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Biological and Medical Applications of Materials and Interfaces

Ordered Co-immobilization of Multienzyme Cascade System with Metal Organic Framework in Membrane: Reduction CO2 to Methanol Dailian Zhu, Shanshi Ao, Huihui Deng, Mei Wang, Cunqi Qin, Juan Zhang, Yanrong Jia, peng ye, and Hua-Gang Ni ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09811 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 18, 2019

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ACS Applied Materials & Interfaces

Ordered Co-immobilization of Multienzyme Cascade System with Metal Organic Framework in Membrane: Reduction CO2 to Methanol Dailian Zhu a, Shanshi Ao a, Huihui Deng a, Mei Wang a, Cunqi Qin a, Juan Zhang b, Yanrong Jia a, Peng Ye a,*, Huagang Ni a,* a

Key Laboratory of Advanced Textile Materials and Manufacturing Technology of

Education Ministry, Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, P.R. China b

School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004,

P.R. China Corresponding author. Email: [email protected]; [email protected] Tel.:+86-571-8684-3691. 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) & reduced pyridine nucleotide (NADH), Formaldehyde

dehydrogenase

(FalDH)

&

GDH

&

NADH

and Alcohol

dehydrogenase (ADH) & GDH & NADH were respectively embedded in ZIF-8 (one kind of metal organic framework) to prepare three kinds of enzymes & coenzymes/ZIF-8

nanocomposites.

Then,

by

the

dead-end

filtration

these

nanocomposites were sequentially located in microporous membrane, which was combined with a pervaporation membrane to timely achieve the separation of product methanol. The incorporation of the pervaporation membrane was helpful to control 1

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reaction direction, and the methanol amount increased from 5.8 ± 0.5 μmol to 6.7 ± 0.8 μmol. The reaction efficiency of immobilized enzymes ordered distribution in membrane was higher than that disordered distribution in membrane, and the methanol amount increased from 6.7 ± 0.8 μmol to 12.6 ± 0.6 μmol. Moreover, it appeared that the introduction of NADH into ZIF-8 enhanced the transformation of CO2 to methanol from 12.6 ± 0.6 μmol to 13.4 ± 0.9 μmol. Over 50 % of their original productivity was retained after 12 h using. This method has wide applicability and can be used in other kinds of multi-enzyme systems. Keywords: multienzyme cascade system (MECS); enzyme immobilization; metal organic framework (MOF); enzyme membrane reactor; reduction CO2 to methanol

 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 2


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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 co-immobilization, due to the orderliness of cascaded reactions, the accurate control of 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 co-localization 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 co-localization of GOx and HRP on Fe3O4 nanoparticles.9 Liu's group reported a precipitation method for the construction of spatially co-localized multi-enzyme 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. Simulating photosynthesis and reduction of CO2 to methanol catalyzed by MECS has been one of the hotpots concerned by researchers. Dave and co-workers firstly 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) and so on）are generally expensive, which has greatly hampered the viability of cofactor-dependent biotransformation for large-scale operations. The 3



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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 mutilenzyme ordered co-immobilization, which enzymes were orderly placed in microporous membrane to simulate MECS in nature.

Firstly,

FDH&GDH&NADH,

FalDH&GDH&NADH

and

ADH&GDH&NADH were respectively embedded in ZIF-8 to prepare three kinds of enzymes&coenzyme/ZIF-8 nanocomposites, in which GDH was used to regenerate NADH.

Afterwards,

these

enzymes&coenzyme/ZIF-8

nanocomposites

were

sequentially located in microporous membrane by the dead-end filtration. The membrane was connected with pervaporation membrane, as show in scheme 1. Finally, this enzyme membrane reactor was employed to reduce CO2 to methanol.

4


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Scheme 1. Ordered co-immobilization of multienzyme cascade system with metal organic framework in Membrane: reduction CO2 to methanol materials and methods.

 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 the other reagents were purchased from Sigma-Aldrich and used without further purification. All the substrate and enzyme solutions were prepared with 0.10 M PBS buffer (pH = 7.0). Commercial PVDF membranes (Haiyan 5



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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 poly (vinylidene fluoride) (PVDF) layer and a nonwoven fabric as support layer, and its aperture was 2 μm. Synthesis of the Enzymes/ZIF-8 nanocomposites23 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 2-methylimidazole water solution (1.25 M, 20 mL) under stirring at 25℃. The mixture then turned milky almost instantly after mixing. After stirring for about 30 min, the mixture was aging for 3 h. Then, the product was collected by centrifuging at 6 000 rpm for 10 min, and washed with deionized water for three times. The product was re-dispersed in deionized water for lyophilization and used for other characterizations. Collect the liquid supernatant to measure the amount of enzyme immobilized by Bradford method. Synthesis of FalDH&GDH/ZIF-8 and ADH&GDH/ZIF-8 nanocomposites was following the same protocol by replacing the multi-enzyme 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 nanocomposite was following the same protocol by adding 5 mg NADH, respectively. Fabrication of Enzyme-membrane: fouling-induced enzyme immobilization 6


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The dead-end filtrations were performed in a pervaporation cell. The descriptions of equipment and procedure can be found in SI （ as shown in Fig. S5 ） . The PVDF membranes were placed on the membrane holder (support layer facing feed, the support layer was ignored due to its very large pore size). The membranes filtered with deionized water for 30 min. For enzyme randomly distribution in 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 were put into the PVDF membrane with 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

FalDH&GDH&NADH/ZIF-8)

and

2

of mg

FalDH&GDH/ZIF-8 of

ADH&GDH/ZIF-8

(or (or

ADH&GDH&NADH/ZIF-8) were respectively put into the PVDF membrane. All the experiments were repeated three times, until no particle could be washed out by deionized water. Collection the filtrate to conduct particle size analysis with dynamic light scattering method (DLS) (as shown in Fig.S3). And this membrane was decorated with PDMS/PVDF membrane by using epoxy resin. The separation layer of PVDF membranes was combined with the PDMS layer of PDMS/PVDF membrane. Acting of Multienzyme Cascade Reaction in solution (Enzymes/ZIF-8 in solution (EMS) and Enzymes&coenzyme/ZIF-8 in solution (ECMS)) 2 mL of 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. And added 4 7



mg

of

FDH&GDH/ZIF-8

FalDH&GDH/ZIF-8

(or

(or

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FDH&GDH&NADH/ZIF-8),

FalDH&GDH&NADH/ZIF-8)

and

4

mg

of

2

mg

of

ADH&GDH/ZIF-8 (or ADH&GDH&NADH/ZIF-8), lasting 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) ) 10 mL of 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 30min) 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. For determining the influence of the flux to the conversion of CO2, adjust pressure 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.

 Results and discussion Synthesis of the enzymes/ZIF-8 nanocomposites and enzymes&conenzyme/ZIF-8 nanocomposites As shown in Fig. 1, the scanning electron microscope (SEM) images of enzymes/ZIF-8 nanocomposites and enzymes&conenzyme/ZIF-8 nanocomposites showed similar morphologies to that of pure ZIF-8. Afterwards, the size of these 8


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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

Fig.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.

9



Fig. 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.

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 (Fig.2), which verified that the incorporation of enzyme and coenzyme did not 10


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affect the crystallinity of ZIF-8.26 The thermal gravity analysis (TGA) in air also confirmed the presence of protein in the composites (Fig. 3). As shown in Fig.3(A), the second-stage decomposition of the composite started from 200 ℃ and finished around 400 ℃, while the pure ZIF-8 crystals had a small amount of weight loss during 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 the 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 could also be attributed to the decomposition of protein molecules. As shown in Fig.3(B), the curves were similar to the curves in Fig.3(A). 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 the decomposition of protein molecules.

11



Fig. 3 TGA curves of Pure ZIF-8, FDH&GDH/ZIF-8, FalDH&GDH/ZIF-8 and ADH&GDH/ZIF-8 composites in air (A); Pure ZIF-8, FDH&GDH&NADH/ZIF-8, FalDH&GDH&NADH/ZIF-8 and ADH&GDH&NADH/ZIF-8 composites in air (B).

Fabrication of enzyme-membrane: fouling-induced enzyme immobilization Fouling-induced enzyme immobilization is an easy method to immobilize enzyme into membrane. Enzyme stability enhanced after immobilization, but it is easy to loss enzyme and difficult to precisely control the orderliness of multienzyme co-immobilization.28 As show in Fig.4, the SEM images demonstrated that the enzymes/ZIF-8 composites were distributed uniformly in the membrane pore. It was obviously that the enzymes/ZIF-8 nanocomposites packed tightly with small spacing. Because of this, the exchange of intermediate products became more ease in MECS and thus the effectiveness of enzyme catalysis got 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 12


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enzymes/ZIF-8 composites from the membrane side with large pore size, and at the same time avoids the loss of the enzymes/ZIF-8 nanocomposites from the other membrane side with small pore size (about 2 μm). From Fig. 4, it could also be found that the membrane pore was irregular and the enzymes/ZIF-8 composites were polyhedral particles. So, 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 oven, and no solid was found in the filtrate, which indicated that the enzymes/MOF nanocomposites were in the membrane.

Fig. 4 SEM image of FDH&GDH/ZIF-8 distributed in PVDF membrane

Catalysis activity of Enzyme-membrane reactor MOFs, which are an emerging class of porous material with tunable pore size, have shown their 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) 13



and ADH&GDH(&NADH) were respectively 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 Fig.5，it was obviously found that the amount of methanol produced by MECS were OECMM > OEMM > DEMM > ECMS > EMS. As shown in Fig.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. And the molecular weight of four enzymes is more than 40 kD, which would minimize the probability of enzymes’ elution. In EMS, these enzymes/ZIF-8 nanocomposites formed a multienzyme cascade system, which catalyzed CO2 to methanol 5.0 ± 0.4 μmol. In ECMS, NADH was also 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 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 reaction system to promote CO2→methanol. In DEMM, as methanol was continuously separated by the pervaporation membrane, the reaction proceeded 14


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towards a positive direction. It was obviously that the amount of methanol produced by MECS were DEMM > EMS. Furthermore, the three kinds of enzymes(&coenzyme)/ZIF-8 nanocomposites were orderly positioned into the membrane pore by dead-end filtration, to achieve the ordered co-immobilization of the MECS. The employment of the alcohol/water separation membrane effectively decreased the concentration of methanol in 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 got 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 pervaporation membrane was employed to timely separate methanol. Both the transfer route of intermediate and reaction direction got more accurately controlled. Thus, in OEMM, the catalysis activity of CO2→methanol significantly enhanced. Besides, introducing NADH into ZIF-8 would also increase the catalysis efficiency of MECS in membrane. It is similar to the result of ECMS, that the catalysis efficiency enhanced after both enzymes and NADH were embedded in ZIF-8.

15



Fig. 5 Methanol production with immobilized enzymes (the value of total flux was 25.30 g·m-2·h-1, the operation time is 6 h).

Effect of operation parameters on the activity of MECS As shown in Fig.6, the amount of methanol firstly increased and then decreased with the increasing of the 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 reaction system, result in the fall of pH value. And it would cause the decrease of enzyme activity including FDH and GDH. Meanwhile, CO2 could change into other 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 So the variation of CO2 concentration 16


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would affect the reaction. When the flux became higher, the intermediates was separated into permeate and the production of methanol was delayed by a low accumulation of the intermediates. During the multi-enzymatic conversion of CO2 to methanol, although the first reaction happened very slowly, the second reaction catalyzed by FaldDH is the real bottleneck. The production of formaldehyde was delayed by a slow accumulation of formic acid from the first reaction.37 Consequently, the amount of methanol got 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.

Fig. 6 Effect of total flux on methanol production by Ordered Enzymes/ZIF-8 in membrane 17



(OEMM) (the operation time is 6 h).

In Fig.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. And for OECMM, when the operation time was 4 h, the methanol concentration reached the maximum. It is common feature in MECS that the reaction maximum appears at a period rather than to start. And 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 enzyme nearby slowly due to the block of the carrier (in this work, it was ZIF-8). And 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 to have a certain amount so that MECS reaction rate increased.37 However, after long time using the enzyme activity decreased and thus the reaction rate became slow. On the other hand, the immobilized enzymes still retains about 50 % of activity after 12 hours which indicated that the enzymes had better stability. In addition, when NADH and enzymes were embedded into ZIF-8, the amount of methanol get improved and the time decreased to obtain maximum total methanol. In this MECS all 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.

18


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Fig. 7 Effect of operation time on methanol production: methanol concentration (A) and methanol amount (B) (the value of total flux was 25.30 g·m-2·h-1).

From Fig. 7(B), 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 that the rates of methanol amount increased slow down.

 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 embed into ZIF-8. Then, the nanocomposites was orderly placed in membrane by the dead-end filtration according to the MECS sequence of the CO2 reduction. By 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. 19



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This method has good operability and universality. Each nanocomposite, in which enzyme 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.

 Acknowledgements 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. 15062095-Y). Supporting Information. PDMS/PVDF Membrane preparation, the pore size distribution and the particle size distribution of nanocomposites, the procedure of fouling-induced enzyme immobilization and enzymatic membrane reactor (EMR) with immobilized enzymes.

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Figure Captions Fig.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. Fig. 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. Fig. 3 TGA curves of Pure ZIF-8, FDH&GDH/ZIF-8, FalDH&GDH/ZIF-8 and ADH&GDH/ZIF-8 composites in air (A); Pure ZIF-8, FDH&GDH&NADH/ZIF-8, FalDH&GDH&NADH/ZIF-8 and ADH&GDH&NADH/ZIF-8 composites in air (B). Fig. 4 SEM image of FDH&GDH/ZIF-8 distributed in PVDF membrane Fig. 5 Methanol production with immobilized enzymes (the value of total flux was 25.30 g·m-2·h-1, the operation time is 6 h). Fig. 6 Effect of total flux on methanol production by Ordered Enzymes/ZIF-8 in membrane (the operation time is 6 h). Fig. 7 Effect of operation time on methanol production (the value of total flux was 25.30 g·m-2·h-1). Scheme 1. Ordered co-immobilization of multienzyme cascade system with metal organic framework in Membrane: reduction CO2 to methanol materials and methods.

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Fig.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. 176x176mm (72 x 72 DPI)


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Fig.1(B) FDH&GDH/ZIF-8 176x176mm (72 x 72 DPI)



Fig.1(C) FalDH&GDH/ZIF-8 176x176mm (72 x 72 DPI)


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Fig.1 (D) ADH&GDH/ZIF-8, 176x176mm (72 x 72 DPI)



Fig.1 (E) FDH&GDH&NADH/ZIF-8 181x181mm (72 x 72 DPI)


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Fig.1 (F) FalDH&GDH&NADH/ZIF-8 173x173mm (72 x 72 DPI)



Fig.1 (G) ADH&GDH&NADH/ZIF-8. 169x169mm (72 x 72 DPI)


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Fig. 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. 231x291mm (300 x 300 DPI)



Fig. 3 TGA curves of Pure ZIF-8, FDH&GDH/ZIF-8, FalDH&GDH/ZIF-8 and ADH&GDH/ZIF-8 composites in air (A); Pure ZIF-8, FDH&GDH&NADH/ZIF-8, FalDH&GDH&NADH/ZIF-8 and ADH&GDH&NADH/ZIF-8 composites in air (B). 272x208mm (300 x 300 DPI)


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Fig. 3 Pure ZIF-8, FDH&GDH&NADH/ZIF-8, FalDH&GDH&NADH/ZIF-8 and ADH&GDH&NADH/ZIF-8 composites in air (B). 272x208mm (300 x 300 DPI)



Fig. 4 SEM image of FDH&GDH/ZIF-8 distributed in PVDF membrane 210x155mm (72 x 72 DPI)


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Fig. 5 Methanol production with immobilized enzymes (the value of total flux was 25.30 g·m-2·h-1, the operation time is 6 h). 272x208mm (300 x 300 DPI)



Fig. 6 Effect of total flux on methanol production by Ordered Enzymes/ZIF-8 in membrane (the operation time is 6 h). 272x208mm (300 x 300 DPI)


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Fig. 7 Effect of operation time on methanol production: methanol concentration (A) and methanol amount (B) (the value of total flux was 25.30 g·m-2·h-1). 272x208mm (300 x 300 DPI)



Fig. 7 Effect of operation time on methanol production: methanol concentration (A) and methanol amount (B) (the value of total flux was 25.30 g·m-2·h-1). 272x208mm (300 x 300 DPI)


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Scheme 1. Ordered co-immobilization of multienzyme cascade system with metal organic framework in Membrane: reduction CO2 to methanol materials and methods. 148x104mm (240 x 240 DPI)


Ordered Co-immobilization of Multienzyme ... - ACS Publications

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