Biosynthese von 4-Hydroxy-2 5-dimethyl-3 - Bina

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Summary
cally to 50 % in pink fruits and 75 % in red fruits. The total aldolase activity also increases
after the pink ripening stage, apparently driven by the increase in cytosol aldolase activity.
Both observations imply that this enzyme functions during fruit ripening. Further evidence
was provided by a cloned cDNA, encoding the cytosolic form of aldolase from strawberry
fruit (SCA1). Expression analyses with both microarrays and RNA gel blots showed that
SCA1 mRNA level increases during strawberry ripening.
Using specific photometric assays, the strawberry protein extract was tested for additional
enzymatic activities relating to the degradation of sugars and sugar phosphates. Activity of
fructose-1,6-diphosphatase, phosphohexose isomerase, glucose-6-phosphate dehydrogenase
and 6-phosphogluconate dehydrogenase were all detected in the extract. Separation
of phosphohexose isomerase and 6-phosphogluconate dehydrogenase isoenzymes was accomplished
employing anion exchange chromatography. All enzymes except phosphohexose
isomerase were found to be ripening induced.
A strawberry fruit cDNA encoding for an alcohol dehydrogenase (ADH) was cloned and
transformed into E. coli BL21 (De 3) pLys bacteria. The isolated and purified expressed
protein exhibits enzymatic activity towards different aldehydes. Acetaldehyde (Km-value
43.5 µM) represented the substrate with the highest conversion rate. In addition, hexanal,
isovaleraldehyde, butanal and propanal were also accepted. The putative involvement of
ADH within the formation of HDMF was excluded by incubation experiments of ADH
with D-fructose-1,6-diphosphate.
An enzyme assay was developed to demonstrate for the first time the biochemical formation
of HDMF in strawberry protein extract and to facilitate the protein purification of the
enzymatic activity. Incubation experiments were performed with strawberry fruit protein
extracts after addition of D-fructose-1,6-diphosphate and the reduction equivalents NADH
and NADPH. HPLC-DAD as well as HPLC-MS analysis confirmed the formation of
HDMF. Characterization of the presumed enzymatic formation allowed the delimitation
towards a purely chemical formation of HDMF. Heat treatment of the extract prior to incubation
led to inhibition of HDMF generation. Successive dilution of the extract with buffer
resulted in a steady decrease in the amount of generated HDMF down to a constant
amount, representing the chemical formation. The temperature optimum was around 35°C,