الفهرس 
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Abstract
The present When the effect of some additives on the production of dextransucrase was studied, maximum enzyme activity was obtained in culture medium consisted of in (g/L): Sucrose, 100; K2HPO4, 3; MgSO4.7H2O, 0.2; yeast extract, 1and wheat, 20.
The study was extend to explore the optimization of Enterococcus faecalisdextransucrase by using statistical based experimental designs:
1- In the first namely Plackett-Burman design, seven independent variables were screened in organized 11 combinations. The activity of dextransucrase is increased to 63.22 U/ml with 1.36 fold when compared to the basal medium.
2- The second namely, Central Composite Design, The coded and levels of the three independent variables investigated at five different levels. The activity of dextransucrase increased to 70.67 U/ml with 1.52 fold when compared to the basal medium.
The second part of this study was extended to partial purification and characterization of the produced dextransucrase from Enterococcus faecalis.
Partial purification of dextransucrase enzyme was achieved by fractional precipitation of crude enzyme by ethanol, acetone and ammonium sulfate. A weak recovery of enzyme from the total enzyme activity was obtained which results to subjecting the enzyme to ultrafiltration and lyophilization.
The third part of this study was the stabilization of the partially purified enzyme by immobilization by using two methods. In this part Enterococcus faecalis dextransucrase
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was immobilized on water insoluble carriers by different immobilization techniques, this include: physical adsorption, covalent binding. Of all the tested immobilization techniques, the highest immobilization yield (94.35 %)and activity (90.23 U/g carrier) were observed by covalent binding to Alg.CMC (2:3) FeCl3 (PEI/GA), respectively. Thus, Alg.CMC (2:3) FeCl3 (PEI/GA)was chosen as an ideal carrier through this study.
About 94.35% of the enzyme activity was retained on the support at 350 μAlg.CMC (2:3) FeCl3 (PEI/GA). The enzyme concentration of270 U was sufficient for achieving maximal enzyme activity with immobilization yield (99.85%) after 16 h. incubation time between the carrier and enzyme. After that the enzyme activity remained constant. The free enzyme had the maximum activity at 35 oC but the immobilized form at 45 oC. At higher temperatures the immobilized enzyme was more active than the free enzyme. The free and immobilized enzyme had an optimum pH of 5.4. The immobilized form showed great increase in thermal and pH stability in compared to the free enzyme. Lineweaver–Burk plot of the free gave Km0.02 mg ml−1, with sucrose, which is lower than the immobilized enzyme (0.384mg ml−1). Also, the Vmax of the free enzyme increased from 40.48 to 64.10 mg ml−1min−1 for the immobilized form.
The effect of various metal salts and ions indicated that, the inhibitory effect of some investigated metal ions was less pronounced with the immobilized enzyme compared with the free enzyme. Thus, immobilization process protected the enzyme against the inhibitory effect of some metal ions.
The effect of different concentrations of NH4Cl and K2HPO4 were tested on the activity of free dextransucrase and resulted in increase in the activity to (107.06 U/ml and 120.06 U/ml, respectively).
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The operational stability of the immobilized enzyme was evaluated using repeated batch process. The retained activity was 100% until 7 cycles where it reached 60% after 15 cycles.
Also the characterization of beads were tested, consists of following and proving the modification and activation process of the carrier beads using thermal stability of the gel, FT-IR, DSC, TGA and SEM.
The fourth part was related to application of the immobilized enzyme for the synthesis of dextran and oligosaccharides. It revealed the enzyme protein concentration 4.78 and 5.78 mg with 6 and 12% sucrose concentration at 30 oC for 16 hours were optimum for the synthesis of dextran of molecular weight 41555 Da, 73378 Da and 117521 daltons with addition of 0.1 g dextran.
The study extend to evaluate the operational stability of the immobilized enzyme for dextran synthesis.
Different ratio of sucrose : maltose were tested for oligosaccharide production at 25 oC for 8 hours. It resulted in maltooligosaccharride production with different DP (3, 4, 5 and 6).
The study also extended to test the antimicrobial activities of nine samples of the produced dextran and oligosaccharides, resulted in all samples had positive effect on the bacteria Staphylococcus aureus and have no activity against E. coli and Candida albicans.
Also the produced samples recorded positive results in prebiotic activity and fibrinolytic activity.vestigation deals with the study of microbial dextransucrase. It included studies aiming to searching for a potent microorganism producing dextransucrase isolated from honey. The studies also included optimization of fermentation parameters for maximal enzyme production and partial purification of the potent enzyme. Moreover, studies on enzyme stabilization by immobilization on different carriers were also included. The comparative study between the properties of the partially purified and immobilized enzyme was also carried out. Finally, The immobilized enzyme used for the production of dextran with different molecular weights and production of oligosaccharides. The biological activities of the products were tested.
The first part of this work was undertaken to investigate the productivity of dextransucrase and levansucrase by different bacterial honey isolates. Of twelve tested microorganisms, the bacteria (isolate 2) was found to be the most potent for dextransucrase production (45.19 U/ml) with no levansucrase production after 24 h. So, it was used throughout the study.
The results are summarized as follow:
The selected dextransucrase producing strain was identified according to its morphological criteria and phylogenetic analysis (16s rRNA) that showed a high level of similarity (99%) with Enterococcus faecalis CGLBL115.
The produced dextran was precipitated by ethanol and then acid hydrolysis was undertaken for the precipitate and the products of hydrolysis were illustrated chromatographically showing glucose only which evidence for presence of dextran only.
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The safety of selected bacteria were undertaken by measuring the antibiotic sensitivity test and found that the bacteria are sensitive to all antibiotics with different concentrations and also the blood haemolysis test (ability to break down the blood) was tested and found that it had no ability to breakdown red blood cells which indicates safe using of bacteria for human products.
Different cultivation parameters were investigated for the optimization of dextransucrase by Enterococcus faecalis. The maximum activity of enzyme (45.19 U/ml) was obtained after 24 hours incubation.
The shaking and static techniques of fermentation were studied and found that shaking technique was the most potent technique for dextransucrase production after 24 hours incubation.
Different concentrations of wheat were added to sucrose in the production medium resulted in 1.5 g/ 50 ml is the most potent concentration for dextransucrase production (45.63 U/ ml).
The effect of different organic (yeast extract, peptone, corn steep liquor, urea, baker’s yeast and soya bean) and inorganic nitrogen sources (sodium nitrate) in the production of dextransucrase by Enterococcus faecalis was also studied. Among all the used nitrogen sources yeast extract achieved the highest dextransucrase activity (45.20 U/ml). Also corn steep liquor concentrations were studied for the production of maximal dextransucrase but still yeast extract is the most potent nitrogen source for dextransucrase production.
Also, Maximal dextransucrase production was observed at 0.3% K2HPO4(46.37 U/ml).