title: Application of mixed modal resin for purification of a fibrinolytic enzyme.
abstract: Recent advances in purification technologies for therapeutic molecules have stirred the research consortium. Mixed mode chromatography, having multiple interactions with the solute molecule, has drawn significant attention due to its overall advantage over traditional ion-exchange and reverse-phase chromatography. Capto adhere, a mixed mode chromatography resin with strong anion-exchange and reverse-phase interaction with solutes, was explored for purification of fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Static and dynamic resin binding study revealed that 30°C temperature, pH 8, and 0.5 mL/min flow rate were optimum for maximum binding of fibrinolytic enzyme. Maximum static dynamic binding and breakthrough capacities for Capto adhere were 249 and 196 U/mL of resin, respectively. Final purification with Sephadex G 100 gel chromatography resulted in 38-fold purity of fibrinolytic enzyme with 39% enzyme recovery. Purified enzyme was further characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis to homogeneity, and molecular mass was found to be around 55-70 kD. Like most of the serine alkaline proteases, purified fibrinolytic enzyme was stable in a temperature range of 25-40°C and pH range of 7-9. Offshoots of our research findings have revealed a broad application area of mixed mode chromatography.
authors.affiliation: a Department of Chemical Engineering , Institute of Chemical Technology , Mumbai , India.
authors.forename: Devchand N
authors.initials: DN
authors.lastname: Avhad
authors.affiliation: a Department of Chemical Engineering , Institute of Chemical Technology , Mumbai , India.
authors.forename: Virendra K
authors.initials: VK
authors.lastname: Rathod
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000379:methods*; D004798:Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D005342:Fibrinolysis; D006863:Hydrogen-Ion Concentration; D008962:Models, Theoretical; D013696:Temperature
chemical_list: D004798:Enzymes
keywords: Bacillus sphaericus; capto adhere; fibrinolytic enzyme; mixed mode chromatography; serine alkaline protease