Screening, Characterization and Production of Thermostable Alpha-Amylase Produced by a Novel Thermophilic Bacillus megaterium Isolated from Pediatric Intensive Care Unit

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 952-960

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Screening, Characterization and Production of

Thermostable Alpha-Amylase Produced by a Novel

Thermophilic Bacillus megaterium Isolated from

Pediatric Intensive Care Unit

Seyyedeh Narjes Abootalebi , Amir Saeed , Ahmad Gholami , Milad Mohkam , Aboozar

Kazemi , Navid Nezafat , Seyyed Mojtaba Mousavi , Seyyed Alireza Hashemi , Eslam

Shorafa²^*

Department of Pediatrics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

Biotchnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan

Department of Mechanical Engineering, Center for Nanofibers and Nanotechnology, National University of Singapore, Singapore

Received: 09/03/2020

Accepted: 13/06/2020

Published: 20/09/2020

Abstract

This study aimed to isolate the thermophilic Bacillus strain capable of producing a high amount of thermo-stable α-amylase.The

screening and isolation of amylase producing bacteria were done on selective media. The identification of bacteria was made using

routine biochemical and molecular 16s rRNA techniques. The amylase activity assay was performed by using dinitrosalicylic acid (DNS)

method, and finally, the optimum temperature, K (Michaelis constant) and maximum rate of reaction (Vmax) of the enzyme was

calculated. Results: The newly thermo-stable amylolytic enzymes of Bacillus megaterium designated AGH01was isolated from pediatric

intensive care unit through a selective enrichment procedure. This isolate was identified based on biochemical and morphological traits

along with 16s rRNA partial sequence analysis. This isolate showed the highest amylolytic activity (19.2 U/mL after 24 h) as compared

to other isolates. The optimum temperature for the enzyme activity was achieved at 90°C and pH 7.0. At this condition, K

for the degradation of starch was 3.36 mg/mL and (Vmax) was 0.177 mM/min. The enzyme was stable at 90°C and 80°C in 33.4% and

5.7% of its original activity at pH 7.0 after 30 minutes and 9.1% and 48% after 60 minutes of incubation, respectively. Bioinformatics

of the enzyme

analysis showed the enzyme had no disulfide bond and had a 35 amino acid signal sequence. The aliphatic index (69.14) and grand

average of hydropathicity index (-0.399) calculated by ProtParam server indicated that this α-amylase may be stable for a wide range of

temperature and has excellent solubility in water.Conclusions: The high-thermal stable enzyme produced by Bacillus megaterium

AGH01 could be considered merit as an excellent alternative source for bioethanol production and pharmaceutical applications.

Moreover, our data provides such beneficial information for a better industrial formulation with proper stability in extreme conditions.

Keywords: Amylase, Amylolytic activity, Bacillus megaterium, Thermo-stability

Introduction¹

and medicinal chemistry(3-6), as well as their extensive usage

in the distilling and brewing industries and starch

saccharification (7, 8). It is necessary to have more thermo-

active and thermostable amylases because these types of

enzyme should be applicable at the high temperature of

liquefaction (80–90 °C) and gelatinization (100–110 °C) to

economize processes (9). Since the geographic region where

the microorganisms are isolated, affect and determine enzyme

behaviour, many attempts have been made to isolate some

potential thermostable amylase producing strains from the

harsh ecological area in the world (10-12). Geothermal sites,

hot spring, compost, and soils were recognized as suitable

habitats for microorganisms which can provide the source of

thermostable enzymes (13). Because of degrading the starch

Amylases take part as an essential class of industrial

enzymes having about 25-33% of the world enzyme market.

Initially, the term amylase was used originally to designate an

extracellular enzyme capable of hydrolyzing α-1,4-glucosidic

linkages in polysaccharides containing three or more glucose

units (1). The enzyme acts on starches, glycogen and

oligosaccharides randomly, leading to liberating reducing

groups of sugars. It has potential applications for a large

number of industrial purposes such as textile, bread and

parchment paper, detergent, food, pharmaceutical and fine-

chemical industries (2). However, with the advent of the new

frontier in biotechnology, the amylase application has also

expanded in numerous other fields such as clinical, analytical

Corresponding authors: (a) Ahmad Gholami, Biotchnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, E-

mail: Gholami@sums.ac.ir, (b) Eslam Shorafa, Biotchnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran,

shorafae@sums.ac.ir. Tel. /fax: +98 711 2426729.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 952-960

constitutes (the highest substrate of these media), amylolytic

bacteria are supposed to be usually available in there(14-18).

Numerous studies have proven that Bacillus sp. found in

natural areas (19-21) (especially those grown at harsh

conditions) was recognized as the leading producer of exotic

amylase with high economic importance (22, 23). This work is

focused on the isolation and identification of a bacterial strain,

which can generate thermophilic and thermo-stable amylase

with excellent characteristics and could be applicable for

commercial purposes.

2.5 Inoculum preparation

Purified bacterial strains were selected based on the

capability to cultivate in the selective medium containing starch

as an individual carbon and energy source at room temperature.

The activation media had the following composition: Soluble

starch (3.0 g/L) and nutrient broth (25 g/L) at pH 7.0. Ten ml

of this activation medium was inoculated by 100µl of 0.5

McFarland of the bacterial strain that grown on starch nutrient

agar plates and incubated for 12 h in a rotary incubator shaker

at 42°C at 140 rpm (28).

.6 Seed culture preparation

A two-day-old culture was used for the preparation of cell

Material and methods

.1 Screening and isolation of microorganisms

suspension. 25 ml of seed medium was added to 250 ml

Erlenmeyer flask. The medium contained soluble starch (2

g/L), peptone (5 g/L) and yeast extract (3 g/L) at pH 7.0. The

flasks were autoclaved at 105 Pa pressure (121 °C) for 20 min

and then gradually cooled at 25°C. Under sterile environments,

During the present investigation, the samples were

collected from pediatric intensive care unit, Namazi hospital in

Shiraz, Iran. One gram of each sample was suspended in 9 mL

of sterile saline (9 g/L NaCl), serially diluted up to 10 and

cultured on starch nutrient agar (25 g/L nutrient agar and 3 g/L

starch at pH 7.0) plate. After 24 h of incubation at 37°C and

further subculturing to obtain pure culture, particular colonies

were isolated, and colonies were measured in diameter.

ml of inoculum was added to the flask, and the flask was

incubated in a rotary incubator shaker (which is kept at 140

rpm) overnight at 37°C.

.7 Production of amylase in the bioreactor system

Enzyme production was carried out using a 10 L bioreactor

.2 Identification of amylase-producing strains

Distinct colonies were identified based on morphological

(

Biotron Inc., South Korea) containing 5L of fermentation

medium. 20% (v/v) microorganism suspension was

transferred from seed culture to the fermentation medium

containing: NaCl (2 g/L), (NH SO (2 g/L), K HPO (2.5

g/L), KH PO (1 g/L), MgSO .7H O (0.01 g/L), FeCl (1 g/L),

CaCl (0.01 g/L), peptone (2 g/L) and soluble starch (2 g/L) at

features like size, colour, elevation, shape, margin, gram stain,

motility, spore stain and cell shape and then, the amylolytic

activity of each isolated strain was measured by adding Gram’s

)

iodine reaction mixture (0.2% KI, 0.2% I ) (24). Colonies with

clear and transparent zone were selected as amylase producing

strains. The selected colonies were purified by replica plating

on starch-nutrient agar slant.

pH 7.0. Subsequently, the bioreactor was set up at 140 rpm at

temperatures ranging from 30 till 90 ºC for 12, 24, 36, 48 and

0 h. After the fermentation period, the cells were discarded by

.3 Selection of desirable enzyme producer strain

In order to select the best thermo-tolerant enzyme producer,

centrifugation, and the Cell-free filtrate was used for enzyme

assay.

the selected bacteria were cultured at different temperatures

30, 37, 42, 45, 50 and 55°C) in the fermentation medium and

incubated at 140 rpm.

(

.8Amylase assay

Amylase assay was performed by using a reaction solution

consisting of 0.5 ml of the substrate (20 g/L soluble starch in

0 mM phosphate buffer pH 7.2 and 0.5 ml of the supernatant

.4 Identification of microorganisms

Various biochemical, physiological and morphological

as a crude enzyme) (8). The reaction was done at 90°C for 5

min. The released sugars from starch were estimated using

dinitrosalicylic acid (DNS) reagent according to the Miller

method. Subsequently, the product was heated to 100°C for 10

min and then cooled. The optical density of samples was taken

at 575 nm in a spectrophotometer (Shimadzu, Japan). The

activity of the enzyme was measured in units (1 unit was

defined as the quantity of enzyme which produces 1mmole

glucose under assay condition.

characterizations of the selected amylase-producing isolates

were determined by using Bergey’s Manual of Determinative

Bacteriology (25). Biochemical tests such as catalase and

Voges-Proskaeur and oxidase test, oxygen requirement and

motility were also studied. Colony morphology such as form,

margin, elevation, colour and diameter (mm) after 24 h were

observed by light microscopy, and finally, the genus of the

selected strain was determined (26). For further identification,

the analysis of 16s rRNA gene sequence was done following

Gholami et al. method (27). Concisely, the bacterial genomic

DNA was extracted by the heat shock method. The universal

.9 Characterization of amylase and kinetic properties

0.25 ml of the diluted crude enzyme was combined with a

prokaryotic (16s rRNA) primers, 5 -ACGGGCGGTGTGTAC-

solution of starch (0.25 mL, 0.02%) at 90°C and the quantity of

resulting glucose over the time was measured. The effects of

temperature, pH and substrate concentration on enzyme

activity were considered, and K and Vmax values of the enzyme

were measured by using double-reciprocal (Lineweaver-Burk)

plot.

as the forward primer and 5 -CAGCCGCGGTAATAC-3 as

the reverse primer was used to amplifying the partial sequence

of 16s rRNA gene of isolates by PCR, which amplify ~ 800

base pair region of the 16s rRNA gene. The PCR products were

purified and then determined by CinnaGen Company (Tehran,

Iran) and then resulting 16s rRNA gene sequences were aligned

and the sequence similarity compared to some known

microorganisms in GenBank database of the National Center

for Biotechnology Information by using Basic Local

Alignment Search Tool (BLAST).

.10 Evaluation of thermo-stability of the enzyme

At 80 and 90 ºC, the stability of the enzyme produced by

Bacillus sp. was assessed at without any additives at pH 7.0,

and the half-life of the enzyme was estimated.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 952-960

.11 Bioinformatics Study of α-amylase

Specific primers for α-amylase was designed according to

at the temperature ranging from 30 to 55°C (Fig. 1). The best

temperature for the highest amylase production of all three

strains is 37°C, and the more increase in temperature the less

production of amylase was achieved. The results showed that

N15 had the highest activity at all temperatures among the three

selected strains.

the available α-amylase nucleotide sequences in the NCBI

database (Forward: 5'-GTGCTAACGTTTCACCGCATC-3'

and Reverse: 5'-CAAGGCCATGCCACCAACCGT-3') and

then sequenced. The multiple sequence alignment (MSA) of

amylase sequences was performed using the CLC free

Workbench, and then the evolutionary tree was constructed by

using the Neighbour Joining algorithm (27, 29). Percentages of

hydrophobic and hydrophilic residues were computed from the

primary structure analysis outcomes. The physicochemical

traits, theoretical isoelectric point (pI), molecular weight,

extinction coefficient, the total number of positive and negative

residues, aliphatic index, instability index and grand average

hydropathy (GRAVY) were calculated using the Expasy’s Prot

Param (http://us.expasy.org/tools/protparam.html) prediction

server. The putative amino acid sequence was inspected for a

signal

sequence

the

SignalP

4.0

server

(

http://www.cbs.dtu.dk/services/SignalP/). The existence of

disulfide bonds was predicted by CYS_REC and DISULFIND

servers (27, 30).

Figure 1: Effect of temperature on amylases production of three

selected strains (N15, N32 and N7) after 24h at pH 7.0 in the

fermentation medium

Results and discussion

It is so necessary to screen bacteria for their amylase

production, which could be a significant source of the enzyme

after pure culture on a large scale. The productions of enzymes

for industrial consistency and cost-effectiveness can be adopted

with the selection of proper strain (31). The preliminary

screening of environmental bacteria has prime importance as

the starch-rich soil and water are the primary sources for

varieties of bacteria as well as their amylase producing activity

.2 Effect of fermentation time on enzyme production

The suitable fermentation time for the highest enzyme

activity was considered. Two strains depicted high enzyme

activities after 24 h [N15 (19.2 U/ml), N32 (16.08 U/ml), and

one of them produced high amylase activity after 36h (N7 (14.6

U/ml)]) (Fig. 2).

(32). Bacteria that produce amylase may be isolated from

locations like vermin compost sites (33), processing firms,

fermenting roots of cassava and naturally fermented alfalfa (34)

and honey processing areas (35) as well as sewage surrounding

mills (36).

At the present investigation, amylase producing strains

were isolated from pediatric intensive care unit in a hospital.

Among fifty-five different colony which produced bright halos

with iodine solution, only three strains produced amylase at

between 10.0 and 20.0 U/ml, and the rest of them produced

amylase activity less than 10.0 U/ml. The high amylase

producing bacteria labelled as strain N15, strain N32, and strain

N7. The halo diameters of the strains N15, N32 and N7, were

1.86, 30.83 and 21.48 mm and their halo diameter to colony

diameter ratios were 6.37, 6.17 and 4.3, respectively (Table 1).

Among the three strains, N15 which was isolated from

pastry waste produced the highest amylase activity (19.8

U/mL) after 24h at pH 7.0 and 90˚C and indicated the highest

value for halo diameter to colony diameter ratio (7.4), which

demonstrated highest amylase activities. Therefore, it was

chosen for additional evaluations.

Figure 2: Effect of fermentation time on amylase production of three

selected strains (N15, N32 and N7) at pH 7.0and at 37°C

However, the growth rate of three strains showed the same

trend and after 12 h reached the maximum growth (Fig. 3).

According to the highest enzyme-producing capability at high

temperature, the strain N15, which was nominated as the best

strain, was further characterized.

.1 Effect of temperature on enzyme production

The effect of temperature on enzyme production was

studied to find the best thermostable enzyme-producing strain,

Table 1: Comparing the amylase activities of three selected strains (N15, N32 and N7), amylolytic power (diameter of clear halo) and

its ratio to colony diameter at pH 7.0 and 90°C after 24 h

Strain

N15

N32

Amylolytic power (mm)

Halo diameter to colony diameter ratio

Amylase activity (U/ml)

7.4

6.0

4.8

19.8

16.3

14.5

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 952-960

Therefore, our result showed that this strain produced more

heat-stable amylase than previous similar works.

Figure 3: Effect of fermentation on the growth of three selected

strains (N15, N32 and N7) at pH 7.0 and 37°C

.3 Identification of the microorganism

In order to identify the genus of the strain, morphological,

Figure 4: Effect of fermentation time on activity of the enzyme

produced by B. megatriumwith soluble starch

cultural and biochemical (Table 2) characteristics were used.

The strain N15 was rod-shaped, Gram-positive, motile, and

with negative results for oxidase test and positive results for

catalase test. According to results, as shown in Table 2, the

strain N15 was similar to those of the genus Bacillus. Finally,

according to the widely used of 16s rRNA gene analysis, we

sequenced the 16s rRNA gene of amylase producing strain

named as AGH01 (16s rRNA accession no. KF437334). The

obtained sequence was blasted against various genomic

databases in NCBI, which indicated 99% homology with B.

megaterium. Therefore, based on perceptions and analysis,

strain AGH01 was classified as B. megaterium AGH01.

.4 Characterization of amylase and kinetic properties

The pattern of the kinetics of the resulting enzyme from B.

megaterium in the first 5 min presented zero-order patterns and

for this reason, in the subsequent investigation, the reaction

time was established on 5 min (Fig. 4). In order to assess the

activity of the enzyme produced by B. megaterium, different

temperatures ranging from 30-95ºCwas studied. The optimal

temperature for amylase activity of this strain was 90 ºC, at pH

Figure 5: The activity of amylase obtained from

B.megatriumat different temperature at pH 7.0

The activity of enzyme achieved from B. megaterium at pH

ranging from 6.0 to 10.0 was assessed. The optimum pH for

activity of amylase was 7.0 (Fig. 6). Enzyme obtains from B.

megaterium displayed 72.1 and 85.2% of its maximum activity

at pH 8.0 and 6.0, respectively.

.0 (Fig. 5). In this study, amylase showed 92.7, 96.4 and

7.0% of its maximal activity at 80, 95 and 85°C, respectively,

at pH 7.0. In contrast, Hayashida et al. obtained the maximum

activity of amylase for B. subtilis 65 at 60°C and pH 6.0 (37).

Table 2: Morphological, cultural and biochemical characteristics of N15

Response of the strain Biochemical test Response of the strain

Pale Anaerobic growth

Properties

Color

Form

Margin

Surface

Opacity

Elevation

Gram staining

Irregular

Moist, shiny

Opaque

Flat

Growth in air

Growth at 55ºC

Growth in 7% NaCl

Haemolysis on blood agar

Lactose fermentation

Hyrolysis of tyrosine

β- haemolysis

Positive

Acid slant, Acid butt,

H2S -, Gas -

Motility

Actively motile

KIA pattern

Diameter of clear halo (mm)

Diameter of colony after 24 h

31.86

Nitrate reduction test

Urease test

(mm)

Shape of vegetative cell

Spore formation

Rod

Positive

Oxidase production

Citrate production

Indole production

Catalase production

Voges-Proskaeur test

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 952-960

was relatively stable at 60 and 70°C for one h, whereas at 80

and 90°C, 12% and 48% of its original activity was lost,

respectively (43). In contrast, amylase obtained from B. subtilis

AX20 in another study exhibited 60% and 35% of maximal

activity at 40 and 70ºC, respectively (44). In comparison to the

above mentioned reports, our findings showed that the enzyme

obtained from B. megaterium AGH01 had superior stability

under the harsh conditions.

Figure 6: The activity of the enzyme obtained from B. megaterium at

different pH at 90°C

Our results were in agreement with other works that

indicated the optimal pH of amylase activity obtained from

various Bacillus spp. such as B. subtilis KIBGE HAS (38),

B.licheniformis (39), and B.amyloliquefaciens (40) and

B.coagulans (41) is neutral. In order to estimate the K and

max, the activity of amylase was measured by the rate of

substrate consumption. The activity became greater than 0.160

mM/min since the concentration of substrate was raised from 5

to 30 mg/mL at pH 7.0 and at 90°C (Fig. 7).

Figure 8: Double-reciprocal plot (Lineweaver-Burkplot) for enzyme

produced by B. megaterium

Figure 7: The activity of the enzyme obtained from B. megaterium

with different concentration of substrate at 90°C and pH 7.0

Figure 9: Evaluation of thermo-stability of amylase produced by B.

megaterium at 80°C, and 90°C without any additives at pH 7.0

The K

of starch was 3.36 mg/ml, and maximum rate of reaction (Vmax

was 0.177 mM/min at pH 7.0 and 90°C (Fig. 8). Inferior values

of K designate superior affinity of the enzyme for the substrate

42). In comparison to previous studies, our research clearly

showed the lower Km values attributed to a higher affinity of

the enzyme (40, 42). Because of variation in obtaining Km and

Vmax values is completely related to the type of substrate and

reaction condition, it is difficult to compare them. However, a

similar work by Dragomirescu et al. showed the Km and Vmax

values of 12.28 mg/ml and 0.82 mM/min at 37°C, respectively,

for B. amyloliquefaciens (40).

(Michaelis constant) of the enzyme for the degradation

3.6 Bioinformatics Study of α-amylase

)

α-amylase gene-specific primers were designated as

detailed in materials and methods section. The sequences

achieved from NCBI was analyzed using the BLASTP search,

which disclosed only one large open reading frame, composed

of 1647 base pairs and 549 amino acid residues. BLAST

searches of the α-amylase protein sequence depicted 89%

identity at the amino acid sequence level with Geobacillus

stearothermophilus. However, it also showed inferior identity

(24-86 %) with other bacterial α-amylase (data not shown). The

amino acid sequence of α-amylase was introduced to CLC free

Workbench software. Then multiple sequence alignment

(

(MSA) (Fig. 10) and phylogenetic tree (Fig. 11) were drawn in

order to reveal the taxonomical variation in α-amylase from

various Bacillus species (45). The multiple sequence alignment

of these protein sequences revealed conserved regions at

different stretches, namely, from 113 to 150, 246 to 274 and

298 to 310. The constructed phylogenetic tree revealed three

distinct clusters (Fig. 11).

.5 Evaluation of enzyme stability

The thermal stability of amylase was assayed without any

additives. After 30 min of incubation at 90°C, amylase obtained

from B. megaterium kept 33.4% of its original activity at pH

activity. In comparison, at 80°C after 30 min of incubation, the

enzyme retained 35.7% of its original activity at pH 7.0, and

after one h, it reduced to 48% of its activity (Fig. 9). Asgher et

al. reported that the amylase produced by B. subtilis JS-2004

.0, and after one hour it decreased to 9.1% of its original