Purification and Molecular Characterization of a Quinoprotein Alcohol Dehydrogenase from Pseudogluconobacter saccharoketogenes IFO 14464

概要

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We have cloned and verified a gene for a novel quinoprotein alcohol dehydrogenase(ADH)from Pseudogluconobacter saccharoketogenes IFO 14464 that has the ability to oxidize L-sorbose to 2-keto-L-gulonic acid(2-KLGA). The enzyme was purified from the soluble fraction of the bacterium and was estimated to be a monomeric protein with a molecular weight of 65kDa from the analyses of SDS-PAGE and gel-filtration chromatography. An open reading frame of 1824 bp for 608 amino acid residues was estimated as the gene for ADH because of the consistency of the calculated molecular mass and the elucidated partial amino acid sequences of the native enzyme. Homology search revealed that the enzyme showed close similarity to quinoprotein alcohol dehydrogenases isolated from Methylobacterium extorquens and Acetobacter aceti, particularly in the tryptophan docking motifs in the α-subunits of those dehydrogenases. The ability to convert L-sorbose to 2-KLGA was found when the lysate of recombinant Escherichia coli DH10B transformed with the gene for ADH was mixed with CaCl_2 and pyrroloquinoline quinone(PQQ). These data indicate that the cloned DNA is the desired gene for the ADH in which CaCl_2 and PQQ are essential for enzymatic activity.
2001-12-25