Production of Trans-Cinnamic Acid by Immobilization of the Bambusa oldhamii BoPAL1 and BoPAL2 Phenylalanine Ammonia-Lyases on Electrospun Nanofibers
Abstract
:1. Introduction
2. Results
2.1. Purification of Recombinant Proteins by Affinity Chromatography
2.2. Optimum Crosslinking Conditions
2.3. Central Composite Design (CCD)-Response Surface Methodology (RSM)
2.4. Surface Morphology
2.5. Temperature and pH Stability of Free and Immobilized BoPAL Proteins
2.6. Kinetic Parameters of Free and Immobilized BoPAL Proteins
2.7. Recyclability, Storage Stability, and Denaturant Tolerance of Free and immobiLized BoPAL Proteins
3. Discussion
4. Materials and Methods
4.1. Reagents
4.2. Expressions of BoPAL1 and BoPAL2 Proteins in Escherichia coli and Pichia pastoris
4.3. Purification of Recombinant BoPAL1 and BoPAL2 Proteins
4.4. Preparation of Nanofibers by Electrospinning Method
4.5. Crosslinking
4.6. Central Composite Design (CCD) Response Surface Methodology (RSM)
4.7. Membrane Surface morphOlogy Characterization Using Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FT-IR)
4.8. PAL Activity Assay
4.9. PAL Enzyme Kinetic
4.10. Biochemical Properties Analysis
4.11. Recyclability and Denaturant Treatment
4.12. Data Analysis and Statistic
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
Abbreviations
ANOVA | analysis of variances |
BMGY | buffered glycerol-complex medium |
BMMY | buffered methanol-complex medium |
CBR | Coomassie Brilliant Blue R-25 |
CCD | central composite design |
CS | chitosan |
FT-IR | Fourier transform infrared spectroscopy |
IPTG | β-D-thiogalactopyranoside |
MD | minimal dextrose |
MM | minimal methanol |
PAL | phenylalanine ammonia-lyase |
PVA | polyvinyl alcohol |
RSM | response surface methodology |
SDS-PAGE | sodium dodecyl sulfate polyacrylamide gel electrophoresis |
TFE-SEM | thermal field emission scanning electron microscope |
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Enzyme | kcat (s−1) | Km (µM) | kcat/Km (s−1 µM−1) | |||
---|---|---|---|---|---|---|
Free | Immobilized | Free | Immobilized | Free | Immobilized | |
eBoPAL1 | 1.01 | 1.21 | 518 | 534 | 1.91 × 10−3 | 2.2 × 10−3 |
eBoPAL2 | 4.02 | 3.99 | 329 | 379 | 1.23 × 10−2 | 1.05 × 10−2 |
pBoPAL1 | 1.03 | 1.25 | 376 | 453 | 2.72 × 10−3 | 3.32 × 10−3 |
pBoPAL2 | 3.81 | 4.01 | 294 | 364 | 1.29 × 10−2 | 1.1 × 10−2 |
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Hong, P.-Y.; Huang, Y.-H.; Lim, G.C.W.; Chen, Y.-P.; Hsiao, C.-J.; Chen, L.-H.; Ciou, J.-Y.; Hsieh, L.-S. Production of Trans-Cinnamic Acid by Immobilization of the Bambusa oldhamii BoPAL1 and BoPAL2 Phenylalanine Ammonia-Lyases on Electrospun Nanofibers. Int. J. Mol. Sci. 2021, 22, 11184. https://doi.org/10.3390/ijms222011184
Hong P-Y, Huang Y-H, Lim GCW, Chen Y-P, Hsiao C-J, Chen L-H, Ciou J-Y, Hsieh L-S. Production of Trans-Cinnamic Acid by Immobilization of the Bambusa oldhamii BoPAL1 and BoPAL2 Phenylalanine Ammonia-Lyases on Electrospun Nanofibers. International Journal of Molecular Sciences. 2021; 22(20):11184. https://doi.org/10.3390/ijms222011184
Chicago/Turabian StyleHong, Pei-Yu, Yi-Hao Huang, GiGi Chin Wen Lim, Yen-Po Chen, Che-Jen Hsiao, Li-Hsien Chen, Jhih-Ying Ciou, and Lu-Sheng Hsieh. 2021. "Production of Trans-Cinnamic Acid by Immobilization of the Bambusa oldhamii BoPAL1 and BoPAL2 Phenylalanine Ammonia-Lyases on Electrospun Nanofibers" International Journal of Molecular Sciences 22, no. 20: 11184. https://doi.org/10.3390/ijms222011184