الفهرس 
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Abstract
1- The present thesis deals with the study of thenno-alkalophilic bacteria
of soil samples representing various localities of Egypt.
2- Thirty five soil samples were collected from five regions of Egypt i.e.
Wady El-Natroon (WN) depression, Port Said salt marshes (PS), Al-
Ameria salt marshes (AS), Mariut Lake (ML) and Qalubyia
Governorate (QG). The samples were collected as possible, from
alkaline and desert regions.
3- The counts ofthermoalkalophilic bacteria in the collected soil samples,
showed that highest counts of thermo-alkalophilic bacteria were
recorded for wadey El-Natroon (WN) region, Port Said (PS) salt
marshes and AI Ameria salt marshes (AS). Moderate counts of thermoalkalophilic
bacteria were recorded for Mariut lake (ML) and Qalubyia
Governorate (QG).
4- 170 Thermo-alkalophilic bacterial isolates were selected from bacterial
flora of the thirty five soil samples.
5- These thermo-alkalophilic bacterial strains were allowed to grow on
growth medium, viz Dox’s - yeast extract -gelatin agar medium
containing 1-2% Na3 P04 for the isolation of thermophilic alkalophilic
proteolytic strains at PH12 and incubation temperature 650C.
6- The incidence of the isolated 170 bacterial strains in relation to their
originating soils was studied. The highest number of thermoalkalophilic
bacterial isolates was recorded in Wady EI-Natroon
depresion (34.~%), followed by Mariut Lakes (19.4%), AI-Ameria salt
marshes (15.9%), Port said salt marshes (15.3%) and Qalubyia
Governorate (14.7%) respectively.
7- A screening program for the proteolytic activities of the 170thermoalkalophilic
bacterial isolates was carried out. This screening
exhibited that 16 isolates were characterized by proteolytic activities at
pH12 and 65°C while the remaining isolates were non-proteolytic. The
most potent isolate which was capable of producing the highest yield
of proteases at 65°C and PHI2 was isolate No. WNI616 B.
8- Identification procedures of the 16 thermo-alkalophilic proteolytic
bacterial isolates were carried out using the international keys. Cells of
these isolates arerod - shaped occur singly, in pairs or in chains. Gram
positive, strict aerobic. Isolates do not produce acetyl methyl carbinol
except isolates Nos 1201, 2305 and 61I produce acetyle methyl
carbinol; isolates Nos 1616B, 1515A, 1201,2305,611 and 3403B
produce acid from D-glucose and D-mannitol but not from D-xylose or
L-arabinose, the remaining isolates do not produce acid. from
D-glycose, D-xylose, L-arabinose or D-mannitol; they hydrolyse
gelatin, casein and starch except isolates Nos. 1515A, 1201,2716 A
and 61I do not hydrolyse casein and isolates Nos. 1616B, 2715A and
2101 do not hydrolyse starch. They degrade tyrosine except isolates
Nos. 3403 K, 1616B, 1515A, 305 B, 2305, 2716 A and 6II do not
degrade tyrosine. All isolates reduce nitrate to nitrite; they produce
indole except isolates Nos. 1616B, 1515A, 2305, 2716 A, and 6II do
not produce indole; they do not produce gas from D-glucose and
nitrate except isolates Nos 3403K, 2305, 2716A and 3403B produce
gas from nitrate. They produce catalase. They are thermophilic, the
growth is’ produced from 45°C to 80°C and no growth is produced at
40°C or below and exhibit optimum growth from 55°C to 65°C. They
are alkalophilic where the growth is produced at pH values from pH7.5
up to pH13.3 and no growth is produced at pH values higher than
pHI3.3 or lower than pH7.5, while isolates Nos 305B and 3503B
exhibited growth from pH8 to pH 13.3 and no growth is produced at
pH lower than pH8 or higher than pH13.3. Isolates give growth with
NaCI concentrations from 2 to 6%.
9- According to Bergey’s Mannual of Systematic Bacteriology (1986) and
other related Keys, All the 16 bacterial isolates belonging to the genus
Bacillus i.e. Bacillus stearothermophi/us (Donk, 1920).
10- A special study has been under taken concerning the productivity of
thermo-alkaline protease(s) by the proteolytic thennoalkalophilic
Bacillus stearothermophilus S- WNI616B isolated from Wady EINatroon
since this strain was found to be the most potent protease(s)
producer.
l l-Factors affecting protease(s) productivity by Bacillus
stearothermophilus S-WN1616B were investigated. The following
data were found to be optimal for a maximal yield ofprotease(s).
a) An inoculum size of 0.5 ml of the bacterial stock suspension (containing
229.3 x 106 cells) was found to be the optimum inoculum for maximum
enzyme yield.
b) The maximal enzyme yield was attained within 30 days incubation
period at 55°C.
c) The optimum incubation temperature at which B. stearothermophilus SWN
161(;8 produced its maximum yield of the extracellular thermoalkaline
protease was at 55°C.
d) The optimum pH value for protease(s) production was found to be at
pHlO with 1% Na3P04.
e) It was found that the most suitable buffer is Borax NaOH at pH 10.2
) ([16.9 ~~ This is followed by carbonate bicarbonate at pHlO.7
’-9 »: ~\.
,lA., I
,p. p-~(--
(282.5 u/ml) then, glycine Na<)H at pHI 0.4 (178.2 u/ml), tris-buffer at
pH9 (126.2 U/ml) and Boric Borax at pH 9.2 (95.7 u/ml) respectively.
f) The maximum amount of protease production was obtained with 3%
NaC!.
g) Introducing different 15 amino acids into the production medium as
organic nitrogen sources instead of NaN03 (as the inorganic basic
source) resulted in the following main data.
I) L-threonine exerted the highest effect ofprotease(s) production (893.4
units I ml) in comparison with NaN03 (126.2 units I ml). This is
followed by DL alanine, L-Cysteine, B-alanine and L-aspartic acid.
II) L-tyrosine, glycine, DL-leu¢ine and L-histidine had ~t ~le~~c.~n
protease production comparable to NaNO,l which exerted the same
yield.
III) L-cystine, DL-phenylalanine, L-asparagine, and DL-Serine exerted
~O% loss .orprote~sen prod~9tivitywhiILL.:glutamic .~<:i~n~~. L- kI £,J :
tryptophane exerted 74% L~~of~en~e production comprable to
NaN03. ,
h) Introducing different nitrogen sources (ammonium molybdate,
anunonium chloride, ammoniian nitrate, ferrous anunonium sulphate,
potassium nitrate, ammoniura dihydrogen phosphate, anunonium
I
sulphate, anunonium oxalate, prea and calcium nitrate). Some of them
. resulted in increasing yield of protease (s) and reached up its maximum
in presence of calcium nitrate. i
I
I) The effect of elimination of on~ or more of ingredients of mineral salts
I of Dox’s medium revealed that production medium containing only tap
water, gelatin and Na3P04 resulted in increasing the yield of
protease(s) than in the presence of any ingredients .
J) Supplying different protein sources (casein, peptone, protease peptone,
tryptone, egg albumine and gelatin) resulted in increased protease
production and reach its maximum with peptone. In absence of protein
source no yield of protease was recorded.
k) The best vitamin, which induced protease production could be arranged
according to the following pantothenic acid (500 ppm), thiamine (250
ppm), L-asco~ic ’?acid(500 ppm), folic acid (250 ppm). Nicotinic acid
(250 ppm) and Riboflavin (250 ppm).
L) Introducing different concentrations of available heavy elements (such
as cupper sulphate cobalt sulphate, zinc sulphate and lead acetate)
resulted in increasing the yield of protease( s) and reached up its
maximum in cases of zin sulphate at 50 p.p.m. but in presence of
c~pper sulphate, cobalt sulphate and lead acetate at all applied
concentrations protease productivity by B. stearothermophilus SWN16l6B
were inhibited in comparison with the control (tap water).
12- The produced thermo-alkaline protease under all the previously
mentioned optimal conditions was subjected to a purification
procedure and the purified enzyme preparation was investigated for
s~e factors affecting its activity while in the purified form.
13- Purification steps included preparation of cell free filterate, ammonium
sulfate fractionation at 60% saturation, dialysis against H2O then
,
aganst pure sucrose crystals, and gel - filtration using sephadex G200
and G100 column chromatographic techniques which increased the
purity of the enzyme up to fourty eight folds with a specific activity of
1267.6 (u/mg. pr//ml).
14- Factors affecting the activity of the purified extracellular enzyme of B.
stearothermophilus S-WN 1616B were investigated. The resulting
data is given in the following:
a) The activity of the purified enzyme increased gradually by increasing
temperature and reached its maximum at 60°C.
b) The optimum pH for a maximum activity of the protease enzyme was
found to be pHIl.
c) The purified enzyme recorded its maximum activity at incubation period
of 62 h. :
d) The optimum concentration of substrate (gelatin) was found to be
(0.5%).
e) Calcium chloride and EDTA exerted the best stimulatory effect on
protease activity at 1.0roM concentration. This was followed by the
stimulation of magnesium sulfate within the range of 1-5 mM and Na-
~ dedocyle benzene sulphonate at 1.0 roM concentration but mercuric
chloride inhibited completely the enzyme activity at all concentrations,
i.e. within the range of 1.0 - 10 roM.
f) The purified enzyme was stable at 60°C and 70°C for 18h and at 80°C it
’retained about 50% of its original activity after ten minutes.
15) The amino acid detected in the purified protease enzyme were as the
following : Isoleucine, leucine, tyrosine, phenylalanine, Histidine,
Lysine, Arginine and glysine.