Xylose utilisation: cloning and characterisation of the xylitol dehydrogenase from Galactocandida mastotermitis.

Abstract

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 degrees 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.