Preventive Nutrition and Food Science

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
권호 표지
DOI QR코드

DOI QR Code

Purification and Characterization of a Fibrinolytic Enzyme from Bacillus pumilus 2.g Isolated from Gembus, an Indonesian Fermented Food

  • Afifah, Diana Nur (Department of Nutrition Science, Diponegoro University) ;
  • Sulchan, Muhammad (Department of Nutrition Science, Diponegoro University) ;
  • Syah, Dahrul (Department of Food Science and Technology, Bogor Agricultural University) ;
  • Yanti, Yanti (Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia) ;
  • Suhartono, Maggy Thenawidjaja (Department of Food Science and Technology, Bogor Agricultural University) ;
  • Kim, Jeong Hwan (Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University)
  • Received : 2014.05.13
  • Accepted : 2014.08.05
  • Published : 2014.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Bacillus pumilus 2.g isolated from gembus, an Indonesian fermented soybean cake, secretes several proteases that have strong fibrinolytic activities. A fibrinolytic enzyme with an apparent molecular weight of 20 kDa was purified from the culture supernatant of B. pumilus 2.g by sequential application of ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic chromatography. The partially purified enzyme was stable between pH 5 and pH 9 and temperature of less than $60^{\circ}C$. Fibrinolytic activity was increased by 5 mM $MgCl_2$ and 5 mM $CaCl_2$ but inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic acid (EDTA). The partially purified enzyme quickly degraded the ${\alpha}$ and ${\beta}$ chains of fibrinogen but was unable to degrade the ${\gamma}$ chain.

Keywords

References

  1. WHO. 2011. Global status report on noncommunicable diseases 2010. World Health Organization, Geneva, Switzerland. p 9-31.
  2. Mathers CD, Loncar D. 2006. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med 3: e442. https://doi.org/10.1371/journal.pmed.0030442
  3. Voet D, Voet JG. 1990. Biochemistry. 2nd ed. John Wiley and Sons, NY, USA. p 1087-1095.
  4. Dobrovolsky AB, Titaeva EV. 2002. The fibrinolysis system: regulation of activity and physiologic functions of its main components. Biochemistry (Mosc) 67: 99-108. https://doi.org/10.1023/A:1013960416302
  5. Blann AD, Landray MJ, Lip GYH. 2002. ABC of antithrombotic therapy: an overview of antithrombotic therapy. BMJ 325: 762. https://doi.org/10.1136/bmj.325.7367.762
  6. Turpie AGG, Chin BSP, Lip GYH. 2002. Venous thromboembolism: treatment strategies. BMJ 325: 948. https://doi.org/10.1136/bmj.325.7370.948
  7. Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. 1987. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia 15: 1110-1111.
  8. Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S. 1993. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto, a popular soybean fermented food in Japan. Biochem Biophys Res Commun 197: 1340-1347. https://doi.org/10.1006/bbrc.1993.2624
  9. Wang C, Du M, Zheng D, Kong F, Zu G, Feng Y. 2009. Purification and characterization of nattokinase from Bacillus subtilis Natto B-12. J Agric Food Chem 57: 9722-9729. https://doi.org/10.1021/jf901861v
  10. Sumi H, Nakajima N, Yatagai C. 1995. A unique strong fibrinolytic enzyme (katsuwokinase) in skipjack "Shiokara," a Japanese traditional fermented food. Comp Biochem Physiol B Biochem Mol Biol 112: 543-547. https://doi.org/10.1016/0305-0491(95)00100-X
  11. Kim WK, Choi KH, Kim YT, Park HH, Choi JY, Lee YS, Oh HI, Kwon IB, Lee SY. 1996. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Appl Environ Microbiol 62: 2482-2488.
  12. Kim GM, Lee AR, Lee KW, Park JY, Chun J, Cha J, Song YS, Kim JH. 2009. Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from Cheonggukjang. J Microbiol Biotechnol 19: 997-1004. https://doi.org/10.4014/jmb.0811.600
  13. Jo HD, Kwon GH, Park JY, Cha J, Song YS, Kim JH. 2011. Cloning and overexpression of aprE3-17 encoding the major fibrinolytic protease of Bacillus licheniformis CH 3-17. Biotechnol Bioprocess Eng 16: 352-359. https://doi.org/10.1007/s12257-010-0328-0
  14. Kim SH, Choi NS. 2000. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from Doen-Jang. Biosci Biotechnol Biochem 64: 1722-1725. https://doi.org/10.1271/bbb.64.1722
  15. Wong AHK, Mine Y. 2004. Novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning. J Agric Food Chem 52: 980-986. https://doi.org/10.1021/jf034535y
  16. Peng Y, Huang Q, Zhang RH, Zhang YZ. 2003. Purification and characterization of a fibrinolytic enzyme produced by Bacillus emyloliquefaciens DC-4 screened from Douchi, a traditional Chinese soybean food. Comp Biochem Physiol B Biochem Mol Biol 134: 45-52. https://doi.org/10.1016/S1096-4959(02)00183-5
  17. Sumi H, Hamada H, Nakanishi K, Hiratani H. 1990. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematol 84: 139-143. https://doi.org/10.1159/000205051
  18. Kuswanto KR. 2004. Industrialization of Tempe production. In Industrialization of Indigenous Fermented Foods, Revised and Expanded. Steinkraus KH, ed. CRC Press, Boca Raton, FL, USA. p 587-635.
  19. Afifah DN, Sulchan M, Syah D, Yanti, Suhartono MT. 2013. Proteolytic and fibrinolytic activities of several microorganisms screened from Red Oncom and Gembus, Indonesian fermented soybean cakes. Abstract No. 1.1 presented at 4th Annual International Symposium on Wellness, Healthy Lifestyle and Nutrition. Yogyakarta, Indonesia.
  20. Jeong SJ, Kwon GH, Chun JY, Kim JS, Park CS, Kwon DY, Kim JH. 2007. Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from cheonggukjang and its expression in protease-deficient Bacillus subtilis strains. J Microbiol Biotechnol 17: 1018-1023.
  21. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 234-254.
  22. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  23. Jo HD, Lee HA, Jeong SJ, Kim JH. 2011. Purification and characterization of a major fibrinolytic enzyme from Bacillus amyloliquefaciens MJ5-41 isolated from meju. J Microbiol Biotechnol 21: 1166-1173. https://doi.org/10.4014/jmb.1106.06008
  24. Hwang KJ, Choi KH, Kim MJ, Park CS, Cha JH. 2007. Purification and characterization of a new fibrinolytic enzyme of Bacillus licheniformis KJ-31, isolated from Korean traditional Jeot-gal. J Microbiol Biotechnol 17: 1469-1476.
  25. Kim HK, Kim GT, Kim DK, Choi WA, Park SH, Jeong YK, Kong IS. 1997. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. KA38 originated from fermented fish. J Ferment Bioeng 84: 307-312. https://doi.org/10.1016/S0922-338X(97)89249-5
  26. Simkhada JR, Mander P, Cho SS, Yoo JC. 2010. A novel fibrinolytic protease from Streptomyces sp. CS684. Process Biochem 45: 88-93. https://doi.org/10.1016/j.procbio.2009.08.010
  27. Yin LJ, Lin HH, Jiang ST. 2010. Bioproperties of potent nattokinase from Bacillus subtilis YJ1. J Agric Food Chem 58: 5737-5742. https://doi.org/10.1021/jf100290h
  28. Gold AM, Fahrney D. 1964 Sulfonyl fluorides as inhibitors of esterases. II. Formation and reactions of phenylmethanesulfonyl $\alpha$-chymotrypsin. Biochemistry 3: 783-791. https://doi.org/10.1021/bi00894a009
  29. Kim SB, Lee DW, Cheigh CI, Choe EA, Lee SJ, Hong YH, Choi HJ, Pyun YR. 2006. Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybeand, Tempeh. J Ind Microbiol Biotechnol 33: 436-444. https://doi.org/10.1007/s10295-006-0085-4

Cited by

  1. Screening and characterization of thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 / Bacillus amyloliquefaciens EGE-B-2d.1’den termostabil fibrinolitik enzimin taranması ve karakterizasyonu vol.41, pp.3, 2016, https://doi.org/10.1515/tjb-2016-0027
  2. An in-depth characterization of the entomopathogenic strain Bacillus pumilus 15.1 reveals that it produces inclusion bodies similar to the parasporal crystals of Bacillus thuringiensis vol.100, pp.8, 2016, https://doi.org/10.1007/s00253-015-7259-9
  3. Screening, production and biochemical characterization of a new fibrinolytic enzyme produced by Streptomyces sp. (Streptomycetaceae) isolated from Amazonian lichens vol.46, pp.3, 2016, https://doi.org/10.1590/1809-4392201600022
  4. Proteomics study of extracellular fibrinolytic proteases from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from Indonesian fermented food vol.55, 2017, https://doi.org/10.1088/1755-1315/55/1/012025
  5. Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in-vitro blood clot lysis potential vol.61, pp.5, 2015, https://doi.org/10.2323/jgam.61.157
  6. Impact of microbial proteases on biotechnological industries vol.33, pp.2, 2017, https://doi.org/10.1080/02648725.2017.1408256
  7. vol.32, pp.1, 2018, https://doi.org/10.1080/08905436.2017.1413986
  8. hydrolyzate vol.116, pp.1755-1315, 2018, https://doi.org/10.1088/1755-1315/116/1/012044
  9. as an alternative of thrombolytic agents vol.131, pp.1755-1315, 2018, https://doi.org/10.1088/1755-1315/131/1/012041
  10. Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil vol.8, pp.2, 2018, https://doi.org/10.1007/s13205-018-1115-4
  11. SRM-001 vol.48, pp.1, 2018, https://doi.org/10.1080/10826068.2017.1387560
  12. Influence Variation of Tempe Gembus (An Indonesian Fermented Food) on Homocysteine and Malondialdehyde of Rats Fed an Atherogenic Diet vol.24, pp.3, 2014, https://doi.org/10.1515/rjdnmd-2017-0026
  13. The Effect of Tempeh Gembus Substitution on Protein Content, Calcium, Protein Digestibility and Organoleptic Quality of Meatballs vol.7, pp.3, 2019, https://doi.org/10.12944/crnfsj.7.3.22
  14. Prospects of fibrinolytic proteases of bacteria from sea cucumber fermentation products as antithrombotic agent vol.28, pp.None, 2014, https://doi.org/10.1051/bioconf/20202802006
  15. Glycemic Index, Starch, and Protein Digestibility in Tempeh Gembus Cookies vol.2020, pp.None, 2014, https://doi.org/10.1155/2020/5903109
  16. The Effects of Tempeh Gembus, an Indonesian Fermented Food, on Lipid Profiles in Women with Hyperlipidemia vol.16, pp.1, 2014, https://doi.org/10.2174/1573401314666180807112549
  17. The Effect of Processed Tempeh Gembus Administration on Blood Glucose in Obese Women vol.519, pp.None, 2014, https://doi.org/10.1088/1755-1315/519/1/012034
  18. Tempeh Gembus Cookies as an Alternative Snack for Adolescent Girls With Obesity vol.4, pp.4, 2014, https://doi.org/10.20473/amnt.v4i4.2020.265-270
  19. Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy vol.8, pp.None, 2014, https://doi.org/10.3389/fmolb.2021.680397
  20. Purification and Characterization of a Thrombolytic Enzyme Produced by a New Strain of Bacillus subtilis vol.31, pp.2, 2014, https://doi.org/10.4014/jmb.2008.08010
  21. Potential of fibrinolytic protease enzyme from tissue of sand sea cucumber (Holothuria scabra) as thrombolysis agent vol.743, pp.1, 2014, https://doi.org/10.1088/1755-1315/743/1/012007
  22. Food-grade protease producing bacteria isolated from Indonesian soybean tempe gembus and red oncom after prolonged fermentation vol.743, pp.1, 2021, https://doi.org/10.1088/1755-1315/743/1/012008
  23. NUTRIENT CONTENT, PROTEIN DIGESTIBILITY, AND ACCEPTABILITY OF SUBSTITUTING TEMPEH GEMBUS NUGGETS WITH TILAPIA FISH vol.16, pp.2, 2021, https://doi.org/10.20473/mgi.v16i2.139-149
  24. Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications vol.13, pp.11, 2021, https://doi.org/10.3390/pharmaceutics13111880