Improvement of Catalytic Activity and Thermostability of Alginate Lyase VxAly7B-CM via Rational Computational Design Strategies
Alginate lyase degrades alginate through the β-elimination mechanism to produce alginate oligosaccharides (AOS) with notable biochemical properties and diverse biological activities. However, its poor thermostability limits large-scale industrial production. In this study, we employed a rational com...
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| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-05-01
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| Series: | Marine Drugs |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1660-3397/23/5/198 |
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| Summary: | Alginate lyase degrades alginate through the β-elimination mechanism to produce alginate oligosaccharides (AOS) with notable biochemical properties and diverse biological activities. However, its poor thermostability limits large-scale industrial production. In this study, we employed a rational computational design strategy combining computer-aided evolutionary coupling analysis and ΔΔG<sub>fold</sub> evaluation to enhance both the thermostability and catalytic activity of the alginate lyase VxAly7B-CM. Among ten single-point mutants, the E188N and S204G mutants exhibited increases in <i>T</i><sub>m</sub> from 47.0 °C to 48.9 °C and 50.2 °C, respectively, with specific activities of 3701.02 U/mg and 2812.01 U/mg at 45 °C. Notably, the combinatorial mutant E188N/S204G demonstrated a Δ<i>T</i><sub>m</sub> of 5 °C and an optimal reaction temperature up to 50 °C, where its specific activity reached 3823.80 U/mg—a 31% increase. Moreover, its half-life at 50 °C was 38.4 h, which is 7.0 times that of the wild-type enzyme. Protein structural analysis and molecular dynamics simulations suggested that the enhanced catalytic performance and thermostability of the E188N/S204G mutant may be attributed to optimized surface charge distribution, strengthened hydrophobic interactions, and increased tertiary structure stability. Overall, our findings provided valuable insights into enzyme stabilization strategies and supported the industrial production of functional AOS. |
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| ISSN: | 1660-3397 |