Performing biocatalysis in nonaqueous solvents is advantageous as it imparts enhanced solubility to hydrophobic substrates and an ability to increase the temperature for shifting reaction equilibrium in the forward direction. In this work, we show the design and development of another class of nonaqueous composite solvent obtained by mixing surface modified enzyme and neat ionic liquid (IL). We systematically probe the interaction and solubility of industrially relevant α-chymotrypsin in its native or surface-bound polymer-surfactant bioconjugated form, with neat protic (N-methyl-2-pyrrolidonium trifluoromethanesulfonate; [NMP][OTf]), or aprotic (1-methyl-3-(4-sulfobutyl)-1H-imidazol-3-ium trifluoromethanesulfonate; [HO₃S(CH₂)₄MIm][OTf]), ILs. Polarized optical micrographs show that the lyophilized powder of native α-chymotrypsin, nCT, does not disperse in either of the neat ILs, however, its polymer surfactant (PS)-coated bioconjugate counterparts, PScCT, in the waterless state, can be well-dispersed and solubilized in the neat [HO₃S(CH₂)₄MIm][OTf]. The solubilization of waterless bioconjugates of PScCT in neat aprotic IL provides a composite liquid, WL-ImPScCT (WL: waterless, Im: [HO₃S(CH₂)₄MIm][OTf]), having a viscosity of 69.6 Pa·s at 25 °C with a shear-thinning behavior, ≈ 15 w/w % α-chymotrypsin, and ≈ 1.2 w/w % residual water content. Detailed secondary structural analysis using circular dichroism and Fourier self-deconvolution on the ATR-FTIR data of WL-ImPScCT liquid reveals retention of the near native secondary structure of α-chymotrypsin. Further, using a combination of fluorescence spectroscopy and electron spray ionization mass spectrometry, we show that scattering of dry and powdered bovine serum albumin (BSA) protein on the WL-ImPScCT composite liquid results in the solubilization of the former, followed by limited proteolysis of BSA by the α-chymotrypsin. Our results, therefore, show the stabilization of α-chymotrypsin in a neat aprotic IL environment to yield a composite liquid, which not only acts as a nonaqueous, nonvolatile, and environmentally benign solvent, but also provides a biocatalytic platform capable of carrying out reactions relevant for biotransformations, food processing, drug delivery, and various other applications.

Anasua Mukhopadhayay

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