Optimization of physical parameters of α amylase producing brevibacillus borostelensis r1 in submerged fermentation

IJRET: International Journal of Research in Engineering and Technology eISSN: 2319-1163 | pISSN: 2321-7308
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Volume: 03 Issue: 01 | Jan-2014, Available @ http://www.ijret.org 517
OPTIMIZATION OF PHYSICAL PARAMETERS OF Α-AMYLASE
PRODUCING BREVIBACILLUS BOROSTELENSIS R1 IN SUBMERGED
FERMENTATION
K.Suribabu1
, T. Lalitha Govardhan2
, K.P.J Hemalatha3
1, 2
Assistant Professor, PG Department of Microbiology and Research Centre, Dr. Lankapalli Bullayya Post-graduate
College, Andhra Pradesh, India, Visakhapatnam-530 013
3
Professor, Department Microbiology, Andhra University, Andhra Pradesh, India
Abstract
Bacteria have been regarded as treasure of many useful enzymes viz., amylases, proteases, lipases, hydrolases and reductases. Among
them amylolytic enzymes have great biotechnological applications and economic exploitations. The production of α-amylases by
fermentation had been thoroughly investigated and shown to be affected by a variety of physicochemical factors, such as the
composition of the growth medium, the type of strain, cell growth, methods of cultivation, inoculum concentration, time of incubation,
pH, temperature, salinity, carbon, nitrogen and mineral sources. The present study was carried out to optimize the α-amylase
production of Brevibacillus borstelensis R1 using ten different media viz., Nutrient broth, Luria Bertain broth, Clarks & Lub medium,
Pikovskaya’s medium, Tendler's non-synthetic medium, Amylase production medium, Soluble starch beef extract medium, Soybean
casein digest medium, Yeast extract peptone dextrose glucose medium and Tryptone glucose beef extract medium. Among these ten
media, Pikovskaya’s (PK) medium proved to be optimal for α-amylase production (1861±17U/ml). The optimized α-amylase
production in PK medium by submerged fermentation (SmF) was subjected to varying physical parameters such as 24hrs incubation
time, 2% inoculum size, 370
C, pH 7.0 and 1% NaCl. Alpha-amylase produced by B.borostelensis R1 have many applications in starch
processing, desizing of textiles, paper sizing, detergent additive, bread improvement, ethanol production, sewage treatment, effluent
treatment and other fermentation processes.
Keywords: Pikovskaya’s medium, incubation time, inoculum size, temperature, pH and salinity.
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1. INTRODUCTION
Screening is the detection and isolation of high-yielding
species from the natural material such as soil, air and water
containing heterogeneous microbial populations. Screening
methods of bacteria include primary screening, secondary
screening and strain improvement [1].
1.1 Primary Screening
Primary screening consists of basic elementary tests required
to detect and to isolate new bacterial species exhibiting the
desired property. Marine microorganisms are known to have a
diverse range of enzymatic activity. Several reports of α-
amylase producing bacteria include: Streptomyces spp. [2],
Lactobacillus spp. [3], Geobacillus stearothermophilus spp. [4]
and Clostridium spp. [5].
1.2 Measurements of Bacterial Growth
Growth is defined as an orderly increase of all cellular
components, with multiplication as a consequence. Death is
the irreversible loss of ability of organism to reproduce itself.
The stages of growth curve are lag phase, log phase
(exponential phase), stationary phase and death phase (decline
phase) [6].
There are four methods of growth measurements:
1.2.1 Direct Microscopic Count (Direct Cell Count
Method)
It helps to count the total number of cells include both living
and dead directly by direct microscopic counts using Petroff-
hauser chamber (nine squares, each 0.1mm deep, volume of
liquid over one square mm is 0.1 cubic mm).
1.2.2 Viable Cell Count Method
It is based on the principle that when material containing
bacteria are cultured, every viable bacterium develops into a
visible colony on a nutrient agar medium.
1.2.3 Turbidity Method
It helps to study the cloudiness of the suspension. Turbidity is
directly proportional to the number of cells. It is quantified

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with the spectrophotometer, which measures the amount of
light transmitted directly through a sample. The cells
suspended in the culture interrupt the passage of light. The
amount of light absorbed through the suspension is measured
as optical density.
1.2.4 Dry Weight Method
It helps to estimate the bacterial growth at regular time
intervals during the incubation period. The growth of the
bacteria is directly proportional to the dry weight harvested.
1.3 Physical Parameters
The physical parameters include pH, temperature and salinity
which influence the production of amylase.
1.3.1 PH
Sudden variations in cytoplasmic pH can harm bacteria by
disrupting the plasma membrane, inhibiting the activity of
enzymes and membrane transport proteins. pH is known to
affect the synthesis, secretion and stability of α-amylase [7].
Selection of a suitable fermentation medium and initial pH is
very important for the enhanced alpha-amylase production [8].
1.3.2 Temperature
High temperatures damage bacteria by denaturing enzymes,
transport carriers, and other proteins. Thermostability is a
desired characteristic of most of the industrial enzymes.
Thermostable α-amylases were produced by mesophilic
species of Bacillus [9]. Therefore, a high value is placed on
extreme thermostability and thermoactivity of the enzymes.
1.3.3 Salinity
Bacteria in marine habitat have modified the structure of
enzymes, ribosomes, and transport proteins which require high
levels of potassium for stability and activity. Halophilic
bacteria are classified according to their salt requirement and
growth pattern. Slight halophiles show optimum growth at 2–
5% NaCl, moderate halophiles at 5–20% NaCl and extreme
halophiles at 20–30% NaCl [10].
2 MATERIALS AND METHODS
2.1 Collection of the Marine Water Samples
Marine water samples were collected from Rushikonda coastal
area of Visakhapatnam, Andhra Pradesh, India. The water
samples were collected in sterile BOD bottles (Borosil) and
brought to the lab, stored in the refrigerator until it was used.
2.2 Primary Screening of α-Amylase Producing
Bacteria
The collected marine water samples were diluted by serial
dilution technique. The diluted samples of 10-4
to 10-6
(0.1ml)
were spreaded with L-shaped glass rod by spread plate
technique on the starch agar plates. After incubation at 370
C
for 24hours, the plates were flooded with Lugol solution (1%
iodine in 2% potassium iodide w/v) [11]. The average cfu/ml,
number of colonies forming clear halo zone of hydrolysis and
zone of starch hydrolysis measured in mm.
2.3 Estimation of Amylase by DNS Method
Maltose produced by the hydrolytic activity of α-amylase on
α-1, 4 linkages present in polysaccharides, reduce 3, 5 dinitro
salicylate to an orange red colored 5-nitro 3-amino salicylate
which can be measured at 520nm. The starch substrate [0.5ml
of 0.5% in 0.1M phosphate buffer (pH 6.8)] was mixed with
1% (0.2ml) NaCl in a test tube and pre incubated at 370
C for
10 minutes. The supernatant collected from the centrifugation
of the production media was used as enzyme source, 0.5ml of
this was added to the reaction mixture. The reaction was
terminated by the addition of 1.0 ml of 3, 5-dinitrosalicylic
acid reagent [1.0 gm DNS in 0.8% NaOH, 60% Na K tartrate]
after incubation at 370
C for 15 minutes. The contents were
mixed well and kept in boiling water bath for 10 minutes.
Then they were cooled and diluted with 10 ml of distilled
H2O. The color developed was read at 520nm. One unit of
enzyme activity was defined as the amount of enzyme that
release 1.0 mmol of reducing sugar (maltose) per minute
under the assay conditions [12].
2.4 Fermentation Media
Optimization of α-amylase production by B. borostelensis R1
was carried out by using ten submerged fermentation media;
Nutrient Broth (NB), Luria Bertain Broth (LB), Clarks and
Lub Medium (CL), Pikovskaya’s Medium (PK), Tendler's
Non-synthetic (TNS) Medium, Amylase Production Media
(APM), Soluble Starch Beef Extract Medium (SB), Soybean
Casein Digest Medium (SCD), Yeast Extract Peptone
Dextrose Glucose Medium (YPDG) and Tryptone Glucose
Beef Extract Medium (TGB) procured from Himedia, India.
The ingredients of ten fermentation media used for the
optimization of α-amylase production were in (g/l) NB (NaCl
5.0, Beef extract 3.0 and Peptic digest of animal tissue 5.0),
LB (Tryptone 10.0, Yeast extract 5.0 and NaCl 10.0), CL
(Glucose 5.0, Peptone 5.0 and K2HPO4 5.0 ), PK (Glucose
10.00, Ca3(PO4)2 5.0, (NH4)2SO4 0.50, MgSO4.7H2O 0.10,
MnSO4.7H2O 0.01, FeSO4 0.01, KCl 0.20 and Yeast extract
0.50), TNS (Tryptone 10.0, Yeast extract 4.0, Sodium citrate
0.5, Ammonium nitrate 1.0, K2HPO4 0.3, MgSO4 0.5 and
Starch 2.0), APM (Starch 2.0, Na2HPO4 3.0, KH2PO4 6.0,
NH4Cl 1.0, CaCl2 0.15, MgSO4.7H2O 0.25, Casein hydrolyte

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0.20 and Yeast Extract 0.10), SB (Soluble Starch 2%, Beef
Extract 1%, Yeast Extract 0.2%, CaCl2 0.02% and
MgSO4.7H2O 0.01 %), SCD (Pancreatic digest of casein
17.00, Soybean meal 3.00, NaCl 5.00, KH2P04 2.50 and
Dextrose 2.50), YPDG (Yeast extract 10.00, Peptone 20.00 ,
Glycerol 30.0ml and Dextrose 1.00), TGB (Tryptone 5.00,
Glucose 3.00 and Beef extract 1.00). The final pH was
adjusted to 7.0 with 0.1N HCl and 0.1N NaOH before
autoclaving.
2.5 Incubation Period
The growth curve of Brevibacillus borstelensis R1was
constructed by using direct microscopic count method, viable
cell count method, turbidity method and dry weight method in
Pikovskaya’s Medium. The α-amylase production during
growth phase (0-30hours) with an interval of 2hrs of B.
borstelensis R1 was assayed by DNS method. The incubation
period with maximum amylase production was determined
during growth cycle.
2.6 Inoculum Size
Two loopful of culture was aseptically inoculated in
Pikovskaya’s Medium. The flask was incubated (370
C) in
rotary shaking incubator at 120rpm, for 24hrs. After
incubation the inoculum optical density was adjusted to 0.9
with sterile distilled water at 600nm in colorimeter. Each ml
contains 1012
cells which were in stationary phase (α-amylase
production phase) as calibrated by growth curve measurement
methods.
The effect of different sizes (1% to 5%) of inoculum on the α-
amylase production of Brevibacillus borstelensis R1 in
Submerged fermentation (SmF) was investigated. The
Erlenmeyer flask was incubated (370
C) in rotary shaking
incubator at 120rpm, for 24hrs.
2.7 Temperature
Hundred ml of the Pikovskaya’s Medium was taken in sterile
erlenmeyer flasks. Inoculum size (2%) of pure culture from
pre-incubated pure strain was inoculated to each of flasks.
Medium was incubated in orbital shaking incubator (120rpm)
at different (40
C, 250
C, 370
C, 500
C and 600
C) temperatures for
24hrs.
2.8 pH
Fermentation medium (Pikovskaya’s Medium) was prepared
at different pH (2.0, 5.0, 7.0, 9.0 and 11.0) by adjusting the pH
with 0.1N HCl and 0.1N NaOH. Hundred ml of different pH
medium were taken in sterile erlenmeyer flasks. Inoculum size
(2%) from preincubated pure strain was inoculated to each of
Erlenmeyer flask. The flasks were incubated in orbital shaking
incubator (120rpm) at 370
C for 24hrs.
2.9 Salinity
Hundred ml of the Pikovskaya’s Medium was taken in
different erlenmeyer flasks. Inoculum size (2%) of pure
culture from preincubated pure strain was inoculated to each
Erlenmeyer flask. Each flask with different concentrations of
NaCl (0.5, 1.0, 1.5, 2.0, and 2.5 % w/v) was incubated in
orbital shaking incubator (120rpm) at 37O
C for 24hrs.
After incubation, the samples were subjected to centrifugation
at 5,000 rpm for 15 minutes at 250
C. The supernatant was
collected in sterile test tubes and the pellet was discarded.
Supernatant (0.5 ml) was used for the amylase assay by DNS
method.
3. RESULTS
3.1 Optimization of the Media
The optimization of media for α-amylase production was
carried out by inoculating B. borostelensis R1 for 24hrs in ten
different submerged fermentation media- Nutrient Broth (NB),
Luria Bertain Broth (LB), Clarks and Lub Medium (CL),
Pikovskaya’s (PK) Medium, Tendler's Non-synthetic Medium
(TNS), Amylase Production Medium (APM), Soluble Starch
Beef Extract Medium (SB), Soybean Casein Digest Medium
(SCD), Yeast Extract peptone Dextrose Glucose Medium
(YPDG) and Tryptone Glucose Beef Extract (TGB) Medium.
The highest production of α-amylase in all media is shown in
Chart -1. The optimal production of amylase (1861±17U/ml)
was observed when R1 strain was subjected to SmF in
Pikovskaya’s (PK) production media.
M e d ia
Enzymeactivity(U/ml)
N
B
L
B
C
L
P
K
T
N
S
A
P
M
S
B
S
C
D
Y
P
D
G
T
G
B
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
1 8 0 0
2 0 0 0
Chart -1: The α-amylase production (U/ml) in various
submerged fermentation (SmF) media; Nutrient broth (NB),
Luria bertain broth (LB), Clarks and lub medium (CL),
Pikovskaya’s medium (PK), Tendler's non-synthetic medium
(TNS), Amylase production medium (APM), Soluble starch
beef extract medium (SB), Soybean casein digest medium
(SCD), Yeast extract peptone glycerol glucose medium
(YPDG) and Tryptone glucose beef extract medium (TGB).

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Y bars indicate the standard deviation of mean value.
**** P < 0.0001 Values differ significantly at p<0.05.
The optimum (1861±17U/ml) α-amylase production was
observed with Pikovskaya’s Medium (PK) and the lowest
production was observed (180±1U/ml) with two media-
Nutrient broth and Luria bertain broth (Chart -1).
3.2 Incubation Period
Measurements of growth curve of B. borstelensis R1 to
determine incubation period: The growth curve of B.
borstelensis R1 at stationary phase was constant from 18-
24hours. It was measured by adopting direct microscopic
count by using Neubauer chamber, 12 log cfu/ml (Chart -2a),
viable cell count method, 12 cfu/ml (Chart -2b), turbidity
method, 0.923±0.012 O.D at 600nm (Chart -2c) and dry
weight method, 1.526667±0.003gm/10ml (Chart -2d). Distinct
phases of the growth curve were observed in each method.
The Rate of Growth (R) measured by direct microscopic
count, viable standard plate count method and turbidity
method was 1.63. The specific growth rate (μ) was 1.65 and
the generation time (G) was 0.61hrs or 36.60minutes.
Time (hours)
LogNumberofCells/ml
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Time (hours)
ViableCount(LogCFU/ml)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Time (hours)
O.Dat600nm
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Time (hours)
Dryweight(gm/10ml)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Chart -2: Growth curve of Brevibacillus borstelensis R1: a, Direct microscopic count method; b, Viable cell count method; c,
Turbidity method and d, Dry weight method.
ba
dc

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**** P < 0.0001 Values differ significantly at p<0.05 with
moderate degree of positive correlation.
The Pikovskaya’s (PK) Medium was found to be the best
among ten fermentation media used. The lag phase (0-2hrs),
log phase (2-18hrs), stationary phase (18-24hrs) and decline
phase followed. The highest α-amylase production
(1,800U/ml) was found during the stationary phase (18-24hrs)
(Chart -3). Amylase production at 24hours was taken as
standard time in all the following experiments.
T im e ( h o u r s )
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
1 8 0 0
2 0 0 0
Chart -3: Effect of incubation period on α-amylase production of Brevibacillus borstelensis R1
**** P < 0.0001 Values differ significantly at p<0.05 with
moderate degree of positive correlation.
3.3 Inoculum size
The Pikovskaya’s (PK) Medium and incubation period (24hrs)
were optimized. The optimum inoculum size was optimized in
PK medium. The effect of different sizes (1% to 5%) of
inoculum on the production of α-amylase by Brevibacillus
borstelensis R1 using submerged fermentation (SmF) was
investigated. The production of amylase was increased with
the increase in the level of inoculum upto 2% (1813±23U/ml).
As the level of inoculum was further increased, the
productivity of amylase was decreased (Chart -4). Thus, the
inoculum level of 2%v/w was found to be optimum for α-
amylase synthesis.
I n o c u l u m s i z e ( % v / w )
1
2
3
4
5
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
1 8 0 0
2 0 0 0
Chart -4: Effect of inoculum size on α-amylase production of Brevibacillus borstelensis R1

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3.4 Temperature
The Pikovskaya’s (PK) Medium, incubation period (24hrs)
and 2% inoculum size were optimized. The optimization of
temperature was carried at different temperatures (40
C, 250
C,
370
C, 500
C & 600
C). The highest amylase activity was
recorded at 370
C (2086±71U/ml) and the lowest at 40
C
(1131±57U/ml) (Chart -5).
T e m p e r a t u r e (
0
C ) o f P ik o v s k a y a 's M e d iu m
4
2
5
3
7
5
0
6
0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
1 8 0 0
2 0 0 0
2 2 0 0
Chart -5: Effect of Temperature on α-amylase production of Brevibacillus borstelensis R1
3.5 pH
The Pikovskaya’s (PK) Medium, incubation period (24hrs),
2% inoculum size and temperature (370
C) were optimized.
The optimization of pH was carried with different pH (2, 5, 7,
9 & 11). The pH was adjusted by using 0.1N HCl and 0.1N
NaOH. The highest amylase activity was recorded at pH 7.0
(2083±56U/ml) and the lowest at pH 11.0 (565±77U/ml)
(Chart -6).
p H o f P i k o v s k a y a 's M e d i u m
2
5
7
9
1
1
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
1 8 0 0
2 0 0 0
2 2 0 0
Chart -6: Effect of pH on α-amylase production of Brevibacillus borstelensis R1

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3.6 Salinity
The Pikovskaya’s (PK) Medium, incubation period (24hrs),
2% inoculum size, temperature (370
C) and pH (7.0) were
optimized. The optimization of salinity at different
concentrations (0.5%, 1.0%, 1.5%, 2.0% & 2.5%) is depicted
in Chart -7. The highest amylase activity was recorded at
salinity of 1.0% (2082±58U/ml) and the lowest at 2.0%
(1121±59 U/ml).
N a C l ( % w / v ) o f P i k o v s k a y a ' s M e d i u m
0
.5
1
.0
1
.5
2
.0
2
.5
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
1 8 0 0
2 0 0 0
2 2 0 0
Chart -7: Effect of salinity on α-amylase production of Brevibacillus borstelensis R1
4. DISCUSSION
The present work was carried out in one medium selected
from ten media to optimize the physical and chemical
parameters. The highest α-amylase production was found in
Pikovskaya’s Medium (PK) and the lowest in Nutrient broth
and Luria bertain broth. The highest α-amylase production
(1,800U/ml) was found during the stationary phase (18-24hrs).
The production of amylase was increased with the increase in
the level of inoculum at 2% (1813±23U/ml). The highest
amylase activity was recorded at 370
C (2086±71U/ml), pH 7.0
(2083±56U/ml) and at salinity 1.0% (2082±58U/ml) and the
lowest at 40
C, pH 11.0 and at salinity 2.0%.
The amylase production after 72hours of growth (Bacillus
subtilis) was maximum (370U/mg). The effects of incubation
period, pH of the medium and incubation temperature were
optimized. The maximum production of enzyme was obtained
at 30°C and pH 7.0 [13]. The production of the enzyme by
Bacillus sp. was maximum at 10hrs after inoculation [14]. The
enzyme production largely dependent on the type of strain,
composition of medium, cell growth, initial pH and thermo
stability [15, 16].Increased incubation period decreased the α-
amylase production. It might be due the depletion of nutrients,
accumulation of toxic byproducts in the medium, proteolysis
of α-amylase and microorganisms [17]. To obtain maximum
enzyme yield, development of a suitable medium and culture
conditions were obligatory [18].
Raj Devi & Yogeesvaran [19] reported that the maximum
enzyme production obtained after 24hrs of incubation in case
of Bacillus amyloliquefaciens and 48hrs of incubation with
Micrococcus halobius. The production of the enzyme was
maximum at 10hrs after inoculation with Bacillus sp. [20].
Maximum growth was shown at 48hrs and amylase activity at
24hrs [21].
In the literature survey the optimum amylase production was
delineated by several authors in different media. In acidic
conditions (pH 4.0 - 6.5) the amylase production was reported
in Bacillus spp. [22].In neutral conditions (pH 6.5 - 7.5) the
amylase production was reported in Bacillus spp. [23]. In
alkaline conditions (pH 7.5 - 11) the α-amylase production
was reported in Bacillus spp. [24].
The production was highest in Pikovskaya’s Medium at 370
C.
The amylase production in different media was reported in
psychrophilic conditions (Temp. 40
C) in Nocardiopsis sp.
7326 [25] and Arthrobacter psychrolactophilus [26].The
zenith amylase production of mesophilic Bacillus spp.
(Temperature 300
-450
C) was communicated by Nagarajan et
al. [27]..

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The amylase production of Brevibacillus borostelensis R1 was
most favorable in Pikovskaya’s medium with augment of 1%
NaCl. The NaCl % source of metal ion was reported to have a
stirring effect on the production of amylase was disclosed in
Bacillus spp. by Ashabil Aygan [28]. Parallel work with
different NaCl concentrations (0.5-1.5%) was carried out in
Bacillus spp. [29]. But subduced effect was reported in
Bacillus sp. [30].
CONCLUSIONS
The present study was carried out to optimize the α-amylase
production of Brevibacillus borstelensis R1 using ten different
media. Among these ten media, Pikovskaya’s (PK) medium
proved to be optimal for α-amylase production
(1861±17U/ml).
The optimized α-amylase production in PK medium by
submerged fermentation (SmF) was subjected to varying
physical parameters such as 24hrs incubation time, 2%
inoculum size, 370
C, pH 7.0 and 1% NaCl).
ACKNOWLEDGEMENTS
We thank Management of Dr.Lankapalli Bullayya College,
Visakhapatnam for the financial support and facilities
provided to make this work possible.
REFERENCES
[1]. Patel AH (2003) Industrial Microbiology, Pub. by Rajiv
Beri for Macmillan India Ltd., pp 15-35.
[2]. Kar S and Ray RC (2008) Statistical optimization of alpha-
amylase production by Streptomyces erumpens MTCC 7317
cells in calcium alginate beads using response surface
methodology Pol. J. Microbiol. 57: 49-57.
[3]. Smita HP, Mnas RS, Shaktimay K, Ramesh CR and Dider
M (2008) Statistical optimization of α-amylase production
from probiotic Lactobacillus plantarum MTCC1407 in
submerged fermentation. Polish J. Microbiol. 57: 149-155.
[4]. Zareian S, Khajeh K, Ranjbar B, Dabirmanesh B, Ghollasi
M and Mollania N (2010) Purification and characterization of
a novel amylopullulanase that converts pullulan to glucose,
maltose, and maltotriose and starch to glucose and maltose.
Enzyme and Microbial Technology 46: 57-63.
[5]. Mrudula S (2010) Optimization of thermostable
amylopullulanase production in solid state fermentation by
Clostridium thermosulfurogenes SVM17 through Plackett-
Burman and response surface methodological approaches.
Malaysian J. of Microbio. 6: 181-195.
[6]. Wulf Crueger and Anneliese Crueer (2004) A textbook of
Industrial Microbiology, Second edition. Panima Publishing
Corporation, pp.1-121.
[7]. Fogarty MW (1983) Microbial Amylases. In; Microbial
Enzymes and Biotechnology, W.M. Fogarty (9 Ed.), Applied
Science Publishers Ltd., London, UK, 1–92.
[8]. Hughes MN and Poole R K (1991) Metal specification
and microbial growth-the hard (and soft) facts. J. Gen.
Microbiol. 137:725-734.
[9]. Burhan A, Nisa U, Gökhan C, Ömer C, Ashabil A and
Osman G (2003) Enzymatic properties of a novel
thermostable, thermophilic, alkaline and chelator resistant
amylase from an alkaliphilic Bacillus sp. Isolate ANT-6. Proc.
Biochem. 38: 1397-1403.
[10]. Mohammed Kuddus and Roohi (2010) Microbial cold-
active α-amylases: From fundamentals to recent
Developments, Cur. Rese. Techno. and Edu. Topics in Appl.
Microbio. and Microb. Biotechn. A.Mendez-vilas (Ed.)
[11]. Amoozegar MA, Malekzadeh F and Malik KA (2003)
Production of amylase by newly isolated moderate halophile,
Halobacillus sp. Strain MA-2. J. Microbio. Meth. 52: 353-359.
[12]. Miller GL (1959) Use of Dinitro salicylic acid reagent
for determination of reducing sugar. Analy. Chem. 31: 426 -
429.
[13]. Harikrishna Yadav, Nanganurum, Narasimhulu,
Korrapatim Satish and Mutyala (2012) Studies of the
production of α-amylase by Bacillus subtilis IOSR J. of Eng.
2(5): 1053-1055.
[14]. Pushpendra Singh, Paras Gupta, Ravindra Singh and
Rajesh Sharma (2012) Factors affecting α- Amylase
Production on Submerged Fermentation by Bacillus sp. Int. J.
of Pharm. & Life Sci. (IJPLS), 3(12): 2243-2246.
[15]. Haq I, Ashraf H, Rani S and Qadeer M A (2002)
Biosynthesis of alpha amylase by chemically treated mutant of
Bacillus subtilis GCBU-20, Pak. J. Biol. Sci. 2: 73-75.
[16]. Haq I, Shamim N, Ashraf H, Ali S and Qadeer
MA(2005) Effect of surfactants on the biosynthesis of alpha-
amylase by Bacillus subtilis GCBM-25. Pak. J. Bot. 37: 373-
379.
[17]. Yang C and Liu W (2004) Purification and properties of
a maltotriose-producing α-amylase from Thermobifida fusca.
Enz. Microbial. Technol. 35: 254-260.
[18]. Ray RC, Kar S and Nayak S (2008) Extracellular α-
amylase production by Bacillus brevis MTCC 7521. Food
Biotechnol. 22: 234-246.
[19]. Raj Devi PK and Yogeesvaran G (1999) Co-expression
of saccharifying alkaline amylase and pullulanase in
Micrococcus halobius OR-1 isolated from tapioca cultivar
soil, Wo. J.Microbiol. Biotechnol. 15: 223-229.
[20]. Pushpendra Singh, Paras Gupta, Ravindra Singh and
Rajesh Sharma (2012) Factors affecting α- Amylase
Production on Submerged Fermentation by Bacillus sp. Int. J.
of Pharm. & Life Sci. (IJPLS), 3(12): 2243-2246.
[21]. Srividya Shivakumar (2012) Co-production of alkaline
protease and amylase of Bacillus sp. Y in solid state
cultivations. Res. J. Biotech. 7 (2): 32-38.
[22]. Asoodeh A, Chamani J and Lagzian M (2010) A novel
thermostable, acidophilic alpha-amylase from a new
thermophilic Bacillus sp. Ferdowsicous isolated from Ferdows
hot mineral spring in Iran: Purification and biochemical
characterization. Int. J. Biol. Macromol. 46: 289–297.

__________________________________________________________________________________________
[23]. Asgher M, Javaid Asad M, Rahman SU and Legge RL
(2007) A thermostable α-amylase from a moderately
thermophilic Bacillus subtilis strain for starch processing. J.
Food Eng. 79: 950-955.
[24]. Saxena L, lyer BK and Ananthanarayan L (2007) Three
phase-partitioning as a novel method for purification of ragi
(Eleusine coracana) bifunctional amylase /protease inhibitor.
Process Biochem . 42:491-595.
[25]. Siddiqui ZS, Shaukat SS and Zaman AU (2006)
Alleviation of salinity-induced dormancy by growth regulators
in wheat seeds. Turk. J. of Bot. 30: 321–330.
[26]. Michael R, Smith AE and James C Zahnley (2005)
Production of amylase by Arthrobacter psychrolactophilus. J.
Ind. Microbiol. Biotechnol. 32: 277–283.
[27]. Nagarajan M, Deborah Paripuranam T and
Umamaheswari S (2010) Efficient production of Alpha–
amylase from agro residues using Bacillus subtilis. J. Chem.
Pharm. Res. 2:442-448.
[28]. Ashabil Aygan, Burhan Arikan, Hatice Korkmaz, Sadik
Dinçer and Ömer Çolak (2008) Highly thermostable and
alkaline α-amylase from a halotolerant alkaliphilic Bacillus sp.
Ab68, Braz. J. of microb. 39:547-553.
[29]. Bal S, Mishra RR, Rath B, Sahu HK and Thatoi HN
(2009) Characterization and extracellular enzyme activity of
predominant marine Bacillus spp. isolated from sea water of
Orissa Coast, India. Malaysian J. of Microbio. 5: 87-93.
[30]. Muhammad Hamad Ashraf (2004) Thesis Studies on the
Biosynthesis of Alpha Amylase by a mutant strain of Bacillus
sp. 398.
BIOGRAPHIES
Dr. K.Suribabu, Assistant Professor, PG
Department of Microbiology and Research
Centre, Dr.Lankapalli Bullayya PG College,
Andhra Pradesh, India,Visakhapatnam-530
013, ksuribabu_sda@yahoo.com
Dr.T.Lalitha Govardhan, Associate
Professor, PG Department of Microbiology
and Research Centre, Dr.Lankapalli Bullayya
Post-graduate College, Andhra Pradesh,
India,Visakhapatnam-530 013,
drlalithagovardhan@gmail.com
Professor K.P.J Hemalatha, Department
Microbiology, Andhra University, Andhra
Pradesh, India,kpjhemalatha@gmail.com

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