الفهرس 
AcknowledgementOnly 14 pages are availabe for public view
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Abstract
In the beginning of this decade the worldwide was interested in the use of thermostable enzymes instead of mesophilic enzyme on the large scale for many of the fields of life (industry, medicine, agriculture…, etc). Thermostable enzymes are gaining wide industrial and biotechnological interest due to the fact that their properties are better suited for harsh industrial processes.
One extremely valuable advantage of conducting biotechnological processes at elevated temperatures is reducing the risk of contamination by common mesophiles. Allowing a higher operation temperature has also a significant influence on the bioavailability and solubility of organic compounds and thereby provides efficient bioremediation. Other values of elevated process temperatures include higher reaction rates due to a decrease in viscosity and an increase in diffusion coefficient of substrates and higher process yield due to increased solubility of substrates and products and favorable equilibrium displacement in endothermic reactions. Such enzymes can also be used as models for the understanding of thermo-stability and thermo-activity, which are useful for protein engineering.
In recent years much interest has been focused on the isolation, purification and characterization of thermostable enzymes (thermozyme). Thermozymes have been isolated mainly from thermophilic micro-organisms, especially thermophilic bacteria which isolated from natural geothermal areas such as: volcanic areas, ocean, submarine hot springs, Solar-heated ponds, biologically-heated composts, deep sediments, rocks and desert.
Thermozymes which have been isolated mainly from thermophilic micro-organisms have many commercial applications because of their overall inherent stability. Thermozymes usually included large groups of most important economical enzymes such as amylases, lipases, pectinase, cellulases and proteases. Amylases have important application in diverse industries such as baking, detergent, medicine, textile, paper, glucose and fructose production, starch processing and waste water treatment. Thus, attempts have been made to isolate a thermophilic microorganism producing thermostable amylases
So, this thesis was focused in the isolation, purification and characterization of starch-degrading enzyme from thermophilic bacteria which was isolated from Egyptian water spring in El-Wahat El-Bahreya, Giza. To achieve the goal, the experimental work was conducted in four parts:
1. In the first part, liquid enrichment isolation procedure using hot water spring sample led to the isolation of 4 isolates that were able to grow at 65oC and showed good amylolytic activities. The most promising bacterial isolate was selected and subjected to molecular identification by using PCR and phylogenetic analysis by sequencing similarity search using BLAST program which revealed 97 % similarity to Geobacillus caldoxylosilyticus. The selected isolated (II) was identified as Geobacillus sp., IS-Mu-1 (QD459002). The biotype Geobacillus sp., IS-Mu-1 (QD459002) was selected as the experimental organism for further studies as it proved to be a true thermophile in addition of being the most active in enzyme production. The phenotypic and biochemical characterization were studied for the selected isolate which was Gram-positive bacilli with terminal endospore. The biochemical study revealed the ability of the isolate to utilize a great number of substrates such as glucose, yeast extract, beef extract, pectin, olive oil, tween 80, casein, gelatin, xylan, dextrin and starch.
2. In the second part, the effect of some factors on the bacterial growth were studied. The most effective factors were starch concentration, yeast extract concentration, temperature of the incubation and pH of the medium. from the obtained results, an optimum medium composition was suggested. The growth and amylase production were also monitored for the selected isolate. A portion (75 ml) of the medium dispensed in 250 ml Erlenmeyer flask was inoculated with 1 ml of 9 hrs old pre-culture. The inoculated flask was incubated for 24 hrs at 200 rpm at 65oC. The results studies revealed that the amylase production was usually associated with the bacterial growth and the maximum production of glucoamylase was appeared at the beginning of stationary phase for the bacterial growth after 7 hrs of incubation and after that time a DROP in enzyme production was noticed.
3. The aim of the third part was production, identification and purification of the culture filtrated (enzyme). Firstly, the culture filtrate was obtained by centrifugation of the bacterial culture (1000 ml) then concentrated to 70.0 ml throughout ultrafiltration system (Amicon system) with filter cut-off 10000 kDa. Secondly, identification of previous culture filtrate by analysis of enzyme end product(s) using TLC technique. The obtained results revealed that the end product of enzyme action on different substrates (amylose, pullulan and starch) was only glucose, and these results led to identify the nature of the purified enzyme as glucoamylase. Finally, acetone (60 %) precipitation was the first stage in purification of glucoamylase, the enzyme was purified to almost 3.50 fold and the total proteins were reduced from 92.43 to 12 mg/ml. The purity of enzyme was reached to 129.36 fold after using the anion exchange column of FPLC system, where the high reduction in the total protein was observed. The maximum purity for the glucoamylase was obtained after elution of the enzyme by sodium acetate and sodium chloride buffer 20 mM, pH 7.0 through gel filtration column. The purity reached to 242.58 fold and the mount of total protein reached to 0.017 % from the origin total protein. The test for the purity, determination of the molecular weight and the zymogram for detection of the amylase activities within the gel were performed by using SDS-PAGE technique. The obtained results revealed the presence of only one single protein band after the purification process with a molecular weight of 59 kDa.
4. In the fourth part of the work, the purified glucoamylase was characterized and different factors were selected to identify the physicochemical properties of the enzyme. The results showed that 60oC was the optimum temperature and the enzyme had thermostability property, about 20 % from its original activity was lost after exposure to 60oC for 60 minutes. Glucoamylase activity losing has positive trend along with incubation time and temperature. The enzyme preferred to work in acidic condition, with a pH optimum of 5.0. Some metals (Mg+2, Mn+2, Zn+2, Cu+2, Ag+2, Al+3 and Co+2 and Hg+) caused strong inhibition for enzyme activity, while Ca+2 ion enhanced the activity. Also, chelating agents (EDTA, sodium citrate, sodium oxalate and sodium tartarate) caused strong inhibition for enzyme activity. The substrate specificity of the purified enzyme was tested towards several substrates with different degrees of polysaccharides chain length. The glucoamylase activity with maltopentaose was better than starch from potato. The activity progressively decreased with increasing the length of the chain. The enzyme kinetic studies using soluble starch as substrate indicated that the enzyme activity was dependent on the concentration of the substrate. Lineweaver Burk analysis revealed that Km value of 8.26 and Vmax value of 3.26 U/mg.