One-pot bi-enzymatic cascade synthesis of puerarin polyfructosides

Article abstract of DOI:10.1016/j.carbpol.2020.116710

Enzymatic glycosylation is an efficient way to increase the water solubility and the bioavailability of flavonoids. Levansucrases from Bacillus subtilis (Bs_SacB), Gluconacetobacter diazotrophicus (Gd_LsdA), Leuconostoc mesenteroides (Lm_LevS) and Zymomonas mobilis (Zm_LevU) were screened for puerarin (daidzein-8-C-glucoside) fructosylation. Gd_LsdA transferred the fructosyl unit of sucrose onto the glucosyl unit of the acceptor forming β-D-fructofuranosyl-(2→6)-puerarin (P1a), while Bs_SacB, Lm_LevS and Zm_LevU synthesized puerarin-4’-O-β-D-fructofuranoside (P1b) and traces of P1a. The Gd_LsdA product P1a was purified and assayed as precursor for the synthesis of puerarin polyfructosides (PPFs). Bs_SacB elongated P1a more competently forming a linear series of water-soluble PPFs reaching at least 21 fructosyl units, as characterized by HPLC-UV-MS, HPSEC and MALDI-TOF-MS. Simultaneous or sequential Gd_LsdA/Bs_SacB reactions yielded PPFs directly from puerarin with the acceptor conversion ranging 82–92 percent. The bi-enzymatic cascade synthesis of PPFs in the same reactor avoided the isolation of the intermediate product P1a and it is appropriate for use at industrial scale.

Full text of DOI:10.1016/j.carbpol.2020.116710

Journal Pre-proof
One-pot bi-enzymatic cascade synthesis of puerarin polyfructosides
˜
´
Gema Nu´nez-Lopez (Investigation) (Writing - original draft),
Sandrine Morel (Conceptualization) (Methodology) (Investigation)
(Visualization)<ce:contributor-role>Writing-review and editing),
´
´
Lazaro Hernandez (Methodology) (Visualization) (Writing - review
and editing), Alexis Musacchio (Data curation) (Formal analysis),
Lorena Amaya-Delgado (Resources) (Funding acquisition), Anne
Gschaedler (Resources) (Funding acquisition), Magali
Remaud-Simeon (Project administration) (Supervision) (Funding
acquisition), Javier Arrizon (Project administration) (Supervision)
(Visualization) (Funding acquisition)
PII:
S0144-8617(20)30884-5
DOI:
https://doi.org/10.1016/j.carbpol.2020.116710
CARP 116710
Reference:
To appear in:
Carbohydrate Polymers
Received Date:
Revised Date:
Accepted Date:
19 May 2020
22 June 2020
29 June 2020
˜
´
´
Please cite this article as: Nu´nez-Lopez G, Morel S, Hernandez L, Musacchio A,
Amaya-Delgado L, Gschaedler A, Remaud-Simeon M, Arrizon J, One-pot bi-enzymatic
cascade synthesis of puerarin polyfructosides, Carbohydrate Polymers (2020),
doi: https://doi.org/10.1016/j.carbpol.2020.116710

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One-pot bi-enzymatic cascade synthesis of puerarin polyfructosides
Gema Núñez-Lópeza,b, Sandrine Morelb, Lázaro Hernándezc, Alexis Musacchioc, Lorena
Amaya-Delgadoa, Anne Gschaedlera, Magali Remaud-Simeonb, Javier Arrizona*
aCentro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C.- Unidad
Zapopan. Camino Arenero 1227, El Bajio del Arenal, 4519, Zapopan, Jal. México.
genunez_al@ciatej.edu.mx, lamaya@ciatej.mx, agschaedler@ciatej.mx, jparrizon@ciatej.mx
bTBI, Université de Toulouse, CNRS, INRAE, INSA, 135 Avenue de Rangueil, 31077 Toulouse,
France. morel@insa-toulouse.fr, remaud@insa-toulouse.fr
cCentro de Ingeniería Genética y Biotecnología (CIGB), Ave. 31 e/158 y 190, Apartado Postal 6162,
Habana 10600, Cuba. lazaro.hernandez@cigb.edu.cu, alexis.musacchio@cigb.edu.cu
*corresponding author: jparrizon@ciatej.mx, tel; +52 (33) 33455200, fax; +52 (33) 33455245
Highligts
 Regioselectivity of fructosyl transfer to puerarin varies between levansucrases
 Bs_SacB synthesizes puerarin-4’-O-β-D-fructofuranoside
 Gd_LsdA
oligofructosides
 Bs_SacB transforms β-D-fructofuranosyl-(26)-puerarin into polyfructosides
 Cascade Gd_LsdA/Bs_SacB reactions yield polyfructosides directly from puerarin
synthesizes
β-D-fructofuranosyl-(26)-puerarin
and
linear
Abstract
Enzymatic glycosylation is an efficient way to increase the water solubility and the
bioavailability of flavonoids. Levansucrases from Bacillus subtilis (Bs_SacB),
1

Gluconacetobacter diazotrophicus (Gd_LsdA), Leuconostoc mesenteroides (Lm_LevS) and
Zymomonas mobilis (Zm_LevU) were screened for puerarin (daidzein-8-C-glucoside)
fructosylation. Gd_LsdA transferred the fructosyl unit of sucrose onto the glucosyl unit of
the acceptor forming β-D-fructofuranosyl-(26)-puerarin (P1a), while Bs_SacB, Lm_LevS
and Zm_LevU synthesized puerarin-4’-O-β-D-fructofuranoside (P1b) and traces of P1a. The
Gd_LsdA product P1a was purified and assayed as precursor for the synthesis of puerarin
polyfructosides (PPFs). Bs_SacB elongated P1a more competently forming a linear series of
water-soluble PPFs reaching at least 21 fructosyl units, as characterized by HPLC-UV-MS,
HPSEC and MALDI-TOF-MS. Simultaneous or sequential Gd_LsdA/Bs_SacB reactions
yielded PPFs directly from puerarin with the acceptor conversion ranging 82-92%. The bi-
enzymatic cascade synthesis of PPFs in the same reactor avoided the isolation of the
intermediate product P1a and it is appropriate for use at industrial scale.
Keywords: fructo-conjugates, fructosyltransferase, flavonoid, levansucrase, puerarin.
1. Introduction
Puerarin (daidzein-8-C-glucoside) is an isoflavone that exhibits vasodilator, anti-oxidant,
anti-cancer, and anti-inflammatory properties. The poor water solubility of puerarin limits its
absorption in vivo, impeding optimal pharmacological applications (Zhou, Zhang, & Peng,
2014). Enzymatic glycosylation has emerged as an efficient way to increase the water
solubility of flavonoids and consequently other physicochemical and biological properties
like stability, bioavailability and bioactivity. The alternative chemical method requires
glycosyl activation and multiple steps of protection/deprotection to control the
regioselectivity, while the enzymatic reaction is regio- and stereoselective, and more friendly
to the environment (Desmet et al., 2015).
In nature, glycosylated forms of flavonoids are produced by Leloir-type glycosyltransferases.
The requirement of nucleotide-activated sugars as donor substrates is a disadvantage for
biotechnological applications. More attractively, glycosyltransferases from Glycoside-
2

Hydrolase (GH) families 13, 70, 32 and 68 transfer the glucosyl or fructosyl moiety of the
cheap and abundant substrate sucrose. GH13 and GH70 enzymes have been widely used for
the in-vitro glucosylation of natural flavonoids (Hofer, 2016; Malbert et al., 2018, 2014;
Slámová, Kapešová, & Valentová, 2018). However, examples of flavonoid fructosylation by
GH32 or GH68 (clan GH-J) enzymes remain scarce (Herrera-González et al., 2017). To our
knowledge, there is no report of enzymatic synthesis of puerarin polyfructosides (PPFs).
Sucrose-transforming fructosyltransferases from plants and fungi (GH32 members) are not
appropriate for the production of PPFs as they synthesize short-chain fructans and their
acceptor range is generally restricted to sugars (Trollope, Görgens, & Volschenk, 2015; Van
Den Ende, Lammens, Van Laere, Schroeven, & Le Roy, 2009). Family GH68 includes
fructosyltransferases from over 500 bacterial and archaeal species. Inulosucrase (EC 2.4.1.9)
and levansucrase (EC 2.4.1.10) produce linear/branched fructan chains elongated via β-
(2→1) and β-(2→6) linkages, respectively. Bacterial inulin and levan contain from two to
thousands fructosyl units. Fructans are water-soluble fibbers with prebiotic effects in humans
and animals. High-molecular-mass levan finds additional applications in several non-food
industries (Öner, Hernández, & Combie, 2016).
Levansucrases catalyse the transfer of the fructosyl unit of sucrose onto a variety of natural
acceptors including water, glucose, fructose, sucrose, and fructans. The double-displacement
(ping-pong) reaction occurs with an overall retention of the anomeric β-configuration of the
fructosyl residue. Variations in size distribution are often observed in the fructans produced
by levansucrases of different origins. For instance, levansucrase from Bacillus subtilis
(Bs_SacB) elongates the products 1-kestotriose and 6-kestotriose using non-processive
(distributive) and processive mechanisms that yield short levans (average mass 7.2 kDa) and
long levans (average mass 2,300 kDa), respectively (Raga-Carbajal et al., 2016; Raga-
Carbajal, López-Munguía, Alvarez, & Olvera, 2018). Levansucrase from Gluconacetobacter
diazotrophicus (Gd_LsdA) accumulates 1-kestotriose, whereas the processive elongation of
6-kestotriose results in levan polymerization (Hernández et al., 1995).
Several GH68 enzymes have shown the ability to fructosylate non-conventional acceptors
such as other sugars, aromatic and aliphatic alcohols, and phenolic compounds (Hernández,
Quintero, & Musacchio, 2019). Enzymatic fructosylation of puerarin was initially attempted
3

in the presence of co-solvents (ethanol or DMSO) that dissolve the isoflavone. By this way,
permeabilized cells of Lysinibacillus fusiformis (Wang et al., 2014) and Microbacterium
oxylans (Yu et al., 2010) synthesized puerarin-7-O-fructoside, while β-fructosidase from
Arthrobacter nicotianae formed mono- and di-β-D-fructofuranosyl-(2→6)-puerarin (Wu,
Chu, Wu, Zhang, & He, 2013). Recently, Núñez-López et al. (2019) reported that Gd_LsdA
fructosylates puerarin and other phenolic compounds in an aqueous medium.
In this study, it was first revealed that the regioselectivity and efficiency of the fructosyl
transfer from sucrose onto puerarin vary among levansucrases of different origins. A second
hypothesis was that PPFs can be produced by the cascade reaction of two levansucrases of
different regioselectivity but complementary transferase activities. Gd_LsdA synthesized the
precursor β-D-fructofuranosyl-(26)-puerarin, which was elongated in a distributive
manner by the more efficient polymerase Bs_SacB yielding a linear series of PPFs with up
to 21 fructosyl units. Then, PPFs were produced directly from natural puerarin in a one-
reactor system using either simultaneous or sequential Gd_LsdA/Bs_SacB reactions. The
incorporation of two or more β(2→6) linked fructosyl units highly enhanced the water
solubility of puerarin.
2. Materials and Methods
2.1 Chemical materials
Puerarin and levan from Erwinia herbicola were supplied by Carbosynth Limited (Compton,
UK), sucrose, dextran standards, dimethylsulfoxyde (DMSO), acetonitrile (ACN) and formic
acid were supplied by Sigma Aldrich Inc. (MO, USA).
2.2 Enzymes and activity determination
Levansucrase from Gluconacetobacter diazotrophicus SRT4 (Gd_LsdA) was purified
according to Hernández et al. 1995. Levansucrase from Bacillus subtilis (Bs_SacB) was
supplied by Ozyme™ (France). Recombinant levansucrases from Zymomonas mobilis subsp.
mobilis ATCC 10988 (Zm_LevU) and Leuconostoc mesenteroides NRRL B-512 F
(Lm_LevS) were produced in Escherichia coli.
4

The enzymatic activity was determined by measuring the reducing sugars released from
sucrose (100 g/L) in 50 mM citrate-phosphate buffer (pH 5.8) using the 3,5-dinitrosalicylic
acid (DNS) method (Sumner & Howell, 1935). The reaction temperature was 42 °C for
Gd_LsdA, Bs_SacB and Zm_LevU), and 30 °C for Lm_LevS. One unit is defined as the
amount of enzyme releasing 1 μmol of glucose per min at initial rates under the described
reaction conditions.
2.3 Synthesis of puerarin fructo-conjugates
P1a and P2a were synthesized by Gd_LsdA (0.1 U/ml) in reaction with 146 mM sucrose and
25 mM puerarin. P1b was produced by Bs_SacB (0.5 U/ml) using 500 mM sucrose and 25
mM puerarin. PPFs were synthesized using either purified P1a as precursor or directly from
puerarin. In the first case, Gd_LsdA (0.5 U/ml) or Bs_SacB (0.5 U/ml) was incubated with
500 mM sucrose and 25 mM P1a. In the second case, two different cascade reactions were
performed: i) Simultaneous enzyme addition: Gd_LsdA and Bs_SacB were mixed prior to
the reaction at rates 0.75-0.75 U/mL (1:1), 0.5-1 U/mL (1:2) and 1-0.5 U/mL (2:1) and
incubated with 500 mM sucrose and 10 mM puerarin, ii) Sequential enzyme addition:
Gd_LsdA (0.1 U/mL) was incubated with sucrose (146 mM) and puerarin (25 mM) for 6 h
and inactivated 5 min at 95 °C, and then Bs_SacB (0.5 U/mL) was added with/without extra
sucrose (292 or 500 mM).
All reactions were in duplicates. Donor and acceptor substrates were mixed in 50 mM citrate-
phosphate buffer (pH 5.8) at 42 °C. After 24 h, the reactions were stopped by heating at 95
°C for 10 min. The acceptor products were quantified by HPLC and the conversion
percentage was determined according to Núñez-López et al. (2019).
2.4 Chromatography analysis
The HPLC-UV-MS analysis of puerarin and its fructo-conjugates was performed on an
Ultimate 3000 series chromatograph equipped with a Dionex 340 UV/VIS detector and
coupled with a simple quadruple mass spectrometer (MSQ Plus, Thermo Scientific). Puerarin
and its fructo-conjugates were detected using UV at 254 nm and separated by a Phenomenex
PFP C18 column (Luna 5µm, 100 A, 250 x 4.6 mm, USA) maintained at 40ºC. Samples were
5

eluted using a mixture of solvent water/formic acid 0.05% (eluent A) and acetonitrile/formic
acid 0.05% (eluent B) by the following gradient (95/5 at 0 min, 75/25 at 16 min, 5/95 during
5 min and finally 95/5 during 10 min) at a flow rate of 1 mL per min. The mass spectrometer
was used in negative and positive mode with a voltage cone at 50, 80 and 110 V, the
temperature of the electrospray ionization (ESI) ion source was 450ºC and the gas carrier
was nitrogen. The mass spectrometer scanned from m/z 100 to 1,500. The data acquisition
and processing were performed using Chromeleon™ 7.2 data systems.
The molar mass (MM) distribution of puerarin fructo-conjugates was determined by high-
pressure size exclusion chromatography (HPSEC) using two columns (Shodex OH-Pak 805
and 802.5) connected in series at 70 °C and equipped with RI and UV detector. Elution was
performed using a solution of 450 mM sodium nitrate and 1% (v/v) ethylene glycol in 50
mM sodium acetate as eluent at a flow rate of 0.25 mL per min for 85 min. MM distribution
was estimated by comparing to the migration of the standards levan from Erwinia herbicola
(1000 kg/mol) and commercial dextrans (3.5, 6, 68 and 400-600 kg/mol).
2.5 Purification of puerarin fructo-conjugates
Puerarin fructo-conjugates were purified by flash chromatography on a Reveleris® Flash
Chromatography System (Grace, USA) with a Reveleris ® C18 column (40 µm, 120 g, Lot
#16052035), using ultra-pure water (eluent A) and acetonitrile (eluent B) under the following
gradient (v/v): 100/0 at 0 min, 75/25 at 11.2 min, 30/70 during 0.9 min and finally keep 100/0
during 1.8 min, the flow rate was 85 mL per min. Sugars eluted with water and puerarin
fructosides of different sizes were recovered with increasing gradient of acetonitrile. The
detection wavelength was set at 254 nm and the evaporative light scattering detector (ELSD)
was set at 20 mV to follow the separation.
2.6 NMR structural characterization
NMR was acquired on an Advance 500 MHz spectrometer (Bruker) operating at 500 MHz
1
for H and 125 MHz for 13C. The data were processed using Topspin 3 Software. All
measurements were performed at 298 K and chemical shifts (supplementary material Table
6

S1) were given in ppm relative to the residual signal of DMSO-d6 (δH ppm : 2.50 ppm, δC
ppm : 39.5 ppm).
2.7 MALDI-TOF-MS analysis of puerarin fructo-conjugates
The MM of puerarin fructo-conjugates was determined by MALDI-TOF-MS in a Waters®
MALDI-Micro MX-TOF Mass spectrometer. The measurements were performed with the
mass spectrometer in positive reflection mode using an accelerating voltage of 12 kV. Mass
spectra were acquired from 400 (m/z) to 3000 (m/z). Samples were dissolved in water (1
mg/mL) and incubated 10 min at 50°C. A 0.5 mL of sample solution was mixed with 0.5 ml
of the matrix solution (2,5-dihydroxybenzoic acid 10 mg/mL in H2O:EtOH 0.5:0.5; v/v) and
a total of 1 µL was applied to a stainless steel sample slide and dried at room temperature.
2.5 Water solubility of isolated puerarin fructo-conjugates
The water solubility of puerarin and puerarin monofructoside (P1a) was determined at 25 °C
as described by Núñez-López et al. (2019). Puerarin difructoside (P2a), puerarin
oligofructosides (POFs, 3-12 fructosyl units, predominantly 3-5) and puerarin
polyfructosides (PPFs, 3-21 fructosyl units, predominantly 12-15) were synthesized from
the precursor P1a and isolated by Flash Chromatography. P2a (1.84 g), POFs (2.01 g) and
PPFs (1.06 g) were mixed with ultrapure water (1 g). Each solution was incubated at 25 °C
and 250 rpm for 2 h and then centrifuged 5 min at 12000g. The very high viscosity of the
medium impeded to evaluate the solubilization of P2a, POFs and PPFs at higher
concentrations.
3. Result and Discussion
3.1 Synthesis of puerarin fructosides by levansucrase from four bacterial species
Levansucrases from Bacillus subtilis (Bs_SacB), Gluconacetobacter diazotrophicus
(Gd_LsdA), Leuconostoc mesenteroides (Lm_LevS), and Zymomonas mobilis (Zm_LevU)
were assayed for their capacity to transfer the fructosyl moiety of sucrose onto puerarin. The
highest percentage of acceptor conversion was achieved by Gd_LsdA (88.7 %) and Bs_SacB
7

(24.8 %) (Table 1). In the case of Lm_LevS and Zm_ LevU, less than 10 % of puerarin was
transformed into products.
Table 1. Fructosylation of puerarin by levansucrases of different origins
Puerarin
Bacteria
Enzyme name
Conversion (%)a
Type of fructosidesb
Bacillus subtilis
Gluconacetobacter
diazotrophicus
Bs_SacB
Gd_LsdA
24.8 ± 0.1
Mono
88.7 ± 0.4
Mono to ≥ penta
Leuconostoc mesenteroides Lm_LevS
7.3 ± 2.2
9.0 ± 2.5
Mono
Mono
Zymomonas mobilis Zm_LevU
Each enzyme (0.5 U/mL) was reacted with sucrose (500 mM) and puerarin (10 mM) in citrate-phosphate buffer
(50 mM, pH 5.8) for 24 h. The reaction temperature was 42 °C except for Lm_LevS (30 °C). All the experiments
were performed in duplicates.
a Puerarin conversion was calculated from the amount of acceptor remaining after the reaction. Conversion
=100*([Puerarin] initial – [Puerarin] final)/ [Puerarin] initial
.
b The type of fructoside is defined according to mass values determined by mass spectrometry.
The acceptor reactions of Bs_SacB, Lm_LevS and Zm_LevU exhibited similar HPLC-UV
chromatograms (Fig. 1A). Puerarin fructosylation resulted in two products, designated as P1a
and P1b, with retention times of 10.36 and 10.19 min, respectively. The molecular ions at
m/z 577.18 (MPuerarin-Fru−H) for P1a and at m/z 577.41 for the most abundant P1b (Fig. 1B)
revealed the synthesis of two monofructoside isomers. By contrast, the HPLC-UV
chromatogram of the Gd_LsdA reaction showed the occurrence of P1a, a major product P2a,
8

and a short series of further elongated fructosides in decreasing amounts (P3a, P4a, P5a and
others) (Fig. 1A). The synthesis of P1a as unique monofructoside highlights the strict
regioselectivity of Gd_LsdA compared to the other tested levansucrases.
Fig. 1. Puerarin fructosylation by the four levansucrases in study. A) Superimposition of
HPLC-UV chromatograms. B) MS chromatograms of puerarin mono-fructosides. Peaks
were identified as P1a and P1b (monofructosides), P2a (difructoside), P3a (trifructoside), P4a
(tetrafructoside), and P5a (pentafructoside). The reaction conditions are described in Table
1.
3.1.1 Structural characterization of products P1a, P2a and P1b
Gd_LsdA and Bs_SacB were used for the preparative synthesis, purification and structural
characterization of the products P1a, P2a and P1b. The chemical structure of P1a, P2a
(supplementary material Fig. S1-S4), and P1b (supplementary materials Fig. S5-S8) was
determined by 1H NMR, 13C NMR and 2D NMR spectroscopy.
P1a was identified as β-D-monofructofuranosyl-(26)-puerarin, confirming that Gd_LsdA
transfers the fructosyl moiety to the O6 position of the glucosyl residue of the isoflavone
(Núñez-López et al. 2019). Levansucrases fructosylate glucose via a β(21) linkage
regenerating sucrose or a β(26) linkage producing the isomer blastose, which is not used
as donor (Homann et al., 2007; Raga-Carbajal et al., 2018). The synthesis of P1a is thus
consistent with the linkage specificity of levansucrases but, with exception of Gd_LsdA, the
presence of the daidzein group prevented the efficient fructosylation of the acceptor at its
glucosyl unit.
The molecular ion at m/z 739 (MPuearin-Fru-Fru−H) for the Gd_LsdA product P2a confirms the
transfer of two fructosyl units to puerarin. The 13C spectrum revealed 33 signals (three
overlapped), of which 13 carbon signals were in the range 110–175 ppm and assigned to
puerarin, and 17 signals were in the range 60-110 ppm and assigned to the three
monosaccharides (two fructose units and one glucose unit). Six additional signals for the
fructosyl unit were identified. Two of these signals with chemical shift at 104.20 and 104.34
ppm characteristic of the carbon atom C2’’’’ and C2’’’ of the fructosyl moiety confirm the
presence of two fructose unit. The HMBC spectrum (supplementary material Fig. S4) of P2a
9

was then used to deduce the position of fructosylation. A long-range correlation between the
carbon atom C2’’’’ of fructose (C 104.20 ppm) and the proton H6’’’ (H 3.55) of the fructose
unit, demonstrate that P2a corresponds to β-D-difructofuranosyl-(26)-puerarin. Notably,
P2a is neither a donor nor an efficient acceptor of Gd_LsdA and thus accumulates in the
reaction mixture after prolonged incubation (see Fig.1).
The 13C spectrum of monofructoside P1b synthesized by Bs_SacB revealed 27 signals (three
overlapped). Twelve signals were in the range 60-110 ppm being characteristic of the
carbons of the fructosyl and glucosyl moieties. The other 13 signals (110-176 ppm) can be
assigned to the isoflavone (supplementary materials Fig. S6). A large downfield shift (+ 6.1)
was observed for carbon C3’ and C5’ and (+ 3.66) for carbon C1’ and an upfield shift (- 2.75)
1
for carbon C4’. The H-NMR spectrum (supplementary material Fig. S5) revealed the
disappearance of the peak at 9.53 ppm, which corresponds to 4’ -OH and a displacement of
the signals at 7.25 ppm (+ 0.45) characteristic of H3’ and H5’, as deduced by comparing to
the spectra of puerarin and P1a (Fig. 2). The position of the attached fructosyl unit was
confirmed using HSQC and HMBC analyses. From these results, P1b was identified as
puerarin-4ꞌ-O-fructofuranoside. The transfer of the fructosyl unit onto the 4’ O-position of
the phenolic cycle of puerarin by the enzymes Bs_SacB, Lm_LevS and Zm_LevU (Fig. 3)
occurred with efficiencies ranging 7-25 %. In previous studies, Lm_LevS (Kang et al., 2009)
and Bs_SacB (Mena-Arizmendi et al., 2011) catalyzed the fructosyl transfer onto the
aromatic compound hydroquinone (benzene-1,4 diol) yielding 4-hydroxyphenyl-β-D-
fructofuranoside, with acceptor conversion below 12 %.
10

Fig. 2. Superimposition of the 1H NMR spectra of puerarin, P1a and P1b
Three positional isomers of puerarin monofructosides have been reported so far. The
synthesis of β-D-fructofuranosyl-(26)-puerarin (Núñez-López et al., 2019; Wu et al.,
2013), puerarin-7-O-β-fructofuranoside (Wang et al., 2014; Yu et al., 2010), and puerarin-
4’-O-β-fructofuranoside (this work) reveals variations in the regioselectivity of puerarin
fructosylation by GH68 enzymes. The occurrence of only β-fructosyl products is in
agreement with the strict stereoselectivity of retaining fructosidases. The 3D-structure
resolution of Gd_LsdA and Bs_SacB in complex with puerarin, as well as molecular docking
simulations and mutagenesis studies would be of interest to study the acceptor interactions
with these two representative levansucrases, and identify the structural determinants at the
origin of their regiospecificity.
11

Fig. 3. Schematic synthesis of puerarin fructosides by Gd_LsdA, Bs_SacB, Lm_LevS and
Zm_LevU.
3.2 Synthesis of puerarin polyfructosides (PPFs) using P1a as acceptor
P1a β-D-monofructofuranosyl-(26)-puerarin that is produced efficiently by Gd_LsdA
was purified by flash chromatography and assayed as a precursor for the synthesis of PPFs
using the same enzyme and the more competent polymerase Bs_SacB. Purified P1a (10 mM)
was incubated with either Gd_LsdA or Bs_SacB at different sucrose concentrations (0.5, 1
or 1.5 M). Bs_SacB transformed 93-95 % of initial P1a regardless of the numeric variation
of donor molecules in the reaction (Table 2). By contrast, the acceptor conversion by
Gd_LsdA (55.2 %) decreased as the sucrose concentration was increased to 1.5 M, a
condition that favors fructosylation over donor hydrolysis. This result reflects a competition
between sucrose and P1a for binding the acceptor subsite. Sucrose is known to be not only
the natural donor but also the preferred acceptor of Gd_LsdA (Hernández et al., 1995).
HPLC-UV chromatograms show remarkable differences in the size distribution of the
puerarin fructo-conjugates synthesized from the acceptor P1a (Fig. 4). Gd_LsdA synthesized
a linear series of puerarin oligofructosides (POFs) (P2a, P3a, P4a, P5a, ...) remaining P2a as
the major product, while Bs_SacB produced PPFs of longer fructan chains (overlapped peaks
12

with retention times 4.2-7.8 min) and at a higher yield. Bs_SacB also synthesized the
monofructoside P1b from remaining traces of puerarin.
Table 2. P1a fructosylation by Gd_LsdA and Bs_SacB at different sucrose concentrations
P1a conversion (%)a
1.0b
Levansucrase
0.5b
1.5b
Gd_LsdA
Bs_SacB
72.1 ± 1.6
65.9 ± 1.6
55.2 ± 1.9
93.2 ± 1.7
95.9 ± 0.1
93.4 ± 0.8
Each enzyme (0.5 U/mL) was incubated with purified P1a (10 mM) and sucrose (0.5-1.5M) in 50 mM phosphate
buffer (pH 5.8) at 42 °C during 24 h under agitation.
aAcceptor conversion =100*([P1a] initial – [P1a] final)/[P1a]initial
bSucrose concentration (M)
In parallel, the course of sucrose transformation into fructans in the presence or absence of
P1a was monitored by HPAEC-PAD (supplementary material Fig. S9). Gd_LsdA
accumulated high levels of 1-kestotriose and 1,1-kestotetraose. The enzyme also synthesized
6-kestotriose and 6,6-kestotetraose, which acted as preferred acceptors for levan
polymerization. By contrast, Bs_SacB produced an array of growing oligosaccharides and a
higher yield of levan polysaccharide. The presence of P1a did not cause a visible variation in
the yield or the spectra of the fructan products synthesized by either enzyme, due to the fact
that compared to the initial concentration of sucrose, the amount of fructosyl units transferred
to P1A is negligible.
The HPLC-UV chromatograms of the Bs_SacB products obtained using puerarin (Fig. 1) or
P1a (Fig. 3) as acceptors show notable variations. Only 25% of the initial puerarin content
was converted into products and the main monofructoside P1b was not further elongated. In
contrast, the enzyme used P1a as a convenient precursor for the synthesis of PPFs with the
conversion value reaching 95 %. The incorporation of a fructosyl unit to the glucosyl unit of
puerarin through the formation of a β(26) linkage appears to fulfill the necessary
requirements for the proper accommodation and effective binding of P1a in the acceptor
subsites of Bs_SacB, Gd_LsdA and probably other levansucrases.
13

14

Fig. 4. HPLC-UV-MS chromatograms of P1a elongation by (A) Gd_LsdA and (B) Bs_SacB.
Each enzyme (0.5 U/mL) was incubated with P1a (10 mM) and sucrose (0.5-1.5M) in 50 mM
phosphate buffer (pH 5.8) at 42 °C during 24 h under agitation. The chromatogram of purified
P1a used as acceptor is shown (C). Puerarin oligofructosides (POFs) produced by Gd_LsdA
comprises P2a (2 fructosyl units) as the major component, followed by decreasing
concentrations of P3a, P4a, P5a and other very small peaks. The linear chains of puerarin
polyfructosides (PPFs) produced by Bs_SacB reach at least 21 fructosyl units being the
predominant range 12-15.
3.2.1 Size estimation of puerarin fructo-conjugates synthesized from precursor P1a
Puerarin fructo-conjugates were synthesized at a preparative scale from sucrose (500 mM)
and P1a (25 mM) by Gd_LsdA and Bs_SacB with conversion efficiencies of 70 % and 73 %,
respectively. The products of the two independent reactions were purified by flash
chromatography and their mass distribution was characterized by HPSEC-RI using dextran
for calibration and MALDI-TOF MS.
The HPSEC chromatogram of the Gd_LsdA products (Fig. 5A) showed a sharp peak
matching to P2a and a shorter but wider peak (retention times 55-65 min) corresponding to
a population of POFs with average molar mass of 1400 g.mol-1 at peak apex. More
interestingly, the Bs_SacB, products (Fig. 5B) were distributed as a small P2a peak and a
larger peak (retention times 52-60 min) corresponding to PPFs with average molar mass of
3500 g.mol-1 at peak apex.
The MALDI-TOF MS analysis of the Gd_LsdA acceptor products (Fig. 5C) revealed a
spectrum of POFs with molecular masses ranging from m/z 601.2 to 2545.8, consistent with
the distributive incorporation of a maximum of 13 fructosyl units. The number average
molecular weight (Mn) and weight average molecular weight (Mw) of POFs synthesized by
Gd_LsdA were 953.1 and 1042.2, respectively. Bs_SacB yielded PPFs of a wider mass range
covering from m/z 601.2 to 3842.2 (Fig 5D). The latter value reveals that the fructan chain
incorporated onto puerarin reached at least 21 fructosyls units. The average values for Mn
and Mw were 1508.4 and 1818.1, respectively.
15

The size distribution of PPFs observed in the HPLC-UV-MS (Fig. 4), HPSEC-RI (Fig. 5 A,
B) and MADI-TOF chromatograms (Fig. 5 C, D) revealed that Bs_SacB polymerizes the
precursor P1a more efficiently than Gd_LsdA, which otherwise tends to accumulate the
immediate product P2a. In both cases, the elongation of the fructan chain occurred via a
distributive mechanism, in which the product dissociates from the enzyme after every
fructosyl transfer.
Fig. 5. Mass distribution of puerarin fructo-conjugates from P1a elongation. A) HPSEC-RI
analysis of puerarin fructo-conjugates synthesized by Gd_LsdA and B) Bs_SacB using
sucrose (500 mM) as donor and purified P1a (25 mM) as acceptor. P2a and dextrans were
used as reference for molar mass determination. The chromatograms corresponding to
Gd_LsdA and Bs_SacB are highlighted in pink and blue, respectively. MALDI-TOF MS
analysis of PPFs produced by C) Gd_LsdA and D) Bs_SacB
Gd_LsdA and Bs_SacB display a single domain 5-bladed β-propeller fold enclosing the
substrate-binding central cavity. In the two enzymes, the donor sucrose accommodates
similarly at the bottom of the deep funnel-shaped pocket (Martínez-Fleites et al., 2005; Meng
16

& Fütterer, 2003), but there are important differences in the predicted area (Bs_SacB 3783
Å2, Gd_LsdA 5773 Å2) and volume (Bs_SacB 6263 Å3, Gd_LsdA 10100 Å3) of their entire
active site cavity (Fig. 6). In this sense, Gd_LsdA offers a wider gate that may facilitate a
more rapid release of the intermediate product P2a to the medium delaying its binding to the
acceptor subsite for the next fructosylation cycles. On the other hand, the 3D structures of
Gd_LsdA and Bs_SacB do not superimpose well at the rim and surface of the active site
pocket (Martínez-Fleites et al., 2005), which include the regions responsible for the acceptor
binding, orientation and elongation (Ortiz-Soto, Porras-Domínguez, Seibel, & López-
Munguía, 2019). Rational mutagenesis of amino acids lining the surface of the active site
cavity of Gd_LsdA may contribute to increase the enzyme affinity for the acceptor P2a. There
is no report on enhanced formation of levan polysaccharide by a mutated variant of Gd_LsdA
or another FOS-yielding levansucrase.
Fig. 6. Representation of the active site cavity in the 3D structure of levansucrases from (A)
Gluconacetobacter diazotrophicus (Gd_LsdA; PDB code 1W18) and (B) Bacillus subtilis
(Bs_SacB; PDB code 1OYG). The surface area (brown) and volume (red) for the entire active
site cavity of Gd_LsdA (5773 Å2, 10100 Å3) and Bs_SacB (3783 Å2, 6263 Å3) were
determined using CavityPlus (Xu et al., 2018), CASTp (Tian, Chen, Lei, Zhao, & Liang,
2018), and UCSF Chimera (Pettersen et al., 2004). The 3D structures are shown as ribbon
diagrams.
17

3.3 One-pot, bi-enzymatic cascade synthesis of PPFs
P1a was the sole monofructoside synthesized from puerarin by Gd_LsdA. The conversion
efficiency of the acceptor reaction was 93% using 146 mM sucrose and 25 mM puerarin in
the absence of co-solvents (Núñez-López et al., 2019). On the other hand, Bs_SacB
fructosylted the precursor P1a to longer chains, compared to Gd_LsdA. In an attempt to avoid
the isolation of the intermediate product P1a, the possibility to produce PPFs directly from
puerarin was evaluated by combining the complementary activities of Gd_LsdA and
Bs_SacB in the same reactor.
The one-pot bi-enzymatic cascade reaction (Fig. 7) was performed through two operating
methods: (i) either by using the two enzymes sequentially or (ii) by using them
simultaneously.
Fig. 7. Schematic synthesis of puerarin polyfructosides by the cascade reaction of Gd_LsdA
and Bs_SacB. The number of fructosyl units (n) in PPFs ranges from 2 to at least 21,
predominantly 12-15.
In the first method, Gd_LsdA initiated the reaction for 6 h (step one) and then Bs_SacB was
incorporated with or without extra sucrose (step two). In the second method, Gd_LsdA and
Bs_SacB were both incorporated in the reactor at the start of the reaction, with different
activity ratios (1:1, 2:1 or 1:2) (Table 3).
18

High percentages of puerarin conversion (82-92%) were achieved, except for the sequential
reaction in which extra fructosyl donor was not supplied at step two. When the reaction was
performed under the same conditions for step one Gd_LsdA (0.1 U.mL-1), sucrose (146 mM)
and puerarin (25 mM) but adding Bs_SacB (0.5 U.mL-1) together with sucrose (292 mM)
the acceptor conversion increased from 38 to 89 % (Table 3).
Table 3. One-pot, bi-enzymatic synthesis of PPFs using puerarin as acceptor
Operating
method
Enzyme
step1
(U/mL)
Sucrose
step 1
(mM)
Acceptor Enzyme
Sucrose
step 2
(mM)
Acceptor
conversion *
(%)
puerarin
(mM)
step 2
(U/mL)
Gd_LsdA 0.1
146
25
Bs_SacB 0.5
0
38.1 ± 0.4
Gd_LsdA 0.1
146
25
Bs_SacB 0.5
292
88.9 ± 0.9
Sequential use
of enzymes
Gd_LsdA 0.1
146
500
25
10
Bs_SacB 0.5
500
-
84.8 ± 0.1
85.9 ± 0.3
Gd_LsdA 0.75
Bs_SacB 0.75
Gd_LsdA 1.0
Bs_SacB 0.5
Gd_LsdA 0.5
Bs_SacB 1.0
-
Simultaneous
use of enzymes
500
500
10
10
-
-
-
-
92.4 ± 0.7
82.2 ± 0.6
Each mixture of donor (sucrose) and acceptor (puerarin) was prepared in 50 mM citrate-phosphate buffer (pH
5.8) and incubated with the enzymes at 42 °C under constant agitation. For the sequential use of enzymes,
Gd_LsdA was reacted with the substrates for 6 h and inactivated 5 min at 100°C (step 1), then Bs_SacB was
incorporated with or without supplementary sucrose (step 2). For the simultaneous use of enzymes, Gd_LsdA
and Bs_SacB were added to the substrate mixture from the beginning of the reaction.
aThe conversion was calculated from the amount of puerarin remaining after 24 h of reaction. Acceptor
conversion = 100*([puerarin] initial – [puerarin] final)/ [puerarin] initial
.
When the two enzymes were incorporated simultaneously in the reactor at the reaction start,
maximum conversion (92.4 %) was obtained with 1 U/mL-1 Gd_LsdA, 0.5 U/mL-1 Bs_SacB,
500 mM sucrose and 10 mM puerarin (Table 3). This value is slightly superior to that
achieved with the best sequential treatment (89 %), which otherwise used ten times less Gd-
LsdA and a closer molar proportion donor/acceptor (439/25). Finally, the one-pot bi-
enzymatic systems (either sequential or simultaneous) were comparable in terms of
conversion with the acceptor reaction performed with Bs_SacB and pure P1a (93.2 %) but
avoided the isolation of the intermediate product P1a, an advantage of prime interest for
biotechnological applications.
19

3.4 Increased water solubility of di-β-D-fructofuranosyl-(2→6)-puerarin (P2a), POFs
and PPFs
In previous experiments, the isomers β-D-fructofuranosyl-(26)-puerarin (P1a) (Núñez-
López et al., 2019) and puerarin-7-O-β-fructofuranoside (Wang et al., 2014) were found to
be 23 and 5.6 times more soluble in water than puerarin, respectively. Those results indicated
that the solubility of puerarin monofructosides is influenced by the position of the fructosyl
moiety. Here, the water solubility of puerarin fructo-conjugates containing linear β(26)-
linked fructan chains of different sizes was investigated. The solubility at 25ºC of P2a (>1840
g/L) and POFs (> 2018 g/L) synthesized by Gd-LsdA and PFFs (>1062 g/L) synthesized by
Bs-SacB was higher than that of P1a (16.2 g/L) and puerarin (0.7 g/L). The high viscosity of
the limpid aqueous solutions of purified P2a, POFs and PPFs impeded to further increase
their concentration until reaching the saturation point. P2a (2 fructosyl units), POFs (3-12
fructosyl units, predominantly 3-5) and PPFs (3-21 fructosyl units, predominantly 12-15)
have an increased number of hydroxyl groups which can form hydrogen bonds with water,
compared to the precursor P1a. Noteworthy, difructosylation of puerarin enabled to improve
by at least 2620-fold the water solubility of the flavone. Our finding reveals that the
solubility, and by consequence the bioavailability, of puerarin can be tremendously enhanced
by attaching two or more fructosyl units via β(26) linkages.
We describe for the first time the enzymatic synthesis of PPFs as novel products using a bi-
enzymatic cascade and demonstrate the interest of conducting one-pot synthesis. In
comparison to puerarin, the increased water solubility of PPFs must enhance their absorption
in vivo, broadening the spectrum of potential pharmacological applications. Additionally, it
will be interesting to investigate if the fructan chain provides PPFs with prebiotic properties.
4. Conclusion
In this study, four levansucrases of different origins were found to fructosylate the non-
conventional acceptor puerarin in an aqueous medium, although with variations in their
regioselectivity and conversion efficiency. The enzyme from Gluconacetobacter
diazotrophicus (Gd_LsdA) efficiently synthesized β-D-fructofuranosyl-(2→6)-puerarin
(P1a), while the three other levansucrases formed puerarin-4’-O-β-D-fructofuranoside (P1b)
20

and only traces of P1a with low conversion rates. Purified P1a was converted into water-
soluble polyfructosides (PPFs) reaching at least 21 fructosyl units by the competent
polymerase activity of Bacillus subtilis levansucrase (Bs_SacB). More attractively,
simultaneous or sequential Gd_LsdA/Bs_SacB reactions yielded PPFs directly from puerarin
in one reactor, using sucrose as a cheap fructosyl donor, and avoiding the isolation of
intermediate precursor P1a. The one-pot bi-enzymatic procedure described in this paper is
appropriate to scale up the cascade production of PPFs, new products with potentially healthy
properties.
Author contributions
Gema Núñez-López: Investigation, Writing original draft
Sandrine Morel: Conceptualization, Methodology, Investigation, Visualization, Writing-
review & editing
Lázaro Hernández: Methodology, Visualization, Writing-review & editing
Alexis Musacchio: Data curation, Formal analysis
Lorena Amaya-Delgado: Resources, Funding acquisition
Anne Gschaedler: Resources, Funding acquisition
Magali Remaud-Simeon: Project administration, Supervision, Funding acquisition in
France.
Javier Arrizon: Project administration, Supervision, Visualization, Funding acquisition in
Mexico.
Conflict of interest
The authors declare that they have no conflict of interest.
Compliance with ethical standards
21

This article does not contain any studies with human participants or animals performed by
any of the authors.
Acknowledgements
We are grateful to Metasys, the Metabolomics & Fluxomics Center at the Toulouse
Biotechnology Institute (Toulouse, France), for NMR experiments. We thank the ICEO
facility dedicated to enzyme screening and discovery, and Part of the Integrated Screening
Platform of Toulouse (PICT, IBiSA) for providing access to HPLC equipment and protein
purification system. Technical assistance provided by Valérie Bourdon of the ICT-FR 2599
(Toulouse, France-ict.ups-tlse.fr). We acknowledge the bilateral project CONACYT-
ANUIES/ECOS-NORD M14A01, the CONACYT project CB-2012-01/000000000181766
for financial support and the CONACYT Ph.D. fellowship 421514.
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