FIBRINOLYTIC ENZYME FROM BACILLUS AMYLOLIQUEFACIENS: OPTIMISATION AND SCALE UP STUDIES
Keywords:
Fibrinolytic enzyme, Bacillus sp, Optimisation, Scale up studies, Commercial prospectsAbstract
Objective: This research work was carried out to identify a potent microorganism, which produced the fibrinolytic enzyme and to optimise the media and growth parameters to achieve the maximal enzyme production for commercial application.
Methods: Microorganisms were isolated from different sources and assayed for fibrinolytic activity. The shortlisted cultures after preliminary screening (casein hydrolysis, blood plate assay and blood clot dissolution) were identified using 16S rRNA amplification method. The media and growth parameters were optimized to achieve the maximal enzyme activity. In-silico studies were carried out to identify the activators and inhibitors of the enzyme.
Results: Two species of Bacillus, namely, Bacillus amyloliquefaciens and Bacillus licheniformis, isolated from spoilt milk and soy flour, respectively, exhibited fibrinolytic activity. In the laboratory scale studies, of these two cultures, B. amyloliquefaciens produced the Fibrinolytic enzyme in higher quantities, 28.98 FU/mL, compared to 26.63 FU/mL in B. licheniformis. The maximal activities were obtained after 72 h. The optimum conditions at laboratory scale for the maximal production of the fibrinolytic enzyme were: pH 7.2, temperature 37 C and agitation 200 rpm. When scale up studies with B. amyloliquefaciens in a 7 L Fermentor were undertaken. The maximal activity obtained was 55.60 FU/mL in 72 h, compared to that of 28.98 FU/mL in shake flask studies. The molecular weight of the enzyme was estimated to be about 38 kDa. In in-silico studies, it was observed that PMSF inhibited the fibrinolytic activity, thereby, confirming this fibrinolytic enzyme is a serine protease (Nattokinase).
Conclusion: The enzyme had exhibited excellent blood clot dissolving property and therefore may be considered for further scale up and commercial exploitation.
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References
Milner M. Nattokinase: Clinical updates-Doctors support its safety and efficacy. FOCUS Allergy Research Group News: Letter Nov; 2008. p. 2-6.
Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. A novel Fibrinolytic enzyme (Nattokinase) in the vegetable cheese Natto; a typical and popular soybean ood in the Japanese diet. Experientia 1987;15:1110-1.
Wang S, Chen H, Liang T, Lin Y. A novel nattokinase produced by Pseudomonas sp TKU015 using shrimp shells as substrate. Process Biochem 2009;44:70-6.
Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Rapp BA, Wheeler DL. Gen Bank. Nucleic Acids Res 2000;28:15–8.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped blast and psiblast: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–402.
Laskowski RA, MacArthur MW, Moss DS, Thornton JM. Procheck: a program to check the stereochemistry of protein structure. J Appl Crystallo 1993;26:283-91.
Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, Liang J. Castp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res 2006;34:116-8.
Greenwood JR, Calkins D, Sullivan AP, Shelley JC. Towards the comprehensive, rapid, and accurate prediction of the favourable tautomeric states of drug-like molecules in aqueous solution. J Comp Aided Mol Des 2010;24:591-604.
Shelley JC, Cholleti A, Frye L, Greenwood JR, Timlin MR, Uchimaya M. Epik: a software program for pKa prediction and protonation state generation for drug-like molecules. J Comp Aided Mol Des 2007;21:681-91.
Coolbear T, Eames CV, Casey Y, Danie RM, Morgan HW. Screening of strains identified as extremely thermophilic bacilli for extracellular proteolytic activity and general property of proteinases from two of the strains. J Appl Bacteriol 1991;71:252.
Deepak V, Kalishwaralal K, Ramakumarpandian S, Babu VS, Senthil kumar SR, Sangailiyandi G. Optimisation of media composition for Nattokinase production by Bacillus subtilis using Response Surface Methodology. Bioresour Technol 2008;99:8170-4.
Bednarski A. Identifying unknown bacteria using biochemical and molecular methods: Washington University at St Louis; 2006. p. 2-6.
Wilson K. Preparation of Genomic DNA from bacteria. Curr Prot Mol Biol 2001;2(4):1-5.
Peng Y, Yang X, Xiao L, Zhang Y. Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis. Res Microbiol 2004;155:167–73.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75.
Yin LJ, Lin H, Jiang ST. Bioproperties of potent Nattokinase from Bacillus subtilis YJ1. J Agri Food Chem 2010;58(9):5737–42.
Sambrook J, Frisch EF, Maniatis T. Molecular Cloning. Cold Spring Harbour Laboratories: New York; 1989.
Sayers EW, Barrett T, Benson DA, Bolton E, Bryant SH, Canese K, Chetvernin V, Church DM, et al. Database resources of the national center for biotechnology information. Nucleic Acids Res 2009;37:216-23.
Ikeda S, Ohsugi T, Sumi H. Activation of fibrinolysis (Nattokinase) induced by dipicolinic acid and related compounds. Food Sci Tech Res 2006;12:152-5.
Margareth BC, Miranda G, Sarachine J. Biological activities of Lupeol. Int J Biomed Pharm Sci 2009;3:46–66.
Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, et al. Glide: a new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracy. J Med Chem 2004;47:1739–49.
Duguid JP. Genus Bacillus. In: College JG, Fraser AG, Marmion BP, Simmons A. (Eds), Mackie and McCartney Practical Medical Microbiology. Churchill Livingstone: New York; 1996;1:317-27.
Al-Jumaily EF, Daweed W, Nadir MI. Production of alkaline protease from local Bacillus stearothermophilus AEAL2 by solid state fermentation. Iraqi J Biotech 2004;3:137-55.
Bhunia B, Dutta D, Chaudhuri S. Selection of suitable carbon, nitrogen and sulphate source for the production of alkaline protease by bacillus licheniformis ncim-2042. Nat Sci Biol 2010;2:56-9.
Kumar G, Takagi H. Microbial alkaline proteases: from a bioindustrial viewpoint. Biotech Adv 1999;17:561-94.
Siraj SG. Comparative studies on production of nattokinase from bacillus subtilis changing the nitrogen sources. MA Thesis, Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore; 2011.
Ellaiah P, Srinivasulu B. Production of extracellular protease by Streptomyces fradiae. Hind Antibiot Bull 1996;38:41-7.
Babu N, Amirtham D, Ramachandran R, Purnima. Morphological, biochemical and molecular characterisation of a non-chitinase protease producing bacteria, its biodegrading effect on shell fish waste and its enzyme kinetics. Elixir Biotech 2013;55:13116-19.
Heinz DW, Priestle JP, Rahuel J, Wilson KS, Gruttee MG. Refined crystal structures of subtilisin novo complex with wild type and two mutant eqlins, Comparison with other serine proteinase inhibitor complexes. J Mol Biol 1991;217:353-71.
Sussman JL, Lin D, Jiang J, Manning NO, Prilusky J, Ritter O, Abola EE. Protein Data Bank (PDB): database of three-dimensional structural information of biological macromolecules. Acta Crystall D Biol Crystallog 1998;54:1078–84.
Sali A, Blundell TL. Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol 1993;234:779-815.
Morris AL, MacArthur MW, Hutchinson EG, Thornton JM. Stereochemical quality of protein structure coordinates. Proteins 1992;12:345-64.
Jorgensen WL, Tirado-Rives J. The OPLS [optimized potentials for liquid simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides and crambin. J Amer Chem Soc 1988;110:1657-66.
Vasantha N, Thompson LD, Rhodes C, Banner CDB, Nagle J, Filpula D. Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. J Bacteriol 1984;59:811–9.