Zhao 2017

www.ejlst.
com European Journal of Lipid Science and Technology
Research Article
Accept e d Article
Immobilization of Candida rugosa lipase on glutaraldehyde-activated
Fe3O4@chitosan as a magnetically separable catalyst for hydrolysis of castor oil†
Running title: Optimized hydrolysis of castor oil
Ke Zhao, Bang Chen, Cong Li, Xing-fu Li, Ke-bin Li* and Ye-hua Shen**
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of
Education, College of Chemistry and Materials Science, Northwest University, Xi’an,
Shaanxi, China
*Corresponding author. Yehua Shen**, Tel.: +86 029 88302635.
E-mail address: nwu-yhshen-lab@vip.163.com;
Kebin Li*, Tel.: +86 132 5988 2543.
E-mail address: kebin68li@163.com
Keywords: magnetic materials, castor oil, hydrolysis, Candida rugosa lipase,
heterogeneous catalyst
†
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
[10.1002/ejlt.201700373].
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: August 29, 2017 / Revised: August 29, 2017 / Accepted: September 27, 2017
www.ejlst.com European Journal of Lipid Science and Technology
Abstract
Accept e d Article
In this study, the catalysed hydrolysis of castor oil by Candida rugosa lipase (CRL)
immobilized on glutaraldehyde-activated magnetic Fe3O4@chitosan was investigated.
Magnetic Fe3O4 was prepared by hydrothermal method and coated with chitosan (CS).
Next, CRL was immobilized on Fe3O4@chitosan using glutaraldehyde as a cross-linking
reagent. The prepared Fe3O4@CS@CRL was confirmed by scanning electron microscopy
(SEM), X-ray powder diffraction (XRD), vibrating sample magnetometry (VSM),
Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA).
The response surface methodology (RSM) based on the Box–Behnken design was used to
evaluate and optimize the hydrolysis reaction variables. The optimum reaction conditions
for the hydrolysis of castor oil by the Fe3O4@CS@CRL heterogeneous catalyst were
found to be a water/oil ratio of 1.60:1, pH of 7.05, reaction temperature of 34 °C, and
lipase concentration of 3.27%; under these conditions, the hydrolysis conversion of castor
oil reached 46.81%. Moreover, the immobilized lipase showed high stability with no
appreciable loss in its activity after three consecutive cycles.
Practical applications: RSM was found to be a useful technique for optimizing

hydrolysis of castor oil. The high conversion of the hydrolysis of castor oil indicates that
the Fe3O4@CS@CRL has potential to be used in preparing ricinoleic acid from castor oil.
Ricinoleic acid have the potential to be used in printing ink as a pigment and dye
disperser, plasticizers, surfactants, lubricants and other valuable products.
Abbreviations: CRL, Candida rugosa lipase; CS, chitosan; Fe3O4@CS, magnetic Fe3O4
coated with chitosan; Fe3O4@CS@CRL, magnetic Fe3O4@CS immobilized Candida
rugosa lipase; SEM, scanning electron microscopy; XRD, X-ray powder diffraction;
VSM, vibrating sample magnetometry; FTIR, fourier transform infrared spectroscopy;
TGA, thermogravimetric analysis; PBS, phosphate buffer solution, RSM, Response
surface methodology.
1. Introduction
Accept e d Article
Castor oil is a pale amber, viscous, non-edible oil that is obtained by extracting or
expressing the seeds of the plant Ricinus communis[1]. India is the world’s largest
exporter of castor oil, with a 70% of share of total exports. Other major producers include
China and Brazil[2]. Castor oil is used as a vital raw material for different types of
products in many chemical industries[3]. The broad and versatile use of castor oil comes
from its main component, ricinoleic acid (C18H34O3, cis-12-hydroxy-9-octadecenoic acid),
which represents approximately 90% of the triglycerides in castor oil[4,5]. Therefore, one
of the most important trends in the use of castor oil is to obtain ricinoleic acid[6]. The
functional groups in ricinoleic acid, namely, hydroxyl group, double bond and carboxyl
group, provide reaction sites for the preparation of a variety of useful industrial
derivatives[7]. To date, numerous studies on the application of ricinoleic acid have been
reported, including in printing ink as a pigment and dye disperser[7], sebacic acid[8],
azelaic acid[5], heptaldehyde[1], plasticizers[9], surfactants[10], lubricants[11] and other
valuable products[2,3].
The conventional methods used to hydrolyse castor oil to ricinoleic acid employs a
saponification reaction followed by acidification, high-temperature and high-pressure
process. However, these processes can give the product an undesired odour and colour
from the unwanted side product ricinoleic acid estolide or from product denaturation
[12-14]. Compared to the conventional methods, castor oil hydrolysis catalysed by lipase
can overcome these drawbacks. The main advantage of lipase-catalysed oil hydrolysis is
that it operates under mild conditions and can produce better quality products[5,7,14]. To
enhance the properties of lipase-catalysed reactions, including the reusability, stability
and recovery, most of the lipases used in industrial processes are immobilized on various
carriers[15,16]. As a result, various different kinds of supports, including synthetic
resins[17], inorganic polymers (mesoporous silica[18] and zeolites[19] etc.), and
synthetic organic polymers[20], have been developed to immobilize lipases.
In recent years, a significant number of magnetic composite materials have been

Accept e d Article
developed for enzyme immobilization[21-23]. The magnetic property of these materials
is of great interest because magnetic separation is quick, simple, eco-friendly and more
effective than filtration or centrifugation[24,25]. However, naked magnetic particles are
not capable of stabilizing enzymes due to their lack of appropriate functional groups. To
overcome this limitation, an additional inorganic or polymeric coating layer is necessary
for binding biomolecules[26].
Chitosan, which is non-toxic, hydrophilic, biocompatible and biodegradable, is one
of the most frequently used materials in the design of modified magnetic particles[27].
The presence of highly reactive amino and hydroxyl groups allows the resulting particles
to be tailored to specific applications. Therefore, chitosan was chosen for modifying
magnetic particles in this study.
In recent decades, numerous studies on the hydrolysis of castor oil using various
lipases have been reported. Puthli et al. reported the hydrolysis of castor oil by a lipase
from Aspergillus oryzae, resulting in 40% conversion in 6 h[5]. Lipases from
Pseudomonas sp. f-B-24[28], castor bean itself[29] and conjugated lipolase[9] were also
used. Candida rugosa lipase (CRL) is an important industrial lipase, and due to its broad
substrate specificity, it has been successfully utilized in a variety of hydrolysis reactions.
Goswami et al. reported the bioconversion of castor oil into ricinoleic acid using CRL
with 63% conversion in 6 h[13]. The results demonstrated that CRL performed better in
the hydrolysis of castor oil than lipases from other sources[21]. Based on this knowledge,
CRL was chosen for this study. Even though the hydrolysis of castor oil using CRL
achieved 63% in 6 h in the studies performed by the Goswami group, the CRL that was
used in their system was not immobilized and could not be reused. Therefore, we
attempted to hydrolyse castor oil using immobilized CRL. To the best of our knowledge,
no studies on the hydrolysis of castor oil using immobilized CRL have been reported.
In this study, a heterogeneous catalyst was developed by immobilizing lipase on a
magnetic material for the hydrolysis of castor oil to ricinoleic acid. To this end, Fe3O4
Accept e d Article
particles were first prepared by hydrothermal method using H2O2 as an oxidizer. Next,
CS and Fe3O4 aqueous slurry were mixed in the appropriate ratio in a reverse-phase
suspension cross-linking method to form Fe3O4@CS. Finally, CRL was immobilized on
Fe3O4@CS using glutaraldehyde as a cross-linking reagent (see Scheme 1). The structure
and magnetic properties of the resultant magnetic particles were characterized by
scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and vibrating
sample magnetometry (VSM). The binding of CS to Fe3O4@CS was confirmed by
Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric analysis (TGA).
Moreover, the Box-Behnken design combined with Response surface methodology (RSM)
was used to optimize the reaction conditions and better understand the relationships
between the reaction variables (water/oil ratio, pH, reaction temperature and lipase
concentration) and the response (hydrolysis conversion). Finally, the repeated use of
Fe3O4@CS@CRL was tested.
2. Materials and methods

2.1. Materials
Candida rugosa lipase (CRL, Type VII, ≥700 unit/mg solid, EC 3.1.1.3) was
purchased from Sigma–Aldrich Co. (Germany). Castor oil was purchased from Jinan
Yutong Chemical Co., Ltd. Chitosan (CS, degree of deacetylation > 95%) was purchased
from Xi’an Zhoudingguo Biotech Co., Ltd. A 25% glutaraldehyde solution was purchased
from Tianjin Tianli Chemical Reagent Co., Ltd. Ferrous sulphate heptahydrate and an
aqueous ammonia solution were purchased from Guangdong Guanghua Sci-Tech Co.,
Ltd. All other chemicals were of analytical grade and used without further purification.
2.2. Immobilization of Candida rugosa lipase on Fe3O4@CS

Accept e d Article
Fe3O4 was prepared by hydrothermal method according to a previously reported
procedure[26]. Magnetic Fe3O4 particles coated with chitosan (Fe3O4@CS) were prepared
following previously reported procedures[26].
The procedure used to prepare Fe3O4@CS@CRL is illustrated in Scheme 1. First,
the primary amino groups of the Fe3O4@CS particles were activated using
glutaraldehyde[30]. The as-prepared Fe3O4@CS particles (0.1 g) were dispersed in 50 mL
of a 5% glutaraldehyde phosphate buffer solution (PBS, 0.02 M, pH 7.0). Subsequently,
the mixture was incubated at room temperature in a shaker for 10 h at 150 rpm. After
incubation, the glutaraldehyde-activated Fe3O4@CS particles were magnetically
separated and washed 2 times with PBS (0.02 M, pH 7.0). A lipase solution (2 mg/mL)
was prepared in 0.02 M PBS (pH 7.0), mixed with 0.1 g of the glutaraldehyde-activated
Fe3O4@CS support and incubated at room temperature at 150 rpm for 5 h. At the end of
the incubation period, the immobilized CRL (Fe3O4@CS@CRL) was magnetically
separated, washed with PBS at pH 7.0 (5×2 mL) to remove excess CRL, and stored at
4 °C prior to use.
2.3. Characterization of the magnetic particles
SEM images were collected using a Carl Zeiss SIGMA microscope. The crystal
structures of Fe3O4 and Fe3O4@CS were obtained using an X-ray diffractometer (XRD,
Bruker D8 advance) with Cu Kα radiation. FTIR spectra of CS, Fe3O4 and Fe3O4@CS
were recorded with KBr discs in the range 4000–400 cm−1 on a Bruker EQUINOX-55
Fourier transform infrared spectrophotometer. Magnetization curves of the dried
magnetic particles were recorded with an MPMS-XL-7 (Quantum Design) magnetometer.
Hysteresis of the magnetization was obtained by changing H between +6000 and −6000
Oe at 25 °C. TGA was carried out on powder samples using a NETZSCH STA 449C
thermogravimetric analyser. Samples weighing between 5 mg and 15 mg were heated
from 33 °C to 700 °C at a rate of 10 °C /min in air.
2.4. Enzymatic hydrolysis of castor oil

Accept e d Article
The hydrolysis of castor oil was carried out in a 100 mL capped flask on a shaking
incubator under ambient conditions. In a typical procedure, 10 g of castor oil, 10 mL of
PBS and the required amount of Fe3O4@CS@CRL were combined in the flask at 25 °C.
After 60 h, the catalyst was carefully filtered by magnetic separation and two layers
formed in the filtrate. The non-aqueous phase was used to determine the percent
hydrolysis of castor oil. The hydrolysis conversion of castor oil was determined according
to published procedures[9]. The liberated fatty acids were titrated with a 0.1 N NaOH
solution using phenolphthalein as an indicator. In addition, the initial acid value of the
castor oil was determined using the same method.
2.5. Response surface experimental design and statistical analysis
RSM is a powerful statistical technique for designing experiments, building models
and optimizing processes. In this study, RSM was applied to study the relationships
between several independent variables and the response[13]. A three-level-four-factor
Box–Behnken design was employed in this study to evaluate the effects of the various
parameters on the hydrolysis of castor oil by Fe3O4@CS@CRL and to determine the
optimum conditions. The factors selected for optimization of the hydrolysis conversion
included the water/oil ratio (X1), pH (X2), reaction temperature (X3) and lipase
concentration (X4). The study ranges included a water/oil ratio of 1:1-3:1, a pH of 6.5-7.5,
a reaction temperature of 30–40 °C, and a lipase concentration of 2-4%. Hydrolysis
conversion was chosen as the response. The actual and coded values of each parameter
are shown in Table 1. The coded values are designated by -1 (minimum), 0 (medium),
and +1 (maximum), and the Box-Behnken experimental design matrix along with the
hydrolysis conversion are provided in Table 2.
The experimental data (Table 2) obtained from the Box–Behnken design were
analysed with the RSM using the following second-order polynomial equation, which
was developed to describe the relationship between the predicted response variable and
the independent variable of the hydrolysis process:

Accept e d Article
k k k k
Y = b 0 + ∑ b i X i + ∑ b ii X i + ∑ ∑b XiX j + e
2
ij (1)
i =1 i =1 i＞＞ j
where Y is the predicted response variable (hydrolysis conversion), b0, bi, bii, and bij are
the intercept, linear, quadratic and interaction constant coefficients, respectively, k is the
number of factors studied and optimized in the experiment, e is the random error and Xi
and Xj are the encoded independent variables. Analysis of variance (ANOVA) and
response surfaces plots were generated using Design-Expert (Version 8.0.6.1) software.
2.6. Reusability assay
To test the stability of Fe3O4@CS@CRL, the Fe3O4@CS@CRL catalyst was
separated magnetically after each run. The used Fe3O4@CS@CRL catalyst was then
washed with tertiary butyl alcohol and PBS (0.02 M, pH 7.0) to remove any substrate or
product that had been retained on the support[30]. The recovered Fe3O4@CS@CRL
catalyst was further used in the next hydrolysis reaction.
3. Results and discussion

3.1. Characteristics of the magnetic particles
Fig. 1 shows the FTIR spectra of Fe3O4 (a), pure CRL (b), chitosan (c), Fe3O4@CS
(d) and Fe3O4@CS@CRL (e). As seen in Fig. 1a, the peak at 568 cm-1 corresponded to
the typical characteristics of Fe-O-Fe in Fe3O4[31]. In the FT-IR spectrum of chitosan
(Fig. 1c), the peak at 1596 cm-1 was due to an N-H bending vibration. The peak at 1380
cm-1 was due to -C-O stretching of the primary alcoholic group in CS. For Fe3O4@CS
(Fig. 1d), a new sharp peak at 570 cm-1 corresponding to the Fe-O stretching vibration
appeared. Compared with the FTIR spectrum of CS (see Curve c), the peak at 1596 cm-1
corresponding to the N-H bending vibration shifted to 1594 cm-1. This shift indicated that
chitosan reacted with glutaraldehyde to form Schiff base[26,30,32]. The results further
indicated that chitosan was coated onto the magnetic Fe3O4 particles through
glutaraldehyde cross-linking[27,33]. A similar result was also reported by Zhu et al. and
Accept e d Article
Qu et al.[31,34]. In the FT-IR spectrum of Fe3O4@CS@CRL (Fig. 1e), a peak at 1658
cm-1 appeared that was assignable to the stretching vibrations of a C=N bond, suggesting
the formation of Schiff base via the reaction between the carbonyl group of
glutaraldehyde and an amine group[30]. The absorption peaks of Fe3O4@CS@CRL at
1095 cm-1 corresponded to the characteristic peaks of lipase, which were observed at
1106 cm-1 in the IR spectrum of pure lipase (see Curve b). Accordingly, CRL was indeed
chemically bound to the surface of the magnetic microspheres.
The structures of Fe3O4 and Fe3O4@CS were characterized by XRD techniques. Fig.
2 shows the XRD patterns of Fe3O4 (a) and Fe3O4@CS (b). For Fe3O4 (see Curve a), six
characteristic peaks occurred at 2θ values of 30.3°, 35.7°, 43.4°, 53.9°, 57.5°, and 63.1°
and were assigned to the (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) planes,
respectively. These peaks are consistent with the JCPDS file (PDF NO.75-0449) and
revealed that the particles consisted of pure Fe3O4 with a spinel structure. For Fe3O4@CS
(see Curve b), the same six characteristic peaks were also observed. This result indicates
that the coating process did not cause a phase change in the Fe3O4 particles[33].
Fig. 3 shows the SEM images used to evaluate the morphological structures of
Fe3O4 and Fe3O4@CS. It can be seen from Fig. 2(a) that the Fe3O4 particles were nearly
spherical or ellipsoidal. The mean particle size was 40 nm, and the magnetic Fe3O4
particles physically aggregated. This could have been caused by the coercive force of the
magnetic particles or the magnetic dipolar interactions between particles[27,35]. The
surface morphology of Fe3O4@CS is shown in Fig. 2(b). After coating with chitosan, the
surface morphology showed little change. The average size of the Fe3O4@CS particles
was 60 nm and physical aggregation was observed. This indicated that the Fe3O4 particles
were thoroughly coated with CS because the Fe3O4 particles were present in the pores of
the CS gel, which caused aggregation of the magnetic nanoparticles[24].
The CS content in the Fe3O4@CS particles was determined using a
thermogravimetric analyser. The thermogravimetric analyses (TGA) of chitosan (a),

Accept e d Article
Fe3O4 (b), and Fe3O4@CS (c) are show in Fig. 4. Pure chitosan (see Curve a) exhibited
slow weight loss from 140 °C to 250 °C that was caused by the decomposition of low
molecular weight species. The thermal decomposition was more pronounced in the region
250 °C to 450 °C due to complex dehydration of the saccharide rings, depolymerization,
and decomposition of the acetylated and deacetylated units of the polymer[32,36]. With
continued heating, a second process characterized by the release of CH4 occurred in the
range 450-700 °C and caused a modest weight loss of 7.5%. This step suggests that
further modification of the material occurs when the structure is almost completely
reduced, causing the production of methane and the consequent formation of a
graphite-like structure via a dehydrogenation mechanism, as suggested in the
literature[36]. The TGA curve of Fe3O4 (see Curve b) showed an approximately 3.5%
weight loss from 33 °C to 700 °C that resulted from the loss of residual water from the
sample. For the Fe3O4@CS particles (see Curve c), below 200 °C, the weight loss was
quite small and could be attributed to the removal of physically and chemically adsorbed
water. When the temperature increased above 500 °C, the weight loss was significant and
was related to the degradation of CS. No significant weight change was observed from
500 °C to 700 °C, implying the presence of only iron oxide within that temperature range.
The total weight loss of the Fe3O4@CS particles is 13.5%. Accordingly, the amount of CS
bound to the surface of the Fe3O4 nanoparticles was 10.0%.
3.2. Magnetic properties of the magnetic materials
The magnetic properties of Fe3O4 (a), Fe3O4@CS (b), and Fe3O4@CS@CRL (c)
were investigated by VSM at room temperature, and the results are displayed in Fig. 5.
The very weak hysteresis observed in Fig. 5 indicated that both the magnetic particles
were nearly superparamagnetic[37]. The saturation magnetization values of Fe3O4,
Fe3O4@CS, and Fe3O4@CS@CRL were found to be 35.1 emu/g (see Curve a), 32.1
emu/g (see Curve b), and 27.3 emu/g (see Curve c), respectively. The saturation
magnetization of Fe3O4@CS@CRL was high enough to facilitate separation from the

Accept e d Article
suspension using a strong external magnet. As seen in Fig. 5, the black Fe3O4@CS@CRL
particles were attracted to the walls of the vial and the supernatant became clear and
transparent within 1 min when an external magnet was used. The good magnetic
properties of Fe3O4@CS@CRL make it favourable for application in bioengineering
fields[38,39].
3.3. Optimization of the reaction conditions using response surface methodology
The Box-Behnken design combined with RSM was used to optimize the hydrolysis
reaction conditions. The parameters used for the hydrolysis reaction were based on the
results obtained of preliminary single-factor experiments. The results of the 29
experiments are shown in Table 2. The relationships between the independent variables
(X1, X2, X3 and X4) and the response (hydrolysis conversion) were established using a
quadratic polynomial equation (Equation 2) where C denotes the predicted hydrolysis
conversion and X1, X2, X3 and X4 are the water/oil ratio, pH, reaction temperature and
lipase concentration, respectively.
C = +45.58 − 3.15X1 + 1.18X 2 — 2.57X 3 + 2.02X 4 − 1.91X1X 2 + 0.33X1X 3 — 1.69X1X 4
(2)
+ 0.88X 2 X 3 − 0.97X 2 X 4 − 1.65X 3 X 4 − 4.80X1 − 7.52X 2 − 7.65X 3 − 5.35X 4
2 2 2 2
The analysis of variance (ANOVA) was performed to evaluate the significance and
adequacy of the model as well as the effects of the significant individual terms and their
interactions with the chosen response. The ANOVA result for the established quadratic
model is presented in Table 3.
The model F-value of 9.14 and P-value <0.0001 implied high significance of the
model. There is only a 0.01% chance that this "Model F-Value" occurred due to noise. An
F-value of 5.52 and a P-value of 0.0571 (P > 0.01) for lack of fit implied that it was not
significant relative to the pure error. The non-significant lack of fit indicates good
predictability of the model[40]. A large R2 value (close to 1) indicates that the model
provides a good fit to the experimental data[41]. An R2 value of 0.9014 was determined
in this study, implying that 90.14% of the experimental data are compatible with the data
Accept e d Article
predicted by the model. This result can be further consolidated into a plot of the predicted
hydrolysis conversion as a function of the experimental values (Fig. 6). The values of the
adjusted regression coefficients (RAdj2 = 0.8028) indicate a high correlation between the
experimentally observed and predicted values and explains any variability in the
response[42]. Moreover, the pure error of the model was very low, indicating good
reproducibility of the obtained data.
In general, P-values below 0.05 indicate that the model terms are significant. The
main effects of the water/oil ratio (X1), reaction temperature (X3), and lipase
concentration (X4) and the second-order effects of the water/oil ratio (X12), pH (X22),
reaction temperature (X32) and lipase concentration (X42) were significant and emerged as
major factors that affected the hydrolysis conversion. In contrast, values greater than
0.1000 indicate that the model terms are not significant, which included the main effect
of the pH (X2) as well as the interaction effects of the water/oil ratio with pH (X1X2), the
water/oil ratio with reaction temperature (X1X3), the water/oil ratio with lipase
concentration (X1X4), pH with reaction temperature (X2X3), pH with lipase concentration
(X2X4) and reaction temperature with lipase concentration (X3X4).
Graphical representations of the regression equation (2) and the 3-D response
surface were obtained using Design-Expert software. Fig. 7 shows these plots to illustrate
the effects of the independent and dependent variables for different fixed parameters.
These plots provide a method of visualizing the relationships between the response and
test variables.
Fig. 7(a) shows the influence of the water/oil ratio (X1) and pH (X2) on the
hydrolysis conversion of castor oil. As shown in Fig. 7(a), the hydrolysis conversion
increased as the pH increased. The hydrolysis conversion reached a maximum at a pH of
7.1 and then decreased in the range 7.1 to 7.5. The optimum pH value of the immobilized
lipase was slightly shifted towards the acidic region (from 7.2 (free CRL) to 7.1
(Fe3O4@CS@CRL)). This might be due different distributions of hydrogen and hydroxyl

Accept e d Article
ions in the area close to the surface and the remainder of the solution, with negative
charges clustering close to the immobilized lipase[43]. A similar shift in the optimum pH
of CRL upon immobilization on glutaraldehyde-activated polyester fiber, PNMA and
glass beads has been reported in the literature[44-46]. In general, the maximum
hydrolysis conversion was observed for a water/oil ratio of 1.5:1-2.0:1 at a pH of
approximately 7.1.
The effect of the water/oil ratio (X1) and reaction temperature (X3) on the hydrolysis
conversion of castor oil is shown in Fig. 7(b). The hydrolysis conversion increased as the
reaction temperature increased. However, an increase in the reaction temperature above
36 °C led to a slight decrease in the hydrolysis conversion due to denaturation of the
enzyme at higher temperature[47]. In general, the maximum hydrolysis conversion was
achieved with a water/oil ratio of 1.5:1-2.0:1 and a moderate reaction temperature.
Fig. 7(c) shows the effect of the water/oil ratio (X1) and lipase concentration (X4) on
the hydrolysis conversion of castor oil. The greater the amount of immobilized lipase
used in the hydrolysis procedure, the higher the hydrolysis conversion. When the lipase
concentration was 3.5%, the hydrolysis conversion reached the maximum. A further
increase in the lipase concentration did not further increase the hydrolysis conversion. In
general, the maximum hydrolysis conversion was achieved with a water/oil ratio of
1.5:1-2.0:1 and a lipase concentration of approximately 3.50%.
Fig. 7(d) shows the effect of the pH (X2) and reaction temperature (X3) on the
hydrolysis conversion of castor oil. An increase in the reaction temperature led to a
remarkable increase in the hydrolysis conversion. Both the reaction temperature and pH
had positive effects on the hydrolysis conversion. Moreover, the pH effect was positive
and smaller than that of the reaction temperature. In general, the maximum hydrolysis
conversion was achieved at a reaction temperature of approximately 36 °C and a pH of
7.1.
Fig. 7(e) shows the effects of pH (X2) and lipase concentration (X4) on the
Accept e d Article
hydrolysis conversion of castor oil. An increase in the lipase concentration led to a
remarkable increase in the hydrolysis conversion. When the lipase concentration was
3.5%, the hydrolysis conversion reached the maximum. A further increase in the lipase
concentration did not further increase the hydrolysis conversion. The pH effect was
positive and smaller than that of the lipase concentration. In general, the maximum
hydrolysis conversion was achieved at a pH of 7.1 and a lipase concentration of
approximately 3.5%.
Fig. 7(f) shows the effect of the reaction temperature (X3) and lipase concentration
(X4) on the hydrolysis conversion of castor oil. Both the reaction temperature and lipase
concentration had a positive effect on the hydrolysis conversion. In general, the
maximum hydrolysis conversion was achieved at a reaction temperature of approximately
36 °C and a lipase concentration of approximately 3.5%.
To determine the optimal conditions for the hydrolysis process, the second-order
polynomial regression equation (2) was solved. The optimum hydrolysis conditions were
as follows: a water/oil ratio of 1.60:1, a pH of 7.05, a reaction temperature of 34 °C, and
a lipase concentration of 3.27%. Under these conditions, the hydrolysis conversion was
46.81%. Confirmatory experiments were performed three times using the predicted
conditions. The observed result (46.53 ± 0.51%) was in close agreement with the
predicted result (46.81%), which indicated that the developed model was accurate and
reliable.
3.4. Reuse of the immobilized CRL
The reusability of an immobilized enzyme is rather important for its practical
application[16]. Herein, a cycle test using Fe3O4@CS@CRL was carried out four times,
and the results are given in Fig. 8. Hydrolysis conversions of 46.18%, 42.08%, 38.67%
and 30.34% were obtained after 1, 2, 3 and 4 cycles, respectively. This demonstrates that
Fe3O4@CS@CRL maintained high reactivity during the first three runs, after which a
slight reduction in the hydrolysis of castor oil was observed in the fourth cycle. This
Accept e d Article
reduction may be due to the irreversible adsorption of substrate and product on the
support after several reuses[16]. Furthermore, after third reuse, a high percent of
hydrolysis was retained (38.67%). The acceptable reusability and stability indicate that
Fe3O4@CS@CRL has potential to be used in bioengineering fields.
4. Conclusions
In this study, CRL was immobilized on magnetic Fe3O4@CS particles and used for
the hydrolysis of castor oil. The properties of magnetic Fe3O4 did not significantly change
upon introduction of the chitosan coating and lipase binding. The saturation
magnetization of Fe3O4@CS@CRL reached 27.3 emu/g, and the particles could be easily
separated from the suspension within 1 min using a strong external magnet. The optimum
conditions for the hydrolysis of castor oil were as follows: a water/oil ratio of 1.60:1, a
pH of 7.05, a reaction temperature of 34 °C, and a lipase concentration of 3.27%. Under
these conditions, the hydrolysis conversion was 46.81%. Fe3O4@CS@CRL showed a
high stability with no appreciable loss in the percent of hydrolysis after three cycles. Due
to the acceptable reusability and stability, Fe3O4@CS@CRL has potential for use in
preparing ricinoleic acid from castor oil.
Acknowledgements
This work was financially supported by the Science and Technology Huimin Plan of
China (No.2012GS610203) and the National Natural Science Foundation of China
(No.21675125).
The authors have declared no conflict of interest.

References
Accept e d Article
[1] Ogunniyi, D.S., Castor oil: a vital industrial raw material, Bioresour Technol. 2006, 163, 1086-1091.
[2] Mutlu, H., Meier, M.A.R., Castor oil as a renewable resource for the chemical industry, Eur J Lipid Sci
Technol. 2010, 112, 10-30.
[3] Akpan, U. G., Jimoh, A., Mohammed, A.D., Extraction, characterization and modification of castor seed
oil, Leonardo Journal of Sciences. 2006, 8, 43-52.
[4] Nezihe, A., Elif, D., Özlem, Y., Microwave heating application to produce dehydrated castor oil, Ind
Eng Chem Res. 2010, 50, 398-403.
[5] Puthli, M.S., Rathod, V.K., Pandit, A.B., Enzymatic hydrolysis of castor oil: process intensification
studies, Biochem Eng J. 2006, 31, 31-41.
[6] Gamayurova, V.S., Zinov’eva, M.E., Tran HT, T., Features of the enzymatic hydrolysis of castor oil,
Catal Ind. 2013, 3, 269-273.
[7] Goswami, D., Sen, R., Basu, J.K., Surfactant enhanced ricinoleic acid production using Candida rugosa
lipase, Bioresour Technol. 2010, 101, 6-13.
[8] Vasishtha, A.K., Trivedi, R.K., Das, G., Sebacic acid and 2-octanol from castor oil, JAm Oil Chem Soc.
1990, 67, 333–337.
[9] Goswami, D., Basu, J.K., Optimization of process variables in castor oil hydrolysis by Candida rugosa
lipase with buffer as dispersion medium, Biotechnol Bioprocess Eng. 2009, 14, 220-224.
[10] Morral-Ruíz, G., Melgar-Lesmes, P., García, M.L., Polyurethane and polyurea nanoparticles based on
polyoxyethylene castor oil derivative surfactant suitable for endovascular applications, Int J Pharm.
2014, 461, 1-13.
[11] Salih, N., Salimon, J., Abdullah, B.M., Thermo-oxidation, friction-reducing and physicochemical
properties of ricinoleic acid based-diester biolubricants, Arabian J Chem. 2017, 10, S2273-S2280.
[12] Khaskheli, A.A., Talpur, F.N., Ashraf, M.A., Monitoring the Rhizopus oryzae lipase catalyzed
hydrolysis of castor oil by ATR-FTIR spectroscopy, J Mol Catal B: Enzym. 2015, 113, 56-61.
[13] Goswami, D., Sen, R., Basu, J.K., Maximization of bioconversion of castor oil into ricinoleic acid by
response surface methodology, Bioresour Technol. 2009, 100, 4067-4073.
[14] Rathod, V.K., Pandit, A.B., Effect of various additives on enzymatic hydrolysis of castor oil. Biochem
Eng J. 2009, 47, 93-99.
[15] Cai, C., Gao, Y., Liu, Y., Immobilization of Candida antarctica lipase B onto SBA-15 and their
application in glycerolysis for diacylglycerols synthesis, Food Chem. 2016, 212, 205-212.
[16] Zheng, M.M., Lu, Y., Dong, L., Immobilization of Candida rugosa lipase on hydrophobic/strong
cation-exchange functional silica particles for biocatalytic synthesis of phytosterol esters, Bioresour
Technol. 2012, 115, 141-146.
[17] Basso, A., Hesseler, M., Serban, S., Hydrophobic microenvironment optimization for efficient
immobilization of lipases on octadecyl functionalised resins, Tetrahedron. 2016, 72, 7323-7328.
[18] Ali, Z., Tian, L., Zhao, P., Immobilization of lipase on mesoporous silica nanoparticles with
hierarchical fibrous pore, J Mol Catal B: Enzym. 2016, 134, 129-135.
[19] Calgaroto, C., Scherer, R.P., Calgaroto, S., Immobilization of porcine pancreatic lipase in zeolite
MCM 22 with different Si/Al ratios, Appl Catal, A. 2011, 394, 101-104.
[20] Sheldon, R.A., van Pelt S, S., Enzyme immobilisation in biocatalysis: why, what and how, Chem Soc
Rev. 2013, 42, 6223-6235.
Accept e d Article
[21] Yong, Y., Bai, Y.X., Li, Y.F., Characterization of Candida rugosa lipase immobilized onto magnetic
microspheres with hydrophilicity, Process Biochem. 2008, 43, 1179-1185.
[22] Zhang, D.H., Yuwen, L.X., Xie, Y.L., Improving immobilization of lipase onto magnetic microspheres
with moderate hydrophobicity/hydrophilicity, Colloids Surf B Biointerfaces. 2012, 89, 73-78.
[23] Motevalizadeh, S.F., Khoobi, M., Sadighi, A., Lipase immobilization onto polyethylenimine coated
magnetic nanoparticles assisted by divalent metal chelated ions, J Mol Catal B: Enzym. 2015, 120,
75-83.
[24] Wu, Y., Wang, Y., Luo, G., In situ preparation of magnetic Fe3O4-chitosan nanoparticles for lipase
immobilization by cross-linking and oxidation in aqueous solution, Bioresour Technol. 2009, 100,
3459-3464.
[25] Zhao, L., Chi, Y., Yuan, Q., Phosphotungstic acid anchored to amino-functionalized core-shell
magnetic mesoporous silica microspheres: a magnetically recoverable nanocomposite with enhanced
photocatalytic activity, J Colloid Interface Sci. 2013, 390, 70-77.
[26] Li, G.Y., Jiang, Y.R., Huang, K.L., Kinetics of adsorption of Saccharomyces cerevisiae mandelated
dehydrogenase on magnetic Fe3O4–chitosan nanoparticles, Colloids Surf, A. 2008, 320, 11-18.
[27] Kuo, C.H., Liu, Y.C., Chang, M.J., Optimum conditions for lipase immobilization on chitosan-coated
Fe3O4 nanoparticles, Carbohydr Polym. 2012, 87, 2538-2545.
[28] Yamamoto, K., Fujiwara, N., The Hydrolysis of Castor Oil Using a Lipase fromPseudomonassp.
f-B-24: Positional and Substrate Specificity of the Enzyme and Optimum Reaction Conditions, Biosci,
Biotech, Bioch. 2014, 59, 1262-1266.
[29] Avelar, M.H.M., Cassimiro, D.M.J., Santo, K.C., Hydrolysis of vegetable oils catalyzed by lipase
extract powder from dormant castor bean seeds, Ind Crops Prod. 2013, 44, 452-458.
[30] Xie, W., Wang, J., Immobilized lipase on magnetic chitosan microspheres for transesterification of
soybean oil, Biomass Bioenerg. 2012, 36, 373-380.
[31] Zhu, A., Yuan, L., Liao, T., Suspension of Fe3O4 nanoparticles stabilized by chitosan and
o-carboxymethylchitosan, Int J Pharm. 2008, 350, 361-368.
[32] Li, G.Y., Jiang, Y.R., Huang, K.L., Preparation and properties of magnetic Fe3O4–chitosan
nanoparticles, J Alloys Compd. 2008, 466, 451-456.
[33] Zhao, D.L., Wang, X.X., Zeng, X.W., Preparation and inductive heating property of Fe3O4–chitosan
composite nanoparticles in an AC magnetic field for localized hyperthermia, J Alloys Compd. 2009,
477, 739-743.
[34] Qu, J., Liu, G., Wang, Y., Preparation of Fe3O4–chitosan nanoparticles used for hyperthermia, Adv
Powder Technol. 2010, 21, 461-467.
[35] Chaichi, M.J., Ehsani, M., A novel glucose sensor based on immobilization of glucose oxidase on the
chitosan-coated Fe3O4 nanoparticles and the luminol–H2O2–gold nanoparticle chemiluminescence
detection system, Sens Actuators, B. 2016, 223, 713-722.
[36] Zulfikar, M.A., Afrita, S., Wahyuningrum, D., Preparation of Fe3O4-chitosan hybrid nano-particles
used for humic acid adsorption, Environ Nanotechnol Monit Manage. 2016, 6, 64-75.
[37] Liu, G., Li, T., Yang, X., Competitive fluorescence assay for specific recognition of atrazine by
magnetic molecularly imprinted polymer based on Fe3O4-chitosan, Carbohydr Polym. 2016, 137,
Accept e d Article
75-81.
[38] Ting, W.J., Tung, K.Y., Giridhar, R., Application of binary immobilized Candida rugosa lipase for
hydrolysis of soybean oil, J Mol Catal B: Enzym. 2006, 42, 32-38.
[39] Zhang, Y., Xu, Q., Zhang, S., Preparation of thiol-modified Fe3O4@SiO2 nanoparticles and their
application for gold recovery from dilute solution, Sep Purif Technol. 2013, 116, 391-397.
[40] Lu, Y., Lu, W., Wang, W., The optimization of aqueous two-phase extraction of lysozyme from crude
hen egg white using response surface methodology, J. Chem Technol Biot. 2013, 88, 415-421.
[41] Khataee, A.R., Karimi, A., Safarpour, M., Europium-doped ZnO as a visible light responsive
nanocatalyst: Sonochemical synthesis, characterization and response surface modeling of
photocatalytic process, Applied Catalysis A: General. 2014, 488, 160-170.
[42] Worapun, I., Pianthong, K., Thaiyasuit, P., Optimization of biodiesel production from crude palm oil
using ultrasonic irradiation assistance and response surface methodology, J. Chem Technol Biot. 2012,
87, 189-197.
[43] Chiou, S.H., Wu, W.T., Immobilization of Candida rugosa lipase on chitosan with activation of the
hydroxyl groups. Biomaterials. 2004, 25, 197-204.
[44] Santos, J.C., Nunes, G.F.M., Moreira, A.B.R., Characterization of Candida rugosa Lipase Immobilized
on Poly(N-methylolacrylamide) and Its Application in Butyl Butyrate Synthesis. Chem Eng Technol.
2007, 30, 1255-1261.
[45] Yilmaz, E., Can, K., Sezgin, M., Immobilization of Candida rugosa lipase on glass beads for
enantioselective hydrolysis of racemic naproxen methyl ester. Bioresour Technol. 2011, 102, 499-506.
[46] Yiğitoğlu, M., Temoçin, Z., Immobilization of Candida rugosa lipase on glutaraldehyde-activated
polyester fiber and its application for hydrolysis of some vegetable oils, J Mol Catal B: Enzym. 2010,
66, 130-135.
[47] Gupta, S., Comparative study on hydrolysis of oils by lipase immobilized biocatalytic PS membranes
using biphasic enzyme membrane reactor. J Environ Chem Eng. 2016, 4, 1797-1809.
Accept e d Article
NH2 NH
H2N NH2 NH
HN
chitosan
NH2 Glutaraldehyde NH
H2N HN
CRL
H2N NH2 NH NH
H2N NH2 NH HN
NH2 NH
NH NH
O O
O O
H H
Fe3O4 Glutaraldehyde Candida rugosa lipase (CRL)
OH OH
OH
O O
HO O HO OH
HO O
OH NH2
NH2 NH2
n
chitosan chain chitosan
Scheme 1. The procedure used for preparation of Fe3O4@CS@CRL.

Legends to Figures
Accept e d Article
Fig. 1. FTIR spectra of Fe3O4 (a), CRL(b), chitosan (c), Fe3O4@CS (d) and
Fe3O4@CS@CRL(e).
Fig. 2. XRD patterns of Fe3O4 (a), and Fe3O4@CS (b).
Fig. 3. SEM micrographs of Fe3O4 (a), and Fe3O4@CS (b).
Fig. 4. Thermogravimetric analysis (TGA) of chitosan (a), Fe3O4 (b), and Fe3O4@CS (c).
Fig. 5. Magnetization curves of Fe3O4 (a), Fe3O4@CS (b), and Fe3O4@CS@CRL (c)
obtained by VSM at 25 °C.
Fig. 6. Plot of actual and predicted values of hydrolysis conversion.
Fig.7. The hydrolysis conversion as a function of hydrolysis parameters (a) the water/oil
ratio with pH (X1-X2); (b) the water/oil ratio with reaction temperature (X1-X3); (c) the
water/oil ratio with lipase concentration (X1-X4); (d) the pH with reaction temperature
(X2-X3); (e) the pH with lipase concentration (X2-X4); (f) the reaction temperature with
lipase concentration (X3-X4).
Fig.8. Recycling studies of Fe3O4@CS@CRL on hydrolysis of castor oil. (Hydrolysis
conditions: stirring speed, 150 rpm; 34 °C; pH 7.05; water/oil ratio of 1.60:1; lipase
concentration of 3.27%, reaction time of 60 h.)
Table 1. Independent variables and levels used for response surface design.
Accept e d Article
Coded levels and actual vaules
Independent variables
-1 0 1
X1 : water/oil ratio 1 2 3
X2 : pH 6.5 7.0 7.5
X3 : Reaction temperature (°C) 30 35 40
X4 : Lipase concentration (%) 2 3 4
Table 2. Response surface Box-Behnken design and results of the experiments for
Accept e d Article
conversion.
Coded levels Hydrolysis

Run
X1 X2 X3 X4 conversion(%)
1 0 0 -1 1 41.04±1.08
2 0 0 1 -1 29.45±0.79
3 0 -1 -1 0 32.97±1.14
4 0 0 0 0 45.46±1.07
5 -1 0 1 0 29.76±0.78
6 -1 0 -1 0 34.12±0.96
7 0 0 1 1 31.43±0.87
8 0 0 0 0 46.04±0.76
9 -1 1 0 0 40.85±0.84
10 0 -1 1 0 25.79±0.67
11 0 -1 0 1 30.57±0.97
12 -1 0 0 1 44.93±1.01
13 1 0 0 1 33.04±0.89
14 0 1 -1 0 34.53±0.92
15 -1 0 0 -1 35.69±1.05
16 0 0 0 0 43.27±0.73
17 1 1 0 0 27.56±0.81
18 0 1 0 1 32.56±0.77
19 0 1 1 0 30.85±0.93
20 0 -1 0 -1 27.67±1.01
21 0 0 -1 -1 32.46±0.67
22 -1 -1 0 0 37.17±0.75
23 0 0 0 0 46.43±0.65
24 1 0 1 0 29.53±0.86
Accept e d Article
25 1 0 0 -1 30.56±1.23
26 0 1 0 -1 33.53±0.77
27 1 0 -1 0 32.56±0.93
28 0 0 0 0 46.71±0.84
29 1 -1 0 0 31.53±0.71
Table 3. ANOVA for the response surface quadratic model.

Accept e d Article
Free Mean
source Sum of squares F-value p-value
degree square
Model 1022.68 14 73.05 9.14 < 0.0001

X1 118.69 1 118.69 14.86 0.0018
X2 16.76 1 16.76 2.10 0.1696
X3 79.41 1 79.41 9.94 0.0071
X4 48.84 1 48.84 6.11 0.0269
X1X2 14.63 1 14.63 1.83 0.1974
X1X3 0.44 1 0.44 0.055 0.8174
X1X4 11.42 1 11.42 1.43 0.2516
X2X3 3.06 1 3.06 0.38 0.5458
X2X4 3.74 1 3.74 0.47 0.5048
X3X4 10.89 1 10.89 1.36 0.2625
X12 149.54 1 149.54 18.72 0.0007
X22 366.46 1 366.46 45.87 < 0.0001
X32 379.87 1 379.87 47.54 < 0.0001
X42 185.50 1 185.50 23.22 0.0003
Residual 111.86 14 7.99

Lack of fit 104.30 10 10.43 5.52 0.0571
Pure Error 7.56 4 1.89
Cor total 1134.54 28
R2=0.9014, RAdj2=0.8028
Accept e d Article
Fig. 1.
Accept e d Article
Fig. 2.
(a) (b)
Accept e d Article
Fig. 3.
Accept e d Article
Fig. 4.
Accept e d Article
Fig. 5.
Accept e d Article
Fig. 6.
Accept e d Article
(a) (b)
(c) (d)
(e) (f)
Accept e d Article
Fig.7.
Accept e d Article
Fig.8.

Zhao 2017

Uploaded by

Copyright:

Available Formats

You might also like

Zhao 2017

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Zhao 2017

Uploaded by

Copyright:

Available Formats

www.ejlst.

com European Journal of Lipid Science and Technology

© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Practical applications: RSM was found to be a useful technique for optimizing

In recent years, a significant number of magnetic composite materials have been

2. Materials and methods

2.2. Immobilization of Candida rugosa lipase on Fe3O4@CS

2.4. Enzymatic hydrolysis of castor oil

the independent variable of the hydrolysis process:

3. Results and discussion

thermogravimetric analyser. The thermogravimetric analyses (TGA) of chitosan (a),

magnetization of Fe3O4@CS@CRL was high enough to facilitate separation from the

(Fe3O4@CS@CRL)). This might be due different distributions of hydrogen and hydroxyl

The authors have declared no conflict of interest.

Scheme 1. The procedure used for preparation of Fe3O4@CS@CRL.

Coded levels Hydrolysis

Table 3. ANOVA for the response surface quadratic model.

Model 1022.68 14 73.05 9.14 < 0.0001

Residual 111.86 14 7.99

You might also like