Cloning and overexpression of the xylitol dehydrogenase gene from Bacillus pallidus and its application to L-xylulose production.
The xylitol dehydrogenase gene (xdh) of Bacillus pallidus was cloned and overexpressed in Escherichia coli using pQE60 vector, for the first time. The open reading frame of 759 bp encoded a 253 amino acid protein with a calculated molecular mass of 27,333 Da. The recombinant xylitol dehydrogenase (XDH) was purified to homogeneity by three-step column chromatography, producing a single SDS-PAGE band of 28 kDa apparent molecular mass. The enzyme exhibited maximal activity at 55 °C in glycine-NaOH buffer pH 11.0, with 66% of initial enzyme activity retained after incubation at 40 °C for 1 h. In further application of the recombinant bacterium to L-xylulose production from xylitol (initial concentration 5%) using a resting cell reaction, 35% L-xylulose was produced within 24 h. This result indicates that this recombinant XDH is applicable in the large-scale production of L-xylulose.
Takata
Goro
G
Rare Sugar Research Center, Kagawa University, Kagawa, Japan. goro@ag.kagawa-u.ac.jp
Poonperm
Wayoon
W
Morimoto
Kenji
K
Izumori
Ken
K
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D050542:D-Xylulose Reductase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli; D007218:Industrial Microbiology / Q000379:methods; D007700:Kinetics; D014996:Xylulose / Q000096:biosynthesis*
D014996:Xylulose; D050542:D-Xylulose Reductase