Research Journal of Agriculture and Biological Sciences, 4(5): 434-446, 2008

© 2008, INSInet Publication

Optimization, Immobilization of Extracellular Alkaline Protease and

Characterization of its Enzymatic Properties
1
Samia A. Ahmed, 2Ramadan A. Al-domany, 3Nefisa M.A. El-Shayeb,
4
Hesham H. Radwan and 5Shireen A. Saleh.

1,3,5
Department of Chemistry of Natural and Microbial Products
National Research Center, Cairo, Egypt.
2,4
Department of Microbiology and Immunology, Faculty of Pharmacy,
Helwan University, Egypt.

Abstract: Streptomyces avermectinus NRRL B-8165 was the most potent for production of extracellular
alkaline protease among 6 strains of Streptomycetes using shaken culture technique (180 rpm) after 72h
at 37°C. Highest yield of extracellular enzyme (3.62 U/ml) was achieved using 7% sucrose and 0.5435%
alkaline extracted soybean as carbon and nitrogen sources, respectively. Addition of wheat bran extract
in concentration of 20 g/L increased alkaline protease production by 24.29% than control. Adding 0.25%
corn oil and MnCl2 (0.1mM) as trace element to the medium caused a slight increase in enzyme activity.
The enzyme was partially purified with 50% acetone and showed 4.1-fold purification. Alkaline protease
was immobilized on different carriers by different methods and its specific activity for casein hydrolysis
was studied. Immobilization of alkaline protease on sponge (covalent binding) had the highest
immobilization yield (86.19%) using glutaraldehyde (0.25% concentration). It was further observed that
thermal stability of the immobilized enzyme was higher compared to free enzyme. The immobilized
enzyme had higher K m (Michaelis constant) and lower Vm ax (Maximum rate of reaction) than the free
enzyme. Activation energy (E A) of the free enzyme was 6.206 Kcal/mol which was higher than the
immobilized enzyme. Half-life (t 1/2 ) of immobilized enzyme was 808.6 and 108.6 at 65 and 70°C,
respectively, which was higher than that of free enzyme. Immobilized enzyme showed the highest
operational stability for up to 15 reuses with 49.74% residual activity. On the other hand, some
applications of enzyme were carried out (dehairing, decomposition of gelatin layers on X-ray films and
detergent stability).

Key words: Alkaline protease, immobilization, Streptomyces, application

INTRODUCTION numerous secondary metabolites and particularly

P roteases are the most important kind of industrial
enzymes[22] and account for about 65% of the
worldwide sale of industrial enzymes in the world
market [21] . Microbial alkaline proteases dominate the
worldwide enzyme market, about two-third share of the
detergent [18] . Applications of alkaline protease are
concentrated in laundry detergents, leather
processin g, b r e wing, food and pharmaceutical
industries[26] . The enzyme should be stable and active
in the presence of typical detergent ingredients for use
in detergent [37]. Microorganisms served as an important
source of proteases mainly due to their shorter
generation time, the ease of bulk production and the
ease of genetic and environmental manipulation [39] .
Streptomyces species are the most industrially
important actinomyces, due to their capacity to produce
antibiotics. The ability of these bacteria to produce
large amounts of enzymes, as proteases with varied
substrate specificities, offers another potentially
interesting use [40] . For industrial applications, the
immobilization of proteases on a solid support can
offer several advantages, included repeated usage of the
enzyme, ease of product separation and improvements
in enzyme stability. Enzyme stabilization will thus
continue to be a key issue in biotechnology.
In the present study, certain environmental and
nutritional parameters controlling the biosynthesis of
extracellular alkaline protease from Streptomyces
avermectinus NRRL B-8165, partial purification and
the immobilization of partially purified enzyme on
different carriers using diff e r e n t m e t hods of
immobilization including physical adsorption, ionic
binding, covalent binding and entrapment were fully

Corresponding Author: N e fis a M . A . El-Shayeb, Department of Chemistry of Natural and Microbial Products National
Research Center, Cairo, Dokki, Egypt.
E-mail: Nefisa_elshayeb@gmail.com
434
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008
investigated. The immobilized enzyme by covalent
binding on sponge had the highest immobilization yield
(86.19% ). The refore, this immobilized enzyme
preparation was used as a typical example for
Streptomyces avermectinus NRRL B-8165 immobilized
alkaline protease and its properties was investigated
and compared with the free one. Also, in this paper,
we present potential applications of extracellular
alkaline protease for different industrial purposes.
MATERIALS AND METHODS
Organism: Streptomyces avermectinus NRRL B-8165
was obtained from Northern Regional Research
Laboratory (NRRL), Peoria, Illinois, USA.
Carriers for Enzyme Immobilization: Polystyrene,
Dowex 50 Wx4 (50-100) mesh, Dowex 1x8 (20-50)
mesh, cellulose triacetate, DEAE-Sephadex A-50,
chitin, agarose, chitosan were from Fluka Company,
Switzerland. Sephadex G-100 was obtained from
Pharmacia fine chemicals, Uppsala, Sweden. DEAE-
cellulose 53 was obtained from Whatman Biosystems
Ltd.
Medium Composition and Cultivation: The basal
medium for liquid cultures consisted of (g/L):
(NH4)2SO 4, 1; KH2PO 4, 0.5; CaCO 3 , 1; NaCl, 1;
MgSO4 , 1; peptone, 1; dextrin (white pract), 30;[2] .
The pH was adjusted to 8.0. The same medium was
also used for inoculum preparation for all the
fermentation experiments. Cultivation was in 250
Erlenmeyer flasks containing 50 ml of sterile medium.
The flasks were inoculated with 2 ml of 24h old
culture. The flasks were incubated at 37°C with
shaking at 180 rpm. The cells were harvested by
centrifugation at 4000 rev/min. The clear culture
filtrates were assayed for enzyme activity.
Assay of Protease Activity: It was determined using
casein as a substrate according to the method reported
by Gessesse and Gashe [17 ] with some modifications and
protease activity was determined as released tyrosine.
One unit of enzyme activity was defined as the amount
of enzyme that librates 1µmole of tyrosine per min
under the above mentioned conditions.
Protein Determination: This was estimated according
to the method of Lowry et al.[32] .
Partial Purification of the Enzyme: This was done by
fractional precipitation using ethanol or acetone or by
salting-out with ammonium sulphate using cell free
culture filtrate of the optimized fermentation medium.
Different acetone concentrations (30-60 %v/v) were
435
tested. The precipitation was done in a sequential
manner. The precipitated fractions were removed by
centrifugation using a refrigerated centrifuge at 4000
rev/min for 15 min. The active fractions were
lyophilized and assayed for caseinolytic activity.
Immobilization Techniques:
Physical Adsorption: It was carried out according to
Woodward (1985), one gram of each carrier was
incubated with the 50% acetone enzyme fraction (66.5
U/g carrier) dissolved in 5 ml glycine-NaOH buffer
(0.1 M, pH 10) overnight at 4°C.
Ionic Binding: It was carried out according to
W oodward[50] in which 0.1 g of the cation or anion
exchanger was equilibrated with glycine-NaOH buffer
(0.1 M, pH 10) and incubated with 1 ml of enzyme
solution (6.65 U) overnight at 4°C.
Covalent Binding: This method was carried out as
follows: One gram of were treated with 5ml of 0.25%
(v/v) glutaraldehyde for 2 h at 30°C. Then washed with
distilled water to remove the excess glutaraldehyde.
The carriers were mixed with 5 ml of enzyme solution
(66.5 U) and incubated overnight at 4°C [3].
Entrapment:
In Agar and Agarose: 10ml of different concentrations
of agar (2, 3 and 4%) or agarose (1, 2 and 3 %)
solutions were mixed with enzyme solution (2.66U).
The mixture was quickly cooled to 4°C and cut into
small cubes[50].
In Ca-alginate Beads: 10ml of different concentrations
of sodium alginate solution (1, 2 and 3%) were mixed
with enzyme solution (2.66U). The entrapment was
carried out by dropping the alginate solution into 0.15
M CaCl2 . The resulting beads (0.5-1.0mm diameter)
were collected[8] .
Thermal Stability: The thermal stability of free and
immobilized protease were tested by incubating the
enzymes in 0.1 M glycine-NaOH buffer (pH 10) at
different temperatures (30-75°C) for different time
intervals (15-120 min) before activity assay.
Effect of the Reaction Time: The enzyme assay was
conducted at 55°C and pH 10 for different time
intervals (5-60 min).
Effect of the Addition of Some Salts of Metal Ions:
Free and immobilized enzymes were incubated, in
absence of substrate, with metal ions in different
concentrations (0.1, 1.0 and 10.0 mM) at 30°C
for 1 h.
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008
Operational Stability of the Immobilized Enzyme:
2.5 grams of immobilized enzyme (wet) containing
about 66.5 U was incubated with substrate in glycine-
NaOH buffer (0.1M at pH 10) at 55°C for 20 min. At
the end of the reaction period, the carrier was squeezed
washed with buffer and the immobilized enzyme was
resuspended in freshly prepared substrate to start a new
run. The wash was assayed for alkaline protease
activity.
Some Applications:
Detergent Stability of Crude Extracellular Alkaline
Protease: This was carried out according to Hadj-Ali
et al.[20] .
Decomposition of Gelatin Layers on X-ray Films:
This was done according to Masui et al.[34] method
with some modifications.
Dehairing Agent: This was done according to Zambare
et al.[51] method.
RESULTS AND DISCUSSION
The ability of 6 Streptomycetes strains to produce
active extracellular alkaline protease was investigated.
They were cultured on the basal medium for different
incubation periods under shaking conditions and in
each case the culture filtrate was assayed for caseinase
activity. The results indicated that Streptomyces
avermectinus NRRL B-8165 was the most potent
producer of extracellular alkaline protease (2.04 U/ml)
after 72 h incubation at 37°C. These results are similar
to those reported by Mohamedin [36] from Streptomyces
ther monitrificans and Gupta et al.[19] from Virgibacillus
pantothenticus for alkaline protease production.
Substitution of the dextrin in the culture medium
by other carbon sources (sucrose, glucose, soluble
starch, lactose, maltose, molasses and casein) indicated
that lactose and casein significantly inhibited the
alkaline protease production. Sucrose proved to be the
most favorable carbon source for the production of
active alkaline protease by Streptomyces avermectinus
NRRL B-8165 (2.61 U/ml). On the other hand,
maltose, molasses, dextrin and glucose gave lower
levels of enzyme productivity compared to sucrose.
These results are similar to that reported by Chi and
Zhao [ 1 4 ] who found that different carbon sources have
different influence on extracellular enzyme production
by different strains. The results indicated that gradual
increase in sucrose concentration from 0.0 to 7.0%
resulted in 12.5-fold increase in alkaline protease
production. Higher levels however showed an adverse
effect on enzyme production. The result is highly
436
compare d t o t h a t found by Kathiresan and
Marivannan [24] who reported the production of alkaline
protease at 5.0% sucrose by Streptomyces sp.
Also, different nitrogen sources in the culture
medium were investigated. This included organic
(yeast extract, backer's yeast, peptone, casein, alkaline
extracted soybean and urea) and inorganic (ammonium
sulphate and ammonium chloride), nitrogen sources
used in the production medium on equal nitrogen basis.
It seemed that the alkaline extracted soybean was more
suitable for the enzyme production than the other
nitrogen sources. Ammonium chloride gave the least
enzyme productivity, while ammonium sulphate
inhibited the enzyme production completely. This result
is similar to that reported by Kaur et al.[25] and
Chauhan and Gupta [11]. They found that organic sources
were better suited to Bacillus sp. for enzyme
production. It has been reported that the effects of a
specific nitrogen supplement on protease production
differ from organism to organism although complex
nitrogen sources are usually used for alkaline protease
production [29] . However, addition of inorganic sources
resulted in decreased production of enzyme. Similar
results also have been obtained with other Bacillus
sp. [ 4 1 ] . In contrast to our results, Sinha and
Satayanarayana [ 4 4 ] and Nascimento and Martins[38]
reported that higher protease production with inorganic
nitrogen sources such as ammonium sulphate and
ammonium nitrate.
Therefore, alkaline extracted soybean was used as
the most suitable nitrogen source for maximal enzyme
production. This result is in agreement with that
obtained by Mabrouk et al.[33]. The study also included
the effect of different concentrations of alkaline
extracted soybean as nitrogen source in the culture
medium. The succeeding experiments were performed
using alkaline extracted soybean at 5.435 g/L
concentration in the culture medium. This concentration
was shown to be the most effective for the enzyme
production (3.62 U/ml).
It is worthy to note that calcium carbonate in the
basal medium was used as a buffering agent.
Therefore, the effect of different levels of CaCO3 was
also investigated. The maximal extracellular alkaline
protease production was obtained when its level was
0.5% g/L. Increasing CaCO 3 level from 0.0 to 0.5 g/L
resulted in 10.44-fold increase in enzyme production.
However, the higher levels showed an adverse effect
on the enzyme production Fig3. In this respect, Laan
and Konings[30] reported that higher concentration of
Ca 2+ ions negatively affected the release of proteinase
from Lactococcus lactis. This indicated that CaCO3 at
0.1% was below the critical level at which the
inhibition of protease occurred.
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008
Some experiments were done in order to explore
the effect of external additives on the enzyme
production. The addition of wheat bran extract and
corn oil was studied. The results showed that wheat
bran extract in concentration of 20 g/L gave alkaline
protease of (5.03 U/ml) with increase in the enzyme
activity by 24.20% than the control.
Also, adding 0.25% corn oil to the medium as a
surfactant led to a little increase in productivity
(1.38%). This result is in agreement with that
obtained by Mabrouk et al.[33] by Bacillus licheniformis
ATCC 21415.
With regard to the effect of addition of some salts
of metal ions indicated that Mn 2+ (1mM) enhanced the
production of extracellular alkaline protease. On the
other hand, Zn 2 + , Fe 3+ and Cu 2+ had adverse effects on
the enzyme production. This result is similar to that
reported by Shafee et al. [ 42] for a Bacillus sp. alkaline
protease.
Production of extracellular alkaline protease by
Streptomyces avermectinus NRRL B-8165 was
considerably affected by the initial pH values. It can be
noticed that pH 7.5 was the most favorable for the
alkaline protease production. Above and below initial
pH values showed a gradual decrease in enzyme
production. This result is similar to those reported by
Banik and Prakash [7] who produced alkaline protease
maximally at pH 7.5 after 72h incubation from
Bacillus cereus.
After optimization of the chemical composition of
the production medium for the biosynthesis of
extracellular alkaline protease by Streptomyces
avermectinus NRRL B-8165, the results indicated that
the culture filtrate possessed extracellular alkaline
protease of 5.22 U/ml. This culture medium was
therefore used in all succeeding work.
Alkaline protease from Streptomyces avermectinus
NRRL B-8165 was partially purified by ethanol,
acetone and ammonium sulphate. The most of
proteolytic activity was recovered at 50% acetone
(21.02) and showed 4.10-fold purification. The results
presented in Table1 points to the most active fractions
produced by ethanol, acetone and ammonium sulphate.
Immobilization of the partially purified extracellular
alkaline protease was studied using the 50% acetone
fraction. This was done using different supports and
different methods of immobilization such as physical
adsorption, ionic binding, covalent binding and
entrapment. The efficiency of enzyme immobilization
was evaluated different parameters including the
retained enzyme activity, the specific activity of the
immobilized enzyme and the loading efficiency
(immobilized activity/g carrier). The immobilization
yield is the key parameter since it represents the
437
general output of the efficiency of the immobilization
process[9] .
The data for immobilization of the extracellular
partially purified alkaline protease by covalent binding
(Fig1) indicated a good immobilization yield with
sponge through a spacer groups (glutaraldehyde) which
sh o w e d go o d lo a d in g e f fic ie n c y a n d go o d
immobilization yield. The good loading efficiency for
the immobilization by covalent binding may be due to
the formation of stable crosslinking between carrier and
enzyme through a spacer group (glutaraldehyde). In
addition, covalent binding through a spacer group
probably increased the surface area of the carrier and
consequently reduced the steric hindrance in the
immediate vicinity of the enzyme molecule. These
results are similar to those reported by Siso et al.[45] .
The results also showed that 0.25% glutaraldehyde was
the best concentration to activate support (sponge) for
giving the highest enzyme immobilization yield (77.47
%). Carrara and Rubiolo [10] reported that the best
concentration of glutaraldehyde was 1% for support
(chitosan) activation. While Tanksale et al.[48] and
Ferreira et al.[16] found the maximum immobilization
yield was obtained with 1.76 and 0.5% glutaraldehyde,
respectively. The decrease in immobilization yield by
increasing glutaraldehyde concentration may be due to
the reticulation among enzyme molecules favored by
the use of a bifunctional molecule and therefore,
affecting the overall activity [16].
The data for the immobilization of alkaline
protease by ionic binding (Fig2) showed low
immobilization yield (11.03%) on DEAE- Sephadex.
On the other hand, the immobilized enzyme on
Amberlite IR-120 showed the highest immobilization
yield (34.25%). Similar results were previously reported
for Bacillus mycoides protease immobilized by ionic
binding on Amberlite IR-120 [1] .
Immobilization of Streptomyces avermectinus
NRRL B-8165 alkaline protease by physical adsorption
(Fig 3) was employed on different carriers including
wool, stone, sponge, loofa, corn pulps, chitin and
cellulose. The immobilization of the enzyme by
physical adsorption on sponge had the highest
immobilization yield (42.36%) and the highest loading
efficiency (37.77 U/g carrier).
The immobilization of the alkaline protease by
entrapment in calcium alginate, agar or agarose was
done using different concentrations (Fig 4). The
results indicated that agarose (1%) gave the highest
immobilization yield (49.79% ). At higher carrier
concentration the immobilization yield decreased.
This may be due to decreased gel porosity with the
increase in gel concentration and consequently the
diffusion limitation was developed. Similar observations
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008
were previously reported for other entrapped
enzymes[31].
From all immobilization methods and carriers, the
enzyme immobilized by covalent binding on sponge
was used as a typical example for immobilized
Streptomyces avermectinus NRRL B-8165 alkaline
protease. The properties were investigated and
compared with those of the free one (Table 2).
The specific activity of immobilized was about
71.1% of the free enzyme which was higher than that
obtained by Mittal et al.[35] with immobilized enzyme
(56% ). while Suh et al.[47] reported that the specific
activity of immobilized enterokinase was 25% .
Investigation of the effect of the reaction time of
immobilized enzyme and free enzyme were 20min and
10min (data not shown). In this respect, Mittal et al.[35]
reported a c a lc iu m a lgin a t e im m o b ilize d
dipeptidylpeptidase which had maximum activity of
90min compared to 60min for the free. They explained
that this was possibly due to limited diffusion of
substrate initially, but once a sufficient amount of
substrate had entered the beads then the enzymatic
activity increased because of the close proximity of
substrate and enzyme.
The optimum temperature of the reaction was not
affected by immobilization process (55°C). This result
is similar to that reported by Abdel-Naby et al.[1] who
reported that both free and immobilized alkaline
protease covalently linked to chitosan had the same
optimum temperature (50°C).
The calculated values of the activation energies for
the free and immobilized enzymes were 6.206 and
5.688 Kcal/mol, respectively. The lower value of
activation energy of the immobilized enzyme compared
to the free enzyme may be attributed to the mass
transfer limitations. Kitano et al.[27] and Allenza et al.[5]
reported that the activation energies of the
immobilized enzymes were lower because the internal
diffusion is the rate limiting step. The decrease of the
activation energy of immobilized protease was also
reported by [16,49].
The rate of heat inactivation of the free and
immobilized enzyme was investigated at 65 and 70°C.
In general, the immobilization process protected the
enzyme against heat inactivation. For example, the
calculated half-live values at 65 and 70°C for the
immobilized enzyme on sponge were 808.6 and 108.6
min, respectively, which were higher than that of the
free enzyme.
The result is higher than that reported by Ahmed
et al.[4] on wool immobilized alkaline protease (t 1 /2 at
60 and 70°C were 100 and 25, respectively). Also,
Silva et al.[43] reported lower half live of immobilized
protease at 60°C (438 min) than our result.
438
The calculated deactivation rate constant showed
that the thermal stability of the immobilized enzyme
was superior to that of free one due to protection
that provided by immobilization to enzyme [28].
The calculated deactivation rate constants at 65 and
70°C for the free enzyme were 4.20 x 10 -3 and 9.33 x
10-3 and for the immobilized enzyme 0.857 x 10-3 and
6.38 x 10-3, respectively.
The investigation of the free and immobilized
enzymes at different pH values showed that the
optimum pH values of the free and immobilized
enzymes were the same (pH 10.0) Table (2). This
result is similar to that reported by Chellapandian and
Sastry[12] who reported that alkaline protease covalently
linked on nylon had optimum pH 8.5 which was
similar to that of free 8.5-9.0.
Thermal stability of the immobilized enzyme was
also studied (Fig 5 & 6). In general, the thermal
stability of the immobilized enzyme was significantly
improved in comparison to the free enzyme. Thus after
heating the enzyme, in the absence of the substrate, the
adverse effect of temperature began with the free
enzyme when incubated at 45°C for 60min and more.
On the other hand, the immobilized enzyme was
slightly affected when heated at 65°C for 90min
leading to loss of only 10.37% of its activity. On
heating free enzyme at 65°C and immobilized at 70°C
and higher, the adverse effect of temperature were
more pronounced in case of free than the immobilized
enzyme. These results agreed with those reported
for immobilization of Bacillus subtilis alkaline
protease by Davidenko [15] . The immobilized enzyme
showed 2-fold increase in thermal stability, compared
to free one, after 2h at 50°C. This result is similar to
Ferreira et al.[16].
Chellapandian and Sastry [12] stated that the increase
in thermal stability of the immobilized protease is due
to reduction in autolysis and this is possibly due to (i)
enzyme-enzyme interaction and (ii) re st ricting
conformational flexibility. The restriction of molecular
flexibility might be due to multipoint covalent
interaction with the matrix.
The effect of the metal salts on the activity of
immobilized and free alkaline protease was investigated
in Tables (3). The activation by metal ions (Ca 2+ , Mg2+ ,
Mn 2+ and Na + ) became more pronounced with the
i m m o b ilize d e n zy m e . O n t h e o t h e r h a n d ,
immobilization protected the enzyme from the
inhibition by some metal ions (Co 2+ , Cu 2+ and Fe 3+ ).
These results similar to Wehidy [49]. On the other hand,
Amaral et al.[6] reported the inhibition of trypsin by
Al3+ , Cu2+ , Zn 2+ and Mg2+ , but immobilization protected
the enzyme to some extent from Ca 2+ and Mg2+
inhibition.
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008

Table 1: Partial purification of Streptomyces avermectinus NRRL B-8165 alkaline protease.

Purification Total protein Total activity Recovered Specific activity Purification
(mg/fraction) (U) activity (%) (U/mg protein) fold
Culture filtrate 247.00 807.69 100 3.27 1
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Ethanol 50% 19.82 77.79 9.63 3.92 1.20
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Acetone 50% 12.65 169.80 21.02 13.42 4.10
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Ammonium sulphate 50% 7.59 55.50 6.87 7.31 2.24

Table 2: Properties of free and immobilized extracellular partially p urified alkaline protease from Streptomyces avermectinus NRRL B-8165
Property Free enzyme Immobilized enzyme
Specific activity (U/mg protein) 13.42 9.54
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Optimum temperature (°C) 55 55
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Optimum pH 10 10
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Activation energy (Kcal/mole) 6.206 5.688
Half life t1 /2 (min) at
65 °C 165.0 808.6
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
70 °C 74.3 108.6
Deactivation rate constant(min-1 ) at
65 °C 4.20 x 10-3 0.857 x 10-3
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
70 °C 9.33 x 10-3 6.38 x 10-3
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Km (mg/ml) 0.59 4.55
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Vm ax (U/mg protein) 16.95 12.50

Table 3: Effect of addition of different concentrations of some metal salt on the ac tivity of free and immobilized alkaline protease of
Streptomyces avermectinus NRRL B-8165.
Metal salt conc. (mM) Relative activity (%)
-------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------
Metal salts Free enzyme Immobilized enzyme
--------------------------------------------------------------- -------------------------------------------------------------------
0.1 mM 0.1 mM 0.1 mM 0.1mM 1 mM 10 mM
Control 100.00 100.00 100.00 100.00 100.00 100.00
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
CaCl2 100.41 106.48 90.97 111.10 119.66 90.25
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
CoCl2 .6H2 O 94.80 90.03 73.42 100.00 93.68 89.07
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
CuCl2 .2H2 O 93.44 100.81 99.74 100.25 100.89 100.50
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
FeCl3 86.72 86.13 84.59 100.55 98.07 90.78
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
LiSO 4 92.72 96.83 101.45 96.62 104.75 123.11
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
MgCl2 .6H2 O 98.02 98.99 98.08 104.50 112.4 159.57
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
MnCl2 .4H2 O 89.78 95.80 116.35 101.08 107.28 134.29
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
KCl 92.35 112.01 114.95 96.46 102.97 119.74
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
NaCl 98.98 100.70 99.64 100.35 139.09 115.66
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
ZnCl2 97.93 100.97 102.50 99.64 98.86 100.86
*Control was carried out without any tested substances.

Lineweaver-Bürk plots of the free enzyme gave Km (maximum reaction rate) of the free enzyme was
(Michaelis constant) of 0.59mg/ml with casein, which 16.95U/mg protein which was 1.36-fold higher than
was 7.7-fold lower than that of the immobilized that of the immobilized (12.5U/mg protein). So, the
enzyme (4.55mg/ml). The calculated value of Vm ax Vm ax value of immobilized enzyme is lower than that

439
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008

Fig. 1: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinus
NRRL B-8165 using covalent binding.

Fig. 2: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinus
NRRL-B-8165 using ionic binding.

440
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008

Fig. 3: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinus
NRRL B-8165 using physical adsorption.

Fig. 4: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinus
NRRL B-8165 using entrapment in agar, agarose and calcium alginate.

441
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008

Fig. 5: Thermal stability of immobilized extracellular partially purified alkaline protease produced by Streptomyces
avermectinus NRRL B-8165.

Fig. 6: Thermal stability of immobilized extracellular partially purified alkaline protease produced by Streptomyces
avermectinus NRRL B-8165.

of the free one [35,43 ] . In this respect, Wehidy [49]
found Km of free was 1.2-fold lower than the
immobilized protease. While Vm a x of free and
immobilized was 7.67 and 7.27 U/mg protein,
respectively.
The operational stability of immobilized enzyme is
one of the most important factors affecting the
utilization of an important enzyme system. Therefore,
the operational stability of the immobilized alkaline
protease was evaluated in repeated batch process
(Fig. 7). The result indicated the durability of the
catalytic activity in repeated use for more than 15
cycles. The retained catalytic activity after being used
for 15 cycles was 50% of the initial activity. In this
respect, it was reported by Chellapandian [13] that the
vermiculite-bound alkaline protease retained 78% of its
original activity after five repeated uses. Our result is
higher than that obtained by, Mittal et al. [ 3 5] when
reused immobilized dipeptidylpeptidase 6 cycles
(retained activity 30%).

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 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008

Table 4: Detergent stability of crude extracellular alkaline protease produced by Streptomyces avermectinus NRRL B-8165.
Time (min)
--------------- Relative activity %
Detergent ---------------------------------------------------------------------------------------------------------------------------------------------------
0 15 30 45 60
Control 100.00 100.00 100.00 100.00 100.00
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Ariel® 100.00 100.00 97.05 86.68 73.46
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Persil color® 100.00 100.00 100.00 98.66 82.03
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Tide® 100.00 85.25 65.42 60.71 53.89
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
X.TRA® 100.00 100.00 100.00 95.38 79.44

Fig. 7: Operational stability of immobilized extracellular partially purified alkaline protease on sponge using
covalent binding produced by Streptomyces avermectinus NRRL B-8165.

Photo 1&2: Decomposition of X-ray films by partially purified extracellular alkaline protease produced by
Streptomyces avermectinus NRRL B-8165. (1) control and (2) experiment.

The overall performance of the immobilized
alkaline protease is promising than the free enzyme.
Thus it is suggested that Streptomyces avermectinus
NRRL B-8165 alkaline protease immobilized on sponge
by covalent binding is suitable for practical application.
In our study, the enzyme compatibility with
commercial detergents was investigated. The enzyme
was most stable with Persil color ® up to 60 min where
it lost only 17.97% of its activity. On the other hand,
the lowest enzyme stability was observed with Tide ® .
However, it lost 20.56% and 26.54% of its activity
after 60 min incubation with X-TRA® and Ariel® ,
respectively at 40°C Table (4). In this respect, Hadj-Ali
et al.[20] reported a detergent stable protease which

443
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008
Photo 3: Dehairing of buffalo hide by partially purified extracellular alkaline protease produced by Streptomyces
avermectinus NRRL B-8165. (1) control, (2) after 12h and (3) after 24h.
retained 65-95% activity after 1h incubation with
commercial detergents at 40°C and Gupta et al.[19]
reported a protease which retained 30-40% activity
after 30min incubation with Ariel® and Surf Exel® but
lost activity after that. On the other hand, it lost only
10-30% activity with other commercial detergents after
2h incubation at 40°C.
In our studying the ability of the partially
purified enzyme to decompose the gelatin layer of
X-ray film in order to recover both silver and
polyethylene tetraphthalate film base within only 15
min (Photo 1&2). This result is similar to Masui
et al.[34] and Karadzic et al.[23]. They reported a
protease that decomposed the gelatin layer on X-
ray film from Bacillus sp. and Pseudomonas
aeruginosa, respectively.
Also, studying the ability of the partially purified
enzyme to dehair buffalo hide was investigated
(Photo 3). The enzyme removed intact hairs completely
from the hide after 24h incubation at 37°C. In this
respect, Zambare et al.[51] reported an alkaline protease
that was applied as a dehairing agent. The enzyme
acted on the epidermis and hair was removed entirely;
the intact hair can be used as a value-added byproduct.
Using enzyme will reduce the time of the process and
yield better quality leather. However, Sivasubramanian
et al.[46] reported that the use of protease in deharing of
goat skins and cow hide revealed complete absence of
fine hair and epidermis and the pelts were whiter than
that dehaired by chemical methods.
At last, it is worthy to note that the results of the
present work point out to the suitability of the strain
Streptomyces avermectinus NRRL B-8165 as a new
promising producer of highly active extracellular
alkaline protea se u sin g inexpensive materials.
Furthermore, enzyme immobilization proved to be a
good technique for increasing enzyme thermostability
and its several reuse.
444
REFERENCES
1. Abdel-Naby, M.A., A.M.S. Ismail, S.A. Ahmed
and A.F. Abdel Fattah, 1998. P roduction and
immobilization of alkaline protease from Bacillus
mycoides. Bioresource Technol., 64: 205-210.
2. Ahmed, S.A., 1994. Biochemical studies on
microbial proteolytic enzymes. MSc. Thesis,
Department of Biochemistry, Faculty of Agric.,
Cairo University, Egypt.
3. Ahmed, S.A., Shousha, W.Gh., E.M.E. Mahdy, H.
Refaat and M.A. Abel-Naby, 2004. Production,
purification, modification and immobilization of
alkaline protease from Bacillus stearothermophilus.
Egypt. J. Biotechnol., 17: 48-59.
4. Ahmed, S.A., S.A. Saleh and A.F. Abdel-
F a t t a h , 2 0 0 7 . S t a b iliza t io n o f B a c i l l u s
Licheniformis ATCC 21415 Alkaline Protease by
Immobilization and Modification. Australian
Journal of Basic and Applied Sciences, 1(3):
313-322.
5. Allenza, P., D.S. Scherl and R.W. Detroy, 1986.
Hydrolysis of xylan by an immobilized xylanase
from Aureobasidium pullulans. Biotechnol. Bioeng.
Symp., 17: 425-423.
6. Amaral, I.P.G., M.G. Carneiro-da-Cunha, L.B.
Carvalho Jr. and R.S. Bezerra, 2006. Fish trypsin
immobilized on ferromagnetic Dacron. Process
Biochem., 41: 1213-1216.
7. Banik, R.M. and M. Prakash, 2004. Laundry
detergent compatibility of the alkaline proteases
from Bacillus cereus. Microbiological Research.
159: 135-140.
8. Bickerstaff, G.F., 1997. Methods in Biotechnology.
In immobilization of enzymes and cells. Humama
Press, Totowa, NJ, 1.
9. Brinbaum, S., 1994. Immobilized enzymes and
cells in biochemical reaction. In: Immobilized
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008
biosystems: theory and practical application.
Blackie Academic and professional.
10. Carrara, C.R. and A. Rubiolo, 1994. The
immobilization of â-galactosidase on chitosan.
Biotechnol. Prog., 10: 220-224.
11. Chauhan, B. and R. Gupta, 2004. Application of
statistical experimental design for optimization of
alkaline protease production from Bacillus sp.
RGR-14. Process Biochem., 39: 2115-2122.
12. Chellapandian, M. and C.A. Sastry, 1996. Covalent
linking of alkaline protease on trichlorotriazine
activated nylon. Bioprocess Engin., 15: 95-98.
13. Chellapandian, M., 1998. P reparation and
characterization of alkaline protease immobilized
on vermiculite. Process Biochem., 33: 169-173.
14. Chi, Z. and S. Zhao, 2003. Optimization of
medium and cultivation conditions for pullulan
production by a new pullulan-producing yeast.
Enzyme Microb. Technol., 33: 206-211.
15. Davidenko, T.I., 1999. Immobilization of alkaline
protease on polysaccharides of microbial origin.
Pharmaceutical Chem. J., 33: 487-489.
16. Ferreira, L., M.A. Ramos, J.S. Dordick and M.H.
Gil, 2003. Influence of different silica derivatives
in the immobilization and stabilization of a
b a c i l l u s l i c h e n i f ormis protease (Subtilisin
Carlsberg). J. Mol. Catal. B: Enzym., 21: 189-199.
17. Gessesse, A. and B.A. Gashe, 1997. Production of
alkaline protease by an alkaliphilic bacteria isolated
from an alkaline soda lake. Biotechnol. Lett.,
19: 479-481.
18. Gupta, M.N. and I. Roy, 2002. Applied
Biocatalysts: An overview. Ind. J. Biochem.
Biophys., 39: 220-228.
19. Gupta, A., B. Joseph, A. Mani and G. Thomas,
2008 . B io sy n t h esis and properties of an
extracellular thermostable serine alkaline protease
from Virgibacillus pantothenticus. World J.
Microbiol. Biotechnol., 24: 237-243.
20. Hadj-Ali, N.E., R. Agrebi, B. Ghorbel-Frikha, A.
Sellami-Kamoun, S. Kanoun and M. Nasri, 2007.
Biochemical and molecular characterization of a
detergent stable alkaline serine-protease from a
newly isolated Bacillus licheniformis NH1. Enzym.
Microb. Technol., 40: 515-523.
21. Johnvesly, B. and G.R. Naik, 2001. Studies on the
production of thermostable alkaline protease from
thermophilic and alkaliphilic Bacillus sp. JB-99 in
a chemically defined medium. Process Biochem.,
37: 139-144.
22. Joo, H.S., C.G. Kumar, G.C. Park, K.T. Kim, S.R.
Paik and C.S. Chang, 2002. Optimization of the
production of an extracellular alkaline protease
from Bacillus Horikoshii. Process Biochem., 38:
155-159.
445
23. Karadzic, I., A. Masui and N. Fujiwara, 2004.
Purification and characterization of a protease from
Pseudomonas aeruginosa grown in cutting oil. J.
Biosci. Bioeng., 98: 145-152.
24. Kathiresan, K. and S. Manivannan, 2007.
Production of alkaline protease by Streptomyces
sp., isolated from coastal mangrove sediment. Res.
J. Environ. Sci., 1: 173-178.
25. Kaur, S., R.M. Vohra, M. Kapoor, Q.K. Beg and
G.S. Hoodal, 2001. Enhanced production and
characterization of a highly thermostable alkaline
protease from Bacillus sp. P-2. W orld J. Microbiol.
Biotechnol., 17: 125-129.
26. Kembhavi, A.A., A. Kulkarni and A. Pant, 1993.
Salt-tolerant and thermostable alkaline protease
from Bacillus subtilis NCIM No.64. Appl.
Biochem. Biotechnol., 38: 83-92.
27. Kitano, H., K. Nakamura and N. Ise, 1982. Kinetic
studies of enzyme immobilized on anionic polymer
lattices. J. Appl. Biochem., 4: 34.
28. Klibanov, A.M., 1979. Enzyme stabilization by
immobilization. Anal. Biochem., 93: 1-2
29. Kumar, C.G. and H. Takagi, 1999. Microbial
alkaline proteases: From a bioindustrial viewpoint.
Biotechnol. Adv., 17: 561-594.
30. Laan, R. and W.N. Konings, 1989. Mechanism of
protienase release from Lactobacillus lactis subsp.
cr emoris Wg2". Appl. Environ. Microbiol., 55(12):
3101-3106.
31. Lamas, E.M., R.M. Berros, V.M. Balcão and F.X.
Malcata, 2001. hydrolysis of whey protein by
protease extracted from Cynara cardunculus and
immobilized into highly activated supports.
Enzyme Microb. Technol., 28: 642-652.
32. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J.
Randall, 1951. Protein measurement with the folin
phenol reagent. J. Biol. Chem., 193: 265-275.
33. Mabrouk, S.S., A.M. Hashem, N.M.A. El-Shayeb,
A.M.S. Ismail and A.F. Abdel-Fattah, 1999.
Optimization of alkaline protease production by
Bacillus licheniformis ATCC 21415. Bioresource
Technol., 69: 155-159.
34. Masui, A., N. Fujiwara, M. Takagi and T.
Imanaka, 1999. Feasibility study for decomposition
of gelatin layers on X-ray film by thermostable
alkaline protease from alkaliphilic Bacillus sp.
Biotechnol. Techniq., 13: 813-815.
35. Mittal, A., S. Khurana, H. Singh and R.C.
Ka mboj, 2005. Cha rac te riz a t ion of
dipeptidylpeptidase IV (DPP IV) immobilized in
Ca alginate beads. Enzyme Microb. Technol.,
37: 318-323.
36. Mohamedin, A.H., 1999. Isolation, identification
and some cultural conditions of a protease-
producing thermophilic Streptomyces strain grown
 Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008
on chicken feather as a substrate. International
Biodeterioration and Biodegradation., 43: 13-21.
37. Najafi, M.F., D. Deobagkar and D. Deobagkar,
2005. Potential application of protease isolated
from Pseudomonas aeruginosa PD100. Elect. J.
Biothechnol., 8(2): 197-203.
38. Nascimento, W .C.A.D. and M.L.L. Martins, 2004.
Production and properties of an extracellular
protease from thermophilic Bacillus sp. Brazilian
J. Microbiol., 35: 91-96.
39. Patel, R., M. Dodia and S.P. Singh, 2005.
Extracellular alkaline protease from a newly
isolated haloalkaliphilic Bacillus sp.: Production
and optimization. Process Biochem., 40: 3569-
3575.
40. Peczynska-Czoch, W. and M. Mordarshi, 1988.
Actinomycetes enzymes. In: Actimomycetes in
biotechnology. London: Academic Press., pp:
219-283.
41. Razak, N.A., M.Y.A. Samad, M. Basri, W.M.Z.Y.
Yunus, K. Ampon and A.B. Salleh, 1994.
Thermostable extracellular protease of Bacillus
stearothermophilus: factors affecting its production.
World J. Microbiol. Biotechnol., 10: 260-263.
42. Shafee, N., S.N. Aris, R.N.Z.A. Rahman, M Basri
a n d A.B. Salleh, 2005. O p t im iza t io n o f
environmental and nutritional conditions for the
production of alkaline protease by a newly isolated
bacterium Bacillus cereus strain 146. J. Appl. Sci.
Res., 1: 1-8.
43. Silva, C.J.S.M., Q. Zhang, J. Shen and A. Cavaco-
Paulo, 2006. Immobilization of proteases with a
water soluble-insoluble reversible polymer for
treatment of wool. Enzyme Microb. Technol.,
39: 634-640.
446
44. Sinha, N. and T. Satayanarayana, 1991. Alkaline
protease by thermophilic Bacillus licheniformis.
Ind. J. Microbiol., 31: 425-430.
45. Siso, M.I.G., M. Graber, J.S. Condoret and D.
Combes, 1990. Effect of diffusional resistance on
the action pattern of immobilized alpha-amylase.
J. Chem. Technol. Biotechnol. 48: 185-200.
46. Sivasubramanian, S., B.M. Manohar, A. Rajaram
and R. Puvanakrishnan, 2008. Ecofriendly lime and
sulfide free enzymatic dehairing of skins and hides
using a bacterial alkaline protease. Chemosphere.,
70: 1015-1024.
47. Suh, C.W., S.H. Park, S.G. Park and E.K. Lee,
2005. Covalent immobilization and solid-phase
refolding of enterokinase for fusion protein
cleavage. Process Biochem., 40: 1755-1762.
48. Tanksale, A., P.M. Chandra, M. Rao and V.
Deshpande, 2001. Immobilization of alkaline
protease from Conidiobolus macrosporus for reuse
and improved thermal stability. Biotechnol. Lett.,
23: 51-54.
49. Wehidy, H.R., 2004. Biochemical studies on
microbial alkaline protease. MSc. Thesis. Science
Biochem., Helwan University, Egypt.
50. Woodward, J., 1985. Immobilized cells and
enzymes: a practical approach. 44, Oxford, IRI,
Press Limited, England.
51. Zambare, V.B., S.S. Nilegaonkar and P.P. Kanekar,
2007. Production of an alkaline protease by
Bacillus cereus MCM B-326 and its application as
a dehairing agent. World J. Microbiol. Biotechnol.,
23: 1569-1574.