Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris
Abstract
:1. Introduction
2. Results and Discussion
2.1. Recombinant Expression of cAlyM and 102C300C
2.2. Purification of Recombinant cAlyM and 102C300C
2.3. Kinetic Parameters of cAlyM and 102C300C
2.4. Enzymatic Properties of cAlyM and 102C300C
2.5. Thermal Stability of cAlyM and 102C300C
2.6. ESI-MS and HPLC Analyses of Degradation Products
3. Materials and Methods
3.1. Strains, Plasmids, and Reagents
3.2. Construction of Alginate Lyase and Mutant Recombinant Plasmid
3.3. Transformation and Colony Screening of P. pastoris
3.4. Purification of Recombinant cAlyM and 102C300C
3.5. Enzymatic Activity Assay
3.6. Kinetic parameters of cAlyM and 102C300C
3.7. Enzymatic Properties of cAlyM and 102C300C
3.8. Thermal Stability of cAlyM and 102C300C
3.9. ESI-MS and HPLC Analyses of Degradation Products
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Enzymes | Step | Total Activity (U) | Total Protein (mg) | Specific Activity (U/mg) | Recovery Rate (%) | Fold | Yield (U/mL) |
---|---|---|---|---|---|---|---|
cAlyM | Fermentation medium | 507.4 ± 8.5 | 3.00 ± 0.06 | 169.1 | 100 | 1 | 33.8 ± 0.57 |
Magnetic beads | 188.4 ± 4.6 | 0.68 ± 0.01 | 277.1 | 37.13 | 1.64 | 26.9 ± 0.66 | |
102C300C | Fermentation medium | 617.6 ± 6.4 | 3.82 ± 0.05 | 161.7 | 100 | 1 | 41.2 ± 0.43 |
Magnetic beads | 237.1 ± 5.1 | 0.95 ± 0.01 | 249.6 | 38.39 | 1.54 | 33.9 ± 0.73 |
Enzyme | Vmax (U/mg) | Km (mg/mL) | kcat (s−1) | kcat/Km (mL/s/mg) |
---|---|---|---|---|
cAlyM | 344.8 ± 5.6 | 1.31 ± 0.21 | 189.1 ± 5.3 | 144.3 |
102C300C | 312.5 ± 10.1 | 1.43 ± 0.20 | 171.3 ± 4.3 | 119.2 |
Enzyme | Origin | Source | Substrate Preference | Optimal Catalytic Temperature | Thermalstability | Specific activity (U/mg) | Reference |
---|---|---|---|---|---|---|---|
Aly08 | Vibrio sp. SY01 | E. coli | Poly G | 45 °C | Retained 48.7% activity at 20 °C for 1 h | 841 | [14] |
Aly7B_Wf | Wenyingzhuangia fucanilytica | E. coli | Poly M | 40 °C | Retained 75% activity at 35 °C for 24 h | 23.24 * | [23] |
AlyH1 | Vibrio furnissii H1 | Native | Poly G | 40 °C | Retained 60% activity at 40 °C for 30 min | 2.40 * | [11] |
FsAlgB | Flammeovirga sp. NJ-04 | E. coli | Poly M and alginate | 40 °C | Retained 80% activity at 40 °C for 30 min | 1760.8 | [15] |
AlgH | Marinimicrobium sp. H1 | E. coli | Poly G | 45 °C | Retained 80% activity at 40 °C for 2 h | 5510 | [13] |
PmC5A | Pseudomonas mendocina DICP-70 | E. coli | Poly G and Poly M | 40 °C | Retained 80% activity at 45 °C for 1 h | N.D. | [16] |
Aly1281 | Pseudoalteromonas carrageenovora ASY5 | E. coli | Poly G | 50 °C | Stable at temperatures lower than 55 °C | 1.15 * | [33] |
Alg17B | BP-2 | Native | Poly M | 45 °C | Retained 10% activity at 45 °C for 1 h | 4036 | [12] |
KJ-2 | Stenotrophomas maltophilia KJ-2 | E. coli | Poly MG | 40 °C | Inactivated at higher than 40 °C for 30 min | 848.3 | [30] |
algA | Pseudomonas sp. E03 | E. coli | Poly M | 30 °C | Retained 50% activity at 50 °C for 30 min | 222 | [28] |
SAGL | Flavobacterium sp. H63 | P. pastoris | Poly M and alginate | 45 °C | Retained 49.0% activity at 50 °C for 72 h | 4044 * | [20] |
AlgC-PL7 | Cobetia sp. NAP1 | E. coli | Poly G and Poly M | 45 °C | Retained 80% activity at 70 °C for 1 h | 30 | [31] |
GLyase | Pseudomonas sp. F6 | Native | Poly G | N.D. | Retained 60% activity at 80 °C for 15 min | 222.8 | [32] |
cAlyM | Microbulbifer sp. Q7 | P. pastoris | Poly G and alginate | 50 °C | Retained 50% activity at 45 °C for 2 h | 277.1 * | This study |
102C300C | Microbulbifer sp. Q7 | P. pastoris | Poly G and alginate | 50 °C | Retained 50% activity at 45 °C for 5.2 h | 249.6 * | This study |
Sample | Peak Area Percentage of G | Peak Area Percentage of M | M/G |
---|---|---|---|
1-fold | 15.05% | 84.95% | 5.61 |
3-fold | 31.17% | 68.83% | 2.08 |
5-fold | 72.91% | 27.09% | 0.37 |
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Yang, S.; Liu, Z.; Fu, X.; Zhu, C.; Kong, Q.; Yang, M.; Mou, H. Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris. Mar. Drugs 2020, 18, 305. https://doi.org/10.3390/md18060305
Yang S, Liu Z, Fu X, Zhu C, Kong Q, Yang M, Mou H. Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris. Marine Drugs. 2020; 18(6):305. https://doi.org/10.3390/md18060305
Chicago/Turabian StyleYang, Suxiao, Zhemin Liu, Xiaodan Fu, Changliang Zhu, Qing Kong, Min Yang, and Haijin Mou. 2020. "Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris" Marine Drugs 18, no. 6: 305. https://doi.org/10.3390/md18060305