One-Pot Biocatalytic Preparation of Enantiopure Unusual α-Amino Acids from α-Hydroxy Acids via a Hydrogen-Borrowing Dual-Enzyme Cascade
Abstract
:1. Introduction
2. Results and Discussion
2.1. Expression and Comparative Enzyme Activity of D-Hydroxy Acid Dehydrogenases
2.2. Rational Engineering of LbMDH
2.3. Site-Saturation Mutagenesis at Residue L243
2.4. Enzyme Characterization and Kinetic Analysis of LbMDH and Variants
2.5. Structure Analysis of LbMDH and Variants
2.6. Secondary Structure and Thermostability Analysis by Circular Dichroism (CD)
2.7. One-Pot Biocatalytic Preparation of UAAs with a Double-Enzyme System
3. Materials and Methods
3.1. Strains, Plasmids, and Materials
3.2. Site-Directed and Structure Analysis of LbMDH
3.3. Expression and Purification of LbMDH and Its Variants
3.4. Enzyme Activity Assays
3.5. Determination of Kinetic Characteristics
3.6. Circular Dichroism for Structure Analysis and Thermal Melts
3.7. Biocatalytic Preparation of UAAs with a Dual-Enzyme Hydrogen-Borrowing Cascade
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Source | Enzyme | D-Mandelic Acid (U/mg) | D-α-Hydroxybutyric Acid (U/mg) | D-α-Hydroxycaproic Acid (U/mg) | Optimum pH | Cofactor |
---|---|---|---|---|---|---|
L. brevis | LbMDH | 243.5 ± 13.5 | 60.2 ± 4.7 | 118.9 ± 10.6 | 10.5 | NAD+ |
E. faecalis | Ef2D2R | 12.4 ± 1.3 | 13.2 ± 3.9 | 10.5 ± 2.4 | 10.5 | NAD+ |
S. aureus | SaDlacDH | 6.5 ± 0.9 | n/a | n/a | 11.0 | NAD+ |
P. aeruginosa | PaDlacDH | 0.9 ± 0.4 | n/a | n/a | 11.5 | NAD+ |
P. aeruginosa | Pa2D2R | n/a | 0.7 ± 0.2 | 0.8 ± 0.4 | 11 | NADP+ |
Substrate | Relative Activity (%) | ||||||
---|---|---|---|---|---|---|---|
WT | L243M | L243W | L243V | L243I | L243F | L243H | |
D-Mandelic acid | 100 | 231.2 ± 5.6 | 42.1 ± 0.8 | 169.5 ± 6.7 | 192.7 ± 2.5 | 50.8 ± 1.9 | 105.2 ± 2.1 |
D-α-Hydroxybutanoic acid | 100 | 90.0 ± 1.3 | 171.0 ± 8.1 | 99.9 ± 4.2 | 107.5 ± 5.3 | 152.9 ± 9.3 | 98.3 ± 3.0 |
D-α-hydroxycaproic acid | 100 | 121.6 ± 3.7 | 85.8 ± 6.0 | 130.8 ± 7.9 | 146.9 ± 7.8 | 93.7 ± 4.1 | 106.6 ± 3.5 |
D-Phenyllactic acid | 100 | 78.5 ± 4.3 | 79.7 ± 3.2 | 67.0 ± 4.8 | 138.8 ± 9.2 | 81.4 ± 3.9 | 92.8 ± 6.2 |
D-Lactic acid | 100 | 92.6 ± 8.9 | 339.1 ± 15.9 | 126.3 ± 6.3 | 75.9 ± 4.4 | 424.2 ± 29.7 | 130.5 ± 5.0 |
Substrate | WT | L243M | ||||
Km | kcat | kcat/Km | Km | kcat | kcat/Km | |
(mM) | (s−1) | (s−1mM−1) | (mM) | (s−1) | (s−1mM−1) | |
NAD+ | 0.25 ± 0.012 | 0.41 ± 0.004 | 1.661 | 0.45 ± 0.071 | 0.42 ± 0.027 | 0.933 |
D-mandelic acid | 3.71 ± 0.031 | 0.44 ± 0.011 | 0.118 | 3.09 ± 0.196 | 0.52 ± 0.090 | 0.168 |
D-α-hydroxybutanoic acid | 31.7 ± 0.580 | 0.22 ± 0.005 | 0.007 | 25.9 ± 0.207 | 0.05 ± 0.006 | 0.002 |
D-α-hydroxycaproic acid | 20.9 ± 0.106 | 0.41 ± 0.037 | 0.02 | 18.7 ± 0.583 | 0.51 ± 0.032 | 0.027 |
D-phenyllactic acid | 47.2 ± 0.237 | 0.16 ± 0.002 | 0.003 | 50.4 ± 1.070 | 0.13 ± 0.028 | 0.003 |
D-lactic acid | 60.4 ± 2.934 | 0.05 ± 0.001 | 0.001 | 61.7 ± 4.535 | 0.08 ± 0.005 | 0.001 |
Substrate | L243W | L243I | ||||
Km | kcat | kcat/Km | Km | kcat | kcat/Km | |
(mM) | (s−1) | (s−1mM−1) | (mM) | (s−1) | (s−1mM−1) | |
NAD+ | 0.72 ± 0.008 | 0.15 ± 0.031 | 0.038 | 0.49 ± 0.014 | 0.61 ± 0.007 | 1.245 |
D-mandelic acid | 5.88 ± 0.119 | 0.13 ± 0.008 | 0.022 | 4.01 ± 0.193 | 0.46 ± 0.005 | 0.114 |
D-α-hydroxybutanoic acid | 9.2 ± 0.470 | 0.11 ± 0.003 | 0.012 | 49.8 ± 1.981 | 0.24 ± 0.073 | 0.005 |
D-α-hydroxycaproic acid | 21.5 ± 0.134 | 0.12 ± 0.022 | 0.006 | 15.8 ± 0.335 | 0.49 ± 0.006 | 0.031 |
D-phenyllactic acid | 49.8 ± 0.576 | 0.10 ± 0.019 | 0.002 | 33.7 ± 0.985 | 0.18 ± 0.038 | 0.005 |
D-lactic acid | 48.2 ± 1.339 | 0.13 ± 0.009 | 0.003 | 65.3 ± 1.805 | 0.09 ± 0.002 | 0.001 |
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Liu, F.; Zhou, J.; Xu, M.; Yang, T.; Shao, M.; Zhang, X.; Rao, Z. One-Pot Biocatalytic Preparation of Enantiopure Unusual α-Amino Acids from α-Hydroxy Acids via a Hydrogen-Borrowing Dual-Enzyme Cascade. Catalysts 2020, 10, 1470. https://doi.org/10.3390/catal10121470
Liu F, Zhou J, Xu M, Yang T, Shao M, Zhang X, Rao Z. One-Pot Biocatalytic Preparation of Enantiopure Unusual α-Amino Acids from α-Hydroxy Acids via a Hydrogen-Borrowing Dual-Enzyme Cascade. Catalysts. 2020; 10(12):1470. https://doi.org/10.3390/catal10121470
Chicago/Turabian StyleLiu, Fei, Junping Zhou, Meijuan Xu, Taowei Yang, Minglong Shao, Xian Zhang, and Zhiming Rao. 2020. "One-Pot Biocatalytic Preparation of Enantiopure Unusual α-Amino Acids from α-Hydroxy Acids via a Hydrogen-Borrowing Dual-Enzyme Cascade" Catalysts 10, no. 12: 1470. https://doi.org/10.3390/catal10121470
APA StyleLiu, F., Zhou, J., Xu, M., Yang, T., Shao, M., Zhang, X., & Rao, Z. (2020). One-Pot Biocatalytic Preparation of Enantiopure Unusual α-Amino Acids from α-Hydroxy Acids via a Hydrogen-Borrowing Dual-Enzyme Cascade. Catalysts, 10(12), 1470. https://doi.org/10.3390/catal10121470