Smitha K.V., Sooraj S. Nath, Kunhi A.A.M. and Pradeep B.V. 2016. Isolation, Production, Purification and Characterization of Fibrinolytic Enzymes Produced by Bacillus altitudinis S-CSR0020. Abstract Book. National symposium on ‘Biosciences and Technology –Recent Developments and Future Prospects’, SIAS-Centre for Scientific Research, Safi Institute of Advanced Study, Vazhayoor, Malappuram, Kerala, p. 52.

ABSTRACT

In the present study soil samples were collected from near slaughter house, isolated by using clot agar was screened as fibrinolytic enzyme producers by observing the halo zone produced and identified by 16S rRNA sequencing as Bacillus altitudinis S-CSR0020 (GenBank:KT369312.1). Experiments revealed that casein was the best substrate with maximum enzyme activity of 790 U/mL. Proteolytic activity was determined by casein agar. Minimal media with fibrin is used for the production of enzyme, and specific activity of enzyme was noted. Optimization of cultural conditions done by RSM, three variable Box-Behnken design for response surface methodology was used to study the combined effect of substrate concentration, broth pH and fermentation temperature on enzyme production over three levels. The final optimized fermentation conditions obtained with RSM were 4 g/L (substrate concentration), 10.5 (broth pH) and 37.5 ̊C (fermentation temperature). The fibrinolytic enzyme with enzyme activity 1020 U/mL partially purified by ammonium sulfate precipitation followed by dialysis showed maximum activity at 80 %, with an enzyme activity of 2292 U/mL. The partially purified fibrinolytic enzyme showed temperature and pH optimal activities at 30 °C and 9 respectively, and stabilities at 40 °C and 7 respectively and the optimum substrate concentration was 2 %, and this enzyme also showed high blood clot lysis activity within 15 minutes, and from this, it is proved as a promising fibrinolytic agent. Although fibrinolytic enzyme showed high activity around pH 7.5-10.5, it showed maximum stability at pH 7 and temperature 40 °C, which is near to body temperature. From this study the Bacillus altitudinis S-CSR0020 has the capacity to produce highly thermostable and high pH active fibrinolytic enzyme which could have broad applications in many industries. 

 

Key words: Bacillus altitudinis , Fibrinolytic activity, Box-Behnken design