Appl Microbiol Biotechnol (2003) 61:323–328
DOI 10.1007/s00253-003-1290-y
ORIGINAL PAPER
A. C. M. M. Aquino · J. A. Jorge · H. F. Terenzi ·
M. L. T. M. Polizeli
Studies on a thermostable a-amylase
from the thermophilic fungus Scytalidium thermophilum
Received: 4 November 2002 / Revised: 11 February 2003 / Accepted: 21 February 2003 / Published online: 22 March 2003

Springer-Verlag 2003
Abstract An a-amylase produced by Scytalidium ther-
mophilum was purified using DEAE-cellulose and CM-
cellulose ion exchange chromatography and Sepharose
6B gel filtration. The purified protein migrated as a single
band in 6% PAGE and 7% SDS-PAGE. The estimated
molecular mass was 36 kDa (SDS-PAGE) and 49 kDa
(Sepharose 6B). Optima of pH and temperature were 6.0
and 60C, respectively. In the absence of substrate the
purified a-amylase was stable for 1 h at 50C and had a
half-life of 12 min at 60C, but was fully stable in the
presence of starch. The enzyme was not activated by
several metal ions tested, including Ca2+ (up to 10 mM),
but HgCl2 and CuCl2 inhibited its activity. The a-amylase
produced by S. thermophilum preferentially hydrolyzed
starch, and to a lesser extent amylopectin, maltose,
amylose and glycogen in that order. The products of
starch hydrolysis (up to 6 h of reaction) analyzed by thin
layer chromatography, showed oligosaccharides such as
maltotrioses, maltotetraoses and maltopentaoses. Maltose
and traces of glucose were formed only after 3 h of
reaction. These results confirm the character of the
enzyme studied to be an a-amylase (1,4-a-glucan
glucanohydrolase).
Introduction
The enzymatic degradation of starch involves the syner-
gistic action of endo- and exo-amylases. One of the most
significant technological advances in starch technology is
the conversion of starch to glucose, and the subsequent
A. C. M. M. Aquino · J. A. Jorge · H. F. Terenzi ·
M. L. T. M. Polizeli ())
Departamento de Biologia, Faculdade de Filosofia,
CiÞncias e Letras de Ribeir¼o Preto,
Universidade de S¼o Paulo, Av. Bandeirantes 3900,
14.040-901 Ribeir¼o Preto S¼o Paulo, Brazil
e-mail: polizeli@ffclrp.usp.br
Tel.: +55-16-6023812
Fax: +55-16-6023666
conversion of glucose into fructose by glucose isomerase
to yield high fructose syrups. Glucose from starch is also
used in the manufacture of citric acid, glutamic acid,
lactic acid, lysine, sorbitol, ethyl alcohol and crystalline
glucose (Pazur et al. 1990).
The most important enzymes for industrial and
biotechnological applications are glucoamylase and a-
amylase (EC 3.2.1.1). a-amylase hydrolyses the internal
a-1,4 glycosidic links in amylose and amylopectin at
random, producing a less viscous solution. There are
numerous industrial and biotechnological applications for
this enzyme (Ito et al. 1998; Nagasaka et al. 1998;
Marchal et al. 1999; Murai et al. 1999; Niehaus et al.
1999; Suresh et al. 1999). a-Amylase is largely obtained
from Bacillus sp., but fungal a-amylases are reportedly
more efficient in saccharifying because they produce
more maltodextrins than the Bacillus sp. enzyme, thus
yielding high concentrations of maltose syrup (Brena et
al. 1996).
Some limiting factors are considered critical for
industrial saccharification processes, such as the poor
stability of amylolytic activities, limited yields of glucose
due to the slow hydrolysis of starch a-1,6 bonds,
formation of condensation products (e.g. a-1,6 isomal-
tooligosaccharides) and most importantly the inhibition of
amylolytic activities by glucose, the end product. In such
cases, microbial enzymes that do not have these charac-
teristics are desirable. In a previous report we described
the purification and characterization of a thermostable
glucoamylase produced by the thermophilic fungus
Scytalidium thermophilum (Aquino et al. 2001). The
present investigation reports the purification and bio-
chemical properties of an extracellular a-amylase activity
from the same microorganism.
Materials and methods
Organism and growth conditions
S. thermophilum 15.1 (= CBS 619.91) was a gift of Dr. G.
Straatsma from the Mushroom Experimental Station, The Nether-
324
lands (Straatsma and Samsom 1993). The mould was maintained at
40C, in slants of solid 4% oatmeal baby food (Quaker) medium.
Conidia from 10-day-old cultures were inoculated into 1,000-ml
Erlenmeyer flasks containing 200 ml of the liquid medium
described by Aquino et al. (2001). The cultures were incubated at
40C without agitation and after 7 days the mycelial pads were
harvested by filtration and the filtrates were saved as a source of
crude extracellular a-amylase.
Enzymatic assays and protein determination
a-Amylase activity was determined either by measuring the
reducing sugar using 3'5-dinitrosalicylic acid (DNS) as described
by Miller (1959), or by the decrease in the amount of starch
(Sigma) in the reaction mixture (Jones and Verner 1967). A 20 ml
volume of enzyme was mixed with 200 ml of 1.0% soluble starch
substrate, in 0.1 M sodium acetate buffer, pH 5.5 at 60C. One unit
of amylase activity was defined as the amount of enzyme that
produced 1 mmol of reducing sugar per minute. Protein was
estimated as described by Lowry et al. (1951) using bovine serum
albumin as the standard.
Enzyme purification
All steps were carried out at 4C. The culture filtrate, dialyzed
against 10 mM sodium acetate buffer, pH 5.5, was applied to a
DEAE-cellulose column (212.7 cm) equilibrated with acetate
buffer and eluted with a linear concentration gradient (0–500 mM)
of sodium chloride in the same buffer. Fractions of 10 ml were
collected at a flow rate of 60 ml h1. Fractions with a-amylase
activity were pooled, dialyzed against 10 mM sodium acetate buffer
(pH 5.5), applied to a CM-cellulose column (210 cm) equilibrated
with the same acetate buffer, and eluted with a linear concentration
gradient (0–1 M) of sodium chloride in acetate buffer. Fractions of
5 ml were collected at a flow rate of 60 ml h1. Fractions with a-
amylase activity were concentrated in a desiccator maintained in a
cold room.
The concentrate (2 ml) was applied to a Sepharose CL-6B gel
filtration column (180 cm) equilibrated and eluted with 50 mM
Tris-HCl buffer, pH 7.5, plus 100 mM KCl. Fractions of 1 ml were
collected at a flow rate of 12.8 ml h1. The a-amylase pooled
fractions were utilized for biochemical characterization and
molecular mass (Mr) determination. Protein molecular mass
standards used were: thyroglobulin (669 kDa), apoferritin
(443 kDa), b-amylase (200 kDa), alcohol dehydrogenase
(150 kDa), bovine serum albumin (66 kDa) and carbonic anhydrase
(29 kDa). The void volume (77 ml) was determined with blue
dextran.
Electrophoresis
To determine the homogeneity and Mr of the purified a-amylase,
the protein was submitted to 7% SDS-PAGE (Laemmli 1970) and
stained with silver (Blum et al. 1987). Non-denaturant (6%) PAGE
Table 1 Summary of the purification steps of the extracellular a-
amylase by Scytalidium thermophilum. n.d. Not determined. Due to
interference of glucoamylase activity in the assay, the contribution
Purification steps Protein Activity
(total mg) (total units)
Filtrate 648.9 715.2
DEAE-cellulose 29.9 8.7
CM-cellulose 36.2 5.9
Concentrate 22.3 5.5
Sepharose 6B 1.5 8.4
was carried out as described by Davis (1964), and stained either
with silver or using the iodine method (Bunni et al. 1989).
Characterization of hydrolysis products
Chromatographic analysis of the reaction end products of a-
amylase activity was carried out using TLC. The reaction mixture
was concentrated and applied (10 ml) on silica gel, and two ascents
chromatography was developed using butanol/ethanol/water (5:3:2)
as the solvent system. Spots were developed by spraying the air-
dried plate with H2SO4 and methanol (1:9) containing 0.2% orcinol
and heating at 100C (Fontana et al. 1988).
Results
Purification of a-amylase
The culture filtrate was applied to a DEAE-cellulose
column as described in Materials and methods. Two
fractions with starch hydrolyzing activity, designated
fractions I and II, were obtained (Fig. 1a). Fraction I did
not bind to the resin and exhibited glucoamylase activity
only. This enzymatic fraction has been characterized by
Aquino et al. (2001).
A minor peak, fraction II, which was retained by the
resin, was eluted with 0.21 M NaCl. Active fractions were
pooled, dialyzed against 10 mM sodium acetate buffer
(pH 5.5), and applied to a CM-cellulose column equili-
brated and eluted as described in Materials and methods.
Again, two peaks with amylolytic activity, designated
fractions IIA and IIB, were eluted (Fig. 1b). Fraction IIA,
which did not bind to the resin, was concentrated and
applied to a Sepharose 6B filtration column equilibrated
and eluted with 50 mM Tris-HCl, pH 7.5, plus 100 mM
KCl. One peak with a-amylase activity was obtained
(Fig. 1c) and this sample was used for biochemical
studies.
Fraction IIB was shown to be a glucoamylase with
properties similar to those of the enzyme characterized by
Aquino et al. (2001) (results not shown). The two
glucoamylases (fraction I and fraction IIB) were probably
the same protein. The fact that they eluted separately and
showed different adsorption on DEAE-cellulose might be
explained on the basis of the evolutionary relationships
among the glucoamylases, as proposed by Coutinho and
Reilly (1997). These authors compared the molecular
structure of glucoamylases from Humicola grisea, Asper-
of a-amylase activity in the crude filtrate could not be determined.
Recovery and purification factor values were based on the activity
recovered after DEAE-cellulose chromatography
Specific activity Yield (%) Purification
[units (mg protein)1] factor
1.1 n.d. n.d.
0.3 100 1
0.2 68 0.6
0.2 63 0.7
5.6 96 18.6

Fig. 1a–c Separation of the amylolytic activity. a DEAE-cellulose
chromatography. b CM-cellulose chromatography. c Sepharose 6B
gel filtration. n Absorbance at 280 nm,
amylolytic activity,
/ NaCl gradient. Enzymatic activity was quantified based on the
amount of reducing sugar (dinitrosalicyclic acid method, DNS)
using starch as the substrate
325
Fig. 2 Thin-layer chromatography of the reaction products of
soluble starch (S) hydrolyzed by the purified a-amylase activity.
Hydrolysis times were 0, 0.5, 1, 2, 3 and 6 h. Standards (St) were a
mixture of 1 mg ml1 of glucose (G1), maltose (G2), maltotriose
(G3), maltotetraose (G4) or maltopentaose (G5)
gillus niger and other fungi and suggested that these
enzymes contain a catalytic domain (CD) and a starch-
binding domain (SBD), connected by an O-glycosylated
linker. Considering that partial or total proteolytic exci-
sion of the SBD results in an enzyme able to degrade
solubilized starch, fraction IIB is likely to be a product of
extracellular proteolysis.
Data from a-amylase purification are summarized in
Table 1. This enzyme was purified approximately 19-fold,
with 96% recovery. Because of the interference of
glucoamylase activity in the assay, the contribution of
a-amylase activity in the crude filtrate could not be
determined. Recovery and purification factor values
(Table 1) were based on the activity recovered after
DEAE-cellulose chromatography.
The purified enzyme preferentially hydrolyzed potato
starch, and exhibited a much lower activity (eight- to
tenfold lower) against amylose or glycogen (data not
shown).
326

Fig. 3 Effect of glucose on the hydrolysis of starch by a-amylase.

Incubation was carried out with 1% soluble starch ( ) or 1% starch
plus 1 M glucose (l). The amount of residual starch was measured
by the iodine method

Fig. 4 Electrophoresis profile. A, 7% SDS-PAGE of the purified a-

amylase (0.2 g of protein). B, 6% PAGE of the purified a-amylase
(0.45 g of protein) developed for a-amylase activity using the
iodine stain and C developed for protein using silver nitrate.
Molecular weight markers (arrows 1–6) were: myosin (205,000); b-
galactosidases (116,000); phosphorylase B (97,400); bovine albu-
min (66,000); egg albumin (45,000) and carbonic anhydrase
(29,000) Fig. 5 Influence of the pH (a), temperature (b) and thermal
inactivation (c) on a-amylase activity. The enzyme was assayed at
various pH values in McIlvaine buffers and at several temperatures.
Thermal inactivation was carried out at 50C ( ), 55C (D), 60C
(h) and 60C (l) protected by 1% starch. The a-amylase activity
was estimated by the DNS method using starch as substrate
 327
Table 2 Effect of metal ions on the activity of the a-amylase Influence of metal ions
produced by S. thermophilum
Compound Relative activity (%) a-Amylases are usually inhibited or activated by ions
(Abou-Zeid 1997; Egas et al. 1998; Azevedo et al. 2000).
1 mM 10 mM
Because it has been reported that a-amylases from several
None 100 100 mesophilic organisms require Ca2+ for activity and/or
BaCl2 88.1 82.3 stability (Vihinen and Mntsl 1989), the effect of Ca2+
CaCl2 108.2 91.8
CuCl2 25.4 4.5 and other ions on the catalytic activity of a-amylase from
HgCl2 15.1 0 S. thermophilum was studied. The enzyme was not
MgCl2 106.1 94.1 activated by any of the metal ions tested, but was
MnCl2 83.2 85.5 inhibited by Hg2+ and Cu2+ (Table 2). Similar results were
NaCl 104.1 110.9
NH4Cl 88.1 90.0 reported with a-amylases from Aspergillus oryzae EI 212
ZnCl2 53.7 7.2 (Kundus and Das 1970), Talaromyces emersonii CBS
814.70 (Bunni et al. 1989), Aspergillus awamori KT-11
(Anindyawati et al. 1998) and Scytalidium sp. (Odibo et
Action mechanism al. 1992).

The classification of the S. thermophilum enzymes as a-

amylase or glucoamylase was based on the products of
starch hydrolysis (Fig. 2). After 30 min of reaction, the
products of fraction IA were mainly oligosaccharides, i.e.
maltotrioses, maltotetraoses and maltopentaoses. After 5 h
of reaction, maltose and traces of glucose were also
formed. These results indicated the endo-amylolytic
character of the enzymatic fraction IA, which was
classified as an a-amylase.
Figure 3 illustrates an important property of the
purified a-amylase from S. thermophilum. This enzyme
was assayed with 1% starch in the absence or in the
presence of 1 M glucose. Residual starch was determined
by iodine method. The low sensitivity to glucose inhibi-
tion of the a-amylase activity was very apparent, i.e. the
rate of starch hydrolysis was practically unaffected by the
presence of glucose.
Determination of Mr
The purified protein migrated as a single polypeptide
under 7% SDS-PAGE (Fig. 4a) and 6%PAGE (Fig. 4b,c).
The Mr estimated by SDS-PAGE and Sepharose CL-6B
gel sieving were 39 kDa and 49 kDa, respectively. The
majority of Mr of other fungal a-amylases are in the range
of 30–70 kDa (Vihinen and Mntsl 1989).
Biochemical properties
Studies on the effect of pH and temperature on the
enzyme activity were carried out utilizing McIlvaine
buffers in the range of pH 4–8, and temperature of 30–
70C. Optima of pH and temperature were estimated at
6.0 and 60C, respectively (Fig. 5a, b). In the absence of
starch the purified enzyme was stable for 1 h at 50C, and
gico
it decayed with a half-life of approximately 25 min at
55C, and 12 min at 60C (Fig. 5c). In the presence of
starch (1%), the enzyme activity was fully stable for 1 h at
60C.
Discussion
The present study adds further information about the
amylolytic complex of the thermophilic fungus S. ther-
mophilum. The principal enzymatic components, glu-
coamylase (Aquino et al. 2001) and a-amylase (present
study), exhibit interesting properties. The two enzymes
are highly thermostable in the presence of starch, most
active at 60ºC and practically insensitive to inhibition by
glucose. These properties may provide a very efficient
way to produce glucose from starch, and justify further
studies on the amylolytic complex of S. thermophilum, for
instance, aiming to optimize the fermentation medium.
Enzymes from thermophilic fungi are economically and
technologically attractive (for a review see Maheshwari et
al. 2000). The potential advantage of using thermostable
a-amylases in the starch biotechnology industry seems
quite evident, nevertheless, although all species of
thermophilic fungi produce and secrete this enzyme
(Maheshwari et al. 2000), only the enzyme from Ther-
momyces lanuginosus (Mishra and Maheshwari 1996;
Nguyen et al. 2002) has been characterized in some detail
up to now. Interestingly, different strains of this fungus
produce different forms of amylolytic enzymes. This may
also be true for other fungal species and this fact,
combined with the differences in methodology employed
in different laboratories, make it difficult to compare the
biotechnological potential of different enzymes. For these
reasons, it would not only be desirable for scientists to
share cultures of thermophilic fungi, as suggested by
Maheshwari et al. (2000), but also to establish a minimum
standardized assay protocol to facilitate comparisons of
data from different laboratories.
Acknowledgements This work was supported by grants from
Funda¼o de Amparo  Pesquisa do Estado de S¼o Paulo (FAPESP)
and Conselho de Desenvolvimento Cient fico e Tecnol
(CNPq). H.F.T., J.A.J. and M.L.T.M.P. are Research Fellows of
CNPq. A.C.M.M.A. was the recipient of a CAPES Fellowship. This
work was part of a Master Dissertation submitted by A.C.M.M.A.
to the Departamento de Biologia, FFCLRP, USP. We thank Ricardo
Alarcon and Mauricio de Oliveira for technical assistance.
328
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