Research article

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

Betzaida Castillo¹ , Vibha Bansal² , Ashok Ganesan³ , Peter Halling³ , Francesco Secundo⁴ , Amaris Ferrer² , Kai Griebenow¹ and Gabriel Barletta²

¹  Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, P.O Box 23346, San Juan, 00931-3346, Puerto Rico

²  Department of Chemistry, University of Puerto Rico at Humacao, CUH Station, Humacao, 00791, Puerto Rico

³  WestCHEM, Department of Pure & Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, UK

⁴  Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, 20131 Milano, Italy

author email corresponding author email

BMC Biotechnology 2006, 6:51doi:10.1186/1472-6750-6-51

Published:	22 December 2006

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-β-cyclodextrin (MβCD). 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 V_max/K_M.

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.