Abstract
This study was performed to investigate the fibrinolytic enzyme-producing potentials of locally isolated soil bacteria and to purify and characterize the fibrinolytic enzyme of the most potent bacterial isolate. Among 40 isolates, the isolate V4 was found to have the highest potential to produce the fibrinolytic enzyme. According to 16 S rRNA sequence analysis, the isolate V4 was identified as Bacillus atrophaeus (GenBank number: OL662991). The optimal parameters for fibrinolytic enzyme production from B. atrophaeus were determined as a skim milk powder concentration of 15 g/L, an initial pH of 7.0, a temperature of 35 °C and an incubation time of 72 h. The molecular weight of the purified enzyme was calculated as 36 kDa. After ammonium sulphate precipitation, ion-exchange chromatography and gel filtration chromotography-based processes, the specific activity of the fibrinolytic enzyme was determined as 6414.7 U/mg. The purified enzyme showed the maximum activity at pH 7.0 and 35 °C. The enzyme was inhibited by PMSF and EDTA. The substrate specifity of the enzyme was in the following order; fibrin (62.3 U/mg) > fibrinogen (46.2 U/mg) > casein (42.1 U/mg) > serum albumin (6.8 U/mg). This is the first report on the fibrinolytic enzyme production potential of the species B. atrophaeus.
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References
Abd-Elaziz AM, Karam EA, Ghanem MM, Moharam ME, Kansoh AL (2020) Production of a novel α-amylase by Bacillus atrophaeus NRC1 isolated from honey: purification and characterization. Int J Biol Macromol 148:292–301. https://doi.org/10.1016/j.ijbiomac.2020.01.120
Adivitiya, Khasa YP (2017) The evolution of recombinant thrombolytics: current status and future directions. Bioengineered 8:331–358. https://doi.org/10.1080/21655979.2016.1229718
Ali AMM, Bavisetty SCB (2020) Purification, physicochemical properties, and statistical optimization of fibrinolytic enzymes especially from fermented foods: a comprehensive review. Int J Biol Macromol 163:1498–1517. https://doi.org/10.1016/j.ijbiomac.2020.07.303
Al Farraj DA, Kumar TSJ, Vijayaraghavan P, Elshikh MS, Alkufeidy RM, Alkubaisi NA, Alshammari MK (2020) Enhanced production, purification and biochemical characterization of therapeutic potential fibrinolytic enzyme from a new Bacillus flexus from marine environment. J King Saud Univ Sci 32:3174–3180. https://doi.org/10.1016/j.jksus.2020.09.004
Altaf F, Wu S, Kasim V (2021) Role of fibrinolytic enzymes in anti-thrombosis therapy. Front Mol Biosci 8:680397. https://doi.org/10.3389/fmolb.2021.680397
Ameri A, Shakibaie M, Faramarzi MA, Ameri A, Amirpour-Rostami S, Rahimi HR, Forootanfar H (2017) Thermoalkalophilic lipase from an extremely halophilic bacterial strain Bacillus atrophaeus FSHM2: purification, biochemical characterization and application. Biocatal Biotransform 35:151–160. https://doi.org/10.1080/10242422.2017.1308494
Anusree M, Swapna K, Aguilar CN, Sabu A (2020) Optimization of process parameters for the enhanced production of fibrinolytic enzyme by a newly isolated marine bacterium. Bioresour Technol Rep 11:100436. https://doi.org/10.1016/j.biteb.2020.100436
Bariş E (2020) Isolation and identification of Bacillus sp. from root soil of the Astragalus gummifer Lab.: obtaining and characterization of α-amylase. ADYU J SCI 10:29–39. https://doi.org/10.37094/adyujsci.680580
Barzkar N, Jahromi ST, Vianello F (2022) Marine microbial fibrinolytic enzymes: an overview of source, production, biochemical properties and thrombolytic activity. Mar Drugs 20:46. https://doi.org/10.3390/md20010046
Cheng Q, Xu F, Hu N, Liu X, Liu Z (2015) A novel Ca2+-dependent alkaline serine-protease (bvsp) from Bacillus sp. With high fibrinolytic activity. J Mol Catal B Enzym 117:69–74. DOI: https://doi.org/10.1016/j.molcatb.2015.04.006
Fayek KI, El-Sayed ST (1980) Purification and properties of a fibrinolytic enzyme from Bacillus subtilis. Z Allg Mıkrobıol 20:375–382. https://doi.org/10.1002/jobm.19800200603
Geneva II, Cuzzo B, Fazili T, Javaid W (2019) Normal body temperature: a systematic review. Open Forum Infect Dis 6:ofz032. https://doi.org/10.1093/ofid/ofz032
Gibbons HS, Broomall SM, McNew LA, Daligault H, Chapman C, Bruce D, Skowronski EW (2011) Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. Globigii, a historical biowarfare simulant. PLoS ONE 6:e17836. https://doi.org/10.1371/journal.pone.0017836
Kelly AM, McAlpine R, Kyle E (2001) Venous pH can safely replace arterial pH in the initial evaluation of patients in the emergency department. Emerg Med J 18:340–342. https://doi.org/10.1136/emj.18.5.340
Kotb E (2018) Microbial fibrinolytic enzyme production and applications. Microbial Functional Foods and Nutraceuticals. John Wiley & Sons Ltd., Hoboken, NJ, USA. https://doi.org/10.1002/9781119048961.ch8
Krishnamurthy A, Belur PD (2018) A novel fibrinolytic serine metalloprotease from the marine Serratia marcescens subsp. sakuensis: purification and characterization. Int J Biol Macromol 112:110–118. https://doi.org/10.1016/j.ijbiomac.2018.01.129
Kumar SS, Haridas M, Sabu A (2018) Process optimization for production of a fibrinolytic enzyme from newly isolated marine bacterium Pseudomonas aeruginosa KU1. Biocatal Agric Biotechnol 14:33–39. https://doi.org/10.1016/j.bcab.2018.02.001
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685
Lowry O, Rosebrough N, Farr AL, Randall R (1951) Protein measurement with the Folin Phenol Reagent. J Biol Chem 193:265–275
Moharam ME, El-Bendary MA, El-Beih F, Easa SMH, Elsoud MMA, Azzam MI, Elgamal NN (2019) Optimization of fibrinolytic enzyme production by newly isolated Bacillus subtilis Egy using central composite design. Biocatal Agric Biotechnol 17:43–50. https://doi.org/10.1016/j.bcab.2018.11.003
Nakamura LK (1989) Taxonomic relationship of black-pigmented Bacillus subtilis strains and a proposal for Bacillus atrophaeus sp. nov. Int J Syst Evol 39:295–300. https://doi.org/10.1099/00207713-39-3-295
Páblo ECS, de Souza FASD, de Barros RC, Marques DAV, Porto ALF, Bezerra RP (2017) Enhanced production of fibrinolytic protease from microalgae Chlorella vulgaris using glycerol and corn steep liquor as nutrient. Ann Microbiol Res 1:9–19
Rajaofera MJN, Wang Y, Dahar GY et al (2019) Volatile organic compounds of Bacillus atrophaeus HAB-5 inhibit the growth of Colletotrichum gloeosporioides. Pestic Biochem Phys 156:170–176. https://doi.org/10.1016/j.pestbp.2019.02.019
Raju EVN, Divakar G (2014) An overview on microbial fibrinolytic proteases. Int J Pharm Sci Res 5(3):643–656. https://doi.org/10.13040/IJPSR.0975-8232.5
Rodríguez M, Marín A, Torres M, Béjar V, Campos M, Sampedro I (2018) Aphicidal activity of surfactants produced by Bacillus atrophaeus L193. Front Microbiol 3114. https://doi.org/10.3389/fmicb.2018.03114
Sella SR, Vandenberghe LP, Soccol CR (2015) Bacillus atrophaeus: main characteristics and biotechnological applications–a review. Crit Rev Biotechnol 35:533–545. https://doi.org/10.3109/07388551.2014.922915
Taneja K, Bajaj BK, Kumar S, Dilbaghi N (2017) Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media. 3 Biotech 7:1–15. https://doi.org/10.1007/s13205-017-0808-4
Taneja K, Kumar Bajaj B, Kumar S, Dilbaghi N (2019) Process optimization for production and purification of novel fibrinolytic enzyme from Stenotrophomonas sp. KG-16-3. Biocatal Biotransform 37:124–138. https://doi.org/10.1080/10242422.2018.1504925
Vijayaraghavan P, Vincent SGP (2014) Statistical optimization of fibrinolytic enzyme production by Pseudoalteromonas sp. IND11 using cow dung substrate by response surface methodology. SpringerPlus 3:1–10. https://doi.org/10.1186/2193-1801-3-60
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. J Ind Microbiol Biotechnol 33:750–758. https://doi.org/10.1007/s10295-006-0111-6
Wang SH, Zhang C, Yang YL, Diao M, Bai MF (2008) Screening of a high fibrinolytic enzyme producing strain and characterization of the fibrinolytic enzyme produced from Bacillus subtilis LD-8547. World J of Microbiol Biotechnol 24:475–482. https://doi.org/10.1007/s11274-007-9496-2
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. Sci Rep 9:1–10. https://doi.org/10.1038/s41598-019-43371-8
Yao Z, Kim JA, Kim JH (2018) Gene cloning, expression, and properties of a fibrinolytic enzyme secreted by Bacillus pumilus bs15 isolated from gul (oyster) jeotgal. Biotechnol Bioprocess Eng 23:293–301. https://doi.org/10.1007/s12257-018-0029-7
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 29:347–356. https://doi.org/10.4014/jmb.1810.10053
Zhang J, Xue Q, Gao H, Lai H, Wang P (2016) Production of lipopeptide biosurfactants by Bacillus atrophaeus 5-2a and their potential use in microbial enhanced oil recovery. Microb Cell Factories 15:1–11. https://doi.org/10.1186/s12934-016-0574-8
Zhang X, Li B, Wang Y, Guo Q, Lu X, Li S, Ma P (2013) Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1. Appl Microbiol Biotechnol 97:9525–9534. https://doi.org/10.1007/s00253-013-5198-x
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 ıts therapeutic efficacy in vivo. Microorganisms 10:843. https://doi.org/10.3390/microorganisms10050843
Zhu W, Wang Y, Yan F, Song R, Li Z, Li Y, Song B (2018) Physical and chemical properties, percutaneous absorption-promoting effects of exopolysaccharide produced by Bacillus atrophaeus WYZ strain. Carbohydr polym 192:52–60. https://doi.org/10.1016/j.carbpol.2018.03.063
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This study was supported by the project (FBG-2020-8779) (Ataturk University, Erzurum, Turkey).
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Ayse Varol, Seyda Albayrak, Hakan Ozkan, Yeliz Demir, Mesut Taskin and Ahmet Adiguzel. The first draft of the manuscript was written by Ahmet Adiguzel and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Varol, A., Albayrak, S., Ozkan, H. et al. Production, purification and characterization of novel fibrinolytic enzyme from Bacillus atrophaeus V4. Biologia 78, 591–600 (2023). https://doi.org/10.1007/s11756-022-01281-7
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DOI: https://doi.org/10.1007/s11756-022-01281-7