Nhóm nghiên cứu Kim và cộng sự (2020) đã tách chiết được 2 enzyme fibrinolytic từ thực phẩm lên men Jotgal Hàn Quốc có tên gọi là JP - I và JP - II [81]. Hai enzyme này có thơng số động học Km trên cơ chất fibrin lần lượt là 2,96 mM và 4,76 mM. Từ thông dữ liệu trên nhân thấy cùng trên cơ chất fibrin thì NK từ chủng R0H1 có hệ số Km xấp xỉ với enzyme JP - I và thấp hơn enzyme JP - II. Như vậy có thể kết luận ái độ của NK từ chủng R0H1 tương tự như enzyme JP - I và cao hơn enzyme JP - II. Ngồi ra NK từ chủng R0H1 có ái độ với fibrin tốt hơn so với NK từ chủng Bacillus sp. KDO - 13 (Km = 110,50 mM) [68].
KẾT LUẬN
1. Hoạt độ NK từ dịch lên men chủng R0H1 trên thiết bị 2 L đạt 158,00 FU/ml sau 13 giờ lên men.
2. Phương án cấp dưỡng trong khoảng thời gian là 5 giờ đem lại hiệu quả tốt nhất: OD 600nm đạt 37,60 và mật độ tế bào 7,90 x 109 CFU/ml tại thời điểm 16 giờ; hoạt độ của NK đạt 351,43 + 8,57 FU/ml sau 22 giờ lên men - tăng lần lượt 2,22 lần và 2,60 lần so với hoạt độ NK trong lên men theo mẻ trên thiết bị và trên bình tam giác
3. Thu hồi enzyme bằng phương pháp lọc dòng ngang đạt hiệu suất 89,70 ± 8,00 % và hoạt độ riêng đạt 2082,23 ± 36,42 FU/mg, enzyme có kích thước khoảng 28 kDa.
4. Enzyme được thu hồi có những đặc tính sau: - Nhiệt độ và pH tối ưu lần lượt là 55oC và 8,50 - Enzyme ổn định trong khoảng 30 - 60oC. - Enzyme tương đối bền ở pH 7,40 và 8,50
- Sự có mặt của 2 ion kim loại Ca2+ và Mg2+ làm tăng hoạt độ enzyme.
- Enzyme có km = 3,08 mM và Vmax = 6,71 nmol/min đối với cơ chất là fibrin.
KIẾN NGHỊ
- Nghiên cứu phương pháp cấp dưỡng kéo dài pha cân bằng để quá trình lên men đạt hiệu quả cao hơn.
TÀI LIỆU THAM KHẢO
1. Unrean, P., et al., Improvement of nattokinase production by Bacillus subtilis using an optimal feed strategy in fed-batch fermentation. Asia-
Pacific Journal of Science and Technology, 2012. 17(5): p. 769-777. 2. Chen, P.T., C.-J. Chiang, and Y.-P. Chao, Medium Optimization for the
Production of Recombinant Nattokinase by Bacillus subtilis Using Response Surface Methodology. Biotechnology Progress, 2007. 23(6): p.
1327-1332.
3. Berenjian, A., et al., Nattokinase production: Medium components and feeding strategy studies. Chemical Industry & Chemical Engineering
Quarterly, 2014. 20(4): p. 541-547.
4. Cho, Y.-H., et al., Production of nattokinase by batch and fed-batch culture
of Bacillus subtilis. New Biotechnology, 2010. 27(4): p. 341-346.
5. Liu, Z., et al., High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters. BMC
Microbiology 2019. 19: p. 89.
6. Venkataraman, D., et al., Medium optimization and immobilization of purified fibrinolytic URAK from Bacillus cereus NK1 on PHB nanoparticles. Enzyme and Microbial Technology 2010. 47: p. 297-304.
7. Zhang, J., et al., High cell density cultivation of a recombinant Bacillus Subtilis for nattokinase production. Authorea Preprints, 2020.
8. Kwon, E.-Y., et al., Production of nattokinase by high cell density fed-batch
culture of Bacillus subtilis. Bioprocess and Biosystems Engineering, 2011.
34(7): p. 789-793.
9. Sumi, H., et al., A novel fibrinolytic enzyme (nattokinase) in the vegetable
cheese Natto; a typical and popular soybean food in the Japanese diet.
Experientia, 1987. 43(10): p. 1110-1111.
10. Chen, H., et al., Nattokinase: A Promising Alternative in Prevention and Treatment of Cardiovascular Diseases. Biomarker insights, 2018. 13: p. 1-
8.
11. Zheng, Z.L., et al., Construction of a 3D model of nattokinase, a novel fibrinolytic enzyme from Bacillus natto. A novel nucleophilic catalytic mechanism for nattokinase. J Mol Graph Model, 2005. 23(4): p. 370-380.
12. Weng, Y., et al., Nattokinase: An oral antithrombotic agent for the prevention of cardiovascular disease. International journal of molecular
sciences, 2017. 18(3): p. 523.
13. Dabbagh, F., et al., Nattokinase: production and application. Applied
Microbiology and Biotechnology, 2014. 98(22): p. 9199-9206.
14. Nagata, C., et al., Dietary soy and natto intake and cardiovascular disease
mortality in Japanese adults: the Takayama study. The American Journal
of Clinical Nutrition, 2017. 105(2): p. 426-431.
15. Huang, Y., et al., Ultra-small and anionic starch nanospheres: Formation
and vitro thrombolytic behavior study. Carbohydrate Polymers, 2013.
96(2): p. 426-434.
16. Fujita, M., et al., Antihypertensive Effects of Continuous Oral Administration of Nattokinase and Its Fragments in Spontaneously Hypertensive Rats. Biological and Pharmaceutical Bulletin, 2011. 34(11):
p. 1696-1701.
17. Sumi, H., et al., Enhancement of the Fibrinolytic Activity in Plasma by Oral
Administration of Nattokinase. Acta Haematologica, 1990. 84(3): p. 139-
143.
18. Fujita, M., et al., Transport of Nattokinase across the Rat Intestinal Tract. Biological & Pharmaceutical Bulletin, 1995. 18(9): p. 1194-1196.
19. Kou, Y., et al., Development of a nattokinase–polysialic acid complex for
advanced tumor treatment. European Journal of Pharmaceutical Sciences,
20. Wu, H., et al., Acute toxicity and genotoxicity evaluations of Nattokinase, a
promising agent for cardiovascular diseases prevention. Regulatory
Toxicology and Pharmacology, 2019. 103: p. 205-209.
21. Huang, Z., et al., Nattokinase Attenuates Retinal Neovascularization Via
Modulation of Nrf2/HO-1 and Glial Activation. Investigative
ophthalmology & visual science, 2021. 62(6): p. 25-25.
22. Lampe, B.J. and J.C. English, Toxicological assessment of nattokinase derived from Bacillus subtilis var. natto. Food and Chemical Toxicology,
2016. 88: p. 87-99.
23. Jensen, G.S., et al., Consumption of nattokinase is associated with reduced
blood pressure and von Willebrand factor, a cardiovascular risk marker: results from a randomized, double-blind, placebo-controlled, multicenter North American clinical trial. Integr Blood Press Control, 2016. 9: p. 95-
104.
24. Kurosawa, Y., et al., A single-dose of oral nattokinase potentiates thrombolysis and anti-coagulation profiles. Scientific reports, 2015. 5: p.
11601.
25. Kim, M.-h., K.-m. Kwon, and K.-s. Kim, Efficacy and Safety of Ultra Nattokinase as a Fibrinolytic Agent: A Randomized, Double-Blind, Placebo-Controlled Study. Korean Journal of Family Practice, 2017. 7(2):
p. 264-270.
26. Maslarov, D. and D. Drenska, Use of Nattokinase in patients with ischemic
stroke and transient ischemic attacks. Neurology and Neuroscience, 2020.
1(2).
27. Fujita, M., et al., Characterization of nattokinase-degraded products from
human fibrinogen or cross-linked fibrin. Fibrinolysis, 1995. 9(3): p. 157-
164.
28. Ali, A.M.M. and S.C.B. Bavisetty, Purification, physicochemical properties, and statistical optimization of fibrinolytic enzymes especially from fermented foods: A comprehensive review. International Journal of
Biological Macromolecules, 2020.
29. Yatagai, C., et al., Nattokinase-promoted tissue plasminogen activator release from human cells. Pathophysiol Haemost Thromb, 2007. 36(5): p.
227-232.
30. Feng, R., et al., Preparation and toxicity evaluation of a novel nattokinase-
tauroursodeoxycholate complex. Asian Journal of Pharmaceutical
Sciences, 2018. 13(2): p. 173-182.
31. Bhatt, P.C., et al., Development of surface-engineered PLGA nanoparticulate-delivery system of Tet1-conjugated nattokinase enzyme for inhibition of Aβ(40) plaques in Alzheimer's disease. International journal of
nanomedicine, 2017. 12: p. 8749-8768.
32. Liu, S., et al., Synthesis of sustained release/controlled release nanoparticles carrying nattokinase and their application in thrombolysis.
Die Pharmazie-An International Journal of Pharmaceutical Sciences, 2021. 76(4): p. 145-149.
33. Huang, M., et al., A nano polymer conjugate for dual drugs sequential release and combined treatment of colon cancer and thrombotic complications. Materials Science and Engineering: C, 2020. 110: p.
110697.
34. Gallelli, G., et al., Data Recorded in Real Life Support the Safety of Nattokinase in Patients with Vascular Diseases. Nutrients, 2021. 13(6).
35. Cai, D., C. Zhu, and S. Chen, Microbial production of nattokinase: current
progress, challenge and prospect. World J Microbiol Biotechnol 2017.
33(5).
36. Inatsu, Y., et al., Characterization of Bacillus subtilis strains in Thua nao,
a traditional fermented soybean food in northern Thailand. Letters in
Applied Microbiology 2006. 43(3): p. 237-242.
37. Wang, S.-L., et al., A novel nattokinase produced by Pseudomonas sp. TKU015 using shrimp shells as substrate. Process Biochemistry, 2009.
38. Kim, W., et al., Purification and characterization of a fibrinolytic enzyme
produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.
Applied and Environmental Microbiology, 1996. 62(7): p. 2482.
39. Liu, J., et al., Optimization of nutritional conditions for nattokinase production by Bacillus natto NLSSE using statistical experimental methods.
Process Biochemistry, 2005. 40(8): p. 2757-2762.
40. Park, C.-S., et al., Identification of fibrinogen-induced nattokinase WRL101
from Bacillus subtilis WRL101 isolated from Doenjang. African Journal of
Microbiology Research, 2013. 7(19): p. 1983-1992.
41. Nie, G., et al., Co-Production of Nattokinase and Poly (³-Glutamic Acid) Under Solid-State Fermentation Using Soybean and Rice Husk. Brazilian
Archives of Biology and Technology, 2015. 58: p. 718-724.
42. Kada, S., et al., Identification of two major ammonia-releasing reactions involved in secondary natto fermentation. Bioscience, Biotechnology, and
Biochemistry, 2008. 72(7): p. 1869-1876.
43. Ni, H., et al., Expression of nattokinase in Escherichia coli and renaturation
of its inclusion body. Journal of Biotechnology, 2016. 231: p. 65-71.
44. Liang, X., et al., Secretory expression of a heterologous nattokinase in Lactococcus lactis. Applied Microbiology and Biotechnology, 2007. 75(1):
p. 95-101.
45. Chen, P.T., Y.-P. Chiang Cj Fau - Chao, and Y.P. Chao, Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis.
Biotechnol Prog, 2007. 23: p. 803-813.
46. Nguyen, T.T., T.D. Quyen, and H.T. Le, Cloning and enhancing production
of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800. Microbial cell factories, 2013. 12: p. 79-79.
47. Reuß, D.R., et al., Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain 3NA. Genome announcements, 2015. 3(2).
48. Ravichandran, S. and V. R, Solid state and submerged fermentation for the
production of bioactive substances: a comparative study. International
Journal of Science and Nature, 2012. 3: p. 480-486.
49. T, P., et al., Glucose-limited fed-batch cultivation of Escherichia coli with
computer-controlled. Biotechnol Bioeng. 22(5): p. 341-346.
50. Yee, L. and H. Blanch, Recombinant protein expression in high cell density
fed-batch cultures of Escherichia coli. Nature Biotechnology, 1992. 10(12):
p. 1550-1556.
51. Ting, T.E., et al., A simple substrate feeding strategy using a pH control trigger in fed-batch fermentation. 2007(0273-2289 (Print)).
52. M, W., et al., Enhancement of oxidative stability of the subtilisin nattokinase by site-directed. Biochim Biophys Acta, 2009. 1794(11): p.
1566-1572.
53. Quoc Tuan, T., et al., Purification and characterization of recombinant nattokinase from Bacillus subtilis. Tạp chí sinh học, 2015. 37: p. 75-84.
54. Xin, X., et al., Purification and characterization of fibrinolytic enzyme from
a bacterium isolated from soil. 3 Biotech, 2018. 8(2): p. 90.
55. Bora, B., et al., The N-terminal-truncated recombinant fibrin(ogen)olytic serine protease improves its functional property, demonstrates in vivo anticoagulant and plasma defibrinogenation activity as well as pre-clinical safety in rodent model. International Journal of Biological Macromolecules,
2018. 111: p. 462-474.
56. Hu, Y., et al., Purification and characterization of a novel, highly potent
fibrinolytic enzyme from Bacillus subtilis DC27 screened from Douchi, a traditional Chinese fermented soybean food. Scientific Reports, 2019. 9(1):
p. 9235.
57. Tian, L., W. Zhou, and Y. Zhang, Construction of a genetically engineered
strain of nattokinase and assessment of its fibrinolytic activity. African
Journal of Microbiology Research, 2019. 13(27): p. 488-499.
58. Yang, H., et al., Genome sequencing, purification, and biochemical characterization of a strongly fibrinolytic enzyme from Bacillus
amyloliquefaciens Jxnuwx-1 isolated from Chinese traditional douchi. The
Journal of General and Applied Microbiology, 2019. 66(3): p. 153-162. 59. Purwaeni, E., C. Riani, and D.S. Retnoningrum, Molecular
Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods. . The Protein Journal, 2020. 39: p. 258-267.
60. Liang, X., et al., Secretory expression of nattokinase from Bacillus subtilis
YF38 in Escherichia coli. Molecular Biotechnology, 2007. 37(3): p. 187-
194.
61. Wang, C., et al., Purification and characterization of nattokinase from Bacillus subtilis Natto B-12. Journal of Agricultural and Food Chemistry,
2009. 57(20): p. 9722-9729.
62. Wang, S.-L., Y.-Y. Wu, and T.-W. Liang, Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. New Biotechnology, 2011. 28(2): p. 196-202.
63. Narasimhan, M.K., et al., Purification, biochemical, and thermal properties
of fibrinolytic enzyme secreted by Bacillus cereus SRM-001. Preparative
Biochemistry & Biotechnology, 2018. 48(1): p. 34-42.
64. Chang, C.-T., et al., Potent Fibrinolytic Enzyme from a Mutant of Bacillus
subtilis IMR-NK1. Journal of agricultural and food chemistry, 2000. 48(8):
p. 3210-3216.
65. Xiao-lan, L., et al., Purification and characterization of a novel fibrinolytic
enzyme from Rhizopus chinensis 12. Applied Microbiology and
Biotechnology, 2005. 67(2): p. 209-214.
66. Jo, H.-D., et al., Purification and characterization of a major fibrinolytic enzyme from Bacillus amyloliquefaciens MJ5-41 isolated from Meju. J
Microbiol Biotechnol, 2011. 21(11): p. 1166-73.
67. L, M. and M. M, Kinetik der Invertinwirkung. Biochem Z, 1913(49): p. 333- 369.
68. Lee, A., et al., Purification and characterization of a fibrinolytic enzyme from Bacillus sp. KDO-13 isolated from soybean paste. Journal of
Microbiology and Biotechnology, 2001. 11: p. 845-852.
69. Garg, R. and B.N. Thorat, Nattokinase purification by three phase partitioning and impact of t-butanol on freeze drying. Separation and
Purification Technology, 2014. 131: p. 19-26.
70. Avhad, D.N. and V.K. Rathod, Application of mixed modal resin for purification of a fibrinolytic enzyme. Preparative Biochemistry &
Biotechnology, 2016. 46(3): p. 222-228.
71. Mesapogu, S., C.M. Jillepalli, and D.K. Arora, Agarose Gel Electrophoresis and Polyacrylamide Gel Electrophoresis: Methods and
Principles, in Analyzing Microbes: Manual of Molecular Biology
Techniques. 2013, Springer Berlin Heidelberg: Berlin, Heidelberg. p. 73-
91.
72. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976. 72(1): p. 248-254.
73. d'Anjou, M.C. and A.J. Daugulis, Mixed-feed exponential feeding for fed-
batch culture of recombinant methylotrophic yeast. Biotechnology Letters,
2000. 22(5): p. 341-346.
74. Xin, X., et al., Development of universal purification protocols for fibrinolytic enzyme-producing bacilli. CyTA - Journal of Food, 2019. 17(1):
p. 112-120.
75. Walker, J.B. and M.E. Nesheim, The molecular weights, mass distribution,
chain composition, and structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin. Journal of Biological
Chemistry, 1999. 274(8): p. 5201-5212.
76. Ren, Y., et al., Biochemical characterization of a fibrinolytic enzyme composed of multiple fragments. Acta Biochim Biophys Sin, 2018. 50(2):
77. Sumaya Ali Hmood*, G.M.A., Optimum conditions for fibrinolytic enzyme
(Nattokinase) production by Bacillus sp. B24 using solid state fermentation.
Iraqi Journal of Science, 2016. 57: p. 1391-1401.
78. Lin, H.T.V., et al., Purification and characterization of nattokinase from cultural filtrate of red alga porphyra dentata fermented by Bacillus subtilis N1. Journal of Marine Science and Technology (Taiwan), 2015. 23: p. 240-
248.
79. Sumaya Ali Hmood, G.M.A., Purification and characterization of
nattokinase produced by local isolate of Bacillus sp. B24. Iraqi Journal of
Biotechnology, 2016. 15(2): p. 93-108.
80. Chang, C.-T., et al., Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis-fermented red bean. Food
Chemistry, 2012. 133: p. 1611–1617.
81. Kim, C., K. Ri, and S. Choe, A novel fibrinolytic enzymes from the Korean
traditional fermented food—Jotgal: Purification and characterization.
PHỤ LỤC
1. Pha hóa chất đo hoạt độ NK
Chuẩn bị cơ chất:
Cơ Fibrin được pha trong 70 mL NaOH 0,1 N. Sử dụng khuấy từ khuấy đến khi tan hết (4 – 6 giờ), chỉnh pH về 7,4 bằng HCl đậm đặc sau đó định mức lên 100 ml bằng nước cất. Phần không tan được loại bỏ nhờ ly tâm 10000 rpm, 10 phút; thu dịch nổi.
Fibrin sau khi pha được bảo quản ở tủ -20ᵒC (bảo quản trong thời gian dài) hoặc 4oC (thường xuyên sử dụng).
Đệm:
Đệm Tris-HCl: 0,1M có bổ sung CaCl2 0,01M
Cân 2,42 g Tris – base hòa tan vào 80 ml nước cất. Chỉnh pH về 7,4. Định mức lên 100 ml bằng nước cất.
Cân 0,222 g CaCl2 hòa tan vào 80 ml nước cất. Chỉnh pH về 7,4. Định mức lên 100 ml bằng nước cất.
Khi sử dụng trộn 2 dung dịch với tỉ lệ 1:1.
Chất dừng phản ứng:
Chất vô hoạt (dừng phản ứng) Trichloroacetic acid (TCA) 1,5 M tương đương 245,07 g/L.
Đường chuẩn tyrosine Phương pháp tiến hành:
Cân 0,001 g Tyrosine pha trong 50 mL HCl 0,2 N, được dung dịch gốc. Từ đó pha lỗng ra các nồng độ từ 5 - 200 µg/mL. Tiến hành so màu ở bước sóng 275 nm và xây dựng đường chuẩn. Theo đường chuẩn sẽ tính được độ tăng độ hấp thụ ở bước sóng 275 nm ứng với tăng nồng độ tyrosine 1µg/mL là hệ số a.
2. Dựng đường chuẩn tyrosine
Tyrosine (µg/ml)
0 5 10 25 50 100 150 200
3. Tính tốn tốc độ cấp dưỡng Các thơng số : Xo = 9,26