الفهرس 
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Abstract
Proteases are one of the highest value commercial enzymes as they have extensive applications in food, pharmaceutical, detergent, dairy industries, meat tenderization, recovery of silver from X-ray film and other applications. Additionally, they serve as important tools in the determination of the structure of proteins and polypeptides. They are physiologically necessary for living organisms. They are ubitiquous, being found in a wide diversity of sources such as plants, animals and microorganisms.
Plasmids pS1, p5.2 that carry the complete alkaline protease gene were used to transform Bacillus subtilis dnaC30 ts mutant cells. This host is known to grow normally at 37°C, however, when the temperature is changed to 48°C (non permissive -temperature) the replication of the chromosome, but not the plasmid, is ceased. Consequently, this system allows the amplification of genes carried on plasmids.
The expression of the cloned genes in B. subtilis dnaC30 host, namely B. subtilis dnaC30 (pS1) and B. subtilis dnaC30 (p5.2) was studied.
The alkaline protease (aprE) gene in bacterial cells harboring (pS1) plasmid started late at approximately two hours after the end of the exponential phase when grown at 37°C. 2.77 fold enhancement in aprE gene expression was achieved when B. subtilis dnaC30 (pS1) cells were grown at 37°C for two hours followed by 24 hours at 48°C. Furthermore, 3.23 fold enhancement in the expression of the aprE gene was achieved when the recombinant B. subtilis dnaC30 (p5.2) cells were grown at 48°C for 24 hours.
It was noticed that the number of colony forming units of the newly constructed cells grown at 48°C was gradually reduced compared to that of the same cells grown at 37°C. This reduction started after 6 hours of incubation at 480C. This could be due to the fact, that replication of the chromosomal DNA was dramatically decreased when cells grown on 48°C.
The stability of the recombinant plasmids (pS1 and p5.2) was examined at different intervals when bacterial cells were grown on PY medium. The plasmid p5.2 was segregationally and structurally very stable for up to 4 days of subculturing, whereas, the (pS1) plasmid was less stable than p5.2 plasmid.
Effect of high copy number of the cloned aprE gene in B. subtilis dnaC30 (p5.2) on growth rate and sporulation frequency was determined. A clear inhibition of sporulation was observed and this could be due to the high copy number of the aprE gene at non-permissive temperature (48°C) in addition to the characteristics of the mutant.
The plasmid copy number (PCN) results indicated that the p5.2 plasmid in bacterial cells when grown at 48°C was increased 4 fold compared to that of the same plasmid when
B. subtilis dnaC30 cells were grown at 37°C.
Additionally, scanning electron microscope (SEM) revealed a significant difference in the cell morphology between B. subtilis dnaC30 (p5.2) cells grown at 48°C and same cells grown at 37°C. Bacterial cells grown at 48°C posses long thin cells compared to those grown at 37°C which had a normal cell morphology that looks like wild-type cells, this feature is mainly due to the effect of the elevated temperature on the mutant cells.
Data concerning temperature shift up/down showed that, shifting the temperature of the culture back to the permissive temperature (37°C, shift down) for 3 or 6 hours would allow cells to restore the chromosomal DNA replication and consequently cells would be saved to continue to produce alkaline protease.
Some optimal conditions for the production of alkaline protease from B. subtilis dnaC30 (p5.2) were determined and it was found that, the baffled or non baffled flasks containing (1XSG) medium supplemented with 2% (v/v) of inoculum size and incubated for 2 hours at 37°C followed by 24 hours incubation at 48°C were the best conditions.
The alkaline protease enzyme that was produced by B. subtilis dnaC30 (p5.2) was purified throughout several purification steps including ammonium sulphate (65% of saturation) followed by dialysis and gel filtration through a Sephadex G-50 column. The fractionation pattern revealed a single peak of protein content that overlapped, to a great degree, with a single peak of proteolytic activity. The overall purification fold was about 39 with 11% recovery.
The predialyzed enzyme obtained from B. subtilis dnaC30 (p5.2) cells grown at 48°C developed an intense colorless band on casein SDS-PAGE (zymogram gel) than that of same cells grown at 37°C. This could be explained by the fact that the proteolytic activity of same cells grown at 48°C is much higher.
The homogeneity of the purified enzyme was confirmed by SDS-PAGE and it was shown a single band indicated that the enzyme was almost purified. The purified enzyme was found to be inhibited by PMSF but not EDTA and this indicates that the majority of the proteolytic activity was in the form of alkaline protease. The molecular weight of the alkaline protease enzyme was determined by SDS-PAGE and it was about 28 KDa.
The optimum pH for enzymatic activity was pH 9.0 and the enzyme was stable at pH values ranging from pH (8-10). The maximum activity was observed at 60°C and the activation energy for conversion of substrate to product was 26.346 J/mole. Concerning thermostability of the enzyme, almost all proteolytic activity was lost when the enzyme was exposed at 60 and 70°C. The alkaline protease was very stable at 50°C for one hour. On the other hand, the enzyme was 100 % stable at 60°C for one hour in the presence of 25mM CaCl2. This would indicate that, bound calcium ions to the enzyme play a key role in the protection against autolysis and thermal denaturation.
Moreover, obtained results showed that the enzymatic activity decreased by pre-incubating the enzyme with heavy metal ions such as CuCl2 and FeSO4. However, the activity was lost when the enzyme was pre-incubated with 2mM of HgCl2. The enzymatic activity was increased as the concentration of Triton X-100 increased and it reached its maximum activity at 2%. This increase in enzyme activity may be due to the effect of the Triton X-100 on the unfolding of the substrate moiety. However, proteolytic activity was not affected by the surfactant Tween-20. Furthermore, SDS at concentration of 0.5% had little effect; while, 2 % SDS inhibited the enzyme activity.
The enzyme kinetics was determined and results showed that, Michaelis-Menten constant Km and Vmax values were 4.513 mg/ml ± 2.086 and 8.288 ± 1.31 U/ml for HPA, respectively.
The ability of the newly constructed bacterial cells harboring p5.2 plasmid to act on keratin-containing materials like feathers was tested. The utilization of chicken feathers by B. subtilis dnaC30 (p5.2) was carried out. The maximal keratinolytic activity was obtained when cultures were grown at 37°C for 7 hours followed by 24 hours at 48°C.
Data obtained, through the present work, would suggest the use of the newly constructed B. subtilis dnaC30 (p5.2) cells in some useful biotechnological applications. The use of temperature sensitive mutation system (dnaC30 system) would improve the alkaline protease production. The digestibility of the keratin-containing materials that yield soluble proteins, peptides and amino acids at considerable level is mainly based on the action of alkaline protease. The produced soluble proteins, peptides and amino acids can promote and/or enhance several new industries in several countries.