12 Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H)
Isolation and Identification of Protease Producing ……….
Isolation and Identification of Protease Producing
Bacterial Strain From Jazan Province, KSA
M. A. Al Abboud
Biology Department, Faculty of Science, Jazan Uninersity, KSA
Abstract
Ten bacterial strains were isolated from hot and salted soil of Jazan region, Kingdom of
Saudi Arabia; the one that had the highest proteolytic activity and growth at high temperature (45 and 50°C respectively) was selected. The potent strain was identified and
determined as Bacillus cereus according to morphological, biochemical tests and 16 S
rRNA gene sequencing. The present study recorded that environmental conditions
played a vital role in a protease production by bacterial isolate. B. cereus was able to
produce of protease at 25, 35 and 45°C. Proteolytic activity was not recorded when the
temperature was the highest at 50°C. The present study revealed that enzyme activity
was enhanced in the presence of NaCl at different concentrations of 2.5 and 5 %. Also,
the strain gave proteolytic activity at pH 7 and 9 more than at pH 3.
Keywords: Bacillus cereus, proteolytic activity, evaluation, optimal conditions
1.INTRODUCTION
Proteases are essential constituents of
all forms of life on earth, including prokaryotes, fungi, plants and animals. Microbial proteases are among the most important hydrolytic enzymes and have been
considered extensively since the advent of
enzymology (Gupta et al., 2002). Variant
of microorganisms were used to produce
protease, but genera of Bacillus were so
far the major important group of enzymes
produced commercially (Ferrero et al.,
1996). The genus Bacillus contains a
number of industrially important species
and an approximately half of the present
commercial production of bulk enzymes
derives from the strains of Bacillus spp.
The Bacillus cereus group is the most
source of commercial alkaline protease
production worldwide (Joo et al., 2002
and Małgorzata et al., 2010). Its several
important applications in routine life affairs like detergent industries, alcohol and
beer production industries, wastewater
treatment, leathering, food industries, biotransformation, debittering of hydrolyzed
ISSN 1658-6050
proteins, oil manufacturing, medical and
pharmaceutical industries and cosmetic/
sanitary industries (Aaslyng et al., 1990;
Bierbaum et al., 1994; Emtiazi et al.,
2005; Beheshti et al., 2009).
Today, proteases account for approximately 40% of the total enzyme sales in
various industrial market sectors (Leisola
et al., 2001; Gupta et al., 2002). Accordingly to Chu (2007) proteases constitute a
class of industrial enzymes, which alone
form approximately 60% of the total
world-wide enzyme production. The most
important applications of protease are
used in laundry detergents. However, proteases are also use in leather processing,
brewing, food and pharmaceutical industries (Abdel-Naby et al., 1998).
Proteases are also useful and important components in biopharmaceutical
products such as contact-lens enzyme
cleaners (Anwar and Saleemuddin, 1997).
The proteolytic enzymes also offer a gentle and selective debridement, supporting
the natural healing process in the successful local management of skin ulcerations
E-mail: mohalabboud@hotmail.com
Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H) 13
M. A. Al Abboud
by the efficient removal of the necrotic
material (Sjodahl et al., 2002).
The production of protease depend on
the type of substrate. Therefore the protease production mainly requires the appropriate substrates. There are many substrates used for protease production,
which include skim milk, milk, peptone,
casein etc. Some of the agricultural
wastes, animal wastes, and plant wastes
are also used as substrates for the production of protease, because they are readily
available and economically very cheap
and also they have high protein content.
Yang et al. (1999) stated they one of the
good substrates used for protease production due to its high protein content. Milk
contains too little free amino acids and
small peptides for sufficient growth of microorganisms. However, it contains 33.5% casein which can be degraded by
proteinases and peptidases. Synthesis of
proteolytic enzyme is therefore an essential requirement for good growth in milk
(Fox, 1981). Recently, the physical factors
affecting the production of protease from
Bacillus cereus was investigated with
Rathakrishnan and Nagarajan (2012 and
2013) where the physiological fermentation factors such as pH (8.0), temperature
(43°C), fermentation time (26 hrs), inoculum level (3.2 ml) and substrate concentration (Agro industrial waste product
groundnut shells 9.6g) were optimized by
statistical analysis using response surface
methodology. The aim of conducted research was to assess proteolytic activity of
Bacillus cereus strains, depending on the
source of proteins in a growth medium
and temperature. The present study was
aimed also to optimize pH, temperature,
and salt concentration for maximum production of protease from Bacillus cereus.
2.MATERIAL AND METHODS
2.1.Source of samples
Soil samples were collected from hot
and salted soil of Jazan province, KSA.
The collected samples were diluted in
sterile saline solution (Hamidreza et al.,
2007). The diluted samples were plated
onto skim milk agar plates containing peptone (0.1% w/v), NaCl (0.5% w/v), agar
(2.0% w/v), and skim milk (10% v/v).
Plates were incubated at 37°C for 24 h. A
clear zone of skim milk hydrolysis gave
an indication of protease producing organisms (Poluri et al., 2003). All the bacterial
colonies were isolated and purified in nutrient agar and screened in skim milk agar
plates.
2.2.Screening for best strain produced
protease
Ten isolates were screening for best
strain produced protease by plate assay using protease specific milk agar medium.
The clear zone diameters were measured
after incubated for 24 h at different temperature ranging from 20 to 60°C by
flooded the plates with mercuric chloride
solution, this method was referred as gelatin clear zone method (Abdel Galil,1992).
Strains were assayed for proteolytic activity and a bacterium strain which had the
most proteolytic activity was selected.
2.3.Identification of protease producing
bacteria
2.3.1.Cultural characterization
The isolates were observed under the
microscope to obtain the colony morphology i.e. colour, shape, size, nature of colony and pigmentation.
2.3.2.Microscopic observation
The bacterial isolates were gram
stained and observed under a high power
magnifying lens in light microscope.
The standard microbiological methods
as described in Bergy s Manual of Systematic Microbiology (Hensyl et al,
1994). Gram reaction, colony morpholo-
14 Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H)
Isolation and Identification of Protease Producing ……….
gy, vegetative cell and spore characteristics were observed from 12h old culture
grown on a rotary shaker at 120 rpm,
30°C.
2.3.3.Physiological and Biochemical
characterization
The physiological and biochemical
characters included: starch hydrolysis,
gelatin liquefaction, indole production, nitrate reduction, urease activity, citrate utilization, production of oxidase, catalase,
methyl red, voges proskauer, tryptophane
deaminase, gelatinase, lysine decarboxylase,
arginine
dihydrolase,
ßgalactosidase and fermentation oxidation
of the following carbon sources (Dglucose, D-mannitol, inositol, D-sorbitol,
rhamnose, D-sucrose, D-melibiose maltose, fructose, inulin and L- arabinose)
were used for identification of bacterial
isolate.
2.4.Identification of bacteria by sequencing of the16S rRNA gene
2.4.1.DNA extraction and PCR Amplification
Genomic DNA was isolated according
to the following procedure. 50 ml LB
broth was inoculated with a single bacterial colony and grown to an absorbance 600
nm of 0.5–1.0 and cells were collected by
centrifugation at 5000 rpm, at 4°C, for 10
min.
The genomic DNA was isolated from
the given organism. Amplification of the
16s rRNA gene was performed using the
universal primers.
F27 (5-AGAGTTTGATCMTGGCTCAG-3) and R1492 (5-TACGGYTACCTTGTTACGACTT-3)(which are targeted to
universally conserved 16S rRNA regions).
PCR was performed as follows in a total volume of 50 µ in a 0.2 ml thin walled
PCR tube.
2.5.Bioinformatics analysis
Sequences were compared to the nonredundant NCBI database by using
BLASTn, with the default settings used to
find the most similar sequence and were
sorted by the E score. A representative sequence of 10 most similar neighbours was
aligned using CLUSTAL W2 for multiple
alignments with the default settings. The
multiplealignment file was then used to
create phylogram using MEGA5 software.
2.6.Qualitative test for protease:
Proteolytic activity of Bacillus cereus
was detected on the basis of appearance of
clear zones around the bacterial colonies.
Luria casein agar (1%) plates and Nutrient
agar supplemented with 5%skim plates
were used for this purpose.
2.7.Optimization of culture conditions
for production of protease:
Different culture conditions, like pH
(3, 4, 5, 6, 7, 9 and 10), temperature (15,
25, 35, 45 and 50°C) and salt concentration (0.0, 2.5, 5.0, 7.5, 10%), were optimized for production of protease from Bacillus cereus. Samples were collected after
24 hrs and centrifuged at 1000 rpm for 30
minutes at 4°C. Supernatant was used as
crude extract. Proteolytic activity was
measured under standard assay conditions.
3.RESULTS AND DISCUSSION
3.1.Screening and isolation of proteolytic bacteria
Bacillus strains are known to produce
and secrete large quantities of extracellular enzymes as well as protease enzyme.
Proteases, is one among the three largest
groups of industrial enzymes. Proteases
from microbial sources are preferred to
the enzymes from plant and animal
sources, since they possess almost all
characteristics desired for their biotechnological applications (Gouda et al., 2006).
In the current study, the proteolytic activities of all strains were assayed using skim
milk agar, and exhibited as diameter of
clear zone. Skim milk agar was the best
for qualitative test of protease as showed
Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H) 15
M. A. Al Abboud
in Fig. (1A). The isolate (No. 1) was out
of the ten isolates, it showed the most proteolytic activity, growing at high temperature 50 °C and therefore was selected for
further study (Table 1). The isolate was
short chains forming, gram-positive and
rod shaped bacteria (Fig.1B). It was determined as Bacillus sp. According to the
biochemical (Table 2) and phylogenic
analysis (Fig. 2). 16S rDNA phylogenetic
assay was performed to determine the
species of the novel isolate and according
to the assay result, it was determined as
B.cereus. Folasade and Ajele (2005) indicated that the proteases secreted by nine
Bacillus strains on medium containing
10% Skim milk agar. Bacillus species are
considered to be the most important
sources of protease (Prakasham et al.,
2006; Rathakrishnan and Nagarajan,
2012). Many authors focused on B. cereus
for protease production (Banik and Prakash, 2004; Mahmoud et al., 2007;
Senthilraja and Saravanakumar, 2011;
Rathakrishnan and Nagarajan, 2013). Phylogenetic analysis of 16 S rRNA sequence
for the isolate revealed that, this strain has
high sequences similarity with Bacillus
cereus (Fig. 2).
Fig. 1. Proteolytic activity of selected isolate (A), Gram positive and rod shape (B)
Table 1. Proteolytic activity and growth of different isolates at different temperatures
No. of isolate
Proteolytic activity
Growth at Different Temperature (°C)
(Clear zone )
30
40
50
1.
+++++
+
+
+
2.
+
+
3.
++
+
+
+
4.
++
+
+
+
5.
+++
+
+
6.
++++
+
+
7.
++
+
+
+
8.
+
+
9.
++
+
+
10.
+
+
+
-
D-Melibiose
α-D-Lactose
-
D-Mannose
-
D-Mannitol
Glycyl-LProline
+
D-Galacturonic
Acid
+
D-Raffinose
-
α-D-Glucose
+
D-Sorbitol
-
Pectin
+
Gelatin
+
+
+
L-Galactonic
Acid Lactone
-
L-Alanine
ND
D-Arabitol
+
D-Fructose
-
D-Maltose
Dextrin
3
Negative
Control
-
2
+
D-Gluconic
Acid
+
L-Arginine
+
myo-Inositol
-
D-Galactose
D-Fucose
+
N-Acetyl-DGlucosamine
-
Gentiobiose
6
-
D-Turanose
8
L-Fucose
ND
L-Rhamnose
-
N-Acetyl-β-D- N-Acetyl-DMannosamine Galactosamine
+
Sucrose
7
Bromo-Succinic
Acid
+
Formic Acid
+
+
Acetic Acid
+
+
D-Saccharic
Acid
-
L-Serine
+
D-Serine
+
Inosine
N-Acetyl
Neuraminic
Acid
-
Stachyose
9
L-Malic Acid
ND
D-Glucose- 6- D-Fructose- 6Glycerol
D-Aspartic Acid
PO4
PO4
+
+
+
L-Glutamic
L-Pyroglutamic
L-Aspartic Acid
L-Histidine
Acid
Acid
+
+
+
+
D-Glucuronic
Glucuronamide Mucic Acid
Quinic Acid
Acid
+
+
ND
ND
+
3-Methyl
Glucose
-
D-Salicin
β-Methyl-DGlucoside
+
+
D-Cellobiose
5
+
D-Trehalose
4
+
p-HydroxyD-Lactic Acid
α-Keto-Glutaric
Phenylacetic Methyl Pyruvate
L-Lactic Acid
Citric Acid
D-Malic Acid
Methyl Ester
Acid
G
Acid
+
+
+
+
+
γ-Aminoα-Hydroxy- β-Hydroxy-D,L- α-Keto-Butyric Acetoacetic
Tween 40
Propionic Acid
Butryric Acid
Butyric Acid
Butyric Acid
Acid
Acid
H
+
ND
ND
+
+
ND, not detected
F
E
D
C
B
A
1
Table 2. Biochemical analysis of proteolytic bacterial isolate (B. cereus)
16 Journal of Jazan University - Applied Sciences Branch
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Isolation and Identification of Protease Producing ……….
Fig. 2. Rooted phylogenetic tree showing the relationship between isolate B. cereus to closely related Bacillus species
Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H) 17
M. A. Al Abboud
18 Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H)
Isolation and Identification of Protease Producing ……….
Fig. 3. Electropherogram Data of B.cereus isolate
Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H) 19
M. A. Al Abboud
The environmental factors play an important role in the enzyme production as
well as bacterial growth. In the present
study, B. cereus was tested for their maximum ability to protease production in
various level of temperature and pH. B.
cereus was able to produce of protease at,
25, 35 and 45°C (Fig 4). On the other
hand, B. cereus was failed to produce protease at 50 °C. Although their growth was
clear compared to growth at low temperature 35 and 45°C (Fig.4). This result was
confirmed that temperature is one of the
most important factors affecting the enzyme production. The enzyme was produced at the pH between 3 and 9 (Fig. 5).
The activity of protease increased at pH 7.
Invariably, Chang et al. (2004) found that
pH 10 was optimum for high alkaline protease production by Bacillus sp. On the
other hand, Esakkiraj et al. (2007) was
pointed out that at pH 7 and 60 °C temperature were optimum for maximum protease production by B. cereus. Other study
was recorded that optimum pH for high
protease production by haloalkalophilic
Bacillus sp., when grown at gelatin broth
medium at pH 8 and 9 (Patel et al., 2005).
The current study may agreement with recent studies where, Ozgur and Nilufer
(2011) stated that maximum growth of
B.cereus was obtained at 30°C and at pH
7.0. The highest protease activity was determined at 30°C temperature and 6.4 pH
conditions and after.
The present study revealed that enzyme activity enhanced in the presence of
NaCl at 2.5 and 5 % (Fig.6). Saxena and
Singh (2011) stated that higher salinity
may promote binding of a hydrophobic
substrate to an enzyme to ensure optimal
folding for enzymatic activity.
Fig. 4. Proteolytic activity of B. cereus at different temperature °C
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Isolation and Identification of Protease Producing ……….
Fig. 5. Proteolytic activity of B. cereus at different pH
Figure 5. Proteolytic activity of B. cereus at different concentrations of NaCl (%)
Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H) 21
M. A. Al Abboud
4.CONCLUSION
The identification of the bacteria was
confirmed by 16S rRNA gene sequencing
studies. The phylogenetic tree revealed
that the cells fit in to an evolutionary cluster comprising members of Bacillus cereus. On the other hand, the present work
shows that, this enzyme needs further
work as extraction, characterization and
purification. This enzyme can promote
several industries with high efficiency
such as meat tenderization, dairy industry,
Pharmaceutical industry, Tanning of
leather, Biodetergent, Bioremediation and
amino acids industry.
REFERENCES
Aaslyng D., Cormsen E., Nordisk M.H.N.
(1990). Mechanistic studies of proteases
and lipases for the detergent industry.
Theor. Tech. Appl., 5: 196-203.
Abdel Galil O.A. (1992). Fermation of
proteases by Aspergillus fumigates and
Pencillium sp. J. king. Saud. University,
4(2): 127-136.
Abdel-Naby M.A., Ismail A.M.S., Ahmed
S.A., Abdel F.A.F. (1998). Production and
immobilization of alkaline protease from
Bacillus mycoides. Bioresour Technology,
64(3): 205-210.
Anwar A. and Saleemuddin M. (1997).
Alkaline-pH acting digestive enzymes of
the polyphagous insect pest Spilosoma
obliqua: stability and potential as detergent additives. Biotechnology and Applied
Biochemistry, 25(1): 43-46.
Banik R.M. and Prakash M. (2004). Laundery detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol Res., 159 (2): 135-140.
Beheshti Maal K, Emtiazi G, Nahvi I
(2009). Production of alkaline protease by
Bacillus cereus and Bacillus polymyxa in
new industrial culture mediums and its
immobilization. African J. Microbiology
Research, 3(9): 491-497.
Bierbaum G, Karutz M, Weuster-Botz D,
Wandrey C (1994). Production of protease
with Bacilluc licheniformis mutants insensitive to repression of exoenzyme biosynthesis. Applied Microbiology Biotechnology, 40(5): 611-617.
Chang C. S., Joo H. S., Ganesh Kumar C.,
Park G. G. and Paik S. R. (2004). Bleach
resistant alkaline protease production by
Bacillus sp. isolated from Korean ploychaete Periserrula leucophryna. Process
Biochemistry, 39: 1441-1447.
Chu W.H. (2007). Optimization of extracellular alkaline protease production
from species of Bacillus. Journal of Industrial Microbiology Biotechnology, 34(3):
241-245.
Emtiazi G, Nahvi I, Beheshti Maal K
(2005). Production and immobilization of
alkaline protease by Bacillus polymyxa
which degrades various proteins. Int. J.
Environ. Stds., 62(1): 101-107.
Esakkiraj P., Immanuel G., Sowmya S.M.,
Iyapparaj P. and Palavesam A. (2009).
Evaluation of protease production ability
of fish gut isolates Bacillus cereus for aqua feed. Food and Bioprocess Technology, 2(4): 383-390.
Ferrero M.A., Castro G.R., Abate C.M.,
Baigori, M.D., Sineriz F. (1996). Thermostable alkaline proteases of Bacillus licheniformis MIR 29: Isolation, production
and characterization. Applied Microbiology Biotechnology, 45: 327-332.
22 Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H)
Isolation and Identification of Protease Producing ……….
Folasade O. M. and Ajele J. O. (2005).
Production dynamic of extracellular protease from Bacillus sp. African Journal of
Biotechnology, 4: 776-779.
Fox P.F. (1981). Proteinases in dairy
technology. Netherland Journal of Milk
Dairy, 35: 233-253.
Gouda M.K. (2006). Optimization and purification of alkaline proteases produced
by marine Bacillus sp. MIG newly isolated from eastern harbor of Alexandria.
Polish Journal of Microbiology, 55(2):
119-126.
Gupta R., Beg Q.K. and Lorenz P. (2002).
Bacterial alkaline proteases: molecular
approaches and industrial applications.
Applied Microbiol Biotechnology, 59(1):
15-32.
Hamidreza F., Mahmoud J., Naser B., Nadia M., Kianoush K. (2007). Production
and purification of a protease from an alkalophilic Bacillus sp. strain isolated from
soil. Iran. J. Biotechnol, 5(2): 110-113.
Hensyl, W. R. (1994). Bergey's Manual Of
Determinative Bacteriology 9th ed. Williams & Wilkins, Baltimore. Philadelphia,
Hong Kong, London, Munich, Sydney, Tokyo. Page 155-156 and group 5, page 179209.
Joo H.S., Kumar C.G., Park G.C., Kim
K.T., Paik S.R. and Chang C.S. (2002).
Optimization of the production of an extra
cellular alkaline protease from Bacillus
horikoshii. Process Biochemistry, 38(2):
155-159.
Leisola M., Jokela J., Pastinen O.,
Turunen O. (2001). Industrial use of enzymes. In: Encyclopedia of Life Support
Systems (EOLSS), EOLSS Publishers Co.
Oxford UK.
Mahmoud M.A.; Al-Agamy M.H.M., ElLoboudy S.S. and Ashour M.S. (2009):
Purification and characterization of neutral protease from soil strain of Bacillus
subtilis. New Egy. J. Microbiology, 22:
13-19.
Małgorzata N., Katarzyna G. and Adam L.
(2010). Proteolytic activity of Bacillus cereus strains. Proceedings of ECOpole,
4(2): 273-277.
Ozgur K. and Nilufer C. (2011). Isolation
of protease producing novel Bacillus cereus and detection of optimal conditions.
African Journal of Biotechnology, 10(7):
1160-1164.
Patel R., Dodia M. and Singh S. P. (2005).
Extracellular alkaline protease from a
newly isolated haloalkaliphilic Bacillus
sp. production and optimization. Process
Biochemistry, 40(11): 3569-3575.
Poluri E.; Kunamneni A. and Davuluri
S.A. (2003). Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. American Association
of Pharmaceutical Scientists. Pharm. Sci.
Technology. Vol. 4(4): 440-448.
Prakasham R.S., Rao C.S., Sarma P.N.
(2006). Green gram husk-an inexpensive
substrate for alkaline protease production
by Bacillus sp. in solid-state fermentation.
Bioresource Technology, 97(13): 14491454.
Rathakrishnan P.
and Nagarajan P.
(2013). Optimization of the production of
protease by Bacillus cereus with response
surface methodology using groundnut
shell. International J. Pharmaceutical,
Journal of Jazan University - Applied Sciences Branch
Vol.3 No.1 Jan. 2014 (Safar 1435 H) 23
M. A. Al Abboud
Chemical and Biological sciences, 3(2):
200-209.
Rathakrishnan P. and Nagarajan P. (2012).
Optimizing factors affecting protease production by a Bacillus cereus using groundnut shell under solid substrate fermentation. International J. Science Inventions
Today, 1 (2): 114-129.
Saxena R. and Singh R. (2011). Characterization of a metallo-protease produced
in solid state fermentation by a newly isolated Bacillus strain. Acta Biologica Szegediensis, 55(1): 13-18.
Senthilraja S. and Saravanakumar K.
(2011). Purification and characterization
of protease from mangroves derived strain
of Bacillus cereus. International Multidisciplinary Research Journal, 1(1): 13-18.
Sjodahl J., Emmer A., Vincent J. and
Roeraade J. (2002). Characterization of
proteinases from Antarctic krill (Euphausia superba). Protein Expression and Purification, 26(1): 153-161.
Yang J., Shih I., Tzeng Y. and Wang S.
(1999). Production and purification of
protease from Bacillus subtilis that can
deproteinize crustacean wastes. Enzyme
Microbial Technology, 26: 406-413.
‫‪24 Journal of Jazan University - Applied Sciences Branch‬‬
‫)‪Vol.3 No.1 Jan. 2014 (Safar 1435 H‬‬
‫‪Isolation and Identification of Protease Producing ……….‬‬
‫عزل وتعريف ساللة بكتيرية منتجة النزيم البروتياز من منطقة‬
‫جازان بالمملكة العربية السعودية‬
‫محمد عبد هللا العبود‬
‫قسم االحياء‪ -‬كلية العلوم‪ -‬جامعة جازان‪ -‬المملكة العربية السعودية‬
‫‪Q‬‬
‫تم عزل عشرة سالالت بكتيرية من التربة الساخنة والمملحة بمنطقة‬
‫جازان بالمملكة العربية السعودية بهدف البحث عن ساللة بكتيرية ذات‬
‫نشاط عالي من انتاجية انزيم البروتياز‪ ،‬ووجد أن أحد العزالت البكتيرية‬
‫لديها القدرة علي انتاج انزيم البروتياز عند درجة حرارة ‪ 45‬درجة مئوية‬
‫‪ ،‬وكان لديها القدرة علي النمو عند ‪ 50‬درجة مئوية ولكن فشلت في انتاج‬
‫االنزيم عند تلك الدرجة‪ ،‬وقد تم تعريف تلك الساللة علي اساس الصفات‬
‫الظاهرية واالختبارات الكيموحيوية ووجد ان الساللة البكتيرية هي ‪16 S‬‬
‫‪ rRNA‬بناء علي التحليل التسلسلي ل باسيلس سيريس‪.‬‬
‫وكشفت هذه الدراسة أن النشاط اإلنزيمي يتحسن في وجود كلوريد‬
‫الصوديوم بتركيزات مختلفة بين ‪ ،٪10 ،2,5‬كما وجد ان النشاط‬
‫االنزيمي عند درجة اس هيدروجيني ‪ 9 ،7‬أفضل من النشاط اإلنزيمي‬
‫عند االس الهيدروجيني ‪.3‬‬
‫كلمات مفتاحية‪ ، Bacillus cereus :‬نشاط الروتييز‪ ،‬تقييم‪ ،‬الظروف المثلى‪.‬‬
‫‪E-mail: mohalabboud@hotmail.com‬‬
‫‪ISSN 1658-6050‬‬