Post on 21-Dec-2016
Glucoamylase (α�1,4�glucan glucohydrolase, EC
3.2.1.3, GlA) is a glucose�producing amylase that cat�
alyzes successive removal of α�D�glucose residues from
the non�reducing end of poly� or oligosaccharides by
cleavage of the 1,4�α�glucosidic bond. Glucoamylases
belong to the 15th family of glycosyl�hydrolases (GH15)
and are well�known enzymes that catalyze hydrolysis of
starch, amylase, amylopectin, pullulan, and maltose with
removal of glucose as a product of enzymatic hydrolysis of
all these substrates. A characteristic feature of GlAs of
different origin is that the higher the degree of substrate
polymerization, the higher the rate of hydrolysis of the
terminal glycosidic bond. It is necessary to note that
many GlAs are capable of hydrolyzing α�1,4� as well as
α�1,6�glucoside bonds provided, in the latter case, the α�
1,4�bond follows an α�1,6�bond [1]. At high glucose con�
centration in the reaction mixture, GlAs may catalyze the
trans�glycosylation reaction with production of maltose,
isomaltose, and other products [2].
GlAs in combination with α�amylases are used for
glucose production, and together with glucoisomeras�
es they are used for production of fructose and glucose�
fructose syrups. Glucose as a final product of enzymatic
hydrolysis of starch�containing substances may be used in
food and alcohol industry, for obtaining products for
microbiological synthesis, and for first�generation biofu�
els. GlAs that possess trans�glycosylase activity can be
utilized in the synthesis of malto�oligosaccharides with
various degrees of polymerization, which are used, for
example, in diabetic diets.
GlAs are widely distributed in nature, and they are
synthesized by many microorganisms. However, GlAs
produced by the fungi Aspergillus awamori and Aspergillus
niger are predominantly used commercially. These
microorganisms secrete extracellular GlAs with molecu�
lar weights of 80 and 140 kDa, respectively [3, 4].
For the past decade, systemic work has been carried
out in the Laboratory of Enzyme Biotechnology in the
Institute of Biochemistry of the Russian Academy of
Sciences (INBI RAS) to develop enzyme preparations
ISSN 0006�2979, Biochemistry (Moscow), 2013, Vol. 78, No. 10, pp. 1180�1189. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © P. V. Volkov, A. M. Rozhkova, M. V. Semenova, I. N. Zorov, A. P. Sinitsyn, 2013, published in Biokhimiya, 2013, Vol. 78, No. 10, pp. 1502�1513.
1180
Abbreviations: a.a., amino acid residue; CF, culture fluid; GFC,
gel�filtration chromatography; GlA, glucoamylase(s); MMD,
molar mass distribution; PNPG, p�nitrophenyl�α�D�glucopy�
ranoside; RS, reducing sugars.
* To whom correspondence should be addressed.
Comparative Study of Biochemical Properties of Glucoamylasesfrom the Filamentous Fungi Penicillium and Aspergillus
P. V. Volkov1*, A. M. Rozhkova1,2, M. V. Semenova1, I. N. Zorov1,2, and A. P. Sinitsyn1,2
1Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33/2, 119071 Moscow,
Russia; fax: +7 (495) 954�2732; E�mail: pvvolkov@mail.ru2Lomonosov Moscow State University, Faculty of Chemistry, Leninsky Gory 1, 119992 Moscow,
Russia; fax: +7 (495) 939�0997; E�mail: oasinitsyna@gmail.com
Received May 17, 2013
Revision received June 21, 2013
Abstract—Here we report the first isolation to homogeneous forms of two glucoamylases from the fungus Penicillium verru�
culosum and their study in comparison with known glucoamylases from Aspergillus awamori and Aspergillus niger. Genes that
encode glucoamylases from P. verruculosum were cloned and expressed in the fungus Penicillium canescens, and the recom�
binant glucoamylases were obtained with subsequent study of their molecular weights, isoelectric points, optimal tempera�
ture and pH values, and stability. The catalytic activities of the recombinant glucoamylases were determined in relation to
soluble potato starch. Changes in molecular mass distribution and content of low molecular weight products during starch
hydrolysis by glucoamylases from P. verruculosum, A. awamori, and A. niger were studied. An exo�depolymerization mech�
anism was established to be the pathway for destruction of starch by the glucoamylases.
DOI: 10.1134/S0006297913100131
Key words: starch�degrading enzymes, glucoamylase, Penicillium canescens, Penicillium verruculosum, Aspergillus niger,
Aspergillus awamori
PROPERTIES OF RECOMBINANT FUNGAL GLUCOAMYLASES 1181
BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
from the fungus Penicillium verruculosum that secretes an
active complex of cellulases [5]. Insufficient amylolytic
activity of glucoamylases has been detected in culture
fluid (CF) of P. verruculosum during cultivation of the
fungus in starch�containing media. To obtain glucoamy�
lases and to study their properties, genes of two secreted
GlAs from P. verruculosum were expressed in a Penicillium
canescens strain under control of the inducible xylanase
promoter.
The goal of the present work was to obtain and char�
acterize new enzyme preparations from a recombinant
strain of P. canescens with heterologous expression of two
GlAs of the fungus P. verruculosum and to compare their
properties with those of glucoamylases from A. awamori
and A. niger, and also to obtain homogenous GlAs of P.
verruculosum and to compare their properties with those
of GlAs from A. niger and A. awamori.
MATERIALS AND METHODS
Strains of microorganisms and enzyme preparations.The micromycete fungus P. canescens RN3�11�7
(niaD–) recipient strain auxotrophic on gene niaD– that
encodes synthesis of nitrate reductase was used for plas�
mid transformation. Genomic DNA of the fungus P. ver�
ruculosum was used as a template for amplification of
genes aglu and gla that encode two GlAs with molecular
weights of 88 and 68 kDa. As a result of plasmid co�trans�
formation of the recipient strain P. canescens RN3�11�7
with plasmid pSTA10 that carries a gene of nitrate reduc�
tase niaD and provides complementation of the gene
niaD–, P. canescens transformants secreting extracellular
heterologous GlA of P. verruculosum, were obtained.
Dry enzyme preparations were obtained by
lyophilization of CF of the recombinant strain P.
canescens D8 with heterologous GlA gene (aglu) from P.
verruculosum with molecular mass of 88 kDa and CF of
recombinant strain P. canescens B2 with heterologous
GlA gene (gla) from P. verruculosum with molecular mass
of 68 kDa. The CF was prepared in 3�liter fermentation
units (Prointech, Russia) in the Center of Collective Use
(CCU) at INBI RAS in medium composed of soybean
husk (4.5%), maize extract (5%), and KH2PO4 (2.5%).
Fermentation was carried out for 144 h at 28°C and
pH 4.5�5.0. The GlAs from A. niger and A. awamori pre�
viously isolated in the Laboratory of Enzyme
Biotechnology of INBI RAS were used for comparison.
Enzyme preparations Glucavamorin G18x (Sibbio�
pharm, Russia; produced by A. awamori) and Glucostar
400L (DYADIC INT., USA; produced by A. niger) were
used as commercial glucoamylase preparations.
Substrates. The following substrates were used to
determine activity of complex and individual enzymes:
soluble potato starch (REACHEM, Russia), soluble
potato amylase (Sigma, USA), soluble potato amy�
lopectin (Serva, Germany), xylan (Sigma), sodium salt of
carboxymethylcellulose (Sigma), maltose (REACHEM),
maltohexaose (Sigma), and also the synthetic substrate p�
nitrophenyl�α�D�glucopyranoside (PNPG) (Sigma).
Isolation and purification of glucoamylases. GlA
preparations from P. canescens were isolated in three
stages: preliminary purification, anion�exchange chro�
matography, and hydrophobic chromatography. The
enzyme preparations that were preliminarily precipitated
by ammonium sulfate (80% saturation at 25°C) were then
dissolved in 0.1 M Na�acetate buffer, pH 5.0. Further, the
preparations were desalted on a column with Biogel�P4
(Bio�Rad, USA) by liquid chromatography using an
ACTA�purifier (GE Healthcare, Sweden) with 0.02 M
bis�Tris/HCl buffer, pH 6.8, as eluent under flow rate
1 ml/min. For anion�exchange chromatography, a
Source 15Q HR 16/5 column (Pharmacia) was used. A
sample containing 100 mg of protein was applied on the
column equilibrated with 0.02 M bis�Tris/HCl buffer,
pH 6.8. Bound protein was eluted by a NaCl concentra�
tion gradient from 0 to 0.4 M under flow rate 5 ml/min
(gradient volume 300 ml). The buffer in collected frac�
tions was replaced on a column with Biogel P4 (BioRad).
Hydrophobic chromatography of fractions that were
active in relation to soluble starch was carried out on a
column with Source 15 Isopropyl (Pharmacia) carrier
equilibrated with 1.7 M (NH4)2SO4 solution in 0.05 M
Na�acetate buffer, pH 5.0. Bound protein was eluted by
buffer containing a linearly decreased concentration of
ammonium sulfate under flow rate 2 ml/min (gradient
volume 240 ml). The collected fractions were desalted on
a column with Sephadex G�25 (Pharmacia) with 0.1 M
Na�acetate buffer, pH 5.0, as eluent at flow rate
0.5 ml/min.
Protein concentration in samples was determined by
the Lowry method with bovine serum albumin as stan�
dard [8] or by absorbance at 280 nm.
Identification of glucoamylases by mass�spectrome�try. The GlAs were identified according to a standard
procedure [6]. A fragment of a protein band after SDS�
PAGE that corresponded to each enzyme was treated by
genetically modified trypsin (Promega, USA) at concen�
tration 5 ng/ml in 0.05 M NH4HCO3. The resulting pep�
tides were extracted with 20% solution of acetonitrile
containing 0.1% trifluoroacetic acid. Mass�spectrometry
of trypsin hydrolysates was performed on an Ultraflex II
TOF/TOF mass spectrometer (Bruker Daltonics GmbH,
Germany) at INBI RAS. The determined weights of pep�
tides were matched with theoretically calculated weights
of putative peptides in accordance with the known amino
acid sequences of the GlAs from P. verruculosum and A.
awamori.
Biochemical characteristics of recombinant andnative glucoamylases. Analytical isoelectrofocusing of
proteins was performed on a Model 111 Cell (BioRad) in
accordance with the user instruction manual for the
1182 VOLKOV et al.
BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
apparatus. Protein electrophoresis under denaturing con�
ditions (in the presence of SDS) was performed in 12%
polyacrylamide gel on Mini Protean device (BioRad).
Protein bands were stained in the gels with Coomassie
Brilliant Blue R�250 (Ferak, Germany). The following
protein kits from Sigma were used: MW�SDS�200 (30�
200 kDa) and IEF�M1A (pI of 2.80�6.55) for SDS�
PAGE and isoelectrofocusing, correspondingly.
Determination of glucoamylase activity. GlA activity
in relation to starch, amylose, and amylopectin was
determined using initial rates of formation of reducing
sugars (RS) by a modification of the Somogyi–Nelson
method [7] and also by the glucose oxidase–peroxidase
method in case of starch and oligosaccharides such as
maltose and maltohexaose [8].
Enzyme activity in relation to PNPG was deter�
mined using the initial rate of p�nitrophenol formation
[9]. A 0.05 M substrate solution in 0.1 M Na�acetate
buffer, pH 5.0, was incubated with the enzyme at 40°C for
10 min. The reaction was stopped by addition of 1 M
solution of Na2CO3. Formation of p�nitrophenol in solu�
tion was determined spectrophotometrically at the wave�
length of 400 nm. Enzyme activity was expressed in inter�
national units per mg of protein (one unit corresponds to
the amount of enzyme that hydrolyzes 1 µmol of substrate
per minute).
Study of kinetic parameters. Kinetic parameters of
the purified GlAs were determined in relation to starch.
To calculate the Michaelis constant (Km), an initial rate of
starch hydrolysis was three times measured at the sub�
strate concentrations from 0.5 to 10 g/liter. Km and Vmax
values were determined using the initial rates of hydroly�
sis in double�reciprocal Lineweaver–Burk coordinates.
Exhaustive hydrolysis of starch, amylopectin, andamylose by glucoamylases. Homogenous enzymes or
enzyme preparation solutions were incubated with starch,
amylopectin, or amylose. Substrates were hydrolyzed for
one day under the following conditions: substrate con�
centration, 5 g/liter; pH 5.0; 0.1 M Na�acetate buffer;
temperature of 50°C. Dosage of enzymes or enzyme
preparations was chosen as 6 units of GlA per gram of dry
substrate. During hydrolysis, samples were taken from the
reaction mixture and then were boiled on a water bath for
5 min to stop the reaction and were then centrifuged to
remove the denatured protein, and the concentration of
RS and glucose was determined in the samples.
Chromatographic determination of composition ofstarch and maltohexaose hydrolysis products. Hydrolytic
products were qualitatively determined with an Agilent
1100 HPLC system (Agilent, USA) with a Coulochem III
electrochemical detector (ESA, USA) on a CarboPac
PA�100 (Dionex, USA) column. Samples were dissolved
in 100 mM NaOH, and isocratic elution mode was used
during the first 10 min with subsequent linear gradient
elution up to 40 mM NaOH during 30 min. The eluent
was detected using a pulsating ammeter (+100 mV for
400 ms, –2000 mV for 20 ms, +600 mV for 30 ms,
–100 mV for 50 ms). The system was calibrated with D�
glucose, D�maltose, and maltohexaose standards to iden�
tify sugars and to determine their concentration in the
analyzed samples.
Changes in molar mass distribution (MMD) of
starch in the course of hydrolysis were determined by
high�pressure gel filtration on a Bio�Gel TSK 30 XL col�
umn (0.78 × 30 cm; Bio�Rad, Japan). A solution of
0.05 M Na�acetate buffer, pH 5.0, containing 0.1 M
NaCl and 0.05% NaN3 (flow rate of 0.5 ml/min) was used
as the eluent. The column was calibrated with dextrans
(20�250 kDa; Pharmacia).
RESULTS AND DISCUSSION
Electrophoretic analysis of enzyme preparations andMALDI�TOF mass spectrometry. Analysis of CF
obtained with the new P. canescens transformants by elec�
trophoresis under denaturing conditions in the presence
of SDS (SDS�PAGE) (Fig. 1) showed notable increase in
intensity of a protein band that corresponds to molecular
weight of about 90 kDa, close to that of GlA of P. verru�
culosum (88 kDa) in the case of the D8 strain in compar�
ison with the recipient strain P. canescens RN3�11�7. In
the case of the B2 strain, an increase in protein band
intensity was observed in the area corresponding
to molecular weight of about 70 kDa (may correspond to
GlA with molecular weight of 68 kDa). The elec�
trophoretic data were then confirmed by determination of
specific activity of GlA in CF of the selected transfor�
mants of 150 and 130 U/ml towards soluble potato starch,
respectively.
Results of electrophoretic analysis of Glucavamorin
G18x and Glucostar 400L preparations obtained from the
fungi A. awamori and A. niger, correspondingly, are shown
on Fig. 1. It is seen that the commercial preparations
contain GlAs with molecular weights of 80 and 140 kDa
(according to the literature data, the existence of two
forms of GlA in these microorganisms is a result of
mRNA�splicing of gla gene [10]).
Protein bands obtained by electrophoresis of CF of
P. canescens D8 and B2 clones (with molecular weights of
88 and 68 kDa) and bands that corresponded to molecu�
lar weights of 80 and 140 kDa (for A. awamori and A.
niger) were treated with trypsin, and the resulting
hydrolysates were subjected to MALDI�TOF mass spec�
trometry. Peptide map analysis along with comparison of
data on molecular weights of peptides obtained experi�
mentally and calculated theoretically confirmed that the
protein bands from P. canescens corresponds to two GlAs
from P. verruculosum, whereas the protein bands
with molecular weights of 80 and 140 kDa in preparations
Glucavamorin G18x and Glucostar 400L correspond to
GlAs from A. awamori and A. niger, respectively. Mass
PROPERTIES OF RECOMBINANT FUNGAL GLUCOAMYLASES 1183
BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
spectrometry of each sample identified more than five
peptides whose amino acid sequences matched those of
the studied GlAs (data not shown), and this allowed
determination that the studied proteins are members of
15th family of glycosylhydrolases.
Using both selected P. canescens strains, D8 and B2,
fermentation was performed in 3�liter fermentation units
and dry enzyme preparations that contained the recombi�
nant GlAs of P. verruculosum with molecular weights of
88 (preparation PCA�AGLU�D8) and 68 kDa (prepara�
tion PCA�GA�B2) were obtained. As seen from Table 1,
all the enzyme preparations (both laboratory and com�
mercial) exhibited activity in relation to potato starch
(1750�3439 U/g for the dry enzyme preparations and
7680�10300 U/ml for the enzyme solution preparations).
Glucavamorin G18x and Glucostar 400L demonstrated
the maximal GlA activity, and this confirmed high con�
tent of the studied proteins in these preparations.
Fig. 1. SDS�PAGE: 1) enzymatic complex secreted by P. canescens recipient strain RN3�11�7; 2) enzymatic complex secreted by P. canescens
strain D8 (88 kDa GlA from P. verruculosum); 3) enzymatic complex secreted by P. canescens strain B2 (68 kDa GlA from P. verruculosum);
4) Glucavamorin G18x; 5) Glucostar 400L.
90
50
36
27
20
118
М 1 2 3 4 М М 5
66
45
36
29
24
116
66
45
36
29
24
116
Enzyme preparation
РСА�AGLU�D8
РСА�GA�B2
Glucavamorin G18x
Glucostar 400L
CMCase
0.6
0.6
0.9
0.2
Table 1. Specific activities of glucoamylase enzyme preparations
xylanase
180
166
1.4
0.06
α�glucosidase
0.01
0.01
0.05
0.03
glucoamylase
6.8
4.1
56.9
35.5
Specific activity, U/mg
1184 VOLKOV et al.
BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
Isolation of glucoamylases from enzyme complexessecreted by P. canescens, A. awamori, and A. niger.Enzyme preparations preliminarily purified from non�
protein admixtures (insoluble substances, carbohydrates,
pigments, etc.) were subjected to anion�exchange chro�
matography on Source 15Q at pH 6.8. In the case of the
P. canescens D8 preparation, the GlA activity in relation
to starch was exhibited by the major fraction eluted at the
beginning part of the main gradient of NaCl (0.095�0.1 M
NaCl; see Fig. 2a), while in case of P. canescens B2 prepa�
ration starch hydrolytic activity was demonstrated by the
major fraction in the middle part of the main gradient
Fig. 2. a) Isolation of 88 kDa GlA from P. verruculosum by anion�exchange chromatography on Source 15Q at pH 6.8. b) Isolation of 68 kDa
GlA from P. verruculosum by anion�exchange chromatography on Source 15Q at pH 6.8. c) SDS�PAGE of purified GlAs: 1) 88 kDa GlA from
P. verruculosum; 2) 68 kDa GlA from P. verruculosum; 3) 80 kDa GlA from A. awamori; 4) 140 kDa GlA from A. awamori; 5) 80 kDa GlA from
A. niger; 6) 140 kDa GlA from A. niger.
2.0
1.8
0.8
0
А280
10 20 30 40 50
a
1.6
1.4
1.2
1.0
0.6
0.40.2
GA 88 kDa
1.0
0.4
0.8
0.6
0.2
0.0
[NaC
l],
M
0.8
0
А280
50 100 150 200 250
b
0.6
0.4
0.2
GA 68 kDa
1.0
0.4
0.8
0.6
0.2
0.0
[NaC
l],
M
300
Eluent volume, ml
М 1 2 М 3 4 5 6
260140
35
10070
50
40
25
20
15
116
66
45
36
29
24
c
PROPERTIES OF RECOMBINANT FUNGAL GLUCOAMYLASES 1185
BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
from 0.15 to 0.18 M NaCl (see Fig. 2b). According to
SDS�PAGE data, GlA�containing fractions of the D8
strain also contained significant amount of contaminat�
ing proteins. So, they were subjected to further separation
by hydrophobic chromatography on a Source 15
Isopropyl column. The GlA from the enzymatic complex
produced by the B2 strain and with molecular weight of
68 kDa (theoretically) and pI 3.0 was almost homoge�
neous (purity degree was more than 90%), so it was not
subjected to further purification. During separation of
GlA(D8)�containing fraction, the major part of the
sought enzyme was detected in the second fraction of an
ammonium sulfate gradient that contained a single pro�
tein (degree of homogeneity >95%) with molecular
weight of 88 kDa and pI 3.5. Figure 2 demonstrates data
of SDS�PAGE of purified recombinant GlA from P. ver�
ruculosum along with purified 80 and 140 kDa (pI 3.0)
GlAs from A. awamori, and also 80 and 140 kDa (pI 3.0)
GlAs from A. niger.
Substrate specificity of homogeneous glucoamylases.The substrate specificities of the homogeneous GlAs are
presented in Table 2. In comparison with the 88 kDa GlA,
the 68 kDa GlA from P. verruculosum exhibited higher
specific towards soluble starch, amylopectin, and amy�
lose – 14.4, 13.5, and 5.4 U/mg protein, respectively.
Moreover, the specific activity of the 88 kDa GlA from P.
verruculosum was 2�3 times higher than that of the 80 kDa
GlA from A. awamori and was quite similar to that of
80 kDa GlA from A. niger. All the studied GlAs (P. verru�
culosum and Aspergillus) exhibited significantly lower
activity towards maltose (relatively high maltase activity
was demonstrated by the 140 kDa GlAs from A. awamori
and A. niger). The activity of glucoamylases from fungi of
the Aspergillus genus towards synthetic substrate PNPG
was weak and did not exceed 0.06 U/mg, whereas P. ver�
ruculosum GlA activity towards this substrate was not
detected.
Mechanism of action of glucoamylase on maltohexa�ose and starch. During maltohexaose hydrolysis by the
68 kDa GlA from P. verruculosum, in addition to glucose,
successive accumulation of first maltopentaose and then
maltotetraose and other low molecular weight products
(maltotriose, maltose) (Fig. 3) was observed. Successive
accumulation of products differed from each other by one
glucose residue, suggesting that the mechanism of action
of the 68 kDa GlA from P. verruculosum is of exo�depoly�
merization type. Similar data were obtained for the
88 kDa GlA from P. verruculosum and also for the GlAs
from A. awamori and A. niger.
Analysis of data on MMD changes under action of
GlA from P. verruculosum, A. awamori, and A. niger on
potato starch obtained by high pressure gel filtration con�
firmed this exo�polymerization mechanism of enzymatic
action. During prolonged hydrolysis of starch by all the
studied GlAs, a similar picture was observed – decrease in
area under the peak corresponding to initial substrate
with no changes in retention time, while the area under
the peak corresponding to low molecular weight product
of the reaction, glucose, increases. Changes in starch
MMD under the action of the 88 kDa GlA from P. verru�
culosum are presented in Fig. 4. HPLC analysis of
exhaustive hydrolysis products showed glucose to be the
only low molecular weight product of reactions catalyzed
by all the GlAs.
Rate and degree of hydrolysis of starch, amylopectin,and amylose by glucoamylases. Dosage of enzymes was
calculated on the basis of GlA activity taken to be equal to
6 U per gram of substrate (starch, amylose, or amy�
140 kDa
18.2
6.8
19.5
2.3
0.05
68 kDa
14.4
5.4
13.5
0.16
0
Substrate
Soluble starch (potato)
Soluble amylose (potato)
Soluble amylopectin (potato)
Maltose
PNPG
140 kDa
19.4
7.1
20.6
2.3
0.05
Table 2. Specific activities of glucoamylases in relation to different substrates
80 kDa
5.0
3.7
5.2
0.87
0.05
80 kDa
2.2
1.6
1.2
0.33
0.06
88 kDa
4.75
3.6
4.8
0.34
0
A. nigerA. awamoriP. verruculosum
Specific activity, U/mg
рН 4.7 30°C
рН 5.0 40°C
1186 VOLKOV et al.
BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
lopectin). The concentration of starch, amylopectin, or
amylose in the reaction mixture was 5 g/liter. The sub�
strates were hydrolyzed in 0.1 M sodium�acetate buffer at
pH 4.7 and 58°C for 24 h.
From the four enzyme preparations studied (PCA�
AGLU�D8, PCA�GA�B2, Glucavamorin G18x, and
Glucostar 400L), PCA�GA�B2 was recognized as the best
on the basis of glucose yield at early stages of potato
starch hydrolysis (for 3 h) (Fig. 5a). The degree of starch
conversion by this preparation was 46%, which was by 4
and 31% higher in comparison with the Glucostar 400L
and Glucavamorin G18x preparations, correspondingly.
During exhaustive starch hydrolysis, the Glucostar 400L
and PCA�AGLU�D8 enzyme preparations were the most
effective. The yield of glucose under the action of these
preparations was by 28 and 17% higher than that under
the action of Glucavamorin G18x.
In hydrolysis of potato amylopectin, PCA�GA�B2
and PCA�AGLU�D8 demonstrated the highest efficiency
in glucose release at initial stages of hydrolysis (3 h). As
seen from Fig. 5b, these preparations provided maximal
degree of exhaustive hydrolysis of amylopectin of 54 and
37%, correspondingly, which on average was higher by 25
and 8%, correspondingly, than that obtained with the use
of the commercial preparation Glucostar 400L, and glu�
cose yield was higher by 28 and 11% than that in case of
the commercial preparation Glucavamorin G18x.
PCA�AGLU and Glucostar 400L enzyme prepara�
tions were the best in hydrolysis of potato amylose both at
early stages and in exhaustive hydrolysis. Degrees of amy�
lose conversion in 3�h hydrolysis by these preparations
were 6.4 and 3.2%, respectively, while in exhaustive
hydrolysis (24 h) they were 8.4 and 5.0%, corresponding�
ly. The results are presented in Fig. 5c.
Kinetic parameters of glucoamylases. Kinetic param�
eters of hydrolysis of starch by all the studied homoge�
Fig. 3. Products of maltohexaose (M6) hydrolysis by the 68 kDa
GlA from P. verruculosum (standards: G, glucose; M, maltose;
M3, maltotriose; M4, maltotetraose; M5, maltopentaose; M6,
maltohexaose). Designations (duration of hydrolysis and % bio�
conversion, respectively): a) standards; b) 10 min – 2%; c)
30 min – 9%; d) 60 min – 23%; e) 100 min – 38%; f) 180 min –
54%; g) 24 h – 100%.
GM
0
a
5 10 15 20
Retention time, min
M3M4 M5
M6
b
c
d
e
f
g
Fig. 4. Changes in MMD of potato starch during enzymatic
hydrolysis with 88 kDa GlA from P. verruculosum. Designations
(duration of hydrolysis): a) starch before hydrolysis; b�f) starch
after hydrolysis for 10 (b), 40 (c), 60 (d), 100 (e), and 180 min
(f); g) glucose (as a standard).
a
10 20
Retention time, min
b
c
d
e
f
g
PROPERTIES OF RECOMBINANT FUNGAL GLUCOAMYLASES 1187
BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
neous glucoamylases are shown in Table 3. The 68�kDa
GlA from P. verruculosum and the 80� and 140�kDa GlAs
from Aspergillus had the highest values of Km (0.4�
1.1 g/liter), while the 88�kDa GlA from P. verruculosum
had much lower Km value of 0.13 g/liter. The highest kcat
values in the case of hydrolysis of starch were observed for
the 68�kDa GlA from P. verruculosum and the two 140�
kDa GlAs from A. niger and A. awamori. For the two 80�
kDa GlAs from A. niger and A. awamori the kcat values
were 5 times smaller, and in case of the 88�kDa GlA from
Fig. 5. a) Kinetics of glucose accumulation during hydrolysis of soluble potato starch. b) Kinetics of glucose accumulation during soluble pota�
to amylopectin hydrolysis. c) Kinetics of glucose accumulation during potato amylase hydrolysis. 1) 68 kDa GlA from P. verruculosum PCA�
GA�B2; 2) homogeneous 68 kDa GlA from P. verruculosum; 3) 88 kDa GlA from P. verruculosum PCA�AGLU�D8; 4) homogeneous 88 kDa
GlA from P. verruculosum; 5) homogeneous 80 kDa GlA from A. awamori; 6) homogeneous 140 kDa GlA from A. awamori; 7) homogeneous
80 kDa GlA from A. niger; 8) homogeneous 140 kDa GlA from A. niger; 9) Glucavamorin G18x; 10) Glucostar 400L.
4.0
3.5
a
3.0
2.5
0 200 400 600 800 1000
Time, min
2.0
1.5
1.0
0.5
12345
678910
12345678910
1200 1400 1600
1
23456
78
9
10
0 200 400 600 800 1000 1200 1400 1600
0 200 400 600 800 1000 1200 1400 1600
4.0
3.5
b
3.0
2.5
2.0
1.5
1.0
0.5
0.40
0.35
c
0.30
0.25
0.20
0.15
0.10
0.05
0.45
[Glu
cose
], g
/lite
r
1188 VOLKOV et al.
BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
Fig. 6. Dependence on pH at 30°C (a) and on temperature at pH 4.7 (b) of activities of studied fungal glucoamylases in relation to soluble
starch: 1) 80�kDa GlA from A. awamori; 2) 140�kDa GlA from A. awamori; 3) 80�kDa GlA from A. niger; 4) 140�kDa GlA from A. niger; 5)
88�kDa GlA from P. verruculosum; 6) 68�kDa GlA from P. verruculosum.
120
100
a
80
60
40
20
0
1
2
3
4
5
6
1.5 2.5 3.5 4.5 5.5 6.5 7.5
рН
bRe
lativ
e a
ctiv
ity,
%
25 45 55 65 75 85 95
Т°C
35
1
2
3
4
5
6
120
100
80
60
40
20
0
PROPERTIES OF RECOMBINANT FUNGAL GLUCOAMYLASES 1189
BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
P. verruculosum – 6.5 times smaller. On the whole, the
ranges of Km and kcat values for the studied GlAs were typ�
ical for these enzymes: 0.13�1.1 g/liter and 79�580 min–1,
correspondingly [11].
pH and temperature dependences of activity of theglucoamylases. Figure 6 depicts the pH profiles of all the
studied GlAs. There are two groups with different pro�
files. One group is represented by two GlAs from P. verru�
culosum, which exhibited maximum activity at pH 4.0 in
relation to soluble starch. These GlAs also exhibited
�50% activity in relation to starch at pH values in the
range from 2.2 to 5.4. Another group is represented by the
GlAs from fungi of the Aspergillus genus that demonstrat�
ed maximum activity in relation to starch at pH 4.0�4.5
and had �50% activity towards starch in the pH range
from 3.0 to 5.0. Temperature optima for the GlAs were
also different. The Aspergillus GlA and the P. verruculo�
sum 88�kDa GlA demonstrated maximum activity at 60�
65°C, while the P. verruculosum 68�kDa GlA was most
active at 55�60°C.
Stability of the glucoamylases. The ability to retain
activity for extended times a high temperatures (40�70oC)
was studied at pH 4.7 according to conditions necessary
for determination of GlA activity (in accordance with the
national standard, GOST 20264.4�89). All the enzymes
retained 100% activity at 40 and 50°C after incubation for
3 h. At temperature of 60°C, the GlAs from A. awamori
and A. niger retained 32�40% activity, while the 88�kDa
GlA from P. verruculosum retained 40% activity, and the
66�kDa GlA from P. verruculosum retained only 20% of
the initial enzymatic activity. Enzymatic activity drasti�
cally decreased with further increase in incubation tem�
perature up to 70°C, and the half�time of inactivation for
all the studied GlAs did not exceed 15 min.
This work was performed under financial support of
the federal program “Research and Development in
Priority Fields of the Scientific and Technological
Complex of Russia for 2007�2012”, state contract No.
16.522.12.2003, May 31, 2011.
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Producing strain
P. verruculosum
P. verruculosum
A. awamori
A. awamori
A. niger
A. niger
Molecular weightof enzyme, kDa
88
68
80
140
80
140
kcat, min–1
79
580
110
500
100
514
Km, g/liter
0.13
0.43
0.96
0.45
1.1
0.73
Table 3. Kinetic parameters of potato starch hydrolysis
catalyzed by glucoamylases from P. verruculosum, A.
niger, and A. awamori