Comparative study of biochemical properties of glucoamylases from the filamentous fungi Penicillium...

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: [email protected] Moscow State University, Faculty of Chemistry, Leninsky Gory 1, 119992 Moscow,

Russia; fax: +7 (495) 939�0997; E�mail: [email protected]

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.


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



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.


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



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.


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



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.


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



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.

REFERENCES

1. Polaina, J., and Maccabe, A. P. (2007) Industrial Enzymes:

Structure, Function and Application, Springer, Dordrecht.

2. Gracheva, I. M., and Krivova, A. Yu. (2000) Technology of

Enzymatic Preparations [in Russian], Elevar, Moscow.

3. Frandsen, T. P., Fierobe, H. P., and Svensson, B. (1999)

Engineering Specificity and Stability in Glucoamylase from

Aspergillus niger in Protein Engineering in Industrial

Biotechnology (Alberghin, L., ed.), Harwood Academic,

Amsterdam, pp. 189�206.

4. Reilly, P. J. (1999) Starch, 51, 269�274.

5. Osipov, D. O., Rozhkova, A. M., Matys, V. J., Koshelev, A.

V., Okunev, O. N., Rubtsova, E. A., Pravilnikov, A.

G., Zorov, I. N., Sinitsyna, O. A., Oveshnikov, I.

N., Davidov, E. R., and Sinitsyn, A. P. (2011) Catalysis in

Industry, 3, 34�40.

6. Gusakov, A. V., Semenova, M. V., and Sinitsyn, A. P. (2010)

Mass�spectrometry (Moscow), 7, 5�20.

7. Sinitsyn, A. P., Chernoglazov, V. M., and Gusakov, A. V.

(1990) in Advances in Science and Technology. Biotechnology

Ser. (Varfolomeeva, S. D., ed.) [in Russian], VINITI,

Moscow, Vol. 25, pp. 30�37.

8. Volkov, P. V., Rozhkova, A. M., Pravilnikov, A. G.,

Andrianov, P. M., Dotsenko, G. S., Bekkarevich, A. O.,

Koshelev, A. V., Okunev, O. N., Zorov, I. N., and Sinitsyn,

A. P. (2012) Appl. Biochem. Microbiol. (Moscow), 48, 66�73.

9. Polygalina, G. V., Cherednichenko, V. S., and Rimareva, L.

V. (2003) Determination of Enzyme Activity [in Russian],

De�Li print, Moscow, pp. 147�169.

10. Nunberg, J. H., Meade, J. H., Cole, G., Lawyer, F. C.,

McCabe, P., Shweickart, V., Tal, R., Wittman, V. P.,

Flatgaard, J. E., and Innis, M. A. (1984) Mol. Cell. Biol., 4,

2306�2315.

11. Siddhartha Kumar, M., Chandana Lakshmi, M. V. V.,

Sridevi, V., and Manasa, M. (2012) IJSID, 2, 74�83.

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

Comparative study of biochemical properties of glucoamylases from the filamentous fungi Penicillium...

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