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).

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.

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.

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.