Bioresource Technology 97 (2006) 12171224

Enzymatic detergent formulation containing amylase

from Aspergillus niger: A comparative study with commercial
detergent formulations
Sydnei Mitidieri
a

a,b,

, Anne Helene Souza Martinelli a, Augusto Schrank

Marilene Henning Vainstein a,d

a,c

Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, CEP 91501-970, Porto Alegre, RS, Brazil
b
Programa de Ps-Graduao em Biologia Celular e Molecular, CEP 91501-970, Porto Alegre, RS, Brazil
c
Departamento de Biologia Molecular e Biotecnologia, Brazil
d
Departamento de Microbiologia, CEP 90050-170, Porto Alegre, RS, Brazil
Received 13 February 2004; received in revised form 5 March 2005; accepted 6 May 2005
Available online 19 August 2005

Abstract
There is a wide range of biotechnological applications for amylases, including the textile, pharmaceutical, food and laundry
industries. Hydrolytic enzymes are 100% biodegradable and enzymatic detergents can achieve eVective cleaning with lukewarm
water. Microorganisms and culture media were tested for amylase production and the best producer was Aspergillus niger L119
(3.9 U ml1 0.2) in submerged culture and its amylase demonstrated excellent activity at 5055 C and pH 4.0, remaining stable at
53 C for up to 200 h. In order to establish the potential uses of this enzyme in detergents, diVerent formulations were tested using the
A. niger amylase extract. Enzyme activity was compared with three commercial formulations. The detergents are used in hospitals to
clean surgical and endoscopy equipment. The presence of amylase in the formulation is because of its action within hospital drainage
system, whether or not it has any function in cleaning the equipment.
2005 Elsevier Ltd. All rights reserved.
Keywords: Amylase; Aspergillus niger; Enzymatic detergents; Detergent formulation

1. Introduction
The majority of industrial enzymes being used currently belong to the hydrolase group, which utilizes several diVerent natural substrates. Detergent industries are
the primary consumers of enzymes, in terms of both volume and value. For years microorganisms have been the
principal source of many diVerent enzymes, which were
*
Corresponding author. Address: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, CEP 91501-970,
Porto Alegre, RS, Brazil. Tel.: +55 51 3316 7765; fax: +55 51 3316
7309.
E-mail address: sydnei@mitidieri.com.br (S. Mitidieri).

0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.05.022

identiWed after extensive research and currently Wnd

their main uses in industrial applications (Bon, 1995).
Several industries employ microbial amylolytic enzymes
and a growing industrial application for them is the
enzymatic conversion of starch into a variety of sugar
solutions (Bon, 1995; Crueger and Crueger, 1984; Classen, 1996). An expanding area in the application of
enzymes is in improving the performance of enzymatic
liquid detergents. Proteases are the Wrst enzyme class
used in the formulation of enzymatic detergent and amylases are the second type of enzymes employed.
The term amylase encompasses a class of enzymes
that occur in a wide variety of organisms, and refers to
-amylases, -amylases and glucoamylases (GAs), which

1218

S. Mitidieri et al. / Bioresource Technology 97 (2006) 12171224

are capable of hydrolyzing starch and glycogen. The

-amylases (1,4--D-glucanohydrolase, EC 3.2.1.1) are
enzymes that randomly catalyze the endoamylolytic
cleavage of -1,4-glucosidic chains in polysaccharides
with three or more -1,4-glucosidic starch chains or
extracts of similar substrates (amylase and V amylopectin). They act by liberating maltose, oligosaccharides and
limit dextrins. The -amylases (EC 3.2.1.2) are exoenzymes that hydrolyze the nonreducing end of starch and
amylopectin chains and glycogen molecules, hydrolyzing
alternate glucosidic chains and consequently resulting in
maltose. The GAs (EC 3.2.1.3) hydrolyze glucose units
from the nonreducing ends in amylase and amylopectin
in a step-by-step manner (Daz et al., 2003). This is a
class of industrial enzymes that accounts for approximately 25% of the world market (Fogarty and Kelly,
1980; Ikram-Ul et al., 2003).
Many species of microorganism have already been
identiWed as good amylase producers. Studies of amylases from bacteria and fungi are well documented (Dey
et al., 1991; Babu and Satyanarayana, 1995; Murado
et al., 1997; Sidhu et al., 1997; Coronado et al., 2000;
Yuguo et al., 2000; Jin et al., 2001; Stamford et al., 2001).
Filamentous fungi are microorganisms that secrete large
amounts of protein in culture medium. Aspergillus niger
has been described as secreting an -amylase (Ramasesh
et al., 1982) and GAs of a number of diVerent molecular
weights (Saha and Zeikus, 1990) in submerged culture.
The use of amylases in detergent formulations is
problematic since the enzyme must oVer stability and an
optimal level of activity in commercially utilized formulations, in the presence of proteases. The present article
describes the production, puriWcation and partial characterization of an amylase produced by A. niger and
establishes a potential use for the enzyme in experimental detergent formulations.

2. Methods
2.1. Microorganisms and culturing conditions
The microorganisms employed for this study were
Aspergillus niger L119, Aspergillus niger GMSA, Aspergillus awamori L107, Bacillus subtilis L119, Bacillus
subtilis ICBS, Bacillus thuringiensis L119, Bacillus sphaericus FF, Bacillus cereus GMSA and Bacillus cereus FF
from the Culture Collection at the Universidade Federal
do Rio Grande do Sul Biotechnology Center. These
microorganisms are now deposited with the Fundao
Andr Tosello collection in Campinas, Brazil.
In order to select amylase-producing microorganisms,
bacteria and Wlamentous fungi were plated onto ISP9
medium (NH4 0.264%/K2HPO4 0.565%/KH2PO4
0.238%/MgSO4 7H2O 0.1%), containing 1% (w/v) starch
and 2% (w/v) agar (pH 7.0) (Shirling and Gottlieb, 1966).

After incubation at a suitable temperature (30 C or

37 C), amylase production ability was observed,
impregnating plates with iodine vapor and analyzing the
pale rings which indicate starch degradation zones
around the colonies. The ratio between the diameter of
the colony and the total colony diameter plus the degradation halo was deWned as an amylase activity index.
The A. niger spores were produced using a standard
inoculum of fungi spores cultivated for 72 h at 30 C in
100 g of rice/1.3 g of meat peptone/30 ml of distilled
water.
The production medium (APM) was prepared using
industrial residues containing soy protein (0.6%) and
starch (1.24%) at pH 6.0. Cultures were grown in 2 l
Erlenmeyer Xasks (400 ml of medium per Xask) at 30 C
and 150 rpm in a G76 Gyrotory water bath shaker
(New Brunswick ScientiWc: Edison, NJ, USA). Flasks
were inoculated with 106 spores ml1 and incubated for
up to 48 h. The culture Wltrate (CF) was separated from
biomass using Whatman no. 1 Wlter paper and dialyzed
against 50 mM of citrate buVer at pH 6.0. A variety of
starch sources were tested with the cultures: wheat Xour,
maize Xour, rice Xour, potato Xour and cassava Xour. At
12-h intervals samples were removed and analyzed for
production of enzymes, proteins, glucose and reducing
sugars.
2.2. Analytical assays
The reducing sugars in Wltrates were determined using
3,5-dinitrosalicylic reagent (DNS) (Benfeld, 1955).
Filtrates (0.1 ml) were mixed with 1 ml of DNS. After
heating to 100 C for 5 min, 2 ml of water was added
and absorption measured at 550 nm with spectrometer
Ultrospec 2000 (Amersham Biosciences, Piscataway, NJ,
USA). The glucose concentration in the culture Wltrates
was determined using the Glucose Enzi Cor (BIO
DIAGNSTICA, Pinhais, PR, Brazil) test kit. Samples
(20 l) were mixed with 2 ml of reagent. Absorption was
measured at 500 nm and a glucose curve was used as a
standard.
Dextrinization activity was determined using the
iodometric experimental method (Fuwa, 1954) with
modiWcations. The reaction mixture was: 40 l of buVer
acetate 0.5 M pH 6.0, 300 l of soluble starch (1% w/v)
and 10 l of the test sample. Final volume was made up
to 0.6 ml with dH2O. After 15 min incubation at 42 C,
200 l of acetic acid 1 M and 200 l of iodine reagent
(iodine 1% in ethanol/potassium iodide 10% and dH2O
1:3:1) and 4 ml of dH2O were added. Measuring the
color (660 nm) produced by the iodinestarch mixture
indicated the quantity of starch remaining after enzyme
hydrolysis. A dextrinizing unit was deWned as the quantity of enzyme that would hydrolyze 1 mg of starch per
minute at pH 6.0 and 42 C. SpeciWc activity was
expressed as U mg1 of protein. The same procedure was

S. Mitidieri et al. / Bioresource Technology 97 (2006) 12171224

used to determine the amylase activity of the commercial

detergents.
Protein measurements were taken by the Bradford
method (Bradford, 1976), using bovine serum albumin
as the standard. They were also determined with absorption at 280 nm.
The eVect of temperature on enzyme activity was
determined at pH 6.0, in the range of 3080 C. In order
to test thermostability, a sample of the enzyme was incubated at 53 C for 200 h and residual activity was tested
as the experiment progressed. Enzyme activity as a function of pH was analyzed using 100 mM citrate buVer for
pH 3.05.0 and 100 mM phosphate buVer for pH 5.78.0.
The pH was tested in this range (3.08.0) because it oscillates from the range at which the enzyme tested is most
active to the pH of the liquid detergent formulations
used in hospitals (7.58.0). The temperature of 53 C was
chosen as being the temperature used in hospital surgical
instrument washing machines.
2.3. Enzyme inhibition and detergent assays
Glucose and maltose (2, 5 and 10 mM, respectively)
were used to test the allosteric inhibition of the enzyme.
The eVects of several diVerent products that often
appear in detergent formulations were also tested:
3,6,9,12,15-pentaoxaheptadecan-1-ol, 17-nonylphenoxy
(Renex 60), polyoxy-1,2-ethanediyl, alpha-4-nonylphenyl-omega-hydroxy (Renex 95), polyethylene glycol
4000 (ATPEG 4000) and 2-propanol. Three commercial
enzymatic detergents that contain amylase (Enzitec,
Endozyme and Enzitec-plus) were used in the comparative tests against the experimental A. niger amylase
formulation. All of the tests of the enzymatic formulations were performed in triplicate and means and standard deviations were calculated.
The RENEX line is made up of tensoactive agents
obtained by means of reacting nonylphenol with ethene
oxide. Depending on the number of units of ethylene
oxide (the degree of ethoxylation), products are obtained
that oVer diVerent values for HLB (hydrophilelipophile
balance), allowing a product to be chosen for every
application. This diVerence in hydrophilelipophile balance can have an inXuence on the conformational structure of the protein, changing its activity.
2.4. Amylase puriWcation
In order to purify the enzyme, A. niger was incubated
at 29 1 C in a New Brunswick 14 l fermenter containing 9 l of APM medium, stirred at 200 rpm and with airXow of 1 volume per volume per minute (vvm). All of
the puriWcation techniques were performed at 4 C. The
supernatant obtained in 23 h of fermentation was precipitated to 80% of saturation with (NH4)2SO4. The precipitate was collected with a Sorvall RC5C centrifuge

1219

(10,000g, 30 min, 4 C) (Sorvall Instruments, Dupont

Co., Wilmington, Del., USA), dissolved in 50 mM Tris
HCl 1 mM pH 8.0 and dialyzed against the same buVer
for 24 h at 4 C. The dialyzed sample was directly applied
to a Q-Sepharose Pharmacia 2.5 21 cm column equilibrated with 50 mM TrisHCl pH 8.0. The column was
washed exhaustively with the equilibrating buVer at a
Xow rate of 2 ml min1 until all unbound proteins had
been removed. The removed proteins were eluted with a
linear gradient from 0 to 1.0 M NaCl in the same buVer.
Fractions (14 ml) were collected and those that exhibited
amylolytic activity were grouped, dialyzed against
50 mM of sodium acetate buVer pH 5.4. The concentrated material was loaded onto a Sephacryl S-100
column (Pharmacia60 2.6 cm), equilibrated and percolated with acetate buVer at a Xow rate of 0.5 ml min1.
Fractions of 10 ml were collected and analyzed. Total
protein was determined during the puriWcation stages
by spectrophotometry at 280 nm or by the Bradford
method (Bradford, 1976). Polyacrylamide gel electrophoresis (SDS-PAGE 12%) added to sodium dodecyl
sulphate was used to verify protein purity and to estimate the puriWed enzymes molecular mass under denaturing conditions.
2.5. Statistical analysis
Statistical analyses were performed using ANOVA
and the Tukey test for an alpha error of 0.05. The SPSS
8.0 for Windows statistical package was used (Inc.,
Headquarters, Chicago, Illinois, USA).

3. Results and discussion

Amylase production is a function of the strains, the
composition of the media and the cultivation methods
employed (Nigam and Singh, 1995). Of the microorganisms tested for amylase production in this study, the best
results were obtained from A. niger L119 (3.9 U mg1
0.2), A. awamori L107 (4.1 U mg1 0.4) and B. subtilis
L119 (2.2 U mg1 0.5). Other microorganisms, such as
A. niger GMSA, B. subtilis ICBS, B. thuringiensis L119,
B. sphaericus FF, B. census GMSA and B. census FF
demonstrated amylase activity indices of less than
2 U mg1. A. niger L119 was chosen for the subsequent
experiments because it had been shown to be an
excellent producer of amylase and spores and a GRAS
organism.
Curves for A. niger growth and amylase production
against time are given in Fig. 1. The initial Wltrate of the
growth culture exhibited a high level of protein as a
result of the soy protein in the medium. The total protein
secreted reduced as the culture progressed, becoming
more accentuated after 12 h. Notwithstanding, a peak in
protein secretion was observed after 26 h of growth

1220

S. Mitidieri et al. / Bioresource Technology 97 (2006) 12171224

Fig. 1. Aspergillus niger L119 growth and amylase secretion kinetics, (a) total protein in culture supernatant; (b) amylase activity quantiWed by the
Fuwa method; (c) remaining starch in medium; (d) reducing sugars quantiWed by the DNS reagent; (e) glucose produced, quantiWed by the ENZI
COLOR kit. A. niger was cultivated in 400 ml of APM medium for 48 h at 30 C in a rotary shaker at 150 rpm. Values are given as mean standard
deviation in triplicate for each point.

together with a peak in amylase secretion (Fig. 1a and b).

Under the conditions described, amylase production
was greatest in cultures after 26 h of the bioprocess, falling progressively to 40 h (Fig. 1b). The maximum amylase activity observed was 18.9 U ml1 min (after 26 h
of growth) although the highest speciWc activity,
133.4 U mg1, took place after 42 h of culturing. Uguru
et al. (1997) observed higher levels of amylase activity

with A. niger, of 16 U mg1, during the early stationary

phase, after 96 h of growth. In our experiment the starch
concentration at the start of fermentation was 1.24%,
but after 20 h of growth all of the soluble starch had
been hydrolyzed (Fig. 1c) and the greatest concentrations of reducing sugars (Fig. 1d) and glucose (Fig. 1e)
were detected. At the start of the microorganisms
growth a progressive increase in free glucose in the

S. Mitidieri et al. / Bioresource Technology 97 (2006) 12171224

medium was demonstrated, peaking between 20 and 24 h

(6 mg ml1) (Fig. 1e). This suggests that the enzyme synthesis does not suVer from any catabolic repression
induced by the substrate (starch) or by the formation of
the end products (maltose and glucose). Starch and, to a
lesser extent, maltose have been described as being the
best inducers of amylase synthesis (Ray et al., 1996; Pandey et al., 2000). In our experiment the maximum statistically signiWcant amylase activity ( D 0.05) was attained
when all of the soluble starch had been degraded
(Fig. 1c). At this point, the totals of free reducing sugars
and glucose present in the culturing medium were similar, suggesting that the enzyme in question may be a glucoamylase (Fig. 1d and e).
The amylase from A. niger exhibited excellent activity
at 5055 C (Fig. 2a). The enzyme maintained more than
50% activity at 53 C for 200 h (Fig. 2b) and the optimum pH was 4.0 (Fig. 2c). As has been observed with
other thermostable enzymes, the inverse correlation
between thermostability and functional eYciency at
room temperature is to be expected (Khajeh et al., 2001).
The use of amylases in detergents is problematic when
formulations have to meet very rigid demands added to

1221

the need for stability and activity. Furthermore, the

enzymatic activities have to resist surfactants and metal
ion chelating agents. The eVects that selected ions had on
amylase activity is shown in Table 1. When Cu2+, Zn2+
or Fe2+ were included in the reaction mixture, enzyme
activity was drastically reduced. Enzyme activity was
not, however, aVected when Mg2+ or Ca2+ were added to
the reaction. The presence of surfactants, such as
RENEX 60 at 1.5%, caused an increase in enzyme
activity, however, this eVect was not observed when
RENEX 95 was added (Table 1). Neither ATPEG nor
2-propanol aVected enzyme activity (up to 10% and 5%,
respectively, Table 1). Glucose and maltose (2, 5 and
10 mM) were used to test for allosteric inhibition of the
amylase, but did not signiWcantly inhibit enzyme activity
(Table 1).
In order to obtain a satisfactory medium for production of industrial enzymes, several starch sources were
tested in shaken cultures. Enzyme production using
starch from cassava, maize, rice or potato was comparable. Using wheat Xour resulted in poorer amylase production (Fig. 3). This could be due to the lower quantity
of starch available in wheat Xour (78% w/w).

Fig. 2. Partial extract of the raw amylase extract secreted by Aspergillus niger L119. EVect of temperature (a); pH (b) and amylase stability (c). Experiments were performed in triplicate in acetate buVer 500 mM, at pH 6.0 and temperature of 42 C. In order to determine optimum pH, citrate buVers
100 mM (pH 3.05.0) and phosphate buVers 100 mM (pH 5.58.5) were used at 42 C. In order to test thermostability, the enzyme extract was incubated at 53 C and aliquots were tested at the temperature intervals shown. All experiments were performed in triplicate. Values given are the mean
for all three experiments. Standard deviations were always less than 10% for each medium.

S. Mitidieri et al. / Bioresource Technology 97 (2006) 12171224

Table 1
The eVects of ions, surfactants, ATPEG 4000, 2-propanol and carbohydrates on the activity of Aspergillus niger amylase
Residual activity (%)a

Assay
Ions (4 mM)
Control
CuSO4
MgSO4
ZnSO4
CaCl2
CaSO4
FeSO4
MgCl2

100.0
3.7
100.2
17.5
87.6
99.9
14.4
97.2

Surfactants
1.5% Renex 60
1.5% Renex 95
1.5% Renex 60 plus 1.5% Renex 95

180.2
101.4
178.7

Glicol polyethylene
1% ATPEG 4000
5% ATPEG 4000
10% ATPEG 4000

98.7
99.3
101.2

2-Propanol
1%
2%
5%

99.3
100.2
96.3

Carbohydrates
2 mM glucose
5 mM glucose
10 mM glucose
2 mM maltose
5 mM maltose
10 mM maltose

98.6
97.6
96.7
97.9
98.7
98.0

a
Results are expressed as percentages of the maximum activity in
the absence of tested substance. Values shown are the mean of three
replicates.

In order to test the potential for using this enzyme in

detergents, an experimental formulation was prepared
with 50 ml of A. niger amylase extract per liter of enzymatic detergent. The activity of this enzyme was compatible with three other commercial formulations against
which it was tested (Table 2).
A summary of the puriWcation steps and also the Wnal
speciWc activity of the puriWed amylase is given in
Table 3. The ammonium sulphate fraction was subjected
to Q-Sepharose, where one peak of amylase activity was
eluted (Fig. 4a). Fractions with amylase activity were
grouped, concentrated, dialyzed and loaded onto a
Sephacryl S-100 gel Wltration column. Homogeneous
amylase eluted with speciWc activity of 6.9 U mg1 of

Amylase activity (U. mL. min-1)

1222

25
a

20
a

15
10
5
0
cassava

corn

wheat flour

rice

potato

Starch fronts

Fig. 3. The eVect of diVerent commercial starch sources on amylase

secretion. Enzyme activity was quantiWed by the modiWed Fuwa
method. Aspergillus niger L119 was cultivated in 125 ml glass bottles
containing 25 ml of medium (soy protein 1% and 1% of each starch
source) for 48 h. Cultures were grown in triplicate. Media followed by
the same letter were not signiWcantly diVerent from each other according to the Tukey test ( D 0.05).
Table 2
Comparison of the amylolytic activity of the experimental formulation
with three commercial detergent formulations
Detergents

U ml1 SD

Enziplus
Enzitec
Endozime
Experimental formulationa

1.91 0.02
1.23 0.04
1.33 0.12
1.42 0.09

a
50 ml l1 of the Aspergillus niger growth medium containing amylase activity. Experiments were performed in triplicate and standard
deviations (SD) are given.

protein (Fig. 4b and Table 3). The apparent molecular

mass of the puriWed amylase was approximately 54 kDa
when determined by gel Wltration and 60 kDa when
assessed by SDS-PAGE (Fig. 5). Other research has
shown that A. niger secretes one -amylase (Ramasesh
et al., 1982) and GAs with variable molecular masses
(Saha and Zeikus, 1990) when cultured. The hydrolytic
action of the puriWed enzyme is shown in Table 4 for
speciWc substrates. Our results suggest that the puriWed
enzyme is a glucoamylase, since it produces single glucose units from the nonreducing ends of amylose and
amylopectin (Daz et al., 2003).
Fungi generally secrete -amylases (dextrinizing
enzymes), although some fungi are capable of secreting
-amylase and -amylase (saccharifying enzymes). Thermomyces lanuginosus produces a glucoamylase in a solid

Table 3
PuriWcation of Aspergillus niger L119 amylase by steps
PuriWcation step

Volume (ml)

Total protein (mg)

Total amylase (U)

SpeciWc amylase
activity (U mg1)

PuriWcation fold factor

Crude extract
Ammonium sulphate
Q-Sepharose
Sephacryl S-100

9000
180
56
373

942
948
1.2
0.7

244.8
101.2
5.8
5.1

260
107
4.8
6.8

1
0.41
18.5
26.2

S. Mitidieri et al. / Bioresource Technology 97 (2006) 12171224

1223

Fig. 4. PuriWcation of Aspergillus niger L119 amylase. (a) Ion-exchange chromatography in Q-Sepharose (2.5 21 cm column). Concentrated sample
was pumped into a column at a Xow rate of 2 ml min1. Fractions of 14 ml were collected and those with amylolytic activity were grouped, concentrated and dialyzed against 50 mM of sodium acetate buVer pH 5.4 loaded to (b) a Sephacryl S-100 column (60 2.6 cm) equilibrated with the same
buVer. Elution was continued with 710 ml of acetate buVer at a Xow rate of 0.5 ml min1. Fractions of 10 ml were collected. Amylolytic activity (- - -);
absorption at 280 nm ().

Table 4
SpeciWcity of the puriWed Aspergillus niger L119 amylase substrate
using DNS, to detect dextrins and -16 ramiWcations and glucose oxidase assay, which detects the glucose produced

Fig. 5. Electrophoretic analysis of puriWed Aspergillus niger L119 amylase in an SDS-PAGE 12% gel. (a) Lane 1: raw extract; lane 2: fraction
36 eluted from Q-Sepharose; (b) grouped fractions from Sephacryl
S-100 and MM, molecular markers (SIGMA 1466 kDa).

stationary culture medium and also in submerged cultures (Nigam and Singh, 1995; Helbert et al., 1996).
Detergents formulated for use in hospitals must be
liquid and are used for washing surgical and endoscopic

Substrate

U (ml min1),
DNS method

U (ml min1), Glucose

oxidase method

Soluble starch
Amylose
Amylopectin

236.8
75.3
182.0

214.3
14.6
117.4

instruments in tanks containing the enzymatic detergent

or in dedicated machines. It is compulsory for all such
formulations to contain amylase, with the objective of
cleaning the hospitals drainage pipes and so the formulation must contain an amylase that resists the formulation of the detergent itself (tensoactive agents, stabilizers
and preservatives), that resists the working temperature
employed to clean instruments, on average 53 C, that
resists the proteolytic attack of the proteases contained
in the formulation and still oVers an optimal level of
activity when Xushed down the drain.
One major problem faced by hospitals is contamination which is often spread by insects in the drains. The
use of microorganisms to keep the drains clean is

1224

S. Mitidieri et al. / Bioresource Technology 97 (2006) 12171224

prohibited in hospital environments and taking advantage of the enzymatic activity of detergents makes the
use of enzymes viable for this purpose.

Acknowledgement
This work was supported by grants and fellowships
from: the Fundao de Amparo Pesquisa do Estado do
Rio Grande do Sul (FAPERGS) and TECFARM Tecnologia Qumica e Farmacutica LTDA. We are grateful to
Dr. Clia Regina Carlini for her critical reading of the
manuscript.
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