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Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 33581−33588
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Ordered Coimmobilization of a Multienzyme Cascade System with a Metal Organic Framework in a Membrane: Reduction of CO2 to Methanol Dailian Zhu,† Shanshi Ao,† Huihui Deng,† Mei Wang,† Cunqi Qin,† Juan Zhang,‡ Yanrong Jia,† Peng Ye,*,† and Huagang Ni*,†
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†
Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Education Ministry, Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, People’s Republic of China ‡ School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, People’s Republic of China S Supporting Information *
ABSTRACT: Enzymatic reduction of CO2 is of great significant, which involves an efficient multienzyme cascade system (MECS). In this work, formate dehydrogenase (FDH), glutamate dehydrogenase (GDH), and reduced pyridine nucleotide (NADH) (FDH&GDH&NADH), formaldehyde dehydrogenase (FalDH), GDH, and NADH (FalDH&GDH&NADH), and alcohol dehydrogenase (ADH), GDH, and NADH (ADH&GDH&NADH) were embedded in ZIF-8 (one kind of metal organic framework) to prepare three kinds of enzymes and coenzymes/ZIF-8 nanocomposites. Then by dead-end filtration these nanocomposites were sequentially located in a microporous membrane, which was combined with a pervaporation membrane to timely achieve the separation of product methanol. Incorporation of the pervaporation membrane was helpful to control reaction direction, and the methanol amount increased from 5.8 ± 0.5 to 6.7 ± 0.8 μmol. The reaction efficiency of an immobilized enzymes-ordered distribution in a membrane was higher than that disordered distribution in the membrane, and the methanol amount increased from 6.7 ± 0.8 to 12.6 ± 0.6 μmol. Moreover, it appeared that introduction of NADH into ZIF-8 enhanced the transformation of CO2 to methanol from 12.6 ± 0.6 to 13.4 ± 0.9 μmol. Over 50% of their original productivity was retained after 12 h of use. This method has wide applicability and can be used in other kinds of multienzyme systems. KEYWORDS: multienzyme cascade system (MECS), enzyme immobilization, metal organic framework (MOF), enzyme membrane reactor, reduction CO2 to methanol
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INTRODUCTION In nature, many biochemical reactions are catalyzed by multienzyme cascade system (MECS) that are constituted of highly ordered assemblies of enzymes. The MECS can accomplish catalysis in a highly efficient way, where the intermediates are transported between the different active sites on enzymes without leaving the MECS.1,2 Cascade reactions are undoubtedly advantageous over classical step-by-step synthesis through eliminating the tedious isolation and purification of reaction intermediates. In addition, higher yields can also be gained, and the atom economy is improved as well. Further benefits of cascade reactions include the possible handling of unstable intermediates and the control and shifting of unfavorable reaction equilibria.3,4 Drawing inspiration from these MECS in nature, researchers have devoted efforts to reconstruct these in vitro with precise design.5−7 In multienzyme coimmobilization, due to the orderliness of cascaded reactions, the accurate control of © 2019 American Chemical Society
positioning and orientation of enzymes needs to be taken into account by selecting appropriate immobilization strategies. For example, Chen et al. designed the construction of metal− organic framework as scaffold for spatial colocalization of glucose oxidase (GOx) and horseradish peroxidase (HRP).8 Garcia adopted a layer-by-layer assembly strategy using biotin−avidin interactions to achieve the desired spatial colocalization of GOx and HRP on Fe3O4 nanoparticles.9 Liu’s group reported a precipitation method for the construction of spatially colocalized multienzyme systems based on inorganic nanocrystal−protein complexes.10 However, it is still difficult to efficiently immobilize more complicated MECS and precisely control the direction of the MECS. Received: June 5, 2019 Accepted: August 16, 2019 Published: August 16, 2019 33581
DOI: 10.1021/acsami.9b09811 ACS Appl. Mater. Interfaces 2019, 11, 33581−33588
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Scheme 1. Ordered Coimmobilization of Multienzyme Cascade System with Metal Organic Framework in Membrane: Reduction CO2 to Methanol Materials and Methods
(ADH&GDH&NADH) were embedded in ZIF-8 to prepare three kinds of (enzymes and coenzyme)/ZIF-8 [(enzymes&oenzyme)/ZIF-8] nanocomposites in which GDH was used to regenerate NADH. Afterward, these enzymes&coenzyme/ ZIF-8 nanocomposites were sequentially located in a microporous membrane by dead-end filtration. The membrane was connected with pervaporation membrane, as shown in Scheme 1. Finally, this enzyme membrane reactor was employed to reduce CO2 to methanol.
Simulating photosynthesis and reduction of CO2 to methanol catalyzed by MECS has been one of the hotspots of concern by researchers. Dave and co-workers first used formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH), and alcohol dehydrogenase (ADH) to reduce CO2 to methanol.11 In this system all three enzymes employ the same cofactor, the reduced form of nicotinamide adenine dinucleotide (NADH), to supply the reducing equivalents required for reaction. Cofactors (like NAD(H), NADP(H), etc.) are generally expensive, which has greatly hampered the viability of cofactor-dependent biotransformation for largescale operations. The regeneration and reuse of cofactor have to be considered for any practical applications.12−14 Metal organic frameworks (MOFs) are porous crystalline organic−inorganic hybrid materials with an architecture quite similar to zeolites but with additional flexibility. MOFs consist of metal-containing nodes and organic ligands linked through coordination bonds.15−17 The very high surface area and pore volume, the ease of pore size tuning, the facile modification on both metal nodes and ligands, and mild synthetic conditions suggest that MOFs can be potent supporting matrices for enzyme immobilization.18−21 Lyu et al. selected a chemically and thermally stable MOF “ZIF-8” as the carrier and further proposed the coordination bonding-based self-assembly of the MOF−enzyme hybrid.22 In this study, we described a strategy of mutilenzymeordered coimmobilization in which enzymes were orderly placed in a microporous membrane to simulate MECS in nature. First, FDH, GDH, and NADH (FDH&GDH&NADH), FalDH, GDH, and NADH (FalDH&GDH&NADH), and ADH, GDH, and NADH
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MATERIAL AND METHODS
Chemicals and Membranes. L-Glutamic dehydrogenase (GDH) from bovine liver, alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, formate dehydrogenase (FDH) from Candida boidinii, and formaldehyde dehydrogenase (FalDH) from Pseudomonas sp. were purchased from Sigma-Aldrich. All of the other reagents were purchased from Sigma-Aldrich and used without further purification. All of the substrate and enzyme solutions were prepared with 0.10 M PBS buffer (pH = 7.0). Commercial PVDF membranes (Haiyan New Oriental Plasticizing Technology Co., Ltd.) were used in this work. CO2 gas (≥99.5%) in a cylinder was purchased from Hangzhou Modern Industrial Gas Co., Ltd. This membrane was composed of a poly(vinylidene fluoride) (PVDF) layer and a nonwoven fabric as support layer, and its aperture was 2 μm. Synthesis of the Enzymes/ZIF-8 Nanocomposites.23 A water solution (1 mL) of FDH (5 mg/mL) and GDH (5 mg/mL) and Zn(NO3)2 water solution (0.31 M, 2 mL) were mixed with 2methylimidazole water solution (1.25 M, 20 mL) under stirring at 25 °C. The mixture then turned milky almost instantly after mixing. After stirring for about 30 min, the mixture was aged for 3 h. Then the product was collected by centrifuging at 6000 rpm for 10 min and washed with deionized water three times. The product was redispersed in deionized water for lyophilization and used for other 33582
DOI: 10.1021/acsami.9b09811 ACS Appl. Mater. Interfaces 2019, 11, 33581−33588
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Figure 1. SEM images of (A) pure ZIF-8, (B) FDH&GDH/ZIF-8, (C) FalDH&GDH/ZIF-8, (D) ADH&GDH/ZIF-8, (E) FDH&GDH&NADH/ZIF-8, (F) FalDH&GDH&NADH/ZIF-8, and (G) ADH&GDH&NADH/ZIF-8. characterizations. The liquid supernatant was collected to measure the amount of enzyme immobilized by the Bradford method. Synthesis of FalDH&GDH/ZIF-8 and ADH&GDH/ZIF-8 nanocomposites was following the same protocol by replacing the multienzyme solution with FalDH water solution (5 mg/mL) and ADH water solution (5 mg/mL), respectively. Synthesis of the Enzymes&Coenzyme/ZIF-8 Nanocomposites. Synthesis of FDH&GDH&NADH/ZIF-8, FalDH&GDH&NADH/ZIF-8, and ADH&GDHNADH/ZIF-8 nanocomposites was following the same protocol by adding 5 mg of NADH. Fabrication of Enzyme−Membrane: Fouling-Induced Enzyme Immobilization. The dead-end filtrations were performed in a pervaporation cell. Descriptions of the equipment and procedures can be found in the SI (as shown in Figure S5). The PVDF membranes were placed on the membrane holder (support layer facing feed, support layer was ignored due to its very large pore size). The membranes were filtered with deionized water for 30 min. For enzyme randomly distribution in the membrane; the enzyme solution containing 4 mg of FDH&GDH/ZIF-8, 4 mg of FalDH&GDH/ ZIF-8, and 2 mg of ADH&GDH/ZIF-8 was put into the PVDF membrane with a 2 μm pore size for the enzyme immobilization operations. For enzyme-ordered distribution in membrane, the three enzyme solutions containing 4 mg of FDH&GDH/ZIF-8 (or FDH&GDH&NADH/ZIF-8), 4 mg of FalDH&GDH/ZIF-8 (or FalDH&GDH&NADH/ZIF-8), and 2 mg of ADH&GDH/ZIF-8 (or ADH&GDH&NADH/ZIF-8) were put into the PVDF membrane. All of the experiments were repeated three times until no particle could be washed out by deionized water. The filtrate was collected to conduct particle size analysis with the dynamic light scattering method (DLS) (as shown in Figure.S3). This membrane was decorated with PDMS/PVDF membrane using epoxy resin. The separation layer of the PVDF membranes was combined with the PDMS layer of the PDMS/PVDF membrane. Acting of Multienzyme Cascade Reaction in Solution (Enzymes/ZIF-8 in Solution (EMS) and Enzymes&Coenzyme/ ZIF-8 in Solution (ECMS)). A 2 mL amount of the reaction mixture containing 10 mM NADH and 4 mM L-glutamate with saturated CO2 (gaseous CO2 was bubbled into solution through a syringe needle for 30 min) was prepared in a 15 mL centrifuge tube covered with Parafilm. A 4 mg amount of FDH&GDH/ZIF-8 (or FDH&GDH&NADH/ZIF-8), 4 mg of FalDH&GDH/ZIF-8 (or FalDH&GDH&NADH/ZIF-8), and 2 mg of ADH&GDH/ZIF-8 (or
ADH&GDH&NADH/ZIF-8) were added, lasting for 6 h for sufficient production of methanol. Acting of Multienzyme Cascade Reaction in Membrane (Disordered Enzymes/ZIF-8 in Membrane (DEMM), Ordered Enzymes/ZIF-8 in Membrane (OEMM), and Ordered Enzymes&Coenzyme/ZIF-8 in Membrane (OECMM)). A 10 mL amount of the reaction mixture containing 10 mM NADH and 4 mM L-glutamate with saturated CO2 (gaseous CO2 was bubbled into solution through a syringe needle for 30 min) was added into the pervaporation cell equipped with 2 μm PVDF membrane, which could separate methanol and water. The reaction lasted 6 h for sufficient production of methanol. To determine the influence of the flux to the conversion of CO2, the pressure was adjusted to get different fluxes. The methanol concentration was determined via gas chromatography (GC) equipped with a flame ionization detector (TCD). All results were repeated three times.
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RESULTS AND DISCUSSION Synthesis of the Enzymes/ZIF-8 Nanocomposites and Enzymes&Conenzyme/ZIF-8 Nanocomposites. As shown in Figure 1, the scanning electron microscopy (SEM) images of enzymes/ZIF-8 nanocomposites and enzymes&conenzyme/ ZIF-8 nanocomposites showed similar morphologies to that of pure ZIF-8. Afterward, the size of these composites ranging from ∼350 to ∼600 nm was obviously bigger than that of the pure ZIF-8 (∼80 nm). Similar results were reported.8,23,24 It seem that the size of the enzyme/ZIF-8 composites was widely distributed due to the rapid nucleation and diverse growth of the crystals of ZIF-8.25 The X-ray diffraction (XRD) patterns of the enzymes/ZIF-8 nanocomposites and enzymes&conenzyme/ZIF-8 nanocomposites agreed well with the pattern of the pure ZIF-8 (Figure 2), which verified that incorporation of enzyme and coenzyme did not affect the crystallinity of ZIF-8.26 Thermal gravity analysis (TGA) in air also confirmed the presence of protein in the composites (Figure 3). As shown in Figure 3A, the second-stage decomposition of the composite started from 200 °C and finished around 400 °C, while the pure ZIF-8 crystals had a small amount of weight loss during 33583
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could also be attributed to decomposition of protein molecules. As shown in Figure 3B, the curves were similar to the curves in Figure 3A. Therefore, during the second stage ∼21 wt % of weight loss occurred for FDH&GDH&NADH/ZIF-8 nanocomposites, ∼21 wt % of weight loss occurred for FalDH&GDH&NADH/ZIF-8 nanocomposites, and ∼16 wt % of weight loss occurred for ADH&GDH&NADH/ZIF-8 nanocomposites, which could also be attributed to decomposition of protein molecules. Fabrication of Enzyme−Membrane: Fouling-Induced Enzyme Immobilization. Fouling-induced enzyme immobilization is an easy method to immobilize enzyme into membrane. Enzyme stability was enhanced after immobilization, but it is easy to lose enzyme and difficult to precisely control the orderliness of multienzyme coimmobilization.28 As shown in Figure 4, the SEM images demonstrated that the enzymes/ZIF-8 composites were distributed uniformly in
Figure 2. XRD patterns of pure ZIF-8, FDH&GDH/ZIF-8, FalDH&GDH/ZIF-8, ADH&GDH/ZIF-8, FDH&GDH&NADH/ ZIF-8, FalDH&GDH&NADH/ZIF-8, and ADH&GDH&NADH/ ZIF-8.
Figure 4. SEM image of FDH&GDH/ZIF-8 distributed in PVDF membrane.
the membrane pore. It was obvious that the enzymes/ZIF-8 nanocomposites packed tightly with small spacing. Because of this the exchange of intermediate products became easier in MECS, and thus, the effectiveness of enzyme catalysis was improved. The membrane used in this experiment is a kind of asymmetric membrane whose pore size of one side is larger than that of the other side. It allows the entrance of the
this temperature range.27 About 14 wt % of weight loss of the nanocomposite occurred during the second stage for FDH&GDH/ZIF-8 nanocomposites, which can be attributed to decomposition of protein molecules. Analogously, during the second stage ∼22 wt % of weight loss occurred for FalDH&GDH/ZIF-8 nanocomposites and ∼22 wt % of weight loss occurred for ADH&GDH/ZIF-8 nanocomposites, which
Figure 3. TGA curves of pure ZIF-8, FDH&GDH/ZIF-8, FalDH&GDH/ZIF-8, and ADH&GDH/ZIF-8 composites in air (A) and pure ZIF-8, FDH&GDH&NADH/ZIF-8, FalDH&GDH&NADH/ZIF-8, and ADH&GDH&NADH/ZIF-8 composites in air (B). 33584
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Figure 5. Methanol production with immobilized enzymes (value of total flux was 25.30 g·m−2·h−1, operation time is 6 h).
embedded into ZIF-8 in that the yield of methanol increased to 5.8 ± 0.5 μmol. This might be due to the closer distance between NADH and enzymes, which greatly reduces the mass transfer resistance and time, thereby improving the activity of the enzyme catalytic reaction. Biocatalytic membranes with enzymes immobilized in industrially manufactured membranes have attracted growing attention.31,32 In a continuous process, product isolation is made simpler with a membrane reactor, which in turn will drive the reaction forward, which has special relevance when the enzymes have a tendency to catalyze the reaction in the reverse way.33 Thus, it is significant to timely separate methanol from the reaction system to promote CO2 → methanol. In DEMM, as methanol was continuously separated by the pervaporation membrane, the reaction proceeded toward a positive direction. It was obvious that the amount of methanol produced by MECS was DEMM > EMS. Furthermore, the three kinds of enzymes(&coenzyme)/ZIF8 nanocomposites were orderly positioned into the membrane pore by dead-end filtration to achieve the ordered coimmobilization of the MECS. Employment of the alcohol/water separation membrane effectively decreased the concentration of methanol in the reaction solution and promoted the reaction of CO2 to methanol. Meanwhile, owing to the ordered packing of the enzymes/ZIF-8 composites in the membrane pore, the transfer routes of intermediates generated by MECS were largely shortened, and thus, the catalytic efficiency of MECS was improved. In fact, multienzymatic pathways in living systems are often segregated into microcompartments or organized as clusters.34 This spatial ordering leads to more stable structures and facilitates substrate channeling between enzymes, thus resulting in increased yields from reactions.35 In OEMM, enzymes/ZIF-8 composites were orderly immobilized according to the MECS sequence of the CO2 reduction and the
enzymes/ZIF-8 composites from the membrane side with a large pore size and at the same time avoids loss of the enzymes/ZIF-8 nanocomposites from the other membrane side with a small pore size (about 2 μm). From Figure 4 it could also be found that the membrane pore was irregular and the enzymes/ZIF-8 composites were polyhedral particles. Thus, when the aqueous solution containing the enzymes/ ZIF-8 nanocomposites was filtered by the membrane, the enzymes/ZIF-8 composites would accumulate in the membrane pore. After filtration the filtrate was collected and dried in an oven, and no solid was found in the filtrate, which indicated that the enzymes/MOF nanocomposites were in the membrane. Catalysis Activity of Enzyme−Membrane Reactor. MOFs, which are an emerging class of porous material with tunable pore size, have shown great promise in the preparation of immobilized enzymes.22,29 In addition, enzymes/MOF composites can be effective at preserving enzyme activity while enforcing a greater degree of stability under catalytically relevant but distinctly abiotic conditions.30 In this work, FDH&GDH(&NADH), FalDH&GDH(&NADH), and ADH&GDH(&NADH) were embedded in ZIF-8 to prepare three kinds of enzymes(&coenzyme)/ZIF-8 nanocomposites in which GDH was used to regenerate NADH. As shown in Figure 5 it was obviously found that the amount of methanol produced by MECS was in the order OECMM > OEMM > DEMM > ECMS > EMS. As shown in Figure S2, the average pore size of these enzymes/ZIF-8 nanocomposites was about 2.2−2.4 nm; NADH (molecular weight is 663 D) could diffuse into these nanocomposites. Also, the molecular weight of four enzymes is more than 40 kDa, which would minimize the probability of enzymes’ elution. In EMS these enzymes/ZIF-8 nanocomposites formed a multienzyme cascade system, which catalyzed CO 2 to methanol, 5.0 ± 0.4 μmol. In ECMS, NADH was also 33585
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form of existence such as carbonic acid, carbonate, and bicarbonate in acidic solution. However, the substrate for the reverse reaction is only CO2(aq).36 Thus, the variation of CO2 concentration would affect the reaction. When the flux became higher, the intermediates were separated into permeate and the production of methanol was delayed by a low accumulation of the intermediates. During the multienzymatic conversion of CO2 to methanol, although the first reaction happened very slowly, the second reaction catalyzed by FaldDH is the real bottleneck. Production of formaldehyde was delayed by a slow accumulation of formic acid from the first reaction.37 Consequently, the amount of methanol reached the maximum as the flux reached in a certain value. With the increase of the flux, the change of the methanol amount was analogous to the change of the methanol concentration. In Figure 7, for OEMM, the methanol concentration first increased and then decreased with the increase of the operation time, and when the operation time was 6 h, the methanol concentration reached the maximum. Also, for OECMM, when the operation time was 4 h, the methanol concentration reached the maximum. It is a common feature in MECS that the reaction maximum appears at a period rather than to start. Also, this phenomenon was noticed in the effect of total flux, which showed that the mass transfer resistance could not be ignored. At the beginning of the reaction CO2 and the intermediate diffused to the enzyme nearby slowly due to blocking of the carrier (in this work, it was ZIF-8). Also, the accumulation rate of intermediate (formic acid and formaldehyde) was a little slow. Hence, the methanol concentration was low in the initial period. As the reaction proceeded, the intermediate reached a certain amount, so that the MECS reaction rate increased.37 However, after a long time using the enzyme activity decreased, and thus, the reaction rate became slow. On the other hand, the immobilized enzymes still retain about 50% of activity after 12 h, which indicated that the enzymes had better stability. In addition, when NADH and enzymes were embedded into ZIF-8, the amount of methanol was improved and the time decreased to obtain maximum total methanol. In this MECS all
pervaporation membrane was employed to timely separate methanol. Both the transfer route of intermediate and the reaction direction were more accurately controlled. Thus, in OEMM, the catalysis activity of CO2 → methanol was significantly enhanced. Besides, introducing NADH into ZIF-8 would also increase the catalysis efficiency of MECS in a membrane. It is similar to the result of ECMS; the catalysis efficiency was enhanced after both enzyme, and NADH were embedded in ZIF-8. Effect of Operation Parameters on the Activity of MECS. As shown in Figure 6, the amount of methanol first
Figure 6. Effect of total flux on methanol production by ordered enzymes/ZIF-8 in membrane (OEMM) (operation time is 6 h).
increased and then decreased with increasing flux, and when the value of total flux was 25.3 g·m−2·h−1, the methanol concentration reached the maximum. When the flux was lower, CO2 and formic acid accumulated in the reaction system, resulting in a decrease of the pH value. Also, it would cause a decrease of the enzyme activity including FDH and GDH. Meanwhile, CO2 could change into another
Figure 7. Effect of operation time on methanol production: methanol concentration (A) and methanol amount (B) (value of total flux was 25.30 g· m−2·h−1). 33586
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(2) Wang, X.; Li, Z.; Shi, J.; Wu, H.; Jiang, Z.; Zhang, W.; Song, X.; Ai, Q. Bioinspired Approach to Multienzyme Cascade System Construction for Efficient Carbon Dioxide Reduction. ACS Catal. 2014, 4 (3), 962−972. (3) Muschiol, J.; Peters, C.; Oberleitner, N.; Mihovilovic, M. D.; Bornscheuer, U. T.; Rudroff, F. Cascade catalysis–strategies and challenges en route to preparative synthetic biology. Chem. Commun. (Cambridge, U. K.) 2015, 51 (27), 5798−811. (4) Schrittwieser, J. H.; Velikogne, S.; Hall, M.; Kroutil, W. Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules. Chem. Rev. 2018, 118 (1), 270−348. (5) Dalal, S.; Kapoor, M.; Gupta, M. N. Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions. J. Mol. Catal. B: Enzym. 2007, 44 (3−4), 128−132. (6) Vinu, A.; Murugesan, V.; Hartmann, M. Adsorption of Lysozyme over Mesoporous Molecular Sieves MCM-41 and SBA-15: Influence of pH and Aluminum Incorporation. J. Phys. Chem. B 2004, 108 (22), 7323−7330. (7) Lian, X.; Chen, Y.-P.; Liu, T.-F.; Zhou, H.-C. Coupling two enzymes into a tandem nanoreactor utilizing a hierarchically structured MOF. Chem. Sci. 2016, 7 (12), 6969−6973. (8) Chen, S.; Wen, L.; Svec, F.; Tan, T.; Lv, Y. Magnetic metal− organic frameworks as scaffolds for spatial co-location and positional assembly of multi-enzyme systems enabling enhanced cascade biocatalysis. RSC Adv. 2017, 7 (34), 21205−21213. (9) Garcia, J.; Zhang, Y.; Taylor, H.; Cespedes, O.; Webb, M. E.; Zhou, D. Multilayer enzyme-coupled magnetic nanoparticles as efficient, reusable biocatalysts and biosensors. Nanoscale 2011, 3 (9), 3721−3730. (10) Li, Z.; Zhang, Y.; Su, Y.; Ouyang, P.; Ge, J.; Liu, Z. Spatial colocalization of multi-enzymes by inorganic nanocrystal-protein complexes. Chem. Commun. (Cambridge, U. K.) 2014, 50 (83), 12465−12468. (11) Obert, R.; Dave, B. C. Enzymatic Conversion of Carbon Dioxide to Methanol: Enhanced Methanol Production in Silica Sol− Gel Matrices. J. Am. Chem. Soc. 1999, 121 (51), 12192−12193. (12) Schall, C. A.; Wiencek, J. M. Stability of nicotinamide adenine dinucleotide immobilized to cyanogen bromide activated agarose. Biotechnol. Bioeng. 1997, 53 (1), 41−48. (13) El-Zahab, B.; Donnelly, D.; Wang, P. Particle-tethered NADH for production of methanol from CO(2) catalyzed by coimmobilized enzymes. Biotechnol. Bioeng. 2008, 99 (3), 508−514. (14) Wang, X.; Li, Z.; Shi, J.; Wu, H.; Jiang, Z.; Zhang, W.; Song, X.; Ai, Q. Bioinspired Approach to Multienzyme Cascade System Construction for Efficient Carbon Dioxide Reduction. ACS Catal. 2014, 4 (3), 962−972. (15) O’Keeffe, M. Design of MOFs and intellectual content in reticular chemistry: a personal view. Chem. Soc. Rev. 2009, 38 (5), 1215−1217. (16) Tranchemontagne, D. J.; Mendoza-Cortes, J. L.; O’Keeffe, M.; Yaghi, O. M. Secondary building units, nets and bonding in the chemistry of metal-organic frameworks. Chem. Soc. Rev. 2009, 38 (5), 1257−1283. (17) Perry, J. J. t.; Perman, J. A.; Zaworotko, M. J. Design and synthesis of metal-organic frameworks using metal-organic polyhedra as supermolecular building blocks. Chem. Soc. Rev. 2009, 38 (5), 1400−1417. (18) Wang, X.; Makal, T. A.; Zhou, H.-C. Protein Immobilization in Metal−Organic Frameworks by Covalent Binding. Aust. J. Chem. 2014, 67 (11), 1629−1631. (19) Gkaniatsou, E.; Sicard, C.; Ricoux, R.; Mahy, J.-P.; Steunou, N.; Serre, C. Metal−organic frameworks: a novel host platform for enzymatic catalysis and detection. Mater. Horiz. 2017, 4 (1), 55−63. (20) Li, P.; Chen, Q.; Wang, T. C.; Vermeulen, N. A.; Mehdi, B. L.; Dohnalkova, A.; Browning, N. D.; Shen, D.; Anderson, R.; GómezGualdrón, D. A.; Cetin, F. M.; Jagiello, J.; Asiri, A. M.; Stoddart, J. F.; Farha, O. K. Hierarchically Engineered Mesoporous Metal-Organic Frameworks toward Cell-free Immobilized Enzyme Systems. Chem. 2018, 4 (5), 1022−1034.
three enzymes employ the same cofactor, NADH, to supply the reducing equivalents required for reaction. The reaction efficiency was improved by reducing mass transfer resistance and time. From Figure 7B it was obvious that with the prolongation of the reaction time, the methanol amount increased but the growth rate gradually decreased and finally reached equilibrium. This was because the rates of methanol amount produced slow down.
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CONCLUSIONS In organisms a variety of enzymes are assembled in a certain order to form a multienzyme cascade system (MECS), which has a very high catalytic activity. How to efficiently mimic the MECS should be one of the most significant research directions in the field of biocatalytic materials. In this work, enzymes and NADH were embedded into ZIF-8. Then the nanocomposites were orderly placed in the membrane by dead-end filtration according to the MECS sequence of the CO2 reduction. Using a pervaporation membrane the product methanol was timely separated from the alcohol/water mixture, which could effectively control the reaction direction and obviously enhance the catalysis efficiency. This method has good operability and universality. Each nanocomposite, in which enzymes are embedded into MOF, can be regarded as a microreactor, which can perform a unit operation independently. In this way, according to the order of MECS in organisms, it is easy to arrange various nanocomposites to form a complex artificial MECS.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b09811. PDMS/PVDF membrane preparation, pore size distribution and particle size distribution of nanocomposites, procedure of fouling-induced enzyme immobilization and enzymatic membrane reactor (EMR) with immobilized enzymes (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. Tel.: +86-571-8684-3691.. *E-mail: [email protected]. Tel.: +86-571-8684-3691.. ORCID
Peng Ye: 0000-0002-4182-4183 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was kindly supported by the National Natural Science Foundation of China (No. 51473148), Public Technology Research Program of Zhejiang Province (No. LGG19E030009 and No. NGF18B070005), and Science Foundation of Zhejiang Sci-Tech University (No. 15062095Y).
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REFERENCES
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ACS Applied Materials & Interfaces (21) Chen, W.-H.; Vázquez-González, M.; Zoabi, A.; Abu-Reziq, R.; Willner, I. Biocatalytic cascades driven by enzymes encapsulated in metal−organic framework nanoparticles. Nat. Catal. 2018, 1 (9), 689−695. (22) Lyu, F.; Zhang, Y.; Zare, R. N.; Ge, J.; Liu, Z. One-pot synthesis of protein-embedded metal-organic frameworks with enhanced biological activities. Nano Lett. 2014, 14 (10), 5761−5765. (23) Wu, X.; Ge, J.; Yang, C.; Hou, M.; Liu, Z. Facile synthesis of multiple enzyme-containing metal-organic frameworks in a biomolecule-friendly environment. Chem. Commun. (Cambridge, U. K.) 2015, 51 (69), 13408−13411. (24) Rafiei, S.; Tangestaninejad, S.; Horcajada, P.; Moghadam, M.; Mirkhani, V.; Mohammadpoor-Baltork, I.; Kardanpour, R.; Zadehahmadi, F. Efficient biodiesel production using a lipase@ZIF67 nanobioreactor. Chem. Eng. J. 2018, 334, 1233−1241. (25) Wu, X.; Yang, C.; Ge, J. Green synthesis of enzyme/metalorganic framework composites with high stability in protein denaturing solvents. Bioresour Bioprocess 2017, 4 (1), 24. (26) Park, K. S.; Ni, Z.; Côté, A. P.; Choi, J. Y.; Huang, R.; UribeRomo, F. J.; Chae, H. K.; O’Keeffe, M.; Yaghi, O. M. Exceptional chemical and thermal stability of zeolitic imidazolate frameworks. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (27), 10186−10191. (27) Pan, Y.; Liu, Y.; Zeng, G.; Zhao, L.; Lai, Z. Rapid synthesis of zeolitic imidazolate framework-8 (ZIF-8) nanocrystals in an aqueous system. Chem. Commun. 2011, 47 (7), 2071−2073. (28) Luo, J.; Meyer, A. S.; Mateiu, R. V.; Kalyani, D.; Pinelo, M. Functionalization of a membrane sublayer using reverse filtration of enzymes and dopamine coating. ACS Appl. Mater. Interfaces 2014, 6 (24), 22894−22904. (29) Ge, J.; Lei, J.; Zare, R. N. Protein−inorganic hybrid nanoflowers. Nat. Nanotechnol. 2012, 7, 428−432. (30) Majewski, M. B.; Howarth, A. J.; Li, P.; Wasielewski, M. R.; Hupp, J. T.; Farha, O. K. Enzyme encapsulation in metal−organic frameworks for applications in catalysis. CrystEngComm 2017, 19 (29), 4082−4091. (31) Sassolas, A.; Blum, L. J.; Leca-Bouvier, B. D. Immobilization strategies to develop enzymatic biosensors. Biotechnol. Adv. 2012, 30 (3), 489−511. (32) Jochems, P.; Satyawali, Y.; Diels, L.; Dejonghe, W. Enzyme immobilization on/in polymeric membranes: status, challenges and perspectives in biocatalytic membrane reactors (BMRs). Green Chem. 2011, 13 (7), 1609−1623. (33) Marpani, F.; Pinelo, M.; Meyer, A. S. Enzymatic conversion of CO2 to CH3 OH via reverse dehydrogenase cascade biocatalysis: Quantitative comparison of efficiencies of immobilized enzyme systems. Biochem. Eng. J. 2017, 127, 217−228. (34) Delebecque, C. J.; Lindner, A. B.; Silver, P. A.; Aldaye, F. A. Organization of Intracellular Reactions with Rationally Designed RNA Assemblies. Science (Washington, DC, U. S.) 2011, 333 (6041), 470−474. (35) You, C.; Myung, S.; Zhang, Y. H. Facilitated substrate channeling in a self-assembled trifunctional enzyme complex. Angew. Chem., Int. Ed. 2012, 51 (35), 8787−8790. (36) Baskaya, F. S.; Zhao, X.; Flickinger, M. C.; Wang, P. Thermodynamic Feasibility of Enzymatic Reduction of Carbon Dioxide to Methanol. Appl. Biochem. Biotechnol. 2010, 162 (2), 391−398. (37) Luo, J.; Meyer, A. S.; Mateiu, R. V.; Pinelo, M. Cascade catalysis in membranes with enzyme immobilization for multienzymatic conversion of CO2 to methanol. New Biotechnol. 2015, 32 (3), 319−327.
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DOI: 10.1021/acsami.9b09811 ACS Appl. Mater. Interfaces 2019, 11, 33581−33588
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Ordered Coimmobilization of a Multienzyme Cascade System with a Metal Organic Framework in a Membrane: Reduction of CO2 to Methanol Dailian Zhu,† Shanshi Ao,† Huihui Deng,† Mei Wang,† Cunqi Qin,† Juan Zhang,‡ Yanrong Jia,† Peng Ye,*,† and Huagang Ni*,†
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†
Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Education Ministry, Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, People’s Republic of China ‡ School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, People’s Republic of China S Supporting Information *
ABSTRACT: Enzymatic reduction of CO2 is of great significant, which involves an efficient multienzyme cascade system (MECS). In this work, formate dehydrogenase (FDH), glutamate dehydrogenase (GDH), and reduced pyridine nucleotide (NADH) (FDH&GDH&NADH), formaldehyde dehydrogenase (FalDH), GDH, and NADH (FalDH&GDH&NADH), and alcohol dehydrogenase (ADH), GDH, and NADH (ADH&GDH&NADH) were embedded in ZIF-8 (one kind of metal organic framework) to prepare three kinds of enzymes and coenzymes/ZIF-8 nanocomposites. Then by dead-end filtration these nanocomposites were sequentially located in a microporous membrane, which was combined with a pervaporation membrane to timely achieve the separation of product methanol. Incorporation of the pervaporation membrane was helpful to control reaction direction, and the methanol amount increased from 5.8 ± 0.5 to 6.7 ± 0.8 μmol. The reaction efficiency of an immobilized enzymes-ordered distribution in a membrane was higher than that disordered distribution in the membrane, and the methanol amount increased from 6.7 ± 0.8 to 12.6 ± 0.6 μmol. Moreover, it appeared that introduction of NADH into ZIF-8 enhanced the transformation of CO2 to methanol from 12.6 ± 0.6 to 13.4 ± 0.9 μmol. Over 50% of their original productivity was retained after 12 h of use. This method has wide applicability and can be used in other kinds of multienzyme systems. KEYWORDS: multienzyme cascade system (MECS), enzyme immobilization, metal organic framework (MOF), enzyme membrane reactor, reduction CO2 to methanol
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INTRODUCTION In nature, many biochemical reactions are catalyzed by multienzyme cascade system (MECS) that are constituted of highly ordered assemblies of enzymes. The MECS can accomplish catalysis in a highly efficient way, where the intermediates are transported between the different active sites on enzymes without leaving the MECS.1,2 Cascade reactions are undoubtedly advantageous over classical step-by-step synthesis through eliminating the tedious isolation and purification of reaction intermediates. In addition, higher yields can also be gained, and the atom economy is improved as well. Further benefits of cascade reactions include the possible handling of unstable intermediates and the control and shifting of unfavorable reaction equilibria.3,4 Drawing inspiration from these MECS in nature, researchers have devoted efforts to reconstruct these in vitro with precise design.5−7 In multienzyme coimmobilization, due to the orderliness of cascaded reactions, the accurate control of © 2019 American Chemical Society
positioning and orientation of enzymes needs to be taken into account by selecting appropriate immobilization strategies. For example, Chen et al. designed the construction of metal− organic framework as scaffold for spatial colocalization of glucose oxidase (GOx) and horseradish peroxidase (HRP).8 Garcia adopted a layer-by-layer assembly strategy using biotin−avidin interactions to achieve the desired spatial colocalization of GOx and HRP on Fe3O4 nanoparticles.9 Liu’s group reported a precipitation method for the construction of spatially colocalized multienzyme systems based on inorganic nanocrystal−protein complexes.10 However, it is still difficult to efficiently immobilize more complicated MECS and precisely control the direction of the MECS. Received: June 5, 2019 Accepted: August 16, 2019 Published: August 16, 2019 33581
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Scheme 1. Ordered Coimmobilization of Multienzyme Cascade System with Metal Organic Framework in Membrane: Reduction CO2 to Methanol Materials and Methods
(ADH&GDH&NADH) were embedded in ZIF-8 to prepare three kinds of (enzymes and coenzyme)/ZIF-8 [(enzymes&oenzyme)/ZIF-8] nanocomposites in which GDH was used to regenerate NADH. Afterward, these enzymes&coenzyme/ ZIF-8 nanocomposites were sequentially located in a microporous membrane by dead-end filtration. The membrane was connected with pervaporation membrane, as shown in Scheme 1. Finally, this enzyme membrane reactor was employed to reduce CO2 to methanol.
Simulating photosynthesis and reduction of CO2 to methanol catalyzed by MECS has been one of the hotspots of concern by researchers. Dave and co-workers first used formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH), and alcohol dehydrogenase (ADH) to reduce CO2 to methanol.11 In this system all three enzymes employ the same cofactor, the reduced form of nicotinamide adenine dinucleotide (NADH), to supply the reducing equivalents required for reaction. Cofactors (like NAD(H), NADP(H), etc.) are generally expensive, which has greatly hampered the viability of cofactor-dependent biotransformation for largescale operations. The regeneration and reuse of cofactor have to be considered for any practical applications.12−14 Metal organic frameworks (MOFs) are porous crystalline organic−inorganic hybrid materials with an architecture quite similar to zeolites but with additional flexibility. MOFs consist of metal-containing nodes and organic ligands linked through coordination bonds.15−17 The very high surface area and pore volume, the ease of pore size tuning, the facile modification on both metal nodes and ligands, and mild synthetic conditions suggest that MOFs can be potent supporting matrices for enzyme immobilization.18−21 Lyu et al. selected a chemically and thermally stable MOF “ZIF-8” as the carrier and further proposed the coordination bonding-based self-assembly of the MOF−enzyme hybrid.22 In this study, we described a strategy of mutilenzymeordered coimmobilization in which enzymes were orderly placed in a microporous membrane to simulate MECS in nature. First, FDH, GDH, and NADH (FDH&GDH&NADH), FalDH, GDH, and NADH (FalDH&GDH&NADH), and ADH, GDH, and NADH
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MATERIAL AND METHODS
Chemicals and Membranes. L-Glutamic dehydrogenase (GDH) from bovine liver, alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, formate dehydrogenase (FDH) from Candida boidinii, and formaldehyde dehydrogenase (FalDH) from Pseudomonas sp. were purchased from Sigma-Aldrich. All of the other reagents were purchased from Sigma-Aldrich and used without further purification. All of the substrate and enzyme solutions were prepared with 0.10 M PBS buffer (pH = 7.0). Commercial PVDF membranes (Haiyan New Oriental Plasticizing Technology Co., Ltd.) were used in this work. CO2 gas (≥99.5%) in a cylinder was purchased from Hangzhou Modern Industrial Gas Co., Ltd. This membrane was composed of a poly(vinylidene fluoride) (PVDF) layer and a nonwoven fabric as support layer, and its aperture was 2 μm. Synthesis of the Enzymes/ZIF-8 Nanocomposites.23 A water solution (1 mL) of FDH (5 mg/mL) and GDH (5 mg/mL) and Zn(NO3)2 water solution (0.31 M, 2 mL) were mixed with 2methylimidazole water solution (1.25 M, 20 mL) under stirring at 25 °C. The mixture then turned milky almost instantly after mixing. After stirring for about 30 min, the mixture was aged for 3 h. Then the product was collected by centrifuging at 6000 rpm for 10 min and washed with deionized water three times. The product was redispersed in deionized water for lyophilization and used for other 33582
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Figure 1. SEM images of (A) pure ZIF-8, (B) FDH&GDH/ZIF-8, (C) FalDH&GDH/ZIF-8, (D) ADH&GDH/ZIF-8, (E) FDH&GDH&NADH/ZIF-8, (F) FalDH&GDH&NADH/ZIF-8, and (G) ADH&GDH&NADH/ZIF-8. characterizations. The liquid supernatant was collected to measure the amount of enzyme immobilized by the Bradford method. Synthesis of FalDH&GDH/ZIF-8 and ADH&GDH/ZIF-8 nanocomposites was following the same protocol by replacing the multienzyme solution with FalDH water solution (5 mg/mL) and ADH water solution (5 mg/mL), respectively. Synthesis of the Enzymes&Coenzyme/ZIF-8 Nanocomposites. Synthesis of FDH&GDH&NADH/ZIF-8, FalDH&GDH&NADH/ZIF-8, and ADH&GDHNADH/ZIF-8 nanocomposites was following the same protocol by adding 5 mg of NADH. Fabrication of Enzyme−Membrane: Fouling-Induced Enzyme Immobilization. The dead-end filtrations were performed in a pervaporation cell. Descriptions of the equipment and procedures can be found in the SI (as shown in Figure S5). The PVDF membranes were placed on the membrane holder (support layer facing feed, support layer was ignored due to its very large pore size). The membranes were filtered with deionized water for 30 min. For enzyme randomly distribution in the membrane; the enzyme solution containing 4 mg of FDH&GDH/ZIF-8, 4 mg of FalDH&GDH/ ZIF-8, and 2 mg of ADH&GDH/ZIF-8 was put into the PVDF membrane with a 2 μm pore size for the enzyme immobilization operations. For enzyme-ordered distribution in membrane, the three enzyme solutions containing 4 mg of FDH&GDH/ZIF-8 (or FDH&GDH&NADH/ZIF-8), 4 mg of FalDH&GDH/ZIF-8 (or FalDH&GDH&NADH/ZIF-8), and 2 mg of ADH&GDH/ZIF-8 (or ADH&GDH&NADH/ZIF-8) were put into the PVDF membrane. All of the experiments were repeated three times until no particle could be washed out by deionized water. The filtrate was collected to conduct particle size analysis with the dynamic light scattering method (DLS) (as shown in Figure.S3). This membrane was decorated with PDMS/PVDF membrane using epoxy resin. The separation layer of the PVDF membranes was combined with the PDMS layer of the PDMS/PVDF membrane. Acting of Multienzyme Cascade Reaction in Solution (Enzymes/ZIF-8 in Solution (EMS) and Enzymes&Coenzyme/ ZIF-8 in Solution (ECMS)). A 2 mL amount of the reaction mixture containing 10 mM NADH and 4 mM L-glutamate with saturated CO2 (gaseous CO2 was bubbled into solution through a syringe needle for 30 min) was prepared in a 15 mL centrifuge tube covered with Parafilm. A 4 mg amount of FDH&GDH/ZIF-8 (or FDH&GDH&NADH/ZIF-8), 4 mg of FalDH&GDH/ZIF-8 (or FalDH&GDH&NADH/ZIF-8), and 2 mg of ADH&GDH/ZIF-8 (or
ADH&GDH&NADH/ZIF-8) were added, lasting for 6 h for sufficient production of methanol. Acting of Multienzyme Cascade Reaction in Membrane (Disordered Enzymes/ZIF-8 in Membrane (DEMM), Ordered Enzymes/ZIF-8 in Membrane (OEMM), and Ordered Enzymes&Coenzyme/ZIF-8 in Membrane (OECMM)). A 10 mL amount of the reaction mixture containing 10 mM NADH and 4 mM L-glutamate with saturated CO2 (gaseous CO2 was bubbled into solution through a syringe needle for 30 min) was added into the pervaporation cell equipped with 2 μm PVDF membrane, which could separate methanol and water. The reaction lasted 6 h for sufficient production of methanol. To determine the influence of the flux to the conversion of CO2, the pressure was adjusted to get different fluxes. The methanol concentration was determined via gas chromatography (GC) equipped with a flame ionization detector (TCD). All results were repeated three times.
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RESULTS AND DISCUSSION Synthesis of the Enzymes/ZIF-8 Nanocomposites and Enzymes&Conenzyme/ZIF-8 Nanocomposites. As shown in Figure 1, the scanning electron microscopy (SEM) images of enzymes/ZIF-8 nanocomposites and enzymes&conenzyme/ ZIF-8 nanocomposites showed similar morphologies to that of pure ZIF-8. Afterward, the size of these composites ranging from ∼350 to ∼600 nm was obviously bigger than that of the pure ZIF-8 (∼80 nm). Similar results were reported.8,23,24 It seem that the size of the enzyme/ZIF-8 composites was widely distributed due to the rapid nucleation and diverse growth of the crystals of ZIF-8.25 The X-ray diffraction (XRD) patterns of the enzymes/ZIF-8 nanocomposites and enzymes&conenzyme/ZIF-8 nanocomposites agreed well with the pattern of the pure ZIF-8 (Figure 2), which verified that incorporation of enzyme and coenzyme did not affect the crystallinity of ZIF-8.26 Thermal gravity analysis (TGA) in air also confirmed the presence of protein in the composites (Figure 3). As shown in Figure 3A, the second-stage decomposition of the composite started from 200 °C and finished around 400 °C, while the pure ZIF-8 crystals had a small amount of weight loss during 33583
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could also be attributed to decomposition of protein molecules. As shown in Figure 3B, the curves were similar to the curves in Figure 3A. Therefore, during the second stage ∼21 wt % of weight loss occurred for FDH&GDH&NADH/ZIF-8 nanocomposites, ∼21 wt % of weight loss occurred for FalDH&GDH&NADH/ZIF-8 nanocomposites, and ∼16 wt % of weight loss occurred for ADH&GDH&NADH/ZIF-8 nanocomposites, which could also be attributed to decomposition of protein molecules. Fabrication of Enzyme−Membrane: Fouling-Induced Enzyme Immobilization. Fouling-induced enzyme immobilization is an easy method to immobilize enzyme into membrane. Enzyme stability was enhanced after immobilization, but it is easy to lose enzyme and difficult to precisely control the orderliness of multienzyme coimmobilization.28 As shown in Figure 4, the SEM images demonstrated that the enzymes/ZIF-8 composites were distributed uniformly in
Figure 2. XRD patterns of pure ZIF-8, FDH&GDH/ZIF-8, FalDH&GDH/ZIF-8, ADH&GDH/ZIF-8, FDH&GDH&NADH/ ZIF-8, FalDH&GDH&NADH/ZIF-8, and ADH&GDH&NADH/ ZIF-8.
Figure 4. SEM image of FDH&GDH/ZIF-8 distributed in PVDF membrane.
the membrane pore. It was obvious that the enzymes/ZIF-8 nanocomposites packed tightly with small spacing. Because of this the exchange of intermediate products became easier in MECS, and thus, the effectiveness of enzyme catalysis was improved. The membrane used in this experiment is a kind of asymmetric membrane whose pore size of one side is larger than that of the other side. It allows the entrance of the
this temperature range.27 About 14 wt % of weight loss of the nanocomposite occurred during the second stage for FDH&GDH/ZIF-8 nanocomposites, which can be attributed to decomposition of protein molecules. Analogously, during the second stage ∼22 wt % of weight loss occurred for FalDH&GDH/ZIF-8 nanocomposites and ∼22 wt % of weight loss occurred for ADH&GDH/ZIF-8 nanocomposites, which
Figure 3. TGA curves of pure ZIF-8, FDH&GDH/ZIF-8, FalDH&GDH/ZIF-8, and ADH&GDH/ZIF-8 composites in air (A) and pure ZIF-8, FDH&GDH&NADH/ZIF-8, FalDH&GDH&NADH/ZIF-8, and ADH&GDH&NADH/ZIF-8 composites in air (B). 33584
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Figure 5. Methanol production with immobilized enzymes (value of total flux was 25.30 g·m−2·h−1, operation time is 6 h).
embedded into ZIF-8 in that the yield of methanol increased to 5.8 ± 0.5 μmol. This might be due to the closer distance between NADH and enzymes, which greatly reduces the mass transfer resistance and time, thereby improving the activity of the enzyme catalytic reaction. Biocatalytic membranes with enzymes immobilized in industrially manufactured membranes have attracted growing attention.31,32 In a continuous process, product isolation is made simpler with a membrane reactor, which in turn will drive the reaction forward, which has special relevance when the enzymes have a tendency to catalyze the reaction in the reverse way.33 Thus, it is significant to timely separate methanol from the reaction system to promote CO2 → methanol. In DEMM, as methanol was continuously separated by the pervaporation membrane, the reaction proceeded toward a positive direction. It was obvious that the amount of methanol produced by MECS was DEMM > EMS. Furthermore, the three kinds of enzymes(&coenzyme)/ZIF8 nanocomposites were orderly positioned into the membrane pore by dead-end filtration to achieve the ordered coimmobilization of the MECS. Employment of the alcohol/water separation membrane effectively decreased the concentration of methanol in the reaction solution and promoted the reaction of CO2 to methanol. Meanwhile, owing to the ordered packing of the enzymes/ZIF-8 composites in the membrane pore, the transfer routes of intermediates generated by MECS were largely shortened, and thus, the catalytic efficiency of MECS was improved. In fact, multienzymatic pathways in living systems are often segregated into microcompartments or organized as clusters.34 This spatial ordering leads to more stable structures and facilitates substrate channeling between enzymes, thus resulting in increased yields from reactions.35 In OEMM, enzymes/ZIF-8 composites were orderly immobilized according to the MECS sequence of the CO2 reduction and the
enzymes/ZIF-8 composites from the membrane side with a large pore size and at the same time avoids loss of the enzymes/ZIF-8 nanocomposites from the other membrane side with a small pore size (about 2 μm). From Figure 4 it could also be found that the membrane pore was irregular and the enzymes/ZIF-8 composites were polyhedral particles. Thus, when the aqueous solution containing the enzymes/ ZIF-8 nanocomposites was filtered by the membrane, the enzymes/ZIF-8 composites would accumulate in the membrane pore. After filtration the filtrate was collected and dried in an oven, and no solid was found in the filtrate, which indicated that the enzymes/MOF nanocomposites were in the membrane. Catalysis Activity of Enzyme−Membrane Reactor. MOFs, which are an emerging class of porous material with tunable pore size, have shown great promise in the preparation of immobilized enzymes.22,29 In addition, enzymes/MOF composites can be effective at preserving enzyme activity while enforcing a greater degree of stability under catalytically relevant but distinctly abiotic conditions.30 In this work, FDH&GDH(&NADH), FalDH&GDH(&NADH), and ADH&GDH(&NADH) were embedded in ZIF-8 to prepare three kinds of enzymes(&coenzyme)/ZIF-8 nanocomposites in which GDH was used to regenerate NADH. As shown in Figure 5 it was obviously found that the amount of methanol produced by MECS was in the order OECMM > OEMM > DEMM > ECMS > EMS. As shown in Figure S2, the average pore size of these enzymes/ZIF-8 nanocomposites was about 2.2−2.4 nm; NADH (molecular weight is 663 D) could diffuse into these nanocomposites. Also, the molecular weight of four enzymes is more than 40 kDa, which would minimize the probability of enzymes’ elution. In EMS these enzymes/ZIF-8 nanocomposites formed a multienzyme cascade system, which catalyzed CO 2 to methanol, 5.0 ± 0.4 μmol. In ECMS, NADH was also 33585
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form of existence such as carbonic acid, carbonate, and bicarbonate in acidic solution. However, the substrate for the reverse reaction is only CO2(aq).36 Thus, the variation of CO2 concentration would affect the reaction. When the flux became higher, the intermediates were separated into permeate and the production of methanol was delayed by a low accumulation of the intermediates. During the multienzymatic conversion of CO2 to methanol, although the first reaction happened very slowly, the second reaction catalyzed by FaldDH is the real bottleneck. Production of formaldehyde was delayed by a slow accumulation of formic acid from the first reaction.37 Consequently, the amount of methanol reached the maximum as the flux reached in a certain value. With the increase of the flux, the change of the methanol amount was analogous to the change of the methanol concentration. In Figure 7, for OEMM, the methanol concentration first increased and then decreased with the increase of the operation time, and when the operation time was 6 h, the methanol concentration reached the maximum. Also, for OECMM, when the operation time was 4 h, the methanol concentration reached the maximum. It is a common feature in MECS that the reaction maximum appears at a period rather than to start. Also, this phenomenon was noticed in the effect of total flux, which showed that the mass transfer resistance could not be ignored. At the beginning of the reaction CO2 and the intermediate diffused to the enzyme nearby slowly due to blocking of the carrier (in this work, it was ZIF-8). Also, the accumulation rate of intermediate (formic acid and formaldehyde) was a little slow. Hence, the methanol concentration was low in the initial period. As the reaction proceeded, the intermediate reached a certain amount, so that the MECS reaction rate increased.37 However, after a long time using the enzyme activity decreased, and thus, the reaction rate became slow. On the other hand, the immobilized enzymes still retain about 50% of activity after 12 h, which indicated that the enzymes had better stability. In addition, when NADH and enzymes were embedded into ZIF-8, the amount of methanol was improved and the time decreased to obtain maximum total methanol. In this MECS all
pervaporation membrane was employed to timely separate methanol. Both the transfer route of intermediate and the reaction direction were more accurately controlled. Thus, in OEMM, the catalysis activity of CO2 → methanol was significantly enhanced. Besides, introducing NADH into ZIF-8 would also increase the catalysis efficiency of MECS in a membrane. It is similar to the result of ECMS; the catalysis efficiency was enhanced after both enzyme, and NADH were embedded in ZIF-8. Effect of Operation Parameters on the Activity of MECS. As shown in Figure 6, the amount of methanol first
Figure 6. Effect of total flux on methanol production by ordered enzymes/ZIF-8 in membrane (OEMM) (operation time is 6 h).
increased and then decreased with increasing flux, and when the value of total flux was 25.3 g·m−2·h−1, the methanol concentration reached the maximum. When the flux was lower, CO2 and formic acid accumulated in the reaction system, resulting in a decrease of the pH value. Also, it would cause a decrease of the enzyme activity including FDH and GDH. Meanwhile, CO2 could change into another
Figure 7. Effect of operation time on methanol production: methanol concentration (A) and methanol amount (B) (value of total flux was 25.30 g· m−2·h−1). 33586
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Research Article
ACS Applied Materials & Interfaces
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three enzymes employ the same cofactor, NADH, to supply the reducing equivalents required for reaction. The reaction efficiency was improved by reducing mass transfer resistance and time. From Figure 7B it was obvious that with the prolongation of the reaction time, the methanol amount increased but the growth rate gradually decreased and finally reached equilibrium. This was because the rates of methanol amount produced slow down.
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CONCLUSIONS In organisms a variety of enzymes are assembled in a certain order to form a multienzyme cascade system (MECS), which has a very high catalytic activity. How to efficiently mimic the MECS should be one of the most significant research directions in the field of biocatalytic materials. In this work, enzymes and NADH were embedded into ZIF-8. Then the nanocomposites were orderly placed in the membrane by dead-end filtration according to the MECS sequence of the CO2 reduction. Using a pervaporation membrane the product methanol was timely separated from the alcohol/water mixture, which could effectively control the reaction direction and obviously enhance the catalysis efficiency. This method has good operability and universality. Each nanocomposite, in which enzymes are embedded into MOF, can be regarded as a microreactor, which can perform a unit operation independently. In this way, according to the order of MECS in organisms, it is easy to arrange various nanocomposites to form a complex artificial MECS.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b09811. PDMS/PVDF membrane preparation, pore size distribution and particle size distribution of nanocomposites, procedure of fouling-induced enzyme immobilization and enzymatic membrane reactor (EMR) with immobilized enzymes (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. Tel.: +86-571-8684-3691.. *E-mail: [email protected]. Tel.: +86-571-8684-3691.. ORCID
Peng Ye: 0000-0002-4182-4183 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was kindly supported by the National Natural Science Foundation of China (No. 51473148), Public Technology Research Program of Zhejiang Province (No. LGG19E030009 and No. NGF18B070005), and Science Foundation of Zhejiang Sci-Tech University (No. 15062095Y).
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DOI: 10.1021/acsami.9b09811 ACS Appl. Mater. Interfaces 2019, 11, 33581−33588