Fluorescence and CD spectroscopic analysis of the α‐chymotrypsin stabilization by the ionic liquid, 1‐ethyl‐3‐methylimidazolium bis[(trifluoromethyl)sulfonyl]amide

Abstract

The stability of alpha-chymotrypsin in the ionic liquid, 1-ethyl-3-methyl-imidizolium bis[(trifluoromethyl)sulfonyl]amide ([emim][NTf2]), was studied at 30 and 50 degrees C and compared with the stability in other liquid media, such as water, 3 M sorbitol, and 1-propanol. The kinetic analysis of the enzyme stability pointed to the clear denaturative effect of 1-propanol, while both 3M sorbitol and [emim][NTf2] displayed a strong stabilizing power. For the first time, it is shown that enzyme stabilization by ionic liquids seems to be related to the associated structural changes of the protein that can be observed by differential scanning calorimetry (DSC) and fluorescence and circular dichroism (CD). The [emim][NTf2] enhanced both the melting temperature and heat capacity of the enzyme compared to the other media assayed. The fluorescence spectra clearly showed the ability of [emim][NTf2] to compact the native structural conformation of alpha-chymotrypsin, preventing the usual thermal unfolding which occurs in other media. Changes in the secondary structure of this beta/beta protein, as quantified by the CD spectra, pointed to the great enhancement (up 40% with respect to that in water) of beta-strands in the presence of the ionic liquid, which reflects its stabilization power.

Lipase‐catalyzed acylation of konjac glucomannan in ionic liquids

“…Recent spectroscopic studies of protein thermostability in ILs have indicated that the significant thermodynamic stabilization effect offered by the ionic liquid seemed to be related to the structural changes of the protein: 31 -33 The fluorescence spectra clearly showed the ability of the ILs to compact the native structural conformation of the enzyme, preventing the usual thermal unfolding which occurs in other media. 31,32 The circular dichroism (CD) spectra also demonstrated changes in the secondary structure of the protein in the presence of the ILs, resulting in a more compact enzyme conformation able to exhibit catalytic activity. 32,33 Moreover, differential scanning calorimetry (DSC) revealed that both the melting temperature and heat capacity of the enzyme were enhanced in the presence of ILs as compared with the other media.…”

“…31,32 The circular dichroism (CD) spectra also demonstrated changes in the secondary structure of the protein in the presence of the ILs, resulting in a more compact enzyme conformation able to exhibit catalytic activity. 32,33 Moreover, differential scanning calorimetry (DSC) revealed that both the melting temperature and heat capacity of the enzyme were enhanced in the presence of ILs as compared with the other media. 32 Thus, the observable improvement in the stability of Novozym 435 in ILs is most probably due to the electrostatic interaction between the ILs and the protein, leading to a more rigid protein, which needs to overcome a higher kinetic barrier to unfold in comparison with the cases when the enzyme is incubated in organic solvents.…”

“…32,33 Moreover, differential scanning calorimetry (DSC) revealed that both the melting temperature and heat capacity of the enzyme were enhanced in the presence of ILs as compared with the other media. 32 Thus, the observable improvement in the stability of Novozym 435 in ILs is most probably due to the electrostatic interaction between the ILs and the protein, leading to a more rigid protein, which needs to overcome a higher kinetic barrier to unfold in comparison with the cases when the enzyme is incubated in organic solvents. 34 Some other factors such as free volume contributions, ionic interactions and confinement effects may also contribute to protein stabilization by ILs.…”

Unexpected improvement in stability and utility of cytochrome c by solution in biocompatible ionic liquids

“…Although traditional ionic liquids are frequently synthesized from imidazolium-based cations and highly fluorinated anions, recent work has shown that ionic liquids can also be formulated from salts (Abbott et al, 2001), sugars (Carter et al, 2004), amino acids (Fukumoto et al, 2005;Tao et al, 2005), and biomolecules (Pernak et al, 2004) that exist in nature, many of which have already been approved as pharmaceutical excipients. Previous work in recent years has shown that enzymes and some proteins can be active in ionic liquids; however, structure is disrupted in many cases (Diego et al, 2004;Laszlo and Compton, 2002;Lau et al, 2004). In a recent report a new family of biocompatible ionic liquids based on the dihydrogen phosphate anion were described (Fujita et al, 2005); these ILs were able to dissolve substantial quantities of cytochrome c (cyt c) and in the solution the protein was shown to retain its structure and activity.…”

(Bio)Catalytic Continuous Flow Processes in scCO2 and/or ILs: Towards Sustainable (Bio)Catalytic Synthetic Platforms

“…They are used for the synthesis of aliphatic and aromatic esters, chiral esters by kinetic resolution of racemic alcohols, carbohydrate esters, polymers, etc. Additionally, the excellent stability displayed by enzymes in water-immiscible ILs for reuse and under high temperatures has been widely described [45]. Concerning medium engineering for biotransformations in ILs, it has been reported that ILs improves activity (e.g.…”