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α,α-Diphenyl-L-prolinol, when coupled to a polymer soluble in organic solvents, gives surprising results for the addition of diethylzinc to aldehydes. For benzaldehyde, the enantiomeric excess strongly depends on the initial substrate ratio: an excess of diethylzinc yields (S)-1-phenylpropanol with up to 80% ee, while an excess of benzaldehyde leads to the (R)-1-phenylpropanol with up to 50% ee. The kinetic properties of the catalyst and the results with other aldehydes are also described. The polymer 1 is a copolymer of octadecyl methacrylate and 2-hydroxyethyl methacrylate.

( S )-2-N,N-dibutylamino-3-butyl-1-[4-(4-Phenylmethoxy)-phenyl]heptan-3-ol 3 based on ( S )-tyrosine has been synthesised. For the diethylzinc addition to both aliphatic and aromatic aldehydes, high enantiomeric excesses up to 95% were obtained.

The development of biotechnological processes using novel two-phase systems based on molten salts known as ionic liquids (ILs) got into the focus of interest. Many new approaches for the beneficial application of the interesting solvent have been published over the last years. ILs bring beneficial properties compared to organic solvents like nonflammability and nonvolatility. There are two possible ways to use the ILs: first, the hydrophobic ones as a substitute for organic solvents in pure two-phase systems with water and second, the hydrophilic ones in aqueous two-phase systems (ATPS). To effectively utilise IL-based two-phase systems or IL-based ATPS in biotechnology, extensive experimental work is required to gain the optimal system parameters to ensure selective extraction of the product of interest. This review will focus on the most actual findings dealing with the basic driving forces for the target extraction in IL-based ATPS as well as presenting some selected examples for the beneficial application of ILs as a substitute for organic solvents. Besides the research focusing on IL-based two-phase systems, the “green aspect” of ILs, due to their negligible vapour pressure, is widely discussed. We will present the newest results concerning ecotoxicity of ILs to get an overview of the state of the art concerning ILs and their utilisation in novel two-phase systems in biotechnology.

The aim of the presented work is the chemoenzymatic synthesis of GDP-mannose (GDP-Man). Mannose-1-phosphate (Man-1-P) is synthesised by a very convenient chemical method and is activated with GTP and recombinant GDP-mannose pyrophosphorylase (GDP-Man PP) to give GDP-Man. The productivity of the process is improved using reaction engineering techniques. Detailed kinetic studies, modelling of the reaction and simulations of different reaction systems revealed that besides a strong product inhibition the cofactor Mg 2+ is a key switch for effective enzymatic synthesis. Using a two-stage cascade of enzyme membrane reactors, GDP-Man could be produced continuously with a space-time yield of 28 g L −1 d −1 and an enzyme consumption of 0.9 U g −1 , which means a six-fold improvement related to batch synthesis.

With the environmental risk assessment (ERA) for genetically modified plants (GMP) the identification of equivalence or differences in the composition of ingredients of GMPs and non-GMPs has to be investigated. The strategy is based upon the idea that traditionally cultivated crops have been used safely for many years, thus every divergence within the compounds of a GMP in contrast to its non-GMP counterpart could refer to a risk and has to be further analysed.The BioOK company offers the opportunity to analyse very different aspects from the ERA of GMPs in one hand. Beside toxicological analysis and the determination of environmental effects via soil analysis, we can look into several plant metabolites to identify substantial equivalence. During the BioOK project a GC MS method for analysing approximately 60 compounds from various substance classes (e.g. sugars, sugar alcohols, amino acids, organic acids, fatty acids, sterols) in only one extraction step was developed. According to the EFSA guidelines the chemical composition of six different potato cultivars in comparison with a genetically modified variety and its non-isogenic counterpart are studied. Thus data from different non-GMPs as a base for the identification of uncommon variations in the substance composition for a GMP are obtained.

The use of biotransformations in the food, pharmaceutical, and chemical industries has increased steadily since the early 1970s. Applications range from synthesis in the milligram scale for precious oligosaccharides up to multiton scale for bulk chemicals either using whole cells or isolated enzymes. When new processes are being developed, the special properties of the biocatalyst must be considered, as well as the basic elements of chemical engineering. There is a strong interaction among the individual aspects, which may be considered for process optimization. The intention of this article is to outline the basic strategy for the development of a biocatalytic step. For some selected examples the beneficial role of biochemical engineering is demonstrated. Keywords: reactor; kinetics; chiral diol; GDP-mannose; enzyme; product inhibition; biotransformation kinetics

Using Escherichia coli K-235 as a production strain in a fed-batch fermentation process with an optimized sorbitol/yeast extract medium, we were able to produce 640 U of CMP-Neu5Ac synthetase in 10 l scale (64 U/l) and 9200 U (total enzyme) in 200 l scale (390 U/kg wet weight). By simple one-step purification procedures, enzyme preparations were obtained that could be used efficiently for the synthesis of CMP-Neu5Ac from CTP and Neu5Ac with over 90% yield, from Neu5Ac, CMP, and ATP or phosphoenolpyruvate by in situ generation of CTP, and from CTP, pyruvate, and ManNAc or GlcNAc by in situ generation of Neu5Ac.

The possibility of using the enzyme (R)-Oxynitrilase in a biphasic lyotropic liquid crystal/dibutylether system has been demonstrated. This reaction system is applicable for the continuous production of (R)-benzaldehydecyanohydrin in a fixed bed reactor. The optical purity was between 94 and 96% ee and independent of the flow rate. The space time yield was maximal (2650 g/(1*d)) at a flow rate of 1.6 ml/min.

Acetonitrile as a solvent used in liquid chromatography/mass spectrometry (LC/MS) of peptides and proteins is a relatively toxic solvent (LD50 oral; rat; 2,460 mg/kg) compared to alternatives like methanol (LD50 oral; rat; 5,628 mg/kg) and acetone (LD50 oral; rat; 5,800 mg/kg). Strategies to minimize its consumption in LC are either to reduce the inner diameter of the column or replace acetonitrile with a suitable alternative. Methanol is often recommended to replace acetonitrile in peptide analysis. In this study however, the main focus lies on another alternative solvent for LC/MS of peptides; acetone. A number of model proteins were tryptically digested and the peptide solutions were analyzed on a linear trap quadrupole (LTQ) mass spectrometer. The performances of acetonitrile, methanol and acetone were compared according to the quality of the chromatograms obtained and identification of the peptides using the BioWorks software developed by Thermo Scientific. In accordance to the elutropic series, acetone was found to significantly reduce the retention times of peptides separated by C18 column material with regard to acetonitrile while methanol led to increased retention times. Acetone was the superior solvent to methanol for most of the tested model proteins reaching similar sequence coverage and numbers of identified peptides as acetonitrile. We therefore propose acetone as an alternative to acetonitrile in LC/MS of peptides.

During the last decades, enzymes became very versatile catalysts for a variety of reactions including natural and unnatural compounds. However, many enzyme-catalysed reactions suffer from diverse restrictions because of limitations related to process parameters or the enzyme. The understanding and overcoming of those undesired side effects is therefore mandatory for the implementation of optimal process parameters. To achieve this aim, various methods from molecular biology and reaction engineering can be employed. By focusing on the hydroxynitrile lyase-catalysed synthesis of enantiopure cyanohydrins, we give an overview of strategies to improve commercially utilized enzymes and to suppress non-enzymatic reactions. Particular emphasis is placed on the necessity to combine approaches from different fields, such as enzyme engineering and reaction engineering.

The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene (cphA(Te)) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphA(Te)) as well as tuber-specific plastidic (pB33-PsbY-cphA(Te)) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphA(Te), both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphA(Te) tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY-cphA(Te) tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.

Kontinuierlich kann die Codimerisierung von Styrol und Ethen mit dendritischen Pd-Komplexen wie 1 in einem Nanofiltrations-Membranreaktor durchgeführt werden. Die Selektivität der Reaktion wird unter kontinuierlichen Bedingungen deutlich erhöht. Monomere Modellkomplexe und dendritische Katalysatoren werden im Satzbetrieb hinsichtlich Aktivität und Selektivität verglichen.

Hydroxynitrile lyases (HNLs) are applied in technical processes for the synthesis of chiral cyanohydrins. Here we describe the thorough characterization of the recently discovered R-hydroxynitrile lyase from Arabidopsis thaliana and its S-selective counterpart from Manihot esculenta (MeHNL) concerning their properties relevant for technical applications. The results are compared to available data of the structurally related S-HNL from Hevea brasiliensis (HbHNL), which is frequently applied in technical processes. Whereas substrate ranges are highly similar for all three enzymes, the stability of MeHNL with respect to higher temperature and low pH-values is superior to the other HNLs with alpha/beta-hydrolase fold. This enhanced stability is supposed to be due to the ability of MeHNL to form tetramers in solution, while HbHNL and AtHNL are dimers. The different inactivation pathways, deduced by means of circular dichroism, tryptophan fluorescence and static light scattering further support these results. Our data suggest different possibilities to stabilize MeHNL and AtHNL for technical applications: whereas the application of crude cell extracts is appropriate for MeHNL, AtHNL is stabilized by addition of polyols. In addition, the molecular reason for the inhibition of MeHNL and HbHNL by acetate could be elucidated, whereas no such inhibition was observed with AtHNL.

The sections in this article are Introduction A Short History of Biotransformations Need for Immobilization Characterization of Immobilized Biocatalysts Reactors for Immobilized Biocatalysts Application of Immobilized Biocatalysts Industrial Application of Immobilized Biocatalysts Production of 7-Aminocephalosporanic Acid and 6-Aminopenicillanic Acid Production of l-Amino Acids Using Aminoacylase Several Selected Processes that Use Lipase in Manufacturing Fine Chemicals Production of Acrylamide by Means of Nitrile Hydratase Production of High-Fructose Corn Syrup Using Xylose Isomerase Production of Lactose-Free Milk Using β-Galactosidase Production of l-Malic Acid Using Fumarase Production of Glycerol-3-Phosphate Using Glycerol Kinase Miscellaneous Industrial Processes Using Immobilized Enzymes Analytical and Medical Applications Waste Treatment Application of Whole Cells (Not Immobilized) in Industrial Biotransformation Process Production of Epoxides Using Monooxygenase Production of Malic Acid Using Fumarase Conclusion and Outlook Keywords: immobilized biocatalysts; immobilization; biocatalysts; biotransformation

The ionic liquid (IL) Ammoeng110 contains cations with oligoethyleneglycol units and was found to be highly effective for the formation of aqueous two-phase systems (ATPS) that can be used for the biocompatible purification of active enzymes. Above critical concentrations of the IL and an inorganic salt in aqueous solution, phase separation takes place resulting in the formation of an IL-enriched upper and a salt-enriched lower phase. For the optimization of the composition of IL-based ATPS with regard to the extraction of catalytically active enzymes, the Box-Wilson method of experimental design was successfully applied; IL-based ATPS proved to be suitable for the purification and stabilization of two different alcohol dehydrogenases (from Lactobacillus brevis and a thermophilic bacterium). Both enzymes were enriched in the IL-containing upper phase resulting in an increase of specific activity by a factor of 2 and 4 respectively. Furthermore, the presence of IL within the system provided the opportunity to combine the extraction process with the performance of enzyme-catalyzed reactions. The IL was found to exhibit a stability improving effect on both enzymes and a solubility enhancing effect on hydrophobic substrates. Thus the conversion and volumetric productivity of ADH catalyzed reduction of acetophenone could be increased significantly.

The application of unusual high pH-values within enzymatic cyanohydrin synthesis has been investigated. Usually enzymatic cyanohydrin synthesis in two-phase systems requires low pH-values within the aqueous phase to suppress the non-enzymatic side reaction. In contrast, we investigated the usage of pH-values above pH 6 by using the highly enantioselective (S)-selective hydroxynitrile lyase from Manihot esculenta. With these unusual reaction conditions also the unfavorable substrate 3-phenoxy-benzaldehyde can be converted by the wild type enzyme with excellent conversion and enantiomeric excess yielding pure (S)-3-phenoxy-benzaldehyde cyanohydrin with an enantiomeric excess of 97%. Although the variant MeHNL–W128A shows a higher activity with respect to this reaction, the enantioselectivity was reduced (85% e.e.(S)). Additionally, a new continuous spectroscopic cyanohydrin assay monitoring the formation of 3-phenoxy-benzaldehyde cyanohydrin was developed.

In this work, a model describing the complete enzyme catalysed synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc) is presented. It includes the combined reaction steps of epimerisation from GlcNAc to N-acetyl-D-mannosamine (ManNAc) and the aldol condensation of ManNAc with sodium pyruvate yielding Neu5Ac. The model is expedient to predict the reaction course for various initial and feed concentrations and therefore to calculate reaction times and yields. The equilibrium constants calculated from the kinetic constants via the Haldane relationship correspond with experimental values very well (0.26 calculated and 0.24 experimental value for the epimerisation, 27.4 l mol(-1) calculated and 28.7 l mol(-1) experimental for the aldol condensation). The actual relevance of the model is shown by a scale-up. Using the model, an optimisation of reaction conditions in consideration of different targets is possible. Exemplarily, it is presented how the optimal ratio of the two enzymes in the reaction can be determined and how the composition of the reaction solution in a fed-batch reactor can be designed to meet downstream processing needs.

Drug-coated balloons (DCB), which have emerged as a therapeutic alternative to drug-eluting stents in percutaneous cardiovascular intervention, are well described with regard to clinical efficacy and safety within a number of clinical studies. In vitro studies elucidating the correlation between coating additive and DCB performance are however rare but considered important for the understanding of DCB requirements and the improvement of established DCB. In this regard, we examined three different DCB-systems, which were developed in former studies based on the ionic liquid cetylpyridinium salicylate, the body-own hydrogel hyaluronic acid and the pharmaceutically well-established hydrogel polyvinylpyrrolidone, considering coating morphology, coating thickness, drug-loss, drug-transfer to the vessel wall, residual drug-concentration on the balloon surface and entire drug-load during simulated use in an in vitro vessel model. Moreover, we investigated particle release of the different DCB during simulated use and determined the influence of the three coatings on the mechanical behavior of the balloon catheter. We could show that coating characteristics can be indeed correlated with the performance of DCB. For instance, paclitaxel incorporation in the matrix can reduce the drug wash-off and benefit a high drug transfer. Additionally, a thin coating with a smooth surface and high but delayed solubility can reduce drug wash-off and decrease particle burden. As a result, we suggest that it is very important to characterize DCB in terms of mentioned properties in vitro in addition to their clinical efficacy in order to better understand their function and provide more data for the clinicians to improve the tool of DCB in coronary angioplasty.

Background: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. Objectives: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. Methods: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. Results: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. Conclusion: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

The three component coupling reaction of aldehydes, phenylacetamide and dienophiles gave the corresponding N -phenylacetamidocyclohexene derivatives in good yields (55–70%) and high regioselectivity. For the first time, enzymatic kinetic resolution of this class of compounds has been achieved. The enantioselective hydrolysis of 4- N -phenylacetylamino- cis -3a,4,7,7a-hexahydro iso indole-1,3-dione derivatives using Penicillin G amidase (PGA) from Escherichia coli gave the remaining enantiomer with ee-values from 30 to 70% at 50% conversion. On the basis of molecular modeling predictions, 1- N -phenylacetylamino-2-cyano-5-cyclohexene derivatives were synthesized. The kinetic resolution resulted in ees up to 99%. The differences of the observed selectivities confirmed the in silico predictions based on the simulation of enzyme–substrate interactions.

Ionic liquids offer new possibilities for the application of solvent engineering to biocatalytic reactions. Although in many cases ionic liquids have simply been used to replace organic solvents, they have often led to improved process performance. Unlike conventional organic solvents, ionic liquids possess no vapor pressure, are able to dissolve many compounds, and can be used to form two-phase systems with many solvents. To date, reactions involving lipases have benefited most from the use of ionic liquids, but the use of ionic liquids with other enzymes and in whole-cell processes has also been described. In some cases, remarkable results with respect to yield, (enantio)selectivity or enzyme stability were observed.

Introduction Reactors for Immobilized Enzymes Characterization of Immobilized Enzymes Selected Examples of Reactions Using Immobilized Enzymes Production of l-Amino Acids Using Amino Acylase Production of 6-Amino Penicillanic Acid and 7-Amino Cephalosporanic Acid Using Amidases Production of High-Fructose Corn Syrup Using Glucose Isomerase Production of Acrylamide Using Nitrile Hydratase Production of Glycerol-3-Phosphate Using Glycerol Kinase Production of (2R,3S)-Methyl-p-Methoxyphenylglycidate Using a Lipase Miscellaneous Processes Using Immobilized Enzymes Nomenclature Bibliography Keywords: amino acylase; enzyme, immobilized; glucose-fructose isomerase; glycerol kinase; lipase; mass transport limitation; membrane; nitrile hydratase; PenG acylase; reactor

Introduction Whole Cells or Isolated Enzymes? Classification of Different Reactors Mass Balances Kinetics Process Development, Step by Step Examples Product Inhibition Overcoming Product Inhibition by Product Extraction Overcoming Product Inhibition by a CSTR Cascade Bibliography Keywords: chiral diol; enzyme; GDP-mannose; kinetics; product inhibition; reactor

Substrate oxidation of aromatic substances by the enzyme laccase followed by a heteromolecular coupling with a co-substrate is a promising possibility for the synthesis of new compounds. To find a suitable reactor for the effective production of new compounds, the laccase-catalysed coupling of 3-(3,4-dihydroxyphenyl)propionic acid with 4-aminobenzoic acid was investigated as a model system. Based on the kinetic parameters, a mathematical model was used to predict the reaction yield and oxygen demand in a discontinuously stirred tank reactor and a continuously operated stirred tank reactor. Membrane processes were used for bubble-free aeration of the system and to recover the soluble enzyme.

The determination of vaporization enthalpies of extremely low volatility ionic liquids is challenging and time consuming due to the low values of vapor pressure. In addition, these liquids tend to decompose even at temperatures where the vapor pressure is still low. Conventional methods for determination of vaporization enthalpies are thus limited to temperatures below the decomposition temperature. Here we present a new method for the determination of vaporization enthalpies of such liquids using differential fast scanning calorimetry. We have developed and proven this method using [EMIm][NTf2] at temperatures of up to 750 K and in different atmospheres. It was demonstrated that evaporation is still the dominating process of mass loss even at such highly elevated temperatures. In addition, since the method allows very high heating rates (up to 10(5) K s(-1)), much higher temperatures can be reached in the measurement of the mass loss rate as compared to common devices without significant decomposition of the ionic liquid. We discuss the advantages and limits of this new method of vaporization enthalpy determination and compare the results with data obtained from established methods.

This article discusses different methods for the recycling of chiral catalysts, including heterogenization of the soluble catalyst on an insoluble inorganic or organic support, membrane filtration of homogeneously soluble catalysts, precipitation, and two-phase systems. In principle, all the methods presented enable the repeated use of a chiral catalyst without loss of activity and/or enantioselectivity. Examples will be given from laboratory and industrial processes, incuding hydrogenations, ketone reductions, epoxidations, dihydroxylations, diethylzinc additions and Diels-Alder reactions catalyzed by chemocatalysts or biocatalysts. Different approaches for cyanation, hydrogenation and epoxidation are compared. Data from industrial processes include the production of metalochlor, the production of (-)-menthol and the production of L-tert-leucine.

Transmetalation of polylithiated, carbosilane (CS) dendrimers functionalized with the potentially terdentate ligand [C6H2(CH2NMe2)(2)-2,6-R-4](-) ( NCN) with NiCl2(PEt3)(2) produced a series of nickel-containing dendrimers [GO]-N-4 (4), [G1]-Ni-12 (5), and [G2]-Ni-36 (7) in moderate to good yields. The metallodendrimers 4, 5, and 7 are catalytically active in the atom-transfer radical addition (ATRA) reaction (Kharasch addition reaction), viz. the 1:1 addition of CCl4 to methyl methacrylate (MMA). The catalytic data were compared to those obtained for the respective mononuclear compound [NiCl(C6H2{CH2NMe2}(2)-2,6-SiMe3-4)] (2). This comparison indicates a fast deactivation for the dendrimer catalysts beyond generation [GO]. The deactivation of [G1]-Ni-12 (5) and [G2]-Ni-36 (7) is caused by irreversible formation of catalytically inactive Ni(III) sites on the periphery of these dendrimers. This hypothesis is supported by results of model studies as well as ESR spectroscopic investigations. Interestingly, the use of two alternative nickelated [G1] dendrimers [G1]*-Ni-12 (11) and [G1]-Ni-8 (15), respectively, in which the distance between the Ni sites is increased, leads to significantly improved catalytic efficiencies which approximate those of the parent derivative 2 and [GO]-Ni-4 (4). Preliminary membrane catalysis experiments with [GO]-Ni-4 (4) and [G1]-Ni-12 (5) show that 5 can be efficiently retained in a membrane reactor system. The X-ray crystal structure of the Ni(III) complex [NiCl2(C6H2{CH2NMe2}(2)-2,6-SiMe3-4)] (16), obtained from the reaction of 2 with CCl4, is also reported.