Synthesis of lutein esters using a novel biocatalyst of: Candida antarctica lipase B covalently immobilized on functionalized graphitic carbon nitride nanosheets

Article abstract of DOI:10.1039/d0ra00563k

Lutein scavenges free radicals and inhibits vision damage caused by photo oxidation, while decomposing easily with light and heat. Its stability and bioavailability can be tremendously improved by lutein ester synthesis. However, green and efficient ester

Full text of DOI:10.1039/d0ra00563k

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PAPER
Synthesis of lutein esters using a novel biocatalyst
of Candida antarctica lipase B covalently
immobilized on functionalized graphitic carbon
nitride nanosheets†
Cite this: RSC Adv., 2020, 10, 8949
a
a
a
a
Jie Shi, *ab Qianchun Denga
Huijuan Shangguan, Shan Zhang, Xin Li, Qi Zhou,
a
and Fenghong Huang
Lutein scavenges free radicals and inhibits vision damage caused by photo oxidation, while decomposing
easily with light and heat. Its stability and bioavailability can be tremendously improved by lutein ester
synthesis. However, green and efficient esterification preparation methods are urgently needed. In this
3 4
study, which used functionalized graphitic carbon nitride nanosheets (g-C N -Ns) as the immobilized
carrier, a novel biocatalyst was designed and prepared to accommodate Candida antarctica lipase B
CALB), considerably enhancing the performance. It was characterized by TEM, XRD, FTIR, XPS, TGA, and
BET to demonstrate successful preparation and then applied to catalyze esterification between lutein
(
Received 18th January 2020
Accepted 22nd February 2020
and succinate anhydride in dimethyl formamide (DMF) solvent resulting in a conversion rate up to 92% at
ꢀ
5
0
C in 60 h, 34% more than free CALB under the same conditions. We believe this is the highest
DOI: 10.1039/d0ra00563k
esterification rate in lutein esters synthesis and it has great potential to facilitate eco-friendly and
efficient preparation.
rsc.li/rsc-advances
can be endowed with different biological activities and physio-
1
. Introduction
2,3,8
logical functions.
Traditionally, the reported approaches for lutein ester
Exhibiting the activity of provitamin A and the advantages of
anti-oxidation, lutein, together with b-carotene, zeaxanthin, preparation are chemical catalysis, solvent extraction, and
8
9
10
lycopene, b-cryptoxanthin, and a-carotene are the six caroten- supercritical carbon dioxide extraction from marigolds,
1,2
11
oids present in human serum, which improve intercellular microalgae, or other plants. However, these methods have
3
gap junction communication and boost the immune system.
several non-negligible shortcomings such as complicated
Especially, lutein has been recognized as a key supplement in processes, solvent pollution, and environmental contamina-
4
the human diet for its capacity to scavenge reactive oxygen tion. Compared to the methods mentioned above, as one new
(
such as the free ROOc, HOc, HOCl) and nitrogen species, and potential method, esterication by lipase shows many advan-
5
delay age-related macular degeneration. Unfortunately, tages such as high efficiency, selectivity, specicity, mild cata-
12
mammals, including humans, cannot synthesize lutein them- lytic media, and environmental friendliness. Unluckily, like
3
selves and must rely on diet for ingestion. Furthermore, due to protein, lipase is high cost, sensitive to the reaction condition,
13
its high degree of unsaturation, lutein is easily decomposed and easily inactivated. Immobilizing lipase on various carriers
6
under light and heat conditions. To improve its stability and in different ways, can promise the function of improving the
7
bioavailability, esterifying the hydroxyl groups at both ends of thermal stability and reusability, leading to lower cost of lipase
1
4,15
lutein by different anhydrides to form lutein diester is consid- application.
However, very few papers have described the
16
17
ered to be an ingenious strategy, in addition, the original lutein enzymatic synthesis of lutein esters. Wang and Hou tried to
catalyze the synthesis of lutein palmitate by Novozyme 435 and
lipase-Pluronic F-127. Nevertheless, in their studies, toluene,
methyl tert-butyl ether (MTBE), or methyl isobutyl ketone
a
Hubei Key Laboratory of Lipid Chemistry and Nutrition, Key Laboratory of Oilseeds
Processing, Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute,
(MIBK) were used as the solvent for lutein esterication, which
Chinese Academy of Agricultural Sciences, Wuhan 430062, China. E-mail: shijie@
are not suitable for food applications. Therefore, the develop-
ment of new immobilized lipases for efficient and green ester-
ication of lutein esters is urgently needed.
caas.cn
b
School of Food and Biological Engineering, Hefei University of Technology, Hefei
2
30009, China
†
Electronic supplementary information (ESI) available: SEM images, the surface
As an essential participator in the immobilization process,
supporting material deeply affects lipase activity. Two-
area, pore width, pore volume of materials and other supplementary data. See
DOI: 10.1039/d0ra00563k
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18
dimensional nanomaterials such as graphene oxide and transformation. According to the results, the maximum
19,20
21
carbon nanosheets
have gained extensive attention due to conversion rate of lutein disuccinate was up to 92% within 60 h.
their large surface area, abundant active sites, and good To the best of our knowledge, this is the rst study about
biocompatibility. As a superior material, 2D carbon nitride immobilizing CALB on g-C N -Ns lutein ester synthesis, and we
3
4
nanosheets exhibit excellent performance in many elds. obtained the highest esterication rate in the literature.
Among the seven phases of carbon nitride, graphitic carbon
nitride is the most stable formation and possesses outstanding
2
. Experimental
22
advantages. Recently, our group rst synthesized g-C
3 4
N -Ns
2.1. Materials
based immobilized Candida rugosa lipase (CRL) and consid-
23
ered it as a highly promising material; however, since the Candida antarctica lipase B (CALB, liquid, enzyme activity 5000
2
ꢁ1
ꢁ1
surface area of g-C N -Ns (74.374 m
g
) and the protein U mL ) was supplied by Beijing Gaoruisen Biological Tech-
3
4
ꢁ1
amount of the immobilized lipase (44.76 mg g ) were not nology Co., Ltd (Beijing, China). Lutein (75%) and succinic
particularly satisfactory, its further application could not be anhydride were purchased from Aladdin (Shanghai, China).
realized.
In this work, we prepared a new bioreactor that utilized NPP, Aldrich, 99%) were obtained from Sigma-Aldrich (St.
polyethyleneimine (PEI)-modied g-C
-Ns as the carrier and Louis, USA). Chromatographically pure methyl alcohol, aceto-
anchored CALB on g-C
-Ns with the assistance of glutaral- nitrile, and acetic acid were purchased from Merck Biotech-
Dicyandiamide (DCDA, 99%) and p-nitrophenylpalmitate (p-
3 4
N
3 4
N
dehyde (GA). The whole procedure is shown in Scheme 1 and nology Co., Ltd (Shanghai, China). The BCA Protein Assay Kit
the design concept can be explained as follows: (1) an easy, low- was obtained from Shanghai Yuanye Biotechnology Co., Ltd
cost thermal polymerization method was chosen to prepare (Shanghai, China). GA solution (25%), dimethyl sulfoxide
bulk graphitic carbon nitride (bulk g-C
formation and good biocompatibility. The van der Waals forces were supplied by Sinopharm Chemical Reagent Co., Ltd
between the two-dimensional planes of the bulk g-C
were (Shanghai, China).
3 4
N
) with a stable (DMSO), chloroform, methyl tert-butyl ether, acetone, and DMF
3 4
N
destroyed by thermal action stripping it into single layers of
graphitic carbon nitride nanosheets and obtaining a large 2.2. Preparation of g-C
surface area that aided the attachment of CALB; (2) to aid water
3 4
N -Ns
The preparation of g-C N -Ns is based on the existing literature
3
4
dispersibility of g-C N -Ns, branched PEI was selected as the
24
3
4
with a slight adjustment to the procedure. First, 3 g of
dicyandiamide was weighed and placed in a 30 mL alumina
crucible, capped, and le a seam. Second, the alumina crucible
was heated to 550 C at 2.3 C min and held at 550 C for 4 h
in a tube furnace. This process was performed in air atmo-
amino modication to adorn the g-C N -Ns surface for the rich
3
4
–
2
NH group. In addition, positively charged branched PEI could
easily be anchored on the negatively charged graphite carbon
nitride nanosheets via electrostatic adherence, and sufficient
ꢀ
ꢀ
ꢁ1
ꢀ
amino groups on the surface of g-C
3
N
4
-Ns-PEI provided
ꢀ
sphere and cooled to room temperature (25 C) naturally. The
numerous binding sites for CALB; (3) as a bridge, GA made it
possible for the oxygen-containing groups (such as carboxyl,
hydroxyl, and carbonyl) on CALB to covalently connect to the
amino groups on the graphite carbon nitride nanosheets,
effectively preventing enzyme leakage during immobilization
and esterication; (4) with good solubility for lutein and suc-
cinic anhydride, non-polluting DMF was selected as the reac-
tion solvent, which induced catalytic efficiency improvement
hard yellow product was bulk g-C N , which was ground into
3
4
powder by using an agate mortar. Third, 1.5 g bulk g-C N4
3
powder was evenly spread on a 50 mL porcelain boat and heated
ꢀ
ꢀ
ꢁ1
to 500 C at 5 C min in a tube furnace, and kept for 2 h in air
followed by cooling naturally. Finally, the light yellow powders
were collected and preserved in a dry environment.
2
.3. Preparation of g-C
The method for the preparation of functionalized g-C
PEI was based on the literature and had been appropriately
3 4
N -Ns-PEI
during esterication. Simultaneously, g-C
3 4
N -Ns-PEI@CALB
3 4
N -Ns by
was well dispersed in DMF, which is benecial to mass
25
modied. One gram of obtained g-C
3 4
N -Ns was dispersed in
20 mL phosphate buffer (PBS) (0.1 M, pH 7), stirred, and soni-
cated for 10 min, and 10 g of PEI was dissolved in 10 mL PBS
buffer (0.1 M, pH 7) by stirring. Successively, the dissolved PEI
was added drop-wise to the prepared PBS buffer, containing g-
C N -Ns. Aer stirring for 48 h at room temperature, the
3
4
mixture was centrifuged and washed by deionized water several
ꢀ
times. Finally, the sediment was dried in a 60 C oven and then
preserved for use.
2
.4. Covalently immobilized CALB on g-C
Ns-PEI-GA@CALB)
3 4
The immobilization of CALB on g-C N -Ns-PEI was described in
3 4 3 4
N -Ns-PEI (g-C N -
3 4
Scheme 1 Schematic representation of the formation of g-C N -Ns-
PEI-GA@CALB.
23
the previous report. There were two steps to immobilize CALB
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3 4 3 4
on g-C N -Ns-PEI. First, 200 mg of g-C N -Ns-PEI was well 2.7. Synthesis of different lutein esters
dispersed in 6.34 mL PBS buffer (0.1 M, pH 7 or other pH value
according to the experimental request) and then 2 mL 25% GA
was added. The mixture was incubated in a shaker with a rotating
In the typical experiment, 140 mg lutein was rst dissolved in
1
5
mL DMF by sonication in a hermetic round-bottom ask and
00 mg succinic anhydride was added to the solution. Aer
ꢀ
speed of 160 rpm and temperature of 55 C. Aer agitating for
3 4
shaking for several minutes, 100 mg immobilized lipase g-C N -
12 h, the sample was separated by vacuum ltration to remove
Ns-PEI-GA@CALB was added. To protect the lutein from oxi-
dization, nitrogen was injected into the area above the liquid to
the PBS and redundant GA and washed by deionized water
ꢀ
several times. Next, the solid was dried in the oven at 60 C.
ꢀ
expel the air. Next, the ask was shaken at 45 C and 160 rpm in
Subsequently, 18 mL PBS and 6 mL CALB liquid were rst mixed
a horizontal shaker for a certain time.
by a shaker for 5 min, then 200 mg of the prepared g-C N -Ns-
3
4
PEI-GA was added. The reaction liquid was gently vibrated at
ꢀ
2.8. Analysis of the conversion rate of lutein disuccinate
160 rpm and in 37 C for 8 h. Next, the material was centrifuged
and washed by deionized water three times, followed by freeze- The esterication rates of lutein monosuccinate and lutein
drying in a vacuum freeze drier. Finally, the immobilized lipase disuccinate were detected by High Performance Liquid Chro-
g-C
3
N
4
-Ns-PEI-GA@CALB was prepared for use. The protein matography (HPLC) analysis. One hundred microliters of
-Ns-PEI-GA@CALB was determined by terminal solution of esterication reaction were taken out and
loading content of g-C
3 4
N
BCA Protein Assay Kit, taking bovine serum albumin as the mixed with 5 mL chromatographically pure methyl alcohol. The
standard. Additionally, the protein loading content of g-C N -Ns- samples could be analyzed by HPLC aer ltration through
3
4
PEI-GA@CALB was calculated using the subtraction method of a 0.22 mm membrane. The HPLC analysis was conducted using
the initial protein content and the supernatant protein content a YMC-Carotenoid S-5 mm (250 ꢂ 4.6 mm I.D.). Mobile phases A
aer immobilization.
and B were methyl alcohol (0.05% acetic acid) and acetonitrile
(0.05% acetic acid), respectively. The conditions of HPLC anal-
2
.5. Characterization of prepared materials
ysis were as follows: the elution condition was binary high-
ꢁ
1
pressure elution, ow velocity was 1 mL min , column
To observe the morphologies of the series materials at different
steps of the experiment, different methods were used for char-
acterization, including UV-vis absorption spectroscopy (DU 800
UV-vis spectrometer), scanning electron microscopy (SEM,
ZEISS MERLIN Compact), transmission electron microscopy
ꢀ
temperature was 40 C, injection volume was 20 mL, and the
absorbance was detected at 450 nm. Finally, the esterication
rates of lutein disuccinate and lutein monosuccinate were
calculated by the area normalization method.
(TEM, Tecnai G2 F30, FEI), Fourier-transform infrared spec-
troscopy (FT-IR, Bruker Tensor 27 FT-IR spectrometer Ther- 2.9. Reusability of g-C -Ns-PEI-GA@CALB
moelectron), X-ray diffraction (XRD, Bruker D8 Advance), X-ray
3 4
The reusability of g-C N -Ns-PEI-GA@CALB was studied by add-
N
3 4
photoelectron spectroscopy (XPS, ESCALAB 250Xi), thermogra-
vimetric analysis (TGA, TAQ50), and Brunauer–Emmett–Teller
ing 10 mg of g-C N -Ns-PEI-GA@CALB to 1 mL DMF solution
3
4
(
containing 140 mg lutein and 500 mg succinic anhydride) and
(BET) analysis (ASAP2020).
ꢀ
reacting the mixture for 24 h at 50 C in the protection of
3 4
nitrogen. Subsequently, g-C N -Ns-PEI-GA@CALB was regained
2
.6. Enzymatic activity assay and thermal stability of g-C N -
by centrifuging the previous reaction solution and discarding the
supernatant, and was used for the next cycle. The relative ester-
ication rates were dened by presuming the rst cycle as 100%.
3
4
Ns-PEI-GA@CALB
The activities of CALB and g-C N -Ns-PEI-GA@CALB were eval-
3
4
ꢀ
uated by hydrolyzing p-NPP in PBS at 37 C for 5 min, which was
conducted in a table concentrator at 160 rpm. The concentra-
tion of the hydrolysis product p-NP (p-nitrophenol) was
measured by UV spectrophotometer at 410 nm. The relative
3
. Results and discussion
3
.1. Synthesis of the g-C N -Ns-PEI-GA@CALB
3 4
activities were dened by presuming the maximum absorbance The synthesis procedure is illustrated in Scheme 1. First, the
value of the free or immobilized lipase catalysis under optimal carrier material g-C -Ns was prepared by a simple “top-down”
N
3 4
conditions as 100%. Thirty milligrams g-C N -Ns-PEI- method, thermal oxidation etching of bulk g-C N in the air,
3
4
3 4
2
GA@CALB (or 30 mL CALB) were mixed with 1 mL PBS (0.1 M, obtaining 2D-nanosheets with a high surface area of 118.858 m
pH 7.0), and then 1 mL 0.5% p-nitrophenylpalmitate ethanol
solution (w/v) was added. The mixture was incubated at 37 C via electrostatic interaction to create abundant functional
ꢁ1
g
. Then, the surface of g-C
3
N
4
was modied by branched PEI
ꢀ
for 5 min at 160 rpm in a shaker and was terminated by adding groups (–NH
2
). Next, a majority of CALB was immobilized by the
(25 g L ) solution. Subsequently, 3 mL of the covalent connection via GA, while some was anchored by PEI
supernatant was obtained by centrifuging the reaction solution. and g-C via electrostatic interactions or physical absorp-
ꢁ1
2
2 3
mL of Na CO
3 4
N
Then the supernate was suitably diluted and detected on an tions, respectively. Finally, to esterify lutein, the two substrates
ultraviolet-visible spectrophotometer at 410 nm. Thermal and g-C N -Ns-PEI-GA@CALB were well dispersed in the DMF
3
4
stabilities of CALB and g-C N -Ns-PEI-GA@CALB were studied solvent. The large surface of g-C N -Ns could provide sufficient
3
4
3 4
by measuring the residual activities of the lipases aer incu- space for substrate access to CALB and ensured the catalytic
bation at different temperatures for 1 h.
performance.
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3 4
.1.1. Characterization of g-C N -Ns-PEI-GA@CALB. The
Paper
3
morphology of prepared materials was investigated by trans-
mission electron microscopy (TEM) and the TEM images of g-
C
3
N
4
-Ns and g-C
3 4
N -Ns-PEI-GA@CALB are exhibited in Fig. 1a
and b, respectively. Aer thermal stripping of the bulk g-C
3 4
N ,
which was formed by thermal polymerization of dicyandiamide,
we retained the 2D g-C N -Ns, which displayed so silk-like
3
4
nano-sized lamellar structures with folds spread on the at
surface. However, aer immobilizing the CALB on g-C -Ns,
3 4
N
many small dark dots appeared on the surface of the silk in the
high-resolution picture of Fig. 1b, revealing that CALB was
Fig. 2 XDR pattern (a) and FT-IR spectra (b) of g-C
3 4 3 4
N -Ns, g-C N -
Ns-PEI, g-C -Ns-PEI-GA and g-C -Ns-PEI-GA@CALB.
N
3 4
3 4
N
successfully connected with g-C
3 4
N -Ns. This conclusion was
consistent with the preparation of a new biocatalyst C
Ns@CRL in our previous report.
3 4
N -
23
C
3
N
4
-Ns successfully. Moreover, comparing the curves of free
To investigate the lattice structures of the materials prepared
in the process of immobilization, XRD analysis was involved. As
shown in Fig. 2a, at 13 and 27 , the similar characteristic
CALB and g-C -Ns-PEI-GA@CALB, the N–H peak of free CALB
3 4
N
ꢁ1
at 3000 cm , weakened aer immobilization, which can be
explained by the successful formation of amide bonds between
CALB and GA.
The composition and content of elements, as well as chem-
ical bonds of the materials were studied by XPS. As shown in
Fig. 3a, the elements of C1s (287.0 eV), N1s (399.0 eV), and O1s
ꢀ
ꢀ
diffraction angles can be observed in the four samples. The
ꢀ
weak low-angle reection (peak 13 ) stem results from the
repeat units tri-s-triazine in the parallel planes lattice, and the
ꢀ
distinct peak at 27 can be attributed to the interlayer diffrac-
tion of the layered structures of g-C N -Ns. A slight decrease is
3
4
(
532.0 eV) are all observed. However, distinct differences can be
noticed for each element in the characteristics and content. In
Fig. 3b, the O1s peaks of g-C -Ns and g-C -Ns-PEI at
32.51 eV and 531.04 eV, respectively, are very small and
ꢀ
ꢀ
observed in peak intensity at 13 and 27 aer PEI adsorption,
which should be attributed to the lower crystallinity of g-C N -
3
4
3
N
4
3 4
N
Ns caused by PEI. Moreover, there is no marked difference in
peak strength of the four sample curves, which indicates that g-
C N -Ns invariably maintains the original crystal structure
3 4
during PEI modication, GA connection, and CALB
immobilization.
5
negligible; however, aer the addition of GA, the intensity
rapidly increases resulting from the oxygen on GA, which shows
that GA successfully links with g-C
O1s peak of g-C -Ns-PEI-GA@CALB increases slightly
compared with the non-enzyme version, and these inconspic-
uous changes may be caused by small quantities of oxygen-
containing functional groups in CALB. In Fig. 3c, aer PEI
3 4
addition, the C1s peak of g-C N -Ns-PEI separates into two
peaks at 284.99 eV and 287.35 eV, respectively. The new peak at
3 4
N -Ns-PEI. Furthermore, the
3 4
N
In addition, we conducted FT-IR spectroscopy to further
verify the functional groups and chemical bonds on g-C N -Ns
3
4
related products, and the result is shown in Fig. 2b. First, the
ꢁ
1
ꢁ1
peaks between 900 cm and 1800 cm are likely attributed to
the stretching and vibration of the C–N skeleton. The details
ꢁ1
suggest that the peaks at 1500–1300 cm are caused by shear
vibration and in-plane exural vibration of N–H in the aromatic
ꢁ
1
structure. However, the peaks in the range of 1300–800 cm
can be ascribed to the stretching and vibration of trigonal C–N(–
C)–C and bridging C–NH–C units. Second, the two peaks
ꢁ1
around 820 cm in the graph originated from the out-of-plane
bending vibration of the H atom on the heptazine ring. This
evidence further proves that PEI, GA, and CALB attach to g-
Fig. 3 XPS spectra (a), high-resolution O1s (b), high-resolution C1s (c)
Fig. 1 TEM images of g-C
b).
N
3 4
-Ns (a) and g-C
N
3 4
-Ns-PEI-GA@CALB and high-resolution N1s (d) of g-C
3 4 3 4 3 4
N -Ns, g-C N -Ns-PEI, g-C N -
(
Ns-PEI-GA and g-C -Ns-PEI-GA@CALB.
3 4
N
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284.99 eV is ascribed to the C–C bonds introduced by PEI.
Moreover, compared with the g-C N -Ns, the peak (288.34 eV,
3
4
2
sp (N) –C]N) is shied to weak binding-energy regions
2
(287.35 eV) due to the electrostatic interaction between
protonated PEI and negative g-C -Ns. Subsequently, aer the
3 4
N
introduction of GA, a new peak located at 286.41 eV appeared,
2
which is assigned to sp C]O in GA and CO–NH formed by the
aldimine condensation between PEI amino and GA aldehyde
groups. Similarly, the deconvoluted peaks of g-C
GA@CALB are displayed at 284.76 eV, 286.19 eV, and
87.71 eV, corresponding to C–C, C]O, or CO–NH and (N)2–
C]N, respectively. These results disclose that PEI, GA, and
CALB are all anchored on the g-C -Ns surface solidly. The N1s
peak of g-C -Ns in Fig. 3d can be separated into two peaks at
98.85 eV and 400.57 eV, which represent the sp hybridization
of C–N]C or N–(C) bonds in tri-s-triazine rings and the very
small amino existing in (C) –NH bonds. Aer the g-C -Ns
3 4
N -Ns-PEI-
Fig. 5 Nitrogen adsorption–desorption isotherms (a) and pore size
distribution (b) of g-C
N
3 4
3 4 3 4
-Ns, g-C N -Ns-PEI, g-C N -Ns-PEI-GA
2
3 4
and g-C N -Ns-PEI-GA@CALB.
3 4
N
3 4
N
ꢀ
6
50 C, which is due to the evaporation of water, dissociation of
2
3
CALB, and pyrolysis of g-C
CALB is successfully connected to g-C
According to the BET gas sorptometry measurement in
Fig. 5a, the prepared g-C -Ns has a large surface area of
18.858 m g , whereas the surface areas of g-C -Ns-PEI, g-
3
N
4
-Ns. The results also proved that
3
3 4
N
-Ns-PEI-GA.
2
3 4
N
modied by PEI, the peak of (C) –NH shied to weaker binding
2
3 4
N
energy by 0.59 eV, indicating that the electrostatic interaction
between PEI and g-C N -Ns improves the electron distribution
2
ꢁ1
1
3 4
N
3
4
2
C N -Ns-PEI-GA, and g-C N -Ns-PEI-GA @CALB are 54.005 m
3
4
3 4
density outside the nitrogen nucleus and the atomic nucleus
weakens the electron binding force and results in the decreased
ꢁ1
2
ꢁ1
2
ꢁ1
g
, 26.947 m g , and 14.163 m g , respectively, exhibiting
a signicant decrease compared with the pure g-C N -Ns (ESI,
3
4
energy intensity. In addition, compared with g-C
3 4
N -Ns-PEI, g-
Table S1†). Due to the large amount of the PEI polymers, which
are attached to the g-C -Ns covering the triazine rings, the
surface area of g-C -Ns decreases sharply. Subsequently, the
covalent connection between GA and CALB leads to a further
decrease in g-C -Ns-PEI surface area. As shown in Fig. 5b, it is
evident that g-C N -Ns has a mixed pore distribution with the
C
3
N
4
-Ns-PEI-GA and g-C -Ns-PEI-GA@CALB showed new
3 4
N
3 4
N
peaks at 398.88 eV and 399.67 eV, respectively, indicating the
formation of NH–CO bonds, by the reaction between GA and
PEI as well as CALB, respectively. Meanwhile, due to the
formation of new amide bonds, the intensity of C–N]C and N–
3 4
N
3 4
N
3
4
(
C)
3
in g-C
3
N
4
-Ns-PEI-GA shied to the weak binding-energy
-Ns-PEI.
size of 3.794 nm, which may originate from the defects and
holes in the layers. Meanwhile, g-C N -Ns-PEI, g-C N -Ns-PEI-
area by 0.93 eV compared with that of g-C
3 4
N
3
4
3 4
With the help of TGA, we observed the weight loss of
GA, and g-C
.060 nm, and 2.897 nm, proving that g-C
intact layer structure. In addition, compared with the g-C
3
N
4
-Ns-PEI-GA@CALB have pores of 3.058 nm,
-Ns maintains its
-Ns
-Ns-PEI-GA@CALB
0.178 cm g ) reduces by 0.266 cm g . The reason may be
different samples during temperature increase in nitrogen
3
3 4
N
ꢀ
atmosphere. In Fig. 4, when the temperature reaches 650 C, the
3 4
N
curve of g-C N -Ns decreases, indicating g-C N -Ns begins to
3
ꢁ1
3
4
3 4
(
3 4
0.444 cm g ), the pore volume of g-C N
degrade at this point, yet the critical temperatures of g-C N -Ns-
3
ꢁ1
3
ꢁ1
3
4
(
ꢀ
PEI and g-C N -Ns-PEI-GA decomposition are 600 C and
5
3
4
that PEI, GA, and CALB attach or migrate to the edges of the
pores on g-C N -Ns, leading to decreased pore volume.
ꢀ
00 C, respectively, demonstrating that the PEI and GA adhere
3
4
to g-C
3
N
4
-Ns successfully. The curve of g-C
3
N
4
-Ns-PEI-
ꢀ
ꢀ
GA@CALB continually decreases between 100 C and 700 C
and has three evident loss weight steps at 100 C, 450 C, and
ꢀ
ꢀ
3.2. Factors in the immobilization of g-C
GA@CALB
3 4
N -Ns-PEI-
3.2.1. Effect of the lipase concentration on protein loading
content and esterication rate. As is understood, the initial
lipase concentration affects the amount of protein adhered to
the materials and the activity. From Fig. 6a, with the increase in
initial lipase concentration in the range of 2–25%, the protein
loading content of g-C N -Ns-PEI-GA@CALB also increases.
3
4
However, the amount decreases when the lipase concentration
reaches 25%. The decline could be explained by that the
aggregation of excessive CALB instead of distribution as
a monolayer at the surface of g-C
3 4
N -Ns in the immobilization,
which is unfavorable to the attachment of CALB on the g-C
3 4
N -
Ns surface. Meanwhile, the esterication rate increases with
lipase concentration, rapidly between 2% and 16% and slowly
between 16% and 34%. It can be inferred that high loading of
CALB has the potential to form lipase dimers and the active
center will be covered, and combined with that the lipases of the
Fig. 4 TGA analysis of g-C
3 4 3 4 3 4
N -Ns, g-C N -Ns-PEI, g-C N -Ns-PEI-
GA and g-C -Ns-PEI-GA@CALB.
3
N
4
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Table 1 Lipase screening for synthesis lutein disuccinate
Conversion
rate (%)
Lipase
CALB
CRL
21.7
5.6
g-C
3
N
4
-Ns-PEI-GA@CALB
62.2
25.3
Novozyme 435
ꢀ
even at 70 C. This phenomenon may be explained by the
covalent bonds formed when immobilization reduces the
conformational exibility of enzyme molecules, which effec-
tively restricts the distortion or damage during temperature
28
elevation.
3.3. Factors in the esterication of lutein and succinic
Fig. 6 Effect of the initial enzyme concentration on the protein
loading content and the esterification rate of lutein disuccinate (a) and anhydride
on the immobilization time (b); effect of pH (c) on protein content and
3
.3.1. Effect of different lipase on the lutein disuccinate
esterification rate and temperature (d) on the activity of free and
immobilized lipase.
conversion rate. The unique conformation of active centre
endows lipase strict selectivity for substrates and reaction
systems. In order to gure out the most suitable lipase for the
synthesis of lutein disuccinate, we selected four different
lipases including CALB, CRL, g-C N -Ns-PEI-GA@CALB and
3 4
Novozyme 435 for the 48 h reaction. The results are shown in
Table 1, the esterication rate of lutein disuccinate catalyzed by
3 4
g-C N -Ns-PEI-GA@CALB is the highest (62.2%) compared with
other lipases. Even for commercial Novozyme 435, the catalytic
performance is not good. Because most of Novozyme 435 oat
on the solvent and cannot fully interact with the substrate.
29
underlying layer cannot interact with substrates, resulting in
26
the low activity and slowly increased esterication rate.
Therefore, 25% is considered the optimum initial concentration
of lipase. As shown in Fig. 6b, the protein loading content
achieved the maximum value in 8 h, then entered the plateau
period, which indicates that the binding sites on the surface of
g-C
3
N
4
-Ns are saturated.
3.2.2. Effect of pH and temperature on the activity of
3.3.2. Effect of different organic solvents on the lutein
lipase. From Fig. 6c we draw the conclusion that the immobi-
lized protein reaches the maximum value at the pH ¼ 7.0,
implying that the neutral environment is conducive to the
disuccinate conversion rate. On account of the solubility of
lutein and succinic anhydride, as well as CALB activity expres-
sion, an optimal medium is indispensable to provide
a moderate environment for esterication. Considering the
solubility of lutein and succinic anhydride, we selected nine
solvents (ESI, Table S2†) to monitor the activity of CALB during
esterication in 48 h. In the presence of CALB, no signicant
reaction occurs between lutein and succinic anhydride when
hexyl alcohol and N-butyl alcohol are involved. On the contrary,
the conversion rate of lutein disuccinate reaches the maximum
of 64.32% when DMF is used as the solvent, followed by chlo-
roform (56.46%) and toluene (47.72%). In conclusion, DMF is
the best candidate for esterication and activity expression of
CALB. The results reveal that as a less toxic and polluting
solvent, DMF is more suitable for enzyme reaction of edible

xation of CALB on g-C N -Ns-PEI-GA, which can be explained
3 4
by electric charge distribution. When the pH value is higher
than the isoelectric point, the net charge of protein is negative;
on the contrary, the protein is positively charged when the pH is
below the isoelectric point. Therefore, CALB can be absorbed on
carriers via electrostatic adsorption due to the negative charge
of CALB (pI ¼ 6) and positively charged PEI (pI ¼ 8) on the
surface, which acts as an auxiliary mean of chemical covalently
immobilized CALB. Furthermore, compared to free CALB, the
conversion rate of lutein disuccinate for immobilized CALB
remains relatively high between pH 5.0 and 8.0, indicating that
g-C
a wide range of pH condition and has good acid–base tolerance.
The carrier material g-C shields and protects CALB from the
deformation and destruction caused by acid or alkali in the
3 4
N -Ns-PEI-GA@CALB can maintain high activity under
16,17
substrates
and can be employed to replace the commonly
3 4
N
used polluted and toxic reagents for lutein esterication.
3.3.3. Effect of the immobilized lipase dosage and
temperature on esterication rate. The effect of immobilized
lipase dosage on the esterication rate of lutein monosuccinate
and lutein disuccinate is illustrated in Fig. 7a. As shown in the
curves, the esterication rate of lutein disuccinate reaches 69%
when the concentration of g-C N -Ns-PEI-GA@CALB is 10 mg
27
PBS. Furthermore, the thermal stability of free CALB and g-
C N -Ns-PEI-GA@CALB are contrasted in Fig. 6d. In the curves,
3
4
both reach the maximum activity at the optimal temperature of
ꢀ
4
5 C. At the higher temperature range, the relative residual
activity of free CALB decreases to 12% on account of the
conformation of the enzyme damages. Conversely, CALB cova-
3
4
ꢁ
1
mL and reduces gradually with increased immobilized lipase
dosage. Nevertheless, when the dosage of CALB exceeds 10 mg
lently binds with g-C
3 4
N -Ns@PEI showing excellent heat-
resistance, with relative residual activity of approximately 69%
ꢁ
1
mL , the superuous solid could not maintain intimate
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esterication rate increases with the addition of substrate and
ꢁ1
the optimum lutein concentration is 0.7 mol mL . This may
explain why the full interaction of CALB and substrate is easily
achieved with the appropriately substrate concentration. For
ꢁ1
higher substrate concentration (>0.8 mol mL ), the esterica-
tion rate signicantly declines to 27% when lutein molarity
ꢁ
1
reaches 1.2 mol mL . Therefore, appropriate concentration of
substrates can promote the reactions, and higher substrate
concentration may induce the augmentation of the media
34
viscosity, lowering catalytic efficiency at high cost.
.3.5. Time course of free and immobilized lipase catalyzed
3
esterication reactions. The time course could explain the
reaction process and mechanism, and liquid chromatography
was used to analyze samples with different reaction time. As
shown in Fig. 8a, with the extended reaction time, the content of
lutein disuccinate and lutein monosuccinate gradually
Fig. 7 Effect of lipase dosage (a) and temperature (b) on the esterifi- increases and decreases, respectively. In consequence, we can
cation; effect of molar ratio (c) and lutein molarity (d) on esterification infer that lutein monosuccinate is formed rst and then
rate.
transforms to lutein disuccinate by esterication. The CALB has
strong stereo-selectivity due to the “pocket” structure of its
active site that has very limited space available compared with
contact with the solvent, which affects the stable reaction
system. Meanwhile, the overdosed biocatalyst may cause the
agglomeration and precipitation of intermediate mono-
other lipases. Lutein is composed of a conjugated long chain of
eighteen carbon atoms with two ionone rings at its ends, which
may sterically hinder its hydroxyl access to the active CALB
pocket. Longer esterication time is required to strengthen the
interaction between the substrate and CALB. In our enzymatic
reaction, the esterication rate of lutein disuccinate catalyzed
by our immobilized lipase is up to 92% within 60 h while by free
lipase is merely 60%, indicating the great performance of g-
succinate resulting in the inaccessibility of g-C
3
N
4
-Ns-PEI-
GA@CALB. Furthermore, the effect of the esterication
temperature on the activity of g-C -Ns-PEI-GA@CALB is dis-
30
3 4
N
played in Fig. 7b. As expected, the esterication rate increases
ꢀ
with temperature from 35 to 50 C and reaches its maximum.
This is because the relative higher temperature elevates the
esterication rate by increasing the solubility of reactants while
enhancing the frequency of effective collisions and affinity
C
3
N
4
-Ns-PEI-GA@CALB in lutein esterication. Immobilized
CALB on the g-C -Ns-PEI-GA can improve thermal stability,
3 4
N
solvent tolerance, and conformational stability of the active
center. However, free CALB tends to be inactivated when it is
directly exposed to solvent DMF at high temperature for a long
time. The results are also consistent with the viewpoints
mentioned above.
31
between the substrates and CALB molecules. However, when
ꢀ
the temperature is above 50 C, a slight decrease in the esteri-

cation rate occurs due to the decomposition of lutein and the
irreversible deactivation of CALB at the high temperature.
.3.4. Effect of lutein molarity and molar ratio on esteri-
3
3
.3.6. Reusability of the immobilized lipase. As shown in
cation rate. Among other important factors, the effect of
concentration and substrate molar ratio were studied. To
explore the optimal molar ratio of lutein and succinic anhy-
dride, the ratios from 1 : 2 to 1 : 128 were designed to perform
the esterication and the results are shown in Fig. 7c. During
this period, the rate of esterication reaches the maximum
around 1 : 16, and is relatively stable in the range of 1 : 16–
the Fig. 8b, the esterication rate decreases slightly with the
cycle times, and it is worth noting that the conversion rate
remains at 84% aer eight cycles, which may benet from the
role of g-C N -Ns. The CALB is rmly bound to carbon nitride by
3 4
covalent attachment, which effectively reduces the lipase
leakage and ensures that g-C N -Ns-PEI-GA@CALB maintains
3
4
high catalytic activity during every cycles. Aer the ninth cycle,
1
: 20, then rapidly decreases above 1 : 32. According to the
32
“Ping-Pong bi-bi” catalytic mechanism, CALB rst forms the

rst transition state with substrate succinic anhydride which as
the acyl donor, and then the hydroxyl group at one end of lutein
acts as nucleophile to deacylate the acylase intermediate to
form a second transition state. Therefore, moderate excessive
succinic anhydride could promote the formation and accumu-
lation of the rst transitional states in the CALB catalysis, and
meanwhile, accelerate entering the second transition state.
When the succinic anhydride is overdosed, the decreased
esterication rate may be attributed to the superuous and
insoluble succinic anhydride in DMF, forming an obstruction
Fig. 8 The time course of the esterification reaction (a); recycling
33
between the dissolved substrates and lipases. In Fig. 7d, the
study of g-C N
3 4
-Ns-PEI-GA@CALB and the first cycle assign 100% (b).
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the esterication rate decreases to 65%, which may be attrib-
dietary
supplements
by
high-performance
liquid
uted to the mechanical loss of g-C N -Ns-PEI-GA@CALB in the
centrifugation and irreversible partial inactivation of CALB
when catalyzes for several cycles at high temperatures.
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Scavenging capacity of marine carotenoids against reactive
oxygen and nitrogen species in a membrane-mimicking
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3
4
4
. Conclusions
6
A. Robert, S. Andeas and C. Reinhold, Effects of heating and
illumination on trans–cis isomerization and degradation of
carotene and lutein in isolated spinach chloroplasts, J.
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In summary, we prepared g-C
3
N
4
-Ns with a large surface area of
2
ꢁ1
118.858 m
g
and g-C N -Ns-PEI-GA@CALB with a high
3
4
ꢁ1
protein loading content of 60 mg g . The immobilized lipase
exhibited excellent catalysis ability in esterifying lutein and
succinic anhydride. The optimal esterication conditions were
7
8
A. Subagio, H. Wakaki and N. Morita, Stability of lutein and
its myristate esters, Biosci., Biotechnol., Biochem., 1999, 63,
1784–1786.
T. K. Dey, I. Maiti, S. Chakraborty, M. Ghosh and P. Dhar,
Enzymatic synthesis of lipophilic lutein-PUFA esters and
assessment of their stabilization potential in EPA-DHA rich
ꢁ1
as follows: molarity of lutein, 0.7 mol mL ; molar ratio of
lutein and succinic anhydride, 1 : 16; dosage of g-C -Ns-PEI-
3 4
N
ꢁ
1
ꢀ
GA@CALB, 10 mg mL ; and temperature, 50 C. Under the
optimum condition, the esterication rate could up to 92%. In
addition, compared to free CALB, the thermo tolerance of g-
C N -Ns-PEI-GA@CALB was greatly enhanced, in which the

sh oil matrix, J. Food Sci. Technol., 2019, 56, 2345–2354.
9
C. M. Cer ´o n, C. Inmaculada, F. J. S ´a nchez, G. F. Aci ´e n,
M. Emilio and M. J. Fern ´a ndez-Sevilla, Recovery of lutein
from Microalgae biomass: development of a process for
scenedesmus almeriensis biomass, J. Agric. Food Chem.,
3
4
lipase activity of the former was 85% of initial activity while the
ꢀ
latter remained only 12% aer incubation at 60 C for 1 h.
3 4
Meanwhile, g-C N -Ns-PEI-GA@CALB presented excellent
2
008, 56, 11761–11766.
0 Y. Gong, S. Plander, H. Xu, B. Simandi and Y. Gao,
Supercritical CO extraction of oleoresin from marigold
Tagetes erecta L.) owers and determination of its
antioxidant components with online HPLC-ABTS assay, J.
Sci. Food Agric., 2011, 91, 2875–2881.
reusability in the catalyzing esterication reaction; for 8 runs
the relative esterication rate was 84%. These results inspire us
to explore more 2D nanomaterials in the design of the carrier
materials and synthesize new structural lipids with different
functions by esterication.
1
2
(
+
1
1
1
1 J. H. Lin, D. J. Lee and J. S. Chang, Lutein production from
biomass: marigold owers versus microalgae, Bioresour.
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3 S. Rohit, C. Yusuf, C. Uttam and Banerjee, Production,
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Conflicts of interest
There are no conicts to declare.
Acknowledgements
We are grateful to the National Science Foundation of China
(no. 31872896 and 31501423), the Young Elite Scientists Spon-
sorship Program by CAST (2017QNRC001), the Agricultural
Science and Technology Innovation Project of Chinese Academy
of Agricultural Sciences (CAAS-ASTIP-2013-OCRI) for nancial
support.
1
4 D. N. Tran and K. J. Balkus, Perspective of recent progress in
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H. Tang, F. Huang and J. Shi, Novel amphiphilic
polyvinylpyrrolidone functionalized silicone particles as
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