Aggregation and Molecular Properties of β-Glucosidase Isoform II in Chayote (Sechium edule)
Abstract
:1. Introduction
2. Results and Discussion
2.1. Preparation of Crude Extract from Sechium edule
2.2. Extraction and Purification of β-Glucosidase II (Chayote Pulp Homogenate)
2.3. Purification of β-Glucosidase (Sechium edule) through Cation Exchange Chromatography
2.4. Purification of β-Glucosidase (Sechium edule) by Gel Filtration
2.5. Purification of Sechium edule β-Glucosidase by Anion Exchange Chromatography and Detection of Molecular Aggregates
2.6. SDS-PAGE Electrophoresis
2.7. Native-PAGE Electrophoresis
2.8. Electrophoretic Analysis in Acetate-Urea pH 4.4
2.9. Analysis in Two-Dimensional Electrophoresis
2.10. Specificity Assays with Artificial Substrates and Kinetic Parameters
2.11. Analysis of Tryptic Peptides
2.12. Molecular Aggregates
3. Experimental
3.1. Preparation of the Modified Crude Extract of Sechium Edule
3.2. Enzyme Purification
3.2.1. Cation Exchange Chromatography
3.2.2. Molecular Exclusion Chromatography
3.2.3. Anionic Exchange Chromatography
3.2.4. Dialysis and Lyophilization
3.3. Determination of Molecular Mass and Identification of Molecular Aggregates in SDS-PAGE
3.3.1. Native-PAGE Electrophoresis
3.3.2. Acetate-Urea Acid Electrophoresis
3.3.3. Two-Dimensional Electrophoresis
3.4. Determination of Partial Amino Acid Sequence Using Nano-LC-ESI-MS/MS
3.5. Bioinformatic Analysis
3.6. Enzymatic Activity
3.7. Determination of Kinetic Parameters
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the β-Glucosidase from chayote (Sechium edule) are available from the authors. |
Fraction | Total Protein (mg) | Activity of β-Glucosidase (U) | Specific Activity (U/mg) | Purification Factor | Yield (%) |
---|---|---|---|---|---|
Crude pulp extract | 1565.58 | 12,037.56 | 7.69 | 1 | 100 |
Glacial acetic acid Protein supernatant | 445.36 | 10,479.30 | 23.53 | 3.05 | 87.05 |
Cationic chromatography CMC | 19.25 | 4276.20 | 222.14 | 28.88 | 36 |
Gel filtration (S-200HR) | 5.107 | 4102.20 | 803.25 | 104.45 | 34 |
Anionic chromatography (QFF) | 4.855 | 4085.90 | 841.58 | 109.36 | 33.90 |
Amino Acids | Res/mol | Residues/100% | |
---|---|---|---|
Val | V | 4 | 2.23 |
Phe | F | 12 | 6.70 |
Gly | G | 14 | 7.82 |
Ser | S | 7 | 3.91 |
Ala | A | 16 | 8.94 |
Tyr | Y | 17 | 9.49 |
Gln | Q | 4 | 2.23 |
Glu | E | 10 | 5.58 |
Asp | D | 12 | 6.70 |
Lys | K | 12 | 6.70 |
Asn | N | 13 | 7.26 |
Ile | I | 10 | 5.58 |
Trp | W | 4 | 2.23 |
Thr | T | 8 | 4.46 |
His | H | 8 | 4.46 |
Pro | P | 7 | 3.91 |
Arg | R | 4 | 2.23 |
Leu | L | 14 | 7.82 |
Met | M | 3 | 1.67 |
Totals | 179 | 100 |
Artificial Chromogenic Specific Substrates |
p-Nitrophenyl-β-d-glucopyranoside |
p-Nitrophenyl-β-d-galactopyranoside |
p-Nitrophenyl-β-d-fucopyranoside |
Non-Specific Substrates |
p-Nitrophenyl-β-d-manopyranoside |
p-Nitrophenyl-β-d-lactopyranoside |
p-Nitrophenyl-β-d-maltopyranoside |
p-Nitrophenyl-α-d-glucopyranoside |
p-Nitrophenyl-β-d-N,N′-diacetylcytotriose |
p-Nitrophenyl-β-d-cellobioside |
p-Nitrophenyl-β-d-N-acetylgalactosamine |
Substrate | Km (mM) | Standard Error Km | kcat (min−1) | Standard Error kcat | 10−3 × kcat/Km (mM−1 min−1) |
---|---|---|---|---|---|
pNPGlc | 4.59 | 0.49 | 10,086 | 1008 | 2197 |
pNPGal | 5.72 | 0.45 | 13,718 | 1070 | 2398 |
pNPFuc | 16.11 | 2.62 | 16,289 | 2638 | 1011 |
Name | Ion | m/z1 | MW 2 (Da) | Sequence | Start-End Sequence % Identical |
---|---|---|---|---|---|
Sebg1 | 1129.01 | 2 | 2256.02 | VFGSASAAYQFEGAAFEDGK | 46-65-Cmbg 100% y Csbg 85% |
Sebg2 | 903.42 | 3 | 2702.26 | NIWDTFTHKHPTR | 68-80-Cmbg 62% y Csbg 54% |
Sebg3 | 942.53 | 2 | 1883.06 | IYDHSDGDVALDQYHR | 81-96-Cmbg 94% y Csbg 81.25% |
Sebg4 | 408.89 | 3 | 1223.67 | YKEDVALMKK | 97-106-Cmbg 90% y Csbg 80% |
Sebg5 | 122.63 | 2 | 2439.26 | EYYNNLINELLANGIQP | 137-153-Cmbg 94% y Csbg 88.23% |
Sebg6 | 1391.63 | 2 | 2781.26 | HWITFNEPWSFSMGGYAQGANAPGR | 199-223-Cmbg 96% y Csbg 88% |
Sebg7 | 538.82 | 2 | 1075.64 | SLPKFSAK | 324-332-Cmbg 75% y Csbg 50% |
Sebg8 | 1164.07 | 2 | 2326.14 | NALDFLGLNYYTANYAK | 338-354-Cmbg 88% y Csbg 76% |
Sebg9 | 1333.14 | 2 | 2664.28 | IYITENGYLEIDGPPFHEMGIADK | 416-439-Cmbg 62.5% y Csbg 62.5% |
Sebg10 | 811.92 | 2 | 1621.84 | KV-YYHDHLYNLR | 440-451- Cmbg 66.6% y Csbg 50% |
Sebg11 | 560.29 | 2 | 1118.58 | FGLTYIDYK | 483-491-Cmbg 100% y Csbg100% |
Sebg12 | 542.57 | 2 | 982.49 | WFENFLKT | 504-511-Cmbg 100% y Csbg 87.5% |
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Cruz Rodríguez, A.; Sánchez Esperanza, F.A.; Pérez-Campos, E.; Hernández-Huerta, M.T.; Pérez-Campos Mayoral, L.; Matias-Cervantes, C.A.; Martínez Barras, A.; Mayoral-Andrade, G.; Santos Pineda, L.Á.; Díaz Barrita, A.J.; et al. Aggregation and Molecular Properties of β-Glucosidase Isoform II in Chayote (Sechium edule). Molecules 2020, 25, 1699. https://doi.org/10.3390/molecules25071699
Cruz Rodríguez A, Sánchez Esperanza FA, Pérez-Campos E, Hernández-Huerta MT, Pérez-Campos Mayoral L, Matias-Cervantes CA, Martínez Barras A, Mayoral-Andrade G, Santos Pineda LÁ, Díaz Barrita AJ, et al. Aggregation and Molecular Properties of β-Glucosidase Isoform II in Chayote (Sechium edule). Molecules. 2020; 25(7):1699. https://doi.org/10.3390/molecules25071699
Chicago/Turabian StyleCruz Rodríguez, Alberto, Fabiola Anaid Sánchez Esperanza, Eduardo Pérez-Campos, María Teresa Hernández-Huerta, Laura Pérez-Campos Mayoral, Carlos Alberto Matias-Cervantes, Alexis Martínez Barras, Gabriel Mayoral-Andrade, Luis Ángel Santos Pineda, Aymara Judith Díaz Barrita, and et al. 2020. "Aggregation and Molecular Properties of β-Glucosidase Isoform II in Chayote (Sechium edule)" Molecules 25, no. 7: 1699. https://doi.org/10.3390/molecules25071699