Folia Microbiol.

39 (5), 392-398 (1994)

Purification and Characterization of a-Amylase

from Aspergillusflavus
S.L. KHO0, A.-A. AMIRUL, M. KAMARUZAMAN, N. NAZALANand M.N. AZIZAN

School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penan~ Malaysia

Received April 7, 1994

ABSTR~C'T. Aspergillus flavus produced approximately 50 U / m L of amylotytic activity when grown in liquid medium with raw
low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one
amylolytie enzyme, identified as an g-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production
of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by
ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sul-
fate -polyaerylamide gel electrophoresis. The purified enzyme had a molar mass of 52-5 + 2.5 kDa with an isoelectrie point at
pH 3-5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum
temperature for the enzyme was 55 ~ and it was stable for 1 h up to 50 ~ The Km and V for gelatinized tapioca starch were
0.5 g/L and 108.67 lamol reducing sugars per mg protein per rain, respectively.

Starch, a major agricultural product, has in recent years gained industrial importance particu-
larly as a raw material in food, beverage, paper and textile industries. However, starch has a great
potential as raw material since glucose which can be derived from starch hydrolysis can be further con-
verted to various value-added products through fermentation.
Among the major starch hydrolyzing enzymes of industrial importance are (i)0c-amylases
(1,4-r 4-glucanohydrolase, EC 3.2.1.1), which hydrolyze cc-1,4 but bypass r linkages,
resulting in the production of maltose and other maltodextrins, and (//) glucoamylases (1,4-~-D-glucan
glucohydrolase, EC 3.2.1.3), which liberate glucose units from the nonreducing ends of starch.
Although the cx-amylase produced by Bacillus subtilis is perhaps one of the most comprehen-
sively studied microbial amylases, an increasing number of amylases from other sources have been iso-
lated and characterized (Melasniemi 1988; Bhella and Altosaar 1984; Campbell 1955).
The present work was undertaken to purify and characterize the ex-amylase produced by
A. flavus grown on raw low-grade tapioca starch as fermentation substrate. It is shown that the extra-
cellular amylolytic activity manifested by the organism is due to the presence of one form of cx-amylase.

MATERIALS AND METHODS

Microorganism. A. flavus LINK was isolated from soil samples obtained from a cassava pro-
cessing factory by plating out suitably diluted soil suspension onto Czapek-Dox agar containing 1 %
raw low-grade cassava starch as the sole carbon source. Amylolytic colonies were identified by the
clearing zones formed after flooding the plates with iodine solution.
Enzyme production. The organism was grown in liquid culture in 100 mL of medium in 500-mL
flasks containing (%, W/V): yeast extract 0.3, KH2PO4 0.1, MgSO4"7H20 0.05, (NH4)2HPO4
1.0 %-N. The pH of the medium was adjusted to 7.5 and sterilized by autoclaving at 121 ~ for 15 min.
Raw low-grade cassava starch, sterilized dry at 160 *C for 2 h, was added to the above mixture to a final
concentration of 15 % (W/V). The flasks were then incubated for 8 d at 30 ~ on a rotary shaker oper-
ating at 3.3 Hz.
Enzyme assay. Enzyme activity was assayed as described by Bernfeld (1955) in 1 mL of reac-
tion mixture containing 20 ~tL of a suitably diluted enzyme preparation, 480 I~L of 0.2 mol/L cit-
rate- phosphate buffer (pH 6.0) and 500 ~tL of 1 % gelatinized tapioca starch. Incubation was carried
out at 55 ~ for 30 rain. The release of reducing groups from starch was measured colorimetrically
using alkaline cupric solutions (Somogyi 1952) with glucose as the calibration standard. Amylase activ-
ity was expressed in nkat (the amount of enzyme liberating one nmol of glucose equivalents from starch
per s).
Purification of a-amylase. Culture supernatant, obtained by centrifuging 6-d-old cultures at
5 000 g for 10 min, was subjected to ammonium sulfate precipitation (50-90 % saturation). Following
dialysis, the redissolved precipitate was applied to a DEAE-Sephadex A50 (Pharmacia, Sweden)
1994 =-AMYLASE FROM A. flavus 393

column (30 x 900 mm) pre-equilibrated with 50 mmol/L phosphate buffer (pH 6.5). The column was
eluted with a linear salt gradient of 0-0.5 mol/L NaCI in the same buffer at a flow rate of 10 mL/h.
The enzyme fractions containing amylolytie activity (27-30) were pooled and concentrated. All the
above procedures were carried out at 4 ~
Analysis. Protein was estimated according to Lowry's method or by measuring the absorbance
at 280 nm. Blue value reduction curve of starch degradation by amylolytic enzymes was plotted as de-
scribed by Fairbairn et al. (1986).
Chromatography. The presence of glucose, maltose, maltotriose and maltotetraose in starch
hydrolyzates was detected by TLC, essentially as described by Shah et al. (1987). The silica gel plates
(0.2 mm thick) were developed in ethyl acetate- acetic acid- methanol- water (12: 3 : 3 : 2; F / F / V / V )
for 75 min. The plates were then dried, sprayed with a solution containing diphenylamine-aniline-
phosphoric acid-ethanol (0.16:0.16:0.85:100; W / V / V / I / ) , and resolved sugar spots were visualized
by incubating the plates at 85 ~ for 10 min.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) was carried out as
described by Laemmli (1970). For the assessment of molar mass, a Pharmacia molar mass kit was used
as the reference standards.
For zymogram staining analysis, native gel electrophoresis and the subsequent activity staining
was done as described by Bunni et al. (1989).
Isoelectric focussing of the enzyme preparation was done as described by Giulian (1984) in
6.5 % polyacrylamide gel containing ampholytes with a pH range of 2.5-5.0 (Pharmalyte; Pharmacia,
Sweden). The pH across the gel was determined with a surface pH probe.

RESULTS

Enzyme production and purification

The growth ofA. flavus was followed by the production of amylolytic enzyme(s), the maximum
titer being approximately 830 nkat/mL after a 12 d growth (Fig. 1). To determine the type of amylolytic

I 6 8 1oo ~ 1 I I
750 !

oIL pH
nkat/mL

500
/
3 $ 6 0 ~ -
2 5
250
i I 4OI~.
20 ~~i

I I I 0 I I l
3 6 9 12 0 10 20 3o t,o
d D,%

Fig. 1. Productionof or-amylase(activityA, nkat/mL) Fig. 2. Action of amylaseson iodine-stainedstarch

in medium containing raw low-grade tapioca starch; (starch degradation D, %; iodine stain S, %); ~ - en-
P - protein content (g/L). zyme from A. flavus, = - A. oryzae =-amylase, 13 -
sweet potato 13-amylase,7 - A. niger amyloglucosi-
dase. The reaction mixture each contained 83 nkat of
enzyme and 1% starch and incubated at pH 6.0 and
37 *C.

enzyme(s) produced, a comparison of the release of reducing sugars versus iodine staining between the
culture extract and commercial preparations of at-amylase (type X-A from Aspergillus oryzae, Sigma),
amyloglucosidase (from Aspergillus niger, Sigma) and 13-amylase (Type 1-B from sweet potato, Sigma)
acting on starch was carried out. The enzyme extract from A. flavus showed a profile similar to that of
the commercial preparation of 0t-amylase from A. oryzae (Fig. 2).
 394 S . L K H O O et a/. Vol. 39

Analysis of the hydrolysis products of the culture extract acting on gelatinized tapioca starch by
TLC showed the presence of a mixture of glucose, maltose and maltotriose (Fig. 3).

GlCl

GIC2.

GIC3'

GtC4 -

C 0 10 20 30 45 75 2h I d
rain

Fig. 3. Chromatographic analysis of tapioca starch digestion by a-amylase; Glcl - glucose, Gle2 - mal-
tose, GIc3 - maltotriose, GIc4 - maltotetraose. The reaction mixture (10 mL) contained 333 nkat en-
zyme and 1% tapioca starch. After incubation at 30 ~ 105JLsampleswere extracted at various time in-
tervals.

The elution profile of the am-

15
monium sulfate precipitated pro-
I I I I I teins (50-90 % saturation) of the
~tkat/mL -4 crude culture extract through
A28c a DEAE-Sephadex A-50 ion-ex-
10 - -3 -0.6 change column (Fig. 4) showed
m01/L
the presence of only one activity
peak (fractions 26-30). This peak
3 2 -0.~; was shown to be electrophoreti-
5 cally homogeneous on S D S -
P A G E and had a molar mass of
1 -0.2
approximately 52.5 _+ 2.5 kDa
(Fig. 5). When electrophoresed
0 0 under nondenaturing conditions
0 8 16 24 32 n 40
and subsequently subjected to
Fig. 4. Elution profile of extracellular e-amylase of A. flavus extract on zymogram-staining procedure, the
DEAE-AS0 IEC; 1 - absorbance at 280 nm (A2ao), 2 - amylolyticactivity preparation showed a single pro-
(ttkat/mL), 3 - NaCI gradient (tool/L); n - fraction number. tein containing amylolytic activity
(Fig. 6). The purification proced-
ure (Table I) yielded a homogeneous preparation of 0t-amylase with a specific activity of 1850 nkat/mg
protein; a purification factor of 13.8 and a yield of 70 %.
1994 a-AMYLASE FROM A. flavus 395

kDa
9 Fig. 5. SDS-PAGE of the
212
extraceilular a-amylase (10 %
170 polyacrylamide gel); lane 1:
kDa low-molar-mass markers (kDa):
116 soya-bean trypsin inhibitor
(213), carbonate dehydratase
97
(31), egg-white ovalbumin (45),
bovine serum albumin (66),
76 phosphorylase b (97); lanes 2
66 and 3: ,,,-amylase preparations
(approx. 25 lag protein); lane 4:
high-molar-mass markers
(kDa): glutamate dehydroge-
53 nase (53), transferrin (76),
45 ~galactosidase (116), macro-
globulin (170), myosin (212).

31

21

1 2 3 4

~- ~ ~" pH 3.5

<9
9 Fig. 6. Zymogram of a-amylase; left: native gel
electrophoresis of a-amylase preparation (appro-
ximately 100 lag protein), r/ght: activity stain of
native gel.
9 Fig. 7. Isoelectric focussing of a-amyl-
ase.
396 S.L. KHOO et al. Vol. 39

Table I. Major steps of purification of ,t-amylase from A. flavus

Total Total Spercific

Step protein activity activity Yield Purification
mg /akat nkat/mg % factor

Supernatant 8890 521 134 100 1

50-90 % (NH4)2SO4 711 414 583 80 4.4
DEAE-Sephadex A-50 98.6 365 1850 70 13.8

Enzyme characteristics
Isoeclectricpoint (pI) of the purified enzyme was found to be 3.5 by PAGE-isoelectric focussing
(Fig. 7).
pH optimum and stability. The 0c-amylase showed optimum activity at pH 6.0 (Fig. 8, left). The
enzyme showed a rather broad stability profile, maintaining 80 % of its maximum activity between pH 6
to 10 with, being most stable at pH 7.0.

I00~- I I I I 'f

B0~

: c

0
i &
2
-- A
/. 6 8 10
I
30
I
/,0
I
50 60 70
pH ~

Fig. 8. Effect of pH (left) and temperature (~ r/g/u) on the activity (A) and stability (S) of g-amylase
(%). pH stability was assessed from the activity remaining after incubating the enzyme for 24 h at the
desired pH. Temperature stability was determined from the activity remaining after incubating the reac-
tion mixture for 1 h at the desired temperature.

Temperature optimum and stability. The cx-amylase displayed optimum activity at 55 ~ (Fig. 8,
right). The enzyme is relatively thermostable with 80 % of the initial activity maintained after incuba-
tion at 50 ~ for 1 h.
Kinetic parameters of the purified enzyme were estimated from Hanes' (s/v vs. s) plot (Fig. 9),
using gelatinized tapioca starch as substrate. The Km and Vvalues obtained were 0.5 g/L and 1108 nkat
reducing sugars per mg protein, respectively.
Effect of metal ions. Among the various metal ions tested, Ca 2+ was found to be the most
effective, enhancing the activity of the 0c-amylase by 90 % (Table II). Most of the other divalent metal
ions tested showed varying degrees of enhancement, heavy metals such as Ag + and Hg 2+ inhibiting en-
zyme activity.
1994 a-AMYLASE FROM A. flavus 397

6
DISCUSSION
sly I I I I
O
I
g/kat The type of amylolytic enzyme(s) was deter-
5 m

mined using the starch-iodine blue value reduc-

4
tion method of Robyt and French (1963) with
commercial preparations of both 0t- and 13-amylas-
3
es and amyloglucosidase as reference. The rapid
! decrease in the blue value with respect to reducing
2 sugars produced indicates that the amylolytic ac-
tivity of A. flavus was due to endo-enzyme(s), act-
1 ing in a similar manner to the reference or-amylase
used. It may be concluded that the major amylo-
I I I I I lytic component was 0c-amylase. This is supported
-1 0 2 /. 6 8 I0 by the production of glucose, maltose, maltotriose
s, g/l_
and maltotetraose as the hydrolysis products of the
Fig. 9. Hanes' plot of or-amylase acting on gelatinized tapi- culture extract acting on various starches.
oca starch. Activity was assayed at various substrate concen- The results of ammonium sulfate precipitation,
trations (s, g/L). IEC and subsequent electrophoretic analyses un-
der both denaturing and non-denaturing condi

tions showed that A.flavus produces

Table II. Effect of metal ions (as salts) on ~-amylase activity
c~-amylase with no other amylolytic
ofA. flavus
components simultanously produced
when cultured under the conditions
described above, confirming the sug- Salt Relative Salt Relative
10 mmol/L activity, % 10 mmol/L activity, %
gestion made earlier. This is in con-
trast to organisms such as Aspergillus
CaCI2 191 ZnCI 124
awamori (Yamasaki et al. 1977) and CuCI2 183 LiCI 104
A. niger (Lineback et al. 1969) which CoCl2 174 None 100
have been reported to produce multi- BaCi2 164 CsCI 93.0
ple types of amylolytic enzymes. MgSO4 147 FeCI2 673
Through the simple purification proce- FeCI 3 143 AgNO 3 29.4
dure described, an electrophoretically MnCl2 129 HgCI2 2.3
homogeneous form of ~t-amylase was
obtained.
The purified enzyme has a molar mass of 52.5 +_ 2.5, which is similar to the values reported for
0t-amylase from A. niger (Arai et al. 1968), .4. awamori (Bhella and Altosaar 1984) and A. oryzae
(Yakubi et al. 1977). The enzyme may be classifed as acid-unstable (Arai et al. 1968; Minoda et al.
1968), with maximum stability manifested at pH 7.0 and optimum activity at pH 6.0 (Fig. 8, left). In this
respect, the or-amylase from A. flavus shows characteristics similar to other 0t-amylases previously
studied, where the optimum pH values generally occur between pH 4.8 to 6.5 and are usually stable in
the pH range of 5.5 to 8.0 (Fogarty and Kelly 1980). The pI of 3.5 obtained for this enzyme also falls
within the range of 3.44-3.75 reported for the acid-stable and acid-unstable forms for the at-amylases
of A. niger, respectively (Arai et al. 1968), and 4.0 for the 0t-amylase from A. oryzae (Yakubi et al.
1977). In addition to the above characteristics, the or-amylase from.4, flavus, similar to most 0t-amylases
studied, is a metallo-enzyme requiring the presence of Ca 2+ ions for maximum activity (Table II). The
enzyme from A. flavus is also relatively thermostable, maintaining 80 % of its original activity when
incubated for 1 h at as high as 50 ~ This characteristic is relatively similar to a-amylases from A. niger
and A. oryzae (Bhella and Altosaar 1984).
It seems that the c~-amylase from A. flavus shows characteristics quite similar to ~-amylases
from other Aspergillus species in its physicochemical properties. However, the relatively high yields,
simple purification procedure, coupled with the high affinity for tapioca starch would make A. flavus
a good source for commercial production of 0c-amylase, particularly for tapioca starch degradation.
;398 S.L. KHOO et al. Vol. 39

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