Physiological studies on carboxymethyl cellulase formation by Aspergillus terreus DSM 826

Physiological studies were conducted to determine the optimum cultural conditions for maximal carboxymethyl cellulase (CMCase) formation by Aspergillus terreus DSM 826. Shaking condition at 150 rpm is favorable for the production of CMCase from rice straw and sugar cane bagasse. The highest enzyme yield was obtained at the third day of incubation at 30ºC for both cases; however CMCase formation occurred at a broad range of pH values, with maximal formation of A. terreus DSM 826 CMCase at pH 4.5 and 5.0 when rice straw and sugar cane bagasse were used as sole carbon source, respectively. Carboxymethyl cellulose (CMC) was found to be a good inducer for CMCase formation in both agricultural wastes with CMC concentrations of 0.5 and 1.0 % (w/v) in case of rice straw and sugar cane bagasse, respectively. High level of enzyme formation was obtained with the addition of ammonium chloride as nitrogen source in both cases and at a concentration of 0.4 % (v/v Tween-80) as an addition to medium containing rice straw. However this addition did not influence the production of CMCase in case of using sugar cane bagasse as carbon source.

Physiological studies on carboxymethyl cellulase formation by Aspergillus terreus DSM 826.

Physiological studies were conducted to determine the optimum cultural conditions for maximal carboxymethyl cellulase (CMCase) formation by Aspergillus terreus DSM 826. Shaking condition at 150 rpm is favorable for the production of CMCase from rice straw and sugar cane bagasse. The highest enzyme yield was obtained at the third day of incubation at 30 °C for both cases; however CMCase formation occurred at a broad range of pH values, with maximal formation of A. terreus DSM 826 CMCase at pH 4.5 and 5.0 when rice straw and sugar cane bagasse were used as sole carbon source, respectively. Carboxymethyl cellulose (CMC) was found to be a good inducer for CMCase formation in both agricultural wastes with CMC concentrations of 0.5 and 1.0 % (w/v) in case of rice straw and sugar cane bagasse, respectively. High level of enzyme formation was obtained with the addition of ammonium chloride as nitrogen source in both cases and at a concentration of 0.4 % (v/v Tween-80) as an addition to medium containing rice straw. However this addition did not influence the production of CMCase in case of using sugar cane bagasse as carbon source.

Purification and properties of an endoglucanase of Aspergillus terreus DSM 826.

Endoglucanase (EG) from A. terreus DSM 826 grown on sugar cane bagasse as a carbon source was purified using acetone fractionation, then a Sepharose-4B chromatographic column, with purification of about 27-fold and 10.5% recovery. The optimum temperature and pH for activity of the purified EG were found to be 50 degrees C and pH 4.8, respectively. The purified enzyme can stand heating up to 50 degrees C for 1 h without apparent loss of activity. However, the enzyme, incubated at 80 degrees C for 5 min, showed about 56% loss of activity. Optimum EG activity was recorded with a citrate buffer system (pH 4.8; 0.05 M). Co2+ (2.5 x 10(-2) M) and Zn2+ (5 x 10(-2) M) were found to activate the purified EG of A. terreus DSM 826 by about 83 and 25%, respectively. On the other hand, Hg2+ inhibited the activity of the purified EG by about 50 and 71% at a concentration of 2.5 x 10(-2) and 5 x 10(-2) M, respectively. Carboxymethyl cellulose was found to be the best substrate for the purified EG, with V(max) values of 4.35 micrpmol min(-1) mg(-1) protein.

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.

Hydrolytic cleavage of purine ribonucleosides in Aspergillus phoenicis.

Cell-free extracts of nitrate-grown Aspergillus phoenicis could catalyze the hydrolytic cleavage of the N-glycosidic bond of inosine, guanosine and adenosine to the corresponding base and ribose by the nucleoside hydrolase. No evidence was obtained concerning the hydrolytic degradation of N-glycosidic bond of pyrimidine ribonucleosides namely cytidine and uridine by the same extracts. Optimum pH and temperature for adenosine, guanosine and inosine hydrolysis were the same at pH 3.5 and 55 degrees C, respectively. Citrate buffer showed the highest hydrolase activity when compared to the analogous activity obtained with the other buffers used. The rate of hydrolysis of the three nucleosides was in the order inosine > guanosine > adenosine. Incubation of extracts at 55 degrees C for 15 minutes caused about 85%, 75% and 62% loss of activity with adenosine, guanosine and inosine respectively. Dialyzing the extract caused a decrease in enzyme activity. Addition of inorganic arsenate to the reaction mixture (containing adenosine, guanosine or inosine) did not affect the amount of ribose liberated. Addition of EDTA at a concentration of 5 x 10(-3) M caused an inhibition of about 50%, however a complete inhibition for enzyme activity was obtained at 10(-2) M EDTA. MgSO4, CoSO4 and ZnSO4 at a final concentration of 5 x 10(-3) M showed activation of ribonucleoside hydrolase.