IN-DEPTH CHARACTERIZATION OF THE PURIFICATION OF ADENOSINE NUCLEOSIDASE FROM ALASKA PEA SEEDS

Abstract

Alaska pea seeds were tested for the presence of multiple enzyme activities from dry seeds and at different germination time. The maximum activity was reached at 48 hours of germination. Uridine nucleosidase had the highest activity of 67 mol/min and the lowest activity was adenosine nucleosidase with 33 mol/min. However, the dry seeds in all three nucleosidases had the lowest activity.

Adenosine nucleosidase is an enzyme catalyzes the hydrolysis reaction of adenosine to adenine and ribose. The enzyme was purified from Alaska pea seeds five days after germination. Ammonium sulfate precipitation with an increment of 10% was the initial purification step. Further purification was carried out using DEAE and Mono Q chromatography. The enzyme was purified after using only one column which was Mono Q ion exchange column with high pure protein. This is due to the ammonium sulfate fractionations during the purification of the enzyme in which the purity of protein enhanced by increasing salt concentration. The molecular weight of the purified enzyme was determined by SDS-PAGE 26,000 daltons with purification fold of 4.2 and a yield of 1.7%. The specific activity for the enzyme was 2.9 x10-2 mol/min/mg. The desired activity was achieved at 4 C and an optimum pH value of 7.2. The activity of the enzyme was examined based on the change of the factors that carried out during the purification procedure.