Investigating Functional and Folding Stability of an Engineered E. coli L-asparaginase Harboring Y176F/S241C Mutations.

Abstract

Purpose: L-asparaginase has been widely recognized as a critical component in the treatment of various types of lymphoproliferative disorders, since its introduction in 1960s. However, its use in some cases leads to allergic reactions rendering the continuation of treatment unfeasible. Thus, the development of L-asparaginase from alternative sources or the production of engineered enzymes have always been considered. This study aimed to produce and evaluate a novel enzyme designed based on the sequence of L-asparaginase from Escherichia coli bacteria with Y176F/S241C mutations.

Methods: The Y176F/S241C mutant L-asparaginase was successfully expressed as the GST-fusion protein in E. coli, and then was subjected to affinity and size exclusion chromatography. The activity of the purified enzyme was determined based on the released ammonia as the result of substrate hydrolysis using Nessler's reagent. Chemical denaturation experiment in the presence of increasing concentration of guanidinium chloride was applied to determine the folding stability of the purified enzyme.

Results: The mutant enzyme was purified with an efficiency of 77-fold but at a low recovery of 0.7%. The determined kinetic parameters Km, Vmax, kcat, specific activity and catalytic efficiency were 13.96 (mM), 2.218 (mM/min), 273.9 (min^-1), 237.8 (IU/mg) and 19.62 (mM^-1 min^-1), respectively. Moreover, unfolding free energy determined by guanidinium chloride induced denaturation for mutated and commercial L-asparaginase enzymes were 8421 J/mol and 5274 J/mol, respectively.

Conclusion: The mutant enzyme showed improved stability over the wild-type. Although the expression level and recovery were low, the mutant L-asparaginase demonstrated promising activity and stability, with potential clinical and industrial applications.