title: Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme.
abstract: By polymerase chain reaction mutagenesis techniques, an NdeI restriction site was introduced at the initiation codon of the mannitol dehydrogenase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4. The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overexpression vector pET24a+ to give plasmid pASFG1. Plasmid pASFG1 was introduced into Escherichia coli BL21(DE3), which was grown in a 1.5-1 bioreactor at 37 degrees C and pH 7.0. Overexpression of MDH in Escherichia coli BL21(DE3) [pASFG1] was determined by enzymatic analysis and sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Under standard growth conditions, E. coli produced considerable amounts of a polypeptide that correlated with MDH in SDS gels, but the activity yield was low. Decreasing the growth temperature to 27 degrees C and omitting pH regulation resulted in a significant increase in the formation of soluble and enzymatically active MDH up to a specific activity of 12.4 U/mg protein and a yield of 26,000 U/l, which corresponds to 0.38 g/l MDH. This was an 87-fold overexpression of MDH compared to that of the natural host R. sphaeroides Si4, and a 236-fold improvement of the volumetric yield. MDH was purified from E. coli BL21(DE3) [pASFG1] with 67% recovery, using ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration. Partial characterization of the recombinant MDH revealed no significant differences to the wild-type enzyme.
authors.affiliation: Lehrstuhl für Angewandte Mikrobiologie, Universität des Saarlandes, Saarbrücken, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Schäfer
authors.forename: M A
authors.initials: MA
authors.lastname: Stein
authors.forename: K H
authors.initials: KH
authors.lastname: Schneider
authors.forename: F
authors.initials: F
authors.lastname: Giffhorn
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008354:Mannitol Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D008354:Mannitol Dehydrogenases