Pedro Lozano

Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis

Four different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for butyl butyrate synthesis catalyzed by free Candida antarctica lipase B at 2% (v/v) water content and 50 °C. Lipase had enhanced synthetic activity in all ionic liquids in comparison with two organic solvents (hexane, and 1-butanol), the enhanced activity being related to the increase in polarity of ionic liquids. The continuous operation of lipase with all the assayed ionic liquids showed over-stabilization of the enzyme. The reuse of free lipase in 1-butyl-3-methylimidazolium hexafluorophosphate in continuous operation cycles showed a half-life time 2300 times greater than that observed when the enzyme was incubated in the absence of substrate (3.2 h), and a selectivity higher than 90%.

Enzymes in neoteric solvents: From one-phase to multiphase systems

Biphasic systems based on neoteric solvents, e.g. ionic liquids (ILs), supercritical carbon dioxide (scCO2), fluorous solvents (FSs) and liquid polymers (LPs), represent interesting alternatives to organic solvents for designing continuous clean biotransformations methods in non-aqueous environments that directly provide pure products. The classical advantages of scCO2– its ability to extract, dissolve and transport chemicals – are complemented by the high catalytic efficiency of enzymes in liquid neoteric solvents (e.g. ILs, LPs or FSs). Enzyme behaviour in scCO2 and ILs, as well as the phase behaviour of ILs/scCO2, are key parameters for carrying out integral green bioprocesses in continuous operation. Experimental approaches, reactor designs and results are discussed in this Critical review.

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.

Effect of polyols on α-chymotrypsin thermostability: a mechanistic analysis of the enzyme stabilization

The influence of the synthetic substrate (N-acetyl-L-tyrosine ethyl ester) and the different polyols (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of alpha-chymotrypsin at 60 degrees C have been studied. The results obtained showed an important stabilizing effect in the presence of both additives. In order to describe the kinetics of enzyme stabilization, the experimental results were analyzed by a four-parameters deactivation model with excellent agreement. In all cases, alpha-chymotrypsin exhibited non-first-order deactivation kinetics, corresponding to a two-step unimolecular mechanism, where the main protective effect of polyols was observed in the first-step of the deactivation profile. Thus, the presence of polyols increased the level of activity stabilization (alpha 1), and decreased the first-order deactivation rate constant (k1). Additionally, the experimental results were analyzed as a function of both, the change in the standard free energy of denaturation (delta(delta Gzero)), and a protective effect, defined as the ratio of alpha-chymotrypsin half-lives (with and without polyols), showing in both cases a clear stabilizing effect of these polyhydroxylic cosolvents for the enzyme. The overall protective effect of polyols was also simultaneously related to their concentration and their water-activity depressing power.

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.