production, purification and charactrization of protease enzyme frombacillus subtilis€¦ · ·...

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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PRODUCTION, PURIFICATION AND CHARACTRIZATION

OF PROTEASE ENZYME FROM BACILLUS SUBTILIS


Botany and Microbiology Department, Faculty of Science, Al-Azhar University,

Assiut branch, P.O. 71542, Assiut, EGYPT,

E-mail; [email protected], fax; +2088-325436

Running title: Protease production, purification and characterization

Key words: Protease, Production, Purification, characterization and Bacillus

subtilis. `

ABSTRACTProduction 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 ºC; 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 ×103/ 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 (NH4) 2 SO4; 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 ºC. 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.

mailto:[email protected]



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INTRODUCTIONProteolytic 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

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 from Bacillus subtilis.



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MATERIALS AND METHODSMicroorganism 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 ºC. a

Loopful of bacterial strain (Bacillus subtilis) were transferred to a tube of sterile

nutrient broth and allowed to grow overnight at 37 ºC before being used to

inoculation. A stock suspension was prepared and adjusted to 7 ×103 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 ºC for 20 min before inoculation. The contents of the

flasks were mixed thoroughly and then incubated for 24 h at 37 ºC) 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 ºC 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 ºC 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



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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 º C.

b. Incubation 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 º 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. Incubation 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 º 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 º 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. Inoculum 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 × 103 cell/ml-1).



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h. Inoculum 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

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:



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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 4ºC 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 5ºC 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 µ” 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 ±1ºC). 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

without disturbing the gel surface and to the reservoir. Fifty fractions were collected

(each of 5 ml-1).



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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 60ºC 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



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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 time’s viz.: 1, 6,

12, 18, 24, 30 and 36 h at 35 ºC 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).

RESULTSProtease 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 ºC was generally more

favorable for protease production as well. However, the temperature below or above

40 ºC 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 × 103



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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 (NH4) 2 SO4 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.

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



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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



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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 ºC. the purified protease activity reached up to 1168.15 units/ml-1. While the

temperature below or above 35 ºC 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).

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.

DISSCUSIONProtease 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.



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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 ºC 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 ºC 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.

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 ºC. Many investigators study the relation of temperatures



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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 ºC. In addition to that, the optimum temperature for protease production was

between 30 and 45 ºC (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 ºC.

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 ºC. However, proteinase

production was not affected by temperature in the range studied (7-45 ºC) (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 ºC

(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 ºC. (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 ºC) 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 º C. Similarly,

Johnvesly et al., (2002), reported that the optimum temperature for protease activity

were 70 º 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 º

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 º C (Braun and Sutherland, 2003).



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Different volumes of of bacteria cells each ml-1 contain of bacterial suspension

contained (7 × 103 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 ×

103 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

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).



15

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 (NH4) 2 SO4 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

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.



16

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



17

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).

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



18

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).

The results indicated that, the optimum incubation temperature for purified

protease enzyme was 35 ºC. the purified protease activity reached up to 1168.15

units/ml-1. While the temperature below or above 35 ºC 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 ºC (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 ºC and had an optimum

activity at 37 ºC.

However, 45°C was the optimum temperature optimum of the extracellular

proteinase (PSCP) produced by Pseudomonas cepacia (Mckevitt, et al, 1989). In



19

addition to that, Lee (2002) reported that, the optimum temperature of purified

protease was ranged from 40 to 50 ºC.

On the other hand, the highest activity of purified extracellular alkaline

protease produced from the alkalophilic bacterium Alcaligenes faecalis were

exhibited at 55°C (Berla and Suseela, 2002). Moreover, Ammar et al, (2003) reported

that, the optimum temperature for thermostable purified protease enzyme was 55 ºC.

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 bacterium Alcaligenes 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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27

Table 1. Purification of protease enzyme produced by Bacillus subtilis

Purification

Steps

Enzyme

volume (ml)

Protease

activity

(units/ml)

Protein

content

(mg/ml)

Total activity

(units/ml)

Total

protein

(mg/ml)

Specific activity

(units/mg,prot/m

l

Purification

folds

Cell free filtrate 200 242.66 0.30 48532 60 808.8 1

Ammonium sulfate

fractionation (40%

(w/v) saturation

200 575.43 0.15 115086 30 3836.2 4.74

Dialysis against

sucrose

15 1258.92 0.30 18883.8 4.5 4196.4 5.18

Sephafdex G-200

column

5 2552.70 0.4 12763.5 2 6381.75 7.87



28



29



30

Bacillus subtilis

-.

Bacillus subtilis .

.0.5 %

30 h.40 ºC pH

7.0phosphate buffer ml-1(7 ×103/ ml-1)

24 h150 ml-1) (

ml-1beef extract and NaCl

lactose(NH4)2 SO4

Valine

.

sephadex G200 filtration

0.5% (w/v)

35 ºC)phosphate buffer (pH 7.0

.24 h.

production, purification and charactrization of protease enzyme frombacillus subtilis€¦ · ·...

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