earticle

영어

Glycerol 3-phosphate dehydrogenase (Gpd) plays a central role in glycerol metabolism. A cDNA encoding NAD+-dependent Gpd was cloned from Candida magnoliae. The cDNA clone is 1606 bp long with an open reading frame encoding a polypeptide of 360 amino acids. The polypeptide shows high homology with published NAD+-dependent eukaryotic Gpd enzymes. The gene coding for Gpd of C. magnoliae (cmGPD) was expressed in recombinant Escherichia coli and purified to homogeneity by one-step affinity chromatography. The expressed cmGpd displayed an apparent molecular weight of 38 kDa. The purified cmGpd exhibited optimal activity at 28℃ and pH 6.5 and has Km of 1.03 mM for dihydroxyacetone phosphate whereas Km for glycerol 3-phosphate is 11.4 mM. When cmGPD was heterologously expressed in a Saccharomyces cerevisiae strain deficient in glycerol synthesis, the glycerol concentration of 3.2 g/L was produced. The experimental results indicated that cmGpd is the principal enzyme involved in glycerol biosynthesis by preferentially providing glycerol 3-phosphate following a mechanism similar to that of S. cerevisiae.