Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 70, Issue 45, Pages 14499-14509Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.2c05712
Keywords
Bacillus licheniformis; L-asparaginase; thermostability; popmusic; molecular dynamics simulations
Funding
- National Natural Science Foundation of China
- [31871742]
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This study successfully enhanced the thermostability of L-asparaginase from Bacillus licheniformis through rational design, screening out two mutants D172W and E207A, with the double mutant D172W/E207A exhibiting remarkable thermostability. The improved thermostable L-asparaginase offers a facile and efficient process for industrial applications, showcasing increased hydrophobicity and decreased flexibility as key factors.
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 degrees C and 5 degrees C higher optimum reaction temperature and melting temperature (Tm) 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.
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