Engineering of Bifunctional Enzymes with Uricase and Peroxidase Activities for Simple and Rapid Quantification of Uric Acid in Biological Samples
Abstract
:1. Introduction
2. Results and Discussion
2.1. Production of Native and Bifunctional Proteins with Uricase and Peroxidase Activities
2.2. Characterization of Enzymatic Activity and Kinetic Parameters
2.3. Optimization of Two-Enzymatic Cascade Reactions for Quantification of Uric Acid
2.4. Uric Acid Calibration Curve
2.5. Method Evaluation and Measurement of Uric Acid from Lyophilized Serum
3. Materials and Methods
3.1. Bacterial Strains, Plasmids and Chemicals
3.2. DNA Manipulations
3.3. Protein Expression and Purification
3.4. Molecular Weight Determination
3.5. Measurement of Enzymatic Activity and Kinetic Parameters
3.6. Investigation of Sequential Reactions Catalyzed by Uricase (CUOX) and Peroxidase (VHb)
3.7. Preparation of a UA Calibration Curve
3.8. Method Evaluation and Quantification of UA from Lyophilized Human Serum
3.9. Molecular Modeling
3.10. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Protein | Molecular Weight (kDa) | Uricase Activity | Peroxidase Activity | ||||||
---|---|---|---|---|---|---|---|---|---|
U/mg | U/μmol | U/mg (%) | U/μmol (%) | U/mg | U/μmol | U/mg (%) | U/μmol (%) | ||
CUOX | 143 | 9.7 ± 0.2 | 1387.1 ± 28.6 | 100 | 100 | - | - | - | - |
VHb | 35 | - | - | - | - | 20.0 ± 0.5 | 700.00 ± 17.5 | 100 | 100 |
CV | 207 | 6.7 ± 0.3 * | 1386.9 ± 71.7 | 69.07 | 99.98 | 0.68 ± 0.1 ** | 140.76 ± 22.4 | 3.40 | 20.10 |
VC | 207 | 5.9 ± 0.1 * | 1221.3 ± 31.1 | 60.82 | 88.04 | 0.54 ± 0.1 ** | 111.78 ± 16.4 | 2.70 | 15.96 |
Protein | Uricase | Peroxidase | ||||
---|---|---|---|---|---|---|
Km (mM) | kcat (s−1) | kcat/Km (mM−1s−1) | (mM) | kcat (s−1) | (mM−1s−1) | |
CUOX | 0.079 | 29.08 | 368.04 | - | - | - |
VHb | - | - | - | 1.84 | 4.72 | 2.56 |
CV | 0.052 | 38.77 | 735.01 | 2.12 | 2.15 | 1.02 |
VC | 0.046 | 46.93 | 1003.83 | 4.04 | 2.02 | 0.50 |
Analytical Method | Uricase/rxn | Peroxidase or Peroxidase-Like/ rxn | Reaction Phase with Optimal Condition (pH/Temp./Time) | Total Reaction Time | Total Volume (μL) | Linear Range (μM) | LOD (μM) | Reference |
---|---|---|---|---|---|---|---|---|
1. Uricase/HRP/ oxPOD and GSH-MQDs | 200 μg * | HRP 20 μg * | 1. pH NR **, 37 °C, 30 min 2. pH 7.2–7.4, 37 °C, time NR ** | >30 min | 2000 | 1.2–100 | 0.2 | [39] |
2. Uricase/ MoS2/TMB | 2.5 μg * | MoS2 0.18 μg | 1. pH 8.5, 35 °C, 15 min 2. pH 4, 50 °C, 60 min | 75 min | 2000 | 0.5–100 | 0.3 | [40] |
3. Uricase/Cu2+/ TMB | 2.5 μg * | Cu2+ 8 μmol | 1. pH 8.5, 35 °C, 15 min 2. pH 4, 45 °C, 60 min | 75 min | 2000 | 1–100 | 0.64 | [41] |
4. Uricase/ Co-Se-nano- crystalline/ 4-AAP/TOPS | 1 U | Co-Se- nanocrystal 260 μg | 1. pH 8.5, 40 °C, 10 min 2. pH 8.5, 40 °C, 30 min | 40 min | 4000 | 2–40 | 0.5 | [42] |
5. Uricase/ MIL53(Fe)/ TMB | 0.1 U | MIL53(Fe) 20 μg | 1. pH 9.0, 37 °C, 15 min 2. pH 4, 55 °C, 40 min | 55 min | 1000 | 4.5–60 | 1.3 | [43] |
6. Uricase/HRP/ 4-AAP/TOOS | 0.4 U | HRP 1.2 U | 1. pH 6.5, 25 °C, 5 min 2. pH 7.2–7.6, 25°C, 20 min | 25 min | 1020 | Up to 34 | 12 | [18] |
7. UOX/VHb/ Amplex Red | 0.05 U | VHb 0.005 U | 1. pH 8.0, 30 °C, 10 min | 10 min | 100 | 2.5–50 | 1 | This study |
Detected (μM) | Added (μM) | Found (μM) | Recovery (%) | RSD (%, n = 5) |
---|---|---|---|---|
327 ± 5.2 | 50 | 374.4 ± 6.1 | 94.8 | 1.63 |
75 | 399.4 ± 7.4 | 96.5 | 1.85 | |
100 | 424.0 ± 8.0 | 97.0 | 1.90 | |
125 | 452.6 ± 10.0 | 100.5 | 2.22 | |
150 | 477.2 ± 10.4 | 100.1 | 2.19 |
Sample | Developed Assay | Automated Analyzer | Relative Error (%) | Average Relative Error (%) |
---|---|---|---|---|
Serum 1 | 285 | 298 | 4.5 | 3.06 |
Serum 2 | 349 | 360 | 3.1 | |
Serum 3 | 466 | 453 | 2.7 | |
Serum 4 | 539 | 525 | 2.5 | |
Serum 5 | 672 | 655 | 2.5 |
Primers | Description | DNA sequence (5′ → 3′) |
---|---|---|
CUOX_FP | for construction of pETDuetCUOX | CGGGATCCCATGTCAACAACGCTCTCATC |
CUOX_RP | CCCAAGCTTTTACAACTTGGTCTTCTCC | |
Remove XhoI CUOX_FP | for removal of XhoI site from CUOX gene | CCACCTTTGCTCTTGAGAACTCTCCATCTG |
Remove XhoI CUOX_RP | CAGATGGAGAGTTCTCAAGAGCAAAGGTGG | |
Remove stop CUOX_FP | for removal of stop codon from CUOX gene | GAGAAGACCAAGTTGAAGCTTGCGGCCGC |
Remove stop CUOX_RP | GCGGCCGCAAGCTTCAACTTGGTCTTCTC | |
Insert HindIII VHb_FP | for insertion of HindIII site to VHb gene | CATCACGTGGATGACGACAAGCTTATGTTAGACCAGCAAACC |
Insert HindIII VHb_RP | GGTTTGCTGGTCTAACATAAGCTTGTCGTCATCCACGTGATG | |
Insert BamHI VHb_FP | for insertion of BamHI site to VHb gene | GCTCAAGCGGTTGAAGGATCCACCGGGCTTCTCCTC |
Insert BamHI VHb_RP | GAGGAGAAGCCCGGTGGATCCTTCAACCGCTTGAGC |
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Phuadraksa, T.; Chittrakanwong, J.; Tullayaprayouch, K.; Onsirisakul, N.; Wichit, S.; Yainoy, S. Engineering of Bifunctional Enzymes with Uricase and Peroxidase Activities for Simple and Rapid Quantification of Uric Acid in Biological Samples. Catalysts 2020, 10, 428. https://doi.org/10.3390/catal10040428
Phuadraksa T, Chittrakanwong J, Tullayaprayouch K, Onsirisakul N, Wichit S, Yainoy S. Engineering of Bifunctional Enzymes with Uricase and Peroxidase Activities for Simple and Rapid Quantification of Uric Acid in Biological Samples. Catalysts. 2020; 10(4):428. https://doi.org/10.3390/catal10040428
Chicago/Turabian StylePhuadraksa, Thanawat, Jurairat Chittrakanwong, Kittitouch Tullayaprayouch, Naruthai Onsirisakul, Sineewanlaya Wichit, and Sakda Yainoy. 2020. "Engineering of Bifunctional Enzymes with Uricase and Peroxidase Activities for Simple and Rapid Quantification of Uric Acid in Biological Samples" Catalysts 10, no. 4: 428. https://doi.org/10.3390/catal10040428