الفهرس 
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Abstract
The present investigation was conducted to study the ability of different marine endophytes to secrete chitinase enzyme and to improve the productivity of this enzyme by different processes. Furthermore, to study the stability of the enzyme and the reusability by immobilization into different carriers and to investigate its importance as inhibitor to the growth of different plant pathogens. Finally, to estimate its toxicity against the harmful mosquito.
1. The first part was correlated with a screening study where
4 different marine sources were used; algae (Green and Red); Black sponge and Soft coral were collected from Hurghada, washed and used to isolate the endophytic fungi which were tested for chitinase production activity. The results showed that among 32 fungal endophytes only eight isolates were promising producers of chitinase enzyme. Penicillium chrysogenum was the superior one where 15.42 U/mL was obtained followed by Aspergillus versicolor (12.11) U/mL, thus Penicillium chrysogenum was chosen for further experimental study.
2. The promising fungus was genetically identified by 18S rRNA (Ribonucleic acid) sequencing technique and the
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results showed that the active strain was mostly related to Penicillium chrysogenum strain MF077263.1 (99% identity). Moreover, the toxicity of the secondary metabolites in the fermentation medium was investigated by HPLC analysis which confirmed that the products of the isolate were free from any kind of toxic substances.
3. The second part is the optimization of the production medium where the isolate was exposed to a range of temperature from (28 to 37) oC and the given data proved that maximum yield of the enzyme (82.7) U/mL was got at 30oC. By studying the time of incubation it was noticed that 7 days of incubation was the most suitable (167.8) U/mL for chitinase activity. The influence of pH is one of the important factors that affect the efficiency of the enzyme. After the incubation of the fungus at different pH values, the acidic medium at pH 3.5 was the most favorable for maximum production of the enzyme where (290.4) U/mL was obtained.
4. By studying the role of different substrates (colloidal chitin, commercial chitin and shrimp crab) on the production of chitinase. The results showed that the commercial chitin was the most recommended substrate for
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the production of the enzyme where 331.8 U/mL was produced followed by colloidal chitin 292.4 U/mL.
5. The third part in this work was the statistical screening for chitinase production, Plackett–Burman Design (PBD) was constructed with 12 runs with different concentrations of nine variables. All the components in the madia exhibited variation of chitinase activity from 320 U/mL to
596 U/mL. The highest chitinase activity in the experimental runs (596) U/mL was produced at high concentration of chitin (7.5 g/L) in the production media while the lowest enzyme activity (320) U/mL was detected at the low concentration (2.5) g/l of chitin in the fermentation medium. The analysis of variance (ANOVA) represents the effect of independent variables on chitinase activity. It was shown that the chitin has the highest influence (p=0.004) followed by tryptone (p=0.009) and yeast extract (p=0.023) given by p˂0.05. The coefficient of determination R 2 of 0.995 in the experimental model means that the model could explain up to 99.5% of the total variations in the system and only 0.5% of the total variations were not explained by the model. The next experiment involved the optimization of the significant factors. The central composite design seeks the optimal
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conditions for a multivariable system with much fewer experiments than a full factorial design method. On the basis of PBD which revealed that three factors, chitin, yeast extract and tryptone had significant influence on chitinase activity.
6. The spore suspension of the fungus was inoculated in different volumes of the fermentation medium from (250 to 2000) mL. The results showed that the activity of the enzyme was slightly decreased by increasing the volume of the fermentation medium from (550 to 476.4) U/mL, but the total enzyme yield was got in the largest volume (2000 mL) and decreased by decreasing the volume of the fermentation medium.
7. Solid state fermentation (SSF). The impact of agro- industrial waste materials was studied by screening of more than 10 sources as a sole nutrient in the fermentation medium of the fungus. A sharp increase in enzyme activity was noticed at all used sources. The most active waste was the combination of wheat bran (4g) and chitin (1g) where the chitinase activity reached 6577.2 U/mL followed by Wheat bran (5g) (4485.4) U/mL, then rice straw (5g) (4300.5) U/mL. Also rice straw (4g) with Chitin (1g) was a
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promising nutrient which showed (3278.9) U/mL activity and chitin (4g) with wheat bran (1g) where (2198.9) U/mL enzyme activity was produced.
8. Production of chitinase by the use of the aforementioned low materials was confirmed by TLC sheet analysis. The results appeared as spots at the same line of the standard N- acetylglucosamine which confirmed the production of exochitinase enzyme in SSF which breakdown the chitin and produced the end product N-acetylglucosamine. This result was confirmed by HPLC analysis where the beak of N-acetylglucosamine in the SSF medium was developed at the same retention time (7.2) min of the standard N- acetylglucosamine.
9. The fourth part in this investigation was the partial purification of the enzyme by the use of two solvents; Acetone and Ethanol by increasing the concentrations from (30 to 90) % where the fold of purification increased at (30 and 60) % to yield 10.5 and 26.6 which indicated the precipitation of two different enzymes (E1 and E2) by acetone. The same result was got by fractional precipitation with Ethanol at (30 and 70) % where the fold of purification for both enzymes reached 4.7 and 3.3.
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10. The fifth section is the immobilization of the crude enzyme (E1) via a covalent binding into the bentonite where 100% immobilization yield was observed and by the addition of cross linkers, the results showed a decrease in the immobilization yield of the enzyme by Glutaraldehyde (GA) and epichlorohydrin (EPi) where (46.9 and 28.4) % immobilization yield was obtained. By immobilization of the enzyme onto Gelatin, the immobilization yield was 73.84%, thus the immobilization onto bentonite was used for further studies.
11. TEM and FTIR spectrum of enzyme immobilized onto clay (modified bentonite) confirmed a complete loading of the enzyme onto the carrier via covalent binding. Process by the presence of the binding groups (–CH2 and –NH2).
12. The sixth section is the characterization of free and immobilized chitinases, maximum temperature for enzyme activity was converted from 50 °C to 55 °C after the immobilization due to protein protection which led it to tolerate the raise of temperature from (45 to 60) oC with a slight decrease in relative activity. The activation energy was calculated for the free and the immobilized form to record 17.46 KJ•mol−1 and 10.55 KJ•mol−1 respectively.
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13. By studying the effect of pH values on free and immobilized enzymes, it was noticed that the free and immobilized enzymes recorded maximum activity; (100%) at pH 5 and 6 respectively. The alkaline pH led to a sharp lost in enzyme activity.
14. The thermal stability study recorded 100% stability for the free and the immobilized form at 50-55ºC and different time intervals (30-120 min.). While the pH profile for both enzyme forms recorded a complete stability at pH 5, 6 at the different times intervals.
15. Thermodynamic parameters were evaluated such as activation energy for denaturation (Ed), half-lives T1/2 and D-values (decimalreductiontime. Ed value for the free and immobilized form were determined to be 97 kJ mol−1 and 101 kJ mol−1 free and immobilized form respectively. The t1/2 for the free and the immobilized form recorded 230, 138, 77 min. and 345, 172, 115 min respectively. The D value for both enzyme forms were 766, 460, 255 min. and 1150, 575, 383 min. First order of thermal deactivation (Kd) of free and immobilized Penicillium chrysogenum chitinase was determined. The result showed that the kd of the immobilized enzyme was lower than the free enzyme. For
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instance, at 50 ºC the Kd of the free and immobilized were 0.0031 (min−1), 0.0022 (min−1) respectively.
The ΔH values were 72.033, 72.47, 72.8 for the free form and 76.033, 76.47, 76.8 for immobilized form respectively.
While the ΔG was calculated as 765, 736, 714 and 737, 743, 721 for free and immobilized form respectively. ΔS recorded negative results in both the free and the immobilized enzyme.
16. The effect of salts on the activity of chitinase was studied. The presented data showed a negative effect of most tested salts on the potency of the free enzyme which decreased the activity to half its initial value. Whiles, other salts e.g. KI, CuSO4, (NH4)2SO4 and CaCO3 caused activation of the immobilized enzyme.
17. Effect of substrate concentration on efficiency of the enzyme showed a gradual increase in enzymes relative activity by increasing the concentration of the substrate. Km was 0.06 and 0.1; Vmax was 10 and 11 for free and immobilized enzymes.
18. Operational stability of immobilized exochitinase exhibited a complete activity (100) % for the first two
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cycles, a slight decrease in enzyme yield (98)% was noticed at the third evaluation. After the fourth cycle, the activity of the enzyme reduced to 50%.
19. The seventh part is the toxicological evaluation against the late 3rd instar larvae of Cx. Pipiensas. LC50 values for 3rd instar larvae of Cx. pipiens after 24 hours were (3.974 and 27.754) ppm for free and immobilized chitinases and after 48 hours were (0.538 and 5.635), respectively. On the other hand, LC90, values after 24 hours showed (2720.886 and15483.16) ppm and after 48 hours (443.004 and 5046.777), respectively. The obtained data indicated that free enzyme was the most potent against Cx. pipiens larvae.
20. The second application is the antifungal activity of the free and immobilized exochitinase. Both enzymes showed a significant toxicity against Curvularia spicifera. A clear inhibition zone (2) cm was noticed for 20 % free enzyme and the inhibition activity of the immobilized enzyme increased gradually by increasing its concentrations (10, 30, 50, 70 and 100) mg.