Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides
Abstract
:1. Introduction
2. Results
2.1. Construction of Recombinant Cells
2.2. Optimization of the Expression Condition of rMaBGA
2.3. Purification of rMaBGA
2.4. Enzymatic Characterization of MaBGA
2.4.1. Effect of pH and Temperature on the Activity of rMaBGA
2.4.2. Thermal Denaturation Kinetic of rMaBGA
2.4.3. Steady-State Kinetic of rMaBGA
2.5. Synthesis of Galactooligosaccharides
2.5.1. Thin-Layer Chromatography Analysis
2.5.2. Characterization of the Products of Transglycosylation
3. Discussion
4. Materials and Methods
4.1. Expression and Purification of rMaBGA
4.1.1. Strains, Plasmids, and Culture Conditions
4.1.2. Construction of Recombinant Strains
4.1.3. Optimization of the Production of rMaBGA
4.1.4. Expression and Purification of rMaBGA
4.1.5. SDS-PAGE Analysis
4.2. Enzymatic Characterization of rMaBGA
4.2.1. Enzyme Activity Assay
4.2.2. Effect of pH and Temperature on the Activity of rMaBGA
4.2.3. Thermal Denaturation Kinetic of rMaBGA
4.2.4. Steady-State Kinetic of rMaBGA
4.3. Synthesis of Galactooligosaccharides
4.3.1. Thin-Layer Chromatography Analysis
4.3.2. Characterization of the Products of Transglycosylation
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Enzyme | Temperature (°C) | kd (h−1) | t1/2 (h) | ΔH (KJ mol−1) | ΔG (KJ mol−1) | ΔS (J mol−1 K−1) |
---|---|---|---|---|---|---|
wtMaBGA | 50 | 0.0433 | 16.00 | 114.03 | 87.75 | 81.35 |
60 | 0.1597 | 4.34 | 113.94 | 86.94 | 81.10 | |
rMaBGA | 50 | 0.0721 | 9.61 | 147.75 | 86.38 | 189.99 |
60 | 0.3879 | 1.79 | 147.66 | 84.48 | 189.74 |
Enzyme | Km (mM) | Vmax (μM min−1) |
---|---|---|
rMaBGA | 6.85 | 64.13 |
wtMaBGA | 14.19 | 1.05 |
Unit | α-glucosyl | β-glucosyl | β-galactosyl-A | β-galactosyl-B |
---|---|---|---|---|
1 | 5.14 | 4.59 | 4.37 | 4.50 |
2 | 3.52 | 3.21 | 3.62 | 3.62 |
3 | 3.79 | 3.42 | 3.84 | 3.70 |
4 | 3.50 | 3.46 | 4.01 | 3.86 |
5 | 3.85 | 3.60 | 3.70 | 3.67 |
6 | 3.75–3.88 | 3.75–3.88 | 3.70–3.85 | 3.70–3.85 |
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Ding, H.; Zhou, L.; Zeng, Q.; Yu, Y.; Chen, B. Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides. Mar. Drugs 2018, 16, 415. https://doi.org/10.3390/md16110415
Ding H, Zhou L, Zeng Q, Yu Y, Chen B. Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides. Marine Drugs. 2018; 16(11):415. https://doi.org/10.3390/md16110415
Chicago/Turabian StyleDing, Haitao, Lili Zhou, Qian Zeng, Yong Yu, and Bo Chen. 2018. "Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides" Marine Drugs 16, no. 11: 415. https://doi.org/10.3390/md16110415