Optimization of alpha-galactosidase production in Streptomyces erythrus.

Physiological studies on Streptomyces erythrus NRRL ISP 5517 grown on fourteen different media have revealed that the enzyme was formed and released in the medium with different levels depending upon the type of the medium and the carbon source used. The results indicate that S. erythrus produced the highest level of extracellular and endocellular enzyme when grown in modified Czapek-Dox's medium (containing 2% D-galactose as the only carbon source). The highest levels of enzyme formation was obtained upon using D-galactose (9.94 Units/ml and 2.92 Units/ml), raffinose (8.87 Units/ml and 2.69 Units/ml) or melibiose (8.14 Units/ml and 2.52 Units/ml) at a final concentration of 2% as inducers for extra- and endocellular enzyme, respectively. With respect to nitrogen sources tested, sodium nitrate produced the highest level of alpha-galactosidase in both fractions optimally at 2.0 g/l. Studies revealed that the extracellular enzyme levels were produced optimally at initial pH in culture of 7.0 and air:medium ratio in flasks corresponding to 1:5 and after 5 days of incubation at 30 degrees C. On testing the effect of the addition of eight leguminous seeds powders (at a final concentration of 2%), it was found that soybean powder gave the highest induction effect. The addition of sodium nitrate at a concentration of 2 g/l to Dox's soybean medium, the adjustment of initial pH value of the medium to 7.0 and the air:medium ratio in flasks to 1:5 for an incubation period of 4 days produced the highest level of extracellular alpha-galactosidase.

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.

Mode and extent of degradation of adenosine and guanosine by extracts of Aspergillus terricola.

The mode of degradation of adenosine by extracts of Aspergillus terricola was suggested to be affected preliminary by adenosine deaminase to inosine and the resulting ribonucleoside was then degraded hydrolytically to give hypoxanthine and ribose. With regard to guanosine, the same extracts could initially catalyze the hydrolytic cleavage of guanosine to guanine and ribose. The resulting base was then deaminated to give xanthine by guanine deaminase. Addition of arsenate to the reaction mixture or dialyzing the extract did not affect the observed hydrolytic activity indicating the absence of phosphorylase activity or phosphorylase-phosphatase activities in the extracts.

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.

Hydrolytic cleavage of purine ribonucleosides by extracts of Aspergillus terricola.

Cell-free extracts of nitrate-grown Aspergillus terricola could catalyze the hydrolytic cleavage of the N-glycosidic bond of adenosine, guanosine and inosine optimally at pH 4 and 50 degrees C. Incubation of the extracts at 60 degrees C for 60 minutes caused about 86%, 67% and 54% loss of activity respectively. The similarities between the pH or the temperatures profiles indicate that the hydrolytic cleavage of these purine ribonucleosides might be effected by one hydrolase. The results obtained indicate the absence of evidence for the involvement of an SH group(s) in the catalytic site. CoSO4 and CuSO4 showed a remarkable inhibiting effect on enzyme activity. The apparent Km values of the ribonucleoside hydrolase for adenosine, guanosine and inosine were calculated from Lineweaver--Burk plots and found to be 20, 22.2 and 10 x 10(-3)M respectively.

Properties of adenosine deaminase in extracts of Asperigillus terricola.

Cell-free extracts of nitrate-grown Aspergillus terricola catalyze the hydrolytic deamination of adenosine to inosine at maximum rate at pH 6.5 and 50 degrees C. Incubation of the extracts at 60 degrees C for 30 minutes caused about 66.7% loss in activity. Results indicated the involvement of SH groups in the catalytic site of adenosine deaminase. Frequent freezing and thawing of the enzyme preparation for three days (3 times) resulted in about 47% loss in activity. The enzyme is also inhibited by EDTA indicating that adenosine deaminase is a metaloenzyme. MgCl2 and CoSO4 had a remarkable activating effect, whereas MnCl2 showed a slight inhibitory effect on enzyme activity. The apparent Km value was calculated for adenosine and found to be 6.66 x 10(-3) M, which indicates the greater affinity of adenosine deaminase for adenosine.