Enzyme activity in liquid lipase melts as a step towards solvent-free biology at 150 °C

Abstract

Water molecules play a number of critical roles in enzyme catalysis, including mass transfer of substrates and products, nucleophilicity and proton transfer at the active site, and solvent shell-mediated dynamics for accessing catalytically competent conformations. The pervasiveness of water in enzymolysis therefore raises the question concerning whether biocatalysis can be undertaken in the absence of a protein hydration shell. Lipase-mediated catalysis has been undertaken with reagent-based solvents and lyophilized powders, but there are no examples of molecularly dispersed enzymes that catalyse reactions at sub-solvation levels within solvent-free melts. Here we describe the synthesis, properties and enzyme activity of self-contained reactive biofluids based on solvent-free melts of lipase-polymer surfactant nanoconjugates. Desiccated substrates in liquid (p-nitrophenyl butyrate) or solid (p-nitrophenyl palmitate) form can be mixed or solubilized, respectively, into the enzyme biofluids, and hydrolysed in the solvent-free state. Significantly, the efficiency of product formation increases as the temperature is raised to 150°C.

High‐Temperature Electrochemistry of a Solvent‐Free Myoglobin Melt

“…[4,11,14] Melts containing LiPF 6 were produced by adding 5, 10, or 20 wt %L iPF 6 to am ethanolic solutiono fS 1 -cMb, followed by freezing in liquid N 2 and lyophilization for 48 ht op roduce dark-brown, low-density powders that were heated to 60 8Cf or 30 min in ac onvection oven to produce dark-red solvent-free viscousl iquids (5 Li-S 1 -cMb, 10 Li-S 1 -cMb, and 20 Li-S 1 -cMb with Li:Mb molar ratios % 23:1, 46:1, and 92:1, respectively). The melts showed no evidenceo fm icrophase separation, indicating that LiPF 6 was homogeneouslyd ispersed in the solvent-free liquids.T hermogravimetric analysis( TGA) profiles for the solvent-free melts with or without the LiPF 6 electrolyte gave water contentso f< 0.1 wt %( see Figure S1 in the Supporting Information).…”

“…Preparation of Solvent-FreeS 1 -cMb Protein Melt With and WithoutL iPF 6 The solvent-free melt of Mb was prepared through at wo-step process, as demonstrated earlier by our group. [2,11,13] Briefly,t he native Mb was cationized by carbodiimide-mediated coupling of the surface-accessible glutamic and aspartic acid residues to DMAPA.…”

“…[1][2][3][4][5][6] These studies are significant because they extend previousm ethodology for accessing the liquid state of inorganic particles [7][8][9] to soft biomolecular nanoscale objectss uch as globular proteins,e nzymes, and viruses. Conjugation of the cationized proteins andp olymer surfactant was performed under aqueousc onditions, and subsequent freeze-drying and thermala nnealing at 60 8Cy ieldedasolvent-free meltc lose to room temperature.…”

“…LiPF 6 is aw idely used electrolyte in lithium-ion batteries, owing to its highs olubility in nonpolars olvents such as propylene carbonate and 1,2-dimethoxyethane, and the inertness of PF 6 À towards reducing metals [16,17] (the use of water for dispersing LiPF 6 is ruled out because of the highly active proton nature of the solvent, which leads to the production of HF). Na, K, or Li salts have also been dispersed in synthetic polymers to produce polymere lectrolytes;n otably,L is alts and polyethylene oxide (PEO) polymer complexesb ehave as solid electrolytes with ah igh ionic conductivity between 60 and 80 8C, [18] which is governed by continuous dynamics in the amorphousr egion of the polymer rather than chargeh opping.…”

Droplet microfluidics: A tool for biology, chemistry and nanotechnology

“…Efforts have been made to increase the stability and catalytic activity of certain enzymes by dissolving them in a non-aqueous solvent [26,27].…”