Work

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

Year: 2026

Type: article

Abstract: 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 (Asp 53 Ser 54 Thr 55 ) was replaced by the mesophilic counterpart from Escherichia coli (Gly Ser Gln). Three mutant variants, two single (Asp 53 Gly and Thr 55 Gln) and one double (Asp 53 Gly–Thr 55 Gln), were generated and recombinantly produced in Escherichia coli . The purified Thr 55 Gln mutant exhibited >20% increase in enzymatic activity, whereas Asp 53 Gly and Asp 53 Gly–Thr 55 Gln mutants showed negligible activity, highlighting the essential catalytic role of Asp 53 . In our previous study, Lys 299 Leu substitution resulted in ~20% increase in enzyme activity. In order to examine the synergic effect of Thr 55 Gln and Lys 299 Leu mutations, a double mutant (Thr 55 Gln–Lys 299 Leu) was generated and purified. The double mutant enzyme displayed a 52% higher activity than the wild-type. When applied for acrylamide mitigation in chapatis, Thr 55 Gln–Lys 299 Leu mutant reduced the acrylamide content by ~50%, compared to ~35% by the wild-type enzyme, demonstrating its potential for industrial applications.

Source: International Journal of Biological Macromolecules

DOI: 10.1016/j.ijbiomac.2026.150819