Characterization of d-galacturonate reductase purified from the psychrophilic yeast species Cryptococcus diffluens

d-Galacturonic acid reductase was purified from a psychrophilic yeast strain of Cryptococcus diffluens, which was isolated from Satho, a traditional alcohol drink in Thailand. This enzyme, named Cd-GalUAR, assimilates d-galacturonic acid and requires NADPH as a cofactor. Cd-GalUAR is about 45 kDa and stable from pH 6.5 to 7.5 and up to 35°C. Its optimum pH and temperature are pH 7.0 and 40°C, respectively. However, 80% of its maximum activity remained at 4°C. The reaction of Cd-GalUAR from d-galacturonic acid produces L-galactonic acid, which was identified by 13C NMR and LC–MS. Three amino acid sequences were determined from trypsin-digested peptides of Cd-GalUAR. Similar sequences are found in many NAD or NADP oxidoreductases, including some d-galacturonate reductases. Our results suggest that Cd-GalUAR is the first d-galacturonate reductase identified in yeast.