Biochimica et Biophysica Acta 1650 (2003) 22 – 29
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Biochemical characterization of Aspergillus awamori exoinulinase:
substrate binding characteristics and regioselectivity of hydrolysis
Anna A. Kulminskaya a, Michael Arand b, Elena V. Eneyskaya a,
Dina R. Ivanen a, Konstantin A. Shabalin a, Sergei M. Shishlyannikov a,
Andrew N. Saveliev c, Olga S. Korneeva d, Kirill N. Neustroev a,*
a
Division of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Russian Academy of Sciences,
Gatchina, Orlova Rosha, St. Petersburg 188350, Russia
b
Institute of Pharmacology and Toxicology, University of Wuerzburg, Germany
c
Biophysics Department, St. Petersburg Technical University, Russia
d
Voronesh State Technological Academy, Russia
Received 4 December 2002; received in revised form 15 April 2003; accepted 13 May 2003
Abstract
1
H-NMR analysis was applied to investigate the hydrolytic activity of Aspergillus awamori inulinase. The obtained NMR signals and
deduced metabolite pattern revealed that the enzyme cleaves off only fructose from inulin and does not possess transglycosylating activity.
Kinetics for the enzyme hydrolysis of inulooligosaccharides with different degree of polymerization (d.p.) were recorded. The enzyme
hydrolyzed both h2,1- as well as h2,6-fructosyl linkages in fructooligosaccharides. From the kcat/Km ratios obtained with
inulooligosaccharides with d.p. from 2 to 7, we deduce that the catalytic site of the inulinase contains at least five fructosyl-binding sites
and can be classified as exo-acting enzyme. Product analysis of inulopentaose and inulohexaose hydrolysis by the Aspergillus inulinase
provided no evidence for a possible multiple-attack mode of action, suggesting that the enzyme acts exclusively as an exoinulinase.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Exoinulinase; Fructooligosaccharide; Fructosyl-binding site; Multiple attack
1. Introduction
Inulin, i.e. 2 ! 1-linked h-D-fructofuranosyl residues
with a terminal glucose unit, and levans consisting predominantly of h2 ! 6-fructosyl-fructose linkages (Fig. 1), belong to a group of naturally occurring fructans comprising
nondigestible fructooligosaccharides, which are commonly
referred to as FOS in the nutrition industry. It was estimated
that about one-third of the total vegetation on earth consists
of plants that contain these carbohydrates. Pure D-fructose
or fructose syrup, which can be easily produced from inulin,
take an important place in human diets today [1,2]. Levantype FOS have a potential as emulsifying, thickening or
stabilizing agents in various food products [3,4]. As such,
fructan-hydrolyzing enzymes, especially from microbial
* Corresponding author. Tel.: +7-81271-32014; fax: +7-81271-32303.
E-mail address: neustk@omrb.pnpi.spb.ru (K.N. Neustroev).
1570-9639/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S1570-9639(03)00187-0
sources, represent an effective tool in fructan digestion
and nowadays find new industrial applications [5,6]. Microbial inulinases can be divided into two classes according
to their action on inulin. Exoinulinases (2,1-h-D-fructan
fructohydrolase, E.C. 3.2.1.80) sequentially cleave off terminal fructose unit from the inulin molecule. Endoinulinase
(2,1-h-D-fructan-fructanohydrolase, E.C.3.2.1.7) degrades
the inulin-type fructans with an endocleavage, producing a
series of fructooligosaccharides [7,8].
In our previous paper [9], we reported that a strain of
Aspergillus awamori var. 2250 produces an extracellular
enzyme during solid-state fermentation converting inulin
and levan to obtain mainly fructose. Based on its DNA
sequence (EMBL accession number AJ315793), the enzyme
was classified to belong to 32 glycoside hydrolase family
Nucleotide Sequence Database (for the classification of
glycoside hydrolase families see http://afmb.cnrs-mrs.fr/
CAZY/db.html) [9]. Despite the numerous reports on isolation and cloning of exoinulinases and exolevanases from
A.A. Kulminskaya et al. / Biochimica et Biophysica Acta 1650 (2003) 22–29
23
Fig. 1. Structures of h2,6-linked fructan (levan) (A) and h2,1-linked fructan (inulin) (B).
fungal and bacterial sources, mainly their enzymatic properties in the hydrolysis of polymeric substrates, inulin and
levan, were described so far [10,11]. Meanwhile, analysis of
the mode of exoinulinase action towards fructooligosaccharides with different degree of polymerization (d.p.) is an
effective tool for subsite mapping of the active site of this
enzyme. Kinetic studies on the enzymatic hydrolysis of
oligomeric substrates with different lengths allowed determination of affinities and number of subsites in the active
center of h-glucanases and glucoamylases from various
sources [12,13]. In the present study, we describe for the
first time details on the mode of action of the exoinulinase
from A. awamori on fructooligosaccharides using 1H-NMR
analysis, thereby estimating the number of fructosyl-binding
sites and judging on a possible multiple attack mode of
action.
2. Materials and methods
2.1. Substrate production
Inulin was purchased from Merck (cat. number 1.04733.
0010; Germany). Levan from Microbacterium levaniformans was a gift from Dr. A. Miasnikov, Danisco Global
Innovation, Finland. Average inulin d.p. was calculated by
1
H-NMR from the ratio (n) of the signals H3 (d = 4.3 –4.17
ppm) in fructose and H1 (d = 5.43 ppm) in glucose residues
following the relation d.p. = n + 1. Inulin used in these
experiments had d.p. = 23.8 F 0.5 and average molecular
mass 3850 F 80 Da. 1-Kestose (h-D-Fruf-(2 ! 1)-h-D-Fruf(2 X 1)-a-D-Glcp) was obtained with the aid of the fructosyltransferase from Aspergillus foetidus according to the
procedure described in Ref. [14]. Inulooligosaccharides with
d.p. from 2 to 4 were produced by digestion of inulin with
Aspergillus ficuum endoinulinase, which was kindly provided by Prof. Chae K.S., Chonbuk National University,
Korea. To produce inulooligosaccharides, 400 mg of inulin
was dissolved in 3.5 ml of 20 mM sodium acetate buffer,
pH 5.0, and incubated with approximately 0.05 U of the
endoinulinase per 1 mg of inulin at 37 jC for 60 min. The
hydrolysate was applied onto a Biogel P2 Super Fine
column from Bio-Rad, USA (10 1200 mm, flow rate 9
ml/h) to separate high molecular weight fractions. Specific
fractions with d.p. from 2 to 4 were rechromatographed on
a Dextro-Pakk cartridge column (8 10 mm) WATO85650
from Millipore-Waters, USA, using isocratic elution in water
(flow rate 1 ml/min) with refractometric detection. To
produce inulooligosaccharides with d.p. 5– 7, inulin (200
mg) was dissolved with 5% (v/v) of TFA and incubated at
40 jC for 30 min. After incubation, the mixture was
neutralized by mixed Dowex MR-3 (Sigma Chemical Co.,
St. Louis, MO) and concentrated by evaporation. Individual
fractions of inulooligosaccharides with d.p. 5 –7 were isolated chromatographically as described above. h-2,6-fructooligosaccarides were produced by digestion of levan by
levanase [15], which was kindly donated by Dr. Miasnikov,
Danisco Global Innovation, Finland. The reaction mixture
consisted of levan (100 mg/ml) in 20 mM sodium acetate
buffer, pH 5.0, and was incubated with approximately
24
A.A. Kulminskaya et al. / Biochimica et Biophysica Acta 1650 (2003) 22–29
0.1 unit of levanase per 1 mg of fructan at 37 jC for 60
min. After incubation, specific fractions of levanooligosaccharides with d.p. 2 – 7 were isolated as described for
inulooligosaccharides. Completion of the hydrolysis and
purity of fructooligosaccharides obtained were tested by
TLC on Kieselgel 60 plates (Merck) with a mobile phase of
butan-1-ol/acetic acid/water (3:1:1, by vol), HPLC on a
Dextro-Pac column as described above and by 1H- and 13CNMR spectroscopy techniques using data on fructooligosaccharides given in Refs. [16,17]. The purity of the
compounds obtained was greater than 95% as judged by
HPLC and NMR analyses.
2.2. Enzyme and enzyme assays
Exoinulinase was isolated from the solid-state culture of
A. awamori var. 2250 according to the procedures described
previously [9]. The concentration of pure exoinulinase was
estimated by UV absorption spectrophotometry at 280 nm
using a specific absorption coefficient of 2.2 l g 1 cm 1.
Purified exoinulinase contained less than 0.01% of contaminating exo- and endoglycosidase activities. Exoinulinase
activity was determined by Somogyi –Nelson measurement
of reducing sugars released from inulin (2.5 mg/ml) in 20
mM sodium acetate buffer, pH 4.5, 37 jC, as described in
Ref. [9]. One unit of enzymatic activity was defined as the
amount of inulinase required to produce 1 Amol of fructose
per minute at pH 4.5, 37 jC, using inulin as the substrate at
a concentration of 2.5 mg/ml. Alternatively, exoinulinase
action on fructooligosaccharides was evaluated from analysis of hydrolytic products by HPLC on a Dextra-Pakk
column using refractometric detection. Products were quantified by integration of the peaks that corresponded to
inulooligosaccharides with a d.p. of 2– 7 or fructose, using
the respective standards. Peak areas were proportional to the
amounts of oligosaccharide loaded for standards up to a d.p.
of 7. Activity of the enzyme in the hydrolysis of sucrose was
measured following the release of glucose by the glucose
oxidase method [18].
Accompanying exoglycosidase activities in purified
exoinulinase were measured using appropriate PNP a/hglycosides as substrates according to Ref. [19]. Contaminating endo-glycosidase activities were measured by Somogyi –Nelson procedure [20] using cellulose, xylan, glucan
from barley, laminarin and galactomannan as substrates.
Possible transglycosylating activity was studied in 50
mM sodium acetate buffer solutions, pH 4.5 and 5.0, 37 jC,
and 10 mM sodium phosphate buffer solutions, pH 6.0 and
7.0, at 37 jC, using in each case 0.01 U of the enzyme and
up to 100 mM of the respective substrate (sucrose, inulotriose, inulotetraose and levanotriose). Aliquots of the
reaction mixtures were taken after different time intervals
and the reaction was terminated by freeze-drying. Then,
samples were analyzed by TLC in a mobile system of
acetone/water (87:13, by vol) or using a Dextro-Pakk
cartridge column.
2.3. Calculation of subsite affinities
Kinetic parameters, Michaelis constants (Km) and catalytic rate constants (kcat) of the exoinulinase during the
hydrolysis of h2,1-fructooligosaccharides of d.p. 2– 7 were
used to determine subsite affinities of the enzyme. Km and
kcat were determined by fitting the initial rate of a substrate
hydrolysis to the Lineweaver – Burk equation. Rates were
determined under the assay conditions described above with
10– 15 different fructo-oligosaccharide concentrations ranging from 0.1 Km to 4 Km. The Michaelis constants were
determined by nonlinear regression analysis [21]. The
equation system developed by Hiromi [22] for exo-acting
enzymes was used as a base for subsite analysis of the
exoinulinase assuming Davies’ nomenclature [23] from 1
to + 1, + 2, etc.:
lnðkcat =Km Þnþ1 lnðkcat =Km Þn ¼ ðAnþ1 =RT Þ
ð1Þ
where Km and kcat are the kinetic parameters of the exoinulinase-catalyzed hydrolysis of h-2,1-fructooligosacharides
of d.p. 2 – 7, and A is the respective subsite affinity.
Assuming that there is only one nonproductive complex,
A 1 can be obtained from the Hiromi dependence of 1/kcat
on exp( An + 1/(RT )) [22]. The value for A1 can be estimated from Eq. (2) based on preliminary calculated affinities A2 – A5:
ðKm Þn ¼
n
X
Ai
exp RT
i¼1
!
55
nþ1
X
Ai
þ exp RT
i¼2
!
:
ð2Þ
þ ...
3. 1H-NMR investigation of the exoinulinase hydrolysis
of inulin
1
H-NMR spectra were recorded with an AMX-500
Bruker spectrometer. Inulin, buffer materials and the enzyme were freeze-dried from D2O twice prior to use.
Reaction was carried out with inulin (5.4 – 20 mM) in 50
mM sodium phosphate buffer containing D2O (pH 6.5).
After the accumulation of the initial spectrum, approx. 10
units of the inulinase was added to complete hydrolysis of
the substrate in 2 – 20 min. The spectra of the inulin
hydrolysis were recorded at 1-min time interval during the
reaction. Concentrations of substrate and products of the
reaction were determined integrating the peaks characteristic
for each compound: the signal at 5.43 ppm corresponds to
H1 proton in glucose residue of inulin and its glucosecontaining fragments, including sucrose; the signal at 5.23
ppm corresponds to H1 proton of a free a-glucose; the
signal at 4.23 ppm corresponds to the H3 proton of the
terminal fructofuranose of inulin; the signal at 4.32 ppm
corresponds to the H3 proton of the fructose residue
following the glucose; the signal at 4.29 ppm corresponds
A.A. Kulminskaya et al. / Biochimica et Biophysica Acta 1650 (2003) 22–29
to H3 protons of inside residues; finally, the signal at 4.28
ppm corresponds to H3 proton of the terminal fructose.
4. Results and discussion
4.1. Mode of action of the exoinulinase
To investigate the mode of exoinulinase action on inulin
as the substrate, 1H-NMR analysis as an effective method
was used. Results of the NMR study of hydrolysis of the
inulin are shown in Fig. 2 where 1H-NMR spectra of the
reaction mixture at the initial and intermediate stages (0 and
5 min, Fig. 2A,B, respectively), and after the hydrolysis is
25
near completion (20 min, Fig. 2C), are given. The weak
signal with the chemical shift around d = 5.23 ppm corresponds to the anomeric proton of a-glucose. The figure
insert shows the time course of the reaction. It demonstrates
that the decrease in the concentration of inulin (squares) is
closely followed by the increase in the concentration of
fructose divided by the d.p. 1 (circles), indicating that
fructose is likely the only major product of the hydrolysis.
Moreover, the absence of signals in the area of 4.3– 4.1 ppm
that are typical for fructooligosaccharides [17] also confirms
this suggestion.
The possible transglycosylating activity of the exoinulinase can be evaluated by the analysis of the product pattern
of inulin hydrolysis. Transglycosylation is most likely to
Fig. 2. 1H-HMR spectra of the inulin hydrolysis at the initial (A) and intermediate (B) stages of the reaction process and after the hydrolysis is near completion
(C). Insert: The time course of inulin hydrolysis catalyzed by exoinulinase: n—inulin degradation; .—formation of fructose during the reaction. The
concentrations of inulin and free glucose in the course of the reaction were calculated by integrating the NMR peaks at 4.23 and 5.23 ppm, respectively. The
fructose concentration was calculated by summing the concentrations of the pyranose and the furanose forms obtained from integrating the peaks at 3.60 and
3.57 ppm, respectively.
26
A.A. Kulminskaya et al. / Biochimica et Biophysica Acta 1650 (2003) 22–29
occur at high substrate concentrations towards the end of the
reaction, because under these conditions the concentration
of potential acceptor molecules is particularly high. The
indicative products of such reaction would be short fructooligosaccharides, the presence of which could be detected
by the recording of differential NMR spectra, which are
differences between spectrum of the reaction mixture at time
t and those at time t0, taking into consideration the degree of
substrate conversion. At an inulin concentration of 20 mM
and a substrate conversion of 80%, no signals at 4.3 – 4.1
ppm that would be characteristic for fructobioside and
fructo-oligosaccharides with higher d.p. [17] were observed.
Instead, the differential 1H-NMR spectra of the reaction
mixture at the intermediate stage of the reaction (1 min,
Fig. 3A), and after the hydrolysis is near completion (20
min, Fig. 3B) closely resemble the spectrum of fructose
(Fig. 3C). Therefore, only fructose and glucose are produced
as the final results of this reaction. Noteworthy, the use of
1
H-NMR analysis of the reaction mixture allows detection
of down to 40 – 60 Amol of an oligosaccharide formed
during the reaction, which corresponds to less than 0.3%
of initial inulin concentration.
The approach introduced here allowed us to employ 1HNMR analysis to study the kinetics of inulin and inulooligosaccharide hydrolysis carried out by the exoinulinase. The
method can be applied as a continuous assay without the
need for termination of the reaction and isolation of inter-
mediate and final products. The sensitivity of NMR analysis
gives an indisputable advantage over TLC, HPTLC and
HPLC methods [7,8] and, moreover, allows to exclude the
presence of more than 1% of contaminating endoinulinase
activity in the hydrolysis of inulin.
Additionally, transglycosylating activity of the exoinulinase was investigated in the hydrolysis of sucrose as a
substrate in a concentration range from 40 up to 200 mM
and at several pH values from 3.5 to 7.0. Despite of 91%
similarity with A. foetidus fructosyltransferase according to
their amino acid sequences [9], the A. awamori exoinulinase
was found not to produce 1-kestose, the product of fructosyltransferase reaction expected to be most prevalent in the
considered substrate concentration range [14], as evidenced
by HPLC analysis on a Dextra-Pac column and TLC
methods. Moreover, inulotriose, inulotetraose and levanotriose investigated as substrates in the transglycosylation
reaction at the concentrations up to 50 mM and pH values
from 3.5 to 7.0 did not yield the predicted corresponding
products, namely inulotetraose, inulopentaose and levanotetraose. Difructosides were also not formed in the exoinulinase reactions when the enzyme was incubated with
fructose in the concentration range of 50 – 200 mM. In all
these analyses, the level of substrate hydrolysis was at least
20– 25%. Therefore, based on these results, we have no
evidence that the exoinulinase from A. awamori 2250
possesses transglycosylating activity.
Fig. 3. The differential 1H-NMR spectra of the reaction mixture of the inulin hydrolysis at the intermediate stage (A) and after the hydrolysis is near completion
(B). The spectrum (C) is the 1H-NMR spectrum of fructose.
A.A. Kulminskaya et al. / Biochimica et Biophysica Acta 1650 (2003) 22–29
27
Fig. 4. (A) Time-dependent HPLC analysis of the exoinulinase-catalyzed hydrolysis of inulopentaose (o); formation of inulotetraose (5); formation of fructose
(E). (B) Time-dependent HPLC analysis of the exoinulinase-catalyzed hydrolysis of inulohexaose (n); formation of inulopentaose (o); and formation of
fructose (E).
4.2. Multiplicity of the exoinulinase hydrolytic action
As it was reported earlier for starch-degrading enzymes,
endo-acting a-amylases [24,25], exo-acting h-amylases
and glucoamylase [26 –28], hydrolysis of polymeric substrate can occur with multiplicity of attack. Here, the
primary product of the enzymic action serves as the
secondary substrate for the next act of the hydrolysis.
After the initial catalytic action, the product can recover
itself on the enzyme surface giving a new productive
enzyme– substrate complex. This can be repeated several
times before the final dissociation of the enzyme– substrate complex. Besides polymeric substrates, starch and
glycogen [24,25], shorter ones, maltooligosaccharides
[27,28], were used to investigate the phenomenon of
multiple-attack mode of action. The concept of multipleattack mode of action was studied with the exoinulinase
from A. awamori using fructooligosaccharides as substrates.
Taking the possible multiplicity of attack into account,
the reaction of fructooligosaccharide hydrolysis effected by
the exoinulinase may occur in two ways:
occurs, k3 becomes greater than zero. Both pathways can
exist in parallel and be of similar relevance if constants k2
and k3 are in the same range. The extent of multiplicity of
attack by the exoinulinase can be calculated using Eq. (3)
developed earlier [29]:
H¼
aN
½SNt ln
½SN0 ð3Þ
where H is the extent of multiplicity of attack, a is the extent
of hydrolysis of the substrate consisting of n fructose units
at the time t, [SN0] and [SNt] are concentrations of the
substrate at the initial point t0 and time t. Hydrolysis of
inulooligosaccharides with d.p. 4 – 7 and levanopentaose
was analyzed by HPLC of the reaction products after
different time intervals (data are given in Fig. 4). The value
for the multiplicity extent H, which was calculated according to Eq. (3), was about 1 for all tested compounds (Table
1). Therefore, multiple-attack mechanism of the exoinulinase towards inulooligosaccharides with d.p. 4 –6 was not
observed.
Table 1
Multiplicity extent of the hydrolysis of fructooligosaccharides by the
exoinulinase
where (Fru)n is a fructooligosaccharide with d.p. = n, E is the
enzyme, k+ 1 and k 1 are dissociation constants, (Fru)n 1 is
a fructooligosaccharide with d.p. = n 1, and Fru is fructose.
Such minimal kinetic scheme can be discussed in case of
the absence of transglycosylation as was proven above for
the exoinulinase described here. If multiplicity of attack
Substrate
Multiplicity extent, H
Inulotetraose
Inulopentaose
Inulohexaose
Inuloheptaose
Levanopentaose
1.05 F 0.04
1.03 F 0.04
1.08 F 0.02
1.07 F 0.02
1.15 F 0.12
28
A.A. Kulminskaya et al. / Biochimica et Biophysica Acta 1650 (2003) 22–29
4.3. Hydrolytic activity and subsite structure of the active
center
Table 3
Kinetic parameters of the hydrolysis of inulooligosaccharides by the
exoinulinase
Previously, we determined kinetic parameters of the
Aspergillus exoinulinase in the hydrolysis of inulin, levan,
stachyose and raffinose [9]. The exoinulinase hydrolyzed
sucrose with Km = 40 mM and kcat = 1150 F 10 s 1. The
specificity of the inulinase action was also investigated in
the hydrolysis of h2,6-fructooligosaccharides (levanooligosaccharides) with d.p. 2– 6. Kinetics of levanooligosaccharide hydrolysis of the exoinulinase followed Michaelis –
Menten kinetics. The values for kcat and Km obtained with
these substrates are given in the Table 2. Rates were
determined under the assay conditions described above with
12 –16 different levanooligosaccharide concentrations ranging from 0.1 Km to 4 Km. The initial rates of hydrolysis
of h2,1-fructooligosaccharides (inulooligosaccharides) with
d.p. 2 – 7 were measured as a function of substrate concentration. Plots of the hydrolytic rates versus oligosaccharide
concentrations were linear at the initial stages of the reaction
and, therefore, obeyed Michaelis –Menten kinetics ranging
from at least 0.1 Km to 4 Km (Table 3). Kinetic analysis
of the inulinase indicates that hydrolytic reactions of inulooligosaccharides with lower d.p. occur with a lower rate.
The catalytic efficiency factor, kcat/Km, also decreases
depending on the d.p. of the substrates in the same way.
The preference of the exoinulinase for h2,1-fructooligosaccharides of increasing chain length suggested that it has an
extended substrate-binding region. This was confirmed by
calculation of binding energies based on kinetic results in
accordance with Hiromi et al. [12,22] and as presented in
Fig. 5. The results indicate that the exoinulinase has an
extended active center consisting of at least five fructosylbinding sites. The highest values for binding energies were
at subsites 1 and + 1. Similar high affinities for subsites
1 and + 1 were reported for retaining barley h-D-glucosidase/(1,4)-h-D-glucan exohydrolase, which belongs to
glycoside hydrolase family 3 [30] and inverting exo-h1,3-glucanase from Trichoderma viride [13]; retaining aglucosidase of family 13 and h-glucosidase of family 1 [31].
Unfortunately, these data do not allow us to speculate about
the relationships between retention/inversion mechanism of
the A. awamori inulinase action and its binding energies in
the particular sites of the active center. However, kinetic and
subsite structure data confirm our earlier suggestion that the
enzyme can be classified as exo-acting inulinase and not hfructofuranosidase [32].
Substrate
Km [mM]
kcat [s 1]
kcat/Km [s/mM]
Inulobiose
Inulotriose
Inulotetraose
Inulopentaose
Inulohexaose
Inuloheptaose
3.72 F 0.19
1.63 F 0.07
0.94 F 0.04
1.08 F 0.02
1.02 F 0.01
1.10 F 0.01
5060 F 250
6210 F 279
6240 F 275
7670 F 153
6900 F 69
6670 F 33
1360 F 250
3810 F 279
6640 F 275
7100 F 153
6760 F 69
6060 F 33
As seen from Tables 2 and 3, the exoinulinase hydrolyzes
levanooligosaccharides and inulooligosaccharides with comparable rate constants suggesting that the enzyme is built to
act similarly well on both h-2,6- as well as h-2,1-fructosyl
bonds. However, the rate of the hydrolysis for levan is much
lower than that for inulin [9]. This may be explained by
differences of space structures of levan and inulin [17].
Another possible explanation might be interactions between
the enzyme surface and polysaccharides (e.g. inulin and
levan) as it is described for the hydrolysis of laminarin by
Candida albicans h-1,3-exoglucanase [33]. It should be
noted that the Km for the hydrolysis of inulin by the
exoinulinase has the value of 0.05 mM [9], which is lower
than the relative constants for the hydrolytic reactions of
inulooligosaccharides with d.p. 5 –7 (Table 2). On the other
hand, the application of Hiromi approaches showed the
presence of at least five binding sites. Similar dependence
was reported earlier for glucoamylase from fungal sources
[12,22] in the hydrolysis of maltooligosaccharides and
Table 2
Kinetic parameters of the hydrolysis of levanooligosaccharides by the
exoinulinase
Substrate
Km [mM]
kcat [s 1]
Levanotriose
Levanotetraose
Levanopentaose
Levanohexaose
0.21 F 0.01
2.74 F 0.14
10.2 F 0.31
1.01 F 0.02
1840 F 92
1990 F 89
575 F 18
570 F 6
Fig. 5. (A) The subsite structure of the exoinulinase. The subsite affinities
Ai are represented by the histogram. (B) Schematic model of the
exoinulinase-binding site: (.) nonreducing terminus glucosyl residue;
(o) glucosyl residues; vertical arrow, catalytic amino acids.
A.A. Kulminskaya et al. / Biochimica et Biophysica Acta 1650 (2003) 22–29
starch. For both enzymes, the presence of additional noncatalytic binding sites for polymeric substrates, glycogen and
starch was well documented [34]. Possibly, the exoinulinase
considered here also has additional binding site for inulin
located near the active center. Structure investigations of the
enzyme, which are in progress now, will allow to answer this
question.
Acknowledgements
The present work was supported in part by grants no. 0304-48756 and 03-04-49741 from the Russian Foundation
for Basic Research, by the grant of Program for Basic
Research in Physico-Chemical Biology from the Presidium
of Russian Academy of Sciences, and a grant from the 6th
Competition –Examination for Young Scientists of RAS on
Fundamental and Applied Researches.
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