Various fungal genotypes (Aspergillus niger NRRL 3135 (AN1), Aspergillus niger NRRL 326 (AN26), Aspergillus terrus F2-Kh (AT) and Mucor racemosus NRRL 3639 (MI)) were studied for their ability to produce phytase and improve the produced enzyme by ethyl-methane sulfonate (EMS) mutagenesis. AN1 showed the highest phytase activity on phytase screening medium supplemented with glucose (PSMG) after 8 days of incubation (reached 1875.40 IU/mL). The phytase activity of AN1 increased with increasing incubation time and the highest value was achieved at 12 days of incubation (2859.33 IU/mL). The exposure of AN1 spore suspension to 200 mM of EMS for different times enhanced the phytase activity and that mutant 20 Mn exhibited the highest phytase activity (reached 4520.5 IU/mL) therefore it was chosen for the next experiment. An in-vitro gas production procedure was carried out to evaluate the impact of using various amounts of laboratory produced phytase (PE) compared with commercial phytase (Axtra® PHY) on nutrients availability of ruminant's ration. Six levels (0, 400, 800, 1200, 1600 and 2000 IU phytase-1Kg dry matter) of phytase enzyme were evaluated with tested ration consisted of 40% berseem hay (BH) and 60% concentrate feed mixture (CFM). In-vitro dry and organic matter degradability (IVDMD and IVOMD), total gas production (GP), short chain fatty acids (SCFA’s) and inorganic phosphorus (Pi) concentration were improved significantly (P<0.05) by phytase addition from the two sources of phytase and the highest significant (P<0.05) values achieved at the level of 1200 IU. Results suggest that phytase activity was influenced by exposure to EMS mutagen compared to the wild type. Also, the produced enzyme source has the ability to improve the utilization efficiency of phytate diets as evidenced by the significant (P<0.05) increase in all tested parameters compared to the commercial source.