Production Of Fibrinolytic Protease Research Articles

Fibrinolytic Protease Production: Impact of Initial pH and Temperature in Solid-State Fermentation by Rhizopus microsporus var. oligosporus FNCC 6010

Background: Fibrinolytic enzyme is one of the cardiovascular disease therapies. Rhizopus microsporus var. oligosporus is microorganism that has been evaluated to produce fibrinolytic protease by fermentation. This study conducted fermentation of helianthi annui semen (sunflower seed) by Rhizopus microsporus var. oligosporus to produce fibrinolytic enzyme. Objective: This study aims to determine the effect of Initial pH and incubation temperature and its optimization in the production of fibrinolytic protease by Rhizopus microsporus var. oligosporus FNCC 6010 in solid-state fermentation on helianthi annui semen (sunflower seed) substrate. Optimum condition was determined by maximum protease and fibrinolytic activity. Method: A crude enzyme of protease fibrinolytic was obtained from the supernatant extract of fermented sunflower seed. Protease activity was measured by the skimmed milk agar (SMA) plate method, and fibrinolytic activity was determined by the fibrin agar plate method. Result: It was found that the starting pH affects both the proteolytic and fibrinolytic activity of enzymes that are produced in fermentation. The starting pH of 5.0 showed higher fibrinolytic and proteolytic activity values compared to the starting pH of 7.0. The incubation temperature 33±1 °C had the higher activity compared to 28±1 °C or 37±1 °C. Conclusion: Initial pH and incubation temperature affect the proteolytic and fibrinolytic activity of crude enzyme extracted from fermented sunflower seed by Rhizopus microsporus var. oligosporus. The optimum condition for producing fibrinolytic protease in the state fermentation method was an initial pH of 5.0 and an incubation temperature of 33±1°C.

Isolation, identification, and optimization of the fibrinolytic protease-producing strains

Cardiovascular complications due to thrombosis have become one of the main causes of death worldwide. The high cost and undesirable side effects of existing thrombolytic agents have led researchers to isolate potential strains that produce fibrinolytic enzymes for therapeutic applications. Fibrinolytic enzymes, especially of microbial origin, are recognized as potential therapeutic candidates for thrombosis. In this study, isolation, identification, and optimization of fibrinolytic protease enzyme-producing strains were performed using fermentative protein sources. Fibrinolytic protease-producing strains were selected by analyzing the isolated strains on skim milk agar medium. The selected strains were examined on blood agar and fibrin plate medium, and the ones showing high enzymatic activity were determined. The strain determined to have the highest activity was identified as Acinetobacter johnsonii TR01 by 16S rRNA analysis. The maximum fibrinolytic protease production of the strain occurred at 60°C and pH 7.0. Under different medium conditions used for enzyme production, fructose was found to be the best carbon source, while yeast extract and peptone were the best nitrogen sources. It was observed that CaCl2, KH2PO4, andMgSO4 components had a negative effect, while MnCl2and ZnC4H6O4 components had a positive effect on enzyme production. The medium composition for maximum enzyme activity (8.30IU/ml) determined by Response Surface Methodology was 14.22g/L fructose, 11.190g/L yeast extract, 14.22g/L peptone, 0.5g/L MnCl2, and 0.5g/L ZnC4H6O4.

Bioprocess for enhanced production of a fibrinolytic protease with high thrombolytic potential from Aspergillus flavus SH71

The diseases related to the cardiovascular system significantly contribute towards global mortality. Dysregulation/malfunctioning of coagulation and fibrinolytic pathways especially under pathophysiological conditions disturbs the homeostasis and causes cardiovascular complications. Thrombosis is one such disorder wherein normal blood flow is obstructed due to an abnormal fibrin clot. Serious issues with the available therapeutics (thrombolytics) viz. fatal side effects, low specificity and efficacy and high cost, motivate the research for discovery of potentially novel, safe, efficacious and cost-effective thrombolytics. In the present study, an effectual fibrin specific fibrinolytic protease (thrombolytic) was produced from a newly isolated fungus Aspergillus flavus SH71 by using low-cost agricultural residues as substrates. Molasses as a sole carbon source and linseed cake as an exclusive nitrogen source supported the protease production of 188.99 U/mL and 215.67 U/mL respectively which were higher than that obtained in control (133.91 U/mL). Also, some other agro-based substrates (orange peel, rice bran, malt, mixed fruit brunch and soymeal) supported higher enzyme titre than control. Furthermore, a bioprocess was developed for fibrinolytic protease production by optimization of process variables i.e. concentration of molasses and linseed cake, medium pH and incubation time based on design of experiments and a substantial yield enhancement was achieved (1.97 folds).

Doehlert Matrix-assisted optimization of Salmonella typhi Vi polysaccharide purification parameters

Enhanced fibrinolytic protease production by Serratia marcescens RSPB11 through Plackett-Burman and response surface methodological approaches Paruchuru Lakshmi Bhargavi, Reddy Shetty Prakasham

In vitro fibrinolytic activity of an enzyme purified from Bacillus amyloliquefaciens strain KJ10 isolated from soybean paste.

A fibrinolytic protease secreting producing Bacillus amyloliquefaciens strain KJ10 was initially screened from the fermented soybean. Maximum productivity was obtained in the culture medium after 40 h incubation, 34 °C incubation temperature at pH 8.0. Fibrinolytic protease production was enhanced in the culture medium with 1% sucrose (3712 ± 52 U/mL), 1% (w/v) yeast extract (3940 ± 28 U/mL) and 0.1% MgSO4 (3687 ± 38 U/mL). Enzyme was purified up to 22.9-fold with 26%recovery after Q-Sepharose HP column chromatography. After three steps purification, enzyme activity was 1606U/mg and SDS-PAGE analysis revealed 29 kDa protein and enzyme band was detected by zymograpy. Enzyme was highly active at pH 8.0, at wide temperature ranges (40 °C − 55 °C) and was activated by Mn2+ (102 ± 3.1%) and Mg2+ (101.4 ± 2.9%) ions. The purified fibrinolytic enzyme was highly specific against N-Suc-Ala-Ala-Pro-Phe-pNA (189 mmol/min/mL) and clot lytic activity reached 28 ± 1.8% within 60 minin vitro. The purified fibrinolytic enzyme showed least erythrocytic lysis activity confirmed safety to prevent various health risks, including hemolytic anemia. Based on this study, administration of fibrinolytic enzyme from B. amyloliquefaciens strain KJ10 is safe for clinical applications.

Production of Fibrinolytic Protease from a Halobacterium Bacillus licheniformis VITLMS Isolated from Marine Sponges of Rameshwaram Coast, India

Background: Marine bacteria serve as excellent sources of therapeutic enzymes, metabolites and natural products, which possess novel therapeutic properties. Increasing death rates due to cardiovascular diseases urge for cost-effective production of the fibrinolytic enzyme. Methods: In this study, marine sponge samples were screened for potent fibrinolytic producing bacteria. The primary screening was done for protease production, and clot lysis activity. The secondary screening was done for casein plasminogen activity and fibrinolytic activity. The strain which had potent fibrinolytic activity among them was further subjected to morphological, biochemical and molecular characterization. Media optimization was carried out to enhance enzyme production. The enzyme produced was subjected to purification using ammonium sulfate precipitation, gel filtration and characterized using HPLC and FTIR analysis. Results: Sponge was identified to be Desmapsamma anchorata. Thirteen bacterial isolates were isolated from the sponge sample. The 16S rRNA sequencing revealed that the potential strain had 99% similarity with Bacillus licheniformis. Amongst the isolates, most were found to be morphologically identical to the Bacillus genus. Gram’s staining and SEM analysis of the potent isolate were performed to identify the spore formation and rod-shaped morphology of the bacteria. The optimal temperature and pH for the production of the enzyme were 37°C and 8, respectively. The carbon source maltose and nitrogen sources were malt extract and yeast extract that were found to be optimal. The optimum incubation time was found to be 4 to 5 days. The crude supernatant was purified with ammonium sulfate precipitation and gel filtration chromatography. The retention time of 11.3 min and the presence of functional groups show the purity of the enzyme. The partially purified enzyme showed 96.4% clot lysis in artificial clot lysis activity. Conclusion: Although the secretion of fibrinolytic enzymes from Bacillus species is not new, based on our investigation, there are no reports regarding Bacillus licheniformis being isolated from marine sponges. However, there are reports of Bacillus licheniformis secreting fibrinolytic enzymes isolated from fermented food samples. This study identifies the marine environment as a potential source of new exploration for drug discovery.

Purification and characterization of fibrinolytic protease from Streptomyces parvulus by polyethylene glycol-phosphate aqueous two-phase system.

Fibrinolytic proteases are a promising alternative in the pharmaceutical industry, they are used in the treatment of cardiovascular diseases, especially thrombosis. Microorganisms are the most interesting source of fibrinolytic proteases. The aim of this study was the production of fibrinolytic protease from Streptomyces parvulus DPUA 1573, the recovery of the protease by aqueous two-phase system and partial biochemical characterization of the enzyme. The aqueous two-phase system was performed according to a 24-full factorial design using polyethylene glycol molar mass, polyethylene glycol concentration, citrate concentration and pH as independent variables. It was analyzed the effect of different ions, surfactants, inhibitors, pH and temperature on enzyme activity. The best conditions for purifying the enzyme were 17.5% polyethylene glycol 8,000, 15% Phosphate and pH 8.0, it was obtained a partition coefficient of 7.33, a yield of 57.49% and a purification factor of 2.10-fold. There was an increase in enzyme activity in the presence of Fe2+ and a decrease in the presence of $\beta$-Mercaptoethanol, phenylmethylsulfonyl fluoride and Iodoacetic acid. The optimum pH was 7.0 and the optimum temperature was 40 ºC. The purified protease exhibited a molecular mass of 41 kDa. The fibrinolytic protease from Streptomyces parvulus proved to be a viable option for the development of a possible drug with fibrinolytic action.

Novel Fibrinolytic Protease Producing Streptomyces radiopugnans VITSD8 from Marine Sponges.

Fibrinolytic enzymes have received more attention due to their medicinal potential for thrombolytic diseases. The aim of this study is to characterize the in vitro fibrinolytic nature of purified protease producing Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifera. Three varieties of sponge were collected from the Rameshwaram Sea coast, Tamil Nadu, India. The fibrinolytic activity of Streptomyces sp. was screened and determined by casein plasminogen plate and fibrin plate methods respectively. The crude caseinolytic protease was purified using ammonium sulfate fractionation, affinity and ion-exchange chromatography. Based on the morphological, biochemical, and molecular characterization, the isolate VITSD8 was confirmed as Streptomyces radiopugnans. Maltose and peptone were found to be the best carbon and nitrogen sources for the production of fibrinolytic protease. The carbon and nitrogen source peptone showed (781 U/mL) enzyme activity. The optimum pH and temperature for fibrinolytic protease production was found to be 7.0 and 33 °C respectively. The purified enzyme showed a maximum specific activity of 3891 U. The blood clot lysis activity was compared with the standard, and it was concluded that a minimum of 0.18 U (10 µL) of purified protease was required to dissolve the blood clot. This is the first report which exploits the fibrinolytic protease activity of Streptomyces radiopugnans VITSD8 extracted from a marine sponge. Hence the investigation suggests a potential benefit of purified fibrinolytic protease which will serve as an excellent clot buster alternative.

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.

Fibrinolytic Protease from Marine Streptomyces rubiginosus VITPSS1.

Fibrinolytic enzymes must currently originate a significant product in the arena of medical research. Very limited studies are stated on fibrinolyticenzyme production from actinomycetes. Streptomyces sp. isolated from marine soil was chosen to optimize its fibrinolytic protease production.16s rRNA sequencing confirmed the isolated potent strain to be Streptomyces rubiginosus VITPSS1. A fibrinolytic protease was then purified from Streptomyces rubiginosus VITPSS1, with the target of producing a cost effective feasible enzyme from a potential actinomycete. SDS-PAGE results exhibited a protein band of about 45 kDa and the fibrinolytic band was detected by zymography. Optimization of physical and nutritional parameters for fibrinolytic protease production from a marine soil isolate Streptomyces strain was done by response surface methodology. The optimal cultural condition for fibrinolytic protease production was obtained with response surface methodology was based on OFAT results, it was inferred that glycerol, Soyabean meal, pH 7.2 and temperature 37°C. The optimization of the production of fibrinolytic protease with response surface methodology bring about two-folds increase in production by Streptomyces rubiginosus VITPSS1. Thus, this study presents its novelty by highlighting the potential of marine Streptomyces as a significant source for fibrinolytic enzyme production.

Enhanced production of fibrinolytic enzyme by a new Xanthomonas oryzae IND3 using low-cost culture medium by response surface methodology.

Cardiovascular diseases (CVDs) cause high mortality throughout the world. Existing fibrinolytic agents are highly expensive and have many side effects. Microbial fibrinolytic enzymes are very much considered as novel therapeutic candidate for the treatment of CVDs. Reports on fibrinolytic enzyme from Xanthomonas sp. is lacking. This study reports fibrinolytic enzymes from Xanthomonas oryzae IND3 as it shows hyperactivity on fibrin-agarose plates. This organism utilized various agro-industrial wastes for enzymes production. Among all, cow dung enhanced more enzyme production, hence it was used as the low-cost substrate for statistical optimization of fibrinolytic protease in Solid state fermentation. Response surface methodology was employed to optimize the factors and enhanced yield by 4-fold. The interactions among the variables, viz, sucrose, yeast extract, and pH of the medium were investigated using Central Composite Design (CCD). The predicted fibrinolytic enzyme activity was 2340 U/g, and the observed fibrinolytic enzyme activity was 2294 ± 12.8 U/g. The fibrinolytic enzyme degraded blood clot in vitro completely. This study is the first report on statistical optimization of fibrinolytic enzyme production in SSF from Xanthomonas sp. The crude extract has immense activity on proteinaceous wastes. The production of fibrinolytic protease using the low-cost substrate could reduce the production cost of enzyme.

Optimization and modeling studies on the production of a new fibrinolytic protease using Streptomyces radiopugnans_VITSD8

The current study demonstrated the possibility of statistical design tools combination with computational tools for optimization of fermentation conditions for enhanced fibrinolytic protease production. The effects of using different carbon and nitrogen sources for protease production by Streptomyces radiopugnans_VITSD8 were examined by a full factorial design method. The incubation time, temperature, pH of the medium, and RPM were assessed by the predictable one factor at a time (OFAT) method. Optimization was carried out using starch and oat meal as carbon source, nitrogen source as peptic and malt extract using Fractional Factorial Design (FFD). The analysis was further continued for medium volume, temperature, initial medium pH, inoculum concentration, high determination co-efficient as (R’-0.965), and lower determination co-efficient of variation (CV-8.19%), which defines a reliable and accurate experimental value. Analysis of variance by the fixed slope effect by temperature and starch; temperature and L-aspargine, temperature and oat meal, temperature and peptic extracts, temperature and pH, temperature and duration of incubation were more vital for protease production at an interactive level. Response surface plots revealed that temperature, starch, and peptic extracts affix critical concerning in temperature. Programming estimated a 28% increase in protease production. Incubation temperature and medium volume portrayed extreme impact among all factor. Starch, peptic and temperature play an important regulatory role in protease production. Optimium temperature for protease production was 33°C. The ratio of carbon and nitrogen sources and pH were the major regulatory factors in protease production by Streptomyces radiopugnans_VITSD8. It demonstrated a 4% noteworthy change in condition. Among all the selected parameters, temperature was the most intuitive factor, demonstrating a notable connection with the type of media and pH, while inoculum fixation had a direct impact on protein production.

Production of fibrinolytic protease from Streptomyces lusitanus isolated from marine sediments

This study aim was to isolate, screen, characterize and optimize marine Streptomyces for fibrinolytic enzyme production. The potent actinomycete isolate was subjected to optimization. The parameters for optimization included pH, temperature, carbon, nitrogen sources. The crude supernatant produced was purified using size exclusion gel filtration chromatography. The optimized parameters for maximum productivity were found to be pH 7, 37°C, maltose and peptone respectively. The molecular weight of the purified enzyme was found to be 21kDa.

Enhanced Production of Fibrinolytic Protease from Microalgae Chlorella Vulgaris using Glycerol and Corn Steep Liquor as Nutrient

According to the World Health Organization (2012), in 2030, almost 3.6 million people will die from Cardio Vascular Diseases (CVDs), mainly from heart disease and stroke. One of the pathophysiological Conditions which causes myocardial infarction, stroke and other CVDs, is the formation of a fibrin clot by the proteolytic action of thrombin on fibrinogen, and consequent accumulation of fibrin in the blood vessels usually leads to thrombosis.

Enhanced fibrinolytic protease production by Serratia marcescens RSPB11 through Plackett-Burman and response surface methodological approaches

A well characterized alkaline metalloprotease enzyme called serralysin with fibrinolytic activity has been reported in the newly isolated Serratia marcescens RSPB11. In view of its potential application in thrombolytic therapy this study has been made for understanding the nutritional parameters requirement needed for production. Therefore, medium components required for the production of serralysin were optimized using a two step statistical approach. Fermentation variables were selected in accordance with the Plackett-Burman design and were further optimized via response surface methodological approach. A total of seven parameters viz., casein, dextrose, KH2PO4, MgSO4, NaCl, CaCl2 and inoculum have been considered for the optimization studies. The statistical model was constructed via central composite design (CCD) using five screened variables (casein, dextrose, KH2PO4, CaCl2 and inoculum size). An overall 51.8% increase in protease production was achieved in the optimized medium as compared with the unoptimized casein medium.With the application of statistical design methodology serralysin production increased significantly with optimized casein medium (23910 U/ml) when compared to yeast extract-peptone medium (5363 U/ml).

Statistical media optimization for enhanced production of fibrinolytic enzyme from newly isolated Proteus penneri SP-20

Abstract Thrombolytic enzymes are invaluable for combating cardiovascular diseases. In this study a thorough isolation resulted, a potent fibrinolytic enzyme producer which was identified as Proteus penneri SP-20 using 16S rRNA sequencing. It secretes extracellular protease possesing fibrinolytic activity which was confirmed through the analysis of fibrin degradation by SDS-PAGE, phase contrast microscopy and fibrin zymography. Response surface methodology is used for media optimization using newly isolated P. penneri SP-20. Plackett Burman (PB) design was employed for selection of critical media constituents while; further optimization was carried out using Central composite rotatable design (CCRD). Casein, yeast extract, maltose and MnCl2·4H2O were critical medium components selected on the basis of PB design. Optimized media components obtained by CCRD were casein; 12.58 g l−1, yeast extract; 17.27 g l−1, maltose; 1.65 g l−1, MnCl2·4H2O; 0.564 g l−1. As compared with initial enzyme activity of 38.19 Units CCRD yielded 796.82 Unit activity, increasing the overall production of fibrinolytic protease by 20.86 fold. Hence the model is reliable for process optimization.

Fibrinolytic protease production by new Streptomyces sp. DPUA 1576 from Amazon lichens

article Background: Streptomyces sp. DPUA 1576 from Amazon lichens was studied to protease and fibrinolytic production. A 2 2 factorial experimental design was applied to optimize its protease enzyme production using two independent variables, namely soybean flour and glucose concentrations. Results: The optimal conditions to obtain high protease production (83.42 U/mL) were 1.26% soybean flour and 1.23% glucose concentration. A polynomial model was fitted to correlate the relationship between the two variables and protease activity. In relation to fibrinolytic activity, the highest activity of 706.5 mm 2 was obtained at 1.7% soybean flour and 1.0% glucose concentration, which was 33% higher than plasmin. Fibrinolytic production was not optimized in the studied conditions. Conclusions: These results show that the optimization of the culture medium can enhance protease production, thus becoming a good process for further research. In addition, Streptomyces sp. DPUA 1576, isolated from Amazon lichens, might be a potential strain for fibrinolytic protease production.

Optimization of fibrinolytic protease production from Bacillus subtilis I-2 using agro-residues

The aim of this work was to study the production of fibrinolytic protease by Bacillus subtilis I-2 on agricultural residues. Molasses substantially enhanced (63%) protease production (652.32 U/mL) than control (398.64 U/mL). Soybean meal supported maximum protease production (797.28 U/mL), followed by malt extract (770.1 U/mL), cotton cake (761.04 U/mL), gelatin (742.92 U/mL) and beef extract (724.8 U/mL). Based on the Plackett-Burman designed experiments, incubation time, soybean meal, mustard cake and molasses were identified as the significant fermentation parameters. Ammonium sulfate precipitation and DEAE sephadex chromatography resulted 4.8-fold purification of protease. Zymography showed the presence of three iso-forms in the partially purified protease preparation, which was confirmed by the SDS-PAGE analysis (42, 48, 60 kDa). Protease exhibited maximum activity at 50oC and at pH 8.0. Significant stability was observed at 30-50oC and at pH 7.0-10.0. Mg2+, Zn2+, Co2+, Ca2+, Mn2+ and Cu2+,EGTA, EDTA and aprotinin severely decreased the enzyme activity.

Integrated Process Production and Extraction of the Fibrinolytic Protease from Bacillus sp. UFPEDA 485

Fibrinolytic proteases are enzymes that degrade fibrin; these enzymes are a promising alternative for thrombolytic therapy, and microorganisms produce them. The aim of this study was to evaluate the optimum conditions for the integrated production and purification of fibrinolytic protease from Bacillus sp. UFPEDA 485. Extractive fermentation was carried out in a culture medium containing soybean flour and by adding polyethylene glycol (PEG) and Na2SO4 according to a 2(3) experimental design. In all assays, the enzyme preferentially partitioned to the bottom phase (K < 1), with an optimum activity of 835 U ml(-1) in the bottom phase (salt-rich phase). The best conditions for extractive fermentation were obtained with 18 % PEG 8000 and 13 % Na2SO4. Characterization showed that it is a metalloprotease, as a strong inhibition-residual activity of 3.13 %-occurred in the presence of ethylenediaminetetraacetic acid. It was also observed that enzymatic activity was stimulated in the presence of ions: CaCl2 (440 %), MgCl2 (440 %), FeSO4 (268 %), and KCl (268 %). The obtained results indicate that the use of a low-cost substrate and the integration of fermentation with an aqueous two-phase system extraction may be an interesting alternative for the production of fibrinolytic protease.

Enhanced production of fibrinolytic protease from Bacillus cereus NS-2 using cotton seed cake as nitrogen source

Abstract Cardiovascular complications due to thrombosis have become one of the major causes of mortality. High cost and fatally undesired side effects associated with the available thrombolytic agents motivated the researchers to investigate potentially better agents for therapeutic applications. In the current study, production of an efficacious fibrinolytic protease from a bacterial isolate Bacillus cereus NS-2 was optimized by employing low-cost agricultural residues as substrates (wheat bran and cotton cake at 1%, w/v, each). Wheat bran supported fibrinolytic protease production (148 U/ml) that was comparable with control (145.5 U/ml on glucose). Cotton cake as nitrogen source enhanced fibrinolytic protease production substantially (71%) as compared to control (315 U/ml vs 184 U/ml). Fibrinolytic protease was partially purified (2.35-fold) by ammonium sulfate precipitation and diethylaminoethyl-sepharose chromatography with the yield of 58.27%. Maximum activity of partially purified fibrinolytic protease was observed at 40 °C and at pH 9. Fibrinolytic protease activity was increased immensely by Fe 2+ (76.6%) and moderately by Ca 2+ , Mn 2+ , Zn 2+ , Cu 2+ and Mg 2+ (29–44%), however, Pb 2+ and Hg 2+ strongly inhibited the protease. B. cereus NS-2 protease showed reasonable stability in presence of Triton X-100 and Tween 20 (relative activity 87 and 80%, respectively) but poor stability in presence SDS (relative activity 29%). Retention of considerable activity (46%) in presence of EDTA reflects that requirement of divalent ions is not absolute for catalysis by B. cereus NS-2 fibrinolytic protease.