Heterologous Expression of a Thermostable α-Glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and Its Potential Application for Isomaltose–Oligosaccharide Synthesis
Abstract
:1. Introduction
2. Results
2.1. Expression and Purification of α-Glucosidase
2.2. Characterization of the Recombinant Enzymes
2.2.1. Temperature Optimum and Thermostability
2.2.2. pH Optimum and Stability
2.2.3. Kinetic Studies
2.2.4. Metal Requirements
2.2.5. Substrate Specificity
2.3. Hydrolysis Activity
2.4. Transglycosylation Activity of Recombinant α-Glucosidase
3. Discussion
4. Materials and Methods
4.1. Cloning and Expression of GSJ in E. coli BL21 (DE3)
4.2. Assay of α-Glucosidase Activity
4.3. Enzyme Preparation and Purification
4.4. Effects of Temperature and pH on the Enzyme Activity and Stability
4.5. Metal Requirement
4.6. Kinetic Parameters
4.7. Substrate Specificity
4.8. Hydrolysis Reaction
4.9. Transglycosylation Reaction
4.10. HPAEC Analysis
4.11. MALDI-TOF Mass Spectrometry
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Purification | Total Protein (mg) | Total Activity (U) | Specific Activity (U/mg) | Purification Fold | Yield (%) |
---|---|---|---|---|---|
Crude enzyme | 90.90 | 3876.1 | 42.64 | 1.0 | 100.0 |
Heat treatment | 25.05 | 3206.9 | 128.0 | 3.0 | 82.7 |
Ni-Sepharose | 10.08 | 2856.0 | 283.3 | 6.6 | 73.7 |
Substrate | Km (mM) | Vmax (U mg−1) | kcat (s−1) | kcat/Km (mM−1s−1) |
---|---|---|---|---|
pNP-α-D-glucopyranoside | 2.321 | 306.3 | 352 | 151.6 |
Substrate | Relative Activity (%) |
---|---|
pNP-α-D-glucopyranoside | 100.0 |
pNP-β-D-glucopyranoside | 4.1 |
pNP-α-L-arabinofuranoside | 6.6 |
pNP-α-L-arabinopyranoside | 0.6 |
pNP-α-D-galactopyranoside | 2.7 |
pNP-β-D-galactopyranoside | 0.0 |
pNP-α-L-rhamnopyranoside | 6.8 |
pNP-β-D-mannopyranoside | 0.0 |
pNP-β-D-xylopyranoside | 3.9 |
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Zhang, F.; Wang, W.; Bah, F.B.M.; Song, C.; Zhou, Y.; Ji, L.; Yuan, Y. Heterologous Expression of a Thermostable α-Glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and Its Potential Application for Isomaltose–Oligosaccharide Synthesis. Molecules 2019, 24, 1413. https://doi.org/10.3390/molecules24071413
Zhang F, Wang W, Bah FBM, Song C, Zhou Y, Ji L, Yuan Y. Heterologous Expression of a Thermostable α-Glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and Its Potential Application for Isomaltose–Oligosaccharide Synthesis. Molecules. 2019; 24(7):1413. https://doi.org/10.3390/molecules24071413
Chicago/Turabian StyleZhang, Fan, Weiyang Wang, Fatoumata Binta Maci Bah, Chengcheng Song, Yifa Zhou, Li Ji, and Ye Yuan. 2019. "Heterologous Expression of a Thermostable α-Glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and Its Potential Application for Isomaltose–Oligosaccharide Synthesis" Molecules 24, no. 7: 1413. https://doi.org/10.3390/molecules24071413