An Enzyme Cascade Synthesis of Vanillin
Abstract
:1. Introduction
2. Results and Discussion
2.1. Screening of the Small Focused CYP102A1 Library
2.2. Screening of Additional CYP102A1 Variants
2.3. Molecular Dynamics Simulation
2.4. Screening of Quadruple CYP102A1 Variants for Substrate Conversion
2.5. In Vitro One-Pot Cascade Reactions
2.6. In Vivo Cascade Reactions
3. Materials and Methods
3.1. Materials
3.2. Plasmids and Strains
3.3. Molecular Biology Techniques and Enzyme Expression
3.4. Determination of Protein Concentration
3.5. In Vitro Biotransformations
3.6. In Vivo Biotransformations
3.7. Sample Treatment
3.8. GC Analysis
3.9. HPLC Analysis
3.10. Molecular Dynamics Simulations
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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CYP102A1 Variant | Product Yield (% of Initial Substrate) | Total Conversion (%) | ||||
---|---|---|---|---|---|---|
2 | 3 | 6 | 9 | 10 | ||
wild-type | – | – | <1 | – | – | <1 |
A328L | <1 | 2.7 ± 0.2 | 26.6 ± 2.5 | <1 | – | 31.0 ± 2.8 |
F87V | <1 | <1 | 12.5 ± 0.2 | <1 | – | 14.9 ± 0.3 |
F87V/A328L | 9.1 ± 0.2 | 1.6 ± 0.1 | 23.2 ± 0.4 | 12.0 ± 0.8 | 13.5 ± 0.8 | 59.3 ± 1.8 |
F87V/A328L/L437I | 20.0 ± 0.9 | <1 | 14.0 ± 0.7 | 7.4 ± 1.3 | 19.3 ± 0.5 | 61.3 ± 2.9 |
R47L/Y51F | <1 | – | 1.5 ± 0.1 | – | – | 1.6 ± 0.1 |
R47L/Y51F/F87V/A328V | 5.0 ± 0.3 | <1 | 24.0 ± 0.8 | 3.0 ± 0.1 | 2.1 ± 0.2 | 34.9 ± 1.4 |
R47L/Y51F/F87V/A328L/L437I | 13.5 ± 0.4 | <1 | 10.1 ± 0.5 | 7.7 ± 0.2 | 17.8 ± 0.8 | 50.4 ± 1.8 |
Enzymes | Reaction Time (h) | Product Yield (% of Initial Substrate) | Total Conversion (%) | |||
---|---|---|---|---|---|---|
2 | 3 | 5 | 6 | |||
A328L R47L/Y51F/F87V/A328V | 2 | 2.2 ± 0.1 | 1.2 ± 0.1 | – | 19.3 ± 1.3 | 24.7 ± 1.6 |
1 + 1 | 1.2 ± 0.2 | 2.4 ± 0.5 | – | 25.6 ± 4.8 | 30.3 ± 5.7 | |
A328L VAO_F454Y | 2 | <1 | <1 | <1 | 19.8 ± 2.7 | 22.9 ± 3.1 |
1 + 1 | 1.0 ± 0.1 | <1 | 1.2 ± 0.1 | 25.0 ± 2.2 | 29.0 ± 2.5 |
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Klaus, T.; Seifert, A.; Häbe, T.; Nestl, B.M.; Hauer, B. An Enzyme Cascade Synthesis of Vanillin. Catalysts 2019, 9, 252. https://doi.org/10.3390/catal9030252
Klaus T, Seifert A, Häbe T, Nestl BM, Hauer B. An Enzyme Cascade Synthesis of Vanillin. Catalysts. 2019; 9(3):252. https://doi.org/10.3390/catal9030252
Chicago/Turabian StyleKlaus, Tobias, Alexander Seifert, Tim Häbe, Bettina M. Nestl, and Bernhard Hauer. 2019. "An Enzyme Cascade Synthesis of Vanillin" Catalysts 9, no. 3: 252. https://doi.org/10.3390/catal9030252