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R E S E A R C H Open Access
Production and optimization of α-amylase
from thermo-halophilic bacteria isolated
from different local marine environments
Eman A. Elmansy
1*
, Mohsen S. Asker
1
, Ebtsam M. El-Kady
1
, Saadia M. Hassanein
2
and Fawkia M. El-Beih
2
Abstract
Background: Amylases are among the most important enzymes which are of great significance for biotechnology
and have almost completely replaced chemical hydrolysis of starch in the starch processing industry. The present
study was concerned with the production and optimization of extracellular α-amylase from Bacillus sp. NRC22017.
Results: The effect of various fermentation conditions on α-amylase production through shake-flask culture was
investigated. Bacterial strain produces α-amylase was isolated from water in Wadi El-Natron. Based on microbiological,
biochemical tests, and 16S rRNA gene sequences, the isolate was identified as Bacillus sp. NRC22017 and was later used
for further studies. Maximum yield of α-amylase is 15.15 ± 0.47 U/ml from Bacillus sp. NRC22017; this strain is characterized
with high temperature and high salinity in cultivated culture, and achieved maximum yield of α-amylase at pH 6.0 with
inoculum size of 500 μl at 45 °C and aerobically incubation period of 72 h. The optimum volume of the fermentation
medium was found to be 20 ml in 100 ml Erlenmeyer flask; the best starch and meat extract plus peptone concentration
that provided the highest enzyme production from Bacillus sp. NRC22017 were found to be 2% and 1.05%
(w/v) respectively.
Conclusion: Enzyme production was higher after optimizing the production conditions as compared to the
basal medium.
Keywords: α-Amylase, Starch hydrolysis, Thermo-halophilic bacteria
Background
Alpha amylase (EC: 3.2.1.1), randomly attack α-1,
4-glycosidic bond of starch, maltodextrins, maltose, and
glucose units were formed as a result of α-amylase ac-
tion (Maity et al. 2015). Alpha amylase has gained a
great attention due to its broad spectrum of applications
and economic merits (Bansode 2010). Currently, amylase
production has reached up to 65% of enzyme market in
the world and is continuously increasing (Abdullah et al.
2014). It has a wide range of applications in starch lique-
faction, paper, desizing of textile fabrics, preparing starch
coatings of paints, removing wallpaper, brewing industry,
sugar induction by the production of sugar syrups, and
pharmaceuticals. Alpha amylase can be produced by mi-
cro- and macroorganisms (Simair et al. 2017). When the
origins are in comparison, amylases from animal and
plant origins have low resistance under acidic, basic, and
high temperature conditions. On the other hand, bacter-
ial and fungal amylases have good stability under such
conditions and have more economic production opera-
tions. So, microbial enzymes have been applied in a large
number of applications.
Recently, starch saccharification, the main use of amyl-
ase, has totally displaced chemical utilization with amyl-
ase enzyme hydrolysis. Saccharification is executed at
raised temperature and thermophilic microorganisms
could be most hopeful candidates for amylase produc-
tion because these strains will produce thermostable
amylase. This is why, still, search for new microbial
strains is continued to achieve industrial requirements
of enzymes.
In addition, amylase is supplemented in local deter-
gents due to high alkaline pH stability needed for indus-
tries (Asad et al. 2011). Moreover, thermophilic amylase
* Correspondence: emmyahmed36@yahoo.com
1
Microbial Biotechnology Department, National Research Centre, El-Tahreer
Street, Dokki, Cairo, Egypt
Full list of author information is available at the end of the article
Bulletin of the National
Research Centr
e
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.
Elmansy et al. Bulletin of the National Research Centre (2018) 42:31
https://doi.org/10.1186/s42269-018-0033-2
is required for other applications in the production of
sweeteners from starch and saccharification of starch for
biochemical production (Castro et al. 1999).
Bacillus genus is famous for alpha-amylase production
and several Bacillus strains such as B.stearothermophilus,
B.subtilis,B.cereus,B.licheniformis,andB.amyloliquefa-
ciens are isolated and screened for amylase production
(Sivaramakrishnan et al. 2006). Some Bacillus strains are
involved in raw starch degradations (Demirkan et al. 2005;
Goyal et al. 2005; Puspasari et al. 2013).
Still, many are being searched to obtain the maximum
output with unique industrial properties. Enzyme pro-
duction could be improved through the optimization of
various physical and nutritional growth parameters; the
important factors that determine the bioprocess are in-
cubation period, temperature, pH, aeration, inoculum
size, carbon, and nitrogen sources. Therefore, in this
study, the effects of physical and nutritional parameters
were investigated for the optimum production of extra-
cellular enzyme α-amylase from Bacillus sp. NRC22017.
Results
Isolation, screening, and identification of amylolytic
bacteria
In the present investigation, a pure strain of Bacillus sp.
NRC22017 was isolated from Wadi El-Natron and we
found bacteria isolated from such places may have better
potential to produce enzyme under adverse conditions.
The isolate was inspected for the qualitative production
of α-amylase on a starch agar plate supplemented with
2% soluble starch using iodine solution (Fig. 1). Finally,
NRC22017 strain was selected as the best amylase pro-
ducer. The promising isolate (NRC22017) was identified
based on its microscopic, morphological, and biochem-
ical characterization and it was proven to be Bacillus sp.
(Table 1).
Fig. 1 Bacterial isolate negative for starch hydrolysis test (a). Zone of clearance around NRC22017 (b) due to starch hydrolysis
Table 1 Cultural and biochemical characteristic of the promising
bacterial isolate
Test Bacillus sp. NRC22017
Morphological characterization
Gram stain Positive
Motility Positive
Spore-former Positive
Appearance of the colony surface Wrinkled
Color Creamy
Elevation Umbonate
Edge Entire
Whole colony Circular
Pigmentation No pigment
Opacity of the bacterial colony Opaque
Biochemical characterization
Starch hydrolysis Positive
Voges-Proskauer Negative
Citrate utilization Negative
Nitrate reduction Negative
Oxidase Positive
Catalase Positive
Acid production from
Glucose Positive
Fructose Positive
Galactose Negative
Lactose Negative
Maltose Negative
Sucrose Positive
Mannose Positive
Xylose Negative
Elmansy et al. Bulletin of the National Research Centre (2018) 42:31 Page 2 of 9
A molecular technique was used to prove and further
confirm the identification of the isolate to the species
level. The partial 16S rDNA sequence was determined
and was compared to the GenBank databases. The iso-
late was identified as Bacillus sp. NRC22017. The se-
quence was submitted to GenBank in NCBI (https://
www.ncbi.nlm.nih.gov/nuccore/KY614074) with the ac-
cession number KY614074. The phylogenetic tree based
on different species of Bacillus was constructed using
neighbor joining method (Fig. 2).
Optimization of α-amylase production conditions
Though different Bacillus species have similar growth
patterns and enzyme profiles, but their optimized condi-
tions vary, depending upon the strain. Optimization of
the process parameters is needed for the improved pro-
duction of the enzyme to make the process cost effect-
ive. Selected strain Bacillus sp. NRC22017 was subjected
to various culture conditions to investigate the optimum
culture conditions for α-amylase production.
Effect of five different fermentation media on alpha
amylase production
Studies on bacterial growth and α-amylase production from
Bacillus sp. NRC22017 in shake-flask cultures were carried
out using five different media at pH 7.0 and incubated for
72hat50°C.ResultsinFig.3illustrated that among the
culture media, the medium 5 exhibited a significant impact
on the microorganism development (2.15 ± 0.067) and en-
zyme production (11.02 ± 0.17 U/ml), while the lowest
growth rate (0.089 ± 0.001) and α-amylase synthesis (3.20 ±
0.12 U/ml) were obtained when medium 3 was used.
Effect of physical parameters on alpha amylase production
Incubation period The results obtained from the incu-
bation time study in submerged culture cleared that bac-
terial growth and α-amylase productivity was gradually
raised with the increment in the incubation period. Incu-
bation for 3 days was the best for both α-amylase prod-
uctivity (11.56 ± 0.20 U/ml) and bacterial growth (2.04 ±
0.09 g/l). The activity of α-amylase was declined by fur-
ther increasing the incubation period. At 5 days of incu-
bation, it was extremely reduced and the minimum
amount of α-amylase was obtained after 5 days of incu-
bation (6.96 ± 0.17 U/ml) (Fig. 4a).
Inoculum size Data in Fig. 4b show the effect of inocu-
lum size (200 to 700 μlv/v) on cell growth and extracellu-
lar amylase secretion. The highest amylase yield was
achieved when 500 μl (14.24 ± 0.2 U/ml) of 24-h-old seeds
culture was inoculated into the fermentation medium.
Incubation temperature Data in Fig. 4c show the effect
of fermentation temperature (40–55 °C) on the synthesis
of amylase by Bacillus sp. NRC22017 grown in fermen-
tation medium containing 20 g/l starch and 14 g/l pep-
tone plus yeast extract at pH 7.0 and incubated for 72 h.
Enzyme concentration increased with incubation
temperature and maximum amylase yield was noted at
Fig. 2 Phylogenetic tree based on the 16S rRNA sequences of isolate and its closest Bacillus sp.
Fig. 3 Production of α-amylase by Bacillus sp. NRC22017 in five
different fermentation media. Data were expressed as mean ±
standard error
Elmansy et al. Bulletin of the National Research Centre (2018) 42:31 Page 3 of 9
a
b
c
d
e
Fig. 4 Effect of incubation period (a), inoculum size (b), incubation temperature (c), medium volume in 100 ml conical flask (d), and different pH
values (e)onα-amylase production by Bacillus sp. NRC22017. Data were expressed as mean ± standard error
Elmansy et al. Bulletin of the National Research Centre (2018) 42:31 Page 4 of 9
45 °C (15.27 ± 0.88 U/ml) and a further increase of
temperature reduced amylase synthesis. The production
of the enzyme was greatly inhibited at 55 °C (5.32 ± 0.36).
Aeration In our study, the highest productivity of
α-amylase was recorded at 20 ml broth medium in 100 ml
conical flask (15.42 ± 0.68 U/ml), and the lowest produc-
tion level was reported at 25 ml broth medium in a con-
ical flask of 100 ml capacity (10.89 ± 1.57 U/ml) (Fig. 4d).
pH In our study, the maximum bacterial growth and
synthesis of α-amylase were found at pH 6.0 (12.00 ±
0.28 U/ml) (Fig. 4e).
Effect of nutritional factors on α-amylase production
Various carbon sources Different carbon sources
(starch, sucrose, fructose, xylose, and maltose) at a con-
centration of 2.0% (w/v)wereindividuallytestedinthe
basal medium at optimal temperature, incubation period,
and pH to observe the effect on enzyme synthesis by Ba-
cillus sp. NRC22017. Out of these carbon sources, maltose
was found to be the best for amylase production (13.74 ±
0.02 U/ml) within 72 h (Fig. 5a). Even though the max-
imum activity of α-amylase enzyme was observed in the
existence of maltose as a carbon source, starch is used for
supplementation in the production process, because it
works as an inexpensive source as compared with maltose.
From the present findings, it was evident that the en-
hancement of α-amylase needs substrates with α-1,4
glycosidic bonds, including starch and maltose.
The influence of starch concentration as a carbon
source on α-amylase production from Bacillus sp.
NRC22017 was clarified in Fig. 5b. The amount of
α-amylase increase until it reaches the highest level at a
starch concentration of 2% (w/v) (12.42 ± 0.06 U/ml),
then it starts to decrease.
Various nitrogen sources The various organic nitrogen
sources impact on bacterial growth and α-amylase pro-
duction from Bacillus sp. NRC22017 was shown in Fig.
5c. Supplementation of yeast extract plus meat extract
to the fermentation medium gave the highest bacterial
growth and enzyme productivity (14.17 ± 0.20 U/ml).
Figure 5d demonstrated that Bacillus sp. NRC22017
gave the highest α-amylase productivity at 1.05% meat
plus yeast extract (15.15 ± 0.47 U/ml).Also, from the ob-
tained data, it was obvious that bacterial growth increase
with increasing nitrogen source concentration and the
highest cell dry weight was obtained at the highest nitro-
gen source concentration (1.4%) (4.67 ± 0.18 g/l), but
α-amylase synthesis decreased with increasing nitrogen
source concentration above the optimum concentration.
Discussion
The initial screening of Bacillus sp. NRC22017 strain re-
vealed zones of hydrolysis on starch agar plates. This
clear zone indicated that NRC22017 α-amylase can
hydrolyze starch.
In the present study, we observed 72 h as the
optimum growth period for the presently reported Bacil-
lus strain and longer incubation period supported less
number of colonies and lower enzyme activity. A pro-
longed incubation period extremely reduced α-amylase
possibly due to the exhaustion of nutrients, death of mi-
croorganisms, gathering of byproducts in the medium
a
b
c
d
Fig. 5 Effect of different carbon sources (a), different concentrations
of starch (b), different nitrogen sources (c), and different concentrations
of yeast extract plus meat extract (d)onα-amylase production by
Bacillus sp. NRC22017. Data were expressed as mean ± standard error
Elmansy et al. Bulletin of the National Research Centre (2018) 42:31 Page 5 of 9
such as toxins, inhibitors, proteolysis of α-amylase by
proteases enzymes, and also the cells may attained the
decline phase and showed diminished amylase synthesis
(Teodoro and Martins 2000; Aiyer 2005). These findings
were closely similar to the results of B.licheniformis
ATCC 12759 (Nurullah 2011) and Bacillus isolates
(Kanimozhi et al. 2014). While, Kaur and Vyas (2012),
Deb et al. (2013), Singh et al. (2016), and Paul et al.
(2017) found that an extended period of incubation be-
yond 48 h did not increase the enzyme production from
Bacillus sp. DLB, B.amyloliquefaciens P-001, Bacillus
sp. strain B-10, and Bacillus sp. MB6, respectively. These
results were in contradiction with our results in the
present study.
Concerning inoculum size, this study proposed that
α-amylase output from Bacillus sp. strain depends on
the growth. Our results are almost in harmony with lit-
erature for enzyme production from different strains
concerning inoculum size. The size of inoculum plays a
notable role in the fermentation rate. It is noteworthy
that there is no precise bacterial inoculum volume suit-
able for amylase production. It can vary from 0.5% for B.
amyloliquefaciens (Haq et al. 2010) to 2.95% (Zambare
2011)for Bacillus sp. and 8% for B.cereus (Sivakumar
et al. 2012).
Temperature is a highly sensitive parameter for
α-amylase productivity and so it needs to be controlled
and this is usually varied from organism to another
(Sivakumar et al. 2011). Temperature can affect an en-
zyme in two ways. One is a direct influence on the reac-
tion rate constant, and the other is in thermal
denaturation of the enzyme at elevated temperatures
(Demirkan et al. 2017). Similarly, Vijayaraghavan et al.
(2015) and Asad et al. (2011) obtained maximum amyl-
ase secretion from Bacillus cereus IND4 and Bacillus sp.
WA21 at 45 °C on starch agar medium, whereas Hasan
et al. (2017) reported maximum amylase production was
at moderate temperature (37 °C) for both Chryseobacter-
ium and Bacillus isolates.
During fermentation, the aeration rate indirectly indi-
cates the dissolved oxygen level in the fermentation
broth. Yegneswaran and Gray (1991) reported that a
high concentration of oxygen had an influencing effect
on microbial growth and enzyme production. The bio-
synthesis of α-amylase was diminished with the increase
in the broth volume; this behavior might probably be-
cause of a reduction in air supply as a result of the
drooping in the agitation rate of medium that happened
with a high volume of fermentation medium (Narang
and Satyanarayana 2001). Riaz et al. (2003) and Dash et
al. (2015) conveyed that the maximum yield of
α-amylase from B.subtilis GCBUCM-25 and B.subtilis
BI19 was gained at 25 and 50 ml of enzyme production
medium in 250 ml Erlenmeyer flask respectively.
Different enzymes have different optimum pH values.
This is the pH value at which the bonds within them are
affected by H
+
and OH
−
ions in a way that the shape of
the active site is the most complementary to the shape
of the substrate. At the ideal pH, the rate of reaction is
optimum. Any alteration in pH above or below the
optimum will quickly cause a decline in the reaction
rate, since more of the enzyme molecules will have ac-
tive sites whose shapes are not (or at least are less) com-
plementary to the shape of their substrate (Demirkan et
al. 2017). Similar result has been recorded for Bacillus
sp. and Brevibacillus borstelensis R1 (Singh et al. 2012,
Suribabu et al. 2014). The production optimization stud-
ies showed that maximum enzyme production by Peni-
cillium notatum IBGE 03 was obtained at pH 5.5
(Ahmed et al. 2015). In neutral conditions (pH 6.5–7.5),
the α-amylase production was reported in Bacillus sp.
(Asgher et al. 2007), and Bacillus sp. NRC12017 (Elkady
et al. 2017). In alkaline conditions (pH 7.5–11.0), the
α-amylase production was reported in Bacillus sp.
(Saxena et al. 2007). It was stated that at high pH, the
metabolic action of bacterium may be suppressed and thus
it inhibits the enzyme production (Ellaiah et al. 2002).
Regarding the effect of different carbon sources on alpha
amylase production by Bacillus sp. NRC22017, results ob-
tained were in agreement with results of NandLal et al.
(2017)inBacillus licheniformis JAR-26; Thippeswamy et
al. (2006)inBacillus species (B
3
) where highest amylase
production (0.464 U/ml) was induced by maltose. Suri-
babu et al. (2014) also found maltose better than other
carbon sources examined for α-amylase production with
Brevibacillus borostelensis R1 under submerged fermenta-
tion. Whereas Elkady et al. (2017) and Gangadharan et al.
(2006)reportedthatBacillus sp. NRC12017 and Bacillus
amyloliquefaciens gave the highest enzyme yield with sol-
uble starch, followed by maltose. Bacillus sp. NRC22017
could grow and produce α-amylase in the presence or ab-
sence of starch from the fermentation medium. Sucrose,
fructose, and starch have almost similar effect on
α-amylase synthesis from Bacillus sp. NRC22017 (12.61 ±
0.72, 12.07 ± 0.05, and 12.01 ± 0.17 U/ml), respectively.
There was a decrease in enzyme production at exces-
sive starch concentration and this might be imputed to
the rapid consumption of starch leading to the release of
toxic metabolic wastes which suppress the growth of
bacteria and α-amylase production. Also, high starch
concentrations caused the broth culture to be more vis-
cous, thus interfering with O
2
transfer resulting in re-
striction of dissolved O
2
required for the microbial
growth. Similarly,Mishra and Behera (2008) found that
raising the starch concentration increased both growth
and α-amylase production by Bacillus strain from kit-
chen wastes and the maximum yield of the enzyme
was reached at a starch concentration of 2%. While
Elmansy et al. Bulletin of the National Research Centre (2018) 42:31 Page 6 of 9
Tiwari et al. (2014)informedthat4%solublestarch
concentration was the best for amylase production by
B.tequilensis RG-01 and above this concentration en-
zyme production was slightly decreased tested.
Nitrogen source is another essential nutrient that is re-
quired by the microorganisms in comparatively larger
amounts. Nitrogen source type and relative concentra-
tion in the growth medium are important for both mi-
crobial growth and amylase production (NandLal et al.
2016). The nitrogen is metabolized to produce primarily
amino acids, nucleic acids, protein, enzymes, and other
cellular components that play a vital role in metabolism.
The decrease in α-amylase production at excess nitrogen
levels could be due to the pH depression or the stimula-
tion of protease enzyme, which repress the amylolytic
activity (Tonkova 2006). Both lower and higher nitrogen
sources levels in the production broth are equally detri-
mental and cause inhibition of the enzyme (Sharma
et al. 2012).
Conclusion
In this study, a combination of physical and chemical
parameters was employed to maximize α-amylase syn-
thesis. By optimizing the incubation conditions of
α-amylase production from Bacillus sp. NRC22017 was
enhanced with 37.63% enzyme yield as compared to
control. Enzyme activities in modified medium and
control medium were 15.15 ± 0.47 U/ml and 11.02 ±
0.17 U/ml, respectively. The results obtained in this
study illustrated that the optimization of culture condi-
tions played a pivotal role in influencing output through
the fermentation bioprocess. Screening of microorgan-
isms with higher α-amylase activities could therefore fa-
cilitate the discovery of novel amylases suitable for new
industrial applications. Purification and characterization
of the enzyme are in progress.
Materials and methods
Sample collection
Samples were collected from water and sediment of
marine and salterns at different locations for isolation of
bacteria. The samples were taken from Rashid, Sidi
Bisher beach at Alexandria, Tiba rose village (El Sāhel
Al-shamali), Hurghada, mangrove tree around the rhizo-
sphere area (Marsa Alam), Safaga, El-Ain Elsokhna
beach, and Wadi El-natron. Samples were collected in
sterile bottles and brought to the lab, stored in the re-
frigerator at 4 °C until it was used.
Isolation and purification of α-amylase producing bacteria
Sample was suspended into 90 ml sterilized saline solu-
tion (0.85%, NaCl) and diluted using the dilution method
(Hayakawa and Nonomura 1987). Then, 100 μl of di-
luted sample (10
−4
–10
−6
) concentrations was placed on
the starch agar plate and spreads with a sterile L-shaped
glass rod (Kanimozhi et al. 2014). The plate was incu-
bated at 50 °C for 24 and 48 h, and single colonies of
different sizes were selected. The colonies were sub-
jected to purification to obtain single pure colonies.
Screening of isolated samples for α-amylase production
All isolates were curried using the starch plate (Moller
et al. 2004). Bacteria were inoculated on starch plates to
test for α-amylase secretion, incubated at 50 °C for
3 days, and stains with an iodine solution (0.5%).
Amylase-positive stains were determined by the presence
of a clear zone of starch hydrolysis around the colony on
the starch plates, while presence of blue color around
the growth indicated negative result (Hollo and Szeiti
1968). The bacterial isolates which produced a clear
zone of hydrolysis in starch agar were selected as
α-amylase producers for subsequent investigation. Se-
lected colonies were maintained on nutrient agar slants
at 4 °C and sub-cultured monthly.
Identification of potent amylase producer
Biochemical, morphological, and physiological character-
istics of the potential producer (isolate number 16) was
determined by adopting standard methods (Bergey and
Holt 1994). The identification was confirmed with
phylogenetic analysis. Briefly, genomic DNA of bacteria
was extracted and universal primer 5′-TCCGTAGGT
GAACTTTGCGG-3′and 5′-TCCTCCGCTTATTGATA
TGC-3′primers was used for the amplification of DNA
(Gardes and Bruns 1993). A single discrete PCR ampli-
con band was observed when resolved on agarose gel.
The PCR amplicon was purified to remove contami-
nants. Sequencing products were resolved on an Applied
Bio-systems model 3730XL automated DNA sequencing
system (Applied BioSystems, USA). Data were submitted
to GenBank database. The DNA sequence was com-
pared to the GenBank database in the national Center
for Biotechnology Information (https://www.ncbi.nlm.
nih.gov/genbank/) using the BLAST program (Tamura
et al. 2011). The sequencing was performed by Lab
Technology Company.
Growth conditions for α-amylase production
To choose a proper culture medium for growth of the
isolated strain and α-amylase production, primarily five
different fermentation media were examined: Medium 1
(g/l): Na
2
HPO
4
6.0, KH
2
PO
4
3.0, NaCl 0.5, MgSO
4
0.24,
CaCl
2
0.01, Peptone 3.0, Starch 10.0 (Burhan et al.
2003). Medium 2 (g/l): Starch 10.0, Yeast extract 2.0,
Peptone 5.0, MgSO
4
0.5, NaCl 0.5, CaCl
2
0.15 (Swain et
al. 2006). Medium 3 (g/l): Starch 20.0, Peptone 0.5,
MgSO
4
.7H
2
O 0.5, NaH
2
PO
4
0.1, (NH
4
)
2
SO
4
0.1, KCl 0.1
(Mishra and Behera 2008). Medium 4 (g/l): Peptone
Elmansy et al. Bulletin of the National Research Centre (2018) 42:31 Page 7 of 9
10.0, Starch 5.0, Beef extract 10.0 (Amoozegar et al.
2003). Medium 5 (g/l): Starch 20, Peptone 10, Yeast ex-
tract 4, MgSO
4
0.5, CaCl
2
0.2 (Kanimozhi et al. 2014).
The pH of the media was adjusted to 7 with 0.1N HCl
and 0.1N NaOH. The media were sterilized and Erlen-
meyer flasks of 100 ml capacity containing 20 ml of cul-
ture medium were inoculated with 1 ml of previously
prepared inoculum and incubated at 50 °C in a rotary
shaker at 140 rpm for 72 h. The samples were harvested
after 72 h and the cells were separated by centrifugation
(5000 rpm for 15 min at 4 °C) in centrifuge (SIGMA
3-18 KS). The cell dry weight (CDW) of culture broth
was measured by harvesting the cells after centrifugation
and drying them at 105 °C to a constant weight.
Enzyme assay
α-Amylase activity was determined by measuring the re-
duction in blue color intensity resulting from enzyme
hydrolysis of starch (Palanivelu 2001). The reaction mix-
ture consisted of 200 μl cell-free supernatant, 250 μlof
soluble starch (1% w/v), and 500 μl phosphate buffer
(0.2 M pH 7) incubated at 50 °C for 30 min. The reac-
tion was stopped by adding 250 μl of 0.1 N HCl and
color was developed by adding 250 μl of iodine solution.
The optical density (OD) of the blue color solution was
determined at 660 nm using (JASCO V-630) spectro-
photometer. One unit (U) of enzyme activity is defined
as the amount of enzyme that hydrolyzes 0.5 mg of
starch per minute under assay optimum conditions. Spe-
cific activity was expressed as units of enzyme activity
per mg of protein. All measurements were performed
three times. The data were expressed as mean ± standard
error of the mean.
Protein determination
Protein content was estimated by Bradford’smethod
(Bradford 1976) using bovine serum albumin as standard.
Factors affecting α-amylase production from Bacillus sp.
NRC22017
Flask cultures were performed in 100 ml Erlenmeyer
flasks containing 20 ml of the medium 5. Factors affect-
ing cell growth and α-amylase production were investi-
gated using one factor at a time method. The optimized
parameters were incubation time (1, 2, 3, 4, and 5 days)
at 50 °C under shaking conditions at 140 rpm, inoculum
size (100, 200, 300, 400, 500, 600, and 700 μl), medium
volume (10, 15, 20, and 25 ml), temperature (40, 45, 50,
and 55 °C), and initial pH of the medium (5.5, 6.0, 6.5,
7.0, 7.5, and 8.0). Studies were also performed to evalu-
ate the influence of different carbon sources (maltose, su-
crose, xylose, fructose, and starch 2% w/v) and different
concentrations of starch (0.5, 1.0, 1.5, 2.0, and 2.5% w/v).
Also different nitrogen sources were used (peptone, yeast
extract, peptone plus yeast extract, yeast plus meat extract,
and peptone plus meat extract 1.4% w/v) at different con-
centrations (0.35, 0.70, 1.05, and 1.40% w/v)ofmeatex-
tract plus yeast extract were tested to get the best one for
α-amylase production. The enzyme activity and protein
concentration were determined.
Acknowledgements
Not applicable.
Funding
This research was a part of MSc that was funded by national research center.
Availability of data and materials
All data generated or analyzed during this study are included in this
published article [and its supplementary information files].
Authors’contributions
This research was extracted from MSc thesis. All authors read and approved
the final manuscript.
Ethics approval and consent to participate
Not applicable (this study does not involve human participants, human data,
or human tissue)
Consent for publication
Not applicable.
Competing interests
Purification and characterization of the enzyme are in progress.
Publisher’sNote
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published maps and institutional affiliations.
Author details
1
Microbial Biotechnology Department, National Research Centre, El-Tahreer
Street, Dokki, Cairo, Egypt.
2
Microbiology Department, Faculty of Science, Ain
Shams University, Cairo, Egypt.
Received: 31 July 2018 Accepted: 19 November 2018
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