E. M. El- Safey and U. M.

Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
1
PRODUCTION, PURIFICATION AND CHARACTRIZATION
OF PROTEASE ENZYME FROMBACI LLUS SUBTI LI S
E. M. El-Safey and U. M. Abdul-Raouf
Botany and Microbiology Department, Faculty of Science, Al-Azhar University,
Assiut branch, P.O. 71542, Assiut, EGYPT,
E-mail; elsafey5@hotmailcom, fax; +2088-325436
Running title: Protease production, purification and characterization
Key words: Protease, Production, Purification, characterization and Bacillus
subtilis. `
ABSTRACT
Production and partial purification of protease enzyme by Bacillus subtilis was the
aim of this study. Bacillus subtilis was allowed to grow in broth culture for purpose of
inducing protease enzyme. Optimal conditions for protease production by Bacillus
subtilis were; an optimum substrate concentrations 0.5 %; optimum incubation period,
30 h.; optimum incubation temperature was 40 C; the optimum pH was 7.0; the best
buffer for production of protease enzyme was phosphate buffer. An optimum
inoculum size was 1 ml
-1
from stock suspension of Bacillus subtilis (7 10
3
/ ml
-1
); an
optimum inoculum age 24 h. 250 ml
-1
was the optimum fermentor (flask) capacity
(aeration); the best-extracted volume 150 ml
-1
. The best broth ingredient was beef
extract and NaCl; An optimum carbon sources was lactose; an optimum nitrogen
source for protease production was (NH
4
)
2
SO
4
; Valine was the best amino acids to
production of protease enzyme; the utilized organic acids, acetic, citric, lactic acid
decreased protease production at different concentrations. The protease enzyme was
purified by ammonium sulfate precipitation and sephadex G 200 filtration. A trial for
the purification of protease resulted in an enzyme with specific activity of 6381.75
(units/mg prot/ml
-1
) with purification folds 7.87 times. The protease activity increased
as the increase in enzyme concentration; optimum substrate concentration (gelatin)
was 0.5% (w/v); an optimum incubation temperature was 35 C. Purified protease
enzyme had a maximum activity at pH 7.0 of phosphate buffer, and the optimum
incubation time was 24 h. Data emphasized the possibility of the production and
purification microbial protease enzyme for application under industrial scale.
INTRODUCTION
Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms,
and are essential for cell growth and differentiation. The extracellular proteases are
commercial value and find multiple applications in various industrial sectors.
Although there are many microbial sources available for producing proteases, only a
few are recognized as commercial producers (Gupta, et al., 2002b). Of these, strains
of Bacillus sp. dominate the industrial sector (Gupta et al., 2002a). Early in 1977,
Priest et al, reported that, the gram-positive, sporeforming bacterium Bacillus subtilis
produces and secretes proteases, esterases, and other kinds of exoenzymes at the end
of the exponential phase of growth. In addition to that, several workers investigated
the production of protease and alkaline protease from Bacillus subtilis (Uchida et al.,
1972; Daguerre et al., 1975; Remeikaite, 1979; Massucco, 1980; Gomaa et al., 1987)
We believe with Andrade et al., (2002) that microorganisms produce a large
variety of enzymes, most of which are made in only small amounts and are involved
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
2
in cellular proteases. Proteolytic enzymes from microorganisms may be located
within the cell (intracellular), cell wall associated (periplasmic), or excreted into the
media (extracelluar) (Kohlmann et al., 1991). Extracellular enzymes are usually
capable of digesting insoluble nutrient materials such as cellulose, protein and starch,
and the digested products are transported into the cell where they are used as nutrients
for growth (Gibb and Strohl, 1987 and Oh, et al., 2000).
Some extracellular enzymes are used in the food, dairy, pharmaceutical, and
textile industries and are produced in large amounts by microbial synthesis (Aleksieva
and Peeva, 2000 and Benslimane et al., 1995). Proteases are one of the most
important group of industrial enzymes and account for nearly 60% of the total enzyme
sale (Brown and Yada, 1991 and Escobar and Barnett, 1993). The major uses of free
proteases occur in dry cleaning, detergents, meat processing, cheese making, silver
recovery from photographic film, production of digestive and certain medical
treatments of inflammation and virulent wounds (Nout and Rombouts, 1990).
In this work, we report the finding of production, purification and
characterization of extracellular protease enzyme isolated fromBacillus subtilis.
MATERIALS AND METHODS
Microorganism and inoculum preparation
A culture of Bacillus subtilis previously isolated from water and identified by
standard method for bacterial identification. Stock cultures were maintained in
nutrient broth medium (Difco) with 70% glycerol, cultures were preserved at -20 C. a
Loopful of bacterial strain (Bacillus subtilis) were transferred to a tube of sterile
nutrient broth and allowed to grow overnight at 37 C before being used to
inoculation. A stock suspension was prepared and adjusted to 7 10
3
cell/ml
-1
.
Fermentation procedure:
Protease crude enzyme was produced by fermentation of the (50 ml
-1
/flask).
The nutrient broth {production medium (PM)} was supplemented with gelatin (10 g)
and then autoclaved at 120 C for 20 min before inoculation. The contents of the
flasks were mixed thoroughly and then incubated for 24 h at 37 C) before enzyme
assay.
Extraction of Protease:
The whole contents of fermented containing protease were filtered through
Whitman No. 1 filter paper to obtain the extracted volume then preserved in the
refrigerator at 4 C as a crude protease filtrate according to Ammar et al (1985).
Enzyme assay:
1. Gelatin clearing zone technique:
The protease enzyme activity was determined as previously mentioned by El-
Safey and Ammar, (2002) briefly, according to gelatin clearing zone (GCZ)
technique of Elwan et al (1986) standardized later by Ammar et al (1998). In this
assay, soluble gelatin (1 % w/v) was emulsified and supplemented with (1.5 %
w/v) Bacto-agar, pH was adjusted as required with proper buffer (e.g. phosphate
buffer at pH 7.0) cups were made (3 cups optimal) in each plate. Equal amounts
(0.1 ml suitable) of extracted enzyme (or enzyme solution) to be assayed were
introduced into each cup. The plates were incubated at 35 C for 24 h., at the end
of incubation time, the plates were flooded with previously prepared Mercuric
chloride (HgCL) in HCL solution (HgCL, 15g and 20 ml of 6N HCL completed to
100 ml
-1
with distilled water) (Cowan, 1974), and the mean diameters of recorded
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
3
clearing zones were calculated. Then expressed in terms of units/ml using a
special standard curve constructed for such a purpose (Ammar et al, 1998).
Parameter controlling protease production
I. Enzyme production
a. Different substrate (gelatin) concentrations
The effect of different gelatin concentrations (g/l
-1
) was performed using 0.05,
0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % (w/v)), then incubated for 24 h at 37 C.
b. I ncubation temperature
Bacillus subtilis was growing on production medium and incubated at
different incubation temperatures viz.: 10, 20, 25, 30, 35, 40, 45, 50, 55 and 60 C
respectively.
c. Different pH values:
The different buffers prepared at different pH values were applied. The
production medium was adjusting using a standard pH meter (model Jenway 3020 pH
meter). Other conditions were taken into consideration.
d. I ncubation period
The effect of incubation period was determined by incubating production
medium for different incubation periods viz. 6, 12, 18, 24, 30, 36, 42, 48 and 72 h at
35 C. Taking other conditions into consideration.
e. Elimination of one or more of the ingredients:
The three ingredients of nutrient broth medium were subjected to a process of
elimination of one or more of the ingredients as shown in Fig 1 (e). Then incubated
for 24 h at 35 C was carried out taking other parameters into consideration.
f. Fermenter (flask) volume:
Growing the bacterium in different volumes of flask viz. 100, 250, 500, 1000
and 2000 ml
-1
performed the effect of flask volume on protease productivity by
Bacillus subtilis.
g. I noculum size:
Eight different inocula sizes of Bacillus subtilis were studied. The spore
suspension was prepared as previously mentioned. Different inocula sizes were
applied viz. 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml
-1
/flask. Each 1 ml
-1
of bacterial
suspension contained (7 10
3
cell/ml
-1
).
h. I noculum age
The effect of inoculum age on protease productivity was carried out by
growing Bacillus subtilis on nutrient broth medium for different incubation times, viz.
6, 12, 18, 24, 30, 36, 42 and 48 h. At the end of each incubation period, a standard
inoculum (of bacterium suspension) was prepared and then transferred into production
medium.
i. Medium volume
The effect of medium volume on the level of protease productivity was
performed using different volumes of production medium viz., 50, 100, 150, 200, 250
and 300 ml
-1
.
j. Different carbon sources:
Different carbon sources were prepared at an equimolecular carbon level
located in (Fig 1, H) was used separately as c-sources in production medium.
Whereas, a control represented by production medium without any carbon source was
performed at the same time.
k. Different nitrogen sources
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
4
Different organic and inorganic nitrogen sources (Fig. 1, I) were added at an
equimolecular nitrogen contents (to that located in sodium nitrate) to production
medium.
L. Different amino acids
This experiments was carried out in order to investigate the effect of different
amino acids on protease production. Different amino acids (Argnine, Cystine,
Glutamic acid, Isolusine, Lysine, Methionine, Aspartic acid, Proline, Phenyl alanine,
Glycine, Valine and Tryptophane) were introduced into production medium.
m. Different organic acids
This experiments was carried out in order to investigate the effect of organic
acids on protease production. Different organic acids including lactic acid, acetic
acid, and citric acid were introduced into production medium.
II. Enzyme purification
a. Enzyme purification
The protease purification steps were described as previously mentioned by El-
Safey, (1994). This included the following steps:
Step 1. Enzyme production and preparation of cell free filtrate
Bacillus subtilis was grown under optimized conditions. The filtrate broth
(crude protease) was collected and centrifuged at 4000 rpm for 15 min at 4C in order
to obtain a cell free filtrate (cff). After performing a test for sterility, 200 ml of the
cell free filtrate (CFF) containing protease were collected and their proteolytic
activities and protein content were determined.
Step 2. Ammonium sulfate fractionation
200 ml
-1
of the crude protease enzyme were first brought to 20% (w/v)
saturation with solid ammonium sulfate (enzyme grade) according to the chart of
Gomori (1955) as mention as Dixon and webb (1964). The precipitated proteins were
regimented by centrifugation for 15 min at 500 min
-1
. The resulted pellet was
dissolved in 5 ml of phosphate buffer at (pH 7.0). The left supernatant was applied
again with ammonium sulfate to achieve 20, 40, 60, 80, and 100% (w/v) saturation.
Both enzyme activity and protein content were determined for each separate fraction.
Step 3. Dialysis against distilled water and buffer
The obtained ammonium sulfate precipitate (in solution) was introduced into
special plastic bag for dialysis against distilled water for 3 h, followed by dialysis
against phosphate buffer at pH 7.0. The obtained protease enzyme preparation was
concentrated against crystals of sucrose and kept in the refrigerator at 5C for further
purification.
Step 4. Application on column chromatographic technique
Preparation of the gel column and the fractionation procedures was determined
as previously mentioned by Ammar (1975). For this purpose, a Pharmacia column
(2.6 7.0 cm) has been used. Sephadex G-200 (Pharmacia, Upsulla, Sweden)
practical size 200 was also used. 0.2 M phosphate buffer was used at pH 6.2 and
the slurry was allowed to swell for 3 d at room temperature ( 22 1C). Sodium
azide (0.02%) was added to prevent any microbial growth. Applying a mixture of blue
dextran 2000 and bromophenol blue determined the void volume.
One ml
-1
of the enzyme preparation sample was applied carefully to the top of
the gel. It was allowed to pass into the gel by running the column. Buffer was added
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
5
without disturbing the gel surface and to the reservoir. Fifty fractions were collected
(each of 5 ml
-1
).
Proteolytic activity and protein content were carried out for each individual
fraction. Sharp peaks of fractions obtained after applying Sephadex G 200 column
were collected and investigated for the properties of the partially purified protease
enzyme.
b. Enzyme activity
The protease enzyme activity was determined as previously mentioned by El-
Safey and Ammar, (2002).
c. Protein determination
The protein content of protease enzyme was determined by the method of
Biuret as mentioned in Chykin, (1966).
d. Determination of the specific activity of protease enzyme
The specific activity of the protease enzyme protein was expressed in terms of
units/mg protein/ml
-1
according the following equation:
Specific activity = enzyme activity / protein content (mg/ml
-1
)
III. Enzyme characterization
Characterization of protease
1. Effect of different enzyme concentrations:
This experiment was performed to investigate the effect of different
concentrations of protease enzyme on their activities. The purified protease enzyme
dilutions were, 0.0075, 0.0150, 0.0300, 0.0600 and 0.120 % (w/v) (mg, protein/ml
-1
)
2. Effect of different substrate concentrations:
This experiment was carried out to study the effect of different substrates
(starch) concentrations on purified protease. Different soluble concentrations (w/v)
were used, viz. 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % respectively.
3. Effect of incubation temperature:
This experiment was performed by incubating protease at different
temperatures viz.: 10, 25, 30, 35, 40, 50 and 60C respectively.
4. Effect of different pH values:
This experiment was planned to investigate the effect of different pH values of
different buffers on purified protease activities. The purified protease was incubated at
different pH values of different buffers. pH measurements were made by a standard
pH meter using a model Jenway 3020 pH meter.
5. Effect of incubation period:
The purified protease was incubated for different incubation times viz.: 1, 6,
12, 18, 24, 30 and 36 h at 35 C respectively.
iv. Statical procedures
In all experiments, the measurements were carried out with duplicated parallel
cultures. The values reported are means S.D. calculated as described by Snedecor
and Cochran (1980).
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
6
RESULTS
Protease production
The extracellular protease enzyme was synthesized by Bacillus subtilis
previous isolated from water. The results obtained in this work revealed the ability of
Bacillus subtilis to produce extracellular protease. Different culture conditions were
used to obtain the maximum levels of protease productivity by B. subtilis.
Fig 1 (A) shows the ability of B. subtilis to utilizing gelatin as a carbon source
and energy material to produce protease enzyme. Interestingly, the results indicted
that B. subtilis exhibited their maximum ability to biosynthesize protease within 30 h.
incubation period.
The effects of different incubation temperatures on protease production were
evaluated. It obvious from the results in fig 1 (B) that 40 C was generally more
favorable for protease production as well. However, the temperature below or above
40 C caused a sharp decrease in protease yield as compared to the optimal
temperature.
Different substrate (gelatin) concentrations were applied for investigated their
effect on protease productivity by Bacillus subtilis. Data (fig 1, C) indicated that the
maximum productivity was attained at a gelatin concentration of 0.5 % (w/v) higher
or lower concentrations resulted in a notable decrease in protease productivity.
Eight different inoculums size represented graphically in fig 1 (D) were
investigated for their effect on productivity of the protease enzyme by Bacillus
subtilit. Our results indicated that the use of 1.0 ml
-1
inoculum volume (7.0 10
3
cell/ml
-1
) gave the highest yield of protease. Higher or lower inoculum sized resulted
in a significant decrease in enzyme productivity.
Fig 1 (E) shown that eight different inoculum age of Bacillus subtilis, while
the best inoculum age for production of proteases enzyme by Bacillus subtilis was 24
h.
Fig. 1 (F) shows the results for protease production of Bacillus subtilis grown
in presence of different fermentor volumes. The highest levels of protease production
were obtained when Bacillus subtilis growing in fermentor (flask) capacity 250 ml
-1
.
An experiment was designed to investigate the effect of different carbon
sources on protease production by Bacillus subtilis. The result in fig 1 (G) shows that
the best carbon sources for protease production was lactose. When the bacillus
subtilis used lactose as a carbon source, the protease production reach to the
maximum. While the other carbon sources gave weak or loss protease production
Fig 1 (H) shows the results of different nitrogen sources in relation to protease
production by Bacillus subtilis. Different organic and inorganic nitrogen source were
used. The best nitrogen source for protease production was (NH
4
)
2
SO
4
with enzyme
level 10.96 units/ml
-1
.
Data recorded in fig 1 (I) show that various amino acids incorporated
separately into production medium in absence of any other nitrogen sources except
gelatin succeeded to promote proteases productivity by Bacillus subtilis. Four amino
acids ( Argnine, glutamic acids, lysine, and valine) out of twelve amino acids under
investigation gave stimulatory effects concerning protease production in comparison
to the control and other amino acids under investigation. However, the best amino
acid for protease production was valine with enzyme productivity 389.04 units/ml
-1
.
The medium volume for Bacillus subtilis growing and protease production
were study. The results cited in fig 1 (J) indicated that the protease production reach
to the maximum at 150 ml
-1
medium volume. Decrease or increase in the medium
volume lead to decease in protease production.
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
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The effect of elimination of the ingredients of production medium on the
productivity of protease enzyme by Bacillus subtilis was undertaken. Data indicated
that, protease reached its maximum productivity 31622.77 units/ml
-1
when both beef
extract and NaCl were introduced into production medium (Fig 1, K).
Different organic acid, lactic, citric and acetic acids were incorporated in
production medium to investigate their effects on protease production by Bacillus
subtilis. The results (Fig 1, L, M, & N) indicated that all organic acids applied have
stimulatory effect to protease production from concentrations 0.1 to 1.5% (w/v) of
lactic acid with enzyme productivity ranged from 177.82 to 4.89 units/ml
-1
0.1 to
0.5% (w/v) of citric acid with enzyme productivity ranged from 165.93 to 3.34
units/ml
-1
, 0.1 to 0.2% (w/v) of acetic acid with enzyme productivity ranged from
16.59 to 2.08 units/ml
-1
. On the other hand, when increase acids concentrations gave
inhibitory effects on production of protease enzyme. When incorporated different
acids to production medium, at 1.5% to 3.0% of lactic acid concentrations there is no
protease production. While at citric acid at 1.5% to 3.0 there is no production of
protease but in case of acetic acid there is no protease productivity at acid
concentrations ranging from 0.1% to 3.
The production medium was adjusted at different pH values of different
buffers. Results (Fig 2, A; B; C; D; E and F) indicated that the best buffer was
phosphate buffer at optimum pH for production of protease was recorded at 7.0. with
177.83 units/ml
-1
. A notable decline in the enzyme productivity occurred at both
higher or lower pH values.
Protease purification
The culture supernatant of Bacillus subtilis containing an initial protease activity
(242.66 units/ml
-1
) was concentrated by ammonium sulfate precipitation. The
optimum ammonium sulfate fractionation was (40% (w/v) saturation) showed the
4.74 or more fold increase in specific activity compared to the unconcentrated
supernatant.
Protease enzyme was purified by ammonium sulfate precipitation and
Sephadex G200 filtration. As shown in table (1) ammonium sulfate precipitation
resulted in specific activity of 3836.2 (units/mg prot/ml
-1
) and purification folds 4.74
times (fig 3, A). The protease was subjected to dialysis against sucrose resulted in
specific activity 4196.4 (units/mg prot/ml
-1
) and purification folds 5.18 times (Table
1).
A trial for the purification of protease enzyme resulted in specific activity of
6381.75 (units/mg prot/ml
-1
) with purification folds 7.87 times (Table 1 and fig 3, B).
Protease characterization
Results in fig (3, C) indicated that as protease concentration increase the
protease activity increase. The protease activity reached to the maximum with
optimum substrate (gelatin) concentration 0.5% (w/v) with enzyme activity 59.56
units/ml
-1
. Increase or decrease of substrate concentration gave the decrease in
protease activity (Fig 3, D).
The effect of temperature on the activity of the purified protease is shown in
figure (3, E). The optimum incubation temperature for purified protease enzyme was
35 C. the purified protease activity reached up to 1168.15 units/ml
-1
. While the
temperature below or above 35 C exhibited lower activities of protease.
The results (fig 3, F) indicated that, as time increase the enzyme activity
increase. The optimum incubation period for protease activity was 24 h (1840.77
units/ml
-1
).
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
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The enzyme activity of the protease was determined at different pH values of
different buffers. As shown in figure 3 (G, H, I, J, K and L) the best buffer was the
phosphate buffer (fig 3, G) and pH values for maximal activity is 7.0 with 851.13
units/ml
-1
.
DISSCUSION
Protease production
The number of enzymes secreted by various strains of Bacillus subtilis includes
amylase, several proteases, levansucrase, RNase, and alkaline phosphotase
(Matsubara, et al., 1958; McConn, et al., 1964; McConn, et al., 1964; Rappaport, et
al., 1965; Boyer and Carlton, 1968; Prestidge, et al., 1971; Higerd, et al., 1972;
Kanamori, et al., 1974; Kunst, et al., 1974; Uehara, et al., 1974; Yoneda, and Maruo.
1975; Millet, et al., 1976; and Manstala, and Zalkin. 1979).
Data presented here show that Bacillus subtilis produces an extracellular
protease. The optimal conditions for protease production have been folly determined
under bench scale fermentation conditions.
Our results indicated that the optimum incubation period for protease
production was 30 h (Fig 1, A). This result is in complete accordance with finding of
many investigators (Vaskivyuk, 1981; Gomaa et al., 1987; and Takami et al., 1989).
In addition to that, Myhara and Skura, (1990) investigated centroid search
optimization of cultural conditions affecting the production of extracellular proteinase
by Pseudomonas fragi ATCC 4973 and reported that the optimum incubation period
for proteinase production by Ps. fragi was incubation time, 38 h.
However, Abdul-Raouf (1990) reported that both Bacillus anthracis, S-44 and
Bacillus cereus var. mycoides, S-98 exhibited their maximum ability to biosynthesize
proteases within 24 h incubation periode since the productivity reached up to 126.09
units/ml
-1
for Bacillus anthracis, S-44 corresponding to 240.45 units/ml
-1
for Bacillus
cereus var. mycoides, S-98 respectively. Moreover, Johnvesly et al., (2002), found
that A high level of extracellular thermostable protease activity was observed after 24
h incubation. In addition to that, a new strain of Streptomyces fradiae was found to be
a potential producer of protease enzyme. The maximum enzyme yield of 930 P.U./ml
-
1
. (about 3-fold increase) was obtained with optimum with 48 hrs. inoculum (Ellaiah
and Srinivasulu 1996).
On the other hand, Kohlmann et al, 1991) found that the detection of
extracellular proteinase was made at 7 days of incubation at 7 C by Pseudomonas
fragi and P. fluorescens. In addition to that, the pseudomonas cultures grew in
refrigerator milk media and produced an extracellular protease during the incubation
period there was an initial lag period following inoculation, as evidenced by little
change in the APC from 0-d to 4-d incubation. The lag period can be explained
because 7 C is lower than the optimum growth temperature (Cousin, 1982).
Moreover in study on the production of proteases and lipases by three strains of
sychrotrophic pseudomonas spp. In whole milk, Stead, (1987) found a short lag
period following inoculation before the growth of cultures. In the same study (Stead,
1987), protease production by P. fluoresenscens and P. fargi began at 10 d of
incubation and increased rapidly throught at 50 d period. .
Different concentrations from gelatin were used. The maximum protease
productivity was attained in the presence of gelatin concentration of 0.5% (w/v).
Abdul- Raouf (1990), reported that the maximum protease productivity was
attained at a gelatin concentration of 1% (w/v) for Bacillus anthracis, S-44
corresponding to 1.5-2 % (w/v) for Bacillus cereus var. mycoides, S-98.
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
9
However, the activities of proteinases in the culture fluid and cellular fractions
of Bacillus intermedius 3-19 grown under various conditions were studied. Production
of these enzymes was maximal on medium containing inorganic phosphate and
gelatin and decreased 2- to 4-fold on medium with glucose and lactate. (Sharipova et
al, 2000).
Our results indicated that the optimum temperature for protease productivity
by Bacillus subtilis was 40 C. Many investigators study the relation of temperatures
and protease production the temperature ranging from 2-70 or more all depends on the
type of organism, the medium conditions and the type of enzyme. Secades, et al.,
(2001), observe the same results that the optimum temperature for an extracellular
protease produced by Flavobacterium psychrophilum was at temperatures between 25
and 40 C. In addition to that, the optimum temperature for protease production was
between 30 and 45 C (Wery, et al., 2003). Jobin and Grenier (2003) investigated the
production of proteases by Streptococcus suis serotype 2 and recoded that the
optimum temperature for protease production ranged from 25 to 42 C.
In view of the data of the other investigators, Growth and extracellular
proteinase production by Enterococcus faecalis subsp. liquefaciens was studied on
several culture media and under different incubation conditions. The optimum
temperature for production of proteinase being at 37 C. However, proteinase
production was not affected by temperature in the range studied (7-45 C) (Garcia de
Fernando, et al., 1991). Moreover, A new strain of Streptomyces fradiae was found to
be a potential producer of protease enzyme. The maximum enzyme yield of 930
P.U./ml. (about 3-fold increase) was obtained with optimum temperature 28 C
(Ellaiah and Srinivasulu 1996). In addition to that, A Pseudomonas sp. produced an
extracellular thermostable protease Growth of the organism and the production of
protease was optimum at 30 C. (Chakraborty R, Srinivasan M., 1992)
On the other hand, under conditions of submerged fermentation of Bacillus
licheniformis strain L-3 in 15-L MBR-Schulzer bioreactor, the maximum production
of proteolytic enzymes was good up to a temperature stability (65 C) Michalik et al.,
(1997). Moreover, Joo, et al., (2003) reported that alkaline protease secreted by
Bacillus clausii of industrial significance at optimum temperature of 60 C. Similarly,
Johnvesly et al., (2002), reported that the optimum temperature for protease activity
were 70 C produced by thermoalkaliphilic Bacillus sp. JB-99.
Moreover, The production of extracellular proteinase by the optimum
temperature for proteinase production by Pseudomonas fragi ATCC 4973 was 12.5
C (Myhara and Skura, 1990). In addition to that, The possibility was examined of
developing a predictive model that combined microbial growth (increase in cellular
number) with extracellular lipolytic and proteolytic enzyme activity of a cocktail of
four strains of Pseudomonas spp. and one strain each of Acinetobacter sp. and
Shewanella putrefaciens. The optimum temperature for enzyme production was
ranged from 2-20 C (Braun and Sutherland, 2003).
Different volumes of of bacteria cells each ml
-1
contain of bacterial suspension
contained (7 10
3
cell/ml
-1
) were used as an inoculum size. The optimum inoculum
size for protease production by Bacillus subtilis was 1.0 ml
-1
inoculum volume (7.0
10
3
cell/ml
-1
). This piece of information revealed that, the optimization of the
inoculum size depends mainly on the growth period allowed (age of colony) for the
applied culture, thus while the best inoculum age for production of proteases enzyme
by Bacillus subtilis was 24 h. inoculum size was optimum for highest protease
enzyme production. To our knowledge, there is poorly published information, about
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
10
the relation of inoculum size, inoculum age and protease production from bacteria.
So, this is considering the first published information in this relation.
The highest levels of protease production were obtained when Bacillus subtilis
growing in fermentor (flask) capacity 250 ml
-1
with 13963.68 units/ml
-1
. Other
investigators, Ellaiah and Srinivasulu (1996) reported that, the protease produced by
Streptomyces fradiae reached to maximum enzyme yield of 930 P.U./ml. (about 3-
fold increase) was obtained with optimum 1:20 medium to flask volume ratio.
Certain carbohydrates were introduced as carbon sources into the production
medium of protease biosynthesis by Bacillus subtilis. Our results indicated that the
lactose was the best carbon source that induced the production of protease by Bacillus
subtilis on production medium and reached to the maximum productivity (926.82
units/ml
-1
). The same finding were reported by Yang, et al., (1999) studied the effect
of carbone sources on the production of protease by Bacillus subtilis growing in
shrimp and crab shell powder medium containing one of the additional carbon
sources; glucose, lactose, carboxymethyl cellulose, D(-) arabinose, D(+)xylose, and
rice bran. They found that protease production was greatly enhanced by the the
addition of lactose or arbinose into the medium and that 1% (w/v) arabinose was the
most effective substrate and concentration for protease production.
On the other hand, Phadatare et al., (1993) evaluated various sugars such as
glucose, ractose, lactose, maltose, sucrose, xylose, and sugar alcohols, glycerol,
mannitol, and sorbitol for their effect on protease production. The results obtained
revealed that sucrose gave maximum protease activity. Moreover, Andrade et al.,
(2002) found that the protease production reached to the maximum when added D-
glucose to the medium especially when used at low concentrations (40g/l).
In contrast, a recent investigation showed that protease from sterptomyces
ambofaciens was detected only after glucose depletion (Benslimane et al., 1995).
Moreover, other investigators reported that glucose has been reported to suppress
protease production (Sen and Satyanarayana, 1993 and Sonnleitner, 1983).
However, Madzak et al., (2000) recoded that the sucrose is good substrate for
production extracellular proteases. Actually, the production of two extracellular
proteases, an endopeptidase and an aminopeptidase, by the marine bacterium Vibrio
SA1 was studied in batch cultures. It was repressed by easily metabolisable carbon
compounds such as glucose, lactate and succinate during growth in peptone media
(Wiersma et al., 1978).
Our results indicated that the best nitrogen source for protease production by
Bacillus subtilis was (NH
4
)
2
SO
4
with enzyme level 10.96 units/ml
-1
.
Several investigators study the effect of nitrogen sources on protease
productivity, Marine Pseudomonas strain 145-2 having the ability to produce
extracellular protease using casein, as the nitrogen and carbon source (Makino, et al,
1981). Nigam et al, (1981), reported that, A strain of Pseudomonas aeruginosa from
soil produced large quantities of extracellular neutral proteinase and could utilize
several organic substances as carbon and nitrogen sources for enzyme production. The
growth media required the presence of a high amount of phosphate when glucose was
the carbon source. The intermediates of citric-acid cycle acids supported the
proteinase production more than any other carbon sources. However, complex
nitrogenous substances supported enzyme production more efficiently. Higher
concentration of casamino acids suppressed the protinase synthesis.
On the other hand, An exocellular protease having the activity of coagulase
was synthesized by Bacillus subtilis var. amyloliquefaciens when the growth medium
contained no nitrogen sources. The removal of a nitrogen source from the medium
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
11
was found to induce the synthesis of exoproteases by washed bacterial cells
(Cherdyntseva, et al, 1982).
The effect of elimination of the ingredients of production medium (PM) on the
productivity of protease enzyme by Bacillus subtilis was undertaken. Data indicated
that, protease reached its maximum productivity 31622.77 units/ml
-1
when both beef
extract and NaCl were introduced into production medium. However, the addition of
only tap water into production medium is sufficient to produce protease enzyme with
productivity up to 177.82 units/ml
-1
.
The results indicated that various amino acids incorporated separately into
production medium in absence of any other nitrogen sources except gelatin succeeded
to promote proteases productivity by Bacillus subtilis. The best amino acid for
protease production was valine with enzyme productivity 389.04 units/ml
-1
.
Our results indicated that all organic acids applied have stimulatory effect to
protease production from concentrations 0.1 to 1.5% (w/v) of lactic acid with enzyme
productivity ranged from 177.82 to 4.89 units/ml
-1
, 0.1 to 0.5% (w/v) of citric acid
with enzyme productivity ranged from 165.93 to 3.34 units/ml
-1
, 0.1 to 0.2% (w/v) of
acetic acid with enzyme productivity ranged from 16.59 to 2.08 units/ml
-1
. On the
other hand, when increase acids concentrations gave inhibitory effects on production
of protease enzyme. When incorporated different acids to production medium, at
1.5% to 3.0% of lactic acid concentrations there is no protease production. While at
citric acid at 1.5% to 3.0 there is no production of protease but in case of acetic acid
there is no protease productivity at acid concentrations ranging from 0.1 to 3% (w/v).
The production medium was adjusted at different pH values of different
buffers. Results indicated that the best pH for production of protease was at phosphate
buffer (pH 7.0.) with protease productivity 177.83 units/ml
-1
. Similarly, the optimal
pH of protease activity produced by Clostridium bifermentans NCTC 2914 was 7.0.
(Macfarlane and Macfarlane, 1992). Moreover, investigated the production of
proteases by S. suis serotype 2. Proteases were identified and characterized using
chromogenic and fluorogenic assays and zymography the optimum pH for all four
proteases was between 6 and 8 (Jobin and Grenier, 2003).
In view of the data of the other investigators, Johnvesly et al, (2002) reported
that, a high level of extra cellular thermostable protease activity produced by
Thermoalkaliphilic Bacillus sp. JB-99 was observed at pH 11. So this enzyme showed
stable activity under alkaline conditions. Moreover, The production and properties of
protease from Bacillus sphaericus strain C3-41. The optimal activities of the protease
were around pH 11.0. The enzyme was stable at pH5.0-12.0. (Sun et al, 1997).
In the other hand, Eighty seven yeast strains representing 34 species isolated
from Parahancornia amapa fruit and associated Drosophila flies collected in the
Brazilian Amazon rain forest, were screened for proteinase production.The proteolytic
activity was tested on pH ranging from 2.0 to 9.0 in correspondence to the pH of the
cultures media in which the yeasts were grown. Greater production occurred in acidic
culture and high activity was observed at pH 3.0, 4.0 and 5.0. (Braga et al., 1998). In
addition to that, A new strain of Streptomyces fradiae was found to be a potential
producer of protease enzyme. The maximum enzyme yield of 930 P.U./ml. (about 3-
fold increase) was obtained with optimum pH. 7.0, (Ellaiah and Srinivasulu 1996).
Purification of protease enzyme
Protease enzyme was purified by ammonium sulfate precipitation and
Sephadex G200 filtration as mentioned by El-Safey and Ammar, (2003).
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
12
A trial for the purification of protease enzyme resulted in specific activity of
6381.75 (units/mg prot/ml
-1
) with purification folds 7.87 times. Similarly, ammonium
sulphate pricipatation and applying sephadex G200 column chromatographic
technique were applied for protease purification resulted in having two proteases (A)
and (B) with specific activity of 229.6 and 286.46 units/mg prot/ml
-1
crrospoding to
purification folds of 55.7 and 69.5 times of the origin respectively (Abdul-Raouf,
1990).
The same method were used for purification of thermostable protease
produced by B. brevis geltinoamylolyticus attacked fish wastes and poultry wastes.
The thermostable protease were purified by applying ammonium sulphate
fractionation and sephadex G200 and G100 column chromatography, where specific
activity 44562.5 units/ml
-1
protien/ml
-1
with purification folds of 8.5 times for
sehpadex G200 and 69017.5 units/ml
-1
protien/ml
-1
with purification folds 13.18 times
for sephadex G100 (Ammar, et al, 2003).
Moreover, an extracellular protease produced from Flavobacterium
psychrophilum (fish pathogen) was purified to electrophoretic homogeneity from the
culture supernatant by using ammonium sulfate precipitation, ion-exchange
chromatography, hydrophobic chromatography, and size exclusion chromatography
(Secades, 2001).
In addition to that, a novel protease, hydrolyzing azocasein, was purified from
the culture supernatant of Yersinia ruckeri.(fish pathogen) Exoprotease. The protease
was purified in a simple two-step procedure involving ammonium sulfate
precipitation and ion-exchange chromatography (Secades and Guijarro, 1999).
However, a protease (protease A) was successfully purified from the extracellular
proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor
trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography
(Lee, 2002). Moreover, extracellular alkaline protease from the alkalophilic bacterium
Alcaligenes faecalis was purified by a combination of ion-exchange and size-
exclusion chromatographic methods, and the purified enzyme had a specific activity
of 563.8 lmol of tyrosine/min per mg of protein conditions (Berla and Suseela, 2002)
On the other hand, an extracellular proteinase (PSCP) produced by
Pseudomonas cepacia was purified from culture supernatants by ammonium sulfate
precipitation, anion exchange chromatography on DEAE-Sephacel, and G200 gel
filtration chromatography (Mckevitt, et al, 1989). Moreover, Extracellular and
membrane-bound proteases produced by Bacillus subtilis YY88 were purified by
ammonium sulfate precipitation, dialyzed, and applied to a DEAE-cellulose column
(Mantsala P. and Howard Z., 1980)
Properties of the purified protease enzyme
Our result indicated that as protease concentration increase the protease
activity increase. This behavior is in accordance with the observations of West, et al.
(1967) who stated that within fairly wide limits the speed of enzyme concentration is
directly proportional to the enzyme concentration. The same finding also reported by
Abd El-Rahman, (1990); El-Safey, (1994); El-Safey and Ammar, (2003).
The protease activity reached to the maximum with optimum substrate
(gelatin) concentration 0.5 % (w/v) with enzyme activity 59.56 units/ml
-1
. Increase or
decrease of substrate concentration gave the decrease in protease activity.
In view of other investigators, 0.1% (w/v) gelatin was the best substrate
concentration for thermostable protease activity produced by B. brevis
geltinoamylolyticus (Ammar, et al, 2003).
E. M. El- Safey and U. M. Abdul-Raouf
International Conferences For Development And The Environment In The Arab World, Assiut Univ., March 23-25, 2004. p 14.
13
The results indicated that, the optimum incubation temperature for purified
protease enzyme was 35 C. the purified protease activity reached up to 1168.15
units/ml
-1
. While the temperature below or above 35 C exhibited lower activities of
protease. Similarly, protease enzyme produced by B. anthracis, S-44 exhibited an
optimum incubation temperature for purified enzyme activity was 35 C (Abdul-
Raouf. 1990). Moreover, Secades and Guijarro, (1999) reported that, a novel protease,
was purified from the culture supernatant of Yersinia ruckeri.(fish pathogen)
Exoprotease. it was more active in the range of 25 to 42 C and had an optimum
activity at 37 C.
However, 45C was the optimum temperature optimum of the extracellular
proteinase (PSCP) produced by Pseudomonas cepacia (Mckevitt, et al, 1989). In
addition to that, Lee (2002) reported that, the optimum temperature of purified
protease was ranged from 40 to 50 C.
On the other hand, the highest activity of purified extracellular alkaline
protease produced from the alkalophilic bacterium Alcaligenes faecalis were
exhibited at 55C (Berla and Suseela, 2002). Moreover, Ammar et al, (2003) reported
that, the optimum temperature for thermostable purified protease enzyme was 55 C.
Results indicated that, as time increase the enzyme activity increase. The
optimum incubation period for protease activity was 24 h (1840.77 units/ml
-1
). In
view of other investigators, Ammar et al, (2003) reported that, the optimum
incubation period for thermostable purified protease enzyme was ranging from 60 to
72 h.
The enzyme activity of the protease was determined at different pH values of
different buffers. The best buffer for protease activity was the phosphate buffer and
pH values for maximal activity is 7.0 with 851.13 units/ml
-1
. Similarly, Abdul-Rouf
(1990) reported that the optimum pH for all purified 4 proteases enzymes in their
reaction mixture was found to be 7.2.
The optimal pH for purified extracellular alkaline protease produced from the
alkalophilic bacteriumAlcaligenes faecalis was 9.0 (Berla and Suseela, 2002)
In addition to that, The optimum pH for extracellular proteinase (PSCP) produced by
Pseudomonas cepacia was 6 (Mckevitt, et al, 1989). On the other hand, the protease
enzyme had an optimum pH of around 8. (Secades and Guijarro, 1999). Moreover,
Lee (2002) reported that, the optimum pH of purified protease was pH 8.
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