Isolation and analysis of vitamin B12 from plant samples.

Based on increased demands of strict vegetarians, an investigation of vitamin B12 content in plant sources, was carried out. The vitamin B12 concentration was determined by RP-HPLC with UV detection, after prior matrix isolation by immunoaffinity chromatography (IAC). Vitamin B12 was extracted in the presence of sodium cyanide, to transform all forms of cobalamin into cyanocobalamin. Diode array detector was used to monitor vitamin B12, after its chromatographic separation under gradient elution with a mobile phase consisting of acetonitrile and trifluoroacetic acid 0.025% (w/v). The method demonstrated excellent linearity with a limit of detection 0.004µg/ml. The method precision was evaluated for plant samples and it was below 0.7% (n=6). Significant amounts of vitamin B12 in plants were detected in Hippophae rhamnoides (37µg/100g dry weight), in Elymus (26µg/100g dry weight) and in Inula helenium (11µg/100g dry weight).

Factors limiting the enzymatic hydrolysis of wheat gluten.

The enzymatic hydrolysis of wheat gluten for the production of seasonings using mixtures of endo- and exopeptidases results in yields typically below 40%. Possible limiting parameters, such as an increasing product inhibition, autopeptidolysis of the enzymes, and lack of cleavage sites, were studied using novel peptidases from Flammulina velutipes or the commercial Flavourzyme preparation. Seven intermittent electrodialysis steps (10 g/L gluten and 10 kaU/mL) for the in situ removal of amino acids minimized the product inhibition. During 16 h, hydrolysis progressed nearly linearly. Compared to the batch control, a 3-fold yield of amino acids released was obtained indicating that an integrated product removal alleviates the problem of product inhibition. Autopeptidolysis, as shown using sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme activity assays, was suppressed with increasing concentrations of competing gluten substrate. Peptidases of F. velutipes showed product inhibition only, whereas a combined effect of product inhibition and lack of cleavage sites was observed for Flavourzyme.

Expression, purification and activity assay of a patchoulol synthase cDNA variant fused to thioredoxin in Escherichia coli.

Probing a cDNA library extracted from Pogostemon cablin (Indian Patchouli) with gene specific primers, a variant of patchoulol synthase PTS (GenBank acc. No.: AY508730) was amplified, cloned, and sequenced. The amino acid sequence deduced from the cloned cDNA exhibited a sequence variation of 3.4% compared to the annotated sequence. The enzyme variant tended to form inclusion bodies when expressed in Escherichia coli. The coding sequence was fused to the T7-tag, His-tag and to thioredoxin. Constructs were expressed in three different E. coli expression strains, with several strain/construct combinations yielding soluble enzyme. By fusion to thioredoxin and careful codon optimization of the eukaryotic sequence, soluble expression could be improved on average by 42% in comparison to an unoptimized, His-tagged construct. The thioredoxin-fused protein was successfully purified using a one-step Co(2+)-IMAC purification procedure. Bioactivity assays using prepared farnesyl diphosphate (FDP) in milliliter-scale batch reactions, showed activity of the fused enzyme even with thioredoxin attached. The product spectrum of the enzyme was compared to patchouli oil standards by GC-MS and the main products were identified. Interestingly, the variant showed a shift in product spectrum with germacrene A being the most abundant product instead of patchouli alcohol. In silico structural modeling shows a possible chemical and structural change in the active site of the enzyme, which might be responsible for the shift in product composition.

Antioxidants in food: mere myth or magic medicine?

The powerful action of antioxidants in preventing premature lipid oxidation in food suggests that the same compounds, when consumed with the daily diet, could unfold antioxidative/anti-aging effects in the human body. Therefore, it has been hypothesized that antioxidants are helpful in preventing various diseases. More detailed chemical and physiological examination of antioxidants shows, however, that the extrapolation of in vitro data to in vivo behavior may be misleading. Indeed, such a procedure fails to take into account the mismatch between most in vitro models (e.g., cell cultures) and in vivo systems. For example, the physiological relevance of pro-oxidative and other physiological activities of antioxidants have been largely underestimated. Actually, contrary to the antioxidant hypothesis, clinical trials testing the health benefits of dietary antioxidants have reported rather mixed or negative results. Many clinical studies have not taken into account the nutrikinetic and nutridynamic nature of antioxidants. Further, oxidative stress is not only an inevitable event in a healthy human cell, but responsible for the functioning of vital metabolic processes, such as insulin signaling and erythropoietin production. In the light of recent physiological studies it appears more advisable to maintain the delicate redox balance of the cell than to interfere with the antioxidant homeostasis by a non-physiological, excessive exogenous supply of antioxidants in healthy humans.

Foaming of proteins: New prospects for enzyme purification processes.

Efficient techniques for the isolation of enzymes from a microbial production culture are required to meet the growing needs of the "White Biotechnologies" for novel catalysts. Traditional protein purification procedures typically comprise multistep operations, which inevitably come along with significant losses of enzyme activity. Foaming offers an alternative minimizing the processing steps, preserving the purification efficiency and decreasing the activity losses all at the same time. This review provides an insight into the foaming process itself and its application in separating enzymes from model systems and from complex media, such as microbial cultures. Examples demonstrate fractionated foaming and the tweezer technique.

Genetic dissection of scent metabolic profiles in diploid rose populations.

The scent of flowers is a very important trait in ornamental roses in terms of both quantity and quality. In cut roses, scented varieties are a rare exception. Although metabolic profiling has identified more than 500 scent volatiles from rose flowers so far, nothing is known about the inheritance of scent in roses. Therefore, we analysed scent volatiles and molecular markers in diploid segregating populations. We resolved the patterns of inheritance of three volatiles (nerol, neryl acetate and geranyl acetate) into single Mendelian traits, and we mapped these as single or oligogenic traits in the rose genome. Three other volatiles (geraniol, beta-citronellol and 2-phenylethanol) displayed quantitative variation in the progeny, and we mapped a total of six QTLs influencing the amounts of these volatiles onto the rose marker map. Because we included known scent related genes and newly generated ESTs for scent volatiles as markers, we were able to link scent related QTLs with putative candidate genes. Our results serve as a starting point for both more detailed analyses of complex scent biosynthetic pathways and the development of markers for marker-assisted breeding of scented rose varieties.

Enzyme immunoassay of testosterone, 17beta-estradiol, and progesterone in perspiration and urine of preadolescents and young adults: exceptional levels in men's axillary perspiration.

Enzyme immunoassays for testosterone, 17beta-estradiol, and progesterone were validated for human facial and axillary perspiration and compared to levels in urine. In study 1, these assays were applied to samples from preadolescent girls and boys and young women and men. Men's axillary perspiration contained substantially higher levels of steroids than seen in other substrates from men or in any sample from women, boys, and girls. Male axillary steroid levels were very variable across individuals, and on average they exceeded levels in facial perspiration by 90-fold for testosterone and 45-fold for estradiol. Men's urinary testosterone also exceeded urinary levels of the other subjects. In study 2, axillary perspiration, urine, and saliva were collected from young men. Substantial axillary levels of testosterone and estradiol were again observed. Correlations of the same hormone among the different substrates were generally very low, except for a small correlation between estradiol levels measured in axillary perspiration and urine in study 2. High unconjugated steroid content in men's axillary excretions could, if absorbed by women during intimacy, be implicated in pheromonal activity.

Degradation of alpha-pinene oxide and [2H7]-2,5,6-trimethyl-hept-(2E)-enoic acid by Pseudomonas fluorescens NCIMB 11761.

When submerged cultured Pseudomonas fluorescens NCIMB 11761 was fed-batch supplemented with alpha-pinene oxide, a rapid formation of 2,6-dimethyl-5-methylene-hept-(2Z)-enal (I) (isonovalal) was observed. Biotransformation and isomerisation of (I) to the (2E)-isomer (II) (novalal) were enhanced by Lewatit OC 1064, a macroporous polystyrene adsorbent. Accelerated isomerisation in the presence of an amino donor (glycine) at pH 7.3 pointed to a merely chemical mechanism. A maximum yield of 48 g of aldehydesl(-1) was achieved, but quantitative analysis of the volatile fraction showed that the molar conversion of the pinene oxide substrate reached no more than 67%. To fill this gap of the mass balance, the acidic fraction was isolated. It contained several compounds which suggested a beta-oxidation-like catabolism starting from 2,6-dimethyl-5-methylene-hept-(2E)-enoic acid (III) (novalic acid). Using [2H7]-2,5,6-dimethyl-hept-(2E)-enoic acid as a conversion substrate and gas chromatography coupled to atomic emission detection and mass spectrometry a degradation pathway via labelled 3,4-dimethylpentenoic and methylpropanoic acids was evidenced. This pathway may play a predominant role in isoprenoid degradation by soil bacteria.

Cleavage of beta,beta-carotene to flavor compounds by fungi.

More than 50 filamentous fungi and yeasts, known for de novo synthesis or biotransformation of mono-, sesqui-, tri-, or tetraterpenes, were screened for their ability to cleave beta,beta-carotene to flavor compounds. Ten strains discolored a beta,beta-carotene-containing growth agar, indicating efficient degradation of beta,beta-carotene. Dihydroactinidiolide was formed as the sole conversion product of beta,beta-carotene in submerged cultures of Ganoderma applanatum, Hypomyces odoratus, Kuehneromyces mutabilis, and Trametes suaveolens. When mycelium-free culture supernatants from five species were applied for the conversions, nearly complete degradation of beta,beta-carotene was observed after 12 h. Carotenoid-derived volatile products were detected in the media of Ischnoderma benzoinum, Marasmius scorodonius, and Trametes versicolor. beta-Ionone proved to be the main metabolite in each case, whereas beta-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed in minor quantities. Using a photometric bleaching test, the beta,beta-carotene cleaving enzyme activities of M. scorodonius were partially characterized.

A labeling study to elucidate the biosynthesis of 4-(4-hydroxyphenyl)-butan-2-one (raspberry ketone) by Nidula niveo-tomentosa.

Submerged cells of the basidiomycete Nidula niveo-tomentosa, a microbial producer of 4-(4-hydroxyphenyl)-butan-2-one, were supplemented with (13)C-labeled L-phenylalanines and with [1-(13)C]glucose. Labeled transformation products were detected by a novel method of analyzing stable isotope-labeled metabolites, gas chromatography (GC) coupled to an atomic emission detector, and by GC-mass spectrometry. A benzoate moiety was side chain elongated according to the poly-beta-keto scheme. The presence of an acetyl coenzyme A-carboxylase inhibitor shifted the spectrum of products to benzyl compounds. Hence, the fungal pathway differs from the one established for plant tissues.

Raspberry ketone from submerged cultured cells of the basidiomycete Nidula niveo-tomentosa.

The basidiomycete Nidula niveo-tomentosa produced 4-(4-hydroxyphenyl)-butan-2-one (raspberry ketone), one of the character impact components of raspberry flavor, and its corresponding alcohol. A systematic attempt was made to improve the productivity of this fungus. Variation of nutrient medium composition, precursor amount, time of supplementation, and cultivation period yielded a 50-fold increase in metabolite concentrations. Raspberry ketone and alcohol were easily isolated from the culture medium by solvent extraction. Glycosidically bound forms or accumulation of raspberry compounds in fungal cells were not detected. This microbial process offers an alternative for the production of natural raspberry flavor.

Intelligent use of NSAIDs--where do we stand?

Non-steroidal anti-inflammatory drugs (NSAIDs) are drugs commonly prescribed for a variety of medical conditions. They are potent pharmacological agents efficacious for inflammatory conditions, but have significant gastrointestinal (GI), renal and haematological toxicity that must not be taken lightly. The newer, more cyclooxygenase-(COX)-2-selective NSAIDs, have no effects on platelet function and little GI toxicity, but do have renal physiological effects. The superiority of one NSAID over another has not been clinically demonstrated in musculoskeletal conditions, nor has the efficacy of NSAIDs in non-inflammatory rheumatic conditions been shown to be better than that of simple analgesics. NSAIDs are indicated for primary therapy of inflammatory rheumatic diseases and the more selective COX-2 agents should be employed as first choice when economically feasible. NSAIDs should not be used indiscriminately for non-inflammatory osteoarthritis or musculoskeletal injuries, particularly in the elderly patient, in whom alternative, less toxic therapy should be sought.

Phenyl propenoic side chain degradation of ferulic acid by Pycnoporus cinnabarinus - elucidation of metabolic pathways using [5-2H]-ferulic acid.

The white-rot fungus Pycnoporous cinnabarinus (DMS-1184) was submerged cultured for 22 days under controlled conditions in a bioreactor. After 6, 9, and 15 days of culture the growth medium was supplemented with [5-2H]-labelled ferulic acid (I). The major phenolic compounds identified labelled were four lignans, the methyl esters of ferulic (I) and vanillic acid (VIII), (E)-coniferyl aldehyde (II), (E)-coniferyl alcohol (III), vanillic acid (VIII), vanillin (IX) and vanillyl alcohol (X). The detection of considerable amounts of labelled 4-hydroxy-3-methoxyacetophenone (VII) in the late growth phase suggested the increasing formation and decarboxylation of free 4-hydroxy-3-methoxybenzoylacetic acid (VI) and, thus, a beta-oxidation-like degradation of ferulic acid (I) or its methyl ester to vanillic acid (VIII). 4-Hydroxy-3-methoxybenzoylacetic acid methyl ester (VI) and 3-hydroxy-(4-hydroxy-3-methoxyphenyl)-propanoic acid methyl ester (V) were synthesised and then identified as metabolites in the culture medium. The fungal degradation of the phenyl propenoic side chain of ferulic acid (I), a principal key step of lignin decomposition, appeared to proceed analogous to fatty acids.

A comparative study on the disintegration of filamentous fungi.

Different methods for cell disintegration were tested for their efficacy on filamentous fungi, including percussion grinding, homogenization using an Ultra-Turrax, chemical treatment and lyophylization. The release of protein from Ganoderma applanatum and Pycnoporus cinnabarinus and the activity of cytoplasmatic glucose-6-phosphate dehydrogenase in the crude extracts were monitored to determine the efficiency of each disintegration technique used. Fungal cells proved to be particularly resistant towards some disintegration methods commonly used for yeasts and bacteria. Best results were obtained using a percussion grinder, if necessary, in combination with an Ultra-Turrax pretreatment.

Purification, characterisation and cDNA sequencing of pyruvate decarboxylase from Zygosaccharomyces bisporus.

Cells of the wild-type yeast strain Zygosaccharomyces bisporus CBS 702 form alpha-hydroxy ketones from aromatic amino acid precursors during fermentation. Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the non-oxidative decarboxylation of pyruvate and other 2-oxo-acids, was purified and characterised. The active enzyme is homotetrameric (alpha4) with a molecular mass of about 244 kDa. Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent Km value 1.73 mM; Hill coefficient 2.10). A cDNA library was screened using a PCR-based procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit). Sequence alignments demonstrate high homologies (> 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.

Generation of odorous acyloins by yeast pyruvate decarboxylases and their occurrence in sherry and soy sauce.

Volatile acyloins (alpha-hydroxy ketones) were obtained by condensing either aldehydes with pyruvate or 2-keto acids with acetaldehyde in a reaction catalyzed by yeast pyruvate decarboxylases (EC 4.1.1.1). Odor qualities and threshold values of 34 acyloins were evaluated, and 23 of them possessed distinct flavor properties. Sherry and soy sauce flavors were analyzed: 2-hydroxy-3-pentanone and 3-hydroxy-2-pentanone were identified in soy sauce for the first time; these and 2-hydroxy-5-methyl-3-hexanone and 3-hydroxy-1-phenyl-2-butanone were isolated from sherry for the first time. The biocatalytic efficiencies of crude pyruvate decarboxylase preparations from Zygosaccharomyces bisporus, Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxianus were compared. Product yields comparable to those of conversions with purified pyruvate decarboxylase demonstrated the suitability of crude enzyme extracts as cost-effective biocatalysts in acyloin formation. Conversion rates of >50% showed that the potential of this type of enzyme to catalyze the formation of aliphatic acyloins has been underestimated before.

Effects of R-(+)-limonene on submerged cultures of the terpene transforming basidiomycete Pleurotus sapidus.

The biotransformation of limonene by the basidiomycete Pleurotus sapidus yielded cis/trans-carveol and carvone as the main products. The transformation period was extended from 4 days after direct addition to 12 days by gas phase addition of the substrate. After 2 days of transformation, 97% of the substrate had accumulated in the mycelium, while only 3% were present in the culture medium. Substrate toxicity led to a decrease of dry matter. Adaptation of the precultures with small amounts of substrate doubled the concentration of carveol and increased the concentration of carvone by a factor of 3-4. Total product concentrations of > 100 mg l-1 were reached.

Biotransformation of citronellol by the basidiomycete Cystoderma carcharias in an aerated-membrane bioreactor.

The basidiomycete Cystoderma carcharias transformed citronellol into 3,7-dimethyl-1,6,7-octanetriol as the main product. 3,7-Dimethyl-6,7-epoxy-1-octanol was identified as important intermediary product of the biotransformation, and the allylic diols 2,6-dimethyl-2-octene-1,8-diol, 3,7-dimethyl-5-octene-1,7-diol and 3,7-dimethyl-7-octene-1,6-diol were found to be minor products. Microbial formation of rose oxide, a flavour-impact component, was observed for the first time. The formation of the main products was inhibited by 70% after addition of 0.1 mmol l-1 cytochrome monooxygenase inhibitors. Formation of 3,7-dimethyl-1,6,7-octanetriol was effective in a bioreactor with aeration over a coil of a hydrophobic microporous polypropene capillary membrane. Production rates of up to 150 mg l-1 day-1 were reached and led to a product concentration of 866 mg l-1 (conversion rate: 52%). The total loss of the added volatile substrate via the exhaust air was 4.5% when this aeration method was used.

Biotechnological production of flavours and fragrances.

The biotechnological generation of natural aroma compounds is rapidly expanding. Aroma chemicals, such as vanillin, benzaldehyde (bitter almond, cherry) and 4-(R)-decanolide (fruity-fatty) are marketed on a scale of several thousand tons per year. Their possible production by single-step biotransformations, bioconversions and de novo synthesis using microorganisms, plant cells or isolated enzymes is shown. The perspectives of bioprocesses for the oxifunctionalisation of lower terpenes by genetically modified organisms and economic aspects are discussed.