Improvement of L-asparaginase thermal stability by regulating enzyme kinetic and thermodynamic states

L-asparaginase (EC 3.5.1.1) needs to endure high temperatures during applications in food processing. We here improved the thermal stability of Bacillus subtilis 168 L-asparaginase by regulating both the kinetic and thermodynamic states of the enzyme. According to the molecular dynamics simulation for L-asparaginase, 10 flexible residues potentially affecting the kinetic stability were individually subjected to saturation mutagenesis, generating two highly stabilized mutants, S180N and D289T. As predicted by Discovery Studio, eight residues of Lasparaginase formed multiple unfavourable charge-charge interactions adversely affecting thermodynamic stability. Single-site saturation mutagenesis on these residues resulted in two thermodynamically stabilized mutants, E260F and E292S. Using these mutants, we constructed a further stabilized quadruple mutant NTFS (S180N/D289T/E260F/E292S). Compared with the wild-type enzyme, NTFS exhibited an 8.1-fold increase in half-life at 65 degrees C and a 5.56 degrees C increase in melting temperature. NTFS also showed a substantial increase in the transition state energy barrier (Delta Delta G(transition) = 5.27 KJ/mol) and a clear decrease in folding free energy (Delta Delta G(folding) = -19.12 kcal/mol) relative to the wild-type enzyme. These results indicated that simultaneous regulation of both the kinetic and thermodynamic states of an enzyme represents an efficient strategy for enhancing thermal stability. The highly stable NTFS described here might be beneficial for industrial application.