A novel strategy to improve the aromatic alcohols tolerance of enzyme for preparative-scale synthesis of natural glycosides

Article abstract of DOI:10.1016/j.catcom.2017.07.025

Aromatic alcohols are liable to result in enzyme inactivation. A novel strategy was established for improving the aromatic alcohol tolerance of β-galactosidase (BMG) from Bacillus megaterium YZ08. The half-life of BMG in 200 mM vanillyl alcohol solution was dramatically increased by 9–123 times with the addition of hydrophilic solvents. In 30% DMSO, the reaction concentration of aromatic alcohol could reach up to 300–400 mM, and 0.85–2.44 g of natural glycosides were successfully obtained in 100 mL-scale. The simple and effective strategy shows potential applications when dealing with the preparative-scale exploitation of enzymatic reactions.

Full text of DOI:10.1016/j.catcom.2017.07.025

Accepted Manuscript
A novel strategy to improve the aromatic alcohols tolerance of
enzyme for preparative-scale synthesis of natural glycosides
Youzhi Zhou, Lu Zhao, Ke Liu, Jinsong Zhang, Jianlin Chu,
Bingfang He
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DOI:
Reference:
S1566-7367(17)30327-8
doi: 10.1016/j.catcom.2017.07.025
CATCOM 5144
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25 July 2017
27 July 2017
Please cite this article as: Youzhi Zhou, Lu Zhao, Ke Liu, Jinsong Zhang, Jianlin Chu,
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ACCEPTED MANUSCRIPT
A novel strategy to improve the aromatic alcohols tolerance of enzyme for
preparative-scale synthesis of natural glycosides
Youzhi Zhoua, Lu Zhaoa, Ke Liua, Jinsong Zhanga, Jianlin Chub, Bingfang Hea,b,*
a. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech
University, China.
b. School of Pharmaceutical Sciences, Nanjing Tech University, China.
* Corresponding author. Tel/Fax: 86-25-58139902.
E-mail: bingfanghe@njtech.edu.cn

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Abstract
Aromatic alcohols are liable to result in enzyme inactivation. A novel strategy
was established for improving the aromatic alcohol tolerance of β-galactosidase
(BMG) from Bacillus megaterium YZ08. The half-life of BMG in 200 mM vanillyl
alcohol solution was dramatically increased by 9–123 times with the addition of
hydrophilic solvents. In 30% DMSO, the reaction concentration of aromatic alcohol
could reach up to 300–400 mM, and 0.85–2.44 g of natural glycosides were
successfully obtained in 100 mL-scale. The simple and effective strategy shows
potential applications when dealing with the preparative-scale exploitation of
enzymatic reactions.
Keywords: Enzyme inactivation; Substrate tolerance; Hydrophilic solvent;
Preparative-scale; Natural glycoside

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1. Introduction
Aromatic alcohols are aglycones of a series of important natural glycosides [1].
They have attracted the attention of food and pharmaceutical industries first of all for
their well-acquainted antioxidant activity [2]. Not only that, they also possess
anti-angiogenic, anti-inflammatory and anti-nociceptive activities [3]. Glycosylation
can improve aromatic alcohols’ pharmaceutical and pharmacokinetic properties, such
as selectivity, bioavailability, water solubility and oxidative stability [4]. More
importantly, some important natural glycosides and their derivatives can be easily
synthesized by the glycosylation of aromatic alcohols. Salidroside [5] and gastrodin [6]
can be efficiently synthesized by using tyrosol and 4-hydroxybenzyl alcohol as
substrates, respectively. For the glycosylation of these compounds, conventional
chemical glycosylation is difficult to implement due to the insurmountable drawbacks
in the multiple steps of protection/deprotection to control regio- and stereoselectivity.
Exquisite selectivity is the hallmark of enzymatic glycosylation. However, majority of
aromatic alcohols significantly inhibit enzyme activity, and the reactant concentration
is generally restricted to relatively low concentrations [7-9].
Several strategies have been reported for reducing the adverse effects of aromatic
alcohol and its derivative on enzymes. One such strategy is adsorbing substrates
through the use of silica gel or cyclodextrins [10, 11]. Bridiau et al. used silica gel to
adsorb aromatic primary alcohols under positive argon pressure away from light; the
substrate concentration reached 250 mM [10]. Cyclodextrins successfully limited the
substrate inhibition of carboxypeptidase A in catalyzing the hydrolysis of

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(S)-2-O-(N-benzoylglycyl)-β-phenyllactate) [11]. Both of these methods relieved the
effect of the substrate on the enzyme by reducing the concentration of substrate free in
solution. This suggests that the catalytic efficiency did not enhance with the increase
in the total concentration of the substrate. A potential alternative is the water-organic
biphasic system [12, 13], in which the substrate is mainly retained in organic phase.
Consequently, the substrate concentration is regulated and minimized around the
enzyme [13]. Nevertheless, the mass-transfer limitation in the case of biphasic system
is a major challenge for glycosylation reaction, because the hydrophilic sugar donor
and the hydrophobic acceptor are in different phases [14]. By contrast, enzymatic
glycosylation in hydrophilic organic solvents presents no mass-transfer limitation and
provides numerous industrially attractive advantages, such as increased solubility of
hydrophobic substrates and reversal of the thermodynamic equilibrium of hydrolysis
reactions [15-17].
In this study, a novel strategy was successfully established for reducing the
inactivation of β-galactosidase (BMG) from Bacillus megatheriumYZ08 caused by
aromatic alcohols. The addition of hydrophilic solvents remarkably enhanced the
stability of BMG in aromatic alcohol solution. To the best of our knowledge, this
paper is the first report on improving the substrate tolerance of enzymes by simply
adding hydrophilic solvents.
2. Experimental
2.1. Materials
2-Nitrophenyl-β-D-galactopyranoside, vanillyl alcohol and other aromatic

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alcohols were purchased from Aladdin (Shanghai, China). One Step Cloning Kit was
purchased from Novoprotein (Shanghai, China). Organic solvents were purchased
from Sinopharm (Shanghai, China).
2.2. Enzyme preparation and activity assay
The preparation of purified BMG and the determination of BMG activity were
conducted as described previously [18].
2.3. Effect of vanillyl alcohol on BMG stability in different systems
The effect of different concentrations of vanillyl alcohol on the activity and
stability of purified BMG was investigated in Na2HPO4/KH2PO4 (50 mM, pH 7.4)
buffer containing 5 mM Mg2+. The stabilizing effect of hydrophilic solvents in 200
mM vanillyl alcohol solution was then detected. The samples were incubated for
appropriate periods and the residual activities were determined as described above.
2.4. Kinetic analysis of galactosylation of vanillyl alcohol catalyzed by purified BMG
The kinetic parameters were determined in the reaction mixture containing
vanillyl alcohol as the substrate and 500 mM lactose in Na2HPO4/KH2PO4 buffer (50
mM, pH 7.4) containing 5 mM MgCl2. The substrate concentration was in the range
of 0.5–5 mM. Purified BMG (final concentration 12.15 μg/mL) was added, and the
reaction was incubated at 35 °C for 10 min, or 20 min. The reaction was stopped by
adding 900 μL methanol to 100 μL reaction solution. The reaction products were
analyzed by HPLC.
2.5. HPLC analysis
The mixture of enzymatic galactosylation was analyzed by RPHPLC on an

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Agilent TC-C18 column (4.6 mm×250 mm, 5 mm) with a flow rate of 0.9 mL/min.
The elution is detailed in supplementary material.
2.6. Preparative-scale synthesis, purification and structure determination of the
products
The reaction mixture (70 mL Na2PO4/KH2PO4 buffer (50 mM, pH 7.4) + 30 mL
DMSO) containing MgCl2 (5 mM), lactose (50 mmol), aromatic alcohols (30–40
mmol), crude enzyme solution (100 U) was prepared in a 250 mL Erlenmeyer flask.
The reaction was conducted at 35 °C with shaking at 200 rpm for 12 h. AB-8
macroporous resin column and flash column chromatography were used to purify the
products. The procedure is detailed in supplementary material. The product structure
was determined by NMR (BrukerAV-400 spectrometer, Switzerland).
3. Results and Discussion
3.1. Effect of aromatic alcohols on BMG
Many aromatic alcohols severely affect the activity of enzymes, including BMG.
The effect of aromatic alcohol on the BMG was analyzed using vanillyl alcohol as the
model substrate. The half-lives of BMG in vanillyl alcohol solution added at different
concentrations were measured (Table 1). As the concentration of vanillyl alcohol was
increased, the half-life of BMG was increasingly shortened. When the concentration
of vanillyl alcohol was only 25 mM, the half-life of BMG was less than 1 hour, which
was only 2.31% of that without addition of vanillyl alcohol. These results indicated
that BMG was unstable in vanillyl alcohol aqueous solution presumably because of
the powerful capability of vanillyl alcohol to seize essential water molecules from

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BMG. Moreover, after 1 h of incubation, a small amount of white precipitate was
observed in the mixture of BMG and 50–200 mM vanillyl alcohol, further reinforcing
BMG inactivation.
3.2. Hydrophilic solvent for improving aromatic alcohol tolerance of BMG
To date, hydrophilic solvents have been rarely used to reduce the effect of
substrates on enzymes. The reason is that hydrophilic solvents are generally
considered toxic to most enzymes because of the powerful capability of such solvents
to seize essential water molecules from protein [19, 20]. Fortunately, BMG is stable in
various 10–30% (v/v) hydrophilic solvents [21]. In addition, BMG is more stable in
aqueous hydrophilic solvent than in aqueous solution [18]. On this basis, the half-lives
of BMG in hydrophilic solvents with different log P values at 10% (v/v)
concentration containing 200 mM vanillyl alcohol were tested (Table 2). There is no
apparent association between stabilizing effect and the log P values of solvents. The
half-lives were markedly improved in all tested aqueous hydrophilic solvents except
for 10% acetonitrile. The ideal results were obtained in 10% DMSO and methyl
alcohol, and the half-life of BMG was increased by 98 and 123 times in the two
solvent systems, respectively. These may be because that the addition of DMSO and
methyl alcohol could significantly enhance the rigidity of BMG, but BMG was
unstable in 10% acetonitrile.
To further elucidate how hydrophilic solvents can protect BMG from destruction
by vanillyl alcohol molecules, the kinetic parameters were determined by using
purified BMG in different solvent systems (Table 3). The vanillyl alcohol

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concentration used to measure the kinetic parameters was kept within 5 mM to
minimize BMG denaturation. Compared with that in the aqueous solution, the Κm
values of purified BMG to vanillyl alcohol in aqueous hydrophilic solvents obviously
increased, demonstrating the decreased affinity of BMG for vanillyl alcohol in
aqueous hydrophilic solvents. The changes in affinity to the substrate suggested that
the addition of hydrophilic solvents reduced the interaction between BMG and
vanillyl alcohol. The κcat values in 10% methanol, ethanol, DMF and acetone were
slightly lower than that in aqueous solution, although BMG was stable in these
solvent systems, which may be because the enhanced rigidity of BMG resulted in the
loss of activity. Surprisingly, κcat value in 10% DMSO was significantly enhanced,
indicating that the addition of DMSO enhanced the activity of BMG. The values of
kinetic parameters were different from solvent to solvent, presumably because the
addition of different solvents induced different conformational changes in the
catalytic active center of BMG. Therefore, the addition of hydrophilic solvents not
only enhanced the rigidity of BMG but also reduced the interaction between BMG
and vanillyl alcohol, and only in 10% DMSO, the enhanced stability of BMG was not
at the cost of enzyme activity.
3.3. Preparative-scale synthesis of natural glycosides at high concentrations
With simple and convenient addition of hydrophilic solvents, the inactivation of
BMG due to vanillyl alcohol could be relieved effectively. Fig. 1 showed the effects
of DMSO and methanol on the conversion at different concentrations of vanillyl
alcohol. As expected, in 10% DMSO and methanol, with the increase of vanillyl

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alcohol concentration, the decreasing trend of conversion became slower compared
with that in aqueous solution. When the concentration of vanillyl alcohol was
increased to 300 mM, the conversion in 10% DMSO and methanol only decreased by
22% and 37%, respectively, while the conversion in aqueous solution dropped by
more than 90%. It is worth noting that the maximum conversion in 10% methanol was
about half of that in aqueous solution and 10% DMSO, which probably caused by the
slight conformational changes in the catalytic active center of BMG in 10% methanol.
Pioneering studies in this field have proven that organic solvents could alter
conformation of enzyme active site [22]. There are several cases in which various
types of enzyme selectivity have been profoundly changed by switching from one
solvent to another, including substrate, enantiomeric, prochiral, regio- and
chemoselectivities [23, 24].
With the further increase of DMSO concentration, the maximum conversion
increased slightly. More importantly, the decreasing trend of conversion became
slower further. In 30% DMSO, about 40% conversion was successfully obtained at
vanillyl alcohol concentration of 400 mM, while the reaction almost could not be
carried out at the same concentration of vanillyl alcohol in aqueous solution.
BMG successfully synthesized a series of important natural glycosides from
vanillyl
alcohol,
4-hydroxybenzyl
alcohol,
3-hydroxybenzyl
alcohol,
2-hydroxybenzyl alcohol, 3-phenyl-2-propene-1-ol, 4-methoxybenzyl alcohol and
4-methoxyphenethyl alcohol, except phenol (Table 4), indicating that BMG is
selective of the alcohol hydroxyl groups. In aqueous solution, when the substrate

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concentration was 10 mM, BMG could effectively catalyze the galactosylation of
most substrates. Under the same substrate concentration, slightly higher conversions
were obtained in 30% DMSO because of the change in the thermodynamic
equilibrium of hydrolysis reaction. As the concentration of aromatic alcohols
increased, the BMG inactivation in aqueous solution intensified. When the substrate
concentration was increased by 30–40 times, all galactosylation reactions in the
aqueous solution became inefficient, although most substrates were completely
dissolved in aqueous solution. By contrast, BMG still effectively catalyzed
galactosylation in 30% DMSO under the same concentration, and 0.85–2.44 g of
natural glycosides were successfully obtained. Bridiau et al. [10] used silica gel to
adsorb aromatic primary alcohols, and the substrate concentration reached 250 mM.
However, adsorption was conducted under positive argon pressure and away from
light. The proposed strategy for improving the aromatic alcohol tolerance of enzymes
by adding hydrophilic solvent is a simple yet effective approach that is feasible
practical applications.
4. Conclusions
BMG inactivation caused by aromatic alcohols was successfully relieved by
adding hydrophilic solvents. The stability of BMG in aromatic alcohol solution was
remarkably enhanced by the simple addition of hydrophilic solvents. The conspicuous
stabilization of BMG in aromatic alcohol solution is beneficial to preparative-scale
synthesis of natural glycoside from aromatic alcohol. In 30% DMSO, the
concentration of aromatic alcohol reached up to 300–400 mM, and 0.85–2.44 g of

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natural glycosides were successfully obtained in 100 mL-scale. Therefore, this
method provides a new idea for relieving the inactivation of enzymes caused by
aromatic alcohols.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(81673321, 21376119, 21506099).
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Figure legends
Fig. 1. Effect of hydrophilic solvent on galactosylation of vanillyl alcohol catalyzed
by BMG. ■, aqueous system; ●, 10% methanol; ▲, 10% DMSO; □, 20% DMSO; ○,
30% DMSO.

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Table 1 Stability of BMG in the presence of vanillyl alcohol in aqueous solution
Vanillyl alcohol concentration (mM)
Half-life (h)
35.9 ± 2.2
6.2 ± 0.3
0
10
25
0.80 ± 0.05
0.43 ± 0.03
0.33 ± 0.03
0.30 ± 0.03
0.22 ± 0.03
0.18 ± 0.02
50
75
100
150
200

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Table 2 Stability of BMG with addition of 200 mM vanillyl alcohol in different
solvent systems
System
log P
Half-life (h)
Without vanillyl alcohol
35.9 ± 2.2
With 200 mM vanillyl alcohol
0.18 ± 0.02
18.7 ± 1.5
Aqueous solution
10% DMSO
-
-1.49
-0.6
-0.27
0.07
0.2
148.9 ± 8.7
66.2 ± 2.8
10% DMF
2.9 ± 0.2
10% Methyl alcohol
10% Ethyl alcohol
10% Acetone
168.3 ± 11.7
152.4 ± 9.1
14 ± 1.1
23.4 ± 1.7
5.6 ± 0.4
1.8 ± 0.1
10% Acetonitrile
0.17
0.93 ± 0.08
0.33 ± 0.03

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Table 3 Effects of media on kinetic parameters for galactosylation of vanillyl alcohol
catalyzed by BMG
Solvent
Κm (mM)
38.1 ± 1.9
87.9 ± 4.2
78.6 ± 6.9
66.7 ± 5.2
72.1 ± 6.3
79.4 ± 7.2
κcat (s-1)
κcat/Κm (s-1 mM-1)
0.29 ± 0.03
0.26 ± 0.02
0.13 ± 0.01
0.12 ± 0.01
0.13 ± 0.01
0.11 ± 0.01
Aqueous solution
10% DMSO
11.1 ± 0.5
23.2 ± 0.8
10.2 ± 0.8
8.3 ± 0.8
9.2 ± 0.8
8.6 ± 0.5
10% DMF
10% Acetone
10% Methyl alcohol
10% Ethyl alcohol

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Table 4 Enzymatic galactosylation of aromatic compounds in aqueous solution and 30%
DMSOa
Substrate
Substrate
concentration
(mM)
Conversions (%)b
Aqueous solution
30 % DMSO system
0
10
0
Phenol
10
44.7 ± 3.9
2.2 ± 0.2
44.2 ± 1.9
3.9 ± 0.6
43.2 ± 3.9
47.2 ± 2.1
39.6 ± 1.7
46.3 ± 2.7
38.8 ± 1.1
45.3 ± 2.9
400
10
Vanillyl alcohol
4-Hydroxybenzyl
alcohol
400
10
400
3.8 ± 0.3
37.1 ± 2.4
3-Hydroxybenzyl
alcohol
10
45.3 ± 1.9
4.7 ± 0.3
43.9 ± 3.3
3.3 ± 0.4
38.1 ± 2.1
2.2 ± 0.3
1.3 ± 0.1
0
48.3 ± 3.6
42.8 ± 2.9
45.5 ± 2.8
37.7 ± 2.9
40.8 ± 3.2
32.2 ± 2.1
30.1 ± 2.1
22.1 ± 1.8
2-Hydroxybenzyl
alcohol
400
10
4-Methoxybenzyl
alcohol
400
10
4-Methoxyphenethyl
alcohol
300
10
3-Phenyl-2-propene-
1-ol
300
aCinnamyl alcohol was insoluble in aqueous solution, but soluble in 30% DMSO, and
the other aromatic alcohols were soluble in aqueous solution and 30% DMSO.
bConversions were calculated by the ratio of the galactosylated aromatic alcohol

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concentration to the initial aromatic alcohol concentration.

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Figure 1

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Graphical abstract

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Highlight
 A novel strategy was established to improve substrate tolerance of
β-galactosidase.
 Hydrophilic solvent was employed to increase the stability of BMG in vanillyl
alcohol solution.
 0.85–2.44 g of natural glycosides were successfully obtained in 100 mL-scale.