Beate Häcker, Andreas Habenicht, Michael Kiess, Ralf Mattes
Xylose Utilisation: Cloning and Characterisation of the
Xylose Reductase from Candida tenuis
Xylose reductases catalyse the initial reaction in the
xylose utilisation pathway, the NAD(P)H+H+ dependent
reduction of xylose to xylitol. In this work, the xylose
reductase gene from Candida tenuis CBS 4435
was cloned and successfully expressed in E. coli.
From the purified and partially sequenced protein
primers were deduced for PCR. The fragment obtained
was used for Southern blot analysis and
screening of a subgenomic library. The clone containing
the open reading frame was sequenced; the gene
consisted of 969 nucleotides coding for a 322 amino
acids protein with a molecular mass of 36 kDa. Putative
regulatory signals were identified with the help
of a Saccharomyces cerevisiae regulatory sequence
database. In order to express the xylose reductase in
E. coli, the gene was placed under positive and negative
control. At low temperatures, the xylose reductase
was expressed in soluble and active form up to about
10% of the soluble protein; with rising temperatures
formation of visible inclusion bodies occurred. In refolding
experiments we were able to recover the major
portion of xylose reductase activity from the pellet
fraction.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 380, 12/1999
Pages: 1395 - 1403
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