Expression And Purification Of Recombined Glycerol-3-pHospHate Dehydrogenase With Two Domains In Dunaliella Salina And Partial Enzymatic Properties

Dunaliella salina is an unicellular green algae with high resistence to salt and extremely strong ablity to adjust osmotic pressure. Glycerol is its main substance for osmotic pressure adjustment. The metabolic pathway for glycerol cycle is a specialized metabolic pathway for osmotic pressure adjustment in the Dunaliella salina. It can promptly respond to salt concentration change in the environment and adjust glycerol concentration to strike a balance for osmotic pressure between inside and outside the cell. Glycerol-3-pHospHate (G-3-P) dehydrogenase is the first enzyme in glycerol cycle, playing an important role in regulation therein, thus of significance for the properties of glycerol-3-pHospHate dehydrogenase in Dunaliella salina.The primary reseach for its properties was conducted, based on optimalizing the expression of recombined glycerol-3-pHospHate dehydrogenase and its purification. The engineering strain BL21(DE3)/pET32-GPD1.9 was induced with 0.3mmol/L IPTG at 30 ℃. The recombined glycerol-3-pHospHate dehydrogenase was in the suspension in the form of soluble protein. It was isolated and purified by the steps of metal chelate chromatograpHy of rough enzyme extract with Chelating F.F., dialysis, cleaving by enterokinase, metal chelate chromatograpHy with Chelating H.P. and so on. All results of SDS-PAGE showed one strip. The GPD (reverse) and GPP specific activities were 192 U/mg and 4440 U/mg respectively. The relative molecular weight was 60 KDa via SDS-PAGE analysis. The optimal temperature for reverse enzyme...