A Novel D-Psicose 3-Epimerase from Halophilic, Anaerobic Iocasia fonsfrigidae and Its Application in Coconut Water
Abstract
:1. Introduction
2. Results
2.1. Amino Acid Sequence Analysis
2.2. Protein Expression and Purification and Allergen Analysis
2.3. pI Values of IfDPEase and Other Ketose 3-Epimerases
2.4. Substrate Specificity
2.5. Enzyme Kinetics
2.6. Effects of pH, Temperature, and Metal Ions on IfDPEase Activity
2.7. Effect of Sodium Chloride on IfDPEase Activity
2.8. Conversion of D-Fructose in Coconut Water into D-Psicose by IfDPEase
3. Discussion
4. Materials and Methods
4.1. Strains and Plasmid
4.2. Chemicals and Reagents
4.3. Alignment and Allergenic Protein Sequence Search
4.4. Phylogenetic Tree of IfDPEase with Other Characterized Members
4.5. Gene Manipulation, Expression, and Purification of Recombinant IfDPEase
4.6. Enzyme Assay
4.7. Substrate Specificity of IfDPEase
4.8. Effects of pH, Temperature, and Metal Ions on IfDPEase Activity
4.9. Effect of Sodium Chloride on IfDPEase Activity
4.10. Prediction of the Isoelectric Point (pI) and Acidic Amino Acids on the Surface of Ketose 3-Epimerase Structures
4.11. Determination of Enzyme Kinetics
4.12. Application of IfDPEase to Coconut Water
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Source | Total Number of Aspartic and Glutamic Acids | Aspartic and Glutamic Acids on the Protein Surface (NetsurfP-3.0) | Aspartic and Glutamic Acids on the Protein Surface (PredictProtein) | Percentage of Aspartic and Glutamic Acids on the Protein Surface (%) | pI | Similarity of IfDPEase Compared to Other 3-Epimerases (%) |
---|---|---|---|---|---|---|
Iocasia fonsfrigidae strain SP3-1 | 40 from 269 aa | 34 | 34 | 85.00 | 4.95 | 100 |
Bacillus sp. KCTC 13219 | 46 from 288 aa | 37 | 37 | 80.43 | 4.98 | 24.32 |
Clostridium cellulolyticum H10 | 43 from 293 aa | 35 | 35 | 81.39 | 5.41 | 23.55 |
Agrobacterium sp. ATCC 31749 | 37 from 289 aa | 27 | 27 | 72.97 | 5.94 | 22.01 |
Ruminococcus sp. | 43 from 291 aa | 32 | 32 | 74.41 | 5.24 | 21.84 |
Agrobacterium tumefaciens | 38 from 289 aa | 24 | 24 | 63.15 | 5.88 | 21.24 |
Rhodobacter sphaeroides | 40 from 295 aa | 33 | 33 | 82.50 | 5.20 | 20.93 |
Treponema primitia ZAS-1 | 41 from 295 aa | 31 | 31 | 75.60 | 5.93 | 20.69 |
Clostridium. scindens 35704 | 47 from 289 aa | 35 | 35 | 74.46 | 5.14 | 20.46 |
Pseudomonas cichorii | 46 from 290 aa | 38 | 38 | 82.60 | 5.21 | 20.38 |
Desmospora sp. 8437 | 49 from 289 aa | 38 | 38 | 77.55 | 5.24 | 20.08 |
Caballeronia fortuita | 44 from 291 aa | 33 | 33 | 75.00 | 5.19 | 20.00 |
Clostridium bolteae | 49 from 301 aa | 39 | 39 | 79.59 | 5.04 | 19.92 |
Dorea sp. CAG317 | 46 from 289 aa | 34 | 34 | 73.91 | 4.99 | 18.92 |
Active Site | I. fonsfrigidae | Bacillus sp. | C. cellulolyticum | C. boltae | A. tumefaciens | P. cichorii | C. fortuita | R. sphaeroides |
---|---|---|---|---|---|---|---|---|
Catalytic site | Glu153 | Glu150 | Glu150 | Glu155 | Glu150 | Glu152 | Glu152 | Glu156 |
Glu247 | Glu244 | Glu244 | Glu256 | Glu244 | Glu246 | Glu246 | Glu250 | |
Metal-coordinating site | Glu153 | Glu150 | Glu150 | Glu162 | Glu150 | Glu152 | Glu152 | Glu156 |
Asp186 | Asp183 | Asp183 | Asp195 | Asp183 | Asp185 | Asp185 | Asp189 | |
His212 | His209 | His209 | His221 | His209 | His211 | His211 | Gln215 | |
Glu247 | Glu244 | Glu244 | Glu256 | Glu244 | Glu246 | Glu246 | Glu250 | |
Binding site on D-fructose for O-1 | Glu153 | Glu150 | Glu150 | Glu162 | Glu150 | Glu152 | Glu152 | Glu156 |
Gly218 | Arg215 | Arg215 | Arg227 | Arg215 | Arg217 | Arg217 | Arg221 | |
O-2 | Asp186 | Asp183 | Asp183 | Asp195 | Asp183 | Asp185 | Asp185 | Asp189 |
His189 | His186 | His186 | His198 | His186 | His188 | His188 | His192 | |
Glu247 | Glu244 | Glu244 | Glu256 | Glu244 | Glu246 | Glu246 | Glu250 | |
O-3 OE1 | Glu153 | Glu150 | Glu150 | Glu162 | Glu150 | Glu152 | Glu152 | Glu156 |
OE2 | Glu153 | Glu150 | Glu150 | Glu162 | Glu150 | Glu152 | Glu152 | Glu156 |
O-4 | His212 | His209 | His209 | His221 | His209 | His211 | His211 | Gln215 |
O-6 | Cys6 | Tyr6 | Tyr6 | Tyr16 | Tyr6 | Phe7 | Phe7 | Ile8 |
Reference | This study | [2] | [11] | [13] | [14] | [15] | [3] | [16] |
Enzyme | Source | Specific Activity (U mg−1) | Epimerization Reaction Ratio (D-Fructose/D-Psicose) | Reference | ||
---|---|---|---|---|---|---|
D-Fructose | D-Psicose | D-Tagatose | ||||
DPEase | I. fonsfrigidae SP3-1 | 67.62 ± 2.10 | 96.35 ± 1.70 | ND | 0.70 | This study |
C. bolteae | 150.70 | 226.90 | 52.70 | 0.66 | [13] | |
Dorea sp. CAG317 | 803.00 | 1310.00 | 64.18 | 0.61 | [17] | |
Ruminococcus sp. | 8.95 | 16.00 | 0.15 | 0.56 | [18] | |
A. tumefaciens | 8.89 | 17.50 | 5.93 | 0.51 | [14] | |
C. cellulolyticum H10 | 287.00 | 595.40 | 29.20 | 0.48 | [19] | |
B. produca | 1.76 | 5.27 | 3.76 | 0.33 | [4] | |
DTEase | P. cichorii | 4.00 | 12.00 | 20.00 | 0.33 | [20] |
C. fortuita | 270.00 | 450.00 | 801.00 | 0.60 | [3] | |
DFEase | R. sphaeroides | 380.70 | 209.90 | 230.80 | 1.81 | [16] |
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Wulansari, S.; Heng, S.; Ketbot, P.; Baramee, S.; Waeonukul, R.; Pason, P.; Ratanakhanokchai, K.; Uke, A.; Kosugi, A.; Tachaapaikoon, C. A Novel D-Psicose 3-Epimerase from Halophilic, Anaerobic Iocasia fonsfrigidae and Its Application in Coconut Water. Int. J. Mol. Sci. 2023, 24, 6394. https://doi.org/10.3390/ijms24076394
Wulansari S, Heng S, Ketbot P, Baramee S, Waeonukul R, Pason P, Ratanakhanokchai K, Uke A, Kosugi A, Tachaapaikoon C. A Novel D-Psicose 3-Epimerase from Halophilic, Anaerobic Iocasia fonsfrigidae and Its Application in Coconut Water. International Journal of Molecular Sciences. 2023; 24(7):6394. https://doi.org/10.3390/ijms24076394
Chicago/Turabian StyleWulansari, Shinta, Sobroney Heng, Prattana Ketbot, Sirilak Baramee, Rattiya Waeonukul, Patthra Pason, Khanok Ratanakhanokchai, Ayaka Uke, Akihiko Kosugi, and Chakrit Tachaapaikoon. 2023. "A Novel D-Psicose 3-Epimerase from Halophilic, Anaerobic Iocasia fonsfrigidae and Its Application in Coconut Water" International Journal of Molecular Sciences 24, no. 7: 6394. https://doi.org/10.3390/ijms24076394