Synthesis of retinyl palmitate catalyzed by Candida sp.99-125 lipase immobilized on fiber-like SBA-15 mesoporous material

Article abstract of DOI:10.1166/jnn.2011.4731

Candida sp.99-125 lipase was suitable for transesterification of fats and oils to produce fatty acid methyl ester. The adsorption of Candida sp.99-125 lipase onto the fiber-like SBA-15 mesoporous material has been studied. The unaltered structural order of the fiber-like SBA-15 before and after the adsorption has been confirmed by FT-IR, SEM and N₂ adsorption. The amount of adsorbed Candida sp.99-125 lipase depends both on the solution pH and reaction time. Good adsorption capacity of Candida sp.99-125 lipase on fiber-like SBA-15 may be due to solution pH from 5.0 to 9.0 especially at 7.0 (93.99 mg enzyme per gram silica is obtained and the activity recovery is 281.05%). A high lipase loading (135.9 mg enzyme per gram silica) was obtained, but it did not produce a proportionate level of catalytic activity. The immobilized Candida sp.99-125 lipase showed increased adaptability in the hydrolysis of p-nitrophenyl acetate compared to free Candida sp.99-125 lipase at pH 5.0-9.0. Meanwhile, the immobilized Candida sp.99-125 lipase showed higher thermal stability than that of free Candida sp.99-125 lipase. And the synthesis of retinyl palmitate in organic solvent with the immobilized Candida sp.99-125 lipase was investigated. The influence factors, such as: the solvent used, the molar ratio and concentrations of substrates, the reaction time and the amount of lipase were studied and optimized. In the conditions of transesterificating 0.164 g retinyl acetate and 0.32 g palmitic acid, 10 mL of solvent hexane, 1:4 of mass ratio of lipase to retinyl acetate, and 6 hours of reaction time, 74.6% of retinyl acetate was converted into retinyl plamitate. Copyright

Full text of DOI:10.1166/jnn.2011.4731

Copyright © 2011 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Nanoscience and Nanotechnology
Vol. 11, 7593–7602, 2011
Synthesis of Retinyl Palmitate Catalyzed by
Candida sp.99-125 Lipase Immobilized on
Fiber-Like SBA-15 Mesoporous Material
Zhu Kai1, Wang Jianqiang2, Wang Yan-hua1, Liu Hui1,
Han Ping-fang1ꢀ∗, and Wei Ping1
1College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology,
Nanjing, 210009, China
2College of Sciences, Nanjing University of Technology, Nanjing, 210009, China
Candida sp.99-125 lipase was suitable for transesterification of fats and oils to produce fatty acid
methyl ester. The adsorption of Candida sp.99-125 lipase onto the fiber-like SBA-15 mesoporous
material has been studied. The unaltered structural order of the fiber-like SBA-15 before and after
the adsorption has been confirmed by FT-IR, SEM and N2 adsorption. The amount of adsorbed
Candida sp.99-125 lipase depends both on the solution pH and reaction time. Good adsorption
capacity of Candida sp.99-125 lipase on fiber-like SBA-15 may be due to solution pH from 5.0 to
9.0 especially at 7.0 (93.99 mg enzyme per gram silica is obtained and the activity recovery is
281.05%). A high lipase loading (135.9 mg enzyme per gram silica) was obtained, but it did not
produce a proportionate level of catalytic activity. The immobilized Candida sp.99-125 lipase showed
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increased adaptability in the hydrolysis of p-nitrophenyl acetate compared to free Candida sp.99-
IP: 186.190.27.212 On: Sat, 13 Feb 2016 12:50:21
125 lipase at pH 5.0–9.0. Meanwhile, the immobilized Candida sp.99-125 lipase showed higher
Copyright: American Scientific Publishers
thermal stability than that of free Candida sp.99-125 lipase. And the synthesis of retinyl palmitate
in organic solvent with the immobilized Candida sp.99-125 lipase was investigated. The influence
factors, such as: the solvent used, the molar ratio and concentrations of substrates, the reaction
time and the amount of lipase were studied and optimized. In the conditions of transesterificating
0.164 g retinyl acetate and 0.32 g palmitic acid, 10 mL of solvent hexane, 1:4 of mass ratio of lipase
to retinyl acetate, and 6 hours of reaction time, 74.6% of retinyl acetate was converted into retinyl
plamitate.
Keywords: Immobilization, Candida sp.99-125 Lipase, SBA-15, Hydrolysis Activity.
1. INTRODUCTION
The immobilization of biomolecules such as proteins
and peptides onto solid supports has attracted much atten-
tion because of its scientific importance, and Candida
sp.99-125 lipase was relevant in many areas, such as
biology, medicine, biotechnology, and food processing.1–4
Because of the interesting properties of lipases, several
studies reported the immobilization of these enzymes
using different protocols: adsorption on hydrophobic sup-
ports, entrapment in gels, and covalent attachment to solid
supports. Immobilization of enzymes by physical proce-
dures such as adsorption on a solid was very attractive
because of its simplicity.8–14 Mesoporous materials have
been a hot topic in last 20 years. However, many papers
about preparation and application of mesoporous materi-
als have been published very recently.15–31 In particular,
use of mesoporous materials and nanoorganized materials
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) were
widely used as versatile biocatalysts in modern organic
chemistry, especially for synthesis of enantioenriched com-
pounds and polymerization reactions, esterification, and
interesterification.1–3 The lipase Candida sp.99-125 has
good esterification ability without positional specificity for
triglycerides and has potential in the synthesis of short
chain esters as flavor compounds or biofuels such as
biodiesel.4–6 In practice, a free Candida sp.99-125 lipase
has inherent problems, such as stability, recovery from the
reaction system, and operational cost. However, immobi-
lized enzymes have the advantages of stability and sepa-
ration and thus can avoid these problems.7
∗Author to whom correspondence should be addressed.
J. Nanosci. Nanotechnol. 2011, Vol. 11, No. 9
1533-4880/2011/11/7593/010
doi:10.1166/jnn.2011.4731
7593

Synthesis of Retinyl Palmitate Catalyzed by Candida sp.99-125 Lipase
Kai et al.
as the supports leads to development of nanosize reactors
and sensors. Among the mesoporous materials, nanoor-
ganized materials have received considerable attention in
the adsorption of large molecules such as enzymes and
vitamins because of their well-ordered pore structure with
high specific surface areas and pore volume. Recently, high
enzyme loading has attracted much attention in the area of
enzyme immobilization.32–34
2. MATERIALS AND METHODS
2.1. Materials
Candida sp.99-125 lipase (68.06 U/mg) was purchased
from Beijing CTA New Century Biotechnology Co. Ltd.
(PR China). The p-nitrophenyl acetate (p-NPB) was
obtained from TCI (Shanghai). Poly(ethylene glycol)-
block-poly(propylene glycol)-block-poly(ethylene glycol)
(P123) was obtained from Sigma-Aldrich in the highest
grade commercially available. Vitamin A acetate (96.5%)
was purchased from Xin Hecheng Biochemical Corp.
(China); palmitic acid was of analytical grade and obtained
from Sinopharm Group Chemical Reagent Co., Ltd.
(China); Vitamin A palmitate (99%) was obtained from
SIGMA Co. Ltd.(USA); Methanol, ethanol, n-propanol,
iso-propanol, butanol, hexane and other reagent were pro-
vided by Sinopharm Chemical Reagent Co., Ltd. All
reagents and solvents were of AR grade.
Vitamin A plays an important role in the control of anti-
aging skin and cancer, et al. Vitamin A and its derivatives
have been widely used in food, cosmetics and drugs.35–39
Vitamin A and retinyl acetate were the most commonly
used in vitamin A series, but they were readily oxi-
dized by light, air, oxidizing agents or heat.40 Now a
amount of researches have focused on the development
of the stable synthesis of retinyl derivatives, in particu-
lar, retinyl palmitate. At present, retinyl ester was man-
ufactured by chemical methods using inorganic alkaline
catalysts at high temperature, and the unstable retinol
and retinyl ester result in the formation of by-products
easily. At the same time, there were high energy con-
sumption, high requirements for equipment and environ-
mental pollution.41ꢀ42 Lipase-catalyzed reactions can avoid
these problems. Lipase-catalyzed reactions were superior
to conventional chemical methods owing to their mild
reaction conditions, high catalytic efficiency and the inher-
ent selectivity of natural catalysts that have been used in
2.2. Preparation of Fiber-Like SBA-15 Mesoporous
Material
The SBA-15, which was prepared in our laboratory, was
prepared using reported procedures.6ꢀ12 The P123 was used
as a surfactant template. The molar composition of the
mixture was 1 TEOS: 0.017, P123:5.7, HCl:173 H2O.
First, 4 g P123 was dissolved in the mixture of 30 g water
and 120 g 2 mol/L HCl aqueous solution, the resulting
solution was stirred at room temperature until the solu-
tion became clear. Then, 8.5 g TEOS was added drop
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the synthesis of a variety of esters, in line with today’s
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“green chemistry” requirement, which has become the
Copyright: American Scientific Publishers
development direction of the synthesis of retinyl esters in
future.
ꢀ
ꢀ
wise into the solution of P123 in acid media at 40 C.
After TEOS was added, the mixture was stirred at 40 C
In this study, we described the preparation of fiber-
like SBA-15 and a method of immobilizing the Candida
sp.99-125 lipase via physical adsorption in the channels
of the fiber-like SBA-15 support. The hydrolytic activity
of the Candida sp.99-125 lipase was determined by fol-
lowing the formation of the product, p-nitrophenol (pNP),
from hydrolysis of p-nitrophenyl acetate. The influences of
buffer pH and time on the amount of Candida sp.99-125
lipase immobilized onto fiber-like SBA-15 were reported.
The thermal stability of immobilized and free Candida
sp.99-125 lipase as well as reuse of immobilized Can-
dida sp.99-125 lipase, the effects of various factors such
as solvents, substrates, immobilized lipase lifetime and
absorbent agent, on esterification were studied. In the
investigation of the transesterification of retinyl actate and
palmitic acid, the author discovered that the immobilized
Candida sp.99-125 lipase enhance the yield of the transes-
terification reaction. This has demonstrated the possibility
of the immobilized Candida sp.99-125 lipase accelerat-
ing enzyme-catalyzed reaction in organic phase. But so
far the immobilized Candida sp.99-125 lipase adopted in
Vitamin A esterification has not been reported. In this
paper, we also have studied the immobilized Candida
sp.99-125 lipase-catalyzed synthesis of retinyl ester in
organic solvent.
for 24 h. This mixture was then transferred into a Teflon-
lined autoclave and heated to and kept at 100 ꢀC for
24 h. Finally, white precipitates were collected, filtered,
and washed with water, air-dried at room temperꢀature, and
ꢀ
calcined at 550 C for 5 h at a heating rate of 1 C·min−1
in a tube furnace to remove the organic template.43ꢀ44
2.3. Immobilization of Lipase on SBA-15
SBA-15 is a kind of pure silica material consisting of SiO2
units with free hydroxyl groups on its surface that can
form hydrogen bonds with the functional groups of the
side chains of amino acids of enzyme. Besides this force, a
weak van der Waals interaction also assists in the enzyme
adsorption on the surface of the support. Hydrophobic
interactions contribute to adsorption by interactions of
hydrophobic patches on the enzyme with the silica net-
work of support. Moreover, Candida sp.99-125 lipase has
a molecular dimension of 3 nm×4 nm×5 nm and hence
the Candida sp.99-125 lipase may enter the pores of fiber-
like SBA-15 (pore size 7.1 nm).
A total of 20 mg of activated SBA-15 was dispersed in
2 ml phosphate buffer solution (pH 7.0, 0.1 mol/L) con-
taining approximately 8 to 24 mg Candida sp.99-125. The
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Kai et al.
Synthesis of Retinyl Palmitate Catalyzed by Candida sp.99-125 Lipase
container with this mixture was put in an ice-water system,
and then the suspensions were separated by centrifugation
and washed with phosphate buffer (pH 7.0, 0.01 mol/mL)
three times. The amount of Candida sp.99-125 immo-
bilized on the SBA-15 was determined by measuring
the concentration of protein in the supernatant using the
Bradford protein assay method.45
2.5. Thermal Stability of Free and Immobilized
Lipase
Thermal stabilities of free and immobilized enzymes were
determined by measurꢀing their residual activities after they
were incubated at 50 C for 8 h.
2.6. Synthesis of Retinyl Palmitate
ꢁCi −Cf ꢂ
The transesterification reactions were conducted in a series
Pa =
×V
(1)
W
of 25 ml screw-caped glass vials. 0.04 g lipase was added
into 10 mL hexane containing 0.164 g retinyl acetate and
0.32 g palmitic acid. The reaction was carried out by agi-
where Pa is the amount of immobilized enzyme onto car-
riers (mg · g−1, mg enzyme per gram silica); Ci and Cf
were the concentration of the enzyme initial and final in
the reaction medium (mg/mL), respectively; V is the vol-
ume of the reaction medium (mL); and W is the weight of
the carriers (g).
ꢀ
tation at 180 oscillations/min and 30 C. These conditions
were used unless otherwise stated. At 1–6 h intervals, the
yield of samples was detemined by HPLC. The yield of
retinyl palmitate was defined as:
Mp
Transesterification yield ꢁ%ꢂ =
×100
(5)
Ma
0
2.4. Activity Assays of Free and Immobilized Lipase
where Mp was the mole of retinyl palmitate in the product,
and Ma0 was the mole of retinyl acetate added.
The activity of free and immobilized Candida sp.99-125
lipase was determined by using p-nitrophenyl acetate as a
substrate according to previously reported assay method.
The reaction mixture (3.0 mL) was composed of 2.7 ml
phosphate buffer (pH 6.5, 0.01 mol/L), enzyme solution
of appropriate dilution (0.15 mL) and appropriate immo-
2.7. Determination of Initial Rate
The transesterification reactions were conducted in a series
of 25 ml screw-caped glass vials. 0.04 g lipase was added
into 10 mL hexane containing 0.164 g vitamin A acetate
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bilized Candida sp.99-125 (approximately 3 to 5 mg)
IP: 186.190.27.212 On: Sat, 13 Feb 2016 12:50:21
where the buffer was used for the blank. The mixture was
and 0.32 g palmitic acid. After 30 min, the yield of sam-
ples was detemined by HPLC. The initial rate of transes-
terification reaction was defined as the increase of retinyl
palmitate in unit time.
Copyright: American Scientific Publishers
ꢀ
incubated for 2 min at 37 C. Reactions were initiated
by adding 0.15 ml of p-nitrophenyl acetate solution and
stopped by addition of acetone (3 mL) after 10 min. The
mixtures were clarified by filtration, and the absorbance
of solution resulting from the release of p-nitrophenol
was measured at 400 nm. One unit of lipase activity was
defined as the amount of enzyme that liberates 1 ꢃmol of
p-nitro-phenol per min. The specific activity is the num-
ber of lipase units per mg of enzyme protein. The cou-
pled yield and the relative activity and were defined as
follows.46
2.8. HPLC Analysis of Retinyl Palmitate
Quantitative analyses of the reactants and the products
were obtained by a HPLC with SCL-10AVP system
(Shimadzu, Japan). The detector was SPD-10AVP UV/V,
which was operated at 364 nm. And a reversed-phase col-
umn (Alltech C18 250 mm × 4ꢄ6 mm, 5 ꢃm) was used.
A 20 ꢃL sample of proper dilution of the reaction mix-
ture was injected. 100% methanol was used as the mobile
Ci −Cf
Coupled yield ꢁ%ꢂ =
×100
(2)
Ci
phase at room temperature at a flow rate of 1 mL·min−1
.
A
Ra ꢁ%ꢂ =
A0
3. RESULTS AND DISCUSSION
×100
(3)
(4)
3.1. Structure of SBA-15 Mesoporous Material by
SEM and TEM
B
Realtive activity ꢁ%ꢂ =
×100
Bm
Figures 1(a) and (b) shows scanning electron micrograph
(SEM) imagesꢀof calcined fiber-like SBA-15 material pre-
pared at 100 C. The SBA-15 has a fiber-like morphol-
ogy, by stacking and coupling of rod-like particles of
which respective length and width were approximately
6 ꢃm and 0.5 ꢃm. Typical TEM images of the fiber-like
SBA-15 were shown in Figures 1(c) and (d). The presence
where Ra is the activity recovery of the immobi-
lized lipase (%), A is the activity of the immobi-
lized enzyme (U·mg−1ꢂ, A0 is the activity of the equal
weight free enzyme (U · mg−1ꢂ, B is the activity of the
free/immobilized enzyme (U·mg−1ꢂ, and Bm is the maxi-
mum activity of the free enzyme in solution (U·mg−1ꢂ.
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Synthesis of Retinyl Palmitate Catalyzed by Candida sp.99-125 Lipase
Kai et al.
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Copyright: American Scientific Publishers
Fig. 1. Scanning electron micrographs of SBA-15 (a) and (b) and transmission electron micrographs of SBA-15 (c) and (d).
of ordered arrays of silica channels with wall thickness
(approximately 3.4 nm) and uniform diameter (approxi-
mately 7.1 nm) were clearly observed, which was fitting
for the entrapment of Candida sp.99-125 lipase molecules
sized 3 nm×4 nm×5 nm.
b
3.2. FTIR Spectra of Immobilization of Candida
a
sp.99-125 lipase
To confirm that Candida sp.99-125 lipase was immobilized
on SBA-15, the Fourier transform infrared (FTIR) spec-
tra of SBA-15, Candida sp.99-125 lipase, and immobi-
lized Candida sp.99-125 lipase were studied. As shown in
Figure 2, the typical Si–O–Si bonds around 1082, 798 and
462 cm−1 were associated with the formation of a con-
densed silica network. The weak band in the range of 940–
960 cm−1 was assigned to non-condensed Si–OH groups.
After Candida sp.99-125 lipase was adsorbed, the intensity
of this band dramatically decreased. The result indicates
that a hydrogen bonding interaction occurred between free
Si–OH groups and the N–H groups of Candida sp.99-125
lipase. Furthermore, absorption bands associated with C–H
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber cm–1
Fig. 2. FT-IR spectra of silica sphere (a) and silica spheres adsorpted
with Candida sp.99-125 Lipase (b).
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Kai et al.
Synthesis of Retinyl Palmitate Catalyzed by Candida sp.99-125 Lipase
stretching (between 2885 and 2995 cm−1ꢂ, C–O stretching
in Table I. According to Table I, a maximal enzyme
(1577 cm−1ꢂ, and C–H deformations (around 1484 cm−1
were observed in the spectra of Candida sp.99-125 lipase
and immobilized Candida sp.99-125 lipase but not in the
spectra of SBA-15. These results confirmed that the Can-
dida sp.99-125 lipase had been successfully immobilized
on the SBA-15.9
ꢂ
activity recovery is about 2 times higher than that of
the free lipase. The activity recovery of the immobi-
lized Candida sp.99-125 lipase increases with enhance-
ment of reaction time and the maximum activity recovery
is 221.05% obtained under immobilization proceeding for
60 min, however, it decreases when the reaction time
is further prolonged. Candida sp.99-125 lipase molecules
form gradually a monolayer adsorption in 60 mins, and
the increase of activity recovery with an increment of
adsorption time can be explained in terms of available
space for the adsorbed protein molecules to optimize
their interactions with the adsorbent surface. However, if
the adsorption time of Candida sp.99-125 lipase is more
than 60 mins, Candida sp.99-125 lipase molecules form
a multilayer adsorption and the overlay and aggregation
of the adsorbed enzyme molecules would increase accord-
ingly, which cause that some of enzymatic activities can-
not be expressed. The adsorbed amount and immobilized
Candida sp.99-125 lipase activity at different time are
shown in Table I. The adsorbed amount of the Candida
sp.99-125 lipase increases with enhancement of immobi-
lization time, and the maximum immobilization percent of
Candida sp.99-125 lipase on fiber-like SBA-15 is up to
90.6%. Moreover, there is no obviously increasing amount
adsorbed of the Candida sp.99-125 lipase after 360 mins.
3.3. N2 Adsorption–Desorption Isotherm
Figure 3 reports the 77 K nitrogen adsorption–desorption
isotherms of the fiber-like SBA-15 material before
and after Candida sp.99-125 lipase was adsorbed. The
isotherms can be classified as type IV and exhibit an
H1-type hysteresis loop at high relative pressure. These
were typical features of hexagonal cylindrical channel
mesoporous materials. Compared to SBA-15, SBA-15
after Candida sp.99-125 lipase adsorption shows a marked
decrease in the specific surface area (from 575.49 m2 ·g−1
to 440.72 m2 ·g−1ꢂ and pore volume (from 0.966 m3 ·g−1
to 0.874 m3 ·g−1ꢂ. The pore size of the SBA-15 before and
after Candida sp.99-125 lipase was adsorbed was around
7.10 nm. Similar results have been reported for the adsorp-
tion of lysozyme, cytochrome c, porcine pancreas lipase,
or CRL (lipase from Candida rugosa) onto MCM-41 and
SBA-15.47–49 The decrease in surface area and pore vol-
ume distribution suggests that the immobilization of Can-
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dida sp.99-125 lipase in the channels of SBA-15 has
At this time, electrostatic repulsion and attraction between
IP: 186.190.27.212 On: Sat, 13 Feb 2016 12:50:21
Copyright: American Scientific Publishers
occurred.
adsorbed proteins reach to the equilibrium. In this case,
lipase immobilization reaches the adsorption equilibrium.
However, only 109.2% of the activity recovery is retained
on the support at 360 mins. The results from Table I show
that higher loadings do not result in significant increase in
activity recovery on support. This may be due to longer
incubation time would imply more enzyme-support links
as a result of multilayer adsorption of enzyme and for-
mation of enzyme aggregates which may decrease the
enzyme activity, as it may prevent the interaction of the lid
with external hydrophobic surfaces. Moreover, the enzyme
immobilized may become a barrier to products and sub-
strates diffusion.
3.4. Effect of Reaction Time on Amount Adsorbed
and Activity Recovery of Immobilized Candida
sp.99-125 Lipase
The effect of reaction time on activity recovery (Ra)
of the immobilized Candida sp.99-125 lipase is shown
700
SBA-15
SBA-15
SBA-15-lipase ADS
SBA-15-lipase DES
ADS
DES
600
500
400
300
200
100
0
Table I. The amount adsorbed and immobilized Candida sp.99-125
lipase activity at different time (pH 7.0 phosphate buffer solution).
Time
(min)
Pa
activitya
(U·(g·carrier)−1
(mg·(g·carrier)−1
ꢂ
IY (%)
ꢂ
Ra (%)
0
0ꢄ0
59ꢄ1
92ꢄ7
121ꢄ1
116ꢄ9
128ꢄ7
135ꢄ9
0ꢄ0
39ꢄ4
61ꢄ8
80ꢄ7
77ꢄ9
85ꢄ8
90ꢄ6
0ꢄ0
6093ꢄ5
0ꢄ0
151ꢄ5
161ꢄ5
221ꢄ1
175ꢄ5
129ꢄ4
109ꢄ2
15
30
60
90
150
360
10188ꢄ7
18211ꢄ6
13959ꢄ6
11333ꢄ7
10100ꢄ3
0.0
0.2
0.4
0.6
0.8
1.0
Relative pressure P/P0
Fig. 3. N2 adsorption–desorption isotherms of SBA-15 before Candida
sp.99-125 lipase was adsorbed.
aThe activity of free Candida sp.99-125 lipase: 68.08 U·mg−1
.
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Synthesis of Retinyl Palmitate Catalyzed by Candida sp.99-125 Lipase
Kai et al.
3.5. Effect of Reaction pH on Amount Adsorbed and
Activity Recovery of Immobilized Candida
sp.99-125 lipase
3.6. Effect of pH on Activity of Free and Immobilized
Candida sp.99-125 lipase
Effects of pH on the enzymatic activity of the free
and immobilized Candida sp.99-125 lipase for hydrol-
ysis of p-nitrophenyl acetate was determined with pH
ranging from 5.0 to 9.0, and the results were shown in
Figure 4. Although the maximum values of relative activity
were observed at pH 8.0 and 7.0 for free and immobi-
lized Candida sp.99-125 lipase, the immobilized Candida
sp.99-125 lipase holds excellent adaptability in a wider pH
range (5.0 to 9.0) compared to free Candida sp.99-125
lipase, and the shape of the enzymatic activity isotherm
of the immobilized Candida sp.99-125 lipase was nor-
mally bell-shaped. It was known that polyionic matrices
cause the partitioning of protons between the bulk phase
and enzyme microenvironment causing a shift in the opti-
mum pH value.50The adaptability should depend on the
method of immobilization, as well as the structure and
charge of the carrier; furthermore, the screen function of
the structure would play an important role in enhancing
the adaptability.51
Table II shows the amount adsorbed of Candida sp.99-125
and immobilized enzyme activity at different pH. It
can be clearly seen from Table II that the amount
adsorbed is significantly affected by the solution pH. The
maximum adsorption amount of Candida sp.99-125 is
96.88 mgg−1 at pH 5.0 while only the activity recovery
is 121.3%. Although the amount adsorbed is 93.29 mg/g
at pH 7.0 which is lower than that at pH 5.0, the activity
recovery is 281.05%. The activity recovery is also clearly
a function of solution pH. It has been reported that the
maximum amount adsorbed of enzyme occurs at a pH near
the pI of the protein–substrate complex and not at the pI of
the protein. The candidates for the dominant driving forces
for the adsorption of Candida sp.99-125 include hydropho-
bic interactions (a kind of van der Waals attraction), elec-
trostatic repulsion and attraction between the amino acid
residues on the surface of Candida sp.99-125 and silanol
groups on the surface of the silica walls of the mesoporous
materials, the intramolecular cohesive attraction and repul-
sion. Hydrophobic interactions are more dominant near the
pI than electrostatic interactions. These hydrophobic inter-
actions originate from
3.7. Effect of Temperature on Activity of the Free and
Immobilized Enzymes
The effect of the reaction temperature on the enzy-
matic activity of the free and immobilized Candida
sp.99-125 lipase was shown in Figure 5. The optimum
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(1) the interaction between the nonpolar side chains of the
IP: 186.190.27.212 On: Sat, 13 Feb 2016 12:50:21
amino acids residues on the surface of Candida sp.99-125
and surface siloxane bridges or
Copyright: American Scientific Publishers
reaction temperatures of free and immobilized Candida
sp.99-125 lipase were 40 ꢀC and 50 ꢀC, respectively.
At other temperatures, the activity of the immobilized
Candida sp.99-125 lipase was higher than that of the free
Candida sp.99-125 lipase. In other words, the immobi-
lized Candida sp.99-125 lipase holds more heat resis-
tance than that of free Candida sp.99-125 lipase between
45 ꢀC and 60 ꢀC. The increasing temperature will be
suitable for adjusting the conformational integrity of the
(2) from the lipase–lipase interactions between the
hydrophobic side chains of neighboring Candida
sp.99-125 molecules adsorbed on the surface of SBA-15.
There is a decrease of the amount adsorbed of Candida
sp.99-125 from pH 5.0 to pH 9.0. This can be inter-
preted as follows: charged amino acid residues on the
protein surface change from positive charges to negative
charges gradually with the increase of pH. Both the surface
of the protein molecule and the silica surface are nega-
tively charged, which creates a strong electrostatic repul-
sion between the enzyme and the absorbent and leads to a
low amount of adsorption.
110
100
90
80
70
60
Table II. The amount adsorbed of Candida sp.99-125 lipase and immo-
bilized enzyme activity at different pH levels (incubation time 1 h).
50
Pa
activitya
40
pH
(mg·(g·carrier)−1
ꢂ
IY (%)
(U (g·carrier)−1
ꢂ
Ra (%)
30
20
10
free lipase
immobilized lipase
5
6
7
8
9
96.88
93.99
93.29
56.28
36.79
96.88
93.99
93.29
56.28
36.79
8000.45
9501.63
17850.00
7100.99
4196.06
121.30
148.49
281.05
185.33
167.53
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
pH
Fig. 4. Variation of enzymatic activity versus medium pH immobilized
aThe activity of free Candida sp.99-125 lipase: 68.08 U·mg−1
.
lipase and free lipase.
7598
J. Nanosci. Nanotechnol. 11, 7593–7602, 2011

Kai et al.
Synthesis of Retinyl Palmitate Catalyzed by Candida sp.99-125 Lipase
free Candida sp.99-125 lipase, the thermal stability of
the immobilized sample was better than that of the free
sample. Thus the mesoporous pores of supports can keep
enzymes from injury resulting from direct exposure envi-
ronmental changes.
100
80
60
40
20
3.9. Effect of Different Lipases in Synthesis of
Retinyl Palmitate
free enzyme
immobilized enzyme
Six lipases from different sources and different specifici-
ties were assayed for synthesizing the vitamin A palmi-
tate, which was shown in Table III. Novozym 435 showed
good activity for the synthesis of retinyl palmitate and
the yield of retinyl palmitate reached 81.7%. More-
over the researches showed that Novozym 435 was the
ideally lipase of ester synthesis. And the immobilized
Candida sp.99-125 lipase on fiber-like SBA-15 materials
was almost as good as Novozym 435.54–59
30
35
40
45
50
55
60
Temperature(ºC)
Fig. 5. Effect of temperature on activity on the free and immobilized
Candida sp.99-125 lipase.
enzyme immobilization by adsorbing to the mesoporous
materials.52ꢀ53
3.10. Effect of Organic Solvent in Synthesis of
Retinyl Palmitate
As the reaction media of enzyme, organic solvents have a
huge effect on enzyme-catalyzed reaction. It can directly
affect the catalytic activity, stability, substrate specificity
of enzyme, the balance and rate of reaction. Laane used
polar parameters (logP) of organic solvents to describe
3.8. Thermal Stability of Free and Immobilized
Lipase
It was well known that enzymes in solution were not stable
and their activities also decrease gradually in the usage. To
investigate the thermal stability of immobilized Candida
sp.99-125 lipase on fiber-like SBA-15 materials, the resid-
ual activities of immobilized Candida sp.99-125 lipase and
free Candida sp.99-125 lipase were measured and com-
pared in Figure 6, when they were incubated at 50 ꢀC. The
residual activity of immobilized Candida sp.99-125 lipase
maintained comparative stability and held over 42% of
the hydrolysis activity at 6 h, whereas, that of the free
Candida sp.99-125 lipase decreased quickly with increas-
ing thermal treatment time and maintained only 25% of
the activity at 4 h. Comparing the immobilized to the
Delivered by Publishing Technoleofgfeycttoo:fMocrMgaansictesroUlvneinvtesrsointythe enzyme reaction (logP
IP: 186.190.27.212 On: Sat, 13 Feb 2016 12:50:21
was the partition coefficient of organic solvent in the
octanol/water system).59 We tested six kinds of commonly
used organic solvents, whose relative polarity and yield
show in Table IV catalyzed by immobilization of Candida
sp.99-125 lipase on SBA-15.
Copyright: American Scientific Publishers
As seen from Table IV, the yield increased while logP
of organic solvents increased under the same other con-
ditions. This further confirmed the Laane’s conclusion,60
hydrophobic solvent was in favor of esterification reaction.
As was seen from the table, hexane was the best solvent,
so we choose hexane as the reaction media in the follow-
ing trials.
100
80
60
40
20
0
free lipase
immobilized lipase
3.11. Effect of Substrates Molar Ratio on Reaction of
Synthesis of Retinyl Palmitate
Lipase-catalyzed transesterification reaction was
a
reversible reaction. To make a thorough reaction, the
Table III. Effects of different lipases on reaction.
Lipase
Maximum yield/%
Porcine pancreatic lipase
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Free Candida sp. lipase
Candida sp. lipase on SBA-15
0
81.7
34.1
28.3
50.3
75.2
0
2
4
6
8
Time(h)
Note: Reaction condition: solvent hexane up to 10 mL, lipase 0.04 g, Time 6 h,
CA = 0ꢄ05 mol/L, CB = 0ꢄ125 mol/L.
Fig. 6. Thermal stability of free and immobilized Candida sp.99-
125 lipase.
J. Nanosci. Nanotechnol. 11, 7593–7602, 2011
7599

Synthesis of Retinyl Palmitate Catalyzed by Candida sp.99-125 Lipase
Table IV. Effect of solvent on reaction.
Kai et al.
100
90
80
70
60
50
40
30
20
10
0
immobilized liase
free lipase
Solvent
logP
yield/%
Butanone
0ꢄ29
0ꢄ5
1ꢄ4
2ꢄ5
3ꢄ5
4ꢄ5
0
0
10ꢄ7
54ꢄ8
74ꢄ2
72ꢄ2
Tetrahydrofuran
Tert-amyl alcohol
Toluene
N-hexane
N-octane
Note: Reaction condition: solvent up to 10 mL, lipase 0.04 g, Time 6 h, CA
0ꢄ05 mol/L, CB = 0ꢄ125 mol/L.
=
reactant must be excess, or removed a certain product
during reaction. In order to improve yield of the retinyl
palmitate, we fixed the amount of retinyl acetate and
altered the amount of palmitic acid, then several different
substrate molar ratios were compared to determine the
optimal molar ratio. The results were shown in Figure 7.
From Figure 7, we can see the yield increase with
the increased molar ratio of palmitic acid and retinyl
acetate. When the molar ratio of palmitic acid and retinyl
acetate was 2.5:1, the yield was the highest of 74.6%.
The yield of esterification got balance when acid contin-
ued to increase, which showed that the substrate concen-
tration around enzyme molecules had reached a balance,
and it had no effect on the reaction balance to continue to
increase the amount of palmitic acid, and adding too much
0
1
2
3
4
5
6
7
8
9
10 11 12
Time (h)
Fig. 8. Effect of reaction time on yield.
Note: Reaction condition: solvent hexane up to 10 mL, lipase 0.04 g,
CA = 0ꢄ05 mol/L, CB = 0ꢄ125 mol/L.
little, and reaction was basically balanced. However, when
the reaction time was more than 10 h, the yield decreased
slightly. It may be that the long time reaction has a certain
negative impact on the structure of retinyl palmitate. That
was the structure of enzyme molecules tended to unreason-
able with long reaction time, so impeded enzyme molecule
itself exercise biological function and seemingly showed
their activity decreased and even inactivated which lead to
acid also caused a waste of raw materials. Therefore, the
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the descending the yield.
IP: 186.190.27.212 On: Sat, 13 Feb 2016 12:50:21
use of substrate molar ratio of 2.5:1 was appropriate.
Copyright: American Scientific Publishers
3.12. Effect of Reaction Time in Synthesis of
4. CONCLUSIONS
Retinyl Palmitate
The adsorption of Candida sp.99-125 lipase on the meso-
porous fiber-like SBA-15 from buffer solutions with
different pH values and times were studied as a model pro-
tein adsorption system. The adsorbed amount depends on
the solution pH and time.61 The optimal pH and tempera-
ture regions of the immobilized Candida sp.99-125 lipase
for ꢀthe hydrolysis of p-nitrophenyl acetate were 5 and
35 C, respectively. Meanwhile, the immobilized Candida
sp.99-125 lipase exhibits much better adaptability com-
pared to frꢀee enzyme at pH 5.0 to 9.0 and at a tempera-
ture of 50 C. The immobilized Candida sp.99-125 lipase
showed higher thermal stability than that of free Candida
sp.99-125 lipase. Screening the enzyme and media, we
found that the immobilized Candida sp.99-125 lipase was
suitable for the reaction of synthesis of retinyl palmitate,
while the best medium was hexane. We also determine
the optimal reaction conditions: 0.164 g retinyl acetate and
0.32 g palmitic acid, 10 mL solvent hexane, 1:4 of mass
ratio of lipase to retinyl acetate, and 6 hours of reaction
time, 74.6% of retinyl acetate was converted into retinyl
plamitate.
We have studied the effect of reaction time on esteri-
fication of retinyl palmitate. The results were shown in
Figure 8, with the increase in reaction time the yield con-
tinued to increase, after 6 h the variation of yield was
100
immobilized liase
90
free lipase
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
Palmitic acid / Retinyl acetate (mol/mol)
Fig. 7. Effect of substrates molar ratio on yield.
Acknowledgments: Thank for the National Basic
Research Program of China (No 2009CB724700), the
Note: (Reaction condition: solvent hexane up to 10 mL, lipase 0.04 g,
Time 6 h, CA = 0ꢄ05 mol/L).
7600
J. Nanosci. Nanotechnol. 11, 7593–7602, 2011

Kai et al.
Synthesis of Retinyl Palmitate Catalyzed by Candida sp.99-125 Lipase
financial support of Youth Foundation of Nanjing Univer-
sity of Technology (39704011), the financial support of
the Innovation Fund for Doctoral Theses and the Nanjing
University of Technology (No. BSCX200811) and the
Foundation of the Nanjing University of Technology (No.
39708006) support.
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Nomenclature
Ra activity recovery of the immobilized lipase, %
A
activity of the immobilized enzyme, U·mg−1
activity of the free/immobilized enzyme, U·mg−1
A0 activity of the equal weight free enzyme, U·mg−1
B
Bm maximum activity of the free enzyme in solution,
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Pa amount of immobilized enzyme onto carriers,
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Ci concentration of the enzyme initial in the reaction
medium, mg/mL
Cf concentration of the enzyme final in the reaction
medium, mg/mL
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V
W
M
volume of the reaction medium, mL
weight of the carriers, g
the mole of retinyl palmitate in the product, mol
Ma0 the mole of retinyl acetate added, mol
31. B. A. Akhoon, S. K. Gupta, and V. Verma, J. Biomed. Nanotechnol.
CA the mole concentration of retinyl acetate, mol/L
CB the mole concentration of palmitic acid, mol/L
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Received: 24 January 2011. Accepted: 17 March 2011.
Delivered by Publishing Technology to: McMaster University
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Copyright: American Scientific Publishers
7602
J. Nanosci. Nanotechnol. 11, 7593–7602, 2011