Novel magnetic cross-linked lipase aggregates for improving the resolution of (R,S)-2-octanol

Article abstract of DOI:10.1002/chir.22411

Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of-NH₂ terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve (R,S)-2-octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates.

Full text of DOI:10.1002/chir.22411

CHIRALITY (2014)
Novel Magnetic Cross-Linked Lipase Aggregates for Improving the
Resolution of (R, S)-2-octanol
*
YING LIU, CHEN GUO, AND CHUN-ZHAO LIU
National Key Laboratory of Biochemical Engineering & Key Laboratory of Green Process Engineering, Institute of Process Engineering, Chinese
Academy of Sciences, Beijing, P.R. China
ABSTRACT
Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing
the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal
groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions,
100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked li-
pase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to effi-
ciently resolve (R, S)-2-octanol, and retained 100% activity and 100% enantioselectivity after
10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity
and 70% enantioselectivity due to insufficient cross-linking. These results provide a great poten-
tial for industrial applications of the magnetic cross-linked lipase aggregates. Chirality 00:000–
000, 2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: Yarrowia lipolytica lipase; magnetic nanoparticles; cross-linked enzyme
aggregates; immobilization; (R, S)-2-octanol; enantioselectivity
INTRODUCTION
developed. For enzymes with a low number of reactive
amino groups on the external surface, the aggregation
may be conducted in the presence of a polymer or macro-
molecules with many reactive amino groups.13 However,
Schiff bases resulting from the reaction of glutaraldehyde
and amino groups are unstable under acidic conditions,
and they have a tendency to break down and regenerate
the aldehyde and amine.14 This may result in the gradual
release of the enzyme into the reaction medium due to
inadequate cross-linking.15 In addition, the CLEAs form
clusters due to the separation of CLEAs from the reaction
medium by centrifugation or filtration. This results in inter-
nal mass-transfer limitations, especially when enzymes act
on macromolecular substrates.13,16,17
Magnetic cross-linked enzyme aggregates (MCLEAs),
which were prepared by the addition of functionalized magne-
tite particles as an additive into enzyme solution, precipitation
of enzyme to form aggregates, and then cross-linking of
enzyme aggregates and particles, can circumvent the
problem of clumping of CLEAs.13 Because of the magnetic
properties of the magnetite particles, the resulting MCLEAs
can be easily controlled and separated from the reaction
medium by the application of a permanent magnet or
magnetic field, eliminating the need of filtration and centrifu-
gation. However, MCLEAs prepared with glutaraldehyde still
cannot solve the problem of the gradual release of the
enzyme into the reaction medium.
Lipases have exhibited capability in the preparation of opti-
cally active single enantiomers for synthesizing a variety of
fine chemicals and pharmaceuticals.1,2 Developing effective
immobilization techniques to stabilize and reuse lipases is
one of the key steps to make the enzymatic process econom-
ically viable.1,3 Immobilization techniques can be divided into
two types: carrier-bound and carrier-free. The disadvantage of
carrier-bound enzymes is the dilution of catalytic activity due
to the introduction of a large proportion of mass (~90-99% of
the total mass), which results in lower volumetric and
space-time yields and lower catalyst productivity. In addition,
immobilization using a solid carrier is generally expensive
and often requires the chemical modification of an inert ma-
trix to allow for covalent coupling of the enzyme.4 Carrier-free
immobilization via cross-linking of the enzyme using bifunc-
tional cross-linking agents is a promising strategy that over-
comes the disadvantage of carrier-bound enzymes.3 Due to
the lack of a support, the enzyme activity is not diluted, and
the specific activity of the resulting biocatalyst is increased.5
Furthermore, an industrial enzymatic process using carrier-
free immobilized enzymes can utilize smaller bioreactors
than the enzymes immobilized on a solid support.2 Among
carrier-free immobilization technologies, cross-linked enzyme
aggregates (CLEAs) technology is attractive for its low cost,
simplicity, and efficiency.6–9 The preparation of CLEAs
consists of precipitating the enzyme and cross-linking the
resulting physical aggregates with glutaraldehyde, a common
cross-linking agent,10,11 via the reaction with the reactive
amino groups (mainly from Lys) on the enzyme surface.
Therefore, immobilization using CLEAs technology is strongly
dependent on the nature of the enzyme in terms of Lys
content. Immobilization by CLEAs technology may not be
highly efficient for electronegative enzymes with few surface
reactive amino groups.12
In this work, in order to overcome the limitations of
CLEAs technology, a novel strategy was developed to pre-
pare MCLEAs by conducting the CLEAs onto magnetic par-
ticles with a high number of –NH2 terminal groups using
*Correspondence to: Chun-Zhao Liu, Professor of Biochemical Engineering,
National Key Laboratory of Biochemical Engineering, Institute of Process
Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China.
E-mail: czliu@ipe.ac.cn
Received for publication 16 June 2014; Accepted 15 October 2014
DOI: 10.1002/chir.22411
Published online in Wiley Online Library
(wileyonlinelibrary.com).
In order to overcome the difficulty of utilizing enzymes
with few surface reactive amino groups, several approaches
for the preparation of CLEAs have been successfully
© 2014 Wiley Periodicals, Inc.

LIU ET AL.
(c) of (R, S)-2-octanol was determined by the decrease of (R, S)-2-octanol.
p-benzoquinone as the cross-linking agent. The prepared
magnetic CLEAs were used as the biocatalyst to improve
the resolution of (R, S)-2-octanol.
The enantiomeric excess (ee) of the product was determined by the peak
areas of the two isomers using Eq. (3). The enantioselectivity (E) was cal-
culated using Eq. (4) according to a previously reported method.24
MATERIALS AND METHODS
R ꢀ S
eepð%Þ ¼
ꢁ100%
(3)
Materials
R þ S
Yarrowia lipolytica lipase (YLL) was kindly donated by Beijing CTA
New Century Biotechnology (China). (R)-2-octanol, (S)-2-octanol, (R, S)-
2-octanol, vinyl acetate, polyethylenimine (PEI), cationic polyacrylamide
(CPAM), polymethylmethacrylate (PMMA), and p-benzoquine were
purchased from Sigma-Aldrich (Shanghai, China). (R)-2-octanol acetate
and (S)-2-octanol acetate were synthesized using the method previously
reported.18 Magnetic particles coated with PEI (Fe3O4-PEI, MP1) and
magnetic particles coated with CPAM (Fe3O4-CPAM, MP2) were
prepared according to our previous reports.19,20 Fe3O4-PMMA with
dendritic –NH2 (MP3) were prepared according to the method previously
reported.21 The other reagents were purchased from Beijing Chemical
Reagents (Beijing, China).
where eep is the ee of the product, R is the concentration of the (R)-enan-
tiomer, and S is the concentration of the (S)-enantiomer.
ln½1 ꢀ cð1 þ eepÞꢂ
E ¼
(4)
ln½1 ꢀ cð1 ꢀ eepÞꢂ
where c is the extent of the conversion of (R, S)-2-octanol.
RESULTS AND DISCUSSION
MCLEAs have emerged as a novel and versatile carrier-free
immobilization technique, involving the precipitation of an
enzyme from an aqueous solution and cross-linking the
aggregates onto the surfaces of the magnetic particles using
a bifunctional reagent.25 Therefore, parameters that alter the
enzyme precipitation and the aggregate cross-linking will
affect the properties of the MCLEAs. The effects of various
parameters during MCLEAs preparation on immobilization
efficiency and activity recovery of MCLEAs were studied.
Preparation of Magnetic Cross-Linked Lipase Aggregates
Magnetic cross-linked lipase aggregates (MCLLAs) preparation involves
two steps: the precipitation of lipase and the subsequent cross-linking of the
enzyme aggregates onto the surfaces of the magnetic particles. In a typical
experimental procedure the magnetite particles (2.5 mg) were mixed with
5 mL of Yarrowia lipolytica lipase in buffer solution (5 mg protein/mL,
0.067 M phosphate buffer, pH 8.0) and shaken for 10 min at 20 °C. Then
20 mL of saturated ammonium sulfate solution was added with stirring at
4 °C for 40 min. After precipitation, p-benzoquinone was added to the sus-
pension up to a final concentration of 5 mM and stirred for 20 h at 20 °C.
After cross-linking, MCLLAs were separated using a magnet, washed three
times with phosphate buffer, and immediately frozen and lyophilized. The
cross-linked lipase aggregates (CLLAs) were prepared without the addition
of magnetic particles as a control.
The nature of YLL and the type and quantity of
magnetic particles
YLL, which belongs to the same gene family as
Thermomyces lanuginosus lipase (TLL), is a lipase with multi-
ple applications in the field of biotechnological processes.26
However, due to its low Lys residue content, CLLAs prepared
using YLL may not be highly effective due to inadequate
cross-linking, which results in low mechanical stability or
even the release of the enzyme during reactions.15 In order
to overcome this, three types of magnetite particles coated
with large amino groups were added to the YLL solution to
prepare MCLLAs via cross-linking of YLL aggregates and
the amino functionalized magnetite particles. The results are
shown in Table 1. All the magnetite particles tested improved
the η and λ of the MCLLAs compared to the control (prepared
The immobilization efficiency (η) of the MCLLAs was calculated
from the difference between the initial concentration of enzyme (C0)
and the concentration remaining after the preparation approach (C1)
using Eq. (1).7
C0 ꢀ C1
ηð%Þ ¼
ꢁ100%
(1)
C0
The activity recovery (λ) was estimated using Eq. (2).
Specific activity of MCLLAs
Specific activity of the same amount of free lipase as that in the MCLLAs
λð%Þ ¼
ꢁ100%
(2)
without magnetite particles). The microparticle, MP2, due to
its low surface area, was coated with fewer amino groups,
which resulted in a lower η compared to the nanoparticles
MP1 and MP3.27 MP3, due to dendritic modifications, has a
much larger number of amino groups (about 0.5 mmol/g, de-
termined via ninhydrin colorimetry.28) on its surface than
MP1. Therefore, the addition of MP3 into the YLL solution
can lead to a high η due to sufficient cross-linking of YLL
aggregates.
The ratio of the magnetic particles to the enzyme (protein)
was an important factor in determining the performance of
the MCLLAs.7 The η and λ of the MCLLAs showed an upward
trend for MP3: YLL mass ratios in the range of 0.02–0.1
(Fig. 1). The increase of this ratio to values higher than 0.1
The specific activity of the lipase was assayed using the p-nitrophenol
method.22
Resolution of (R, S)-2-octanol Catalyzed by MCLLAs
The resolution of (R, S)-2-octanol catalyzed by MCLLAs was conducted
as in our previous report.23 The reaction solvent was the mixture of ace-
tone and carbon tetrachloride, v/v =3:7. Reusability of the MCLLAs was
studied in batch operations under the same conditions. After each batch
reaction, the activity and enantioselectivity of the MCLLAs were mea-
sured and expressed as the residual activity and enantioselectivity by tak-
ing the activity and enantioselectivity of the first batch as 100%.
Analysis
The (R, S)-2-octanol and product were analyzed using gas chromatogra-
phy (GC) using a previously reported method.23 The extent of conversion
Chirality DOI 10.1002/chir

NOVEL MAGNETIC CROSS-LINKED LIPASE AGGREGATES
TABLE 1. MCLLAs prepared with different magnetic particles
Magnetic particles
MCLLAs
Entry
Type
Average diameter
Structure of covering layer
-
η (%)
γ (%)
1a
2
-
-
42
70
3
2
85
91
5
3
MP1
12 nm
3
4
MP2
MP3
58 μm
51
3
2
87
5
200 nm
100
120
3
aWithout the addition of magnetic particles.
150
120
90
60
30
0
150
120
90
60
30
0
Immobilization efficiency
Activity recovery
4
SO
)
2
4
Acetone
Ethanol
NH
Tert-butyl
alcohol
2-Propanol
(
Acetonitrile
Fig. 1. Effect of the ratio of MP3 to YLL on the immobilization efficiency
and activity recovery of the MCLLAs.
Fig. 2. Effect of the precipitation agent on the immobilization efficiency
and activity recovery of the MCLLAs.
had no additional effect on η, but resulted in a decrease in λ
(Fig. 1). This was most likely due to excessive cross-linking
(due to excess MP3), which resulted in larger particle sizes
of the prepared MCLLAs and a loss of the ability of the inner
YLLs to form complexes with substrates. Therefore, a ratio of
MP3 to YLL of 0.1 was used in further experiments.
and structural properties of the enzyme which causes the
optimal precipitant to vary from one enzyme to another.25
The amount of precipitant also affected the performance of
the MCLLAs (Fig. 3). The λ in the YLL CLEAs increased with
increasing (NH4)2SO4 saturation of the enzyme solution dur-
ing precipitation, which was in agreement with Candida
rugosa lipase29 and tyrosinase.30 When the volume ratio of
(NH4)2SO4 solution to YLL solution increased to 4, "hyper
activation" (greater than 100% λ), was observed due to the
Type and Quantity of the Precipitant
YLL was precipitated using different precipitation agents,
such as (NH4)2SO4, ethanol, acetone, acetonitrile, 2-propanol,
and tert-butyl alcohol. The experimental data are presented in
Figure 2. (NH4)2SO4 was the most successful agent for the
precipitation of YLL and led to high η and λ of the prepared
MCLLAs. All the organic solvents used as precipitants
affected the activity of YLL to a different degree. The optimal
precipitant of YLL of (NH4)2SO4 is in contrast to the optimal
precipitant for the lipase from Aspergillus niger, which is di-
methyl carbonate.7 This is due to the different biochemical
formation of more structured and fine-grained CLEAs.31
A
further increase in the ratio of the (NH4)2SO4 solution to
the YLL solution resulted in a decrease in λ due to the larger
particle size of the MCLLAs.
Nature and Amount of the Cross-Linking Agent and
Cross-Linking Time
p-Benzoquinone was used as the cross-linking agent be-
cause it reacts with the amino or hydroxyl groups of YLL
Chirality DOI 10.1002/chir

LIU ET AL.
150
120
90
60
30
0
150
120
90
60
30
0
enzyme’s flexibility with high η and λ. However, the opti-
Immobilization efficiency
Activity recovery
mum concentration is strongly dependent on the functional
groups (type and amount), which vary from one enzyme to
another. This is confirmed by other studies.1,7,13
Cross-linking is a reaction; therefore, the time required to
obtain the maximum λ is very important.32 The MCLLAs of
YLL were prepared using varying cross-linking times (4, 8,
12, 20, and 24 h). The results (presented in Fig. 5) showed
that 8 h was required for complete cross-linking. And at the
same time, the maximum λ, 139%, was achieved.
Resolution of (R, S)-2-octanol Catalyzed by the MCLLAs
0
1
2
3
4
5
6
The newly synthesized MCLLAs of YLL were used to cata-
lyze the resolution of (R, S)-2-octanol. The enzymatic activity
and enantioselectivity of the MCLLAs were 48.5 ( 0.2)
μmol/g/min and 850 ( 5), respectively, which were higher
than those (29.8 0.3 μmol/g/min, 500 2) of the CLLAs,
and also higher than those of the magnetite-immobilized
YLL and free YLL catalyzed under the same conditions. The
ee of the product obtained by MCLLAs catalysis was 99.1%,
which was higher than that (98.2%) of the product obtained
by CLLAs catalysis.23 The enhanced activity and enantio-
selectivity of MCLLAs may be partly due to the formation of
the more structured and fine-grained CLEAs mentioned
above and partly to the addition of the magnetic particles with
a high number of –NH2 terminal groups during the prepara-
tion of MCLLAs. Similar to BAS as additive (during the immo-
bilization of formate dehydrogenase from Candida boidinii via
CLEAs33), the magnetic particles may also act as a proteic
feeder, preventing from excess cross-linking of important
amino acid residues of YLL.
As with other immobilized enzymes, reusability is ex-
tremely important for potential industrial applications.25 The
reusability of the MCLLAs of YLL was studied in batch oper-
ations at optimum reaction conditions for a fixed reaction
time. After each batch reaction, the enzyme activity and
enantioselectivity in the MCLLAs were determined and
expressed as the residual activity and enantioselectivity by
setting the activity and enantioselectivity of the first batch
as 100%. The results (Fig. 6) showed that almost no activity
and enantioselectivity in YLL in MCLLAs were lost after 10 cy-
cles of use, while they decreased continuously when CLEAs
(prepared without MP3) were tested over 5 cycles. These re-
sults suggested that the loss in YLL activity and
enantioselectivity in CLEAs was due to the leaking of YLL
Fig. 3. Effect of the amount of (NH4)2SO4 on the immobilization efficiency
and activity recovery of the MCLLAs.
and the amino of MP3 and forms stable MCLLAs due to the
formation of C–O and C–N bonds tolerant to pH variation.1
This circumvents the existing problems of glutaraldehyde,
which cross-links the amino groups of enzyme aggregates
via the formation of unsaturated bonds and Schiff bases,
which are unstable.
An important parameter in the preparation of the MCLLAs
was the amount of p-benzoquinone; this influenced the η and
λ of the resulting MCLLAs (Fig. 4). The concentration of
p-benzoquinone was clearly optimal at 4 mM. The η and
λ of the MCLLAs prepared with increasing concentrations
of p-benzoquinone increased to a maximum value and
then λ decreased while η reached a plateau. This was be-
cause at lower p-benzoquinone concentrations, insufficient
cross-linking occurred. On the one hand, portions of the
YLL were not cross-linked and washed into the supernatant,
resulting in a lower η. On the other hand, the unstable
MCLLAs that resulted from the insufficient cross-linking
released free YLL into the reaction medium, resulting in a
lower λ. When the p-benzoquinone concentration was high,
excessive cross-linking occurred, resulting in a loss of YLL’s
flexibility, which is crucial for enzyme activity. In addition,
steric hindrance due to excessive cross-linking may have
resulted in the rigidification of YLL and prevented the
substrate from reaching the active site. Therefore, for an en-
zyme, there is an optimum cross-linking agent concentration
at which sufficient cross-linking occurs while maintaining the
150
120
90
60
30
0
150
120
90
60
30
0
150
120
90
60
30
0
150
120
90
60
30
0
Immobilization efficiency
Activity recovery
Immobilization efficiency
Activity recovery
0
2
4
6
8
10
12
0
8
16
24
32
The concentration of cross agent (mM)
Cross-linking time (h)
Fig. 4. Effect of the amount of cross-linking agent on the immobilization ef-
ficiency and activity recovery of the MCLLAs.
Fig. 5. Effect of cross-linking time on the immobilization efficiency and ac-
tivity recovery of the MCLLAs.
Chirality DOI 10.1002/chir

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Fabrication of MCLLAs using p-benzoquinone as the cross-
linking agent in the presence of Fe3O4-PMMA with dendritic
–NH2 is an efficient strategy for YLL immobilization. The
MCLLAs of YLL, which were synthesized under optimum
conditions, were able to catalyze the efficient resolution of
(R, S)-2-octanol, and demonstrated excellent reusability.
These results suggest that the synthesized MCLLAs of YLL
have potential for industrial applications in the preparation
of optically active single enantiomers.
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ACKNOWLEDGMENTS
The work was financially supported by the National Basic
Research Program of China (No. 2013CB733600), the National
Natural Science Foundation of China (No. 21176241), and the
Key Deployment Program of Chinese Academy of Sciences
(KSZD-EW-Z-015). We thank Dr. Amanda Stiles (the Chinese
Academy of Sciences Fellowships for Young International
Scientists, No. 2011Y1GA01) for helping edit the article.
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