Factors affecting D-galactonate degradation by extracts of Aspergillus niger.

Biochemical studies on the degradation of D-galactonate by cell-free extracts of Aspergillus niger indicated that the pH value and temperature optima were 8.0 and 7.5 and 40 degrees C and 50 degrees C for the two enzymes responsible for this degradation namely D-galactonate dehydratase and 2-keto-3-deoxy-D-galactonate (KDGal) aldolase respectively. The effects of the nature of the buffer substance, buffer molarity and enzyme concentration were also studied. Thermal stability behaviour studies show that D-galactonate dehydratase was stable at 40 degrees C for 60 minutes and about 41%, 80% and 90% of enzyme activity were lost by exposing the extracts to 50 degrees C, 60 degrees C and 70 degrees C, respectively, for the same period. However, exposing the extracts to 70 degrees C after 60 minutes caused a complete inhibition for KDGal aldolase activity and a gradual decrease in activity was noticed by incubation the extracts at 60 degrees C. The results of freezing and thawing treatment indicated that KDGal aldolase was more stable than D-galactonate dehydratase in this respect, as only 27% of enzyme activity was lost after 5 days of storage at -5 degrees C. Dialyzing the extracts significantly affects KDGal formation from D-galactonate. Results obtained also indicated the non-requirement of metal ions for activation of KDGal aldolase. On the other, hand D-galactonate dehydratase has a requirement for Mg++ and Mn++, however ZnSO4 and HgCl2 caused a complete inhibition of the enzymatic activity of this enzyme.

Evidence for a non-phosphorylated route of galactose breakdown in cell-free extracts of Aspergillus niger.

Aspergillus niger could utilize D-galactose as sole source of carbon. Cell-free extracts of D-galactose-grown mycelia were able to catalyze the oxidation of D-galactose to D-galactonic acid-gamma-lactone (GalA-gamma-lact) in the presence of NAD, followed by the appearance of 2-keto-3-deoxy-D-galactonate (KDGal), pyruvate and glyceraldehyde. From 10 &mgr;moles only 6.6 &mgr;moles of GalA-gamma-lact were disappeared after 60 min of reaction indicating the presence of GalA-gamma-lactonase. Identification of GalA-gamma-lact was achieved by ascending paper chromatography. KDGal, pyruvate and glyceraldehyde were also chromatographically identified in the reaction mixture containing D-galactonate which suggests that D-galactonate is degraded into pyruvate and glyceraldehyde via the intermediate formation of KDGal. Such reactions are supposed to be catalyzed by an inducible D-galactonate dehydratase and a constitutive KDGal aldolase. The amount of KDGal, pyruvate and glyceraldehyde were found to be almost equivalent and the equilibrium of the reaction being toward the formation of KDGal. The apparent equilibrium constant (K(eq)) was calculated and found to be 0.5 x 10(-3) M. Results also proved the reversibility of the reaction catalyzed by KDGal aldolase of A. niger. In the light of the findings obtained from the degradation of D-galactose by cell-free extracts of A. niger grown on D-galactose and D-galactonate a nonphosphorolytic pathway was suggested to be operative for the degradation of D-galactose in extracts of A. niger.

Properties of enzymes involved in D-galactonate catabolism in fungi.

Two enzymes catalyze the two step reactions in the D-galactonate nonphosphorolytic catabolic pathway of Aspergillus terreus, namely D-galactonate dehydratase and 2-keto-3-deoxy-D-galactonate (KDGal) aldolase. Maximum enzyme activities were obtained at 40 degrees C and pH 8.0 or at 50 degrees C and pH 7.5 for these two enzymes, respectively. Stability of the two enzymes under different conditions was investigated. From a Lineweaver-Burk plot of the reciprocal of initial velocities and substrate concentrations, apparent Km values were calculated for D-galactonate, pyruvate and glyceraldehyde and found to be 8.33, 14.28 and 5.55 mM, respectively, in crude cell-free extracts. Results indicated the requirement of magnesium cation for D-galactonate dehydratase activity at an initial concentrations of 10(-2) M. The presence of Mg2+ in the reaction mixture seems to induce greatly the fitness of the dehydratase with D-galactonate as no activity could be detected with 24-h dialyzed extract in the absence of magnesium cation.