Kinetic Study and Modeling of Wild-Type and Recombinant Broccoli Myrosinase Produced in E. coli and S. cerevisiae as a Function of Substrate Concentration, Temperature, and pH
Abstract
:1. Introduction
2. Enzyme Kinetics Models
2.1. Substrate-Dependent Models
2.2. Temperature-Dependent Models
2.3. pH-Dependent Models
3. Results and Discussion
3.1. Enzyme Kinetics in Terms of Substrate Concentration
3.2. Effect of Temperature on Myrosinase Kinetics
3.3. Effect of pH on Myrosinases Kinetics
4. Material and Methods
4.1. Myrosinase Production and Isolation
4.2. Myrosinase Kinetics
4.3. Modeling
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Model | Source | Kinetic Parameters | |||||||
---|---|---|---|---|---|---|---|---|---|
Vmax (µmol/min) | Km (µM) | Ks (µM) | Ki (M) | β | α | R2 (%) | RMSE | ||
MM (Equation (1)) | BMYR | 0.76 | 0.07 | - | - | - | - | 94.7 | 0.08 |
EMYR | 0.42 | 0.04 | - | - | - | - | 99.1 | 0.02 | |
SMYR | 1.33 | 0.04 | - | - | - | - | 98.9 | 0.07 | |
SIMM (Equation (2)) | BMYR | 0.96 | 0.09 | - | 1.01 | - | - | 96.0 | 0.07 |
EMYR | 0.42 | 0.04 | - | 160.00 | - | - | 99.1 | 0.02 | |
SMYR | 1.75 | 0.07 | - | 1.01 | - | - | 99.0 | 0.08 | |
TBSI (Equation (3)) | BMYR | 143.02 | - | 19.88 | 88.58 | 0.10 | 50.93 | 98.4 | 0.05 |
EMYR | 124.46 | - | 33.68 | 62.00 | 0.10 | 93.43 | 97.2 | 0.05 | |
SMYR | 65.184 | - | 4.83 | 87.48 | 0.11 | 14.13 | 97.6 | 0.14 |
Gene | Source | Expression System | Km (mM) | Vmax (μmol/min) | Reference |
---|---|---|---|---|---|
CpTGG1 | Carica papaya | P. pastoris | 2.83 | 59.9 | [38] |
CpTGG2 | Carica papaya | P. pastoris | 2.24 | 24.3 | [39] |
TGG4 | Arabidopsis thaliana | P. pastoris | 0.25 | 12.2 | [40] |
TGG5 | Arabidopsis thaliana | P. pastoris | 0.55 | 48.1 | [40] |
TGG1 | Arabidopsis thaliana | P. pastoris | 0.05 | 2.30 | [40] |
Myr1 | Brassica napus | P. pastoris | 1.00 | 15.0 | [17] |
Myr | Armoracia rusticana | - | 0.13 | 0.62 | [41] |
Myr | Brassica oleracea | - | 0.07 | 0.76 | [20] |
Myr | Brassica oleracea | E. coli | 0.04 | 0.42 | [22] |
Myr | Brassica oleracea | S. cerevisiae | 0.04 | 1.33 | [22] |
Model | Source | Parameters | |||||||
---|---|---|---|---|---|---|---|---|---|
Prieto (Equation (5)) | n | a | b | R2 (%) | RMSE | ||||
BMYR | 5.76 | 2.83 × 10−7 | 0.17 | 98.6 | 0.03 | ||||
EMYR | 6.35 | 1.19 × 10−7 | 0.22 | 96.3 | 0.04 | ||||
SMYR | 12.72 | 2.05 × 10−8 | 0.40 | 84.8 | 0.16 | ||||
Briere (Equation (6)) | α | m | Tmin (°C) | Tmax (°C) | |||||
BMYR | 8.0 × 10−6 | 0.18 | 8 | 104 | 97.1 | 0.04 | |||
EMYR | 2.0 × 10−6 | 0.16 | 12 | 87 | 95.8 | 0.09 | |||
SMYR | 1.4 × 10−1 | 0.14 | 19 | 73 | 95.8 | 0.06 | |||
Schoolfield (Equation (7)) | ∆H_H (KJ/mol) | ∆H_L (KJ/mol) | ∆H_A (KJ/mol) | n25 (d−1) | T1/2_L (°C) | T1/2_H (°C) | |||
BMYR | 93.46 | 182.34 | 19.29 | 0.26 | 19.45 | 67.48 | 99.0 | 0.06 | |
EMYR | 127.85 | 62.80 | 14.67 | 0.26 | 17.50 | 35.57 | 94.0 | 0.09 | |
SMYR | 251.21 | 523.35 | 41.87 | 0.82 | 22.00 | 37.00 | 92.2 | 0.14 |
Enzyme | Ea (kJ/mol) Activation (10–30 °C) | Vmax (30 °C) (µmol/min) | R2 (%) | Ea (kJ/mol) Inactivation (30–70 °C) | V (60 °C) (µmol/min) | V (70 °C) (µmol/min) | R2 (%) |
---|---|---|---|---|---|---|---|
EMYR | −34.1 | 0.44 | 86.6 | 76.1 | 0.04 ± 0.00 | 0.01 ± 0.00 | 89.3 |
BMYR | −83.5 | 0.65 | 97.0 | 52.8 | 0.18 ± 0.01 | 0.07 ± 0.01 | 92.6 |
SMYR | −288.1 | 1.20 | 88.9 | 72.2 | 0.16 ± 0.03 | 0.04 ± 0.02 | 98.4 |
Model | Source | Parameters | ||||||
---|---|---|---|---|---|---|---|---|
Briere (Equation (8)) | α (×106) | m | pH_min | pH_max | R2 | RMSE | ||
BMYR | 9.56 × 10−6 | 1.0 · 10−1 | 1 | 15 | 98.3 | 0.24 | ||
EMYR | 4.84 × 10−5 | 1.0 · 10−1 | 1 | 14 | 98.5 | 0.24 | ||
SMYR | 1.3 × 10−1 | 1.7 · 10−1 | 1 | 14 | 82.5 | 0.27 | ||
Jurado (Equation (12)) | Vmax (U/mg) | pkES1 | pkES2 | |||||
BMYR | 2.90 | 3.4 | 5.5 | 97.2 | 0.34 | |||
EMYR | 3.10 | 3.1 | 5.1 | 96.0 | 0.40 | |||
SMYR | 1.17 | 4.2 | 7.3 | 90.8 | 0.21 | |||
Prieto (Equation (9)) | b0 | b2 | b22 | b33 | b44 | |||
BMYR | −9.13 | 10.65 | −3.13 | 0.36 | −0.01 | 98.8 | 0.17 | |
EMYR | −11.59 | 14.12 | −4.51 | 0.57 | −0.03 | 98.5 | 0.23 | |
SMYR | −14.98 | 15.64 | −5.27 | 0.74 | −0.04 | 96.8 | 0.13 |
Specific Activity at 30° (U/mg) | ||
---|---|---|
pH 7.0 | pH 3.0 | |
BMYR | 0.50 | 3.30 |
EMYR | 0.38 | 3.60 |
SMYR | 1.00 | 1.45 |
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Jiménez, A.; Castillo, A.; Mahn, A. Kinetic Study and Modeling of Wild-Type and Recombinant Broccoli Myrosinase Produced in E. coli and S. cerevisiae as a Function of Substrate Concentration, Temperature, and pH. Catalysts 2022, 12, 683. https://doi.org/10.3390/catal12070683
Jiménez A, Castillo A, Mahn A. Kinetic Study and Modeling of Wild-Type and Recombinant Broccoli Myrosinase Produced in E. coli and S. cerevisiae as a Function of Substrate Concentration, Temperature, and pH. Catalysts. 2022; 12(7):683. https://doi.org/10.3390/catal12070683
Chicago/Turabian StyleJiménez, Adielis, Antonio Castillo, and Andrea Mahn. 2022. "Kinetic Study and Modeling of Wild-Type and Recombinant Broccoli Myrosinase Produced in E. coli and S. cerevisiae as a Function of Substrate Concentration, Temperature, and pH" Catalysts 12, no. 7: 683. https://doi.org/10.3390/catal12070683