New Ionic Liquids with Buffering and Chelating Abilities for Enzyme Engineering

Ionic liquids (ILs) with buffering and chelating abilities were designed and synthesized on the basis of ethylenediaminetetraacetic acid (EDTA) for the development of buffered enzymatic IL systems and for enzymatic reaction in heavy metal containing aqueous system. Transesterification activity of Candida antarctica lipase B dissolved in the hydroxyl-functionalized IL was buffer dependent. High activity and outstanding stability was obtained with the buffered enzymatic IL systems for the transesterification. In heavy metal containing aqueous system, EDTA IL buffers as Hg 2+ chelators protected horseradish peroxidase (HRP) against Hg 2+ -induced denaturation and precipitation. Higher pH favored the protection, while at lower pH the protection di-minished. We can conclude that the new ILs possess both buffering and chelating abilities.


Introduction
Enzymes catalyze a wide variety of reactions best in heavy-metal-free aqueous environments and at physiological pH with exquisite selectivity and stereospecificity [1]. Thus, an appropriate buffer is needed to closely regulate the ionization state of ionizable groups of the enzyme in both aqueous and non-aqueous media. We have synthesized a new class of ionic liquids (ILs) with buffering behaviour that are referred to as IL buffers, which can be used for control of ionization state of enzymes in non-aqueous media [2]. Remarkable buffer dependence of the catalytic activities has been observed in hydrogenation of olefins [2] and selective hydrogenation of unsaturated aldehyde [3] in organic solvents and in Advances in Bioscience and Biotechnology ILs. Organic solvents are widely used with enzymes to improve the solubility of hydrophobic reactants and/or products and to shift reaction equilibria from hydrolysis toward synthesis [4]- [9]. With the emergence of ILs, the use of ILs as a new type of non-aqueous medium may offer a convenient solution to both the solvent emission and the catalyst recycling problem [5] [9]- [22].
Increasing water contamination by heavy metals has prompted investigations to find ways to clean the environment and also to understand the mechanisms leading to metal toxicity, among which is enzyme inhibition. This inhibition was most often attributed to the reaction of the metal ions with the thiol groups of cysteine residues of the enzyme, resulting in the formation of mercaptides [23] [24].
It was reported that peroxidases are inhibited by heavy metal ions at higher concentrations [25] and Hg 2+ ion is listed as the most effective inhibitor [26] [27]. Konyaeva et al. reported that Hg 2+ chelators like EDTA can protect hemoglobin against Hg 2+ -induced denaturation and precipitation [28].
Like most biological buffers in use today, IL buffers were developed only for keeping the pH of a solution constant, which cannot protect enzymes against metal-induced denaturation and precipitation. We then designed and synthesized new ILs with both buffering and chelating abilities for enzymatic reactions in heavy metal containing aqueous system and in IL system.

Materials
Candida antarctica lipase B (CALB, 10.9 U•mg −1 ), horseradish peroxidase (HRP, 150 U•mg −1 ), bis-tris-propane (BTP), guaiacol and ethyl butyrate were purchased from Sigma-Aldrich. Ethyl butyrate and n-butanol were analytical reagents and were dried by 3A molecular sieves before use. All other chemicals and reagents were of analytical grade.    All assays were carried out at 298 K using a UV-Vis spectrophotometer (Unico, UV2800) which cell was connected to a thermostat.

Titration Profiles of EDTA
The titration profile of EDTA with [BMIM]OH in water expressed 3 buffering-like regions, which are centered at pH 3.90, 6.50 and 8.50, respectively ( Figure 1).

Synthesis and Characterization of EDTA IL Buffer
The structure of EDTA IL buffers were shown in Scheme 1. The IL buffers were synthesized as previously reported [2] [11] and characterized by NMR. The characterization data of EDTA IL at pH 3.90 and pH 9.80 are consistent with the expected compositions and structures. The pH, the dilution value, and buffer values of EDTA IL buffers were summarized in Table 1. It can be seen that the ILs synthesized possess buffering ability and can be used for controlling the ionization of enzymes in both aqueous and non-aqueous media.

Enzymatic Transesterification in the Presence of EDTA IL Buffer
Lipase-mediated transesterification is one of the economically viable clean technology for flavor ester production [29].  Figure 2), indicating that the buffer was responsible for the rate enhancement.
The above results showed that the lipase activity is greatly affected by the IL and IL buffer. This is because the ionization constant of ionizable groups of the lipase is greatly affected by the solvent [30]. Thomazeau

HRP Activity in Mercury Containing Aqueous System
Oxidative stress causes uncontrolled oxidation, resulting in the progressive deterioration and the collapse of organs and systems in the living organisms [32].
Peroxidases are important detoxifying enzymes serving to rid cells of excess G. N. Ou, B. Y. He    Figure 3 showed that EDTA IL buffers as Hg 2+ chelators protected HRP against Hg 2+ -induced denaturation and precipitation. Higher pH favored the protection, while at lower pH the protection diminished. Because the concentration of the deprotonated conjugate base of EDTA increases with the increase of pH of solution, its subsequent increased binding to Hg 2+ causes the protection of HRP against Hg 2+ -induced denaturation. On the contrary, there is almost no Hg 2+ chelators in BTP buffer system, thereby, Hg 2+ inhibition occurs. This chelating effect undoubtedly explained the difference in Figure 3 and Figure 4.

Conclusion
In summary, ILs based on EDTA were synthesized by neutralization of [BMIM]OH with EDTA acid for the development of buffered enzymatic IL systems and for enzymatic reaction in heavy metal containing aqueous system. In buffered [C 2 OHMIM][BF 4 ], transesterification activity of CALB is buffer dependent and CALB is stable during recycles. In EDTA IL buffer aqueous system, EDTA as Hg 2+ chelators protected HRP against Hg 2+ -induced denaturation and precipitation. Higher pH favored the protection, while at lower pH the protection diminished. We can conclude that the new ILs possess both buffering and chelating abilities and can be used for enzymatic applications.

Data Availability
The data used to support the findings of this study are included within the article.