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 Virginia’s College at Wise Wise, VA, 24293(USA) . 5 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 categories—physical 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, 5 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 5 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 nature—applying 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. O’Keeffe, 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. Gándara, E. GutiérrezPuebla, M. A. Camblor, Adv. Mater. 2011, 23, 5283. 40[6] D. I. Fried, F. J. Brieler, M. Fröba, 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.