Production Of Fibrinolytic Enzyme Research Articles

Process optimization for the production of fibrinolytic enzyme by a novel marine fungus – Penicillium steckii KU1

The demand for microbial thrombolytic agents is alarmingly increasing, and the discovery of novel microbial species with significant level of fibrinolytic enzyme production are required to cope up with the current circumstances. Surprisingly, marine fungi remain largely unexplored in this regard, presenting an untapped potential for the production of fibrinolytic enzymes.A novel marine fungal strain (Penicillium steckii KU1) was isolated successfully from Kappil beach, Kerala. At the beginning Czapek-dox media was used for the production of fibrinolytic enzyme. Statistical optimization of the enzyme production was achieved initially by Plackett-Burman design (PB). Interactive effect of various process parameters was further scrutinized by Central Composite Design (CCD). Incubation time, inoculum size and concentration of casein were shown significant contribution towards production. Statistical optimization, resulted in a tenfold increment (454.49 ± 14.66 U/mL) [optimized conditions - 30 °C, pH 7, casein 2.7 % (w/v), inoculum 5.75 million spores/mL and 115 h incubation], against the predicted value 441.42 U/mL.

Fibrinolytic Enzyme from Green Microalgae: A New Potential Drug for Thrombolytic Therapy?

Thrombosis is characterized by the pathological formation of fibrin clots within a blood vessel, leading to the obstruction of blood flow. Fibrinolytic enzymes from microorganisms have been shown to be more efficient and safer in dissolving clots. Then, this study aimed to evaluate the cell growth and fibrinolytic enzyme production of Tetradesmus obliquus under different cultivation conditions. T. obliquus grew under autotrophic and mixotrophic conditions using different concentrations of corn steep liquor (0.25 ≤ CSL ≤ 4.00%). The cells were concentrated and lysed via two different methods (sonication or homogenization) to trigger the release of the enzyme. It was precipitated via acetone or ammonium sulfate additions and purified using ion exchange chromatography. The highest biomass productivity (Px = 130 ± 12.8 mg∙L−1day−1), specific growth rate (µmax = 0.17 ± 0.00 day−1), and fibrinolytic activity (391 ± 40.0 U∙mg−1) was achieved on a mixotrophic cultivation at a 0.25% CSL concentration. The results showed that the homogenizing method had better performance in the release of enzyme, and the precipitation with acetone obtained the highest fibrinolytic activity (567 ± 49.3 U∙mg−1). The purified enzyme showed a specific activity of 1221 ± 31 U∙mg−1 and a molecular mass of 97 kDa. So, the fibrinolytic enzyme from T. obliquus had higher activity when compared to the other fibrinolytic enzymes, being a potential source for the development of therapeutic agents in thrombosis treatment. Additional studies are needed to investigate the biochemical properties and biological profile of this enzyme.

Thrombolytic and anticoagulant efficiencies of purified fibrinolytic enzyme produced from Cochliobolus hawaiiensis under solid-state fermentation.

Cochliobolus hawaiiensis Alcorn Assiut University Mycological Centre 8606 was chosen from the screened 20 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 (39.7U/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 (405U/mg protein) on sugarcane bagasse, incubation period of 5 days, moisture level of 100%, initial pH of salt basal medium 7.8, incubation temperature at 35°C, and supplementation of the salt basal medium with corn steep liquor (80％, v/v). 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 by ammonium sulfate (70%), gel filtration, and ion-exchange columns chromatography caused a great increase in its specific activity to 2581.6U/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°C. Significant pH stability was recorded at pH 6.6-7.2, and thermal stability was recorded at 33-41°C. The enzyme showed the highest affinity toward fibrin, with Vmax of 240U/mL and an apparent Km value of 47.61mmol. Mg2+ and Ca2+ moderately induced fibrinolytic activity, whereas Cu2+ and Zn2+ greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non-metalloprotease. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplatelet 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.

Modeling and in-vivo evaluation of fibrinolytic enzyme produced by Bacillus subtilis Egy under solid state fermentation

Blood clot formation increases cases of myocardial infarction (AMI) and stroke, thus urges directing much research works for treatment and prevention of the causes. One of these directions is the microbial production of fibrinolytic enzymes as thrombolytic agents. In the current work, Bacillus subtilis Egy has been used for enzyme production under solid state fermentation. Among twelve nutrient meals in addition to wheat bran as a control fodder yeast yielded the highest enzyme activity reaching 114U/g. Applying statistical model for optimization of enzyme production revealed that 3.6%, fodder yeast; 40%, moisture content; 6 days, incubation period and 2%, inoculum size were the optimum conditions for maximum fibrinolytic enzyme production (141.02 U/g) by Bacillus subtilis Egy under solid-state fermentation The model was significant and data were experimentally validated. The produced fibrinolytic enzyme was evaluated for in vitro and in vivo cytotoxicity. In-vivo examination of the enzyme resulted in no mortality during the first 24 h after treatment. After 14 days, the results revealed no significant changes detected in hematological parameters (RBCs, MCV, hemoglobin except WBCs which showed an increase for both sexes. Histopathological examination of liver and kidney of rats received oral and subcutaneous treatments showed normal architecture. The data showed the applicability of the produced enzyme for the treatment of blood clot with no significant effect on living cells or on physiological functions.

Screening of a Novel Fibrinolytic Enzyme-Producing Streptomyces from a Hyper-Arid Area and Optimization of Its Fibrinolytic Enzyme Production

Fibrinolytic enzymes are a kind of proteolytic enzymes that can hydrolyze fibrin and dissolve blood clots. They could be used as a therapeutic agent for treating thrombosis. It is important for the treatment of cardiovascular disease to find and develop new thrombolytic drugs. In order to explore new fibrinolytic enzymes, a strain named 214L-11 with protease and fibrinolytic enzyme activity, which was isolated from the Flaming Mountain of Xinjiang Province, was screened using the skimmed milk plate, the blood powder agarose plate and the fibrin plate methods. Phylogenetic analyses showed that strain 214L-11 shared the highest similarity with Streptomyces fumanus NBRC 13042T (98.88%), which indicated that it represented a potential novel species in the Streptomyces genus. The fibrinolytic enzyme produced by 214L-11 displayed thrombolytic and anticoagulant activities, and it could degrade a single specific protein in the thrombus, thereby destroying the thrombus structure. The fermentation medium optimized through response surface methodology was 15 g/L soluble starch, g/L KNO3 0.58, 0.43 g/L peptone, 0.01 g/L FeSO4·7H2O, 0.5 g/L MgSO4·7H2O, 0.2 g/L Mn2+, 0.5 g/L NaCl and 1 L distilled water, pH 8, and the maximum amount of fibrinolytic enzyme produced by strain 214L-11 in the optimal fermentation medium was 1255.3 FU/mL. Overall, the fibrinolytic enzyme-producing strain was screened from the Flaming Mountain of Xinjiang for the first time, which provided a basis for further research and the development of new efficient and safe hemolytic drugs.

Production of Fibrinolytic Enzyme by Soil Actinobacteria

Thrombotic diseases are increasingly becoming among the prominent causes of death across the globe. Researchers are now turning attention towards fibrinolytic enzymes as potential alternative treatment for thrombolytic diseases. This present study focused on the production of extracellular fibrinolytic enzyme from soil Actinobacteria and evaluation of its hydrolytic activity on blood clot. The Actinobacteria was isolated from compost soil of semi-arid climate in Nigeria. Bacterial isolation was achieved using starch casein agar. Potent fibrinolytic enzyme producing Actinobacteria were identified and subjected to enzyme production using submerged fermentation method. The interactive effects of incubation time, temperature, pH and media components on enzyme production were analysed. Extracellular fibrinolytic enzyme produced by the selected Actinobacteria was partially purified by ammonium sulphate precipitation and subsequently assayed for blood clot lysis activity. Results of these studies indicated that fibrinolytic enzyme was produced optimally at pH 8 and temperature of 40 oC after 72 hour of fermentation. Partially purified fibrinolytic enzyme was able to degrade blood clot comparable to the positive control. These results shows that soil Actinobacteria of unexplored semi-arid climate of Nigeria present a prospect in search of novel microorganisms with potentials in the production of fibrinolytic enzyme that can serve as an alternative blood clot buster in treating thrombolytic diseases.

Isolation, production, and application of fibrinolytic enzyme from fermented food sources: a review

Isolation, production, and application of fibrinolytic enzyme from fermented food sources: a review

Cost-effective fibrinolytic enzyme production by microalga Dunaliella tertiolecta using medium supplemented with corn steep liquor.

A fibrinolytic enzyme from the microalga Dunaliella tertiolecta was produced under mixotrophic conditions using different corn steep liquor (CSL) concentrations ( 0 ≤ CLS ≤ 0.75%), purified using a combination of salting out and ion-exchange chromatography, and then biochemical characterized. Cultivation of this microalga using 0.5% CSL led to the highest maximum cell concentration (1.960±0.010 mg L-1) and cell productivity (0.140g L-1 day-1), besides a high fibrinolytic activity of the extract obtained by the homogenization method (102 ±1 U mL-1). The enzyme extracted from the microalgal biomass was 5-fold purified with a 20% yield and was found to have a specific activity of 670 U mg-1. The enzyme, whose molecular weight determined by fibrin zymography was 10 kDa, was shown to be stable at pH 3.0-9.0 and up to 70°C with optimal pH and temperature values of 8.0 and 50°C, respectively. When compared to other fibrinolytic enzymes, this protease stood out for its high fibrinolytic activity, which was enhanced by Fe2+, inhibited by Zn2+, Cu2+, Mg2+, and Ca2+, and strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that it belongs to the serine metalloprotease family. Moreover, thanks to its thermal stability, the enzyme may be easily preserved and activated under high-temperature conditions.

Production, purification and characterization of novel fibrinolytic enzyme from Bacillus atrophaeus V4

Production, purification and characterization of novel fibrinolytic enzyme from Bacillus atrophaeus V4

Cultivation conditions and biochemical characterization of the proteolytic enzymes with fibrinolytic action obtained from mushrooms in the last ten years

Fibrinolytic enzymes are proteases that are capable of degrading the fibrin mesh present in blood clots and can be obtained through fungal extracts. Thus, the objective of this paper was to seek out and compare all the information regarding the production and biochemical characterization of fibrinolytic enzymes of mushroom species published in papers in the last ten years. The research was carried out in eight national and international electronic databases using the terms: “fungal enzymes”, “fibrinolytic Proteases”, “thrombosis” and “mushrooms”. The results obtained were analyzed according to each step. In the first stage, the titles and abstracts of all papers were analyzed independently; in the second stage, all papers were read and those that did not meet the inclusion criteria were excluded. Then, the remaining papers were reviewed and the data were tabulated and compared. As a result, it was found that enzymes with fibrinolytic action were obtained from eight species of mushrooms in the period of this research: P. sajor-cashew, P. ferulae, P. ostreatus, L. shimeji, A. polytricha, H. erinaceum, C. comatus and C. militaris. The enzymes were identified as protease SPPs, metalloproteases, serine proteases and serine metalloproteases. The methods used to evaluate fibrinolytic activity were the fibrin plate method and/or the formation of artificial thrombus through thrombin and fibrinogen. The molecular mass of these enzymes ranged from ~18 to 66 kDa and, for the biochemical characterization of the extracts, the pH ranged from 4.0 to 9.5 and the optimum temperature from 25 to 70 ºC. Via the review, it was observed that few articles were published on the subject in question, which made it necessary to carry out more studies to discover the biotechnological properties of mushrooms and define their applications in the most diverse sectors of industries, not only in commercial species of mushrooms, but also in native species.

In vitro thrombolytic potential of fibrinolytic enzyme from Brevibacterium sp. isolated from the root of the plant, Aloe castellorum

Cardiovascular diseases (CVDs) like stroke and heart attack are leading causes of mortality worldwide, resulting in about 30% of deaths. The proportion in the Kingdom of Saudi Arabia was 46%. The commercial thrombolytic agents are thus associated with a range of side effects, while bacterially derived fibrinolytic enzymes have no or little side effects. Hence, there is an urgent need to identify novel fibrinolytic enzymes to treat or prevent CVDs. An endophytic bacterial strain producing fibrinolytic enzyme was isolated from the root of the plant Aloe castellorum. Enzyme production was enhanced through a conventional method and a statistical approach. The in vitro lytic activity of blood clots was also assessed. Endangered plant of Aloe castellorum roots, researchers isolated an endophytic strain producing fibrinolytic enzymes. It was identified as Brevibacterium sp. The culture medium was also optimized by a conventional method to screen the factors significant for successful culture. Fibrinolytic enzyme production peaked when Brevibacterium sp. was cultured in the presence of maltose (613 ± 12 U/mL), followed by starch (576 ± 13 U/mL). Among the selected nitrogen sources, yeast extract (642 ± 5.9 U/mL) and beef extract (610 ± 13 U/mL) enhanced enzyme yield relative to that in the control (487 ± 7.2 U/mL). Ionic sources such as Mg2+ (672 ± 10.3 U/mL) and Ca2+ (605 ± 12.3 U/mL) showed enhanced fibrinolytic enzyme production relative to the control (405 ± 13.2 U/mL). Upon employing a two-level full factorial design for examining the significant factors, it was revealed that maltose, yeast extract, and Mg2+ were important (p < 0.05). To enhance thrombolytic agent production using central composite design and response surface methodologies. The fibrinolytic enzyme obtained from Brevibacterium sp. has great potential to lyse blood clots in vitro.

Utilization of sugarcane bagasse for enhancement production of fibrinolytic enzyme using statistical approach

The purpose of this investigation was to produce fibrinolytic enzyme by utilizing sugarcane bagasse as low cost substrate through solid state fermentation in an effective way. The potential producer of microorganism, Alcaligenes aquatilis PJS_1 was isolated and identified from slaughter house soil samples. In small scale level, higher production of microbial fibrinolytic enzyme was observed when the conditions provided like Glucose for carbon, Casein for nitrogen and (NH 4 ) 2 SO 4 for mineral sources when maintained at 60 h with 100% moisture. The sugarcane bagasse medium with glucose and casein was found to have optimum production when incubated for 60 h at 35°C. The culture conditions were optimized to improve the production of fibrinolytic enzyme (3832 U ml −1 ) from Alcaligenes aquatilis PJS_1 through statistical approach. Blood clotting assay was performed to determine its anticoagulant property. The fibrinolytic property of the synthesised enzyme was confirmed when it dissolved blood clot sample effectively. Thus, the enzyme it could be used in medicine to produce as a thrombolytic agent.

Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications.

Cardiac disorders such as acute myocardial infarction, embolism and stroke are primarily attributed to excessive fibrin accumulation in the blood vessels, usually consequential in thrombosis. Numerous methodologies including the use of anti-coagulants, anti-platelet drugs, surgical operations and fibrinolytic enzymes are employed for the dissolution of fibrin clots and hence ameliorate thrombosis. Microbial fibrinolytic enzymes have attracted much more attention in the management of cardiovascular disorders than typical anti-thrombotic strategies because of the undesirable after-effects and high expense of the latter. Fibrinolytic enzymes such as plasminogen activators and plasmin-like proteins hydrolyse thrombi with high efficacy with no significant after-effects and can be cost effectively produced on a large scale with a short generation time. However, the hunt for novel fibrinolytic enzymes necessitates complex purification stages, physiochemical and structural-functional attributes, which provide an insight into their mechanism of action. Besides, strain improvement and molecular technologies such as cloning, overexpression and the construction of genetically modified strains for the enhanced production of fibrinolytic enzymes significantly improve their thrombolytic potential. In addition, the unconventional applicability of some fibrinolytic enzymes paves their way for protein hydrolysis in addition to fibrin/thrombi, blood pressure regulation, anti-microbials, detergent additives for blood stain removal, preventing dental caries, anti-inflammatory and mucolytic expectorant agents. Therefore, this review article encompasses the production, biochemical/structure-function properties, thrombolytic potential and other surplus applications of microbial fibrinolytic enzymes.

Substantial improvement of protease production using statistical optimization and Downstream approach from the strain Bacillus toyonensis VKB5 sp., isolated from the host Actinidia delicosa

Endophytes are microbes that reside inside the plant without affecting the host. A highly active species of extracellular protease production is Bacillus species. This study aims at optimization and partial purification of fibrinolytic protease enzyme production from the isolate Bacillus toyonensis VKB5 sp. isolated from the fruit Actinidia delicosa. Optimization studies on medium components show that the enzyme production can be achieved in alkaline conditions with nutrient sources mannitol, yeast extract, ammonium chloride. The statistical optimization studies using Plackett-Burman and Response surface methodology determines that the interaction of yeast extract with the mannitol and ammonium chloride enhances the enzyme production. Studies on purification stages describe that the protease purified was 7.4 – fold purer that the crude and 68.9% enzyme was recovered comparing the crude. The molecular weight determined was found to be 21.9 kDa using SDS-PAGE. The purity of the protease was analyzed using HPLC. Further studies on the effect of temperature, pH, inhibitors, detergents and metal ions confirm that the enzyme purified can be alkaline serine protease that withstands thermal condition up to 60°C.

Enhanced Production of Fibrinolytic Enzyme from Pseudomonas aeruginosa by Optimization Media Components

Fibrinolytic enzyme is factor that lysis fibrin clots. This fibrinolytic factor has prospective use to treat cardiovascular diseases, such as stroke and heart attack. Cardiovascular diseases attracted worldwide attention for their elevation morbidity and mortality. Expensive cost and fatally undesired side effects associated with the available fibrinolytic agents to treat these diseases stimulated the researchers to investigate potentially better agents for curative applications. In the current investigation, fibrinolytic enzyme production from Pseudomonas aeruginosa isolated from injuries of wounds and burns patients. Parameters for the promoted production of the enzyme under minimal production media were optimized. It comprised carbon source (glucose), Nitrogen source (Yeast extract), Fibrinogen concentration (0.5 %), inoculum size (1 %), temperature (37°C), and PH (7). Enhanced fibrinolytic enzyme activity (136.2 IU/ml) was obtained after optimization Medium Components compared with that obtained with the minimal medium (60.2 IU/ml) which is 2.2 times higher than the same under non optimized production conditions. Media optimization researches for enhancement of fibrinolytic enzyme production from Pseudomonas aeruginosa in Iraq has not been performed so far. This may be the first study to optimization media for the production of fibrinolytic enzyme from Pseudomonas aeruginosa. The importance of this study lies in the enhancing the production of the fibrinolytic enzyme with high activity using these bacteria.

Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy

Thrombosis, a major cause of deaths in this modern era responsible for 31% of all global deaths reported by WHO in 2017, is due to the aggregation of fibrin in blood vessels which leads to myocardial infarction or other cardiovascular diseases (CVDs). Classical agents such as anti-platelet, anti-coagulant drugs or other enzymes used for thrombosis treatment at present could leads to unwanted side effects including bleeding complication, hemorrhage and allergy. Furthermore, their high cost is a burden for patients, especially for those from low and middle-income countries. Hence, there is an urgent need to develop novel and low-cost drugs for thrombosis treatment. Fibrinolytic enzymes, including plasmin like proteins such as proteases, nattokinase, and lumbrokinase, as well as plasminogen activators such as urokinase plasminogen activator, and tissue-type plasminogen activator, could eliminate thrombi with high efficacy rate and do not have significant drawbacks by directly degrading the fibrin. Furthermore, they could be produced with high-yield and in a cost-effective manner from microorganisms as well as other sources. Hence, they have been considered as potential compounds for thrombosis therapy. Herein, we will discuss about natural mechanism of fibrinolysis and thrombus formation, the production of fibrinolytic enzymes from different sources and their application as drugs for thrombosis therapy.

Optimization of Cultural Conditions for Maximum Production of Fibrinolytic Enzymes from the Local Marine Bacterial Isolates and Evaluation of their Wound Healing and Clot Dissolving Properties

Fibrinolytic enzymes find necessary applications to treat and prevent cardiovascular diseases. In this study, optimal conditions for enhancing the production of fibrinolytic enzyme from local marine bacterial strains were evaluated. The present study also focuses on screening of wound healing efficacy of the isolated fibrinolytic enzymes.Various physical parameters such as temperature, pH, incubation time and medium components viz. inoculum size, substrate (nitrogen and carbon) concentrations were optimized. A cultivation medium was designed using optimized conditions for mass production of fibrinolytic enzyme and specific activity of enzyme was analyzed. The maximum enzyme production was observed at 37 °C temperature, 8.0 pH,substrate concentration with 3 ml inoculum size and 32 h. of incubation time. Among the different carbon sources tested, Mannitol showed maximum enzyme activity i.e 538 U/ml. yeast extract was found to be the best nitrogen source with an enzyme activity of 498 U/ml. The best substrate for the production fibrinolytic enzyme was found to be kernelwith high activity of 1056U/ml. The crude enzyme displayed potent activity and digested blood clot completely in in vitro condition and exhibited potent activity on wound healing property in macrophages.

Increase of a Fibrinolytic Enzyme Production through Promoter Replacement of aprE3-5 from Bacillus subtilis CH3-5.

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (Pcry3A, P10, PSG1, PsrfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P10 promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

Isolation, production and application of fibrinolytic enzyme from fermented food sources

The accumulation of the fibrin in bacterial fibrinolytic enzymes finds applications to treat and prevent cardiovascular diseases which fail in hemostasis that leads to the formation of undesirable blood clots in the blood vessels leading to condition called thrombosis. The fibrinolytic enzymes from food grade organisms are useful for thrombolytic therapy. Conventional thrombolytic agents such as streptokinase, nattokinase etc. Nattokinase is one such fibrinolytic enzyme with a wide range of applications in Pharmaceutical industry, health care and medicine etc. Hence, potent blood-clot dissolving protein used for the treatment of cardiovascular diseases is produced by the bacterium Bacillus subtilis during the fermentation of soybeans to produce Natto. The health benefits of some fermented foods are synthesis of nutrients, prevention of cancer, diabetes due to presence of functional microorganisms, which possess probiotics properties, antimicrobial, antioxidant, etc. The first report of fibrinolytic enzyme production of cow dung used as a cheap substrate from Bacillus species in SSF has been given earlier. This review describes different isolation methods which enable the screening and selection of promising organisms for industrial production. The purification and properties of these fibrinolytic proteases is discussed, and the use of fibrinolytic enzyme. In order to obtain Bacillus species producing fibrinolytic enzymes, the fermented food sample such as sprouted grain and processed grain etc were used. The heat tolerant isolates initially were selected for catalase test. Fibrinolytic activity of the selected isolates was determined by using Fibrin plate assay. From the above work, it can be concluded that the fibrinolytic enzyme produced by Bacillus from fermented food samples had the ability to degrade the fibrin and hence can be used for functional food formulation.

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)

BackgroundAs a main drug for diseased thrombus, some clinically used thrombolytic agents have various disadvantages, safer novel thrombolytic agents are of great demand. This study aimed to achieve high and efficient production of a fibrinolytic enzyme with superior enzymatic properties, by a combination strategy of codon optimization, gene dosage and process optimization in Komagataella phaffii (K. phaffii).ResultsAfter codon optimization, the fibase from a marine Bacillus subtilis was expressed and secreted in K. phaffii GS115. Recombinant strains harboring different copies of the fib gene (fib-nc) were successfully obtained via Geneticin (0.25–4 mg/ml) screening on minimal dextrose selection plates and assessment via real-time quantitative PCR. The respective levels of fibase produced by strains expressing fib-5.4c, fib-6c, fib-8c, fib-9c, and fib-12c were 4428, 5781, 7323, 7930, and 2472 U/ml. Levels increased as the copy number increased from 4 to 9, but decreased dramatically at copy number 12. After high cell density fermentation optimization, the highest fibase activity of the strain expressing fib-9c was 7930 U/ml in a shake flask and increased to 12,690 U/ml after 3 days of continuous culture in a 5-L fermenter, which is one of the highest levels of production reported. The recombinant fibase was maximally active at pH 9.0 and 45 °C, and was remarkably stable at pH levels ranging from 5 to 10 and temperatures up to 50 °C. As a metal-dependent serine protease, fibase did not cause hemolysis in vitro and preferentially degraded fibrin directly.ConclusionsThe combination of codon optimization, gene dosage, and process optimization described herein could be used for the expression of other therapeutic proteins difficult to express. The characteristics of the recombinant fibase suggest that it has potential applications for thrombosis prevention and therapy.