الفهرس 
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Abstract
The present study was carried out to produce gluconic acid by fermentation using the most potent fungal species among the tested microorganisms. Also, studying some physiological and biochemical factors for obtaining the optimal production of GA. Utilization of some wastes for GA production as more economic carbon source than glucose for GA production. Partial purification of glucose oxidase and its immobilization on suitable carrier. Finally, studying some properties of free and immobilized glucose oxidase.
The present work was conducted according to the following plan:
1. Survey and collection of relevant literatures.
2. Screening experiments to choose the most potent fungal sp. producing GA.
3. Studying the physiological and biochemical factors affecting GA production.
4. Utilization of some wastes for the production of the acid.
5. Partial purification of glucose oxidase and studying some properties of the enzyme.
6. Immobilization of partially purified glucose oxidase.
To achieve this goal:
• Studies were made on 9 microbial cultures which manifested large variations in capacity of gluconic acid production by using Liu et al. (1999) medium having the following composition (g/l): glucose, 100; K2HPO4, 0.35; Urea, 0.55; MgSO4.7H2O, 0.15 and CaCO3, 30.
• Penicillium funiculosum AUMC 302 was selected as the experimental fungus throughout this work since it gave the highest yield of gluconic acid (5.21 g/100ml GA) after 5 days fermentation on the previous medium.
• Different experiments were conducted to study the effect of some physiological and biochemical factors on GA production.
• Gluconic acid produced by P. funiculosum was markedly affected by the type of fermentation. The submerged technique was superior to the static technique.
• The effect of different fermentation periods was studied where GA production gradually increased with the increase of the fermentation period till it reached maximum (5.8 g/100ml GA) after 4 days of fermentation then it began to decline with time.
• The effect of the composition of the fermentation medium was studied where 4 different fermentation media were used. The maximum GA production (5.8 g/100ml GA) was recorded by Liu et al. (1999) medium, having the following composition (g/l): glucose, 100; K2HPO4, 0.35; Urea, 0.55; MgSO4.7H2O, 0.15 and CaCO3, 30.
• The effect of substitution of glucose in the basal fermentation medium by other carbon sources namely; mannose, galactose, fructose, lactose and sucrose was studied. Yet, glucose was proved to be the most suitable carbon source.
• The effect of different glucose concentrations on GA production was examined. The production increased with increasing glucose concentration reaching a maximum (7.39 g/100ml GA) at 12.5 g/100ml after which it decreased.
• The effect of substitution of urea in the basal fermentation medium by other nitrogen sources was studied. Different nitrogen sources in the culture medium were investigated on equivalent nitrogen basis. These included organic (yeast extract, beef extract, casein, peptone, corn steep liquor, malt extract and soy bean) and inorganic (sodium nitrate, ammonium sulphate and di-ammonium hydrogen phosphate) nitrogen sources. Among these nitrogen sources, yeast extract afforded maximal GA production (7.65 g/100ml GA).
• The GA production increased with increasing yeast extract concentration till it reached a maximum value with the concentration 0.7 g/100ml of yeast extract (8.21 g/100ml GA), then it began to decrease.
• The effect of inoculum size on GA production was conducted by testing different inoculum sizes (1, 3, 5, 7 and 9 ml/100ml fermentation medium). Inoculum size 3ml/100ml fermentation medium showed maximum production of GA (8.30 g/100ml GA).
• Gluconic acid production by P. funiculosum was influenced by pH of the fermentation medium. Different initial and buffering pH values were tested. Initial pH values [4, 4.5(control), 5, 6, 7, 8 and 9 using 1N NaOH or 1N HCl] and buffering pH values [5, 6, 7 and 8 using phosphate buffer]. Initial pH value 5 showed maximum GA production (8.56 g/100ml GA).
• Changing MgSO4.7H2O concentration had an effect on GA production.
• Changing K2HPO4 concentration had an effect on GA production.
• The influence of some additives (sodium chloride, ferrous sulphate, manganese chloride and zinc sulphate, methanol, soy oil and hydrogen peroxide) was tested. All the tested additives had inhibitory effect on GA production except sodium chloride and all the tested levels of hydrogen peroxide. Hydrogen peroxide (1.6 ml/100ml fermentation medium) showed the greatest activating influence on GA production (9.30 g/100ml GA).
• Utilization of some wastes (glucose syrup, sugarcane and beet molasses) for GA production instead of glucose was also studied in which glucose syrup was superior among the tested wastes.
• Partial purification of the crude glucose oxidase produced by P. funiculosum was carried out by fractional precipitation with ammonium sulphate. The fraction obtained at 60-80 % ammonium sulphate saturation showed the highest specific enzyme activity and purification fold (40.71 U/mg protein & 6.76 fold, respectively).
• The partially purified glucose oxidase was immobilized by entrapment using different concentrations (1-4%) of sodium alginate as carriers. The enzyme immobilized by entrapment in sodium alginate (3 %) showed the highest immobilization yield of 94.11 %.
• Comparative studies between the properties of the free and immobilized enzymes were carried out. The optimum pH value for the free and immobilized glucose oxidase enzymes was 5.6. pH stability of immobilized enzyme was significantly improved by immobilization process. The immobilized enzyme was more stable at acidic and nearly alkaline pH values than the free one. The immobilized glucose oxidase enzyme was optimally active at the same temperature of the free partially purified enzyme (30°C). The thermal stability of the immobilized enzyme was significantly improved by the immobilization process. The immobilized enzyme was significantly more stable than free enzyme at different temperatures.
• Different concentrations of substrate (glucose) were tested. Both immobilized and free enzymes were optimally active at glucose concentration (1 g%).
• The operational stability of the immobilized enzyme was evaluated in repeated batch processes. The retained activity after being used for 12 cycles was 45.4 % of the original activity of the immobilized enzyme.