Sixty different bacterial isolates were isolated from soil sample collected from four different collected from four different locations in the city of Baghdad. Capable of degrading cellulose at 37C A selective medium containing carboxymethylcellulose as the sole carbon source was used in the isolation. The sixty isolates were re-cultivated for purification on Gauza agar medium with carboxymethylcellulose as the sole carbon source. Forty two potential Strcpiomyccs isolates were selected according to their morphological appearance which included, the color of aerial and substrate mycelia, secretion of soluble pigments beside their antimicrobial activities against other bacteria present with them in the isolation plate. These morphological features were consequently used as well as the microscopic features that included, the morphology of the spore chain and the number of spores for the primary classification of the isolates into thirty seven cellulolytic Streptomyces isolates. To assess the cellulolytic activity of these isolates, they were tested using the sensitive assay of Congo red reagent. All of the isolates showed different diameters of clearance zones around the colonies. These results were confirmed using a quantitative assay with the 3,5-dinitrosalicylic acid reagent to assess the liberation of glucose from the carboxymethylcellulose. by the carboxymethylcellulase enzyme (s) action. Two isolates showed the highest cellulolytic activity and were subjected tofurther biochemical and morphological characterization assays tor species identification and from which they showed an ability to utilize different carbon sources, produce antimicrobial agents as well as their resistance to different concentration of sodium chloride and to different tvpes of antibiotics. Accordingly, one isolate was classified as streptomyces coelicolor. However, no species was assigned to the other isolate. Although, the two selected isolates showed promising characteristic, the unclassified one was selected for further enzymological studies due to it's characteristic antimicrobial activity in inhibiting the growth of test microorganisms such as Escherichia coll, Pseudomonas aeroginosag Staphylococcus aiireus and Candida albicans_and it's resistance to a wide range of antibiotics such as: Amoxicillin, Erythromycin, Kanamycin, Tetracycline and Rifampicin. Carboxymethylcellulase enzyme production by the selected Streptomyces isolate was measured by the ability of the extracellular supernatant to degrade the carboxymethylcellulose and produce sugar detected by the 3,5- dinitrosalicylic acid reagent-Maximum enzyme yields were obtained after 96 hours of incubation at 37°C for 7 days. Subsequent work was carried out concerning the conditions for ^enzyme production and a pH of (7.5) and a temperature of 45°C were to be optimum. In addition, Carboxymethylcellulase enzyme production was activated in the presence of different concentrations of CaCl2 and MgSO4. Beside, the activity was inhibited in the presence glucose in the growth medium which might indicate a catabolic Repression. The celluar location of the enzyme activity was studied too and cellular fractions were assayed including the extracellular extracelluisr. Supernatant, the cell free extract and the cellular precipitate after sonication. It appeared that the celuloytic enzymatic activity was only extracellular. However, the possibility of die presence of intracelluiar cellulolvlic enzymes wasn't excluded, sines the cellulase enzyme could be in a complex form which might be inhibited by sonication and cell disruption.
An attempt was made to improve the production of the carboxymethylcellulase enzyme. The mutagenic agent (Nitrosoguanidin-NTG) was used and a mutant was isolated according to the large diameter of clearance zone compared with the wild type measured using the Congo red reagent. This higher activity was also confirmed using the 3,5-dinitrosalicylic acid rocedure. However, the mutant could not be differentiated from the wild type morphologically. According to the all carried up experiments and obtained results it appeared that the selected Streptomyces isolate was not included within the culture collection of Streptomyces species in the Department of Biotechnology of Saddam University. Although, the species of the selected Streptomyces isolate wasn't allocated but it was believed that this isolate could be used in environmental biotechnology as a useful tool to solve some of the environmental contamination problems associated with paper and plant materials wastes.