Design, production and biological evaluation of new variant of L-asparaginase from E.coli

Abstract

L-asparaginase is an enzyme that is used to produce antineoplastic drugs. The medicinal usage of L-asparaginase may be hazardous due to allergic responses and toxicities, necessitating the development of bio-better L-asparaginase. Diverse techniques, including directed evolution, random point mutations, chain extension, and site-directed mutagenesis are used to improve protein stability. Aims: (i) designing novel L-asparaginase enzyme with improved biopharmaceutical properties (ii) producing the designed enzyme and evaluating its activity. Methods: 21 important L-asparaginase mutations and their associated functional significances were collected from the literature. The identified mutations were used to generate a list of L-asparaginase mutants with all possible combinations of up to 4 mutations guided by identified mutations. Three-dimensional structural models of all 7546 proposed L-asparaginase mutants were generated and their in-silico stabilities were calculated. The most promising mutant was selected for designing a genetic construct suitable for expression and purification of L-asparaginase in bacterial expression system. The designed genetic construct was purchased, amplified and transformed into E.coli expression host. The crude protein sample was tested for in-vitro enzymatic activity using spectroscopic method. Results: A computational study predicted that Y176F/S241C double mutation of E.coli L-asparaginase may increase its folding stability. The DNA sequence coding for the designed enzyme inserted in bacterial expression vector was used to produce recombinant L-asparaginase in two different E. coli strains. The levels of enzyme expressed in both strains are qualitatively equal, but it seems that L-asparaginase produced in PlysS strain contains a pelB signal sequence, whereas those expressed in Origami strain are mainly without the signal peptide. Functionality assay showed that the designed recombinant ASNase was active. Conclusions: A novel functional double mutant L-asparaginase with predicted improved enzymatic properties was designed and produced. Based on the findings of this study, the identified enzyme needs to be further evaluated for its possible use in pharmaceutical and industrial applications.