Cloning expression and characterization of a thermostable exopolygalacturonase from Thermotoga maritima

Article abstract of DOI:10.1016/S0008-6215(03)00165-4

A gene encoding for a thermostable exopolygalacturonase (exo-PG) from hyperthermophilic Thermotoga maritima has been cloned into a T7 expression vector and expressed in Escherichia coli. The gene encoded a polypeptide of 454 residues with a molecular mass of 51,304 Da. The recombinant enzyme was purified to homogeneity by heat treatment and nickel affinity chromatography. The thermostable enzyme had maximum of hydrolytic activity for polygalacturonate at 95°C, pH 6.0 and retains 90% of activity after heating at 90°C for 5 h. Study of the catalytic activity of the exopolygalacturonase, investigated by means of ¹H NMR spectroscopy revealed an inversion of configuration during hydrolysis of α-(1→4)-galacturonic linkage.

Full text of DOI:10.1016/S0008-6215(03)00165-4

Carbohydrate Research 338 (2003) 1333ꢀ1337
/
www.elsevier.com/locate/carres
Note
Cloning expression and characterization of a thermostable
exopolygalacturonase from Thermotoga maritima
Judica e¨ l Parisot, Virginie Langlois, Vehary Sakanyan, Claude Rabiller*
Unit e´ de Recherches en Biocatalyse (unit e´ CNRS 2230), Facult e´ des Sciences et des Techniques, 2, rue de la Houssini e` re, BP, 92208 F-44322 Nantes,
France
Received 7 February 2003; received in revised form 31 March 2003; accepted 1 April 2003
Abstract
A gene encoding for a thermostable exopolygalacturonase (exo-PG) from hyperthermophilic Thermotoga maritima has been
cloned into a T7 expression vector and expressed in Escherichia coli. The gene encoded a polypeptide of 454 residues with a
molecular mass of 51,304 Da. The recombinant enzyme was purified to homogeneity by heat treatment and nickel affinity
chromatography. The thermostable enzyme had maximum of hydrolytic activity for polygalacturonate at 95 8C, pH 6.0 and retains
1
9
0% of activity after heating at 90 8C for 5 h. Study of the catalytic activity of the exopolygalacturonase, investigated by means of H
NMR spectroscopy revealed an inversion of configuration during hydrolysis of a-(10
/4)-galacturonic linkage.
#
2003 Elsevier Science Ltd. All rights reserved.
Keywords: Exopolygalacturonase; Thermostable enzyme; Thermotoga maritima; Inversion of configuration mechanism
1
. Introduction
chanism exhibited by glucuronidase seems different
since endo b-polyglucuronidase (family 79) or b-glucur-
onidase (family 2) were recognized as retaining enzymes
and thus were able to catalyse regioselective synthesis of
Poly[(10
/
4)-a-
D-galacturonates] are degradated by two
types of enzymes. On one hand lyases (EC 4.2.2.)
catalyse a b-4,5-elimination reaction leading to an
unsaturated 4,5-glycuronate moiety. For instance, exo-
polygalacturonate lyases are able to catalyze this
elimination at the terminal reducing part of the poly-
galacturonates either at the first, at the second or at the
8
ꢀ10
glycuronic linkage.
enzymes since they are commonly used as processing
Nevertheless, PGs are important
1
tools in food industries. So far, several exo-PGs have
1
1
2ꢀ20
been purified and studied,
but few have been cloned
2
1
and overexpressed. The need of new exo-PGs activities
is important for food industry applications and parti-
cularly from the thermostability point of view. For this
reason, we have chosen Thermotoga maritima as a
source assuming that metabolism of this hyperthermo-
philic bacterium might reflect the evolutionary adapta-
tion to extreme environmental conditions. Besides, a
better solubility of substrates at high temperatures and a
longer operational stability of the corresponding enzyme
might be a crucial prerequisite for performing the
catalytic synthesis with a thermostable biocatalyst. A
nucleotidic sequence (Tm0437) coding for a putative
1
ꢀ5
third galacturonyl bond.
polygalacturonases (endo-PGs, Poly[(10
turonide] glycanhydrolase EC 3.2.1.15) and exo-PGs
hydrolyse the (104) bond between two a-galacturonic
acid (GalA) residues. Exo-PGs EC 3.2.1.67 and EC
.2.1.82 release GalA and dimeric GalA, respectively.
On the other hand, endo-
/
4)-a- -galac-
D
/
3
On the basis of their sequence, PGs as well as
rhamnogalacturonases have been classified in family
6
8 of glycosidases. Most of these enzymes have been
2
described as ‘inverting’ enzymes. Conversely, the me-
7
2
2
exo-PG has been identified in the T. maritima genome.
*
Corresponding author. Tel.: ꢁ
51-125637.
E-mail address: claude.rabiller@chimbio.univ-nantes.fr (C.
Rabiller).
/
33-251-125732; fax: ꢁ33-
/
Following this indication, we have cloned and over-
expressed the corresponding gene in Escherichia coli
cells. The enzyme thus obtained had an a-exogalactur-
2
0008-6215/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00165-4

1334
J. Parisot et al. / Carbohydrate Research 338 (2003) 1333ꢀ1337
/
onase activity. The aim of this paper is to present the
biochemical properties of this thermostable hydrolase.
2
. Results and discussion
2.1. Cloning of the exopolygalacturonase gene from T.
maritima
Fig. 2. Thermal stability of exopolygalacturonase from T.
maritima.
PCR allowed the genesis of a 1.5 kb DNA fragment
covering a region with exo-PG gene from the T.
maritima MSB8 strain and its cloning into a pCR4-
TOPO vector (Invitrogen). The coding sequence for exo-
2
.3. Biochemical properties of exo-PG
2.3.1. Thermal stability of exo-PG. Fig. 2 shows the
PG was subcloned into a pET21d(ꢁ) vector and the
/
variation of the enzymatic activity over a 5 h incubation
period at various temperatures. At temperatures up to
9
resulting plasmid pETpgTm was transformed into E.
coli BL21(DE3) cells.
0 and below 100 8C, the enzymatic activity remained
constant; at Tꢂ100 8C, the enzyme lost 50% of its
/
initial activity within 30 min.
2.2. Expression and purification of the
exopolygalacturonase gene from T. maritima
The variation versus temperature of the initial rate of
the hydrolysis reaction catalyzed by T. maritima exo-
polygalacturonase was determined under conditions of
Bacterial cells were grown at 30 8C followed by a 6 h
cultivation at the same temperature after addition of
the initial rate. A maximum was obtained at Tꢂ
/
95 8C
(Fig. 3). Obviously, the curve shown in Fig. 3 is the sum
of two phenomena: the increase of the reaction rate with
temperature and the partial inactivation of the enzyme.
isopropyl 1-thio-b-D-galactopyranoside (IPTG) in order
to induce the expression of a functionally active enzyme.
The overexpressed exo-PG was purified in two steps.
Heating the extracts of E. coli BL21(DE3)/pETpgTm
cells at 70 8C for 1 h allowed to denaturate the majority
of proteins of the mesophilic host. A partially purified
protein was then subjected to chromatography on a
At T ꢀ95 8C, the rate of enzyme inactivation becomes
/
important thus leading to an overall decrease of the
activity.
2.3.2. Optimum pH of exo-PG. At pH values below 4.5
2
ꢁ
Ni /nitriloacetic resin. The purified protein of 51 kDa
was detected by SDS-PAGE that corresponds to the
deduced molecular mass 51,304 Da of a Tm0437 ORF
from T. maritima (Fig. 1).
or above 7.5, no appreciable activity could be detected.
As seen Fig. 4, a maximum activity was obtained at pH
6
in 0.1 M acetate buffer. In 0.1 M phosphate or in 0.1
M citrate/0.2 M phosphate buffers, the maximum
activities, obtained for pH 5.8 with the later, were lower
than that measured in acetate buffer, suggesting an
inhibitory effect by phosphate ions.
2.3.3. Determination of the selectivity and the
stereoselectivityofexo-PG.Theendproductsofenzymatic
hydrolysis of polygalacturonic acid by exo-PG were
Fig. 1. Purification steps of T. maritima exopolygalacturonase
from E. coli BL21(DE3)/ETpgTm cells. A, crude extracts of E.
coli BL21(DE3)/ETpgTm cells; B, crude extracts of E. coli
BL21(DE3)/ETpgTm cells after IPTG induction; C, protein
Fig. 3. Variation with temperature of the initial rate of the
hydrolysis reaction catalyzed by T. maritima exopolygalactur-
onase.
2
ꢁ
purified on Ni /NTA resin; D, reference molecular mass
proteins (Da).

J. Parisot et al. / Carbohydrate Research 338 (2003) 1333ꢀ
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1337
1335
mechanism is conserved in the sequence-based families
2
of glycoside hydrolases.
3
In conclusion, the putative ‘inverting’ exo-PG from T.
maritima was cloned and overexpressed in E. coli. This
very thermostable hydrolase which present a maximum
activity at pH 6, constitutes a powerfull tool for food
industry applications.
3. Experimental
3
.1. DNA isolation and cloning
Fig. 4. Variation of the activity of T. maritima exopolygalac-
turonase as a function of pH (j, acetate buffer 0.1 M; ',
phosphate buffer 0.1 M; m, citric acid 0.1 Mꢁ
/
dibasic sodium
Chromosomal DNA of T. maritima MSB8 was isolated
4
as described by Dimova with minor modifications.
The T. maritima DNA region containing a putative PG
gene was amplified by PCR with oligonucleotide pri-
2
phosphate 0.2 M buffer).
analysed using thin layer chromatography (TLC).
Galacturonic acid was the only product detected during
hydrolysis, demonstrating that this enzyme is an EC
mers
5?-CTGCACTAGCTAGTACC
and
TTCTCCAGA-GGCATGTC. Thirty cycles of PCR
5?-
3
.2.1.67 exopolygalacturonase (Scheme 1). A complete
absence of digalacturonic acid and of DPB6 was noticed
on the TLC plates. Exo-PG also catalyses hydrolysis of
polygalacturonic acid of 2BDPB6. These results
indicate that -galacturonic acid was released from the
non-reducing end of polygalacturonate. Other possible
substrates like pNP-a- -galacturonic acid and oNP-a-
amplification with Goldstar Taq DNA polymerase
/
(
4
2.5 U) were carried out in a 50 mL solution containing
00 mM dNTPS each, 20 pmol each primers and 200 ng
/
/
genomic DNA template. The PCR product (1.5 kb) was
cloned using the TOPO TA Cloning kit (Invitrogen).
Each cycle consisted of heating at 94 8C for 1 min, 48 8C
for 1 min and 72 8C for 1 min 30 s. Next, the PG coding
sequence was amplified using two flanking primers 5?-
D
D
D-
galacturonicacidwerenotsubstratesforthisenzyme. This
resultsuggeststhatexo-PGmayhaveatleasttworesidues-
binding subsites and thus requires at least two carbox-
ylates for substrate recognition.
ATGA/CCATGG
/
AAGAACTGGCAAAAAAG-3? and
5
?-CCG
/
CTCGAG/
TTTCAGCAGGCTTACCTTC-3?,
carrying additionally NcoI or XhoI restriction sites.
PCR Amplification 30 cycles with Goldstar Taq DNA
polymerase (2.5 U) was carried out in a 50 mL reaction
containing 400 mM dNTPS each, 20 pmol each primers
and 10 ng plasmidic DNA template. Each cycle con-
sisted of heating at 94 8C for 1 min, 50 8C for 1 min and
In order to determine the mode of action of the exo-
PG, we have followed hydrolysis of digalacturonate by
1
means of H NMR in situ. Monitoring of the reaction
revealed that the released monomer II had the b-
configuration [d(H-1II)ꢂ
/
4.56 ppm, J1,2ꢂ
7.9 Hz) (see
/
Fig. 5). The slow apparition of a H-1 resonance for a-
galacturonic acid at 5.28 ppm is due to the mutarotation
of the b-anomer of galacturonic acid II and to monomer
I (Scheme 1). This experiment shows that exo-PG
induces the synthesis of the b-anomer of galacturonic
acid from the hydrolysis of oligogalacturonates and
consequently is an ‘inverting’ glycanase. This result
confirms many earlier observations that the molecular
7
2 8C for 1 min 30 s. The PCR product was purified
using the QIAquick PCR purification kit (Qiagen) and
digested with NcoI and XhoI restriction enzymes. A
band of the correct size predicted for the digested PG
gene was purified by electrophoresis and ligated into the
T7 expression vector pET21d(ꢁ) (Novagen) that al-
lowed the creation of a His-tag at the C-terminal
extremity of the protein.
/
Scheme 1. Stereoselectivity of the hydrolysis reaction catalyzed by exo-PG from T. maritima.

1336
J. Parisot et al. / Carbohydrate Research 338 (2003) 1333ꢀ1337
/
Fig. 5. Proton NMR spectra (500 MHz, solvent D
maritima exo-PG for 5 min (B). The chemical shifts are quoted from the H trimethysilyl resonance of DSS.
2
O, pH 6.0, 25 8C) of pure digalacturonate (A) and after incubation with T.
1
3
.2. Protein overexpression and purification
and the supernatant was loaded on a column containing
3
2
mL Ni /NTA resin (Quiagen). The column was first
ꢁ
The E. coli BL21(DE3) strain harbouring the cloned T.
maritima PG gene in pET21d(ꢁ) was grown in LB with
00 mg/mL ampicillin at 30 8C to OD600 0.8 and
incubated further with isopropyl b- -thiogalactopyra-
washed twice with 10 mL of the described buffer except
the concentration of imidazole which was 50 mM. The
protein attached to the resin was eluted with the same
buffer containing 250 mM imidazole and the collected
/
1
D
noside (IPTG, 1 mM) for 6 h. The cell culture (500 mL)
was centrifuged (2500g, 15 min) and resuspended in 10
mL of 50 mM sodium phosphate buffer (pH 8.0)
containing NaCl (300 mM) and imidazole (10 mM).
The cells were lysed by ultra-sound Raytheon 250 W
apparatus, centrifuged (18,000g, 15 min) and the clear
lysate was incubated at 70 8C for 1 h in order to
denature E. coli proteins. After centrifugation at
protein was dialysed against 10 mM TrisꢀHCl buffer,
pH 8.0. The purified PG protein was analyzed by SDS-
PAGE (12% polyacrylamide gel, 0.1% SDS, 375 mM
/
TrisꢀHCl buffer pH 8.8) at a constant voltage (120 V).
/
The gel was coloured with Coomassie blue G-250 (Bio-
Rad). Protein concentrations were determined using
Bradford method. The reactant (50 mL) was added to
200 mL of the sample on microplates. The optical density
was measured at 620 nm after 10 min incubation at
1
8,000 g for 15 min at 4 8C, the debris were discarded

J. Parisot et al. / Carbohydrate Research 338 (2003) 1333ꢀ
/
1337
1337
room temperature. The protein concentration was
calculated from a calibration curve obtained with bovine
serum albumin (Sigma).
Recherche Scientifique for financially supporting this
work.
3.3. Biochemical analysis of T. maritima
exopolygalacturonase
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/
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3
/
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/
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.3.3. Determination of the optimal temperature and pH.
/
The activity of the exo-PG was measured in the standard
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/
/
/
3.4. Determination of the selectivity and catalytic
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/
1
1
/
The analysis of hydrolysis products was followed by
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/
1
2
2
/
565.
6
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/
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