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Alkaline protease production by an actinomycete MA1-1 isolated from marine

sediments

Article  in  Annals of Microbiology · March 2007

DOI: 10.1007/BF03175053

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Annals of Microbiology, 57 (1) 000-000 (2007) PR

Alkaline protease production by an actinomycete MA1-1 isolated

from marine sediments

E. Esin HAMEŞ-KOCABAŞ1*, Ataç UZEL2

1Science and Technology Center, Ege University, Bornova Campus, 35100, Izmir; 2Ege University Faculty of Science, Department of
Biology, Basic and Industrial Microbiology Section, Bornova Campus, 35100, Izmir, Turkey

Received 4 August 2006 / Accepted 22 November 2006

Abstract - Alkaliphilic actinomycetes isolated from sediment samples of the Izmir Gulf, Turkey were studied for the production of pro-
tease activity. Strain MA1-1 was selected as a good alkaline protease producer as measured by the clear zone diameter by the hydrol-
ysis of skim-milk and casein. The alkaline protease production from the marine alkaliphilic actinomycete MA1-1 was studied by using
different carbon and nitrogen sources in medium containing glycerol, peptone, KCl, MgSO4, K2HPO4, and trace elements at 30 ºC for
72 h. Among the different carbon and nitrogen sources, fructose, starch, maltose, D(+) glucose, yeast extract, malt extract, beef extract
and peptone provided higher production of protease. Starch was also found to be effective for growth and enzyme production with high-
est specific activity at 699 U mg-1. Purification was achieved by adsorption on Diaion HP 20 which resulted in a recovery rate of 68%
with a specific activity of 7618 U mg-1 protein and 40-fold purification. The optimum pH and temperature of the partially purified pro-
tease were determined as pH 9.0 and 50 ºC, but high activity was also observed at pH 8.0-13.0 and 35-50 ºC. The inhibition profile
exhibited by phenylmethylsulphonyl fluoride (PMSF) showed that this enzyme belongs to the serine-protease group.

Key words: alkaline protease, alkaliphilic, halophilic, actinomycete, marine sediments.

INTRODUCTION Actinomycetes are representative of terrestrial microor-

Proteases are physiologically necessary for living organisms.
They are found in a wide diversity of sources such as plants,
animals, and microorganisms (Rao et al., 1998). On the
other hand, protease constitutes one of the most important
groups of industrial enzymes, accounting for at least 25% of
the total enzyme sales, with two-thirds of the proteases pro-
duced commercially being of microbial origin (Mehta et al.,
2006). Microorganisms are an attractive source of proteas-
es as they can be cultured in large quantities in a relatively
short time by established fermentation methods, and as
they can be made to produce an abundant, regular supply
of the desired product. Bacteria are the most dominant
group of alkaline protease producers with the genus Bacillus
being the most prominent source (Gupta et al., 2002; Ban
et al., 2003; Rahman et al., 2003). Several fungi have also
been reported to produce extracellular alkaline proteases
(Coral et al., 2003). However, very few studies exist on the
alkaline protease producing alkalophilic actinomycetes
(Kumar and Takagi, 1999). Among actinomycetes, strains
of Streptomyces are the preferred source (Petinate et al.,
1999; Gupta et al., 2002). While most studies on actino-
mycetes have focused on antibiotic production, only a few
reports are on their enzymes (Kumar and Takagi, 1999;
Mehta et al., 2006).
* Corresponding author. Phone: +90 232. 343 4400;
Fax: +90 232. 374 4289; E-mail: esin.kocabas@ege.edu.tr
ganisms and usually are isolated from soils. Compared to
terrestrial actinomycetes, however, very little work has
been conducted on marine actinomycetes. The marine envi-
ronment is considerably different from the terrestrial envi-
ronment, and therefore, it needs to be explored and exploit-
ed for new biological products (Imada, 2005).
This paper describes the isolation of an alkaline protease
producing actinomycete from marine sediments. The deter-
mination of optimal conditions for protease production and
partial characterisation of the enzyme.
MATERIALS AND METHODS
Microorganism isolation. The extracellular alkaline pro-
tease producer strain MA1-1 was selected as a good pro-
tease producer among the isolates from marine sediment of
the Izmir Gulf, Turkey. Samples were collected from 25
meter depth in a sterile 50 ml screw cap containers by scuba
diving. The sediment was dried overnight in a laminar flow
hood and serially diluted with sterile seawater. The diluted
sample was vortex mixed, allowed to settle for a few min-
utes, and 0.1 ml supernatant inoculated onto the surface of
an agar plate and spread with an alcohol-sterilised glass
rod. The actinomycetes strains were isolated at 30 ºC in a
medium containing (g/l): starch (Sigma) 10.0, yeast extract
(Oxoid) 4.0, peptone (Oxoid) 2.0, agar (Oxoid) 1.5%, sea
water 1000 ml, pH 10.5 (Jensen et al., 2005). A qualitative
screening for the proteolytic activity of the isolates was indi-
2 E.E. Hameş-Kocabaş and A. Uzel
cated by growth and clear zones appearance on isolation
medium (prepared with distilled water) supplemented with
skim-milk (Oxoid) and casein (Sigma) respectively (1%,
w/v). The colonies surrounded by clear zones caused by the
hydrolysis of skim-milk and casein were evaluated as a pro-
tease producers.
Growth conditions and enzyme production. Strain
MA1-1 was cultured in Glycerol-peptone-salt medium
(GlyPep) consisting of (g/l): glycerol (Panreac) 20.0, pep-
tone (Oxoid) 4.5, KCl 0.3, MgSO4 0.5, K2HPO4 2.0, and 1 ml
trace element solution (1 mg/ml of each CuSO4·5H2O,
MnSO4·7H2O, ZnSO4·7H2O, CaCl2·2H2O) (Petinate et al.,
1999). The pH of the medium was adjusted to 10.5 before
steam sterilisation and the culture was grown in 500 ml of
Erlenmeyer flasks containing 100 ml medium. The flasks
were inoculated with 5% (v/v) of 48 h old culture and were
incubated on a rotary incubator shaker at 30 ºC with agita-
tion (180 rpm) for 120 h. Aliquots (5 ml) of culture medium
were collected daily and centrifuged at 9000 rpm for 10 min
to collect biomass. Cells were washed with 0.85% w/v NaCl
solution and lyophilised for biomass determination and the
supernatant was examined for total protein content and
enzyme activity.
The effect of various carbon and nitrogen sources on
the enzyme production was tested by using GlyPep medi-
um consisting glycerol and peptone as carbon and nitrogen
sources. The composition of the medium was changed by
replacing the original C and N sources. The culture was
grown in 250 ml of Erlenmeyer flasks containing 50 ml
medium were inoculated with 5% (v/v) of 48 h old culture
and the flasks incubated at 30 ºC with 180 rpm agitation
for 72 h. The carbon source glycerol was replaced with
various carbon sources (1% w/v) such as, D(+) glucose,
lactose, starch, D(+) raffinose, D(+) mannose, maltose,
sucrose, fructose and D(+) xylose (Sigma). And the nitro-
gen source peptone was also replaced with various nitro-
gen sources (0.45% w/v) such as NaNO3, KNO3, NH4Cl,
NH4NO3, urea (Merck), yeast extract, beef extract, malt
extract, gelatine, peptone (Oxoid) and casamino acids
(Sigma).
Protease assay. The activity of alkaline protease in the
cell free supernatant was measured by modified method of
Takami et al. (1989). Alkaline protease activity was deter-
mined using casein as a substrate at a concentration of
0.6% w/v in 50 mM glycine-NaCl-NaOH buffer (pH 10.5).
The assay was carried out routinely in a mixture containing
0.50 ml of a suitably diluted enzyme solution and 2.5 ml
casein solution. After incubation for 20 min at 30 ºC the
reaction was terminated by the addition of 2.5 ml 0.44 M
trichloroacetic acid (TCA) solution. After 10 min the mixture
was centrifuged at 8000 rpm for 10 min. An aliquot of 0.5
ml of supernatant was mixed with 2.5 ml of 0.5 M Na2CO3
and 0.5 ml of Folin-Ciocalteu’s phenol solution and kept for
30 min at room temperature. The optical densities of the
solutions were determined with respect to the sample
blanks at 660 nm using Helios Unicam spectrophotometer.
Three sets of experiments were carried out for the protease
assay. One unit of proteolytic activity was defined as the
amount of the enzyme resulting in the release of 1 µg of
tyrosine per minute at 30 ºC under the defined assay con-
ditions.
Protein content. The protein concentration of the samples
was determined at 595 nm by the method of Bradford
(1976).
Partial characterisation of the enzyme. Diaion HP 20 is
a polymeric resin based on styrene-divinylbenzene with
mean pore size of 0.3 nm and surface area of 500 m2 g-1.
The resin is widely used in a broad range of applications.
Diaion HP 20 was added to the cell free supernatant to a final
concentration of 5% (w/v), and stirred vigorously at 250
rpm with mechanical stirrer for 4 h, according to the method
previously described (Joo et al., 2001). The resin was recov-
ered by suction filtration, and then eluted with buffer A (0.05
M Tris-HCl, pH 8.5) containing 20% v/v acetone. The eluted
enzyme was then precipitated with two volumes of cold ace-
tone (-20 ºC) and collected after overnight standing in a cold
chamber. The precipitate enzyme was re-dissolved in buffer
A and stored as aliquots at –70 ºC for further use.
Effect of pH, temperature and phenylmethylsulphonyl
fluoride (PMSF) on activity. The pH optimum of the par-
tially purified protease was determined at 30 ºC in different
buffer systems of 50 mmol l-1 each: acetate (pH 4-6), phos-
phate (pH 7-8), Tris-HCl (pH 9-10), glycine-NaOH (pH 10.5
-11) and KCl-NaOH (pH 13) with pH values ranging from 4.0
to 13.0.
The temperature optimum was evaluated by incubation
of partially purified protease for 20 min in 50 mmol l-1
glycine-NaCl-NaOH (pH 10.5) at different temperatures
ranging from 20 to 60 ºC and measuring the activity as
described earlier.
The effect of PMSF was investigated to further charac-
terise the enzyme. The enzyme was pre-incubated with the
PMSF (1 mM) for 3, 5, 15, 20 min at 30 ºC. After incubation
the residual activity (%) was determined.
Statistical analysis. ANOVA analysis was performed with
SPSS 10.0 software for comparison the effect of different
carbon and nitrogen sources on the enzyme production.
Significance (p) was set as 0.01.
7 20,0
Enzyme activity (U/ml)
6
16,0
Dry weight (g/l)
5
12,0
4
3 8,0
2
4,0
1
0 0,0
0 50 100 150 200
Time (h)
FIG. 1 - Time course of growth and protease production of strain
MA1-1. (]) Cell growth, (p) enzyme activity.
Ann. Microbiol., 57 (1), 000-000 (2007) 3

TABLE 1 - Biomass, total protein, enzyme and specific activities of strain MA1-1 at different carbon and nitrogen sources at 30 ºC for
72 hours

Sources Lyophilised biomass Total protein Enzyme activity Specific activity

(g l-1) (mg ml-1) (U ml-1) (U mg-1)

None 0.73 0.01 02.97 ± 0.32 224 ± 4.0

D (+) Glucose 2.07 0.03 11.35 ± 0.27 341 ± 7.0
Carbon sources (1% w/v)

Lactose 1.13 0.01 03.41 ± 0.21 258 ± 8.0

Starch 2.77 0.02 12.76 ± 0.52 699 ± 7.5
D (+) Raffinose 0.40 0.01 02.58 ± 0.32 195 ± 6.0
D (+) Mannose 2.30 0.03 07.39 ± 0.38 222 ± 7.0
Maltose 2.87 0.04 11.79 ± 0.72 272 ± 9.0
Sucrose 0.53 0.01 01.57 ± 0.27 191 ± 3.0
Fructose 2.40 0.03 13.73 ± 0.14 413 ± 3.5
D (+) Xylose 1.43 0.02 05.18 ± 0.62 284 ± 9.0
Glycerol 1.50 0.04 07.24 ± 0.23 189 ± 9.0

None NG NG NG NG
NaNO3 NG NG NG NG
Nitrogen sources (0.45% w/v)

KNO3 NG NG NG NG
NH4Cl NG NG NG NG
Urea NG NG NG NG
NH4NO3 NG NG NG NG
Yeast extract 3.40 0.05 09.42 ± 0.37 177 ± 7.0
Beefextract 2.90 0.06 09.30 ± 0.30 147 ± 6.0
Malt extract 0.80 0.01 02.57 ± 0.23 194 ± 4.0
Gelatine 2.33 0.03 03.27 ± 0.23 116 ± 5.0
Casamino acid 1.27 0.02 02.12 ± 0.12 091 ± 2.0
Peptone 1.50 0.04 07.24 ± 0.13 189 ± 2.5

NG: No growth

RESULTS AND DISCUSSION
In recent years many researchs are focused to undiscovered
marine habitats as a new source of microroganisms. This
work describes the production, characterisation and partial
purification of an alkaline protease from a marine actino-
mycete strain MA 1-1.
Growth properties of strain MA1-1
The results of the zone diameter and clearance on skim-milk
were similar to those on casein. The strain MA1-1 was
selected as a good alkaline protease producer and used in
all further investigations. Strain MA1-1 was able to grow up
to 45 ºC, with an optimum 30 ºC. It was able to grow over
a wide range of initial pH (4.0-12.0) and it requires an alka-
line pH of 8.0-11.0 for growth. The cells were able to grow
in the presence of 9% (w/v) NaCl indicating that it was a
moderate halophilic actinomycete.
Protease production by the strain MA1-1 was first
recorded at the early exponential growth phase (15.7 U ml-1
at 24 h) and then increased slightly in the stationary phase
(18.1 U ml-1 at 72 h) (Fig. 1). Maximum enzyme production
was detected at the late exponential phase of growth. Mehta
et al. (2006) also reported that the protease production by
Streptomyces sp. was detected at the beginning of the late
exponential phase.
Effect of carbon and nitrogen sources on the protease
production
The carbon source glycerol was replaced with various car-
bon sources (1% w/v). Samples were removed after 72 h
and proteolytic activities and protein content of culture
supernatants were then assayed. Strain MA1-1 grew on all
carbon sources except sucrose and raffinose. The produc-
tion of protease varied with each carbon sources (Table 1).
Presence of fructose, starch, maltose and D(+) glucose
provided higher production of protease. We have also
noticed that among these carbon sources, highest specific
activity was obtained using starch (Fig. 2). Replacement of
the glycerol with sucrose and raffinose did not effect spe-
cific activity significantly (p > 0.01) but replacement with
fructose, starch, maltose, glucose, lactose, mannose and
xylose were provided significant increase on the enzyme
production (p < 0.01).
The effect of the nitrogen source on extracellular
enzyme secretion was also studied by replacing peptone in
the GlyPep medium. Samples were removed after 72 h and
proteolytic activities and protein content of culture super-
natants were assayed. Lack of nitrogen source and use of
inorganic nitrogen compounds inhibited growth.
The specific activity of protease production with starch
was 699 U mg-1 ± 7.50 protein. This value is higher than
the other carbon sources. Among nitrogen sources, yeast
4 E.E. Hameş-Kocabaş and A. Uzel

800

700

Specific activity (U/mg)

600

500

400

300

200

100

se
ne

se

ch

se

se

se

se

l
ro
os

os
no
co

no

to

to

lo
ar
No

ce
ct

cr

Xy
al

uc
St
lu

ffi

an

ly
La

Su
M
G

Fr

G
Ra

)
(+
)
(+

)
)

(+
(+

D
D

D
D

FIG. 2 - Specific activities of strain MA1-1 at different carbon sources. Cells were grown in
GlyPep medium at 30 ºC for 72 h.

TABLE 2 - Partial enzyme purification with Daion HP20

Enzyme Total units Protein (mg) Specific activity Purification fold Yield
(U) (mg) (U mg-1) (no.) (%)

Crude 10860 57 190.5 1 100.0

HP20 7390 0.97 7618 40 68

extract and beef extract seems to be good nitrogen sources
for enzyme secretion, while other inorganic nitrogen com-
pounds and urea inhibited growth (Table 1 and Fig. 2).
Partial purification and characterisation of enzyme
After partial purification by using Diaion HP20, protease
recovery was 68%, with a specific activity of 7618 U mg-1
and 40 purification fold (Table 2). Joo et al. (2001) found
that serine protease from the Perriserrula leucophryna was
purified 56.7 fold using Diaion HP20 for four hours. Petinate
et al. (1999) reported that extracellular proteinase produced
by Streptomyces cyaneus was purified 22 fold using an
aprotinin-agarose affinity chromatography.
The optimum pH and temperature of the protease were
obtain at pH 9.0 and 50 ºC, but it also gave high activity
the pH range and temperature, 8.0-13.0 pH and 35-50 ºC
respectively. The results are expressed as percentage of
residual activity, taking into account the activity deter-
mined maximum (Fig. 3). The protease which originated
from marine actinomycetes was active in alkali pH and
broad range of temperature. This enzyme activity had a
broad pH range with an optimum at 9-11. Considering
these properties, strain MA1-1 protease could find potential
application in the detergent and leather tanning industries.
The optimum pH and temperature values of the enzyme
are in agreement with previous report that described alka-
line proteases obtained from Streptomyces species
(Petinate et al., 1999).
Strain MA1-1 protease was partially inhibited by PMSF
(Fig. 4). The strain MA1-1 protease was mostly inactivated
by PMSF which indicates that this enzyme is a serine pro-
tease. Serine proteases are of considerable interest, in view
of their activity and stability at alkaline pH, because of this
properties they have applications in many industries (Gupta
et al., 2002).
Proteases represent the most important groups of indus-
trial enzymes and one of the three largest groups (Arbige
and Pitcher 1989; Rao et al., 1998). Microbial proteases are
mostly produced from Bacillus species for commercial use.
Although Bacillus strains are of considerable industrial
importance (Kumar and Takagi, 1999; Gupta et al., 2002),
other groups of producers may have similar potential.
Enzyme technology and application area of the proteases
continue to develop. Therefore there is always a need for
new and robust enzymes. Actinomycetes are an alternative
source for proteases, especially which harbour marine habi-
tats are not well explored until now. Alkaline protease activ-
ity of the strain MA1-1 has a wide pH range in alkaline area
with a very little decrease in activity. Since the strain MA1-
1 is also halophilic, this enzyme could be useful in industri-
al applications where a protease activity requires alkaline
and high salt conditions. According to the best of our knowl-
edge, this is the first report on protease activity of a
halophilic alkaliphilic actinomycete isolated from marine
sediments in Turkey.
Ann. Microbiol., 57 (1), 000-000 (2007) 5

A
100

Relative activity (%)

80
120
Relative activity (%)

100 60

80
40
60
20
40

20 0
0 05 10 15 20 25
3 4 5 6 7 8 9 10 11 12 13 14 Time (min)
pH

FIG. 4 - Effects of PMSF on enzyme activity. Maximal enzyme

activities were set as 100% relative activity.

120 Gupta R., Beg Q.K., Lorenz P. (2002). Bacterial alkaline proteas-
Relative activity (%)

es: molecular approaches and industrial applications. Appl.

100
80
60
40
20
0
15 25
Temperature (ºC)
FIG. 3 - Effects of (A) pH and (B) temperature on enzyme activity.
Maximal enzyme activities were set as 100% relative
activity.
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