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Abstract The greater dependency of cancer cells on L-methionine for growth compared to normal cells indicates that depletion of Lmethionine using L-methioninase is effective for cancer therapy. Accordingly, the exploitation of L-methioninase as a promising enzyme for anticancer therapy necessitates the search for new isolates producing L-methioninase. Therefore, the present study focused on screening of different yeast isolates (locally isolated from soil, marine water or some cheese products) for their ability to produce L-methioninase (the subject of this thesis). Optimization of cultural and nutritional conditions required for maximum enzyme formation by the most promising yeast isolate was studied. Moreover, purification and characterization of L-methioninase from CFE of the selected yeast isolate were investigated. In addition, the antitumor activity of the purified enzyme was evaluated. The results obtained in this study could be summarized as follows: 1- Screening studies showed that out of 50 yeast isolates tested, Isolate No.5 was the most promising isolate which showed the highest L-methioninase formation. 2- According to identification and taxonomical studies, isolate No. 5 was identified as Candida tropicalis. 3- Among various treatments used to release the intracellular L-methioninase from yeast cells, treatment of yeast cells with N-butanol proved to be the most efficient method for obtaining maximum yield of the enzyme followed in descending order by grinding with sea sand and freezing and thawing methods. 4- On studying some factors affecting crude L-methioninase activity, it was found that the optimum temperature was at 45°C and the highest enzyme activity was obtained at pH 6.5 using 0.075 M sodium citrate buffer. 5- Results concerning optimization of cultural and nutritional conditions required for maximum L-methioninase formation could be summarized as follows: · Among different media tested, Modified medium proved to be the most suitable for L-methioninase formation. · Two days incubation period at 30°C was correlated with maximum L-methioninase formation. · Optimum intitial pH of growth medium was observed at 5.7 adjusted by 0.04M potassium phosphate buffer. Among different carbon sources tested in the growth medium, glucose at 4% final concentration supported highest level of L-methioninase formation followed by ethanol at 1% final concentration. · Regarding the effect of different nitrogen sources on growth and L-methioninase formation, the highest enzyme formation was obtained in the presence of peptone followed by yeast extract at final concentration of 0.08% N-base. On the other hand, addition of inorganic nitrogen sources was not suitable for obtaining high levels of both growth and enzyme formation. · L-methioninase formation was found to be affected by the presence of some amino acids in the growth medium. The enzyme formation was stimulated in the presence of arginine, serine, proline or alanine. On the other hand, a sever inhibition of enzyme formation was noticed in the presence of cysteine and asparagines. · Addition of some surfactants Tween 80, Tween 60 and Dimethylsulphoxide to the growth medium was correlated with a slight increase of both growth and enzyme formation. Purification of L-methioninase from cell free extract of Candida tropicalis was accomplished in three purification steps which involved heating at 60°C for 10 min followed by anion-exchange chromatography and Gel filtration on G- 200 column. from this purification scheme, the enzyme was purified by 43.19-fold, with an activity yield of 27.98 %. The homogeneity of the purified enzyme was demonstrated by appearance of a single band on SDS-page. In addition, the molecular weight of the purified enzyme was estimated to be 46 kDa. 7- characterization and biochemical properties of purified Lmethioninase from Candida tropicalis were investigated and the results obtained are summarized as follows: · Maximum enzyme activity was observed at temperatures ranging from 45 to 55°C. Furthermore, the enzyme was thermostable up to 55°C for 15min in the absence of its substrate. · Optimal pH for enzyme activity was found to be 6.5 adjusted by sodium citrate buffer (0.075 M). The pH stability of the purified enzyme was observed at pH ranging from 5.5 to 7.5. · The substrate specificity of L-methioninase revealed that the enzyme had highest specificity towards L-methionine as a standard substrate. However, the enzyme showed activity towards cysteine and D-methionine. The apparent Km value of the enzyme for L-methionine was calculated to be 0.5 mM. · Concerning the effect of activators and inhibitors on Lmethioninase activity, the enzyme was significantly activated in the presence of Na+, Ni+2 & K. While Cr+2, Zn+2, Fe+ had an inhibitory effect on enzyme activity. In addition, the inhibitory effect of metal chelating agents (EDTA) on enzyme activity ensuring the metallic nature of this enzyme. Complete inhibition of enzyme activity was noticed in the presence of Cu+2, Cd+2 and Iodoacetate. · The cytotoxic effects of pure L-methioninase on the growth of three human tumor cell lines namely liver (HEPG2), breast (MCF7) and colon (HCT-116) showed significant antitumor efficiency of L-methioninase against HEPG2 and MCF7 cancer cell lines. Moreover, cytotoxic effect of the enzyme was increased with increasing enzyme concentration. |