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Abstract Cyclodextrins (CDs) are cyclic homogeneous non-reducing oligosaccharides consisting of 6 to 12 glucose units joined by α-1, 4- glucosidic linkages. Generally, the most common available CDs to be synthesized are composed of 6, 7 and 8 glucose units named α-, β-, and γ- cyclodextrins, respectively. The interior of CDs is hyDROPhobic and its surface is hyDROPhilic. Due to this structure, CDs can form an inclusion complex with various organic and inorganic molecules which can change the physicochemical properties of the guest molecule; thus increasing their water solubility and stability. These properties made CDs became increasingly important as molecular encapsulator for industrial application particularly in food, dairy products, pharmaceutical and cosmetics industry as stabilizing agents, emulsifiers and antioxidants. CDs are synthesized from starch by the cyclization reaction of cyclodextrin glucanotransferase which converts starch to cyclodextrin through cyclization reaction. Cyclodextrin glucanotransferase can be found in several bacterial specially Bacillus species. The main objective of the present work is isolation and screening of bacterial strains from Egyptian soil that have the ability to produce cyclodextrin glucanotransferase used in cyclodextrin production. This study had focused on the optimization of the fermentation conditions, purifying and characterization of the purified enzyme. Summary 88 The experimental results in this study clearly showed that:- 1- Fifteen soil samples were collected from rice, wheat, corn, sweet potato and potato fields in shebeen El-kanater (El Kalubia governorate: Egypt). 2- from these samples, eight alkalophilic bacterial colonies were isolated they were able to hydrolyze starch. They were screened for their ability to produce cyclodextrin glucanotransferase. 3- Of the tested bacterial isolates, two isolates (from potato field) were able to produced cyclodextrin glucanotransferase based on the formation of yellowish zones around their colonies in the plates. 4- One isolate exhibit sufficient productivity of CGTase was identified as alkalophilic Bacillus subtilis by Micro-log system, it was chosen for enzyme production using shake flasks. 5- Production of cyclodextrin glucanotransferase by Bacillus subtilis was investigated in the standard medium at 37ºC, pH 10.5, and inoculum size 2% of the culture under shaken condition of 200 rpm. 6- With the aim of enhancement of enzyme production, the optimal nutritional condition affecting the activity were studied by tested some carbon sources in the nutritional media such as rice starch, corn starch, sweet potato, potato starch, dextrin and glucose. 7- Rice starch was the best carbon source for maximum enzyme production at 2.5 g/L. An increase in rice starch concentration above this, led to a decrease in enzyme production. While, glucose had no effect on enzyme production. 8- It was also found that 5g/L peptone combined with 5g/L yeast extract were the most favorable nitrogen sources for maximum enzyme production. Summary 89 9- To improve the CGTase production, some affecting factors were tested such as incubation temperature, pH and incubation period. The result showed that the best factors were 37ºC incubation temperature at pH 10.5 and incubation period 24 h. 10-The fermentation process was carried out in a bioreactor and from this process, the biomass, produced glucose concentration, starch consumed, and decrease in pH and also cyclodextrin production were calculated. 11-Three precipitating agents were used for employing the partial purification of the CGTase, namely acetone, ethanol and ammonium sulphate. Acetone precipitation afforded 46.6% recovered activity and 10.5 U total activity. On the other hand, ethanol precipitation afforded 40.4% recovered activity and 9.1 U total activity. While, ammonium sulphate afforded 11% recovered activity and 2.48 U total activity of the crude CGTase. 12-The previous results showed that acetone at 50% was the most promising treatment with the highest recovered activity. Further purification of 50% acetone fraction of CGTase was preceded chromatographically as follows. A- Gel filtration through sephadex G-100 This purification step resulted in 24% recovery of the applied protein with 162 U total activity and exhibited 38-fold purification. B- Ion exchange chromatography on DEAE-cellulose DE52 Further purification was done through DEAE-cellulose DE52. Elution with 0.1 M, 0.2 M and 0.3 M NaCl in 10 mM Tris-HCl buffer, pH 8.0. The major pure protein was separated in a component which contains Summary 90 all total activity 52 U, specific activity 57.7 U/mg and 7.7% recovered activity. 13- Checking of CGTase purity was conducted in a disc poly acrylamide gel electrophoresis where it migrated as a defined protein band with molecular weight 74.1 kDa. 14- Some properties of the purified CGTase were studied, the results revealed that the purified CGTase could reach its maximum activity at optimal pH and temperature 7.0 (using 10 mM Tris-HCl buffer) and 60ºC, respectively. 15- The purified CGTase exhibited the thermal stability at 50ºC for 1 h. At the elevated temperature to 70ºC, the residual activity decreased to 17%. When the temperature increased to 90ºC, the enzyme loss 55% of its activity. The enzyme was stable from pH 7.0 to 8.0. 16- To study the effect of activator and inhibitor substances on the purified CGTase, Cu2+ and Fe2+ ions activated the enzyme by 105 % and 109 %, respectively. While, Ca2+, Mn2+ and Cd2+ had no influence on the enzyme activity. The enzyme was slightly inhibited by Ba2+, and strongly inhibited with Mg2+, Zn2+, Al3+ and K+. 17- Km and V max. values of the purified CGTase in the reaction mixture were 2.2 mg/ml and 7.8 mg β cyclodextrin /ml/min, respectively. The purified enzyme showed a relatively high affinity for the soluble starch. |