Supramolecular stabilization of acid tolerant l-arabinose isomerase from Lactobacillus sakei

Article abstract of DOI:10.1039/c1cc14930j

l-Arabinose isomerase stability is a crucial criterion for the industrial application of this biocatalyst. Noria and NoriaPG are capable of increasing the l-arabinose isomerase stability not only at high temperatures but also at low pH. Such results highlight, for the first time, the use of the Noria series of molecules for protein stabilization and activation.

Full text of DOI:10.1039/c1cc14930j

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COMMUNICATION
Supramolecular stabilization of acid tolerant L-arabinose isomerase from
Lactobacillus sakeiw
Said Jebors,a Yannick Tauran,a Nushin Aghajari,b Samira Boudebbouze,c
Emmanuelle Maguin,c Richard Haser,b Anthony W. Coleman*a and Moez Rhimi*b
Received 9th August 2011, Accepted 11th October 2011
DOI: 10.1039/c1cc14930j
L-Arabinose isomerase stability is a crucial criterion for the
industrial application of this biocatalyst. Noria and NoriaPG
are capable of increasing the L-arabinose isomerase stability not
only at high temperatures but also at low pH. Such results
highlight, for the first time, the use of the Noria series of
molecules for protein stabilization and activation.
The interactions between supramolecular systems and various
proteins have been widely reported with reviews by Perret and
Coleman,9–11 Kalchenko et al.,12 and Djedaini-Pilard et al.13
Uses range from cryptophane-Xenon cage structures for
NMR studies,14 through destabilization and refolding of
proteins by the cyclodextrins,15 to protein sensing,16 and
stabilization17 by the calix[n]arenes. A recently synthesized
class of supramolecules, the Noria, name derived from their
geometry resembling a water wheel are interesting as they
present an almost spherical shape covered by hydrophilic
phenolic groups.18
L-Arabinose isomerase (L-AI) belongs to the EC 5.3.1.4 family
and is able to convert L-arabinose into L-ribulose within living
systems.1 The same enzyme is also known as D-galactose
isomerase due to its capacity to isomerize D-galactose into
D-tagatose in vitro.2,3 In agro-food industry D-tagatose has
gained great interest as sweetener.4 Indeed this ketohexose has
the taste and natural properties of sucrose, but it is an anti-
hyperglycemiant factor known to reduce symptoms associated
with Type 2 diabetes.4,5 Moreover, D-tagatose has not only a
very low calorific value of 1.5 kcal gÀ1 (only 30% of the energy
content of sucrose), but it also promotes weight loss.6 All the
above properties are very attractive for industrial scale utilisation;
however, the use of this natural sugar is limited due to its cost.
For this reason considerable work has focused on cost-effective
production methods. The most profitable production procedure
described until now is based on the use of L-AI.7 Previously
reported L-AIs have a tetrameric functional arrangement
except that of E. coli which displays a hexameric structure.8
The main factor limiting the industrial application of this
biocatalyst is its stability under operating conditions consisting
of high temperatures and low pH.7 Therefore, stabilization of
this enzyme under such conditions is of considerable
importance.
In this communication we report the synthesis of a novel
derivative of Noria called NoriaPG. The role of Noria and
NoriaPG on the stabilization of L. sakei L-AI has been explored.
We have shown that the L-AI stability at low pH and high
temperatures is highly increased by these molecules, which is of
great interest for the use at industrial scale (Scheme 1).
Noria was synthesized, by acid catalysed condensation of
pentandial with resorcinol, as previously reported by Kudo et al.18
NoriaPG was synthesised, using pyrogallol instead of resorcinol,
as described in the ESI.w
The IR spectrum shows a total absence of the CQO stretching
band at 1700 cmÀ1 implying complete cyclization. The MALDI-
TOF mass spectrum shows a single peak at 1900.3 Daltons for
the M + H+ cation (Fig. S1, ESIw). Lactobacillus sakei L-AI was
produced in E. coli and then purified.19 The biochemical
characterization of the enzyme was performed as described (ESIw).
The study of the enzyme behaviour at different temperatures
confirmed what has been previously reported concerning the
a
´
LMI CNRS UMR 5615, Universite de Lyon 1, Villeurbanne,
F69622, France. E-mail: antony.coleman@adm.univ-lyon 1.fr;
Tel: +33 4 4243 1027
b Laboratory for Biocrystallography and Structural Biology of
´
Therapeutic Targets, ‘‘Bases Moleculaires et Structurales des
Systemes Infectieux’’ (BMSSI) UMR5086-CNRS/Universite de
`
´
´
Lyon 1, Institut de Biologie et Chimie des Proteines FR3302, 7
Passage du Vercors, F-69367 Lyon cedex 07, France.
E-mail: m.rhimi@ibcp.fr, rhimimoez@yahoo.fr;
Fax: +33 4 72 72 26 16; Tel: +33 4 72 72 26 34
c Institut National de la Recherche Agronomique, UMR 1319 Micalis,
F-78350 Jouy-en-Josas, France
Scheme 1 Molecular structures of Noria (left) and its pyrogallol
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc14930j
derivative NoriaPG (right).
c
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Chem. Commun., 2011, 47, 12307–12309 12307

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suggest that this arises from Noria and especially NoriaPG
stabilizing the enzyme, at low pH and high temperatures.
Enzyme thermostability studies showed that the enzyme was
fully stable at temperatures until 35 1C after 24 h (data not
shown). After 2 h at 40 and 45 1C the enzyme maintained only
95% and 60% of its initial activity, respectively, and at 50 1C the
half-life was evaluated to be 80 min (Fig. 2A). At higher
temperatures the enzyme was rapidly inactivated (data not shown).
Fig. 1 Temperature (A) and pH (B) activity profiles of the protein
supplemented with Mg2+ and Mn2+ (E), Noria (’) and NoriaPG
(m). Activities at optimal-pH and -temperature were defined as 100%
and correspond to 218 U mgÀ1. Error bars represent the standard
deviation from three separate experiments.
implication of magnesium and manganese ions on the
enhancement of the L. sakei activity.19 In fact, the enzyme
activity was increased nearly 1.8 fold after addition of 0.8 mM
Mg2+ and 0.8 mM Mn2+. Analysis of the L-AI activity
profiles at different temperatures demonstrated that at low
temperatures the addition of Noria or NoriaPG did not
significantly influence the enzyme activity (Fig. 1A). Conversely,
at higher temperatures including those over 35 1C, the relative
activity values are clearly improved in the presence of Noria
and NoriaPG, with the highest relative activity obtained in the
case of the enzyme supplemented with NoriaPG (Fig. 1A). At
55 1C the L. sakei L-AI has relative activities of 47%, 59% and
66% for the protein supplemented with divalent ions, Noria
and NoriaPG, respectively. These data suggest that Noria and
NoriaPG seem to be involved in modulating the enzyme
activity only at high temperatures.
Investigation of the enzyme activity at different pH values
disclosed that Noria and NoriaPG do not affect the enzyme pH
profiles at pH’s higher than 5.0 (Fig. 1B). Nevertheless, at lower
pH values these compounds increase the enzyme activity when
compared to the free enzyme. Noticeably, the highest relative
activities were again achieved by using NoriaPG (Fig. 1B).
Thus, both Noria and NoriaPG appear to improve L-AI
activity both at high temperatures and at low pH. Our results
Fig. 2 Thermostability profiles of (A) the free L. sakei L-AI, (B) in
the presence of 1 mM Noria and (C) 1 mM NoriaPG: (’) 35 1C, (E)
40 1C, (m) 45 1C and (K) 50 1C. The initial activity was defined as
100% and corresponds to 218 U mgÀ1 at pH 5.0. Error bars represent
the standard deviation from three separate experiments.
c
12308 Chem. Commun., 2011, 47, 12307–12309
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Table 1 Effect of addition of Noria and NoriaPG on the L-AI pH
stability. Here are presented the half-lives in hours. Noria and
NoriaPG were added at a final concentration of 1 mM. WA denotes
measurement without addition of Noria and NoriaPG
(Table 1). At pH values above 5.0 and after addition of Noria
and NoriaPG, the obtained half-lives were equal to those
found in the absence of these compounds (Table 1). These
trends showed that at high pH the Noria and NoriaPG did not
participate in the stabilization of L. sakei L-AI but do so at low
pH values.
pH
WA
Noria
NoriaPG
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
33 Æ 0.1
36 Æ 0.2
42 Æ 0.4
45 Æ 0.3
49 Æ 0.7
48 Æ 0.6
47 Æ 0.5
43 Æ 0.8
37 Æ 0.4
40 Æ 0.2
44 Æ 0.4
47 Æ 0.1
50 Æ 0.6
52 Æ 0.8
47 Æ 0.4
47 Æ 0.2
43 Æ 0.6
37 Æ 0.3
43 Æ 0.1
46 Æ 0.5
50 Æ 0.9
53 Æ 0.6
54 Æ 0.7
47 Æ 0.5
48 Æ 0.3
43 Æ 0.2
37 Æ 0.4
In conclusion, for the first time, we report here the use of
Noria and NoriaPG as stabilizers for macromolecules having
industrial potential. The use of these interesting components in
the case of the food-grade L. sakei L-AI results in an increase
of the enzyme stability at low pH and high temperatures. This
perfectly matches with the industrial application conditions
which promotes the use of this biocatalyst. In our further work
we are investigating the use of Noria and NoriaPG as
stabilizing agents for other agrofood and pharmaceutical
applications.
Addition of 0.8 mM Mg2+ and 0.8 mM Mn2+ enhanced the
enzyme thermostability, see ESI.w In fact, in the presence of
these divalent ions the enzyme retains 88% and 78% of its
activity after 2 h at 45 and 50 1C, respectively (Fig. S2, ESIw).
These results confirm the role of divalent ions on the stabili-
zation of the L. sakei L-AI.19 Addition of Noria and NoriaPG
did not affect the enzyme thermostability at temperatures up
to 35 1C (Fig. 2B and C). However, at higher temperatures
including 40, 45 and 55 1C the enzyme thermostability is
highly improved (Fig. 2B and C). These data confirm, as
observed for the metal ions, that Noria and NoriaPG did
not influence the enzyme activity but its stability.
Notes and references
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Therefore, we further explored the effect of Noria and
NoriaPG concentration on the thermostability (Fig. S3, ESIw).
This study demonstrates that for its maximal thermostability
the L. sakei L-AI requires 1 mM of Noria and 1 mM NoriaPG
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of divalent ions demonstrating that the derivative NoriaPG
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NoriaPG in the presence of 0.8 mM Mg2+ and 0.8 mM Mn2+ leads
to increased stabilization of the L. sakei L-AI (Fig. S4, ESIw).
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compounds do not act in the same way as divalent cations.
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active and acidotolerant L-AI at high temperatures and low
pH (Fig. S5, ESIw). This gains more importance when one
considers that for industrial applications it will be of use to
stabilize the biocatalyst as long as possible.
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Investigation of pH stability revealed that addition of Noria
and NoriaPG enhanced the half-lives at pH below 5.0
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12307–12309 12309