الفهرس 
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Abstract
The present study was carried out to achieve
~wo main objectives
The first object was to evaluate the possibili ty of
utilization of sugar beet industry by products
(beet pulp, and beet molasses) for production of some
microbial enzymes (pectinases).
The second goal of the present work deals with the
evaluation of fungal biomass of Myrothecium verrucaria
(involved In pectinases production) as a possible
source of protein.
The whole frame of the work and the findings
obtained can be summarized in the following:
1. Screaning studies were carried out using nine strains
of M. verrucaria, as to select the suitable strain
for production of
obtained from
pectinases.
different
These cultures were
microbial cultures
coJlections in etherland, United Kingdom, Canada,
United State of America and Egypt, for production
of pectinases. Modified Czapek-- Dox media were
used in the screening studies with the replacement
glucose, apple pectin or beet pulp at 1% final
concentration.
2. The results obtained revealed that M. verruca ria
CBS 28846, CBS 17627 and Ls-NRC could produce high
levels of pectolytic activities on Czapek Dox
medium containing beet pulp as a carbon source
whereas the rest of strains tested exhibited feeble
or no pectolytic activity at all. On the other
hand all tested strains yielded very low or no
activity in media containing glucose or apple pectin
as carbon sources.
3. The highly active pectolytic strains of M. verrucaria
were subjected to a comparative study as to find
out the type of pectolytic enzymes produced. The
results demonstrated that the observed pectolytic
activi ties are due to the presence of endo
polygalacturonase enzymes; no pectin lyase acti vi ty
could be detected.
4. Further characterization polygalacturonase (PG)
produced revealed that the PG activities of the
tested strains namely CBS 28846, CBS 17627 and
Ls-NRC of M. verrucaria belong to the endo type
under all medium: air ratios tested in the
as judged by their effect on viscosity and release
of reducing groups.
5. Physiological studies on formation of endo-PG enzymes
by the active pectolytic strains of M. verrucar ia
grown in beet pulp
the
containing medium have
demonstrated that optimization of growth
conditions of the fungal cultures for production
of high levels of pectolytic activities could be
as follows:
a) With respect to the incubation period it was found
that 3-4 days of incubation is optimal for endo-
PG production from M. verrucaria CBS 28846 and
CBS 17627 whereas 8 days were required for strain
Ls-NRC to obtain high levels of enzyme under study.
b) Using M. verrucaria strains CBS 28846 and CBS 17627
experimental flasks the static cultures gave better
enzyme yields as compared to shake cultures. This
finding was more
Air: mediam
accentuated at
ratio namely
the highest tested
54:6. However
high levels of PG activities were also produced
under the shaking conditions although the enzyme
yield was less than in case of static cultures.
activities (14.3 and 15.2% RV respectively)
wi th those obtained with beet pulp (65.8
endo-PG
compared
and 53%
observed non homogeneity of the cultures grown under
the shaking conditions, may be another factor affects
the level of produced PG activities side by side
with the aeration level.
c) The optimum
production
for strain
initial pH values of the medi urn for
of PG acti vi ty was In
CBS 28846 and 2.6-4.6
the
for
range 3-5.2
strain CBS
17627 of M. verrucaria. However the tested strains
produced appreciable amounts of PG acti vi ties over
a wide range of the
2.6-7. In spite of
value of the medium
in the acidic side.
initial pH values ranged from
the variation of initial pH
the pH at harvest was always
d) Various carbon sources were tested with respect
to their effect on PG formation. These included
arabinose xylose,
Galacturo!1ic
fructose,
lactose,
glucose galactose,
malatose. Sucrose,
Galacturonic acid induced much lower
raff inose, cellulose, pectin, starch beet molasses,
and beet pulp. The beet pulp proved to be est
carbon source for both test strains CBS, 28846
and CBS 17627 of M. verrucaria.
affected by the
s i z e 0 f 4 % ( V/v )
attaining highest
test strains.
size of inoculum.
could be employed
activities with
Thus inoculum
favourably for
respect to the
RV) using M. verrucaria CBS 28846 and CBS 17627
respectively.
exhibi ted no
On the
inducing
other
effect
hand the apple pectin
for endo-PG formation
by M. verrucaria~ The other carbon sources gave
no or feeble endo-PG activi ties.
e) No marked differences could be detected within
the enzyme levels produced at 1-4 % (W/V) of beet
pulp In the growth medium. However the lowest
concentration (0.5% W/V) gave the lowest activities.
f) The levels of PG activi ties produced was slighthly
nitrate, ammonium sulphate,
tested, namely sodium
ammonium chloride,
g) Various nitrogen sources were
urea, peptone and yest extract.
In general no drastic effect for the type of nitrogen
source could be detected on PG acti vi ties proudced.
However, ammonium sulphate supported the formation
of slighthly higher levels of PG activities in
both tested strains.
6. Kinetics studies of crude endo- PG of M. verrucar ia
in a CBS 28846 and CBS 17627 revealed the following
properties:
a) The optimum temprature for PG activity was 60°C
for both CBS 28846 and CBS 17627 strains of M.
verrucaria. Moreover the two enzymes
different heat stability reaching 60 and
15 minutes respectively.
exhibited
50°C for
b) With respect to enzyme concentration linear activity
response was obtained with enzyme concentation
up to 0.048 mg protein/ml for both CBS 28846 and
CBS 17627. The activity increased at higher protein
concentrations but not in linear manner.
c) For both
CBS 17627,
enzymes of M. verrucaria CBS 28846 and
a linear response of reaction rate with
reaction time was obtained up to 10 minutes.
the reaction progressed with time but not
strictly linear manner for about 15 minutes.
Then
reaction reached steady
took
state and leveling
The
off
for reaction rate place after 35-40 minutes
of the incubation time.
d) A linear increase in enzyme acti vi ty was observed
by increasing subs. rate concentration (apple pectin)
up to 0.8% w/v pectin concentration, for crude
enzymes of M. verrucaria CBS 28846 and CBS 17627. The reaction rates leveled off at higher substrate
concentrations 2% and 1.2% W/V of pectin for both
strains respectively).
e) The optimum pH values for enzyme activity were
the range between 3-5 and 4-5 for the enzymes
obtained from both fungal strains tested.
7. Partial purification of PG enzyme of M. verrucaria
CBS 28846 was carried out using fractional
precipi tation with acetone, and gel filtration
on sephadex GIOO cou1mn. Electrophoresis technique
was used to show the degree of purity.
The acetone fractionation yielded enzyme fractions
of different purity, some fractions gave about
6 and 9 fold of purification. The highest
purification fold (about 38 times) was obtained
in a t.wo-stage acetone fraction through which the
appropriate fraction from the first stage was
refractionated for obtaining a higher level of
enzyme purity. Yet the enzyme was unstable. Thus
when applied on sephadex GIOO column, only feeble
activity was obtained in the resultant sephadex
fractions. However the gel filtration gave partially
purified enzyme of about 26 fold purification using some other acetone fractions.
The gel electrophoresis on polyacylamide gel showed
that the enzyme obtained from the sephadex column
is only partially purified.
8. Kinetics studies of partially purified enzyme of
M. verrucaria CBS 28846 showed that:
a) The optimum temprature for PG acti vity was as that
of crude enzyme i.e 60°C. The enzyme exhibted thermal
stability up to 60°C. Then a sharp decrease of
enzyme activity was observed at higher tempratures.
b) With respect to the enzyme concentration the enzyme
exihibted a linear response in reaction rate to
protein concentration at least up to 0.03 mg/ml.
The activity incr ased at higher protein
concentration but not in linear manner.
c) The reaction rate progressed with time in an
approximately linear manner at least up to 10 minutes
(viscometerically measured). w ilst the reducing
group determination showed a reaction linearity
at least for 15 min. Upon extending the reaction
time the rate of reduction of viscosity and release
of reducing groups were relatively slow during
the following five minutes bebore leveling off at longer reaction time.
d) With respect to the substrate concentration showed
a linear increase of the reaction rate up to 0.8 %
substrat (Apple pectin) concentration.
e) The optimum pH value was 4.5, then a sharp drop
of reaction rate was noted by increasing the pH
values of the reaction mixture. At’pH 7 no detectable
activity was obtained.
9. The chemical and biological evaluation of the fungal
biomass of M.verrucaria showed that the amino acid
methionine was revealed the first limiting amino
acid. Histidine and threonine were the second and
the third I1mi ting amino acids re spec t”i.v1e:;~The other
essential amino acids are present in actquate amounts
and exceeded their conterpart amounts in the FAa
pattern, (1973). On the other hand the animal feeding
experiment indicated that the fungal biomass of
M. verrucaria was palatable, digistable and enhanced
the growth and gain in weight of the test animal
(rats). The nutritional evaluation parameters namely
PER, AD and TD, BV, NPU and up proved that this
kind of protein could be considered of high quality.