Full Papers
doi.org/10.1002/cctc.202100481
ChemCatChem
with the calix-enzyme complex in the structure of Fe3O4@Calix-
ZIF-8@CRL and its conformation is well preserved, and con-
sequently, denaturation and enzyme leakage might be very
low.[43,44] Significantly improved MOF-biocatalyst performance,
as well as increased recyclability, shows that it can be utilized in
very various industrial applications.[45]
Characterization
The vibrational spectra of biocomposites were recorded on a Bruker
Fourier Transform Infrared FTIR. Transmission electron microscopy
(TEM, FEI Company- Tecnai TMG2 Spirit / Biotwin, USA) and
scanning electron microscopy (SEM, Jeol, JSM5310, Japan) was
identified for particle size and surface morphology of the samples.
The spectroscopic measurements were performed by Shimadzu UV-
1700 Pharma spectrophotometer. X-ray diffractions (XRD, Bruker D8
Advance) were used to investigate crystal structures of the
biocomposite. Surface distribution of Energy Dispersive X-Ray
Analysis (EDX) elements was examined. Thermogravimetric analysis
(TGA) was carried out on a TGA Perkin-Elmer Puris thermogravi-
Conclusions
The new magnetic MOF biocatalyst was successfully fabricated,
immobilized lipase and utilized for enantioselective hydrolysis
of (R,S)-naproxen methyl ester. The magnetic MOF biocatalyst
was prepared by interacting with Calix, CRL, and Fe3O4 nano-
particles and then reacted with imidazole and Zn+2 by co-
precipitate. It exhibited approximately 2.88-fold enhancement
in hydrolytic activity of Fe3O4@Calix-ZIF-8@CRL. Additionally,
the reusability of Fe3O4@Calix-ZIF-8@CRL is also important for
economical use of the lipase, which is very simple due to its
magnetic feature. It was observed that the immobilized enzyme
(Fe3O4@Calix-ZIF-8@CRL) retained its initial activity by 65% after
the 7th repeated use. In enantioselective enzymatic hydrolysis
of (R,S)-naproxen methyl ester, the Fe3O4@Calix-ZIF-8@CRL was
utilized as an efficient biocatalyst. It was observed that
Fe3O4@Calix-ZIF-8@CRL has excellent enantioselectivity and
conversion (X= 49%; E=371) compared to immobilized lipase
without calixarene derivative (Fe3O4@ZIF-8@CRL) (X= 22%; E=
131). Therefore, the immobilization of lipase on magnetic MOF
suggestions a simple and inexpensive way for the pharmaceut-
ical industry of significant differences in the biological activities
of the enantiomers.
°
metric analyzer in the 0–600 C range under nitrogen atmosphere
°
(heating rate 10 C/min). The samples were analyzed by High
Performance Liquid Chromatography (HPLC, Agilent 1200 Series)
and enantiomeric excess (ee) was determined by using a Chiralcel
ODÀ H column.
Determination of lipase activity and stability
The catalytic activities of free lipase, Fe3O4@ZIF-8@CRL and
Fe3O4@Calix-ZIF-8@CRL were determined by hydrolysis of p-NPP.[1–4]
It is found from the 405 nm absorbance of p-nitrophenol (p-NP)
released using UV-visible spectrophotometer. 1 Unit (U) of lipase is
the amount of lipase required to hydrolyze 1 μmol p-NPP per
minute. Bradford method was used to calculate the protein amount
of immobilized lipases.[47]
The activities of free and immobilized enzymes were measured at
various pH (4.0–9.0) and temperature (30–60 C). In order to
examine their thermal stability, enzyme activities were determined
by keeping immobilized lipase solutions at 60 C and different time
intervals (20–120 minutes). The immobilized lipases were washed
with deionized water and easily separated by magnetic filtration,
and the enzyme activity was measured after each use. All experi-
ments were repeated three times.
°
°
Kinetic constants determination
Experimental Section
Catalytic activities Fe3O4@ZIF-8@CRL and Fe3O4@Calix-ZIF-8@CRL
were exanimated at various p-nitrophenyl palmitate (p-NPP)
concentrations. Kinetic parameters (Vmax and Km) were determined.
Chemicals
Zinc nitrate hexahydrate (Zn(NO3)2 ·6H2O), 2- methylimidazole,
Candida rugosa lipase (CRL, type VII), p-nitrophenyl palmitate (p-
NPP), Iron(II) chloride tetrahydrate (FeCl2 ·4H2O), Iron(III) chloride
hexahydrate (FeCl3 ·6H2O), Bradford reagent, S-naproxen were
supplied from Sigma and Merck.
Enantioselective hydrolysis
The preparation of racemic naproxen methyl ester was carried out
according to previously described method.[48] Enantioselective
hydrolysis reactions were carried out in aqueous buffer solution/
isooctane reaction system. A solution of racemic Naproxen methyl
ester (20 mM) in 2 mL isooctane was added to 2 mL of buffer
containing immobilized lipases, the mixture was stirred at 150 rpm
in the incubator. Samples taken from the isooctane phase after
24 hours were analyzed by HPLC to calculate conversion and
enantioselectivity. Besides, the effect of immobilized lipases on
enantioselectivity was determined by incubation at different pH
and temperatures. It was also performed reusability of immobilized
lipases.
Preparation of Fe3O4@ZIF-8@CRL and Fe3O4@Calix-ZIF-8@CRL
Fe3O4 nanoparticles and tetracarboxylic acid derivatives of calix[4]
arene were synthesized according to our previous studies [29–
31,33,46]. Zn(NO3)2.6H2O (92.5 mg/mL) and 2-methylimidazole
(4.1 g) was mixed into an aqueous solution at room temperature
for 10 minutes. Then, Calix (20 mg) were added to CRL solution
(100 mg) and after 10 minutes incubation and Fe3O4 nanoparticles
(50 mg) were added and mixed for 10 minutes. The mixture was
added to the 2-methylimidazole solution and incubated at room
temperature for 30 minutes, after which the mixture turned a milky
°
brown color. The solution was then stored overnight at 4 C, and
after 24 hours it was washed three times with deionized water and
centrifuged (2028×g, 5 minutes). The immobilized lipases were Acknowledgments
dried by lyophilization.
We would like to thank the Research Foundation of Selcuk
University (SUBAP-Grant Number:20201054) for the financial
ChemCatChem 2021, 13, 1–9
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