الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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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.