Synthesis of lutein esters by using a reusable lipase-pluronic conjugate as the catalyst

Article abstract of DOI:10.1007/s10562-015-1597-1

An enzymatic route to synthesize lutein esters was investigated using a lipase-Pluronic conjugate as the catalyst. For the synthesis of lutein palmitate, the enzymatic process gave a conversion of 85 % for lutein and a product purity of 96 % at 50 °C within 12 h, whereas the free lipase catalyzed process achieved a conversion below 10 %. To demonstrate the generality of this enzymatic process, lutein laurate, lutein myristate and lutein stearate were also synthesized achieving conversions of 73.8, 88.5, 84.4 % respectively. Moreover, the lipase-Pluronic conjugate can be reused for batches in the synthesis of lutein esters. As demonstrated by this study, the enzyme-Pluronic conjugate holds great promise for practical applications in industrial biocatalysis. Graphical Abstract An enzymatic approach to synthesize lutein esters was investigated using a lipase-Pluronic conjugate as the catalyst.

Full text of DOI:10.1007/s10562-015-1597-1

Catal Lett
DOI 10.1007/s10562-015-1597-1
Synthesis of Lutein Esters by Using a Reusable Lipase-Pluronic
Conjugate as the Catalyst
Miao Hou1 Rui Wang1 Xiaoling Wu1 Yifei Zhang1 Jun Ge1 Zheng Liu1
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•
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Received: 23 June 2015 / Accepted: 16 July 2015
Ó Springer Science+Business Media New York 2015
Abstract An enzymatic route to synthesize lutein esters was
investigated using a lipase-Pluronic conjugate as the catalyst.
For the synthesis of lutein palmitate, the enzymatic process
gave a conversion of 85 % for lutein and a product purity of
96 % at 50 °C within 12 h, whereas the free lipase catalyzed
process achieved a conversion below 10 %. To demonstrate
the generality of this enzymatic process, lutein laurate, lutein
myristate and lutein stearate were also synthesized achieving
conversions of 73.8, 88.5, 84.4 % respectively. Moreover, the
lipase-Pluronic conjugate can be reused for batches in the
synthesis of lutein esters. As demonstrated by this study, the
enzyme-Pluronic conjugate holds great promise for practical
applications in industrial biocatalysis.
Keywords Enzymatic catalysis Á Green chemistry Á
Nanostructure
1 Introduction
Lutein esters are widely applied as food colorant to poultry
feed and are recognized as essential human dietary sup-
plements because they can inhibit age-related macular
degeneration (AMD), protect against oxidative and blue-
light damage in the retina, and are responsible for central
and high acuity vision [1–4]. The lutein esters have higher
bioavailability for administration and higher stability for
storage than free lutein molecules [5]. Industrial production
of lutein esters are suffered from limitations including
excess chemical wastes, high energy consumption and
laborious purification steps.
Graphical Abstract An enzymatic approach to synthesize
lutein esters was investigated using a lipase-Pluronic con-
jugate as the catalyst.
The esterification catalyzed by lipase offers an oppor-
tunity to overcome the above-mentioned problems. How-
ever, the lipase-catalyzed esterification needs to be carried
out in organic solvents in which most enzymes display
much lower activities than in aqueous solution mainly due
to the formation of agglomeration of the insoluble enzyme
molecules in organic solvents making enzyme less acces-
sible to substrates.
Nanostructured enzyme catalysts [6–9] such as
enzyme nanoparticles [10, 11], enzyme nanogels [12–17],
flower-like enzyme-inorganic hybrid crystals [18–21],
enzyme-metal-organic framework hybrid composites [22]
and enzyme–polymer conjugates [23–25] have been
demonstrated as effective chemical ways to re-engineer
enzyme catalysts for improved activity and stability. For
example, conjugation with an amphiphilic polymer such
as Pluronic can enhance the solubilization of enzyme in
organic solvents and therefore increase the apparent
& Jun Ge
junge@mail.tsinghua.edu.cn
1
Key Lab for Industrial Biocatalysis, Ministry of Education,
Department of Chemical Engineering, Tsinghua University,
Beijing, China
123

M. Hou et al.
enzymatic activity by orders of magnitudes, as compared
to its native counterpart. Taking the advantage of the
temperature responsiveness in organic media, the enzyme
conjugates can be readily recovered by a temperature-
induced precipitation.
dissolved in phosphate buffer (10 mM, pH 7.0), mixed
with CALB (5–10 mg/mL) in phosphate buffer solution
(the molar ratio of the aldehyde group to the amine group
of protein is 1.1:1). NaCNBH3 (10 % weight of Pluronic)
was then added to the mixture after 2 h reaction and
reacted for 15 h at room temperature to reduce the Schiff
base, followed by dialysis against phosphate buffer solu-
tion (10 mM, pH 7.0). Finally, the powder of CALB-
Pluronic conjugate was obtained by lyophilization.
The microstructure and morphology of the products
were investigated by scanning transmission electron
microscopy (TEM, JEOL JEM-2010) with an accelerating
voltage of 120 kV. The dry powder of the conjugate was
dissolved in toluene (10 lg/mL of protein concentration).
10 lL of this solution was placed on the carbon-coated
grid. After the toluene evaporated, 10 lL of sodium
phosphotungstate aqueous solution (1 %, pH 7.0) was
applied to stain the sample. The sample was dried for
24 h at room temperature before making TEM
measurements.
In this study, an enzymatic synthesis route for lutein
esters was investigated using a lipase-Pluronic conjugate as
the catalyst (Fig. 1). The conjugate was synthesized by
attaching lipase with a commercially available polymer
Pluronic F-127. In the experiment, Pluronic F-127 (POH)
was first oxidized with Dess–Martin periodinane, to con-
vert the hydroxyl end-groups into aldehyde functionalities.
Then the aldehyde-derived Pluronic (PCHO) was cova-
lently conjugated to the lysines of CALB by the formation
of a Schiff base, followed by a reduction with NaCNBH3 to
convert the imine bond to a stable secondary amine bond.
The conjugate was soluble in organic solvents at the
reaction temperature and displays a high apparent activity.
After reaction, the reaction mixture was cooled down to a
low temperature at which the enzyme conjugate was pre-
cipitated for reuse.
The hydrolytic activity of CALB and CALB-Pluronic
conjugate was determined using 4-nitrophenyl butyrate (p-
NPB) as the substrate. Briefly, p-NPB was first dissolved in
acetone and then diluted with phosphate buffer (10 mM,
pH 7.0) containing 1.25 % (w/v) Triton X-100, giving a
final concentration of 0.5 mM. The reaction was started by
adding 50 of enzyme solution (50 in 10 mM phosphate
buffer, pH 7.0) to 950 of substrate solution and the increase
of absorbance was detected at 348 nm using UV/Vis
spectrophotometer.
2 Experimental
Candida antarctic lipase B (CALB), p-nitrophenyl buty-
rate, PluronicÒ RF-127, Dess–Martin periodinane, and
racemic phenylglycine methyl ester were purchased from
Sigma-Aldrich. Methyl tertiary butyl ether and 4-methyl-2-
pentanone were purchased from Alfa Aesar. Vinyl laurate,
vinyl myristate, vinyl stearate and vinyl palmitate were
purchased from J&K Chemical.
The activity of CALB and CALB-Pluronic conjugate in
organic solvents was determined by transesterification of
hexanoic acid and n-butyl alcohol. During a run, dry power
of native CALB or CALB-Pluronic conjugate with the
same protein content of 0.5 mg was added into 5 mL of
toluene solution containing 0.1 mol/L of fatty acid and
0.1 mol/L of alcohol. The reaction mixture was shaked at
40 °C and 200 rpm in a horizontal shaker. At specific
times, the reaction was terminated by diluting with 10 mL
of ethanol/acetone (1:1, v/v). The remaining free fatty acid
The conjugation of Pluronic-F127 to Candida antarctica
lipase B (CALB) was started by generating aldehyde
groups on Pluronic-F127 by Dess–Martin periodinane (the
molar ratio of Dess–Martin to Pluronic F-127 was 5:1) as
reported elsewhere. Then solvent was evaporated and the
obtained viscous solution was filtered with diethyl ether
and then dried under vacuum at room temperature. The
obtained aldehyded-functionalized Pluronic was then
Fig. 1 Esterification of lutein
and vinyl dipalmitate in organic
media catalyzed by lipase-
Pluronic conjugate
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Synthesis of Lutein Esters by Using a Reusable Lipase-Pluronic Conjugate as the Catalyst
in the reaction mixture was determined by titration with
0.05 mol/L of NaOH using phenolphthalein as the indica-
tor. Conversion of 1 lmol of fatty acid per minute in the
assay conditions was defined as one enzyme unit activity
(U/mg).
In a typical experiment, 50 mg of lutein and 250 mg of
vinyl dipalmitate was added in 5 mL of methyl tertiary
butyl ether (MTBE) in a hermetic round-bottom flask,
followed by the addition of 50 mg of Novo 435 (or CALB-
Pluronic with same protein content) to start the reaction. At
specific times, sample was taken for HPLC analysis. HPLC
analysis was used to measure the conversion of lutein
catalyzed by CALB-Pluronic conjugate and free CALB. It
is carried out on a SHIMADZU HPLC system by using a
ChiralcelÒ OD column (250 4.6 mm, 5). The mobile phase
A was the mixture of hexane and isopropanol (1:3, v/v) and
the mobile phase B was methyl tertiary butyl ether as the
gradient elution phase. The analysis was performed at an
aggregate rate 1 mL/min, and the absorbance was detected
at 445 and 450 nm. NMR and FT-IR were used to prove
the existence of lutein dipalmitate.
3 Results and Discussion
Fig. 2 a Activities of CALB-Pluronic conjugate compared to native
lipase; b gel electrophoresis (CALB: native, CALB-P: conjugate) and
TEM image of the CALB-Pluronic conjugate
The hydrolytic activity of the CALB-Pluronic conjugate
determined with 4-nitrophenyl butyrate (p-NPB) as sub-
strate was *106 % with reference to free CALB (Fig. 2a).
A very weak band of free CALB appeared in the lane of
CALB-Pluronic conjugate in the gel electrophoresis
(Fig. 2b), indicating that most protein was conjugated with
polymer and the conjugation had a relatively high yield.
The number of lysines modified with Pluronic was mea-
sured by a fluorometric assay using standard method [2].
The result indicated that each single enzyme was conju-
gated with *6 Pluronic molecules. Transmission electron
microscopy (TEM) image showed the conjugate appeared
as nanospheres with diameters from 10 to 50 nm (Fig. 2b).
The catalytic performance of CALB-Pluronic conjugate
in the esterification of lutein and palmitic acid vinyl ester
was investigated in comparison with free CALB. In a
typical experiment, 50 mg of lutein and 250 mg of vinyl
dipalmitate were added in 5 mL of methyl tertiary butyl
ether (MTBE). Then, 42 mg of the CALB-Pluronic con-
jugate (or free CALB containing the equivalent amount of
protein) was added to the mixture, followed by stirring at
250 rpm, 50 °C. At intervals, samples were taken for
HPLC analysis to determine the conversion of substrate. As
shown in Fig. 3, the reaction catalyzed by CALB-Pluronic
conjugate reached a conversion of 85.1 % in 12 h while
that by free CALB was below 10 %.
isobutyl ketone (MIBK) are the favorable solvents for the
enzymatic reaction, giving 85.1 and 68.2 % of conversion
at 50 °C respectively. The favorable temperature for the
enzymatic catalysis in MIBK is 60 °C. The conversion at
70 °C falls below 10 %, due to the decomposition of lutein
at high temperature. The favorable temperature for reaction
in MTBE is 50 °C. Higher temperature was not attempted
because the boiling point of MTBE is at 55.2 °C.
At the optimized condition, the reaction catalyzed by
CALB-Pluronic nanoconjugate at 50 °C reached a con-
version of *85 % in 12 h in MTBE, about eightfold
higher than that obtained with free CALB as the catalyst.
Calculated from the initial reaction rate, the apparent
activity of the conjugate was about 12-fold higher than that
of free CALB.
TheHRMSdataoftheproducthasbeenreportedelsewhere
[26]. The FT-IR spectrum of the product showed character-
istic absorption bands at 3305 cm-1 (OH stretch),
2850–2950 cm-1 (aliphatic CH stretch), 1580 cm-1 (C=C
stretching) cm-1, discernible in that of lutein. An extra IR
signaloftheproductat1732 cm-1, ascribabletothepalmitoyl
carbonyl group, indicated the existence of lutein dipalmitate
(Fig. 4). The product was further confirmed by the NMR. 1H-
NMR (600 MHz, CDCl3): d (ppm) = 6.62 (m, 4H), 6.36 (dd,
J = 14.9, 5.8 Hz, 2H), 6.25 (d, J = 8.7 Hz, 2H), 6.19–6.03
The effects of reaction parameters on the conversion
were examined. As shown in Table 1, MTBE and methyl
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M. Hou et al.
Fig. 4 FT-IR spectra of lutein and purified product lutein dipalmitate
Fig. 3 Conversions of lutein catalyzed by CALB-Pluronic conjugate
and free CALB in MTBE
Table 1 Effects of solvents and temperature on CALB-Pluronic
conjugate catalysed reaction
Condition
Conversion (%)
Temperature (°C)
MTBE
MIBK
Chloroform
Acetone
30
50
60
31.4
85.1
–
52.7
68.2
79.8
–
17.8
–
–
21.8
–
Reaction condition: 50 mg lutein, 250 mg vinyl dipalmitate in 5 mL
solvent, 42 mg CALB-Pluronic conjugate (containing 5 mg protein),
12 h. Conversions were determined by HPLC analysis
(m, 5H), 5.48 (s, 1H), 5.43 (dd, J = 15.5, 9.7 Hz, 1H), 5.33 (s,
1H), 5.13–5.01 (m, 1H), 2.40 (m, 3H), 2.28 (t, J = 7.8 Hz,
4H), 2.11 (dd, J = 16.8, 9.5 Hz, 1H), 1.98–1.95 (s,
J = 4.1 Hz, 9H), 1.90 (s, 3H), 1.84 (dd, J = 13.9, 6.0 Hz,
1H), 1.77 (m, 1H), 1.72 (s, 3H), 1.65 (s, 3H), 1.64–1.55 (m,
5H), 1.44 (dd, J = 13.8, 5.2 Hz, 1H), 1.40–1.20 (m, 48H),
1.11 (s, 3H), 1.07 (s, 3H), 1.0 (s, 3H), 0.88 (9H).
Fig. 5 a Conversions of lutein for the consecutive batches catalyzed
by CALB-Pluronic conjugate. b Photos showing CALB-Pluronic
nanoconjugate dispersed in the reaction mixture and precipitated from
the mixture after reaction
Scale-up experiments catalyzed by the CALB-Pluronic
conjugate were carried out in 20 mL MTBE with the input of
0.16 g enzyme catalyst, 0.2 g lutein and 1 g of vinyl
dipalmitate. Afterconductingtheenzymaticcatalysisat50 °C
for 12 h, a lutein conversion of about 80 % of was obtained.
The reusability of CALB-Pluronic conjugate in the
consecutive batches was examined. After each batch (in
MTBE, 50 °C, 11 h) the mixture was cooled from 50 to
0 °C to recover the catalyst. Then new reactants and sol-
vent were added to the precipitated catalyst for the subse-
quent batch. In a typical experiment, 75 mg of CALB-
Pluronic conjugate (containing 9 mg of protein) was
applied to catalyze the conversion of lutein (50 mg of
lutein and 250 mg of vinyl dipalmitate in every batch) to
lutein ester in MTBE for 5 batches. An average conversion
over 60 % was obtained for these batches (Fig. 5a).
Figure 3b shows the CALB-Pluronic conjugate dispersed
well in the reaction mixture at 50 °C and precipitated from
the mixture after reaction at 0 °C. The main reason for
activity lost during the recycling use might be the deacti-
vation of enzyme in organic solvents.
Compared with commercial immobilized lipase Novo
435 [27], which reached 83 % conversion of lutein at
60 °C for 8 h, the lipase-Pluronic conjugate showed similar
catalytic performance with a 85 % conversion at 50 °C for
12 h. In addition, the conjugate gave a comparable
reusability to Novo 435. The high stability and easy
recycling of the catalyst hold great promise for the lipase-
Pluronic conjugate in the production of lutein esters.
The synthesis of different lutein esters using the CALB-
Pluronic conjugate as the catalyst was also investigated. At
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Synthesis of Lutein Esters by Using a Reusable Lipase-Pluronic Conjugate as the Catalyst
Table 2 Conversions of enzymatic reactions by using other long-
chain carboxylic acid vinyl esters
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In summary, we reported a CALB-Pluronic conjugate cat-
alyzed esterification of lutein ester. The CALB-Pluronic
conjugate displayed higher apparent activity compared to
free CALB. At an optimized condition, the conversion of the
reaction catalyzed by conjugate reached 85.1 % for 12 h at
50 °C in MTBE. The conjugate can be readily recovered by a
temperature-induced precipitation and displayed similar
activity once applied to a subsequent run. Lutein esters using
different long-chain fatty acids as substrate also get high
conversions. This study shows the lipase-Pluronic conjugate
is promising for the production of lutein esters.
27. Wang R, Hou M, Zhang Y, Ge J, Liu Z (2015) Catal Lett
145:995–999
Acknowledgments This work was supported by the National High
Technology Research and Development Program (‘‘863’’ Program) of
China under the Grant Number of 2014AA020507, the National
Natural Science Foundation of China under the Grant Number of
21206082, Tsinghua University Initiative Scientific Research Pro-
gram under the Grant Number of 20131089191.
123