Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent
Abstract
:1. Introduction
- The poor solubility of 2-PB in aqueous solutions.
- The necessity for obtaining an inert support after the immobilization process, reducing the remaining aldehydes to hydroxyls, or blocking them with other substances.
- The ability of the 2-PB to reduce all the Schiff bases that are formed between the enzyme and the support. 2-PB molecules, which are much larger molecules than the hydrides, have to go through the region that lies between the enzyme and the support to reduce all the Schiff base (the width of this region is approximately 2 nm) [25].
2. Results
2.1. Immobilization of PGA, DAAO, ADH2, GlyDH, and Xys1Δ on Glyoxyl-Activated Agarose
2.2. Reduction of the Immobilized Enzymes with 2-Picoline Borane
2.3. Comparison of Immobilized Enzymes Reduced with Borohydride and 2-Picoline Borane: Expressed Activity and Thermal Stability
2.4. Evaluation of the Correlation between Thermal Stability and Number of Enzyme–Support
2.5. Immobilization–Stabilization of a Bacterial Mandelate Dehydrogenase: Optimization of the Eduction Step
3. Materials and Methods
3.1. Materials
3.2. Methods
3.2.1. Protein Production
3.2.2. Enzymatic Assays
3.2.3. Support Immobilization Preparation
3.2.4. Protein Immobilization
Reduction of Schiff Base with Sodium Borohydride
Reduction of Schiff Bases with the 2-Picoline Borane Complex
3.2.5. Thermal Inactivation Assays
3.2.6. Estimation of the Lysine Residues Involved in the Immobilization of the Enzyme on the GA Support
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Enzyme | Conjugate 1 | Immobilization Yield (%) 5 | Expressed Activity before Reduction Step (%) 6 | Expressed Activity after Reduction Step (%) 7 | Half-Life (h) 8 | Stability Factor 9 |
---|---|---|---|---|---|---|
PGA (dimer) | GA-B 2 | 97 | 43 | 43 | 70 | 1 |
GA-G 3 | 48.6 | 6 | 0.09 | |||
GA-E 4 | 48.6 | 1 | 0.01 | |||
DAAO (dimer) | GA-B | 99 | 25 | 20.7 | 15 | 1 |
GA-G | 25 | 3 | 0.2 | |||
GA-E | 27.5 | 1.5 | 0.1 | |||
GlyDH (octamer) | GA-B | 100 | 35 | 35 | 10 | 1 |
GA-G | 25.5 | 11 | 1.1 | |||
GA-E | 53.5 | 1 | 0.1 | |||
ADH2 (dimer) | GA-B | 98 | 65 | 69.5 | 4.5 | 1 |
GA-G | 87.7 | 20 | 4.4 | |||
GA-E | 107 | 18 | 4 | |||
Xys1Δ (monomer) | GA-B | 75 | 60 | 60 | 3.5 | 1 |
GA-G | 53 | 5 | 1.4 | |||
GA-E | 66 | 2.5 | 0.7 |
Enzyme | GA-B 1 | GA-G 2 |
---|---|---|
PGA | 7 | 9 |
DAAO | 4 | 5 |
Xys1Δ | 5 | 4 |
Conjugate | ManDH-GA-B 1 | ManDH-GA-E 2 | ManDH-GA-G1 3 | ManDH-GA-G2 4 | ManDH-GA-G3 5 |
---|---|---|---|---|---|
Immobilization yield (%) 6 | 99 | 99 | 99 | 99 | 99 |
Expressed activity before reduction step (%) 7 | 15 | 14 | 17 | 50 | 39 |
Recovered activity after reduction step (%) 8 | 1.5 | 14 | 16.5 | 16 | 19.5 |
Half-life (h) 9 | N.D. | 19 | 221 | 43 | 82 |
Stabilization factor 10 | N.D. | 31 | 357 | 69 | 132 |
Enzyme | Buffer | Substrate 1 | Temperature (°C) 2 | Wavelength (nm) |
---|---|---|---|---|
PGA | 50 mM sodium phosphate pH 7 | 0.15 mM NIPAB | 25 | 405 |
DAAO 3 | 50 mM sodium phosphate pH 7.5 | 10 mM D-phenylalanine | 25 | 450 |
ADH2 | 50 mM sodium phosphate pH 7 | 10 mM 2,2,2-trifluoroacetophenone and 0.25 mM NADH | 65 | 340 |
GlyDH | 50 mM sodium phosphate pH 7 | 100 mM glycerol and 2.5 mM NAD+ | 25 | 340 |
ManDH | 0.1 M potassium phosphate pH 7 | 1.5 mM phenylglyoxylate and 0.25 mM NADH | 30 | 340 |
Enzyme | Immobilization Time (h) | Immobilization Buffer | Offered Protein per g of Support (mg) | Temperature (°C) |
---|---|---|---|---|
PGA | 1 | 0.1 M sodium bicarbonate pH 10, 0.1 M phenylacetic and 25% glycerol | 5 | 25 |
DAAO | 1 | 0.1 M sodium bicarbonate buffer pH 10 | 2.7 | 25 |
ADH2 | 6 | 0.1 M sodium bicarbonate buffer pH 10 | 2 | 25 |
GlyDH | 3 | 0.1 M sodium bicarbonate buffer pH 10 | 2.5 | 25 |
Xys1Δ | 24 | 0.1 M sodium bicarbonate buffer pH 10 | 10 | 4 |
ManDH | 1–3 | 0.1 M potassium bicarbonate buffer pH 10 and 40% trehalose | 0.5 | 4–25 |
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H. Orrego, A.; Romero-Fernández, M.; Millán-Linares, M.D.C.; Yust, M.D.M.; Guisán, J.M.; Rocha-Martin, J. Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent. Catalysts 2018, 8, 333. https://doi.org/10.3390/catal8080333
H. Orrego A, Romero-Fernández M, Millán-Linares MDC, Yust MDM, Guisán JM, Rocha-Martin J. Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent. Catalysts. 2018; 8(8):333. https://doi.org/10.3390/catal8080333
Chicago/Turabian StyleH. Orrego, Alejandro, Maria Romero-Fernández, María Del Carmen Millán-Linares, María Del Mar Yust, José M. Guisán, and Javier Rocha-Martin. 2018. "Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent" Catalysts 8, no. 8: 333. https://doi.org/10.3390/catal8080333