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Some properties of thermostable α-amylase of four isolates of Bacillus

licheniformis

Article  in  Nusantara Bioscience · September 2015

DOI: 10.13057/nusbiosci/n070205

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N U S AN T AR A BIO S C IEN C E ISSN: 2087-3948
Vol. 7, No. 2, pp. 90-95 E-ISSN: 2087-3956
November 2015 DOI: 10.13057/nusbiosci/n070205

Some properties of thermostable α-amylase of four isolates of Bacillus

licheniformis

ELHAM S. DAWOOD1, SIRAG A. IBRAHIM2, SAIFELDIN A.F. EL-NAGERABI3,

1
Department of Life Science, Faculty of Education, Nile Valley University, Atbara, Sudan.
2
Department of Botany, Faculty of Science, University of Khartoum, Khartoum, Sudan
3
Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, P.O. Box 33, Birkat Al Mouz-616, Nizwa, Oman,
Tel. +968-96365051, Fax. +968-25443050, e-mail: [email protected]

Manuscript received: 7 June 2015. Revision accepted: 9 September 2015.

Abstract. Dawood ES, Ibrahim SA, El-Nagerabi SAF. 2015. Some properties of thermostable α-amylase of four isolates of Bacillus
licheniformis. Nusantara Bioscience 7: 90-95. Amylases are one of the most important enzymes in the food industry and biotechnology.
The present investigation was concerned with the production, purification and partial characterization of extracellular amylase from
endogenous Sudanese Bacillus licheniformis isolates namely SUDK1, SUDK2, SUDK4 and SUDO. The extracted enzyme was partially
purified using DEAD Sephadex A-25 gel filtration and the enzymatic reaction product was identified using thin layer chromatography.
The results revealed that DEAE-Sephadex is an excellent and effective purification method and the activity of the partially purified
enzymes (83.343-121.755 U/mg protein) compared to the crude extracts (4.626-7.250 U/mg protein) with increase of up to 16.8-18.3
folds. Amylase enzymes hydrolyze starch forming various maltooligosaccharides, such as maltose as major products and were identified
as α- amylases. The enzymes activity was optimum at 60-70°C and were active up to 90°C with residual activity of only 30-50%. All
enzymes were stable between pH 6.0-9.0 with optimum activity at pH 7.0. The enzymes were stable and retained nearly all of their
initial activities at -20°C in the end of 24 weeks and lost less than 60% of their initial activities at 4°C after 8 weeks. The Km values for
this enzyme were 1.25-2.0 mg/mL which showed high affinity and needs only small amount of substrate to be saturated. Therefore,
these α-amylases were thermostable at a wide range of temperature and pH values rendering them useful properties in food, feed,
textiles and relevant biotechnological industries.

Keywords: α-Amylases, Bacillus licheniformis, enzymes, DEAD Sephadex, partial purification, thermostable

INTRODUCTION many researchers (Gupta et al. 2008; Liu and Xu 2008; Chi
et al. 2009). Although there are many microbial sources for
Microbial enzymes are widely used in in industrial amylases production, only few investigations were carried
processes due to their low cost, large productivity, on Bacillus subtilis, Bacillus licheniformis and Bacillus
chemical stability, eco-friendly, plasticity and vast amyloliquifaciens as commercial producers of these
availability. Enzyme kinetics and thermostability are the enzymes (Saxena et al. 2007). Bacillus strains were the
most important characteristics for their uses in food and most important bacteria producing about 60% of
feed production (Burhan et al. 2003; Mishra and Behera commercially available enzymes (Burhan et al. 2003).
2008; Deb et al. 2013). The emphasis was directed to the Therefore, production and yield improvement of α-
study of the thermotolerant microorganisms as an excellent amylases and consequent cost reduction depend mainly on
source of novel thermostable enzymes. There is an evident the selection of the efficient strains, the optimization of the
increase of their uses in different food, beverage, textile, physico-chemical factors, kinetic studies and the
leather, paper industries, wastes treatment and biochemical characterization at different temperature and
biodegradation of organic materials to biofuel (Yandri et al. pH levels (Saito 1973; Machaiah and Vakil 1981; Violet
2010). Of these enzymes, α-amylases (EC3.2.1.1, 1,4-a-D- and Meunier 1989; Aiyer 2004; Dutta et al. 2006;
glucan-glucanohydrolyase) are extracellular enzymes Mohamed et al. 2010; Enemchukwu et al. 2013; Miao et al.
which hydrolyze starch into a range of products such as 2013). Besides, each application of α-amylase in different
glucose and maltose or specific maltooligosaccharide or industry requires unique properties with respect to
mixed maltooligosaccharides (Aiyer 2004; Hashim et al. specificity and thermostability (Konsula and Liakopoulou-
2004; Messaoud et al. 2004; Kathiresan and Manivannan Kyriakides 2004). Although, the endogenous micro-
2006). Although α- amylases can be derived from several organisms are the most important biological source for
sources such as plants, human saliva, pancreatic juice, enzyme production, nonetheless, enzymes used in the local
breast milk, blood serum, and liver, the enzymes from industries were commonly imported which represent
microbial sources are preferred (Kathiresan and economic burden for the country.
Manivannan 2006; Enemchukwu et al. 2013). Therefore, this study was designed to develop proper
Main properties of amylases from bacteria, yeast and technique of enzyme isolation, purification and
other fungi sources have been reported and described by characterization of purified extracellular α-amylase enzyme
 DAWOOD et al. – α-amylase from Bacillus licheniformis isolates 91

from local Bacillus licheniformis isolates namely SUDKI, Identification of the enzyme digests
SUDK2, SUDK3, SUDK7 and SUDO. This will effectively The products of starch hydrolysis by the partially
contribute to the microbial food industry, textile and related purified enzyme were identified with thin layer
biotechnology. chromatography using n-butanol:ethanol:water in a ratio of
4:2.2:2 as a solvent and maltose and glucose as standard
sugars as described by Trevelyan et al. (1950).
MATERIALS AND METHODS
Effect of temperature and pH on the enzyme activity
Inoculation of the bacterial isolates For testing the effects of temperature and pH on the
The bacterial isolates of B. licheniformis used in this enzyme activity, the test was carried out at different
investigation were inoculated on broth containing 10 g of incubation temperature of between 40-90ºC and pH range
peptone (g/L) for all isolates except isolate SUDK2 which of 4-12. The enzyme activity was measured at the end of
needs 10 g malt extract; dipotassium hydrogen phosphate 3 incubation time.
g, magnesium sulphate hydrated 1 g and 0.5 g soluble
starch. The cultures were grown on a rotary shaker (200 Storability of the enzyme
rev./min) at 50˚C for 24 hours. The cultures were then For the evaluation of the effect of the storage conditions
centrifuged in a refrigerated centrifuge and the supernatants on the enzyme stability, six fractions of 5 mL each from
were collected and used for enzyme assay and study. A partially purified enzyme were taken in sample bottles.
modified peptone-malt extract medium used for amylase Three of these were stored at 4ºC in a refrigerator and the
production contained (g/L) peptone 10 g for all isolates rest were stored in a freezer at -20ºC. Every week the
except SUDK2 which needs 10 g of malt extract), enzyme activity was assayed under both conditions. The
dipotassium hydrogen phosphate 3 g, magnesium sulphate Change in absorbance was measured using a
hydrated 1 g and starch 5 g (for all isolates except B. spectrophotometer and the residual activity was calculated.
licheniformis SUDO which needs 2 g ). The cultures were
grown on a rotary shaker (200 rev/min) at 50˚C for 24
hours. RESULTS AND DISCUSSION

Amylase enzymes assay Many bacterial isolates produce extracellular amylase

Enzyme assay was estimated using dinitrosalicyclic enzymes as a result of starch fermentation (Aiyer 2004;
acid (DNSA) method (Miller 1959). The reaction mixtures Hashim et al. 2004; Messaoud et al. 2004; Kathiresan and
consist of 0.5 mL of substrate solution (1% soluble starch Manivannan 2006). Various methods were adopted for
in 0.05 M phosphate buffer, pH7.0) and 0.5 mL of the cell purification of enzymes such as column chromatography,
free extract. The reaction mixture was incubated at 30°C ion exchange chromatography, ammonium sulphate, starch
for 3 min and the reaction was terminated by the addition absorption, DEAE-cellulose treatment, RESOURCE,s
of 1 mL of dinitrosalicyclic (DNSA) reagent. The mixture column, organic solvent fractionation, Sephadex G-1100
was heated at 100°C for 5 min, cooled, and the optical gel filtration, CM-sephadex column chromatography, and
density was measured with spectrophotometer adjusted at CM-cellulose column chromatography (Saito 1973;
540 nm. One unit of amylase activity is expressed as one Machaiah et al. 1981; Dey et al. 2002; Adeyanju et al.
mg of maltose liberated per mL of culture supernatant at 2007; Ul-Haq et al. 2010). The enzyme from B.
30°C and pH 7.0. Protein content of the enzyme solution licheniformis NCIB 6346 was purified 30-fold by ion-
was assayed by Bradford method (Bradford 1976). exchange chromatography and was then characterized. It
had an endo-action on starch yielding maltopentose as the
Purification of the enzyme major product, and was identified as an -amylase
The crude enzyme preparations from the culture (Morgan and Priest 2008). The activity of -amylase
filtrates of B. licheniformis SUDK1, SUDK2, SUDK4, and enzyme from mutant B. licheniformis EMS-6 purified up to
SUDO were applied separately to 1.8×20 cm column of homogeneity level by ammonium sulphate and ion-
DEAE-Sephadex A-25 equilibrated with 0.05 M sodium exchange chromatography using fast protein liquid
phosphate buffer of pH 7.0, when the sample completely chromatography (FPLC) system was increased by 4-5 time
entered the resin, one bed volume of the equilibrating while the yield was found to be 40.4% and the purification
buffer was passed three times through the column until the fold by RESOURCE-S was recorded to be 3.58 (Ul-Haq et
unbound proteins were removed. The enzyme was eluted al. 2010). The enzyme was purified 126-fold by starch
with a linear gradient of sodium chloride (0-0.4 M) in 200 absorption, DEAE-cellulose treatment, and CM-cellulose
mL of sodium phosphate buffer (0.05 M and pH 7.0) with column chromatography (Saito 1973). In the present study,
the aid of gradient mixer. The flow rate was adjusted to 1 the degradation products of starch hydrolysis by partially
mL per minute and the 200 mL of eluents were collected purified amylase enzymes from three isolates of B.
into 40 tubes (1×7 cm) using an automatic circular fraction licheniformis (SUDK1, SUDK4 and SUDO) and identified
collector. The enzyme activity and protein concentration by thin layer chromatography (TLC) were mainly
were similarly determined in each fraction as described maltooligosaccharides and maltose while SUDK2 isolate
above. produced maltose and glucose (Figure 1) as reported in
92 N U S A N T A R A B I O S C I E N C E 7 (2): 90-95, November 2015

Table 1. Effect of partial purification of amylase enzyme activity and total protein contents of different B. licheniformis isolates

Bacillus Step Volume Specific activity Total protein Total Yield Purification
isolates (mL) (U/mg protein) (mg) activity (%) (Fold)
SUDK1 Crude enzyme 300 7.250 116.4 843.9 100 1
DEAE-Sephadex 30 121.755 4.7 566.2 67.1 16.8
SUDK2 Crude enzyme 200 4.626 94.0 434.8 100 1
DEAE-Sephadex 30 83.343 4.2 350.0 80.5 18.0
SUDK4 Crude enzyme 300 6.973 115.8 795.9 100 1
DEAE-Sephadex 30 120.842 4.8 581.3 73.0 17.3
SUDKO Crude enzyme 300 5.238 124.5 652.1 100 1
DEAE-Sephadex 25 95.883 5.2 493.8 75.7 18.3

range of purified -amylase from B. licheniformis EMS-6

for hydrolysis of soluble starch was found to be at 60-70°C
(Duy and Fitter 2005; Ul-Haq et al. 2010). Maximum
activities of -amylase from saliva samples of adult
smokers and no smokers were obtained at an optimum
temperature of 40°C (Enemchukwu et al. 2013). The
enzyme was stable for at least six month at 25°C (Sampson
et al. 1981). The enzyme activity in the serum of
pancreatitis patients was very stable at 37-40°C (Mohhmod
2010; Amutha and Priya 2011) and optimum at 50°C (Kim
et al. 1992). The enzyme activity from Huai yam powder
was established for hydrolysis reaction under the
Figure 1. Thin layer chromatograms of the starch hydrolysis
temperature range of 40-70°C (Miao et al. 2013). The
products by -amylases from different B. licheniformis isolates.
G: Glucose standard; M: Maltose standard; K-O: Samples
enzyme was stable at 25-60°C (Saito 1973). The
deactivation mechanism involves the existence of a
temperature-dependent intermediate form (Violet and
similar investigations (Kumar et al. 2010). The specific Meunier 1989). Partially purified -amylase enzyme from
activity of partially purified amylases using DEAE- freshwater zooplankton Heliodiaptmus viduus (Gurney)
sephadex from all isolated increased by 16.8-18.3 folds showed activity up to 70°C and optimum at 30°C, and
over the crude extract. The total protein content decreased inactive at 60°C after 2 hrs and at 70°C after one hour
from 94.0-124.5 mg for crude extract to 4.7-5.2 mg for (Dutta et al. 2006). In the present study, the optimum
partially purified extract, and the yield percentages of all activity of the enzymes was recorded at 70°C except for
amylases varied in the range of 67.1- 80.5% (Table 1). SUDK2 isolate where the optimum activity occurred at
These investigations show that DEAE-Sephadex is an 60°C and the enzyme was active up to 90°C with a residual
excellent exchanger for these enzymes and effectiveness activity of only 30-50% (Figure 2) as concluded by many
purification method comparable to other methods as researchers using different microorganism (Sampson et al.
suggested in similar investigations (Al-Quadan et al. 2009). 1981; Violet and Meunier 1989; Duy and Fitter 2005;
Thermodynamic and kinetics parameters provide a Dutta et al. 2006; Mohhmod 2010; Ul-Haq et al. 2010;
detailed mechanism for many chemical and biological Amutha and Priya 2011; Enemchukwu et al. 2013; Miao et
reactions (Tanaka and Hoshino 2003; Ul-Haq et al. 2010). al. 2013). The amylase enzymes isolated from four B.
The purified -amylase from B. licheniformis NCIB 6346 licheniformis isolates and stored at -20ºC retained more
strain was stable at pH 7.0 and 10.0 and was maximally than 90% of the original activity during 24 weeks of
active at 70-90°C and pH 7 as thermostable enzyme storage, while at 4ºC all the enzymes showed gradual loss
(Morgan and Priest 2008). Maximum enzyme production of their activities and lost 60% of their activities after 8
from B. amyloliquefaciens P-001 with initial pH 7 at 40°C, weeks (Figure 4-7) as reported in similar studies carried on
optimum pH 6.5 and 60°C and retained about 73% of its B. circulanus an B. subtilis (Adeyanju et al. 2007) and the
activity at 50°C (Deb et al. 2013). The pH 4.9 and 40°C enzyme showed broad temperature range of between 40-
were optimum for amylase from B. circulans GRS 313 70°C (Mahdavi et al. 2010).
which was stable at 60°C and pH range of 5.0-8.0 (Dey et The amylase enzyme exhibited activity at a wide range
al. 2002). The temperature profile of -amylase from B. of pH, optimum being pH 9.0 which is desirable
cereus isolated from acidic soil showed a very broad characteristic for its application in detergents as additive
temperature range of between 10-70°C and optimum at and textile desizing (Aiyer 2004). Poor enzyme stability
50°C which is different from those known Bacillus under standard conditions of pH and temperature evidently
amylases (Mahdavi et al. 2010). The optimum temperature affect the yield of hydrolysis products (Riaz et al. 2007).
 DAWOOD et al. – α-amylase from Bacillus licheniformis isolates 93

Figure 2. Effect of different temperature on -amylase activity Figure 5. Effect of storage temperature on -amylase activity
from different isolates of Bacillus licheniformis from Bacillus licheniformis SUDK2

Figure 3. Effect of pH on -amylase activity from different Figure 6. Effect of storage temperature on -amylase activity
isolates of Bacillus licheniformis from -amylase activity from Bacillus licheniformis SUDK4

Figure 4. Effect of storage temperature on -amylase activity Figure 7. Effect of storage temperature on -amylase activity
from -amylase activity from Bacillus licheniformis SUDK1 from -amylase activity from Bacillus licheniformis SUDO
94 N U S A N T A R A B I O S C I E N C E 7 (2): 90-95, November 2015

The amylase was stable over a wide pH range of between ACKNOWLEDGEMENTS

4.5 and 9.0, and optimum at pH 7.0 (Ul-Haq et al. 2010).
The enzyme activity in the serum of pancreatitis patients We thank the Department of Life Science, Faculty of
was very stable under pH 7.0-7.6 (Mohhmod 2010). Education, Nile Valley University, Department of Botany,
Maximum activity for saliva amylase was obtained at an Faculty of Science, University of Khartoum Sudan, and
apparent pH of 7.0 (Enemchukwu et al. 2013). Enzyme Department of Biological Sciences and Chemistry, College
from freshwater zooplankton, Heliodiaptomus viduus was of Arts and Sciences, University of Nizwa for providing
active at pH 3.5-8.5 (Dutta et al. 2006). Either acidic or space and facilities. We thank the University of Nizwa
alkaline conditions significantly affect the growth and Writing Center for proof reading the English of this
enzyme production by B. subtilis (Amutha and Priya 2011). manuscript.
The amylase was stable at pH 6.0 and 11.0 at 25°C, and
below 60°C and pH 8.0 (Saito 1973). It was active over a
pH range of 6.0-8.0 at 50°C (Kim et al. 1992). The - REFERENCES
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