The oxidative
D-xylose pathway, i.e. Dahms pathway, can be utilised to produce from cheap biomass raw material useful chemical intermediates. In vitro metabolic pathways offer a fast way to study the rate-limiting steps and find the most suitable
enzymes for each reaction. We have constructed here in vitro multi-
enzyme cascades leading from
D-xylose or D-xylonolactone to
ethylene glycol,
glycolic acid and
lactic acid, and use simple spectrophotometric assays for the read-out of the efficiency of these pathways. Based on our earlier results, we focussed particularly on the less studied xylonolactone ring opening (hydrolysis) reaction. The bacterial Caulobacter crescentus lactonase (Cc XylC), was shown to be a
metal-dependent
enzyme clearly improving the formation of
D-xylonic acid at pH range from 6 to 8. The following
dehydration reaction by the ILVD/EDD family
D-xylonate dehydratase is a rate-limiting step in the pathway, and an effort was made to screen for novel
enolase family D-xylonate
dehydratases, however, no suitable replacing
enzymes were found for this reaction. Concerning the oxidation of
glycolaldehyde to
glycolic acid, several
enzyme candidates were also tested. Both Escherichia coli
aldehyde dehydrogenase (Ec AldA) and Azospirillum brasilense α-ketoglutarate semialdehyde
dehydrogenase (Ab AraE) proved to be suitable
enzymes for this reaction.