الفهرس 
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Abstract
Sixteen proteolytic thermophilic bacteria were isolated from soil at Fayoum Governorate. The isolates were screened for their proteolytic activity on skim milk agar medium; the diameter of hydrolysis zone was the measurements from the level of proteolytic activity. The most active isolates were selected for the fermentation experiments and the determination of their productivity in the submerged culture. The seven selected isolates were used in fermentation experiments and the proteolytic activity was determined after 48 hrs. The enzyme yields obtained in the fermentation medium were corresponding to the proteolytic level recorded by the diameter of hydrolysis zone on the skim milk agar medium. That indicates a positive relationship between the amount of the enzyme and its spreading in the skim milk agar medium. According to the results from the fermentation experiments, strains S-5, S-8 and S-9 which gave the highest enzymatic yields were chosen for studying the best environmental conditions for the enzyme production. The three isolates were identified based on morphological, biochemical and 16S rRNA gene sequencing analysis, isolates S-5 was identified as Brevibacillus panacihumi; isolates S-8 and S-9 were identified as Bacillus aerius. The environmental conditions such as pH, temperature and fermentation period were studied for the highest production of enzymatic yield. The results showed that, the highest enzymatic yield was obtained in the fermentation medium at pH 7.0 for Bacillus stearothermophilus ATCC7953 and Brevibacillus panacihumi S-5 while at pH 8.0 for both Bacillus aerius S-8 and Bacillus aerius S-9.The fermentation temperatures at 40, 50 and 60 °C showed that the optimum incubation temperature for the enzyme synthesis was 50°C. However, the incubation period required for maximum accumulation of protease was 72 hrs. Glucose was the best carbon source for protease production followed by dextrin, sucrose, maltose and glycerol, respectively for Bacillus stearothermophilus ATCC7953, Bacillus aerius S-8 and Bacillus aerius S-9. However, dextrin as well as glucose gave the higher enzymatic activity than other carbon sources used for Brevibacillus panacihumi S-5. Also, the maximum enzyme production was recorded in the presence of 1.5 % tryptone for all tested bacterial strains while, 1.0 % yeast extract secured the
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highest enzymatic activity for Bacillus stearothermophilus ATCC7953and
Brevibacillus panacihumi S-5. However, control which contain 0.5% yeast
extract seemed to be the most suitable concentration for Bacillus aerius S-8
and Bacillus aerius S-9. Rice straw media resulted in somewhat higher
enzymatic yields as those in media containing cane sugar bagasse. In addition,
extraction of the enzyme by precipitation method was carried out with the
organic solvents, acetone, ethanol and isopropanol at different concentrations
(fermentation liquor /solvent v/v). Also, the enzyme was extracted by salting
out using different concentrations of ammonium sulfate ranging from 30 to
80 % (w/v). Therefore, of ammonium sulfate gave better results than that
obtained by organic solvents for all tested bacterial strains. The best
concentration for enzyme precipitation seemed to be between 70% and 80%
saturation of ammonium sulfate science the highest specific activity and
recovery % were at 80% for all tested bacterial strains. Analysis of molecular
mass of the partially purified enzyme was carried out by SDS-PAGE which
revealed four protein bands with different molecular weights ranged from 75 to
20 KDa. These bands were varied in molecular weight (63, 48, 30 and 25
KDa).
The enzyme was active in a broad temperature range between 40 °C and
100 °C with an optimum at 70 °C and the maximum activity was at pH 7.0 for
Bacillus sterothermophilus ATCC 7053, while at pH 8.0 for the other three
tested bacterial strains. In the presence of Ca-ions, the thermal stability of the
enzyme was considerably enhanced with no apparent loss of enzyme after 45
minutes for Bacillus aerius S-8 and 30 minutes for the other three tested
bacterial strains.