Thermostability Improvement of L-Asparaginase from <i>Acinetobacter soli</i> via Consensus-Designed Cysteine Residue Substitution
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 <i>Acinetobacter soli</i> L-asparaginase (AsA...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-10-01
|
| Series: | Molecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1420-3049/27/19/6670 |
| _version_ | 1797477838638546944 |
|---|---|
| author | Linshu Jiao Huibing Chi Bingjie Xia Zhaoxin Lu Xiaomei Bie Haizhen Zhao Fengxia Lu Meirong Chen |
| author_facet | Linshu Jiao Huibing Chi Bingjie Xia Zhaoxin Lu Xiaomei Bie Haizhen Zhao Fengxia Lu Meirong Chen |
| author_sort | Linshu Jiao |
| collection | DOAJ |
| description | 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 <i>Acinetobacter soli</i> 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 °C, an over 34-fold increase compared with that of the wild-type. Its melting temperature (<i>T</i><sub>m</sub>) value reaches 62.3 °C, which is 7.1 °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. |
| first_indexed | 2024-03-09T21:23:25Z |
| format | Article |
| id | doaj.art-22282297de984853911a50865462c822 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T21:23:25Z |
| publishDate | 2022-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-22282297de984853911a50865462c8222023-11-23T21:15:19ZengMDPI AGMolecules1420-30492022-10-012719667010.3390/molecules27196670Thermostability Improvement of L-Asparaginase from <i>Acinetobacter soli</i> via Consensus-Designed Cysteine Residue SubstitutionLinshu Jiao0Huibing Chi1Bingjie Xia2Zhaoxin Lu3Xiaomei Bie4Haizhen Zhao5Fengxia Lu6Meirong Chen7College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaTo 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 <i>Acinetobacter soli</i> 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 °C, an over 34-fold increase compared with that of the wild-type. Its melting temperature (<i>T</i><sub>m</sub>) value reaches 62.3 °C, which is 7.1 °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.https://www.mdpi.com/1420-3049/27/19/6670L-asparaginase<i>Acinetobacter soli</i>thermostabilityconsensus designcysteine substitutionacrylamide inhibition |
| spellingShingle | Linshu Jiao Huibing Chi Bingjie Xia Zhaoxin Lu Xiaomei Bie Haizhen Zhao Fengxia Lu Meirong Chen Thermostability Improvement of L-Asparaginase from <i>Acinetobacter soli</i> via Consensus-Designed Cysteine Residue Substitution Molecules L-asparaginase <i>Acinetobacter soli</i> thermostability consensus design cysteine substitution acrylamide inhibition |
| title | Thermostability Improvement of L-Asparaginase from <i>Acinetobacter soli</i> via Consensus-Designed Cysteine Residue Substitution |
| title_full | Thermostability Improvement of L-Asparaginase from <i>Acinetobacter soli</i> via Consensus-Designed Cysteine Residue Substitution |
| title_fullStr | Thermostability Improvement of L-Asparaginase from <i>Acinetobacter soli</i> via Consensus-Designed Cysteine Residue Substitution |
| title_full_unstemmed | Thermostability Improvement of L-Asparaginase from <i>Acinetobacter soli</i> via Consensus-Designed Cysteine Residue Substitution |
| title_short | Thermostability Improvement of L-Asparaginase from <i>Acinetobacter soli</i> via Consensus-Designed Cysteine Residue Substitution |
| title_sort | thermostability improvement of l asparaginase from i acinetobacter soli i via consensus designed cysteine residue substitution |
| topic | L-asparaginase <i>Acinetobacter soli</i> thermostability consensus design cysteine substitution acrylamide inhibition |
| url | https://www.mdpi.com/1420-3049/27/19/6670 |
| work_keys_str_mv | AT linshujiao thermostabilityimprovementoflasparaginasefromiacinetobactersoliiviaconsensusdesignedcysteineresiduesubstitution AT huibingchi thermostabilityimprovementoflasparaginasefromiacinetobactersoliiviaconsensusdesignedcysteineresiduesubstitution AT bingjiexia thermostabilityimprovementoflasparaginasefromiacinetobactersoliiviaconsensusdesignedcysteineresiduesubstitution AT zhaoxinlu thermostabilityimprovementoflasparaginasefromiacinetobactersoliiviaconsensusdesignedcysteineresiduesubstitution AT xiaomeibie thermostabilityimprovementoflasparaginasefromiacinetobactersoliiviaconsensusdesignedcysteineresiduesubstitution AT haizhenzhao thermostabilityimprovementoflasparaginasefromiacinetobactersoliiviaconsensusdesignedcysteineresiduesubstitution AT fengxialu thermostabilityimprovementoflasparaginasefromiacinetobactersoliiviaconsensusdesignedcysteineresiduesubstitution AT meirongchen thermostabilityimprovementoflasparaginasefromiacinetobactersoliiviaconsensusdesignedcysteineresiduesubstitution |