Biocatalytic Synthesis of Calycosin-7-O-β-D-Glucoside with Uridine Diphosphate–Glucose Regeneration System
Abstract
:1. Introduction
2. Results and Discussion
2.1. Regioselective Glucosylation of Calycosin and Formononetin by UGT88E18
2.2. Kinetic Analysis of UGT88E18 Toward Formononetin and Calycosin
2.3. Optimization of UGT88E18–SuSy Cascade Reaction for Cy7G Synthesis
2.4. Fed-Batch Synthesis of Cy7G by UGT88E18–SuSy Cascade Reaction
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. Expression and Purification of Recombinant Proteins
3.3. Enzyme Activity
3.4. Kinetic Parameters of UGT88E18
3.5. Optimization of UGT88E18–SuSy Cascade Reaction
3.6. Fed-Batch Synthesis of Cy7G
3.7. Purification and Structural Elucidation of the Glucosylated Products
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Substrate | Km (μM) | kcat (s−1) | kcat/ Km (s−1 M−1) |
---|---|---|---|
Calycosin | 18.67 ± 1.36 | 7.67 ± 0.13 | 4.11 × 105 |
Formononetin | 30.64 ± 2.30 | 5.39 ± 0.11 | 1.76 × 105 |
Entry | UGT88E18 (mU mL−1) | SuSy (mU mL−1) | Cy7G (mM) |
---|---|---|---|
1 | 50 | 150 | 1.30 |
2 | 100 | 150 | 2.56 |
3 | 150 | 150 | 3.35 |
4 | 200 | 150 | 3.78 |
5 | 200 | 50 | 1.82 |
6 | 200 | 100 | 3.31 |
7 | 200 | 150 | 3.75 |
8 | 200 | 200 | 3.80 |
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Hu, Y.; Min, J.; Qu, Y.; Zhang, X.; Zhang, J.; Yu, X.; Dai, L. Biocatalytic Synthesis of Calycosin-7-O-β-D-Glucoside with Uridine Diphosphate–Glucose Regeneration System. Catalysts 2020, 10, 258. https://doi.org/10.3390/catal10020258
Hu Y, Min J, Qu Y, Zhang X, Zhang J, Yu X, Dai L. Biocatalytic Synthesis of Calycosin-7-O-β-D-Glucoside with Uridine Diphosphate–Glucose Regeneration System. Catalysts. 2020; 10(2):258. https://doi.org/10.3390/catal10020258
Chicago/Turabian StyleHu, Yumei, Jian Min, Yingying Qu, Xiao Zhang, Juankun Zhang, Xuejing Yu, and Longhai Dai. 2020. "Biocatalytic Synthesis of Calycosin-7-O-β-D-Glucoside with Uridine Diphosphate–Glucose Regeneration System" Catalysts 10, no. 2: 258. https://doi.org/10.3390/catal10020258