47. M. R. Okamura-Matsui. pp. production. Nucleotide sequence of the subtilisin NAT gene.. Sera. T. Vol. H. 189-259.. Vol. Y. No.33. M. Vol.71.. J. Sumi. Fujii. Research in Microbiology. & Sumi. Nakajima. M. Journal of Pharmacological Sciences.2. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi. X. X. Hamada. 1869-1871. Optimization of submerged culture conditions for the mycelial growth and exo-biopolymer production by Cordyceps militaris. B. J. Takemura. 247-251. Y. pp. Yang. Nakamura. Kim. Mihara. Lee.. pp.. S. No. Vol.. The American Journal of Cardiology.. (1993). Kim. S.57. The fibrinolytic activity of a novel protease derived from a tempeh producing fungus.. & Zhang. R. aprN. & Zhang.. (1999). Y. (2005). L. R. & Hiratani.. Hwang. Norris. pp. Shah.. Best Practice & Research. (2001). S. Blood coagulation.11. 1726-1730. 152-156. W. (2007). Vol. H.69. of Bacillus subtilis (natto). 2184-2189. K. Z.. Characterization of potent fibrinolytic enzymes in earthworm. Johdo. Rude. 369-383. Paul-Labrador. Q. pp. No. Applied Microbiology & Biotechnolgy.1. Y. M. Bioscience Biotechnology and Biochemistry. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. H. S. S. K.84. pp.3..71. Huang. & Ohsugi. H. K.. Y. M. Part B. pp..3.7. and Biochemistry.56. S. J. Y.9. Choi. Zhang.1.. No.. Biochemistry & Molecular Biology. O. & Zhang. Zhu. Noda. Comparative Biochemistry and Physiology. L. No. (2007). Maeda. pp. H.. 126-132. N. S. P. K. Vol. Yang. No. Kim. H. Nakanishi.134. 45-52. H. properties. pp.. S. No.. M. Omura. & Kaul. Biotechnology. No. T. & Ichishima. a traditional Chinese soybean food. (1992). Ikeda.. Z. Acta Haematologica. 139-143. No..2.. pp. 2214-2222. Journal of Bioscience and Bioengineering. Bioscience. 30-32. 76-81. K. P. D. J. A newly derived protein from Bacillus subtilis natto with both antithrombotic and fibrinolytic effects. No.3. Purification and characterization of a fibrinolytic protease from a culture supernatant of Flammulina velutipes mycelia. C. & Nakamura. S. E. Biotechnology. No. Peng..com . pp. S. Vol. No. Letters in Applied Microbiology. C. H. & Tokudome. J.92.. (2003).

Part B. Vol. A unique strong fibrinolytic enzyme (katsuwokinase) in skipjack "Shiokara.4. S... S. Ng.. K. C. pp. Vol.intechopen.3. Takada.46.3. C. (2010). & Mine. V. & Yatagai.. M. L. 269-281. Yamada. W. a local edible mushroom. B.. pp.. 164-168. K. No. Sun. Journal of Agricultural and Food Chemistry. Wong. New directions in thrombolytic therapy. pp. S. Mihara. & Andersson. pp. Vol. H.-H. pp.1. Y. www. Vol. W. BLB. D. K. Wong. (2002). No.. Goldfarb. No.. pp.25. & Cheung. H. Blood Reviews. F. K." a Japanese traditional fermented food. (2003). Anti-inflammatory effect of the polysaccharides of Golden needle mushroom in burned rats. Wu. D.24. Lai. Whiss. 1-31.-W.. No. Vol. Novel fibrinolytic enzyme in fermented shrimp paste.-M.57. Naemura. D. A. pp. 1110-1111. K. 543-547. Vol. Toombs... X. Y.3.-C. A. P. & Doi. Y. T. R. pp. Proceedings of the National Academy of Sciences of the United States of America.2.. M. Kubo. Thrombin generation and fibrin clot structure.1. R.. Chu. 407-416.-X.-Q. No. Immunomodulatory activities of mushroom sclerotial polysaccharides. S. No. & Arai. (2011). No. & Chang.. The physiological aspects of fibrinolysis. W. T. Takada. & Chang. J. (2007). Crystal structure of earthworm fibrinolytic enzyme component a: revealing the structural determinants of its dual fibrinolytic activity.13. Applied Microbiology & Biotechnology. Thrombosis Research. No. T. Purification and characterization of fibrinolytic alkaline protease from Fusarium sp.com . H. No.. H.81.2. International Journal of Biological Macromolecules. No. Duan. Yamamoto. K.. A.10.4. & Muraki.. X. Zhang. Vol. (1995).1.. No. Testing various herbs for antithrombotic effect.321. pp. Nakajima.5. 980-986. Gui.-N. a traditional Asian fermented seasoning. Vol. (1984). Hwang. & Mourao..-K.. No. H. E. Y. 331-338. & Cen. Vol. 151-158. C.. Vol. X. L...74. pp.52. Liang.10. Tang.. A. (2005). W. T. & Nakamura. Y. C. No. D. & Tang.21. L. H. Vol. 647-650. Recombinant streptokinase production by fed-batch cultivation of Escherichia coli. H. Ueda.3. The nontoxic mushroom Auricularia auricula contains a polysaccharide with anticoagulant activity mediated by antithrombin.. a typical and popular soybean food in the Japanese diet.. The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo.-J. C. pp. (2004).-A. pp.. (1994). Current Opinion of Pharmacology.. Vol. N.21. Vol. No.1. (1999). Experientia. Immunomodulatory and antitumor activities of a polysaccharide-peptide complex from a mycelial culture of Tricholoma sp. 131-142. M. Vol. Blood Coagulation Fibrinolysis.112. S. T. Gu.2. Vol. Y. Food Hydrocolloids. R. 57-68.. H. Liu. 150-158. G. Huang. X. Vol.. Biochemistry & Molecular Biology.5. pp. No. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto. T. Liu. Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression. 1184-1188. Hamada. T. A. (2001).-Q. D. Wong. pp. Sumi. Tsushima. T. (2007). Ooi. S. Journal of Molecular Biology. (1995). Yoon. 100-103.112. A. Juneja. Wang. No. P.43. Thrombosis Research. (1987). P.. R. Yamashita. Life Science. Zhang.362 Protein Structure Sumi. Miyatake. 580-587. Price. Enzyme and Microbial Technology. H. & Urano. Z. Pyun. Wolberg. Nutrition..76. pp. (2002). pp. Comparative Biochemistry and Physiology.-R. J. Liu. Jiang. Yu. J.

In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. Shanghai.). 2012 Published in print edition April. Eshel Faraggi (Ed. feel free to copy and paste the following: Chung-Lun Lu and Shiu-Nan Chen (2012).65. and in the current state of affairs.com/books/protein-structure/fibrinolytic-enzymes-from-medicinal-mushrooms InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka. April. Fibrinolytic Enzymes from Medicinal Mushrooms. Available from: http://www. and only within the last several decades did the proteins play a pivotal role in this existence. and probably even before. Only relatively recently. InTech. we hope. Office Block. Protein Structure. Dr. 2012 Since the dawn of recorded history.Protein Structure Edited by Dr. Eshel Faraggi ISBN 978-953-51-0555-8 Hard cover. did this grasp yield anything of substance. The expert meanwhile.com InTech China Unit 405. Yan An Road (West). China Phone: +86-21-62489820 Fax: +86-21-62489821 . How to reference In order to correctly reference this scholarly work. The aim is that a non-expert can gain some appreciation for the intricacies involved. 200040.intechopen. 396 pages Publisher InTech Published online 20. Hotel Equatorial Shanghai No. ISBN: 978-953-51-0555-8. can gain a deeper understanding of the topic. Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen. men and women have been grasping at the mechanisms by which they themselves exist.