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Production and Enzyme Activity of an

Extracellular Protease from Aspergillus Niger
and Bacillus Subtilis

Article · March 2009

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International Journal of Biotechnology and Biochemistry
ISSN 0973-2691 Volume 5 Number 3 (2009) pp. 335–342
© Research India Publications
http://www.ripublication.com/ijbb.htm

Production and Enzyme Activity of an Extracellular

Protease from Aspergillus Niger and Bacillus Subtilis

R.S. Pagare, A.M. Ramdasi, S.R. Khandelwal,

M.O. Lokhande and B. A. Aglave*

Department of Biotechnology,
Institute of Life Sciences, H.P.T. Arts and R.Y.K. Science College,
Nashik, Maharashtra, 422 005, India
*Correspondence address: Email: ba_aglave@rediffmail.com

Abstract

An extracellular protease enzyme production from Aspergillus niger and

Bacillus subtilis in culture medium supplemented with glucose and trisodium
citrate as a substrate. These microorganisms could grow up to 60°C within a
broad pH range of 5 to 10 with an optimal growth temperature and pH at 60°C
and 8.0 respectively. For the three Bacillus subtelis was shown better growth
at temperature 50°C and pH 9. Similarly Aspergillus niger was well cultured
at temperature 60°C and pH 8. Maximum protease enzyme activity was
recorded at temperature 60°C (26.1±0.07 Uml-1min-1) and pH 8 (26.0±0.07
Uml-1min-1). The results showed that Bacillus species under study are good
producers of extracellular protease at high temperature in alkaline medium.

Keywords: Protease, Trisodium citrate, Glucose, enzyme activity.

Introduction
Proteases are essential constituents of all forms of life on earth including prokaryotes,
fungi, plants and animals. They are most important industrial enzymes occupying
nearly 60% of the enzyme sales, obtained from microbial, plant and animal sources
(Godfrey and Reichelt, 1983). The alkaline proteases are highly exploited enzymes in
food processing, leather, detergent, pharmaceutical, diagnostics, waste management,
silver recovery medical purposes as well as feeds and chemical industries (Ward,
1985; Kumar and Tyagi,1999; Pastor et al., 2001). The protease enzyme constitutes
two thirds of total enzymes used in various industries and this dominance in the
industrial market is expected to increase by the year 2005 (Gupta et al., 2002). Of all
proteases, alkaline proteases produced by Bacillus species are of great importance in
336 R.S. Pagare et al

detergent industry due to their high thermostability and pH stability. For production of
enzyme for industrial use, isolation and characterization of new promising strains
using cheap carbon and nitrogen source is a continuous process (Parekh et al., 2002).
The success of microbial proteases in food and other biotechnological systems
could be attributed to the broad biochemical diversity of the microorganisms, to the
genetic manipulation of the organisms and the improvement of the techniques in the
enzyme production, purification and characterization. Information pertaining to the
rates of enzyme activity regulation (activation and inactivation of enzymes) and their
functional efficiency constitutes the kinetic data, which determines the way in which
the reaction occurs. Enzyme activity rates are often heavily influenced principally by
conditions such as substrate, activator, inhibitor, enzyme concentration, pH, redox
potential, temperature, etc. Studies on protease production from Bacillus sp. has been
studued by Manachini et al., 1988; Takami et al., 1989; Takami et al., 1990; Mabrouk
et al., 1999.
Even though most commercial proteases originated from microorganisms
belonging to the genus Bacillus, fungi exhibit a wider variety of proteases than
bacteria. Furthermore, fungi are normally GRAS (generally regarded as safe) strains
and they produce extracellular enzymes, which are easier to be recovered from
fermentation broth [Sandya et al., 2005]. The microbial proteases of Aspergillus
species, in particular, have been studied in detail since they are known for their
capacity to secrete high levels of enzymes in their growth environment. Several of
these secreted enzymes, produced in a large-scale submerged fermentation, have been
widely used in the food and beverage industry for decades [Biesebeke et al., 2005].

Material and methods

Microorganism and culture conditions:
Aspergillus niger and Bacillus subtilis was obtained from NCIM (National Center for
Industrial Microorganism), National Chemical Laboratory, Pune. Pure starter culture
of Aspergillus niger were cultured on the potato dextrose agar medium and the pure
starter culture of Bacillus subtilis was maintained on nutrient agar medium of pH 7.0-
7.2. The nutrient agar mediums were incorporated with peptone (0.1g); NaCl (0.5g);
agar (2g) and skim milk (1%). Both culture were maintained by the incubation at the
temperature 40C. The subculture of the microorganism was done by strict plate
method technique. The experiment was set to check the growth of culture and
production of protease at different temperature (40-1000C) and pH (3-10).

Enzyme production medium

The composition of production medium for Bacillus subtilis used was peptone (0.1g);
NaCl (0.5g); Agar (2g) and skim milk (1%) in one liter. The pH was adjusted to 7.0-
7.2 with Na2CO3 followed by media was sterilized by autoclaving at 1210C for 25
minutes. Peptone was sterilized separately and aseptically added to the flask
containing the liquid medium, after cooling. The medium (50ml in 250ml conical
flask) was inoculated with 1ml of an overnight culture of Bacillus subtilis and
incubated at 500C in a rotary shaker operated at 150 rpm for 12hr. at time intervals;
Production and Enzyme Activity of an Extracellular Protease 337

the turbidity of the culture was determined by measuring the increase in optical
density at 450 nm with a spectrophotometer.
The composition of production medium for Aspergillus niger used was (NH4)2SO4
(0.1g); MgSO4.7H2O (0.5g); KH2PO4 (0.5g); FeSO4.7H2O (0.0005g); Glucose (2 g)
and Jowar seeds (1g) were used as a substrate. The pH of medium was adjusted to 5.0
and autoclaved at 1210C for 25min. The liquid medium was inoculated with overnight
grown Aspergillus culture. Fermentation duration was examined for 24 to 120 hrs.
The culture flask was kept on rotary shaker operated at 300 rpm at 280C. Submerged
fermentation and solid state fermentation technique was used for the production of
amylase.

Product recovery and purification

Protease is an extracellular enzyme so its recovery is quite easy. After incubation the
production medium was centrifuged at 12,200 rpm for 15 min to separate the cells.
The supernatant was collected as it contained the crude enzyme and stored at 40C till
further use.

Dialysis for purification

In the dialysis of protease enzyme dry membrane was used. The dialysis buffer was
ingraded with sodium bicarbonate, EDTA, NaCl, KCl MgCl2, Glycerin, Tris HCl and
DDT. Tubing method was used for dialysis. Both the types of enzymes viz. bacterial
and fungal were purified using this technique. The pure enzyme was recovered from
the membrane and stored at 40C till further use. As the enzyme was a coloured
solution, optical density of both crude and pure enzymes were taken at 660 nm, in
order to confirm the enzyme purification.

Enzyme assay
Casein solution of 2% (1 ml) was incubated with 0.1 ml of enzyme solution and 0.9
ml of sodium phosphate buffer (pH 7) for 10 minutes at 400 C. The reaction was
stopped using 10% TCA solution. After 20 minutes the mixture was centrifuged
10,000 rpm for 5 minutes. The colour intensity of supernatant was read at 280 nm.
The enzyme activity was calculated from standard curve of L-Tyrosine. The serial
increase in concentration of tyrosine (10-100 µg/ml) was read at 280 nm for the
standard graph. Enzyme activity was depends on the temperature, particular pH,
substrate concentration and the active site of enzyme. In present investigation,
protease activity was checked at different temperature (50-1000C) as well as varied
pH range (3-10) of phosphate buffer (mixture of monobasic and dibasic phosphate
buffer).

Statistical Analysis
The experiments were set up with minimum three replicate per treatment and
experiments were repeated at least three times. The experimental conditions were kept
constant with respect to the treatment. Data were analysed by Analysis of Variance
(ANOVA) to detect the significant difference between means. Means are compared
Duncan's Multiple Range Test (DMRT).
338 R.S. Pagare et al

Result and Discussion

Effect of culture conditions of enzyme secretion
The Growth pattern of Basillus subtilis and Aspergillus niger and protease production
was observed for 12 hours in liquid medium with trisodium citrate as a carbon source.
Among Basillus subtilis and Aspergillus niger, A. niger shows very fast growth and
reached a maximum in 11 hour and then began to fall. These results tells that the
protease enzyme production was directly linked to the culture being metabolically
active.
The supplementation of culture medium with a solution of metal traces improved
substantially the growth of Bacillus and Aspergillus as well as enzyme production.
These suggest that the requirement of some metal ion for protease production by the
microorganism. Earlier Nascimento and Martins (1996) and Jansen et al., 1994
reported the requirement of metal ion for the production of protease in the culture of
microorganism. Freewro et al., 1996 reported the use of trisodium citrate along with
MgSO4, CaCl2, MnSO4, and ZnSO4, for protease production by Basillus
licheniformis.
In the culture media of Basillus subtilis trisodium citrate and for Aspergillus niger
casein was used which are better carbon source for the carbon source. These results
are in similar to results of the Nascimento and Martins (1996) and Johnvesly and
Nailk (2001).

Effect of pH and temperature on culturing of the microorganism

A range of pH 3 to 10 as well as the vaiable temperature incubation from 40 -1000C
were used to study the effect of these parameter on the growth of Basillus subtilis and
Aspergillus niger. Optimum pH were found to be 9 and 8 respectively (Table 2). Wu
et al., (2006) reported the optimum pH for the culture and production of protease in A.
terrus. Zeikus (1979) reported that majority of the thermophilic bacilli are found to
grow at pH and temperature range of 5.8-8.0 and 50-65°C, respectively. According to
Kumar and Takagi (1999), because of the close relationship between protease
synthesis and the utilization of nitrogenous compounds, pH variations during
fermentation may provide kinetic information about the protease production, such as
the start and end of the protease production period. These results suggest that the
required alkaline pH is required for the growth of both Basillus subtilis and
Aspergillus niger. Similarly the incubation temperature also affect on the culturing of
the organism. The optimum temperature for the growth are 50 and 600C respectively
(Table1).

Effect of pH on protease activity

In the range of pH (3-10) which were used to check the optimum pH for the protease
activity was 8. The slightly alkaline pH shows maximum activity which is 26
Uml-1min-1. Nascimento and Martins (1996) and Sookkheo et al., (2000) were earlier
reported that the optimum pH for protease activity was 7.0 to 8.5. According to Borris
(1987) alkaline protease production is found to be maximum at pH 9-13. Dutta and
Banerjee (2006) reported that 7 pH is required for the maximum protease activity in
Pseudomonas.
Production and Enzyme Activity of an Extracellular Protease 339

Effect of temperature on protease activity

The protease activity were analyzed at different temperatures ranging from 50-1000C
at a constant pH at 7.5. Enzyme activity was observed increasing upto 600C. A
reduction in protease activity was observed at above 600C. Horikoshi (1990) and
Banerjee et al., (1999) reported the optimum temperature for protease activity was
600C. These results are supports to the present work.

Table 1: Growth of Bassilus subtilis and Asergillus niger after incubation at different
temperature.

Temperature (0C) Bassilus subtilis Asergillus niger

40 + +
50 + +++ ++
60 +++ ++++
70 + ++
80 + ++
90 + +
100 + +

+ sign indicate growth of the microorganism

+ and ++: Small Zone; +++: Moderate zone; ++++: largest zone

Table 2: Growth of Bassilus subtilis and Asergillus niger after incubation at different
pH

pH Bassilus subtilis Asergillus niger

3 + +
4 + +
5 + +
6 ++ ++
7 ++ + ++
8 +++ ++++
9 + + ++ ++
10 + +

+ sign indicate growth of the microorganism

+ and ++: Small Zone; +++: Moderate zone; ++++: largest zone
340 R.S. Pagare et al

Table 3: Enzyme activity at different pH

pH Enzyme activity
(Uml-1min-1)
3 22.4 ± 0.00
4 23.0 ± 0.01
5 24.0 ± 0.03
6 24.8 ± 0.06
7 25.1 ± 0.09
8 26.0 ± 0.07
9 23.0 ± 0.09
10 20.7 ± 0.02

The values represent the Mean ± SE Calculated on three independent experiments.

Each based on minimum 3 replicates. Values followed by the same letter were not
significantly different at 5% level (DMRT).

Table 4: Enzyme activity at different temperature

Temperature Enzyme activity

(0C) (U/ml-1min-1)
500C 24.9 ± 0.09
600C 26.1 ± 0.07
700C 25.2 ± 0.03
800C 24 ± 0.04
900C 23.3 ± 0.06
1000C 21 ± 0.01

The values represent the Mean ± SE Calculated on three independent experiments.

Each based on minimum 3 replicates. Values followed by the same letter were not
significantly different at 5% level (DMRT).

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