Fast multipoint immobilization of lipase through chirall-proline on a MOF as a chiral bioreactor

Article abstract of DOI:10.1039/d0dt04081a

In this paper, we describe the facile preparation of a chiral catalyst by the combination of the amino acid,l-proline (Pro), and the enzyme, porcine pancreas lipase (PPL), immobilized on a microporous metal-organic framework (PPL-Pro@MOF). The multipoint

Full text of DOI:10.1039/d0dt04081a

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Fast multipoint immobilization of lipase through
chiral L-proline on a MOF as a chiral bioreactor†
Cite this: Dalton Trans., 2021, 50,
1866
a
a
a
a
Stephen Lirio,‡ Yung-Han Shih,‡ Pamela Berilyn So,
Li-Hao Liu,
a
b
c
a
Yun-Ting Yen, Shuhei Furukawa, * Wan-Ling Liu,* Hsi-Ya Huang *§ and
d
Chia-Her Lin
*
In this paper, we describe the facile preparation of a chiral catalyst by the combination of the amino acid,
L-proline (Pro), and the enzyme, porcine pancreas lipase (PPL), immobilized on a microporous metal–
organic framework (PPL-Pro@MOF). The multipoint immobilization of PPL onto the MOF is made possible
with the aid of Pro, which also provided a chiral environment for enhanced enantioselectivity. The appli-
cation of the microporous MOF is pivotal in maintaining the catalytic activity of PPL, wherein it prevented
the leaching of Pro during the catalytic reaction, leading to the enhanced activity of PPL. The prepared
biocatalyst was applied in asymmetric carbon–carbon bond formation, demonstrating the potential of
this simple approach for chemical transformations.
Received 29th November 2020,
Accepted 30th December 2020
DOI: 10.1039/d0dt04081a
rsc.li/dalton
1
0
amino acid derivatives greatly enhanced the selectivity of cat-
alysts for asymmetric reactions. In particular, doping porcine
Introduction
Biocatalysts are valuable tools in organic synthesis and con- pancreas lipase (PPL) with L-proline (Pro) was found to be
1
1
sidered to be green and more sustainable alternatives com- useful for asymmetric catalysis,
via non-covalent inter-
1
pared to their synthetic counterparts. Even though there is a actions, and showed potential for the synthesis of polyfunctio-
specific enzyme suited for every natural compound, several nalized 4H-pyran. A previous report also suggested that the
enzymes are classified as promiscuous or may also catalyze presence of L-proline could accelerate enzymatic activity.
1
0
1
2
other reactions. Upon exploration of biocatalytic promiscuous Such induction shows that it could work in a cooperative
enzymes reacting with unnatural substrates for alternative fashion. However, one major drawback of this approach is the
2
9
chemical transformations, some demonstrated the catalysis reusability of the biocatalyst and the long reaction time.
3
–6
of C–C or C–heteroatom bond formation.
enzymes require long reaction times (24–144 h) and the yields cals is increasing. With this, the need for the development of
and enantioselectivities (ee) are still low. Thus, improvement efficient asymmetric catalysts is also increasing.
However, these Globally, the demand for high-value enantiopure fine chemi-
1
3
of their biocatalytic efficiencies still remains a challenging
Heterogeneous catalysts have attracted considerable atten-
task. Several notable strategies by inducing or doping the tion due to their efficient chemical transformations. In
7
–9
enzymes with metal ions
(e.g. artificial metalloenzymes) or general, an enzyme immobilized on a solid support would
exhibit enhanced catalytic performance compared to its free
counterpart. The application of heterogeneous catalysts pro-
a
Department of Chemistry, Chung Yuan Christian University, No. 200, Zhongbei Rd.,
Zhongli Dist., Taoyuan 32023, Taiwan
vides several advantages such as ease of separation, cost-effec-
tiveness, and recyclability. Metal–organic frameworks
b
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University,
1
4,15
Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
(MOFs),
which are constructed from clusters of metal ions
c
College of Science, Chung Yuan Christian University, No. 200, Zhongbei Rd.,
and organic ligands, are a class of well-ordered crystalline
structure materials that have gained popularity over the past
years due to their potential applications. Their unique pro-
perties, such as high surface area, tunable pore size, and
Zhongli Dist., Taoyuan 32023, Taiwan
d
Department of Chemistry, National Taiwan Normal University, National Taiwan
Normal University, No. 88, Sec. 4, Ting-Chow Rd., Taipei, 11676, Taiwan.
E-mail: chiaher@ntnu.edu.tw
†
Electronic supplementary information (ESI) available: Synthesis of MOFs and thermal and chemical stability, revealed higher loading
1
6
experimental procedures (section S1), optimization and characterization of the capacities for enzymes
as compared to other porous
bioreactor (section S2), comparison with other literature reports (section S3),
and characterization of the aldol product (section S4)]. See DOI: 10.1039/
d0dt04081a
17
materials. To date, several strategies have been reported for
the immobilization of enzymes using MOFs, which include
1
8–26
27–33
34,35
encapsulation,
adsorption,
covalent bonding,
and
‡
These authors contributed equally.
Deceased August 22, 2017.
3
6,37
§
a dye-tagging strategy.
Although most of these methods
1866 | Dalton Trans., 2021, 50, 1866–1873
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bestowed enhanced catalytic activities, some processes of
immobilization are rather time consuming and complicated.
Some methods also require sophisticated approaches to
immobilize the enzymes or by complementary matching of the
1
9–21
enzymes with MOF pores.
MOFs with inherent activity for enantioselective catalysis
38
were achieved by direct synthesis of homochiral MOFs.
However, extensive efforts are needed for the synthesis of these
types of homochiral MOFs, which possibly limits their practical
use. Postsynthetic modification of achiral frameworks for the
preparation of homochiral MOFs using chiral templates contain-
ing enantiopure ligands or imparting chiral molecules
Scheme 1 Schematic representation of the (a) immobilization of PPL
and Pro on a microporous MOF and (b) aldol reaction for the as-pre-
pared biocatalyst.
(L-proline) onto the MOFs has also been reported. However,
homochiral MOFs showed poor to moderate enantioselectivity
39,40
and may require long reaction times (∼27 to 72 h).
As an
alternative, our previous study showed that PPL can be directly
immobilized via adsorption using organic solvent and applied
for the biosynthesis of warfarin. We also demonstrated an
efficient multipoint immobilization of enzymes by conjugating
Results and discussion
Screening of MOFs for PPL immobilization
30
3
0
fluorescent dyes on several MOFs, which was attributed to In our previous report, we demonstrated the application of
hydrogen bonding and strong π–π interactions that govern the aluminum MOF MIL-53(Al) (Al(OH)(1,4-BDC), BDC = 1,4-ben-
host–guest attractions between the dye and the organic linkers zenedicarboxylate) for PPL immobilization. In this study, we
37
in the MOFs. Therefore, utilizing the versatile characteristics of further explore other aluminum based MOFs including
MOFs may lead to the development of several novel bioreactors.
MOF-1,4-NDC (Al(OH)(1,4-NDC), NDC = 1,4-naphthalenedicar-
In this study, we present a facile strategy to prepare a hetero- boxylate) and DUT-5 (Al(OH)(BPDC), BPDC = 4,4′-biphenyldi-
geneous biocatalyst with enhanced catalytic activity of the carboxylate), prior to the immobilization and application of
immobilized PPL. Our strategy is to confine the Pro within the PPL-Pro@MOFs in chemical transformations. Since our pre-
microporous MOF and allow it to interact with the immobilized vious report showed that MIL-53(Al) displayed its potential
PPL. We hypothesize that since Pro (encapsulated) is in close support for PPL immobilization, we first optimized the para-
proximity to the immobilized PPL (adsorbed), it is highly poss- meters affecting the catalytic efficiency for C–C bond for-
ible that the Pro could freely interact with the adsorbed enzyme mation (Fig. S1†). These parameters include the amount of
without being released into the reaction media. Therefore, the PPL, water concentration, MOF amount, reaction time, and
presence of Pro would provide a continuous enhanced catalytic reaction temperature (see the ESI† for details). It has been
activity on the active site of PPL, which could be beneficial for reported that different solid supports with different surface
several cycles. Since Pro could provide intermolecular hydrogen structures and properties (e.g. hydrophobic effect) could lead
4
2
bonding and hydrophobic interactions, leading to the protec- to the opening of the active site of PPL. Since MOFs have
41
tion of the structural integrity of the enzyme, we propose that different functionalities, we explored several aluminum-based
4
3
its presence would enhance the activity of PPL, as well as the MOFs for PPL immobilization and evaluated their catalytic
adsorption efficiency onto the MOF through multipoint inter- performances (Fig. S2†). As shown in Fig. S2,† different
action of PPL and Pro. Furthermore, the microporous MOF PPL@MOFs showed significant effects on both yield and ee.
could act as both a reservoir and a solid support. Therefore, PPL@DUT-5(Al) displayed the highest yield of the aldol
selecting the suitable MOF is pivotal in immobilizing Pro product (27% yield), while PPL@MOF-1,4-NDC(Al) had the
(encapsulation) and PPL (surface adsorption).
highest ee, which could be attributed to the hydrophobic
Herein, we co-immobilized PPL and Pro onto MOF-1,4-NDC organic ligand of MOF-1,4-NDC(Al) (composed of naphtha-
4
4
(
Al) (NDC = 1,4-naphthalenedicarboxylic acid) via adsorption/ lene), which helps in exposing the lid of PPL (Fig. S2†). In
co-precipitation (Scheme 1a). It should also be noted that PPL contrast, MIL-53(Al) and DUT-5(Al), which were constructed
is a bulky molecule and rather difficult to immobilize in the from benzenedicarboxylic acid (BBDC) or biphenyl-4,4′-dicar-
MOF. Thus, with the aid of Pro, through high interaction with boxylic acid (BPDC) as organic ligands, presented lower ees.
PPL, it could be anchored onto microporous MOF crystals. Despite the presence of two aromatic rings for DUT-5(Al), it
Such an approach would generate an efficient heterogeneous should be noted that naphthalene is more hydrophobic than
biocatalyst in which the amino acids are retained within BPDC. These results also demonstrate that different MOFs had
the microporous MOFs and are capable of maintaining the different loading efficiencies for the PPL enzyme in which
catalytic activity of the PPL. The optimization of PPL-Pro MOF-1,4-NDC(Al) afforded the highest loading efficiency and
immobilized on MOF-1,4-NDC(Al) (hereafter denoted as are consistent with the catalytic results (Fig. S2†). This is
PPL-Pro@MOF) was investigated for asymmetric carbon– mainly due to the high π–π and hydrophobic interactions
carbon bond formation (Scheme 1b).
between the organic ligand and PPL enzyme. Thus, MOF-1,4-
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NDC(Al) was chosen as the solid support in the successive distribution shows that MOF-1,4-NDC(Al) exhibits a micro-
experiments.
porous structure wherein Pro can fit in but hinders the
entrance of bulky PPL. To verify this, Pro alone was immobi-
lized in MOF-1,4-NDC(Al) and it indeed showed a decrease in
the pore volume (Fig. S4†), indicating that Pro is able to insert
into the micropores of the MOF. Furthermore, the dis-
appearance of the 0.53 nm pore after PPL-Pro immobilization
in the MOF suggests that the micropores of the structure were
blocked by the presence of the enzyme.
Immobilization and characterization of PPL-Pro@MIL-1,4-
NDC(Al)
Among the MOFs used as solid supports, PPL@MOF-1,4-NDC
(Al) presented the best conversion and selectivity. Thus, the
co-immobilization of Pro and PPL was carried out on MOF-1,4-
NDC(Al). The stability of PPL-Pro using the immobilizing
solvent based on the fluorescence experiment shows the effect
of Pro on the conformation of PPL (Fig. S3†). As shown, no sig-
nificant change in the fluorescence spectra was observed after
Pro addition, suggesting that the PPL was not denatured. This
is also similar to a previous report in which the presence of
Pro could prevent the denaturation of the PPL enzyme. An
increase in fluorescence intensity also suggests the interaction
of Pro and PPL through inter- and intramolecular hydrogen
bonding, which prevented the denaturation of the tertiary
structure of the enzyme. Furthermore, Pro could also serve
as a protecting agent in maintaining the structural integrity of
the enzyme when exposed to organic solvents.
The powder XRD pattern of PPL-Pro@MOF-1,4-NDC(Al)
revealed the retained intrinsic crystalline structure of pristine
MOF-1,4-NDC(Al), indicating that the MOF was preserved after
PPL-Pro immobilization (Fig. 1a). The CO2(g) sorption isotherm
Several characterization techniques were also employed,
such as scanning electron microscopy (SEM, Fig. 2) and
thermogravimetric analysis (TGA, Fig. S5†). In particular, the
TGA curve of PPL-Pro@MOF-1,4-NDC(Al) shows the presence
of Pro and PPL with thermal degradations at 200 °C (3.8% wt
loss) and 350 °C (16.3% wt loss), respectively (Fig. S5b†).
SEM-EDS and elemental analysis revealed the homogeneous
dispersion of PPL-Pro, while the increase in nitrogen content
indicated the presence of Pro (Fig. S6 and Table S2†). FTIR
analysis of PPL-Pro@MOF-1,4-NDC(Al) showed strong absorp-
1
2
4
1
tion bands at 1627 cm− and 1412 cm , which can be
ascribed to the N–H stretching vibration especially for the
strong absorption bands of Pro and PPL (1659 cm− , N–H moi-
eties) (Fig. 3). The aromatic CvC stretching vibrations
observed at 1592 and 1468 cm− correspond to the aromatic
ring of the 1,4-NDC ligand in MOF-1,4-NDC(Al). Interestingly,
1
−1
1
3
1
1
2
−1
FTIR analysis revealed the presence of the amide I region
displayed a BET surface area of 416 m g for MOF-1,4-NDC
Al) and the decrease in the adsorption–desorption isotherm
1
(
1600–1700 cm− ) for the immobilized PPL-Pro, which denotes
(
the secondary structural features of the enzyme. Fig. S6† pro-
vides a comparison using the second-derivative method for
pristine PPL and immobilized PPL-Pro onto MOF-1,4-NDC(Al).
The slight change in the magnitude of the α-sheet and β-helix
contents in the secondary structure for PPL is due to its
adsorption on the MOF surface. As shown in Fig. S7,† similar
secondary structural features of the backbone peptides were
observed after the immobilization of PPL-Pro. However, direct
correlation to qualitatively determine and evaluate the magni-
tude of the changes in α-sheet and β-helix contents was unsuc-
cessful. This is due to the small amount of enzyme adsorbed
onto the MOF surface. Furthermore, the results of circular
dichroism spectroscopy could not be established due to the
interference of MOFs (i.e., organic ligands). This drawback is
similar to that in a previous report on enzymes adsorbed onto
denotes the immobilization of PPL-Pro (Fig. 1b). The pore size
4
5
single-walled nanotubes.
Fig. 1 (a) PXRD patterns and (b) CO2(g) sorption isotherms (inset: pore
size distribution) of pristine MOF-1,4-NDC(Al) and PPL-Pro@MOF-1,4-
NDC(Al).
Fig. 2 SEM images of (a) pristine MOF-1,4-NDC(Al) and (b)
PPL-Pro@MOF-1,4-NDC(Al) (inset: optical image of PPL-Pro@MOF-1,4-
NDC(Al)) at 10k magnification.
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PPL displayed lower ee, which can be associated with the
leaching of Pro, therefore causing the prevention of its inter-
action with PPL. In contrast, simultaneous immobilization of
PPL and Pro enhances the PPL activity by almost ∼2.4 fold as
the Pro concentration is further increased to 60% mmol with
high loading efficiency in MOF-1,4-NDC(Al) (Fig. S9†). This
could be associated with electrostatic interactions and hydro-
5
1
gen bonding among Pro, PPL, and the MOF. The loading
capacity of MOF-1,4-NDC(Al) was determined to be 0.291 mg
PPL per mg carrier (Fig. S10 and 11†).
The above results demonstrated that the presence of Pro
enhances PPL activity and provides higher adsorption
efficiency for MOF-1,4-NDC(Al). However, the origin of the
catalytic effect for PPL–Pro is yet to be reported. Therefore, we
used another enzyme, bovine serum albumin (BSA), and co-
catalyzed it with D- or L-Pro to evaluate the catalytic effect of
PPL (Table S4†). The combination of L-Pro and BSA exhibited
poor yield and ee compared to in-solution PPL-Pro. A similar
result was also obtained after co-catalyzing with D-pro and PPL,
confirming that the origin of the catalytic effect of the enzyme
is affected by Pro. The synergistic effect of L-pro and PPL led to
moderate yield and ee. These results show that the presence of
L-pro plays a pivotal role in enhancing the selectivity of the
PPL enzyme.
Fig. 3 (a) FTIR spectra of PPL, Pro, and PPL-Pro@MOF-1,4-NDC. (b)
Scale (x-axis) for FTIR spectra was adjusted from 1025 to 1800 cm−
1
.
We also evaluated the potential of PPL-Pro@MOF-1,4-NDC
Al) based on its recyclability. A sharp decrease in both yield
Application of PPL-Pro@MOF-1,4-NDC(Al)
(
To assess the potential of PPL-Pro@MOF-1,4-NDC(Al) as a and ee was observed after the second cycle, which could be
green and renewable catalyst, asymmetric aldol reaction was ascribed to the immobilizing solvent (e.g., DMSO and metha-
carried out in this study (Scheme 1b). The catalytic efficiency nol) (Fig. S12†). Thus, we changed the immobilizing solvent to
1
1
of PPL-Pro@MOF-1,4-NDC(Al) was also optimized in which DMSO/acetone in which the Pro was found to be soluble.
several parameters affecting the yield and ee were evaluated The immobilized PPL-Pro underwent co-precipitation due to
Fig. S8†). The results indicated that the presence of Pro the presence of acetone. Interestingly, PPL-Pro@MOF-1,4-NDC
(
further enhanced the activity of the immobilized PPL. Free (Al) proved to be reusable and maintained the conversion and
PPL demonstrated 17% yield and 36% ee while the Pro doped ee even after 10 catalytic cycles (∼70% average yield and ∼65%
PPL (free PPL-Pro) showed almost a 3-fold and 2-fold increase average ee) (Fig. 4). In contrast, Pro@MOF-1,4-NDC(Al) can
in the yield and ee, respectively (Fig. S8a†). This is likely due to only be recycled up to four times and it drastically declined in
the electrostatic interactions between Pro and the oxyanion the succeeding cycles due to the leaching of Pro, as confirmed
hole of PPL during the course of immobilization, leading to by TGA (Fig. S13†). Besides, the TGA curve of
1
0
the enhancement of ee. Besides, the immobilized Pro (in the PPL-Pro@MOF-1,4-NDC(Al) confirms the presence of PPL-Pro
4
6
th
pore of MOF-1,4-NDC(Al) (0.53 nm) ) contributed towards even after the 6 cycle, which further supports the significance
enhancing the loading efficiency of the MOF for PPL immobil- of microporous MOF-1,4-NDC(Al) in preventing the leaching of
ization via electrostatic interactions. This is also similar to pre- Pro, maintaining its close proximity to PPL for interfacial acti-
4
7,48
vious reports suggesting that the presence of metal ions,
dynamers, or amino acids
vation. The reproducibility of PPL-Pro@MOF-1,4-NDC(Al)
can provide non-covalent using different batches of MOF-1,4-NDC(Al) also afforded com-
interactions (e.g. hydrogen bonding) in the active site of the parable yields and ees (Fig. S14 and 15†).
4
9
10,12,50
enzyme, leading to its interfacial activation. Since the immo-
To demonstrate the significance of microporous MOF-1,4-
bilization of PPL-Pro was carried out simultaneously, we evalu- NDC(Al) in preventing the leaching of Pro, various solid sup-
ated different immobilization techniques to enhance the ports with different pore sizes to immobilize PPL-Pro were
loading efficiency of MOF-1,4-NDC(Al) via sequential immobil- employed. Proline, with molecular dimensions of 0.47 × 0.24
3
ization (Fig. S8c†). However, changing the immobilization × 0.23 nm , conveniently fits the 0.53 nm pores of MOF-1,4-
method for PPL-Pro on MOF-1,4-NDC(Al) exhibited some NDC(Al). As PPL is a bulky molecule (4.6 × 2.6 × 1.1 nm
decrease in ee when either the Pro or PPL was first immobi- molecular dimensions) and rather difficult to immobilize in
lized, whereas simultaneous immobilization of PPL and Pro microporous MOFs, it could be anchored stably onto the
still afforded the highest yield and ee. This result suggests that MOF, through its high interaction with Pro. As depicted in
sequential immobilization reduces the interaction between the Fig. 5, PPL-Pro@SBA-15 (∼5 nm pore size) showed a signifi-
Pro and PPL. For instance, immobilizing the Pro followed by cant decline in the yield and ee, suggesting the leaching of
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Fig. 5 Comparison of (a) aldol product yield and (b) enantioselectivity
using different solid supports with varying pore sizes.
Table 1 Comparison of the aldol product and enantioselectivity using
different porous supports with varying pore sizes
Fig. 4 Conversion and enantioselectivity of the aldol product using
PPL-Pro@MOF-1,4-NDC(Al) under multiple catalytic cycles.
b
c
%
product yield (ee )
a
Pore size
(nm)
st
nd
rd
th
th
Support
1
2
3
4
5
MOF-1,4-NDC(Al) 0.53
72 (68) 76 (67) 74 (65) 75 (66) 66 (65)
73 (63) 65 (62) 63 (62) 56 (63) 47 (63)
77 (63) 73 (62) 61 (62) 56 (63) 51 (63)
78 (62) 70 (61) 64 (61) 62 (62) 54 (64)
71 (64) 59 (63) 49 (62) 41 (56) 33 (51)
PPL and Pro after several cycles (Table 1). Meanwhile, other MIL-53(Al)
0.85
MIL-100(Al)
0.8, 1, 2
0.7–0.8
5
solid supports with different pore sizes displayed lower yields
NaY
SBA-15
and ees. This result further reveals the significance of pore
size and hydrophobic MOFs. Unlike the MOF-1,4-NDC(Al),
MOFs containing different pore sizes displayed a significant
decrease in yield, which could be associated with the
leaching of Pro, thus lowering the catalytic activity of the
immobilized PPL. For instance, PPL-Pro@MIL-53(Al) and
PPL-Pro@MIL-100(Al) displayed lower ees compared to
a
b
Pore size of the porous support. The percent yield was calculated
c
based on silica gel chromatography. The ee was calculated based on
HPLC.
PPL-Pro@MOF-1,4-NDC(Al). This could be attributed to the time of the latter was 168 h. Meanwhile, the promiscuous
presence of hydrophobic ligands constructed from MOF-1,4- enzyme catalyzed aldol reaction showed varying yields and
NDC(Al). Collation of these results proved that microporous ees (Table S5†). The reaction took up to 46–170 hours unlike
MOFs could serve as both a reservoir and a solid support for the prepared PPL-Pro@MOF-1,4-NDC(Al) with only 24 h reac-
Pro, which provides continuous catalytic activity for the tion time. Overall, the application of PPL-Pro@MOF-1,4-NDC
immobilized PPL.
(Al) for asymmetric catalysis is rather energy efficient and
In comparison with the reported MOFs for asymmetric environment friendly. This further expands the simplicity of
catalysis of aldol reactions, their reactions are rather time improving the selectivity of promiscuous enzymes for asym-
consuming (168 h) with poor to moderate ee and the prepa- metric catalysis application. It should also be noted that
ration is labor-intensive, as depicted in Table S4.† In con- given the high number of possible interactions, as well as
trast, the prepared biocatalyst PPL-Pro@MOF-1,4-NDC(Al) the synergistic effects among the MOF, PPL, and Pro, we
demonstrated better ee than the Al-MIL-101-NH-Gly-Pro cata- believe that another study to address this area will be
lyst using the same model compound. Besides, the reaction interesting.
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For porcine pancrease lipase (PPL) immobilized on a MOF
Conclusion
(
hereafter denoted as PPL@MOF), 10 mg of MOF were dis-
In summary, we have demonstrated for the first time the suc- persed in 1 mL of DMSO : MeOH or DMSO : acetone (1 : 1, v/v)
cessful co-immobilization of PPL and Pro on microporous and 10 mg of PPL were added into a vial. The mixture was vor-
MOF-1,4-NDC(Al) and its application as a heterogeneous bio- texed for 1 h at room temperature and centrifuged for 5 min at
catalyst for asymmetric aldol reaction. The microporous MOF 10 000 rpm. The supernatant was removed and washed with
could be used as a reservoir for Pro, which provided continu- MeOH or acetone to remove the excess liquid to afford
ous enhanced catalytic activity for the PPL enzyme. In PPL@MOF biocatalysts. For the co-immobilization of L-proline
addition, Pro showed a significant effect on the loading (Pro) and PPL on MOF (denoted as PPL-Pro@MOF), 6.8 mg of
efficiency of PPL on the MOF. This shows that microporous Pro was added to PPL following the abovementioned pro-
MOFs could pave the way for the immobilization of several co- cedure. For Pro immobilization on the MOF (denoted as
factor dependent enzymes for chemical processes. This could Pro@MOF), 6.8 mg of Pro was directly immobilized using the
be done by entrapping the co-factors into the microporous above procedure.
MOFs and subsequently immobilizing the enzyme. We believe
that this work would contribute to overcoming some of the
Characterization
challenges encountered in biocatalysis such as operational A scanning electron microscope model JSM-7600F (JEOL,
cost and reusability. Therefore, this simple approach could Japan) was used for morphology observation while powder
open up new perspectives towards the development of cheap X-ray diffraction (PXRD) patterns were recorded using a Bruker
and green heterogeneous biocatalysts. Investigation using the D8 Advance ECO model X-ray diffractometer equipment
proposed approach by encapsulating both the amino acid and (Bruker Daltonics). The infrared spectrum was recorded using
the enzyme in exploring other chemical transformations is of an FT-IR 4200 model spectroscopy system (Jasco, Japan). A Tri-
great interest.
star 3000 model surface area analysis equipment from
Micromeritics (Norcross, GA, USA) was employed for surface
area measurement of the MOFs and L-proline-lipase immobi-
lized on MOF-1,4-NDC(Al) (PPL-Pro@MOF). The separation of
the aldol product was carried out on an Agilent 1100 series
HPLC system (Japan) using a Daicel Chiralpak AS-H column
with a mobile phase of n-hexane : IPA (6 : 4, v/v) at a flow rate
Experimental
Materials
All the reagents used were of analytical grade and used
without further purification: porcine pancreatic lipase (Sigma;
Lot #SLBC9250 V), hexane and methanol (J.T Baker, USA), di-
methylsulfoxide (DMSO) and 2-propanol (IPA) (Tedia, USA),
−1
of 1.0 mL min and UV detection at 254 nm. All fluorescence
experiments were performed using a Hitachi system (F-7000,
Hitachi, Japan), while the UV-Vis experiments were performed
using a Shimadzu system (UV-2550, Shimadzu, Japan).
4
-nitrobenzaldehyde (Acros organics, USA), acetone (Echo
Chemical Co., Ltd, Taiwan), ethyl acetate (Merck, USA), anhy-
drous magnesium sulfate (Showa Chemical Industry Co. Ltd, Catalytic reaction studies of PPL@MOF and PPL-Pro@MOF
Japan), silica gel 60 (0.063–0.2 mm) (Merck, USA), zeolite Y,
sodium (NaY) (Alfa Aesar, USA), and SBA-15 (5 nm) were syn-
The aldol reaction was conducted according to a previously
reported paper. The reaction was initiated using PPL@MOF
4
thesized according to the published literature via microwave-
or PPL-Pro@MOF, 0.12 mmol 4-nitrobenzaldehyde, 475 µL of
acetone (6.5 mmol, excess) and 25 µL of water. The reaction
mixture was gently stirred at room temperature for 24 h.
Afterwards, the biocatalysts were recovered by centrifugation.
To the supernatant, 1 mL of water was added with and
extracted using ethyl acetate (EtOAc) (3 × 1 mL). The
obtained organic phase was dried over anhydrous mag-
nesium sulfate and the solvent was evaporated under vacuum
at 45 °C. The crude material was purified using silica gel
chromatography with an organic solvent of hexane : EtOAc
5
2
assisted synthesis.
Synthesis of MOF-1,4-NDC(Al)
MOF-1,4-NDC(Al) was synthesized using a microwaved-assisted
4
6
reaction according to the published literature. In brief, a
mixture of aluminum nitrate nonahydrate (Al(NO ) ·9H O,
3
3
2
375.1 mg), 1,4-naphthalenedicarboxylic acid (1,4-NDC:
1
08 mg), and 10 mL of deionized H O was placed in a 100 mL
2
Teflon autoclave in a microwave oven. The mixture was heated
at 180 °C for 30 min. The resulting light yellow powdered
sample was collected by filtration, washed with D.I. water and
dried in the oven at 70 °C for 24 h. MOF-1,4-NDC(Al) was
heated at 180 °C for 12 under vacuum prior to use. The com-
plete details of the syntheses of other MOFs are found in the
ESI.†
(
4 : 1, v/v). The product was characterized by NMR spec-
troscopy (Fig. S16 and 17†). The enantioselectivity of the
aldol product was determined using an HPLC Agilent 1100
series system equipped with a Diacel Chiralpak AS-H column
(
Fig. S18†).
Preparation of the biocatalyst
Conflicts of interest
The preparation of the biocatalyst was carried out following
our previously published paper with slight modifications.
3
0
There are no conflicts to declare.
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Dalton Trans., 2021, 50, 1866–1873 | 1871

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4 S. Patra, T. Hidalgo Crespo, A. Permyakova, C. Sicard,
C. Serre, A. Chausse, N. Steunou and L. Legrand, J. Mater.
Chem. B, 2015, 3, 8983–8992.
Acknowledgements
The authors would like to thank the Ministry of Science and
Technology, Taiwan, for financial support with grants MOST 25 F.-K. Shieh, S.-C. Wang, C.-I. Yen, C.-C. Wu, S. Dutta,
1
07-2113-M-003-017-MY2 and MOST 107-2628-M-003-005-MY3.
L.-Y. Chou, J. V. Morabito, P. Hu, M.-H. Hsu, K. C. W. Wu
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