الفهرس 
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Abstract
The present investigation deals with, in general, isolation, studying of fungi inhabitant in honey and parameters affecting their growth. Also, it includes comparative studies between the enzymes produced from these strains and enzymes present in honey. In addition, the study aiming at searching the potent enzymes produced from the honey strains and optimization of fermentation parameters for maximal enzyme production, and partial purification of the potent enzyme. Moreover, the immobilization of enzyme was studied on different carriers. The comparative study between the properties of the partial purified and immobilized enzyme was also carried out. The results are summarized as follows:
(1) Initial steps of this work resulted in isolation of two strains from Yemen mountain honey. Morphological and genetic identification studies for both strains have indicated that the isolate are Aspergillus niger and Aspergillus awomori. Studies of 18S rDNA segment of both isolates were submitted to Genbank. After homology searching against the Genbank, the sequences of isolate A. niger was found to shear 96% similarity with those of A. niger EM77 (KF774181). Whereas, isolate 2 awomori was found to share 95% similarity with those of A. awamori EM66 (KF774180). The Random Amplified Polymorphic DNA (RAPD) was implemented by using 10 primers, a total of different reproducible RAPD markers were generated from the strain genomes the RAPD technique indicated coincidence between fungus A. niger EM77 (KF774181) and fungus A. awamori EM66 (KF774180) that is 51%. As for comparative study between honey strain and other same strain isolated from soil, results showed coincidence between fungus A. niger EM77 (KF774181) and A. niger isolated from soil was 58%. The coincidence between fungi A. awamori EM66 (KF774180) and A. awamori isolated from soil was 50%.
(2a) The second part of the study is aiming at exploring the parameters effecting fungal growth. The effect of temperature degree was studied, and the most suitable temperature for growth of both strains was 30°C. The influence of the initial pH value of the fermentation medium showed that maximum activity was obtained at initial pH value of 7. In addition, the effect of different carbon sources was investigated, showing that maltose was the most effective carbon source for the growth of both strains, followed by pectin then cellulose. Effect of nitrogen source was also studied, and the best organic nitrogen source was casein for both strains and ammonium chloride as inorganic nitrogen source. The effect of inoculum heating on both strains growth was studied by exposure the inocula at different degrees of temperature (40, 50, 60, 70 & 80˚C) for different times (10, 20 & 40 min.) The results indicated that both strains have grown after exposure at 70˚C for 40 min. with 10mg/50 ml in A. niger EM77 (KF774181) and 20 mg/50ml for A. awomori EM66 (KF774180). Investigating whether both fungi could grow under saline conditions, the results showed that both of them were characterized by the halo-tolerant feature; and found that A. niger EM77 (KF774181) and A. awamori EM66 (KF774180) could grow in the presence of 10 and 12 % NaCl, respectively.
(2b) Then, some biochemical & antimicrobial studies were carried out on both strains. Results showed that both strains have antimicrobial activity against Candida albicans, C. pseudotropicalis, and Pseudomonas sp. In addition, antioxidant activity was assessed by the reducing power assay; both straains exhibited great antioxidant activities and showed 82 & 83% antioxidant activities for A. niger EM77 (KF774181) and A. awamori EM66 (KF774180), respectively. Some enzymatic activities were investigated; both strains were able to produce different amounts of enzymes of different applications. The most pronounced activities were noticed with invertase, pectinases, levansucrase and chitinase. Comparing both strains investigated, A. niger EM77 (KF774181) was the best one regarding production of enzymes including invertase; therefore, it was selected to continue the studies with it throughout this work.
(3) The third part of the present study is aiming at investigating the effect of some physiological factors on invertase production by A. niger EM77 (KF774181). In the following series of experiments, unless otherwise stated, fermentation was carried out under solid-state fermentation for 72h at 30ºC.
Thus, the highest production of invertase occurred with wheat bran as carbon source (69.7 U/g). In addition, the effect of its concentration was studied and the maximum production of invertase attained by A. niger EM77 (KF774181) was 101.9 U/g at 5 gm of wheat bran.
Investigating the effect of incubation period showed that the maximum activity of enzyme (84.9 U/g) were obtained after 3 days of incubation period. The influence of initial pH value of the fermentation medium showed that maximum activity (194.7 U/g) was obtained at initial pH value of 5.5. In addition, studying the effect of temperature on invertase production showed that the maximum production of invertase was at 30°C (194.7 U/g).The effect of inoculum size on enzyme production was studied and the optimum activity was detected at 2 ml per flask (114.6 U/g).
In addition, the effect of aeration on the production of invertase was investigated; and the maximum production of invertase (114.7 U/g) was with conical flask 250 ml. The effect of different carbon source on the production of invertase was studied; sucrose supported the highest level of invertase production (144.4 U/g). Also, the effect of sucrose concentration was studied and 1% (w/v) sucrose concentration has supported the highest production of invertase. Moreover, the effect of different nitrogen sources on invertase production was also studied, and the results revealed that ammonium sulfate was a good nitrogen source for the production of invertase (158.5 U/g). Also, the effect of NH4SO4 concentration was studied; and it was found that 0.15% (w/v) was the optimum concentration for maximum invertase production (158.5 U/g).
Besides, the effect of various divalent cations (10 mM) on invertase production by A. niger EM77 (KF774181) was investigated. As for Hg, it inhibited the activity of invertase (48.7%). Whereas, Magnesium and Calcium activated the enzyme to 115.7 & 103 % respectively.
(4) The fourth part of this study was dealing with partial purification of invertase enzyme. It was conducted by fractional precipitation of crude enzyme by organic solvents (ethanol and acetone), and salting out by ammonium sulfate. The fraction precipitated at 40-80 ethanol showed the highest specific activity (6.03) and fold purification. Also, the immobilization yield was (69.69%).
(5) The fifth part of this study was the immobilization of invertase enzyme produced by A. niger EM77 (KF774181) on different carriers namely Polyvinyl alcohol (PVA) sponge, Polyvinyl Alcohol sponge + Agar (PVAG), Luffa (L), Luffa+agar (LG), pumic, rice straw, and agar capsule. Results indicated that the highest immobilization yield 70.2 % and activity 70.4 U were observed with PVAG sponge. On the other hand, re-operation stability of the immobilized invertase was tested, and the results showed that the immobilized enzyme was successfully reused 12 cycles. The enzyme kept its completed stability for 4 cycles (100%), which indicate the high stability of immobilized enzyme. On the other hand, at cycle number 12 the immobilized enzyme lost 29% of its original activity. Moreover, the effect of Time loading of invertase on (PVAG) was studied and the results showed that the highest activity of immobilized enzyme (70%) has got after 24h of enzyme loading at 4ºC. The effect of amount (in units) of enzyme loaded on PVAG was also studied; and it was found that the highest activity of immobilized enzyme (74.7 %) was got at 173.5 U of enzyme loaded after 72h at 4ºC.
(6) Comparison between properties of free and immobilized invertase enzyme is the sixth part of this study. Thus, the activity of free and immobilized invertase enzyme was assayed at various incubation times. Both free and immobilized invertase had maximum activity after 15 minutes.
Concerning the effect of temperature, both free and immobilized invertase had maximum activity at 50ºC. As for thermal stability of free and immobilized invertase, data showed, in general, that the immobilized enzyme was more stable than the free one. Since, at 60ºC, the immobilized enzyme retained 100% of its original activity after 60 min. while, the free one retains 40% only of its original at the same temperature. On the other hand, at 70˚C, the immobilized enzyme retained about 78.2% of its original activity after 60 min while, on contrary, the free one was completely inactivated under the same conditions.
Also, the effect of pH of free and immobilized invertase was studied. The results indicated that both free and immobilized invertase enzyme had maximum activity at 5.2 pH. As for the effect of pH stability of free and immobilized invertase, results showed that as the pH increase the enzyme activity decrease. This result was more pronounced in case of free than the immobilized one.
As for effect of metals on free and immobilized invertase activity, results indicated that HgCl2, MgSO4, CaCL2, FeSO4, CuSO4, KCl and PbNO2 had inhibitory effects on both of them. On the contrary, MnSO4, Na2SO4 and NaCl had stimulating effects on both activities. Effect of NaCl concentrations on free and immobilized invertase was also studied; and the results indicated that both free and immobilized enzyme were characterized by extreme halophilic property, since the highest enzyme activity was obtained at between 3.5 – 5.0 M and the enzyme activity work with good efficiency up to 6 M.