BIOCHEMICAL AND BIOTECHNOLOGICAL STUDIES FOR SOME MICROORGANISMS

Abstract

In order to obtain microorganisms for degradation of the agricultural wastes, six native Egyptian fungal strains were isolated, morphologically identified and screened for cellulose biodegradation potential which was determined as endoglucanase or as carboximethylcellulase (CMCase) producers. The most promising isolate (Aspergillus terreus) with cellulolytic activity 2.26 U/mL was selected for molecular characterizations based on sequencing of internal transcribed spacer (ITS). The result confirmed that the strain is 99.8% homology with A. terreus. Further optimization experiments revealed that 35ºC is the optimum temperature for cellulase production and raised the enzyme activity up to 3.19 U/mL. Out of two organic nitrogen sources; peptone and yeast extract, the peptone at concentration 6g/l was found to be the optimum nitrogen source for cellulase production with the highest activity 4 U/mL. Whereas, the different four carbon sources: microcellulose, corn stalks, wheat straw and rice straw showed significant differences in the enzyme activity with values 11.07, 9.68, 7.87 and 3.71 U/mL, respectively at pH 3. The maximum enzyme activity was recorded to be within 5-7 days of incubation, depending on the type of carbon sources. Eventually, the optimization of different incubation conditions raised cellulolytic activity from 2.26 U/mL up to 11.18 U/mL. Then the enzyme was submitted to three purification steps. Ammonium sulfate fractionation raised the specific activity from 394 U/mg of the crude enzyme up to 574 U/mg. The next step was dialysis, which elevated the specific activity to 895 U/mg. The last step was gel filtration using Sephadex G-25. The Sephadex increased the specific activity to 1262.7 U/mg. To confirm the enzyme purity, SDS-PAGE electrophoresis was carried out and the enzyme appeared as a single band on the gel with molecular weight ~66kDa.