الفهرس 
MushroomOnly 14 pages are availabe for public view
 |  | from | 131 |  |  |
| | | from | 131 | | |
Abstract
Optimization of growth media components and the cultural conditions
was carried out by using statistical factorial design L18 (21x37). 18 media
were constructed with different composition and conditions.
2- In all media the fibrinolytic activity (F), proteolytic activity (P), F/P
ratio, protein and specific activities were determined.
3- The highest mycelial extracellular fibrinolytic activity and high F/P ratio
were obtained when P. ostreatus was cultured on medium (2) composed
of (g/100ml): fibrin, 0.2; yeast extract, 1.5; glucose, 1.0; MgSO4, 0.1
and K2HPO4, 0.20; inoculums, 7.0 discs; initial pH, 6.0; Temperature,
35.0°C and incubation period 7 days.
4- Highest proteolytic with low F/P ratio was achieved on medium (6)
composed of (g/100ml): fibrin, 0.3; yeast extract, 1.0; glucose, 1.0;
MgSO4, 0.1 and K2HPO4, 0.20; inoculums, 5.0 discs; initial pH, 7.0;
Temperature, 45.0°C and incubation period 7 days.
5- Based on the results of the factorial design experiment, extra medium
was constructed to contain the optimal levels of all factors for
fibrinolytic activity only. This medium was signaled as modified
medium. Promising increase in fibrinolytic enzyme production exceeded
that gained in medium (2) by 1.2 fold was observed with higher F/P
ratio. Since this medium was selected as the optimum one for
purification, characterization and molecular studies.
6- The extracellular fibrinolytic enzyme from P. ostreatus mycelium was
purified to homogeneity using ammonium sulphate precipitation,
DEAE-cellulose column and gel filtration on Sephadex G-100 column.
7- Three separate peaks of fibrinolytic activity were detected by DEAEcellulose,
named FA, FB and FC. The composite sample of the three
active peaks when applied on Sephadex G-100 showed one fraction
Summary
90
peak with high fibrinolytic activity with 69% recovery and 2.5
purification fold.
8- The purified fibrinolytic enzyme (PoFR) from P. ostreatus mycelium
showed a single band using SDS-PAGE. The apparent molecular mass
was estimated to be 44 kDa.
9- Characterization of the pure fibrinolytic enzyme was done to include the
effect of pH, temperature, metal ions, protease inhibitors, substrate
specificity and amidolytic activity, in addition to Km and Vmax
determination.
10- The optimum pH of the fibrinolytic enzyme was observed at pH 6. The
enzyme was highly active at pH range 5-8 at 37°C for 1 h but below or
above this range the enzyme activity decreased rapidly.
11- The optimum temperature of the fibrinolytic enzyme PoFR from P.
ostreatus was 35°C. The enzyme activity was stable between 30-40°C.
The enzyme remain active but with decreasing values up to 75°C.
12- The effect of metal ions indicated that the fibrinolytic enzyme
completely inhibited by Hg2+ and partially inhibited by Cu2+, Ni2+ and
Al3+. It strongly activated by Zn2+, Mg2+, Ca2+, K+ and Fe2+ in decreasing
order.
13- EDTA and EGTA strongly inhibited the fibrinolytic activity of PoFR.
The enzyme was also inhibited by serine protease inhibitors PMSF and
aprotinin. The cysteine protease inhibitor TLCK and aspartic acid
protease inhibitor pepstatin A exerted weak effect on the enzyme
activity.
14- The purified fibrinolytic enzyme (PoFR) from P. ostreatus was appeared
to be serine metalloprotease.
15- The fibrinolytic enzyme showed high specificity towards fibrin. This
direct action toward fibrin degradation suggests it could be used as an
Summary
91
effective thrombolytic agent. The enzyme was specific also to
fibrinogen and gelatin but it could not hydrolyse casein, egg albumin
and elastin.
16- The amidolytic activity for several synthetic substrate of PoFR showed
that the strongest fibrinolytic activity was exhibited for the substrate NSuccinyl-
Ala-Ala-Pro-Phe-pNA for (subtilisin and chymotrypsin). The
enzyme also shows high activity for N-benzoyl-Phe-Val-Arg-pNA for
(trypsin and thrombin). So the fibrinolytic purified enzyme from P.
ostreatus mycelium is considering chymotrypsin or subtilisin
metalloprotease.
17- The effect of different concentration of the substrate (N-Succinyl-Ala-
Ala-Pro-Phe-pNA) was studied. The Km and Vmax were determined to
be 0.35 mM and 21 U ml-1, respectively.
18- Molecular study and sequence analysis of the gene encoding fibrinolytic
enzyme (PoFR) from P. ostreatus were investigated. The resulting
cDNA showed one fragment about 845 bp. The cDNA fragment was
transformed into E. coli (α-DH5) competent cell.
19- Two screening procedures were done to detect recombinant clones
harboring the specific gene encoding fibrinolytic enzyme.
20- The 1st procedure achieved by PCR screening using pair of specific
primers which indicated the same predicted size of cDNA (about 845
bp).
21- The 2nd procedure was done by restriction digestion where the plasmid
from transformant bacteria was prepared and digested by restriction
enzyme. This procedure indicated also that a DNA fragment of about
845 bp was released and separated by agarose gel electrophoresis.
22- The DNA sequence revealed that the putative mature fibrinolytic gene
exhibited a high sequence homology (95%) with fibrinolytic enzyme
Summary
92
isolated from P. ostreatus fruiting bodies (GenBank Accession No.
AY640032.1).
23- The sequence analysis of the fibrinolytic gene showed that it contains an
Open Reading Frame (ORF) of 845 bp with start code ATG and encodes
281 amino acids. The deduced amino acids indicated dissimilarities to
other mushrooms and Bacillus producing fibrinolytic enzymes. It
showed high homology (90%) with fibrinolytic enzyme isolated from P.
ostreatus fruiting bodies (GenBank Accession No. AAU94648.1).
24- The fibrinolytic enzyme from P. ostreatus mycelium has an extended
zinc-binding consensus sequence which indicated that it is from
metzincin family of metalloprotease.
25- In vitro assay showed the direct action and the high specificity of PoFR
toward fibrin clot.
26- In vivo assay of the thrombolytic activity of the purified enzyme (PoFR)
in P. ostreatus showed that the enzyme exerted fibrinolysis in male and
female mice which predicted by decrease in the coagulation marker
hematocrit percentage and prolonged prothrombin time (PT) and
thrombin time (TT). This indicated that PoFR has anticoagulating
activity in addition to fibrin clot degradation