MANNITOL DEHYDROGENASE FROM RHODOBACTER-SPHAEROIDES-SI4 - SUBCLONING,OVEREXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

By polymerase chain reaction mutagenesis techniques, an NdeI restricti on site was introduced at the initiation codon of the mannitol dehydro genase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4. The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overe xpression vector pET24a+ to give plasmid pASFG1. Plasmid pASFG1 was in troduced into Escherichia coli BL21(DE3), which was grown in a 1.5-1 b ioreactor at 37 degrees C and pH 7.0. Overexpression of MDH in Escheri chia 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 y ield was low. Decreasing the growth temperature to 27 degrees C and om itting pH regulation resulted in a significant increase in the formati on of soluble and enzymatically active MDH up to a specific activity o f 12.4 U/mg protein and a yield of 26000 U/l, which corresponds to 0.3 8 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 characterizati on of the recombinant MDH revealed no significant differences to the w ild-type enzyme.