Purification and characterization of fibrinolytic protease from Bacillus amyloliquefaciens MCC2606 and analysis of fibrin degradation product by MS/MS

A serine protease with preference for fibrin protein was purified and characterized from Bacillus amyloliquefaciens MCC2606, isolated from dosa batter. The protease was purified using ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The degradation activity of the protease toward the fibrin was significantly higher compared with other protein substrates in the study. The molecular weight of the CFR15-protease was estimated to be 32?kDa based on SDS-PAGE. The purified enzyme exhibited both fibrinolytic and fibrinogenolytic activity. The optimum pH and temperature for the activity of the enzyme was found to be 10.5 and 45°C. A significant inhibition was seen with the protease inhibitors phenyl methyl sulphonyl fluoride and ethylene diamine tetra acetic acid and the activity of the enzyme was enhanced in presence of Mn²⁺. There was an observed increase in vitro activated partial thromboplastin time and prothrombin time of both time and dose dependent study. Among the four chains of fibrin, the β-chain of fibrin appears to be the primary component and site susceptible for CFR15-protease in early action as indicated by MS/MS analysis of initial degradation products. These results indicated that the CFR15-protease have the potential to be an effective fibrinolytic agent.     

Thrombolytic,anticoagulant and antiplatelet activities of codiase,a bi-functional fibrinolytic enzyme from Codium fragile

Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. In this study, codiase, a new bi-functional fibrinolytic serine protease having thrombolytic, anticoagulant, and antiplatelet activities was purified from marine green alga, Codium fragile. The molecular weight of the enzyme was estimated to be 48.9 kDa by SDS-PAGE, and mass spectrometry. Fibrin zymography analysis showed an active band with similar molecular weight. The N-terminal sequence was found to be APKASTDQTLPL, which is different from that of other known fibrinolytic enzymes. Codiase displayed maximum activity at 30 °C and pH 6.0, and the activity was inhibited by Zn²⁺ and Fe²⁺. Moreover, the enzyme activity was strongly inhibited by serine protease inhibitor such as PMSF. Codiase exhibited high specificity for the substrate S-2288, and the K_m and V_max values for this substrate were found to be 0.24 mM and 79 U/ml respectively. Fibrin plate assays revealed that it was able to hydrolyze fibrin clot either directly or by activation of plasminogen. Codiase effectively hydrolyzed fibrin and fibrinogen, preferentially degrading α- and Aα chains, followed by γ–γ, and γ-chains. However, it provoked slower degradation of Bβ and β-chains. The structural change of fibrin clot and fibrinogen by codiase was also detected by FTIR-ATR spectroscopy analysis. In vitro and in vivo studies revealed that codiase reduces thrombosis in concentration-dependent manner. Codiase was found to prolong activated partial thromboplastin time (APTT), and prothrombin time (PT). PFA-100 studies showed that codiase prolonged the closure time (CT) of citrated whole human blood. These favorable antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to mice and NIH-3T3 cells, make it a potential agent for thrombolytic therapy.     

Enzymatic properties and identification of a fibrinolytic serine protease purified from Bacillus subtilis DC33

A novel fibrinolytic enzyme subtilisin FS33 was purified from Bacillus subtilis DC33, isolated from a traditional flavour-rich food in China. The purified subtilisin FS33 was a single chain protein with a molecular mass of 30 kDa measured by SDS-PAGE. After activated SDS-PAGE, the enzyme band exhibited strong fibrinolytic activity on the fibrin plate. Subtilisin FS33 was temperature-stable below 60°C over the pH range 5–12, with a maximum activity at pH 8.0, but the activity completely disappeared after 10 min above 65°C. The NH₂-terminal amino acid sequence of the enzyme was different from that of other known fibrinolytic enzymes, such as NK, CK, SMCE, KA38, subtilisin E, subtilisin DFE and Katsuwokinase. The amidolytic activities of subtilisin FS33 were inhibited completely by phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI). EDTA did not affect the enzyme activity, and none of the ions tested activated the activity. Therefore, the enzyme was thought to be a subtilisin-like serine protease. The enzyme degraded the Bβ-chains of fibrin(ogen) very rapidly and then degraded the Aα-chain and at least five fragments from fibrin(ogen) were obtained after hydrolysis. Subtilisin FS33 was also able to cleave blood clots in the absence of endogenous fibrinolytic factors.     

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS–PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), β-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bβ-chains and Aα-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.     

Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12

A novel fibrinolytic enzyme from Rhizopus chinensis 12 was purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange, and gel filtration chromatography. The purification protocol resulted in a 893-fold purification of the enzyme, with a final yield of 42.6%. The apparent molecular weight of the enzyme was 18.0 kDa, determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 16.6 kDa by gel filtration chromatography, which revealed a monomeric form of the enzyme. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5±0.1. The enzyme hydrolyzed fibrin. It cleaved the , , and chains of fibrinogen simultaneously, and it also hydrolyzed casein and N-succinyl-Ala-Ala-Pro-Phe-pNA. The enzyme had an optimal temperature of 45°C, and an optimal pH of 10.5. EDTA, PCMB, and PMSF inhibited the activity of the enzyme, and SBTI, Lys, TPCK, and Aprotinine had no obvious inhibition, which suggested that the activity center of the enzyme had hydrosulfuryl and metal. The first 12 amino acids of the N-terminal sequence of the enzyme were S-V-S-E-I-Q-L-M-H-N-L-G and had no homology with that of other fibrinolytic enzyme from other microbes.     

Chemical coupling of a monoclonal antisurfactant protein-B antibody to human urokinase for targeting surfactant-incorporating alveolar fibrin

Intraalveolar fibrin formation is a common histopathological finding in acute inflammatory and chronic interstitial lung diseases. Incorporation of hydrophobic surfactant components into polymerizing fibrin results in a severe loss of surface activity, altered mechanical and structural clot properties, and a reduced susceptibility toward fibrinolytic degradation. Such events have been implicated in atelectasis formation, impairment of gas exchange, and provocation of fibroproliferative changes. In an effort to address the unique features of alveolar fibrin, we designed a hybrid molecule consisting of a monoclonal antibody against surfactant protein SP-B (8B5E) and the catalytic domain of urokinase (B-chain), which was termed MABUC. The urokinase B-chain was prepared by limited reduction of human two-chain-urokinase and subsequent affinity purification and coupled to the antibody using a heterobifunctional cross-linker. Purification of the chimeric protein included gel filtration chromatography and affinity chromatography. An ELISA-like microtiter plate assay, based on the immunological detection of the SP-B moiety and the fibrinolytic activity of the u-PA domain, was developed for the detection of the hybrid molecule. Chromogenic substrate assays, (125)I-based fibrin plate assays, and active site titration were performed to analyze the specific fibrinolytic activity of the conjugate. MABUC was found to fully retain the ability of SP-B binding and the fibrinolytic activity of u-PA. In addition, MABUC was noted to be 1.5-2-fold more effective in the dissolution of surfactant embedding clots and to be approximately 3-fold more resistant against PAI-1, the predominant fibrinolysis inhibitor in the alveolar compartment, as compared to the native u-PA. The superiority of MABUC was particularly prominent (>5-fold efficacy) when investigating clot material incorporating both PAI-1 and surfactant, as a mimicry of alveolar fibrin. We conclude that urokinase and 8B5E can be cross-linked chemically, thus yielding a fibrinolytic enzyme with enhanced substrate specifity for surfactant-containing clots and higher PAI-1 resistance as compared to native u-PA.     

Identification of a fibrinolytic enzyme by <Emphasis Type="Italic">Bacillus vallismortis</Emphasis> and its potential as a bacteriolytic enzyme against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

A potent fibrinolytic enzyme-producing bacterium was isolated from the traditional Korean condiment Chungkook-jang and identified as Bacillus vallismortis Ace02. The extracellular fibrinolytic enzyme was purified with a 18% recovery of activity from supernatant cultures using CM-Sepharose column chromatography and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme was 757 kFU mg⁻¹. Its molecular mass was about 28 kDa and the initial amino acids of the N-terminal sequence were AQSVPYGVSQ. The full amino acid sequence of fibrinolytic enzyme Ace02 corresponded with bacteriolytic enzyme, L27, from Bacillus licheniformis, which has strong lytic activity against Streptococcus mutans, a major causative strain of dental caries. This suggests that the purified enzyme should be used for prevention of dental caries as well as being an effective thrombolytic agent.     

可口革囊星虫体内纤溶酶的初步研究

目的从可口革囊星虫中寻找到纤溶酶,并对其进行初步研究。方法采用匀浆、抽提离心、Sephacryl S-300凝胶过滤等方法对可口革囊星虫纤溶酶初步分离,用纤维蛋白平板法和合成发色底物法检测其纤溶活性,并用该酶进行体外溶血凝块实验。结果可口革囊星虫内脏中存在纤溶酶。此酶既直接降解纤维蛋白又间接激活纤溶酶原,它对体外血凝块有明显溶解作用。结论可口革囊星虫内脏中存在着一种既具直接降解纤维蛋白作用又具激活纤溶酶原作用的纤溶酶。     

Screening of a high fibrinolytic enzyme producing strain and characterization of the fibrinolytic enzyme produced from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> LD-8547

A fibrinolytic enzyme producing strain Bacillus subtilis LD-8 was isolated from douchi, a traditional Chinese soybean-fermented food. After mutagenesis treatments by UV, NTG (N-methyl-N′-nitroso-N-nitroso-guanidine) and γ-radiation, a high fibrinolytic enzyme producing strain B. subtilis LD-8547 was obtained. Under optimum condition, LD-8547 was able to yield the average fibrinolytic activity of 4220 U/mL in 15 L fermenter. The strong fibrin-specific enzyme was purified from supernatant of B. subtilis LD-8547 culture broth using the combination of various steps. The optimal temperature and pH value of this fibrinolytic enzyme were 50 °C and 8.0, respectively. The molecular weight was about 30 kDa measured by SDS-PAGE. The amidolytic activity of this fibrinolytic enzyme was inhibited completely by 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), but EDTA and EGTA did not affect the enzyme activity. The apparent K _m and V _max values were 0.521 mmol/L and 0.049 mmol/min, respectively. In vitro assays revealed that the enzyme could catalyze blood clot lysis effectively, indicating that this enzyme could be a useful thrombolytic agent.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Effects of Some Antibiotics on Human Erythrocyte 6-Phosphogluconate Dehydrogenase: An in vitro and in vivo Study

The in vitro and in vivo effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase were investigated. Human erythrocyte 6-phosphogluconate dehydrogenase was purified with ammonium sulphate precipitation, 2′,5′ ADP-Sepharose 4B affinity and gel filtration chromatography. Some antibiotics (netilmicin sulphate, cefepime, amikacin, isepamycin, chloramphenicol, ceftazidim, teicoplanin, ampicillin, ofloxacin, levofloxacin, cefotaxime, penicillin G, gentamicin sulphate, ciprofloxacin) inhibited enzyme activity in vitro but others (cefozin, decefin, streptomycin, combisid, and meronem) were devoid of inhibitory effects. For the drugs having low IC50 values (netilmicin sulphate and cefepime), in vivo studies were performed in rats. Netilmicin sulphate at 15-mg/kg inhibited enzyme activity significantly (p < 0.001) 1h, 2h, and 3h after dosing and cefepime at 200-mg/kg very significantly (p < 0.001) inhibited the enzyme 1 h and 2 h after dosing. Netilmicin sulphate and cefepime inhibited rat erythrocyte 6-phosphogluconate dehydrogenase both in vivo and in-vitro.     

Effect of Pepsin Inhibitor (S-PI) on the Growth of Rhodotorula glutinis No. K-24

A tissue-type transglutaminase (TGase) was purified from liver tissue of the red sea bream, Pagrus major, by ion-exchange chromatography and heparin-Sepharose affinity chromatography. Its activity was assessed using a fluorometric assay to measure the incorporation of monodansylcadaverine into N,N′-dimethyl casein. The molecular mass of purified TGase was estimated to be 78kDa by SDS–polyacrylamide gel electrophoresis. The enzyme required Ca²⁺ to express its activity, although 10 mM Sr²⁺ also activated the enzyme fully. TGase activity was maximal at pH 9.0–9.5, and the enzyme was strongly inhibited by sulfhydryl reagents. The purified enzyme catalyzed the cross-linking of myosin heavy chain obtained from Alaska pollack, resulting in gelation of an actomyosin solution. The partial amino acid sequence of this fish TGase showed divisionally significant similarity to TGase from guinea pig liver.     

Biochemical analysis of a fibrinolytic enzyme purified from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain A1

A fibrinolytic enzyme from Bacillus subtilis strain Al was purified by chromatographic methods, including DEAE Sephadex A-50 column chromatography and Sephadex G-50 column gel filtration. The purified enzyme consisted of a monomeric subunit and was estimated to be approximately 28 kDa in size by SDS-PAGE. The specific activity of the fibrinolytic enzyme was 1632-fold higher than that of the crude enzyme extract. The fibrinolytic activity of the purified enzyme was approximately 0.62 and 1.33 U/ml in plasminogen-free and plasminogen-rich fibrin plates, respectively. Protease inhibitors PMSF, DIFP, chymostatin, and TPCK reduced the fibrinolytic activity of the enzyme to 13.7, 35.7, 15.7, and 23.3%, respectively. This result suggests that the enzyme purified from B. subtilis strain Al was a chymotrypsin-like serine protease. In addition, the optimum temperature and pH range of the fibrinolytic enzyme were 50°C and 6.0–10.0, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as Q-T-G-G-S-I-I-D-P-I-N-G-Y-N, which was highly distinguished from other known fibrinolytic enzymes. Thus, these results suggest a fibrinolytic enzyme as a novel thrombolytic agent from B. subtilis strain Al.     

Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004

A fibrinolytic enzyme (SFE1) from Streptomyces sp. XZNUM 00004 was purified to electrophoretic homogeneity with the methods including ammonium sulfate precipitation, polyacrylamide gel, DEAE-Sepharose Fast Flow anion exchange and gel-filtration chromatography. The molecular weight of SFE1 was estimated to be 20 kDa by SDS-PAGE, fibrin zymography, and gel filtration chromatography. The isoelectric point was 4.9. K (m) and V (max) values were 0.96 mg/ml and 181.8 unit/ml, respectively. It was very stable at pH 5.0-8.0 and below 65 °C. The optimum pH for enzyme activity was 7.8. The optimum temperature was 35 °C. The fibrinolytic activity of SFE1 was enhanced by Na(+), K(+), Mn(2+), Mg(2+), Zn(2+) and Co(2+). Conversely, Cu(2+) showed strong inhibition. Furthermore, the fibrinolytic activity was strongly inhibited by PMSF, and partly inhibited by EDTA and EGTA. SFE1 rapidly hydrolyzed the Aα-chain of fibrinogen, followed by the Bβ-chain and finally the γ-chain. The first 15 amino acids of the N-terminal sequence were APITLSQGHVDVVDI. Additionally, SFE1 directly digested fibrin and not by plasminogen activators in vitro. SFE1 can be further developed as a potential candidate for thrombolytic therapy.     

Verticase： a Fibrinolytic Enzyme Produced by Verticillium sp, Tj33, an Endophyte of Trachelospermum jasminoides

Plant endophytes are among the most important resources of biologically active metabolites. Twenty-three endophyte strains residing in Trachelospermum jasminoides were cultivated in vitro with the cultures assayed for the fibrinolytic substance production. As a result, the culture of VerticUlium sp. Tj33 was shown to be the most active. A fibrinolytic enzyme designated as verticase was subsequently purified from the supernatant of Verticillium sp. culture broth by a combination of DEAE-52, Sephadex G-75 and hydrophobic column chromatographies. Verticase, with its molecular mass of 31 kDa and pl of 8.5, was demonstrated to be homogeneous by sodium dodecyl suIfate-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Verticase is an enzyme that hydrolyzes fibrin directly without activation of plaminogen. It was stable in a broad pH range from 4 through to 11 with the optimal reaction pH value and temperature shown to be around 9-10 and 50-60℃, respectively. The fibrinolytic activity of verticase was severely inhibited by phenylmethylsulfony fluoride, indicating that verticase was a serine protease.     

Purification and characterization of a novel fibrinolytic protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. CPCC 480097

A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF) was 8.1, and the optimum temperature and pH value were 45°C and 8.5, respectively. Fu-P cleaved the α-chain of fibrin (ogen) with high efficiency, and the β-chain and γ-γ (γ-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF, and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S–S-G-T-P-A-T-I-R-V-L-V–V and showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic therapy and in thrombosis prevention.     

A fibrinolytic enzyme from the medicinal mushroom Cordyceps militaris

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and 37 degrees , respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin alpha-chain followed by the gamma-gamma chains. It also hydrolyzed the beta-chain, but more slowly. The Aalpha, Bbeta, and gamma chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by Cu2+ and Co2+, but enhanced by the additions of Ca2+ and Mg2+ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it 's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.     

Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo

The effects of streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, and cefodizime on the enzyme activity of glutathione reductase (GR) were studied using human and rat erythrocyte GR enzymes in in vitro and in vivo studies, respectively. The enzyme was purified 5,342-fold from human erythrocytes in a yield of 29% with 50.75?U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies, and rat erythrocyte hemolysate was used in the in vivo studies. In the in vitro studies, I₅₀ and K_i values were 12.179?mM and 6.5123±4.1139?mM for cefotaxime, and 1.682?mM and 0.7446±0.2216?mM for cefodizime, respectively, showing the inhibition effects on the purified enzyme. Inhibition types were noncompetitive for cefotaxime and competitive for cefodizime. In the in vivo studies, 300?mg/kg cefotaxime and 1000?mg/kg cefodizime when administered to rats inhibited enzyme activity during the first 2?h (p<0.01). Cefotaxime led to increased enzyme activity at 4?h (p<0.05), but neither cefotaxime nor cefodizime had any significant inhibition or activation effects over 6?h (p>0.05).     

Production and properties of fibrinolytic enzyme in solid state cultures of Fusarium pallidoroseum

Summary Ten local fungal isolates were screened for their ability to produce extracellular fibrinolytic enzyme activity in solid state and skaken cultures. Fusarium pallidoroseum was the most active in wheat bran solid cultures. Maximum activity was obtained at 25 °C and 50% moisture content. Addition of 2% casein to the culture increased the activity by 1.7-folds. The 65% ammonium sulphate fraction showed the highest fibrinolytic activity it was further purified by gel filtration on Sephadex G-100 followed by rechromatography of the most active peak on DEAE-cellulose. The pure enzyme was highly active on human fibrin and showed an optimum reaction temperature of 40 °C and pH 7. The enzyme was relatively sensitive to heat treatment at 55 °C and strongly inhibited by EDTA, it restored its activity by adding cobalt ions to the reaction.     

Direct acting anti-thrombotic serine protease from brown seaweed Costaria costata

A direct acting antithrombotic serine protease (CCP) was purified from brown seaweed Costaria costata. CCP was a monomeric protease with molecular mass of 60,547.598 daltons as determined by mass spectrometry. The N-terminal sequence of CCP was SCNSCLDKVDADGLN. Proteolytic activity was inhibited by PMSF and APMSF. CCP exhibited high amidolytic activity toward substrate S-2251 with apparent K_m and V_max values were 14.5 μM and 183.5 U/ml respectively. Fibrin plate and fibrin zymography results revealed that CCP was able to degrade fibrin clots directly. It specifically hydrolyzed Aα and α and Bβ and β chains followed by γ and γ–γ chains of human fibrinogen and fibrin respectively. Cleavage of fibrin clot and fibrinogen was emphasized by observing the alteration of secondary structure using FTIR spectroscopy. Morphological alteration of fibrin clot was also evidenced by fluorescent microscopic observation. CCP reduced thrombus effectively in vitro. In vivo observation showed that it prevented/decreased thrombus formation in carrageenan-induced mice tail model. CCP prolonged activated partial thromboplastin time (APTT) and had little effect on prothrombin time (PT). Platelet function analyzer (PFA-100) tests showed that CCP prolonged closure time (CT). These data suggest that CCP could have therapeutic potential for the treatment of thrombosis.