Production Purification and Characterization of Fibrinolytic Enzyme from Marine Bacterium Fictibacillus sp strain SKA27 and in vivo evaluation of its thrombolytic activity

Abstract

Intravascular thrombosis of major blood vessels restricts blood flow to vital organs newlineand is the leading cause of cardiovascular deaths around the world. Unlike newlineanticoagulants, fibrinolytic enzymes can rapidly restore the blood flow in the newlinethrombotic arteries and veins by dissolving the thrombus. In particular, highly efficient newlinefibrinolytic agents from marine microorganisms have the ability to combat the rise in newlinemortality and morbidity from thrombosis. newlineThe present study reports the optimized production and purification of an extremely newlineactive fibrinolytic enzyme from newly isolated marine bacterium Fictibacillus sp. strain newlineSKA27. Extract of wheat bran was used for submerged fermentation, which enhanced newlinefibrinolytic enzyme production combined with yeast extract and CaCl2. The newlineconcentration of medium components was optimized through response surface newlinemethodology (RSM) and a backpropagation multilayer artificial neural network with a newline3-5-1 topology was trained to predict the enzyme activity. The prediction and newlineoptimization capabilities of regression and artificial neural network (ANN) were newlinecompared by analyzing root mean square error (RMSE), coefficient of determination newline(R2), standard error of prediction (SEP) and average absolute deviation (AAD). ANN newlinedisplayed a higher proficiency in prediction with an R2 value of 0.99 and RMSE of 2.0 newlinecompared to 0.98 R2 and 48.9 RMSE of the regression model. ANN linked Genetic newlinealgorithm (GA) resulted in better optimization of the medium components to achieve newlinehigh enzyme activity of 4175.41 U/mL, which was 1.8 fold higher than the unoptimized newlinemedium. Further, the fibrinolytic enzyme was purified to electrophoretic homogeneity newlinewith an apparent molecular weight of 28 KDa on SDS PAGE. The characterization newlinestudies on the purified enzyme revealed the enzyme to be an alkaline protease, which newlineexhibited broad pH and temperature stability, with a pH range of 5-11 and heat newlineresistance up to 60 °C for 1 h and up to 50 °C for 90 min.