RESUMO
Aim: The aims of the present study were to screen different filamentous fungi for extracellular cellulases production and to optimize solid-state fermentation medium and culture conditions to enhance cellulases production. Study Design: Using agro-industrial waste as raw material for the production of cellulases by a hyper cellulase producing fungus and evaluating the influence of various parameters to design a suitable SSF process for cellulase production. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between January 2013 and October 2013. Methodology: Different filamentous fungi were grown and maintained on potato dextrose agar slants at 28ºC for 7 days. The spores were washed down by distilled water. Then, 2.0 ml aliquots were used to inoculate 250 ml Erlenmeyer flasks, containing rice straw as the only carbon source. The inoculated flasks were incubated for 5 days at 28ºC. The enzymes were extracted by mixing homogenously the fermented substrate with 50 ml citrate phosphate buffer (0.1 M, pH 5.0) and agitated (150 rpm) for 1 hr. Pooled extracts were centrifuged at 5000 rpm for 15min and the clear supernatant was used as a source of extracellular enzyme. Results: Aspergillus oryzae NRRL 3484 exhibited relatively higher cellulases production. The optimum incubation period, temperature, and initial moisture level were reported on the 7th day, at 28°C, and 70%, respectively. Peptone proved to be the suitable nitrogen source followed by yeast extract, while pH 5.0 was ideal for cellulases production. Conclusion: Using ligninolytic fungi, including their enzymes, may be one potential alternative to provide a more practical and environmental-friendly approach for enhancing the nutritive value of rice straw. Moreover, the application of ligninolytic fungi or their enzymes combined with chemical pre-treatments to rice straw may be an alternative way to shorten the period of the incubation times and (or) decrease the amount of chemicals, effecting some synergy.
RESUMO
A non specific acid phosphatase from Aspergillus oryzae NRRL447 catalyzes the phosphate hydrolysis from nicotinamide adenine dinucleotide forming nicotinamide riboside, adenosine and Pi as the final products of the reaction. The enzyme was purified to homogeneity by a sequential treatment of acetone fractionation, DEAE-cellulose chromatography and gel filtration chromatography. The enzyme was purified 400-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 52 kDa. The enzyme displayed maximum activity at pH 5.0 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg2+, Ca2+ whereas inhibited strongly by F-, Mo04 -, Cu2+ and Fe2+. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km for NAD+ was 6.25 x 10-4 M.