الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
This work included the selection of the best local fungal isolate for β-mannanase production under optimal conditions and using selected fungal biomass which produced as byproduct form β-mannanase production process as absorbent agent for chelation of some heavy metal ions especially Cu+2 from contaminated aqueous solutions.
Initial experiment was carried out for screening of some local fungal isolates to select the highest an extracellular β-mannanase producing strain in both static and shaking cultures. Screened isolates at the static fermentation achieved higher β- mannanase production than shaking one.
Among forty fungal isolates, the isolate NRC3 recorded the highest specific activity (13.82 U/mg) with β-mannanase activity (27.64 Um l-1) and protein content (2.00 mg/ml), this isolate was identified by Molecular technique (18 S rRNA) as Aspergillus tamarii NRC3. Negative result of aflatoxins test was obtained in A. tamarii NRC3 culture filtrate. So, it was used throughout the study.
Optimization of the fermentation parameters for maximal β- mannanase activity was performed. The effect of different carbohydrate sources and natural substrates on the productivity of β-mannanase by A. tamarii NRC3 was investigated. Locust bean gum (1%) recorded the best substrate for β-mannanase production (30.89 Um l-1) while sodium nitrate (0.2%) as a nitrogen source gave the highest β-mannanase activity (31.00 Uml-1). The optimum initial pH for the best A. tamarii NRC3 β-mannanase activity was pH (5). Inoculum’s age (6 days) and inoculum’s size (2spore/ml x107) of A. tamarii NRC3 recorded the β-mannanase activity of 31.67Uml-1. The favorable volume of production medium gave the best β-mannanase activity (31.70 Uml-1) was 50 ml. Seven days of static incubation period and temperature (25 ºC) were the the optimum factors for the highest β-mannanase activity (31.75 UmL-1; 31.88 Uml-1, respectively). Surfactants (tween 20 and tween 80) hadn’t a positive effect on mannanase production and A. tamarii NRC3 couldn’t grow at presence of SDS.
Stabilization of mannanase production by immobilized A. tamarii NRC3 cells was studied. In this part A. tamarii NRC3 cells were immobilized on water insoluble carriers by different matrixes these included agar, agarose, gelatin, ca-alginate, and polyacrylamide. Of all the tested matrixes, agarose (3%) was found to be the suitable matrix to produce mannanase enzyme by immobilized A. tamarii NRC3 cells with a powerful effectiveness factor (1.01 Cimmo/Cfree). 5 x103spore/block and 3g were the optimum initial cell concentration loaded in agarose matrix and block weight, respectively. 90 min., was the favorable curing time for stability of agarose blocks. The immobilized cells obtained under the optimal conditions were repeatedly used for number of successive cycles, and their activity was retained for about 9 cycles. The total β-mannanase yield by using immobilized cells for nine cycles was 277.50 U/ml, while from free cells for one cycle was 44.33 U/ml. The normal volumetric productivity with the immobilized cells was 1.65 U/ml/h, while it was 0.26 U/ml/h with free cells.
The purification studies of extracellular β-mannanase from Aspergillus tamarii NRC3 was carried out. Purified enzyme with 4.97 purification fold and 15.46% enzyme recovery was obtained from efficiency purification three steps including acetone precipitation followed by two chromatographic procedures using ion-exchange chromatography(DEAE-cellulose column) and gel filtration chromatography (Sephadex G-100 column). The molecular mass of the purified enzyme was determined by polyacrylamide gel electrophoresis (SDS-PAGE) technique and it was estimated to be approximately 80 kDa.
The purified enzyme appeared to be rich with high concentration of glutamic acid (3.98 %) and aspartic acid (3.39 %) among other amino acids at amino acid analysis test.
The purified Aspergillus tamarii NRC3 β-mannanase was optimally active and had the best conformation at pH 6.0 and 40°C. The purified enzyme kept stable at pH range of 5.0–6.5 while it’s thermostable was up to 60°C for 5 min. The Km and Vmax values of purified β-mannanase were found to be about 0.10 mM, and 16.10 μmol/ml/min, respectively, with an isoelectric point at pH 4.2.
The inhibition effect of some metal ions and some chemical reagent on purified enzyme activity especially Hg+2 which made a complete inhibition referred to Aspergillus tamarii NRC3 β-mannanase was not a metalloenzyme and had cysteine residues presence at or near the active site and play an important role in the substrate binding of the enzyme.
The DP value of product performed from hydrolysis process of LBG by Aspergillus tamarii NRC3 β-mannanase was ~3.5 so based on that DP value we concluded that MOS had formed.
TLC for assessing the action mode of the enzyme for MOS production gave various products were including mannobiose (M2), mannotriose (M3) and mannotertrose (M4), and mannopentaose (M5) and these confirmed that Aspergillus tamarii NRC3 β-mannanase was an endo-type-enzyme.
Results of evaluation of possible prebiotic potential of the (MOS) showed that MOS had an enhancement effect on the growth of Lactobacillus sp. (L. rhamnosus, L. reuteri, and L. acidophilus) and inhibition effect on the tested pathogenic strains growth indicated that MOS had prebiotic potential.
Investigation of kinetic and mechanism of Cu2+ uptake by Aspergillus tamarii NRC3 biomass which formed as a by-product from mannanase production process illustrated that Aspergillus tamarii NRC3 biomass play a successful and an efficiency role for removal, recovery and reloading of some heavy metal ions especially copper (II) from aqueous solutions and an industrial effluent.
The initial Cu+2concentration had a role on the Cu+2 uptake by fungal biomass where amount of Cu+2 uptake by Aspergillus tamarii NRC3 biomass, increased as the initial concentration increased and the removal was efficient ( 92.31%) in solution containing up to 80 ppm Cu+2. The rate of Cu+2uptake from Cu+2solution proceeded rapidly and it appeared to be virtually complete during the initial 5min (92%), the maximum uptake of Cu+2 was appeared at 30°C, pH 5, biomass concentration 5g w/w. On the other hand, the fungal biomass was able to remove considerable proportion of Pb2+, Co+2, Ni2+, Fe+3, and Cr3+ in addition to Cu2+. The uptake of Cu+2 with pretreated biomass by boiling with water, soaking in 5% KOH or soaking in 1.85x10-5 NaN3 indicating that uptake of Cu+2 by fungal biomass was independent of cellular metabolism. Recovery of the sorbed metal ions by desorbing agents and the potential reuse of the regenerated biomass in metal ions uptake (reloading) showed that the greatest efficiency of desorption was observed with the use of 0.1N HCl (Cu+2,61.23%; Pb2+,69.22%; Co+2, 37.03%; Ni2+, 57.65%; Fe+3, 43.53%; and Cr3+, 51.48%) and reusability of spent adsorbent can be assessed by its adsorption performance in successive adsorption- desorption operation. The effluent from the Egyptian Company for leather tanning contained Cr as the major heavy metal pollutant beside minor traces of Cu, Pb, Ni, Fe, and Co was used on small scale experiment for evaluation of the fungal biomass efficiency in metal ions. where the efficiency of Cu2+ and Co2+ removal was high (90.94%, and 60.0%, respectively) followed by Ni2+, Fe3+, Pb2+, and Cr3+ (40.0, 34.47, 29.13 and 11.45%, respectively) also, the biomass treatment resulted in the removal of the color and the pungent odour from the effluent and the biomass itself gained the coloration of the effluent.