Endo- and Exo-Inulinases: Enzyme-Substrate Interaction and Rational Immobilization
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Endo- and Exo-Inulinases: Enzyme-Substrate Interaction
and Rational Immobilization
Alessandra Basso and Patrizia Spizzo
Laboratory of Applied and Computational Biocatalysis, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste,
Piazzale Europa, Trieste 1-34127, Italy
SPRIN s.r.l., c/o Università degli Studi di Trieste, Piazzale Europa, Trieste 1-34127, Italy
Valerio Ferrario
Laboratory of Applied and Computational Biocatalysis, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste,
Piazzale Europa, Trieste 1-34127, Italy
Lorena Knapic
Laboratory of Applied and Computational Biocatalysis, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste,
Piazzale Europa, Trieste 1-34127, Italy
SPRIN s.r.l., c/o Università degli Studi di Trieste, Piazzale Europa, Trieste 1-34127, Italy
Nina Savko
Laboratory of Applied and Computational Biocatalysis, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste,
Piazzale Europa, Trieste 1-34127, Italy
Paolo Braiuca
Laboratory of Applied and Computational Biocatalysis, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste,
Piazzale Europa, Trieste 1-34127, Italy
SPRIN s.r.l., c/o Università degli Studi di Trieste, Piazzale Europa, Trieste 1-34127, Italy
Cynthia Ebert
Laboratory of Applied and Computational Biocatalysis, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste,
Piazzale Europa, Trieste 1-34127, Italy
Emanuele Ricca and Vincenza Calabrò
Dipartimento di Ingegneria Chimica e dei Materiali, Università della Calabria, via P. Bucci, Edificio Cubo 44A,
Arcavacata di Rende 87036, Italy
Lucia Gardossi
Laboratory of Applied and Computational Biocatalysis, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste,
Piazzale Europa, Trieste 1-34127, Italy
DOI 10.1002/btpr.334
Published online November 25, 2009 in Wiley InterScience (www.interscience.wiley.com).
Three-dimensional models of exoinulinase from Bacillus stearothermophilus and endoinulinase
from Aspergillus niger were built up by means of homology modeling. The crystal structure of exoi-
nulinase from Aspergillus awamori was used as a template, which is the sole structure of inulinase
resolved so far. Docking and molecular dynamics simulations were performed to investigate the dif-
ferences between the two inulinases in terms of substrate selectivity. The analysis of the structural
differences between the two inulinases provided the basis for the explanation of their different
regio-selectivity and for the understanding of enzyme-substrate interactions. Surface analysis was
performed to point out structural features that can affect the efficiency of enzymes also after immo-
bilization. The computational analysis of the three-dimensional models proved to be an effective
tool for acquiring information and allowed to formulate an optimal immobilized biocatalyst even
more active that the native one, thus enabling the full exploitation of the catalytic potential of these
enzymes. V C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 397–405, 2010.
Keywords: Endo-inulinase, exo-inulinase, inulin, molecular modeling, docking, homology
modeling, immobilization
Additional Supporting Information may be found in the online
version of this article.
Correspondence concerning this article should be addressed to L.
Gardossi at gardossi@units.it.
C 2009 American Institute of Chemical Engineers
V 397398 Biotechnol. Prog., 2010, Vol. 26, No. 2
Introduction
Microbial inulinases belong to an important class of indus-
trial enzymes that have gained increasing attention in the
recent years. Inulinases can be produced by a series of
microorganisms, including fungi, yeasts, and bacteria.1
Inulinases are hydrolytic enzymes able to hydrolyse inulin,
a natural storage polymer found widely in plants such as
chicory, artichoke and banana. Inulin is a polydisperse fruc-
tan with the degree of polymerization that typically ranges
from 2 to 60, but may be higher. The fructosyl units in inu-
lin are linked by b (2,1) linkages and the resulting polymer
chain terminates with a glucose residue. Inulin is used as an
indigestible soluble dietary fiber and thickener in foods; it is
a potential source of fructose. Fructose is significantly
sweeter than the table sugar sucrose and glucose,2 it is not
associated with obesity, caries, atherosclerosis and diabetes.3
There are two different subclasses of inulinase,4 endo- and
exo-inulinase: exoinulinase (EC 3.2.1.80) hydrolyses the ter-
minal fructose from the inulin chain, whereas endoinulinase
(EC 3.2.1.7) reduces the long chain of inulin into smaller ol-
igosaccharides, which are similar to fructooligosaccharides.5
In the present study we have investigated the inulinases pres-
ent in the commercial preparation Fructozyme L from Novo-
zymes, which is a mixture of 10% of endoinulinase from
Aspergillus niger and 90% of exoinulinase from Bacillus
stearothermophilus. The three dimensional models of the Figure 1. Superimposition of the structural models of A. awa-
two enzymes were constructed by homology modeling, using mori exoinulinase (black backbone) from PDB
the structure of the exoinulinase from Aspergillus awamori (1Y4W) and the two generated structures: exoinuli-
as a template.6 The models allowed to draw the picture of nase from B. stearothermophilus (main differences in
dotted gray) and endoinulinase from A. niger (main
how the two enzymes recognize inulin and to explain their differences in gray).
inherent different regio-selectivity. Finally, superficial fea- The Glu residue of A. niger endoinulinase, responsible for the
tures of the proteins were also studied by using different nucleophilic attack, is represented in gray CPK mode. B. stear-
computational methods and the commercial enzymatic prepa- otermophilus exoinulinase and A. niger endoinulinase are ho-
mologous with A. awamori exoinulinase for 37% and 34%
ration was experimentally tested after immobilization on two respectively on the basis of their primary structure.
different supports and under various conditions.
Results and Discussion
Starting from the sequences of exoinulinase from B. stear-
Construction of the three-dimensional models othermophilus and endoinulinase from A. niger, published on
Commercial preparation of Fructozyme L from Novo- UniProtKB,8 their three-dimensional structures were gener-
zymes is a mixture of 10% of endoinulinase from Aspergil- ated by homology modeling. Bacillus stearotermophilus
lus niger (EC 3.2.1.7) and 90% of exoinulinase from exoinulinase is 37% homologous with Aspergillus awamori
Bacillus stearothermophilus (EC 3.2.1.80). The study of the exoinulinase and 34% homologous with Aspergillus niger
three-dimensional structures of these two enzymes was per- endoinulinase on the basis of their primary structure.
formed with two principal objectives: firstly to understand As mentioned earlier, exoinulinase structure from A. awa-
the different selectivities of these two enzymes; secondly to mori (PDB ID:1Y4W) was used as the template structure
acquire information on the structural features of the surface and the generated models were analyzed with Ramachandran
of the two proteins which might be crucial for developing map and compared by consensus analysis (see ‘‘Experimental
efficient optimization protocols. section’’ for details); as Bacillus stearotermophilus exoinuli-
The crystal structures of Aspergillus niger endoinulinase nase is concerned 85.13% of the residues are in the core
and Bacillus stearothermophilus exoinulinase are not avail- region of the Ramachandran map, this percentage is 86.61%
able in PDB (Protein Data Bank)7 so that the structure of for the model of A. niger endoinulinase, while all the other
these two enzymes had to be calculated by homology model- residues lie inside the allowed region of the map. Bond
ing. For this purpose, the structure with the PDB code lengths, angles and dihedral were within acceptable parame-
1Y4W of exoinulinase from Aspergillus awamori was used ter ranges, after models refinement and no atom clashes
as template for the calculation of both models. As a matter could be found.
of fact, three different structures of exoinulinase from This high score of Ramachandran maps indicate the qual-
Aspergillus awamori are the sole 3-D structures of inulinases ity of the generated structures (see Supporting Information).
available in PDB and 1Y4W has the highest resolution It must be underlined that the comparison of the structures
(1.55 Å). A second structure, 1Y9M, having a lower resolu- of all the three inulinases (A. awamori exoinulinase, B.
tion (1.89 Å) was used as a reference model for all the sub- stearothermophilus exoinulinase and A. niger endoinulinase)
sequent docking studies because of the presence of a shows a high degree of similarity in the structural domain
fructose molecule co-crystallized in the active site.6 opposite to the active site region, as reported in Figure 1.Biotechnol. Prog., 2010, Vol. 26, No. 2 399
Figure 2. Exoinulinase from B. stearothermophilus: the active
site is highlighted in surface mode and the 1-kestose
is docked inside. Figure 3. Endoinulinase from A. niger with the funnel domain
highlighted in surface mode with a six unit fructose
polymer docked inside.
As expected, the main differences among the structures
were found in the catalytic site of the enzymes. The two
exoinulinases share common structural features, presenting
Asp residue (Asp24 in the B. stearothermophilus enzyme,
Asp41 in the A. awamori one) that is supposed to be respon-
sible of the nucleophilic attack.6 In the active site of the
endoinulinase the Asp is replaced by a Glu 43 residue.
Because of the large size of the natural substrates, the cat-
alytic site of the endoinulinase is quite wide and accounts
about 90 of the 516 residues. On the contrary, the active site
of the exoinulinase, which acts on the terminal residue of
the polymer, is smaller and it includes 42 of the 493 total
aminoacids.
An interesting common feature among the three structures
is the presence of the Arg-Asp-Pro motif (RDP), a sequence
that is preserved also in other classes of enzymes like fructo-
syl-transferases or invertases. The presence of this motif is
supposed to be important for the recognition of the pyranosi-
dic ring and it has been reported to be responsible for the
enzyme specificity towards fructopyranosidic residues.6
Enzyme-inulin interaction: docking and molecular
dynamics simulations
Molecular docking9 and molecular dynamics10 were
applied to the study of the interactions between inulin and
the active site of the two inulinases.
The different structural organization of the active sites of
these two enzymes appears evident by comparing the two Figure 4. The exoinulinase from B. stearothermophilus: (a) dis-
homology models and it offers the basis for the explanation tance of the anomeric carbon of the 1-kestose from
of their different regio-selectivity. The exoinulinase displays Asp24 during the dynamics trajectory and (b) dis-
a hole-shaped binding pocket with the RDP motif located at tance of the glycosidic oxygen of the 1-kestose and the
stabilizing Glu203 during the dynamics trajectory.
the bottom, whereas the endoinulinase presents a funnel-
shaped active site, with the RDP motif in the middle. This
causes a deep static and dynamic difference in the interac- molecule present in the active site of the 1Y9M PDB struc-
tion with inulin, that translates into their different regio- ture were used as a reference for the scoring of the obtained
selectivity. conformations. The selected conformation was chosen as the
The enzyme-substrate interactions were studied by subject- initial state for subsequent molecular dynamics simulations
ing the enzyme-substrate complexes to 2ns molecular dy- (see following section).
namics simulations. In the case of the exoinulinase the The simulation of the endoinulinase was more complex,
complex (Figure 2) was calculated by docking 1-kestose since there is no crystal structure available presenting a
[composed by two fructose molecules linked via b(1,2)-gly- sugar molecule docked inside the active site. Therefore a
cosidic bond]11 by means of a molecular simulation based fructose hexamer, composed by six fructose units linked via
on AlphaPMI algorithm. The coordinates of the fructose b(1,2)-glycosidic bonds, was sketched and manually placed400 Biotechnol. Prog., 2010, Vol. 26, No. 2
into the funnel shaped active site. The substrate conforma- The stabilization of the complex is significantly driven by
tion was manually modified to improve the fitting with the the interaction between the glycosidic oxygen of the 1-kes-
active site shape. The manually placed substrate was then tose and Glu203. The monitoring of this interaction during
used as starting conformation for the docking simulation, the trajectory pointed out a dynamic switch between two
similarly to what has been done in the case of exoinulinase. conformations, which correspond to a distance of 3.5 Å (sta-
The conformation obtained from the docking procedure bilized conformation) and 5.5 Å (de-stabilized conformation)
(Figure 3) was used as initial state of the molecular dynam- respectively (Figure 4b). This conformational switch is prob-
ics simulation. ably crucial for the release of the product from the active
site after the enzymatic catalysis and it also limits product
inhibition effects.
Molecular dynamics of the enzyme-substrates complexes
The endoinulinase shows a significantly different dynamic
The two enzyme-substrate complexes were used as start- behavior: although the thermodynamic equilibrium is
ing conformations for 2 ns molecular dynamics simulations reached after a few ps, the enzyme toggles between two
based on AMBER94 force field12 and implicit solvent states during the entire equilibrium phase of the dynamics.
treatment. The first conformation of the funnel domain corresponds to
The exoinulinase system reached the equilibrium after 500 the closed/inactive state whereas the second conformation
picoseconds (ps) and remained stable during the rest of the corresponds to the open/active conformation (Figures 5 and
molecular dynamics trajectory. As it can be seen in Figure 6). This conformational change is most probably related to
4a, there are two major different states of the system during the mechanism of substrate/product acceptance and release,
the first 50 ps of the simulation. Initially the substrate reor- although it does not alter the interaction of the reactive part
ients its position in the pocket by increasing the distance of the substrate with the active site. The stabilization of the
between the anomeric carbon of the 1-kestose and the active glycosidic oxygen of the substrate by hydrogen bond forma-
residue Asp24 (from 6 to 14Å). Afterwards, this distance is tion with Glu233 appears similar to the stabilization that
progressively reduced and after 500 ps, the complex encoun- takes place in the exoinulinase, as it can be seen comparing
ters a stabilization in the range of 4.5–6.5 Å (Figure 4a). distances variations during the dynamics (Figures 4b and 7).
From the analysis of the RDP motif during the molecular
dynamics trajectories, it appeared evident that, besides a sin-
gle H-bond with the substrate, these three residues do not
have a relevant direct role in the enzyme-substrate interac-
tions either in the exo- and endoindulinase. Nevertheless,
they establish a complex network of interactions with several
other residues of the enzymes’ active site, playing a major
role in maintaining its shape and therefore indirectly respon-
sible of the chemo-selectivity of the enzyme (see Supporting
Information Figure 4).
Surface analysis
A detailed GRID13 analysis of the surface of the enzymes
was performed to map the distribution of the hydrophilic and
Figure 5. The endoinulinase from A. niger: the opened and hydrophobic zones. The analysis showed a neat hydrophilic
closed conformation of the funnel constituting the
active site, measured as a function of the distance character in both enzymes surfaces, even if more marked in
between a carbons of Trp67 and Glu173, two resi- endoinulinase from A. niger. This is in line with features
dues on the opposite side of the funnel. already observed for other hydrolases acting in aqueous
environments.14
Figure 6. A. niger endoinulinase: conformational change of the funnel domain in surface mode showing the closed active site, on the
left, and the open conformation, on the right, with the substrate (fructose hexamer) docked inside, represented in a licorice
mode.Biotechnol. Prog., 2010, Vol. 26, No. 2 401
Looking into detail at the catalytic sites of both enzymes, immobilization processes. Twenty-six lysine residues were
an increase of hydrophobicity can be noticed in the proxim- found to be on the surface of exoinulinase, none of them are
ity of the entrance of the active sites, which might have the close to the active site. Eleven lysine residues can be found
function to avoid an excessive stabilization of the enzyme- on the endoinulinase surface, only one being located near
substrate complex and favor the release of the hydrolysis the active site (Figure 8).
products (Figure 8). In the perspective of developing an immobilization proto-
N-glycosylation sites were also investigated. N-Glycosyla- col, the enzymes seem to be optimal for the covalent attach
tion of proteins takes place in the correspondence of the to functionalized polymeric carrier. None of the lysine resi-
Asn-X-Ser/Thr sequence15 through the linking of the Asn to dues of both inulinases can significantly affect the access to
two GlcNAc residues.16 the active site or the dynamic behavior of the structural
The Fuzzpro algorithm of the EBI SRS set of bioinfor- motifs involved in the catalysis.
matic tools17 was used to identify N-glycosylation sites. Therefore two methacrylic polymers carrying amino and
Exoinulinase and endoinulinase present 4 and 5 potential epoxy functional groups have been chosen for covalent
immobilization, namely SepabeadsV EC-HA and SepabeadsV
R R
sites of glycosylation respectively, as showed in Figure 9.
All the resulting glycosylation sites are actually located on EC-EP.
the protein surface. Their relative positions on the surface
are very similar, also near the active sites.
Immobilization
The analysis of the surface was also extended to the
superficial lysines, that can be potentially used in covalent The immobilization studies were performed using the
commercial preparation of inulinases Fructozyme L from
Novozymes. The commercial preparation was characterized,
in terms of protein content and specific activity (release of
fructose, see Experimental Section). Moreover, it was eval-
uated that the enzymatic preparation loose 90% of activity
after 1 h of incubation at 65 C.
The use of the enzyme in productive industrial process is
often limited by the low stability of the enzyme preparation
and by the difficulty to recover and reuse the biocatalyst; a
typical approach for improving both aspects is represented
by immobilization, which can be performed according to
various types of techniques with the concomitant advantages
and disadvantages.18,19 In this study the enzymes were cova-
lently immobilized on methacrylic supports, namely amino
and epoxy Sepabeads (SepabeadsV EC-HA and SepabeadsV
R R
Figure 7. Endoinulinase from A. niger: distance of the glyco- EC-EP, produced by Resindion S.r.l., Mitsubishi Chem.
sidic oxygen of the 1-kestose and the stabilizing Corp.), which are solid resin employed for enzyme immobi-
Glu233 during the dynamics trajectory. lization on industrial scale.
Figure 8. Grid profile of B. stereatermophilus exoinulinase (a) and A. niger endoinulinase (b): in crossed zones the hydrophobic areas
of the enzyme surface and in light gray the hydrophilic areas of the enzyme surface.
The active site area is highlighted in the circle.402 Biotechnol. Prog., 2010, Vol. 26, No. 2
Figure 9. Exoinulinase from B. stearothermophilus (a) and endoinulinase from A. niger (b): the active site is represented in surface-
transparent mode, the superficial lysines in space-filling mode and the glycosilation sites as big beads.
Table 1. Properties of the Immobilized Preparations of Fructozyme Table 2. Characteristics of the Preparations on 100 g Scale of
L on Sepabeads Immobilized Fructozyme L
Bound Water Activity Bound Water Activity
Support* Proteins† % Content %†,‡ U/gdry§ Support Proteins %* Content %† U/gdry‡
Sepabeads EC-EP 75 71 204 Sepabeads EC-EP 77 71 205
Sepabeads EC-HA 76 69 300 Sepabeads EC-HA 82 67 285
* Immobilization was carried out in KPi buffer (KH2PO4/K2HPO4 * Determined by Pierce method, using inulinase solution for calibra-
buffer 1.25 M, pH 8.0; see Experimental Section), starting from 250U of tion curve.22
†
enzyme per gram of dry support. The water content of preparation was evaluated by drying the sam-
†
Determined by Pierce method, using inulinase solution for calibra- ples at 110 C for 6h.
tion curve.22 ‡
Hydrolytic activity measured by hydrolysis of inulin.
‡
The water content of preparations was evaluated by drying the sam-
ples at 110 C for 6 h.
§
Hydrolytic activity measured as release of fructose from hydrolysis the bulky inulin towards the anchored enzymes (see Support-
of inulin. ing Information).
The excellent activity of the immobilized inulinases was
also confirmed by reproducing the protocols on 100 g scale
Immobilization on epoxy supports proceeds through direct (Table 2) and by assaying their efficiency in a 500 mL bio-
nucleophilic attack of e-amino groups of lysines to the epoxy reactor (see Experimental Section).
groups, whereas immobilization on amino supports requires The fructose production was monitored for the initial 30
a preactivation by glutaraldehyde.11,20,21 hours of the reactions (Figure 10). Using 1 gwet of both the
Immobilizations were performed at pH 8.0 using in all the immobilized preparations (corresponding to 59U for the
experiments 250 enzyme Units of native preparation per dry Sepabeads EC-EP preparation and 94U for Sepabeads EC-
gram of immobilization polymer (Table 1) HA) in 24 h the fructose released is above the 90% (Sepa-
As indicated by results in both cases the expressed activity beads EC-EP, gray empty squares) and 80% (Sepabeads EC-
of the immobilized biocatalysts was very high and in the HA, gray empty circles) of the maximum theoretical amount
case of Sepabeads EC-HA the immobilization efficiency is obtainable from the type of inulin employed in the
even [100%. These data suggest that upon immobilization experiments.
the enzymes assume optimal orientations and conformations, As shown in Figure 10, reaction rates with Sepabeads EC-
which facilitate the inulin access to the active sites as well HA preparation was slightly faster (v0 ¼ 1.49 lmol U1
as the prompt release of the product into the bulk medium. min1, black circles) than with Sepabeads EC-EP (v0 ¼ 0.71
This is particularly important since in many cases the immo- lmol U1 min1, black squares).
bilization causes a decrease of the activity of the enzyme
due to unfavorable conformational distortions or occlusion of
Conclusions
the active sites.19 Moreover, diffusion limitations can occur
when enzymes are anchored into the pores of the supports. The combination of computational and experimental
Indeed, when the inulinases were marked with fluorescein investigations described in this article shed light on the prop-
and analyzed by fluorescent microscopy it was evident that erties of an enzymatic preparation of industrial relevance.
the protein was present exclusively on the first external layer Homology modeling methods allowed to construct the first
of the beads (data not shown), thus favoring the diffusion of three-dimensional structures of exoinulinase from BacillusBiotechnol. Prog., 2010, Vol. 26, No. 2 403
The immobilized protein was rinsed with KPi buffer (0.02
M, pH 8.0, ratio support/buffer 1/4 w/v). Adsorbed proteins
were desorbed by adding 0.5 M NaCl in KPi buffer (0.02 M
pH 8.00, ratio support/buffer 1/4 w/v) and then by stirring
for 45 min. The preparations were rinsed with KPi buffer
(0.02 M, pH 8.0, ratio support/buffer 1/4 w/v), finally with
sodium acetate buffer (0.1 M, pH 5.0) and the activity was
checked. The immobilized enzymes were stored at 4 C.
Immobilization on amino SepabeadsV
R
Amino SepabeadsV were pre-activated with glutaraldehyde
R
(2% v/v in KPi buffer, 0.02M, pH 8.0, support/buffer ratio
Figure 10. Kinetic profile and yield of the reaction of inulin
hydrolysis catalyzed by Fructozyme L immobilized of 1/4 w/v) for 60 min at 25 C and then washed twice with
on Sepabeads EC-EP (black squares: kinetic profile, the same buffer.
gray empty squares: yield) and EC-HA (black The immobilization was carried out at 25 C and 40 rpm
circles: kinetic profile, gray empty circles: yield).
in a blood rotator in KH2PO4/K2HPO4 KPi buffer (0.02 M,
Conditions: T ¼ 50 C, [inulin]0 ¼ 2.12 mmol L1, yield is
calculated on the basis of the maximum achievable fructose
pH 8.0). 77 U of Fructozyme L per gram of wet polymer
concentration ¼ 59, 36 mmol L1 (medium degree of poly- were used with a support/buffer ratio of 1/4 w/v. The immo-
merization of the inulin ¼ 28 fructose unit). bilization was carried out under stirring for 19 h in KPi
buffer (0.02 M, pH 8.0). The solution was filtered and the
liquid phase was recovered for protein determination. The
stearothermophilus and endoinulinase from Aspergillus immobilized protein was washed with KPi buffer (0.02 M,
niger, allowing the analysis of their different regio-selectiv- pH 8.0, ratio support/buffer 1/4 w/v). Adsorbed proteins
ity. The information gathered by the computational investi- were desorbed by adding 0.5 M NaCl in KPi buffer (0.02 M
gation not only led to a more detailed comprehension of the pH 8.00, ratio support/buffer of 1/4 w/v) and then by stirring
catalytic machinery but also allowed to rationalize the exper- for 45 min. The preparations were rinsed with KPi buffer
imental strategies for immobilizing the biocatalysts. (0.02 M, pH 8.0, ratio support/buffer 1/4 w/v), finally with
In conclusion, this investigation would like to show an sodium acetate buffer (0.1 M, pH 5.0) and the activity was
example of how computational methods can be used as a tool checked. The immobilized enzymes were stored at 4 C.
not only for understanding enzyme-substrate interactions but
also for developing efficient biocatalysts of practical use, thus
making biocatalysis less empiric and more rational based. Activity assay
The assay is based on the hydrolysis of inulin into fruc-
Experimental Section tose. A 10 g/L solution of inulin (average P.M. 4554 Da) in
acetate buffer (0.1 M, pH 5.0) was maintained at 50 C. The
Materials native or immobilized enzyme was added to the solution of
Fructozyme L was from Novozymes (see Supporting inulin and the suspension was stirred for a variable time
Information). from 15 to 30 minutes. To stop the reaction, 300 lL of
Immobilization carriers: SepabeadsV are methacrylic poly- NaOH 1 M were added to the suspension of native enzyme,
R
mers produced and commercialized by Resindion S.r.l. (Mit- whereas the immobilized enzyme was filtered off. The solu-
subishi Chem. Corp., Milan, Italy), supplied with a water tion was cooled down to 25 C and analyzed by HPLC. The
content of about 70% w/w. SepabeadsV with epoxy groups analysis was performed at isocratic conditions, 75% acetoni-
R
(Sepabeads EC-EP) or amino groups (Sepabeads EC-HA) trile and 25% water, using an integrated system Shimadzu
were used in the present work. associated to refractometer RID-6A. An Aphera NH2 Poly-
Chemicals: all chemicals were purchased from Sigma– mer column (polyamine-bonded polymeric gel column) 5 l,
Aldrich and were used without any further purification. 250 4.6 mm (Astec) was used. Rt fructose: 8 min. The
activity was calculated on the basis of a calibration curve.
The medium degree of polymerization of the inulin used
in all experiments is of 28 fructose unit. Activity ¼ lmol fructose/(min g dry)
Spectroscopy: UV measurements were performed with a The activity of Fructozyme L was 851 U per g of prepara-
Lambda 20 UV/Vis Perkin–Elmer spectrophotometer. tion or 39 U per mg of protein.
Immobilization on epoxy SepabeadsV
R
Protein determination
All the immobilization procedures were performed loading
250 U/gdry of enzymatic native preparation. The immobiliza- The protein content of Fructozyme L (25.17 mg/mL) was
tion was carried out at 25 C and 40 rpm in a blood rotator determined by using bicinchoninic acid kit (SIGMA)—Pierce
in KH2PO4/K2HPO4 KPi buffer (KH2PO4/K2HPO4 buffer method, using BSA as standard protein.
1.25 M, pH 8.0). 77 U of Fructozyme L per gram of wet The percentage of bound enzyme was determined by the
polymer were used with a support/buffer ratio of 1/4 w/v. difference between the concentration of the native protein
The immobilization proceeded for 18 h, under constant stir- before immobilization and in the filtrates after immobiliza-
ring, then for 20 h without stirring. The solution was filtered tion. The protein amount was determined by Pierce method,
and the liquid phase was recovered for protein determination. using inulinase solution for calibration curve.22404 Biotechnol. Prog., 2010, Vol. 26, No. 2
Determination of water content score of the MOE and one model for each enzyme was cho-
The water content of each preparation was evaluated by sen on the basis of the Z score. The quality of the generated
drying the samples at 110 C for 6 h on aluminum dishes and models was assessed by the ‘‘protein report’’ tool of the
by determining the difference in weight between the wet and MOE program and Ramachandran map (see Supporting
the dried sample. Information).
The final structures were refined by manual corrections
and energy minimizations, during which the minimization
Stability software used three derivative methods successively: steepest
descent, conjugated gradient and truncated Newton. The
A solution of Fructozyme L, diluted in acetate buffer (0.1
AMBER94 force field was used. Refinement of the model
M, pH 5.0), was divided into aliquots of 2.5 mL each. Each
proceeded up until the obtained protein quality was compara-
sample was thermostatted at 65 C for a time from 0 to 90
ble to a medium/high resolution structure.
minutes. Each solution was cooled down to 25 C and the re-
sidual activity was evaluated. The resulting models were compared by means of the
‘‘protein consensus’’ tool of the MOE package utilized to
identify regions with conserved structures.
Hydrolysis of inulin catalyzed by the immobilized inulinase
Batch tests were run in a system (Applikon, The Nether-
lands) consisting of: a 1.5 L glass vessel, a six-blade impel- Docking and molecular dynamics simulation
ler driven by an electric motor (Stirrer Motor Assembly Water molecules and all glycosides, unless the fructose
P100) controlled by a stirrer controller (P100, ADI 1032), present in the active site of the enzyme, were removed from
pH and temperature sensors, a jacket for temperature control the structure of A. awamori exoinulinase 1Y9G (retrieved
by means of a Bio Controller (ADI 1030). from PDB). The maintained fructose residue was trans-
The reaction volume was 500 mL, pH was kept to 5.0 by formed into a dimer in the case of exoinulinases and into a
means of an acetate buffer and its values were constantly hexamer in case of the endoinulinase. The molecules
measured; the temperature value was 50 C, enzyme loading obtained functioned as substrates for the respective enzymes,
Eo ¼ 1 gwet of both the immobilized preparations, that corre- subsequently used to perform the docking calculations, sub-
sponds to 94U for the Sepabeads EC-EP preparation and strates were defined in the same force-field of the rest of the
59U for the other one (Sepabeads EC-HA); and substrate system.
(inulin) initial concentration So ¼ 10 g/L (2.12 mM). The substrates were placed manually into the active sites
Reaction was quenched by separating the enzyme from and the docking procedure was performed taking into
the reacting mixture by means of a 90 lm filter (iron steel account an area of 12 Å around the substrate. The Alpha
filter) and samples were immediately analyzed. PMI algorithm was used for the placement procedure in
association with the Depth HB scoring function. Two-hun-
dred conformations having the highest score were conserved.
Computational studies The final spatial conformation was chosen evaluating the
All the calculations were performed on a dual Xeon Linux score and the orientation of the substrate in respect to the
workstation. catalytic residues of the active site.
Homology modeling, molecular dynamics simulations, The areas included in a radius of 12 Å and 5 Å around
energy minimizations, and docking were calculated by using the substrate for the two exoinulinases and the endoinulinase
the Molecular Operating Environment (MOE) version respectively were minimized for 9000 steps totally, using
2006.08 software. For GRID’s MIF calculation the GRID 22 three derivative methods successively: steepest descent, con-
Linux edition package was used. jugated gradient and truncated Newton. The AMBER94
force field12 was used for the definition of the entire system.
Molecular dynamics simulations of the same enzyme areas
Homology model generation were set up for 2000 ps at 300 K in a NTV environment,
using AMBER94 force field. The rest of the structure was
The aminoacid sequence of A. niger endoinulinase and
maintained constrained.
B. stearothermophilus exoinulinase (accession number
O74641_ASPNG and Q8GI55_BACST respectively in
UniProtKB) were taken from the EBI SRS server by using
the BlastP algorithm23,24 query instrument. Surface analysis
The chain alignment was performed using as template the Water molecules were removed from the crystallographic
structure of A. awamori exoinulinase which was retrieved structure 1Y4W, the three-dimensional matrices of interac-
from PDB (1Y4W), with the align algorithm of the MOE tion energies (MIFs) were calculated by using the GRID
program by using the Blosum 30 amino acid substitution ma- methods with WATER and DRY probes. For each structure
trix with a tree-based method,25 visually verified and man- the calculation was performed simulating a grid with 0.5 Å
ually corrected. knots distance. The calculated MIFs were visualized with the
The construction of the three-dimensional models was car- program Gview setting 4.00 kcal for WATER interactions
ried out with the MOE homology modeling module calculat- and 0.25 kcal for DRY interactions.
ing 10 intermediate models for each enzyme which were The glycosylation sites in the protein sequences were
coarsely minimized maintaining as template the 1Y4W struc- identified by using the protein motifs searching tool FUZZ-
ture, purged from water molecules and glycosides. The PRO as available in the EBI SRS server. The standard ‘‘N-
obtained structures were ranked by the structure quality Z any-S/T’’ pattern was used as a query.Biotechnol. Prog., 2010, Vol. 26, No. 2 405
Acknowledgments 13. Goodford PJ. A computational procedure for determining ener-
getically favorable binding sites on biologically important mac-
The authors are grateful to Resindion S.r.l. (Mitsubishi romolecules. J Med Chem. 1985;28:849–857.
Chem. Corp., Milan, Italy) for providing immobilization car- 14. Basso A, Braiuca P, Cantone S, Ebert C, Linda P, Spizzo P,
riers and Dr. Laura Ciccarelli for precious technical advice. Caimi P, Hanefeld H, Degrassi G, Gardossi L. In Silico analysis
of enzyme surface and glycosylation effect as a tool for efficient
covalent immobilization of CalB and PGA on SepabeadsV. Adv
R
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