الفهرس 
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Abstract
L-Asparaginase (L-asparagine amino hydrolase, E.C.3.5.1.1) is an important enzyme that converts L-asparagine to L-aspartate and ammonia via hydrolysis reaction. Microbial L-asparaginase had gained significant importance due to its use in biosensors development and its application in the food and healthcare industries. L-Asparaginase was classified among the highly important biomedical and biotechnological groups of enzymes since it constituted 40 % of the whole global enzyme sales and one-third of the global demand as anticancer agent. This study reported the isolation of some yeast isolates from different sources, screening for L-asparaginase production using qualitative and quantitative assay, optimization of the production, purification of the produced enzyme, immobilization, characterization, and anticancer assay of the produced L-asparaginase.
►Seventeen yeast isolates were isolated from different sources including (Egyptian clover flower honey, Nigerian honey, banana peel, yoghurt, grape, sugar-cane bagasse, and soil).
►Twenty yeast isolates were assayed for the qualitative production of L-asparaginase using the rapid plate assay; the isolated seventeen isolates in addition to three yeast isolates were purchased from Northern Regional Research Laboratory (NRRL).
►The results revealed that only sixteen yeast isolates showed a pink zone around their colonies in the qualitative assay. Hence, they were considered as L-asparaginase producers. The most potent L-asparaginase producer showed the highest pink zone diameter was a yeast isolate (Ba) isolated from banana peel.
►All yeast isolates (the sixteen isolates) having a pink zone around their colonies in the qualitative assay was assayed for the quantitative determination of L-asparaginase using submerged fermentation.
►The results revealed that the yeast isolate (Ba) isolated from banana peel was also the most potent L-asparaginase producer having the highest specific activity. Hence, it was chosen for further studies.
►The yeast isolates were identified morphologically using CMA description and the identification was further confirmed using molecular identification for the most potent yeast isolate and Vitek 2 system for other yeast isolates.
►The most potent yeast isolate (Ba) isolated from banana peel was identified as Kodamaea ohmeri ANOMY based on the analysis of a part of the gene that encodes the D1/D2 domain of the nuclear large subunit (26S) ribosomal DNA.
►Other five yeast isolates were identified using Vitek 2 system. Yoghurt isolate (Y) was identified as Candida krusei, sugar-cane bagasse isolate (B2) was identified as Candida tropicalis, sugar-cane bagasse isolate (B1) was identified as Candida famata, grape isolate (G) was identified as Cryptococcus laurentii, and Nigerian honey isolate (Sh15) was identified as Candida guilliermondii.
►Kodamaea ohmeri ANOMY was inoculated into ten different fermentation media with different compositions. The results revealed that the L-asparagine fermentation medium with a composition of (g/L): L-asparagine (10.0), KCl (0.52), K2HPO4 (1.52), MgSO4.7H2O (0.52), and NaNO3 (0.30), was the optimum fermentation medium which yielding the highest L-asparaginase specific activity.
►This medium was optimized using the classical one factor-at-a time (OFAT) method. The results demonstrated that the elimination of K2HPO4 from the L-asparagine fermentation medium increased L-asparaginase production.
►The physical factors affecting the production of L-asparaginase by Kodamaea ohmeri ANOMY were optimized. The results revealed that the inoculation of the production medium with an inoculum of 36 h age at a temperature of 30℃ with an agitation speed of 150 rpm were the most preferable physical conditions for the production of L-asparaginase by Kodamaea ohmeri ANOMY.
►Based on the results obtained from the OFAT method, four important medium components for the production of L-asparaginase including; L-asparagine, NaNO3, MgSO4.7H2O, and KCl were determined with operating conditions such as the incubation period, inoculum size, medium volume, and pH using the PBD. The statistical method was aimed to screen the effect of different levels of the most influenced factors on the production process using a few experimental runs.
►The results of the PBD revealed that among the medium constituents; L-asparagine and MgSO4.7H2O had high F-values (Fisher’s F-test) and low P-values (P < 0.05) were the most significant factors for L-asparaginase production followed by NaNO3 and KCl. Whereas, among the operating conditions, the incubation period and medium volume were the most significant factor for L-asparaginase production followed by inoculum size and pH within their tested limits.
►Box-Behnken design (BBD) was used to investigate the interactive effects among the selected factors. The four most important factors (the incubation period, inoculum size, L-asparagine, and medium volume) were investigated in 27 experimental trials.
►The production medium for the maximum L-asparaginase specific activity (8500±578U/mg protein) was as follows (g/L): L-asparagine (7.50), NaNO3 (0.50), MgSO4.7H2O (0.80), KCl (0.80) associated with an incubation period of 5 days, a medium volume of 68 mL/250 mL Erlenmeyer flask, inoculum size of 5.60 %, and pH (7.0).
►The optimization process increased L-asparaginase production by 2.78-fold compared to the non-optimized medium.
►The produced L-asparaginase from Kodamaea ohmeri ANOMY was purified and eluted as a single protein after two purification steps using ammonium sulphate precipitation (80% saturation) followed by gel filtration chromatography using a Sephadex G-100 column.
►Kodamaea ohmeri ANOMY L-asparaginase was purified to 17-fold with a yield of 60% and a specific activity of 144476 U/mg protein.
►The molecular weight of the purified L-asparaginase from Kodamaea ohmeri ANOMY was determined by SDS-PAGE analysis, a single distinct band was observed. Using the standard molecular weight markers, the molecular weight of Kodamaea ohmeri ANOMY purified L-asparaginase was found to be 66 KDa.
►A total of twenty-eight carriers were prepared and applied for the immobilization of the purified L-asparaginase from Kodamaea ohmeri ANOMY using encapsulation and covalent binding immobilization methods. The results revealed that the Alg. carrier was the optimum carrier having the highest L-asparaginase activity and also, the highest operation stability (8 consecutive runs). Moreover, the immobilized L-asparaginase maintained more than 35% of its original activity after 6 consecutive runs.
►To improve the activity of the immobilized L-asparaginase on the Alg. carrier, it was found that the gel beads with a size of 450 µm and the addition of Mg2+ with a concentration of 4 mM increasing the activity of the immobilized L-asparaginase on the Alg. carrier.
►The influence of various metal ions on the activity of the purified L-asparaginase was evaluated. The results revealed that only the presence of Mg2+ increased L-asparaginase activity by 15.32%. While, the presence of Zn2+, Ni2+, Co2+, Li+, Na+, Ca2+, Fe2+, K+, Cu2+, Mn2+, and Hg2+ decreased L-asparaginase activity.
►The effect of the inhibitors and/or activators on the activity of the purified L-asparaginase was studied. It was found that EDTA and PMSF displayed no effect on the activity of L-asparaginase of Kodamaea ohmeri ANOMY. L-Asparaginase activity was reduced in the presence of SDS, urea, sodium azide, and triton X-100. In contrast, in the presence of non-ionic surfactants (Tween-80 and Tween-20), and β-ME L-asparaginase activity was enhanced by 11.23, 10.59, and 6.97 %, respectively.
►The effect of pH on the activity of the purified free and immobilized L-asparaginase was studied using different pH values. The results revealed that both the purified free and immobilized L-asparaginase was active over a wide pH range, their activities gradually increased up to pH 7.0. Both exhibited the maximum L-asparaginase activity at pH 7.0 (634.50±62.93 U/mL and 789.96±28.34 U/g carrier, respectively). However, at higher pH values, the activity of both the free and immobilized L-asparaginase decreased. At pH (10), the immobilized L-asparaginase retained about 74% of its activity while the free L-asparaginase retained about 59% of its activity.
►The effect of the temperature on the activity of the purified free and immobilized L-asparaginase was studied using different reaction temperatures. The results revealed that both the purified free and immobilized L-asparaginase was active over a wide temperature range. The free L-asparaginase exhibited the maximum activity at a temperature of 37°C (632.50±17.68 U/mL) and retained about 45% of its activity at a temperature of 70°C. While the immobilized L-asparaginase exhibited the maximum activity at a temperature of 45°C (793.62±9.19 U/g carrier) and retained about 40% of its activity at a temperature of 80°C.
►The Michaelis-Mentenʹs constant (Km) and maximal velocity (Vmax) of the purified L-asparaginase from Kodamaea ohmeri ANOMY were determined by Lineweaver-Burk plot analysis using various concentrations of L-asparagine. The results revealed that the values of the Km and Vmax were 1.0303 mM and 303.0303 U/mL, respectively.
►The anticancer activity of the purified L-asparaginase from Kodamaea ohmeri ANOMY was evaluated toward MCF-7, HepG-2, and HCT-116 cells using the cell morphology technique. The resulting toxicity of L-asparaginase against the three cell types was dose-dependent since the gradual increase in the dose of L-asparaginase showed a gradual inhibition of cell growth.
►L-Asparaginase inhibited the growth of HepG-2, MCF-7, and HCT-116 cells at a concentration of 20, 50, and 60 µL, respectively.