Lasse Greiner

Ionic liquids in biotechnology: applications and perspectives for biotransformations

Ionic liquids are considered as an alternative to organic solvents for catalysis. The literature in this field is reviewed with focus on advantageous use of ionic liquids in biocatalysis and biotransformations. The overview reveals that the exploration and mapping of ionic liquids with respect to biocatalysis is still sketchy. It is apparent that advantages can be gained in view of activity, stability and selectivity. Furthermore, integration of reaction and separation has a high potential in the field. The review presents quantitative data on the productivities, space–time yields, as well as stability as far as they can be extracted from the literature.

Benzoylformate Decarboxylase from Pseudomonas putida as Stable Catalyst for the Synthesis of Chiral 2-Hydroxy Ketones

The thiamin diphosphate- and Mg2+-dependent enzyme benzoylformate decarboxylase (BFD) from Pseudomonas putida was characterized with respect to its suitability to catalyze the formation of chiral 2-hydroxy ketones in a benzoin-condensation type reaction. Carboligation constitutes a side reaction of BFD, whereas the predominant physiological task of the enzyme is the non-oxidative decarboxylation of benzoylformate. For this purpose the enzyme was obtained in sufficient purity from Pseudomonas putida cells in a one-step purification using anion-exchange chromatography. To facilitate the access to pure BFD for kinetical studies, stability investigations, and synthetical applications, the coding gene was cloned into a vector allowing the expression of a hexahistidine fusion protein. The recombinant enzyme shows distinct activity maxima for the decarboxylation and the carboligation beside a pronounced stability in a broad pH and temperature range. The enzyme accepts a wide range of donor aldehyde substrates which are ligated to acetaldehyde as an acceptor in mostly high optical purities. The enantioselectivity of the carboligation was found to be a function of the reaction temperature, the substitution pattern of the donor aldehyde and, most significantly, of the concentration of the donor aldehyde substrate. Our data are consistent with a mechanistical model based on the X-ray crystallographic data of BFD. Furthermore we present a simple way to increase the enantiomeric excess of (S)-2-hydroxy-1-phenyl-propanone from 90% to 95% by skillful choice of the reaction parameters. Enzymatic synthesis with BFD are performed best in a continuously operated enzyme membrane reactor. Thus, we have established a new enzyme tool comprising a vast applicability for stereoselective synthesis.

Practical applications of hydrogenase I from Pyrococcus furiosus for NADPH generation and regeneration

The soluble hydrogenase I (H-2:NADP(+) oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P furiosus hydrogenase I preparation (PF H(2)ase I) has been used as biocatalyst in the enzymatic production and regeneration of beta-1,4-nicotinamide adenindinucleotide phosphate, reduced form (NADPH), utilizing cheap molecular hydrogen and forming protons as the only side-product. Any excess of dihydrogen can be removed easily. It could be demonstrated, that this hyperthermophilic hydrogenase exhibits a high stability under reaction conditions. Generation as well as regeneration of NADPH were performed in batch and repetitive batch experiments with recyclisation of the biocatalyst. In two repetitive batch-series 6.2 g l(-1) NADPH could be produced with a total turnover number (ttn: mol produced NADPH/mol consumed enzyme) of 10,000. Utilizing the thermophilic NADPH-dependent alcohol dehydrogenase from Thermoanaerobium spec. (ADH M) coupled to the PF H(2)ase I in situ NADPH-regenerating system, two prochiral model substrates, acetophenone and (2S)-hydroxy-1-phenyl-propanone (HPP), were quantitatively reduced to the corresponding (S)-alcohol and (1R,2S)-diol. An e.e. >99.5% and d.e. >98%, respectively, with total turnover numbers (ttn: mol product/mol consumed cofactor NADP(+)) of 100 and 160 could be reached

Gaining pH-control in water/carbon dioxide biphasic systems

Utilizing the biphasic system water/CO2 as a reaction and extraction medium is of rising importance, because of its advantages as a green solvent combination. The problem of predictable pH control in the aqueous phase has to be addressed in view of optimized reaction conditions. The control is possible up to a pH of approximately 6 by means of buffer salts, and the resulting pH can be predicted by an algebraic equation.

Substrate dependent synergetic and antagonistic interaction of ammonium halide and polyoxometalate catalysts in the synthesis of cyclic carbonates from oleochemical epoxides and CO2

Organic halides and polyoxometalates (POMs) were studied as catalyst systems for the insertion of carbon dioxide into epoxides originating from plant oils as biogenic feedstock. In the formation of methyl oleate carbonate, synergistic rate acceleration and increased cis-selectivity were observed using ammonium halide and transition metal substituted silicotungstate POMs catalysts in a combined system. Mechanistic insight into the cooperative action was gained by kinetic measurements and analysis of the stereochemical outcome of the reaction. For poly-epoxidised oleochemicals as substrates, the simple ammonium halide catalyst gave better performance as side reactions were encountered with the POM-containing system.

Screening of new solvents for artemisinin extraction process using ab initio methodology

The solubility of artemisinin in a range of conventional and novel solvents was evaluated using the COSMO-RS approach, and verified experimentally as well as against literature data. The computational method was improved by calibrating against a limited set of experimental data, enhancing the accuracy of the calculations. The optimised method was shown to be in reasonable agreement with experimental data; however, lack of reliable experimental data is identified as an issue. Several novel solvents perceived as green alternatives to conventional solvents were targeted and shown to offer good solubility of artemisinin. Extraction from Artemisia annua by carbonate solvents was experimentally verified.

Nanoparticle catalysed oxidation of sulfides to sulfones by in situ generated H2O2 in supercritical carbon dioxide/water biphasic medium

In a one-pot reaction, hydrogen peroxide generated from H(2) and O(2) on a Pd catalyst was utilised as oxidant for the TiO(2) catalyzed conversion of a sulfide to a sulfone. This transformation, where two different nanoparticle catalysts were employed in a supercritical carbon dioxide/water biphasic system, demonstrates the potential of compartmentalising catalytic processes in consecutive reactions.

New aspects for biomass processing with ionic liquids: towards the isolation of pharmaceutically active betulin

By utilising ionic liquids the pharmaceutically active triterpene betulin can be extracted from biomass with significantly improved extraction yield and purity. The recovery of the ionic liquid 1-ethyl-3-methylimidazolium acetate via azeotropic distillation of EtOH/H2O was successfully demonstrated.

Continuous catalytic Friedel–Crafts acylation in the biphasic medium of an ionic liquid and supercritical carbon dioxide

Immobilisation of catalytically-active metal salts in ionic liquids, with extraction by supercritical carbon dioxide, affords continuous Friedel-Crafts acylation, with in situ-recycling of the catalyst.

Ionic Liquids as Performance Additives for Electroenzymatic Syntheses

Electroenzymatic syntheses combine oxidoreductase-catalysed reactions with electrochemical reactant supply. The use of ionic liquids as performance additives can contribute to overcoming existing limitations of these syntheses. Here, we report on the influence of different water-miscible ionic liquids on critical parameters such as conductivity, biocatalyst activity and stability or substrate solubility for three typical electroenzymatic syntheses. In these investigations promising ionic liquids were identified and have been used as additives for batch electrolyses on preparative scale for the three electroenzymatic systems. It was possible to improve the space-time-yield for the electrochemical regeneration of NADPH by a factor of three. For an amino acid oxidase catalysed resolution of a methionine racemate with ferrocene-mediated electrochemical regeneration of the enzyme-bound cofactor FAD a 50% increase in space time yield and 140% increase in catalyst utilisation (TTN) were achieved. Furthermore, for the chloroperoxidase-catalysed synthesis of (R)-phenylmethylsulfoxide with electrochemical generation of the required cosubstrate H2O2 the space time yield and the catalyst utilisation were improved by a factor of up to 4.2 depending on the ionic liquids used.