Breeding Of Dihydroxyacetone-Producing Strains And Studies On The Enzymatic Characterization Of Glycerol Dehydrogenase

Dihydroxyacetone is a kind of important chemical materials, which has been widely applied in chemistry, pharmacy, food and so on. More than 20 strains capable of producing dihydroxyacetone from glycerol were isolated from 4 different natural environment samples by using two detection methods. The strain 6-8 which could grow on medium containing glycerol as sole carbon source had a higher converting capability. Under a better culture, the highest DHA production of the strain 6-8 reached 6.2 g/L. In addition to general morphological and biochemical characteristics, the strain 6-8 was identified by 16S rDNA sequence and systematic analysis. The results showed that 16S rDNA sequence of the strain 6-8 had similarity of 99.7% with Acinetobacter sp. B8-22 suggesting that the strain 6-8 is one of subspecies of Acinetobacter sp. The experiment was used to optimiaze parameters of the fermentation condition of Acinetobacter sp. 6-8. The maximal yield of DHA (7.21 g/L) could be obtained when the concentrations of glycerol, mannitol, yeast extract, and corn steep liquor were set at 10.0%, 2.0%, 0.5%, and 0.2%. In this condition the production of DHA was enhanced by 9.8% than that of the prior to optimization.A method to determine dihydroxyacetone (DHA) in fermentation broth was developed by high performance liquid chromatography (HPLC). DHA was separated on an Alltima C18( 5μm,250×4.6 mm ). The mobile phase was 0.5% methanol solution (pH adjusted to 3.0 with H3PO4), the flow-rate was 1.0 mL/min and the detective wavelength was 203 nm. The detection limits of DHA was 0.1 g/L～10.0 g/L. 6.2 g/L DHA in the fermentation broth was detected by HPLC method, which was in agreement with the result by spectrophotometric method. The method in this paper was simple, rapid and applicable for DHA determination in the fermentation broth. The method was applicable for DHA determination in the fermentation process.The dhaD gene encoding glycerol dehydrogenase (GDH) from Klebsiella sp. was amplified and inserted into expression vector pET-28a(+), the plasmid pET-28a-dhaD was constructed and introduced into Escherichia coli BL21 (DE3).SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21. Then GDH was purified by Ni-NTA affinity chromatography, the result showed a single band about 39 kDa on SDS-PAGE gel, and the specified activity was about 156 U/mg, the special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%. The optimum reaction pH was 11.0, and the GDH activity have little change when incubated in the buffer of pH 7.0~11.0. The optimum reactive temperature was 30℃,and the GDH was more stable on the temperature of 25℃~45℃. The Km value was 0.54 mmol/L and Vmax was 0.49μmol/(mL·min) in the glycerol. The production of dihydroxyacetone from glycerol was 1.4 g/L by whole cell catalysed conversion of recombinant E.coli BL21.