Amylase 2017; 1: 50–58
Research Article
Mario Carrasco, Jennifer Alcaíno, Víctor Cifuentes, Marcelo Baeza*
Purification and characterization of a novel
α-glucosidase from an Antarctic yeast Dioszegia
fristingensis isolate
DOI 10.1515/amylase-2017-0005
Received March 23, 2017; accepted May 18, 2017
Abstract: Starch hydrolyzing enzymes, amylases, are
important commercial enzymes used in several productive
areas. A current tendency is to find amylases with high
catalytic activity at 20-40°C, to generate products that
work well at low temperatures, such as detergents, and
for energy saving resources in industrial processes. In this
work, an α-glucosidase secreted by the cold-adapted yeast
Dioszegia fristingensis was purified and biochemically
characterized. The effect of physicochemical parameters
on the enzyme activity was evaluated. According to our
results, the amylolytic enzyme secreted by D. fristingensis
is a monomeric α-glucosidase of about 30 kDa that
displayed the highest activity at 37-40°C and at pH 5.5-6.5,
in the presence of 10 mM CaCl2. The enzyme exhibited a
Michaelis-Menten kinetics with Km and kcat of 1.3 g/L and
15 min-1, respectively.
Keywords: starch hydrolysis; cold-adapted α-glucosidase;
Dioszegia fristingensis; Antarctic yeast.
Abbreviations: DNSA, dinitrosalicylic acid; E-pNP-G7,
ethylidene-4-nitrophenyl-α-d-maltoheptaoside; kcat,
catalytic rate constant; Km, Michaelis-Menten constant;
4-NPGP, 4-nitrophenyl-α-d-glucopyranoside; SDS, sodium
dodecyl sulphate; SDS-PAGE, sodium dodecyl sulphate
polyacrylamide gel electrophoresis; Vmax, maximum
velocity.
*Corresponding author: Marcelo Baeza, Departamento de Ciencias
Ecológicas, Facultad de Ciencias, Universidad de Chile. Las Palmeras
3425, Casilla 653, Santiago, Chile; E-mail: mbaeza@u.uchile.cl
Mario Carrasco, Jennifer Alcaíno, Víctor Cifuentes, Departamento de
Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile. Las
Palmeras 3425, Casilla 653, Santiago, Chile
© 2017 Mario Carrasco, et al., published by De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
Open Access
1 Introduction
Humans have been using enzymes in the production
of food and beverages from ancient times but without
knowing its existence. Only since the 19th century the
role and importance of enzymes for application in
diverse industrial markets, such as food and beverages,
detergents, animal feed, biofuels, leather, and textile
has been acknowledged [1,2]. Currently, industrial
enzymes comprise a well-established global market,
estimated to reach a value of $6.3 billion in 2021 [3]. In
the last decades, a growing interest in enzymes produced
by microorganisms living in cold environments can be
recognized, which is due to their high catalytic activity
at middle or low temperatures that allow, among other
properties, energy saving in productive processes [4-6].
Examples of these attractive enzymes are the cold-active
amylases, lipases, proteases, cellulases, pectinases, and
esterases, as they are applied in food, wine, textile, and
detergent industries [7,8]. Amylases are used as additives
in detergent formulations, in wool treatment, and in the
production of biofuels from starch-rich wastes, besides
having an increasing importance in the fields of clinical,
medicinal and analytical chemistries [9-11]. A requisite
for an efficient microbial production of biofuels from
raw starch wastes is the complete degradation of this
polysaccharide, a task that is currently accomplished
by the addition of α-amylase and α-glucosidase, or
glucoamylase enzymes during the fermentative process
[12-14].
Amylases are classified as endoamylases or
exoamylases. Endoamylases cleave the inner α-1,4-
glycosidic bonds of the amylose or amylopectin
chain, while exoamylases cleave the α-1,4-glycosidic
linkages or both the α-1,4- and α-1,6-glycosidic bonds.
α-Amylase (EC 3.2.1.1) is a well-studied endoamylase that
disrupts the α-1,4-glycosidic linkages generating as end
products oligosaccharides with varying length with an
α-configuration and α-limit dextrins, which constitute
  Novel α-glucosidase secreted by psychrotolerant yeast D. fristingensis
branched oligosaccharides. The exoamylase β-amylase
(EC 3.2.1.2) catalyzes the hydrolysis of the second α-1,4-
glycosidic bonds from the non-reducing end of starch,
generating as end products β-maltose and α-limit
dextrin. The exoamylases glucoamylase (EC 3.2.1.3) and
α-glucosidase (EC 3.2.1.20) hydrolyze both the α-1,4- and
α-1,6-glycosidic bonds from the non-reducing end of the
starch molecule, generating exclusively β-d-glucose
as the end product. These last enzymes differ in their
substrate preference, i.e. glucoamylase hydrolyzes best
long-chain polysaccharides, whereas α-glucosidase short
maltooligosaccharides [15-17].
The α-amylase is the most characterized starch
degrading enzyme, which is used in baking and textile
industries, and in starch saccharification [18,19]. The
commercially available α-amylases have limited activity
at low pHs and temperatures, exhibiting usually Ca2+
dependence for their activity [20]. For its part, several
α-glucosidases and glucoamylases from mesophilic
bacteria, fungi and yeasts have been described, which, in
general, display optimal activity at temperatures up to 45°C
and at acidic pH [21-23]. The mostly used α-glucosidases and
glucoamylases in industrial processes have been obtained
from Aspergillus species with major activity displayed at
temperatures between 45-60°C [13,24,25]. Currently, the
search for novel microbial sources of amylases with high
activity at low temperatures, for energy-saving, gains
importance. Furthermore, these cold-active amylases also
have specific properties that make them compatible with
application in different fields, such as acid stability for bio-
pulping and compatibility with detergent components.
Yeasts thriving in cold environments (i.e. cold-adapted
yeasts) represent a promising source for cold-active
amylolytic enzymes since they, using the available carbon
sources, secrete hydrolytic enzymes, which have potential
for biotechnological and industrial applications [26-29].
An α-amylase and a glucoamylase were described from
Candida antarctica (now Moesziomyces antarcticus) CBS
6678 [30], both enzymes being monomeric glycoproteins
that preferentially hydrolyzed high-molecular-mass
substrates. The optimal pH and temperature for activity
of these glucoamylase and α-amylase were 4.2 and 57°C,
and 4.2 and 62°C, respectively [30]. An amylase produced
by the yeast Clavispora lusitaniae isolated from vegetables
of a market of Bhopal (India) had higher activity at pH
11 and 42°C, maintaining a 42% of its activity at 4°C and
showed no dependency on Ca2+ for activity and stability.
This enzyme was prosed as a good candidate to be applied
in detergents because it retained nearly 80% of activity
after 2 h of exposing to sodium dodecyl sulphate, Tween-
80 and Triton X-100, H2O2 and NaClO3 [31]. The Antarctic
 51
fungus Tetracladium sp. secreted a glucoamylase that is a
glycosylated enzyme of approximately 84 kDa displaying
best soluble starch degrading activity at 30°C and pH
6.0, and exhibited also no dependency on calcium for its
activity [32].
Several Antarctic yeast species able to growth in
media with soluble starch as unique carbon source and
displaying extracellular soluble starch hydrolyzing
activity have been reported [33,34]. From these yeasts, an
isolate identified as Dioszegia fristingensis displayed the
highest starch hydrolyzing activity.
The aim of this work was therefore to purify and
biochemically characterize the amylolytic enzyme
secreted by D. fristingensis, determining the optimal
conditions for enzyme activity, thermal stability and
kinetics parameters.
2 Materials and methods
2.1 Culture conditions
D. fristingensis isolate T9Df1 was routinely grown at 15°C
in YM medium (yeast extract 0.3%, malt extract 0.3%,
peptone 0.5%) supplemented with 1% glucose (YM-G).
For protein purification purposes, the YM medium was
supplemented with 1% soluble starch (Sigma-Aldrich
Corporation, St Louis, USA) (YM-S). Semisolid media
were prepared by the addition of agar at 1.5%, before
autoclaving at 121°C for 20 min.
2.2 Extracellular protein purification
Three hundred mL of yeast cultures at late exponential
phase of growth were centrifuged at 7,000×g for 10 min
at 4°C and the supernatant was filtered through a sterile
0.45-μm pore size polyvinylidene fluoride membrane
(Millipore, Billerica, MA, USA). The proteins from the cell-
free supernatants were differentially precipitated using
ammonium sulphate at 20, 40, 60 and 80% of saturation.
In each step of precipitation, the sample was incubated on
ice for 2 h and centrifuged at 10,000×g at 4°C for 15 min.
The pellet was suspended in 2 mL of potassium phosphate
buffer (20 mM and pH 7.0) and then samples were desalted
using a HiTrap Desalting column (GE, Schenectady, New
York, USA). Then, 2 mL protein samples were loaded onto
an ion-exchange SP FF 16/10 column (General Electrics,
New York, USA) equilibrated with 20 mM sodium
phosphate buffer at a flow rate of 1 mL/min and attached
to an Akta Prime purification system (General Electrics,
New York, USA). The bounded proteins were eluted with
52   M. Carrasco, et al.
a 50 mL NaCl gradient from 0 to 1 M and fractions of 1 mL
were collected. When necessary, fraction samples were
pooled and concentrated at 1,000×g at 4°C using Amicon
filters with a 3 kDa molecular weight cut-off.
2.3 Methods for protein analysis
Proteins were quantified using the BCA Kit Assay (Thermo
Scientific, IL, USA) according to the manufacturer’s
instructions and visualized by sodium dodecyl sulpate
polyacrylamide gel electrophoresis (SDS-PAGE). The
relative molecular weight in reducing and non-reducing
conditions was estimated from SDS-PAGE gels and
from gel filtration, respectively. The PageRuler Plus
Prestained Protein Ladder (Thermo Scientific, IL, USA)
and Gel filtration standard (Biorad, CA, USA) were
used as commercial protein reference standards. The
existence of protein glycosylation was determined using
the Pierce Glycoprotein Staining Kit (Thermo Scientific,
IL, USA) according to the manufacturer’s instructions.
Protein characterization by peptide mass fingerprinting
was performed at the Centre for Functional Genomics
(University at Albany, New York) and analyzed by Mascot;
only results having a score greater than 54 (P < 0.05) were
considered statistically significant [35].
2.4 Analysis of the amylolytic activity
The enzyme activity on soluble starch was measured as
the liberation of reducing sugars, which were quantified
by the dinitrosalicylic acid (DNSA) method [36]. Briefly,
50 µL of 10 g/L soluble starch was mixed with 50 µL of
the protein sample and incubated for 1 h. Then, 100 µL
of DNSA solution (1.6% NaOH, 30% sodium potassium
tartrate, 1% 3,5-DNSA) were added, mixed, incubated for
10 min at 100°C followed by 5 min on ice. The absorbance
at 540 nm was measured in 96 well microplates using
an Epoch 2 microplate reader (Biotek Instruments Inc.,
Winooski, VT, USA). Values were normalized by the
amount of protein present in each sample. The released
glucose from soluble starch was quantified with the
d-Glucose Assay Kit (Megazyme, IL, USA), which was
used according to the manufacturer’s instructions. To
determine the specificity of the amylolytic enzyme, the
chromogenic substrates ethylidene-4-nitrophenyl-α-d-
maltoheptaoside (E-pNP-G7; Abnova, Taipei, Taiwan)
and 4-nitrophenyl-α-d-glucopyranoside (4-NPGP; Sigma-
Aldrich Corporation, St Louis, USA) were used.
2.5 Analysis of optimal conditions and
kinetic parameters for the amylolytic activity
The effect of temperature, pH, soluble starch concentration,
CaCl2 and MgCl2 on the activity of the amylolytic enzyme
was evaluated using a Placket-Burman design (Table
1). The complete progression curve was determined in
each trial, and the reaction velocity values were used to
calculate the effect on enzyme activity of the mentioned
physicochemical factors. The optimal temperature and
pH for the enzyme activity was determined by applying
a response surface methodology. The kinetic parameters
were determined using the optimal temperature and
pH conditions indicated in the previous step. In these
assays, 500 µL of the amylase purified solution at 1.5 µg/
mL was incubated with 500 µL of different concentration
of soluble starch. At different times, 50 µL of the reaction
were taken and assayed by the DNSA method as described
before. The initial reaction velocities at different soluble
starch concentrations were calculated and the Km and
Vmax values were estimated applying the Hill non-linear
fitting method. The kcat value was determined once these
values were known. To evaluate the irreversible thermal
inactivation, purified enzyme samples were incubated at
different temperatures from 4°C to 50°C during 1-8 h. In
each case, the reaction velocity was calculated.
Table 1. Influence of different parameters on the activity of
α-glucosidase.a
Factor High Low Effect
Temperature (oC) 37 25 0.6
pH 7 5 1.9
Soluble starch (g/L) 5 0.5 1.8
CaCl2 (mM) 10 0* 8.2
MgCl2 (mM) 10 0* 0.6
a
The effect corresponds to ratio between influence of each para-
meters and experimental error. The asterisk means no addition of
compounds.
3 Results
3.1 Enzyme purification and characterization
The proteins profiles and amylolytic activity were
determined in fractions collected from anionic-exchange
chromatography. As shown in Figure 1, two peaks centred
at fractions 35 (peak 1) and 87 (peak 2) were observed,
and the amylolytic activity was only detected in samples
from fractions corresponding to peak 2 (Fig. 1A). When
  Novel α-glucosidase secreted by psychrotolerant yeast D. fristingensis  53

these fractions were analyzed by SDS-PAGE, a single 32
kDa protein band was observed (Fig. 1A). This enzyme
secreted by D. fristingensis would not be glycosylated
as demonstrated in Figure 1B,C. The relative molecular
weight under non-reducing conditions determined
by gel filtration chromatography was 30 kDa, which
is similar to the value determined under reducing
conditions on SDS-PAGE suggesting that the amylolytic
enzyme secreted by D. fristingensis is a monomer. The
purified amylolytic enzyme was analyzed by peptide
mass fingerprinting and 10 of the 54 obtained peptides
displayed hits for putative or uncharacterized proteins
from Saccharomyces cerevisiae in the Mascot report, but
no matches were found for any known starch degrading
enzyme (Fig. S1).

Figure 1. Purification of the amylolytic enzyme from D. fristingensis. (A) Protein samples were loaded onto a SP FF 16/10 column equilibra-
ted with 20 mM sodium phosphate buffer, pH 7.0, with a flow rate of 1.0 mL/min and NaCl gradient from 0 to 1 M (discontinuous line). The
absorbance values at 280 nm (continuous line) and amylase activity (dotted line) were measured. SDS-PAGE of fractions from both peaks
observed at 280 nm (peak 1: fractions 20, 25, 30, 35, and 40; peak 2: fraction 87), M: protein marker. (B,C), SDS-PAGE stained with Coomas-
sie blue or a glycoprotein staining kit, respectively. The sample of the amylolytic enzyme from D. fristingensis (lanes 1 and 4), and positive
(horse peroxidase, lanes 3 and 6) and negative (soybean trypsin inhibitor, lanes 2 and 5) controls for glycosylation. The arrow indicates the
protein band associated to amylolytic activity.
54   M. Carrasco, et al.
3.2 Determination of enzyme substrate
specificity
The enzyme specificity of the amylolytic enzyme secreted
by D. fristingensis was evaluated using E-pNP-G7 and the
4-NPGP as substrates in the reactions. These compounds
are specific substrates of α-amylases and α-glucosidases,
respectively. As shown in Figure 2A, the D. fristingensis
enzyme was able to hydrolyze 4-NPGP, but not E-pNP-
G7, indicating that this enzyme is an α-glucosidase. The
enzyme reaction was also performed using 5 g/L soluble
starch as substrate. According to results (Fig. 2B), terminal
glucose was released from soluble starch in the enzyme
reaction, which is characteristic of α-glucosidases, while
α-amylase release different oligosaccharides.
Figure 2. Substrate specificity of the amylolytic enzyme from D. fris-
tingensis. (A) Enzymatic reactions performed using E-pNP-G7 (open
symbols) or 4-NPGP (filled symbols) as substrates. The release
of p-nitrophenol was measured at 405 nm. Squares, amylolytic
enzyme from D. fristingensis; open circles, commercial α-amylase
(Amylase assay kit, Abnova, China); filled circles, commercial
α-glucosidase (d-glucose/d-fructose determination Kit, Megazyme,
USA); triangles, negative control (no enzyme added). (B) Glucose
released from soluble starch along time by the amylolytic enzyme
from D. fristingensis.
3.3 Influence of physical-chemical parame-
ters on α-glucosidase activity
The influence of temperature and pH, and the
concentration of Ca2+, Mg2+ and soluble starch on enzyme
activity was estimated using a two-level placket-Burman
design. In each trial, the reaction progress was followed
by the release of reducing sugars, until no increment was
observed. The reaction velocities were calculated from
the slopes of each curve to estimate the effect of each
variable on the α-glucosidase activity (Table 1). Under
the ranges used of each factor in the analyses, CaCl2
concentration showed to be the factor that most affected
the α-glucosidase activity, followed by pH and soluble
starch concentration. Therefore, α-glucosidase activity
was measured at different CaCl2 concentrations, showing
a 200% increment of the reaction velocity at 10 mM CaCl2,
and 170% increment at both 20 and 40 mM CaCl2 (Fig.
3) when compared to the value obtained in the absence
of CaCl2. No α-glucosidase activity was observed at 100
mM CaCl2. According to these results, the subsequent
α-glucosidase activity assays were performed using buffer
supplemented with 10 mM CaCl2. To find the pH and
temperature values, at which the α-glucosidase exhibited
the highest hydrolytic activity on soluble starch, a central
composite design of two levels was applied [37]. At 1 h
of reaction, the α-glucosidase secreted by D. fristingensis
displayed the highest activity at 37-40°C and at pH 5.5-6.5
(Fig. 4).
Figure 3. CaCl2 influence on the α-glucosidase activity. Each reaction
was performed with 5 g/L of soluble starch and variable concentra-
tions of CaCl2: 0 mM (squares), 10 mM (circles), 20 mM (triangles),
40 mM (rhombs) and 100 mM (stars). The release of reducing sugars
was quantified by the DNSA method.
  Novel α-glucosidase secreted by psychrotolerant yeast D. fristingensis  55

The effect of temperature on α-glucosidase activity
and stability using soluble starch as substrate was
evaluated with or without the addition of 10 mM CaCl2.
In the presence of CaCl2, the enzyme activity decreased
at temperatures below 37°C, retaining 55% and 32% of its
maximum activity when it was assayed at 30°C and 22°C,
respectively. When CaCl2 was not added, the α-glucosidase
activity decreased at lower temperatures but this decrease
was less pronounced than in the previous conditions. In
the presence of CaCl2, only a slight α-glucosidase activity
decrease was observed at 45°C and 50°C, retaining over
90% of its maximum activity. Instead, a more pronounce
decrease of the α-glucosidase activity was observed at
45°C and 50°C without the addition of CaCl2.
3.4 Thermal inactivation of D. fristingensis
α-glucosidase

The enzyme remained highly stable after 1 h of incubation

Figure 4. Effect of pH and temperature on the α-glucosidase activity.
Central composite design of the enzyme reactions using pH and
temperature as variables. Enzyme reactions were followed by the
release of reducing sugar measured by the DNSA method until no
linear increase in the absorbance values at 540 nm was observed.
The response surface plot shows the calculated reactions velocities
(colour scale) at each condition.
at temperatures from 4 to 37°C, before performing the
activity assay at its optimal conditions. Similar results were
obtained when the assays were performed with or without
the addition of CaCl2. An abrupt reduction of the activity
was observed when samples were incubated for 1 h at
temperatures ≥ 40°C, retaining only 25% of its maximum
activity after incubation at 50°C, but this reduction was
more pronounced when no CaCl2 was added (Fig. 5A). The
stability of the enzyme at longer times of incubation at
different temperatures is shown in Figure 5B. The enzyme
showed to be very stable after incubation at 22°C, keeping
up to 90% of its maximum activity even after 8 h of
incubation. At 30°C and 40°C, the α-glucosidase activity
displayed almost a linear decrease along time, with half-
life of 7.0 h and 5.6 h, respectively. At 50°C the decrease
in activity was abrupt in the first 2 h of incubation (30%
residual activity), and then a linear decay was observed
until 8 h of incubation, reaching 15% residual activity.

Figure 5. Temperature and calcium influence on the α-glucosidase activity. Assays were performed for 1 h with soluble starch as substrate
at pH 6.0, in presence (closed symbols) or absence (open symbols) of 10 mM CaCl2, and followed by the release of reducing sugar. In each
curve, the residual % of activity relative to maximum activity is shown. (A) Squares, assays performed at different temperatures; circles,
assays performed at 37°C after incubation of enzyme samples for 1 h at different temperatures. (B) Assays performed at 37°C after the
enzyme samples were incubated at 22°C (squares), 30°C (circles), 40°C (triangles) and 50°C (rhombs) for different times.
56   M. Carrasco, et al.
3.5 Determination of kinetics parameters
The kinetics of α-glucosidase from D. fristingensis using
soluble starch as substrate was analyzed in steady-state
experiments. As shown in Figure 6, the enzyme displayed
a Michaelis-Menten kinetics, and the values for Km and kcat
were of 1.3 g/L and 15 min–1, respectively.
4 Discussion
The soluble starch degrading enzyme secreted by D.
fristingensis T9Df1 was purified and, according to substrate
specificity assays, it corresponded to a 30 kDa monomeric
α-glucosidase. The reported molecular weight for fungal
amylolytic enzymes is wide variable ranging from 27 to
250 kDa, and the majority of them are glycosylated [13,38].
Glycosylation is a posttranslational modification present in
almost all excreted eukaryotic polypeptides and important
for protein exporting, folding and biological activity [39].
In Cryptococcus flavus, the N-glycosylation is important
for the export of the α-amylase Amy1, but not for its
enzymatic activity [40]. Similarly, no difference in stability
towards proteolytic degradation, heat, and acid pH was
observed in the glycosylated and the non-glycosylated
form of an α-amylase produced by Aspergillus oryzae [41].
According to our results, the α-glucosidase secreted by D.
fristingensis is non-glycosylated; however, the assay used
was based in periodic acid-Schiff method that specifically
detects glycosylated proteins having sialic acid and other
oxidizable carbohydrate groups. On the other hand, there
is a possibility that the enzyme has few glycosylation sites
and therefore low staining intensity [42]. The presence of
10 mM CaCl2, pH 5.5-6.0 and temperature between 37-40ºC
were determined as conditions for the best activity of the
α-glucosidase secreted by D. fristingensis, which retained
a 60% of the optimal activity at 30°C, and a 30% at both
10°C and 4°C. Most of other microbial α-amylases and
glucoamylases exhibit their best activity at 40°C, and
have lower optimal pH (average 4.5) for optimal activity
[20]. The cold-active α-amylase produced by Antarctic
bacterium Pseudoalteromonas haloplanktis is best active
at 25°C and pH 7.0 [43], whereas the one from the yeast
Candida antarctica (now Moesziomyces antarcticus)
displayed optimal activity at 57°C and pH 4.2 [30].
The influence of CaCl2 on the activity of α-glucosidase
from D. fristingensis varied at different temperatures, while
CaCl2 improved the activity at temperatures above 37oC,
the contrary effect was observed at lower temperatures.
Also, the residual activity after 1-h incubation at different
temperatures was higher in the presence of CaCl2, but
Figure 6. Steady-state kinetics. Enzyme reactions were carried out
with 0.04 µg of the purified α-glucosidase and different concentra-
tions of soluble starch as substrate, at 37°C and pH 6.0.
only at temperatures higher than 37°C. These results
suggest that Ca2+ improves both the activity and stability
of the α-glucosidase, but only at higher temperatures. In
general, it has been reported that the presence of calcium
increases the activity and thermostability of amylases
[44], e.g. for the glucoamylase produced by Aspergillus
phoenicis [45]. A clue characteristic of cold-active enzymes
is that they have higher Km values than their mesophilic
or thermophilic counterparts [46,47]. Although D.
fristingensis is a psychrotolerant yeast having an optimal
growth temperature at 15°C, its secreted α-glucosidase
exhibit the best activity at 37-40°C having its Km value
on soluble starch of 1.3 g/L. This Km value is in the range
typical for microbial α-amylases and glucoamylases
described to be best active at 40-50°C, but is lower than
that for the enzymes with optimal activity above 60°C
[13,48-50]. In relation to thermal inactivation of the
α-glucosidase from D. fristingensis, its decimal reduction
time at 30°C, 40°C and 50°C was 12.5, 10.5 and 7 h,
respectively. Comparatively, this is much lower than seen
for a glucoamylase from Colletotrichum sp. KCP1, which
showed a good stability at 30-50oC with decimal reduction
time values higher than 17 h within that temperature
interval [51]. The decimal reduction time value of the
D. fristingensis α-glucosidase was approximately 70 h.
However, this is only a preliminary characterization and
additional thermodynamic studies must be performed
to understand the enzyme thermal behaviour and its
activity-stability trade-off [47].
Finally, according to the properties of the α-glucosidase
secreted by D. fristingensis T9Df1, this enzyme could be a
good candidate to be applied in processes, such as the
  Novel α-glucosidase secreted by psychrotolerant yeast D. fristingensis
simultaneous saccharification and fermentation of raw
starch, and clarification of beers and juices [52,53].
Acknowledgments: This study was financially supported
through FONDECYT grant 1130333.
Conflict of interest: The authors declare that they have
no conflicts of interest with the contents of this article.
Author contributions: MB conceived the study; MC
preformed the experiments; MC and MB analyzed the
results; MC, MB, JA and VC drafted the manuscript. All
authors read and approved the final manuscript.
References
[1] Sarmiento F., Peralta R., Blamey J.M., Cold and hot
extremozymes: industrial relevance and current trends, Front.
Bioeng. Biotechnol., 2015, 3, 148.
[2] Cherry J.R., Fidantsef A.L., Directed evolution of industrial
enzymes: an update, Curr. Opin. Biotechnol., 2003, 14,
438–443.
[3] BCC Research, Report overview, In: Global Markets for Enzymes
in Industrial Applications, BIO030J, 2017, 1–5.
[4] Siddiqui K.S., Cavicchioli R., Cold-adapted enzymes, Annu. Rev.
Biochem., 2006, 75, 403–433.
[5] Gerday C., Aittaleb M., Bentahir M., Chessa J.P., Claverie P.,
Collins T., et al., Cold-adapted enzymes: from fundamentals to
biotechnology, Trends Biotechnol., 2000, 18, 103–107.
[6] Siddiqui K.S., Some like it hot, some like it cold: temperature
dependent biotechnological applications and improvements in
extremophilic enzymes, Biotechnol. Adv., 2015, 33, 1912–1922.
[7] Buzzini P., Branda E., Goretti M., Turchetti B., Psychrophilic
yeasts from worldwide glacial habitats: diversity, adaptation
strategies and biotechnological potential, FEMS Microbiol.
Ecol., 2012, 82, 217–241.
[8] Białkowska A., Turkiewicz M., Miscellaneous cold-active yeast
enzymes of industrial importance, pp. 377–395, In: Buzzini
P., Margesin R. (Eds.), Cold-adapted Yeasts: Biodiversity,
Adaptation Strategies and Biotechnological Significance,
Springer, Berlin, Heidelberg, 2014.
[9] Gurung N., Ray S., Bose S., Rai V., A broader view: microbial
enzymes and their relevance in industries, medicine, and
beyond, Biomed Res. Int., 2013, 2013, 329121.
[10] Uthumporn U., Shariffa Y.N., Karim A.A., Hydrolysis of native
and heat-treated starches at sub-gelatinization temperature
using granular starch hydrolyzing enzyme, Appl. Biochem.
Biotechnol., 2012, 166, 1167–1182.
[11] Kuddus M., Roohi, Arif J.M., Ramteke W.P., An overview of
cold-active microbial α-amylase: adaptation strategies and
biotechnological potentials, Biotechnology, 2011, 10, 246–258.
[12] van Zyl W.H., Bloom M., Viktor M.J., Engineering yeasts for
raw starch conversion, Appl. Microbiol. Biotechnol., 2012, 95,
1377–1388.
 57
[13] Kumar P., Satyanarayana T., Microbial glucoamylases: charac-
teristics and applications, Crit. Rev. Biotechnol., 2009, 29,
225–255.
[14] Božić N., Lončar N., Slavić M.Š., Vujčić Z., Raw starch degrading
α-amylases: an unsolved riddle, Amylase, 2017, 1, 12–25.
[15] Janecek S., Sevcik J., The evolution of starch-binding domain,
FEBS Lett., 1999, 456, 119–125.
[16] Horvathova V., Janecek S., Sturdik E., Amylolytic enzymes:
molecular aspects of their properties, Gen. Physiol. Biophys.,
2001, 20, 7–32.
[17] van der Maarel M.J.E.C., van der Veen B., Uitdehaag J.C.M.,
Leemhuis H., Dijkhuizen L., Properties and applications
of starch-converting enzymes of the α-amylase family, J.
Biotechnol., 2002, 94, 137–155.
[18] Janecek S., Svensson B., MacGregor E.A., α-Amylase: an
enzyme specificity found in various families of glycoside
hydrolases, Cell. Mol. Life Sci., 2014, 71, 1149–1170.
[19] Kuddus M., Roohi, Microbial cold-active α-amylases: from
fundamentals to recent developments, pp. 1265–1276, In:
Menedez-Vilas A. (Ed.), Current Research, Technology and
Education Topics in Applied Microbiology and Microbial
Biotechnology, Vol. 2, Formatex Research Center, Bajadoz,
2010.
[20] Sharma A., Satyanarayana T., Microbial acid-stable α-amylases:
characteristics, genetic engineering and applications, Process
Biochem., 2013, 48, 201–211.
[21] El-Fallal A., Abou M., El-Sayed A., Omar N, Starch and microbial
α-amylases: from concepts to biotechnological applications,
pp. 459–488, In: Chang C.F. (Ed.), Carbohydrates – Compre-
hensive Studies on Glycobiology and Glycotechnology, InTech,
2012.
[22] Pandey A., Nigam P., Soccol C.R., Soccol V.T., Singh D.,
Mohan R., Advances in microbial amylases, Biotechnol. Appl.
Biochem., 2000, 31, 135–152.
[23] Gupta R., Gigras P., Mohapatra H., Goswami V.K., Chauhan B.,
Microbial α-amylases: a biotechnological perspective, Process
Biochem., 2003, 38, 1599–1616.
[24] Michelin M., Ruller R., Ward R.J., Moraes L.A., Jorge J.A., Terenzi
H.F., et al., Purification and biochemical characterization of
a thermostable extracellular glucoamylase produced by the
thermotolerant fungus Paecilomyces variotii, J. Ind. Microbiol.
Biotechnol., 2008, 35, 17–25.
[25] Aquino A.C.M.M., Jorge J.A., Terenzi H.F., Polizeli M.L.T.M.,
Thermostable glucose-tolerant glucoamylase produced by
the thermophilic fungus Scytalidium thermophilum, Folia
Microbiol., 2001, 46, 11–16.
[26] Alcaíno J., Cifuentes V., Baeza M., Physiological adaptations
of yeasts living in cold environments and their potential
applications, World J. Microbiol. Biotechnol., 2015, 31,
1467–1473.
[27] Cavicchioli R., Charlton T., Ertan H., Mohd Omar S., Siddiqui
K.S., Williams T.J., Biotechnological uses of enzymes from
psychrophiles, Microb. Biotechnol., 2011, 4, 449–460.
[28] Feller G., Psychrophilic enzymes: from folding to function and
biotechnology, Scientifica (Cairo), 2013, 2013, 512840.
[29] Gerday C., Fundamentals of cold-active enzymes, pp. 325–350,
In: Buzzini P., Margesin R. (Eds.), Cold-adapted Yeasts:
Biodiversity, Adaptation Strategies and Biotechnological
Significance, Springer, Berlin Heidelberg, 2014.
58   M. Carrasco, et al.
[30] De Mot R., Verachtert H., Purification and characterization
of extracellular α-amylase and glucoamylase from the yeast
Candida antarctica CBS 6678, Eur. J. Biochem., 1987, 164,
643–654.
[31] Ranjan K., Lone M.A., Sahay S., Detergent compatible
cold-active alkaline amylases from Clavispora lusitaniae CB13,
J. Microbiol. Biotechnol. Food Sci., 2016, 5, 306–310.
[32] Carrasco M., Alcaíno J., Cifuentes V., Baeza M., Purification and
characterization of a novel cold adapted fungal glucoamylase,
Microb. Cell Fact., 2017, 16, 75.
[33] Carrasco M., Villarreal P., Barahona S., Alcaíno J., Cifuentes
V., Baeza M., Screening and characterization of amylase and
cellulase activities in psychrotolerant yeasts, BMC Microbiol.,
2016, 16, 21.
[34] Carrasco M., Rozas J.M., Barahona S., Alcaíno J., Cifuentes
V., Baeza M., Diversity and extracellular enzymatic activities
of yeasts isolated from King George Island, the sub-Antarctic
region, BMC Microbiol., 2012, 12, 251.
[35] Perkins D.N., Pappin D.J., Creasy D.M., Cottrell J.S., Probability-
based protein identification by searching sequence databases
using mass spectrometry data, Electrophoresis, 1999, 20,
3551–3567.
[36] Miller G.L., Use of dinitrosalicylic acid reagent for the
measurement of reducing sugar, Anal. Chem., 1959, 31,
426–428.
[37] Box G.E.P., Hunter J.S., Multi-factor experimental designs for
exploring response surfaces, Annals Math. Statist., 1957, 28,
195–241.
[38] da Silva T.M., Fungal amylases: applications and functional
properties, pp. 152–172, In: Polizeli M.L.T.M., Rai M. (Eds.),
Fungal Enzymes, CRC Press, Boca Raton, London, New York,
2014.
[39] Demain A.L., Vaishnav P., Production of recombinant proteins
by microbes and higher organisms, Biotechnol. Adv., 2009, 27,
297–306.
[40] de Barros M.C., do Nascimento Silva R., Ramada M.H.,
Galdino A.S., de Moraes L.M., Torres F.A., et al., The influence
of N-glycosylation on biochemical properties of Amy1, an
α-amylase from the yeast Cryptococcus flavus, Carbohydr. Res.,
2009, 344, 1682–1686.
[41] Eriksen S.H., Jensen B., Olsen J., Effect of N-linked
glycosylation on secretion, activity, and stability of α-amylase
from Aspergillus oryzae, Curr. Microbiol., 1998, 37, 117–122.
[42] Roth Z., Yehezkel G., Khalaila I., Identification and quanti-
fication of protein glycosylation, Int. J. Carbohydr. Chem., 2012,
2012, 640923.
[43] Feller G., Lonhienne T., Deroanne C., Libioulle C., Van Beeumen
J., Gerday C., Purification, characterization, and nucleotide
sequence of the thermolabile α-amylase from the antarctic
psychrotroph Alteromonas haloplanctis A23, J. Biol. Chem.,
1992, 267, 5217–5221.
[44] Vihinen M., Mäntsälä P., Microbial amylolytic enzyme, Crit. Rev.
Biochem. Mol. Biol., 1989, 24, 329–418.
[45] Benassi V.M., Pasin T.M., Facchini F.D., Jorge J.A., Teixeira
de Moraes Polizeli M.L., A novel glucoamylase activated by
manganese and calcium produced in submerged fermentation
by Aspergillus phoenicis, J. Basic Microbiol., 2014, 54,
333–339.
[46] Feller G., Gerday C., Catalysis and low temperature: molecular
adaptations, pp. 88–121, In: Gerday C., Glansdorff N. (Eds.),
Extremophiles (Life under extreme environmental Condition),
Vol. 2, EOLSS Publishers, Oxford, 2003.
[47] Siddiqui K.S., Defying the activity-stability trade-off in
enzymes: taking advantage of entropy to enhance activity and
thermostability, Crit. Rev. Biotechnol., 2017, 37, 309–322.
[48] Jeon H.Y., Kim N.R., Lee H.W., Choi H.J., Choung W.J., Koo Y.S.,
et al., Characterization of a novel maltose-forming α-amylase
from Lactobacillus plantarum subsp. plantarum ST-III, J. Agric.
Food Chem., 2016, 64, 2307–2314.
[49] Wang S., Jeyaseelan J., Liu Y., Qin W., Characterization
and optimization of amylase production in WangLB, a
high amylase-producing strain of Bacillus, Appl. Biochem.
Biotechnol., 2016, 180, 136–151.
[50] Mehta D., Satyanarayana T., Bacterial and archaeal α-amylases:
diversity and amelioration of the desirable characteristics for
industrial applications, Front. Microbiol., 2016, 7, 1129.
[51] Prajapati V.S., Trivedi U.B., Patel K.C., Kinetic and thermo-
dynamic characterization of glucoamylase from Colletotrichum
sp. KCP1, Indian J. Microbiol., 2014, 54, 87–93.
[52] Cinelli B.A., Castilho L.R., Freire D.M.G., Castro A.M., A brief
review on the emerging technology of ethanol production by
cold hydrolysis of raw starch, Fuel, 2015, 150, 721–729.
[53] Singh R., Mittal A., Kumar M., Kumar P., Amylases: a note on
current applications, Int. Res. J. Biol. Sci., 2016, 5, 27–32.
Supplemental Material: The online version of this article
(DOI: 10.1515/amylase-2017-0005) offers supplementary material.