title: Ultrasound assisted three phase partitioning of a fibrinolytic enzyme.
abstract: The present investigation is aimed at ultrasound assisted three phase partitioning (UATPP) of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Three phase partitioning integrates the concentration and partial purification step of downstream processing of a biomolecule. Three phase system is formed with simultaneous addition of ammonium sulfate to crude broth and followed by t-butanol. UATPP of a fibrinolytic enzyme was studied by varying different process parameters such as ammonium sulfate saturation concentration, pH, broth to t-butanol ratio, temperature, ultrasound frequency, ultrasonication power, and duty cycle. The optimized parameters yielding maximum purity of 16.15-fold of fibrinolytic enzyme with 65% recovery comprised of 80% ammonium sulfate saturation, pH 9, temperature 30 °C, broth to t-butanol ratio 0.5 (v/v), at 25 kHz frequency and 150 W ultrasonication power with 40% duty cycle for 5 min irradiation time. SDS PAGE analysis of partitioned enzyme shows partial purification with a molecular weight in the range of 55-70 kDa. Enhanced mass transfer of UATPP resulted in higher fold purity of fibrinolytic enzyme with reduced time of operation from 1 h to 5 min as compared to conventional TPP. Outcome of our findings highlighted the use of UATPP as an efficient biosepartion technique.
authors.affiliation: Department of Chemical Engineering, Institute of Chemical Technology, Matunga (E), Mumbai 400019, India.
authors.forename: Devchand N
authors.initials: DN
authors.lastname: Avhad
authors.forename: Sonali S
authors.initials: SS
authors.lastname: Niphadkar
authors.forename: Virendra K
authors.initials: VK
authors.lastname: Rathod
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology*; D000440:Butanols / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D005342:Fibrinolysis; D006863:Hydrogen-Ion Concentration; D013696:Temperature; D014465:Ultrasonics
chemical_list: D000440:Butanols; D000645:Ammonium Sulfate
keywords: Bacillus sphaericus; Fibrinolytic enzyme; Mass transfer; SDS PAGE; Ultrasound assisted three phase partitioning