The effect of imidazolium cations on the structure and activity of the Candida antarctica Lipase B enzyme in ionic liquids.

In order to understand how cations affect the structural changes and enzyme activity of Lipase B from Candida antarctica, we performed all-atom molecular dynamics simulations of CALB in four types of ionic liquids (ILs) with varying sizes of imidazolium cations and correlated these results with the experimentally determined CALB activity. The imidazolium cations under study differ in the alkyl tail length in the following order: [Emim](+) < [Bmim](+) < [Hmim](+) < [Omim](+). We observed that the best enzyme activity and structural stability of CALB are obtained in [Bmim][TfO] and [Hmim][TfO]. In contrast, in [Emim][TfO], bonding of [TfO](-) to LYS-290 disrupts the interactions between LYS-290 and ILE-285, which leads to a closed catalytic gate conformation with low accessibility of substrates to the catalytic triad. In [Omim][TfO], strong hydrophobic interactions between [Omim](+) and LEU-278 result in a significant loss of the secondary structure of the α-10 helix and cause the exposure of the catalytic triad to ILs, which affects the stability of the catalytic triad and consequently deteriorates the enzyme activity. Overall, our study indicates that a high ion coordination number ([Emim][TfO]) or the presence of a long hydrophobic tail ([Omim][TfO]) can facilitate ion-protein interactions that cause structural distortions and a decrease in CALB enzyme activity in ILs.

Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids.

The effects of various refolding additives, including metal cofactors, organic co-solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well-known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion-containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca(2+) and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3-fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea-containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor-containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.