Carbohydrate Research 345 (2010) 2348–2353

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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres

Purification and characterization of a thermostable a-amylase produced

by the fungus Paecilomyces variotii
Michele Michelin a, Tony M. Silva a, Vivian M. Benassi b, Simone C. Peixoto-Nogueira b,
Luiz Alberto B. Moraes c, Juliana M. Leão a, João A. Jorge a, Héctor F. Terenzi a,
Maria de Lourdes T. M. Polizeli a,⇑
a
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900,
Monte Alegre, Ribeirão Preto-SP, 14040-901, Brazil
b
Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900,
Monte Alegre, Ribeirão Preto-SP, 14040-901, Brazil
c
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900,
Monte Alegre, Ribeirão Preto-SP, 14040-901, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: An a-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatog-
Received 2 June 2010 raphy, followed by Sephadex G-100 gel filtration and electroelution. The a-amylase showed a molecular
Received in revised form 17 August 2010 mass of 75 kDa (SDS–PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respec-
Accepted 19 August 2010
tively. The enzyme was stable for 1 h at 55 °C, showing a t50 of 53 min at 60 °C. Starch protected the
Available online 25 August 2010
enzyme against thermal inactivation. The a-amylase was more stable in alkaline pH. It was activated
mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The
Keywords:
enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The Km of
Paecilomyces variotii
a-Amylase a-amylase on ReagenÒ and SigmaÒ starches were 4.3 and 6.2 mg/mL, respectively. The products of starch
Purification hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino
Electroelution acid sequence of the enzyme presented similarity to a-amylases from Bacillus sp. These results confirmed
Thermostability that the studied enzyme was an a-amylase ((1?4)-a-glucan glucanohydrolase).
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The a-amylases have the widest range of industrial applica-

Amylases are one of the most important families of enzymes in
biotechnology.1,2 The enzymatic conversion of starch into different
sugars represents an important area of industrial enzyme usage
with great potential for growth, once a variety of industries employ
microbial amylases.3,4 These enzymes have almost completely re-
placed the chemical hydrolysis in the starch processing industry.1,5
The enzymatic hydrolysis of starch involves the synergistic action
of endo-amylases (a-amylases), exo-amylases (b-amylase and
glucoamylase), debranching enzymes (pullulanase), and other
enzymes.6,7 The most important amylases for industrial and biotech-
nological applications are glucoamylases and a-amylases.8 a-Amy-
lase (endo (1?4)-a-D-glucan glucohydrolase: EC 3.2.1.1) randomly
cleaves the internal a-(1?4)-glycosidic linkages, resulting in oligo-
saccharides of varying chain length with an a-configuration and
a-limited dextrins as products, which are further hydrolyzed to
glucose by exo-acting amylases and other amylolytic enzymes.1,5,9
⇑ Corresponding author. Tel.: +55 16 3602 4680; fax: +55 16 3602 4886.
E-mail address: polizeli@ffclrp.usp.br (M.LT.M. Polizeli).
tions,1,2 including brewing, baking, textile, and detergent.1,5,10 In
an earlier report, we described a thermostable glucoamylase pro-
duced by the thermotolerant fungus Paecilomyces variotii.11 The
present investigation reports the purification and biochemical
properties of an a-amylase from the same mold. This enzyme
has interesting characteristics, such as high stability at tempera-
ture and acid-neutral pH. Together, P. variotii a-amylase and gluco-
amylase might act synergistically in industrial processes of starch.
2. Results and discussion
2.1. Purification of a-amylase
The purification scheme is shown in Figure 1. The dialyzed cul-
ture filtrate was applied to a DEAE-cellulose column. A single pool
containing starch-hydrolyzing activity was retained by the resin
and eluted at, approximately, 0.55 M NaCl. After pooling the activ-
ity fractions, the concentrated sample was applied to a Sephadex
G-100 column. Two fractions with amylolytic activity were
obtained (Fractions I and II). Fraction I, containing a-amylase activ-
ity, was submitted to an electroelution procedure and this sample

0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.08.013
 M. Michelin et al. / Carbohydrate Research 345 (2010) 2348–2353 2349

Culture filtrate A B C M kDa

- 205
Dialysis with 10 mM Tris-HCl buffer, pH 7.5
- 116

Filtrate dialyzed - 97.4

- 66
DEAE Cellulose
ion-exchange chromatography
- 45

Amylase fraction

- 29
Dialysis and lyophilization

Sephadex G-100 gel filtration

α-Amylase fraction Glucoamylase fraction
Dialysis and lyophilization
Purified glucoamylase
(Michelin et al., 2008)
Electroelution
Purified α-amylase
Figure 1. Purification protocol of extracellular amylolytic enzymes (a-amylase and
glucoamylase) from P. variotii.
was used for biochemical studies. Fraction II, which showed
glucoamylase activity, has been previously characterized by Mich-
elin et al.11 Data from a-amylase purification are summarized in
Table 1. This enzyme was purified approximately 63-fold, with
8.9% recovery.
2.2. Physicochemical properties of a-amylase from P. variotii
The purification protocol resulted in a pure protein, yielding a
single band on PAGE (Fig. 2A and B) with 75 kDa, as estimated by
SDS–PAGE (Fig. 2C). Most of the microbial a-amylases have molec-
ular masses around 50–60 kDa.5,6 However, Zenin and Park12
reported an a-amylase of Paecilomyces sp. having molecular mass
of 69 kDa; therefore, amylases from Paecilomyces apparently
present higher molecular mass than the majority of a-amylase of
the other microorganisms. The pI of P. variotii a-amylase was 4.5.
This enzyme was a glycoprotein with 23% carbohydrate; for other
a-amylases this represents about 10%.5,6
Figure 2. Electrophoresis analysis of the purified a-amylase: (A) 7% PAGE of the
purified a-amylase developed for protein using silver nitrate; (B) developed for a-
amylase activity using iodine solution; (C) 8% SDS–PAGE of the purified a-amylase
stained by silver. (M) Molecular mass patterns in 8% SDS–PAGE stained-silver:
myosin (205 kDa), b-galactosidase (116 kDa), phosphorylase B (97.4 kDa), bovine
serum albumin (66 kDa), egg albumin (45 kDa), carbonic anhydrase (29 kDa).
2.3. TLC of hydrolysis products
The classification of P. variotii enzyme as a-amylase or glucoam-
ylase was based mainly on the products of starch hydrolysis. After
a reaction period of 0.5–24 h, the a-amylase products formed
against starch were mainly maltose and maltotrioses; traces of
glucose were also formed (Fig. 3). These results indicated the
endo-amylolytic character of the enzyme, which was classified as
an a-amylase.
2.4. Effect of temperature and pH on a-amylase activity and
stability
The optimum temperature of the a-amylase was evaluated by
measuring the amylase activity at different temperatures
(30–70 °C) in 100 mM sodium acetate, pH 4.0, while the enzyme’s
optimum pH was determined by varying the pH of the reaction mix-
tures using McIlvaine buffer (pH 2.5–8.0). The enzyme presented
temperature optimum at 60 °C (Fig. 4A), and the pH optimum was
4.0 (Fig. 4B). a-Amylases from most bacteria and fungi are known
to have pH optima in the acid to neutral range,1,5,13 and this enzyme
does not appear to be exceptional.
The temperature effect over amylase stability was determined
by measuring the residual activity after 5, 15, 30, and 60 min of
preincubation (in the absence of substrate) at temperatures of
50, 55, 60, 65, and 70 °C. The enzyme was very stable at 50 and
55 °C for 1 h, and retained more than 60% activity after 30 min at
60 °C (Fig. 4C). Thus, the a-amylase seemed to be quite resistant
to temperature, a favorable characteristic in industrial processes
of brewing and food industries.10,14

Table 1
Summary of the purification steps of the extracellular a-amylase of P. variotii

Purification steps Volume (ml) Protein (total mg) Activity (total U) Specific activity (U/mg prot) Yield (%) Purification factor (-fold)
Filtrate 410 847.7 8212.3 9.7 100 1
Dialyzed filtrate 420 356.7 10004.0 28.0 121.8 2.9
DEAE-cellulose 70 24.5 1703.1 69.5 20.7 7.2
Sephadex G-100 28 9.3 754.2 81.1 9.2 8.4
Electroelution 31 1.2 735.0 612.5 8.9 63.1
2350 M. Michelin et al. / Carbohydrate Research 345 (2010) 2348–2353
Figure 3. Thin-layer chromatography of the reaction products of soluble starch
hydrolyzed by the purified a-amylase. Hydrolysis times were 0, 0.5, 1, 2, and 24 h.
Standards (St) were a mixture of 1 mg ml1 of glucose (G1), maltose (G2),
maltotriose (G3), maltotetraose (G4), and maltopentaose (G5).
It is known that many factors may contribute to thermostabil-
ity, including the presence of calcium, substrate, and other stabiliz-
ers.5,6 In relation to the thermostability using 1% (w/v) starch in
100 mM sodium acetate buffer (pH 4.0) as protector agent, the en-
zyme was incubated at 60 °C, for a period of 5–120 min. It was ob-
served that starch acted as an agent stabilizer, its protective effect
was observed in periods higher than 40 min, increasing its stability
up to 30% (Fig. 4D). Two nonexcluding explanations may be sug-
gested. One is that when the enzyme is incubated in the presence
of a substrate its denaturation is retarded, because the complex en-
zyme–substrate is more stable than the enzyme alone.11,15 The sec-
ond explanation is that the high concentration of the nonreducing
end of starch would act by protecting the enzyme against thermal
denaturation.11,16 However, the stabilizing effect of the substrate,
in this case, would have a double origin. This is because the starch
generally contains some calcium ion as impurity, therefore when
linked to the substrate, the enzyme conformation would be more
rigid and stable against denaturant conditions. Stabilizer effect of
starch was observed in some microorganisms, such as Scytalidium
thermophilum.8
The effect of pH was also investigated. The enzyme was prein-
cubated at low temperature and different pH for 60 min. After that
the residual enzyme activity was assayed with 200 mM sodium
acetate (pH 4.0) and it was verified that the enzyme was stable
at all pH tested, retaining more than 70% of its original activity in
the range of 5.0–8.0, for 1 h (Fig. 4E). a-Amylases are generally sta-
ble in a wide pH range from 4 to 11;5,6,16–18 however, a-amylases
with stability in a narrow range have also been reported.5
The use of enzymes with optimum activity at low pH values and
good stability at pH 5.0 is very important, considering industrial
applications.10,19 The use of liquefying amylases, active and stable,
around the saccharification pH is attractive for industry, if their use
could avoid or reduce the employment of acid to reduce the pH
from liquefying to saccharifying range, and also simplify the proce-
dures during downstream processing. Further, the use of a-amy-
lases that operate at lower pH values reduces the formation of
some by-products, such as maltulose, which are usually produced
at higher operation pH.10,20 Thus, this enzyme could be tried in
synergism with glucoamylase for the complete hydrolysis of
starch.
2.5. Substrate hydrolysis
The purified enzyme hydrolyzed preferentially starch from
SigmaÒ (100%) and from ReagenÒ (94%), amylose (74%), and amy-
lopectin (65%) and exhibited a much lower activity against maltose
(21%), maltotriose (16%), maltotetraose (24%), maltopentaose
(21%), glycogen (10%), and b-cyclodextrin (8%). Sucrose, trehalose,
and a-cyclodextrin did not serve as substrates for the purified
a-amylase (data not shown). As holds true for the other enzymes,
the substrate specificity of a-amylases varies from microorganism
to microorganism. In general, a-amylases display the highest spec-
ificity toward starch, followed by amylose, amylopectin, cyclodex-
trin, glycogen, and maltotriose.5
2.6. Kinetic studies
The kinetic parameters of purified a-amylase were studied with
its more favorable substrates, which were starch from SigmaÒ and
ReagenÒ, amylose, and amylopectin. The Kcat/Km values obtained
(Table 2) demonstrated that the preferred substrates were starch
from Reagen and Sigma, amylose, and amylopectin, in that order.
2.7. Effects of salts and some other compounds on enzyme
activities
a-Amylases are usually inhibited or activated by ions,21–23 and
it has been reported that a-amylases of various mesophilic organ-
isms require calcium for activity or stability.6 Thus, the effect of
calcium and other composts on the catalytic activity of a-amylase
from P. variotii was studied.
Salts, EDTA (end concentration of 1, 2.5, 5, and 10 mM), and b-
mercaptoethanol (1 and 10 mM) were assayed with previously dia-
lyzed enzyme against distilled water and 1% (w/v) EDTA. Next, the
enzyme was incubated with 1% (w/v) starch (from SigmaÒ) in
100 mM sodium acetate (pH 4.0), at 60 °C. The control for the enzy-
matic assay was carried out in the absence of these compounds.
The enzyme was activated by all of the salts tested, mainly cal-
cium (34%), cobalt (41%), and manganese chlorates (47%) at
2.5 mM concentration (Table 3). However, when the enzyme was
dialyzed against EDTA and tested with these salts in concentration
of 1 mM, the activation of calcium and cobalt chlorates were 65%
and 60%, respectively, and 31% for manganese chlorate (data not
shown). These results suggested that calcium chlorate was bound
to the enzyme before dialysis with EDTA, and this is possible since
the affinity of calcium to a-amylase is stronger than other ions,
being that little amounts of calcium contaminants in reagents
and/or glassworks can present false results. According to some
authors, it is still not clear if calcium can be replaced by other cat-
ions.5,6 However, the results obtained with cobalt chlorate suggest
that this salt can be a strong candidate. On the other hand, a-amy-
lase activity was inhibited by several compounds, mainly by EDTA
and heavy metals, such as aluminum, lead, iron, copper, zinc, mer-
cury, some chlorates, and phosphates. Many cations, especially
heavy metal ions, sulfydryl group reagents, N-bromosuccinimide,
p-hydroxyl mercuribenzoic acid, iodoacetate, BSA, EDTA, and
EGTA, are known to inhibit a-amylases.5
The inhibition produced by EDTA confirmed that a-amylase of
P. variotii is a metalloenzyme, and that it contains at least one cal-
cium ion activating or stabilizing the enzyme. Therefore, one of the
 M. Michelin et al. / Carbohydrate Research 345 (2010) 2348–2353 2351

100 A 100 B
80 80

Activity (%)
Activity (%)

60
60

40
40
20
20
0
0
30 40 50 60 70 2 3 4 5 6 7 8

Temperature ( C) o pH

100 C 100 D

80

Residual Activity (%)

Residual Activity (%)

60
10
40

20

1 0
0 10 20 30 40 50 60 0 20 40 60 80 100 120
Time (min) Time (min)

100 E
Residual Activity (%)

10

1
2 3 4 5 6 7 8
pH

Figure 4. Influence of temperature and pH on the a-amylase activities and stabilities. Optima temperature (A) and pH (B) of reaction. Thermal inactivation (in the absence of
substrate) carried out at 50 °C (4), 55 °C (d), 60 °C (s), 65 °C (j) or 70 °C (h) (C) and at 60 °C in the presence (h) or absence (j) of starch (D). pH stability (E). The a-amylase
activity was estimated by the DNS method using starch as substrate.

Table 2 a-amylases inhibited the enzyme of Aspergillus oryzae EI 21224 and

Kinetic parameters of the a-amylase produced by P. variotii
Schwanniomyces castellii25 in high concentrations.
Substrate Km (mg ml1) Vmax (U/mg) Kcat (s1) Kcat/Km Some heavy metal ions also inhibit the a-amylases from Asper-
Starch (from ReagenÒ) 4.3 (±0.15) 701.7 (±6.75) 42.1 9.8 gillus awamori KT-11,26 Talaromyces emersonii CBS 814.70,27 Scytal-
Starch (from SigmaÒ) 6.2 (±0.32) 854.4 (±8.35) 51.2 8.3 idium sp.,28 S. thermophilum,8 and Thermomyces lanuginosus.29 The
Amylose 7.9 (±0.45) 759.2 (±5.75) 45.6 5.8 chelant EDTA (10 mM) inhibited the activity of a-amylase from T.
Amylopectin 13.1 (±0.55) 757.6 (±7.10) 45.5 3.5 lanuginosus by around 60%, suggesting that calcium is required
The enzyme was incubated with 0–30 mg ml1 soluble starch, amylopectin, or for enzyme activity.30
amylose in 100 mM sodium acetate (pH 4.0), at 60 °C.
2.8. Partial amino acid sequence of the a-amylase
conserved regions of a-amylases probably is responsible for calcium
binding, as suggested by Gupta et al.5 In the presence of calcium, a- Amino acid analysis showed 13 a-amylase residues from P. var-
amylases are much more thermostable and more protected from iotii, that is, HAQTGIENMVGFR. Using a database search (http://
proteolytic degradation.5,6 However, calcium that stabilizes several www.ncbi.nih.gov/BLAST) the segment shows 100% of homology
2352 M. Michelin et al. / Carbohydrate Research 345 (2010) 2348–2353

Table 3 reducing sugar per minute. Protein concentration was measured

Effect of salts, EDTA and b-mercaptoethanol on the activity of the a-amylase according to Lowry et al.33 using bovine serum albumin as a
produced from P. variotii
standard.
Compound Relative activity (%)
1 mM 2.5 mM 5 mM 10 mM 3.4. Enzyme purification
None 100 100 100 100
AlCl3 50.6 24.7 18.8 13.5 All the steps were carried out at 4 °C. The culture filtrate was
BaCl2 123.8 107.1 116.5 115.9
b-Mercaptoethanol 108.2 nd nd 114.4
dialyzed overnight against 10 mM Tris–HCl buffer, pH 7.5, and ap-
CaCl2 110.1 133.9 133.4 110.1 plied to a DEAE-cellulose ion exchange column (20  140 mm)
CoCl2 130.6 141.4 137.6 124.8 previously equilibrated with the same buffer and eluted with
CuCl2 85.9 82.4 71.2 54.7 400 mL of a linear concentration gradient (0–1 M) of sodium chlo-
EDTA 62.4 47.7 40.6 26.5
ride in the same buffer. Fractions were collected and monitored for
FeCl3 76.5 50.0 21.2 13.5
HgCl2 100.0 92.9 78.2 77.7 protein (OD280nm) and for amylase activity. Fractions containing a-
KCl 97.1 88.2 84.7 58.8 amylase activity were pooled, dialyzed against distilled water,
KH2PO4 85.3 70.0 42.9 34.7 lyophilized, and suspended in 2 mL of 100 mM sodium acetate buf-
MgCl2 81.2 75.3 71.8 70.0 fer, pH 5.0, plus 150 mM of sodium chloride. This sample was ap-
MnCl2 138.0 147.0 131.7 115.2
NaCl 68.8 70.6 75.3 102.4
plied to a Sephadex G-100 gel-filtration column (15  510 mm)
NaBr 108.8 92.4 79.4 61.2 equilibrated and eluted with the same buffer. Fractions with a-
NaH2PO4 70.0 63.0 53.5 52.4 amylase activity were pooled, dialyzed against distilled water,
NH4Cl 87.7 78.8 40.0 28.8 lyophilized, and suspended in 1 mL of distilled water in order to
NH4F 117.4 102.3 94.7 78.8
go through an electroelution procedure. The sample was applied
Pb(C2H3O2)2 74.1 52.9 42.9 14.1
ZnCl2 94.7 80.0 75.9 62.4 on the top of 6% polyacrylamide gel and eluted with 50 mM Tris
HCl plus 36 mM glycine buffer, pH 8.9, according to Davis.34 The
nd: not determined.
electrophoresis was performed at 400 V and 10 mA. The a-amylase
The dialyzed enzyme was incubated with 1% (w/v) starch (SigmaÒ) in 100 mM
sodium acetate (pH 4.0), containing salts, EDTA or b-mercaptoethanol at 60 °C.
pooled fractions were utilized for biochemical characterization.
Control was considered to the enzymatic assay carried out in the absence of these
composts.
3.5. Electrophoresis and enzymatic activities in gel

Nondenaturing PAGE (7%) was used to determine homogeneity

with sequences of a-amylases purified from Bacillus sp. This obser-
vation supports the suggestion that the purified enzyme is an a-
amylase. Altogether, these results led us to conclude that this
organism is a good model to study the biochemical basis of ther-
mostable enzymes as well as a rich source of potentially useful
glycosidase.
3. Experimental
3.1. Microorganism
The P. variotii strain used in the present study was isolated in
the laboratory from guava leaves (Psidium guajava) in decomposi-
tion, collected in Pereira Barreto—São Paulo—Brazil. The fungus
was identified by the ‘André Tosello Foundation’ and was main-
tained on slants of PDA medium, grown at 30 °C for seven days
and stored at 4 °C until use.
3.2. Culture conditions
of the enzyme protein according to Davis34 and sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was used
to determine the molecular mass of the purified enzyme under
denaturing conditions using 8% acrylamide gel, as described by
Laemmli.35 The protein was silver stained as described by Blum
et al.36 The molecular mass markers were: myosin (205 kDa), b-
galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum
albumin (66 kDa), egg white albumin (45 kDa), and carbonic anhy-
drase (29 kDa).
To detect the amylase activity in gel, 1% (w/v) starch-polymer-
ized nondenaturing PAGE gels were submerged in 100 mM sodium
acetate buffer (pH 4.0) and incubated at 60 °C for 30 min. The band
with amylase activity was revealed by staining it with iodine solu-
tion (10 mM I2 in 14 mM KI). Isoelectric focusing was carried out
according to O’Farrel et al.37 using Pharmalite (pH 3.0–10.0).
3.6. Determination of carbohydrate content and analysis of
hydrolysis products

A volume of 200 mL of liquid medium described by Rizzatti
et al.31 using 1.5% QuakerÒ oatmeal as carbon source was inocu-
lated with 1  106 spores/mL (final concentration) and incubated
at 30 °C without agitation for six days. After that, the mycelial pads
were removed by filtration, and the filtrate was saved as the source
of crude extracellular amylase.
3.3. Amylase activity and protein content
Total neutral carbohydrate was quantified by the phenol-sulfu-
ric acid method of Dubois et al.38 using D-mannose as a standard.
The purified amylase was incubated with 1% starch in 100 mM so-
dium acetate buffer (pH 4.0) at 60 °C. Samples were removed after
0.5, 1, 2, and 24 h incubation, and hydrolysis was stopped by heat-
ing the samples in boiling water for 3 min. The products were de-
tected by thin-layer chromatography (DC-Alufolien Kieselgel 60,
Merck), as described by Fontana et al.39

Amylase activity was determined by measuring the production
of reducing sugar from starch using 3,5-dinitrosalicylic acid (DNS)
as described by Miller.32 The reaction system consisted of 200 lL of
1.0% soluble starch in 0.1 M sodium acetate buffer, pH 4.0, and
200 lL of enzyme. After a 10 min incubation at 60 °C, aliquots of
100 lL were added into 100 lL of DNS. One unit of amylase activity
was defined as the amount of enzyme that produced 1 lmol of
3.7. Substrate hydrolysis
Starch from SigmaÒ and ReagenÒ, amylose, amylopectin, malt-
ose, maltotriose, maltotetraose, maltopentaose, glycogen, a-cyclo-
dextrin, b-cyclodextrin, sucrose, and trehalose at 1 mg/mL were
dissolved in 100 mM sodium acetate buffer, pH 4.0, and assayed
at 60 °C.
 M. Michelin et al. / Carbohydrate Research 345 (2010) 2348–2353
3.8. Determination of kinetic parameters
The Km and Vmax values for the pure enzyme were determined
by incubating the enzyme with 0–30 mg/mL soluble starch,
amylopectin, and amylose in 100 mM sodium acetate (pH 4.0), at
60 °C. The data obtained were fitted to a standard Michaelis–Men-
ten model. The values were calculated from Hanes plots.40 The
efficiency of the substrate utilization was estimated based on
Vmax/Km.41
3.9. Peptide sequencing by mass spectrometry (ESI-MS/MS)
Coomassie-stained protein bands were excised from the gel, in-
gel digested with trypsin (sequencing grade porcine trypsin, Pro-
mega), according to the University of California, San Francisco
(UCSF) Mass Spectrometry Facility in-gel digestion procedure
(http://donatello.ucsf.edu/ingel.html), and subjected to ESI-MS/
MS. Analysis was performed in a Q-Tof (Micromass) coupled to a
CapLC (Waters) chromatographic system. The tryptic peptides
were purified using a Waters Opti-Pak C18 trap column. The
trapped peptides were eluted using a water/acetonitrile 0.1% (v/
v) formic acid gradient and separated by a 75 mL i.d. capillary col-
umn home-pack with C18 silica. Data were acquired in data-
dependent mode, and multiplied charged ions were subjected to
MS/MS experiments. The MS/MS spectra were processed using
MAXENT 3 (Micromass), and manually sequenced using the PEPSEQ
program (Micromass). The primary sequence was analyzed using
the BLAST database (http://www.ncbi.nih.gov/BLAST).
3.10. Reproducibility
All results are the means of at least three independent
experiments.
Acknowledgments
This work was supported by grants from Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP) and Conselho de Desen-
volvimento Científico e Tecnológico (CNPq). J.A.J. and M.L.T.M.P.
are Research Fellows of CNPq. MM was recipient of CAPES Fellow-
ship. This work was part of a master’s dissertation submitted by
MM to the Departamento de Biologia—FFCLRP—USP. We thank Ri-
cardo Alarcon and Mauricio de Oliveira for their technical
assistance.
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