Cytidine deaminase activity in Penicillium politans NRC-510.

Cell-free extracts of nitrate-grown Penicillium politans NRC-510 catalyzes the hydrolytic deamination of cytidine to uridine. Uridine was chromatographically identified in cell-free extracts. The enzyme exhibited optimum pH and temperature activities at 6.5 and 80 degrees C respectively. Thermal stability experiments indicated that the enzyme restored its activity at 80 degrees C for at least 60 minutes. When cell-free extracts were incubated at 90 degrees C for 5 minutes enzyme activity was inhibited by about 33%. The involvement of sulfhydryl group(s) in the catalytic site of the enzyme was shown. HgCl2 (5 x 10(-3) M) and CuSO4 (10(-2) M) caused a complete inhibition of enzyme activity. Ethylene diamine tetraacetate at a concentration of 5 x 10(-3) M and 10(-2) M inhibited the enzyme as well. Whereas, MgCl2, CoSO2 and MnCl2 had a remarkable activating effect. Dialysis of the cell-free extracts resulted to an increase in enzyme activity by about 30%. To our knowledge the thermophilic nature of the cytidine deaminase of P. politans NRC-510 is unique.

Deamination of adenosine by extracts of Penicillium politans NRC-510.

Cell-free extracts of nitrate-grown Penicillium politans NRC-510 could catalyze the hydrolytic deamination of adenosine to inosine maximally at pH 6.0 and 45 degrees C. However the same extracts could not catalyze the N-glycosidic bond cleavage of adenosine at pH 4.0, 6.0 and 8.0. Incubation of the extracts at 55 degrees C for 30 minutes caused about 31% loss in activity whereas incubation of the extracts at 60 degrees C for 15 minutes caused a complete loss of enzyme activity. Results indicated the absence of the involvement of sulfhydryl groups in the catalytic site of adenosine deaminase. The enzyme is inhibited by ethylene diamine tetraacetate indicating that adenosine deaminase is a metalloenzyme. MnCl2 and MgCl2 had a remarkable activating effect, whereas HgCl2, CaCl2 and ZnSO4 showed an inhibitory effect on enzyme activity. Dialyzing the extracts for 24 hours significantly increase deaminase activity by about 33%. The apparent K(m) value was calculated for adenosine and found to be 3.63 x 10(-3) M, which indicates high affinity of adenosine deaminase for its substrate adenosine.

Some properties of two aldolases in extracts of Aspergillus oryzae.

Fructose 1,6-diphosphate (FDP) aldolase and 2-keto-3-deoxy-D-gluconate (KDG) aldolase the two key enzymes of Embden-Meyerhof-Parnas (EMP) and the nonphosphorolytic Entner-Doudoroff (ED) pathways respectively, were identified in cell-free extracts of four Aspergillus oryzae strains grown on D-glucose as sole source of carbon. A. oryzae NRRL 3435 gave the highest enzymatic activity for the two enzymes and selected for further studies. Studies on the properties of the two key enzymes indicated that the optimum conditions for the activities of FDP aldolase and KDG aldolases occurred at pH 8.5, 45 degrees C and pH 8.0, 55 degrees C, respectively. Tris-acetate buffer and phosphate buffer showed the highest enzymatic activity for these two enzymes respectively. KDG aldolase was stable at 55 degrees C for 60 minutes however FDP aldolase was found to be less stable above 45 degrees C. On the other hand the two aldolases showed a high degree of stability towards frequent freezing and thawing. Dialysis of the extracts caused a decrease in the enzymatic activity of KDG aldolase, and an increase in FDP aldolase activity. The addition of ethylene diamine tetraacetate to the crude extracts caused an inhibition of KDG aldolase, whileas FDP aldolase was not affected. Addition of MnCl(2), CoSO(4), MgCl(2) and ZnSO(4) to the dialyzed extracts increased the activity of KDG aldolase by 67%, 54%, 61% and 37%, respectively. On the other hand the addition of some metal salts caused an inhibition of FDP aldolase. The results obtained indicate the absence of evidence for the involvement of sulfhydryl groups in the catalytic sites of the two aldolases.