الفهرس 
CyclodextrinsOnly 14 pages are availabe for public view
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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.
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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.