Protein Immobilization in Metal-Organic Frameworks by

Covalent Binding
Xuan Wang, Trevor A. Makal and Hong-Cai Zhou*
5
Abstract: Metal-organic Frameworks (MOFs), possessing a well-defined system of pores, demonstrate extensive
potential serving as a platform in biological catalysis. Successful immobilization of enzymes in a MOF system
retains the enzymatic activity, renders the active site more accessible to the substrate, and promises recyclability for
reuse and solvent adaptability in a broad range of working conditions. This highlight describes enzyme
immobilization on MOFs via covalent binding and its significance.

10

Enzymatic catalysis, featuring high reactivity, selectivity, and specificity, has been
extensively studied for chemical, pharmaceutical, and food industries. In commercial use, the
enzyme can be easily denatured by factors such as temperature, pH, and physical damage,
resulting in loss of activity. Therefore, the successful industrial application of biological catalysis
highly relies on the ability to stabilize these enzymes and proteins in an unnatural environment
while retaining functionality and activity. [1]
To solve the stability issue, studies have focused on incorporation of catalytically-active
centers of enzymes into synthetic materials or immobilization of the enzyme in organic or
inorganic structures. Incorporation of active centers into synthetic materials would commonly
result in the need for extensive enzyme engineering. Meanwhile, the latter strategy is extremely
appealing to immobilize enzymes on gels, organic microparticles, nonporous and porous
inorganic supports, as materials may be tailored prior to incorporation of enzymes. In particular,
immobilization of enzymes in the cavities of porous materials presents an unprecedented
opportunity to achieve better efficiency by employing design principles inspired by nature in
synthetic systems. [1, 2] The preliminary step to the use of biocatalysts in industrial application is
to establish a solid support system.
In this context, porous materials with high surface area, tunable but uniform pores, as well
as thermal- and water-stability can serve as platforms for enzyme immobilization. In the past
decade, metal-organic frameworks (MOFs) have emerged as ideal candidates for building
heterogeneous catalytic systems owing to the vast diversity of structures, rich palettes of
functionalities, large pore openings, tunable pore sizes, and permanent porosity. [2-4] Unlike
traditional porous materials, such as zeolites, activated carbons, and mesoporous silica, MOFs
offer the opportunity of tuning the pore sizes and geometries, and chemical composition of the
surfaces to instill desired properties by modifying metal/ligand combinations. This tunability of
pore sizes allows for targeting of specific enzyme candidate. MOFs also prove attractive as they

15

20

25

30

35

[*]

Xuan Wang, Prof. H.-C. Zhou

Department of Chemistry, Texas A&M University
College Station, TX 77843 (USA)
E-mail: zhou@chem.tamu.edu
Homepage: http://www.chem.tamu.edu/rgroup/zhou/
Dr. Trevor A. Makal
Department of Natural Sciences, The University of Virginias College at Wise
Wise, VA, 24293(USA)

10

15

20

25

30

35

may be easily modified with organic functional groups, which may alter the isoelectric points of
the enzyme and consequent electrostatic interactions between the framework and the enzyme. [1]
Plus, contriving the modification of the surface of MOF with hydrophobic/hydrophilic groups
would not only facilitate the substrate accessibility to the immobilized enzyme and bring about a
high enzyme-substrate ratio but also enhance the water-stability of the system. The rapid
development of MOFs has provided a valuable opportunity to develop advanced porous sorbent
systems for efficiently supporting enzymes with 3-D highly ordered architectures.
Although a few catalytically active proteins have been successfully introduced into porous
MOFs, the limited stability of most MOFs precludes their application in aqueous media and
results in poor reactivity and specificity under working conditions. [3] The stability of a
heterogeneous catalytic system remains crucial. The interaction between the immobilized
enzyme and the supporting media greatly affects the entire catalysis. Finding an enzyme-host
interaction of sufficient strength, which prevents leaching of the enzyme while maintaining
activity, remains a daunting challenge. Novel techniques developed for enzyme immobilization
are divided into five categoriesphysical adsorption, microencapsulation, matrix entrapment,
cross-linking, and covalent binding. [5] Immobilized enzymes held through only physical
adsorption tend to demonstrate poor stability. However, covalent binding between the support
and enzyme could efficiently improve the stability. The presence of a covalent bond introduces
such a physical barrier that will rigidify the quaternary structure of enzyme while decreasing its
mobility and consequently fixing the enzyme. [6] As a result, enzyme leaching from the pores will
be minimized, and the entire enzyme-support system can be stabilized under extreme conditions.

Scheme 1: Trypsin Immobilization onto Cr-Based MOFs via covalent binding. ((Reprinted with permission from Ref. [7], copyright 2012 John
Wiley and Sons).

Within this avenue of research, researchers are taking interest in using a covalent bond for
enzyme immobilization. Trypsin, a common protease that catalyzes protein digestion and
cleavage of peptides, has been used widely in biotechnological processes. However, the long
digestion time of free trypsin (about 18 hours) restricts its commercial application. Lin and
coworkers have adapted novel MOFs as effective hosts to improve the efficiency of catalytic
activity of trypsin. [7] Two strategies were applied to develop this novel enzyme immobilizing
system: 1) activation of the free carboxylic acid of MIL-88B(Cr) (MIL = Materials of Institut
Lavoisier) with dicyclohexylcarbodiimide (DCC) and 2) functionalization of MIL-88B(Cr) with
amine (Scheme 1). No obvious alteration of MOF constituents resulting from immobilization of
trypsin during binding was observed from SEM, FT-IR, and powder X-ray diffraction studies,
which indicated the well-maintained crystalline structure of the MOF. The subsequent protein
digestion analysis of trypsin-MIL-88B(Cr)-NH2 system demonstrated 69% efficiency, as
compared to 60% for free trypsin in solution during a 2-minute digestion. In comparison studies,

10

15

20

25

30

35

40

45

poor protein digestion activities were observed in attempts to immobilize trypsin without DCC
coupling or in the absence of amine groups. It was proposed that the DCC coupling agent
efficiently enhanced trypsin immobilization, whereas the plentitude of amine functional groups
improved the immobilization and protein digestion via hydrogen bonding by tuning the
hydrophilicity and bio-compatibility of the MOF surface. Furthermore, the trypsin-MIL88B(Cr)-NH2 system exhibited nearly the same digestion efficiency over four cycles. The
stability was ascribed to the rigidity of the framework because of the large swelling effect in
organic solvent and the immiscibility of chromium(III) in polar solvent. Overall, the trypsinMIL-88B(Cr)-NH2 system exhibited exemplary proteolysis performance over free enzyme
digestion in terms of digestion efficiency, reusability, and stability.
In a similar approach, Lin and coworkers recently developed an advanced strategy without
chemical modification of the MOF. [8] They fabricated a new aluminum based MOF, CYCU-4
(CYCU=Chung Yuan Christian University), as the solid support. The desolvated CYCU-4
displayed a transformation from a micro- to mesoporous structure upon soaking in organic
solvent or aqueous solution, which led to an increase of enzyme loading along with greater
accessibility for substrates. They also coupled fluorescein isothiocyanate (FITC), as fluorescent
binding agent, with trypsin before immobilization onto the framework. In this case, the
previously required surface modification in the MIL-88B(Cr)-NH2 system was avoided, which
consequently resulted in more time-efficient immobilization. After simple vortex performed as
driving force, this trypsin-FITC@MOF biocatalyst exhibited commendable catalytic
performance over the free trypsin digestion and significantly decreased the digestion time from
hours to minutes. In addition, this bioreactor exhibited nearly the same digestion efficiencies
over five cycles. The specific host-guest interaction between FITC and the micropore and the
confinement effect of the mesopores on trypsin enhanced the stability of the entire trypsinFITC@MOF system, resulting in high biocatalytic activity and increased reusability.
It is disputable that if the trypsin is really being trapped in the interior surface of both MOFs,
since trypsin is larger than the pore sizes of the two MOFs. However, the strategy to bridge
trypsin onto the surface of MOF via a covalent bond worked efficiently to maintain the activity
and recyclability in both two cases. This technique will likely be useful in future enzyme
immobilization in MOFs with larger pores which may accommodate enzymes. Summing up the
aforementioned results, immobilization of enzymes in MOFs with the aid of a covalent bond has
been adapted as a high-performance biocatalyts to achieve high enzyme-to-substrate ratio,
reusability, efficient catalytic activity, and short reaction time. The use of a covalent bond
anchors enzyme in the surface of MOF and provides strong enzyme-MOF interaction, which
typically results in both high enzyme loading capacity and the stabilization of the enzyme.
Furthermore, the covalent bond physically segregates the enzyme from the surface of MOF so
that the enzyme still maintains a level of flexibility necessary to retain activity. In short, covalent
binding plays a significant part in enhancing the enzymatic activity and stabilizing the
heterogeneous biocatalyst, with MOFs serving as the ideal enzyme host.
The rapid development of MOFs in enzymatic catalysis should allow their extension to
therapeutic and diagnostic uses. Unfortunately, work on covalent binding of enzyme
immobilization is still very rare. Combined with traditional forces for physical adsorption, such
as ionic interaction, van der Waals interaction and hydrogen bonding, more efforts should be
dedicated to design the proper binding for specific enzymes, which will not only maintain the
enzymatic activity but also optimize the catalytic performance under mild conditions. However,
the field of enzyme immobilization in MOFs is still young, and there are a lot of challenges

10

15

20

25

down the road: 1) a big and complex enzyme requires a stable, at least water-stable, mesoporous
MOF if being immobilized, while such MOFs are quite scanty. 2) The maximum amount of
enzyme loading, significantly affected by pH value, ionic strength, surface characteristics, and
adsorption conditions, remains a problem. 3) As advanced biocatalysis, such as nitrogen fixation
by nitrogenase, increase in complexity, new technologies are absolutely necessary to be
developed for such enzymes to effectively perform in artificial supporting media, like MOFs.
Herein, multi-point bindings could be applied to these cofactor-assisted biocatalysts to create
robust systems inside of MOFs. 4) Due to the tunable pore size of MOFs, the future investigation
on enzyme-MOF bioreactors is full of promises for substrate selectivity and product purification.
5) With the aid of advanced characterization techniques, immobilization of enzymes by
deliberately designed MOFs shows great potential in the study of catalytic mechanisms, as well.
As the world is faced with population explosion and energy crisis, efficient conversions of
abundant natural resources into consumable products, such as the conversion of nitrogen to
ammonia, water splitting to produce hydrogen, and conversion of methane into liquid fuel, are in
urgent demand. To solve these fundamental issues, one of the best methods is to learn from
natureapplying bio-machinery in industrial production. The key step for this is to stabilize the
enzymes, which may have great potential with the advent of covalent binding of enzymes inside
of MOFs.
Acknowledgements:
This working was supported by the Center for Gas Separations Relevant to Clean Energy
Technologies, an Energy Frontier Research Center funded by the U. S. Department of Energy,
Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001015.

References:
[1] S. Hudson, J. Cooney, E. Magner, Angew. Chem., Int. Ed. 2008, 47, 8582.
[2] Z.-Y. Gu, J. Park, A. Raiff, Z. Wei, H.-C. Zhou, Chem. Cat. Chem. 2014, 6, 67.
[3] (a) V. Lykourinou, Y. Chen, X.-S. Wang, L. Meng, T. Hoang, L.-J. Ming, R. L. Musselman, S.
Ma, J. Am. Chem. Soc. 2011, 133, 10382.
30
(b) Y. Chen, V. Lykourinou, C. Vetromile, T. Hoang, L.-J. Ming, R. Larsen, S. Ma, J. Am. Chem.
Soc. 2012, 134, 13188.
(c) Y. Chen, V. Lykourinou, T. Hoang, L.-J. Ming, S. Ma, Inorg. Chem. 2012, 51, 9156.
[4] (a) G. Frey, Chem. Soc. Rev. 2008, 37, 191.
(b) S. Kitagawa, R. Kitaura, S.-i. Noro, Angew. Chem. Int. Ed. 2004, 43, 2334.
35
(c) M. OKeeffe, O. M. Yaghi, Chem. Rev. 2012, 112, 675.
(d) S. T. Meek, J. A. Greathouse, M. D. Allendorf, Adv. Mater. 2011, 23, 249.
(e) H.-C. Zhou, J. R. Long, O. M. Yaghi, Chem. Rev. 2012, 112, 673.
[5] M. E. Medina, A. E. Platero-Prats, N. Snejko, A. Rojas, A. Monge, F. Gndara, E. GutirrezPuebla, M. A. Camblor, Adv. Mater. 2011, 23, 5283.
40[6] D. I. Fried, F. J. Brieler, M. Frba, ChemCatChem 2013, 5, 862.
[7]Y.-H. Shih, S.-H. Lo, N.-S. Yang, B. Singco, Y.-J. Cheng, C.-Y. Wu, I.-H. Chang, H.-Y. Huang,
C.-H. Lin, ChemPlusChem 2012, 77, 982.
[8] W. Liu, S. Lo, B. Singco, C. Yang, H. Huang, C.-H. Lin, J. Mater. Chem. B 2013, 1, 928.