تكامل مستقيم آنزيم اسپارژينازII بمنظور بكارگيري در حلالهاي آلي

Directed evolution of L-asparaginase ІІ enzyme for applied in organic solvents

L-asparaginase is a biological enzyme that catalyzes conversion of asparagine to aspartate and concomitant production of ammonia. This enzyme has been used extensively as major therapeutic treatment against blood cancer. Our aim in this project has been to use directed evolution to improve activity of L-asparaginase ІІ enzyme (ASNaseІІ) in the organic solvents (nonbiological medium). In the first step, ASNase ІІ gene from E-coli was cloned using PCR methodology and the cloned gene was confirmed using restriction enzyme analysis and DNA sequencing. The expression of ASNase was confirmed using SDS-PAGE electrophoresis and western blotting. For the selection of functional mutants a colorimetric assay was optimized using phenol red assay. Our future work include identification of optimized enzyme mutants for conversion of asparagine to aspartate in nonbiological media.