Journal
MOLECULES
Volume 27, Issue 19, Pages -Publisher
MDPI
DOI: 10.3390/molecules27196670
Keywords
L-asparaginase; Acinetobacter soli; thermostability; consensus design; cysteine substitution; acrylamide inhibition
Funding
- National Natural Science Foundation of China [31871742]
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The thermostability of Acinetobacter soli L-asparaginase was improved by mutating two non-conserved cysteine residues, resulting in enhanced efficiency in food pre-processing.
To extend the application range of L-asparaginase in food pre-processing, the thermostability improvement of the enzyme is essential. Herein, two non-conserved cysteine residues with easily oxidized free sulfhydryl groups, Cys8 and Cys283, of Acinetobacter soli L-asparaginase (AsA) were screened out via consensus design. After saturation mutagenesis and combinatorial mutation, the mutant C8Y/C283Q with highly improved thermostability was obtained with a half-life of 361.6 min at 40 degrees C, an over 34-fold increase compared with that of the wild-type. Its melting temperature (T-m) value reaches 62.3 degrees C, which is 7.1 degrees C higher than that of the wild-type. Molecular dynamics simulation and structure analysis revealed the formation of new hydrogen bonds of Gln283 and the aromatic interaction of Tyr8 formed with adjacent residues, resulting in enhanced thermostability. The improvement in the thermostability of L-asparaginase could efficiently enhance its effect on acrylamide inhibition; the contents of acrylamide in potato chips were efficiently reduced by 86.50% after a mutant C8Y/C283Q treatment, which was significantly higher than the 59.05% reduction after the AsA wild-type treatment. In addition, the investigation of the mechanism behind the enhanced thermostability of AsA could further direct the modification of L-asparaginases for expanding their clinical and industrial applications.
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