Biochimica et Biophysica Acta 1750 (2005) 61 – 68

http://www.elsevier.com/locate/bba

Deglycosylation of glucoamylase from Aspergillus niger:

Effects on structure, activity and stability
Javad Jafari-Aghdama, Khosro Khajehb, Bijan Ranjbarb, Mohsen Nemat-Gorgania,c,*
a
Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran
b
Department of Biochemistry, Faculty of Basic Science, Tarbiat Modarres University, P.O. Box 14115-175, Tehran, Iran
c
Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA

Received 28 December 2004; received in revised form 17 March 2005; accepted 22 March 2005
Available online 15 April 2005

Abstract

A comparative structure – function study was performed to establish possible roles of carbohydrates in stabilization of glycoproteins, using
glucoamylase (GA) as a model system. In addition to kinetic properties, stability toward elevated temperatures, extremes of pH, high salt
concentrations together with circular dichroism, intrinsic/extrinsic fluorescence studies, proteolysis and affinity for interaction with
hydrophobic ligands were investigated. Related to all the main properties examined, with one exception, glycosylation provided
improvement in functional characteristics of the enzyme, especially in relation to its thermostability. Results are explained in terms of
provision of stabilizing intermolecular interactions by the sugar molecules. The improvement in protein rigidity together with reduction of
surface hydrophobicity appear to be especially important in relation to prevention of aggregation, an important mechanism of irreversible
thermoinactivation, occurring at elevated temperatures.
D 2005 Elsevier B.V. All rights reserved.

Keywords: Glucoamylase; Irreversible thermoinactivation; Aggregation; Refolding; Deglycosylation

1. Introduction
Glycosylation is one of the major naturally occurring
modifications of the covalent structure of proteins. Most
secretary proteins become glycosylated as soon as the
growing polypeptide chains enter the endoplasmic reticulum,
before the final native-like folded state is attained. Accor-
dingly, the reasons for the occurrence of such events and their
consequences in relation to structure and function of proteins
have been investigated extensively. There are two different
types of protein glycosylation: O-glycosylation at hydroxyl
groups of serine and threonine residues and N-glycosylation
at asparagine residues in the consensus sequence of Asn-
X-Ser/Thr. The biological functions of glycoproteins are well
established but the role that carbohydrates play in these
* Corresponding author. Institute of Biochemistry and Biophysics,
University of Tehran, P.O. Box 13145-1384, Tehran, Iran. Tel.: +1 650
812 1961; fax: +1 650 812 1975.
E-mail address: mohsenn@stanford.edu (M. Nemat-Gorgani).
1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2005.03.011
functions is, for the most part, unclear with contradictory
reports frequently observed in the literature [1– 6]. Glucoa-
mylase, also known as amyloglucosidase-EC 3. 2. 1. 3, is an
exo-amylase which removes glucose from the non-reducing
ends of starch and a variety of other carbohydrate polymers
and oligomers [7]. The enzyme extracted from the fungus
Aspergillus niger has various industrial applications and is
extensively employed in hydrolysis of starch, production of
glucose, high fructose syrups and in alcohol fermentation
[8,9]. It is believed to break down a (1, 4) bonds more
rapidly than a (1, 6). Although in the case of a (1, 6) bonds,
the rate of activity is only 0.2% of a (1, 4), even this amount
of activity is considered sufficient for the purpose of
industrial applications [10 –13]. The enzyme is produced
by a variety of microorganisms, with the ones extracted from
Aspergillus niger, Aspergillus awamori and Rhizopus oryzae
being considered as most important [14]. All enzymes within
glucoamylase family consist of a catalytic domain and a
starch-binding domain which are attached to each other by
means of an O-glycosylation linker [10]. The enzyme
62 J. Jafari-Aghdam et al. / Biochimica et Biophysica Acta 1750 (2005) 61 – 68
contains a very specific carbohydrate region consisting of 30
chains in the form of di- or trisaccharides (mainly mannose)
[15]. In the present study, GA was chosen as a model
glycoprotein and a comparative study was performed using
the native protein structure and a deglycosylated preparation
obtained by the use of a-mannosidase. Results indicate that
upon deglycosylation, flexibility of the enzyme is enhanced
and its thermostability diminished. Studies on irreversible
thermoinactivation combined with elucidation of the struc-
tural properties of the two forms suggest that loss of sugar
molecules result in exposure of hydrophobic residues in the
protein molecule, thereby facilitating aggregation of the
more flexible structure at high temperatures.
2. Materials and methods
2.1. Materials
Glucoamylase from Aspergillus niger, a-mannosidase,
m-aminophenylboronic acid, concanavalinA-S4B and sub-
tilisin were obtained from Sigma (St. Louis, MO, USA).
All other chemicals were obtained from Merck
(Darmstadt, Germany) and were reagent grade.
2.2. Determination of enzymatic activity and protein
concentration
Glucoamylase was assayed colorimetrically at room
temperature, using soluble starch as substrate in 16 mM
sodium acetate, pH 4.8. Concentration of reducing sugars
obtained from the catalyzed reaction was measured by the
dinitrosalicylic acid method according to Bernfeld [16].
Protein concentration was determined by the Lowry et al.
[17] and Bradford methods [18].
2.3. Deglycosylation of glucoamylase
Glucoamylase was treated with a-mannosidase using a
0.1-M sodium acetate buffer (pH 4.5), containing 0.1 mM
ZnCl2 and 0.1% (V/V) h-mercaptoethanol, at 37-C for 24 h.
Three units of a-mannosidase were used for each 1 mg of
GA. The extent of carbohydrate removal was tested by the
use of SDS-PAGE as described earlier [15,19,20,21]. The
deglycosylated form showed enhanced mobility, presu-
mably due to higher extents of SDS-binding [22]. This
was in accord with earlier reports demonstrating a prepon-
derance of carbohydrate moieties in hydrophobic regions of
glycoproteins [15]. A greater affinity of the deglycosylated
form for interaction with hydrophobic adsorbents further
supported this conclusion (Jafari-Aghdam, J. and Nemat-
Gorgani, M., unpublished data). All attempts involving use
of size-exclusion chromatography failed in separating the
two forms. Also the fraction of the mannosidase treated
preparation which did not bind to ConA Sepharose 4B was
not homogeneous on the gel. However, the flow through
from a m-aminophenylboronic acid affinity column [23]
provided a sharp band corresponding to the deglycosylated
form (Fig. 1). This preparation was subsequently used as the
deglycosylated form in the present work.
2.4. Fluorescence measurements
Fluorescence studies was carried out on a Perkin-Elmer
luminescence spectrometer LS 50 B. Intrinsic fluorescence
was determined using 50 Ag/ml protein and an excitation
wavelength of 280 nm. Emission spectra were recorded
between 300 and 400 nm. Extrinsic fluorescence studies
were carried out as outlined earlier [24 – 26], using ANS (8-
anilino-1-naphthalene-sulfonate) as a fluorescence probe.
All experiments were carried out at 25-C with ANS and
protein concentrations of 50 AM and 50 Ag/ml, respectively,
in 16 mM sodium acetate buffer. An excitation wavelength
of 350 nm was used.
2.5. Fluorescence quenching
Fluorescence quenching was carried out by the addition
of 2 M acrylamide to protein solutions (50 Ag/ml).
Quenching data were analyzed in terms of the Stern-Volmer
constant, K sv, which was calculated from the ratio of the
unquenched and the quenched fluorescence intensities,
F o/F, using the relationship F o/F = 1+K sv [Q].
Here Q is the molar concentration of the quencher [27].
The intrinsic protein fluorescence F was corrected for the
acrylamide inner filter effect, f, the latter being defined as
f = 10
q[Q]/2, using an extinction coefficient q of 4.3 M
1
cm
1 for acrylamide at 280 nm.
2.6. Proteolytic cleavage
Proteolytic cleavage of native and deglycosylated forms
of GA was performed by treating the enzyme (0.1 mg/ml)
Fig. 1. SDS-PAGE of native and deglycosylated GA. Lanes: 1, native; 2, a-
mannosidase; 3, GA digest consisting of native, deglycosylated forms and
a-mannosidase; 4, unbound fraction of GA digest on ConA-S4B; 5,
unbound fraction of GA digest on m-aminophenylboronic acid affinity
matrix. Further details are provided in Materials and methods.
 J. Jafari-Aghdam et al. / Biochimica et Biophysica Acta 1750 (2005) 61 – 68
with various concentrations of subtilisin, using 50 mM
borate, 10 mM CaCl2, pH 10. Digestion was carried out at
45-C for 6 h and required volumes were removed from the
reaction mixtures for SDS-PAGE.
2.7. Circular dichroism (CD) measurements
CD measurements were conducted using a Jasco (Tokyo
Japan) J-715 spectropolarimeter equipped with a thermo-
statically-controlled cell holder. Tm measurement was done
using a CD spectropolarimeter (Jasco J-715) employing a
protein concentration of 0.1 mg/ml. Thermal unfolding was
monitored by recording the change of the CD signal at 222
nm as a function of sample temperature. The rate of increase
in temperature was adjusted at 1-C/min.
2.8. Heat stability
Native or deglycosylated enzyme preparations (0.1 mg/
ml) in sodium acetate buffer were incubated at different
temperatures. At regular intervals, samples were removed
cooled on ice and the remaining activity determined.
Activity of the same enzyme solution kept on ice throughout
the procedure was considered as control (100%).
2.9. pH stability determination
A mixed buffer containing acetate, Caps and Tris, each
at 40 mM concentration and adjusted to the indicated pH
was used. The procedure involved use of 100 Al of free
and deglycosylated forms (0.1 mg/ml) which were added
to 400 Al of the buffer and incubated for 2 h at 25-C. This
was followed by addition of 50 Al of each of the samples
to 450 Al of 16 mM acetate buffer, pH 4.8 which were
then left for 30 min at room temperature. Activity
determination was subsequently carried out in the usual
manner.
2.10. Aggregation measurements
The extent of aggregation was determined by measuring
turbidity at 415 nm as reported earlier [28 –30]. The protein
solution (0.1 mg/ml in 16 mM sodium acetate) was
incubated at the desired temperature. The increase in
absorbance (apparent) against time was plotted using a
Shimadzu (UV-160) spectrophotometer.
2.11. Determination of deamidation
Production of ammonia during thermoinactivation of
GA was determined by incubating samples of the
enzyme in 16 mM sodium acetate buffer and different
pH in sealed tubes, at 70-C for 15 min. The tubes were
then cooled, opened and the amount of dissolved
ammonia was determined enzymatically using glutamate
dehydrogenase [27,31].
63
3. Results and discussion
It is an accepted fact that protein sequence ultimately
determines its assigned function. To this end, information on
the three-dimensional structure of the macromolecule is
necessary in order to unequivocally determine the specific
contribution of each of its constituent components to its
functional property as a whole. Several roles have been
suggested for the carbohydrate moieties of glycoproteins
among which stabilization of protein conformation [15],
protection from proteolysis [3,5] participation in cell –cell
interactions and protein folding [32] are a few examples.
However, none of these roles was consistently demonstrated
for all glycoproteins. Despite many efforts directed toward
elucidation of the role of carbohydrates in glycoproteins, the
mechanism of stabilization by glycosylation is not entirely
clear. In the present study, GA was treated with alpha-
mannosidase to provide a deglycosylated preparation which
was compared with the native form in relation to a number of
structure – function properties.
3.1. Structure and function of native and deglycosylated GA
No significant changes were observed in the V max, K m,
activation energy (E a) and pH optima of native and
deglycosylated GA (Table 1). These results indicate that
deglycosylation does not affect the active site nor the
mechanism of catalysis. In addition, no detectable differ-
ences in the far-UV CD spectra of the two forms were found
(Fig. 2), thus suggesting that the secondary structure of the
native protein is maintained upon loss of sugar molecules.
This has also been observed for a GA from a different
source than the one used in the present investigation [19].
Circular dichroism spectra (far-UV) of the native and
carbohydrate-depleted GA with endo-B-N-acetylglucosami-
nidase were found to be identical [19].
Another approach taken in this investigation involved
intrinsic fluorescence studies. As indicated in Fig. 3,
enhancement of fluorescence was observed upon deglycosy-
lation. This would result in a different pattern of conforma-
tional changes in the protein structure with respect to
deglycosylation, followed by alteration of the microenviron-
ment of excitable tryptophan residues, as supported by an
obvious change in the fluorescence intensity (Fig. 3). The
Table 1
Kinetic parameters, Stern – Volmer constant and ammonia production for
native and deglycosylated GA
pH(optim.) K m V max Ea Ksv Ammoniaa
GA 4.8 1.2 T 0.1 10 T 0.2 0.0414 0.0235 20 T 1.5
Deglycosylated 4.8 1.1 T 0.2 9.7 T 0.3 0.0397 0.0248 26 T 2.5
GA
K m (mg/ml) and V max (Amol/min) were determined at 25-C using different
concentrations of starch in 16 mM acetate sodium, pH 4.8, by performing the
assays at least in triplicates. K sv and E a are in mol
1 and kcal mol
1,
respectively. For further details, please see Materials and methods.
a
Amol/L in 70-C (10 min)
64 J. Jafari-Aghdam et al. / Biochimica et Biophysica Acta 1750 (2005) 61 – 68

application is dependent on its thermostability. Thermal

stabilities of native and deglycosylated forms of the enzyme
determined at 70-C and three different pH values are
depicted in Fig. 4. As indicated, the native structure is
clearly more stable than the deglycosylated form confirming
the role of carbohydrate moieties in thermostabilization of
the protein structure. A reduction of thermal stability upon
removal of carbohydrate was also evident at other temper-
atures (60 – 65– 75 –80-C, results not shown).
Proteins unfold both reversibly and irreversibly, as
represented by:
N$U!I
where N is the native form of the protein, U and I are the
reversibly- and irreversibly-unfolded forms, respectively
Fig. 2. Far-UV spectra of native and deglycosylated forms of glucoamylase.
[31,42,46]. I arises from modification of U through events
For details, please see Materials and methods.
such as aggregation, covalent modification, peptide bond
hydrolysis and autooxidation [31,34 –37]. There are many
results of fluorescence quenching experiments allow us to
examples in the literature where deglycosylation has clearly
assess the relative solvent exposure of different types of
altered the UYI reaction [38 – 40]. A GA from Rhizopus
fluorophores. The more exposed a fluorophore is, the more
niveus was found to be thermally less stable upon
effective a collisional quencher will be in reducing the
deglycosylation [19], as also observed for the GA used in
fluorescence intensity displayed by that molecule [27,33,34].
the present study. This was shown by both of these proteins
The Stern –Volmer plot for quenching of intrinsic protein
in spite of the fact that the carbohydrate moieties are O-
fluorescence by acrylamide at pH 4.5 was prepared and the
linked in GA from Aspergillus niger and N-linked in the
K sv values were determined (Table 1). The data suggest that
Rhizopus niveus enzyme, and that the two proteins are
the aromatic amino acids are exposed to similar extents in
significantly different in relation to their amino acid and
native and deglycosylated forms.
carbohydrate composition [41]. Removal of carbohydrates
from human liver a-l-fucosidase did not affect its catalytic
3.2. Effect of deglycosylation on the irreversible
activity, or its gross conformation [20].
thermoinactivation
3.2.1. Aggregation
A clear understanding of operational stability constitutes
A substantially higher degree of aggregation was
an important goal in enzyme technology today. Since
observed at pH 3.0 and 4.8, upon deglycosylation (Fig. 5).
temperature is an important physical variable in enzyme-
This is in line with improvement of surface hydrophobicity
catalyzed reactions, the efforts aimed at elucidation of
of the protein structure as suggested by enhancement of
structure –stability relationships in enzymes on their stabi-
ANS fluorescence (Fig. 6) and increase in affinity for
lization have been focused mostly on thermostability. This is
especially true for an enzyme such as GA whose potential

Fig. 4. Irreversible thermoinactivation of native and deglycosylated forms

of glucoamylase at 70-C: Native (?), deglycosylated (>) at pH 3; native
Fig. 3. Intrinsic fluorescence of native (N) and deglycosylated (D) (h), deglycosylated (g) at pH 4.8; native (r), deglycosylated (‚) at pH 8.
glucoamylase. Details are described in Materials and methods. Further details are described under Materials and methods.
 J. Jafari-Aghdam et al. / Biochimica et Biophysica Acta 1750 (2005) 61 – 68
Fig. 5. Aggregation of native and deglycosylated forms of glucoamylase at
70-C: Native (?), deglycosylated (>) at pH 3; native (h), deglycosylated
(g) at pH 4.8; native (r), deglycosylated (‚) at pH 8. For further details,
please see Materials and methods.
interaction of deglycosylated form with hydrophobic matri-
ces. ANS is essentially non-fluorescent in aqueous solution,
whereas its fluorescence increases in a hydrophobic
environment.
A lower degree of aggregation for the native enzyme
would be expected from the presence of hydrophilic
sugar components providing repulsive interactions, loss of
which could cause aggregation (Fig. 5). Other reasons for
enhancement of aggregation are related to a higher
proportion of beta-sheet structure of the starch binding
site [10]. It has been suggested that the beta structures in
glycoproteins show a high tendency toward aggregation
and that upon deglycosylation they will be more exposed
to the surface [42]. These observations, combined with
the fact that sugar molecules are abundant at the
hydrophobic regions of the protein molecule, would
provide the reasons for the higher aggregation observed
upon deglycosylation (Fig. 5). Lack of any detectable
aggregation at alkaline pH may be explained in terms of
Fig. 6. ANS Fluorescence of native (N) and deglycosylated (D) forms of
glucoamylase. Details are described in Materials and methods.
65
repulsive interactions due to a significant difference
between the pH at which the experiment was carried
out and the pI of the protein (3.5 –3.7), including those
contributed by negatively-charged aspartic acid residues
[43,44]. Accordingly, inclusion of MgCl2 or CaCl2 at
different concentrations was found to enhance this
process (data not shown). These observations clearly
establish that aggregation, an important mechanism of
irreversible thermoinactivation, may indeed be expected
to occur more extensively for the deglycosylated enzyme
in the course of thermoinactivation.
There are a number of reports in the literature
describing enhancement of aggregation upon deglycosyla-
tion. For example, glucose oxidase from Aspergillus niger
and yeast invertase are less soluble and more prone to
aggregation after deglycosylation [39]. Also, glycosylation
has been shown to improve the solubility of unfolded or
partially folded invertase molecules from yeast, leading to
suppression of aggregation [45]. Many recombinant
versions of human proteins including a-antitrypsin (a-
AT) undergo aggregation upon storage. This is attributed to
lack of carbohydrates in the recombinant protein structures
[46,47].
3.2.2. Deamidation
Deamidation of Gln and Asn is an important event which
may be involved in thermoinactivation of a protein [48], and
which is dependent on pH [49]. Results presented in Table 1
indicate no significant differences in the two forms. This
was to be expected since it has been reported that
deamidation of Asn and Gln is enhanced if these residues
are placed before or after a Ser or a Thr residue in the
protein structure [50]. In the process of deglycosylation,
sugar molecules are released from Ser or Thr of GA.
Fig. 7. Cleavage of Asp-X bonds in native (1Y6) and deglycosylated
(7Y12) forms of glucoamylase (0.1 mg/ml) at pH 3, 4.8 and 8 (as indicated
in the Figure), before (B) and after (A) heating for 10 min at 70-C. A mixed
buffer containing acetate, Caps and Tris, each at 40 mM concentration and
adjusted at the three pH values was used. SDS-PAGE of samples was
performed in the usual manner.
66 J. Jafari-Aghdam et al. / Biochimica et Biophysica Acta 1750 (2005) 61 – 68

Fig. 10. Effect of polyols (20%) on aggregation of glucoamylase at 70-C:

native (h) and deglycosylated (g) forms in the absence of polyols; native
(r) and deglycosylated (‚) forms in the presence of mannose; native (?)
Fig. 8. pH stability of native (h) and deglycosylated (g) forms of
and deglycosylated (>) forms in the presence of trehalose.
glucoamylase. For details, please see Materials and methods.

3.3. pH stability of native and deglycosylated forms of GA

However, none of the Asn or Gln residues in this enzyme is
The deglycosylated form clearly showed higher stabi-
found adjacent to a Thr or a Ser.
lities in alkaline conditions (Fig. 8). Accordingly, while the
native form lost almost all activity at pH 12, about half of
3.2.3. Cleavage of Asp-X bonds
the activity was recovered for the deglycosylated protein.
Asp-X bonds are known to be very labile at elevated
temperatures [51]. Cleavage of these bonds may therefore
3.4. Irreversible thermoinactivation and the effect of polyols
be another cause of thermoinactivation in a protein
molecule [49,51]. As indicated (Fig. 7), treatment of
Irreversible thermoinactivation and aggregation of native
the two forms of GA result in breaking up of the
and deglycosylated forms of GA in the presence of 20%
structure, significantly more for the deglycosylated
(W/V) of mannose and trehalose are depicted in Figs. 9 and
enzyme than for the native form, in line with previous
10. As shown, these polyols afford protection for both
reports [e.g., 37]. These results were not unexpected
forms, presumably by being preferentially excluded from
since Asp is abundantly found in GA and predominantly
the protein surface [52]. Sorbitol behaved similarly. It has
at the protein surface [10,37]. Furthermore, loss of sugar
been suggested that these additives may strengthen hydro-
molecules may occur with a concomitant loss of a
phobic interactions among non-polar amino acid residues,
number of structurally-important hydrogen bonds between
thereby making them more resistant to unfolding and
the sugar molecules themselves or the sugar molecules
thermal denaturation. The cohesive force of sugars respon-
and protein backbone structure, thereby leading to
sible for the increase in the surface tension of water is also
enhanced flexibility.
suggested to be an important factor. Accordingly, prefer-

Fig. 9. Effect of polyols (20%) on irreversible thermoinactivation of

glucoamylase at 70-C: native (h) and deglycosylated (g) forms in the Fig. 11. Proteolytic cleavage of native (1Y4) and deglycosylated (5Y8)
absence of polyols; native (r) and deglycosylated (‚) forms in the forms of glucoamylase (0.5 mg/ml) by subtilisin, used at zero (1 and 5), 0.3
presence of mannose; native (?) and deglycosylated (>) forms in the (2 and 6), 0.6 (3 and 7) and 1 (4 and 8) mg/ml. Further details are described
presence of trehalose. in Materials and methods.
 J. Jafari-Aghdam et al. / Biochimica et Biophysica Acta 1750 (2005) 61 – 68
ential interaction of proteins with solvent components, in
aqueous sugar systems, may take place concomitant with
stabilization [53].
3.5. Proteolytic cleavage by subtilisin
As indicated in Fig. 11, the deglycosylated form was
more sensitive to proteolysis by subtilisin than the native
enzyme. This may be due to a direct hindrance of approach
provided by the sugar molecules and/or enhanced flexibility
as a result of deglycosylation. While deglycosylation did not
affect the stability of saposin B [54] toward proteolysis, hen
ovomucoid and low-density-lipoprotein receptor were found
more sensitive toward proteolytic digestion [55] upon
deglycosylation.
In conclusion, results presented in this communication
provide some mechanistic features related to the role of
carbohydrate moieties in stabilization of a glycoprotein.
They also suggest that this property cannot be generalized
and each observation should be analyzed in terms of specific
structural property in relation to the function exhibited by
the protein molecule.
Acknowledgement
This work was supported by a grant from the Research
Council, University of Tehran.
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