Rational engineering S1' substrate binding pocket to enhance substrate specificity and catalytic activity of thermal-stable keratinase for efficient keratin degradation
Copyright © 2024 Elsevier B.V. All rights reserved..
This study reports the rational engineering of the S1' substrate-binding pocket of a thermally-stable keratinase from Pseudomonas aeruginosa 4-3 (4-3Ker) to improve substrate specificity to typical keratinase (K/C > 0.5) and catalytic activity without compromising thermal stability for efficient keratin degradation. Of 10 chosen mutation hotspots in the S1' substrate-binding pocket, the top three mutations M128R, A138V, and V142I showing the best catalytic activity and substrate specificity were identified. Their double and triple combinatorial mutants synergistically overcame limitations of single mutants, fabricating an excellent M128R/A138V/V142I triple mutant which displayed a 1.21-fold increase in keratin catalytic activity, 1.10-fold enhancement in keratin/casein activity ratio, and a 3.13 °C increase in half-inactivation temperature compared to 4-3Ker. Molecular dynamics simulations revealed enhanced flexibility of critical amino acid residues at the substrate access tunnel, improved global protein rigidity, and heightened hydrophobicity within the active site likely underpinned the increased catalytic activity and substrate specificity. Additionally, the triple mutant improved the feather degradation rate by 32.86 % over the wild-type, far exceeding commercial keratinase in substrate specificity and thermal stability. This study exemplified engineering a typical keratinase with enhanced substrate specificity, catalytic activity, and thermal stability from thermally-stable 4-3Ker, providing a more robust tool for feather degradation.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:263 |
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| Enthalten in: |
International journal of biological macromolecules - 263(2024), Pt 1 vom: 07. März, Seite 130688 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Pei, Xiao-Dong [VerfasserIn] |
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| Links: |
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| Themen: |
68238-35-7 |
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| Anmerkungen: |
Date Completed 27.03.2024 Date Revised 27.03.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.ijbiomac.2024.130688 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Copyright © 2024 Elsevier B.V. All rights reserved. | ||
| 520 | |a This study reports the rational engineering of the S1' substrate-binding pocket of a thermally-stable keratinase from Pseudomonas aeruginosa 4-3 (4-3Ker) to improve substrate specificity to typical keratinase (K/C > 0.5) and catalytic activity without compromising thermal stability for efficient keratin degradation. Of 10 chosen mutation hotspots in the S1' substrate-binding pocket, the top three mutations M128R, A138V, and V142I showing the best catalytic activity and substrate specificity were identified. Their double and triple combinatorial mutants synergistically overcame limitations of single mutants, fabricating an excellent M128R/A138V/V142I triple mutant which displayed a 1.21-fold increase in keratin catalytic activity, 1.10-fold enhancement in keratin/casein activity ratio, and a 3.13 °C increase in half-inactivation temperature compared to 4-3Ker. Molecular dynamics simulations revealed enhanced flexibility of critical amino acid residues at the substrate access tunnel, improved global protein rigidity, and heightened hydrophobicity within the active site likely underpinned the increased catalytic activity and substrate specificity. Additionally, the triple mutant improved the feather degradation rate by 32.86 % over the wild-type, far exceeding commercial keratinase in substrate specificity and thermal stability. This study exemplified engineering a typical keratinase with enhanced substrate specificity, catalytic activity, and thermal stability from thermally-stable 4-3Ker, providing a more robust tool for feather degradation | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Keratinase | |
| 650 | 4 | |a Substrate specificity | |
| 650 | 4 | |a Thermal stability | |
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| 700 | 1 | |a Jiao, Dao-Quan |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Fan |e verfasserin |4 aut | |
| 700 | 1 | |a He, Yi-Ning |e verfasserin |4 aut | |
| 700 | 1 | |a Wu, Qing-Ling |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Xiao-Ling |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Cheng-Hua |e verfasserin |4 aut | |
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