A novel enzymatic method for the synthesis of methyl 6-O-acetyl-α-D-glucopyranoside using a fermented solid containing lipases produced by Burkholderia contaminans LTEB11

Article abstract of DOI:10.1016/j.procbio.2018.07.023

Chemical methods for the regioselective modification of carbohydrates require several steps that involve the addition and removal of protective groups. Enzymatic methods are promising since they can give high selectivity in a single step. We developed a new method for acetylation of methyl-α-D-glucopyranoside with vinyl acetate, the reaction being catalyzed by a fermented solid containing lipases from Burkholderia contaminans LTEB11. The main product of the synthesis, methyl 6-O-acetyl-α-D-glucopyranoside (6-OAc-α-MetGlc), was identified by NMR and quantified by HPLC. The conversion of methyl-α-D-glucopyranoside to 6-OAc-α-MetGlc was evaluated at laboratory scale (4 mL) in different solvent systems. The conversion increased with increasing hydrophobicity of the solvent, with the highest conversion, 65% in 72 h, being obtained in the solvent-free system. This system was scaled up to a 1-L reaction medium. A conversion of 76% in 72 h was obtained, with a calculated yield of 1.05 g (4.43 mmol) of 6-OAc-α-MetGlc. This is the largest amount of 6-OAc-α-MetGlc (in moles) produced by enzymatic catalysis that has been reported in the literature to date. These results suggest that the fermented solid produced by B. contaminans LTEB11, which is a low-cost biocatalyst, could potentially be used for the industrial production of esters of α-D-glucose.

Full text of DOI:10.1016/j.procbio.2018.07.023

Accepted Manuscript
Title: A novel enzymatic method for the synthesis of methyl
6-O-acetyl-␣-D-glucopyranoside using a fermented solid
containing lipases produced by Burkholderia contaminans
LTEB11
Authors: Maribel Cordero Villalobos, Alan Guilherme
Gonc¸alves, Miguel Daniel Noseda, David Alexander Mitchell,
Nadia Krieger
PII:
DOI:
Reference:
S1359-5113(18)30182-X
https://doi.org/10.1016/j.procbio.2018.07.023
PRBI 11410
To appear in:
Process Biochemistry
Received date:
Revised date:
Accepted date:
4-2-2018
16-7-2018
30-7-2018
Please cite this article as: Villalobos MC, Gonc¸alves AG, Noseda MD, Mitchell DA,
Krieger N, A novel enzymatic method for the synthesis of methyl 6-O-acetyl-␣-
<ce:small-caps>d</ce:small-caps>-glucopyranosideusingafermentedsolidcontaining
lipases produced by Burkholderia contaminans LTEB11, Process Biochemistry (2018),
https://doi.org/10.1016/j.procbio.2018.07.023
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A novel enzymatic method for the synthesis of methyl 6-O-acetyl-α-D-
glucopyranoside using a fermented solid containing lipases produced by
Burkholderia contaminans LTEB11
Maribel Cordero Villalobosa, Alan Guilherme Gonçalvesb, Miguel Daniel Nosedad, David
Alexander Mitchellc, Nadia Kriegera*
aDepartamento de Química, Universidade Federal do Paraná, Cx. P. 19081 Centro Politécnico, Curitiba
81531-980, Paraná, Brazil
bDepartamento de Farmácia, Universidade Federal do Paraná. Av. Lothario Meissner, 3400, Jardim
Botânico, Curitiba 80210-170, Paraná, Brazil
cDepartamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046
Centro Politécnico, Curitiba 81531-980, Paraná, Brazil
*Corresponding author.
E-mail address: nkrieger@ufpr.br (N. Krieger).
Graphical abstract
1

Highlights
 A novel method is developed for the enzymatic acetylation of methyl-α-D-glucopyranoside
 The catalyst is a fermented solid containing lipases of Burkholderia contaminans LTEB11
 The acetylation was regioselective for C-6, producing methyl 6-O-acetyl-α-D-glucopyranoside
 The process was scaled-up to 1-L of reaction medium in a solvent-free system
 A conversion of 76% was achieved at the 1-L scale, corresponding to 1.05 g of product
ABSTRACT
Chemical methods for the regioselective modification of carbohydrates require several steps that involve
the addition and removal of protective groups. Enzymatic methods are promising since they can give
high selectivity in a single step. We developed a new method for acetylation of methyl-α-D-
glucopyranoside with vinyl acetate, the reaction being catalyzed by a fermented solid containing lipases
2

from Burkholderia contaminans LTEB11. The main product of the synthesis, methyl 6-O-acetyl-α-D-
glucopyranoside (6-OAc-α-MetGlc), was identified by NMR and quantified by HPLC. The conversion
of methyl-α-D-glucopyranoside to 6-OAc-α-MetGlc was evaluated at laboratory scale (4 mL) in
different solvent systems. The conversion increased with increasing hydrophobicity of the solvent, with
the highest conversion, 65% in 72 h, being obtained in the solvent-free system. This system was scaled
up to a 1-L reaction medium. A conversion of 76% in 72 h was obtained, with a calculated yield of 1.05
g (4.43 mmol) of 6-OAc-α-MetGlc. This is the largest amount of 6-OAc-α-MetGlc (in moles) produced
by enzymatic catalysis that has been reported in the literature to date. These results suggest that the
fermented solid produced by B. contaminans LTEB11, which is a low-cost biocatalyst, could potentially
be used for the industrial production of esters of α-D-glucose.
Keywords: Burkholderia contaminans LTEB11, fermented solids, regioselective acetylation, methyl-α-
D-glucopyranoside, solvent-free system, scale-up
1. Introduction
Sugar esters are commonly used as monomeric building blocks for the chemical synthesis of
oligosaccharides. Once synthesized, the oligosaccharides can be used to produce glycoconjugates, which
are biopolymers formed by covalent bonds between a carbohydrate and another biomolecule, such as a
protein or a lipid [1-2].
Glycoconjugates play important roles in cells, often being involved in molecular recognition. As such,
they have biotechnological applications. For example, native glycoconjugates have been isolated and
3

used for the treatment of cancer and also to design vaccines against parasitic diseases, such as malaria,
leishmaniosis and Chagas disease, bacterial infections, such as tuberculosis, and viral infections, such as
AIDS (acquired immunodeficiency syndrome). However, in the production of vaccines, the synthetic
route has the potential to give higher yields and purer products than those obtained through extraction of
the native glycoconjugate directly from the pathogen [3].
The synthetic route for preparing sugar esters as the building blocks for glycoconjugates can be either
chemical or enzymatic. The traditional chemical method has the disadvantage of producing multiple
isomers, because the sugar molecules contain several hydroxyl groups that are chemically very similar.
The desired isomer must then be separated from the others; this is challenging due to the chemical
similarity of the isomers. The production of multiple isomers can be overcome by introducing protective
groups in key positions of the monosaccharide and removing them after the reaction, however, this
involves many steps. On the other hand, the enzymatic synthesis of sugar esters using lipases (E.C.
3.1.1.3) has the potential to provide selective acylation of polyfunctional substrates such as
monosaccharides, without the need for protective groups [4-6]. This simplifies not only the acylation
reaction itself, but also the downstream processing, as the production of undesirable side products is
minimized. Many lipases are readily available commercially in free or immobilized forms [7-8]. These
commercial lipases are often active and stable in the water-restricted media that are used in synthesis
reactions. However, their high cost has been a barrier, limiting industrial applications [9].
The cost of producing lipases can be reduced by using solid-state fermentation (SSF). This technique
offers advantages over traditional submerged fermentation processes, such as the use of lower-cost
substrates, lower energy requirements, higher product yields and less wastewater production.
Furthermore, after the fermentation, the so-called “fermented solids”, which contain naturally
4

immobilized lipases, can be dried and then added directly to the reaction medium. This eliminates the
need for enzyme purification and immobilization, two steps that account for a large portion of the cost of
producing lipases by submerged fermentation. Our group has successfully used SSF of various agro-
industrial residues to produce dry fermented solids (DFS) containing bacterial or fungal lipases. These
DFS have been used in the synthesis of alkyl esters (biodiesel) by esterification and transesterification in
solvent-free systems [10-16], in the hydrolysis of pectin to produce D-galacturonic acid [17], in the
production of structured lipids [18] and in the treatment of wastewaters contaminated with lipids [19].
This work reports a simple, regioselective and cost-effective enzymatic method in which dry fermented
solids produced by Burkholderia contaminans LTEB11 are used to catalyze the synthesis of methyl 6-O-
acetyl-α-D-glucopyranoside (6-OAc-α-MetGlc), a carbohydrate-derivative that can be used as a building
block for the synthesis of pharmaceutically active compounds. The effects of various organic solvents on
esterification reaction yields at laboratory scale were investigated. Over 1 g of 6-OAc-α-MetGlc was
produced at preparative scale, showing the feasibility of the direct use of DFS in this process.
2. Materials and methods
2.1. Reagents and raw materials
Anhydrous α-D-glucose (96% purity), 1-ethyl-3-methylimidazolium methanesulfonate ([emim][MeSO3])
and methyl per-O-acetyl-α-D-glucopyranoside standard were purchased from Sigma-Aldrich (São Paulo,
Brazil). The cationic resin Amberlite IR-120 was purchased from Labsynth (Diadema, São Paulo,
Brazil). Sulfuric acid was HPLC quality and purchased from Tedia (Fairfield, Ohio, USA). The organic
solvents and all other reagents were of analytical grade from Vetec (Duque das Caixas, Río de Janeiro
5

Brasil). Lipase from Candida antarctica B (CALB, NOVOZYM 435) was a kind gift of Novozymes
Latin America Ltda (Araucaria, Paraná, Brazil). Sugarcane bagasse (SB) was kindly donated by Usina
de Álcool Melhoramentos (Jussara, Paraná, Brazil) and sunflower seeds were purchased in a local
market.
2.2. Microorganism
B. contaminans LTEB11 (originally isolated as B. cepacia LTEB11 and previously characterized as B.
lata CPQBA 515-12 DRM0) was maintained in Luria Bertani (LB) broth with 40% (v/v) glycerol at -18
°C. A stock culture was transferred to an LB agar plate and incubated for 48 h at 29 °C. Isolated colonies
were transferred to 30 mL of LB medium in a 250-mL Erlenmeyer flask and then incubated on a rotary
shaker at 29 °C and 200 rpm for 8-10 h. This gave an optical density at 600 nm of 0.6-0.8, which
represents mid-exponential phase. This culture broth was used as inoculum for the SSF [12].
2.3. Chemical synthesis of the substrate
The substrate methyl-α-D-glucopyranoside (α-MetGlc) was prepared by chemical synthesis from
anhydrous α-D-glucose (23.2 g) with methanol (60 mL) and Amberlite IR-120 (5.8 g). All reagents
remained in suspension under magnetic stirring with reflux at 65 °C for 24 h, according to Bollenback et
al. [20]. α-MetGlc was then purified by several crystallization steps and the white crystals were washed
with cold methanol. The substrate purity was 95%, determined by HPLC and NMR analysis.
2.4. Purification of the main product of the enzymatic synthesis
6

After enzymatic synthesis, the reaction medium was vacuum-filtered through Whatman nᵒ 1 filter paper,
washed with methanol (3 × 20 mL) and then concentrated under vacuum. Samples of the filtrate were
then analyzed by thin-layer chromatography (TLC) on silica gel 60 plates (particle size 220-440 mesh)
(Sigma-Aldrich, Jurubatuba, São Paulo, Brazil) using ethyl acetate/methanol 9:1 (v/v) as eluent. Spots
were developed by spraying the plates with 10% (v/v) ethanolic orcinol solution in sulfuric acid,
followed by heating at 105 °C.
The main product of the synthesis was purified by flash chromatography, carried out with silica gel 60
(Sigma-Aldrich, Jurubatuba, São Paulo, Brazil) using ethyl acetate/methanol 9:1 (v/v) as eluent. The
collected fractions were analyzed by TLC. The main product was further purified by preparative-scale
TLC under the same conditions and identified by NMR analysis.
2.5. Solid-state fermentation
Three different substrate mixtures were tested to produce fermented solids with high lipase activity. The
first substrate mixture was the one used by Soares et al. [12]. This solid substrate contained 10 g of a
mixture of sugarcane bagasse and sunflower seed (1:1 w/w, on a dry basis). Prior to incubation, the solid
substrate was wetted with phosphate buffer (0.1 mol L-1, pH 7.0) to obtain a moisture content of 75%
w/w on a wet basis. The fermented solid produced with this substrate mixture is hereafter labeled DFS1.
The second substrate mixture contained the same solid substrates as DFS1, the difference being that the
solids were wetted using a micronutrient solution containing (per L): 4 g urea, 5 g lactose, 5 g K2HPO4,
1 g MgSO4·7H2O and 4 mL of oligo-element solution. The oligo-element solution contained (g L-1):
7

MnCl2·4H2O, 1.98; CoSO4·7H2O, 2.81; CaCl2·2H2O, 1.47; CuCl2·2H2O, 0.17; ZnSO4·7H2O, 0.29; and
EDTA, 10 [21]. The fermented solid produced with this substrate mixture is hereafter labeled DFS2.
The third substrate mixture consisted of 5 g of sugarcane bagasse, which was moistened with the same
solution as that used for DFS2. The sunflower seed meal used to produce DFS2 was replaced with
soybean oil (20% w/w, on a dry basis). The fermented solid produced with this substrate mixture is
hereafter labeled DFS3.
SSF was done in 500-mL Erlenmeyer flasks, which were plugged with cotton wool and autoclaved at
121 °C for 20 min. After cooling, the substrates were inoculated with 1 mL of inoculum and incubated at
29 °C for 72 h. After incubation, the fermented solids were dried with air at room temperature, in a
column filled with activated silica, to a moisture content of 8-10% (w/w on a wet basis) and stored at 4
°C [12]. The moisture content of the fermented solids was determined in an infrared moisture balance
(Gehaka IV 2000, Real Parque, São Paulo, Brazil).
The fermented solids produced as described above (DFS1, DFS2, DFS3) were screened for use in
further experiments, based on their hydrolytic and esterification activities and their ability to catalyze the
acetylation of α-MetGlc.
2.6. Determination of lipase activity in the fermented solids
The hydrolytic activities of the dry fermented solids against olive oil were determined by the titrimetric
method with a pH-STAT (Metrohm, Herisau, Switzerland), with the pH maintained at 7.0 through the
addition of 0.05 mmol L-1 NaOH. The substrate emulsion consisted of 67 mmol L-1 of commercial extra
8

virgin olive oil (Gallo, Lisboa, Portugal), 3% (w/v) gum arabic, 2 mmol L-1 CaCl2, 2.5 mmol L-1 Tris-
HCl and 150 mmol L-1 NaCl, dispersed in distilled water [22]. It was emulsified with a blender at high
speed, initially for 5 min and then for an additional 1 min immediately before use. DFS samples (100
mg) were added to 20 mL of the emulsion under magnetic stirring (300 rpm) at 40 °C, and the reaction
was followed for 5 min. One unit (U) of hydrolytic activity corresponds to the release of 1 μmol of fatty
acid per minute, under the assay conditions, with the activity being calculated from the initial rate of the
reaction. The hydrolytic activity was expressed per gram of fermented solid (i.e. as U g-1).
Esterification activity was determined based on ethyl-oleate synthesis, using 25-mL Erlenmeyer flasks in
an orbital shaker at 40 ºC and 250 rpm. The reaction medium (5 mL) contained n-hexane, ethanol (210
mmol L-1) and oleic acid (70 mmol L-1). The reaction was started with the addition of 110 mg of the
appropriate DFS. At each sampling time, a 100-μL sample of the mixture was collected and analyzed for
residual free fatty acids by the Lowry-Tinsley method [23]. Calibration curves were constructed using
oleic acid. The yield of the reaction in terms of ester production was calculated based on the
consumption of free fatty acids and reported as a percentage of the initial concentration of free fatty
acids. There was no reaction in a control flask in which enzyme was not added. The esterification
activity was calculated from the initial rate of the reaction and was expressed per gram of fermented
solid (i.e. as U g-1, where 1 U corresponds to the consumption of 1 μmol of fatty acid per minute).
Both hydrolytic and esterification activities were expressed as the means of triplicates; the errors are the
standard errors of the mean.
2.7. Screening for acetylation of α-MetGlc
9

Initially, DFS1, DFS2, DFS3 were screened for their ability to acetylate α-MetGlc in a solvent-free
medium. Erlenmeyer flasks (50 mL) containing 15 mg (0.08 mmol) of α-MetGlc (95% purity), 1.65 g of
the appropriate DFS and 20 mL (218 mmol) of vinyl acetate were incubated for 72 h at 40 °C and 250
rpm. The mass of DFS was kept at 1.65 g because this was the maximum amount of solids that could be
added to the reaction medium without causing problems. Higher amounts made the medium too viscous,
hampering mixing and decreasing the reproducibility of the results. All reactions were done in triplicate.
The conversion was calculated based on the disappearance of α-MetGlc, as measured by HPLC. It
should be noted that, after the main product 6-OAc-α-MetGlc was obtained in solvent-free medium (as
described in this section) and purified (as described in Section 2.4), it was used to determine the
conversions in the remaining experiments.
2.8. Stability of the dry fermented solids in different reaction media
DFS3 was chosen as the best catalyst to continue the experiments. The stability of DFS3 was evaluated
by measuring its hydrolytic activity before and after incubation in the following reaction systems: (a)
solvent-free system (i.e. only α-MetGlc and vinyl acetate), (b) α-MetGlc plus a 1:1 (v/v) mixture of
vinyl acetate and DMF, (c) α-MetGlc plus a 1:0.9:0.1 (v/v/v) mixture of vinyl acetate, [emim][MeSO3]
and DMF. For these tests, 5-mL screw-capped bottles containing 200 mg of DFS3, 4.5 mg of α-MetGlc
and 4 mL of reaction medium were incubated on a rotary shaker at 250 rpm and 40 °C for up to 24 h.
After incubation, DFS3 was filtered and its hydrolytic activity was measured. The residual activity was
expressed as a percentage of the activity of fresh DFS3 determined under the same conditions.
2.9. Determination of the solubility of α-MetGlc in the reaction media
10

The solubility of α-MetGlc was determined by incubating 30 mg of α-MetGlc and 1.4 g of autoclaved
DFS3 in the following reaction media: (a) 20 mL of vinyl acetate, (b) 10 mL of vinyl acetate and 10 mL
of DMF, (c) 10 mL of vinyl acetate and 10 mL of acetone, (d) 10 mL of vinyl acetate and 10 mL of a
mixture 9:1 (v/v) of [emim][MeSO3]:DMF. The experiments were done in Erlenmeyer flasks, at 40 °C
and 250 rpm, for 24 h. The solubility of the substrate was calculated from the concentration of α-MetGlc
(mmol L-1) in the organic phase, as determined by HPLC. The calibration curve had α-MetGlc
concentrations from 0.10 to 5.00 mg mL-1.
2.10. Effect of the reaction medium on the enzymatic synthesis
Reactions were done in 5-mL screw-capped bottles containing 4 mL of reaction medium. In addition to
4.5 mg (0.02 mmol) of α-MetGlc, the reaction media contained (a) only vinyl acetate (solvent-free
medium), (b) 1:1 (v/v) vinyl acetate:DMF and (c) 1:0.9:0.1 (v/v/v) vinyl acetate:[emim][MeSO3]:DMF.
Each bottle received 200 mg of DFS3. The bottles were incubated at 40 °C and 250 rpm for 72 h. For
each medium, a control experiment was done using autoclaved fermented solids.
For the characterization of the main product, a sample of solvent-free medium (60 mL) was purified by
flash chromatography in a silica column and identified by NMR (section 2.13). After identification of
the main product, the quantifications of the subsequent experiments were carried out by HPLC analyses
using purified 6-OAc-α-MetGlc (90% purity), according to Eq. (1):
expFD
C = C
× 100%
(1)
Ct
11

where C is the percentage conversion of α-MetGlc to 6-OAc-α-MetGlc, Cexp is the ester concentration
(mg L-1) in the sample, FD is the dilution factor and Ct is the theoretical maximum ester concentration,
calculated for the limiting substrate (mg L-1).
For the study of the kinetics of acetylation in different reaction media, 100-μL samples were taken at
fixed intervals and analyzed by HPLC. The log P values for mixtures of the solvents (log Pmix) were
calculated from the values of the mole fraction (Xi) and log P (log Pi) of each component “i” of the
mixture [24], according to Eq. (2):
log Pmix = ∑ (Xi log Pi)
(2)
For comparison, the enzymatic synthesis of 6-OAc-α-MetGlc was also evaluated using a commercial
lipase (CALB, Novozym 435). This experiment was done with 4.5 mg (0.02 mmol) of α-MetGlc, 2.27
mg of CALB and 4 mL of reaction medium in 5-mL screw-capped bottles, with the same incubation
conditions as described above for DFS3.
2.11. Scale-up of the enzymatic synthesis of 6-OAc-α-MetGlc
The scale of 6-OAc-α-MetGlc production was increased 250-fold. The experiment was carried out in a
screw-capped bottle (1 L) that contained 1.125 g (5.8 mmol) of α-MetGlc, 52.5 g of DFS3 and 1 L (10.9
mol) of vinyl acetate. This mixture was incubated for 8 days at 40 °C and 250 rpm. For the kinetic study,
500-μL samples of the reaction medium were withdrawn at fixed intervals and analyzed by HPLC. The
yield after 8 days was calculated from the results of HPLC analysis of triplicate samples of the reaction
medium.
12

2.12. HPLC analysis
The purities and concentrations of the substrate α-MetGlc and the main product 6-OAc-α-MetGlc were
determined by HPLC with an Agilent 1260 Infinity Bio-Inert Quaternary LC System (Agilent
Technologies, TX, USA) with a refractive index detector (RID) at 40 °C, using a Hi-plex Ca column
(250 × 4.6 mm, Agilent Technologies, Waldbronn, Germany) operated at 65 °C. An aqueous solution of
H2SO4 (0.005 mol L-1) was used as the mobile phase at 0.6 mL min-1. The injection volume was 20 µL
and the analysis time was 30 min. Data acquisition and peak purity evaluation were done with the Chem
Station Open Lab LC® software.
The retention times of α-MetGlc (95% purity) and 6-OAc-α-MetGlc (90% purity) were 11.7 and 16.1
min, respectively. These purified compounds were used as standards for preparing calibration curves.
2.13. NMR analysis and data
NMR analyses were conducted at 400 MHz in a Bruker AV600 spectrometer (Bruker Co,
Massachusetts, USA). The purified substrate (α-MetGlc), prepared as described in section 2.3, and the
purified main product (6-OAc-α-MetGlc), prepared as described in section 2.10, were each dissolved in
500 μL CDCl3, for 1H, HSQC and HMBC analyses. The spectra were obtained using tetramethylsilane
(TMS) as an internal standard.
1H-NMR for α-MetGlc (400 MHz, D2O and acetone-d6): 3.35 (1H, dd, J1=10.00 Hz, J2=0.70 Hz, H-4),
3.37 (3H, s, OCH3), 3.52 (1H, dd, J1=3.80, J2=9.50 Hz, H-2), 3.60 (1H, ddd, J1=2.30 Hz, J2=5.50 Hz,
13

J3=10.00 H-5), 3.62 (1H, t, J1=9.50 Hz, J2=10.00 Hz, H-3), 3.71 (1H, dd, J1=5.50, J2=12.20, Hz, H-6'),
3.83 (1H, dd, J1=2.30, J2=12.20, Hz, H-6''), 4.76 (1H, d, J=3.80 Hz, H-1).
1H-NMR for 6-OAc-α-MetGlc (400 MHz, CDCl3): 2.11 (3H, s, CH3CO), 3.37 (1H, dd, J1=10.26 Hz,
J2=3.73 Hz, H-5), 3.43 (3H, s, OCH3), 3.55 (1H, dd, J1=2.69, J2=3.45 Hz, H-2), 3.73 (1H, dd, J1=10.26
Hz, J2=10.17 Hz, H-4), 3.75 (2H, d, J1=3.73 Hz, H-6), 3.75 (1H, dd, J1=10.17 Hz, J2=3.45 Hz, H-3),
4.77 (1H, d, J=2.69 Hz, H-1).
3. Results
3.1. Selection of the fermented solid
Three different substrate mixtures were used to produce DFS using B. contaminans. Each DFS was
analyzed for hydrolytic activity (using olive oil), esterification activity (ethyl oleate synthesis) and the
ability to acetylate α-MetGlc with vinyl acetate in a solvent-free system. The conversions were
calculated from the disappearance of α-MetGlc by HPLC.
DFS3 gave the highest hydrolytic activity (160 ± 9 U g-1), the highest esterification activity (7.5 ± 0.8 U
g-1) and also the highest conversion of α-MetGlc at 72 h (71%) (Table 1). Compared to DFS1, the
fermented solid that had been used previously in our laboratory, the hydrolytic and esterification
activities of DFS3 are 1.5-fold and 1.25-fold higher, respectively [12].
3.2. Purification of the acetylation product
14

The three fermented solids (DFS1, DFS2, DFS3) were used to catalyze the acetylation of α-MetGlc in a
solvent-free medium. After 72 h, the reaction medium was removed by filtration and analyzed by TLC.
In all three reactions, two main compounds appeared, with Rf values of 0.3 and 0.5. The compound with
the Rf of 0.3 was more abundant and is referred to, hereafter, as “the main product”.
The main product obtained with DSF3 was purified by flash chromatography, using ethyl acetate/methanol
9:1 (v/v) as eluent, with the fractions collected during the process being monitored by TLC. After this first
step of purification, the main product was the major peak in the chromatogram, with 83% purity. The peak
was isolated and further purified using preparative-scale TLC, which gave a purity of 90% for the main
product. This purified main product was then used as a standard in the calibration curves for determination
of conversions in the remaining experiments.
After purification, the main product of the reaction catalyzed by DFS3 was identified, by NMR analysis,
as 6-OAc-α-MetGlc. This shows that the lipases in DFS3 have a high selectivity for esterifying the C-6
hydroxyl group of α-MetGlc (Fig. 1).
3.3. Effect of the reaction medium on the stability of the hydrolytic activity of DFS3
The stability of the hydrolytic activity of DFS3 was evaluated in the solvent-free system and in the
reaction mixtures indicated in section 2.8. The residual hydrolytic activity of DFS3 was not affected
significantly after 24 h of incubation in the solvent-free system. In the media with 1:1 vinyl acetate:DMF
and 1:0.9:0.1 vinyl acetate:[emim][MeSO3]:DMF, the hydrolytic activity decreased 47% and 71%,
respectively, after 24 h of incubation (Fig. 2).
15

3.4. Determination of the solubility of the substrate
The solubility of the substrate α-MetGlc in the reaction medium is a very important variable for
increasing the reaction rate: the higher the solubility, the greater the substrate concentration in the
medium in contact with the enzyme. Highly polar solvents are commonly used in the synthesis of sugar
esters because they dissolve carbohydrates well, but they have the disadvantage of increasing the rate of
denaturation of lipases [25, 26]. It is, therefore, necessary to find solvents that not only solubilize the
substrate but also protect the enzyme activity.
With this purpose, the solubility of α-MetGlc was determined in a reaction medium containing only the
acyl donor vinyl acetate (i.e. in the solvent-free medium), in mixtures of vinyl acetate with organic
solvents (DMF and acetone) and in a mixture of vinyl acetate with an ionic liquid ([emim][MeSO3]) and
DMF. The highest solubility of α-MetGlc was obtained with the mixtures vinyl acetate:DMF (7.23
mmol L-1) and vinyl acetate:[emim][MeSO3]:DMF (6.65 mmol L-1), which solubilized 92% and 84%,
respectively, of the initial mass of α-MetGlc (Table 2). As expected, the ability to solubilize α-MetGlc
increased linearly as the log P value of the medium decreased.
3.5. Effect of the reaction medium on the enzymatic synthesis of 6-OAc-α-MetGlc
DFS3 was used to study the acetylation of α-MetGlc in the presence of (i) pure vinyl acetate (i.e.
solvent-free medium), (ii) vinyl acetate:DMF (1:1) and (iii) vinyl acetate:[emim][MeSO3]:DMF
(1:0.9:0.1) (Table 3). After 4 h, the highest percentage conversion of α-MetGlc to 6-OAc-α-MetGlc was
obtained with vinyl acetate:DMF (45%). This high conversion is likely due to the high solubility of α-
16

MetGlc in this reaction medium (Table 2). At this time, no product was detected in the other two media.
However, after 72 h, the conversion was the highest (65%) in the solvent-free system. With vinyl
acetate:[emim][MeSO3]:DMF, there was still no product. Surprisingly, with vinyl acetate:DMF the
conversion decreased from 45% at 4 h to only 6% at 72 h. This decrease is probably due to further
acetylation of 6-OAc-α-MetGlc to produce another acetylated compound. It should be noted that, in the
experiment reported above in Table 1, DFS3 did produce a secondary product (see Fig. S1 and Fig. S2
Supplementary data).
3.6. Kinetics of acetylation in different reaction media
The two reaction media that produced the best results in Table 3 were chosen to follow the kinetics of
the acetylation of α-MetGlc by DFS3: the solvent-free medium (i.e. containing only vinyl acetate and α-
MetGlc) and α-MetGlc dissolved in vinyl acetate:DMF (Fig. 3). CALB was also used to catalyze the
reaction in solvent-free medium. In this medium, CALB gave 99% conversion in 48 h while DFS3 gave
65% conversion in 72 h. In the vinyl acetate:DMF medium, DFS gave a conversion of 45% at 4 h; after
this point, the conversion decreased to 6% at 72 h, confirming the results shown in Table 3 for this
reaction medium.
3.7. Increasing the scale of the enzymatic synthesis
The solvent-free system was chosen for the scale-up of the acetylation reaction, based on the good
conversion and stability of DFS3 in this medium obtained in the previous experiments. Those
experiments were done in vials containing 4 mL of reaction medium. In this set of experiments, a
volume of 1 L was used, with the ratio of the mass of DFS3 per volume of reaction medium being
17

maintained constant. This experiment gave a conversion of 76% after 72 h at 40°C (Fig. 4). From this
result, it is possible to calculate that 1.05 g of 6-OAc-α-MetGlc had been produced. This indicates that
the synthesis process optimized in this work may be easily scaled up with good yields.
4. Discussion
The current work represents the first time that a sugar ester (in this case, 6-OAc-α-MetGlc) has been
produced using a fermented solid containing lipases. It demonstrates not only that the synthesis is
possible at a small laboratory scale (4 mL), but also that it is possible to produce over 1 g of the product,
using a 1-L reaction medium. This represents the highest quantity of a sugar ester, in terms of moles,
reported in the literature to date (Table 4). The production of high amounts of 6-OAc-α-MetGlc with a
cheap biocatalyst in a solvent-free medium becomes even more important considering the high value of
commercial 6-OAc-α-MetGlc ($1300 g-1, Labsynth, Diadema, São Paulo, Brazil).
The main product obtained in this work was acetylated in the C-6 position. This preference is similar to
that of Novozym 435 (Table 4) [25, 27]. It is probable that the regioselectivity for C-6 is favored by the
lower steric hindrance of the C-6 position, in comparison to the other hydroxyl-groups in the glucose
molecule, given that the C-6 hydroxyl-group is primary [25-28]. Nevertheless, other factors also
influence the regioselectivity of lipases, such as the size, hydrophobicity and configuration of the
anomeric substituents in the carbohydrate and the nature of the organic solvent [30].
One of the major challenges in acylation of carbohydrates is to find a reaction medium that not only
maintains enzyme activity but also dissolves both the carbohydrate (which is polar) and acyl donor
substrates (which have moderate polarity). Sugar acylation reactions have traditionally been done in
18

solvents such as DMF or pyridine, but these solvents are toxic and therefore not particularly convenient
to use [31]. Solvent-free media or environmentally friendly solvents, such as tertiary alcohols or ketones,
have been used, but they dissolve the sugar only partially [32]. In the current work, the substrate α-
MetGlc had higher solubility in solvents with negative log P values (Table 2). Nevertheless, the highest
conversion (72%) was achieved in the solvent-free medium, in which α-MetGlc had a very low
solubility. The other three media, which contained polar solvents or ionic liquids, had a more deleterious
effect on the lipase activity than did the solvent-free medium. The major component of the solvent-free
medium, vinyl acetate, has a log P value of 0.73 (Table 2) and is probably not able to remove the water
of the solvation shell of the protein; removal of this shell by polar solvents favors denaturation of
lipases. Also, since it is somewhat hydrophobic, vinyl acetate might interact with the hydrophobic amino
acid residues of the lid that covers the catalytic site of the lipase, preserving it in its open conformation
and thereby allowing more activity [33, 34].
DFS3 gave poor results in the presence of the ionic liquid [emim][MeSO3]. The physicochemical
properties of ionic liquids depend on the structures of both the cation and the anion, therefore, as with
organic solvents, different ionic liquids may either stabilize or destabilize the lipase [35]. Two results
illustrate this for Novozyme 435: [emim][BF4] gave good results when used as a reaction medium for
the esterification of glucose with vinyl acetate [26] but [emim][MeSO3] was the best for the acylation of
maltose with linoleic acid, compared to acetone and DMF [25]. Our own results were different: no
product was formed when [emim][MeSO3] was used in the reaction medium containing vinyl acetate
and α-MetGlc. Since DFS3 had low residual hydrolytic activity after incubation in this reaction medium,
it is probable that [emim][MeSO3] denatures the lipases present in DFS3. Since [emim][MeSO3] is more
polar than [emim][BF4], it could interact with the solvation layer of the enzyme more strongly,
denaturing it. Beyond this, the methane sulfate ion in [emim][MeSO3] is nucleophilic; it can interact
19

more strongly with positively charged amino acids of the protein, favoring deactivation [35]. These
results show that each new biocatalyst requires a new study to choose the most appropriate medium for
sugar ester synthesis.
5. Conclusions
The current work presents a method for the synthesis of 6-OAc-α-MetGlc that uses dry fermented solids
with naturally immobilized lipases produced by B. contaminans. Using a solvent-free medium
containing vinyl acetate and the substrate α-MetGlc, the conversion was 76% in 72 h, with a calculated
yield of 1.05 g of the product 6-OAc-α-MetGlc. The system has two features that have the potential to
reduce the costs of 6-OAc-α-MetGlc production significantly. Firstly, the biocatalyst can be produced
cheaply by solid-state fermentation followed by a simple air drying. Secondly, the use of a solvent-free
system for the regioselective acetylation of α-MetGlc is not only less expensive in itself, but also
simplifies the downstream processing and purification steps compared to systems that use expensive
reaction media, such as ionic liquids, and solvents that are toxic, such as pyridine, dimethyl sulfoxide or
DMF. These characteristics, together with the regioselectivity of the biocatalyst and the high value of the
product, render the process economically attractive.
Acknowledgements
This research was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e
Tecnológico, Grant No: 441015/2016-6), a Brazilian government agency for the advancement of science
and technology. Research scholarships were granted to David Mitchell and Nadia Krieger by CNPq, and
to Maribel Cordero by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), a
20

Brazilian government agency for the development of personnel in higher education, in conjunction with
the Organization of American States (OAS) and the Coimbra Group of Brazilian Universities (CGBU).
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FIGURE LEGENDS
Fig. 1. Regioselective acetylation of methyl-α-D-glucopyranoside with vinyl acetate catalyzed by
fermented solid containing lipases of Burkholderia contaminans.
Fig. 2. Stability of the hydrolytic activity of the fermented solid (DFS3) after incubation in different
reaction media. Key: () α-MetGlc plus vinyl acetate (solvent-free); (●) α-MetGlc plus a 1:1 (v/v)
mixture of vinyl acetate and DMF; () α-MetGlc plus a 1:0.9:0.1 (v/v/v) mixture of vinyl acetate,
[emim][MeSO3] and DMF. Conditions: 4.5 mg of α-MetGlc (purity 95%), 200 mg of dry DFS3 in 4 mL
of reaction medium, incubated at 40 °C and 250 rpm for 72 h. Activities were determined relative to an
25

initial activity of 160 U g-1 measured by the titrimetric method, using olive oil as the substrate. Values
plotted represent the mean of triplicate experiments ± the standard error of the mean. When the error
bars do not appear, they are smaller than the symbols.
Fig. 3. Effect of reaction medium on the acetylation of methyl-α-D-glucopyranoside with vinyl acetate
catalyzed by different lipases. Key: (●) DFS3 in solvent-free systema; (○) DFS3 in DMF:vinyl acetateb;
() CALB in solvent-free systemc. The reactions contained 4.5 mg of α-MetGlc (0.02 mmol, purity
95%) and: a200 mg of dry DFS3 in 4 mL of vinyl acetate; b200 mg of dry DFS3 in 4 mL of 1:1 (v/v)
vinyl acetate:DMF and c2.27 mg of CALB in 4 mL of vinyl acetate. All reactions were incubated at 40
°C and 250 rpm for 72 h. Conversions were determined by HPLC. Values plotted represent the mean of
triplicate experiments ± the standard error of the mean. When the error bars do not appear, they are
smaller than the symbol.
Fig. 4. Kinetics of acetylation of methyl-α-D-glucopyranoside with vinyl acetate using DFS3 in the
solvent-free system in different volumes of reaction medium. Key: () 1-L of reaction medium for 12
days; (●) 20 mL of reaction medium for 72 h; (×) 4 mL of reaction medium for 72 h. Reaction
conditions: 40 °C and 250 rpm. Conversions determined by HPLC. Values plotted represent the mean of
triplicate experiments ± the standard error of the mean. When the error bars do not appear, they are
smaller than the symbol.
26

27

28

Table 1
Effect of the culture medium on the production of lipases by Burkholderia contaminans LTEB11 in
solid-state fermentation.
Hydrolytic activity
(U g-1)
Esterification activity
(U g-1)
Conversion of
Dry Fermented Solida
α-MetGlc (%)
DFS1
DFS2
DFS3
110 ± 8
6.5 ± 0.4
59
66
71
128 ± 10
4.6 ± 1.1
160 ± 9
7.5 ± 0.8
aDFS1: Sugarcane bagasse & sunflower seeds, 1:1, w/w; moistened with phosphate buffer solution. DFS2: Sugarcane bagasse
& sunflower seeds, 1:1, w/w. DFS3: Sugarcane bagasse with 20% (w/w) of soybean oil. DFS2 and DFS3 moistened with
micronutrient solution. Values reported represent the mean of triplicate experiments ± the standard error of the mean.
Table 2
Solubilities of methyl-α-D-glucopyranoside in different reaction systems.
Solubility of α-MetGlc
Reaction medium
log P of solvent mixture
(mmol L-1)c
Vinyl acetate
0.15
2.42
7.23
6.65
0.73
0.25
Vinyl acetate:Acetonea
Vinyl acetate:DMFa
-0.15
-0.14
Vinyl acetate:[emim][MeSO3]:DMFb
a10 mL of vinyl acetate and 10 mL of each solvent.
b10 mL of vinyl acetate, 9 mL [emim][MeSO3] and 1 mL DMF.
29