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A Novel Fibrinolytic Enzyme from Bacillus Sphaericus MTCC 3672: Optimization and Purification Studies

Devchand N. Avhad, Swapnil S. Vanjari, virendra Rathod

Abstract


A novel extracellular fibrinolytic enzyme was produced from Bacillus sphaericus MTCC 3672 for dissolving blood clots. Optimized fermentation parameters achieved by one factor at a time approach had demonstrated 2.85 fold increase in fibrinolytic activity i.e. from 3.5*104 U/l (basal media) to 9.98*104 U/l after 24 h of incubation in submerged fermentation. Statistical screening of six independent nutritional variables such as, glucose, yeast extract, NaCl, MgCl2, MnCl2, CaCl2 was studied using Plackett-Burman design. Amongst six variables, yeast extract was found to be significant factor affecting yield of a fibrinolytic enzyme. Furthermore, growth kinetics of biomass formation, enzyme production, and substrate utilization was evaluated by unstructured kinetic models and various biokinetic parameters such as µmax (0.37 1/h), Pr (0.12 1/h), YP/S (7.74 U/g) and YX/S (3.32 g/g) were determined. In purification step, ultrafiltered broth was purified with DEAE Cellulose anion exchange chromatography and Sephadex G100 gel filtration chromatography with 10 fold purity. In vitro fibrin clot degradation study had revealed significant breakdown of fibrin clot. The fibrinolytic activity of purified enzyme (7.5 mm) was found to be matching with marketed Nattolife® (8.0 mm) (nattokinase) and superior to Thromboflux® (0 mm) (streptokinase) formulations. Hence, efficient formulation containing purified fibrinolytic agent can be used for medical emergencies viz. myocardial infarction and deep vein thrombosis.


Keywords


Fibrinolytic enzyme; Bacillus sphaericus; Plackett-Burman design; Kinetic Models; DEAE Cellulose

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References


Yong P, Yang X, Zhang Y. Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. Appl Microbiol Biotechnol. 2005, 69:126-132

Balaraman K, Prabakaran G. Production & purification of a fibrinolytic enzyme (thrombinase) from Bacillus sphaericus. Indian J Med Res. 2007, 126:459-464

Chitte RR, Dey S. Potent fibrinolytic enzyme from thermophilic Streptomyces megasporus strain SD5. Lett Appl Microbiol. 2000, 31:405-410

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 food in the Japanese diet. Experienti. 1987, 15:1110-1111

Zhang RH, Xiao L, Pen Y, Wang HY, Bai F, Zhang YZ. Gene expression and characteristics of a novel fibrinolytic enzyme (subtilisin DFE) in Escherichia coli. Lett Appl Microbiol. 2005, 41:190-195

Myers PS, Yousten AA. Localization of mosquito larva toxin of Bacillus sphaericus 1593. Appl Environ Microbiol. 1980, 39 (6):1205-1211

Mahadevan PS, Mahadevan S, Sekar SC, Babu SS. Novel thrombolytic enzyme and process of its preparation. United patent application publication, 2009, US 2009/0285793A1

Prabakaran G, Hoti L. Egg yolk enhances early sporulation and toxicity of Bacillus sphaericus H5a5b for small-scale production of a mosquito control agent. Acta Trop. 2008, 108:50-53

Plackett RL, Burman JP. The design of optimum multifactorial experiment. Biometrika. 1946, 33:305-325

Li E, Yousten AA. Metalloprotease from Bacillus thuringiensis. Applied Microbiology. 1975, 30:354-361

Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 1976, 72:248-254

Jayani RS, Sukla, SK, Gupta R. Screening of bacterial strains for polygalacturonase activity: Its production by Bacillus sphaericus (MTCC 7542). Enzyme Res. 2010, doi:10.4061/2010/306785

Mercier P, Yerushalmi L, Rouleau D, Dochain D. Kinetics Glucose of Lactic Acid Fermentation on and Corn by Lactobacillus amylophilus. J Chem Technol Biot. 1992, 55:111-121

Wang CT, Ji BP, Nout BLR, Li PL, Ji H, Chen LF. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC 33, isolated from Chinese traditional Douchi. J Ind Microbiol Biotechnol. 2006, 33:750-758

Mahajan PM, Gokhale SV, Lele SS. Production of Nattokinase Using Bacillus natto NRRL 3666: Media Optimization, Scale Up, and Kinetic Modeling. Food Sci Biotechnol. 2010, 19(6):1593-1603

Choi NS, Kim BY, Lee GY, Yoon KS, Han KY, Kim SH. Relationship Between Acrylamide Concentration and Enzymatic Activity in An Improved Single Fibrin Zymogram Gel System. J Biochem Mol Biol. 2002, 35:236-238

Chen PT, Chiang CJ, Chao YP. Medium Optimization for the Production of Recombinant Nattokinase by Bacillus subtilis Using Response Surface Methodology. Biotechnol Prog. 2007, 23:1327-1332

Prabakaran G, Balaraman K, Hoti SL, Manonmani AM. A cost- effective medium for the large- scale production of Bacillus sphaericus H5a5b (VCRC B42) for mosquito control. Biol Control. 2007, 41:379-383

Cho YH, Song JY, Kim KM, Kim MK, Lee IY, Kim SB, Kim HS, Han NS, Lee B, Kim BS. Production of nattokinase by batch and fed-batch culture of Bacillus subtilis. New Biotechnol. 2010, 27:341-346

Haji M, Kanoun S, Nasri M, Gharsallah NJ. Purification and characterization of an alkaline serine –protease produced by a new isolated Aspergillus clavatus ES1. Process Biochem. 2007, 42:791-797

Mahajan PM, Nayak S, Lele SS. Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: Media optimization, purification and characterization. J Biosci Bioeng. 2012, 113:307-314


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