Enzymatic deglycosylation of flavonoids in deep eutectic solvents-aqueous mixtures: Paving the way for sustainable flavonoid chemistry

Article abstract of DOI:10.1016/j.molcatb.2016.04.010

The low solubility of glycosylated flavonoids represents a hurdle to conduct efficient enzymatic deglycosylations in aqueous media. To overcome this drawback, environmentally-unfriendly dimethylsulfoxide (DMSO) is typically used as co-solvent. Using a specific diglycosidase from Acremonium sp. DSM24697 for the deglycosylation of the rutinosylated flavonoid (hesperidin) as model reaction, this communication explores the use of (non-hazardous and biodegradable) deep eutectic solvents (DESs) as co-solvents in flavonoid biocatalysis. The enzymatic deglycosylation was observed when DES composed of choline chloride and glycerol or ethylene-glycol was used at proportions of up to 40% (DES-Buffer, v/v), displaying a promising framework to combine enhanced flavonoid solubilities and high enzymatic activities. The deglycosylation activity significantly increased when the single DES components - glycerol and ethylene-glycol - were added (e.g. 140% of enzyme activity at glycerol at 40% v/v), whereas deleterious effects were observed when choline chloride was solely added, presumably due to its chaotropic effect. Future research opportunities may be envisaged in the genetic design to evolve more robust biocatalysts, and in tailoring DES to deliver more enzyme-compatible solvents.

Full text of DOI:10.1016/j.molcatb.2016.04.010

Accepted Manuscript
Title: Enzymatic deglycosylation of flavonoids in deep
eutectic solvents − aqueous mixtures: Paving the way for
sustainable flavonoid chemistry
Author: Gisela Weiz Lucas Braun Rosana Lopez Pablo
´
´
Domınguez de Marıa Javier D. Breccia
PII:
S1381-1177(16)30068-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.04.010
MOLCAB 3358
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
19-12-2015
4-4-2016
26-4-2016
Please cite this article as: Gisela Weiz, Lucas Braun, Rosana Lopez, Pablo
´
´
Domınguez de Marıa, Javier D.Breccia, Enzymatic deglycosylation of
flavonoids in deep eutectic solvents − aqueous mixtures: Paving the way for
sustainable flavonoid chemistry, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2016.04.010
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Enzymatic deglycosylation of flavonoids in deep eutectic solvents – aqueous mixtures:
Paving the way for sustainable flavonoid chemistry
Gisela Weiza, Lucas Brauna, Rosana Lopeza, Pablo Domínguez de Maríab, Javier D. Brecciaa*
a: INCITAP - CONICET (National Scientific and Technical Research Council). Department
of Chemistry, Faculty of Natural Sciences, National University of La Pampa (UNLPam), Av.
Uruguay 151, (6300) Santa Rosa, La Pampa, Argentina
b: Sustainable Momentum, SL. Ap. Correos 3517. 35004 Las Palmas de Gran Canaria.
Canary Islands, Spain.
*Corresponding author: javierbreccia@gmail.com
Phone number: +54-02954-245220 ext. 7426
1

Highlights
Flavonoids deglycosylation > production of hesperetin > Acremonium sp. DSM24697 >
diglycosidase > DES > DES-components > higher efficiency for deglycosylation
2

Abstract
The low solubility of glycosylated flavonoids represents a hurdle to conduct efficient
enzymatic deglycosylations in aqueous media. To overcome this drawback, environmentally-
unfriendly dimethylsulfoxide (DMSO) is typically used as co-solvent. Using a specific
diglycosidase from Acremonium sp. DSM24697 for the deglycosylation of the rutinosylated
flavonoid (hesperidin) as model reaction, this communication explores the use of (non-
hazardous and biodegradable) deep eutectic solvents (DESs) as co-solvents in flavonoid
biocatalysis. The enzymatic deglycosylation was observed when DES composed of choline
chloride and glycerol or ethylene-glycol were used at proportions of up to 40 % (DES-Buffer,
v/v), displaying a promising framework to combine enhanced flavonoid solubilities and high
enzymatic activities. The deglycosylation activity significantly increased when the single
DES components – glycerol and ethylene-glycol – were added (e.g. 140 % of enzyme activity
at glycerol at 40 % v/v), whereas deleterious effects were observed when choline chloride
was solely added, presumably due to its chaotropic effect. Future research opportunities may
be envisaged in the genetic design to evolve more robust biocatalysts, and in tailoring DES to
deliver more enzyme-compatible solvents.
Key words: Flavanone glycoside, glycerol, ethylene glycol, DES, Acremonium sp.
DSM24697
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GRAPHICALABSTRACT
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Flavonoid Chemistry with
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Flavanone glycosides are secondary metabolites that occur widely in citrus species,
influencing citrus fruit production and processing. The deglycosylation of different flavonoid
glycosides represents a necessary step in food technology for de-bittering and clarifying fruit
juices [1]. Likewise, several articles have reported biological activities for diverse de-
glycosylated flavanones [2,3]. Particularly hesperetin – the aglycone of flavonoid hesperidin
(see Scheme 1) –, displays analgesic, anti-inflammatory, and antioxidant properties, and it
can also be used as building block for the production of dyes and sweeteners [3]. Despite its
importance, however, to date only few bioprocesses efficiently hydrolyzing hesperidin to
afford hesperetin have been disclosed [4,5]. They typically involve sequential steps catalyzed
by two mono-glycosidases (-L-rhamnosidase (EC 3.2.1.40) and β-glucosidase (EC
3.2.1.21)), releasing rhamnose and glucose in each step, respectively. Remarkably, a single-
deglycosylating-step procedure using the specific diglycosidase 6-O--rhamnosyl--
glucosidase (EC 3.2.1.168) – directly rendering the disaccharide rutinose and hesperetin – has
been recently described by our research group [6]. The enzyme is a fungal glycoprotein (Mw
42 kDa; pI 5.7) – produced by Acremonium sp. DSM24697 – reported to have a higher
affinity to the substrate hesperidin (Km 1.77 mM) in comparison with the substrate hesperidin
methylchalcone (Km 8.73 mM) [6]. This diglycosidase also enables the transglycosylation of
rutinosyl units using the flavonoids hesperidin or hesperidin methyl-chalcone as rutinose
donor [7].
To pave the way for an efficient biocatalytic process for hesperetin in particular – and
with a broader perspective, for flavonoids in general –, the substrate solubilization in aqueous
media represents a challenge, given the low solubility of flavonoids in such conditions. On
this basis, the use of enzyme-compatible (and acceptable for food industry) co-solvents to
enable proper dissolution of organic compounds would be of utmost importance. Yet, to our
knowledge only dimethylsulfoxide (DMSO) has been assessed for enzyme catalysis in
flavonoid-based reactions [7-10]. Albeit DMSO leads to excellent enzymatic activities and
5

outstanding solubilizing properties, the use of DMSO poses considerable disadvantages
regarding enzyme stability in flavonoid chemistry [9] as well as when scale-up and
downstream processing steps are envisioned (e.g. wastewater formation, energy-demanding
distillations, loss of products, etc.) [8,11,12]. In this respect, the introduction of less
hazardous co-solvents to provide straightforward work-up procedures appears as a promising
research line for flavonoid chemistry.
In the quest for novel tuneable alternatives, the use of deep-eutectic-solvents (DES) is
envisaged. DES result from the complexation of quaternary ammonium salts (hydrogen
acceptor: HA, e.g. choline chloride) with hydrogen bond donors (HBD) such as amines,
amides, alcohols or carboxylic acids. The reduction of anion-cation electrostatic forces
decrease the freezing point of the mixture rendering environmental friendly tuneable solvents
[13-16]. Being in most cases non-hazardous and biodegradable, the use of DES for substrates
of low water solubility and for controlling water concentration in reaction mixtures have
endower their application in biocatalysis [17], with outstanding examples for hydrolases [18],
lyases [19], and even whole-cells containing oxidoreductases [20]. To our knowledge the use
of DES in glycoside hydrolases has not been assessed so far, with the only exception of
genetically-designed cellulases performing cellulose hydrolysis in seawater and DES-aqueous
mixtures [21], or, for ionic liquids, using some imidazolium-based solvents for disaccharide
synthesis [22]. Actually, DES have been claimed as the “natural solvents” able to dissolve
molecules in nature (e.g. flavonoids, with outstanding solubilities of 20-90 g Kg-1) [23], with
potential extractive features to purify them from plant extracts [24]. Conclusively, identifying
enzyme-compatible DES that could also be efficiently applied for flavonoid chemistry would
represent an important step for the future implementation of these technologies at industrial
level on a sustainable manner. On this basis, in this communication the use of DES for the
deglycosylation of hesperidin is reported for the first time.
The model reaction of study is depicted in Scheme 1. As prototypical DES for
6

biocatalysis [25], choline chloride was chosen as the quaternary ammonium salt, and was
combined with glycerol, ethylene-glycol and urea as hydrogen-bond donors (all of them at
1:2 mol:mol). The neat DES displayed excellent solubilizing capabilities for hesperidin,
dissolving up to 90 mM, fully consistent with previous literature for DES and flavonoids
[23,24], as well as for other ionic liquids [26,27].
In a first set of experiments, the enzymatic one-step deglycosylation of hesperidin was
assessed at different DES-buffer proportions (v/v), using 6-O--rhamnosyl--glucosidase as
the biocatalyst (Scheme 1). Results are depicted in Figure 1. As it can be observed, DES
displayed considerable effects on the enzymatic glycosidase activity. Especially when urea
was used as hydrogen bond donor (ChCl:Ur DES), no enzymatic activities were already
detected at DES proportions of ca. 40 % (v/v), suggesting the unfolding of the enzyme.
However, DES formed with polyols as hydrogen bond donors (ethylene-glycol and glycerol)
resulted in better co-solvents for the biocatalytic approach. More specifically, the enzyme
displayed 95 % activity at ChCl:Gly media with DES proportions of up to 40 % (v/v).
Triggered by the observed results, studies using the DES components separately as
co-solvents for the model reaction were conducted. When using DES as co-solvents in
aqueous solutions, the existence of DES “clusters” – keeping its structure even at low
concentrations –, has been recently suggested [25]. Thus, DES might keep some structural
basis even at bulk aqueous solutions, being the actual (synergetic) responsible for the effect
on biocatalysis, rather than their separate components [25]. Results when choline chloride,
glycerol, and ethylene glycol were used as co-solvents are shown in Figure 2. Choline
chloride led to a deleterious effect for the glycosidase even at low additions (At proportions
higher than 70 % w/v choline chloride is not soluble in water and was thus not measured
anymore). Remarkably, both polyols exhibited unexpected improvements of the enzymatic
activity. Hesperetin production was raised up by 140 % (3.74 ± 0.44 U/mL) at 40 % v/v
glycerol. Since transglycosylation of glycerol leading to rutinose-based glycerides could be
7

an alternative explanation for the higher activity, the side reaction would enable access to
potentially interesting new compounds [28]. A pilot work in these conditions (40 % v/v
glycerol, a suspension of 1.8 mM hesperidin (1.1 g/L) and enzyme 1.55 U/mL, 1 h, 60ºC), led
to a yield of 0.34 g hesperetin, accounting for 62 % substrate conversion, in contrast with the
26 % substrate conversion achieved when reactions were conducted in aqueous
environments. Hence, the catalysis could also be favored by the stabilizing effect exerted by
polyols [29]. Results suggest that an enhancement of hesperidin solubility would favor the
catalysis up to the point where high viscosity of the media started to be adverse for the
enzymatic process, presumably due to mass transfer limitations. Thus, the lower viscosity of
the DESs in comparison with the single components – glycerol or ethylene-glycol – might be
counteracted by the deleterious effect of the choline cation [26,31]. Quaternary ammonium
cations are chaotropic ions (Hofmeister series); therefore, a negative effect can be expected
on the enzyme activity [31]. Moreover, the interactions between choline chloride and
hesperidin should not be neglected. Given that DES may be tailored according to actual
(synthetic) necessities, it seems plausible to design novel polyol-based DES bearing more
enzyme-compatible quaternary salts for glycosidases and flavonoid dissolution.
In summary, this communication has successfully explored the use of DES as co-
solvents in flavonoid biocatalysis. Different choline chloride-based DES can be used for
biocatalytic processes using wild-type glycosidases in practical frameworks of 30-40 % DES
(v/v). Given the industrial importance of flavonoids, and the prospective (environmental)
advantages that the use of these co-solvents may have – when compared to more
conventional and broadly used co-solvents such as DMSO – the use of DES open possibilities
to combine biocatalysis with food industry, by using widely acceptable derivatives (such as
glycerol). As lines for future research, the genetic design of robust biocatalysts that may be
more active and stable at higher DES proportions may be a plausible option to increase
substrate solubility. Furthermore, another approach would be to tailor novel DES with better
8

structural properties for glycosidases, to broaden the positive effects of polyols and
diminishing the deleterious influence caused by choline chloride.
Acknowledgements
This work was supported by the National University of La Pampa (UNLPam), the
National Council of Scientific and Technical Research (CONICET), and the National Agency
for Science and Technology Promotion (ANPCyT) of Argentina.
Conflicts of interest
The authors declare that they have no conflict of interest.
Experimental
Production and purification of 6-O-α-rhamnosyl-β-glucosidase.- The α-rhamnosyl-β-
glucosidase was purified from the culture supernatant of Acremonium sp. DSM24697 as
described before [6]. The procedure includes salt precipitation and hydrophobic interaction
chromatography (Butyl-agarose) to yield 30.4% of the initial activity and purified 45.7 fold.
The product was desalted and freeze-dried before storing at -18 °C.
Synthesis and characterization of DESs.- Choline (CH):urea at molar ratio (1:2), with
40 rpm stirring blade at 90 °C for 1 h. CH: glycerol: molar ratio (1:2) without stirring at 70
°C for 30 minutes. CH:ethylene-glycol molar ratio (1:2) without agitation immersed 1 minute
at 80 °C.
Enzyme assay.- For quantification of 6-O-α-rhamnosyl-β-glucosidase activity, each
reaction contained 950 µL varying concentrations of the different DESs or DESs constituents,
1.8 mM hesperidin in 5 mM sodium phosphate buffer pH 6.0 and 50 µL enzyme solution and
the reaction was carried out for 1 h at 60 °C. For transglycosylation reactions, 5-100 %
vol/vol DES, choline, glycerol and ethylene-glycol were added to the reaction mixture. The
9

products of enzymatic reaction were analyzed by thin layer chromatography (Silicagel 60 W)
using ethyl-acetate/2-propanol/water (3:2:2) as mobile phase and stained with anthrone
reagent. The images were analyzed according to previous protocol reported by us [6]. The
product (hesperetin) was quantified by HPLC using a KONIK-500-A series HPLC system
attached to a KONIK UVIS 200 detector. The column was a reversed-phase Kromasil®
Akzo-Nobel 100-5C18 (25 cm length, 4.6 mm internal diameter, pore size 100 Å, particle
size: 5 μm). The elution consisted of an isocratic flow of 50 % v/v methanol and 50 % v/v
water at a flow rate of 1.0 mL/min at 40°C. Peak areas and extinction coefficient were
calculated from chromatograms of authentic standards detected at 285 nm. Hesperetin
extraction was performed twice placing samples (100 µL) with 400 µL of 50 % v/v methanol
and 50 % v/v DMSO for 1 h at 50°C. Then, samples were centrifuged (10000 rpm, 10 min),
the supernatants were suitably diluted in elution solvent. Triplicate samples were used. An
enzyme unit was defined as the amount of enzyme required to produce 1 µmol of hesperetin
per minute.
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Legends of figures
Scheme 1. One-step deglycosylation of hesperidin to afford hesperetin catalyzed by 6-O-α-
rhamnosyl-β-glucosidase.
Figure 1. Effect of DES concentration on the enzymatic activity for the deglycosylation of
hesperidin by α-ramnosyl-β-glucosidase. See experimental part for reaction conditions. One
hundred percent activity corresponded to 1.21 ± 0.17 U/mL, 1.30 ± 0.11 U/mL and 1.43 ±
0.10 U/mL for ChCl:EG, ChCl:Gly and ChCl:Ur respectively.
Figure 2. Effect of polyols and choline chloride on the activity of α-rhamnosyl-β-glucosidase.
One hundred percent activity corresponded to 1.55 ± 0.15 U/mL, 1.24 ± 0.19 U/mL and 3.93
± 0.70 U/mL for Gly, EG and ChCl respectively.
15

OH
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Hesperidin
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