Statistical Optimization of Cold Adapted á-amylase Production by Free and... Nocardiopsis aegyptia

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Statistical Optimization of Cold Adapted á-amylase Production by Free and... Nocardiopsis aegyptia
Journal of Applied Scienes Research, 5(3): 286-292, 2009
© 2009, INSInet Publication
Statistical Optimization of Cold Adapted á-amylase Production by Free and Immobilized
Cells of Nocardiopsis aegyptia
Abou-Elela G.M., Nermeen A. El-Sersy, and Wefky S.H.
National Institute of Oceanography and Fisheries, Microbiology lab. Environmental Division,
Alexandria, Egypt
Abstract: á-Amylase production has a wide range of applications in many industries such as baking,
brewing, ethanolic production, wine and textile industries. The cold adapted á-amylase production from
Nocardiopsis aegyptia was dependent on the fermentation conditions. Plackett-Burman statistical design
was used to optimize culture conditions and evaluate the most significant variables affecting enzyme
production. Potassium nitrate concentration (1.5 g/l) and inoculum size (1.5 ml /50 ml medium) were
positively affecting the production which was increased up to 1.12 fold. The experimental results showed
that optimum temperature and pH values were 25°C and 5.0 respectively. Immobilized Nocardiopsis
aegyptia cells on luffa pulp achieved it´s highest productivity (2255 unit) after 2 days of incubation this
yield increased up to 1.16 fold.
Key words: á-amylase-Nocardiopsis aegyptia - Statistical Optimization- immobilization
as saline soils or marine sediments [9 ,2 ] . The nutritional
and environmental conditions have a great influence on
production of enzymes. In order to develop an efficient
production of enzymes, knowledge regarding the
environmental factors affecting this process needs to be
well identified. Experimental designs are excellent
techniques for optimization of culture conditions to
achieve optimal production [7 ,2 4 ,3 ] . and was recently used
for the production of amylase by actinomycetes [1 3] .
Modification of biotechnology and processes, using
immobilized biocatalysts, has recently gained the
attention of many biotechnologists. Application of
immobilized enzymes or whole cells is advantageous,
because such biocatalysts display better operational
stability[1 0 ] . and higher efficiency of
catalysis [2 2] .
Immobilization of whole cells for extracellular
enzyme production offers several advantages i.e.
the ease to separate cell mass from the liquid
culture for possible reuse, facilities continuous
operation over a prolonged period, enhances reactor
productivity [2 6 ] .
In the present study screening of different
actinomycetes isolates has been carried out to explore
the possibility of amylase production. In addition
fermentation parameters were optimized by applying
Plackett-Burman experimental design. Moreover, cell
immobilization technique was carried out aiming for
maximum amylase production.
Amylase constitute one of the important groups of
enzymes that are used in a wide range of starch
industries i.e. baking, brewing, starch liquefaction and
distillery. Amylase has numerous biotechnological
applications in the production of syrups containing
oligosaccharides, maltose and glucose. Another product
from starch hydrolysis by amylases is dextrin, which is
important in food processing as viscosity improver,
filter or ingredient. In the textile industry, amylase is
used in resizing process to degrade starch from
clothing materials [1 1 ,3 1 ].
Several amylolytic enzymes, with different
specificities can contribute to starch degradation. á
amylases are widely distributed in microorganisms.
Industrial á amylases are produced by bacteria and
fungi, e.g. Bacillus subtilis, B. licheniform is,
Aspergillus oryzae, A. niger, Micrococcus halobius, etc.
Production of amylases was also reported by some
strains of actinomycetes [1 2 ,1 3 ] . in addition to plants, and
Actinomycetes are one of the most investigated
groups because they constitute a potencial source of
b io techno lo gically interesting sub sta n c e s[1 7 ].
Nocardiopsis strains are distributed ubiquitously in the
environment[1 5 ] . They are frequently isolated from
habitats with moderate to high salt concentrations such
Corresponding Author: Nermeen A. El-Sersy, Associate prof. National Institute of Oceanography and Fisheries,
Microbiology lab. Environmental Division, Alanfushy Qayt bay, Alexandria, Egypt.
Tel: 0106620217
E-mail: [email protected]
J. Appl. Sci. Res., 5(3): 286-292, 2009
W here Exi is the variable main effect, and Mi+,
Mi- are the enzyme activity (units) in the trials, where
the independent variable was present in high and low
concentrations, respectively, and N is the number of
trials divided by 2. Statistical t-values for equal
unpaired samples were calculated using Microsoft
Excel to determine the variable significance.
M icro-organisms: All tested organisms were isolated
from aquatic sources. Nocardiopsis aegyptia was
isolated from marine sediment of Abu Qir Bay on the
western seashore, Alexandria, Egypt [ 2 5 ] . while
Streptoverticillum morookaense, Streptomyces globosus,
Streptom yces ruber, Streptom yces alanosinicus,
Streptomyces gancidicus, and Nocardia brasiliansis,
were isolated from Burullus lake sediments [1 ].
Effect of PH on Amylase Production by N.aegyptia:
The optimized culture medium was adjusted at different
pHs (4.0-8.0). The pH measurements were carried out
with Beckman digital pH meter using a glass electrode.
The pHs of 4.0-6.0 was maintained with acetate buffer
(0.2M) while pH 6.0-8.0 were achieved with phosphate
buffer (0.1M) [1 3 ].
Screening for á-amylase Production by Different
Actinomycetes Strains: The organisms were inoculated
on starch nitrate culture medium plates [1 9 ]. contained
(gl-1 ): soluble starch, 20.0 ; KNO 3,1.0; K 2HPO 4,0.5;
MgSO 4 .7H 2 O, 0.5 ; FeSO 4 .7H 2 O, 0.05; and agar, 20 ;
sea water or lake water 1L. Incubation at 30°C was
carried out for 7 days after which the plates were
flooded with Gram's iodine solution (0.1% I 2 and 1%
KI) and the colonies with the largest halo-forming zone
were chosen for further investigations [1 8].
Effect of Immobilization Technique on á-amylase
Production by N.aegyptia: Immobilization was carried
out by adsorption of N. aegyptia cells on different solid
porous supports, 1.5 ml of bacterial suspension were
added to 100 ml sterile flasks containing 50 ml of
optimized culture medium and five grams of porous
support materials (Luffa pulp, sponge and pumice).
Luffa pulp and sponge were cut to small pieces,
washed several times with water before use. The size
of pumice particles was around 0.5 cm in diameter.
The flasks were then shacked slowly at 120 rpm. After
different time intervals of incubation (2-6) days,
am ylase production was estim ated. R e p e a te d
fermentation of immobilized cells in shacked flasks up
to 6 th run carried out in the modified starch nitrate
Effect of Incubation Period on á-amylase Activity
of Nocardiopsis aegyptia: Nocardiopsis eagyptia was
investigated for amylase production at different
incubation periods. Amylase production was estimated
daily for 8 days using the method described by [3 0 ].
Inoculum size was 1ml (10 5 CFU/ml)
Enzyme Assay: Amylase assay was based on the
reduction in blue colour intensity resulting from
enzymatic hydrolysis of starch and formation of starchiodine complex [3 0 ] . The reaction mixture consisted of
0.2 ml enzyme (cell free supernatant), 0.25 ml of
starch solution and 0.5 ml of phosphate buffer (0.1 M,
pH 6). Incubated at 50 º C for 10 min. The reaction
was stopped by adding 0.25 ml of 0.1 N HCl and the
colour was developed by adding 0.25 ml of I/KI
solution (2% KI in 0.2% I). The optical density (O.D.)
of the colour solution was determined using a UV-Vis
Spectrometer at 690 nm. One unit of the enzyme
activity is defined as the quantity of enzyme that
causes 0.01% reduction of blue colour intensity of
starch iodine solution at 50 º C in one min. per ml [3 0 ].
Screening for Extra-cellular Amylase Production:
Among the tested isolates for their ability to degrade
starch, Nocardiopsis aegyptia showed the largest
degradation zone as shown in Table 1. It was found
that degradation zone of Nocardiopsis aegyptia were 30
mm, in spite of, the diameter of that zone equal that
produced by Streptomyces rubber, Streptomyces
alanosinicus and Nocardia brasiliansis, but it was
noticed that, the diameter of Nocardiopsis aegyptia
colony was the lowest (12 mm)..
Optimization of Growth Culture Conditions Using
Plackett Burman Experimental Design: The PlackettBurman design [2 1 ,3 2 ] . was applied to reflect the relative
importance of various environmental factors involved
in the production of á-amylase by Nocardiopsis
aegyptia. For each variable a high (+) and low (-)
levels were tested. The examined variables in this
experiment and their levels are shown in Table 2.
Eight different trials were performed in duplicates.
Rows in Table 3. represent the different trials (Row no.
9 represents the basal control). The main effect of each
variable was determined with the following equation:
Exi = (Mi+ – Mi-) / N
Determination of the Optimum Incubation Period:
The optimum incubation period for á-amylase
production by Nocardiopsis aegyptia was after 6 days
representing (1949 units) of productivity as shown in
(Figure 1), no significant difference in the productivity
between 6 th and 7 th day (1940 units).The productivity
decreased significantly at the 8 th day (1800 units).
J. Appl. Sci. Res., 5(3): 286-292, 2009
Table 1: Screening of different actinom ycetes isolates for starch
degradation after 7 days of incubation.
Colony diam eter
Zonediam eter
(m m )
(m m )
Streptoverticillum m orookaense 13
Streptom yces globosus
Streptom yces ruber
Streptom yces alanosinicus
Streptom yces gancidicus
Nocardia brasiliansis
Nocardiopsis aegyptia
Fig. 1: Effect of incubation periods
production by N. aegyptia.
significant variables. Their interaction was illustrated in
Figure 3, which showed amazing increase of enzyme
production, indicating direct relationship between these
two factors.
Table 2: Independent variables affecting á-am ylase production and
their levels in the Plackett-Burm an design
Sym bol
KN O 3 (g/l)
M g SO 4 (g/l)
K 2 H PO 4 ((g/l)
Sea water conc.
>100% *
FeSO 4 ((g/l)
Inoculum size (m l)
Tem perature (o C)
*150 m l of sea water was concentrated by evaporation to 100 m l
Table 3: The applied Plackett- Burm an experim ental design for
seven cultural variables.
Factor Level
----------------------------------------------------------------------------KN M g K2 SW Fe
Enzym e production
(U nits)
on amylase
Optimization of Amylase Production Using Plakett
-Burman Design: This design was applied with nine
different fermentation conditions as shown in Table 3.
All experiments were performed in duplicates and the
averages of results (enzyme activity expressed in
production units) are presented as the response in Table
3. The main effect of each variable on enzyme
production as well as t-values were estimated for each
independent variable as shown in Table 4 and
graphically presented in Figure 2.
Results in Figure 2 indicated that the presence of
high levels of KNO 3, Mg SO 4, sea water concentration
and inoculum size in the growth medium affects
enzyme production positively. On the other hand, the
presence of K 2 HPO 4 , FeSO 4 and temperature at their
lowest levels would result in high enzyme production.
According to the obtained results, it can be
predicted that the optimum medium composition for áamylase production by Nocardiopsis aegyptiae is as
follows gl-1 : KNO 3 , 1.5; Mg SO 4, 0.75; K 2HPO 4, 0.25;
FeSO 4 , 0.025; concentrated sea water ( >100%);
adjusted to pH 7 and inoculum size 1.5 ml for each 50
ml medium, incubated for 7 days at 25 o C. In order to
evaluate the accuracy of the applied Plackett-Burman
statistical design, a verification experiment was applied
to compare between the predicted optimum levels of
independent variables and the basal condition settings.
It was found that the production of the enzyme
(expressed in units) increased to (2187) unit with (1.12)
fold increase when compared to it's production under
the basal conditions. On the basis of the calculated ttest (Table 4) KNO 3 and inoculum size were positive
Table 4: Statistical analysis of the Plackett- B urm an experim ental
M ain Effect
KN O 3
M g SO 4
K 2 H PO 4
Sea water
FeSO 4
Inoculum size
Tem perature
*t-value significant at the 1% level = 3.70
t-value significant at the 5% level = 2.45
t-value significant at the 10% level = 1.94
t-value significant at the 20% level = 1.37
Standard t-values are obtained from statistical m ethods [4 ] .
Fig. 2: Elucidation of fermentation conditions affecting
á-amylase production by N. aegyptia.
J. Appl. Sci. Res., 5(3): 286-292, 2009
fermentation of immobilized cells up to 6 th run carried
out in modified starch nitrate medium showed that N.
aegyptia were physiologically active.
Table 5: á-am ylase production by cells of N.aegyptia
Im m obilizing substrate
Pum ice
Enzym e production(unit)
Discussion: Amylases have most widely been reported
to occur in microorganisms, although they are also
found in plants and animals. Two major classes of
amylases have been identified in microorganisms,
namely á -amylase and Glucoamylase [2 3 ]. á -Amylase
may be derived from several bacteria, yeasts and fungi.
Bacterial amylase, however, is generally preferred over
fungal amylase due to several characteristic advantages
that it offers. Strain of Aspergillus sp and Bacillus sp
mainly Bacillus amyloliquefaciens and B.licheniformis,
are e m p lo ye d fo r com m ercial applications [ 6 ] .
Thermostable á- amylases are generally preferred as
their application minimizes contamination risk and
reduces reaction time, thus providing considerable
energy saving. Hydrolysis carried out at higher
temperature also minimizes polymerization of Dglucose to Isomaltose.
Commercial production of amylases is carried out
in various steps, essentially because the environmental
factors required for the optimum growth for
microorganism being employed factors required for the
production of enzymes. These parameters include
nutrient supplementation, pH of the medium, osmotic
relationship, and degree of aeration, temperature and
the control of contamination during fermentation.
Maintaining the purity of the medium is also a very
important factor, especially when the fermentation is
carried out under aerobic conditions.
Actinomycetes are Gram positive bacteria with a
high G+C content, have the capacity to produce a vast
array of secondary metabolites and extracellular
proteins. The latter comprise many hydrolytic enzymes
such as amylases, cellulases, chitinases, and xylanases
allowing actinomycetes to grow on polymeric
Here we first report the production of amylase
secreted by Nocardiopsis aegyptia that was isolated as
novel species from marine sediments [2 5] .
In the present study, Plackett-Burman design was
applied which was successfully employed in enzyme
production and other optimization experiments [3 2 ,7 ,8 ].
Results revealed that concentration of potassium nitrate
and inoculum size are the
most highly positive
significant factors which affect enzyme production by
Nocardiopsis aegyptia More over, using the high
settings of potassium nitrate, inoculum size, magnesium
Fig. 3: Inoculum size and K NO 3 concentrations
response as function of á-amylase production.
Effect of different pHs on á -amylase production:
The data presented in fig.4, showed that, production of
the enzyme required acidic conditions, and maximum
yield (2255 unit) was achieved at pH 5, i.e., at pH 5,
the production of á -amylase was 16% high, above and
lower this value, the yield decreased. The lowest
production was estimated at pH 8 (1400 unit).
Fig. 4: Effect of
á -amylase
Effect of different immobilizing substrates on á amylase production: Living cells of N.aegyptia were
sub jected to im mo b ilization using adsorption
techniques. Adsorption was carried out using luffa
pulp, pumice and sponge as supporting materials. The
results in (table 5) showed that, no increase in enzyme
production by using immobilized cells and no
significant differences between the estimated values
resulted from using different substrates.
On the other hand,estimation á-amylase production
by using of luffa pulp immobilized cells at different
time intervals (2-6 days) showed that, the highest
productivity (2255 unit) was achieved after only 2days.
J. Appl. Sci. Res., 5(3): 286-292, 2009
sulphate and concentrated sea water,would positively
affect production by Nocardiopsis aegyptia while the
using of low settings of dipotassium hydrogen
phosphate, ferrous sulphate and temperature, would also
promote enzyme production.. Therefore, results
concluded that to achieve the highest productivity by
No card iop sis aeg yp tia , the o p tim um m edium
composition should be as follows: (g l-1 ) Starch, 20;
sulphate,0.75;dipotassium hydrogen phosphate, 0.25;
ferrous sulphate, 0.025; concentrated sea water, with
inoculum size 1.5 ml (for 50 ml medium) at 25 o C .
Under such conditions, the enzyme production by
Nocardiopsis aegyptia was 2187 unit i.e (1.12 fold
increase) than that obtained using the basal growth
medium. Narayana and Vijayalakshmi.[2 0] reported that,
among the studied carbon sources used for amylase
production starch was found to be the best substrate,
showing maximum enzyme activity K uo and
Hartman[1 6 ] . showed that thermoactinomyces vulgaris
produces best yields of á – amylase when starch or
maltose is used as a carbon source. Simpson and
McCoy,[2 8 ] . studied the influence of incubation period
on á – amylase produced by Streptomyces albidoflavus.
The production of á – amylase began after 24 h of
cultivation and reached to peak levels after 48 h and
declined there after. The highest yield has been found
after 52 to 56h while in this study the maximum
production achieved after 6 days of incubation and
after applying the immobilization techniques, the
maximum productivity (2255 units) was achieved after
2 days.
K uo
H artm an [ 1 6 ] .
thermoactinomyces vulgaris synthesized amylase most
rapidly at pH values ranging from 6.5 and 7.5 and that
amylase inactivation occurred rapidly if pH rose above
7 .5 . A m yla se p ro d uctio n b y S trep to m yces
aureofaciens77 has been increased gradually as the
initial pH values ascend from 5 to 7 . Shatta, et al., [2 7 ].
found that á – amylase are generally stable at pH
ranged from 5.5 to 8.0 and optimal activity of á –
amylase occurs between pH 4.8 and 6.5 these results
agreed with our results where maximum production
achieved at pH 5 and the yield increased to (2255 unit)
i.e (1.16) fold increase.
Only two reports on production of á – amylase
from Nocardiopsis species up to now Stamford, et al.
. showed the highest activity at 70 º C and pH 5.0
while the second study by[3 3 ] . on production of cold
adapted á – amylase by Nocardiopsis species 7326
gave its highest activity at low temperature(35 ºC) and
stability at alkaline pH(8), which also permitted its
biotechnological applications in various studies. For
example, it could be applied as a detergent additive, as
a resizing agent in textile processing, and in food
industry. Also Narayana and Vijayalakshmi[2 0 ] . reported
that the optimum temperature for maximum enzyme
activity was found to be at 30 º C and enzyme stable up
to 35º C and at higher temperature, the enzyme activity
decreased sharply and most purified enzymes lose
activity rapidly above 50 º C. It is amazing that our
strain N.aegyptia achieved the highest productivity at
lower temperature (25 º C) in acidic conditions (pH 5.0).
Immobilizated cells for enzyme production has the
advantage of higher reaction velocity due to higher
cell denisty, enhanced yield and cell viability for
several cycles of operations[2 6 ,1 4 ] . W ith this respect, in
this study, the maximum yield was achieved after 2
days by applying the immobilization techniques in this
study. Immobilization of whole cells may allow
repeated operations[5 ] . and is a strategy for protecting
cells from shear forces during fermentation [1 3 ]. Our
results showed that , the immobilized cells were still
physiologically active up to the 6 th cycle.
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