원문정보
초록
영어
UDP-galactose-4-epimerase (GalE, EC 5.1.3.2) catalyzes the 4-epimerization for free monosaccharides such as galactose, glucose, fructose, tagatose, psicose and sorbose in a low activity while it catalyzes the reaction for nucleic acid-activated sugar in a high activity. To enhance the 4-epimerization activity for free monosaccharides, random mutations were introduced into GalE by error-prone PCR and clones showing furctose 4-epimerization activity higher than 115% were selected. Two clones carrying five mutation points (D58E, N100S, P193S, I196N and T317S) were selected among 3,060 mutant clones. To investigate the effect of the mutation points on the desired characteristics, 5 mutant clones carrying single mutation were constructed and the variant proteins were characterized after purifications using Ni+ affinity chromatography. The variant D58E was found to contribute the twice as much activity enhancement (21 nmol/mg-protein) while the other variants showed less than 15% contribution. The possible reason of the activity enhancement by the Asp58 substitution into Glu58 is discussed based on the 3D-structure.