Engineering Tk1656, a highly active l-asparaginase from Thermococcus kodakarensis, for enhanced activity and stability

l-Asparaginases catalyze the hydrolysis of l-asparagine to l-aspartic acid and ammonia. These enzymes have potential applications in therapeutics and food industry. Tk1656, a highly active and thermostable l-asparaginase from Thermococcus kodakarensis, has been proved effective in selective killing of acute lymphocytic leukemia cells and in reducing acrylamide formation in baked and fried foods. However, it displayed 20 % increase in activity at 85 °C. H158S substitution resulted in >5 °C increase in the optimal temperature. Similarly, a mesophilic-like mutation L56D, resulted in >5-fold increase in activity at pH 7.0 and 37 °C compared to that of the wild-type enzyme. The substrate specificity of the mutant variants remained unchanged. These results demonstrate that L56D and K299L variants of Tk1656 are the potent enzymes for therapeutics and acrylamide mitigation applications, respectively.