Examination of thrombolytic and anticoagulant activities of purified fibrinolytic enzyme extracted from Cochliobolus hawaiiensis under solid state fermentation

doi:10.21203/rs.3.rs-2503121/v1

Download PDF

Research Article

Examination of thrombolytic and anticoagulant activities of purified fibrinolytic enzyme extracted from Cochliobolus hawaiiensis under solid state fermentation

https://doi.org/10.21203/rs.3.rs-2503121/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Cochliobolus hawaiiensis Alcorn AUMC 8606 was chosen from the screened twenty fungal species as the potent producer of fibrinolytic enzyme on skimmed-milk agar plates. The greatest enzyme yield was attained when the submerged fermentation (SmF) conditions were optimized, and it was around (39.7 U/mg protein). Moreover, Upon optimization of fibrinolytic enzyme production under solid state fermentation (SSF), the maximum productivity of fibrinolytic enzyme was greatly increased recorded a bout (405 U/mg protein) on sugar cane bagasse. The yield of fibrinolytic enzyme by C. hawaiiensis under SSF was higher than that of SmF with about 10.20 fold. The purification procedures of fibrinolytic enzyme caused a great increase in its specific activity to 2581.6 U/mg protein with an overall yield of 55.89%, 6.37 purification fold and molecular weight of 35kDa. Maximal activity was recorded at pH 7 and 37^oC. The enzyme showed the highest affinity towards Fibrin, with V_max of 240 U/ml and an apparent Km value of 47.61 mmol. Mg²⁺ and Ca²⁺ moderately induced fibrinolytic activity, while Cu²⁺ and Zn²⁺greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non metalloprotease due to the great suppression in its activity by using phenylmethylsulfonyl fluoride and thylenediamine-tetraacetat, respectively. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplaetlet aggregation activities by completely prevention and dissolution of the blood clot which confirmed by microscopic examination and amelioration of blood coagulation assays. These findings suggested that the produced fibrinolytic enzyme is a promising agent in management of blood coagulation disorders

Fibrinolytic enzyme

thrombolytic efficiency

anticoagulant efficiency

purification

Cochliobolus hawaiiensis

sugar cane bagasse

In order to keep the vascular system fluid and allow for the devolvement of solid blood clots in the occurrence of blood vessel injury, the body has interrelated complex systems that are essential for maintaining homeostasis (Chapin and Hajjar 2015). A thrombus is a blood clot made up of fibrin, platelets and cellular remnants that have been trapped inside a vascular lumen (Mousa 2010). Fibrin is the fundamental protein constituent of blood clots and, is often created from fibrinogen by the activity of the thrombin enzyme within blood arteries that have been damaged by disease or trauma (Kattula et al. 2017). This thrombus can form as a result of disturbances in the anticoagulation process that prevent the blood from flowing freely, which typically results in myocardial infection, rheumatoid arthritis (RA), deep venous thrombosis (DVT), ischemic strokes and a group of cardiovascular diseases (CVDs) linked primarily with the heart and blood vessels (Seo et al. 2018). Fibrin can be degraded by fibrinolytic (thrombolytic) agents such as plasmin, which is often activated from the non active plasminogen by a tissue-type plasminogen activator (tPA) and urokinase (Wang et al. 2011).

Additionally, bacterial sources of streptokinase and staphylokinase have been well regarded and utilized as thrombolytic drugs (Peetermans et al. 2014). However, these agents have a number of drawbacks, including high costs for clinical usage, poor stability and specificity, a short half-life, allergic responses and clot recurrence at the site of persistent thrombosis (Vaidya et al. 2012). Due to these undesirable side effects, researchers are constantly looking for ways to improve the efficacy and specificity of fibrinolytic treatments that are safe and efficient at breaking down fibrin. In recent decades, interest in microbial fibrinolytic enzymes has increased significantly on a global scale (Mahajan et al. 2012). Several fibrinolytic enzymes have been successively extracted from animals, plants, microbes and fermented foods (Ali and Bavisetty 2020).

Microorganisms continue to be the primary source for enzyme production due to mass culture viability, substrate specificity and low production costs. Microbial sources from fermented foods have become quite important especially in the commercial production of fibrinolytic enzymes due to their advantageous qualities, such as high substrate specificity, cheap production cost and high yield rates (Vijayaraghavan et al. 2019). Additionally, fibrinolytic enzyme from microorganisms have been assured for their negligible negative impacts in patients, particularly those with inborn drawbacks (Ali and Bavisetty 2020).

As a result, several microbiological sources, such as actinomycetes, bacteria, algae, and even mushrooms have yielded fibrinolytic enzymes (Kotb et al. 2015). Less fibrinolytic strains of filamentous fungi have been mentioned in literature. Fibrinolytic enzymes were previously extracted from A. oryzae (Shirasaka et al. 2012), Aspergillus brasiliensis (Kotb et al. 2015), Bionectria ochroleuca and Cladosporium cladosporioides (Rovati et al. 2010), Cochliobolus lunatus (Abdel-Fattah and Ismail 1984), Fusarium oxysporum (Tao et al. 1998), Fusarium pallidoroseum (El-Aassar 1995), Fusarium sp. (Wu et al. 2009; Ueda et al. 2007), Mucor subtilissimus (Nascimento et al. 2016), Oidiodendron flavum (Tharwat 2006), Penicillium chrysogenum (El-Aassar et al. 1990), Paecilomyces tenuipes, (Choi et al. 2013), Rhizopus microspores(Zhang et al. 2015), Rhizopus chinensis (Xiao-Lan et al. 2005) and Tricholoma saponaceum (Kim and Kim 2001).

Temperature and pH changes can greatly affect the sensitivity of fibrinolytic enzymes. The activity of fibrinolytic enzymes is markedly reduced in the presence of the acidic environment of the stomach (Abbas et al. 2019). In addition, fibrinolytic enzymes are macromolecular substances that lose their activity after prolonged storage. Consequently, it is difficult to produce fibrinolytic enzymes for commercial application in medicine. As a result, fibrinolytic enzymes must be commercially available in order to be economically viable. For this reason, many scientists have used different biotechnological procedures such as gene cloning and mutations to reach a maximum amount of enzyme in pure state and good stability(Yao et al. 2019). In parallel, some researchers have focused on viable development, promising a cost-effective bioprocess and increased yield by using agro-industrial residues as substrates (Abu-Tahon and Isaac 2016).

From a physiological standpoint, SSF has the capacity to produce enzymes. Numerous advantages come with this system, including high volumetric production, significantly greater product concentration, decreased effluent generation, and a requirement for basic fermentation equipment (Sharma et al. 1991). Moreover, bounded amino acids (solid substrates) are more stable chemically than free ones, therefore, the former are preferred for broad technological application as substrates for bulk production of economically valuable compounds (El-Sayed et al. 2012). Practically no comprehensive studies on the potential of filamentous fungi for fibrinolytic enzymes production. Consequently, in this research, the possibility of Cochliobolus hawaiiensis AUMC 8606 for production of fibrinolytic enzymes was optimized under submerged (SmF) and solid state fermentation (SSF).

Chemicals

Folin reagent, soya bean meal, corn steep liquor, Sephadex G-75 and diethyl amino ethyl cellulose (DEAE-Cellulose) were bought from Sigma-Aldrich (St. Louis, Mo). Cane and beet molasses were obtained from Hawamdiah and Kafr-El-Sheikh sugar factories, respectively. Crude whey was collected from Kafr-El-Sheikh sugar and Hawamdiah factories, respectively. Crude whey was gathered from the Kaluobia rural areas and the other agro-industrial byproducts were collected locally. The remaining substances were of analytical grade.

Isolation and screening of Fibrinolytic enzyme producing fungi

Fungi utilized in this research were previously isolated from several Saudi Arabian Kingdom soil samples and identified by Assiut University Mycological Centre (AUMC). The fungi were subjected to rapid assay of proteolysis by zone of clearance on skimmed-milk agar plates (g/l): sucrose, 30.0; NaNO₃, 3.0; KH₂PO_4,3.0; yeast extract, 1.0; KCl, 0.5; MgSO₄·7H₂O, 0.5; skimmed milk, 100; agar, 20.0; pH 7.0, and incubated at 30^oC for 7 days. Then the fungi which recorded the highest clear zones were exposed to spectrophotometric fibrinolytic enzyme assay under the same previous medium without addition of agar in triplicate sets of 250 ml Erlenmeyer conical flasks containing 50 ml of sterilized medium. Each flask was inoculated with 1.0 ml of fresh spore suspension (1×10⁵ spore/ml)obtained from 7-days old cultures and incubated under the same previous conditions. Then, the fungus with the highest production was chosen for further examination.

Fibrinolytic enzyme production under submerged culture conditions (SmF)

Fibrinolytic enzyme production under SmF by Cochliobolus hawaiiensis was optimized using modified liquid medium as previously reported (Hua et al. 2008). Triplicate sets of 250 ml Erlenmeyer flasks containing 50 ml of sterilized medium containing (g/l): Peptone, 15; glucose, 15; NaH₂PO₄, 1; Na₂HPO₄, 6 and MgSO₄, 0.5. The medium was adjusted to pH 7.0 using 0.1 N NaOH, inoculated with 1.0 ml of fresh spore suspension (1×10⁵ spore/ml)obtained from 7-days old cultures of C. hawaiiensis and incubated at agitation speed of 150 rpm and 30°C for 4 days. The cultures were then filtered through Whatman No. 1 filter paper and the supernatant was centrifuged at 4000 rpm for 5 min, then the enzyme activity and total protein were measured.

Enzyme production under solid-state fermentation (SSF)

Rice bran,wheat bran, shrimp shell powder, crab shell powder, corn cobs, sugar cane bagasse and soya bean meal and were investigated for Fibrinolytic enzyme production under SSF by C. hawaiiensis. In each 250 ml Erlenmeyer conical flasks, 5 g of each dried substrate was moistened with 2 ml of the previously adjusted SmF medium. After autoclaving, the medium was inoculated with 1 ml of fungal spore suspension of 7-days old culture of C. hawaiiensis and incubated at 30°C for five days. The fermented substrate was thoroughly mixed with 50 ml of 0.1 M phosphate buffer (pH 7) using a rotary shaker at 3 x g for 30 min. Each flask's whole contents were filtered then centrifuged at 1252 x g at 4^oC for 15 min. The clear supernatant was employed as the crude enzyme preparation.

Fibrinolytic enzyme assay

The enzyme activity was measured by the method of Anson (Anson 1938) with a few modifications. The reaction mixture consists of 1 ml of 1.2% of bovine fibrinogen solution in 0.1 M phosphate buffer (pH 7) and 1 ml cell-free filtrate. The reaction mixture was incubated for 2 h at 37^oC. The reaction was then stopped by addition of 2 ml of 10% (w/v) trichloroacetic acid. This was followed by centrifugation and assaying the solubilized protein in the supernatant. One unit of fibrinolytic activity (U) was defined as the amount of enzyme needed to liberate 1 μg of L-tyrosine/ml/min at 37^oC.

Protein determination

The protein content of the enzyme preparations was evaluated by the method of (Lowry 1951). All determinations were made in triplicates and the mean values are shown.

Treatment of beet and cane molasses

Beet and cane molasses were prepared as descried previously (Abu-Tahon and Isaac 2016). Molasses were analyzed for carbohydrates content, nitrogen and minerals by the Services Central Lab, National Research Centre Cairo-Egypt.

Crab and shrimp shell powder

Shrimp and crab shells were gathered, cleaned many times in warm water, then in distilled water, and allowed to air dry for 24 hours at 60^oC in an oven. Then, it was crushed to fine particles.

Optimization of fibrinolytic enzyme production parameters under SmF.

Fibrinolytic enzyme production parameters were optimized under SmF conditions. Many carbon sources, specially, fructose, mannitol, sucrose, maltose, lactose, and soluble starch were added to the medium at equimolecular weight to carbon of glucose of previous medium. The peptone-free medium was supplemented with various nitrogen sources, particularly, yeast extract, casein, beef extract, ammonium chloride, ammonium sulphate and sodium nitrate. The supplemental nitrogen sources were in equimolecular weight to nitrogen of peptone. Moreover, the impact of agitation speed (125, 150, 175, 200 and 225 rpm) was considered.

Optimization of fibrinolytic enzyme production parameters under SSF

The SSF was also optimized through various parameters including moisture level of substrate (40, 60, 80, 100, 120, 140 and 160%, v/w), size of inoculum (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 ml of spores suspension), initial pH of basal medium (6.6, 7.0, 7.4, 7.8, 8.2, 8.6, and 9) and the incubation temperature (30, 35, 40, 45, and 50 ^oC). The impact of supplementation of the basal medium with different liquid by-products (cane molasses, beet molasses, corn steep liquor and whey) as additives (40%, v/v) and effect of different concentrations of steep liquor（20: 100％, v/v) were also ascertained.

Enzyme purification

Enzyme purification was started by precipitation of 500 ml of crude enzyme preparation (CEP) with (NH₄)₂SO₄, ethanol, acetone or iso-propanol after being at 4°C over night. Protein precipitates recorded the highest enzyme activity were then fractionated on Sephadex G-75 column. After elution active fractions loaded on DEAE-Cellulose column then eluted with gradient of 0-0.8 M NaCl dissolved in the corresponding buffer. The attained enzyme was lyophilized for further examinations.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SDS-PAGE was completed by (Laemmli 1970) and the proteins were detected by Coomassie Brilliant Blue R250 staining.

Determination of kinetic parameters

The optimum pH of the enzyme activity was investigated at 37 ^oC in various buffers pH (5.4-8.2). The pH stability was estimated by calculating its remaining activity for different periods with different pH values. Optimum temperature of activity was determined by incubating enzyme at different temperatures extending from 29 to 45^oC in 0.1 M phosphate buffer (pH 7). Thermostability of the enzyme was monitored at various temperatures for different periods.

Effect of different metal ions and various inhibitors on fibrinolytic enzyme activity.

The effect of some metal ions (i.e. K⁺, Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Zn²⁺, Mn²⁺ and Hg²⁺), ethylenediamine-tetraacetat (EDTA), phenylmethylsulfonyl fluoride (PMSF) and iodoacetate, on the enzyme activity was tested. The enzyme was pre-incubated with these components (metal ions were added as chlorides) for 15 min at 5 mM, and then the relative enzyme activity was calculated.

Substrate specificity of purified fibrinolytic enzyme

Various protein substances (1.2%) were used, namely, fibrin, fibrinogen, casein and serum albumin. Moreover, the purified enzyme was incubated with gradual concentrations of fibrin and Michaelis constant (Km) and maximum velocity (V_max) of enzyme activity were calculated by linear regression from Lineweaver-Burk plot (Lineweaver and Burk 1934).

Assessment of thrombolytic activity percentage

Thrombolytic activity percentage was measured according to the method described by (Zhou et al. 2022) with minor modifications. 100 μL of 0.25 M CaCl₂ was added to 1.0 ml of buffalo blood containing 3.8% sodium citrate, and let to clot at room temperature for 1 h. Aliquots of 500 μL with various concentrations of purified fibrinolytic enzyme (100-220 U/ml protein) were incubated with 0.1 g blood clot at 37^oC for 10 h. To determine the phosphate buffer's impact (pH 7) as a positive control and distilled water as a negative control, a clot was added to phosphate buffer and distilled water under the same previous conditions, then the thrombolytic activity of all groups was calculated using the following equation

Visual anticoagulant and thrombolytic activities of purified fbrinolytic enzyme

The anticoagulation activity of fibrinolytic enzyme was monitored in both Eppendorf tubes and microscope slide. In Eppendorf tubes, 0.5 ml of fresh buffalo blood was mixed with different concentration of purified enzyme (180 and 200U/ml). The blood in the other tubes mixed with EDTA (positive control) or CaCl₂ (negative control). The experiment was performed at room temperature and the visual monitoring of the blood clot formation was done throughout time, and anticoagulation was checked by inverting the tubes. Each tube was also examined microscopically under a light microscope. The thrombolytic activity of was investigated according to the method of (Harish et al. 2015) with some modification. On the slides, fresh buffalo blood was applied and allowed to coagulate Then, purified enzyme solution (200 U/ml) was applied on to the slides and allowed to stand for 5 min. Both visually and by the use of a light microscope, the blood clot dissociation was detected

Determination of antiplatelet activity

The effect of fibrinolytic enzyme on ADP-induced platelet aggregation was evaluated using the method described by (Xia et al. 2012). The platelet number was determined by blood cell count and platelet-rich plasma (PRP), and wash platelet (WP) was adjusted at 3 x 10⁸ cells/ml by platelet-poor plasma (PPP) and HEPES-Tyrode’s buffer, respectively. Various concentrations of purified enzyme (100-220 U/ml proteins) were incubated with 300 μL PRP or WP at 37^oC for 10 min. Then ADP (5 μM) was added, and platelet aggregation was recorded using an aggregometer (Chrono-Log Corporation, Havertown, USA). Additionally, to evaluate the maximum platelet aggregation of WP, the fibrinogen was mixed into WP to achieve a final concentration of 0.3 g/l. For the control, the sample was induced by ADP in the presence of distilled water (negative control) and phosphate buffer (positive control).

Determination of anticoagulation activity in Vitro

The method used to study the anticoagulant activity of the purified firbrinolytic enzyme is described by (Doley and Mukherjee 2003). Various concentrations of the purified enzyme solution were preincubated with 300 μL of PPP for 10 min at 37 ^oC. The effect of the enzyme on activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and FIB level in plasma was determined using a coagulometer (STA Compact, Diagnostica stago S.A.S, France). For the control, the sample was induced by ADP in the presence of distilled water (negative control) and phosphate buffer (positive control).

Statistical Analysis

The obtained data were statistically analyzed with SPSS. Results were considered highly significant, significant, or non-significant, where p ≤ 0.01, p ≤ 0.05 and p > 0.05, and represented by HS, S and NS, respectively. The data were statistically compared to the highest value obtained, which was marked as ●.

Screening of fungal isolates for Fibrinolytic enzyme producers

According to zone of clearance on skimmed-milk agar plates, twenty fungal species were evaluated for their ability to produce fibrinolytic enzyme belonged to nine genera: namely, Aspergillus, Cochliobolus, Cunninghamella, Emericella, Fusarium, Paecilomyces, Penicillium, Trichoderma and Rhizopus (data not shown). Based on the maximum zone diameter, Cochliobolus hawaiiensis recorded a zone diameter of (3.6 ± 0.15 cm) and diameter colony of (2.6 ± 0.11 cm), followed by Penicillium chrysogenum with a zone diameter of (2.7 ±0.13 cm) and diameter colony of (2.1 ±0.25 cm), followed by Rhizopus stolonifer with a zone of (1.7 ±0.31 cm ) and diameter colony of ( 1.4 ±0.22 cm ), then Fusarium solani with a zone of (1.1 ±0.19 cm) and diameter colony of ( 0.9 ±0.11 cm ). Then the four isolates with the highest clear zones were subjected to fibrinolytic enzyme assay under the same previous medium without agar. Our results showed that fibrinolytic enzyme activity of Cochliobolus hawaiiensis, Penicillium chrysogenum, Rhizopus stolonifer and Fusarium solani recorded 35.43 ±0.83, 23.44 ±1.13, 20.34 ±1.53 and 11.29± 1.17 U/ml, respectively. Based on the maximum zone diameter and fibrinolytic enzyme production, Cochliobolus hawaiiensis Alcorn AUMC 8606 was recorded as the most potent fungus for fibrinolytic enzyme production. Hence, this isolate was selected for the subsequent experimentation under SmF.

Optimization of Fibrinolytic enzyme production from C. hawaiiensis

The environmental and cultural factors have an impact on the production of microbial enzymes. In this regard, a number of tests were conducted to ascertain the impact of certain circumstances on the produced enzyme of C. hawaiiensis AUMC 8606. Under SmF on the previously mentioned medium, the fibrinolytic enzyme activity recorded (46.49 ± 1.2 U/ml) with a protein content (4.42 ± 0.27 mg/ml) and specific activity (10.52 ± 0.32 U/mg protein).

The effect of various carbon sources, was investigated (Table 1). They were separately introduced to the fermentation medium as the only carbon source in equimolecular weight to carbon of glucose. The maximum fibrinolytic enzyme production was increased to (22.34 ± 1.09 U/mg protein) with sucrose. While the lowest enzyme activity was recorded with soluble starch (3.88 ± 0.29 U/mg protein). Moreover, a set of organic and inorganic nitrogen sources were separately added as a sole nitrogen source to the fermentation medium. Table 1 showed that the inorganic sources of nitrogen caused an enhancement in the production of the enzyme reached the maximum productivity with ammonium sulphate (28.9 ± 1.04 U/mg protein).

Table 1

Impact of various parameters on Fibrinolytic enzyme production by from *Cochliobolus hawaiiensis* on submerged culture conditions
Carbon source	In equimolecular weight to Glucose	Glucose	Mannitol	Fructose	Sucrose	Lactose	Maltose	Soluble starch
Carbon source	Specific Enzyme activity (U/mg)	10.52 ± 0.32●	7.55 ± 2.09 S	6.88 ± 0.08 HS	22.34 ± 1.09 HS	5.00 ± 0.21 HS	13.32 ± 0.99 S	3.88 ± 0.29 HS
Nitrogen source	In equimolecular weight to peptone	Peptone	Beef extract	Yeast extract	Casein	NaNO₃	Ammonium Chloride	Ammonium sulphate
Nitrogen source	Specific Enzyme activity (U/mg)	22.34 ± 1.09●	18.55 ± 0.08 HS	19.66 ± 1.41 S	16.0 ± 0.45 HS	24.7 ± 2.19 NS	23.8 ± 1.09 NS	28.9 ± 1.04 HS
Agitation speed	rpm	125	150	175	200	225
Agitation speed	Specific Enzyme activity (U/mg)	25.6 ± 1.70 S	28.9 ± 1.04●	32.7 ± 3.70 NS	39.7 ± 1.10 HS	22.6 ± 0.89 HS

While, the sources of organic nitrogen did not have any stimulating effects compared to the control medium. Our results showed that increasing the agitation velocity resulted in increasing the productivity of fibrinolytic enzyme by C. hawaiiensis reached the maximum accumulation at 200 rpm (39.7 ± 1.1 U/mg protein, Table 1). While increasing the shaking velocity above this rang resulted in decreasing the production of enzyme reached 22.6 ± 0.89 U/mg protein at 225 rpm.

Optimization of fibrinolytic enzyme production under SSF from C. hawaiiensis

The fibrinolytic enzyme production parameters by C. hawaiiensis were optimized under SSF. The impact of using different agro-industrial by-products namely, Wheat bran, rice bran, shrimp shell powder, crab shell powder, corn cobs, sugar cane bagasse and soya bean meal was investigated. Table 2 showed that the maximum yield of fibrinolytic enzyme by C. hawaiiensis was obtained with sugar cane bagasse (66.98 ± 0.29 U/ml) with a protein content (0.49 mg/ml) and specific activity (136.7 ± 3.51 U/mg protein), followed by wheat bran (105.6 ± 2.21 U/mg protein), soya bean meal (69.84 ± 1.55 U/mg protein) and corn cobs (44.76 ± 1.82 U/mg protein). While, the other agro-industrial products recorded low enzyme production.

Table 2

Impact of various parameters on Fibrinolytic enzyme production by from *Cochliobolus hawaiiensis* on solid state fermentation
Agro-industrial residues	5g/flask	Wheat bran	Rice bran	shrimp shell powder	Crab shell powder l	Corn cobs	Sugar cane bagasse	Soya bean meal
Agro-industrial residues	Specific Enzyme activity (U/mg protein)	105.6 ± 2.21 HS	33.88 ± 3.15 HS	22.76 ± 2.06 HS	18.1 ± 2.20 HS	44.76 ± 1.82 HS	136.7 ± 3.51●	69.84 ± 1.55 HS
Moisture level	(%, v/w)	40	60	80	100	120	140	160
Moisture level	Specific Enzyme activity (U/mg protein)	136.7 ± 3.51●	155.3 ± 3.59 HS	170.8 ± 2.53 HS	210.66 ± 3.59 HS	178.8 ± 4.30 HS	110.7 ± 4.74 HS	89.1 ± 3.19 HS
Inoculum size	ml	0.5	1	1.5	2	2.5	3
Inoculum size	Specific Enzyme activity (U/mg protein)	155.94 ± 1.78 HS	210.66 ± 3.59●	220.8 ± 6.29 S	280.6 ± 3.58 HS	271 ± 4.11 NS	240 ± 3.18 HS
Initial pH	pH	6.6	7	7.4	7.8	8.2	8.6	9
Initial pH	Specific Enzyme activity (U/mg protein)	276.6 ± 5.01 NS	280.6 ± 3.58●	294.6 ± 3.31 HS	326.54 ± 4.01 HS	302.3 ± 3.91 HS	288.2 ± 4.41 S	258.3 ± 3.51 HS
Incubation temperature	^oC	30	35	40	45	50
Incubation temperature	Specific Enzyme activity (U/mg protein)	326.54 ± 4.01●	345.21 ± 2.75 HS	320.22 ± 3.38 S	278.7 ± 3.11 HS	198.98 ± 2.27 HS
Additives	40%, (v/v)	Without additives	Corn steep liquor	Cane molass	Beet molass	Whey
Additives	Specific Enzyme activity (U/mg protein)	345.21 ± 2.75●	377.89 ± 3.71 HS	340.33 ± 3.11 NS	338.53 ± 2.51 S	343.28 ± 3.44 NS
Corn steep liquor concentrat- ions	(% ,v/v)	20	40	60	80	100
Corn steep liquor concentrat- ions	Specific Enzyme activity (U/mg protein)	362.8 ± 2.75 S	377.89 ± 3.71●	391.7 ± 5.66 S	405 ± ± 4.51 HS	380.8 ± 2.55 NS

The impact of gradual moisture level of substrate (40, 60, 80, 100, 120, 140 and 160%, v/w) on fibrinolytic enzyme production was investigated (Table 2). Results showed that gradual increase in enzyme production with increasing the moisture level of the substrate reaching the maximum activity (210.66 ± 3.59 U/mg protein) at a moisture level (100%, v/w). Moreover, the extra increase than 100%, w/v caused an inhibition in enzyme production by C. hawaiiensis. As shown in Table 2 The maximum fibrinolytic enzyme production was gained when the culture medium was inoculated with 2 ml of inoculum containing approximately 2×10⁵ spore/ ml (280.6 ± 3.58 U/mg protein).With an additional increase in inoculum size above 2.5 ml, enzyme productivity significantly decreased reached about (240 U/ml, ± 3.18 U/mg protein) at inoculum size of 3.0 ml.

The effect of initial pH of basal medium on fibrinolytic enzyme production by C. hawaiiensis was also investigated. Table 2 revealed that the productivity of fibrinolytic enzyme increased with increasing the alkalinity of the medium to reach maximum yield at pH 7.8 (326.54 ± 4.01 U/mg protein). Regarding the impact of incubation temperature on the production of enzyme, our results revealed that maximum enzyme production was obtained by C. hawaiiensis at 37^oC (345.21 ± 2.1 U/mg protein, Table 2) and any increase above this degree caused an inhibition in enzyme production.

Several by-products (corn steep liquor, whey, cane molasses and beet molasses were added separately to the basal medium by (40%, v/v) as additives to enhance fibrinolytic enzyme production. As shown in Table 2, corn steep liquor recorded stimulatory effect on enzyme production (377.89 ± 3.71 U/mg protein).While, the other additives has no stimulatory effect on enzyme production. In addition, increase the concentration of corn steep liquor to (80%, v/v) resulted in increasing the enzyme yield to (405 ± 4.51 U/mg protein).

Purification of fibrinolytic enzyme from C. hawaiiensis

The purification summary of fibrinolytic enzyme from the crude enzyme preparation (CEP) of C. hawaiiensis under SSF was collected in Table 3. The outcomes demonstrated that the fibrinolytic enzyme was partially purified by fractional precipitation with saturated ammonium sulphate (50–90%). Among all the fractions, the 70% ammonium sulphate fraction showed the highest fibrinolytic enzyme recovered activity of 82.48% with specific activity of 579.06 U/mg protein and 1.42 purification fold. After the precipitation process, the semipurified fibrinolytic enzyme was injected onto the Sephadex G-75 gel filtration column. The enzyme activity of the most active fractions (number 16 to 22) from the gel filtration column recorded a yield recovery of 73.73and 1165.7 U/mg protein with 2.87 purification fold. The semi-purified enzyme fractions were transferred to a diethyl amino ethyl cellulose ion-exchange chromatography (DEAE-Cellulose) column. The fractions (21–27) showed a recovery of 55.89% with specific activity of 2581.6 U/mg protein and 6.37 purification fold.

Table 3

Summary of purification of fibrinolytic enzyme from *C. hawaiiensis* under SSF
Purification step	Total activity (U)	Total protein (mg)	Specific activity (U/mg protein)	Yield (%)	Purification fold
CEP	105300	260	405	100.0	1.00
Ammonium sulphate (70%)	86860	150	579.06	82.48	1.42
Sephadex G-75	77640	66.6	1165.7	73.73	2.87
DEAE-Cellulose	58861	22.8	2581.6	55.89	6.37

Determination of enzyme purity of purified fibrinolytic enzyme from C. hawaiiensis

The SDS-PAGE electrophoresis of the purified fibrinolytic enzyme, confirming the existence of a single protein band (Fig. 1). The molecular mass of detected band was 35 KDs.

Influence of pH on enzyme activity and stability of purified fibrinolytic enzyme from C. hawaiiensis

The results revealed that the purified enzyme showed a great activity at pH 6.6–7.4 with a maximum at pH 7. On each side of this pH range, the enzyme activity was shown to decrease (Fig. 2A). The impact of pH on the enzyme stability showed that it retained full activity after one hour of incubation at pH 7 and about 80% of the original activity after one hour of incubation at pH 6.6 or 7.4 (Fig. 2B). Also, the enzyme retained about 65% of its original after 30 min of incubation at pH 7.8

Impact of temperature on enzyme activity and stability of purified fibrinolytic enzyme from C. hawaiiensis

Maximum activity of purified C. hawaiiensis fibrinolytic enzyme was recorded at a great activity at temperature range 33–41 ^oC with a maximum at 37^oC (Fig. 2C). A progressive decline in enzyme activity was seen at higher temperatures. The results showed that the purified enzyme retained about 82% of its activity at 41^oC for 60 min and retained about 65% of its original activity at 45^oC for 30 min (Fig. 2D).

Effect of different metal ions and inhibitors on of purified fibrinolytic enzyme from C. hawaiiensis

The effect of some metal ions which act as activators or inhibitors on C. hawaiiensis fibrinolytic enzyme activity was tested in Table 4. The results showed that Mg²⁺ and Ca²⁺ moderately induced fibrinolytic activity by 26.8% and 22.4%, respectively compared to control. Moreover, K⁺ slightly enhanced the fibrinolytic activity by 5.4% % compared to control. However, the enzyme activity was greatly suppressed in the presence of Cu²⁺and Zn²⁺ which inhibited the fibrionlytic enzyme by 59.9% and 44.89%, respectively compared to control. Moreover, the other ions Co²⁺, Hg²⁺ and Mn²⁺ caused considerable suppression in fibrinolytic activity of the produced enzyme. Fibrinolytic enzyme was significantly inhibited by (PMSF) retaining only about 18.65%. While, the activity of enzyme was apparently not affected by EDTA and iodoacetate.

Table 4

**Effect of some chemical components (metal ions and enzyme inhibitors) on activity of purified fibrinolytic enzyme from** *C. hawaiiensi*s
Components	Relative enzyme activity (%)
Control	100 ±1.10 HS ●
Mg²⁺	126.8 ± 2.66 HS
Ca²⁺	122.4 ± 3.16 HS
K⁺	105.43 ± 3.16 S
Co²⁺	74.87 ± 5.17 HS
Cu	40.16 ± 1.16 HS
Zn²⁺	55.11 ± 2.23 HS
Hg²⁺	74.10 ± 1.56 HS
Mn²⁺	80.66 ± 2.16 HS
Enzyme inhibitors
EDTA	100 ± 1.06 HS
PMSF	18.65 ± 2.33 HS
Iodoacetate	96.76 ± 3.56 NS
*metal ions were added as chlorides

Substrate Specificity Of Purified Fibrinolytic Enzyme

The substrate specificities of C. hawaiiensis fibrinolytic enzyme are showed in Fig. 3. The enzyme showed the highest affinity towards Fibrin (240 ± 2.2 U/ml) followed by fibrinogen (200 ± 2.4 U/ml) and casein (144 ± 3.1 U/ml). While albumin had the enzyme's lowest level of activity (44 ± 3.4 U/ml). V_max of the purified enzyme was calculated to be 240U/ml. Km value was calculated from the Lineweaver-Burk plot of reciprocals of initial velocities and fibrin concentrations to be 47.61 mmol (data not shown).

Assessment of thrombolytic activity percentage of purified fibrinolytic enzyme from C. hawaiiensis

The thrombolysis ability of C. hawaiiensis purified fibrinolytic enzyme was evaluated with clots from buffalo whole blood (Fig. 4). Our results showed that the rate of thrombolytic activity of both negative and positive control was negligible and also there was no significant difference between their values. To evaluate the thrombolytic activity of different concentrations of purified enzyme, the obtained results were statistically compared to positive phosphate buffer control. Fibrinolytic enzyme showed thrombolysis activity on the blood clots in a dose-dependent action. With increasing the enzyme concentration, the rate of blood clot lysis was increased reached to the complete lysis at 200 U/ml of purified enzyme.

Visual anticoagulant and thrombolytic activities of purified fibrinolytic enzyme from C. hawaiiensis

Tolerable biomedical relevance of the C. hawaiiensis purified enzyme was examined by investigating its ability to act as an anticoagulant agent and prevent blood clots, and the capability to dissolve blood clots when applied as thrombolytic agent. The effect of purified enzyme as anticoagulant on buffalo blood was shown in Fig. 5A. The results showed that coagulation occurred in the negative control Eppendorf tubes which contain only the blood samples within 15 minutes (5A-1). While, the addition of EDTA to Eppendorf tubes (positive control) prevented the formation of clot up to 24 h (5A-2). Moreover, the two concentrations of purified enzyme (180 and 200 U/ml) prevent the coagulation in blood samples to up to 24 h with different percentage as shown in Fig. 5A-3 and Fig. 5A-4, respectively. Furthermore, the purified enzyme displayed potent activity in dissolution of preformed blood clots within 5 min (Fig. <link rid="fig7">5</link>A-5). Moreover, the optical microscopic micrographs showed clear dispersion of the blood clot by using the purified enzyme (200 U/ml) compared to the other treatments (Fig. 5B-4).

The Thrombolytic activity of purified fibrinolytic enzyme on buffalo blood was shown in Fig. 5C. Coagulation occurred in the negative control blood sample which left alone on the slide blood completely clotted within ten minutes (Fig. 5C-1). While the coagulation did not observed in the Fig. 5C-2 and Fig. 5C-3 up to 12 h and 8 h respectively, where EDTA and purified enzyme (200 U/ml) were added to the blood samples, respectively. Furthermore, the purified enzyme (200 U/ml) displayed potent activities in dissolution of previously formed blood clots within 5 min. Furthermore, the optical microscopic micrographs showed clear dispersion of the blood clot by using the purified enzyme (200 U/ml) compared to the slides of other treatments (Fig. 5D-3)

Determination of antiplatelet and anticoagulation activity of purified fibrinolytic enzyme from C. hawaiiensis

Figure 6 showed that ADP succeeded in inducing platelet aggregation in PRP and WP. Aggregation rate in PRP and WP of the phosphate buffer experiment (positive control) was 50% and 65% respectively and distilled water (negative control) was 52% and 63%, respectively with no significant difference between positive and negative control values. To evaluate aggregation rate of different concentrations of purified enzyme, the obtained results were statistically compared to positive phosphate buffer control. In PRP, a gradual increase in fibrinolytic enzyme concentrations (100: 220 U/ml), caused a significant decrease in plate aggregation percentage to 22% at enzyme concentration of 220U/ml, compared with positive control (Fig. 6A). In WP, gradual increasing in fibrinolytic enzyme concentration resulted in a great reduction in plate aggregation percentage reached the maximal value 12% with enzyme concentration of 220 U/ml compared with positive control (Fig. 6B).

Our results (Fig. 7) showed that, APTT, PT, TT, and FIB recorded (45.7 s ± 1.31, 2.2 ± 0.21, 20.6 s ± 2.32 and 3.90 ± 0.23 g/l, respectively in positive control groups and recorded (43.8 s ± 1.53, 2.1 ± 0.23, 19.8 s ± 1.54 and 3.73 ± 0.99 g/l, respectively in negative control groups with no significant difference between positive and negative control groups. To evaluate anticoagulation activity of different concentrations of purified enzyme, the obtained results were statistically compared to positive phosphate buffer control. The APPT (Fig. 7A) and PT (Fig. 7B) showed a significant increase in their values with gradual increase of enzyme concertinos reaching the maximum value of (73.7 s ± 2.23 and 3.8 ± 0.24, respectively) at enzyme concentrations of 180 and 200 U/ml, respectively compared with control groups. Meanwhile, Fibrinolytic enzyme showed no significant increase in TT for the first five concentrations (100: 180 U/ml, Fig. 7C), while, a significant increase of 33.1 s ± 2.13 occurred at 220 U/ml in compared with control. Also, the content of FIB was significantly reduced to 2.05 ± 0.16 g/l with a concentration of 220 U/ml of fibrinolytic enzyme compared with control (Fig. 7D).

Although urokinase injections intravenously are a common fibrinolytic medicine, the enzyme is pricey and patients may suffer from negative side effects such bleeding complications, acute coronary reocclusion and resistance to reperfusion (Bode et al. 1996). So, Numerous fungi were examined for their ability to produce fibrinases. In our study, C. hawaiiensis Alcorn AUMC 8606 showed maximum fibrinolytic enzyme compared with other fungal strains. Similar results were obtained by (Abdel-Fattah and Ismail 1984), who recorded that C. lunatus was the potent funus for fibrinolytic enzyme production.

Submerged culture conditions (SmF) has been employed primarily for fungal enzyme synthesis because it has several advantages, such as well-controlled process parameters, enhanced mass transfer, attainment of an oxygen delivery mechanism and better extracellular enzyme recovery (Stoykov et al. 2015). The production of fibrinolytic enzyme under SmF has been previously recorded (Pan et al. 2019; Wu et al. 2019).

The carbon source is the most crucial raw material for microbial fermentation since it provides the necessary microbial components and the fundamental material for energy production (Lin et al. 2014). Study on the effect of carbon sources on enzyme production by C. hawaiiensis showed that fibrinolytic enzyme production was maximally attained with sucrose. Similar results were previously recorded for the production of fibrinolytic enzyme (Wu et al. 2019; Vijayaraghavan et al. 2019). Nitrogen sources are crucial for microbial fermentation, as they are used to synthesize proteins, nucleic acids and other substances necessary for microbial development (Yu et al. 2008). Ammonium sulphate exerted the greatest increase in the production of enzymes when compared to other nitrogen sources. The present results were in line with that recorded for fibrinolytic enzyme production by Aspergillus brasiliensis (Kotb et al. 2015). In contrast to our result, El-Aassar (El-Aassar 1995) reported that organic nitrogen sources such as casein was the preferred nitrogen source for fibrinolytic enzyme synthesis by Penicillium chrysogenum.

In SmF, The efficient mixing of oxygen, heat, and nutrients in a fermenter is made easier by agitation, and the spreading of air into tiny bubbles reduces the clumping of fungal cells, increases the gas-liquid contact area, and raises the concentration of dissolved oxygen(Pan et al. 2019). Our results showed that the enzyme activity increased with increasing the agitation speeds reached its maximum productivity at 200 rpm. Similar result was obtained by (Wu et al. 2019) who recorded that 200 rpm is the beast agitation velocity for production of fibrinolytic enzyme by Bacillus subtilis. Moreover, our result recoded noticeable decrease in enzyme production at higher velocities. This may be explained by the fact that excessive agitation uses more energy, produces heterogeneous mixing and creates pressure, which can harm delicate microorganisms and interfere with the production of certain products(Zhou et al. 2018).

The use of solid state fermentation (SSF), which can take place in the absence of free water and so create an environment comparable to the chosen microbes natural habitat, was recommended as the best method for microbial fermentation(Holmström and Kjelleberg 1999). Additionally, compared to submerged fermentation, this approach offers several benefits, including easy substrate consumption, reduced pollution, and high yield. Our findings demonstrated that sugarcane bagasse is a useful agricultural waste for the synthesis of fibrinolytic enzyme by C. hawaiiensis. Our results were in coincidence with that reported the sugarcane bagasse as the best agro-industrial product for maximum fibrinolytic enzyme production from Bacillus sp and Alcaligenes aquatilis, respectively reported by (Hmood and Aziz 2016; Prabhu et al. 2021), respectively.

Moisture content is a significant factor in SSF since the moisture of the medium determines the rate of growth and product yield (Viniegra-González et al. 2003). Maximal production of fibrinolytic enzyme by C. hawaiiensis was recorded at moisture level of 100 %, w/v. The same moisture level was recorded previously recorded for the production of fibrinolytic enzyme from Bacillus sp.(Vijayaraghavan et al. 2017) However, lower moisture level (71%) was recorded also for proteolytic enzyme production using wheat bran by Aspergillus terreus (Abu-Tahon et al. 2020), and higher moisture level 800% was registered as the optimum for maximal production of protease using dates from Thermomyces lanuginosus (Ghareib et al. 2014). Lower moisture levels causes a decrease in nutrients solubility of substrates, a higher water tension and lower degree of substrate swelling (Zadražil and Brunnert 1981). Additionally, increased moisture contents were recorded to stimulate the growth of aerial mycelium and decrease porosity, stickiness, gas volume, and gas exchange during fermentation (Lekha and Lonsane 1994).

The unique physical conditions for growth are one of the main drawbacks of SSF, but they can be partially overcome by choosing an inoculum size that will allow the spores or mycelial fragments to initially colonize most of the substrate particles without crowding them or competing with them for the available nutrients (Abdullah et al. 1985). The maximum productivity of C. hawaiiensis fibrinolytic enzyme was achieved with 2 ml of spore suspension. The decrease that was seen with larger inoculums sizes could be due to the shortage of nutrients available for the larger biomass and faster growth of the culture (Hesseltine 1976). Influence of inoculum size on fibrinolytic enzyme production in SSF has previously been reported (Tao et al. 1997; Wu et al. 2019; Vijayaraghavan et al. 2017).

The culture initial pH has an impact on the enzymatic reactions and the transportation of numerous components by the cell membrane (Ellaiah et al. 2002). The maximal productivity of C. hawaiiensis fibrinolytic enzyme was reported at pH 7.8 and this finding was in agree with one that recorded the same initial pH of fibrinolytic enzyme production from Bacillus cereus (Biji et al. 2016). The fermentation temperature is a variable that affects the cellular metabolism and the collision of reaction rates (Elias et al. 2014). The maximal fibrinolytic enzyme production occurred at 37 ^oC. Similar results were recorded for the maximal production of fibrinolytic enzyme from Xanthomonas oryzae under SSF occurred at 37 ^oC (Vijayaraghavan et al. 2017). Higher incubation temperature significantly reduced the synthesis of enzymes this may be because higher temperature alters the transport activity or saturation level of soluble chemicals and solvents in the cells and generates a aggregation of toxin (Auesukaree 2017).

The supplementation of the cultures of C. hawaiiensis with different by products was also investigated. The induction effect of corn steep liquor on fibrinolytic enzyme production was also recorded for maximal production of fibrinoltic enzyme from Bacillus subtilis (Wu et al. 2019). Corn steep liquor is a by-product of the production of corn starch and can provide important nutrients (e.g., amino acids, proteins, vitamins, minerals and trace elements) for the growth of microbes(Zeng et al. 2018). Additionally, corn steep liquor was rich in amino acids, polypeptides, and B-group vitamins; it was a good supply of nitrogen for the majority of microbes (Yu et al. 2008; Cardinal and Hedrick 1948).

The most common method for concentrating and recovering protein from a biological mixture is precipitation, which also plays a crucial part in the purifying process (Ellaiah et al. 2002). The highest fibrinolytic enzyme activity was recorded with ammonium sulphate 70%. Ammonium sulphate (70%) was previously reported as the best precipitated agent in purification of fibrinolytic enzyme from different microbial sources(Tharwat 2006). Moreover, gel filtration by Sephadex G-75 in our purification process recorded an increase in the purity and activity of the separated enzyme. The same degree of cross linking of Sephadex G-75 is also used in purification of fibrinolytic enzyme from Paecilomyces tenuipes and Rhizopus microspores (Kim et al. 2011) and (Zhang et al. 2015), respectively.

The appearance purified fibrinolytic enzyme form C. hawaiiensis as a single band on denaturing gel with a molecular mass of 35 kDa ensures the homogeneity and purity of the purified enzyme. These results are in harmony with previous findings on other fibrinolytic enzyme from different fungal sources with the same molecular weights (Osmolovskiy et al. 2017; Popova et al. 2021; Kornienko et al. 2021).

The purified fibrinolytic enzyme form C. hawaiiensis was found to have good activity at pH ranges from pH 6.6 to 7.4 with the maximum at pH 7. These results are in agreement with the previous findings on another fibrinolytic enzyme from Rhizopus microspores (Zhang et al. 2015), Aspergillus carbonarius (Afini et al. 2016) and Neurospora sitophila(Deng et al. 2018).

The purified fibrinolytic enzyme form C. hawaiiensis was found to have good activity at reaction temperature ranges from 33 to 39 ^oC with the maximum at 37 ^oC. Above this temperature gradual decrease in enzyme activity was recorded and this may attributed to denaturation of the enzyme subunits by the effect of high heat (Dias and Weimer 1998). Our results were in complete accordance with that recorded maximal fibrinolytic activity at temperature range (37: 40 ^oC) for Aspergillus fumigates (Larcher et al. 1992), Aspergillus ustus (Popova et al. 2021), C. lunatus (Abdel-Fattah and Ismail 1984) and Rhizopus microspores(Zhang et al. 2015). Meanwhile, a higher optimum temperature for fibrinolytic activity was also recorded at 45 ^oC from Aspergillus brasiliensis (Chimbekujwo et al. 2020), 49 ^oC from Neurospora sitophila (Deng et al. 2018) and 50 ^oC from Fusarium sp (Ueda et al. 2007).

The purified fibrinolytic enzyme was enhanced by Mg²⁺ and Ca²⁺. These results were in accordance with that reported for Rhizopus microsporus and Neurospora sitophila (Zhang et al. 2015) and (Deng et al. 2018), respectively. In general, the motivational action of metal ions may be ascribed to their stabilizing impact on the conformational structure, the protection of the enzyme from autoproteolysis and heat denaturation (Secades et al. 2001). Also the purified Fibrinolytic enzyme activity was inhibited by Cu²⁺, Zn²⁺, Hg²⁺, Mn²⁺ and Co²⁺. Similar results were recorded for fibrinolytic enzyme activity from Paecilomyces tenuipes(Kim et al. 2011), Rhizopus microspores (Zhang et al. 2015) and Neurospora sitophila (Deng et al. 2018). The fact that PMSF (serine protease inhibitor) caused a great inhibitory effect on fibrinolytic enzyme activity suggested that serine or threonine hydroxyl groups are involved in the binding of substrate to the purified enzyme. PMSF also inhibited the fibrinolytic enzyme activities from different fungal strains (Osmolovskiy et al. 2017; Popova et al. 2021; Galiakberova et al. 2021). The absence of significant effect of a thiol reducing agent such as iodoacetate on fibrinolytic enzyme activity by was a great evidence for the absence of -SH group in the active sites of enzyme .Our results are in complete agreement with that recorded by (Xin et al. 2018). Complete lack of inhibitory effect of EDTA (Metalloprtoease inhibitor) on enzyme activity also suggests that C. hawaiiensis Fibrinolytic enzyme is a non metalloprotease enzyme. Similar results were reported their fibrinolytic enzyme as non metalloproteases such as those produced from Cordyceps militaris (Katrolia et al. 2020) and micromycete Sarocladium (Kornienko et al. 2021).

The high affinity of C. hawaiiensis fibrinolytic enzyme to fibrin as a substrate was also recorded for Aspergillus brasiliensis (Kotb et al. 2015), Aspergillus carbonarius (Afini et al. 2016) and and Aspergillus flavus(Galiakberova et al. 2021). The Michaelis constant (Km value) of the purified enzyme was calculated to be 47.61 mmol and the same value is recorded for the fibrinolytic enzyme from Bacillus subtilis(Wang et al. 2006).

The thrombolytic activity was confirmed by our results, where 200 U/ml completely dissolve the blood clot. The ability of fibrinolytic to lyses the blood clot was previously reported (Ahamed et al. 2022; Zhou et al. 2022). Controlling blood coagulation disorders is necessary since blood clots have been linked to a number of health challenges, including cardiovascular diseases, injuries, autoimmune diseases, allergic reactions and cancer (Lateef et al. 2018). Both visual and microscopic examinations evidenced that C. hawaiiensis fibrinolytic enzyme in this study acted as anticoagulant and thrombolytic agent by inhibiting the clot formation in blood collected in Eppendorf tubes or on slides. The optical microscopic micrographs revealed the effective dispersion of disc-like red blood cells due to reaction of blood clot with the purified enzyme. The results are in line with some previous reports on the anticoagulant and thrombolytic potentials of fibrinolytic enzymes (Ahamed et al. 2022; Krishnamurthy et al. 2018; Lateef et al. 2018).

Platelet aggregation is crucial for physiological hemostasis and thrombosis so, inhibiting platelet aggregation can lower the risk of bleeding and enhancing its suitability for therapeutic applications (Ruggeri and Mendolicchio 2007). Our results showed a considerable effect of purified fibrinolytic enzyme in antiplatelet aggregation activity. The effect of fibrinolytic enzyme in reduction of plate aggregation percentage in WP is stronger than in PRP, this may be attributed to the hydrolysis of fibrinolytic enzyme to other proteins in plasma in case of PRP (Zhou et al. 2022). Also, the inhibition of plate aggregation percentage in WP may be a clear evidence for the direct effect of fibrinolytic enzyme on platelet membrane receptors or integrins to reduce the aggregation rate (Zhou et al. 2022).

The coagulation screen, which includes the APTT, prothrombin time (PT), and fibrinogen assay, is frequently used in clinical settings to diagnose bleeding or clotting problems (Triplett 2000). APTT and PPT were used to measure the activity of the extrinsic and intrinsic pathway of the clotting cascade, respectively (Rountree et al. 2021). Our results showed a significant increase in APPT, PT and TT with gradual increase in fibrinolytic enzyme concentrations. Our results in agree with those recorded for fibrinolytic enzyme obtained from Bacillus velezensis (Zhou et al. 2022)

A locally isolated fungal strain of C. hawaiiensis was used for the production of fibrinolytic enzyme in both a submerged and solid culture conditions. But, the yield of fibrinolytic enzyme in SSF was 10.20 fold higher than SmF. The enzyme showed the maximum activity at pH 7 and 37 ^oC and these conditions are suitable for working in further prospective biomedical relevance. The purified enzyme was categorized as serine and non metalloprotease. Finally, the practical potential of the purified fibrinolytic enzyme as thrombolytic and antiplatelet aggregation agents was realized by the prevention and dissolution of the induced blood clot in compare with control groups. Also, the purified enzyme showed a significant efficiency in amelioration of blood coagulation measurements (APTT, PT, TT, and FIB). Therefore, the produced fibrinolytic enzyme from C. hawaiiensis is a promising candidate in management of cardiovascular diseases.

Acknowledgment

The authors gladly recognize that this research work was approved and supported by the grant no. (SCAR-2022-11-1704) from the Deanship of Scientific Research at Northern Border University, Arar, KSA

Conflicts of Interest

The authors indicated no potential conflicts of interest

Funding

This work was supported by deanship of Scientific Research at Northern Border University, Arar, KSA, under grant no. (SCAR-2022-11-1704).

Abbas M, Hussain T, Arshad M, Ansari AR, Irshad A, Nisar J, Hussain F, Masood N, Nazir A, Iqbal M (2019) Wound healing potential of curcumin cross-linked chitosan/polyvinyl alcohol. International journal of biological macromolecules 140:871-876. https://doi.org/10.1016/j.ijbiomac.2019.08.153
Abdel‐Fattah AF, Ismail AMS (1984) Preparation and properties of fibrinolytic enzymes produced by Cochliobolus lunatus. Biotechnology and bioengineering 26 (1):37-40. https://doi.org/10.1002/bit.260260108
Abdullah A, Tengerdy R, Murphy V (1985) Optimization of solid substrate fermentation of wheat straw. Biotechnology and bioengineering 27 (1):20-27. https://doi.org/10.1002/bit.260270104
Abu-Tahon MA, Arafat HH, Isaac GS (2020) Laundry detergent compatibility and dehairing efficiency of alkaline thermostable protease produced from Aspergillus terreus under solid-state fermentation. Journal of Oleo Science 69 (3):241-254. https://doi.org/10.5650/jos.ess19315
Abu-Tahon MA, Isaac GS (2016) Comparative study of a new alkaline L-methioninase production by Aspergillus ustus AUMC 10151 in submerged and solid-state fermentation. Brazilian Archives of Biology and Technology 59. https://doi.org/10.1590/1678-4324-2016150484
Afini A, Sooraj S, Smitha K, Kunhi A (2016) Production and partial characterization of fibrinolytic enzyme from a soil isolate Aspergillus carbonarius S-CSR-0007. Int J Pharm Pharm Sci 8:142-148. https://doi.org/10.22159/ijpps.2016v8i12.15069
Ahamed NA, Arif IA, Al-Rashed S, Panneerselvam A, Ambikapathy V (2022) In vitro thrombolytic potential of fibrinolytic enzyme from Brevibacterium sp. isolated from the root of the plant, Aloe castellorum. Journal of King Saud University-Science 34 (3):101868. https://doi.org/10.1016/j.jksus.2022.101868
Ali AMM, Bavisetty SCB (2020) Purification, physicochemical properties, and statistical optimization of fibrinolytic enzymes especially from fermented foods: a comprehensive review. International Journal of Biological Macromolecules 163:1498-1517. https://doi.org/10.1016/j.ijbiomac.2020.07.303
Anson ML (1938) The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. The Journal of general physiology 22 (1):79. https://doi.org/10.1085/jgp.22.1.79
Auesukaree C (2017) Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered during ethanol fermentation. Journal of bioscience and bioengineering 124 (2):133-142. https://doi.org/10.1016/j.jbiosc.2017.03.009
Biji GD, Arun A, Muthulakshmi E, Vijayaraghavan P, Arasu MV, Al-Dhabi NA (2016) Bio-prospecting of cuttle fish waste and cow dung for the production of fibrinolytic enzyme from Bacillus cereus IND5 in solid state fermentation. 3 Biotech 6 (2):1-13. https://doi.org/10.1007/s13205-016-0553-0
Bode C, Runge M, Smalling R (1996) The future of thrombolysis in the treatment of acute myocardial infarction. European heart journal 17 (suppl_E):55-60. https://doi.org/10.1093/eurheartj/17.suppl_E.55
Cardinal EV, Hedrick LR (1948) Microbiological assay of corn steep liquor for amino acid content. Journal of Biological Chemistry 172 (2):609-612. https://doi.org/10.1016/S0021-9258(19]52747-8
Chapin JC, Hajjar KA (2015) Fibrinolysis and the control of blood coagulation. Blood reviews 29 (1):17-24. https://doi.org/10.1016/j.blre.2014.09.003
Chimbekujwo KI, Ja'afaru MI, Adeyemo OM (2020) Purification, characterization and optimization conditions of protease produced by Aspergillus brasiliensis strain BCW2. Scientific African 8:e00398. https://doi.org/10.1016/j.sciaf.2020.e00398
Choi B-S, Sapkota K, Choi J-H, Shin C-h, Kim S, Kim S-J (2013) Herinase: a novel bi-functional fibrinolytic protease from the monkey head mushroom, Hericium erinaceum. Applied biochemistry and biotechnology 170 (3):609-622. https://doi.org/10.1007/s12010-013-0206-2
Deng Y, Liu X, Katrolia P, Kopparapu NK, Zheng X (2018) A dual-function chymotrypsin-like serine protease with plasminogen activation and fibrinolytic activities from the GRAS fungus, Neurospora sitophila. International journal of biological macromolecules 109:1338-1343. https://doi.org/10.1016/j.ijbiomac.2017.11.142
Dias B, Weimer B (1998) Purification and Characterization ofl-Methionine γ-Lyase from Brevibacterium linens BL2. Applied and Environmental Microbiology 64 (9):3327-3331. https://doi.org/10.1128/AEM.64.9.3327-3331.1998
Doley R, Mukherjee AK (2003) Purification and characterization of an anticoagulant phospholipase A2 from Indian monocled cobra (Naja kaouthia) venom. Toxicon 41 (1):81-91. https://doi.org/10.1016/S0041-0101(02]00213-1
El-Aassar S, El-Badry H, Abdel-Fattah A (1990) The biosynthesis of proteases with fibrinolytic activity in immobilized cultures of Penicillium chrysogenum H9. Applied microbiology and biotechnology 33 (1):26-30. https://doi.org/10.1007/BF00170564
El-Aassar SA (1995) Production and properties of fibrinolytic enzyme in solid state cultures of Fusarium pallidoroseum. Biotechnology letters 17 (9):943-948. https://doi.org/10.1007/BF00127431
El-Sayed AS, Shindia AA, Zaher Y (2012) L-Amino acid oxidase from filamentous fungi: screening and optimization. Annals of microbiology 62 (2):773-784. https://doi.org/10.1007/s13213-011-0318-2
Elias M, Wieczorek G, Rosenne S, Tawfik DS (2014) The universality of enzymatic rate–temperature dependency. Trends in Biochemical Sciences 39 (1):1-7. https://doi.org/10.1016/j.tibs.2013.11.001
Ellaiah P, Srinivasulu B, Adinarayana K (2002) A review on microbial alkaline proteases.
Galiakberova A, Bednenko D, Kreyer V, Osmolovskiy A, Egorov N (2021) Formation and Properties of the Extracellular Proteinase of Aspergillus flavus O-1 Micromycete Active against Fibrillar Proteins. Applied Biochemistry and Microbiology 57 (5):586-593. https://doi.org/10.1134/S0003683821050069
Ghareib M, Fawzi EM, Aldossary NA (2014) Thermostable alkaline protease from Thermomyces lanuginosus: optimization, purification and characterization. Annals of microbiology 64 (2):859-867. https://doi.org/10.1007/s13213-013-0725-7
Harish B, Uppuluri KB, Anbazhagan V (2015) Synthesis of fibrinolytic active silver nanoparticle using wheat bran xylan as a reducing and stabilizing agent. Carbohydrate polymers 132:104-110. https://doi.org/10.1016/j.carbpol.2015.06.069
Hesseltine C (1976) Production of fungal spores as inocula for oriental fermented foods. Dev Ind Microbiol 17:101-115
Hmood SA, Aziz GM (2016) Optimum conditions for fibrinolytic enzyme (Nattokinase) production by Bacillus sp. B24 using solid state fermentation. Iraqi Journal of Science 57 (2C):1391-1401
Holmström C, Kjelleberg S (1999) Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. FEMS microbiology ecology 30 (4):285-293https://doi.org/10.1111/j.1574-6941.1999.tb00656.x
Hua Y, Jiang B, Mine Y, Mu W (2008) Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste. Journal of Agricultural and Food Chemistry 56 (4):1451-1457. https://doi.org/10.1021/jf0713410
Katrolia P, Liu X, Zhao Y, Kopparapu NK, Zheng X (2020) Gene cloning, expression and homology modeling of first fibrinolytic enzyme from mushroom (Cordyceps militaris). International journal of biological macromolecules 146:897-906. https://doi.org/10.1016/j.ijbiomac.2019.09.212
Kattula S, Byrnes JR, Wolberg AS (2017) Fibrinogen and fibrin in hemostasis and thrombosis. Arteriosclerosis, thrombosis, and vascular biology 37 (3):e13-e21. https://doi.org/10.1161/ATVBAHA.117.308564
Kim HC, Choi B-S, Sapkota K, Kim S, Lee HJ, Yoo JC, Kim S-J (2011) Purification and characterization of a novel, highly potent fibrinolytic enzyme from Paecilomyces tenuipes. Process Biochemistry 46 (8):1545-1553. https://doi.org/10.1271/bbb.65.356
Kim J-H, Kim YS (2001) Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum. Bioscience, biotechnology, and biochemistry 65 (2):356-362. https://doi.org/10.1016/j.procbio.2011.04.005
Kornienko E, Osmolovskiy A, Kreyer V, Baranova N, Kotova I, Egorov N (2021) Characteristics and properties of the complex of proteolytic enzymes of the thrombolytic action of the micromycete Sarocladium strictum. Applied Biochemistry and Microbiology 57 (1):57-64. https://doi.org/10.1134/S0003683821010129
Kotb E, Helal GE-DA, Edries FM (2015) Screening for fibrinolytic filamentous fungi and enzymatic properties of the most potent producer, Aspergillus brasiliensis AUMC 9735. Biologia 70 (12):1565-1574. https://doi.org/10.1515/biolog-2015-0192
Krishnamurthy A, Belur PD, Subramanya SB (2018) Methods available to assess therapeutic potential of fibrinolytic enzymes of microbial origin: a review. Journal of Analytical Science and Technology 9 (1):1-11. https://doi.org/10.1186/s40543-018-0143-3
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature 227 (5259):680-685. https://doi.org/10.1038/227680a0
Larcher G, Bouchara J-P, Annaix V, Symoens F, Chabasse D, Tronchin G (1992) Purification and characterization of a fibrinogenolytic serine proteinase from Aspergillus fumigatus culture filtrate. FEBS letters 308 (1):65-69. https://doi.org/10.1016/0014-5793(92]81052-N
Lateef A, Ojo SA, Elegbede JA, Akinola PO, Akanni EO (2018) Nanomedical applications of nanoparticles for blood coagulation disorders. Environmental nanotechnology:243-277. https://doi.org/10.1007/978-3-319-76090-2_8
Lekha P, Lonsane B (1994) Comparative titres, location and properties of tannin acyl hydrolase produced by Aspergillus niger PKL 104 in solid-state, liquid surface an submerged fermentations. Process Biochemistry 29 (6):497-503. https://doi.org/10.1016/0032-9592(94]85019-4
Lin H-TV, Hwang P-A, Lin T-C, Tsai G-J (2014) Production of Bacillus subtilis-fermented red alga Porphyra dentata suspension with fibrinolytic and immune-enhancing activities. Bioscience, Biotechnology, and Biochemistry 78 (6):1074-1081. https://doi.org/10.1080/09168451.2014.915726
Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. Journal of the American chemical society 56 (3):658-666. https://doi.org/10.1021/ja01318a036
Lowry OH (1951) Protein measurement with the Folin phenol reagent. J biol Chem 193:265-275
Mahajan PM, Nayak S, Lele SS (2012) Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: Media optimization, purification and characterization. Journal of bioscience and bioengineering 113 (3):307-314. https://doi.org/10.1016/j.jbiosc.2011.10.023
Mousa SA (2010) In vivo models for the evaluation of antithrombotics and thrombolytics. In: Anticoagulants, Antiplatelets, and Thrombolytics. Springer, pp 29-107. https://doi.org/10.1007/978-1-60761-803-4
Nascimento TP, Sales AE, Porto CS, Brandão RMP, de Campos-Takaki GM, Teixeira JAC, Porto TS, Porto ALF, Converti A (2016) Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate). Journal of Chromatography B 1025:16-24. https://doi.org/10.1016/j.jchromb.2016.04.046
Osmolovskiy A, Kreier V, Baranova N, Egorov N (2017) Properties of extracellular plasmin-like proteases of Aspergillus ochraceus micromycete. Applied Biochemistry and Microbiology 53 (4):429-434. https://doi.org/10.1134/S000368381704010X
Pan S, Chen G, Wu R, Cao X, Liang Z (2019) Non-sterile submerged fermentation of fibrinolytic enzyme by marine Bacillus subtilis harboring antibacterial activity with starvation strategy. Frontiers in microbiology 10:1025. https://doi.org/10.3389/fmicb.2019.01025
Peetermans M, Vanassche T, Liesenborghs L, Claes J, Vande Velde G, Kwiecinksi J, Jin T, De Geest B, Hoylaerts MF, Lijnen RH (2014) Plasminogen activation by staphylokinase enhances local spreading of S. aureus in skin infections. BMC microbiology 14 (1):1-12. https://doi.org/10.1186/s12866-014-0310-7
Popova E, Kreyer V, Komarevtsev S, Shabunin S, Osmolovskiy A (2021) Properties of extracellular proteinase of the micromycete Aspergillus ustus 1 and its high activity during fibrillary-proteins hydrolysis. Applied Biochemistry and Microbiology 57 (2):200-205. https://doi.org/10.1134/S0003683821020125
Prabhu N, Gajendran T, Karthikadevi S, Archana A, Arthe R (2021) Utilization of sugarcane bagasse for enhancement production of fibrinolytic enzyme using statistical approach. Cleaner Engineering and Technology 5:100269. https://doi.org/10.1016/j.clet.2021.100269
Rountree KM, Yaker Z, Lopez PP (2021) Partial thromboplastin time. In: StatPearls [Internet]. StatPearls Publishing,
Rovati JI, Delgado OD, Figueroa LI, Fariña JI (2010) A novel source of fibrinolytic activity: Bionectria sp., an unconventional enzyme-producing fungus isolated from Las Yungas rainforest (Tucumán, Argentina). World Journal of Microbiology and Biotechnology 26 (1):55-62. https://doi.org/10.1007/s11274-009-0142-z
Ruggeri ZM, Mendolicchio GL (2007) Adhesion mechanisms in platelet function. Circulation research 100 (12):1673-1685. https://doi.org/10.1161/01.RES.0000267878.97021.ab
Secades P, Alvarez B, Guijarro J (2001) Purification and characterization of a psychrophilic, calcium-induced, growth-phase-dependent metalloprotease from the fish pathogen Flavobacterium psychrophilum. Applied and environmental microbiology 67 (6):2436-2444. https://doi.org/10.1128/AEM.67.6.2436-2444.2001
Seo J, Al-Hilal TA, Jee J-G, Kim Y-L, Kim H-J, Lee B-H, Kim S, Kim I-S (2018) A targeted ferritin-microplasmin based thrombolytic nanocage selectively dissolves blood clots. Nanomedicine: Nanotechnology, Biology and Medicine 14 (3):633-642. https://doi.org/10.1016/j.nano.2017.12.022
Sharma D, Niwas S, Behera B (1991) Solid state fermentation of bagasse for the production of cellulase enzyme from cellulolytic fungi and extent of simultaneous production of reducing sugars in the fermenter. Journal of Microbial Biotechnology 6 (1):7-14
Shirasaka N, Naitou M, Okamura K, Fukuta Y, Terashita T, Kusuda M (2012) Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3. Mycoscience 53 (5):354-364. https://doi.org/10.1007/S10267-011-0179-3
Stoykov YM, Pavlov AI, Krastanov AI (2015) Chitinase biotechnology: production, purification, and application. Engineering in Life Sciences 15 (1):30-38. https://doi.org/10.1002/elsc.201400173
Tao S, Peng L, Beihui L, Deming L, Zuohu L (1997) Solid state fermentation of rice chaff for fibrinolytic enzyme production by Fusarium oxysporum. Biotechnology Letters 19 (5):465-467. https://doi.org/10.1023/A:1018352328874
Tao S, Peng L, Beihui L, Deming L, Zuohu L (1998) Successive cultivation of Fusarium oxysporum on rice chaff for economic production of fibrinolytic enzyme. Bioprocess Engineering 18 (5):379-381. https://doi.org/10.1007/PL00008997
Tharwat NA (2006) Purification and biochemical characterization of fibrinolytic enzyme produced by thermophilic fungus Oidiodendron flavum. Biotechnology 5 (2):160-165. https://doi.org/10.3923/biotech.2006.160.165
Triplett DA (2000) Coagulation and bleeding disorders: review and update. Clinical chemistry 46 (8):1260-1269. https://doi.org/10.1093/clinchem/46.8.1260
Ueda M, Kubo T, Miyatake K, Nakamura T (2007) Purification and characterization of fibrinolytic alkaline protease from Fusarium sp. BLB. Applied microbiology and biotechnology 74 (2):331-338. https://doi.org/10.1007/s00253-006-0621-1
Vaidya B, Agrawal G, Vyas SP (2012) Functionalized carriers for the improved delivery of plasminogen activators. International journal of pharmaceutics 424 (1-2):1-11. https://doi.org/10.1016/j.ijpharm.2011.12.032
Vijayaraghavan P, Arasu MV, Rajan RA, Al-Dhabi N (2019) Enhanced production of fibrinolytic enzyme by a new Xanthomonas oryzae IND3 using low-cost culture medium by response surface methodology. Saudi Journal of Biological Sciences 26 (2):217-224. https://doi.org/10.1016/j.sjbs.2018.08.029
Vijayaraghavan P, Rajendran P, Prakash Vincent SG, Arun A, Abdullah Al-Dhabi N, Valan Arasu M, Young Kwon O, Kim YO (2017) Novel sequential screening and enhanced production of fibrinolytic enzyme by Bacillus sp. IND12 using response surface methodology in solid-state fermentation. BioMed Research International 2017. https://doi.org/10.1155/2017/3909657
Viniegra-González G, Favela-Torres E, Aguilar CN, de Jesus Rómero-Gomez S, Dıaz-Godınez G, Augur C (2003) Advantages of fungal enzyme production in solid state over liquid fermentation systems. Biochemical Engineering Journal 13 (2-3):157-167. https://doi.org/10.1016/S1369-703X(02]00128-6
Wang CT, Ji BP, Li B, Nout R, Li PL, Ji H, Chen LF (2006) Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. Journal of Industrial Microbiology and Biotechnology 33 (9):750-758. https://doi.org/10.1007/s10295-006-0111-6
Wang S-L, Wu Y-Y, Liang T-W (2011) Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. New biotechnology 28 (2):196-202. https://doi.org/10.1016/j.nbt.2010.09.003
Wu B, Wu L, Ruan L, Ge M, Chen D (2009) Screening of endophytic fungi with antithrombotic activity and identification of a bioactive metabolite from the endophytic fungal strain CPCC 480097. Current microbiology 58 (5):522-527. https://doi.org/10.1007/s00284-009-9361-7
Wu R, Chen G, Pan S, Zeng J, Liang Z (2019) Cost-effective fibrinolytic enzyme production by Bacillus subtilis WR350 using medium supplemented with corn steep powder and sucrose. Scientific reports 9 (1):1-10. https://doi.org/10.1038/s41598-019-43371-8
Xia Q, Wang X, Xu D-J, Chen X-H, Chen F-H (2012) Inhibition of platelet aggregation by curdione from Curcuma wenyujin essential Oil. Thrombosis research 130 (3):409-414. https://doi.org/10.1016/j.thromres.2012.04.005
Xiao-Lan L, Lian-Xiang D, Fu-Ping L, Xi-Qun Z, Jing X (2005) Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12. Applied microbiology and biotechnology 67 (2):209-214. https://doi.org/10.1007/s00253-004-1846-5
Xin X, Ambati RR, Cai Z, Lei B (2018) Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil. 3 Biotech 8 (2):1-8. https://doi.org/10.1007/s13205-018-1115-4
Yao Z, Kim JA, Kim JH (2019) Characterization of a fibrinolytic enzyme secreted by Bacillus velezensis BS2 isolated from sea squirt jeotgal. J Microbiol Biotechnol. 2019, 29(3):347-356. https://doi.org/10.4014/jmb.1810.10053
Yu L, Lei T, Ren X, Pei X, Feng Y (2008) Response surface optimization of l-(+)-lactic acid production using corn steep liquor as an alternative nitrogen source by Lactobacillus rhamnosus CGMCC 1466. Biochemical Engineering Journal 39 (3):496-502. https://doi.org/10.1016/j.bej.2007.11.008
Zadražil F, Brunnert H (1981) Investigation of physical parameters important for the solid state fermentation of straw by white rot fungi. European journal of applied microbiology and biotechnology 11 (3):183-188. https://doi.org/10.1007/BF00511259
Zeng W, Chen G, Wu Y, Dong M, Zhang B, Liang Z (2018) Nonsterilized fermentative production of poly‐γ‐glutamic acid from cassava starch and corn steep powder by a thermophilic Bacillus subtilis. Journal of Chemical Technology & Biotechnology 93 (10):2917-2924. https://doi.org/10.1002/jctb.5646
Zhang S, Wang Y, Zhang N, Sun Z, Shi Y, Cao X, Wang H (2015) Purification and Characterisation of a Fibrinolytic Enzyme from Rhizopus microsporus var. tuberosus. Food Technology and Biotechnology 53 (2):243-248. https://doi.org/10.17113/ftb.53.02.15.3874
Zhou Y, Chen H, Yu B, Chen G, Liang Z (2022) Purification and Characterization of a Fibrinolytic Enzyme from Marine Bacillus velezensis Z01 and Assessment of Its Therapeutic Efficacy In Vivo. Microorganisms 10 (5):843. https://doi.org/10.3390/microorganisms10050843
Zhou Y, Han L-R, He H-W, Sang B, Yu D-L, Feng J-T, Zhang X (2018) Effects of agitation, aeration and temperature on production of a novel glycoprotein GP-1 by Streptomyces kanasenisi ZX01 and scale-up based on volumetric oxygen transfer coefficient. Molecules 23 (1):125. https://doi.org/10.3390/molecules23010125

No competing interests reported.

graphicalabstract.png

Download PDF

Version 1

posted

You are reading this latest preprint version

Examination of thrombolytic and anticoagulant activities of purified fibrinolytic enzyme extracted from Cochliobolus hawaiiensis under solid state fermentation

Status:

Version 1

Abstract

Figures

Introduction

Materials & Methods

Results

Screening of fungal isolates for Fibrinolytic enzyme producers

Substrate Specificity Of Purified Fibrinolytic Enzyme

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1