Indian Journal of Biochemistry & Biophysics
Vol. 36, June 1999, pp. 150-157
Characterization of a thermostable a-amylase from a thermophilic Streptomyces
megasporus strain SD 12
Sabita Dey· and Shubha 0 Agarwalt
*Divi sio n of Microbial Sciences, Agharkar Research Institute, G.G . Agarkar Road, Pune 411004, and
tMicrobiology Department, AbasahebGarware College, Pune 411004
Received 9 November 1998; revised 8 March 1999
An extracellular a-amylase (I ,4-a D-glucan glucan hydrolase; EC 3.2. 1.1 ) was isolated from the cell free broth of
Streptomyces megasporus SDI2 grown in. glucose, soluble starch and raw starch. The enzyme was purified 55-fold with a
specific activity of 847.33 U mg" of protein and with a yield of 36% activity. The apparent molecular mass of the enzyme
was 97 kDa, as estimated by SDS-PAGE. The pI of the enzyme was 5.4 and it was stable at a pH range of 5.5 to 8.5 with an
optimum pH 6. The enzyme was stable upto 85°C with a half life of 60 min . With soluble starch as substrate the enzyme
exhibited a Km and kCaJ value of 4.4 mg mi" and 2335 U min" mg" of protein respectively . The major end products of starch
hydrolysis were maltotriose and maltose depending on th e incubation period. The production of the enzyme with
ag ricultural wastes as substrates was 643 to 804 U min" mg" of protein in submerged fermentation whereas solid state
fermentation could produce only 206 U min" mg" of protein .
Thermophilic
microorganIsms
exhibit
highly
thermostable macromolecules ' . An <x-amylase from
Bacillus stearothermophilus, the first thermostable
protein characterized, was found to have a low
molecular weight and an unusual tertiary structure 2 .
Despite the prevalence of starch hydrolyzing enzymes
in actinomycetes, relatively few have been studied in
great detail. Recently a number of <x-amylases have
been reported In actinomycetes, principally In
mesophilic
streptomycetes
with
interesting
properties). Most of the amylases from mesophilic
streptomycetes produced maltose as predominant
product from starch except <x -amylase from
Streptomyces hygroscopicus which could degrade
4
starch to maltotriose and maltose . Similarly other
Streptomyces strains produced mostly extracellular
amylase induced by maltose or malt extract but not by
. 6
7
starch)' . Hyslop et al. reported that Streptomyces
aureofacience produced both extracellular and cell
bound amylases to hydrolyze starch into maltose,
maltotnose and dextrin. Thermostable <x -amylases
were reported from Thermoactinomyces 8 and
Bacillus'J , but an <x -amylase with a high temperature
optima from Streptomy ces is not reported yet. In the
present communication, isolation and characterization
*Corres ponding author
Te l: 35435 7. Fax : 91-020-351542
E. mail : dey.dipan @axccss.ne t.in
of a thermostable <x-amylase from a thermophilic
Streptomyces is discussed.
Materials and Methods
All buffers, enzyme substrates and other reagents,
both inorganic and organic were purchased from the
local market and all standard proteins and critical
reagents purchased from Sigma Chemicals, St. Louis,
USA.
Organism and growth condition
Streptomyces megasporus SDI2, isolated from a
hot spring of Western Maharashtra, was identified by
cell wall chemotaxonomy' O and bacterial identifier!! .
The organism was grown in a synthetic medium, pH
7.2, containing glucose (1 %), peptone (0.5 %), yeast
extract (0.5 %), NaCI (0 .5% ) and CaCI 2 (0 .2%) in a
shaker incubator at 55°C for 18 hr (the stationary
phase). The cells were harvested by centrifugation
[10000 g for 15 min at 4°C] and the cell free·
supernatant was used as the crude extra cellular
enzyme to determine the a-amylase activity.
Agricultural wastes such as com cob, wheat bran and
defatted coconut cake were used as substrates for the
production of the enzyme.
To determine the cell bound <x -amylase, the cell
mass was washed and sonicated in phosphate buffer,
pH 6.0, centrifuged at 15000 g to remove the debris
DEY & AGARW AL; CHARACTERIZATION OF THERMOSTABLE AMYLASE FROM S. MEGASPORUS
151
and the clear supernatant was used as enzyme
solution.
the purified amylase was estimated usmg standard
markers.
En zyme assay
Isoelectric point determination
Analytical isoelectric focussing was performed in
the 10% slab gel system containing 1 ml ampholyte
with a range of 3-10 (Sigma Chemical Co, St. Louis)
and electrophoretically homogenous protein. Proteins
were stained with Coomassie brilliant blue R 250 and
the isoelectric point of the purified amylase was
estimated from its position relative to those of
standard pI markers [amyloglucosidase (3 .6 kDa),
glucose oxidase (4.2 kDa) , ~lactoglobulin (5 .1 kDa) ,
carbonic anhydrase (5.9 kDa), and myoglobin (7.2
kDa) obtained from Sigma Chemical Co.
Amylase acti vity was determined by measuring the
reducing sugar formed by the enzymatic hydrolysis of
soluble starch . The reaction mixture consisted 100 III
o f 1% (w/v) soluble starch in phosphate buffer, pH
6.0 containing 0 .2% CaCh and the mixture was
incubated at 55°C for 30 min after addition of 100 III
enzyme solution. The amount of reducing sugar
generated was quantified by dinitro salicylic acid
l3
method
aga inst control without enzyme. The
production of reducing sugar was linear throughout
the 30 min assay period.
One unit of amylase activity was defined as the
amount of enzyme that liberated 1 Il mole of maltose
per min. Several other substrates were tested under
the same condition s for relati ve enzyme activity.
When maltose was used as substrate, the amount of
glucose liberated was quantified by glucoseperox idase method 14. Protein concentration was
ls
estimated by the method of Lowry et al. with
lysozy me as standard .
Effect ofpH
The reaction mixtures contained various buffers
e.g. succinate buffer (PH 4 to 6), phosphate buffer
(PH 6-7) and Tris-HCI (PH 7.5-12) and I % soluble
starch as substrate . To test the pH stability, the
purified enzyme was diluted 10-fold in the above
buffers and incubated at 55 °C for 1 hr and then the
diluted enzyme was assayed at pH 6 as described
above.
Purificatiun of amylase
The cell free supernatant was concentrated using
ultra filter (Centriplus, Amicon Mr cutoff 10000) to
use as crude amylase preparation. The crude protein
was prec ipitated by 70% saturation of ammonium
sul fa te and kept at 4°C for 4 hr. The precipitate was
collected by centri fugation (12000 g for 15 min at
4°C) and resuspended in 10 ml of 100 rnM phosphate
buffe r pH 6.0. The sample was loaded onto a
Sephadex G2 5 (S igma, USA) column (2 x 18 cm) and
eluted with the same buffer. The desalted sample was
then applied to a column (2 x 18 cm) of DEAE
cellulose and eluted at a flow rate of 0 .5 ml min· 1
usi ng NaC I gradi ent (0 .1 M to I M) . At each step the
amylase acti vity was determined .
Electrophoresis and molecular mass determination
Na tive PAGE I 6 was performed with a slab gel
e lec trophoresis system (Genei) using 10% gel
l7
foll ow ed by post-electrophbretic reacti vation using
starch gel to detec t the amylase acti vity. SDS-PAGE I 8
was carried out after heating the sampl e at ·IOO°C for
5 min, in 0 .1 M T ris-HC l, containing 2.5% SDS and
0.00 I % bromophenol blue. Proteins were stained with
Coomass le blue R250 . The relative molecular mass of
Effect of temperature
The enzyme samples were incubated for I hr at
different temperatures ranging from 20°C to 75 °C in
phosphate buffer, pH 6.0 . The optimum temperature
was also determined by using buffers of different pH
(4-12) in the reaction mixture. The thermal stability of
the enzyme was monitored by exposing the dilute
enzyme solutions in phosphate buffer pH 6 to
different temperatures ranging from 40°C to 90°C
Effects of metals and other chemicals
Enzyme assays were performed in presence of
various metals ( I mM) or other reagents ( 10 mM) and
the chloride salts of all metals were used except lead
suI fate. The residual activi ty was measured after
preincubation of the enzyme in the reacti on buffer
containin g different metals and other compounds
separately at 20°e. The relative activity of the
enzyme was compared with that in buffer containing
substrate onl y. The response of dinitro salicylic acid
reagent to starch was determined in the presence of
each metal or reagent as control s.
Iodine number
Iodine number of S. megasporus <x-amylase was
19
determined by Yoo ' s method by measuring the
152
INDIAN J. BIOCHEM. BIOPIHYS., VOL. 36, JUNE 1999
reduction of blue colour of iodine starch complex at
675 nm.
Kinetic determination
Initial rates of starch hydrolysis was determined at
various substrate concentrations (0.5 mg to 10 mg).
The reaction mixture contained 0.09 Ilg of purified
enzyme mrl . The initial velocity of the reaction was
determined in 0.01 M phosphate buffer. The kinetic
constants Km and Vrna x w~re' estimated by the method
2
of Lineweaver and Burk °:while kcat (kinetic constant)
was determined by the method of Englard and
)1
·
S mger- .
Results
Purification of amy lase
S. mega5porus SO I 2, when grown in medium
containing starch as carbon source, secreted 90% of
the total amylase produced, into the culture medium
while 10% activity remained associated with cell.
However, when grown in medium contammg 1%
cheap agricultural wastes as the carbon source, only
45% enzyme was extraceHular and, 55% of the
amylase activity was cell associated. The cell free
broth from both media could hydrolyze soluble starch
into maltotriose (10 min) and maltose (30 min)
depending upon the incubation time. This could be
detected in the paper chromatogram as faint band so
further quantitative work was not possible. Similar
observations were reported earlier also 22 .23 . The crude
and concentrated culture supernatant contained about
26.4 U of amylase activity rnrl as determined by the
assay method. After ammonium sulfate precipitation.
and desalting the enzyme preparation exhibited 293.5
U mrl and 305 .6 U mrl of amylase activities
respectively. The enzyme was purified to apparent
homogeneity by
a
single
amon
exchange
chromatography on OEAE cellulose. The elution
profile (inset Fig. 1) showed a single peak of amylase
with an activity of 5253.4 U mrl . The protein was
purified to apparent homogeneity as judged by SOS2
3
6
116_
S'2
84-.
S'O
58
~
4·8
~
Crt
0
4·6
-oJ
4·4
4·2
40
36
29
i
2
4
6
8
10
12
II I
Relative Mobility (em)
Fi g. I- Estimation of molecul ar weight ofoc-amylase.from S. megasporus by SDS-PAGE and from relative mobility of the protein along
with marker proteins. (Lane I, molecular weight markers p-galactosidase (116 kDa), fructose-6-pho sphate kinase (84 kDa), serum
albumin (66 kDa), pyruvate kinase (58 kDa) ovalbumin (45 kDa), b etic dehydrogenase (36 kDa). Lane 2, crude a-amylase. Lane 3,
purified a -am ylase. A, oc -amylase; I, 36 kDa; 2, 45 kDa; 3, 58 kDa; 4, 66 kDa; 5, 116 kDa. Inset: Purification of S. megasporus amylase
by DEAE ce ll ulose ion··exchange chromatography. The fraction size was 0.5 ml. The oc-amylase eluted from the column at 0.5 M NaCI
(6-6). en zyme activity: (0-0), protein concentration]
DEY & AGARWAL: CHARACTERIZATION OF THERMOSTABLE AMYLASE FROM S MEGASPORUS
153
Table I-Purification of cx:-amylase from S megasporus SD 12
Fraction
Total activity
(U/I)
Specific activity
.y ield
(mgl I)
(U /mg)
(%)
Purification
(Fold)
172
170
112
108
62
2640
2695
2935
3056
52534
15 .35
15 .85
26 .31
28.32
847.33
100
98 .84
65 .12
62.74
36
I
1.03
1.71
1. 84
55.20
Total protein
Culture broth
Concentrated broth
(N H4 hS04 pptn
SephadexG -25-1 20
DEAE Ce llul ose
2·5
(A)
(B)
.2 ·5
2
M~
o
2'0
)(
1·5
>-
>
~
l'
<{
~--~----.----.----.---.---~--~~ O·5~-r~--.--r~r-.--r-'r-~
5
6
7
pH
8
9
10
11
30 35 1.0 45 50 55 60 65 70 7S
Temp
°c
Fig. 2-Effcc t of p H on S ./ll egasporus cx: -amylase [A , optimum acti vity in 100 mM phosphate buffer pH 6.0; B, stability was measured
in different buffe rs as menti oned in the text]
PAGE . A s ummary of purification is presented in
Table I . This protocol yielded a pure a-amylase with
a speci fic activity of 847 .32 U mg·) of protein .
Overall , the specific activity increased about 55-fold
w ith 36% yiel d of activity and 39% yield of protein.
With com cob , wheat bran and defatted coconut cake
in water, pH 6.0 S. megasporus produced 694.80,
643.94 a nd 804.96 U min-) m g- ) of protein.
Physical properties
The a ppa rent molecular mass of the purified
S. megaspol'us a m y lase was 65 kDa as estimated by
ge l permeation chro:natography (data not shown).
However, the molecul ar ma ss estimated by SDSPAGE was 97 kDa and the gel calibration graph
revea led a lso a s ing le band of purified enzyme in
monom eric form, with a n approximate molecular
m ass of 97 kDa (Fig. I) . The isoelectric poi'nt was
found to b e 5.4 b y PAG E-i soelectric focusing.
Influ ence a/pH
T he influence of pH on enzyme activity and
stabi lity is shown in Fig . 2 A , B. oc -Amylase showed
optimal activity at pH 6 .0 and was reasonably stable
over a broad pH range of 5.5 to 8.5 with a relative
activity of 60 to 70% respectively.
Th ermal stability
The optimum temperature for the enzyme was
60°C but more than 90% of the activity was present at
a temperature rang ing from 55 °C to 70°C (Fig. 3A,
B) . At 85°C the enzyme retained 50% activity after
1hr exposure which showed the stability of the
en zyme at highe r temperatures. The half life of the
enzyme at 70°C was 2 hr and at 80°C it was 112 hr
approximately.
Cataly tic properties
The substrate specifici.ty of the purified oc -amy lase
was studied with starch and other polysaccharides as
substrates. The actual and re lative activities are li sted
in Table 2 . The enzyme did not have any detectabl e
activity on cyclodextrin, pull ulan and dextran . The
enzyme showed significant activity not only towards
starch, but raw starch in com cob, wheat bran and
defatted coconut cake also. The only detectable
product was maltose after an incubation period of 30
154
INDIAN 1. BIOCHEM. BIOPHYS., VOL. 36, JUNE 1999
100 (A)
(8)
-
lOO'......&-e---e-~__..L"\
~
!...7S
~
75
>
~ 50
50
2
5
7
6
8
9
10
1.0
so
pH
60
70
Temp ·c
80
90
Fig. 3--Effect of temperature on the activity, (A); and stability, (IB) of S. megasporus oc-amylase [The optimum temperature was
measured using phosphate buffer pH 6.0 and .the stability was determined at 60°C after preincubation of the enzyme at various
temperatures for I hr in the same buffer]
Table 2-Production of maltose by s. megasporus oc-amylase in
presence of different substrates after 30 min incubation period
Substrate
Glucose, soluble starch
Insoluble starch
Cyclodextrin, pullulan, dextran
Com .c ob powder
Wheat bran
Defatted coconut cake
Defatted soy and mustard cake
Activity
(%)
Actual activity
(U/mg)
100
90
847.32
762.59
82
76
95
694.80
643 .96
804.96
Maltotriose was onl y detected in the paper chromatogram as faint
band when soluble starch was used as substrate with an incubation
period of 10 min
min but when the enzyme was incubated for only 10
min, then traces of maltotriose also appeared. The
apparent Km and k:a! values at 55°C were 4.4 mg mrl
and 2335 U min<' mg of protein<' (equivalent to 2996
mole of reducing sugar.s<1 mole of enzyme<I).
The followin g metal ions, at a final concentration
of 1 mM each had slight inhibitory effect on the
2+ C 1+
enzyme-Mg-7+ , Mn-J + , Ba-1 + , Fe-" +, Zn,
u- except
Ag2+, Hg2' and EDTA, at a concentration of 10 mM,
which exhibited 22%, 15%, and 12% activity
respectively. But Ca 2+ increased the activity by 10%.
2-Mercaptoethanol or dithiothreitol had little effect on
the activ ity but p-chloromercuribenzoate (PCMB I
mM) inhibited nearly 90% activity which could be
reversed by the addition of 10 mM dithiothreitol
(Table 3).
Table 3--Effect of different metals (I mM) and other chemicals
(10 mM) on th e activity of a-amylase of S. megasponls SD 12
Metals/Chemicals
Control
Calcium chloride:
Magnesium chloride
Barium chloride
Zinc chloride
Cupric chloride
Cobalt chioride
Lead sulfate
Silver chloride
Ferrous chloride
Manganese chloride
EDTA
2-Mercaptoethanol
Dithiothreitol
Mercuric chloride
p-Chloromercuri benzoate
(PCMB)
PCMB + dithiothreitol
Activity
(U mg<! of protein)
Relative
activity
847.33
932.06
593.13
508.93
466.03
610.78
296.57
423.67
194.89
525 .34
381.30
127.95
652.44
762 .60
101.68
68 .79
100
110
70
60
55
745.65
88
72
35
50
23
62
45
15
77
90
12
8
For estimation of iodine number (Fig. 4) it was
observed that initially OD increased from 0.15 to 0.3
upto 7.5 min, followed by reduction of OD and within
15 min the OD came down to original value and
within 20 min only less than 16% colour remained.
The increase of blue colour at the initial stage was due
to the reaction of substrate with iodine which was
immediately followed by reduction by the enzyme
activity.
155
DEY & AGARWAL: CHARACTERlZATION OF THERMOSTABLE AMYLASE FROM S MEGASPORUS
Table 4---(:omparison of the properties of oc-amylases from different sources
Organism
Optimum
pH
Temp.
pI
M.W.
kDa
Substrate
End product
Soluble
starch
Soluble and raw
starch
Raw starch and
pullulan
Raw starch and
and pullulan
Soluble
starch
Soluble
starch
Soluble
starch
Glucose, soluble
and raw starch
Glucose, maltotriose
and maltotetrose
Pentose, isomaltose
and isomaltotriose
Maltose and polymers
Thermostabi Iity
-Ca 2+
+CaL+
°c
Therm oactinomyces
7.0
80
Thermoactinomyces
vulgari/!
Clostridium Sp43
4.5
70
5.2
5
60-70
NO
NO
Penicillium eXJlGflsum 44
4.5
60
3.9
63
Streptomyces lim osu/ 5
7.0
35
Streptomyces
aureo!acience/ 6
Streptomyces spraecax 47
5.6
40
6.0
47
6.0
60
Sp4 1
Streptomyces mega5p orus
SDI2
47
40
97
5. 1
0.35
OJ
--00
0· 25
0 ·2
0
0
0.15
0.1
0.05
0
0
5
10
15
20
25
30
35
Minutes
Fig. 4--lodine number of a -amylase
Properties of oc-amylase from S. fnegasporus were
compared with the ava ilable data (Table 4) from other
sources. oc-Amylases from Th ermoaclinomyces and
Clostridium are thermostable but oc-amylases from
Streptomyces are active upto 40°C. The oc-amylase
from S. megasporus is active up to 85°C with a half
life of I hr.
Discussion
oc -Amylase isolated from S. megasporus grown on
so luble sta rch could be purified approximately
Polymers
Maltose
Maltose
Maltose
Maltose and
maltotriose (trace)
+
+
55-fold with a yield of 36% with an apparent purity as
demonstrated by SDS-PAGE and isoelectric focusing.
The pI value was similar to those found in other
24
bacteria • On gel filtration the molecular mass was 66
kDa unless 4 M urea was included in the buffer the
molecular mass was estimated to be 96 kDa. The
enzyme may interact with agarose gel, resulting in
retardation of its mobility and thus, an error could
take place. In presence of 4 M urea, such interaction,
may be neutralized. This type of discrepancy in
molecular mass determined by two methods are
25
reported for several microbial amylases . The pH
optima for oc-amylase activity (PH 5.5-6.5) and
stability (PH 5-8.5) was similar to values reported for
26
27
most bacterial and yeast amylases.
The pH dependence curve reflected ionization of
the residues required in a protonated state, the two
pKs being close, i.e. 5.5 and 6.5. As the normal pH
range of ionizable groups in proteins are known , the
pK range of 5.5 -6.5 of oc-amylase possibly suggests
the presence of histidine imidazole as ionizable group.
The thennostability upto 85°C is reported in enzymes
28
of Thermoactinomycetes . oc-Amylases from other
microbes and yeast are stable upto 50°C (ref. 29).
Calcium may help in stabilization of protein or
refolding into the active form 30·. While comparing the
Km and k ca\ of various amylases variations in the
source of starch , method of preparation as well as the
conditions of enzyme assay should be taken into
consideration . However, the apparent Km of
S. megasp qrus oc-amylase for soluble starch (4.4
INDIAN 1. BIOCHEM. BIOPHYS., VOL. 36, JUNE 1999
156
mg/ml) was within the range of 0.35 to 4.3 mg/ml as
reported 3) for various oc-amylases. The kcat value of
the enzyme obtained from other bacteria (35 U/rng to
403 U/mg for potato starch)32 was less than that of kcat
of S. megasporus oc-amylase even at 55°C. It should
be noted that at higher temperature kcat is lesser than
at lower temperature 33. It was reported 33 that
temperature optima do not reflect the physical
property of an enzyme but rather it is a function of the
assay conditions. Thus kcat value of the enzyme might
not be significantly high as the optimum temperature
for enzyme activity and optimum temperature for
growth of S. megasporus were same. Experiments to
purify and characterize the cell associated amylase
produced by S. megasporus grown on defatted oil
cake are in progress. Thermolabile amylases produce
maltose/ maltotriose 34 but with thermostable enzymes,
polymerization of the end product formed in larger
amount through pansyl transfer to dextrose 35 produce
maltotetrose or maltopentose. Conversion of polymers
to maltose takes place by synthetic steps. The enzyme
from S. megasporus on the other hand produced
maltose as end product depending on the incubation
period. Wako et at. 36 reported an isoenzyme along
with oc-amylase but S. megasporus amylase did not
contain any isoenzyme. It was observed by Teramato
et at. 37 that oc-amylase lost its viability to digest raw
starch in presence of pronase. S. megasporus could
produce traces of protease under the specified
conditions but the digestion of raw starch was not
hampered. On the contrary the proteinase helpe:d in
liberating to starch molecule from the protein part of
the agricultural wastes. The enzyme possibly
remained bound with the raw starch granules38 or
acted synergistically on raw starch by acting on the
peripheral and then penetrating deeper into the
granule for further attack39 as reported in yeast by
40
Laluce and Mattoon .
It
can be
concluded
that thermophilic
S. megasporus SD 12 produces an extracellular
thermostable oc-amylase which can hydrolyze starch,
both soluble and raw, to maltose at 55°C. With
soluble starch 90% of the enzyme was extracellular
but with raw starch 55% remained cell bound. The
enzyme is stable at a broad range of pH and
temperature . The product of starch hydrolysis was
maltose but in early incubation period traces of
maltotriose was noted.
2
3
4
5
ilifu~
6
7
8
9
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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