Biochemical insights into the key residue substituents of an L-asparaginase from Thermococcus kodakarensis and their application in acrylamide mitigation

L-Asparaginases are enzymes of biotechnological importance due to their applications in anticancer therapy and acrylamide mitigation. Identification of key residues is crucial for developing improved variants. A conserved triad, Asp Ser Thr, is found in the characterized thermophilic L-asparaginases. In order to evaluate structure-function relationships, this conserved triad in the L-asparaginase of Thermococcus kodakarensis (Asp⁵³ Ser⁵⁴ Thr⁵⁵) was replaced by the mesophilic counterpart from Escherichia coli (Gly Ser Gln). Three mutant variants, two single (Asp⁵³Gly and Thr⁵⁵Gln) and one double (Asp⁵³Gly-Thr⁵⁵Gln), were generated and recombinantly produced in Escherichia coli. The purified Thr⁵⁵Gln mutant exhibited >20% increase in enzymatic activity, whereas Asp⁵³Gly and Asp⁵³Gly-Thr⁵⁵Gln mutants showed negligible activity, highlighting the essential catalytic role of Asp⁵³. In our previous study, Lys²⁹⁹Leu substitution resulted in ~20% increase in enzyme activity. In order to examine the synergic effect of Thr⁵⁵Gln and Lys²⁹⁹Leu mutations, a double mutant (Thr⁵⁵Gln-Lys²⁹⁹Leu) was generated and purified. The double mutant enzyme displayed a 52% higher activity than the wild-type. When applied for acrylamide mitigation in chapatis, Thr⁵⁵Gln-Lys²⁹⁹Leu mutant reduced the acrylamide content by ~50%, compared to ~35% by the wild-type enzyme, demonstrating its potential for industrial applications.