Transient accumulation of gamma-butyrolactone during degradation of bis(4-chloro-n-butyl) ether by diethylether-grown Rhodococcus sp. strain DTB.

Rhodococcus sp. strain DTB (DSM 44534) grows aerobically on diethylether as sole source of carbon and energy. Dense cell suspension experiments showed that the induced ether-cleaving enzyme system attacks a broad range of ethers like tetrahydrofuran, phenetole and chlorinated alkylethers including Calpha-substituted alkylethers. Identification of metabolites revealed that degradation of the ethers started by an initial attack of the ether bond. Diethylether-grown cells degraded bis(4-chloro-n-butyl) ether via an initial ether scission followed by the transient accumulation of gamma-butyrolactone as intermediate at nearly stoichiometric concentrations.

Purification and characterization of two enone reductases from Saccharomyces cerevisiae.

Two enone reductases catalyzing irreversibly the enantioselective reduction of alpha,beta-unsaturated carbonyls have been purified 165-fold and 257-fold, respectively, from the cytosolic fraction of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, blue Sepharose CL-6B chromatography and Superdex 200 preparation-grade filtration. One enzyme (E I) was NADPH-dependent, showed a molecular mass of 75 kDa and decomposed under denaturing electrophoretic conditions into two subunits of 34 kDa and 37 kDa. The other enzyme (E II) was NADH linked and the molecular mass estimated by means of Superdex 200 preparation-grade filtration, was 130 kDa. The enzyme decomposed into subunits of 56 kDa and 64 kDa on SDS/PAGE. Both enzymes were most active at pH 4.8 and accepted 1-octen-3-one, 1-hexen-3-one, 3-alken-2-ones, 4-alken-3-ones, 2-cyclohexen-1-ones, 2-alkenals, 2,4-alkadienals, 2-phenyl-2-alkenals, and 2-alkyl-2-alkenals as substrates. Both enzymes showed their highest activities with 1-hexen-3-one and 1-octen-3-one, respectively. The Km values for 1-octen-3-one were estimated as 0.54 mM (E I) and 0.20 mM (E II), respectively. Both enzymes catalyzed the enantioselective reduction of cis- and trans-2-phenyl-2-butenal into (R)-2-phenylbutanal.

Stereospecific metabolism of isomeric epoxyoctadecanoic acids in the lactone-producing yeast Sporidiobolus salmonicolor.

The metabolic course of four isomeric epoxyfatty acids derived from oleic-, elaidic-, (Z)-, and (E)-vaccenic acids in the lactone-producing yeast, Sporidiobolus salmonicolor, was studied by using the deuterium-labeled precursors. Dihydroxy-, hydroxyoxo-, and hydroxy fatty acids as well as gamma-lactones were identified as metabolic intermediates. Quantitative analysis of the label content and estimation of the enantiomeric composition of the lactones established that, in the first step, the racemic epoxyfatty acids were enantiospecifically hydrolyzed by an epoxide hydrolase. During the subsequent metabolism, the stereochemical orientation of the hydroxy groups of the dihydroxyfatty acids were modified by an oxidation/reduction step.

New Melanoidin-like Maillard Polymers from 2-Deoxypentoses.

In the 2-deoxy-D-ribose/methyl 4-aminobutyrate Maillard system a trapped N-substituted 2-(hydroxymethyl)pyrrole is one of the major products. However, nontrapped representatives of this type of compound were hitherto not found in other Maillard model systems, indicating their extraordinary reactivity. Model experiments with 2-deoxy-D-ribose/methylamine enabled the detection of N-methyl-2-(hydroxymethyl)pyrrole (1) and some derived linear oligomers (2, 3) as minor components. Consequently, 1 was synthesized and its oligomerization studied under very mild acidic conditions. The deformylated dimeric bis(N-methyl-2-pyrrolyl)methane (2) and trimeric N-methyl-2,5-bis(N-methyl-2-pyrrolylmethyl)pyrrole (3) were characterized by GC/MS and NMR. Higher regular oligomers up to 6 N-methyl-2-pyrrolylmethyl units as well as corresponding dehydro-oligomers up to 12 units were identified by MALDI-TOF-MS. A complementary experiment starting with N-methyl-2-hydroxy[(13)C]methylpyrrole ([(13)CH(2)OH]-1) confirmed the structure and the oligomerization pathway. The possible significance of this type of model oligomer for the melanoidin formation in Maillard reactions is discussed. The antioxidative activity of the isolated dimer and trimer was tested in Fe(III)-thiocyanate and DPPH assays.

Light from Maillard reaction: photon counting, emission spectrum, photography and visual perception.

Several authors have reported on high-sensitivity measurement of oxygen-dependent low-level chemiluminescence (CL) from Maillard reactions (MR), i.e. nonenzymatic amino-carbonyl reactions between reducing sugars and amino acids (also referred to as nonenzymatic browning). Here we report for the first time, that light from Maillard reactions can be seen by the human eye and also can be photographed. In parallel with visual perception and photography CL was monitored by means of a CL-detection programme of a liquid scintillation counter (LSC, single photon rate counting). CL emission spectrum was recorded by a monochromator-microchannel plate photomultiplier arrangement. CL intensity from reaction of 6-aminocaproic acid with D-ribose (200 mg each) in 5 mL H2O at pH 11 at 95 degrees C was high enough for visual perception after adaptation to absolute darkness. Reaction in dimethylsulphoxide (DMSO) exhibited strongly enhanced CL (10 mg each in 5 mL were sufficient for visual detection) and could be photographed (15 minutes' exposure, ASA 6400); all characteristics of Maillard specific CL (O2-dependence, no CL from nonreducing sugars, inhibition by sulphur compounds) remained. Visual detection of CL and measurement by LSC were in full concordance. The CL emission spectrum showed two broad peaks at around 500 nm and 695 nm. Fluorescence emission of the brown reaction mixture matched the blue-green part of the CL emission spectrum. Emission of visible light during Maillard reactions may partly originate from oxygen-dependent generation of excited states and energy transfer to simultaneously formed fluorescent products of the browning reaction.

Purification and properties of two oxidoreductases catalyzing the enantioselective reduction of diacetyl and other diketones from baker's yeast.

The NADPH-linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enantioselective reduction of diacetyl (2,3-butanedione) to (S)- and (R)-acetoin (3-hydroxy-2-butanone) [so-called (S)- and (R)-diacetyl reductases] (EC 1.1.1.5) which have been isolated to apparent electrophoretical purity. The clean-up procedures comprising streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B column chromatography, affinity chromatography on Matrex Gel Red A and Superose 6 prep grade filtration led to 120-fold and 368-fold purifications, respectively. The relative molecular mass of the (R)-diacetyl reductase, estimated by means of HPLC filtration on Zorbax GF 250 and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was 36,000. The (R)-enzyme was most active at pH 6.4 and accepted in addition to diacetyl C5-, C6-2,3-diketones, 1,2-cyclohexanedione, 2-oxo aldehydes and short-chain 2- and 3-oxo esters as substrates. The enzyme was characterized by high enantioselectivity and regiospecificity. The Km values for diacetyl and 2,3-pentanedione were determined as 2.0 mM. The Mr of the (S)-diacetyl reductase was determined as 75,000 by means of HPLC filtration of Zorbax GF 250. The enzyme decomposed into subunits of Mr 48,000 and 24,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The optimum pH was 6.9. The purified (S)-enzyme reduced stereospecifically a broad spectrum of substrates, comprising 2,3-, 2,4- and 2,5-diketones, 2-oxo aldehydes, 1,2-cyclohexanedione and methyl ketones as well as 3-, 4- and 5-oxo esters. The 2,3- and 2,4-diketones are transformed to the corresponding (S)-2-hydroxy ketones; 2,5-hexanedione, however, was reduced to (S,S)-2,5-hexanediol. The Km values for diacetyl and 2,3-pentanedione were estimated as 2.3 and 1.5 mM, respectively. Further characterization of the (S)-diacetyl reductase revealed that it is identical with the so-called '(S)-enzyme', involved in the enantioselective reduction of 3-, 4- and 5-oxo esters in baker's yeast.

Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae.

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diastereomeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C5-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (Mr) of the enzyme was estimated as 140,000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of Mr 35,000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast.

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.

[Phenols in roasted coffees of different varieties. I. (author's transl)]. / Flüchtige Phenole in Röstkaffees verschiedener Sorten. I.

Thirty phenols were isolated from roasted coffee by distillation-extraction, separated from other constituents by absorption-chromatography, and characterized by means of GC-MS. Robusta coffee contained the largest amount of phenols, followed by Arabusta (Côte d'Ivoire) and Arabica. Quantity and type of phenols depends on variety as well as on the roasting conditions.

[Diphenols and caramel compounds in roasted coffees of different varieties. II. (author's transl)]. / Diphenole und Caramelkomponenten in Röstkaffees verschiedener Sorten. II.

Eleven diphenols and seven caramel compounds were extracted from roasted coffee with pentane/ether (1 + 1), silylated with BSTFA, characterized, and semiquantified by GC-MS. Seven phenols and five caramel components were identified for the first time in roasted coffee. Robusta coffee contained the highest amounts of phenols and maltol. Arabica showed higher amounts of furaneol (2.5-dimethyl-4-hydroxy-3(2H)-furanone), isomaltol, 5-hydroxymaltol, and 5-hydroxy-5.6-dihydromaltol.

[Determination of aroma substances in Spalter hops by gaschromatography-mass spectrometry (author's transl)]. / Gaschromatographisch-massenspektrometrische Untersuchung der Aromastoffe von Spalter Hopfen.

The volatiles of Spalter hops were concentrated by destillation-extraction, separated by liquid solid chromatography and characterized by capillary gas chromatography-mass spectrometry. 125 constituents (terpenes, sesquiterpenes, esters, alcohols, acids) were identified and semiquantified.

[Volatile phenolic components in beer, smoked beer, and sherry (author's transl)]. / Flüchtige phenolische Verbindungen in Bier, Rauchbier und Sherry

Volatile phenolic compounds of beer, wort, smoked beer, and sherry were enriched, separated, identified, and quantified by means of the methods of column chromatography, gas chromatography, and mass spectrometry. The analyzed foods are significantly varying in the composition and quantity of the phenolic components. Four origins of phenolics in food are shown by these results.

[Gas chromatographic-mass spectrometric investigations on the formation of phenolic and aromatic hydrocarbons in food (author's transl)].

Phenolic compounds and aromatic hydrocarbons are components in many foods and often typical flavoring substances for example of roasted and smoked products. They are also of toxicological importance. By means of model reactions we have investigated their formation by thermal fragmentation of cinnamic, p-coumaric, ferulic and sinapic acids. The products of these reactions were determined by gas chromatography and identified by mass spectrometry or infrared spectroscopy. Product ratio and mass spectra are given. Barley was roasted in a similar manner, and the components formed were identified. Column chromatography and preparative gas chromatography were used for preliminary separation.