Andreas Habenicht, Hassan Motejadded, Michael Kiess, Angelika Wegerer, Ralf Mattes
Xylose Utilisation: Cloning and Characterisation of the
Xylitol Dehydrogenase from Galactocandida mastotermitis
We cloned and successfully expressed the gene for
xylitol dehydrogenase from Galactocandida mastotermitis
in Escherichia coli. The amino acid sequence
revealed that the enzyme belongs to the superfamily of
zinc containing, medium-chain alcohol dehydrogenases.
The enzyme catalyses the second step in the
xylose utilising pathway converting xylose to xylulosephosphate.
Xylulose-phosphate is further degraded
by the transaldolase and transketolase reactions of
the pentose phosphate pathway.
The purified xylitol dehydrogenase from G. mastotermitis
was subjected to partial amino acid sequence
analysis. The resulting amino acid information was
then used to construct oligonucleotide probes for
PCR amplification. The PCR product was used to
screen a genomic library. The identified xdh gene includes
one short intron at its 5? end. Putative regulatory
signals were identified with the help of Saccharomyces
cerevisiae regulatory sequence databases.
An intronless xdh transcript, cloned by RT-PCR, was
actively expressed in pBTac1 at 37°C to approximately
8% of the soluble E. coli protein. Furthermore, the
kinetic parameters were determined and conditions
were found to stabilise the soluble and active protein.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 380, 12/1999
Pages: 1405 - 1411
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