Mannitol-2-dehydrogenase (EC 1.1.1.67) of Leuconostoc pseudomesenteroides ATCC 12291 catalyzing the NADH-dependent reduction of d-fructose to d-mannitol was purified to homogeneity. Native mannitol-2-dehydrogenase has a molecular mass of 155 kDa as determined by gel filtration chromatography. In SDS-PAGE, a single band appeared corresponding to a molecular mass of 43 kDa which indicated that the enzyme was composed of four identical subunits. Enzyme activity was completely inhibited by EDTA and could be restored by zinc ions, but not by Mn(2+) or Mg(2+) which demonstrated that zinc is a cofactor. Purified mannitol-2-dehydrogenase exhibited a maximal specific activity of 400 micromol fructose reduced min(-1) x (mg protein)(-1), using NADH as electron donor. The enzyme showed a high substrate specificity for d-fructose and d-mannitol, however it accepted NADPH as a cofactor with 32% activity ( V(max)) relative to NADPH (100%). The mdh gene, encoding mannitol-2-dehydrogenase, was identified by hybridization with a degenerate gene probe complementary to the nucleotide sequence encoding the first eight N-terminal amino acids of the enzyme. The mdh gene was cloned on a 4.2-kb DNA fragment, subcloned, and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 1017 bp, encoding a protein of 338 amino acids with a predicted molecular mass of 36.0 kDa. Plasmid-encoded mdh was functionally expressed, with 70 U/mg of cell-free protein in E. coli. Multiple sequence alignments showed that mannitol-2-dehydrogenase was affiliated with members of the Zn(2+)-containing medium-chain alcohol/polyol dehydrogenase/reductase protein family (MDR).
Isothermal compressibilities have been determined from density
measurements of the liquid mixtures of
1,2-dichloroethane, of trans-1,2-dichloroethene, and of
cis-1,2-dichloroethene + heptane, + decane,
+
dodecane, + tetradecane, + hexadecane, and +
2,2,4-trimethylpentane at pressures from (0.1 to 10) MPa
and 293.15 K. The compressibilities were calculated from a fit of
the molar volumes to a modified Tait
equation. The data could also be correlated surprisingly well by a
new linear p−V−T
relationship
suggested in the literature recently. This correlation makes it
possible to judge the quality of the
measurements.
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