Lipase Encapsulation onto ZIF-8: A Comparison between Biocatalysts Obtained at Low and High Zinc/2-Methylimidazole Molar Ratio in Aqueous Medium

Article abstract of DOI:10.1002/cctc.201701984

Lipase AK from Pseudomonas fluorescens and Lipase RM from Rhizomucor miehei were encapsulated into a zeolite imidazolate framework (ZIF-8) by a “one-pot” synthesis to obtain AK@ZIF-8 and RM@ZIF-8 biocatalysts. The effect of a high (1:40) and low (1:4) Zn/

Full text of DOI:10.1002/cctc.201701984

Accepted Article
Title: Lipases encapsulation onto ZIF-8. A comparison between
biocatalysts obtained at low and high zinc:2methyl-imidazole
molar ratio in aqueous medium
Authors: Federica Pitzalis, Cristina Carucci, Maryam Naseri, Lida
Fotouhi, Edmond Magner, and Andrea Salis
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemCatChem 10.1002/cctc.201701984
Link to VoR: http://dx.doi.org/10.1002/cctc.201701984
A Journal of
www.chemcatchem.org

ChemCatChem
10.1002/cctc.201701984
FULL PAPER
Lipases encapsulation onto ZIF-8. A comparison between
biocatalysts obtained at low and high zinc:2-methylimidazole
molar ratio in aqueous medium
†[a]
†[b]
[a,c]
[c]
[b]
Federica Pitzalis ,Cristina Carucci , Maryam Naseri , Lida Fotouhi , Edmond Magner* , Andrea
[a]
Salis*
Abstract: Lipase AK from Pseudomonas fluorescens, and Lipase
RM from Rhizomucor miehei were encapsulated into a zeolite
imidazolate framework (ZIF-8) via “one-pot” synthesis to obtain
AK@ZIF-8 and RM@ZIF-8 biocatalysts. The effect of a high (1:40)
and low (1:4) Zn:2-methylimidazole molar ratio on the biocatalysts
synthesis was investigated. The different Zn:L ratios affected both
the surface area, the loading and the specific activity of the
biocatalysts. Samples synthesised using high Zn:L ratio had high
values of surface area whereas those obtained using low Zn:L ratio
had higher loadings and specific activities. The decrease of pH (from
(ZIFs) are an important MOF subclass containing a zeolite-like
2
+
2+
crystalline structure where Zn (or Co ) ions are coordinated by
[
11–15]
imidazole derivatives forming
a
tetrahedral network.
Systematic change of the organic ligand or the metal ion results
in a variety of 3D structures, similar to those of natural
[
16]
zeolites.
solvothermal,
microwave-assisted,
Among these, water synthesis is facile, rapid, and low cost.
Moreover, this is a very suitable way to obtain enzyme@ZIF
ZIFs can be prepared in several ways, i.e. by
[
15–17]
[18]
[19]
mechano-chemical,
or hydrothermal
sonochemical,
synthetic methods.
[
20]
[12]
[12]
[
21–23]
2+
11.6 to 9.4) during the synthesis at high Zn:L ratio produced ZIF-8
conjugates.
ZIF-8 is a metal organic framework where Zn
samples with features similar to those observed for low Zn:L ratio
samples. The low Zn:L (1:4) ratio AK@ZIF-8 biocatalyst retained
ions are coordinated by 2-methylimidazole (2-MIM) to form
[15]
rhombic dodecahedral structures.
These structures are often
9
9% activity after storage for 15 days at 5°C and 40% activity after 5
referred to as "SOD-like" due to their similarity to those of the
natural zeolite "sodalite". Although water synthesis is
reaction cycles.
[
21–
predominantly used for the immobilisation of single enzymes
2
4]
[25]
or to obtain bienzymatic systems, some critical aspects of
the mechanism of formation remain unknown. The mechanism
1. Introduction
[
20]
[12]
[26]
of crystallization,
porosity, and the surface area
of pure
ZIFs prepared in aqueous media are strongly influenced by the
Enzymes are generally active under mild conditions, i.e. room
2
+
[1]
molar ratio between Zn ions and the alkyl-imidazole ligand, as
temperature and atmospheric pressure, conditions that enable
the development of environmentally friendly 'green' industrial
processes. However, in contrast to conventional chemical
catalysts, enzymes are expensive and unstable in e.g. non
aqueous solutions and at extremes of pH. Immobilisation can
[
26]
well as by the water content.
Moreover, the addition of the
enzyme to the reaction mixture during crystal nucleation can
[
22,24]
affect the crystal shape and morphology
and, consequently,
[
22,24]
the activity of the obtained immobilised biocatalyst.
[
2]
Lipases (E.C. 3.1.1.3 triacylglycerol acylhydrolases) are one of
the most widely used enzymes for biotechnological applications,
overcome these disadvantages.
When an enzyme is
immobilised on a solid support it generally retains its catalytic
activity, enhances its stability, and can be reused for several
[
27–29]
[28,30,31]
such as the production of biodiesel,
as well as in the
[
32]
[33,34]
[
3, 4]
food,
and pharmaceutical
industries. To the best of our
reaction cycles.
In the past 15 years ordered mesoporous
knowledge, only a limited number of works investigating the
catalytic performance of lipases immobilised on MOFs in
silicates have widely been used for enzyme immobilisation due
[
5–9]
to their textural and structural features.
More recently, metal
[
34–36]
general
and on ZIFs in particular, have been reported. Very
organic frameworks (MOFs) have emerged as a possible new
family of enzyme supports. MOFs, first synthesised by Yaghi et
recently, Gascòn et al. immobilised lipase B from Candida
[37]
[
10]
antarctica on Basolite F-300 like materials.
Cao et al.
al.
consist of metal ions coordinated with organic ligands to
measured the catalytic activity of Bacillus subtilis lipase
form a porous 3D network. Zeolite imidazolate frameworks
[
35]
immobilised on Cu-BTC MOF.
He et al. immobilised a
thermophilic lipase from Alcaligenes sp. on ZIF-8, through a
[
38]
mechanochemical synthetic method.
Cheong et al.
†
[
a]
F. Pitzalis , Prof, Andrea Salis*
immobilised Burkholderia cepacia lipase in amino-functionalised
Department of Chemical and Geological Sciences, University of Cagliari,
Cittadella Universitaria, SS 554 bivio Sestu, 09042 Monserrato (CA)
ZIF-8 through physical adsorption, and measured both the
[
39]
(
Italy)
hydrolytic and transesterification activity.
Shi et al.
E-mail: asalis@unica.it
immobilised the lipase from Candida rugosa on ZIF-8
synthesised via a solvothermal method using methanol as the
†
[
b]
C. Carucci , Prof. E. Magner*
Department of Chemical Sciences, Synthesis and Solid State
Pharmaceutical Centre and Bernal Institute, University of Limerick,
Limerick, V94 T9PX, Ireland
[
40]
solvent.
Liang et al. immobilised several enzymes on ZIF-8,
[24]
including a lipase (origin not specified). Lyu et al. reported the
in-situ immobilisation of lipase from Thermomyces lanuginosus
on ZIF-8, using methanol as solvent and polyvinylpyrrolidone as
a protective coating for the enzymes.
prepared a hierarchical magnetic ZIF-8/UiO-66/Fe
E-mail: edmond.magner@ul.ie
c] M. Naseri, Prof. L. Fotouhi
[
Department of Chemistry, Alzahra University, Tehran, Iran
E-mail: lfotouhi@alzahra.ac.ir
[
41]
Finally, Huo et al.
support for
3
42]
O
4
†
[
These authors contributed equally to this work, *corresponding authors.
the encapsulation of Candida antarctica lipase B.
Supporting information for this article is given via a link at the end of
the document.
In this work the 'one-pot' water synthesis of ZIF-8 and the
encapsulation of lipases from Pseudomonas fluorescens (lipase
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201701984
FULL PAPER
AK) and Rhizomucor miehei (lipase RM), to obtain AK@ZIF-8
The XRD patterns of ZIF-8HR and lipase@ZIF-8HR (Figure 1A)
and RM@ZIF-8 biocatalysts, is reported. These lipases are well
are in good agreement with those of a ZIF-8 simulated pattern
[
43–46]
[52,53]
characterized.
Lipase RM has a molecular mass of 32kDa,
reported in the literature.
The diffractograms (Figure 1B) of
[
47]
with an isoelectric point, pI, of 3.8. Lipase AK has a molecular
mass of 33kDa and a pI = 4. These lipases are very promising
enzymes due to their good activity toward triglyceride
ZIF-8LR and lipase@ZIF-8LR samples have different patterns.
XRD patterns for ZIF-8 materials synthesised with various 2-MIM
concentrations were previously observed in a variety of studies.
Kida et al. observed the occurrence of a crystalline ZIF-8 SOD-
like structure with high molar Zn:L ratios (1:40; 1:60; 1:80; 1:100),
while, at lower ratios (Zn:L ratio 1:20) they observed the
[
48]
[
47,49–51]
transesterification for biodiesel production.
While they
have mainly been immobilized on polymers of silica supports, to
the best of our knowledge, no data is available about the
immobilization of AK and RM lipases on ZIF-8.Here, two
occurrence of mixed phases associated with the formation of
2
+
[26]
stoichiometric ratios of Zn and the 2-MIM ligand (L) i.e.
Zn(OH)
2
, Zn(OH)NO
3
, and Zn
5
(OH)
8
(NO
3
)
2
(H
2
O)
2
.
Shi et al.
[
25]
[24]
Zn:L=1:40
and Zn:L=1:4
were used in previous reports.
synthesised a ZIF-8 sample (Zn:L ratio of 1:4) that had an XRD
[
54]
The Zn:L ratio affects both the texture of ZIF-8 and the activity of
the obtained lipase@ZIF-8 biocatalyst. A similar effect on ZIF-8
structure and catalytic activity was obtained by changing the pH
at which the biocatalysts were prepared. Finally, the storage
stability and the ability to recycle of the most active lipase@ZIF-
pattern which was assigned to an unknown mixed phase while
Liang et al. obtained a ZIF-8 sample with a typical SOD-like
[24]
crystalline structure at a Zn:L ratio of 1:4. The structure of ZIF-
8 is affected by the presence of encapsulated protein. At a Zn:L
ratio of 1:4 different morphologies were observed for a wide
range of proteins (i.e. nanoflowers for horseradish peroxidase,
8
biocatalysts were examined.
[24]
nanostars for trypsin etc.).
Similarly, Cui et al. observed a
variety of XRD patterns and crystal morphologies for
catalase@ZIF-8 samples obtained with Zn:L ratios in the range
2. Results and Discussion
[
22]
1:1 - 1:20. The concentration of protein concentration also
affects the structure of ZIF-8. The peak intensity decreased with
2.1 Characterisation of ZIF-8 and lipase@ZIF-8 samples
[
24]
increasing concentration of BSA.
The XRD patterns of
AK@ZIF-8 and RM@ZIF-8 samples showed different peak
intensities (Figure 1A-B) suggesting an interaction between the
ZIF-8 and lipase@ZIF-8 samples were synthesised by using two
2
+
different Zn :2-MIM (Zn:L) molar ratios. Samples named ZIF-
HR and lipase@ZIF-8HR were obtained by using a high Zn:L
[37,55]
enzyme molecules and the ZIF-8 material.
These findings
8
[
25]
demonstrate that the formation of pure ZIF-8 and lipase@ZIF-8
structures in aqueous solution is a complex process.
(
1:40) molar ratio, whereas samples ZIF-8LR and lipase@ZIF-
[
24]
8LR were obtained by using a low Zn:L (1:4)
molar ratio. All
samples were characterised using a range of techniques i.e. X-
ray diffraction (XRD), porosimetry, scanning electron microscopy
Table 1.Textural parameters obtained by the N
2
physisorption analysis of ZIF-
8
and lipase@ZIF-8 samples
(
SEM), Fourier transform infrared spectroscopy (FTIR) and
Sample
Zn:L
Ratio (m²g )
S
BET
Pore volume
thermogravimetric analysis (TGA).
-
1
3 -1
(cm g )
ZIF-8HR
AK@ZIF-8HR
RM@ZIF-8HR
ZIF-8LR
AK@ZIF-8LR
RM@ZIF-8LR
1:40
1:40
1:40
1:4
1:4
1:4
919
615
790
79
4.4
30
n.a.
39
n.a.
0.50
0.049
0.22
Figures 1C-D show the textural characterisation of ZIF-8 and
lipase@ZIF-8 samples performed by nitrogen physisorption. A
ZIF-8HR sample displayed a typical type I isotherm associated
with a microporous material (Figure 1 C) with a high surface
2
area of 919 m /g (Table 1).The small hysteresis present at p/p
0
>
0.8 may be due to the presence of mesopores in ZIF-8HR as well
as in the lipase@ZIF-8HR samples. This was previously
[
22]
observed for other enzyme@ZIF-8.
The decrease in surface
area for lipase@ZIF-8 (Table 1) is likely due to enzyme
[22,24,37,38]
encapsulation into the ZIF-8 structure.
The
N
2
adsorption/desorption isotherms obtained for the samples
synthesised with the low Zn:L (1:4) ratio (Figure 1D), are
significantly different. Low ratios of Zn:2-MIM ligand result in low
surface areas (Table 1). This is consistent with the work of Kida
et al. who investigated the textural parameters of ZIF-8 samples
Figure 1. XRD patterns of ZIF-8 and lipase@ZIF-8 prepared using a Zn:L
ratio of A) 1:40 and B) 1:4. N
2
adsorption and desorption isotherms of ZIF-8,
[
26]
AK@ZIF-8 and RM@ZIF-8 prepared using a Zn:L ratio of C) 1:40 and D) 1:4.
at different Zn:L ratios over the range 1:100 - 1:20.
They
2
observed surface areas of over 1500 m /g for SOD-type ZIF-8 at
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201701984
FULL PAPER
[
12]
high Zn:L ratios (from 1:40 to 1:100) and lower values of surface
the ZIF-8 structure. In the case of the samples synthesised at
a low (1:4) ratio of Zn:L, the curves in Figure 2D follow different
trends. ZIF-8LR sample has a small mass loss 4.7 % up to 500°C,
similar to that of ZIF-8HR. The steep mass loss above 500 °C is
likely due to the thermal decomposition of the structure (Table 2).
For the lipase@ZIF-8LR samples the initial mass loss (T <
130°C) is associated with the loss of water. Between 230-500°C,
the decomposition of the enzymes occurs with mass losses of
2
[26]
areas (from 1300 to 120 m /g) for the molar ratio 1:20.
Scanning electron microscopy (SEM) images of ZIF-8, AK@ZIF-
, and RM@ZIF-8 synthesised at a Zn:L=1:40 ratio and the
8
corresponding samples synthesised at a Zn:L=1:4 ratio are
shown in Figures S1A-C and Figure S1D-F (see Supporting
Information file), respectively. Changes in particle size and
morphology were observed in the presence and absence of
enzyme. Figure 2 displays the FTIR spectra of the pure ZIF-8
and the lipase@ZIF-8 samples obtained at high (Figure 2A) and
low (Figure 2B) Zn:L ratio. For ZIF-8, the C=N stretching mode
7.3% and 11.4% for AK@ZIF-8LR and
respectively. The diversity in shape in Figures 2C and 2D are
due to the different synthesis conditions.
RM@ZIF-8LR,
[
22,26,37,57,58]
-
1
-1
shows a band at 1584 cm . The band at 1420 cm is associated
[
14]
with a stretching mode of imidazole ring. A strong band at 760
Table 2. Mass loss (∆m) of ZIF-8 and lipase@ZIF-8 samples at high
(HR=1:40) and low (LR=1:4) Zn:L ratios.
-1
cm is due to out of plane bending of the imidazole aromatic ring,
whereas the in-plane bending of the ring produces bands in the
-1
-1 [14]
Sample
ZIF-8HR
AK@ZIF-8HR
RM@ZIF-8HR
ZIF-8LR
m (%)
T<230°C
18.8
Δm (%)
30°C <T<500°C
Δm (%)
T>500°C
76.1
range 900 cm - 1350 cm . Finally, the characteristic band at
2
-1
[14,56]
4
21 cm is associated with the Zn-N stretching mode.
For
5.1
17.0
15.3
4.7
-
1
lipase@ZIF-8 samples the bands in the range 1644-1648 cm ,
due to the C=O stretching of the amide I, confirm the successful
encapsulation of lipase molecules in the ZIF-8.
21.5
23.3
61.6
36.6
54.4
0.7
AK@ZIF-8LR
RM@ZIF-8LR
29.3
60.0
7.3
11.4
63.4
15.0
2
.2 Loading and activity of AK@ZIF-8 and RM@ZIF-8
biocatalysts
Protein loading (Figure 3 A,B) and enzyme activity (Figure 3
C,D) of the lipase@ZIF-8 samples were quantified by the
[
59]
[60,61]
Bradford and p-nitrophenyl butyrate assays,
respectively.
Lipase@ZIF-8 samples obtained at a Zn:L ratio 1:4 had higher
protein loadings than those obtained at a ratio of 1:40. The
loadings were 138±13 mg/g (AK@ZIF-8LR), 77±12 mg/g
(
(
RM@ZIF-8LR), 40±10 mg/g (AK@ZIF-8HR), and 12±6 mg/g
RM@ZIF-8HR).
Figure 2. FTIR spectra of ZIF-8 and lipase@ZIF-8, A) high Zn:L (1:40) ratio;
B) low Zn:L (1:4) ratio. TGA curves of ZIF-8 and lipase@ZIF-8 prepared by
using the C) high Zn:L ratio, and D) low Zn:L ratio.
Thermogravimetric analysis (TGA) data show (Figure 2C-D) that
ZIF-8HR and lipase@ZIF-8HR samples are stable up to 140°C.
Both materials displayed a similar mass loss of ca. 20 % (see
Table 2) in the temperature range140-230°C (Figure 2C),
[
12]
associated with the removal of water and unreacted reagents.
At higher temperatures, in the range 230°C - 500°C, a small
mass loss was observed for pure ZIF-8HR (5.1 %), while
lipase@ZIF-8HR biocomposites showed
a
gradual and
continuous decomposition, with ∆m of 17.0 % and 15.3 % for
AK@ZIF-8HR and RM@ZIF-8HR, respectively (Table 2). This
mass loss is probably due to the decomposition of enzymes
Figure 3. Comparison A) enzyme loading, B) encapsulation efficiency, C)
enzymatic activity, and D) specific activity (PNP= p-nitrophenol) of lipase ZIF-8
prepared at Zn:L ratios of 1:40 and 1:4.
[25,38]
encapsulated in the ZIF-8 network.
At temperature greater
than 500°C the mass loss is likely due to the decomposition of
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201701984
FULL PAPER
Moreover, the samples obtained using a ratio of 1:4 resulted in a
very high encapsulation efficiency (EE%, the amount of
immobilised protein relative to the total protein amount in the
immobilising solution) (Figure 3B). Lipase RM in particular was
completely immobilised in the ZIF-8LR material, with an EE% of
Zn:L=1:40, the highest surface area was achieved at pH 10.2 for
2
2
AK@ZIF-8HR (618 m /g) and RM@ZIF-8HR (923 m /g) samples.
Conversely, at pH 9.4 the surface areas of AK@ZIF-8HR (33
2
2
m /g) and RM@ZIF-8HR (43 m /g) were similar to those of the
2
corresponding biocatalysts obtained at Zn:L=1:4 (4.4 m /g and
30 m /g). These data show that, in addition to pH, the type of
2
99% while, with lipase AK, an EE% of 78% was observed. The
encapsulation efficiency for samples obtained by Zn:L=1:40
ratios was 14% and 75% for AK@ZIF-8HR and lipase RM@ZIF-
enzyme influences the properties of lipase@ZIF-8.
8HR respectively (Figure 3B). The catalytic activities of
lipase@ZIF-8 samples are shown in Figures 3C-D. Specific
-1
-1
activities (molPNP min mgprotein = U/mg) were 84.5±0.3 U/mg
(
AK@ZIF-8LR), 75.8±0.4 U/mg (RM@ZIF-8LR), 52.2±2 U/mg
(
AK@ZIF-8HR) and 24.9±0.7 U/mg (RM@ZIF-8HR). Although
samples obtained at the low Zn:L (1:4) molar ratio had low
surface areas (Table 1), they resulted in higher loadings,
encapsulation efficiencies and specific activities than samples
synthesised with a high (1:40) ratio. This means that lipases are
better adapted to the ZIF-8LR than to ZIF-8HR structures.
2.3 Effect of pH on lipase@ZIF-8HR samples
The two procedures used to synthesise ZIF-8 and lipase@ZIF-8
samples differ in terms of Zn:L ratio and in the pH of the
synthesis solution. The pH was 11.6 at a Zn:L ratio of 1:40 and
10.6 for a ratio of 1:4. This pH difference might be responsible of
at least part of the observed differences of surface area, loading
and catalytic activity between the two types of samples. Thus,
samples at a Zn:L=1:40 were prepared, but changing the pH of
the synthesis solution (from 11.6 to 10.2, 9.8, 9.4) by adding HCl.
The effect of the selected pH on surface area, protein loading,
encapsulation efficiency and specific activity of the lipase@ZIF-
2
Figure 4. N ads-desorption isotherms and comparison among surface areas
obtained at different pH conditions of synthesis. A) AK@ZIF-8 and B)
RM@ZIF-8, obtained by means of a Zn:L=1:40 controlling the pH during the
synthesis. and; C) influence of pH on the surface area of all lipase@ZIF-8
samples at all synthesis conditions tested.
The effect of the synthesis pH on protein loading and catalytic
activity was then investigated. Figure 5A shows that protein
loading increases by decreasing the pH of the synthesis solution.
This applies to both lipases, although the loading is always
higher for lipase RM than for lipase AK. At pH 9.4 a loading of
8HR biocatalysts was then examined. As shown in Figure 4, a
change of pH of the synthesis solution (Zn:L=1:40) affects the
surface area of lipase@ZIF-8HR samples. Although the
isotherms of the samples obtained at different pH values have
similar shapes, the amount of adsorbed nitrogen decreases at
decreasing pH for AK@ZIF-8HR (Figure 4A) resulting in a
decrease in the surface area (Figure 4C). On the contrary, for
RM lipase the highest surface area was obtained with samples
synthesised at pH 10.2. At pH 9.4 the surface area decreased
dramatically for both AK@ZIF-8HR and RM@ZIF-8HR. At this pH
the isotherms are similar to those obtained for the samples with
Zn:L=1:4 ratio (Figure 1C), prepared at a pH of 10.6. These data
clearly show the effect of pH on the surface area of lipase@ZIF-
102±10 mg/g and 55±6 mg/g was obtained for RM@ZIF-8HR and
AK@ZIF-8HR samples, respectively. It should be noticed that the
highest protein loading obtained at pH 9.4 is lower than that
obtained with a Zn:L ratio of 1:4 (Figure 3A). Similarly, the
highest encapsulation efficiency was reached for the samples
obtained by the Zn:L=1:40 ratio at pH 9.4. EE% was 71% for
RM@ZIF-8HR and only 34% for AK@ZIF-8HR. These values were,
however, lower than those obtained for Zn:L= 1:4 ratio (Figure
S3B).
[
62]
8
HR samples. The pK
a
of 2-methylimidazole is 7.85. As the pH
of 2-
of the ligand solution is lowered to values closer to the pK
a
MIM, the concentration of the protonated form of the ligand
increases. This may affect the formation of the structure of
lipase@ZIF-8HR samples. At pH 9 no solid sample could be
collected by centrifugation after a synthesis time of 24 h.
Moreover, the isotherms obtained at a ratio Zn:L=1:4 (Figure 1D)
are similar to those obtained at a ratio of Zn:L=1:40 at pH 9.4
(
Figures 4A and 4B) suggesting that a decrease of pH produces
an effect similar to the decrease of the ratio of Zn:L. That
hypothesis is supported by the data reported in Figure 4C, and
in Table S1, where the comparison among surface areas
obtained with the two synthesis conditions is shown. At ratio
Figure 5. Lipase@ZIF-8 biocatalyst.
obtained with Zn:L=1:40 ratio at different synthesis solution pH. A) Loading
and B) catalytic activity.
A comparison among the samples
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201701984
FULL PAPER
The specific activities of the immobilised biocatalysts obtained
by means of a Zn:L=1:40 ratio at different pH are shown in
Figure 5B. AK@ZIF-8HR samples increased their specific activity
by decreasing the synthesis pH from 11.6 to pH 9.4. The specific
activity measured for RM@ZIF-8HR instead, was always lower
than that of AK@ZIF-8HR in the investigated pH range. Moreover
the specific activity was about zero for RM@ZIF-8HR samples
synthesised at pH 9.4 (Figure 5B). It has been reported that
After 5 days storage of the immobilised lipases at two different
temperatures, they obtained an activity retention of 20.3%
(37°C) and 6.0% (60°C). The operational stability to recycling of
the two biocatalysts was then investigated (Figure 6B).
RM@ZIF-8LR biocatalyst retained only a 30% of its initial activity
in the second reaction cycle, and became fully inactive in the
third cycle. Instead, AK@ZIF-8LR biocatalyst displayed a better
performance, since it could be reused 5 times with activity
[
47]
th
Mucor miehei lipases display the maximal activity at pH 8.
retention of 40% at the 5 reaction cycle (Figure 6B). He et al.
Thus, we expect RM lipase to be less active in highly alkaline
conditions (pH > 9.4) required for ZIF-8 formation. From these
results we may argue that pH affects the synthesis of ZIF-8 and
the consequent biocatalyst in a manner that cannot be ignored.
A final comparison between the effect of pH and that of Zn:L
ratio deserves notice. For the synthesis at Zn:L=1:4 ratio -
whose initial pH of ligand solution was 10.6 - the biocatalysts are
generally more active than those obtained at different pH tested
at Zn:L=1:40 ratio (see par. 2.2). This suggests that the
concentration of the 2-MIM ligand plays a more important role
than pH on affecting the specific activity of the lipase@ZIF-8
biocatalysts.
could recycle their biocatalyst 10 times with 75.7% retention of
th
[38]
activity after the 10 reaction cycle. Hence, although our and
He et al. experimental conditions were not exactly the same,
AK@ZIF-8LR was less stable to recycling but more stable to
storage than Alcaligenes lipase immobilised on ZIF-8. From
these measurements it is difficult to discern the cause of the
observed activity loss. Nonetheless, due to the fact that the
enzyme is encapsulated within ZIF-8 structure it is more likely
that deactivation rather than leaching is the cause of the
observed trends.
3.
Conclusions
2.4 Stability of lipase@ZIF-8LR biocatalysts
Two synthetic methods differing for the ratio between zinc ion
and 2-MIM ligand were compared for the preparation of
The stability to store and to reuse an enzyme is a key issue for
the development of a suitable immobilised biocatalyst. Free
enzymes are generally quite expensive and unstable particularly
in terms of long-term storage and harsh reaction conditions such
as extremes of temperature or pH. Hence, a high storage
stability allows the design of a large-scale biocatalyst production,
while recycling allows for a reduction of operational costs. Figure
[
25]
lipase@ZIF-8 biocatalysts. A high Zn:L (1:40) ratio and a low
[
24]
(
1:4) ratio gave lipase@ZIF-8HR and lipase@ZIF-8LR samples,
respectively. The structural and textural features of ZIF-8 and
lipase@ZIF-8 samples are both affected by Zn:L ratio. ZIF-8HR
samples had very high surface areas compared with ZIF-8LR
samples (Table 1). However, the latter samples had higher
enzyme loadings and higher specific activities (Figure 3). The
effect of decreasing the solution pH during the synthesis of
lipase@ZIF-8HR samples was then investigated. The obtained
samples had lower surface areas (Figure 4) but higher loadings
and specific activities (Figure 5), mimicking the behaviour of
lipase@ZIF-8LR samples. This observation, to the best of our
knowledge, had not already been reported. AK@ZIF-8LR and
RM@ZIF-8LR retained high levels of activity after 15 days
storage at 4°C. Finally, AK@ZIF-8LR could be recycled 5 times
6
shows the storage stability and the recycling of lipase@ZIF-8LR
biocatalysts (Zn:L=1:4 ratio). These samples were chosen
because, respect to those synthesised with the Zn:L=1:40 ratio,
they reached the highest enzyme loading and specific activity.
The biocatalysts were stored at 4°C for 15 days. An activity
retention of 88% and 99% was obtained for RM@ZIF-8LR and
AK@ZIF-8LR, respectively (Figure 6A). This demonstrates that,
the investigated storage conditions do not significantly affect the
relative activity of both biocatalysts. He et al. investigated the
long-term stability and recycling of the thermophilic lipase from
th
with activity retention of 40% at the 5 reaction cycle. This last
[
38]
biocatalyst might be a good candidate for further studies as, for
Alcaligenes sp. immobilised on ZIF-8.
[50]
example, the synthesis of biodiesel.
Experimental Section
4.1 Chemicals
Lipase AK from Pseudomonas fluorescens (LAKGO751641)
was purchased from Amano Enzymes, Japan. Lipase RM (2000
-1
U g ) from Rhizomucor Miehei fungi was purchased from Sigma.
Both lipases were used without any further purification. p-
nitrophenyl butyrate, zinc nitrate hexahydrate (98%), 2-
methylimidazole (99%), 2-propanol, bovine serum albumin (BSA,
Figure 6. Stability and recycling of lipase ZIF-8LR (Zn:L=1:4 ratio) biocatalyst.
A) residual activity of the catalyst after 15 days and B) recycling.
98%), Bradford reagent, sodium phosphate dibasic (ACS Grade,
≥
99%), magnesium nitrate (ACS Grade, ≥99%), were purchased
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201701984
FULL PAPER
from Sigma-Aldrich. Sodium phosphate monobasic monohydrate
means of a TGA 4000 Perkin Elmer. The temperature range was
from 30 °C to 900 °C, the ramp rate selected was 10 °C per min,
(
≥99%) was purchased from J.T. Backer.
−
1
under nitrogen flow (flow rate=20 mL min ). Scanning electron
microscopy (SEM) analysis was performed at various
magnifications, by a HITACHI SU-70 equipment at 10 kV.
Fourier transform infrared (FTIR) analysis was conducted with a
Bruker Tensor 27 spectrophotometer, equipped with a diamond-
ATR accessory and a DTGS detector. A number of 256 scans at
4
.2 Synthesis of ZIF-8 and lipase@ZIF-8 samples with a low
Zn:2-MIM ratio (Zn:L=1:4).
[
24]
For the preparation of ZIF-8,
aqueous solution of zinc nitrate (40mM corresponding to 0.60 g
and 2 mmol of Zn(NO ×6H O) was quickly added under stirring
a volume of 50 mL of an
−1
3
)
2
2
a resolution of 2 cm were averaged from wave number 4000 to
−
to 50 mL of an aqueous solution of 2-methylimidazole (160 mM
corresponding to 0.66 g and 8 mmol, pH 10.6). The reaction
mixture was aged overnight at 25°C. Then the ZIF-8 sample was
collected by centrifugation and washed three times with milliQ
water. For the preparation of lipase@ZIF-8 biocatalyst, 0.4 g of
lipase AK or 8mL (corresponding to 9.2 g of the commercial
liquid preparation) of lipase RM were dissolved in the 2-
methylimidaziole aqueous solution (160 mM). The resulting
lipase/2-methylimidazole solution (pH=10.2) was quickly mixed
under stirring to 50 mL the 40 mM zinc nitrate solution. Then the
synthesis of Lipase@ZIF-8 was carried out with the same
procedure followed for ZIF-8 sample (Zn:L=1:4).
400 cm 1. An ASAP 2020 (Micromeritics), was used to
investigate the textural characteristics of all the samples. The N
2
adsorption/desorption isotherms were recorded at 77 K. Before
the analysis, ZIF-8 (lipase@ZIF-8) samples were out-gassed
under vacuum for 12 h at 200°C (120°C). The Brunauer-
[
63]
[64]
Emmett-Teller (BET), and the Barret-Joyner-Halenda (BJH)
methods were used to determine specific surface area, the pore
volume and the pore size distribution.
4.5 Determination of enzyme loading and encapsulation
efficiency
Protein loading and the encapsulation efficiency of lipase@ZIF-8
samples were calculated by measuring the protein concentration
in the initial and final immobilising solutions. The protein
concentration was obtained spectrophotometrically (= 595 nm)
4
.3 Synthesis of ZIF-8 and lipase@ZIF-8 samples with a high
Zn:2-MIM ratio (Zn:L=1:40).
[
59]
[
12,25]
through the Bradford Assay.
Encapsulation efficiency (EE%)
For the preparation of ZIF-8,
0.60 g of zinc nitrate (2mmol)
is the percent ratio between the amount of immobilised protein
and the amount of protein in the immobilising solution:
were dissolved in 4 g of milliQ water and quickly added under
stirring to 40 g of an aqueous solution containing 6.63 g (80
mmol) of 2-methylimidazole. The reaction mixture was left under
continuous stirring for 10 minutes. Then the solution was aged
overnight at 25°C. The ZIF-8 sample was recovered by
centrifugation and washed three times with milliQ water. For the
preparation of lipase@ZIF-8, 0.4 g of lipase AK or 8mL of lipase
RM (corresponding to 9.2 g of the commercial liquid preparation)
were dissolved into 40 g of an aqueous solution containing 6.63
f 0
EE% = (1 – [P] / [P] ) × 100%
Where [P]
concentrations in the immobilising solution.
0
and [P]
f
are the initial and the final protein
[
65]
Protein loading (mgprotein/gZIF-8) is the amount of immobilised
[
66,67]
protein per g of support:
g
(80 mmol) of 2-methylimidazole aqueous solution. The
resulting lipase/2-methylimidazole solution (pH=11.2) was
quickly mixed under stirring to 4 g of the zinc nitrate solution.
Then the synthesis of Lipase@ZIF-8 was carried out with the
same procedure followed for ZIF-8 sample (Zn:L=1:40).The
effect of pH on the synthesis of lipase@ZIF-8 samples was also
investigated. To this purpose 4 additional lipase@ZIF-8 samples
were prepared by decreasing the pH of 2-methylimidazole
solution - before the addition of the lipases - from the initial value
Where, [P]
in the initial, final and washing solutions; V
initial and final volumes of the immobilising solution; and V
is the volume of washing solution; mZIF-8 is the mass (g) of ZIF-8
support.
0
, [P]
f
and [P]
w
are the protein concentrations (mg/mL)
and V (mL) are the
(mL)
0
f
w
11.6 to 10.2, 9.8, 9.4 and 9.0, by means of HCl addition. Then
the synthesis proceeded as described above.
4.4 Characterization of ZIF-8 samples
4.6 Determination of biocatalysts activity and stability
A X’PERT Pro PANalytical diffractometer was used for X-ray
diffraction (XRD) experiments with Cu-Kα radiation. The data
were collected with a two theta step size of 0.02° from 5 to 50°
and an accumulation time 20 s. The experimental XRD patterns
were compared (Fig. 1A,B) with simulated patterns obtained
The activity of free and immobilised lipases was measured by
means of Varian Cary 60 UV-VIS spectrophotometer,
equipped with an optical fibre probe with the p-nitro
a
[60,68,69]
phenylbutyrate (PNPB )assay.
AK and RM) was measured by adding 5 μL of the lipase
aqueous solution to 2 mL of 0.1 M phosphate buffer pH 7, 200
The activity of free lipases
(
[
53]
using a deposited cif.file
and plotted with Mercury software.
Thermogravimetric analysis (TGA) analysis was carried out by
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201701984
FULL PAPER
Cheetham, T. Friščić, Angew. Chemie - Int. Ed. 2010, 49, 9640–
μL of PNPB (0.02 M in 2-propanol) solution and 200 μL of 2-
propanol. The activity of immobilised lipases was measured by
adding 5 mg of lipase@ZIF-8 samples to 5 mL of 0.1 M
phosphate buffer pH 7, 500 μL of PNPB solution and 500 μL of
9643.
[
[
19]
20]
H. Y. Cho, J. Kim, S. N. Kim, W. S. Ahn, Microporous Mesoporous
Mater. 2013, 169, 180–184.
Q. Bao, Y. Lou, T. Xing, J. Chen, Inorg. Chem. Commun. 2013, 37,
170–173.
2
-propanol. The (free and immobilised) lipases catalyse the
[21]
K. Liang, C. J. Coghlan, S. G. Bell, C. Doonan, P. Falcaro, Chem.
Commun. 2016, 52, 473–476.
J. Cui, Y. Feng, T. Lin, Z. Tan, C. Zhong, S. Jia, ACS Appl. Mater.
Interfaces 2017, 9, 10587–10594.
E. Gkaniatsou, C. Sicard, R. Ricoux, J.-P. Mahy, N. Steunou, C.
Serre, Mater. Horiz. 2017, 4, 55–63.
K. Liang, R. Ricco, C. M. Doherty, M. J. Styles, S. Bell, N. Kirby, S.
Mudie, D. Haylock, A. J. Hill, C. J. Doonan, et al., Nat. Commun.
formation of p-nitrophenol which was quantified at =400 nm
[
[
[
22]
23]
24]
-1
-1
(
molar extinction coefficient of p-nitrophenol, =9396.1 M cm )
at room temperature. The lipase activity (mol p-nitrophenol ×
-1
min ) was corrected by subtracting the amount of p-nitrophenol
formed in the absence of lipases. Storage and recycling
stabilities were carried out by means of the PNPB assay, as
described above. For storage tests, the wet biocatalyst was
stored at 4°C and dried under vacuum the day before the activity
measurements. For recycling tests, the used biocatalyst (5 mg)
was recovered by centrifugation and washed with 1 mL of 0.1 M
phosphate buffer pH 7 before the addition of the fresh substrate
solution. All activity and stability measurements were carried out
at least in triplicate.
2015, 6, 7240.
[
[
25]
26]
X. Wu, J. Ge, C. Yang, M. Hou, Z. Liu, Chem. Commun. 2015, 51,
13408–13411.
K. Kida, M. Okita, K. Fujita, S. Tanaka, Y. Miyake, CrystEngComm
2013, 15, 1794.
[27]
W. Shuai, R. K. Das, M. Naghdi, S. K. Brar, M. Verma, Biotechnol.
Appl. Biochem. 2017, 64, 496–508.
Z. Amini, Z. Ilham, H. C. Ong, H. Mazaheri, W. H. Chen, Energy
Convers. Manag. 2017, 141, 339–353.
[
[
[
[
28]
29]
30]
31]
M. L. Verma, M. Puri, C. J. Barrow, Crit. Rev. Biotechnol. 2014,
8551, 1–12.
A. Salis, M. S. Bhattacharyya, M. Monduzzi, V. Solinas, J. Mol.
Catal. B Enzym. 2009, 57, 262–269.
A. Salis, M. Pinna, M. Monduzzi, V. Solinas, J. Mol. Catal. B Enzym.
2008, 54, 19–26.
Acknowledgements
[32]
J. Trbojevic Ivic, D. Velickovic, A. Dimitrijevic, D. Bezbradica, V.
Dragacevic, M. Gavrovic Jankulovic, N. Milosavic, J. Sci. Food Agric.
2
016, 4281–4287.
AS thanks FIR 2017 and Fondazione di Sardegna and Regione
Autonoma Sardegna (F72F16003070002) and MIUR (FFABR
[33]
P. Choudhury, B. Bhunia, Biopharm J. 2015, 1, 41–47.
W.-L. Liu, N.-S. Yang, Y.-T. Chen, S. Lirio, C.-Y. Wu, C.-H. Lin, H.-Y.
Huang, Chem. - A Eur. J. 2015, 21, 115–119.
Y. Cao, Z. Wu, T. Wang, Y. Xiao, Q. Huo, Y. Liu, Dalt. Trans. 2016,
45, 6998–7003.
S. Jung, Y. Kim, S.-J. Kim, T.-H. Kwon, S. Huh, S. Park, Chem.
Commun. (Camb). 2011, 47, 2904–2906.
V. Gascón, C. Carucci, M. B. Jiménez, R. M. Blanco, M. Sánchez-
Sánchez, E. Magner, ChemCatChem 2017, 9, 1182–1186.
H. He, H. Han, H. Shi, Y. Tian, F. Sun, Y. Song, Q. Li, G. Zhu, ACS
Appl. Mater. Interfaces 2016, 8, 24517–24524.
[
[
[
34]
35]
36]
2017) for financial support. FP is grateful to the "Agenzia delle
Dogane e dei Monopoli" for funding her PhD. Synthesis and
Solid State Pharmaceutical Centre (SSPC), funded by Science
Foundation Ireland (SFI) under grant 12/RC/2275 is thanked for
financial support. This work was also supported by the Iran
National Science Foundation: INSF (Grant no. 93043395).
[37]
[
[
[
[
[
[
38]
39]
40]
41]
42]
43]
L.-Z. Cheong, Y. Wei, H. Wang, Z. Wang, X. Su, C. Shen, J.
Nanoparticle Res. 2017, 19, 280.
J. Shi, X. Wang, S. Zhang, L. Tang, Z. Jiang, J. Mater. Chem. B
Keywords: Lipase•encapsulation•biocatalysis•ZIF 8•MOF
2
016, 4, 2654–2661.
F. Lyu, Y. Zhang, R. N. Zare, J. Ge, Z. Liu, Nano Lett. 2014, 14,
761–5765.
J. Huo, J. Aguilera-Sigalat, S. El-Hankari, D. Bradshaw, Chem. Sci.
015, 6, 1938–1943.
[
[
[
[
1]
2]
3]
4]
S. Jemli, D. Ayadi-Zouari, H. Ben Hlima, S. Bejar, Crit. Rev.
Biotechnol. 2016, 36, 246–258.
R. DiCosimo, J. McAuliffe, A. J. Poulose, G. Bohlmann, Chem. Soc.
Rev. 2013, 42, 6437.
J. M. Blamey, F. Fischer, H.-P. Meyer, F. Sarmiento, M. Zinn, in
Biotechnol. Microb. Enzym., Elsevier, 2017, pp. 347–403.
I. Eş, J. D. G. Vieira, A. C. Amaral, Appl. Microbiol. Biotechnol. 2015,
5
2
A. M. Brzozowski, U. Derewenda, Z. S. Derewenda, G. G. Dodson,
D. M. Lawson, J. P. Turkenburg, F. Bjorkling, B. Huge-Jensen, S. A.
Patkar, L. Thim, Nature 1991, 351, 491–494.
[
[
[
44]
45]
46]
M. Adamczak, W. Bednarski, Process Biochem. 2004, 39, 1347–
99, 2065–2082.
1361.
[
[
[
5]
6]
7]
E. Magner, Chem. Soc. Rev. 2013, 42, 6213.
C. Angkawidjaja, D. ju You, H. Matsumura, K. Kuwahara, Y. Koga,
K. Takano, S. Kanaya, FEBS Lett. 2007, 581, 5060–5064.
G. Fernández-Lorente, J. M. Palomo, M. Fuentes, C. Mateo, J. M.
Guisán, R. Fernández-Lafuente, Biotechnol. Bioeng. 2003, 82, 232–
M. Hartmann, X. Kostrov, Chem. Soc. Rev. 2013, 42, 6277.
N. Carlsson, H. Gustafsson, C. Thörn, L. Olsson, K. Holmberg, B.
Åkerman, Adv. Colloid Interface Sci. 2014, 205, 339–360.
A. Salis, L. Medda, F. Cugia, M. Monduzzi, Colloids Surf. B.
Biointerfaces 2016, 137, 77–90.
[
[
8]
9]
237.
[
47]
H. Gustafsson, E. M. Johansson, A. Barrabino, M. Odén, K.
Holmberg, Colloids Surf. B. Biointerfaces 2012, 100, 22–30.
Y. Kojima, S. Shimizu, J. Biosci. Bioeng. 2003, 96, 219–226.
M. Garmroodi, M. Mohammadi, A. Ramazani, M. Ashjari, J.
Mohammadi, B. Sabour, M. Yousefi, Int. J. Biol. Macromol. 2016, 86,
M. Piras, A. Salis, M. Piludu, D. Steri, M. Monduzzi, Chem.
Commun. 2011, 47, 7338.
[
[
48]
49]
[
[
10]
11]
O. M. Yaghi, H. Li, J. Am. Chem. Soc. 1995, 117, 10401–10402.
W. Morris, C. J. Stevens, R. E. Taylor, C. Dybowski, O. M. Yaghi, M.
A. Garcia-Garibay, J. Phys. Chem. C 2012, 116, 13307–13312.
Y. Pan, Y. Liu, G. Zeng, L. Zhao, Z. Lai, Chem. Commun. 2011, 47,
208–215.
[
[
[
[
12]
13]
14]
15]
[
[
[
50]
51]
52]
A. Salis, M. F. Casula, M. S. Bhattacharyya, M. Pinna, V. Solinas, M.
Monduzzi, ChemCatChem 2010, 2, 322–329.
A. Salis, M. Pinna, M. Monduzzi, V. Solinas, J. Biotechnol. 2005,
2071.
T. D. Bennett, D. A. Keen, J. C. Tan, E. R. Barney, A. L. Goodwin, A.
K. Cheetham, Angew. Chemie - Int. Ed. 2011, 50, 3067–3071.
Y. Hu, H. Kazemian, S. Rohani, Y. Huang, Y. Song, Chem.
Commun. (Camb). 2011, 47, 12694–6.
K. S. Park, Z. Ni, A. P. Cô Té, J. Y. Choi, R. Huang, F. J. Uribe-
Romo, H. K. Chae, M. O ’keeffe, O. M. Yaghi, Proc. Natl. Acad. Sci.
U. S. A. 2006, 103, 10186.
119, 291–299.
D. W. Lewis, A. R. Ruiz-Salvador, A. Gomez, L. M. Rodriguez-
Albelo, F.-X. Coudert, B. Slater, A. K. Cheetham, C. Mellot-
Draznieks, CrystEngComm 2009, 11, 2272–2276.
D. Fairen-Jimenez, S. A. Moggach, M. T. Wharmby, P. A. Wright, S.
Parsons, T. Dꢀren, J. Am. Chem. Soc. 2011, 133, 8900–8902.
Q. Shi, Z. Chen, Z. Song, J. Li, J. Dong, Angew. Chemie - Int. Ed.
[
[
[
53]
54]
55]
[
[
[
16]
17]
18]
S. Bhattacharjee, M.-S. Jang, H.-J. Kwon, W.-S. Ahn, Catal. Surv.
from Asia 2014, 18, 101–127.
S. K. Nune, P. K. Thallapally, A. Dohnalkova, C. Wang, J. Liu, G. J.
Exarhos, Chem. Commun. 2010, 46, 4878–80.
2011, 50, 672–675.
V. Gascón, E. Castro-Miguel, M. Díaz-García, R. M. Blanco, M.
Sanchez-Sanchez, J. Chem. Technol. Biotechnol. 2017, 92, 2583–
P. J. Beldon, L. Fábián, R. S. Stein, A. Thirumurugan, A. K.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201701984
FULL PAPER
2593.
[
[
[
56]
57]
58]
M. Jian, B. Liu, G. Zhang, R. Liu, X. Zhang, Colloids Surfaces A
Physicochem. Eng. Asp. 2015, 465, 67–76.
P. Chulkaivalsucharit, X. Wu, J. Ge, RSC Adv. 2015, 5, 101293–
101296.
M. Sanchez-Sanchez, I. de Asua, D. Ruano, K. Diaz, Cryst. Growth
Des. 2015, 15, 4498–4506.
[
[
59]
60]
M.M.Bradford, Anal. Biochem. 1976, 72, 248–254.
F. Beisson, A. Tiss, C. Rivière, R. Verger, Eur. J. Lipid Sci. Technol.
2
000, 102, 133–153.
S. H. Sterri, B. A. Johnsen, F. Fonnum, Biochem. Pharmacol. 1985,
4, 2779–2785.
[
61]
3
[
[
62]
63]
B. Lenarcik, P. Ojczenasz, J.heterocyclic Chem. 2002, 39, 287.
S. Brunauer, P. H. Emmett, E. Teller, J. Am. Chem. Soc. 1938, 60,
3
09–319.
E. P. Barrett, L. G. Joyner, P. P. Halenda, J. Am. Chem. Soc. 1951,
3, 373–380.
S. Yang, J. Chen, D. Zhao, D. Han, X. Chen, Int. J. Pharm. 2012,
34, 155–160.
D. Steri, M. Monduzzi, A. Salis, Microporous Mesoporous Mater.
013, 170, 164–172.
F. Pitzalis, M. Monduzzi, A. Salis, Microporous Mesoporous Mater.
017, 241, 145–154.
[
[
[
[
[
64]
65]
66]
67]
68]
7
4
2
2
J. Amoah, S. H. Ho, S. Hama, A. Yoshida, A. Nakanishi, T.
Hasunuma, C. Ogino, A. Kondo, Bioresour. Technol. 2016, 211,
224–230.
[
69]
D. Gilham, R. Lehner, Methods 2005, 36, 139–147.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201701984
FULL PAPER
FULL PAPER
Rhizomucor miehei and
Pseudomonas fluorescens lipases
were encapsulated on ZIF-8
support via a “one-pot” aqueous
synthesis. The effect of two different
Zinc:Ligand molar ratios on enzyme
loading and activity was investigated.
†
[a]
†[b]
Federica Pitzalis ,Cristina Carucci
,
[
a,c]
[c]
Maryam Naseri , Lida Fotouhi ,
[
b]
[a]
Edmond Magner* , Andrea Salis*
Page No. – Page No.
Lipases encapsulation onto ZIF-8. A
comparison between biocatalysts
obtained at low and high zinc:2-
methylimidazole molar ratio in
aqueous medium
This article is protected by copyright. All rights reserved.