H. Cheng et al.
ProcessBiochemistryxxx(xxxx)xxx–xxx
chemical processes, so far very few studies have been presented re-
garding enzymatic synthesis of polyphenolic compounds. Our study is
the first one to demonstrate the use of tyrosinase CLEAs as a new bio-
catalyst for the synthesis of the above 3 polyphenol products, while no
reports in literature have been given yet about biocatalytic production
of 3′-hydroxypterostilbene.
Meanwhile, stimulated by our recent findings that deep eutectic
solvents (DESs) can viably activate and stabilize enzymes when added
as additives in the reaction system [25,26], we surmised that introdu-
cing a DES into CLEAs might trigger the immobilized enzyme more
active or more stable. Here a DES is a new type of ‘green’ nonaqueous
solvent that can be easily prepared by mixing an ammonium salt (such
as choline chloride) with a hydrogen-bond donor (HBD, such as urea) at
a specified molar ratio (for a review see [27]). It has currently attracted
widespread academic and industrial interests with a broad range of
applications. Therefore this study was also attempted to testify whether
incorporating DES into tyrosinase CLEAs during their preparation
would exert positive effects on the catalytic performance of the im-
mobilized enzyme and on the synthesis of the polyphenol products.
2.4. Activity assays for tyrosinase CLEAs
The activity of tyrosinase CLEAs in aqueous solution was de-
termined spectrophotometrically by following the oxidation of L-DOPA
to dopachrome [23,28]. Typically, 7.5 mg CLEAs were added to
20.0 mL Na phosphate buffer (50 mM, pH 6.0) containing 0.8 mM L-
DOPA in a capped test tube to start the reaction, which was carried out
in a shaking incubator at 25 °C with agitation of 200 rpm. All tests
throughout this study were triplicated with standard deviations not
greater than 5%.
2.5. Reaction to produce 3,4-dihydroxyphenylacetic acid (DHPAA) from 4-
hydroxyphenylacetic acid (HPAA)
A typical reaction was started by addition of 15.0 mg of tyrosinase
CLEAs into 20.0 mL of Na phosphate buffer (50 mM, pH 6.0) containing
10.0 mM HPAA and 15.0 mM L-ascorbic acid in a 50 mL conical flask,
which was placed in a shaking incubator with agitation of 200 rpm at
25 °C. Periodically, a 0.15 mL sample was taken which was then four
times diluted with the buffer before subjected to HPLC analysis as de-
scribed below.
2. Materials and methods
2.6. Reaction to produce piceatannol (Pic) from resveratrol (Res)
2.1. Materials
The reaction was normally carried out in 2.0 mL phosphate buffer
(50 mM, pH 6.0) containing 20.0 mM Res, 40.0 mM L-ascorbic acid and
25% (v/v) DMSO (for pre-dissolving Res) in a shaking incubator with
agitation of 220 rpm at 30 °C. 10.0 mg of tyrosinase CLEAs was added to
start the reaction, and every one hour a 0.1 mL sample was taken,
which was then 20 times diluted with 50% (v/v) acetonitrile aqueous
solution before being subjected to HPLC analysis as described below.
Fresh mushrooms were obtained from a local supermarket in
Shenzhen, China. 3,4-Dihydroxyl-phenylalanine (L-DOPA) was pur-
chased from Sigma–Aldrich China Inc. 4-Hydroxyphenylacetic acid
(HPAA), 3,4-dihydroxyphenylacetic acid (DHPAA), resveratrol (Res)
and piceatannol (Pic) were of all HPLC grade from Tokyo Chemical
Industry Co., Ltd. Pterostilbene (PS) and 3′-hydroxypterostilbene (HPS)
were both kindly provided by School of Pharmaceutical Sciences, Sun
Yat-Sen University, China. Choline acetate (ChAc, 99%) was purchased
from ShangHai Cheng Jie Chemical Co. Ltd. Choline chloride (ChCl)
and all other reagents used were of analytical grade from local manu-
facturers in China.
2.7. Reaction to produce 3′-hydroxypterostilbene (HPS) from pterostilbene
(PS)
A substrate solution containing 20.0 mM PS, 40.0 mM L-ascorbic
acid and 50% (v/v) DMSO (for pre-dissolving PS) was prepared in
phosphate buffer (50 mM, pH 6.0). The reaction was conducted by
adding 10.0 mg CLEAs to 2.0 mL of the substrate solution, which was
placed in a capped test tube in a shaking incubator with agitation of
220 rpm at 25 °C. At intervals, a 20 μL sample was taken and 20 times
diluted with 50% (v/v) acetonitrile aqueous solution before being
subjected to HPLC analysis as described below.
2.2. DES preparation
A DES was prepared by mixing an ammonium salt (ChCl or ChAc)
and a hydrogen-bond donor (urea, glycerol, acetamide, or ethylene
glycol, respectively abbreviated as U, G, A or EG) at a molar ratio of 1:2,
1:1 or 2:1, following the procedures described in [25,26]. The con-
centration of a DES in aqueous solution refers to the molar concentra-
tion of the cholinium salt involved in the DES. It is worth noting that
the term of DES is sometimes misunderstood. Strictly speaking, mixing
the two components at a certain molar ratio may result in a room
temperature liquid but does not necessarily make the system DEEP
EUTECTIC, while only the composition leading to the LOWEST melting
or freezing point is called DEEP eutectic. Here in this paper, following
the general use in other literature, the term DES is still used referring to
those ammonium salt-hydrogen bond donor mixtures which are liquid
under ambient temperature.
2.8. HPLC analysis
HPLC analysis of the substrates and products of the above three
synthetic reactions was performed on a Shimadzu LC-20AT HPLC
system equipped with an SPD-20A UV/Vis detector and
a
150 × 4.6 mm, 5 μm inertsil ODS-SP column (GL Sciences Inc. Japan).
The detailed conditions used for HPLC analysis of the three reactions
are listed in Table S1.
3. Results and discussion
3.1. Confirmation of the products produced from the three enzymatic ortho-
2.3. Preparation of tyrosinase CLEAs with and without DES incorporation
hydroxylation reactions
Tyrosinase CLEAs were prepared from fresh mushrooms by pre-
cipitating the enzyme with ammonium sulfate and subsequent cross-
linking with glutaraldehyde, following the procedures described in
[23]. Typically, the enzyme solution (40 mL) was mixed with 10 mL of
sodium phosphate buffer (50 mM, pH 6.0, in the presence or absence of
0.5 M of a DES), to which ammonium sulfate (to reach a final 50%
saturation) and 1.0 mL of 25 wt% glutaraldehyde were successively
added.
The three synthetic reactions were monitored by HPLC analysis. As
shown in Fig. 1, samples removed from the reaction mixtures presented
not only the peaks for the substrates (HPAA, Res and PS) but also new
ones that match the authentic products (DHPAA, Pic and HPS) re-
spectively, and these product peaks kept rising as the reactions went on,
thus confirming the formation of the products. No peaks for L-ascorbic
acid were observed in Fig. 1(B and C) simply because this reducing
agent does not have absorbance at the detection wavelengths of 320 nm
3