Glucansucrases from lactic acid bacteria as biocatalysts for multi-ring catechol glucosylation

Article abstract of DOI:10.1080/10242422.2020.1784882

Catechins are the major group of bioactive flavanols in green tea and cacao. 17 glucansucrase-active strains were identified from a set of 41 lactic acid bacteria, which were able to glucosylate (+)-catechin in a non-natural acceptor reaction. In total cell free extracts of 12 Leuconostoc and 5 Weissella strains were active on catechin and also 8 cell fractions exhibited catechin glucosylation activity. Six enzymes were selected for further evaluation and enriched up to 37 fold in yields of at least 40%. Glucansucrase of L. citreum DSM 5577 was the most efficient biocatalyst for (+)-catechin transformation with conversions of >40% after 24 h. NMR analysis of the major reaction product confirmed the (+)-catechin-4′-O-α-d-glucoside. Only L. kimchi B-65337 produced a second catechin monoglucoside. Four out of six glucansucrases glucosylated esculetin and all enzymes were active on haematoxylin. Glucansucrases of L. citreum DSM 5577, L. kimchi B-65337 and W. beninensis DSM 22752 were the best suited biocatalysts with conversions of >30% for esculetin and >60% for haematoxylin. W. beninensis DSM 22752 glucansucrase produced 89% haematoxylin glucosides without process optimization. L. kimchi B-65337 and W. beninensis DSM 22752 synthesized >40% diglucosides with the bifunctional haematoxylin. NMR analysis of the purified esculetin products confirmed formation of the 6-O-α-d- and 7-O-α-d-glucosides. Also two haematoxylin monoglucosides were identified as the 9-O-α-d- and 3-O-α-d-glucosides.

Full text of DOI:10.1080/10242422.2020.1784882

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Glucansucrases from lactic acid bacteria as
biocatalysts for multi-ring catechol glucosylation
Johannes Nolte, Alexander Kempa, Matthias Hochgürtel & Ulrich Schörken
To cite this article: Johannes Nolte, Alexander Kempa, Matthias Hochgürtel & Ulrich Schörken
(2020): Glucansucrases from lactic acid bacteria as biocatalysts for multi-ring catechol
glucosylation, Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2020.1784882
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2020.1784882
RESEARCH ARTICLE
Glucansucrases from lactic acid bacteria as biocatalysts for multi-ring
catechol glucosylation
a,c
a
a
Johannes Noltea,b, Alexander Kempa , Matthias Hochgurtel and Ulrich Schorken
€
€
a
b
€
Faculty of Applied Natural Sciences, TH Koln – Campus Leverkusen, Leverkusen, Germany; Vetter Pharma-Fertigung GmbH & Co.
c
€
KG, Ravensburg, Germany; R&R Extrakte GmbH, Koln, Germany
ABSTRACT
ARTICLE HISTORY
Received 16 September 2019
Revised 27 November 2019
Accepted 12 June 2020
Catechins are the major group of bioactive flavanols in green tea and cacao. 17 glucansucrase-
active strains were identified from a set of 41 lactic acid bacteria, which were able to glucosylate
(þ)-catechin in a non-natural acceptor reaction. In total cell free extracts of 12 Leuconostoc and
5 Weissella strains were active on catechin and also 8 cell fractions exhibited catechin glucosyla-
tion activity. Six enzymes were selected for further evaluation and enriched up to 37 fold in
yields of at least 40%. Glucansucrase of L. citreum DSM 5577 was the most efficient biocatalyst
for (þ)-catechin transformation with conversions of >40% after 24 h. NMR analysis of the major
reaction product confirmed the (þ)-catechin-40-O-a-D-glucoside. Only L. kimchi B-65337 produced
a second catechin monoglucoside. Four out of six glucansucrases glucosylated esculetin and all
enzymes were active on haematoxylin. Glucansucrases of L. citreum DSM 5577, L. kimchi B-65337
and W. beninensis DSM 22752 were the best suited biocatalysts with conversions of >30% for
esculetin and >60% for haematoxylin. W. beninensis DSM 22752 glucansucrase produced 89%
haematoxylin glucosides without process optimization. L. kimchi B-65337 and W. beninensis DSM
22752 synthesized >40% diglucosides with the bifunctional haematoxylin. NMR analysis of the
purified esculetin products confirmed formation of the 6-O-a-D- and 7-O-a-D-glucosides. Also
two haematoxylin monoglucosides were identified as the 9-O-a-D- and 3-O-a-D-glucosides.
KEYWORDS
Glucansucrase; glucosyla-
tion; acceptor reaction;
catechin; esculetin;
haematoxylin
1. Introduction
represents an attractive green alternative to the chem-
ical process (de Roode et al. 2003; Desmet et al. 2012).
Glucansucrases (EC 2.4.1.5) are particularly interest-
ing in this respect due to utilization of readily avail-
able sucrose as cost efficient donor substrate (Andre
et al. 2010; Meng et al. 2016) and process stability e.g.
in the presence of organic solvents (Girard and Legoy
1999). Glucansucrases of family GH70 (Lombard et al.
2014) are high molecular weight enzymes of 160 kDa
on average, which are found in lactic acid bacteria
and enable these organisms to form extracellular
homopolysaccharides from sucrose. They are either
cell-bound or secreted to the outer medium (van
Hijum et al. 2006; Meng et al. 2016), from which they
can be isolated and purified by different methods
(Majumder et al. 2007). The enrichment of native glu-
cansucrases from culture supernatant by polyethylene
glycol (PEG) phase partitioning is considered the most
effective procedure for glucansucrase isolation (Shukla
et al. 2010; Mohan Rao and Goyal 2013). This method
makes use of the tendency of glucansucrases to
Natural biologically active polyphenols like the flava-
nol (þ)-catechin are interesting products for dietary
supplements, cosmetics and the pharmaceutical indus-
try (Nadim et al. 2014; Quideau et al. 2011; Sauer and
Plauth 2017). Catechins possess antioxidative, anti-
inflammatory, anti-thrombotic and anti-microbial prop-
erties (Jochmann et al. 2008; Brown et al. 2011; Deka
and Vita 2011), which have been utilized in traditional
medicine for a long time (Taylor et al. 2005). To pro-
tect these polyphenols from oxidation, to increase
their water solubility and to enhance their bioavailabil-
ity covalent attachment of a glycosyl-residue as nat-
ꢀ
ural protective group was proposed (Kren 2008;
Desmet et al. 2012; Thuan and Sohng 2013; Nadim
et al. 2014; Xu et al. 2016). However; site-specific
chemical glucosylation is a complex task, which
requires multi-step protective group chemistry.
Therefore biocatalytic glucosylation of polyphenols
€
€
Faculty of Applied Natural Sciences, TH Koln – Campus Leverkusen, CHEMPARK E39,
CONTACT Ulrich Schorken
ulrich.schoerken@th-koeln.de
Kaiser-Wilhelm-Allee, Leverkusen, 51368, Germany
Supplemental data for this article can be accessed here.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
J. NOLTE ET AL.
aggregate with glucan in solution and leads to a
selective co-precipitation of glucansucrases and glucan
(Paul et al. 1984). Additionally, a stabilizing effect on
the enzymes by both PEG and glucan has been
described (Miller and Robyt 1984).
marine natural products, which exhibit strong anti-
inflammatory properties and are used in skin care cos-
meceuticals (Newman and Cragg 2004). Haematoxylin
is a natural compound extracted from Haematoxylon
campechianum, which is used in histology as a dye
and interestingly was proposed as a natural sweetener
(Arnoldi et al. 1995).
The non-natural acceptor side reaction of glucansu-
crases was originally shown with (þ)-catechin by
Nakahara et al. (1995). Further glucosylation studies
with different flavonoids revealed that a catechol-
motif is the favoured glucosylation site and the
hydroxyl group in para-position (40-O-glycoside) is pre-
ferred over the hydroxyl group in meta-position to the
aromatic ring side chain (30-O-glycoside) (Bertrand
et al. 2006; Moon et al. 2007; Woo et al. 2012).
Additionally some glucosylation of the non-catecholic
7-OH was observed with catechin as substrate and
multiple glucosylations of the flavonoids were also
detected (Meulenbeld et al. 1999; Bertrand et al. 2006;
Overwin et al. 2015). Glucansucrases acceptor reaction
activities strongly depend on the structure of the fla-
vonoid substrates. Flavanes and flavanones with a cat-
echol motif were better acceptors than the structurally
analogous flavones, while isoflavones were poor sub-
strates (Overwin et al. 2015, 2016).
2. Materials and methods
2.1. Bacterial strains, chemicals and
substrate synthesis
Forty-one Leuconostoc and Weissella strains obtained
from DSMZ, USDA, Agroscope, Sacco S.r.l., Ernst
€
Bocker GmbH and Moguntia Food Group AG were
used in this study. All strains were maintained at
ꢀ80 ꢁC as 20% (v/v) glycerol stocks obtained from sin-
gle colonies and propagated in De Man, Rogosa and
Sharpe (MRS) medium containing 2% (w/v) glucose at
30 ꢁC. (þ)-Catechin was obtained from Sigma-Aldrich;
esculetin and haematoxylin were from Alfa Aesar and
carnosic acid from TCI. Pseudopterosine analogon
5,6,6a,7,8,9-hexahydro-4H-phenalene-1,2-diol
was synthesized in multistep
(rac-5)
synthesis
a
Studies concerning polyphenol glucosylation were
limited to enzymes from a few Streptococcus species,
Leuconostoc mesenteroides and Lactobacillus reuteri for
a long time, though the two aerotolerant genera
Leuconostoc and Weissella are rich sources of largely
unexploited glucansucrases (Bounaix et al. 2009). Thus
the glucosylation potential of glucansucrases from
these genera was analysed by our group in more
detail with the antioxidant caffeic acid and structural
analogs as well as with biologically active nordihydro-
guaiaretic acid, the major lignan of the creosote bush
Larrea tridentata (Nolte et al. 2019a, 2019b). Several
glucansucrase positive strains were identified and we
were able to show that the glucosylation of natural
catechols is a widespread ability of this class of
enzymes. Here we present the glucosylation of (þ)-cat-
echin with soluble and cell-bound glucansucrases iso-
lated from Leuconostoc and Weissella strains.
Furthermore we analysed the substrate spectrum of
selected PEG-enriched glucansucrases from six strains
with other multi-ring compounds including the cou-
marine esculetin, the abietane diterpene carnosic acid,
the bifunctional indenochromene haematoxylin and a
synthetic pseudopterosine analogon. Esculetin and
carnosic acid possess antioxidant properties (Bubols
et al. 2013) and esculin, the b-D-glucoside of esculetin,
is used in cosmetics as a bioactive sunscreen ingredi-
ent (Lee et al. 2007). Pseudopterosins are glycosylated
(Supplementary Figure S1) starting from commercially
available 6,7-dimethoxy-1-tetralone.
2.2. Acceptor substrate characterization
LogP values of acceptor substrates were calculated
with the programme Marvin Sketch and three-dimen-
sional models of acceptor substrates were visualized
with the MolView web application (http://molview.org/),
which uses PubChem data or CORINA for three-
dimensional structure generation.
2.3. Strain cultivation and preparation of
supernatant and cell fractions
Bacterial cultures from glycerol stocks were grown for
15 hours in 2 mL pre-culture containing MRS media
with 2% (w/v) glucose at 30 ꢁC, 288 rpm in 48 well
round plates covered with an air permeable sealing
tape in an 80% humidified atmosphere using a
R
V
BioLector micro bioreactor system. For cultivation of
Weissella strains, the medium was supplemented with
0.1% (w/v) cysteine hydrochloride. Cells from the pre-
cultures were centrifuged (4 ꢁC, 15 min at 2800ꢂg),
washed once with sodium acetate buffer, pH 5.4, and
suspended in 2 mL main culture media (MRS) supple-
mented with 2% (w/v) sucrose to a final OD(600 nm) of
R
V
0.25. Main cultures were incubated in the Biolector

BIOCATALYSIS AND BIOTRANSFORMATION
3
system at 25 ꢁC and 288 rpm for 48 hours. The bacter-
ial culture was centrifuged (4 ꢁC, 2800ꢂg, 20 min) to
separate cells from supernatant. The cell fraction was
washed three times with 20 mM sodium acetate, pH
5.4. After that the cells were frozen in liquid nitrogen
and lyophilized. The cells were then stored at room
temperature until further use.
glucansucrases to a final activity of 1.0 U mLꢀ1 and
samples were taken periodically for further analysis.
For isolation of polyphenol glucosides the reaction
was scaled up to 14mL and was run for 24h under
essentially the same reaction conditions as described
above (1 U mLꢀ1
)
glucansucrase activity, 200mM
sucrose, 10mM acceptor substrate, pH 5.4, 30ꢁC). The
reaction was stopped by addition of ethanol, which
was also used for glucan precipitation. Ethanol was
evaporated from the glucoside containing samples
under reduced pressure and the samples were lyophi-
lized to remove traces of ethanol. The polyphenol glu-
cosides were purified by preparative HPLC with an
Interchim puriflash 4250/250 system equipped with a
Kromasil 100 C18 column (20 ꢂ 250 mm, 5 mm) and a
PDA detector. The separation was run isocratically with
water and acetonitrile supplemented with 0.2% (v/v)
formic acid at a flow-rate of 8 mL minꢀ1. 10% of aceto-
nitrile were used for separation of all polyphenol gluco-
sides except for carnosic acid, which afforded 30%
acetonitrile due to higher hydrophobicity. Glucoside
containing fractions were pooled and lyophilized.
2.4. Concentration of glucansucrases from shake
flask culture supernatants
Pre-cultures of selected strains were grown in a vol-
ume of 15 mL and main cultures in a volume of
200 mL in shake flasks at 100 rpm essentially according
R
V
to the Biolector micro-scale method described in 2.3.
After harvesting of the main cultures glucansucrases
were isolated according to the method of Paul et al.
(1986). An aqueous (50% w/v) polyethylene glycol
4000 solution was added under continuous stirring to
the supernatants to reach a final concentration of 25%
(w/v). The mixtures were centrifuged at 2800ꢂg, 4 ꢁC
for 15 min and the precipitates containing glucan and
glucansucrase were suspended in 20 mM sodium acet-
ate, pH 5.4. The PEG precipitation was repeated twice
and the final precipitates were dissolved in 20 mM
sodium acetate, 0.45 mM CaCl2, pH 5.4 containing pro-
tease inhibitor cocktail (EDTA-free, Roche) and stored
at 4 ꢁC. Glucansucrase enrichment was calculated on
the basis of volumetric glucansucrase activities and
isolated glucansucrase yields were calculated as:
2.6. Glucansucrase activity and
carbohydrate analysis
Twenty-five microliters of glucansucrase preparation in
an appropriate dilution was mixed with 475 mL of
assay buffer containing 20 mM sodium acetate with a
pH of 5.4, 292 mM sucrose, 0.45 mM sodium chloride
and 0.03 mM dextran. The mixtures were incubated at
30 ꢁC for 2 hours and samples were taken periodically
and supplemented with an equal amount of dinitrosa-
licylic acid solution to stop the reaction. Samples were
then heated at 95 ꢁC for five minutes and cooled
down. 50 mL of the sample was diluted with 200 mL of
water and the absorbance was determined at 540 nm
with a multiplate reader (Spectra Max 190, Molecular
Devices, San Jose, CA, USA). The quantification was
done based on a fructose calibration curve and one
unit corresponds to the release of 1 mmol of fructose
per minute under the given reaction conditions.
Sucrose, fructose and glucose were analysed with
an enzymatic kit from R-Biopharm GmbH according to
the manufacturer’s protocol. Glucose-6-phosphate is
quantified by the NADþ-dependent oxidation to 6-
phospho-gluconate catalysed by glucose-6-phosphate
dehydrogenase and analysis at 340 nm. Glucose is
phosphorylated by hexokinase to glucose-6-phosphate
whereas fructose is transformed to glucose-6-phos-
phate by hexokinase and phosphoglucose isomerase.
Sucrose is hydrolysed to glucose and fructose by
b-fructosidase and both monosaccharides are
total units PEG fraction
total units supernatant
ð Þ
yield % ¼
ꢂ 100
2.5. Biocatalytical glucosylation and glycoside
purification
Glucosylation mixtures contained 200 mM sucrose and
40 mM (þ)-catechin in 20 mM sodium acetate, pH 5.4
supplemented with 0.45 mM CaCl2 and 15% (v/v)
dimethyl sulfoxide (DMSO). Initial catechin glucosyla-
tions were done in a scale of 25 mL and were started
by addition of 13.75 mL of glucansucrase containing
supernatant or cells obtained from 55 mL of cell sus-
pension and incubated statically for 24 hours at 30 ꢁC.
The reaction was stopped by addition of 9 vol. of etha-
nol (ꢀ20 ꢁC). Samples were vortexed for 20 s and pre-
cipitated glucans were removed by centrifugation at
3300ꢂg for 20 min at 4 ꢁC according to the method of
Overwin et al. (2015) before further analysis. Reactions
with PEG concentrated glucansucrases were done
accordingly with 10 or 40 mM of polyphenol acceptor
substrates. The reaction was started by addition of

4
J. NOLTE ET AL.
transformed into glucose-6-phosphate as described
above. The assay was scaled down to 200 mL and ana-
lysed in microplate format as described in Vermeir
et al. (2007). Analyses were performed with a Spectra
Max 190 multiplate reader at 340 nm and the molar
extinction coefficient of NAD(P)H at 340 nm of
6.22 mMꢀ1 cmꢀ1 was used for quantification.
glucansucrase producers (Bounaix et al. 2009). In a
previous screening with a Biolector microfermentation
system, we detected 19 glucansucrase positive strains
from a set of 25 Leuconostoc and 16 Weissella strains
(Nolte et al. 2019a, 2019b). These strains were ana-
lysed for their (þ)-catechin glucosylation potential
employing LC-UV analysis for glucoside quantification
coupled with LC-MS for peak assignment (Figure 1).
Most strains produced one major catechin monogluco-
side with an assigned mass of m/z ¼ 451. Only L. kim-
chi B-65337 produced significant amounts of a second
monoglucoside, which was clearly separated in HPLC
analysis (Figure 1(b)).
In the basic screening cells were separated from the
supernatant fraction (Supplementary Figure S2) and
were analysed separately for their glucosylation ability.
Catechin was successfully transformed with 17 out of 19
glucansucrase positive supernatants (Table 1) displaying
the widespread ability of glucansucrases for this non-
natural acceptor reaction. Similarly caffeic acid was glu-
cosylated by all glucansucrase active fractions in our
previous studies (Nolte et al. 2019a), whereas the more
hydrophobic bifunctional catechol nordihydroguaiaretic
acid was only accepted by 6 out of 19 glucansucrases
(Nolte et al. 2019b). Supernatants of L. citreum DSM
5577, L. kimchi B-65337, L. pseudomesenteroides DSM
20193 and B-65331 performed best with glucoside con-
versions exceeding 10%. These four strains as well as L.
pseudomesenteroides B-65334 and Weissella beninensis
DSM 22752 were chosen for further studies.
2.7. Chromatographic and spectroscopic analysis
of glucosides
LC-UV was carried out with a Thermo Scientific Accela
system equipped with an Accela 80Hz PDA detector
and a Hitachi LaChrom II C18 reversed phase column
(4.6ꢂ 250 mm, 5 mm) as described before (Nolte et al.
2019a, 2019b). A linear gradient with D2.1% minꢀ1 of
water (eluent A) and acetonitrile (eluent B, starting con-
centration 10%) each supplemented with 0.1% (v/v) for-
mic acid was used for the separation and a wavelength
of 280 nm was used for polyphenol and polyphenol
glucoside detection. According to Meulenbeld et al.
(1999) the molar absorption coefficients of the aromatic
ring systems were used for quantification of substrates
and corresponding glycosides. Conversion of acceptor
substrates was calculated as:
ð Þ
conversion % ¼
P
peak area polyphenol glucosides
P
Þꢂ100
ðpeak area polyphenol aglyconþ
peak area polyphenol glucosides
It is known that glucansucrases exist both in soluble
and cell-associated form (van Hijum et al. 2006; Meng
et al. 2016), but cell-associated glucansucrases have not
been investigated in terms of non-carbohydrate acceptor
glucosylation before. Analysis of cell fractions revealed
that glucansucrase activity was present in several strains
and that catechin glucosylation was possible as a whole
Mass spectrometric analyses were performed using
a Shimadzu LC-30AD with a SPD M20A diode array
detector and a LCMS-2020 single quadrupole mass
spectrometer with electrospray ionization (ESI-MS)
equipped with the same column (Nolte et al. 2019a,
2019b). The mass spectrometer was run in positive
and negative scan mode in the m/z range from 150 to
1000 at 10,000 u sꢀ1 and an event time of 0.1 s.
1H- und 13 C-NMR spectra including 2 D-NMR
experiments (COSY, HMBC, HSQC, NOESY) were
recorded with a 400 MHz Bruker AscendTM 400 and
analysed with MestReNova (Mestrelab Research S.L.) as
described before (Nolte et al. 2019a, 2019b).
Methanol-d4 was used as solvent for all NMR analyses.
cell biotransformation with
8
strains (Table 1).
Surprisingly supernatant and cell-associated glucosylation
did not necessarily coincide. Cells of L. fallax DSM 20189
and L. suionicum DSM 20241 performed best with con-
versions of 17 and 10%. These initial data may serve as a
starting point for a more detailed investigation of whole
cell glucosylation with Leuconostoc. Advantages could be
the simple and efficient preparation of the whole-cell bio-
catalyst, the easy separation and the GRAS status (gener-
ally regarded as safe) of most lactic acid bacteria strains.
3. Results and discussion
3.1. Catechin glucosylation potential of
glucansucrase positive lactic acid bacteria
3.2. Enrichment of selected glucansucrases and
comparative catechin glucosylation
Glucan synthesizing lactic acid bacteria of the
genera Leuconostoc and Weissella are well known
For the preparation of glucansucrases, the culture vol-
ume was increased to 200 mL and glucansucrases

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 1. LC-UV chromatogram with LC-MS peak assignment obtained from reaction of (þ)-catechin and sucrose with glucansu-
crase containing supernatant fractions of strain DSM 5577 (A) and B-65337 (B) from initial screening trials (25 ml scale, 200 mM
sucrose, 40 mM catechin, pH 5.4, incubation at 30 ꢁC for 24 h).
located in the supernatant were enriched by an aqueous
two-phase extraction using 50% (w/v) PEG 4000 solution
with three repetitions to remove undesired contamina-
tions (Supplementary Figure S2, Table 2). All glucansu-
crases were successfully enriched by factors of 6.4 for
L. pseudomesenteroides B-65334 to over 37 for W. beni-
nensis with isolated yields of at least 40%. These prepa-
rations were used for all further glucosylation studies.
Comparative (þ)-catechin glucosylation with 1 U mLꢀ1
of enriched glucansucrase confirmed the data from the

6
J. NOLTE ET AL.
Table 1. Catechin transformation with supernatants and cell fractions of glucansucrase positive strains.
Supernatant
Cell fraction
)
Strain
Activity (U mLꢀ1
)
Conversion (%)
Activity (U mLꢀ1
Conversion (%)
L. citreum DSM 201881
0.66 0.07
1.48 0.16
0.12 0.07
1.46 0.37
0.35 0.06
0.31 0.15
0.15 0.03
1.74 0.21
0.77 0.12
0.30 0.03
1.03 0.30
4
29
5
22
5
3
1
9
13
9
12
4
0.60 0.20
0.66 0.26
0.18 0.05
na
0.19 0.06
0.13 0.03
0.19 0.05
1.14 0.49
0.22 0.07
0.02 0.02
0.06 0.02
0.07 0.05
0.08 0.03
na
4
<1
17
na
<1
nd
nd
10
nd
nd
nd
nd
3
L. citreum DSM 55771
L. fallax DSM 201891
L. kimchi B-653372
L. mesenteroides FAM 224883
L. mesenteroides LN274
L. mesenteroides dextranicum DSM 462162
L. mesenteroides suionicum DSM 202412
L. pseudomesenteroides B-653312
L. pseudomesenteroides B-653342
L. pseudomesenteroides DSM 201931
L. pseudomesenteroides LN234
W. beninensis DSM 227521
W. cibaria DSM 158781
0.18
0
0.19 0.06
0.16 0.05
0.37 0.03
0.40 0.06
0.58 0.04
5
4
3
5
3
W. confusa BSM 11935
0.22 0.04
na
0.15 0.05
2
W. confusa BSM 13905
W. confusa WS014
3
<1
1
2
3
4
€
DSMZ, ARS culture collection (USDA), Agroscope, Sacco S.r.L, Ernst Bocker GmbH; na: no glucansucrase activity; nd: no glucosylated product detected
in HPLC analysis.
Table 2. Enrichment of glucansucrases from culture supernatants of selected bacterial strains.
Activity supernatant
Total activity
supernatant (U)
Activity PEG-fraction
Total activity PEG-
fraction (U)
Isolated
yield (PEG)
Strain
(U mLꢀ1
)
[U mLꢀ1
]
L. citreum DSM 5577
L. kimchi B-65337
L. pseudomesenteroides DSM 20193
L. pseudomesenteroides B-65331
L. pseudomesenteroides B-65334
W. beninensis DSM 22752
1.3
1.5
0.5
0.5
0.3
0.11
260.0
308.0
94.0
108.0
60.0
19.0
10.2
4.6
6.5
1.9
227.4
87 %
40 %
92 %
72 %
105 %
65 %
121.8
86.2
78.0
63.0
14.4
22.0
4.1
initial screening with supernatant fractions (Figure 2(A)).
Glucansucrase from L. kimchi B-65337 produced two
monoglucosylated products, while all other glucansu-
crases only formed one catechin monoglucoside. The
best strain for catechin glucosylation was L. citreum DSM
5577 with a catechin conversion of approximately 40%
after 9 h. In a 24 h kinetic study the formation of catechin
glucosides was monitored simultaneously with sucrose
transformation and monosaccharide formation (Figure
2(B)). Transformation of sucrose led to a predominant
fructose release with only very low glucose contents.
Thus contaminating enzyme activities like e.g. invertase
and glucansucrase catalysed hydrolysis were negligible
during catechin glucosylations. Comparison of initial
decrease of sucrose to catechin glucoside formation illus-
trates that natural glucan formation is the preferred reac-
tion of the DSM 5577 glucansucrase. The decrease in
catechin formation seems not to be an equilibrium phe-
nomenon but is caused by a fast drop of sucrose con-
centration. Thus an improvement of (þ)-catechin
transformation should be possible by optimization of
reaction conditions. In a design of experiments approach
to optimize process conditions we were able to increase
nordihydroguaiaretic acid (NDGA) glucosylation from 39%
to >90% with glucansucrase from L. pseudomesenteroides
DSM 20193 (Nolte et al. 2019b).
The major catechin monoglucoside was isolated by
preparative LC and analysed with NMR (Table 3). A
downshift of the 50 proton in comparison to the agly-
con, a NOESY coupling of the anomeric proton to the
50 proton and a coupling constant of 3.7 Hz for the
anomeric proton confirmed the (þ)-catechin-40-O-a-D-
glucoside. Our data are in agreement with the NMR
analysis of Nakahara et al. (1995) and confirm that the
40-OH of flavonoid B-rings is the preferred glucosyla-
tion site of glucansucrases (Klingel et al. 2019).
Literature data on flavanol glucosylation suggest that
the second (þ)-catechin monoglucoside found as
minor product with Leuconostoc kimchi B-65337 is
either the 30-O-a-D-glucoside or the 7-O-a-D-glucosides
(Meulenbeld et al. 1999; Bertrand et al. 2006; Overwin
et al. 2015).
3.3. Glucosylation of multi-ring catechols
Esculetin was converted by glucansucrases of DSM
5577, DSM 22752, B-65337 and B-65331 (Table 4).
Esculetin glucosideconversions of DSM 5577 and DSM

BIOCATALYSIS AND BIOTRANSFORMATION
7
Figure 2. (A) Analysis of (þ)-catechin glucosylation with 1 U mLꢀ1 glucansucrase for 9 hours (light grey bars: (þ)-catechin-40-glu-
coside; dark grey bars: other (þ)-catechin-glucoside); (B) Analysis of catechin and carbohydrate transformation with L. citreum
DSM 5577 glucansucrase (straight line, ᭜¼ (þ)-catechin; straight line, ᭢¼ (þ)-catechin monoglucoside, dotted line, ᭞ ¼ sucrose,
dotted line, ^ ¼ fructose).
Table 3. NMR data and structure of (þ)-catechin-40-O-a-D-glucoside in comparison to the aglycon.
(þ)-Catechin
dC (ppm)
dH (ppm)
dC (ppm)
dH (ppm)
2
3
4
82.8
68.8
28.5
4.56 (d, J ¼ 7.5 Hz, 1H)
82.5
68.8
28.0
4.61 (d, J ¼ 7.4 Hz, 1H)
3.98 (td, J ¼ 7.9, 5.4 Hz, 1H)
2.51 (dd, J ¼ 16.1, 8.2 Hz, 1H)
2.85 (dd, J ¼ 16.1, 5.4 Hz, 1H)
3.98 (td, J ¼ 7.8, 5.4 Hz, 1H)
2.51 (dd, J ¼ 16.2, 8.1 Hz, 1H)
2.83 (dd, J ¼ 16.2, 5.4 Hz, 1H)
4a/5
6
100.8/157.6
100.7/157.6
96.2
95.5
5.93 (d, J ¼ 2.3 Hz, 1H)
96.3
95.5
5.93 (d, J ¼ 2.3 Hz, 1H)
8
5.85 (d, J ¼ 2.3 Hz, 1H)
5.86 (d, J ¼ 2.3 Hz, 1H)
7/8a/10
20
157.8/156.9/132.2
157.9/156.8/136.3
115.2
6.84 (d, J ¼ 1.9 Hz, 1H)
115.9
6.91 (d, J ¼ 2.1 Hz, 1H)
30/40
50
146.2/146.2
148.6/146.2
116.0
120.0
6.76 (d, J ¼ 8.2 Hz, 1H)
118.8
119.9
101.4
73.5
74.9
71.4
7.25 (d, J ¼ 8.3 Hz, 1H)
6.82 (dd, J ¼ 8.3, 2.1 Hz, 1H)
5.33 (d, J ¼ 3.7 Hz, 1H)
3.58 (dd, J ¼ 9.7, 3.7 Hz, 1H)
3.87 (t, J ¼ 9.7, 9.0 Hz, 1H)
3.43 (t, J ¼ 9.6 Hz, 1H)
3.83 ꢀ 3.66 (m, 1H)
60
6.72 (dd, J ¼ 8.2, 1.9 Hz, 1H)
Glc 1
Glc 2
Glc 3
Glc 4
Glc 5
Glc 6
NOESY
74.5
62.4
3.83 ꢀ 3.66 (m, 2H)
Glucose H1 couples with (þ)-catechin H50

8
J. NOLTE ET AL.
Table 4. Glucosylation of esculetin and haematoxylin with enriched glucansucrases.
Compound/biocatalyst
20193
65334
22752
5577
65337
65331
Esculetin
Conversion (%)
Product distribution
Mono- : Diglucoside
Haematoxylin
–
–
43
>99:1
46
>99:1
36
>99:1
3
>99:1
Conversion (%)
Product distribution
Mono- :Diglucoside
48
>99:1
54
>99:1
89
54:46
62
99:1
69
57:43
45
96:4
Figure 3. LC-UV chromatograms with mass assignment from LC-MS obtained from reaction products of esculetin conversion with
DSM 5577 (A) and haematoxylin conversion with B-65337 glucansucrase (B) from trials with PEG-enriched glucansucrases (1 U
mLꢀ1, 200 mM sucrose, 10 mM acceptor substrate, pH 5.4, incubation at 30 ꢁC for 24 h).

BIOCATALYSIS AND BIOTRANSFORMATION
9
Table 5. NMR data and structures of esculetin-O-a-D-glucosides.
dC (ppm)
dH (ppm)
dC (ppm)
dH (ppm)
2
ꢃ
3
4
112.9
146.0
6.22 (d, J ¼ 9.4 Hz, 1H)
7.83 (d, J ¼ 9.5 Hz, 1H)
114.7
146.0
6.29 (d, J ¼ 9.5 Hz, 1H)
7.83 (d, J ¼ 9.5 Hz, 1H)
2
2
ꢃ
ꢃ
2/4a
5
6/7/8a
8
Glc 1
Glc 2
Glc 3
Glc 4
Glc 5
Glc 6
NOESY
162.3/112.9
162.3/112.9
117.0
7.53 (s, 1H)
153.1/151.3/151.3
6.79 (s, 1H)
114.9
7.05 (s, 1H)
153.1/151.3/148.5
7.34 (s, 1H)
104.8
101.6
73.4
74.8
71.5
75.0
62.6
105.5
100.5
73.4
74.8
71.5
75.0
62.5
5.41 (d, J ¼ 3.7 Hz, 1H)
3.62 (dd, J ¼ 9.7, 3.6 Hz, 1H)
3.87 (t, J ¼ 9.2 Hz, 1H)
3.40 (t, J ¼ 9.3 Hz)
5.58 (d, J ¼ 3.6 Hz, 1H)
3.62 (dd, J ¼ 9.7, 3.6 Hz, 1H)
3.87 (t, J ¼ 9.2 Hz, 1H)
3.40 (t, J ¼ 9.3 Hz)
3.78 ꢀ 3.69 (m, 1H)
3.73 ꢀ 3.69 (m, 1H)
3.78 ꢀ 3.69 (m, 1H)
3.73 ꢀ 3.69 (m, 2H)
Glucose H1 couples with esculetin H5
Glucose H1 couples with esculetin H8
22752 were higher than with (þ)-catechin. Only a sin-
gle product peak with significant fronting was
detected in LC-UV analysis and hardly any oligogluco-
sides were formed (Figure 3(a)). NMR analysis of the
product at m/z ¼ 339 confirmed the formation of two
glucosides (Table 5), which could not be separated by
preparative HPLC. Coupling constants of 3.6 and
3.7 Hz for the anomeric protons confirmed the a-con-
figuration. Discrimination between protons 5 and 8
adjacent to the catechol motif was possible by select-
ive NOESY coupling of proton 5 with proton 4.
Downfield shifts of 0.6 ppm for proton 5 of glucoside
1 and 0.59 for proton 8 of glucoside 2 as well as cor-
responding NOESY couplings confirmed the structures
as esculetin-6-O-a-D-glucoside and esculetin-7-O-a-D-
glucoside (Table 5). According to proton intensities in
1H-NMR analysis esculetin-6-O-a-D-glucoside was the
major product in transformation with L. citreum
DSM 5577.
The large indenochromene haematoxylin was glu-
cosylated by all glucansucrases tested with exception-
ally high conversions of up to 89% (Table 4).
Glucansucrases from DSM 20193, B-65331 and DSM
5577 exhibited one main product peak in LC-UV/MS
analysis (m/z ¼ 463) corresponding to a mono-gluco-
side, whereas W. beninensis DSM 22752 and L. kimchi
B-65337 also showed the formation of significant
amounts of a haematoxylin diglucoside (Figure 3(b)).
Structural analysis of the monoglucoside from
biotransformation using L. citreum 5577 glucansu-
crases revealed the existence of two haematoxylin glu-
coside isomers. The major reaction product was
identified as haematoxylin-9-O-a-D-glucoside, the
minor product as haematoxylin-3-O-a-D-glucoside by
NMR spectroscopy (Table 6). The characteristic proton
signal of H2 compared to the aglycon determines the
glucosylation site on position 3 in the minor product.
This result is supported by the NOESY correlation
between H2 and the anomeric proton at 5.30 ppm.
The haematoxylin-9-O-a-D-glucoside was identified by
the HMBC interaction of H8 with C7, the shift of H8 in
comparison to the aglycon and the NOESY correlation
between proton H8 and H7. In analogy to results
obtained with the bifunctional catechol nordihydro-
guaiaretic acid (NDGA, Nolte et al. 2019b) the haema-
toxylin
diglucosides
are
most
probable
monoglucosylated at both ends. Though multiple glu-
cosylation at one hydroxyl group is possible, single
glucosylation at each catechol seemed to
be preferred.
Analysis of the acceptor substrates revealed that
the three-dimensional structures of (þ)-catechin,
haematoxylin and the previously published NDGA
(Nolte et al. 2019b) are similar. As outlined in Figure 4,
all molecules are bended and side chains pointed to
the front, when the catechol ring was positioned in
the plain with the upper hydroxyl group as major glu-
cosylation
site.
Haematoxylin
possesses
four

10
J. NOLTE ET AL.
Table 6. NMR data and structures of haematoxylin-O-a-D-glucosides.
dC (ppm)
121.22
dH (ppm)
dC (ppm)
dH (ppm)
1
2
6.74 (d, J ¼ 8.4 Hz, 0.9 Hz)
6.51 (d, J ¼ 8.4 Hz)
121.45
112.46
145.3/137.4/143.6
71.1
6.84 (d, J ¼ 8.6 Hz, 0.9 Hz)
6.95 (d, J ¼ 8.6 Hz)
109.98
3/4/4a
6
145.8/134.5/143.6
71.1
3.74 (d, J ¼ 11.6 Hz, 1H)
3.74 (d, J ¼ 11.6 Hz, 1H)
4.06 ꢀ 4.02 (m, 1H)
4.06 ꢀ 4.02 (m, 1H)
6a/7a
7
78.2/136.9 or 131.5
78.0/141.6 or 131.3
42.9
2.87 (d, J ¼ 15.8 Hz, 1H)
3.06 (d, J ¼ 15.8 Hz, 1H)
7.08 (s)
42.9
112.9
112.4
2.81 (d, J ¼ 15.7 Hz, 1H)
3.01 (d, J ¼ 15.7 Hz, 1H)
6.60 (s)
8
9/10
11
11a/c
11b
Glc 1
Glc 2
Glc 3
Glc 4
Glc 5
Glc 6
NOESY
116.3
113.3
148.0/145.8
145.4/145.6
6.80 (s)
136.9 or 131.5/116.0
4.01 (s)
6.72 (s)
141.6 or 131.3/120.3
ꢃ
ꢃ
51.5 7
51.3 7
101.7
4.01 (s)
101.9
5.19 (d, J ¼ 3.7 Hz, 1H)
5.3 (d, J ¼ 3.7 Hz, 1H)
ꢃ
ꢃ
ꢃ
ꢃ
73.6 8
3.57–3.54 (m, 1H) 9
73.4 8
3.57–3.54 (m, 1H) 9
ꢃ
ꢃ
ꢃ
ꢃ
74.6 10
3.90–3.78 (m, 1H) 11
3.49–3.36 (m, 1H)
74.5 10
3.90–3.78 (m, 1H) 11
ꢃ
ꢃ
71.4 12
71.4 12
3.49–3.36 (m, 1H)
ꢃ
ꢃ
ꢃ
ꢃ
74.9 13
3.80–3.78 14 (m, 1H)
74.8 13
3.80–3.78 14 (m, 1H)
ꢃ
ꢃ
65.5 15
3.90–3.78
65.5 15
3.90–3.78 (m,
Glucose H1 couples with haematoxylin H8
Glucose H1 couples with haematoxylin H2
structurally divergent hydroxyls and only the two
hydroxyls, which resulted in a similar structure overlay,
were glucosylated. Similarly the symmetric NDGA con-
tains two different hydroxyls at each catechol and
only the one at position 4 or 40 was glucosylated. In
modelling studies using the crystal structure of amylo-
sucrase co-crystallized with acceptor maltoheptaose
(PDB: 1MW0), it was shown, that superimposition with
good fit was possible with the flavonoid acceptor sub-
strate luteolin and that docking studies allowed gluco-
sylation of the 4-OH position (Malbert et al. 2014).
Though luteolin is slightly less bended than catechin,
haematoxylin and NDGA, molecule size and overall
structure are similar and an equally good fit with a
glucansucrase active site can be envisaged.
the active site of glucansucrases. No glucosylation of
carnosic acid and the pseudopterosin analogon
5,6,6a,7,8,9-hexahydro-4H-phenalene-1,2-diol could be
observed. One reason may be the lower polarity of
the compounds (logP 5.1 and 3.6) in comparison to
esculetin (logP 1.2) and haematoxylin (logP 1.5).
However, NDGA was glucosylated by several glucansu-
crases despite its hydrophobicity (logP 4.8). Thus,
structural issues may better explain the absence of
glucosylation activity. Both substrates are more bulky
and differ from the three-dimensional structure of the
other acceptor substrates significantly (Figure 4).
Conclusions
Our results demonstrate the widespread acceptance of
(þ)-catechin as non-natural substrate of glucansucrases
from the genera Leuconostoc and Weissella. The utiliza-
tion of cells as whole cell glucosylation biocatalysts for
catechin transformation was shown for the first time.
New glycosides of esculetin and haematoxylin were
synthesized and the structures were elucidated.
The structure of esculetin is planar and resembles
that of previously published caffeic acid (Nolte et al.
2019a) in size (Figure 4). Caffeic acid was found to be
a very good acceptor substrate for glucansucrases,
whereas esculetin was only accepted by 4 glucansu-
crases. Most probably the more flexible side chain of
caffeic acid allows a more favourable positioning in

BIOCATALYSIS AND BIOTRANSFORMATION
11
Figure 4. Two- and three-dimensional structures of acceptor substrates tested in this study in comparison to caffeic acid and nor-
dihydroguaiaretic acid, in all 3-dimensional displays glucosylation sites are the upper left hydroxyl groups marked by the respect-
ive number, oxygens are coloured black; (A) (þ)-catechin, (B) haematoxylin, (C) nordihydroguaiaretic acid, (D) esculetin, (E) caffeic
acid, (F) carnosic acid, (G) pseudopterosin analogon 5,6,6a,7,8,9-hexahydro-4H-phenalene-1,2-diol.
Acknowledgements
Esculetin glucosides may be utilized as cosmetic bioac-
tives and haematoxylin glucosides may be developed
as new sweeteners for food applications.
The authors thank the companies Sacco S.r.L., Moguntia
€
GmbH and Ernst Bocker GmbH & Co. KG as well as the ARS

12
J. NOLTE ET AL.
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Disclosure statement
The authors have declared no conflict of interest.
Funding
This work was supported by the NRW Ministry of Innovation,
Science and Research within the program FH Struktur.
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