الفهرس 
Material and methodsOnly 14 pages are availabe for public view
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Abstract
L-Asparaginase (L-asparagine amidohydrolase, Ee. 3.5. 1. 1)
isolated from several biological sources has been shown- to posses
antitumor activity. Therefore, an attempt was made search for new
L-asparaginase (s) from microbial source via brown pigmented
streptomycetes.
The present study deals with the following main topics: -
1) Twenty-eight brown-pigmented streptomycetes were isolated
from different fertile Egyptian soil has different asparaginolytic
activities. Therefore, the L-asparaginase activity of these isolates
was studied. Results showed that the highest activity was exhibited
by isolate FS-39 as compared with the activity of other isolates,
being 3.391U\ml culture.
2) The cultural, morphological and physiological characteristics of
the selected isolate (FS-39) were studied using the media and the
methods of the International Streptomyces Project (ISP).
3) The streptomycetes isolate FS-39 was identified according to
Kuster (1972) and Bergey’s manual (1974) as Streptomyces
phaeochromogenes.
4) Studying the factors affecting L-asparaginase production by
S. phaeochromogenes revealed the following results: -
i- Maximal L-asparaginase production could be secured using
glycerol-asparagine medium at 7dl day of incubation period,
under static culture condition.
ii- 0.20/0L-asparagine and 2.0% glycerol (w/v) proved to be the
concentrations giving maximal yield of L-asparaginase
(9.1IU\ml culture).
iii- Fe2+ (0.00010/0) is essential for the biosynthesis of
L-asparaginase.
iv- 0.1% (w/v) yeast extract was the optimum concentration for
maximal L-asparaginase production.
v- Studying the effect of initial pH, incubation temperature and
age size of inocula on L-asparaginase production, results
revealed that, the maximal yield of L-asparaginase from
S. phaeochromogenes FS-39 could be attained under the
following condition: - By growing it on glycerol-L-asparagineyeast
extract (GAY) medium which was initially adjusted to
pH7.0, inoculated by 2% (v/v) of homogenized spore
suspension (containing approximately 3.2x 107 spores/ml) of
3 days old culture on starch nitrate medium and incubated at
30°C.
5) Purification of L-asparaginase produced by Streptomyces
phaeochromogenes FS-39: - Precipitation of the enzyme from cell
free culture carried out by organic solvents and ammonium sulfate
at different saturation. In such precipitation it was found that the
precipitation by ammonium sulfate at 50-70% saturation was more
than and better purification from addition of other ammonium
sulfate saturation and from addition of organic solvents (ethanol or
acetone).
Trials were made for a further purification of enzyme by
dissolving the precipitate in 0.05M borate buffer (pH8.5), then after,
adsorption chromatography on DEAE-Cellulose column was
applied. The L-asparaginase activity was detected only in fractions
number (30-36). The active fractions were pooled together. Enzyme
activity and protein contents were determined. Pooled fractions
were subjected again to ammonium sulfate (50-70% saturation),
then precipitated and dialyzed overnight against the same buffer.
The dialyzed enzyme was applied to Sephadex G-200 column
chromatography. The L-asparaginase activity was detected only in
fractions numbers 27-31. The active fractions were pooled together
and assayed for enzyme activity and protein content then after,
subjected to precipitation by 50-70% saturation ammonium sulfate.
The precipitate was collected and redissolved in 10ml of borate
buffer at pH8.5.
The L-asparaginase of S. phaeochromogenes was purified
67.2 fold with overall yield of 17.7% of the original activity and
specific activity 179.41IU/mg protein.
6) Characteristics of L-asparaginase (ES. 3. 5. 1. 1): -
Effect of temperature, thermal stability, pH and pH stability on
the activity of L-asparaginase of S. phaeochromogenes FS-39 were
studied. Data revealed that the optimal temperature for maximal
activity was 35°C and its activity did not loose during 180min. at
35°C. Also, results revealed that the optimum pH value for the
activity of L-asparaginase was 8.5 and the enzyme was found to be
stable in pH range from 8.0 to 9.0 after l80min. treatment at 30°C.
* The enzyme was shown to be stable when stored at 4°C for 7 daysand
for 4 days at lab. temperature (20-40°C) in borate buffer at
pH8.5.
* The effect of some metal ions and some inhibitors on
S. phaeochromogenes FS-39 L-asparaginase activity was tested.
Results revealed that the activity of enzyme was slightly stimulated
by addition of MgS04 and FeS04.7H20 at concentration 1.0 and
lO.OmM. ZnCh.7H20, MnS04.4H20 and KMnS04caused slightly
inhibitory effect on enzyme activity at concentration of 0.1, 1.0 and
10.0mM. CUS04, AgN03, Iodine, L-cysteine and L-aspartic acid at
concentration of O.lmM, Iodoacetic acid at 1.0mM caused
moderately inhibition on enzyme activity. While CUS04,HgCh,
AgN03, KeN, Iodine, L-cysteine and L-aspartic acid at
concentration of 1.0 and 10.0mMhave highly inhibitory effect on
the enzyme activity.