Lipase from pseudomonas cepacia immobilized into ZIF-8 as bio-catalyst for enantioselective hydrolysis and transesterification

Article abstract of DOI:10.1016/j.procbio.2020.12.017

Enzyme immobilization in MOFs offers retained enzyme integrity and activity, enhanced stability, and reduced leaching. In this work, Pseudomonas cepacia lipase (PCL) was successfully immobilized into the Zeolitic imidazolate framework-8 (ZIF-8) by physical adsorption. The amounts of PCL in the immobilized enzyme (PCL?ZIF-8) was determined to be 16.7 % (200.5 mg PCL/g ZIF-8). Furthermore, the immobilized enzyme was applied as efficient bio-catalyst for enantioselective hydrolysis of 2-phenylpropionic acid (2-PPA) ester enantiomers and enantioselective transesterification of 1-phenylethanol enantiomers. The enzymatic activity of the immobilized enzyme was three times more than that of free PCL in enantioselective hydrolysis system, and the enantiomeric excess was maintained above 99 %. There was no significant difference in enzyme activity between immobilized PCL and free PCL in transesterification system. In addition, the immobilized enzyme showed good reusability in both hydrolysis (30.57 % of initial activity, 4 cycle) and transesterification reaction systems (64.38 % of initial activity, 6 cycle).

Full text of DOI:10.1016/j.procbio.2020.12.017

Process Biochemistry 102 (2021) 132–140
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Lipase from pseudomonas cepacia immobilized into ZIF-8 as bio-catalyst for
enantioselective hydrolysis and transesterification
Jian Ou, Xin Yuan, Yu Liu*, Panliang Zhang, Weifeng Xu, Kewen Tang*
Department of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang, 414006, Hunan, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Enzyme immobilization in MOFs offers retained enzyme integrity and activity, enhanced stability, and reduced
leaching. In this work, Pseudomonas cepacia lipase (PCL) was successfully immobilized into the Zeolitic imida-
zolate framework-8 (ZIF-8) by physical adsorption. The amounts of PCL in the immobilized enzyme (PCL@ZIF-8)
was determined to be 16.7 % (200.5 mg PCL/g ZIF-8). Furthermore, the immobilized enzyme was applied as
efficient bio-catalyst for enantioselective hydrolysis of 2-phenylpropionic acid (2-PPA) ester enantiomers and
enantioselective transesterification of 1-phenylethanol enantiomers. The enzymatic activity of the immobilized
enzyme was three times more than that of free PCL in enantioselective hydrolysis system, and the enantiomeric
excess was maintained above 99 %. There was no significant difference in enzyme activity between immobilized
PCL and free PCL in transesterification system. In addition, the immobilized enzyme showed good reusability in
both hydrolysis (30.57 % of initial activity, 4 cycle) and transesterification reaction systems (64.38 % of initial
activity, 6 cycle).
ZIF-8
Immobilization
Pseudomonas cepacian lipase
Enzymatic reaction
Chiral resolution
1. Introduction
various 2-arylpropionic acid derivatives. Therefore, the acquisition of
optically pure 2-PPA enantiomer is significant in pharmaceutical field.
Chiral drugs play an important role in the global pharmaceutical
industry. The pharmacological actions of chiral drug are achieved by a
strict chiral matching between the drug enantiomer and the macro-
molecule in the human body. Thus, there are significant differences in
the absorption, distribution, pharmacological activity, metabolic pro-
cesses and toxicity of different enantiomers of chiral drugs. In most
cases, these differences result in one enantiomer being efficacious, while
another enantiomer is ineffective or even has toxic side effects [1–3].
The resolution of enantiomers is a crucial work for improving the ac-
tivity of chiral drugs and reducing the toxicity and metabolic burden of
the human body [4].
Chiral alcohols, which have wide application value in medicine, are an
important class of synthetic precursors with stable physicochemical
properties. Chiral alcohol can be used to synthesize a variety of drugs for
the treatment of diseases such as cardiovascular and hypertension [7].
1-Phenylethanol is an important optically active substance. (S)-1-phe-
nylethanol can be used to synthesize sertraline for the treatment of
depression, as well as drugs for asthma and immune enhancement.
(R)-1-phenylethanol can be used to synthesize drugs that inhibit
cholesterol absorption [8]. Therefore, the preparation of a single 1-phe-
nylethanol enantiomer is of great significance in the pharmaceutical
industry. In recent years, researchers have obtained single enantiomers
by a variety of separation methods [9]. The enzymatic kinetic resolution
has emerged as an attractive method [10].
2-Arylpropionic acid derivatives are a class of important non-
steroidal anti-inflammatory drugs (NSAIDs), which have favorable
analgesic, antipyretic and anti-inflammatory effects [5]. In general, the
activity of (S)-enantiomer is much higher than that of (R)-enantiomer.
For example, the anti-inflammatory and analgesic effect of (S)-ibuprofen
is more than 100 times in comparison with (R)-ibuprofen. Likewise,
(S)-flubuprofen is 30 times higher than (R)-flubuprofen [6]. Therefore,
the optically pure drugs are preferred in clinical treatment. 2-PPA en-
antiomers are common pharmaceutical intermediate for the synthesis of
Lipases (triacylglycerol acyl ester hydrolases, EC 3.1.1.3) have been
universally applied enzymes for the resolution of chiral drugs, which has
the characteristics of wide substrate specificity, high regioselectivity,
and enantioselectivity [11]. A structural feature that is common to most
lipases is the presence of a so-called “lid”, composed of one or even two
α
-helix peptides, that covers the active site of the lipase. This poly-
peptide chain may fully isolate the lipase from the reaction medium
* Corresponding authors.
E-mail addresses: lyxtmj_613@163.com (Y. Liu), tangkewen@sina.com (K. Tang).
https://doi.org/10.1016/j.procbio.2020.12.017
Received 20 August 2020; Received in revised form 18 December 2020; Accepted 19 December 2020
Available online 25 December 2020
1359-5113/© 2020 Elsevier Ltd. All rights reserved.

J. Ou et al.
Process Biochemistry 102 (2021) 132–140
[12–14]. Lipase from pseudomonas cepacia has molecular weight
ranging between 28 and 33 kDa and average molecular diameter of
4ꢀ 5 nm. Lipases from different groups have distincts properties. PCL has
an optimal temperature at 50 ℃ and optimal pH of 7.0. Compared with
other lipase, PCL is stable in organic solvents, with some exception
stimulation or inhibition [15]. PCL is widely applied in enantioselctive
hydrolysis, esterification and transesterification reaction due to high
substrate specificity and enantioselectivity [16]. Nevertheless, PCL
mainly exists in the form of enzyme powder in the market, which limits
their repeated use in industrial processes. Generally, immobilization of
lipase is one of the effective strategies to solve these problems, such as
high cost, difficult recovery and low stability [17]. Immobilizing en-
zymes on a solid support can improve enzyme stability as well as make
them ease of separation and recovery while maintaining selectivity and
activity [18]. Up to now, immobilization techniques have been devel-
oped maturely, such as physical adsorption [19], covalent linkage [20],
pore entrapment [21] and crosslinking [22]. In addition to immobili-
zation techniques, the choice of solid supports is also extremely
important for the immobilization of enzymes.
β-cyclodextrin (HP-β-CD) (purity > 99 %) was bought from Shandong
New Fine Chemical Co., Ltd. (Shandong, China). Phosphoric acid, acetic
acid, triethylamine and disodium hydrogen phosphate dodecahydrate
were purchased from Huihong Reagent Co., Ltd. (Shanghai, China). Zinc
nitrate hexahydrate (Zn(NO3)2•6H2O) was bought from Shanghai
Aladdin Bio-Chem Technology Co. Ltd. (Shanghai, China). N, N-dime-
thylformamide (DMF) (purity > 99.5 %) was acquired from Shanghai
Titan Scientific Co., Ltd (Shanghai, China). Sodium dodecyl sulfate
(SDS) (purity > 99 %), p-nitrophenyl palmitate (p-NPP) (purity > 98 %),
p-nitrophenol (p-NP) (purity > 99 %) were purchased from Sigma-
Aldrich (USA). Solvent for chromatography was of HPLC grade. All
other chemicals were of analytical-reagent grade.
2.2. HPLC analysis
The analysis of (R)-2-PPA and (S)-2-PPA were performed by high
performance liquid chromatography (HPLC) composed of binary pump
system (Waters 1525). An Inertsil ODS-3 column (250 mm × 4.6 mm,
5 μm) was employed. The wavelength of UV/visible detector (Waters
Metal-organic frameworks (MOFs), a class of novel porous materials,
which are consist of organic ligands and inorganic metal nodes through
coordination bonds [23]. Because of the diversity of organic ligands and
metal ions as well as the ways of their combination, MOFs materials
have a variety of types, which have been counted to more than 20,000
kinds. MOFs have been given increasing attention due to their diverse
crystal structure, controllable pore size, high porosity and specific sur-
face area [24]. According to functional requirements, MOFs can be
practically applied in many aspects through post-modification and
structural adjustment of the pores and structures, including adsorption
separation [25], photoelectric induction [26], biotechnology [27] and
others. Immobilization of enzymes on less uniform solid support typi-
cally results in low protein loading efficiency [28], low stability at
elevated temperatures, and enzymatic leaching. MOFs have good
adjustability, crystallinity and uniformity. Therefore, MOFs may be su-
perior to other commonly used porous materials for immobilizing pro-
teins and enzymes, such as sol-gel, zeolite and mesoporous silicon-ica
supports. Zeolitic imidazolate frameworks (ZIF) are a class of promising
supports for enzyme immobilization in various MOFs [29], because of
their easy synthesis, excellent stability and negligible cytotoxicity [30].
In the past few years, MOFs have been used as a popular support for
immobilization of enzymes, and excellent results have been achieved in
catalytic applications [31]. However, there is rarely report on enantio-
selective catalyzed resolution of chiral enantiomers by using the
immobilized enzymes as bio-catalyst.
2489, U. S. A) was 230 nm. The mobile phase was consisting of meth-
anol and aqueous solution (containing 25 mmol/L HP-β-CD and 0.5 %
acetic acid) at the volume ratio of 20:80 (adjusted to pH = 4.0 with
triethylamine) [32]. The flow rate of the mobile phase and the column
temperature were maintained at 1.0 mL/min and 298 K, respectively.
The injection volume of each sample was 10 μL. The retention time of
(R)-2-PPA and (S)-2-PPA were 24.10 and 27.17 min, respectively. The
(R, S)-2-PPA esters were monitored by HPLC, and the mobile phase was
composed of acetonitrile and water at the volume ratio of 70:30. Other
chromatographic conditions are the same as (R, S)-2-PPA.
In the enantioselective hydrolysis of (R, S)-2-PPA ester, the enan-
tiomeric excess of (S)-2-PPA (eep) and conversion rate (cR) of (R)-2-PPA
were calculated as follows:
[PPAS] ꢀ [PPAR]
eeP
=
× 100%
(1)
[PPAS] + [PPAR]
[PPAR]
[SR]0
cR
=
× 100%
(2)
where [PPAS] and [PPAR] are the concentrations of (S)-2-PPA and (R)-2-
PPA after the reaction, respectively; [SR]o represents initial concentra-
tion of (S)-2-PPA ester.
The remaining 1-phenylethanol enantiomers in reaction system were
analyzed by HPLC employing a Waters e2695 series apparatus. The
column was a Chiralcel® OJ-RH column (250 mm × 4.6 mm, 5 μm,
In this work, Pseudomonas cepacia lipase was immobilized into ZIF-8
(hereafter denoted PCL@ ZIF-8) by using physical adsorption. The
structure of ZIF-8 and PCL@ ZIF-8 was characterized by powder X-ray
diffraction spectrometry (PXRD). Besides, the ZIF-8 and PCL@ ZIF-8
were further characterized by Fourier Transform Infrared Spectros-
copy (FTIR), N2 adsorption-desorption methods, and thermogravimetric
analysis (TGA) to confirm the immobilization of PCL into ZIF-8.
PCL@ZIF-8 was used as bio-catalyst for enantioselective hydrolysis of
2-PPA ester enantiomers and transesterification of 1-phenylethanol en-
antiomers to evaluate the catalytic activity and selectivity. In addition,
the stability of immobilized enzyme was evaluated by investigating
reusability.
Japan). The wavelength of photodiode array detector (Waters 2998) was
210 nm, and the column temperature was 298 K. The mobile phase was
a 20:80 (v/v) mixture of acetonitrile and water. The flow rate was
0.5 mL/min, and the injection volume was set at 10 μL. The retention
time of (S)-1-phenylethanol was less than that of (R)-1-phenylethanol.
In the enantioselective transesterification of 1-phenylethanol enan-
tiomers, the enantiomeric excess of the product (eep) was determined by
the enantiomeric excess of the substrate (ees) and total conversion rate
(c).
(
)
ee
c
eeP
=
S ꢀ eeS × 100%
(3)
where
2. Materials and methods
[A(S)] ꢀ [A(R)
[A(S)] + [A(R)
]
]
eeS
=
× 100%
(4)
(5)
2.1. Materials
(
)
Lipase from Pseudomonas cepacia (PCL, 100,000 U/g) was acquired
from Amano Enzyme Inc. (Nagoya, Japan). (R, S)-2-PPA (purity > 99
%), 1-phenylethanol (purity > 98 %) and 2-Methylimidazole (purity >
98 %) were obtained from Adamas Reagent Co., Ltd. (Shanghai, China).
(R, S)-2-PPA esters were prepared in the laboratory. Hydroxypropyl-
[A(S)] + [A(R)
[A(S)]0 + [A(R)
]
]
c = 1 ꢀ
× 100%
0
where [A(S)] and [A(R)] represent the substrate concentration of (S)-1-
phenylethanol and (R)-1-phenylethanol after reaction, respectively;
133

J. Ou et al.
Process Biochemistry 102 (2021) 132–140
[A(S) 0
]
and [A(R) 0
]
represent the initial concentration of (S)-1-phenyl-
bio-catalyst and 10 mmol/L (R, S)-2-PPA ester as the reaction substrate.
Both free PCL and PCL@ZIF-8 are identical in enzyme content. The
temperature and stirring speed of thermostatic reactor (IKA®RCT CV
S025, Werke GmbH & Co. KG) were maintained at 328 K and 400 rpm,
respectively. After a certain period of reaction, the reaction mixture was
filtered to obtain a sample. HPLC detected the concentrations of prod-
ucts. Kinetic resolution of 2-PPA enantiomers by the enzymatic is dis-
played in Fig. 1.
ethanol and (R)-1-phenylethanol, respectively.
2.3. Preparation and characterization of the immobilized lipase
Preparation of ZIF-8: The ZIF-8 was prepared according to the
literature [33]. In brief, 2-methylimidazole (7.5 mmol) and Zn
(NO3)2•6H2O (2.5 mmol) were mixed into the DMF (600 mL) solution in
the round-bottomed flask. When the mixture turns out a homogeneous
phase, the round-bottomed flask was placed in an oil bath at 413 K one
day. After cooled to ambient temperature, the mixture was rinsed three
times with methanol (3 × 100 mL), then brownish-black solid product
was obtained after drying 12 h in a vacuum oven at 353 K. The structure
of ZIF-8 was characterized by powder X-ray diffraction spectrometry
(PXRD) and scanning electron microscope (SEM).
Enzymatic transesterification: Enzymatic transesterification of 1-
phenylethanol was carried out in a 25 mL glass tube with a screw seal.
The hybrid media was composed as follows: 3 mL of hexane as reaction
medium, 5 mg free PCL and 25 mg PCL@ZIF-8 as bio-catalyst,
20 mmol/L 1-phenylethanol and 100 mmol/L acetoxyethylene as the
reaction substrate. The reaction temperature and agitation speed in
thermostatic reactor were kept at 310 K and 500 rpm, respectively. The
sample was obtained by filtering the enzyme out of the reaction solution.
HPLC detected the products concentrations. The reaction mechanism of
enzymatic transesterification was shown in Fig. 2.
Immobilization of lipase: 400 mg ZIF-8 and 160 mg lipase PS were
dispersed in 80 mL deionized water to form a mixture. The mixture was
put in the Thermostatic oscillator at 298 K for 24 h. Lipase PS immo-
bilized into ZIF-8 (PCL@ZIF-8) was rinsed with deionized water three
times. Finally, the PCL@ZIF-8 after washing was dried by freeze dryer
for 12 h. The chemical composition of PCL@ZIF-8 was analyzed by the
Fourier Transform Infrared Spectroscopy (FTIR).
2.6. Reusability of PCL@ZIF-8
The reusability of PCL@ZIF-8 was studied by reusing 4 times for
enantioselective hydrolysis of (R, S)-2-PPA ester and 6 times for enan-
tioselective transesterification of 1-phenylethanol. In the hydrolysis of
(R, S)-2-PPA ester, the PCL@ZIF-8 was recovered by filtration and
washed with 0.1 mol/L PBS (pH = 6.0) after every cycle. In the trans-
esterification of 1-phenylethanol, the PCL@ZIF-8 was washed with
hexane (3 × 30 mL) after every cycle.
2.4. Assay of enzyme activity and protein assay
The enzyme activities in the immobilization process were deter-
mined with the substrate of p-nitrophenyl palmitate (p-NPP). In 50 mM
sodium phosphate at pH 7.5 and 25 ℃, the concentration of p-nitro-
phenol (p-NP) produced by lipase catalyzed hydrolysis of 0.2 mM p-NPP
was determined by UV spectrophotometer at 410 nm. One international
unit of activity (U) was defined as the amount of enzyme that produce
3. Result and discussion
1 μmol NP per minute under the conditions described previously. In
3.1. Characterization of immobilized lipase
order to facilitate the investigation of the influence of variables on the
activity of immobilized enzymes. Take the highest value of enzyme ac-
tivity in the same group of experiments as 100 %.
For the preparation of the immobilized lipase, ZIF-8 was first syn-
thesized, and then Pseudomonas cepacia lipase was immobilized into ZIF-
8 by physical adsorption (Fig. 3).
The immobilization efficiency was deuced by BCA (bicinchoninic
acid) method, using bovine serum albumin (BSA) as standard. Analyze
the protein concentration in the enzyme solution with microplate
reader. The immobilized yield of lipase was indirectly determined by
comparing the protein concentration difference between the enzyme
solution provided before immobilization and the filtrate separated after
immobilization. The immobilized yield (IY) was described by Eq. (6).
The structures of synthesized ZIF-8 and PCL@ZIF-8 were character-
ized by PXRD. The PXRD patterns reveal that the three typical charac-
teristic peaks at 7.36◦, 12.7◦, and 18◦ were matched well with the
theoretical calculational values (Fig. 4a). There is no significant differ-
ence concerning the crystal structure and crystallinity between the ZIF-8
and PCL@ZIF-8 samples, confirming that the ZIF-8 material was suc-
cessfully synthesized and the PCL@ZIF-8 host could still maintain its
framework integrity. SEM were also used to explore morphology. The
SEM images show that the morphology of synthesized PCL@ZIF-8
composite is the same as pure ZIF-8 (Fig. 4c and d), indicating that
the immobilization process of lipase would not affect the surface
morphology of ZIF-8. In order to ascertain that PCL is indeed adsorbed
by ZIF-8, the ZIF-8 and PCL@ZIF-8 samples were examined by FTIR. The
FTIR results of ZIF-8 ranging from 4000ꢀ 400 cmꢀ 1 are shown in Fig. 4b.
The absorption bands at 600ꢀ 900 cm-1 may be due to the stretch of
C0 ꢀ C
IY(%) =
1 × 100%
(6)
C0
C0 and C1 are the protein concentration of lipase solution initially and
finally, respectively.
2.5. Catalytic performances study
Preparation of (R, S)-2-PPA ester: (R, S)-2-PPA and isobutanol with
equivalent molar were dissolved in toluene for esterification reactions,
with p-toluenesulfonic acid as catalyst. After stirring for one night at
383 K, (R, S)-2-PPA esters were synthesized. The saturated NaHCO3
solution (3 × 40 mL) was added to remove the excess acid in mixture
solution. Then, distilled water (3 × 40 mL) was used to clean above (R,
S)-2-PPA esters to neutral. The anhydrous MgSO4 was applied to dry
separated organic phase for 12 h, and then the solid was filtered. Finally,
the toluene was removed by evaporation with lower pressure, and the
yellow liquid product was obtained, that is (R, S)-2-PPA ester. In the end,
HPLC analysis was used to confirm the synthesized products.
aromatic C H, and 3000–3200 cm-1 can be attributed to the stretching
–
–
of aliphatic C H by methylimidazole linker [34]. The absorption bands
900ꢀ 1250 cmꢀ 1 correspond to the C N bond stretching. The bands at
–
1350ꢀ 1500 cmꢀ 1 are caused by the stretching of entire methyl-
imidazole ring. The peak at 1580 cmꢀ 1 corresponds to C N stretching
–
–
vibration. Besides, the FTIR spectra of PCL@ZIF-8 demonstrated
Enzymatic hydrolysis: Enzymatic hydrolysis reaction was performed
in a 10 mL glass tube with a screw seal. The hybrid media was composed
as follows: 2 mL of phosphate buffer solution (PBS, 0.1 mol/L, pH = 6.0)
as reaction medium, 2.5 mg/mL free PCL or 12.5 mg/mL PCL@ZIF-8 as
Fig. 1. Enantioselective hydrolysis of (R, S)-2-phenylpropionic acid ((R, S)-2-
PPA) ester catalyzed by the enzyme.
134

J. Ou et al.
Process Biochemistry 102 (2021) 132–140
deeper drop above 303 ℃ compared to pure ZIF-8, which is attributed to
the decomposition of the PCL in PCL@ZIF-8. Compared with the
remaining amount of both ZIF-8 and PCL@ZIF-8, the content of PCL in
the PCL@ZIF-8 was estimated to be about 16.7 % (200.5 mg PCL/g
ZIF-8). From Table S2, compared with other types of supports, such as
silica, Silica gel, cellulose, hydroxyapatite and others [40–45], ZIF-8 as
immobilization carrier has a higher immobilized capacity due to the
high specific surface area and suitable voids.
3.2. Free PCL immobilized into ZIF-8
The effect of immobilization time on immobilized lipase activity and
lipase loading amount are shown in Fig. S1. In the initial stage of the
reaction, with the extension of the immobilization time, the immobilized
lipase activity and lipase loading amount increased rapidly. The relative
activity of immobilized lipase and lipase loading amount reached the
maximum at 24 h. However, the lipase activity decreased with the
enhancement of the immobilization time. It may be due to the weak
interaction between free PCL and ZIF-8. Sodium dodecyl sulfate (SDS) is
used as a desorbent for the enzyme-MOF complex. After the desorption
experiments of PCL@MOF, the catalytic performance of immobilized
lipase was rarely maintained only 10 % relative activity, indicating that
the lipase is mainly immobilized in the cavity of the MOFs in the form of
physical adsorption. Lipase relies on weak van der waals forces and
hydrogen bonds to attach to the cavity of the MOFs. A part of free PCL
was separated from ZIF-8 after long time soaking. As shown in Fig. S1, it
is proposed to control the immobilization time at 24 h.
Fig. 2. Enantioselective transesterification of 1-phenylethanol catalyzed
by lipase.
Since the chemical nature of lipases are proteins, their performances
are easily affected by temperature. The effect of temperature on
immobilized lipase activity and lipase loading amount were investigated
in the range of 5 ℃ to 65 ℃. As shown in Fig. S2, the optimal incubation
temperature is 25 ℃. The immobilized amount of lipase did not obvi-
ously change with the change of temperature. The immobilized lipase
activity increases as the temperature increases within a certain range,
and then gradually decreased with further temperature increases. The
reason for this phenomenon is that temperature has a dual effect on li-
pases. Increasing temperature can increase the lipase loading amount,
but it will also deform the enzyme and lose its activity after a certain
temperature.
Fig. 3. Synthesis of the Zeolitic imidazolate framework-8 (ZIF-8) and tentative
mechanism for the translocation of free Pseudomonas cepacia lipase (PCL) into
the cavities of ZIF-8.
3.3. Catalytic performance of immobilized lipase
characteristics peaks of PCL at 1660 cmꢀ 1, which belongs to C
O
–
–
3.3.1. Hydrolytic activities of free PCL and PCL@ZIF-8
stretching vibration. Therefore, it was confirmed that Pseudomonas
cepacia lipase was stabilized into ZIF-8 by adsorption. To further verify
the immobilization of lipase, N2 adsorption-desorption isotherms of
ZIF-8 and PCL@ZIF-8 were investigated using the surface area analyzer
ASAP-2020 at 77 K. The N2 adsorption-desorption isotherms of ZIF-8
and PCL@ZIF-8 show a characteristic Type Ⅳ sorption behavior
(Fig. 4e and f) [35]. After adsorption of PCL into ZIF-8, the porosity of
ZIF-8 was dramatically decreased due to the residence of enzymes in
pores [36]. The BET surface area of ZIF-8 was reduced from 1431 m2/g
to 1253 m2/g (Table S1). In addition, the t-plot micropore area of ZIF-8
was reduced from 1262 m2/g to 1100 m2/g. It was inferred from these
observations that a large amount of PCL was adsorbed inside the pores of
ZIF-8 (Fig. 3), and an amount of PCL was adsorbed on the surface of
ZIF-8 [37]. The pore size distribution of ZIF-8 and PCL@ZIF-8 are shown
in Fig. 4e and f, respectively. Compared with ZIF-8, the mesoporous pore
number of PCL@ZIF-8 decreased obviously. From Table S1, the total
pore volume of ZIF-8 and PCL@ZIF-8 are 1.2991 cm3/g and
1.2145 cm3/g, respectively. PCL@ZIF-8 has a smaller total pore volume
compared to ZIF-8 due to the embedded PCL molecules, which is
consistent with the results reported in the literature [38].
Catalytic experiments were carried out for free PCL and PCL@ZIF-8
to evaluate the activities and enantioselectivities. The schematic dia-
gram of enantioselective catalyzed hydrolysis of 2-PPA ester was dis-
played in Fig. 5 by using free PCL and PCL@ZIF-8 as catalyst. The
activities and enantioselectivities for free PCL and PCL@ZIF-8 were
estimated by the conversion rate of (S)-2-PPA ester and enantiomeric
excess of (S)-2-PPA for enantioselective hydrolysis reaction. The curves
of conversion rate and enantiomeric excess at different times were
shown in Fig. 6. It was observed from Fig. 6 and Table 1 that free PCL
and PCL@ZIF-8 indicated the same enantiomeric excess of product at
the tested times, showing that the immobilization PCL into ZIF-8
maintains the initial enantioselectivity of the free enzyme. We also
found that PCL@ZIF-8 demonstrated a faster initial rate of 3.96 × 10ꢀ 4
mM/s than that of 1.32 × 10ꢀ 4 mM/s for free PCL as derived from the
slope in the first 2 h. In the enantioselective hydrolysis reaction, the
catalytic efficiency of the immobilized enzyme is three times that of the
free enzyme. After 28 h (Fig. 6 and Table 1), the conversion rate of (S)-2-
PPA ester is up to 92 % for PCL@ZIF-8, while it is only 54 % for free PCL.
In the same amount of PCL, the immobilization of lipase can signifi-
cantly enhance the activity of enzyme and improve the initial rate of
enantioselective hydrolysis reaction.
The content of PCL adsorbed into ZIF-8 was determined by ther-
mogravimetric analysis (TGA) method [39]. The TGA curves of ZIF-8
and PCL@ZIF-8 are shown in Fig. 4g. The PCL@ZIF-8 sample shows a
By immobilizing PCL into ZIF-8, high selectivity for PCL was not only
maintained, but also activity was greatly enhanced. After
135

J. Ou et al.
Process Biochemistry 102 (2021) 132–140
Fig. 4. Characterization: (a) PXRD patterns of simulated Zeolitic imidazolate framework-8 (ZIF-8), as-synthesized ZIF-8, and free Pseudomonas cepacia lipase
immobilized into ZIF-8 (PCL@ZIF-8); (b) FTIR spectra of ZIF-8 and PCL@ZIF-8; SEM images of (c) ZIF-8 and (d) PCL@ZIF-8; Nitrogen adsorption-desorption iso-
therms and Pore-size distribution of (e) ZIF-8 and (f) PCL@ZIF-8; (g) TGA curves of ZIF-8 and PCL@ZIF-8.
136

J. Ou et al.
Process Biochemistry 102 (2021) 132–140
Fig. 5. Schematic diagram of enantioselective hydrolysis of 2-phenylpropionic acid (2-PPA) ester catalyzed by free Pseudomonas cepacia lipase (PCL) and free
Pseudomonas cepacia lipase immobilized into ZIF-8 (PCL@ZIF-8).
more easily contact with the enzyme to form a complex. Free PCL is a
hydrolytic enzyme that is easily soluble in water. Enantioselective hy-
drolysis of 2-PPA ester enantiomers by free PCL catalyzed was carried
out by liquid-liquid interfacial reaction. Therefore, PCL@ZIF-8 has
stronger activity than free PCL. Fig. S3 shows that the enantioselective
hydrolysis reaction catalyzed by PCL @ ZIF-8 with an optical purity of
99.62 %.
3.3.2. Transesterification activities of free PCL and PCL@ZIF-8
Fig.
7 shows the reaction process of enantioselective trans-
esterification of 1-phenylethanol enantiomers by free PCL and
PCL@ZIF-8 catalyzed. The activities for free PCL and PCL@ZIF-8 were
estimated by the conversion rate of (R, S)-1-phenylethanol for enan-
tioselective transesterification reaction (Fig. 8). It was observed from
Fig. 8 that there is no significant difference in the transesterification
activity of free PCL and PCL@ZIF-8. At the beginning, free PCL has
higher transesterification activity than PCL@ZIF-8, However, the
transesterification activity of free PCL decreased significantly after 4 h.
This phenomenon is consistent with literature reports [46]. Free PCL is
not soluble in the organic phase but is suspended in the organic phase,
which is prone to agglomeration in the reaction process. However,
PCL@ZIF-8 can effectively prevent the agglomeration of PCL due to the
protection of the ZIF-8 with excellent dispersion properties, and still
maintain a high transesterification activity. Fig. S4 indicates that the
optical purity of the reaction product is up to 99.47 % for enantiose-
lective transesterification reaction by PCL@ZIF-8 catalyzed.
Fig. 6. Hydrolysis activity and selectivity of the free Pseudomonas cepacia lipase
(PCL) and free Pseudomonas cepacia lipase immobilized into ZIF-8 (PCL@ZIF-8).
Conditions: 10 mmol/L (R, S)-2-phenylpropionic acid ester, 2.5 mg/mL free
PCL or 12.5 mg/mL PCL@ZIF-8, pH = 6.0, T =328 K.
Table 1
Summary of hydrolytic activity and selectivity of free Pseudomonas cepacia lipase
(PCL) and free Pseudomonas cepacia lipase immobilized into ZIF-8 (PCL@ZIF-8).
PCL
PCL@ ZIF-8
Rate (mM/s)a
eep (%)b
1.32 × 10ꢀ 4
3.9 × 10ꢀ 4
99.46
99.62
cs (%)c
54.225
92.327
a
Rate of (S)-2-phenylpropionic acid ((S)-2-PPA) ester calculated from the first
3.3.3. Reusability of PCL@ZIF-8
2 h.
b
As well known, free PCL is easily soluble in water and agglomerates
in organic solvents, making it difficult to reuse. Fortunately, one of the
most prominent advantages of the immobilization technology is to
improve the reusability of lipase. To investigate the recyclability of
PCL@ZIF-8, the enzyme activity and enantioselectivity were determined
at different cycles.
Final eep after 24 h.
c
Final cs after 24 h. Conditions: 10 mmol/L (R, S)-2-PPA ester, 2.5 mg/mL
free PCL or 12.5 mg/mL PCL@ZIF-8, pH = 6.0, T =328 K.
immobilization of PCL into ZIF-8, the BET surface area and the t-plot
micropore area of PCL@ZIF-8 were still up to 1253 m2/g and 1100 m2/
g, respectively. 2-PPA ester is difficult to dissolve in water, while the
high BET surface area of PCL@ZIF-8 can easily adsorb 2-PPA ester into
the cavity of the enzyme-MOF composite material, making the substrate
Fig. 9 exhibits the recyclability of PCL@ZIF-8 in enantioselective
hydrolysis reaction. As the cycle number of PCL@ZIF-8 increases, the
conversion rate of (S)-2-PPA ester decreases gradually, but enantiomeric
137

J. Ou et al.
Process Biochemistry 102 (2021) 132–140
Fig. 7. Schematic diagram of enantioselective transesterification of 1-phenylethanol catalyzed by free Pseudomonas cepacia lipase (PCL) and free Pseudomonas cepacia
lipase immobilized into ZIF-8 (PCL@ZIF-8).
Fig. 10. Transesterification activity and selectivity of free Pseudomonas cepacia
lipase immobilized into ZIF-8 (PCL@ZIF-8) during the recycling use. Condi-
tions: 20 mmol/L (R, S)-1-phenylethanol, 100 mmol/L vinyl acetate, 25 mg
PCL@ZIF-8, 3 mL hexane, T =310 K, 10 h.
Fig. 8. Transesterification activity of the Pseudomonas cepacia lipase (PCL) and
free Pseudomonas cepacia lipase immobilized into ZIF-8 (PCL@ZIF-8). Condi-
tions: 20 mmol/L (R, S)-1-phenylethanol, 100 mmol/L vinyl acetate, 5 mg free
PCL or 25 mg PCL@ZIF-8, 3 mL hexane, T =310 K.
increase of conversion rate was the formation of imprinted enzymes
[47]. From the third cycle to the sixth cycle, it was observed that the
residual activity gradually declined from 96.48% to 56.01%. Decrease in
reusability occurred due to the following reasons: (1) Immersion time in
water for too long caused the structure to partially collapse and lipase
was separated from ZIF-8. (2) Electrostatic interaction between enzyme
and support was destroyed to result in desorption of the immobilized
enzyme, including hydrophobic interaction, intermolecular force,
hydrogen bonding force, and others. (3) The denaturation of lipase
during the reaction process and the diffusion limitation by substrate
caused the decay of immobilized enzyme activity [48,49].
In the past decade, some literatures have also reported on the reus-
ability of immobilized PCL in various catalytic reactions [50–55].
However, different characteristics of carrier materials and different
types of catalytic reactions have a great influence on the reusability of
the immobilized enzyme. It was observed from Table S3 that immobi-
lized PCL can be reused 4–8 times in the catalytic reaction. Compared
with other immobilized PCL, the PCL@ZIF-8 also exhibits excellent
reusability, maintaining the six cycles. In two reaction system, the re-
covery rate of the PCL@ZIF-8 was more than 90 % during the recycling
use. The advantage of ZIF-8 as a solid carrier for the preparation of
immobilized enzymes is that it exhibits high catalytic activity and
reusability while maintaining the initial enantioselectivity of the free
enzyme. On the other hand, the high specific surface area of ZIF-8 can
enhance the adsorption of insoluble substrates, resulting in an increase
of reaction rate.
Fig. 9. Hydrolysis activity and selectivity of free Pseudomonas cepacia lipase
immobilized into ZIF-8 (PCL@ZIF-8) during the recycling use. Conditions:
10 mmol/L substrate, 12.5 mg/mL PCL@ZIF-8, pH = 6.0, T =328 K, t =28 h.
excess of (S)-2-PPA remains almost constant. PCL@ZIF-8 still remained
30.57 % of the initial hydrolysis activity at the fourth cycle. The decay of
activity for PCL@ZIF-8 originated from leaching of the PCL immobilized
into ZIF-8 during multiple cycles. At the same time, we also investigated
the transesterification activity of PCL@ZIF-8 at different cycles. As
shown in Fig. 10, the conversion rate of (R)-1-phenylethanol first in-
creases and then decreases. The conversion rate of (R)-1-phenylethanol
increased at the second cycle, and we speculated that the reason for the
138

J. Ou et al.
Process Biochemistry 102 (2021) 132–140
[8] A.P. de Los Ríos, F. van Rantwijk, R.A. Sheldon, Effective resolution of 1-phenyl
ethanol by candida antarctica lipase B catalysed acylation with vinyl acetate in
protic ionic liquids (PILs), Green Chem. 14 (2012) 1584–1588, https://doi.org/
10.1039/C2GC35196J.
4. Conclusions
In this work, the free PCL was successfully immobilized into the
water-stable material ZIF-8 by physical adsorption. Characterization
results confirmed that the majority of PCL was adsorbed inside the pores
of ZIF-8, and an amount of PCL was adsorbed on the surface of ZIF-8. The
PCL@ZIF-8 as effective bio-catalyst was applied for enantioselective
enzymatic hydrolysis of (R, S)-2-PPA ester and transesterification of 1-
phenylethanol enantiomers. In the catalytic experiments, PCL@ZIF-8
exhibited more excellent enzymatic catalytic performance compared
with free PCL. The hydrolysis activity of the immobilized enzyme was
three times more than free PCL, and the hydrolysis selectivity was
maintained above 99 %. In addition, the PCL@ZIF-8 showed good
reusability in both hydrolysis (30.57 % of initial activity, 4 cycle) and
transesterification reaction systems (64.38 % of initial activity, 6 cycle).
MOFs, which possess simple synthesis, variety and stability, is a kind of
potential immobilized carrier for the immobilization of enzymes in the
industrial application.
[9] K. Kawakami, M. Ueno, Y. Oda, Application of a burkholderia cepacia lipase-
immobilized silica monolith micro-bioreactor to continuous-flow kinetic resolution
for transesterification of (R, S)-1-phenylethanol, Process Biochem. 47 (2012)
147–150, https://doi.org/10.1016/j.procbio.2011.09.017.
¨
[10] O. Verho, J.-E. Backvall, Chemoenzymatic dynamic kinetic resolution: a powerful
tool for the preparation of enantiomerically pure alcohols and amines, J. Am.
Chem. Soc. 137 (2015) 3996–4009, https://doi.org/10.1021/jacs.5b01031.
[11] G.A.I. Moustafa, Y. Oki, S. Akai, Lipase-catalyzed dynamic kinetic resolution of C1-
and C2- symmetric racemic axially chiral 2,2’-dihydroxy-1,1’-biaryls, Angew.
Chem. Int. Ed. 130 (2018) 10435–10439, https://doi.org/10.1002/
anie.201804161.
´
[12] R.C. Rodrigues, J.J. Virgen-Ortíz, J.C.S. dos Santos, A. Berenguer-Murcia, A.
R. Alcantara, O. Barbosa, C. Ortiz, R. Fernandez-Lafuente, Immobilization of
lipases on hydrophobic supports: immobilization mechanism, advantages,
problems, and solutions, Biotechnol. Adv. 37 (2019) 746–770, https://doi.org/
10.1016/j.biotechadv.2019.04.003.
[13] S. Arana-Pea, N.S. Rios, D. Carballares, L.R.B. Goncalves, R. Fernandez-Lafuente,
Immobilization of lipases via interfacial activation on hydrophobic supports:
production of biocatalysts libraries by altering the immobilization conditions,
Catal. Today 362 (2021) 130–140, https://doi.org/10.1016/j.cattod.2020.03.059.
´
[14] R.R.C. Monteiro, J.J. Virgen-Ortiz, A. Berenguer-Murcia, T.N. da Rocha, J.C.S. dos
´
Santos, A.R. Alcantara, R. Fernandez-Lafuente, Biotechnological relevance of the
CRediT authorship contribution statement
lipase A from Candida antarctica, Catal. Today 362 (2021) 141–154, https://doi.
org/10.1016/j.cattod.2020.03.026.
Jian Ou: Methodology, Investigation, Data curation, Writing -
original draft. Xin Yuan: Investigation, Writing - review & editing. Yu
Liu: Writing - review & editing. Panliang Zhang: Investigation. Wei-
feng Xu: Investigation. Kewen Tang: Conceptualization, Project
administration, Writing - review & editing.
[15] D. Romano, F. Bonomi, M.C. de Mattos, T. de Sousa Fonseca, M. da C.F. de Oliveira,
F. Molinari, Esterases as stereoselective biocatalysts, Biotechnol. Adv. 33 (2015)
547–565, https://doi.org/10.1016/j.biotechadv.2015.01.006.
[16] N.S. Rios, B.B. Pinheiro, M.P. Pinheiro, R.M. Bezerra, J.C.S. dos Santos, L.R.
B. Goncalves, Biotechnological potential of lipases from Pseudomonas: sources,
properties and applications, Process Biochem. 75 (2018) 99–120, https://doi.org/
10.1016/j.procbio.2018.09.003.
´
´
[17] R.C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres, R. Fernandez-Lafuente,
Modifying enzyme activity and selectivity by immobilization, Chem. Soc. Rev. 42
(2013) 6290–6307, https://doi.org/10.1039/C2CS35231A.
Declaration of Competing Interest
[18] S. Hudson, J. Cooney, E. Magner, Proteins in mesoporous silicates, Angew. Chem.
Int. Ed. 47 (2008) 8582–8594, https://doi.org/10.1002/anie.200705238.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
´
[19] S. Patra, T.H. Crespo, A. Permyakova, C. Sicard, C. Serre, A. Chausse, N. Steunou,
L. Legrand, Design of metal organic framework-enzyme based bioelectrodes as a
novel and highly sensitive biosensing platform, J. Mater. Chem. B 3 (2015)
8983–8992, https://doi.org/10.1039/C5TB01412C.
Acknowledgments
[20] X. Yuan, Y. Liu, F. Cao, P. Zhang, J. Ou, K. Tang, Immobilization of lipase onto
metal-organic frameworks for enantioselective hydrolysis and transesterification,
AIChE J. 66 (2020), e16292, https://doi.org/10.1002/aic.16292.
[21] Y. Chen, V. Lykourinou, T. Hoang, L.-J. Ming, S. Ma, Size-selective biocatalysis of
myoglobin immobilized into a mesoporous metal-organic framework with
hierarchical pore sizes, Inorg. Chem. 51 (2012) 9156–9158, https://doi.org/
10.1021/ic301280n.
This work was supported by the National Natural Science Foundation
of China (No. 21676077), Innovation Research Group Project of Natural
Science Foundation of Hunan Province (No. 2020JJ1004) and Hunan
Provincial Innovation Foundation for Postgraduate (No. CX20190915).
[22] A.S. Sahutoglu, C. Akgul, Immobilisation of aspergillus oryzae α-amylase and
aspergillus niger glucoamylase enzymes as cross-linked enzyme aggregates, Chem.
Pap. 69 (2015) 433–439, https://doi.org/10.1515/chempap-2015-0031.
[23] X. Qiu, W. Zhong, C. Bai, Y. Li, Encapsulation of a metal-organic polyhedral in the
pores of a metal-organic framework, J. Am. Chem. Soc. 138 (2016) 1138–1141,
https://doi.org/10.1021/jacs.5b12189.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.procbio.2020.12.017.
[24] K. Shen, L. Zhang, X. Chen, L. Liu, D. Zhang, Y. Han, J. Chen, J. Long, R. Luque,
Y. Li, B. Chen, Ordered macro-microporous metal-organic framework single
crystals, Science 359 (2018) 206–210, https://doi.org/10.1126/science.aao3403.
[25] X. Zhou, W. Huang, J. Shi, Z. Zhao, Q. Xia, Y. Li, H. Wang, Z. Li, A novel MOF/
graphene oxide composite GrO@MIL-101 with high adsorption capacity for
acetone, J. Mater. Chem. A 2 (2014) 4722–4730, https://doi.org/10.1039/
C3TA15086K.
References
[1] I. Boussouar, Q. Chen, X. Chen, Y. Zhang, F. Zhang, D. Tian, H.S. White, H. Li,
Single nanochannel platform for detecting chiral, Anal. Chem. 89 (2017)
1110–1116, https://doi.org/10.1021/acs.analchem.6b02682.
[26] Q. Gong, Z. Hu, B.J. Deibert, T.J. Emge, S.J. Teat, D. Banerjee, B. Mussman, N.
D. Rudd, J. Li, Solution processable MOF yellow phosphor with exceptionally high
quantum efficiency, J. Am. Chem. Soc. 136 (2014) 16724–16727, https://doi.org/
10.1021/ja509446h.
[2] Y. Wang, S. Zhuo, J. Hou, W. Li, Y. Ji, Construction of β-cyclodextrin covalent
organic framework modified chiral stationary phase for chiral separation, ACS
Appl. Mater. Interfaces 11 (2019) 48363–48369, https://doi.org/10.1021/
acsami.9b16720.
[27] J. Yao, Y. Liu, J. Wang, Q. Jiang, D. She, H. Guo, N. Suna, Z. Pang, C. Deng,
W. Yang, S. Shen, On-demand CO release for amplification of chemotherapy by
MOF functionalized magnetic carbon nanoparticles with NIR irradiation,
Biomaterials 195 (2019) 51–62, https://doi.org/10.1016/j.
´
´
[3] J. Navarro-Sanchez, A.I. Argente-García, Y. Moliner-Martínez, D. Roca-Sanjuan,
´
D. Antypov, P. Campíns-Falco, M.J. Rosseinsky, C. Martí-Gastaldo, Peptide metal-
organic frameworks for enantioselective separation of chiral drugs, J. Am. Chem.
Soc. 139 (2017) 4294–4297, https://doi.org/10.1021/jacs.7b00280.
[4] S. Bhatia, A. Kamaruddin, S.L. Wei, Chiral resolution of racemic ibuprofen ester in
an enzymatic membrane reactor, J. Membr. Sci. 247 (2005) 185–200, https://doi.
org/10.1016/j.memsci.2004.09.019.
biomaterials.2018.12.029.
[28] K. Yasutaka, Y. Takato, K. Takashi, M. Kohsuke, Y. Hiromi, Enhancement in
adsorption and catalytic activity of enzymes immobilized on phosphorus- and
calcium-modified MCM-41, J. Phys. Chem. B 115 (2011) 10335–10345, https://
doi.org/10.1021/jp203632g.
[5] I. Bhushan, R. Saraswat, P. Gupta, B.A. Shah, Enantioselective resolution of 2-
arylpropionic acid derivatives employing immobilization of lipase from bacillus
subtilis strain kakrayal_1 (BSK-L), Bioresource Technol. 269 (2018) 581–585,
https://doi.org/10.1016/j.biortech.2018.08.123.
[29] H. He, H. Han, H. Shi, Y. Tian, F. Sun, Y. Song, Q. Li, G. Zhu, Construction of
thermophilic lipase-embedded metal-organic frameworks via biomimetic
mineralization: a biocatalyst for ester hydrolysis and kinetic resolution, ACS Appl.
Mater. Interfaces 8 (2016) 24517–24524, https://doi.org/10.1021/
acsami.6b05538.
[6] K. Tang, L. Song, Y. Liu, Separation of flurbiprofen enantiomers by biphasic
recognition chiral extraction, Chem. Eng. J. 158 (2010) 411–417, https://doi.org/
10.1016/j.cej.2010.01.009.
[30] M. Niknam Shahrak, M. Ghahramaninezhad, M. Eydifarash, Zeolitic imidazolate
framework-8 for efficient adsorption and removal of Cr(VI) ions from aqueous
solution, Environ. Sci. Pollut. Res. 24 (2017) 9624–9634, https://doi.org/10.1007/
s11356-017-8577-5.
`
´
[7] J.F. Collados, R. Sola, S.R. Harutyunyan, B. Macia, Catalytic synthesis of
enantiopure chiral alcohols via addition of grignard reagents to carbonyl
compounds, ACS Catal. 6 (2016) 1952–1970, https://doi.org/10.1021/
acscatal.5b02832.
139

J. Ou et al.
Process Biochemistry 102 (2021) 132–140
[31] V. Lykourinou, Y. Chen, X.-S. Wang, L. Meng, T. Hoang, L.-J. Ming, R.
L. Musselman, S. Ma, Immobilization of MP-11 into a mesoporous metal-organic
framework, MP-11@mesoMOF: a new platform for enzymatic catalysis, J. Am.
Chem. Soc. 133 (2011) 10382–10385, https://doi.org/10.1021/ja2038003.
[32] S. Tong, Y. Zheng, J. Yan, Application and comparison of high performance liquid
chromatography and high speed counter-current chromatography in
enantioseparation of (±)-2-phenylpropionic acid, J. Chromatogr. A 1281 (2013)
79–86, https://doi.org/10.1016/j.chroma.2013.01.056.
[43] L. Li, P.W. Dyer, H.C. Greenwell, Biodiesel production via trans-esterification using
pseudomonas cepacia immobilized on cellulosic polyurethane, ACS Omega 3
(2018) 6804–6811, https://doi.org/10.1021/acsomega.8b00110.
[44] G.S. Dias, P.T. Bandeir, S. Jaerger, L. Piovan, D.A. Mitchell, F. Wypych, N. Krieger,
Immobilization of pseudomonas cepacia lipase on layered double hydroxide of Zn/
Al-Cl for kinetic resolution of rac-1-phenylethanol, Enzyme Microb. Technol. 130
(2019), 109365, https://doi.org/10.1016/j.enzmictec.2019.109365.
[45] D. Kumar, T. Das, B.S. Giri, E.R. Rene, B. Verma, Biodiesel production from hybrid
non-edible oil using bio-support beads immobilized with lipase from pseudomonas
cepacian, Fuel 255 (2019), 115501, https://doi.org/10.1016/j.fuel.2019.115801.
[46] Y.-C. Xie, H.-Z. Liu, J.-Y. Chen, Effect of water content on enzyme activity and
enantioselectivity of lipase-catalyzed esterification of racemic ibuprofen in organic
solvents, Ann. NY. Acad. Sci. 864 (1998) 570–575, https://doi.org/10.1111/
j.1749-6632.1998.tb10383.x.
[33] L. Jiang, W. Zhang, C. Luo, D. Cheng, J. Zhu, Adsorption toward trivalent rare
earth element from aqueous solution by zeolitic imidazolate frameworks, Ind. Eng.
Chem. Res. 55 (2016) 6365–6372, https://doi.org/10.1021/acs.iecr.6b00422.
[34] W.-L. Liu, S.-H. Lo, B. Singco, C.-C. Yang, H.-Y. Huang, C.-H. Lin, Novel
trypsin–FITC@MOF bioreactor efficiently catalyzes protein digestion, J. Mater.
Chem. B 1 (2013) 928–932, https://doi.org/10.1039/C3TB00257H.
[35] M. He, J. Yao, Q. Liu, K. Wang, F. Chen, H. Wang, Facile synthesis of zeolitic
imidazolate framework-8 from a concentrated aqueous solution, Microporous
Mesoporous Mater. 184 (2014) 55–60, https://doi.org/10.1016/j.
[47] A.M. Klibanov, What is remembered and why? Nature 374 (1995) 596, https://doi.
org/10.1038/374596a0.
[48] V. Stepankova, S. Bidmanova, T. Koudelakova, Z. Prokop, R. Chaloupkova,
J. Damborsky, Strategies for stabilization of enzymes in organic solvents, ACS
Catal. 3 (2013) 2823–2836, https://doi.org/10.1021/cs400684x.
micromeso.2013.10.003.
[36] X. Lian, Y.-P. Chen, T.-F. Liu, H.-C. Zhou, Coupling two enzymes into a tandem
nanoreactor utilizing a hierarchically structured MOF, Chem. Sci. 7 (2016)
6969–6973, https://doi.org/10.1039/c6sc01438k.
[49] Z. Zhong, S. Pang, Y. Wu, S. Jiang, J. Ouyang, Synthesis and characterization of
mesoporous Cu-MOF for laccase immobilization, J. Chem. Technol. Biotechnol. 92
(2017) 1841–1847, https://doi.org/10.1002/jctb.5189.
[37] L.-Z. Cheong, Y. Wei, H. Wang, Z. Wang, X. Su, C. Shen, Facile fabrication of a
stable and recyclable lipase@amine-functionalized ZIF-8 nanoparticles for esters
hydrolysis and transesterification, J. Nanopart. Res. 19 (2017) 280, https://doi.
org/10.1007/s11051-017-3979-3.
[50] P. Xue, X.H. Yan, Z. Wang, Lipase immobilized on HOOC-MCF: a highly
enantioselective catalyst for transesterification resolution of (R, S)-1-
phenylethanol, Chin. Chem. Lett. 8 (2007) 929–932, https://doi.org/10.1016/j.
cclet.2007.06.005.
[38] F.-K. Shieh, S.-C. Wang, C.-I. Yen, C.-C. Wu, S. Dutta, L.-Y. Chou, J.V. Morabito,
P. Hu, M.-H. Hsu, K.C.-W. Wu, C.-K. Tsung, Imparting functionality to biocatalysts
via embedding enzymes into nanoporous materials by a de novo approach: size-
selective sheltering of catalase in metal-organic framework microcrystals, J. Am.
Chem. Soc. 137 (2015) 4276–4279, https://doi.org/10.1021/ja513058h.
[39] X. Mu, J. Qiao, L. Qi, P. Dong, H. Ma, Poly(2-vinyl-4,4-dimethylazlactone)-
functionalized magnetic nanoparticles as carriers for enzyme immobilization and
its application, ACS Appl. Mater. Interfaces 6 (2014) 21346–21354, https://doi.
org/10.1021/am5063025.
[51] H. Han, Y. Zhou, S. Li, Y. Wang, X.Z. Kong, Immobilization of lipase from
Pseudomonas fluorescens on porous polyurea and its application in kinetic
resolution of racemic 1-phenylethanol, ACS Appl. Mater. Interfaces 8 (2016)
25714–25724, https://doi.org/10.1021/acsami.6b07979.
[52] M. Li, H. Shan, L. Zhou, Y. Yin, Z. Li, Novel bioreactor for resolution of (R, S)-1-
phenylethanol using the functional conducting polymer and ionic liquid with
excellent catalytic activity and stability, J. Chem. Technol. Biotechnol. 88 (2013)
2091–2097, https://doi.org/10.1002/jctb.4077.
[40] C. Jiang, C. Cheng, M. Hao, H. Wang, Z. Wang, C. Shen, L.-Z. Cheong, Enhanced
catalytic stability of lipase immobilized on oxidized and disulfide-rich eggshell
membrane for esters hydrolysis and transesterification, Int. J. Biol. Macromol. 105
(2017) 1328–1336, https://doi.org/10.1016/j.ijbiomac.2017.07.166.
[41] R. Abdulla, P. Ravindra, Immobilized burkholderia cepacia lipase for biodiesel
production from crude Jatropha curcas L. oil, Biomass Bioenerg. 56 (2013) 8–13,
https://doi.org/10.1016/j.biombioe.2013.04.010.
[53] W. Bai, Y.-J. Yang, X. Tao, J.-F. Chen, T.-W. Tan, Immobilization of lipase on
aminopropyl-grafted mesoporous silica nanotubes for the resolution of (R, S)-1-
phenylethanol, J. Mol. Catal. B-Enzym. 76 (2012) 82–88, https://doi.org/10.1016/
j.molcatb.2011.11.005.
[54] X. Li, S. Huang, L. Xu, Y. Yan, Improving activity and enantioselectivity of lipase
via immobilization on macroporous resin for resolution of racemic 1-
phenylethanol in non-aqueous medium, BMC Biotechnol. 13 (2013) 92, https://
doi.org/10.1186/1472-6750-13-92.
[42] G.-C. Chen, I.-C. Kuan, J.-R. Hong, B.-H. Tsai, S.-L. Lee, C.-Y. Yu, Activity
enhancement and stabilization of lipase from pseudomonas cepacia in
polyallylamine-mediated biomimetic silica, Biotechnol. Lett. 33 (2011) 525–529,
https://doi.org/10.1007/s10529-010-0451-1.
[55] I. Hoffmann, V.D. Silva, M.D.G. Nascimento, Enantioselective resolution of (R, S)-
1-phenylethanol catalyzed by lipases immobilized in starch films, J. Brazil. Chem.
Soc. 22 (2011) 1559–1567, https://doi.org/10.1590/S0103-50532011000800021.
140