Production Of Fibrinolytic Enzyme Research Articles

Production of Fibrinolytic Enzyme from Bacillus amyloliquefaciens by Fermentation of Chickpeas, with the Evaluation of the Anticoagulant and Antioxidant Properties of Chickpeas

To develop safe and cheap thrombolytic agents, a fibrinolytic enzyme productive strain of LSSE-62 was isolated from Chinese soybean paste. This strain was identified as Bacillus amyloliquefaciens by 16S rDNA sequence analysis. Nucleotide and amino acid sequence analysis showed that this fibrinolytic enzyme was identical to subtilisin DJ-4. Chickpeas were used as the substrate for fibrinolytic enzyme production from B. amyloliquefaciens in solid-state fermentation. Under the optimized conditions (34 °C and 50% initial moisture content), the fibrinolytic activity of fermented chickpeas reached 39.28 fibrin degradation units (FU)/g. Additionally, the fermented chickpeas showed anticoagulant activity, and the purified anticoagulant component showed higher anticoagulant activity than heparin sodium. After fermentation, the total phenolic and total flavonoid contents increased by 222 and 71%, respectively, and then the antioxidant activities were improved significantly. This study provided a novel method for the preparation of multifunctional food of chickpeas or raw materials for the preparation of functional food additives and potential drugs.

Isolation, production, purification, assay and characterization of fibrinolytic enzymes (Nattokinase, Streptokinase and Urokinase) from bacterial sources

Nattokinase, Streptokinase and Urokinase are novel fibrinolytic enzymes which are isolated from Bacillus subtilis, β-haemolytic Streptococci and urine sample . The fibrinolytic enzyme Nattokinase, Streptokinase and Urokinase was purified from supernatant of Bacillus subtilis, β-haemolytic Streptococci and recombinant E.coli containing short fragment genomic DNA of Pseudomonas sp. Culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH of Nattokinase, Streptokinase and Urokinase were 37-55°C and 9, 27-37°C and 7 and 55°C and 9, respectively. The molecular weight of Nattokinase, Streptokinase and Urokinase was approximately 28 kDa, 47 kDa and 34 kDa, respectively, as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The caseinolytic activity of Nattokinase, Streptokinase and Urokinase were 576.73 U, 467.73 U and 785.73 U, respectively, while fibrinolytic activity achieved by fibrin plate method were 10 U, 5 U and 15 U, respectively.

Fibrinolytic Serine Protease Isolation from Bacillus amyloliquefaciens An6 Grown on Mirabilis jalapa Tuber Powders

In this study, Mirabilis jalapa tuber powder (MJTP) was used as a new complex organic substrate for the growth and production of fibrinolytic enzymes by a newly isolated Bacillus amyloliquefaciens An6. Maximum protease activity (1,057 U/ml) with casein as a substrate was obtained when the strain was grown in medium containing (grams per liter) MJTP 30, yeast extract 6, CaCl(2) 1, K(2)HPO(4) 0.1, and K(2)HPO(4) 0.1. The strain was also found to grow and produce extracellular proteases in a medium containing only MJTP, indicating that it can obtain its carbon, nitrogen, and salts requirements directly from MJTP. The B. amyloliquefaciens An6 fibrinase (BAF1) was partially purified, and fibrinolytic activity was assayed in a test tube with an artificial fibrin clot. The molecular weight of the partially purified BAF1 fibrinolytic protease was estimated to be 30 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for the caseinolytic activity were 60 degrees C and 9.0, respectively. The enzyme was highly stable from pH 6.0 to 11.0 and retained 62% of its initial activity after 1 h incubation at 50 degrees C. However, the enzyme was inactivated at higher temperatures. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, suggesting that BAF1 is a serine protease.

A novel source of fibrinolytic activity: Bionectria 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 (Tucuman, 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.

Production of Fibrinolytic Enzyme by Streptmyces Rimosus at Conditions of Nitrogen Limitation

The kinetic of the enzyme production of Streptomyces rimosus, a producer of exoproteases was investigated in conditions of nitrogen limitation.The maxima of fibrinolytic and caseinolytic activities by Streptomyces rimosus were reached at 84 h, respectively 96 h. Values of activities were increased 5-fold with those on initial medium. The ultrastructure changes were followed. In the earlier hours an aggregation of ribosomes in cells was established. Large membranes and numerous electron- transparent structures were found. The obtained results indicated close connection between cell status of the producer, its enzyme productivity and an ability of the strain to survive at conditions of nitrogen limitation.

청국장에서 분리한 Bacillus licheniformis HK-12의 혈전용해활성과 프로테옴 분석

자연발효된 청국장으로부터 혈전용해활성을 가지는 세균 Bacillus licheniformis HK-12를 농화분리하여, 배양기간 동안 생산된 혈전용해활성을 가지는 단백질에 대해 프로테옴 분석을 실시하였다. B. licheniformis HK-12를 액체 영양배지에 접종하여 얻어진 배양상등액을 피브린 평판법(fibrin plate method)을 사용하여 효소활성을 측정하였다. 그 결과, HK-12의 혈전용해 활성은 대조구인 plasmin보다 약 2.3배정도 높은 활성을 나타내었다. 효소는 배양상등액을 ammonium sulfate 침전, DEAE-cellulose chromatography, Sephadex chromatography 등을 수행하여 분리정제하였으며, 정제된 혈전용해효소의 분자량은 SDS-PAGE를 통해 약 23 kDa로 측정되었다. 배양시간에 따른 HK-6의 세포외 단백질의 변화를 2-D PAGE 분석을 통하여 분석하였다. 그 결과 36시간배양 후에 가장 현저하게 유도된 spot #1을 분리하였으며, MALDI-TOF MS를 이용하여 단백질 동정을 실시한 결과, 유도된 단백질의 아미노산 서열은 <TEX>$^1EKKIEKYREEEORLK^{15}$</TEX>으로서, serine protein kinase (PrkA) (AAU22526)로 확인되었다. The strain HK-12 was enriched and isolated from naturally fermented soybean for the production of fibrinolytic enzyme and the proteome of this enzyme induced during the incubation period was analyzed. The activity of fibrinolytic enzyme derived from supernatants of the HK-12 culture was performed by fibrin plate method for solid fibrinolytic activity. As the result, the fibrinolytic activity of HK-12 grown on the nutrient agar media was about 2.3 times greater than that of plasmin used as standard. The purified enzyme was prepared by a series of purification process including ammonium sulfate precipitation, DEAE-cellulose, Sephadex chromatography. The molecular weight of the enzyme was determined to approximately 23kDa with SDS-PAGE. In order to examine which strain HK-12 proteins increased or decreased during the incubation period, 2-DE analysis was performed. Protein spot #1 significantly expressed on the 2-DE gel of bacteria cultivated for 36-hrs was analysed. As the result of protein sequence analysis using MALDI-TOF MS, one protein was identified as serine protein kinase (PrkA).

Optimization of fibrinolytic enzyme production by Bacillus subtilis DC-2 in aqueous two-phase system (poly-ethylene glycol 4000 and sodium sulfate)

The central composite rotable design (CCRD) was used to determine optimal conditions for fibrinolytic enzyme production by Bacillus subtilis DC-2 in poly-ethylene glycol 4000 (PEG 4000) and sodium sulfate (Na 2SO 4) aqueous two-phase system (ATPS). PEG 4000 and Na 2SO 4 concentration, fermentation time and temperature, and pH were selected as variables to evaluate the fibrinolytic activity in PEG phase. Using response surface methodology (RSM), a second-order polynomial equation was obtained by multiple regression analysis. The predicted maximal fibrinolytic activity in PEG phase was 1241.02 IU/ml with 9.05% PEG 4000 concentration, 5.06% Na 2SO 4 concentration, 118.77 h fermentation time, 37.57 °C fermentation temperature and pH 6.52. The validity of the response model was verified by a good agreement between predicted and experimental results. The fibrinolytic activity obtained from experimental results in PEG phase (1223.61 IU/ml) was higher than that produced in homogeneous fermentation (1165.58 IU/ml).

Bacillus firmus NA-1 균주와 Bacillus subtilis G7-D 균주를 이용한 발효비지의 기능성

일본 청국장에서 분리된 Bacillus firmus NA-1과 재래청국장에서 분리한 Bacillus subtilis GT-D를 이용하여 전지활성 생 대두미세분말(MFS) 첨가 및 발효시간에 따른 비지 발효물의 혈전용해효소, 가수분해효소, 점질물, 펩타이드 생산 및 풍미개선 효과를 알아보았다. 84%의 수분함량을 포함하는 비지에 대두미세분말을 10, 15, 20% 첨가함으로써 78, 74, 70%로 수분함량을 감소시킬 수 있으며, 발효시간이 경과할수록 tyrosine함량이 증가하였으며, B. firmus NA-1 균주보다는 B. subtilis GT-D 균주를 사용하는 경우에 높은 값을 보였다. 발효비지의 점조도는 B. firmus NA-1 균주를 이용한 발효물이 높은 값을 보였으며, 두 균주 모두 MFS 20%를 첨가한 후 26시간 발효한 발효물에서 2.18, 0.35 ㎩ㆍsⁿ로 가장 높은 점조도 값을 나타내었다. 혈전용해효소 역시 B. firmus NA-1 균주를 이용한 비지청국장에서 10%이상의 높은 활성을 보였으며, 발효 22시간까지 증가하다가 26시간부터는 큰 변화가 없거나 감소하는 경향을 보였다. 따라서 20%의 MFS를 첨가한 후 42℃에서 22시간 동안 발효하는 것이 발효취 생성을 최소화하는 조건이라 사료되었다. 비지청국장의 동결건조는 청국장 냄새 및 수분함량을 6%수준으로 줄일 수 있었으며, 혈전용해효소의 활성을 포함하였다. B. firmus NA-1 균주를 이용한 시료에서는 615 unit/g의 가장 높은 protease의 활성을 보였으며, B. subtilis GT-D 균주를 이용한 시료에서는 1903 ㎎%의 tyrosine 함량 및 180 unit/g의 α-amylase의 활성을 나타내었다.

Production of Fibrinolytic Enzyme from Soybean Grits Fermented byBacillus firmusNA-1

Bacillus firmus NA-1 producing fibrinolytic enzyme was isolated from Japanese traditional fermented soybean (Natto). Seed starter was cultured in 5% soy milk prepared with micronized soybean powder. To optimize the production of fibrinolytic enzyme, the soybean grits were mixed with 1 volume of water and sterilized at 121 degrees C for 5 minutes, and then used as the medium for solid-state fermentation at 42 degrees C. The fibrinolytic enzyme activity of the fermented grits was 1,120 plasmin units/100 g (wet weight) after fermentation for 24 hours. The water extract of the fermented grits showed the highest viscosity after fermentation for 12 hours. However, the tyrosine content was the highest (962 mg%) after fermentation for 60 hours. The color of raw soybean grit was affected by heat treatment. The activity of fibrinolytic enzyme was stable after freezing-drying, but was completely destroyed by heating at 70 degrees C for 10 minutes. The color of soybean grit was greatly darkened by increasing fermentation time. Soybean grits were completely converted into valuable functional ingredients containing fibrinolytic enzyme, peptide, and mucilage by the solid-state fermentation.

Optimization of the Production of Fibrinolytic Enzyme from Bacillus firmus NA-1 in Fermented Soybeans

Bacillus strains capable of producing fibrinolytic enzyme were isolated from traditional fermented Korean soybean paste and Japanese fermented soybean (Natto). Among the 16 strains, a selected Bacillus sp. was identified as Bacillus firmus, with 80.7% homology, by API kit analysis. Seed starter of B. firmus NA-1 was prepared with 5% soymilk prepared from micronized soybean powder. To produce fibrinolytic enzyme by B. firmus NA-1 the liquid culture was performed with NB broth (pH 7.0) fortified with 1% galactose, 0.1% tryptone, and 0.5% K₂HPO₄, by shaking with 180 rpm at 37℃. Fibrinolytic enzyme activity reached the highest value at 7.8 unit/mL (plasmin unit) after fermentation for 72 hr. The crude fibrinolytic enzyme showed higher relative activity in the range of pH 7.0～9.0. The activity of crude fibrinolytic enzyme was well maintained even after concentration by the vacuum evaporation at 50℃ for 1 hr.

Fibrinolytic Enzyme Production by Bacillus subtilis KH-4 Isolated from Deonjang

A strong fibrin-specific fibrinolytic enzyme was produced from Bacillus subtilis KH-4 isolated from Deonjang, a Korean fermented soybean paste similar to Japanese miso. The addition of glucose as a carbon source resulted in the highest levels of caseinolytic and fibrinolytic activities. Likewise, the addition of yeast extract as the nitrogen source resulted in the highest caseinolytic and fibrinolytic activities (3473.2 unit and 47.4 munit, respectively). It was observed that out of all metal ion sources only calcium (chloride) enhanced caseinolytic and fibrinolytic activities, with increases of 4949.3 unit and 58.2 unit/mg, respectively. The optimal temperature for the production of the enzyme was found to be 40℃ in the optimal medium (glucose 20 g, yeast extract 5 g, CaCl₂ 1 g, and NaCl 2 g). The maximum fibrinolytic activity was observed at the late stationary phase. B. subtilis KH-4 produced a fibrinolytic enzyme at 40℃, after 30 h growth, which increased up to 54 h and then remained constant. These results suggest that Deonjang has potential as a source of physiologically active anti-thromotic enzymes.

Streptomyces sp. JK-20유래 혈전용해효소의 생산조건

토양으로 부터 혈전용해효소를 생산하는 방선균을 분리하여, 본 방선균이 생산하는 혈전용해효소의 최적 생산 조건을 검토하였다. 생산균주는 회색의 기균사를 생성하며 약 0.6~0.8<TEX>$\times$</TEX>0.4~0.8<TEX>$\mu\textrm{m}$</TEX> 크기의 원통형 포자를 가지며 포자의 표면은 매끈하며, 약 10~40개의 포자가 고리를 이루고 있는 형태를 나타내었다. 이상의 결과로 본 균주는 Sfreptomyces 속으로 추정되어 Streptomyces sp. JK-20로 명명하였다. 본 균주의 생리학적 특성으로 20~32<TEX>$^{\circ}C$</TEX>의 온도에서 생육 가능하며 최적 생육 온도 및 pH는 24<TEX>$^{\circ}C$</TEX> 및 pH 6이었다. 본 균주의 혈전용해효소의 생산을 위한 최적 생산 조건을 검토한 결과 탄소원으로 1% xylose였고 질소원은 0.5% yeast extract, 0.5% polypeptone, 금속염은 0.1% MgSO<TEX>$_4$</TEX>.7<TEX>$H_2O$</TEX>였으며 인산염은 0.1% NaH<TEX>$_2$</TEX>Po<TEX>$_4$</TEX>.2<TEX>$H_2O$</TEX>이었다. 본 생산 균주를 최적 배지 조건에서 배양하였을 때 배양 4일째에 혈전용해효소의 생산력이 가장 높았다. An actinomycetes which produces fibrinolytic enzyme was isolated from soil. Characteristics of the isolated strain and the optimal conditions for the productions of fibrinolytic enzyme were summarized as follows; The fibrinolytic enzyme production strain generates gray airmycelium and had about 0.6~0.8<TEX>$\times$</TEX>0.4~0.8<TEX>${\mu}{\textrm}{m}$</TEX> cylindrical spore, smooth surface and formed spore chain of 10~40 spores. We have identified this strain as Streptomyces sp. JK-20. This strain was able to grow up at 20~32<TEX>$^{\circ}C$</TEX> and its optimum growth temperature and pH was 24<TEX>$^{\circ}C$</TEX> and pH 6.0, respectively. The optimal conditions for porducing fibrinolytic enzyme; carbon source, nitrogen source, metal ions and phosphorous sources was 1% xylose, 0.5% yeast extract, 0.5% polypepton, 0.1% MgSO<TEX>$_4$</TEX>.7<TEX>$H_2O$</TEX> and 0.1% NaH<TEX>$_2$</TEX>PO<TEX>$_4$</TEX>.2<TEX>$H_2O$</TEX>, respectively. This strain showed the highest productivity of fibrinolytic enzyme after the fourth day under such optimal culture conditions.

Successive cultivation of Fusarium oxysporum on rice chaff for economic production of fibrinolytic enzyme

Production of fibrinolytic enzyme by successive solid state cultivation of Fusarium oxysporum on rice chaff (RC) was studied. From recycling 20 g dry RC six times by pure batch culture, an overall of 23200IU fibrinolytic enzyme was obtained. A total of 27400IU was obtained when the same amount of RC was used for six cycles with repeated batch fermentation employing immobilized whole cells, which also resulted in 30% (w/w) loss by weight of the substrate, while only 8% (w/w) of RC was degraded in the initial pure batch fermentation.

New solid-state fermentation process for repeated batch production of fibrinolytic enzyme by Fusarium oxysporum

A novel solid-state fermentation system using rice chaff as carrier/substrate impregnated with mineral solution was developed in order to produce, repeatedly, an industrially useful enzyme. Fusarium oxysporum and a fibrinolytic enzyme was used as the model microorganism and target product, respectively. The dry spores were pneumatically inoculated and incubated for 96 hours, resulting in immobilized F. oxysporum cells on moist substrate. The ability of the immobilized culture to perform a long-term operation was confirmed by repeated batch fermentation. Production of the enzyme continued for 31.5 days and total fibrinolytic enzyme activity reached 154500 IU/litre bulk volume.

Solid state fermentation of rice chaff for fibrinolytic enzyme production by Fusarium oxysporum

Rice chaff was used as the substrate for the production of fibrinolytic enzyme by Fusarium oxysporum in solid state fermentation. The optimized moisture content of the medium was 30 - 50% (v/w). An inoculum of 5 - 10% (v/v) and an average particle size of 400 μm for the substrate were optimum for productivity and enzyme activity of 80 IU per ml filtrate was achieved after 96 hours of fermentation.

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

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.

Production and some properties of fibrinolytic enzymes from Fusarium oxysporum N.R.C.1

A mixture of yeast extract and peptone in the culture medium was the most favourable for the production of active fibrinolytic enzyme by Fusarium oxysporum N.R.C.1. Potassium dihydrogen phosphate had stimulating effect, while glucose, sucrose, lactose, ribose and soluble starch had adverse effect on enzyme productivity. The optimum of the fibrinolytic enzyme activity was at pH 7.0.

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH4)2SO4 and soybean flour as nitrogen sources and KH2PO4 at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.

Phorbol ester stimulates macrophage invasion of fibrin matrices.

Macrophages migrate through a fibrin-rich extracellular matrix in chronic inflammation, wound healing, and other pathophysiological processes. To investigate the factors that might influence the ability of mononuclear phagocytes to invade fibrin matrices, we cultured macrophage-like P388D1 cells as well as resident and thioglycollate-elicited mouse peritoneal macrophages on three-dimensional fibrin gels, and we examined the effect of agents known to stimulate a variety of macrophage functions, including the production of fibrinolytic enzymes. Cells grown on fibrin gels under control conditions, as well as cells treated with either bacterial lipopolysaccharide or concanavalin A, remained confined to the gel surface. In contrast, the tumor promoter 4 beta-phorbol 12-myristate 13-acetate (PMA) induced both P388D1 cells and peritoneal macrophages to invade the underlying fibrin matrix. The invasive behavior of PMA-treated P388D1 cells was not affected by protease inhibitors of various specificities. These results demonstrate that certain exogenous signals can profoundly modify the ability of macrophages to migrate through fibrin matrices.