László Gubicza

Enzymatic synthesis of ethyl lactate was studied in organic solvents and in ionic liquids to determine optimal media for the reaction, and to investigate the effect of water content on the ester yield. Experiments proved that Cyphos 202 ionic liquid is the best solvent affording the highest ethyl lactate yields. Furthermore, 20 times less enzyme sufficed to carry out the reaction in this ionic liquid compared to organic solvents; ionic liquid could be applied as solvent. Using water removal, the ester yield decreased since a side reaction, dimerization of lactic acid, occurred as well. In contrast to these results, without water removal, the produced water was partly consumed by the decomposition of lactoyllactic acid to free lactic acid, increasing thus the substrate concentration of the mixture and enhancing the ester yield.

Microwave heating was found to have a beneficial effect on the enzymatic esterification of lactic acid (LA) with ethanol in Cyphos 202 ionic liquid (IL) medium. Microwave irradiation caused hydrolysis of lactoyllactic acid (the linear dimer of LA) during the reaction, thus, as a result, more lactic acid was available as substrate and a higher ester yield was achieved in the esterification.

The enzyme catalytic esterification reaction of acetic acid and ethanol in [bmim]PF₆ ionic liquid was studied to manufacture natural ethyl acetate by a continuous waste-free process. The optimal parameters of the reaction were determined in batch experiments, and then a continuous set-up was constructed. Ethyl acetate and water formed in the bioreactor were removed by a double pervaporation system using hydrophobic and hydrophilic membranes, respectively. Acetic acid and ethanol were added as they were consumed, thus steady state concentrations in the reactor were maintained and continuous operation for 72 hours long was ensured without any activity loss of the enzyme.

The ability of ILs (ionic liquids) to provide an environment of increased stability and, in this way, improve the recyclability of enzymes has been studied. The description of this phenomenon is not easy; there are several approaches for explanation. In this mini-review, the results from different research groups are summarized, with the aim of explaining the strong stability effect of ILs on several enzymes. Spectroscopic methods (e.g. fluorescence and CD, IR spectroscopy, mass spectroscopy and NMR) and investigations of polarity and kosmotropicity of ions are promising methods. Since higher stability means that we may be able to reuse enzymes more times, the recyclability of enzymes was also in the focus. From this point of view, the advantages and disadvantages of applying monophasic or biphasic systems are discussed too, presenting the coupled techniques as well.

In this work, the lipase-catalyzed synthesis of i-amyl oleate was performed in a reversed micellar system of a cationic surfactant, CPC. The influence of the RM-system constituents on the biocatalysis characteristics (initial esterification rate and conversion extent) was studied and discussed in terms of the RM-structure. The initial water content in the RM-system (the water to surfactant mole ratio, Wo), affected both reaction parameters in a bell-shaped manner, with the maximum depending on the alcohol amount. In general the optimal Wo-values remained between 34 ?40 for a wide range of the initial substrate ratio. The polarity (logP) and the structure of the organic solvent used as the RM-continuum strongly affected the kinetic parameters. A linear function of the initial rate on arg(logP) was established for the alkanes up to n-C1O. The highest rates were measured using a medium of cycle or branched alkanes. The esterification under inhibiting concentrations of the substrates could be intensified by a small change in the initial Wo, thus improving the enzyme RM-accommodation and manipulating the effective concentrations of both substrates around the lipase. In the studied RM-system of 0.115 mol dm-3 CPC, the maximal initial rate of the lipase-catalyzed i-amyl oleate synthesis was found to be limited at ~340 ?mol min-1 g-1.

Glucoamylase was produced extracellularly by fermentation of strain Aspergillus awamori, which had been genetically modified to have high-level glucoamylase activity. Initial experiments showed that the enzyme deactivated quickly, with a half-life of less than 6 days even stored at 5°C. A possible reason for the rapid deactivation was the presence of proteases, attacking and degrading the glucoamylase. Therefore a liquid protease inhibitor cocktail (Sigma, USA) was selected and applied to enhance the stability of the enzyme. The activity of the enzyme (stored at 5°C) measured by the Schoorl-method with starch as substrate showed that the cocktail was effective with the enzyme maintaining 95% of its initial storage activity for almost one year. The enzyme preparation has been used for starch hydrolysis in a flat-sheet membrane bioreactor at 60°C to manufacture glucose solution and its operation stability extended by using the cocktail.

Ethanol recovery from aqueous fermentation broth by extraction using oleic acid with simultaneous esterification by lipase enzyme was studied. To determine the optimal conditions for the complex process, the ternary system was characterised; binodal curves and tie lines of (ethanol+oleic acid+water) system were determined. Enzymatic esterification of ethanol and oleic acid was carried out and resulted in higher than 50% conversion with simultaneous reduction of ethanol content in the broth. Finally, the effect of the ester product (ethyl oleate) on the distribution of ethanol was determined.u

The application of ionic liquids as solvents for the enantioselective esterification of (R,S)-2-chloropropanoic acid with butan-1-ol using Candida rugosa lipase is reported. The role of water produced during the reaction and controlling of the water activity with pervaporation was studied. The enantioselective esterification of (R,S)-2-chloropropanoic acid with butan-1-ol in different organic solvents and ionic liquids was studied. The reaction was effectively catalysed by Candida rugosa lipase. From the ionic solvents investigated, the best results were achieved in [bmim]PF₆. The presence of water had a strong effect on the activity of the lipase. Since in esterification reactions with acid and alcohol equimolar amounts of water are formed, the excess of water was removed and the water activity was kept constant using a pervaporation system without any additives. At the optimum water activity Candida rugosa lipase showed high thermal stability in [bmim]PF₆ and it could be reused for at least five recycles with only a small lost of activity.

Thermostability of Candida rugosa lipase in organic solvents and ionic liquids was studied. During our experiments the aim was to determine if the conversion degree and enzyme half-life are changed, i.e. how to tolerate the enzyme the elevated temperature in various solvents. It was found that the enzyme kept its activity and enantioselectivity much better in ionic liquids than in the traditional organic solvents.