Teresa De Diego

Criteria to Design Green Enzymatic Processes in Ionic Liquid/Supercritical Carbon Dioxide Systems

Five different ionic liquids (ILs) based on quaternary ammonium cations, with functional side chains ((3‐hydroxypropyl)‐trimethyl‐, (3‐cyanopropyl)‐trimethyl‐, butyl‐trimethyl‐, (5‐cyanopentyl)‐trimethyl‐ and hexyl‐trimethyl‐) associated with the same anion (bis(trifluoromethane)sulfonyl amide)), were synthesized, and their suitability for Candida antarctica lipase B (CALB)‐catalyzed ester synthesis in IL/supercritical carbon dioxide (scCO2) biphasic systems was assayed. Catalytic efficiency of the system has been analyzed as a function of both enzyme properties and mass‐transfer phenomena criteria. First, the suitability of these ILs as enzymic reaction media was tested for the kinetic resolution of rac‐phenylethanol. All ILs were found to be suitable media for enzyme catalysis, the best catalytic parameter (5.3 U/mg specific activity, 94.9% selectivity) being obtained for the (5‐cyanopentyl)‐trimethylammonium. Second, enzyme stability in all of the ILs was studied at 50 °C over a period of 50 days, and data were analyzed by a two‐step kinetic deactivation model. All of the ILs were shown to act as stabilizing agents with respect to hexane, producing an increase in the free energy of deactivation (to 25 kJ/mol protein) and an improvement in the half‐life time of the enzyme (2000‐fold), which agrees with the observed increased hydrophobicity of the cation alkyl side chain (measured by Hansenapos;s solubility parameter, δ). By using two different CALB‐IL systems with different hydrophobicity in the cation, continuous processes to synthesize six different short chain alkyl esters (butyl acetate, butyl propionate, butyl butyrate, hexyl propionate, hexyl butyrate, and octyl propionate) in scCO2 at 10 MPa and 50 °C were carried out. Both rate‐limiting parameters (synthetic activity and scCO2–ILs mass‐transfer phenomena) were related with the δ‐parameter of the ILs‐alkyl chain and reagents.

Lipase Catalysis in Ionic Liquids and Supercritical Carbon Dioxide at 150 °C

Free and immobilized Candida antarctica lipase B dispersed in ionic liquids (1‐ethyl‐3‐methylimidazolium bistriflimide and 1‐buthyl‐3‐methylimidazolium bistriflimide) were used as catalyst for the continuous kinetic resolution of rac‐1‐phenylethanol in supercritical carbon dioxide at 120 and 150 °C and 10 MPa. Excellent activity, stability and enantioselectivity levels were recorded in continuous operation.

On the nature of ionic liquids and their effects on lipases that catalyze ester synthesis

Free and immobilized lipases from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) were used as catalysts in the synthesis of butyl propionate by transesterification in reaction media consisting in nine different ionic liquids. Enzyme activities were clearly dependent on the nature of the ions, the results being improving as the alkyl chain length of the imidazolium cation increased, and as a function of the type of anion ([PF(6)], [BF(4)] or [ethylsulphate]). The best synthetic activity (655.5U/mg protein at 40 degrees C) was obtained when free CALB were assayed in the water-miscible IL cocosalkyl pentaethoxy methyl ammonium methosulfate ([CPMA][MS]), and was clearly related with the water content of the medium. The synthetic activity of free CALB in [CPMA][MS] was enhanced with the increase in temperature, while practically no effect was obtained for TLL. The ability of free CALB to synthesize aliphatic esters of different alkyl chain lengths, using different alkyl vinyl esters and 1-alkanols as substrates, was also studied in [CPMA][MS], the best results (4500U/mg protein) being obtained for the synthesis of hexyl butyrate.

Continuous green biocatalytic processes using ionic liquids and supercritical carbon dioxide

Soluble Candida antarctica lipase B dissolved in ionic liquids showed good synthetic activity, enantioselectivity and operational stability in supercritical carbon dioxide for both butyl butyrate synthesis and the kinetic resolution of 1-phenylethanol processes by transesterification.

Dynamic structure–function relationships in enzyme stabilization by ionic liquids

The stability of α-chymotrypsin and Candida antarctica lipase B (CALB) in two ionic liquids (i.e. 1-ethyl-3-methyl-imidazolium, bis[(trifluoromethyl)sulfonyl]imide [emim] [NTf2], and butyl-trimethylamonium bis[(trifluoromethyl)sulfonyl]imide [btma] [NTf2]) has been studied. Both enzymes were strongly stabilized by the ionic liquids, the respective half-life times increasing 96 and 1660 times, with respect to those obtained in classical organic solvents such as 1-propanol and hexane, respectively. The stabilization of both enzymes by ionic liquids may be related to the associated structural changes of proteins that they can be observed by both fluorescence and circular dichroism spectroscopic studies.

Ionic liquids improve citronellyl ester synthesis catalyzed by immobilized Candida antarctica lipase B in solvent-free media

Several citronellyl esters (acetate, propionate, butyrate, caprate and laurate) were synthesized by immobilized Candida antarctica lipase B (Novozym) in high yields (>99%) using equimolar mixtures of citronellol and alkyl vinyl ester as substrates in solvent-free medium. The best results were obtained for citronellyl butyrate synthesis (17.4 µmol min−1 mg IME−1) at 70 °C, which could be improved up to two-fold by coating the biocatalyst particles with alkyl imidazolium-based ionic liquids, which favoured partitioning of the substrate and product molecules.

A recyclable enzymatic biodiesel production process in ionic liquids

Immobilized Candida antarctica lipase B suspended in ionic liquids containing long alkyl-chain cations showed excellent synthetic activity and operational stability for biodiesel production. The interest of this process lies in the possibility of recycling the biocatalyst and the easy separation of the biodiesel from the reaction mixture. The ionic liquids used, 1-hexadecyl-3-methylimidazolium triflimide ([C(16)MIM][NTf(2)]) and 1-octadecyl-3-methylimidazolium triflimide ([C(18)MIM][NTf(2)]), produced homogeneous systems at the start of the reaction and, at the end of the same, formed a three-phase system, allowing the selective extraction of the products using straightforward separation techniques, and the recycling of both the ionic liquid and the enzyme. These are very important advantages which may be found useful in environmentally friendly production conditions.

Regulation of bacterial physiology by lysine acetylation of proteins.

Post-translational modification of proteins is a reversible mechanism of cellular adaptation to changing environmental conditions. In eukaryotes, the physiological relevance of N-ɛ-lysine protein acetylation is well demonstrated. In recent times, important roles in the regulation of metabolic processes in bacteria are being uncovered, adding complexity to cellular regulatory networks. The aim of this mini-review is to sum up the current state-of-the-art in the regulation of bacterial physiology by protein acetylation. Current knowledge on the molecular biology aspects of known bacterial protein acetyltransferases and deacetylases will be summarized. Protein acetylation in Escherichia coli, Salmonella enterica, Bacillus subtilis, Rhodopseudomonas palustris and Mycobacterium tuberculosis, will be explained in the light of their physiological relevance. Progress in the elucidation of bacterial acetylomes and the emerging understanding of chemical acylation mechanisms will be discussed together with their regulatory and evolutionary implications. Fundamental molecular studies detailing this recently discovered regulatory mechanism pave the way for their prospective application for the construction of synthetic regulation networks.

Supported Ionic Liquid-Like Phases (SILLPs) for enzymatic processes: Continuous KR and DKR in SILLP–scCO2 systems

The immobilisation of Candida antarctica lipase B (CALB) onto SILLPs has been studied in order to obtain the maximum activity, selectivity and stability of the resulting supported biocatalyst for the KR of rac-1-phenylethanol by flow processes using scCO2. This involves the use of hydrophilic SILLPs (chloride anion) with high loadings of IL moieties and CALB. The combination of the immobilized biocatalyst with an acidic zeolite has allowed us to carry out DKR processes. Excellent results in terms of yield (92%) and enantioselectivity (>99.9% ee) are obtained by the use of a “one-pot” reactor containing a mixture of both catalyst particles under scCO2 flow conditions (50 °C, 10 MPa), resulting in an environmentally friendly process with high productivity and enantioselectivity.

Long term continuous chemoenzymatic dynamic kinetic resolution of rac-1-phenylethanol using ionic liquids and supercritical carbon dioxide

The long term continuous dynamic kinetic resolution (DKR) of rac-phenylethanol in IL/scCO2 biphasic systems was carried out by simultaneously using immobilized lipase (Novozym 435) and acidic zeolite catalysts at 50 °C and 100 bar, providing good yields (up to 98.0%) for R-phenylethyl propionate with excellent enantioselectivity (up to 97.3% ee) and without any activity loss during 14 days of operation.