Neat Ionic liquid and α-Chymotrypsin-Polymer Surfactant Conjugate-Based Biocatalytic Solvent

Abstract

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; [HO 3 S­(CH 2 ) 4 MIm]­[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 [HO 3 S­(CH 2 ) 4 MIm]­[OTf]. The solubilization of waterless bioconjugates of PScCT in neat aprotic IL provides a composite liquid, WL-ImPScCT (WL: waterless, Im: [HO 3 S­(CH 2 ) 4 MIm]­[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.

Dispersion and Interaction of Charged Fluorescent Dyes in Protein‐Polymer Surfactant‐based Non‐Aqueous Liquid

“…Overall, this work provides valuable insights for the dissolution of both specific and non‐specific binding dyes in the non‐aqueous protein polymer conjugated liquid/solvent matrix. Moreover, our recent studies have shown an another class of neat ionic liquid and enzyme‐polymer surfactant bio‐conjugate based bio‐catalytic solvent medium . It can be envisaged that in conjunction with ionic liquids, the current study of dissolving and dispersing various dyes in bioconjugates can pave a path forward for design and development of solid state electrolytes and lasing dye media for potential applications.…”

“…Methods : Stock solutions of Rh6G and FL were prepared by dilution of concentrated Rh6G and FL dissolved in water and concentration of Rh6G and FL were determined from respective molar extinction coefficient of ϵ 526 nm =81,000 M − 1 cm −1[69] and ϵ 490 nm =76,000 M −1 cm −1 . Stock solution of native BSA was prepared in 10 mM pH 7.4 phosphate buffer and concentrations of nBSA and PSpBSA sol were determined from molar extinction coefficient of ϵ 280 nm =43,824 M −1 cm −1 . UV‐Vis measurements were done using Cary Varian 100 spectrophotometer, steady‐state emission spectra were recorded using Varian Eclipse Fluorescence spectrophotometer and bright field optical micrographs were taken using Olympus BH2 microscope and temperature‐dependent microscopic studies were performed using Linkam stage (TP 94 and LNP).…”

Applications of Ionic Liquids in Whole-Cell and Isolated Enzyme Biocatalysis

“…The modification enhanced the solubility of the enzyme in neat protic and neat aprotic ionic liquids (N-methyl-2-pyrrolidonium trifluoromethanesulfonate; [NMP][OTf]), or (1-methyl-3-(4-sulfobutyl)-1H- imidazol-3-ium trifluoromethanesulfonate; [HO 3 S(CH 2 ) 4 MIM][OTf]) respectively and retained a similar β-sheet structure to the native protein in aqueous solution. The unmodified enzyme was insoluble in the ILs [ 95 ].…”

Preparation and application of solvent-free liquid proteins with enhanced thermal and anhydrous stabilities

“…Mukhopadhayay et al also demonstrated that ionic liquids could be used to improve the activity of solvent-free liquid α-chymotrypsin, further evidence to the effectiveness of the strategy. 78…”