Improving Rutin Biotransformation Efficiency of α-L-Rhamnosidase from Bacteroides thetaiotaomicron VPI-5482 via Targeted Mutagenesis Focused on General Acid Motif
Abstract
:1. Introduction
2. Results and Discussion
2.1. Structural and Sequence Analyses of General Acid Motif
2.2. Screening of the Best Mutants with Higher Catalytic Activity
2.3. Biotransformation of Rutin by BtRha78A Wild Type and Its Best Mutants
2.4. Enzyme Steady-State Kinetics for BtRha78A Wild Type and Its Best Mutants on Rutin
2.5. Scale-Up Reaction for Production of Isoquercetin by BtRha78A V338A
3. Materials and Methods
3.1. Bacterial Strains, Enzymes, and Reagents
3.2. Construction, Over-Expression, and Purification of BtRha78A Site-Directed Mutants
3.3. Activity Evaluation of BtRha78A Site-Directed Mutants on Rutin by Using Whole Cells
3.4. Biotransformation of Rutin by BtRha78A Wild Type and Its Best Mutants
3.5. Enzyme Steady-State Kinetics for BtRha78A Wild Type and Its Best Mutants on Rutin
3.6. Scale-Up Reaction for Production of Isoquercetin
3.7. High-Performance Liquid Chromatography (HPLC)
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Enzymes | Vmax (µmol min−1 mg−1) | kcat (s−1) | KM (mM) | kcat/KM (s−1 M−1) |
---|---|---|---|---|
Wild type | 0.40 ± 0.01 | 0.57 ± 0.01 | 0.57 ± 0.03 | 1000.0 |
V338A | 0.43 ± 0.01 | 0.62 ± 0.01 | 0.48 ± 0.03 | 1291.7 |
V338I | 0.39 ± 0.02 | 0.56 ± 0.03 | 0.86 ± 0.09 | 651.2 |
S340A | 0.29 ± 0.01 | 0.42 ± 0.01 | 0.64 ± 0.03 | 656.3 |
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Li, B.-C.; Li, X.; Peng, B.; Wu, B.; Li, X.; Ding, G.-B. Improving Rutin Biotransformation Efficiency of α-L-Rhamnosidase from Bacteroides thetaiotaomicron VPI-5482 via Targeted Mutagenesis Focused on General Acid Motif. Catalysts 2024, 14, 501. https://doi.org/10.3390/catal14080501
Li B-C, Li X, Peng B, Wu B, Li X, Ding G-B. Improving Rutin Biotransformation Efficiency of α-L-Rhamnosidase from Bacteroides thetaiotaomicron VPI-5482 via Targeted Mutagenesis Focused on General Acid Motif. Catalysts. 2024; 14(8):501. https://doi.org/10.3390/catal14080501
Chicago/Turabian StyleLi, Bin-Chun, Xue Li, Bo Peng, Bingbing Wu, Xinfeng Li, and Guo-Bin Ding. 2024. "Improving Rutin Biotransformation Efficiency of α-L-Rhamnosidase from Bacteroides thetaiotaomicron VPI-5482 via Targeted Mutagenesis Focused on General Acid Motif" Catalysts 14, no. 8: 501. https://doi.org/10.3390/catal14080501