Process Biochemistry 111 (2021) 102–108

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

An optimization approach to the bioconversion of flour mill waste to

α-amylase enzyme by Aspergillus oryzae
Mauricio Braia a, b, *, Ignacio Cabezudo c, Virginia Lis Barrera a, Pablo Anselmi a,
María-Rocío Meini a, Diana Romanini a
a
Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas
y Farmacéuticas, Universidad Nacional de Rosario (UNR), Rosario, Argentina
b
INGEBIO, Facultad de Química e Ingeniería del Rosario, Pontificia Universidad Católica Argentina (UCA), Rosario, Argentina
c
Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario and Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Rosario, Argentina

A R T I C L E I N F O A B S T R A C T

Keywords: Alpha-amylase is one of the most employed enzymes in the food industry because of its capacity to degrade
agro-industrial waste starch, improving the organoleptic and nutritional properties of food products. Thus, it is very important to
fermentation develop novel industrial production processes for its production. A submerged fermentation process using
experimental design
Aspergillus oryzae was optimised to modulate the most important factors affecting alpha-amylase production.
fractional precipitation
hydrophobic interaction chromatography
Flour mill waste, an abundant worldwide agro-industrial residue, was used as substrate due to its starch-rich
composition. This residue was able to avoid catabolite repression during the fermentation, acting as a slow-
release substrate. The process optimization led to a maximum yield of alpha-amylase production of 14076 ±
2346 U/L. The secretome of the fungi in the tested conditions was analysed by LC-MS, showing that two isoforms
of alpha-amylase (amy-1 and amy-3) were produced. Finally, a two-step process was developed to purify alpha-
amylase, consisting of fractional precipitation using (NH4)2SO4 followed by hydrophobic interaction chroma­
tography. The purification allowed a four-time concentration of alpha-amylase, with an eleven-time purification
factor and 65 % recovery and a reduction of the proteolytic activity to 14%.

1. Introduction
Alpha-amylase (AMY) is a glycoprotein present in most living or­
ganisms, including prokaryotes such as Bacillus species and eukaryotic
organisms, from fungi to plants and mammals [1,2]. This enzyme hy­
drolyses the glycosidic bonds of amylose and amylopectin molecules to
render a mixture of dextrins and fermentable sugars such as D-glucose
[3]. Because of its capacity to degrade starch, AMY is widely used in
several industrial areas to produce enzymatic detergents and bioethanol,
among other products. Moreover, in the food industry, this enzyme is
used to produce fermented foods and beverages such as bread and beer
to improve their organoleptic properties by increasing the fermentable
sugar content [4,5].
The production of AMY using filamentous fungi has been studied
both in solid-state and submerged fermentations using inducing sugars
alone or combined with wheat bran [6,7]. Nevertheless, flour mill waste
(FMW), a starch-rich residue from the production of wheat flour that is
produced in large quantities in China, India, Argentina, USA and other
wheat producers, has scarcely been employed as substrate. As the carbon
source may represent 80% of the cost of the culture medium, employing
agro-industrial wastes as substrates can drastically reduce the produc­
tion costs. In addition, it might act as a slow-release substrate [8],
helping to reduce catabolite repression exerted by simple sugars [9].
Aspergillus oryzae is considered one of the best producers of AMY and
the substances resulting from its culture are considered GRAS (generally
recognized as safe). The AMY from A. oryzae (E.C. 3.2.1.1) is a globular,
monomeric protein belonging to the glycoside hydrolase family 13 [10].
Its molecular weight is 55 kDa and its isoelectric point is around 4.50. Its
optimum temperature may varies from 35 ◦ C to 55 ◦ C (depending on the
strain) and the range of pH of stability is between 5.00 and 8.00 [11].
We have previously tested the application of combinations of soy­
bean husk and FMW as substrate for solid-state fermentation production
of α-amylase by A. oryzae [12]. Although being a promising strategy, the
number of factors to be controlled increase exponentially when dealing

* Corresponding author at: Faculty of Chemistry and Engineering. Catholic University of Argentina, Av. Pellegrini 3314 (S2002QEO), Rosario, Argentina.
E-mail address: braia@conicet-rosario.gov.ar (M. Braia).

https://doi.org/10.1016/j.procbio.2021.07.019
Received 30 January 2021; Received in revised form 22 June 2021; Accepted 24 July 2021
Available online 26 July 2021
1359-5113/© 2021 Published by Elsevier Ltd.
M. Braia et al.
with its optimization for industrial application in solid state, compared
to submerged fermentation [13].
In the present study we use flour mill waste (FMW) as substrate for
submerged fermentation of A. oryzae, a system that has less factors and is
easier handling for optimization purposes. To the best of our knowledge,
there are no studies on submerged fermentation using flour mill waste
(FMW) as substrate. We aimed to develop a process to produce and
purify AMY based on submerged fermentation of A. oryzae using FMW as
a low-cost and slow-release substrate. For purification, we propose to
use fractional precipitation and hydrophobic interaction chromatog­
raphy, taking advantage of the high superficial hydrophobicity of AMY
[14].
2. MATERIALS AND METHODS
2.1. Microorganism, inoculum preparation and substrate
Aspergillus oryzae NRRL695 was used to produce AMY in submerged
fermentation, the strain was purchased from the Agricultural Research
Service Culture Collection. The inoculum was prepared by culturing
A. oryzae in papa dextrose agar at 30 ◦ C for 7 days. Then, the culture was
harvested by adding 10 mL of 20% (w/w) glycerol solution and
magnetically stirred for 30 min. Finally, 1 mL aliquots of the spore
suspension were stocked at − 20 ◦ C until using. The concentration of
spores in the inoculum was measured using a Neubauer counting
chamber.
Flour mill waste (FMW) was kindly donated by Molinos Río de la
Plata S.A., Argentina`s largest branded food products company, that
owns 188,000 acres of prime land to produce wheat. FMW was ground
with a laboratory mill and sieved using a laboratory test sieve with a
mesh size of 840 μm. Then, the FMW was fractioned and autoclaved at
121 ◦ C for 25 min.
2.2. Statistical experimental design for the screening and optimisation of
AMY production
In the screening phase, a design of experiments with half-fractional
factorial design was prepared to determine the level of significance of
six factors on two responses, consisting in 32 total cultures [15]. The
following factors were evaluated in two levels: FMW/water ratio (1-10
g/L), KH2PO4 (2-10 g/L), KNO3 (5-10 g/L), incubation temperature
(25-45 ◦ C), incubation time (2-5 days), and inoculum size (103-106
conidia/mL). The responses were AMY concentration and protease
concentration. To assess their significance, the p-value of each factor
was compared to an α value = 0.05. Design matrix and experimental
results for AMY and protease as responses are shown in Results.
In the optimisation phase, response surface methodology (RSM) was
used to optimise the culture conditions for AMY production. A
Box–Behnken design was prepared using the following factors: FMW/
water ratio, incubation time, temperature, and inoculum size. This
design consisted of 27 cultures, including three central points. The
higher limit of the incubation time factor was increased to 8 days, and
the temperature interval was set to 20–40 ◦ C. The non-significant fac­
tors, KH2PO4 y KNO3 were fixed at 6 g/L, and 7.5 g/L respectively.
Design matrix and experimental results for AMY and protease concen­
trations as the response variables are shown in Results. The experi­
mental data was subjected to a full quadratic regression fitting,
including a stepwise method which removes the least significant term
for each step. The exploration of the optimal region was done by
applying the individual desirability function method to maximize AMY
production.
The model obtained was validated by comparison of the experi­
mental results with the predicted values for optimised experimental
conditions. Minitab 17 software (Minitab Inc.) was used for all experi­
mental design purposes [16].
103
Process Biochemistry 111 (2021) 102–108
2.3. Analysis of protein samples by SDS-PAGE and LC-MS
The purity of AMY at each purification step was analysed by SDS-
PAGE, using 10% stacking gel and 13% resolving gel. After running,
the gel was stained with colloidal Coomasie Brillant Blue G250 (Sigma
Aldrich) [17]. The 1X working solution is prepared by diluting the
Coomasie Brillant Blue G250 concentrate in 800 mL of deionized water.
Prior to staining, 4 parts of methanol must be combined with one part of
1X working solution.
LC-MS was performed to identify all the proteins that A. oryzae was
capable to secrete in the optimised culture conditions. Protein sample
spots were cut out from the gel and treated with 20 mM DTT and 20 mM
iodoacetamide prior tryptic digestion. LC-MS analysis was performed
using the Thermo Scientific Q ExactiveTM Hybrid Quadrupole-Orbi­
trapTM Mass Spectrometer. The software Proteome Discoverer 2.1 was
used to analyze the LC-MS spectra and the genome of Aspergillus oryzae
42149/RIB40 (Yellow koji mold) was used as database to identify the
proteins in the filtered broth. The analysis of all the samples was per­
formed at the “Centro de Estudios Químicos y Biológicos por Espec­
trometría de Masa” (CEQUIBIEM) in Buenos Aires, Argentina.
2.4. Purification of AMY by fractional precipitation with (NH4)2SO4
After submerged fermentation of A. oryzae, the broth was filtered to
recover the AMY in the liquid media and to test fractional precipitation
as a first step of a purification protocol. A solution of 2.5 g/L commercial
AMY (Sigma-Aldrich) was used to study appropriate conditions of
fractional precipitation, such as saturation, pH and incubation time.
Then, these conditions were applied to the precipitation of AMY in the
culture filtrate. Solid (NH4)2SO4 or 4 M solution was added to 10 mL of
the filtrate at pH 4.50 and 4 ◦ C to reach the desire saturation (20, 40, 60,
80 % (w/v)). The system was magnetically stirred at low speed and the
pH was controlled and corrected to 4.50 (the isoelectric point of AMY)
with 4 N NaOH if necessary. After complete dissolution of (NH4)2SO4,
the system was centrifuged at 9500 x g for 10 min at 4 ◦ C. Then, the
precipitate containing AMY was dissolved with 50 mM potassium
phosphate buffer at pH 6.00 and the supernatant pH was risen to 6.00
with 4 N NaOH. The catalytic activity of AMY and the concentration of
total proteins were measured in both supernatant and precipitate phases
to determine the yield and the purification factors of each step. Finally,
the best conditions to purify AMY were applied to 160 mL filtered broth
with a catalytic activity of AMY of 10557 U/L. After centrifugation, the
precipitate was re-dissolved with 10 mL of 50 mM potassium phosphate
buffer pH 6.00. This solution was used for further purification using
hydrophobic interaction chromatography (HIC).
2.5. Purification of AMY by HIC
Experiments were performed using an AKTA prime plus FPLC system
with a 1 mL HiTrap™ phenyl HP column (0.7 x 2.5 cm), at room tem­
perature. Separation of AMY was performed by adding 500 μL of the
redissolved precipitate to the column and elution was performed by
linear gradient elution of (NH4)2SO4 using 50 mM potassium phosphate
buffer pH 6.00 at a flow rate of 1 mL/min. Fractions of 500 μL were
collected every 1 minute, absorbance at 280 nm and catalytic activity of
AMY in each fraction collected was measured.
2.6. Evaluation of the thermal stability of purified AMY
After purification of AMY from the culture broth, its thermal stability
was evaluated. Different solutions of the purified enzyme were prepared
by diluting it in 50 mM potassium phosphate buffer pH 6.00 and incu­
bated at 30, 40, 50, 60 and 70 ◦ C for 60 minutes. Then, the catalytic
activity of AMY was measured and plotted against temperature to
determine the range of temperatures where the enzyme is stable.
M. Braia et al.
2.7. Purification table
All the results of the purification steps were summarized in a puri­
fication table that includes all the data needed to calculate the yield (Y
%) and the purification factor (PF) of AMY after each step, as shown in
equations 1 and 2.
total catalytic activity of AMY (units) at each step
Y(%) = x 100 (1)
total catalytic activity of AMY initially
specific activity of AMY at each step
PF = (2)
specific activity of AMY initially
2.8. Quantification of the catalytic activity of AMY
The catalytic activity of AMY was determined using the chromogenic
substrate 2-chloro-p-nitrophenyl-α-D-maltotrioside (CNP-G3). The
enzyme AMY specifically hydrolyses this substrate rendering 2-chloro-p-
nitrophenol (CNP), a compound with a maximum absorption wave­
length of 405 nm and a molar extinction coefficient of 12.9 mM-1 cm-1.
AMY and CNP-G3 (0.23 mM) were mixed in 50 mM potassium phos­
phate buffer pH 6.00. The absorbance at 405 nm was monitored for 180
seconds and plotted against time. The experimental data was fitted to a
simple linear regression model and the slope (m) of the fitted line was
used to calculate the units of activity of AMY as shown in the equation 3.
One unit of activity was defined as the amount of AMY needed to pro­
duce 1 μmol of CNP in 1 minute at 37 ◦ C.
( )
U total volume
catalytic activity = m x 77, 52 x (3)
L sample volume
A value of 1 U of this method correspond to 1173 U of the traditional
method that employs starch as substrate and the 3,5-dinitrosalicylic acid
(DNS) for determination of reducing sugar [18], where one enzyme unit
is defined as the amount of enzyme producing 1 μmol of reducing sugar
per min in the described conditions [19]. All of the AMY values obtained
were expressed as in the Miller method.
2.9. Quantification of the concentration of protease and total proteins
The proteolytic activity was measured using azocasein as substrate
[20]. AMY sample and 50 mM potassium phosphate buffer pH 6.00 were
mixed and incubated for 5 min at 37 ◦ C before adding azocasein to a
final concentration of 1 % (w/v) and incubating the mixture for addi­
tional 30 min. Then, 10 % (w/v) TCA was added; the sample was
incubated for 10 min and centrifuged at 9500 x g for 5 min. Finally, the
supernatant was mixed with 4 N NaOH and absorbance was measured at
440 nm. A calibration curve was prepared using trypsin solutions of
known concentration. The concentration of protease in the samples was
expressed as g/L trypsin equivalents.
Total protein content was measured by the bicinchoninic acid (BCA)
method [21]. The AMY sample was mixed with BCA and incubated for
30 min at 37 ◦ C. Then, absorbance at 562 nm was measured and used to
calculate the concentration of total proteins, using a calibration curve
made with AMY solutions of known concentration.
3. RESULTS AND DISCUSSION
3.1. Screening of the factors involved in the production of AMY by
A. oryzae using submerged fermentation on FMW
To develop an AMY production process from the broths of A. oryzae
in FMW-based cultures, the experimental factor conditions were
screened. An experimental design allowed for the analysis of indepen­
dent factors and their interactions. The initial values for the factors in­
tervals were based on fermentations using other substrates as carbon
source [22,23]. In the screening phase, the aim was to maximise AMY
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Process Biochemistry 111 (2021) 102–108
production and minimize protease production, since proteases could
degrade and destabilize AMY. For screening purposes, a half-fractional
factorial design was chosen, consisting of 32 experiments. It was a
representative subset of the full factorial design, with a resolution of the
same order. The design included the following six factors: FMW (1-10
g/L), KH2PO4 (2-10 g/L), KNO3 (5-10 g/L), temperature (25-45 ◦ C), time
(2-5 days) and inoculum concentration (103-106 conidia/mL). Table 1
shows the design of experiments and the results obtained for AMY and
protease catalytic activities.
A factorial regression was performed to assess the significance of the
factors in the responses. Fig. 1 shows the normal probability plots
indicating the influence of the factors on the production of AMY and
proteases. We found that time, FMW, and temperature significantly
affected the production of AMY. The carbon source was reported as one
of the most influential variables to produce AMY employing starch or
maltose as inducers but usually a repression is observed at high con­
centration of the inducer, because of catabolite repression [24]. At all
the FMW/water ratios tested, a positive effect in AMY production was
observed. FMW is a rich source of starch, but in contrast to the com­
mercial isolated starch, the complexity provided by the FMW structure
might lead to a slow release of the monomeric/oligomeric carbohy­
drates, preventing the catabolite repression effect.
The inoculum factor was in the limit of significance, but it was kept
among the factors used in the optimisation phase. Temperature has been
reported as one of the most important factors on AMY production [25].
Nevertheless, a raise in temperature to 45 ◦ C could have resulted lethal
for this strain of fungus, since the biomass production was very low in
cultures at that temperature. Therefore, the higher temperature was
reduced to 40 ◦ C in the optimisation phase.
The concentrations of KH2PO4 and KNO3 were not significant in the
production of AMY in the concentrations evaluated. However, in the
literature the role of the carbon/nitrogen ratio for protein synthesis as
well as the regulatory role of phosphorus in the synthesis of primary and
secondary metabolites in microorganisms was highlighted [22,23,26].
Since FMW can provide nutrients other than carbon, as it is composed of
carbohydrates (60 – 75%), proteins (9.60 - 18.6%), phosphorus (0.9 -
1.5%), and other essential micronutrients and phytochemicals [27].
In general, the conditions that favoured the production of AMY also
favoured the production of protease in the cultures (Table 1). In fact, a
good correlation was found between responses (R2 = 0.8627). Under the
conditions tested, it was not feasible to design a process that favoured
the production of AMY over proteases, therefore we aimed to design an
efficient separation strategy for AMY.
3.2. Optimisation and validation
A Box-Behnken design was used in the optimisation of fermentation
factors, comprising 27 experiments which included three central points.
The factors were studied at three levels and the concentration of KH2PO4
and KNO3, which were determined as not significant factors for AMY
production, were set in the centre of their screening concentrations: 6 g/
L and 7.5 g/L. The factors FMW, time, temperature and inoculum and
the double interactions FMW*FMW, temperature*temperature,
FMW*time and time*inoculum were significant in the production of
AMY (p-value <0.05), validating the conclusions of the screening phase.
The experimental design and the results obtained are detailed in
Table SI.
AMY activity response was modelled considering only those signifi­
cant factors, resulting in a model that was significant (p-value <0.05)
with an R2 = 0.9181, an adjusted R2 = 0.8771 and a prediction R2 =
0.7838, meaning that the model has a fairly good ability to predict the
outcome of a fermentation. The equation 4 is the equation that allows
predicting AMY values at any given conditions within the limits of the
design:
M. Braia et al. Process Biochemistry 111 (2021) 102–108

Table 1
Experimental design and responses values of the screening phase.
Culture KH2PO4 (g/L) FMW (g/L) Time (days) Temperature (ºC) KNO3 (g/L) Inoculum (conidia/mL) AMY (U/L) Protease (g/L)
3
1 10 1 5 45 10 10 9 -
2 2 1 2 25 5 103 10 -
3 10 1 5 25 5 103 847 0.01
4 10 10 5 25 5 106 8509 0.06
5 10 10 2 25 10 106 4324 0.05
6 10 10 5 45 5 103 38 0.01
7 10 10 2 45 10 103 303 -
8 2 10 5 45 10 103 185 0.01
9 2 1 5 25 10 103 526 -
10 2 10 5 45 5 106 843 0
11 2 10 2 25 5 106 3022 0.02
12 10 1 2 45 5 103 126 -
13 2 1 2 45 5 106 172 -
14 2 10 2 25 10 103 1269 0.01
15 2 1 2 45 10 103 366 -
16 10 1 2 25 5 106 462 -
17 10 1 5 45 5 106 198 -
18 2 10 5 25 5 103 1128 0.01
19 10 10 5 25 10 103 8802 0.08
20 2 1 5 25 5 106 1151 -
21 2 10 2 45 5 103 222 0.01
22 2 10 2 45 10 106 330 -
23 2 10 5 25 10 106 7165 0.03
24 2 1 5 45 5 103 48 0.01
25 10 10 2 25 5 103 1852 0.02
26 10 1 2 45 10 106 674 -
27 10 10 2 45 5 106 11 -
28 2 1 2 25 10 106 92 0.01
29 2 1 5 45 10 106 31 0.01
30 10 1 5 25 10 106 1255 -
31 10 10 5 45 10 106 2290 0.02
32 10 1 2 25 10 103 28 -

Fig. 1. The normal probability plots show the importance of the factors (and their interactions) for both A) AMY and B) protease responses.

( )
U This plot shows that the maximum production of AMY occurs around 28
AMY = − 29362 + 432∗FMW + 272∗time + 1905∗T
L ◦
C, which is consistent to the optimum temperature growth of A. oryzae
+ 0.00766∗inoculum − 61.0∗FMW 2 − 33.38∗T 2 [28] and that lower inoculum favours AMY production.
Finally, the model to produce AMY was validated by preparing a
+ 199.6∗FMW∗time − 0.001127∗ time∗inoculum (4)
culture under the optimised conditions in triplicate. Under these con­
According to this equation, the optimal fermentation conditions to ditions, the model predicted a concentration of AMY equal to 13724 U/
maximize AMY production were 10 g/L of FMW, 8 days, 28 ◦ C and 103 L, with a 95% of confidence that the value obtained will be between
conidia of A. oryzae per mL. For an improved visualisation and under­ 11097 and 16352 U/L AMY activities. The model’s ability to predict the
standing on how factors affect each response, contour plots are showed production of AMY was validated: the experimental AMY activity
in Fig. 2. FMW/water ratio showed a marked effect on AMY production average was 14076 ± 2346 U/L, representing 35% of the total protein in
and at any time, the response increased with an increase in FMW/water the broth.
ratio (Fig. 2a). Also, it was observed that an increase in fermentation
time generally produced an increase in the production of the enzyme. In
Fig. 2b, AMY activity was plotted against inoculum and temperature.

105
M. Braia et al. Process Biochemistry 111 (2021) 102–108

3.3. Analysis of protein samples by SDS-PAGE and LC-MS

To identify the most abundant proteins that A. oryzae secreted to the

medium under the proposed optimised conditions, LC-MS experiments
were performed. This enabled us to know all the proteins that might be
produced along with AMY, focusing on proteases. FMW is not only a
starch-rich residue but also a protein-rich residue and so it is very
difficult to evaluate the fermentation broth since many peptides were
released during fermentation. Therefore, in the LC-MS analysis a broth
obtained from a submerged fermentation using a simple carbon source
(glycerol) was prepared using all the other optimized parameters.
Three spots observed in the SDS-PAGE gel were used to identify the
proteins secreted by A. oryzae. A total of 40 proteins were detected and
50% of them were identified, which are showed in Table 2.
The only intracellular protein identified in the secretome was dihy­
drolipoyl dehydrogenase, probably due to secretion via a non-classical
pathway (or less likely, cell lysis). The most abundant enzymes were
two isoforms of AMY (type-1,2 and type-3) followed by glucoamylase.
These confirm the high capacity of A. oryzae for degrading starch, as it
produces complementary amylases of different types. Also, various types
of proteases were found in the secretome: leucine aminopeptidase A,
extracellular metalloproteinase and alkaline protease 1 were highly
abundant. Cell-wall degrading enzymes were also identified in the
secretome, including mannosyl-oligosaccharide alpha-1,2-mannosidase
1B, endo-beta-1,4-glucanase and beta-glucosidase A. Other extracellular
enzymes identified were S-adenosylmethionine synthase, ribonuclease
T2 and catalase B.

3.4. Purification of AMY by fractional precipitation followed by

hydrophobic interaction chromatography

Fig. 2. A) Contour graphs for AMY activity response vs FMW and time, and B)
AMY vs T and inoculum.
Prior to test the recovery of the enzyme of interest from the broth by
fractional precipitation, the technique was assayed using commercial
AMY. At the isoelectric point, pH 4.5, AMY precipitated at 60 and 80 %
(w/v) saturation, and the highest yield was obtained at 80 %(w/v)
saturation. The same conditions were applied to precipitate AMY from
the filtered broth. Since the yield of AMY at 40 % (w/v) was low but the
amount of precipitate was high, fractional precipitation was tested be­
tween 40 and 80 % (w/v) saturation. The results are shown in Table 3a),
including the precipitation of proteases and total protein.
The precipitate at 40 % (w/v) (NH4)2SO4 had low amounts of AMY,

Table 2
Proteins identified by LC-MS in the secretome of A. oryzae
Protein Gen Sum PEP Score Matching Coverage (%) Theoretical pI* Molecular Mass (KDa)
peptides *

Alpha-amylase A type-3 amy3 220.536 32 68 4.52 54.8

Alpha-amylase A type-1/2 amy1 207.716 31 68 4.48 54.8
Glucoamylase glaA 76.558 18 44 4.99 65.5
Leucine aminopeptidase A lapA 63.385 13 39 5.03 41.4
Extracellular metalloproteinase NpI 38.399 10 25 5.07 69.2
Alkaline protease 1 alp1 37.834 8 20 5.95 42.6
Probable leucine aminopeptidase 2 lap2 27.944 9 28 5.03 41.1
Mannosyl-oligosaccharide alpha-1,2-mannosidase mns1B 23.456 7 15 5.00 56.6
1B
Dipeptidase AO090023000428 19.096 6 23 5.49 42.0
Beta-cyclopiazonate dehydrogenase cpaO 16.782 8 29 4.81 51.0
Probable glucan endo-1,3-beta-glucosidase eglC 15.518 4 10 4.72 46.7
Dihydrolipoyl dehydrogenase AO090011000486 14.692 6 17 7.64 54.6
Ribonuclease T2 rntB 11.735 4 18 5.02 30.7
Dipeptidyl-peptidase 5 AO090011000795- 11.317 6 11 4.66 80.3
A
Neutral protease 2 AO090010000493 10.28 3 12 5.13 37.5
Endo-beta-1,4-glucanase celB 8.984 2 7 4.96 44.4
Probable beta-glucosidase A bglA 8.493 4 7 4.86 93.4
S-adenosylmethionine synthase AO090038000357 6.102 3 9 5.59 42.5
Catalase B catB 5.293 2 3 5.29 79.9
Carboxypeptidase AO090012000706 4.471 2 5 4.70 52.6
*
Parameters were determined with the ProtParam tool – ExPASy [30] for the most abundant proteins in the extract.

106
M. Braia et al. Process Biochemistry 111 (2021) 102–108

Table 3
Purification of AMY by the two-step method.
A) Fractional precipitation steps with NH4SO4.

Fraction AMY (%) Protease (%) Total protein (%)

Precipitate 40 % 0.7 ± 0.1 0.8 ± 0.2 5.0 ± 0.4

Supernatant 80 % 21 ± 2 4.8 ± 0.8 83 ± 1
Precipitate 80 % 78 ± 3 91 ± 3 11.0 ± 0.2

B) Purification of AMY by precipitation followed by HIC chromatography

Purification step Volume (mL) Total activity (U) Total protein (mg) Specific activity (U/mg) Purification factor Yield (%)

Culture Broth 160.0 1642 ± 6 110.00 ± 0.01 15 ± 6 1 100

Precipitation 10.0 1279 ± 4.101 11.7 ± 0.2 109 ± 5 7.3 ± 0.3 78 ± 2
HIC 40.0 1067 ± 2.101 6.6 ± 0.5 163 ± 1.101 11 ± 1 65 ± 2

protease and total protein, indicating that the contaminant proteins

were not separated from AMY. Nevertheless, other contaminants such as
cellular debris were separated, allowing the clarification of AMY.
Table 3a) shows that almost all proteases and AMY are in the 80 % (w/v)
(NH4)2SO4 precipitate but the total protein content diminished to 11%.
The physicochemical properties of A. oryzae proteases, such as molec­
ular weight and isoelectric point, are similar to those of AMY (Table 2),
therefore it is very difficult to separate them using precipitation. It is
worth noting that the precipitate could be re-dissolved with 10 mL of 50
mM potassium phosphate buffer pH 6.00, achieving a concentration of
16 times for AMY. Therefore, even if protease was not separated, other
proteins were separated from AMY.
AMY has a very high superficial hydrophobicity, therefore hydro­
phobic interaction chromatography (HIC) was used for its purification
[14]. Usually, a precipitate obtained from fractional precipitation has
high concentration of salt, therefore, a desalting step is required if a
subsequent ion-exchange or size-exclusion chromatography step is
employed. In contrast, no additional steps are required for further pu­
rification by HIC [29].
The separation of AMY was achieved at pH 6.00, at a flow rate of 1
mL/min. The analysis of enzymatic activity of fractions collected cor­
responding to each peak of the chromatogram, in Figure SI a), showed
that AMY was collected between 27 and 32 min, with the highest AMY
activity at 28.5 min. Some proteolytic activity was found in fractions Fig. 3. Stability of AMY at 30, 40, 50, 60 and 70 ◦ C. Buffer 50 mM potassium
collected around AMY’s peak. Despite that, a purified AMY fraction phosphate buffer pH 6.00.
could be collected around 28.5 min. This second step allowed purifying
AMY 4 times and recovering 83% of the precipitate AMY activity with 4. CONCLUSION
only 14% of the proteolytic activity of the precipitate. In the SDS-PAGE,
in Figure SI b), HIC samples showed that the AMY band only appeared in This is the first report about production of alpha-amylase by
line 7 (purified AMY fraction). No other bands were detected in this line, A. oryzae submerged fermentation using low-cost flour mill waste me­
indicating that the contaminant proteins are present at concentrations dium. Experimental design led to an increase in AMY production. A
below the limits of detection. The purification protocol, in Table 3b), novel two-step purification protocol was developed to separate AMY
showed a global recovery of 65 % of AMY activity with a purification after fermentation, including fractional precipitation and hydrophobic
factor of 11. Though the recovery decreased slightly, the purification interaction chromatography. This technique was applied for the first
factor of this procedure was increased in respect to our previous results time for the isolation of AMY from proteases. Previous dialysis step was
employing size-exclusion chromatography as a single step to purify AMY not required.
[12]. Proteases present in the filtered broth are the main contaminant Flour mill waste can be a substrate for similar strains, and the puri­
because of its hydrolytic activity, HIC was an effective step in further fication could be applied, for the preparations of AMY, protease or other
purifying AMY which allowed to eliminate 86% of the proteolytic tailor-made enzymatic cocktails in the future.
activity.

Declaration of Competing Interest

3.5. Evaluation of the thermal stability of purified AMY

The authors report no declarations of interest.

Fig. 3 shows the plot of catalytic activity of AMY vs temperature. It
can be seen that the maximum stability is at 30 ◦ C, losing 10 % of the
activity at 40 ◦ C and 63 % at 50 ◦ C. At temperatures over 60 ◦ C AMY is
completely inactivated probably due to the loss of its native structure.
These findings agree with thermal stability results in the literature [31,
32].
Acknowledgement
Ignacio Cabezudo and Pablo Anselmi thank CONICET for their fel­
lowships; Mauricio Braia, María-Rocío Meini and Diana Romanini are
CONICET researchers. The authors would like to thank Dra. Silvia
Moreno from CEQUIBIEM for performing the analysis of protein samples
by LC-MS. This work was supported by Agencia Nacional de Promoción

107
M. Braia et al. Process Biochemistry 111 (2021) 102–108

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