Activity assessment of microbial fibrinolytic enzymes

Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.     

食品纤溶酶研究概况

溶栓疗法是血栓性疾病安全有效的治疗手段,开发安全高效、廉价的新型纤溶酶对于预防与治疗血栓性疾病具有重要意义。近年来,在许多亚洲传统发酵食品中如日本纳豆、韩国大豆酱、中国豆豉、发酵虾酱等中均发现有丰富的纤溶酶资源。本文重点介绍传统发酵食品中纤溶酶的研究概况及其开发前景。     

Process optimization for production and purification of novel fibrinolytic enzyme from Stenotrophomonas sp. KG-16-3

Intravascular thrombosis is a major cardiovascular complication responsible for high mortality worldwide. Existing thrombolytic agents are expensive and have various side effects. As a consequence, researchers continue to search for better thrombolytic agents. Fibrinolytic proteases especially those of microbial origin are considered as potential therapeutic candidates for thrombosis. The current study reports fibrinolytic protease from a bacterial isolate Stenotrophomonas sp. KG-16-3, as it exhibits high fibrinolytic activity on fibrin agarose plate. Studies on fibrinolytic protease from Stenotrophomonas sp. are lacking. So, a detailed study was conducted for the production and purification of fibrinolytic protease. Optimizing process parameters using the Design of Experiments method enhanced the yield by 1.5-fold. The fibrinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography resulting in 7.1-fold purification and 16.7% yield with specific activity of 383.8?U/mg. The purified enzyme exhibited higher fibrinolytic activity than plasmin and had a molecular weight of 39?kDa. Optimal activity of the enzyme was observed at 50?°C and pH 10. The enzyme exhibited stability up to 60?°C, over pH 7–10 and in the presence of different metal ions and solvents. The activity of the enzyme was significantly reduced in the presence of phenylmethyl sulfonyl fluoride, iodoacetic acid and 1,10-phenanthroline, suggesting that the enzyme belonged to the serine–cysteine metalloprotease category. The present study is the first ever report on the Design of Experiments based optimization of fermentation conditions for the production of fibrinolytic protease from Stenotrophomonas sp.     

Purification,biochemical, and thermal properties of fibrinolytic enzyme secreted by Bacillus cereus SRM-001

The discovery of microbial fibrinolytic enzymes is essential to treat cardiovascular diseases. This study reports the discovery of a fibrinolytic enzyme secreted by Bacillus cereus SRM-001, a microorganism isolated from the soil of a chicken waste-dump yard. The B. cereus SRM-001 was cultured and the secreted fibrinolytic enzyme purified to show that it is a ～28 kDa protein. The purified enzyme was characterized for its kinetics, biochemical and thermal properties to show that it possesses properties similar to plasmin. A HPLC-MS/MS analysis of trypsin digested protein indicated that the fibrinolytic enzyme shared close sequence homology with serine proteases reported for other Bacillus sp. The results show that the B. cereus SRM-001 secreted enzyme is a ～28 kDa serine protease that possesses fibrinolytic potential.     

The biotechnological potential of fibrinolytic enzymes in the dissolution of endogenous blood thrombi

Formation of endogenous thrombi in blood vessels is one of the leading causes of death in our modern life. According to data provided by the World Health Organization (WHO) in 2000, heart diseases are responsible for 29% of the total mortality rate in the world. For this, a tremendous amount of research has been done in the area of prevention and treatment of these diseases. The classical therapy of these thrombi relies upon the use of antiplatelets, anticoagulants, or even surgeries. Relatively recently, the fibrinolytic enzymes produced by microorganisms, snakes, earthworms, insects, plants, and other organisms are being successfully used in the treatment of blood clots, especially with regard to the direct dissolving action on fibrin in tandem with less cost and side effects in comparison with the first‐generation thrombolytic agents, streptokinase and urokinase. Furthermore, recombinant DNA technology has succeeded in improving and decreasing the undesirable effects of the first generation of enzymes. Recombinant PAs or rt‐PAs like alteplase, retelase, saruplase, tenecteplase, lanoteplase, and desmoteplase became available in the drug markets with advantages of less binding loci with PAI‐1 to avoid degradation while providing faster and more complete reperfusion in a greater number of patients with less risk of bleeding and intracranial hemorrhage. This review is the first to cover all the natural and recombinant thrombolytic agents used in enzyme therapy. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:656–672, 2014     

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.     

Purification of serine protease from polychaeta, <Emphasis Type="Italic">Lumbrineris nipponica</Emphasis>, and assessment of its fibrinolytic activity

Ischemic stroke and cardiovascular disease can occur from blockage of blood vessels by fibrin clots formed naturally in the body. Therapeutic drugs of anticoagulant or thrombolytic agents have been studied; however, various problems have been reported such as side effects and low efficacy. Thus, development of new candidates that are more effective and safe is necessary. The objective of this study is to evaluate fibrinolytic activity, anti-coagulation, and characterization of serine protease purified from Lumbrineris nipponica, polychaeta, for new thrombolytic agents. In the present study, we isolated and identified a new fibrinolytic serine protease from L. nipponica. The N-terminal sequence of the identified serine protease was EAMMDLADQLEQSLN, which is not homologous with any known serine protease. The size of the purified serine protease was 28 kDa, and the protein purification yield was 12.7%. The optimal enzyme activity was observed at 50°C and pH 2.0. A fibrin plate assay confirmed that indirect fibrinolytic activity of the purified serine protease was higher than that of urokinase-PA, whereas direct fibrinolytic activity, which causes bleeding side effects, was relatively low. The serine protease did not induce any cytotoxicity toward the endothelial cell line. In addition, anticoagulant activity was verified by an in vivo DVT animal model system. These results suggest that serine protease purified from L. nipponica has the potential to be an alternative fibrinolytic agent for the treatment of thrombosis and use in various biomedical applications.     

嵌合体纤溶酶的研究进展

嵌合体纤溶酶是采用基因重组技术或化学偶联的方法将纤溶酶与其它的功能多肽(舍有酶原结构域,具有抗凝血酶、抗血小板聚集活性,能特异识别纤维蛋白的多肽)结合起来所形成的嵌合体蛋白质．它尽可能地保留了分子中每一组分的生物活性,增强了纤溶酶的溶栓功效,较大程度地克服了临床溶栓药物在某些方面的不足,是目前溶栓药物研究领域中的热点。     

Detection of a unique fibrinolytic enzyme in <Emphasis Type="Italic">Aeromonas</Emphasis> sp. JH1

A fibrinolytic enzyme was found in a Gram-negative bacterium, Aeromonas sp. JH1. SDS-PAGE and fibrinzymography showed that it was a 36 kDa, monomeric protein. Of note, the enzyme was highly specific for fibrinogen molecules and the hydrolysis rate of fibrinogen subunits was highest for α, β, and γ chains in that order. The first 15 amino acids of N-terminal sequence were X-D-A-T-G-P-G-G-N-V-X-T-G-K-Y, which was distinguishable from other fibrinolytic enzymes. The optimum pH and temperature of the enzyme were approximately 8.0 and 40°C, respectively. Therefore, these results provide a fibrinolytic enzyme with potent thrombolytic activity from the Aeromonas genus.     

Purification,biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala–Ala–Pro–Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu²⁺, Mg²⁺, and Fe²⁺. The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

     

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.     

钜齿远蚓(Amynthas dancataa)纤溶酶的分离纯化及其若干性质

 以野生型钷齿远蚓为材料,组织匀浆后,经生理盐水抽提,硫酸铵分级沉淀,葡聚糖凝胶过滤和DEAE离子交换层析,得到两种纯的蚯蚓溶酶,具有强烈的溶解纤维蛋白的作用。它们都是糖蛋白,非寡聚酶,分子量分别为23,000、40,000。测定了一个酶的氨基酸组成,它对某些底物的作用,被一些抑制剂抑制的程度,说明它是练氨酸蛋白酶类、胰蛋白酶类酶     

A novel source of fibrinolytic activity: <Emphasis Type="Italic">Bionectria</Emphasis> sp., an unconventional enzyme-producing fungus isolated from Las Yungas rainforest (Tucumán,Argentina)

Fibrinolytic enzyme production was evaluated in fungal specimens isolated from the sub-tropical Las Yungas Pedemontana forest (Tucumán, Argentina). Proteolytic and fibrinolytic activities were evaluated in freeze-thaw crude extracts from 230 fungal isolates on 1% w/v skimmed-milk or 0.25% w/v fibrin-agar plates, respectively. Proteolytic activity was positive in 62% of the isolates, whilst only three of them were able to produce extracellular fibrinolytic enzymes on solid nutritive medium. Fibrinolytic-positive extracts were able to degrade fibrin clots in a direct plasminogen-independent way. Selected isolates were identified by sequencing the 26S rDNA D1/D2 domain. Isolates LY 4.1 and LY 4.4 showed a 99.9% similarity with Bionectria ochroleuca, while LY 4.2 showed a 99.9% identity with Cladosporium cladosporioides. Under submerged culture conditions, LY 4.1 and LY 4.4 were able to excrete fibrinolytic enzymes, reaching a maximum at 120 h of cultivation of 100.2 and 107.9 U/ml in plasmin-equivalent units, respectively. Fibrinolytic enzyme production could be scaled-up to fermenter scale reaching similar values. Fibrin zymography showed that fibrinolytic activity was associated with ~173-, 153- and 80-kDa protein fractions. Extracellular fibrinolytic enzymes from Bionectria species may be potentially related to pathogenesis mechanisms, as already demonstrated for serine-proteases from the nematicidal anamorph Clonostachys rosea. This work reveals the potential of Bionectria strains as an unconventional and unexplored production alternative to already known thrombolytic agents. The value of Las Yungas forests as a reservoir of fungal species with promising biotechnological value could be also highlighted.     

蚯蚓纤溶酶分子生物学进展

蚯蚓纤溶酶是近年发现的一种新型的溶解血栓物质,属丝氨酸蛋白酶,不同种属的蚯蚓中均可分离到,具纤溶活性和溶栓活性。有较好的热稳定性,多为单体酶,多数兼有纤溶活性和纤溶酶原激活活性。不同种属的蚯蚓分离的纤溶酶性质上有一定差别。已获得多种纤溶酶的N端序列及部分核酸序列,相互之间及与某些蛋白酶之间有一定的同源性。纤溶酶通过降解目的蛋白的特定位点而起作用 。     

Exploring microbial bioactive molecules from Western Ghats,India

Western Ghats are designated as world heritage sites and global biodiversity hotspots. Microbial diversity in this forest area has been largely neglected and is getting attention since the end of the last millennium. In this review, we have studied and organized various microorganisms from Western Ghats and their diversity, important characteristics and potential biotechnological applications. Microorganisms from Western Ghats have been explored individually for potential bioactive molecules. While most of the microorganisms were analyzed for antimicrobial activities, there have been studies on microbial promotion of plant growth. The microbes analyzed included from aquatics, soil, rhizosphere, phyllosphere and even as endophytes. There have been microorganisms which have shown antioxidant and anti-cancerous activities. There are microorganisms capable of degrading plant wastes and also xenobiotics. Microorganisms capable of producing industrially important enzymes have also been reported. The present review explores largely neglected microbial diversity with respect to their ability to produce potential bioactive biomolecules.     

Comparative study of proteolytic enzyme preparations of the actinomycete Streptomyces spheroides and its mutant

Preparations of proteolytic enzymes with high fibrinolytic and thrombolytic activities were isolated from the cultural broth filtrates of Streptomyces spheroides and its mutant by precipitation with ammonium sulfate, dialysis and freeze-drying. The fibrinolytic and thrombolytic activities of the enzymes produced by the mutant are 5.6 and 6 times higher, respectively, than those of the parent strain. Moreover, the enzymes of the mutant have an elevated stability in the alkaline pH region, the optimum of their activities is also shifted to the alkaline pH region, they are more resistant to the action of blood plasma inhibitors and more sensitive to the action of high temperatures. As was shown using isoelectric focusing in a sucrose density gradient, the preparations of both the parent and mutant strains contain several proteolytic enzymes (or their groups) with acid, neutral and alkaline isoelectric points.     

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.     

Characteristics of the development and biosynthetic activity of a mixed culture of microorganisms

The growth of microbial populations and their biosynthesis of proteinases with the fibrinolytic activity were studied in an artificial ecosystem composed of a strain producing the enzymes (Nocardia minima 1) and a zero strain (Arthrobacter citreus VKM 654). The microorganisms in the association were shown to interact in terms of amensalism via metabolites if the enzyme-producing strain dominated.     

Expression of cholera toxin B subunit–lumbrokinase in edible sunflower seeds–the use of transmucosal carrier to enhance its fusion protein's effect on protection of rats and mice against thrombosis

Lumbrokinase (LK) is a group of serine proteases with strong fibrinolytic and thrombolytic activities and is useful for treating diseases caused by thrombus. Cholera toxin B subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. We investigate here the application of CTB as a transmucosal carrier in enhancing its fusion protein‐LKs effect to protect rats against thrombosis. Thus, in this study, CTB‐LK fusion gene separated by a furin cleavage site was expressed in seeds of Helianthus annuus L. The activity of recombinant protein in seeds of transgenic sunflower was confirmed by Western blot analysis, fibrin plate assays and G_M1‐ganglioside ELISA. The thrombosis model of rats and mice revealed that the oral administration of peeled seeds of sunflower expressing CTB‐LK had a more significant anti‐thrombotic effect on animals compared with that administration of peeled seeds of sunflower expressing LK. It is possible to conclude that CTB can successfully enhance its fusion protein to be absorbed in rats or mice thrombosis model. The use of CTB as a transmucosal carrier in the delivery of transgenic plant‐derived oral therapeutic proteins was supported. In addition, for the purpose of that recombinant CTB‐LK was designed for oral administration, thus the expression of CTB‐LK in edible sunflower seeds eliminated the need for downstream processing of proteins. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1029–1039, 2014     

Highly potent fibrinolytic serine protease from Streptomyces

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.