DOI: http://dx.doi.org/10.18273/revion.v30n2-2017007
Artículos de Investigación Científica y Tecnológica
Obtaining
and evaluation of enzymatic extract from Aspergillus
spp. by saccharification of sour cassava starch liqued
Obtención
y evaluación de un extracto enzimático de Aspergillus
spp. para la sacarificación de almidón de yuca licuado
Recolha
e avaliação de um extracto de enzima de Aspergillus spp. para a sacarificação
da mandioca amido liquefeito
Andrés M. Rueda1
Luis J. Rueda2
Daniel Molina V.1,2
Clara I. Sánchez3*
1Grupo de Investigación en Bioquímica y Microbiología, Universidad
Industrial de Santander, Bucaramanga, Santander, Colombia.
2Escuela de Química, Universidad Industrial de Santander,
Bucaramanga, Santander, Colombia.
3Escuela de Microbiología. Universidad Industrial de
Santander, Bucaramanga, Santander, Colombia.
Abstract
The saccharification enzymes are more
expensive than liquefaction enzymes for that reason are sought strategies that
allow the supply of these enzymes at low cost. The objectives of this study
were to evolve saccharolytic enzymatic extracts from native strains, to select
an extract and to determine the best variables for the production of glucose
syrups from liquefied bitter cassava starch.Thirteen mushrooms isolated from
sour cassava (Manihot sp). were
evaluated for their saccharolytic activity, hydrolysis of maltose, glucose
production and biomass formation in submerged fermentation. Aspergillus A1 was selected because it
had the highest volumetric activity (0.09UI.L-1). During solid-state
fermentation, the presence of proteins was corroborated by electrophoresis
SDS-PAGE. Through various experiments, the best experimental condition were pH
4.0, agitation 0r.p.m and temperature 55.0°C; the effect of cofactors Cu2+,
K+, Mg2+ and Na+ was evaluated, Mg2+
increases the activity in 1.32UImg-1 (32.4%). The thermal stability
at 55.0°C is 120 minutes. Finally, the saccharolytic capacity of the enzymatic
extract was confirmed using liquefied cassava starch.
Keywords: Glucoamylase, Aspergillus, Saccharification, Syrup Glucose.
Resumen
Comercialmente las enzimas de
sacarificación son más costosas que las de licuefacción, por tal razón se
buscan estrategias que permitan el suministro de estas enzimas a bajo costo.
Los objetivos de este estudio fueron evaluar extractos enzimáticos
sacarolíticos a partir de cepas nativas, seleccionar un extracto y determinar
las mejores variables para la producción de jarabes de glucosa. Para ello, en
trece hongos filamentosos aislados de yuca amarga Manihot sp. fueron evaluadas la actividad sacarolítica, la
hidrólisis de maltosa, la producción de glucosa y la formación de biomasa bajo
condiciones de fermentación sumergida. El aislamiento identificado como Aspergillus A1 fue seleccionado por
presentar la más alta actividad volumétrica (0,09UI.L-1). Durante la
fermentación en estado sólido se corroboró la presencia de proteínas mediante
el método de electroforesis SDS-PAGE y se evidenció una banda de mayor
intensidad con peso molecular entre 60 y 70kDa.
Para el extracto enzimático de Aspergillus
A1 se determinó que las mejores condiciones experimentales de sacarificación,
con el uso de maltosa como sustrato, fueron pH 4,0, temperatura 55,0°C, y sin
agitación. Igualmente en la evaluación del efecto de los cofactores Cu2+,
K+, Mg2+ y Na+ en concentraciones de 1 mM, se
observó que todos incrementan la actividad enzimática especialmente el Mg2+
, el cual la aumenta en 1,32 UI.mg-1 (32,4%). La estabilidad
térmica de la proteína a 55,0°C fue de 120 minutos. La capacidad sacarolítica
del extracto enzimático fue confirmada usando como sustrato almidón de yuca
amarga licuado.
Palabras clave:
Glucoamilasa, Aspergillus, Sacarifcación, jarabes de glucosa.
Resumo
Comercialmente as enzimas de
sacarificação são mais custosas do que as de liquefação, por esta razão
procuram-se estratégias que permitam fornecer este tipo de enzimas a baixo
custo. Os objetivos deste estúdio foram avaliar extratos enzimáticos
sacarolīticos de cepas nativas, selecionar um extrato e determinar as melhores
variáveis para a produção de xaropes de glicose. Para isto, de treze fungos
filamentosos isolados de mandioca amarga Manihot
sp., foram avaliadas a atividade sacarolítica, a hidrólise de maltose, a
produção de glicose e a formação de biomassa em condições de fermentação
submersa. O isolamento Aspergillus A1
foi selecionado por apresentar a maior atividade volumétrica (0,091UI.L-1).
Durante a fermentação em superfície a presença de proteínas foi confirmada pelo
método de eletroforese de SDS-PAGE junto com uma banda de maior intensidade com
peso molecular entre os 60 e 70kDa. Para os preparados enzimáticos de Aspergillus A1 determinou-se que as
melhores condições experimentais de sacarificação usando maltose como substrato
foram pH 4,0, temperatura de 55,0°C, sem agitação. Igualmente na avaliação do
efeito dos cofatores Cu2+, K+, Mg2+ e Na+ em
concentrações de 1 mM, observou-se que todos incrementam a atividade enzimática
principalmente o Mg2+ em 1,32UI.mg-1 (32,4%) em relação
ao controle. A estabilidade térmica da proteína de 55,0°C é de 120 minutos. A
capacidade sacarolítica do extrato enzimático foi confirmada usando substrato
hidrolisado de mandioca amarga.
Palavras-chave: Glucoamilase, Aspergillus, Sacarificação, xarope de glicosa.
Fecha
Recepción: 20 de diciembre de 2016
Fecha
Aceptación: 11 de agosto de 2017
Introduction
World
cassava production has increased in the last years, reaching 270.2 million of
tons by 2014 [1]. Starch from sour cassava is a cheap source to produce
ethanol. Starch pretreatment process,
involve the breaking of amylose and amylopectin by enzymes, being these the
costliest step in the ethanol production from starch, around 47% of total
value, mainly by the enzymes price [2]. One example is the use of saccharolytic
enzymes for the hydrolysis of starch in obtaining fermentable syrups for
bio-ethanol production, which reduces time and generates fewer residues
compared with traditional acid hydrolysis [3]. Enzymatic extracts are
commercially available for liquefaction and saccharification of corn and
sorghum starch, highcost raw materials that compromise food security. However,
for cassava starch, which is not suitable for human consumption, no commercial
enzymes for hydrolysis exist in the market. In addition, sour cassavas abound
greatly in Latin America. Glucose syrups can be obtained via chemical or enzymatic
hydrolysis of starch [4]. In chemical hydrolysis, the use of acids increases
the cost of production, unwanted products are formed, and wastes can pollute
the environment. In contrast, enzymatic hydrolysis is a cheaper alternative
that produces less pollution. However, due to the complexity of the structure
of starch, it is necessary to incorporate heat treatment and the use of
different enzymes to ensure the highest conversion of this polymer to glucose
[5].
The
enzymatic hydrolysis of starch is carried out in two steps: liquefaction and
saccharification. In the first step, α-amylase (EC 3.2.1.1) is used to make
cuts at random into the internal structure of starch due to endo-enzyme
activity, generating many products such as pullulan, dextrin and glucose. The
pullulan and dextrin are not viable to be used in the alcoholic fermentation
process because they are not absorbed by fermenting microorganisms.
Saccharification can occur by two enzymes: pullulanase (EC 3.2.1.41), which
only breaks α-1.6 bonds and produces mostly dextrins, disaccharides such as
maltose and a very small amount of glucose; and glucoamylase (EC 3.2.1.3 and EC
3.2.1.20), which hydrolyzes α -1,4 glycosidic terminal bonds of dextrins from
starch liquefaction, forming mainly glucose. The pullulanases are produced
mostly by bacteria, and glucoamylases are produced by fungi [6]. Enzymatic
saccharification is expensive and thermally unstable. The enzymatic process is
performed at high temperatures because this improves solubility, decreases
viscosity of the starch, reduces microbial contamination, decreases the process
time and reduces production costs [7]. The bio-fuel industry seeks to replace
the enzymes used in the industrial processes of starch hydrolysis with enzymes
having higher thermostability [8]. These enzymes can be obtained from wild type
microorganisms because in some cases, native enzymes exhibit better catalytic
activity than commercial enzymes [9].
For
production of enzymes with saccharolytic activity from filamentous fungi, the
genera Aspergillus and Rizhopus have been the most studied using solid state fermentation (SSF)
[10]. Native fungal enzymes also have been shown to viably be used for glucose
obtaining on an industrial scale.
The aim of this project was the
isolation, identification and selection of native filamentous fungi with
saccharolytic activity, and to obtain an enzymatic extract from the optimal
isolation to be used in glucose syrup production from maltose and cassava
starch.
Materials
and methods
For primary
isolation of fungi, sour cassava (Manihot
sp) was collected with evident fungal colonization from two farms in rural
Barrancabermeja, Santander, Colombia. Ten grams of sour cassava was chopped,
homogenized and diluted in 90ml of sterile saline solution. Each suspension
from cassava homogenized dilutions were taken to obtain fungal colonies by
surface culture. Sub-cultures were made to obtain axenic strains from the
isolations at 25°C [11]. The culture media used contained maltose (5g.L-1)
as the only carbon source, rose bengal to restrict microbial growth and
chloramphenicol (0.1g.L-1) as a broad-spectrum antibiotic.
Genus of
Isolated fungi were identified by macro- and microscopic characteristics using
taxonomic keys. The culture media for identification was potato dextrose agar
(PDA).
Submerged
fermentation (SF) was performed in flasks of 250mL in sterile conditions, each
flask contained 50mL of basal media in deionized water with the following per
L: maltose (5), KH2PO4 (5), MgSO4.7H2O
(5), peptone (2) and yeast extract (1). SF was made to determine maltose
consumption and production of glucose, protein and biomass. Each flask was
adjusted to a final concentration of 105 propagules mL-1. Each batch of SSF isolations had twenty-five
flasks, the fermentation was held with agitation at 150rpm and incubate at 25˚C
for 72 hours. Every eight hours, one flask and two replicates were taken to
obtain the enzymatic extracts, the biomass was separated from the broth using
filtration through a Büchner funnel with Whatman No. 1 filter paper, and the
enzymatic extract was obtained by vacuum.
A first
screening of the enzymatic extract was performed by evaluating maltose
hydrolysis and glucose production using 3,5-dinitrosalicylic acid (DNS) [12]
and glucose oxidase (Biosystems®) [13], respectively. Volumetric productivity
was calculated based on glucose production. The maltose concentration was
calculated via subtraction of the concentration of reducing sugars and glucose.
The production of glucose, protein by Bradford method [14], and biomass by dry
weight and the hydrolysis of maltose were determined in a second screening. The
isolation with the best results was named Aspergillus
A1. The enzymatic volumetric activity (UI.L-1) of the enzymatic
extract from this isolation was quantified (Figure 1). The enzymatic volumetric
activity (UI.L-1) was defined as micromoles of glucose produced per
ml in one minute at 50°C and 200rpm.
To
increase the protein concentration from the enzymatic extract, SSF of Aspergillus A1 was performed in a column
bioreactor with a lignocellulosic support sterilized at 121°C for 15min. The
column bioreactor was filled with 8cm of support and 150ml of broth. The crude
enzymatic extract was obtained by pressing the lignocellulosic polymer,
followed by centrifugation at 10000 × g for 15 minutes and finally filtering
(Millipore, cellulose 0.45µm).
Electrophoresis
SDS-PAGE was performed on enzymatic extracts obtained by SSF of Aspergillus A1 to determine the molecular
weight of the proteins present in the extract. A Fermentas PageRuler of 10 to
200kDa was used as a molecular weight standard. Samples were run at 120 volts
in 12% polyacrylamide gels using Coomassie blue as the dye.
Analysis of variance was performed to
establish the highest values of enzymatic activity of the enzymatic extract
from Aspergillus A1 at a pH 3-9, a
temperature of 30-90°C and an agitation of 0-600rpm. The results of this
analysis were used to determine the best experimental conditions for maltose
hydrolysis. The independent variables were pH 4.0; 4.5 and 5.0; 16mmol.L-1
buffer sodium acetate and 0,1molL-1 citric acid; temperatures of 55,
60 and 65°C; and agitation at 0, 200 and 400rpm. The experiments were performed
in tubes containing 1mL of enzymatic extract and 1mL of maltose at a
concentration of 5mgmL-1.
The response variable was the glucose
concentration versus time. Each assay was performed in triplicate, and the
results were analyzed using Statgraphics software.
The effect of 1mmolL-1 aliquots of
cofactors Cu2+, K+, Mg2+ and Na+ on enzymatic extract activity was determined
per the methodology of Bhatti et al. [15]. This was performed based on the
optimized conditions found in the experimental design. The thermal stability of
the enzymatic extract was also evaluated at 55°C through glucose quantification
every 15 minutes for 3 hours.
Finally, the saccharolytic activity of
the selected enzymatic extract was evaluated using the optimized parameters for
the best cofactor on fermentable syrups obtained from liquefied cassava starch.
Liquefied cassava starch was obtained according to the methodology described by
Ruiz et al. [16].
Results and
Discussion
From
samples of sour cassava, it was possible to obtain 13 fungal isolations
belonging to the genera Fusarium,
Penicillum, Geotrichum, Scopularipsis and Aspergillus, and all the
isolations showed saccharolytic activity. The two isolations with the best volumetric
productivity were Aspergillus A1 and Aspergillus A12 with 0.07 and 0.05gL-1,
respectively. The isolation that showed the best production of glucose from
maltose was Aspergillus A1 (1.3gL-1)
between 50 and 60 hours of fermentation. This agrees with the highest
volumetric enzymatic activity from extract obtained at the same time, despite a
low protein concentration, as shown in Figure 1. Based on the volumetric
activity, glucose production from maltose hydrolysis was confirmed in all
fungal isolations, with the fungi of genera Aspergillus
having the best productivity. The previous results confirm that the Aspergillus genus had the highest
glucose syrup production by submerged fermentation [17,18]. Authors such as
Riaz et al [19]. and O’Brien et al [20], have suggested several
species of Aspergillus, including A. awamori, A. foetidus, A. niger, A. oryzae, A. terreus, for glucose syrups obtaining from polysaccharides.
The
enzymatic extracts obtained from submerged fermentation of Aspergillus A1 did not reach a higher concentration of protein
(0.05g.L-1). This contrasted with SSF, where it was possible to
obtain concentrations greater than 0.05g.L-1.
The
increase in protein concentration in the enzymatic extract was possible with
the change from submerged fermentation to solid fermentation with a
lignocellulose support. With this variation, the protein concentration reached
0.06g.L -1. This is consistent with the work of Rodríguez et al [21], and Singhania et al [22], who concluded that use of SSF for obtaining enzymatic extracts is
better than the concentrations achieved by submerged fermentation. The
electrophoresis SDS-PAGE results of enzymatic extracts obtained from SSF of Aspergillus A1 showed 11 bands with
different molecular weights. The band with a molecular weight between 60 and
70kDa had the highest intensity, as shown in Figure 2.
Figure
2. Polyacrylamide
gel electrophoresis of enzymatic extract from Aspergillus A1 obtained by SSF. Line 1 Molecular weight marker;
Lines 2 and 3 enzymatic extract from Aspergillus
A1 from two different bioreactors. Coomassie brilliant blue G-250 staining.
The bands
observed in gels from SDS-PAGE obtained from different SSF of enzymatic
extracts from Aspergillus A1 were not
different from each other, guaranteeing the reproducibility of SSF, as shown in
Figure 2. The band with the highest intensity occurred between 60 and 70kDa.
These results corroborate those of Norouzian et al.,8 who established the molecular weights of fungal
glucoamylases to be within a range of 48 to 90kDa. da Silva et al [23] and Bagheri et al [24] reported the molecular weight
of glucoamylase from Aspergillus niveus
to be 76kDa.
Analysis
of variance showed that the best saccharolytic activity of the extract at pH
values between 4 and 5, agitation between 200 and 600rpm and a temperature of
60°C. By using these values, it was possible to define the variables for the
experimental design matrix.
As can be
seen in Figure 3, the best enzymatic extract activity from Aspergillus A1 with respect to pH occurs at 20 minutes, (4.4UI.mg-1)
at a pH between 4 and 5 and at 60°C. Under these conditions, the activity
reached 3.5µmol.L-1. These results are comparable to those of
Bagheri et al [24], who reported that
the highest activity of glucoamylase occurs at a pH between 4.5 and 6.5 and at
temperatures between 50 and 70°C.
Figure 3. Glucoamylase activity under the effect of different values
of pH 3 – 6 (A) and temperatura 40–70°C (B), using maltose to concentration
5mg.mL-1 like a substrate. (black bars) glucoamylase activity at 20min; (grey
bars) glucoamylase activity at 40min and (white bars) glucoamylase activity at
60min.
The
highest values of enzymatic activity and glucose obtained from maltose were at
pH 4 (4.4UI.mg-1 and 6,8µmolmL-1, respectively) and a temperature of 60°C
(5.6UI.mg-1 and 3.5µmol.mL-1, respectively), as shown in
Figure 3.
From
these experimental design results, seven effects were found with a P value
lower than 0,05 and confidence level of 95%. The results indicate that there
was little or no interaction between the temperature and agitation. This is
contrary to what is normally observed regarding interactions of pH/ temperature
and pH/agitation, with the latter typically being the most significant.
The
polynomial obtained for glucoamylase activity (Equation 1) showed an R-squared
value of 89.1% and an adjusted R-squared value of 87.7%, which could explain
the variability in enzymatic activity with a standard error of 0.4% and an absolute
error of 0.3%.
From the
mathematical model obtained to predict enzymatic activity, pH and pH2 were the
most influential factors. From the response surface and surface contour (Figure
4) it can be concluded that the best saccharolytic activity obtained by the
enzymatic extract was at an agitation of 0rpm. Enzymatic activity was increased
when the agitation increased to 4rpm at an average temperature of 55°C [25].
Figure 4. Response surface estimated of the
combined effects of pH and temperature of enzymatic extract from Aspergillus
A1, without agitation, 5mgmL-1 of maltose concentration at 15 minutes of
reaction
Enzymatic cofactor
evaluation showed that all cofactors evaluated had a positive influence on enzymatic
activity. The Mg2+ ion was found to increase enzymatic activity by 32%,
resulting in a value of 4.15UI.mg-1. Figure 5 shows that the use of metallic
ions such as enzymatic cofactors increases enzymatic activity compared to the
control. Mg+ was the most influential factor, resulting in a 32.4% increase in
enzymatic activity. This result agrees with that reported by Bhatti et al [15]
and Benassi et al [26], who found a 176% increase in enzymatic activity using
manganese ions in glucoamylase from Fusarium phoenicis.
Figure 5. Effects of various metal ions at 1mmol concentration on
glucoamylase activity, (black bar) control.
The
thermal stability assay showed three periods of variation. The first 30 minutes
of enzymatic activity peaked at 5.6UI.mg-1, followed by a decrease
by 22%, and then the activity remained stable until 120 minutes. The enzymatic
activity finally decreased to an average of 56% at the completion of the study.
The half-life determined for the
enzymatic extract from Aspergillus A1
was 60 minutes of incubation at 60°C, which was a similar time to that obtained
by Sutthirak et al [27], who
determined that the greatest decrease in enzyme activity occurred after two
hours (Figure 6).
Figure
6. Thermal
stability of enzymatic extract from Aspergillus
A1, 5mg.ml-1 maltose concentration.
In
saccharification assays of liquefied cassava starch, it has been found that
saccharolytic activity has a glucose/enzymatic activity ratio of 0.9 for
enzymatic extracts from Aspergillus
A1. This is lower than that obtained by Ruiz et al [16], who found a value of 3.7 for purified commercial
glucoamylase Spirizyme ® Fuel.
From 13
fungal isolations identified from decomposed cassava, the Aspergillus genera had the highest saccharolytic activity. From this
group, it was possible to obtain an enzymatic extract with glucoamylase enzyme
activity.
The SSF
make it possible to obtain a higher protein concentration in enzymatic extracts
from Aspergillus A1 for
quantification and observation of the proteins by Bradford assay and
electrophoresis, respectively.
The best
parameters for maltose hydrolysis by enzymatic extracts from Aspergillus A1 were a pH of 4.0; a
temperature of 55°C without agitation and 1 mmol.L -1 of Mg2+
cofactor.
It was
confirmed that the enzymatic extract from native Aspergillus sp. could undergo saccharification of liquefied cassava
starch.
Acknowledgements
The
authors would like to thank the Ministry of Agricultura y Desarrollo Rural of Colombia
(Project No. 026-2007D3321-639-07), the Dirección de Investigaciones de la
Facultad de Salud de la Universidad Industrial de Santander for providing
funding for this project (Project No. 5645)
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Cita: Rueda AM, Rueda LJ, Molina DR, Sánchez CI. Obtaining and
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