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On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg.

Castillo B, Bansal V, Ganesan A, Halling P, Secundo F, Ferrer A, Griebenow K, Barletta G - BMC Biotechnol. (2006)

Bottom Line: Additionally, the hydration level of the enzyme does not seem to affect its stability.Incubation was also accompanied by a substantial decrease in Vmax/KM.These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Puerto Rico at Humacao, CUH Station, Humacao, 00791, Puerto Rico. betzaida.castillo@yahoo.com <betzaida.castillo@yahoo.com>

ABSTRACT

Background: Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.

Results: As model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-beta-cyclodextrin (MbetaCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM.

Conclusion: These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.

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Far and near UV circular dichroism spectra of fresh and incubated lyophilized and co-lyophilized preparations of subtilisin C. CD spectra are shown for samples freshly suspended in 1,4-dioxane (solid lines) and after further incubation for 4 days (dotted lines)(A) Far UV CD of lyophilized subtilisin C. (B) Near UV CD of lyophilized subtilisin C. (C) Far UV CD of subtilisin C. co-lyophilized with methyl-β-cyclodextrin. (D) Near UV CD of subtilisin C. co-lyophilized with methyl-β-cyclodextrin.
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Figure 3: Far and near UV circular dichroism spectra of fresh and incubated lyophilized and co-lyophilized preparations of subtilisin C. CD spectra are shown for samples freshly suspended in 1,4-dioxane (solid lines) and after further incubation for 4 days (dotted lines)(A) Far UV CD of lyophilized subtilisin C. (B) Near UV CD of lyophilized subtilisin C. (C) Far UV CD of subtilisin C. co-lyophilized with methyl-β-cyclodextrin. (D) Near UV CD of subtilisin C. co-lyophilized with methyl-β-cyclodextrin.

Mentions: To further probe changes in structure, including the tertiary structure, we used circular dichroism spectroscopy (CD). Spectra were recorded for both the lyophilized and the co-lyophilized powders before and after incubation in 1,4-dioxane for 4 days (Figure 3). The near UV CD of neither enzyme preparation was influenced by incubation, illustrating that the enzyme tertiary structure in both preparations remains unaffected. The far UV CD of the co-lyophilized enzyme is also unaffected, but the lyophilized powder shows a reduction in ellipticity, which was reproducible. It is unusual for a protein to show spectral changes in the far UV but not in the near UV regions, because this implies a change in secondary structure without change in tertiary structure. However, far UV CD of particulate samples is significantly affected by absorption flattening, for which a semi-empirical correction may be made [27]. The correction involves one adjustable parameter per sample, and it is possible to choose values such that the spectra for the fresh and incubated samples are the same within experimental error. However, this must remain tentative, because here we have no independent method to estimate the correction parameters. A change in absorption flattening would also be most likely to result from a change in particle structure, and none was observed in the SEM imaging studies. The spectra of the colyophilized preparation shows higher ellipticity than the lyophilized powder (Figure 3). This is most likely due to a difference in absorption flattening because the particles are different in nature. The co-lyophilized preparation was observed to disperse in dioxane better than the lyophilized powder did. It is also possible that association with chiral β-methyl cyclodextrin (the additive) induces increased CD from the protein peptide bonds.


On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg.

Castillo B, Bansal V, Ganesan A, Halling P, Secundo F, Ferrer A, Griebenow K, Barletta G - BMC Biotechnol. (2006)

Far and near UV circular dichroism spectra of fresh and incubated lyophilized and co-lyophilized preparations of subtilisin C. CD spectra are shown for samples freshly suspended in 1,4-dioxane (solid lines) and after further incubation for 4 days (dotted lines)(A) Far UV CD of lyophilized subtilisin C. (B) Near UV CD of lyophilized subtilisin C. (C) Far UV CD of subtilisin C. co-lyophilized with methyl-β-cyclodextrin. (D) Near UV CD of subtilisin C. co-lyophilized with methyl-β-cyclodextrin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1764882&req=5

Figure 3: Far and near UV circular dichroism spectra of fresh and incubated lyophilized and co-lyophilized preparations of subtilisin C. CD spectra are shown for samples freshly suspended in 1,4-dioxane (solid lines) and after further incubation for 4 days (dotted lines)(A) Far UV CD of lyophilized subtilisin C. (B) Near UV CD of lyophilized subtilisin C. (C) Far UV CD of subtilisin C. co-lyophilized with methyl-β-cyclodextrin. (D) Near UV CD of subtilisin C. co-lyophilized with methyl-β-cyclodextrin.
Mentions: To further probe changes in structure, including the tertiary structure, we used circular dichroism spectroscopy (CD). Spectra were recorded for both the lyophilized and the co-lyophilized powders before and after incubation in 1,4-dioxane for 4 days (Figure 3). The near UV CD of neither enzyme preparation was influenced by incubation, illustrating that the enzyme tertiary structure in both preparations remains unaffected. The far UV CD of the co-lyophilized enzyme is also unaffected, but the lyophilized powder shows a reduction in ellipticity, which was reproducible. It is unusual for a protein to show spectral changes in the far UV but not in the near UV regions, because this implies a change in secondary structure without change in tertiary structure. However, far UV CD of particulate samples is significantly affected by absorption flattening, for which a semi-empirical correction may be made [27]. The correction involves one adjustable parameter per sample, and it is possible to choose values such that the spectra for the fresh and incubated samples are the same within experimental error. However, this must remain tentative, because here we have no independent method to estimate the correction parameters. A change in absorption flattening would also be most likely to result from a change in particle structure, and none was observed in the SEM imaging studies. The spectra of the colyophilized preparation shows higher ellipticity than the lyophilized powder (Figure 3). This is most likely due to a difference in absorption flattening because the particles are different in nature. The co-lyophilized preparation was observed to disperse in dioxane better than the lyophilized powder did. It is also possible that association with chiral β-methyl cyclodextrin (the additive) induces increased CD from the protein peptide bonds.

Bottom Line: Additionally, the hydration level of the enzyme does not seem to affect its stability.Incubation was also accompanied by a substantial decrease in Vmax/KM.These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Puerto Rico at Humacao, CUH Station, Humacao, 00791, Puerto Rico. betzaida.castillo@yahoo.com <betzaida.castillo@yahoo.com>

ABSTRACT

Background: Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.

Results: As model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-beta-cyclodextrin (MbetaCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM.

Conclusion: These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.

Show MeSH
Related in: MedlinePlus