Enhanced Thermostability of <i>Pseudomonas nitroreducens</i> Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis
Vanillin has many applications in industries. Isoeugenol monooxygenase (IEM) can catalyze the oxidation of isoeugenol to vanillin in the presence of oxygen under mild conditions. However, the low thermal stability of IEM limits its practical application in the biosynthesis of natural vanillin. Herei...
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MDPI AG
2021-09-01
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| Series: | Catalysts |
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| Online Access: | https://www.mdpi.com/2073-4344/11/10/1199 |
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| author | Xin-Yi Lu Xiao-Mei Wu Bao-Di Ma Yi Xu |
| author_facet | Xin-Yi Lu Xiao-Mei Wu Bao-Di Ma Yi Xu |
| author_sort | Xin-Yi Lu |
| collection | DOAJ |
| description | Vanillin has many applications in industries. Isoeugenol monooxygenase (IEM) can catalyze the oxidation of isoeugenol to vanillin in the presence of oxygen under mild conditions. However, the low thermal stability of IEM limits its practical application in the biosynthesis of natural vanillin. Herein, two rational strategies were combined to improve the thermostability of IEM from <i>Pseudomonas nitroreducens</i> Jin1. Two variants (K83R and K95R) with better thermostability and one mutant (G398A) with higher activity were identified from twenty candidates based on the Surface Residue Replacement method. According to the Consensus Mutagenesis method, one mutant (I352R) with better thermostability and another mutant (L273F) with higher activity were also identified from nine candidates. After combinatorial mutation, a triple mutant K83R/K95R/L273F with the best thermostability and catalytic efficiency was generated. Compared with the wild-type IEM, the thermal inactivation half-lives (<i>t</i><sub>1/2</sub>) of K83R/K95R/L273F at 25 °C, 30 °C, and 35 °C increased 2.9-fold, 11.9-fold, and 24.7-fold, respectively. Simultaneously, it also exhibited a 4.8-fold increase in <i>k</i><sub>cat</sub>, leading to a 1.2-fold increase in catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>). When the whole cell of K83R/K95R/L273F was applied to the biotransformation of isoeugenol on preparative scale, the vanillin concentration reached 240.1 mM with space-time yield of 109.6 g/L/d, and vanillin was achieved in 77.6% isolated yield and >99% purity. |
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| language | English |
| last_indexed | 2024-03-10T06:40:23Z |
| publishDate | 2021-09-01 |
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| series | Catalysts |
| spelling | doaj.art-51ce701cb84c4d4089fc31ab152a57572023-11-22T17:43:45ZengMDPI AGCatalysts2073-43442021-09-011110119910.3390/catal11101199Enhanced Thermostability of <i>Pseudomonas nitroreducens</i> Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus MutagenesisXin-Yi Lu0Xiao-Mei Wu1Bao-Di Ma2Yi Xu3School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, ChinaSchool of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, ChinaSchool of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, ChinaSchool of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, ChinaVanillin has many applications in industries. Isoeugenol monooxygenase (IEM) can catalyze the oxidation of isoeugenol to vanillin in the presence of oxygen under mild conditions. However, the low thermal stability of IEM limits its practical application in the biosynthesis of natural vanillin. Herein, two rational strategies were combined to improve the thermostability of IEM from <i>Pseudomonas nitroreducens</i> Jin1. Two variants (K83R and K95R) with better thermostability and one mutant (G398A) with higher activity were identified from twenty candidates based on the Surface Residue Replacement method. According to the Consensus Mutagenesis method, one mutant (I352R) with better thermostability and another mutant (L273F) with higher activity were also identified from nine candidates. After combinatorial mutation, a triple mutant K83R/K95R/L273F with the best thermostability and catalytic efficiency was generated. Compared with the wild-type IEM, the thermal inactivation half-lives (<i>t</i><sub>1/2</sub>) of K83R/K95R/L273F at 25 °C, 30 °C, and 35 °C increased 2.9-fold, 11.9-fold, and 24.7-fold, respectively. Simultaneously, it also exhibited a 4.8-fold increase in <i>k</i><sub>cat</sub>, leading to a 1.2-fold increase in catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>). When the whole cell of K83R/K95R/L273F was applied to the biotransformation of isoeugenol on preparative scale, the vanillin concentration reached 240.1 mM with space-time yield of 109.6 g/L/d, and vanillin was achieved in 77.6% isolated yield and >99% purity.https://www.mdpi.com/2073-4344/11/10/1199vanillinisoeugenol monooxygenasethermostabilitysurface residue replacementconsensus mutagenesis |
| spellingShingle | Xin-Yi Lu Xiao-Mei Wu Bao-Di Ma Yi Xu Enhanced Thermostability of <i>Pseudomonas nitroreducens</i> Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis Catalysts vanillin isoeugenol monooxygenase thermostability surface residue replacement consensus mutagenesis |
| title | Enhanced Thermostability of <i>Pseudomonas nitroreducens</i> Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis |
| title_full | Enhanced Thermostability of <i>Pseudomonas nitroreducens</i> Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis |
| title_fullStr | Enhanced Thermostability of <i>Pseudomonas nitroreducens</i> Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis |
| title_full_unstemmed | Enhanced Thermostability of <i>Pseudomonas nitroreducens</i> Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis |
| title_short | Enhanced Thermostability of <i>Pseudomonas nitroreducens</i> Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis |
| title_sort | enhanced thermostability of i pseudomonas nitroreducens i isoeugenol monooxygenase by the combinatorial strategy of surface residue replacement and consensus mutagenesis |
| topic | vanillin isoeugenol monooxygenase thermostability surface residue replacement consensus mutagenesis |
| url | https://www.mdpi.com/2073-4344/11/10/1199 |
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