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

We cloned and successfully expressed the gene for xylitol dehydrogenase fro m Galactocandida mastotermitis in Escherichia coli. The amino acid sequence revealed that the enzyme belongs to the superfamily of zinc containing, me dium-chain alcohol dehydrogenases. The enzyme catalyses the second step in the xylose utilising pathway converting xylose to xylulose-phosphate. Xylul ose-phosphate is further degraded by the transaldolase and transketolase re actions of the pentose phosphate pathway. The purified xylitol dehydrogenase from G. mastotermitis was subjected to p artial amino acid sequence analysis. The resulting amino acid information w as 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 i dentified with the help of Saccharomyces cerevisiae regulatory sequence dat abases. An intronless xdh transcript, cloned by RT-PCR, was actively expres sed in pBTac1 at 37 degrees C to approximately 8% of the soluble E. coli pr otein. Furthermore, the kinetic parameters were determined and conditions w ere found to stabilise the soluble and active protein.