الفهرس 
Introduction & Aim of the workOnly 14 pages are availabe for public view
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Abstract
The present study began by the isolation and purification of the local alkalophilic bacterial colonies from cultivated and non-cultivated soil samples collected from El-Khalubia governoate region as follows. Abu-Zaabal village, El-Gabal El-Asfar, El-Khanka village and El-Nasr pharmaceutical company byproduct region, these soil samples were near by the around industrial factories. Dilution plate technique was used for isolating alkalophilic bacterial colonies. Three types of media were used for such a purpose, DEV-Gelatin, SATO-agar and Casin-Peptone Soy Meal-peptone agar.
Forty three bacterial isolates were isolated. The potentiality of using agar plates amended with a gelatin (0.4%) as a substrate for alkaline protease production. The investigation was accomplished by visual of white precipitate as indication for the negatively alkaline protease producer. The most potent producer isolate was known as ENPC.34, which showed growth and produce the highest alkaline protease activity (4392ug/ml) when determined chemically, in presence of standard curve of protein.
Certain identification methods were carried out on the isolate ENPC.34 which were conducted according to the manual methods for General Bacteriology and other taxonomic characteristics was confirmed by the “Biological Control of B.F on V, F project” at plant pathology Reseach Institue ARC at Giza Egypt according to the identification program (Biology & Microgiology 34.01A). the present isolate was suggested to be a member of Aureobacterium terregens, thus given the name Aureobacterium terregens ENPC.34
Optimization for the production of alkaline protease by Aureobacterium terregens ENPC.14 under the following environmental conditions:
The optimum environmental conditions of alkaline protease production such as pH, 10- 10.5; incubation time, 96 hours and incubation temperature was 37⸰C.
The nutritional requirements of maximum alkaline protease production by Aureobacterium terregens ENPC.34 1.5% (w/v) glucose represent the best carbon source and a mixture of soy bean meal and Corn-Steep Liquor as (3:2, w/v), respectively were found to be the most favorable nitrogen source. Phosphate as (0.5%, w/v) K2HPO4 (5mg); and MnCl2. 6H2o (10mg). With 5% of bacterial seed culture as the best inoculum size and under the optimal environmental, nutritional conditions. The laboratory bench- frementor was used for alkaline protease production by Aureobacterium terregens ENPC.34, on the large scale. The rate of aeration and other physical factors were controlled by Microprocessor. Maximum activity using shaking flasks reached 28000 µg/ml.
The crude extract of alkaline protease from Aureobacterium terregens ENPC.34, was precipitated by saturated ammonium sulfate (70%). The precipitate was re-dissolved in a Tris/HCl buffer (pH, 8.8) as a dialysis solution.
The purification of alkaline protease from Aureobacterium terregens ENPC.14, By using of DEAE-Cellulose and C.M-Cellulose columns with final recovery of about 36.9% and 9.41 fold purification.
Homogeneity of alkaline protease from Aureobacterium terregens ENPC.14, was carried out by using SDS-Polyacrylamide gel electrophoresis (10%), and give only one band which revealed the homogeneity of the enzyme.
The molecular weight of the purified alkaline protease from Aureobacterium terregens ENPC.34 was detected by using SDS-Poly acralamide Gel Electrophoresis which corresponds to 23.000 daltons as a molecular weight.
Kinetics of alkaline protease from Aureobacterium terregens ENPC.34 was studied and showed that the enzyme is active at pH (10-10.5) and stable at pH (6-12), when incubated at 37 ◦C for 1 hour in the presence of 5mM of Cacl2. The optimum temperature of the enzyme activity was recorded at 60◦C in the absence of Cacl2, while in the presence of CaCl2, the enzyme was stable at 70◦C, accordingly, alkaline protease from Aureobacterium erregens ENPC.34 was stable at higher degree of temperatures.
The effect of different inhibitors on the activity the of purified alkaline protease from Aureobacterium terregens ENPC.34 revealed that the enzyme was not affected by certain inhibitors as Ethylenediamine tetraacetic acid (EDTA), polyoxyethylene sorbitan monolaurate (Tween20), I sooctylphenyl polyethoxy a lcohole (Triton x 100) and P. Chloro mercuric benzoate (PCMB). On the other hand certain inhibitors as sodium lauryl sulfate (SLS), Benzylkonium Chloride (BKCI) were slightly affected, but by using Di isopropyl fluorophosphates (DFP) and Alkyl benzene sulfonate (ABS), showed the enyme was more inhibited.
the amino acids composition of the purified enzyme was determined, and indicate that the enzyme is rich in glutamic and content methionine, threonine, cysteine and poor in its content of phenyl alanine and lysine among the basic amino acids residues.
Also studying the stability of enzyme with some commercial detergents, which indicate that the enzyme is quit stable in the presence of Arial, Percil, Omo, Lang and Biocleana.
Finally we could isolate and purify a new alkalophilic bacteria which given the name Aureobacterium terregens ENPC.34 which could produce alkaline protase with activity about 28000 µg/ml by using a low cost media.
We can use the produced enzyme in many application industries such as, detergent, leather, and photographic.