الفهرس 
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Abstract
Summary
The present study investigated the chitinolytic activity throughout the following steps:
1. Survey of different isolated microorganisms for their chitinolytic activity and selection of the most potent isolate. from all tested microorganisms, the honey isolate, Aspergillus awamori EM66 was the highest producer of chitinase (270.1& 0.55( mU/ml for exo and endochitinase respectively) after six days incubation period at 30°C and was chosen for further investigation.
2. Optimization of chitinase production by Aspergillus awamori EM66 was carried out through using (RSM) response surface methodology which included first, Plaket-Burman design in which fifteen variables had been studied to determine the significant factors for chitinase production. Of all the tested variables, chitin, MnSO4, CaCl2, FeSO4, NaNO3, K2HPO4, CuSO4 and incubation period showed positive effect on exochitinase activity where as wheat flour, soya bean, glucose, pH, MgSO4, ZnSO4 and dried insect, were contributed negatively. The exochitinase production using the Plaket-Burman design scored 2045 mU/ml which presented about 7.57 fold increase in the enzyme activity. Then the variables showing confidence level above 98% in the Plackett–Burman design were selected for further optimization using the central composite design (CCD) to investigate the optimal concentration for each, they are (Chitin, MnSO4 and CaCl2). An overall 22.2 - fold increase in exochitinase activity was being achieved after application
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of RSM. This reflects the necessity and value of optimization process.
Concerning the endochitinase optimization, CuSO4, glucose, Soya bean, CaCl2, K2HPO4, MgSO4, MnSO4, ZnSO4, chitin and incubation period showed positive effect on endochitinase activity whereas Dried insect, NaNO3, FeSO4, pH and wheat flour were contributed negatively. The enzyme activity measurement using this technique was 2.38 mU/ml, which presented about 4.33-fold increase in the enzyme activity. The most significant medium components (glucose, soya bean and CuSO4) showing 98% confidence level in the endochitinase Plackett–Burman design, were further investigated for their optimal concentration using (CCD). An overall 10.2 - fold increase in endochitinase activity was achieved after application of RSM. This reflects the necessity and value of optimization process
3. Partial purification of the exochitinase using different methods including fractionation by ethanol, acetone and salting out with ammonium sulphate. The ethanol fraction at 30% concentration had the highest purification fold about 18 times and undergone the following investigations.
4. Immobilization of the partially purified enzyme using entrapment and covalent bond methods was carried out. Of the tried methods, immobilization on K-carragenan gel beads using covalent bond technique had the highest immobilization yield about 93%.
5. The optimal incubation time for the assay was 10 minutes for both enzyme forms (free and immobilized form).
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6. Optimization of the immobilization process which included optimization of loading time and protein capacity was carried out. The optimal loading time was 18 hours with about 97% immobilization yield, the enzyme 1:2 concentration scored 100% immobilization yield.
7. characterization of both forms of the enzyme in a comparative study had been carried out which included first, optimum temperature, which achieved 35 and 45°C for free and immobilized form respectively.
8. The hydrogen ion concentration at which maximal enzyme activity took place was pH 5.0 using Na acetate buffer for both enzyme forms.
9. Thermal stability of free and immobilized enzyme had been studied; both forms retain their complete activity at temperatures ranging from 35 to55°C up to 120 min incubation period. A relatively slight decrease in the activity of both enzyme forms was recorded after 120 min of exposure at 60 & 65 °C, the free and the immobilized enzyme retained about (85 ,75%) and (86 , 76%) of their activity at these temperatures respectively. At 70 and 75°C, the effect of immobilization was clear, as there was complete loss in the partial pure enzyme activity where as the immobilized form remained about 71% of its activity at 70°C after 120 min and kept half of its activity at 75°C for 60 min.
10. The enzyme kinetics (Km and Vmax) had been calculated with the aid of Lineweaver-Burk plot figure. The apparent Km values of the enzyme was found to be 0.33 & 2.0 mg/ml and V max as 13.33& 40.0 U/mg /min for free and immobilized form respectively. Maximum enzyme activity occurred at 2.0 and 1.5 mg concentration for partial pure and immobilized enzyme respectively.
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11. The effect of metals showed that, from all metals tested K+ had the highest stimulatory effect on the partially purified enzyme recording only about 22% increase in activity. On the other hand, Mn2+ had a higher stimulatory effect (77%) towards the immobilized form. Mercurey had inhibitory effect on both forms of exochitinase and the enzyme retained only about half of its activity. The other metals tested also resulted in varied stimulation effect especially in case of the immobilized form.
12. The effect of NaCl study showed that, enzyme activity was stimulated by NaCl addition and maximum increase occurred at 0.75M concentration with about 16 and 20% increase in partially purified and immobilized enzyme respectively. Moreover, the enzyme showed great stability against the gradual increase in NaCl concentration, since it realized complete activity until 5M concentration, after that a gradual and not sharp decrease in enzyme activity occurred.
13. The immobilized enzyme showed a great reusability character, the enzyme retained its complete activity up to 28 cycles, after which a gradual decrease in activity occurred.
14. The immobilization process afforded the enzyme with great shelf stability, the enzyme retained its complete activity along the period of 12 weeks at 4˚C and both forms keep their complete stability at freezing degree for more than twelve months.
15. The free form of Asperigillus awamori EM66 exochitinase showed great antifungal activity against Fusarium oxysporum which causes the wilt disease for different plants after only 48h incubation. On the other
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hand, the enzyme had no effect on the other plant pathogenic fungi, (Asperigillus niger, Fusarium solani, Sclerotium rolfsii and Alternaria sp), tested under this experiment, which clears the specificity of this enzyme towards some pathogens only.
16. Application of the enzyme in the biocontrol field had been tested. The enzyme had a great effect on both larva and pupal stage of the three types of lepidopteran pests (the black cutworm, Agrotis ipsilon and the Egyptian cotton leaf worm, Spodoptera littoralis and the great wax moth, Galleria mellonella). The enzyme effect was more severe on Galleria mellonella, the great wax moth recording about 84% mortality for the larva and 8% for pupal scoring total mortality about 92%. 86.67% is the total mortality in Spodoptera littoralis while less effect was noticed at Agrotis ipsilon which reached to 65.67%.
The new isolate A. awamori EM66 is suggested as a new potential candidate for exochitinase enzyme production, which has many biotechnological applications