Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

Abstract

L-Asparaginase has gained much attention for effectively treating acute lymphoblastic leukemia (ALL) and mitigating carcinogenic acrylamide in fried foods. Due to high-dose dependence for clinical treatment and low mitigation efficiency for thermal food processes caused by poor thermal stability, a method to achieve thermostable L-asparaginase has become a critical bottleneck. In this study, a rational design including free energy combined with structural and conservative analyses was applied to engineer the thermostability of L-asparaginase from Bacillus licheniformis (BlAsnase). Two enhanced thermostability mutants D172W and E207A were screened out by site-directed saturation mutagenesis. The double mutant D172W/E207A exhibited highly remarkable thermostability with a 65.8-fold longer half-life at 55 °C and 5 °C higher optimum reaction temperature and melting temperature (T m ) than those of wild-type BlAsnase. Further, secondary structure, sequence, molecular dynamics (MD), and 3D-structure analysis revealed that the excellent thermostability of the mutant D172W/E207A was on account of increased hydrophobicity and decreased flexibility, highly rigid structure, hydrophobic interactions, and favorable electrostatic potential. As the first report of rationally designing L-asparaginase with improved thermostability from B. licheniformis, this study offers a facile and efficient process to improve the thermostability of L-asparaginase for industrial applications.

Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from Streptomyces rameus L2001 by Rigidifying Flexible Regions in Loop Regions of the N-Terminus

“…In addition, the surface net charge of the enzyme has a significant influence on its thermal unfolding and refolding behavior . Increasing the surface net charge by altering the distribution of the surface charge of proteins can enhance the thermal stability of proteins. ,, In the present study, Mut1 and Mut2 constructed by combinational mutations showed significant changes in the surface electrostatic potential in the loop2, loop3, and loop4 regions (Figure ), resulting in an increase in the net surface charge of the mutants in comparison with that of wild-type XynA (Table ). A higher surface net charge can promote reversible refolding by preventing xylanase aggregation.…”

Residue Effect-Guided Design: Engineering of S. Solfataricus β-Glycosidase to Enhance Its Thermostability and Bioproduction of Ginsenoside Compound K

“…For example, a BlAsnase variant obtained by site-directed saturation mutagenesis improved its thermal stability by 65.8-fold at 55 °C. 22 Similarly, a thermostable cellobiose CtCel6 from Thermus thermophilus was rationally engineered using site-directed mutagenesis based on structure to give a variant with a halflife (t 1/2 ) increased by 1.42-fold and 2.40-fold at 80 and 90 °C, respectively. 23 Rational and semirational design strategies have been used to identify mutations at five beneficial sites of βglycosidase BglY of T. thermophilus, which were then recombined in variant HF5 to increase the t 1/2 by 4.7-fold at 93 °C.…”

“…Approaches such as directed evolution, rational design, and computer-aided design have proven to be effective strategies for engineering thermophilic enzymes. For example, a BlAsnase variant obtained by site-directed saturation mutagenesis improved its thermal stability by 65.8-fold at 55 °C . Similarly, a thermostable cellobiose CtCel6 from Thermus thermophilus was rationally engineered using site-directed mutagenesis based on structure to give a variant with a half-life ( t 1/2 ) increased by 1.42-fold and 2.40-fold at 80 and 90 °C, respectively .…”

Enhanced Thermostability of Geobacillus stearothermophilus α-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement

“…The enzyme thermostability is crucial for their kinetic stability and robustness . Various strategies have been employed to enhance enzyme thermostability, including the introduction of disulfide bonds, proline modification, salt bridge formation, and charge on the protein surface .…”

“…The enzyme thermostability is crucial for their kinetic stability and robustness. 13 Various strategies have been employed to enhance enzyme thermostability, including the introduction of disulfide bonds, 14 proline modification, 15 salt bridge formation, 16 and charge on the protein surface. 17 Among these strategies, the incorporation of disulfide bonds has gained significant attention due to their ability to reduce the entropy of the unfolded state, 18 thus improving protein thermostability.…”