# Diversity of Potential (Bio)Technological Applications of Amino Acid-Based Ionic Liquids
## 2 Bioapplications of AA ILs: An Overview
### Stability and Activity of Enzymes and Proteins  
#### 2.5.1 AA-ILs as Media for Biocatalytic Reactions  
A comprehensive study of eighteen [Ch][AA] ILs showed that they all improved the water solubility of phytosterols (\(\beta\)-sitosterol, stigmasterol, campesterol, and brassicasterol) [82]. With the exception of [Ch][Trp], little to no effect on cell membrane integrity and the activity of _Mycobacterium_ sp. MB 3683 was observed at concentrations up to 4%. Furthermore, in the presence of 1% [Ch][Asp], [Ch][Glu], or [Ch][Gln], the yield of androst-4-ene-3,17-dione, catalytically converted from phytosterols by whole cells of Mycobacterium sp., was higher compared to IL-free media [82]. On the other hand, [PERSON] et al. observed that the addition of [Ch][Gly] slightly increased the molecular volume of bromelain, causing partial unfolding compared to the PBS medium (control), leading to a loss of its compact structure and increased flexibility [83]. This is attributed to the formation of effective hydrogen bonds between the glycine anions and the peptide chains of the protein. However, the addition of [Ch][Gly] at concentrations up to 0.1 M had no significant effect on the hydrolytic activity of bromelain [83]. Similarly, a slight increase in hydrolytic activity of bromelain was observed in the presence of [Bmim][Gly] [84]. In the case of bromelain, interactions involving the AA anion appear to be predominant and have a stronger effect on its conformational stability and catalytic activity than those involving the cation [83,84]. In contrast, [PERSON] et al. reported a 30% increase in the hydrolytic activity of papain in the presence of 1 mM [Ch][Trp], whereas the addition of [N\({}_{2222}\)][Trp] at the same concentration resulted in a 15% decrease in the hydrolytic activity of papain [85]. However, when the concentration of either IL was increased even slightly, papain was almost completely inactivated due to the disruption of disulfide bonds in the papain molecule and subsequent unfolding of the protein. The IL-induced unfolding of papain was shown to be concentration-dependent [85]. Interestingly, the hydrolytic activity of papain was enhanced by 2-fold and 1.4-fold, respectively, when immobilized on gold nanoparticles (AuNPs) via [Ch][Trp] or [N\({}_{2222}\)][Trp] [86]. In addition, thermal stability was maintained, and secondary structure rearrangements without unfolding were observed for the supported papain molecules. Furthermore, AuNP/[Ch][Trp] showed a higher loading capacity for papain compared to AuNP/[N\({}_{2222}\)][Trp] [86]. In contrast, all 20 amino acid ILs, including mono- and bis-salts containing TEA cation and various anions, inhibited the enzymatic activities of proteolytic enzymes such as papain, bromelain, or subtilisin when added as co-solvents in a casein hydrolysis reaction [26]. The inhibition constants were estimated to be in the micromolar to millimolar range, depending on the specificity and type of IL anion. Lipases are the second largest group of industrially used enzymes after proteases. Therefore, efforts are being made to improve their stability and catalytic activity in order to increase the yield of the target product and minimize production costs. We tested the effect of two large series of amino acid ILs containing either the [Emim] or [Ch] cation on the hydrolytic activity of a lipase from _Candida rugosa_ (CRL) [87]. At concentrations above 0.025 mM, both series showed a clear trend of decreasing activity, with the influence of anions ranked as follows: Leu > Trp \(\geq\) Thr > Val \(\geq\) Met > Ile > Gly, with the effect being stronger for the [Emim] series. Additionally, all ILs induced an increase in random coils, disordered structures, and antiparallel \(\beta\)-sheets [87]. On the other hand, AA-based ILs with a symmetrical tetraalkylammonium cation seem to have a positive effect on the catalytic activity of lipases. [PERSON] et al.