Biotransformation of procyanidins by a purified fungal dioxygenase: Identification and characterization of the products using mass spectrometry

Article abstract of DOI:10.1016/j.procbio.2010.02.019

Procyanidins commonly known as condensed tannins are a type of polyphenol with wide abundance naturally. They are commonly known as potent anti-oxidants with powerful free radical scavenging activity as well as anti-tumor-promoting activity. Little is known about the enzymatic mechanisms/pathways involved in the microbial biotransformation of these polyphenolic molecules. The extracellular enzyme, dioxygenase produced by Aspergillus fumigatus was used as in vitro tools to study the degradation pathway of a model procyanidin dimer, namely procyanidin B2. The enzyme was purified to homogeneity by a two step process of anion-exchange chromatography coupled with FPLC followed by gel-filtration chromatography coupled with HPLC and the molecular mass estimated. In addition, the different biotransformed products resulted from the dioxygenase action on PB2 were purified using Reversed-Phase-High Performance Liquid Chromatography prior to their identification and characterization by structural elucidation using Electrospray Ionization-Mass Spectrometry. Subsequently, the mechanism of dioxygenase action on procyanidin dimer was defined.

Full text of DOI:10.1016/j.procbio.2010.02.019

Process Biochemistry 45 (2010) 904–913
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Biotransformation of procyanidins by a purified fungal dioxygenase:
Identification and characterization of the products using mass spectrometry
a
b
c
c
a
Krishnankutty Roopesh , Sylvain Guyot , Abdulhameed Sabu , M. Haridas , Perraud Gaime Isabelle ,
Sevastianos Roussos , Christopher Augura
IMEP-IRD-UMR-193, Ecologie Microbienne et Biotechnologies, Case 441, Université Paul Cézanne, FST St Jérôme, Avenue Escadrille Normandie-Niemen,
a,∗
a
F-13397, Marseille Cedex 20, France
b
INRA, UR117, Unité de Recherches Cidricoles, Biotransformation des Fruits et Légumes, Le Rheu, F-35653, France
Department of Biotechnology and Microbiology, School of Life Sciences, Palayad campus, Kannur University, Kannur 670661, Kerala, India
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Procyanidins commonly known as condensed tannins are a type of polyphenol with wide abundance nat-
urally. They are commonly known as potent anti-oxidants with powerful free radical scavenging activity
as well as anti-tumor-promoting activity. Little is known about the enzymatic mechanisms/pathways
involved in the microbial biotransformation of these polyphenolic molecules. The extracellular enzyme,
dioxygenase produced by Aspergillus fumigatus was used as in vitro tools to study the degradation pathway
of a model procyanidin dimer, namely procyanidin B2. The enzyme was purified to homogeneity by a two
step process of anion-exchange chromatography coupled with FPLC followed by gel-filtration chromatog-
raphy coupled with HPLC and the molecular mass estimated. In addition, the different biotransformed
products resulted from the dioxygenase action on PB2 were purified using Reversed-Phase-High Perfor-
mance Liquid Chromatography prior to their identification and characterization by structural elucidation
using Electrospray Ionization-Mass Spectrometry. Subsequently, the mechanism of dioxygenase action
on procyanidin dimer was defined.
Received 24 August 2009
Received in revised form 5 January 2010
Accepted 11 February 2010
Keywords:
Procyanidins
Aspergillus fumigatus
Dioxygenase
Purification
Reversed-Phase High Performance Liquid
Chromatography
Electrospray Ionization-Mass Spectrometry
©
2010 Elsevier Ltd. All rights reserved.
1
. Introduction
Also, little is known about the enzymes involved in the degradation
pathway of these oligomeric procyanidins. The ability to modulate
or biotransform these molecules could have significant applications
in the field of food biotechnology (fruit juice clarification, control
of palatability, digestibility of feedstock, etc.).
Procyanidins (PCs) or condensed tannins are phenolic com-
pounds with pronounced biological activities found in many plants,
in wide variety of fresh fruits, foods and beverages [1]. PCs are
known for their ability to bind strongly to proteins, reducing sig-
nificantly the nutritional value of the feed especially in animal
diets [2]. Recently, procyanidins have attracted interest because
of their antioxidant properties [3,4]; also increasing evidence for
oligomers in a wide range of active assays has attracted attention
to their structural elucidation [5]. Owing to the complexity of the
oligomers, most studies have focused on specific dimeric procyani-
dins. PCs are known to inhibit microbial growth [6]. Nevertheless,
certain microorganisms mainly bacteria are able to grow on pro-
cyanidins by using it as a sole carbon source [7]. However, only the
information on PCs degradation by fungi is scarce mainly due to
lack of commonly available substrates. Mean time, there are certain
reports mentioning the ability of filamentous fungi to degrade these
complex molecules [8,9]. Moreover, the enzymatic mechanisms
have not been established as standards are scarce and expensive.
Natural PCs are oligomers and polymers of flavan-3-ol units
such as (+)-catechin or (−)-epicatechin [10] mostly linked by C4-
C8 or C4-C6 inter-flavan bond (Fig. 1). In 1994, Nguz et al. [11]
reported for the first time the degradation of PCs by Penicillium.
PCs are characterized by their average degree of polymerization
(DPn) that corresponds to the average number of flavanol units
making up the polymer and it is measured as the molar ratio
between all units (terminal + extension) and terminal units. The
ratio obtained after procyanidin de-polymerization in the presence
of benzyl thioether (thiolysis) can be quantified by HPLC analysis of
the thiolysis medium [12,13]. Also, the use of thiolysis-based tech-
nique has allowed the characterization and quantification of the
constitutive units of procyanidins in apples and apple products [14].
In recent years, the coupling of liquid chromatography–mass spec-
trometry (LC–MS) with electrospray ionization (ESI) techniques
has been proven to be a powerful tool for the characterization
of PC structures. Once identified, PC structures can be elucidated
through the study of fragment ions obtained by multiple fragment
∗ Corresponding author. Tel.: +33 491 289103; fax: +33 491 288668.
E-mail address: s.roussos@univ-cezanne.fr (S. Roussos).
n
mass spectrometry (MS ) [15]. In addition, more recently, LC–MS
1
359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.02.019

K. Roopesh et al. / Process Biochemistry 45 (2010) 904–913
905
Fig. 1. General structures of procyanidins: (A) (+)-catechin and (−)-epicatechin monomers, (B) procyanidin B2 dimer, (C) proyanidin oligomers with C4-C8 linkage (adapted
from Contreras-Domínguez et al., 2006).
and LC–MS–MS was shown to be well adapted to the analysis of
polyphenol oxidation products resulting from chemical or enzy-
matic oxidation [16]. Also, there are reports stating that catechol
2.2. Culture medium
Solid-State Culturing (SSC) was employed for production of the enzyme. Zero
point three (0.3) gram of Polyurethane foam (PUF) was weighed into 100 mL
Erlenmeyer flask, cotton plugged and sterilized. Nutrient (basic) solution [19] was
prepared by dissolving (in g L ) (NH ) SO 1.0; KH PO 1.3; Na HPO4 0.12; MgSO4
0.3; urea 0.3; glucose 2.0 in distilled water, supplemented with 0.1% of oligo-element
1
,2-dioxygenase and catechol 2,3-dioxygenase are some of the key
−
1
enzymes involved in the catabolism of monocyclic aromatic com-
pounds [17]. In a previous research work [18], the main degradation
product resulting from the action of an oxygenase from Aspergillus
fumigatus on procyanidin B2 was identified.
4 2 4 2 4 2
solution (FeSO4·7H2O 5.0; MnSO4·H20 1.6; ZnSO4·7H2O 1.4; CaCl2 2.0); prepared in
distilled water. The pH of the oligo-element solution was adjusted to 6.0 using NaOH
◦
(
3.0 M) and sterilized at 121 C, 15 psi for 20 min.
The aim of the present study was to obtain a complete under-
standing of the different steps involved in microbial degradation
of condensed tannins (PCs) through the study of key enzymes
involved in the degradation process. The extracellular enzyme,
dioxygenase produced by A. fumigatus was used as in vitro tools
to study the degradation pathway of a model procyanidin dimer,
namely procyanidin B2 [(−)-epicatechin-(4␤-8)-(−)-epicatechin].
The enzyme was purified to homogeneity and the different bio-
transformed products obtained after the catalytic action of the
enzyme on Procyanidin B2 (PB2) dimer were purified using liquid
chromatography, subsequently their structures were elucidated by
electrospray ionization-mass spectrometry (ESI-MS) and finally the
mechanism of enzyme action was defined.
2.3. Induced enzyme production
PCs with DPn of 9 were obtained from a cider apple freeze-dried powder (Marie
Ménard variety) by aqueous acetone and solid phase extraction [20]. This was used
as a carbon source for A. fumigatus growth and to induce the dioxygenase enzyme
production. Purified PCs dissolved in sterile nutrient (basic) solution were added
asepticallytothePUF (solid support) usingasyringe filter (PVDF- 0.22 ␮m, Millipore)
to reach a final concentration of 2.0 g L . Inoculation was done with A. fumiga-
tus spore suspension (respecting the final moisture content –85 ± 2%). The flasks
were then shaken (in upright position, tapped at the bottom) gently to homoge-
−
1
◦
nize the culture medium. Fermentation was carried out at 30 C for 36 h. All of the
experiments were performed under strict sterile conditions and in triplicate.
2
.4. Extracellular extract
The fermented medium was mixed gently using a sterile spatula and the con-
tents were squeezed through a sterile syringe (50 mL, Millipore) using hand pressure
2.
Materials and methods
to get the extracellular enzyme extract. The extract was collected in clean storage
2
.1. Microorganism and inoculum preparation
vials, a portion of it stored at 4 ◦C was used for enzymatic as well as other assays and
◦
the remainder was frozen (at −80 C) until further use.
The filamentous fungus A. fumigatus MC 8 (wild strain isolate, IRD Laboratory
◦
culture collection, France) was cultured on Potato Dextrose Agar (Sigma) at 30
C
2
.5. Procyanidin removal from extracellular extract
for spore production. Six days old spores were harvested with 30 mL of sterile dis-
tilled water containing 0.01% (v/v) Tween 80 under controlled agitation. The spore
suspension (5 × 10 spores/mL) obtained was used as inoculum of culture medium.
The presence of non-biotransformed (residual) PCs in the extracellular enzyme
extract is responsible for interferences with different types of assays. For instance, it
7

9
06
K. Roopesh et al. / Process Biochemistry 45 (2010) 904–913
an hour and then cooled at room temperature for 15 min. Color formation was mon-
itored with a spectrophotometer set at 550 nm. PCs were quantified by comparison
to a PC standard curve.
2
.8. Bradford (micro) assay for total soluble protein content
The total soluble protein content of the extracellular extract before and after
the addition of ampholyte was quantified by using the method of Bradford [22].
The extracellular extract (100 ␮L) was diluted to a final volume of 1000 ␮L using
Milli-Q ultrapure water to which 200 ␮L of concentrated Bradford dye (Bio-Rad)
was added. The solution mix was vortexed well and incubated at room temperature
for 30 min. Thereafter, the samples were measured at 595 nm against a reagent
blank (containing procyanidins) in a spectrophotometer. Several dilutions of protein
−
1
standard (Bovine Serum Albumin, Sigma) from 1 to 25 ␮g mL concentration were
also prepared and read at the same wavelength. The OD595 versus concentrations
of standards plotted, served as the standard curve in determining the unknown
concentrations.
2
.9. Iso-electric focusing (IEF)
The pI of the enzyme was determined by using the technique of iso-electric
focusing. Forty milliliters of the extracellular enzyme extract mixed with 1.0 mL of
ampholyte (3/10, Bio-Rad) solution was loaded into Rotofor [Preparative Iso-Electric
Focusing cell (IEF); Bio-Rad] system under controlled manner. A power pack con-
nected to the system delivered 12 W to the IEF cell. The whole unit was connected
◦
to a cooling system and was maintained at 4 C during the entire run. The concen-
trated and separated samples were harvested as (2.0 mL × 20) different fractions
from the IEF cell using a harvesting kit under vacuum. The pH of each of the frac-
tions obtained was read immediately. Subsequently, the fractions were assayed for
dioxygenase activity as well as the total soluble protein content.
Fig. 2. SDS-PAGE of purified dioxygenase from A. fumigatus MC8. Lane 1: standard
proteins of different molecular weight (205.0–24.0 kDa); Lane 2: purified (freeze-
dried sample after gel filtration) dioxygenase run in SDS-PAGE (10%) and stained
with commassie blue.
2
.10. Purification of extracellular dioxygenase
All steps in the purification were carried out at 0–4 C unless otherwise
◦
mentioned. Chromatography columns were carried out using Fast Protein Liquid
Chromatography (FPLC) system [Bio-Rad, Germany] as well as High Performance
Liquid Chromatography system [Agilent Technologies, USA]. The supernatant
obtained after procyanidin precipitation from the extracellular extract using
ampholyte (3/10) solution was applied to UNO
(Bioscale Mini UNOsphere Q cartridge – 5.0 mL, BioRad) pre-equilibrated with
0.1 M acetate buffer, pH 5.0 (buffer A) and extensively washed using the same buffer.
Proteins in the column were eluted using a linear NaCl gradient up to 2.0 M in buffer
A using BioLogic DuoFlow FPLC system (BioRad). Fractions with dioxygenase activ-
ity were pooled, desalted by dialyzing against Milli-Q water at 4 C and freeze-dried
(Heto Power Dry LL1500 Freeze Dryer, Thermo Electron Corporation, U.S.A.).
leads to heavy non-specific interactions with chromatographic materials (especially
solid phases) during enzyme(s) purification processes. As an attempt to exempt the
residual procyanidins from the extracellular extract, ampholyte (Bio-Lyte® 3/10,
Bio-Rad) was added to the crude enzyme extract (0.24%, v/v) with the objective
of precipitating selectively procyanidins and at the same time retaining enzyme
TM
Q6 anion-exchange column
TM
TM
◦
activity. The mixture was kept at 4 C for 1 h and then centrifuged at 12,000 rpm
for 20 min. The supernatant was collected and assayed for dioxygenase activity. The
total procyanidin content of the extracellular enzyme extract before and after the
addition of ampholyte solution was also quantified.
◦
2.6. Enzyme assay
2.10.1. Determination of molecular mass
The molecular mass of the purified protein was estimated by gel filtration chro-
matography using Bio-Sil® SEC-125 gel filtration column coupled with HPLC. The
freeze-dried samples (obtained after anion-exchange chromatography) were re-
suspended in Tris–NaCl buffer, pH 8.0 (0.1 M NaCl in 25 mM Tris) and loaded on to
Bio-Sil® SEC-125 gel filtration column (300 mm × 7.8 mm) using Agilent HPLC sys-
tem (Agilent Technologies, U.S.A.) pre-equilibrated with Tris–NaCl buffer, pH 8.0.
Proteins were eluted with an isocratic elution using Tris–NaCl buffer, pH 8.0. Frac-
tion(s) were purified from HPLC with respect to appearance of the absorption peaks
at 280 nm for proteins. The fraction (peak) with dioxygenase activity was separated,
desalted (as described above) and freeze-dried. The subunit molecular weight of
the protein was determined by Sodium Dodecyl Sulfate-Polyacrylamide Gel Elec-
trophoresis (SDS-PAGE). Electrophoresis using 10% (v/v) polyacrylamide gels was
performed as described by Laemmli [23].
The dioxygenase activity was estimated by the ability of the extracellular
enzyme extract to degrade/bio-transform the specific dimeric substrate procyani-
din B2 (PB2) [Fumakoshi, Japan]. The assay mixture contained 50 ␮L of PB2 solution
(
1.0 mM), 100 ␮L of phosphate buffer, pH 6.0 (0.2 M) and 100 ␮L of the extracellu-
◦
lar enzyme extract. The reaction mixture was vortexed and incubated at 30 C for
6
0 min. Ten % of TriChloro Acetic acid (TCA) in water was used to stop the reaction.
The assay mixture was then centrifuged (micro-centrifuge, Millipore) at 15,000 rpm
for 10 min. The supernatant was passed through micro filter (0.22 ␮m, PTFE, Milli-
pore) into small glass vials for High Performance Liquid Chromatography (HPLC)
analysis. The dioxygenase activity was determined by HPLC by following the disap-
pearance of the PC dimer PB2 (peak) at 280 nm. A substrate blank was also prepared
and analyzed against the reaction mixtures. Standard concentrations of PB2 from
1
.0 to 5.0 mM were also prepared and analyzed in the HPLC. One unit of activity was
−
1
defined as the amount of enzyme that catalysed the disappearance of 1 ␮mol min
of PB2 at 30 C.
2.11. Reversed-phase HPLC analysis
◦
The dioxygenase activity was quantified using HPLC by following the disappear-
ance of the PC dimer PB2 at 280 nm. The HPLC apparatus was equipped with a HP
quaternary gradient pump 1200 series (Agilent Technologies, U.S.A.), an autosam-
2.7. Total procyanidin determination
Total PCs were estimated as reported by Gessiner and Steiner [21]. The reaction
pler and a photo diode array detector. The enzymatically cleaved products were
TM
mixture contained 7.0 mL of butanol–HCl taken in a glass tube (15 ml), with 500 ␮L
separated on an Atlantis
dC18 column (250 mm × 4.6 mm, Waters Co., U.S.A.). A
◦
of extracellular extract added to it. The reaction mixture was then heated at 95 C for
binary gradient was applied (eluent A (%): acetic acid (2), Water (98); eluent B (%):
Table 1
Purification scheme for dioxygenase from Aspergillus fumigatus MC8.
Fraction
Volume (mL)
Protein (mg/mL)
Specific activity (Units/mg)
Purification fold
Recovery/yield (%)
Extracellular (crude) extract
Extracellular extract (after PC
precipitation using ampholyte)
75.0
74.3
2.77
2.91
1.37
1.51
1
1
100
92.0
TM
Eluate UNO Q column
5.0
2.5
3.35
0.73
11.53
53.0
8.4
38.7
83.3
35.2
Eluate Bio-Sil® SEC-125 column

K. Roopesh et al. / Process Biochemistry 45 (2010) 904–913
907
Fig. 3. UV 280 nm trace of the Reversed-Phase HPLC showing PB2 and its biotransformed products together with their elucidated structures (with their corresponding
pseudo-molecular ion m/z values in the negative mode mass spectrometry).
Acetonitrile (100); initial, 7% B; 0–15 min, 45% B linear; 15–20 min, 50% B linear;
Thermofinnigan), Diode array detector (UV6000Lp, Thermofinnigan) and data were
processed by Xcalibur® version 1.2 software. Samples were separated on a Zorbax
Eclipse XDB C18 column (2.1 mm × 150 mm, 3.5 mm, Agilent Technologies, U.S.A.).
A binary gradient was applied (eluent A, 0.1% formic acid in water and eluent B,
−1
2
0–40 min, 100% B linear, at a flow rate of 1.0 ml min , followed by reconditioning
®
and recalibration of the column. Data were processed by using Chemstation 2D
software.
0
.1% formic acid in acetonitrile): initial, 3% B; 0–5 min, 9% B linear; 5–15 min, 16% B
−
1
2.12. Purification of PB2 and the biotransformed products for analytical purposes
linear; 15–45 min, 50% B linear, at a flow rate of 0.2 ml min . Full scan signals were
recorded within the m/z range of 50–2000. Thiolysis was carried out according to the
procedure described by Guyot et al. [24]. In brief, 2.0 mL of anhydrous MeOH were
added to freeze-dried biotransformed PB2 samples (≈3.0 mg). The reaction was car-
ried out in glass tubes (250 ␮L) as follows: 50 ␮L of sample mixed with 100 ␮L of
benzylthioether solution (5% in anhydrous MeOH) and 50 ␮L HCl (0.4N) solution. The
Purification of PB2 and the enzymatically cleaved products were performed by
HPLC. The running and elution conditions were identical to those described above.
Peaks corresponding to residual PB2 and those of the major degraded products
obtained were collected separately (in total 4 peaks). Samples from about 50 runs
in HPLC were collected to obtain sufficient quantity of the products used for ana-
lytical purposes. For each of the runs 3.0 ␮g of biotransformed PB2 were injected
into HPLC column. Excess acetonitrile and acetic acid were eliminated by rota-
◦
mixture was incubated in a water bath at 40 C for 30 min. For the thiolysis reaction
on both PB2 and enzyme-treated PB2 (biotransformed), samples were diluted in
anhydrous MeOH and thiolysis was carried out as described just above. The reac-
tion was stopped by transferring samples to an ice bath. HPLC-ESI-MS analyses of
the thiolyzed products were also carried out as described above.
◦
evaporation at 35 C and the residues were freeze-dried. The samples were then
dissolved in MeOH (methanol) and filtered through a 0.45 mm Teflon membrane
(
PTFE, Millipore) and analyzed by Reversed-Phase High-Performance Liquid Chro-
matography/Electrospray Mass Spectrometry (RP-HPLC-ESI-MS). In addition, these
samples were also subjected to thiolysis and the products were analyzed by RP-
HPLC-ESI-MS.
3. Results and discussion
2
.13. HPLC-ESI-MS analysis of the biotransformed and thiolyzed products of PB2
The microbial synthesis of procyanidin biotransforming enzyme
was induced by growing A. fumigatus MC 8 on solid medium con-
taining cider apple procyanidins (DPn: 9) for 36 h. The extra-cellular
enzyme was extracted from the fermented medium, purified and
this purified dioxygenase enzyme was used to study the biotrans-
formation of procyanidin B2. The biotransformed products were
analyzed by LC–MS with or without combination of thiolysis reac-
tion.
The mass spectrometry (MS) apparatus was an LCQ Deca ion trap spectrome-
ter (Thermofinnigan, San Jose, CA, USA) equipped with an electrospray ionization
−
1
source. All MS experiments were carried out in the negative mode [M−H] . Nitro-
gen was used as nebulizing gas. For LC–MS, the source parameters were: spray
voltage 3.7 kV; sheath gas 65 arbitrary units; auxiliary gas 10 arbitrary units; cap-
illary temperature 250 C. Helium was used as dampening gas. Auto Gain Control
mode was used to optimize injection time. ESI-MS was coupled to an HPLC system:
HP binary gradient Pump 1100 series (Agilent Technologies), autosampler (Surveyor,
◦

9
08
K. Roopesh et al. / Process Biochemistry 45 (2010) 904–913
Scheme 1. Proposed fragmentation pathway of the ion (m/z) 641 from MS2 analysis.
3.1. Procyanidin removal from extracellular extract
peak was found to have dioxygenase activity, separating the pro-
tein of our interest from rest of the contaminants (proteins). Hence,
the first eluted peak (peak at 6.2 min) was purified, dialyzed and
freeze-dried. The freeze-dried sample was dissolved in Tris–NaCl
buffer and re-eluted on the same gel-filtration column to achieve
homogeneity. Single peak was observed after gel filtration-HPLC
indicating its homogeneity. A set of standard molecular weight
protein markers (Bio-Rad’s gel filtration standards, range: 1350 to
670,000 Da) were also injected into the gel filtration column to
obtain the retention time (RT) of the standard proteins. In com-
parison with the standard protein markers, the molecular mass of
the purified dioxygenase was estimated to be 117 kilo Dalton (kDa)
under non-denaturing conditions. About 40-fold purification of the
protein was achieved with a yield of 35% (Table 1).
Ampholyte solution (3/10) was found to be much efficient in
removing the PCs (residual) present in the extracellular extract
than the classical technique of polyphenol precipitation using
polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP). PC
precipitation was achieved by the addition of ampholyte solu-
tion, resulting in about 94% of its removal from the extracellular
extract retaining 92% of the enzyme activity indicating that the co-
precipitation of protein with procyanidins were much less. In the
case of PVP or PVPP it is the stronger affinity of these insoluble
molecules towards the polyphenols makes them precipitate from
a mixture while, the underlying mechanism of polyphenol precip-
itation by ampholytes is not known and is yet to be discovered as
this is the first report on it.
3.2.1. Molecular mass
3.2. Dioxygenase purification
SDS-PAGE of the purified enzyme indicated the presence of
a single band. The molecular weight of the purified dioxygenase
was calculated to be ∼39 kDa from the log plot obtained from the
relative mobility of the standard proteins in SDS-PAGE (Fig. 2).
However, molecular mass of the intact protein was found to be
117 kDa after gel filtration chromatography. This suggests that the
enzyme is a trimer. This is the first report of a trimeric dioxygenase
being purified from A. fumigatus MC8.
Iso-electric focusing using the system of rotofor resulted in
obtaining the pI of the enzyme which was found to be 3.5. Hence,
for further purification studies anion-exchange chromatography
was selected. Out of six different fractions obtained from anion-
exchange chromatography using Uno Q6 (5.0 mL) cartridge, only
three fractions (over lapped peaks) exhibited dioxygenase activity.
These three fractions were pooled together, desalted by dialysis
◦
against Milli-Q water (at 4 C) and freeze-dried. The sample was
3.3. Characterization of the biotransformed products of PB2
◦
then re-suspended in Tris–NaCl buffer (at 4.0 C), pH 8.0 and run
on HPLC-gel-filtration column to exclude out the proteins based
on their size. The elution profile obtained was with a single peak
clearly separated and eluted out at 6.2 min, followed by a bunch
of peaks that eluted out of column after 8 min. The first eluted
The products formed after the enzymatic cleavage of PB2 by
the action of dioxygenase were analyzed using HPLC. Chromato-
graphic profile could be considered as a way to characterize the
cleaved products: it informs on the polarity of the products (for

K. Roopesh et al. / Process Biochemistry 45 (2010) 904–913
909
Scheme 2. Proposed fragmentation pathway of the ion (m/z) 609 from MS2 analysis.
instance, in the present case with the enzymatically cleaved PB2,
we can observe that all the cleaved products were eluted before
PB2 indicating that they are more polar which is in accordance
with their structures containing carboxyl groups). Fig. 3 shows the
of the pseudo-molecular ion of PB2 is at m/z 577 in negative
mode.
The MS spectrum of compound PB2-X2 showed a pseudo-
−
molecular ion [M−H] at m/z 641 (Fig. 3) corresponding to an
(
UV 280 nm) HPLC profile of PB2 and the biotransformed products.
increase of 64 atomic mass unit (amu) compared to the m/z of
PB2 pseudo-molecular ion. This mass increase indicated that PB2-
X2 has integrated 4 additional oxygen atoms in its structure in
comparison to PB2, which is in accordance with a dioxygenase
intra-diol cleavage of two catechol rings. This suggested that both
the terminal and the extension units of the dimeric molecule were
oxidized. It could be noted that oxidation of the catechol ring into
a (5-Oxo-tetrahydro-furan-2-yl)-acetic acid group can lead to the
formation of a new asymmetric carbon which was not asymmet-
ric before oxidation of the catechol group which means that two
or four diastereoisomers for each of the compounds 641 and 609
could be potentially formed.
◦
After enzymatic reaction for 60 min at 30 C, pH 6.0, with PB2 as
substrate and the extracellular extract (enzyme) from A. fumiga-
tus, four different (major) peaks were observed in the HPLC profile.
Among them, three major peaks named PB2-X, PB2-X , and PB2-
2
X3 were eluted before PB2 showing RT at 11.6, 12.5 and 18.1 min,
respectively (Fig. 3). They were purified by HPLC and analyzed
by electrospray ionization-mass spectrometry. The corresponding
−
molecular ions [M−H] in the negative mode for each of these
peaks were at m/z 609, 641 and 609, respectively. PB2 had a RT of
2
3.61 min with a corresponding molecular ion at m/z 577 (Fig. 3).
The major products obtained after the enzymatic cleavage of
PB2: PB2-X, PB2-X , PB2-X3 were identified and characterized by
The full MS spectrum of compound PB2-X3 was very similar to
that of PB2-X, showing a pseudo-molecular ion at m/z 609 (Fig. 3).
Nevertheless, its RT was clearly longer than that of PB2-X which
indicated it is probably less polar. Therefore, our first hypothesis
was that this compound was the isomeric form of PB2-X with an
intra-diol catechol ring cleavage located at the extension unit of the
dimer PB2 instead of at the terminal unit.
2
mass spectrometry (Fig. 3). The MS spectrum of the compound PB2-
X showed an ion at m/z 609. This compound was already identified
in a previous published work [18]. Compared to PB2 structure, PB2-
X corresponds to a product with a structural modification of PB2
where the catechol ring of the terminal unit has been oxidized
by the intra-diol dioxygenase action and finally converted into a
non phenolic moiety corresponding to a (5-Oxo-tetrahydro-furan-
To determine the structures of PB2-X2 and PB2-X3 and also to
localize the respective oxidized moieties involved in degradation,
2
-yl)-acetic acid group (Fig. 3). For comparison, the MS spectrum

9
10
K. Roopesh et al. / Process Biochemistry 45 (2010) 904–913
Fig. 4. MS spectrum of the thiolytic reaction product of PB2. The ions generated are at m/z 411 [(−)-epicatechin benzylthioether] and 289 [(−)-epicatechin].
these two peaks were purified by HPLC and analyzed by multiple
fragmentation mass spectrometry (MS ) and thiolysis.
The first one, with m/z 565 corresponded to a decarboxylation (loss
of 44 amu) and was also observed for PB2-X [18]. Interestingly, the
two other fragments (i.e. at m/z 319 and 407) were not observed
on PB2-X MSn spectra. A direct cleavage of the inter-flavan bond
between extension unit and terminal unit of the ion at m/z 565 lead
to the formation of the fragment ion at m/z 319 which is in accor-
dance with the structure of extension unit that has been oxidized
by the enzyme (Scheme 2).
n
.4. MS2 fragmentation study of the molecular ions of PB2-X2 at
m/z 641 and PB2-X3 at m/z 609
3
n
Recently, Contreras-Domínguez et al. [18] showed the MS frag-
mentation scheme of PB2-X. Several fragmentation pathways were
elucidated: (a) the loss of ring B through retro-DielseAlder (rDA) fis-
sion [25]; (b) quinone methide formation from direct cleavage of
interflavan bond [26]; and (c) loss of ring A by heterocyclic ring
Another ion at m/z 407 was also observed. This indicated a loss of
1
84 amu (atomic mass unit) corresponding to the B ring elimination
of the extension unit as a consequence of retro-DielseAlder (rDA)
fragmentation of the heterocyclic C-ring. The resulting fragment at
m/z 425 (possible intermediate) was not detected probably because
it is immediately converted into the fragment at m/z 407 after the
loss of a water molecule.
fission [27].
Scheme 1 shows the different fragments obtained from MS2
analysis of the pseudo-molecular ion of PB2-X2 at m/z 641. Four
main fragment ions (II–V) were observed at m/z 623, 597, 439 and
The main fragments from PB2-X2 & PB2-X3 observed from MS2
experiments were wholly consistent with an enzymatic oxidative
degradation of the B ring (catechol ring) of either the terminal
unit or the extension unit or even the catechol rings at both
the units. However, MS fragmentations of these two molecules
do not allow distinguishing between two hypotheses concern-
ing the structure of the oxidized catechol rings of PB2: does it
correspond to a dienedioic acid structure, or is it a lactone [i.e.
3
19. The loss of 44 by decarboxylation produced the fragment at
m/z 597, while the loss of a water molecule led to the fragment at
m/z 623. Besides, the cleavage of the C ring (oxidized) of the exten-
sion unit through retro-DielseAlder (rDA) fission led to the ion at
m/z 457 (not detected), subsequently losing one water molecule
from this fragment resulted in another fragment at m/z 439.
Scheme 2 presents the possible fragmentation pathway resulted
from MS2 study of the molecular ion of PB2-X3 at m/z 609. Three
(5-Oxo-tetrahydro-furan-2-yl)-acetic acid group] as observed for
main fragment ions (II–IV) were observed at m/z 565, 319 and 407.
Table 2
The fragments obtained after MS and MS2 analysis of each of the four major peaks from the HPLC profile.
Peak (product) name
Retention time (min)
UV lambda max (nm)
MS fragment [M−H]−
MS2 fragments
PB2-X
PB2-X2
PB2-X3
PB2
11.6
12.5
18.1
23.6
270
270
270
280
609
641
609
577
565, 457, 439
597, 623, 439, 319
565, 407, 319
559, 451, 425, 289

K. Roopesh et al. / Process Biochemistry 45 (2010) 904–913
911
Scheme 3. Proposed MS fragmentation of PB2 dimer formed from thiolysis reaction.
PB2-X product [18]. Therefore, in order to complete the structural
3.5. MS fragmentation study of the thiolysis reaction products of
PB2, PB2-X2 and PB2-X3
characterizations; PB2, PB2-X2 & PB2-X3 were subjected to thioly-
sis reaction and the products were analyzed by HPLC-UV-ESI-MS.
The fragments obtained from multiple fragmentation mass spec-
The thiolysis reaction has been used frequently for the char-
acterization of procyanidins [28]. The reaction occurs when
procyanidins are heated in the presence of acid and benzyl mer-
n
trometry (MS ) of the four main degraded products are illustrated
in Table 2.
Scheme 4. Proposed MS fragmentation of PB2-X2 and PB2-X3 (oxidized products) formed from thiolysis reaction.

9
12
K. Roopesh et al. / Process Biochemistry 45 (2010) 904–913
Fig. 5. The proposed dioxygenase reaction mechanism on PB2 & resulting oxidized products.
captan (also known as toluene-␣-thiol, phenylmethane thiol)
and corresponds to the acidic cleavage of the inter-flavan link-
age of procyanidins. Terminal units are liberated in their free
epicatechin form whereas extension units are liberated as a fla-
vanyl carbocation immediately converted into the corresponding
benzylthioether adduct [29]. In principle, this reaction permits
determination of both chain length and composition of a pro-
cyanidin molecule when thiolysed products are analyzed by HPLC.
Problems arise if the procyanidin molecule analysed is heteroge-
neous or if the thiolysis reaction is incomplete [30]. Freeze-dried
samples of PB2, PB2-X2 and PB2-X3 were subjected to the thioly-
sis reaction as described by Guyot et al. [14]. The reaction products
were then analyzed by HPLC coupled with UV-Visible and MS detec-
tion. As expected, analysis of the thiolysis products of PB2 revealed
two reaction products [peak 1 (RT - 19.3), m/z 289 and peak 2 (RT
transformed products. The enzyme was identified as a dioxygenase.
It was purified to homogeneity and the various degraded products
obtained after its catalytic activity with the dimeric substrate PB2
were characterized by mass spectroscopy. The isoelectric point (pI)
of the enzyme was determined by chromatofocusing and found to
be 3.5. A sequential strategy to purify the enzyme involving anion-
exchange chromatography followed by gel-filtration was devised
and performed. The purified dioxygenase enzyme had a molecu-
lar weight of 117 kilo Daltons (kDa) in non-denaturing conditions.
The SDS-PAGE analysis indicated that the purified protein was a
trimer. The MS spectrum of the identified and characterized prod-
ucts showed ions at m/z 609 and 641 that corresponded to the
products obtained by the opening of the catechol ring of either
the terminal unit or the extension unit and the catechol rings of
both the units of PB2 dimer via intra-diol dioxygenase cleavage.
The pseudo-molecular ion of PB2 had m/z 577 in negative mode.
-
42.2), m/z 411, Fig. 4]. These two products were thus identified as
2
(
−)-epicatechin and (−)-epicatechin benzylthioether (Scheme 3).
Detailed MS analysis following thiolysis experiments resulted in
When PB2-X (m/z 641) & PB2-X3 (m/z 609) were submitted to
the structural elucidation of each of the biotransformed products.
Subsequently, the mechanism of dioxygenase catalytic action was
devised paving the way towards the novelty of the present work,
as it is for the first time a dioxygenase reaction mechanism on a
procyanidin dimer is being reported.
2
LC–MS after thiolytic reaction both of them resulted in getting a
common peak at fragment at m/z 443, which is the oxidized exten-
sion unit (dioxygenase action) coupled with the benzylthioether.
The three other fragments detected were, one at m/z 289, identified
as the terminal unit released from pseudo-molecular ion m/z 609;
second one m/z 321 and the third one at m/z 335 formed from the
pseudo-molecular ion of m/z 641 (Scheme 4). In comparison with
earlier works dealing with the oxidation of monomeric catechin
Acknowledgments
KR thanks Département Soutien et Formation des communautés
scientifiques du sud (DSF), Institut de Recherche pour le Développe-
ment (IRD), France for the PhD fellowship. KR is grateful to Dr.
Sridharan Vellaiswamy, Postdoctoral researcher, Faculty of Phar-
macy, University Complutense of Madrid, Spain for the helping
hand offered in interpreting the results from mass spectrometry
as well as elucidating the structures. The article is dedicated to the
loving memory of Dr. Christopher AUGUR, Research officer, IRD,
France.
[
31] the fragment with m/z 335 was identified as the methylated
form of the oxidative derivative of epicatechin. Methylation of the
carboxylic group can occur during thiolysis because reaction con-
ditions (HCl-MeOH) are favorable to esterification by methanol.
Similarly (unmethylated) oxidation derivatives of epicatechin have
also been structurally elucidated. Hence, the fragment with m/z 321
was identified to be the unmethylated oxidized epictaechin. Fan et
al. [32] identified similar molecules termed viniferone B or C [31] or
n
(
−)-epicatechin lactone. It was therefore confirmed, following MS
studies that the enzyme dioxygenase from A. fumigatus is capable
of modifying the catechol ring of either the extension unit (upper
moiety) or the terminal unit (lower moiety) or even both the units
of PB2. Subsequently these results paved the way for elucidating
the mechanism of dioxygenase catalytic action (Fig. 5).
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