RSC Advances PAPER Simultaneous CO2 reduction and NADH Cite this: RSC Adv., 2022, 12, 31142 regeneration using formate and glycerol dehydrogenase enzymes co-immobilized on modified natural zeolite† Clarissa Cocuzza,a Giuseppe Pietricola,a Ilaria Zonca,a Melodj Dosa,a Oscar Romero, c Tonia Tommasi,a Valentina Cauda, a Debora Fino,a Carminna Ottone *b and Marco Piumetti *a In this work, the co-immobilization of formate dehydrogenase (FDH) and glycerol dehydrogenase (GlyDH) enzymes is proposed to reduce CO2 into formic acid, an important chemical intermediate. The reduction of carbon dioxide is carried out by FDH to obtain formic acid, simultaneously, the GlyDH regenerated the nicotinamide cofactor in the reduced form (NADH) by the oxidation of glycerol into dihydroxyacetone. Natural zeolite was selected as immobilization support given its good properties and low cost. The natural zeolite was modified with subsequent acid-alkaline attacks to obtain a mesostructurization of the clinoptilolite. The two enzymes were co-immobilized on clinoptilolite, previously hetero-functionalized with amino and glyoxyl groups. The distribution of the enzymes was confirmed by fluorescence microscopy analysis. Furthermore, a great increase in the retained activity for the formate dehydrogenase enzyme was noted, passing from 18% to 89%, when the mesostructured clinoptilolite was used as support. The immobilization yield of formate dehydrogenase and glycerol dehydrogenase is around 100% with all the supports studied. The promising results suggest a possible development of this procedure in enzyme immobilization and biocatalysis. The biocatalysts were characterized to find the Received 3rd June 2022 Accepted 24th October 2022 optimal pH and temperature. Furthermore, a thermal stability test at 50 °C was carried out on both enzymes, in free and immobilized forms. Finally, it was shown that the biocatalyst is effective in reducing DOI: 10.1039/d2ra03459j CO2, both by using the cofactor in the reduced form (NADH) or the oxidized form (NAD+), obtaining rsc.li/rsc-advances NADH through the regeneration with glycerol in this latter case. considered a reagent to produce organic compounds such as 1 Introduction urea and salicylic acid.3 As a result, the catalytic reduction of In the last two centuries, research in catalysis was focused on CO2 into high-value-added products or chemical intermediates pursuing greener and more sustainable technologies for the to decrease its high concentration in the atmosphere has been abatement of atmospheric contaminants and the formation of proposed in recent studies.1,4 high-value-added molecules from waste.1,2 Carbon dioxide Mimicking the natural photosynthetic mechanism is a viable (CO2) is the predominant greenhouse gas generated both from route, it is considered an alternative to the more energy- anthropogenic and natural sources, and its emissions are real- consuming classical heterogeneous catalysis, alongside istically impossible to nullify. On the other hand, CO2 is exploiting biological systems, namely microbial metabolic involved in several industrial processes, and it could be pathways and enzyme-mediated processes.3,4 Using cellular systems such as bacteria or fungi lead to cheaper processes, decreasing both the operative and the downstream costs. a Department of Applied Science and Technology, Politecnico di Torino, Corso Duca However, they require more stringent operative conditions degli Abruzzi 24, I-10129 Turin, Italy. E-mail: marco.piumetti@polito.it; Tel: +39 regarding temperature, pH, aeration, and nutrient supply.2 On 011 0904753 b the other hand, the production of enzymes is generally more Escuela de Ingenierı́a Bioquı́mica, Ponticia Universidad Católica de Valparaı́so, Av. Brasil 2085, Valparaı́so, Chile. E-mail: carminna.ottone@pucv.cl; Tel: +56 32 2372018 expensive, but since they are single proteins catalyzing single c Bioprocess Engineering and Applied Biocatalysis Group, Departament of Chemical, reactions, they provide an incomparable selectivity to the Biological and Enviromental Engineering, Universitat Autònoma de Barcelona, desired product, making the purication process easier and 08193, Spain cheaper. Over the years, various enzymes, mainly in the classes † Electronic supplementary information (ESI) available. See DOI: of oxidoreductases and lyases, have been investigated to yield https://doi.org/10.1039/d2ra03459j 31142 | RSC Adv., 2022, 12, 31142–31155 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances carbon-based chemicals with higher value, including methane, zeolites, present good features and are very suitable for enzyme methanol, and carbohydrates.5 immobilization, although in some cases it is necessary to Obert and Kuwabata developed the rst enzymatic cascade change the zeolites' morphology due to their reduced pore reactions to reduce CO2 into methanol.6,7 The process proposed size.15–17 Natural zeolites such as clinoptilolite can be used as by Obert seems to be very promising and entails three dehy- a valid alternative to synthetic ones due to their low cost. drogenases to work as an assembly line, with the consumption Furthermore, they have the advantage of being materials that of three moles of reduced nicotinamide adenine dinucleotide are widespread in different parts of the world.18,19 Their porous cofactor (NADH) for a mole of methanol produced.6 The structure can be improved to satisfy the working parameters of mechanism mainly consists of the reverse oxidation of meth- a specic process, for example, through a mesostructuring anol: the rst enzyme, formate dehydrogenase (FDH, EC procedure.20 In this way, it is possible to minimize the mass 1.2.1.2), carries out the reduction of carbon dioxide into formic transfer phenomena, caused by too small porosities, and acid; then a second oxidoreductase, formaldehyde dehydroge- increase enzymatic stability. In particular, zeolites are well- nase (FaldDH, EC 1.2.1.46), converts formate into the corre- suited for the covalent immobilization technique, which sponding aldehyde; eventually, alcohol dehydrogenase (ADH, ensures the proper holding of the protein, minimizing the risk EC 1.1.1.1) reduces formaldehyde into methanol.3,6 To obtain of leakage.21 Zeolites can be hetero-functionalized, with glyoxyl better performance, previous studies in the literature suggest and amino groups, to perform a two-step covalent immobili- using carbonic anhydrase to increase the CO2 solubilization in zation strategy: the rst step at neutral pH enhances the the reaction medium, before performing the cascade reactions immobilization yield by ionic interactions without damaging previously described.8 The rst step of this cascade reaction is the activity of the enzyme; the second step at basic pH allows the studied in this paper, using the enzyme FDH co-immobilized multipoint covalent interactions.22 Since the immobilization of with glycerol dehydrogenase (GlyDH, EC 1.1.1.6). The second enzymes over solid supports involves strong bonds between the enzyme is necessary to perform a cofactor regeneration during support and specic amino acids, this strategy has to be care- the CO2 reduction reaction. fully optimized to avoid changes in the conformation of the In fact, in addition to the problems derived from the active site of the protein.23 The multipoint covalent strategy, expensive and burdensome production of the enzymes, and the despite increasing the rigidity of the immobilized enzymes, stable maintenance of their three-dimensional structure, the makes them more stable in the presence of distorting high cost of the cofactor is another critical issue to scale up this reagents.24,25 process to the industrial level.5,9 As far as the regeneration of In this work, the Candida boidinii FDH enzyme is used along cofactor NADH is concerned, it requires the delivery of electrons with the geobacillus stearothermophilus GlyDH enzyme to to the oxidized form of the molecule. Different approaches have investigate the reduction of CO2 to formic acid, the rst step of been designed and studied, such as electrochemical, photo- the cascade reactions proposed by Obert and Kuwabata.6,7 For chemical, and biochemical processes.10–12 The exploitation of the rst time to our knowledge, both the GlyDH and FDH solar energy or electricity from renewable sources may be enzymes were co-immobilized covalently on natural zeolite to a promising solution for greener applications. However, by perform the CO2 reduction and simultaneously the regenera- using enzymes good efficiency and selectivity have been ob- tion of the NADH cofactor. The clinoptilolite was modied with tained. Furthermore, it is not necessary to use expensive subsequent acid and alkaline attacks to alter its morphology mediators, such as molecules based on noble metals.5 By using making it more suitable for enzyme immobilization.26 Two the glycerol dehydrogenase enzyme, it is possible to regenerate different processes of mesostructuring were tested. The rst one the NADH cofactor13 and at the same time, the organic waste involved an ion exchange of the original cations of the frame- from the biodiesel industry can be valorized. This enzyme work before the mesostructuring process. Instead, the second performs the oxidation of glycerol to dihydroxyacetone using used the natural zeolite with the native cations originally one mole of the oxidized cofactor, returning the reduced form occupying the cavities of the framework. Subsequently, the NADH. This reaction has several advantages: the oxidized form zeolites were hetero-functionalized, and nally, FDH and of the cofactor (NAD+) is regenerated to NADH and dihydroxy- GlyDH were co-immobilized on the support. acetone (DHA) is obtained as a co-product, DHA has a market The co-immobilization of the two enzymes on the support value of about twenty-ve times the value of glycerol.14 Finally, decreases the delay of the regeneration of the cofactor in its there is the possibility to valorize a by-product of the biodiesel reduced form (NADH) compared to a similar process with FDH industry, which can potentially be almost cost-free. and GlyDH immobilized on separate particles, increasing the Furthermore, the immobilization of enzymes will allow the efficiency of the CO2 reduction. With FDH and GlyDH separately re-using of them, lowering their cost of application. The supported, the cofactor would be diluted in a medium and immobilization process is a critical point because the environ- would need more time to migrate from one enzyme to the other. ment in which the reactions occur signicantly affects the On the other hand, the co-immobilization strategy also has global performance of the enzyme: the connement into some drawbacks, for example, ones related to the different materials with specic morphologies and the co-presence of stability conditions of distinct enzymes. This is another reason other enzymes in the surroundings are two conditions that can why it is important to nd operative parameters suitable for modify the behavior of the enzyme.15 Among all the possible both enzymes.27 Another important aspect is the distribution of materials, inorganic porous supports, such as silica and the FDH and GlyDH on the surface of the support; this can be © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31142–31155 | 31143 RSC Advances Paper investigated with uorescence microscopy. The biocatalyst then the hydrolysis with sulphuric acid, with the opening of the (enzyme immobilized on support) was also characterized ring structure to form diols, and nally the oxidation of the through the retained activity (Ract) and immobilization yield latter with sodium periodate to yield glyoxyl groups. For each (IY). In addition, the optimum pH and temperature were eval- sample, 1 g of zeolite was put into a balloon with GPTMS and uated as well as the stability factor obtained aer the immobi- APTES, both 5% v/v, in 30 mL of toluene at 105 °C for 5 h. To lization. Finally, the biocatalyst was tested to carry out the CO2 avoid the evaporation of the solvent, the glass round-bottom reduction reaction through FDH. The CO2 reduction was per- ask was connected to a water-cooling column. Then, the formed using the oxidized form of the nicotinamide cofactor solid was ltered and thoroughly washed with acetone and (NAD+) and GlyDH enzyme to regenerate the cofactor, thus water. Hydrolysis of epoxy groups was carried out by putting the conrming the regeneration process of NADH. powder in contact with 30 mL of sulphuric acid in a concentra- tion of 0.1 M at 85 °C for 2 h. Again, it followed a step of ltration and deep washing with abundant distilled water. The 2 Materials and methods last stage of functionalization was the oxidation of diol groups 2.1 Materials by 30 mL of sodium periodate aqueous solution 0.1 M at room (3-Glycidyloxypropyl)trimethoxysilane (GPTMS, $98%), (3-ami- temperature for 2 h more, followed by one last ltration and nopropyl)triethoxysilane (APTES, $98%), sulfuric acid ($98%), washing with distilled water and phosphate buffer 25 mM pH 7 sodium borohydride ($98%), sodium hydroxide (97%), toluene to remove periodate ions from the surface. The quantication of (99.8%) and hydrochloric acid (37%) were supplied from Sigma- glyoxyl groups on the surface of the materials was performed by Aldrich. Sodium formate, sodium metaperiodate, and glycerol a back-titration method, as reported by Guisan.32 The moles (86%) were purchased from Merck. Formate dehydrogenase were calculated by exploiting eqn (1)   (FDH) from Candida boidinii with a protein concentration of 25 molgly 1 Absfinal ¼ VIO4  ½IO4  in 1   1000 (1)  1 mg mL−1 (according to Bradford assay28) was purchased msup msup Absinit from Megazyme. NAD+ (99.6%) was supplied from PanReach AppliChem. Base Clinoptilolite (B-Clino) was supplied by Zeo- where the amount of glyoxyl groups are expressed in mmol, msup lado (Greece). Glycerol dehydrogenase (GlyDH) from Geobacillus the mass of support subjected to functionalization (g), VIO4− is stearothermophilus was produced and puried as previously the volume of the NaIO4 solution (mL), [IO4−] is the concen- reported.29 tration of IO4− ions in the latter (mmol mL−1), and Absinit and Absnal are the values of absorbance measured, for the super- natant at the beginning and at the end of the oxidation, at 2.2 Methods 420 nm. The functionalization resulted to be efficient for all 2.2.1 Preparation of mesostructured zeolites. The two supports. All zeolites have a functionalization yield of around samples of mesostructured clinoptilolite (M-Clino and M-Na- 300 mmol g−1 (in detail: B-Clino 273 mmol g−1; M-Clino 332 mmol Clino) were prepared by adapting the method reported by g−1; M-Na-Clino 304 mmol g−1). Moradi et al.30 Clinoptilolite with a Si/Al ratio in the range of 4.0 2.2.3 Activity assay. 50 mL of FDH solution (0.05 mg mL−1 to 5.3 was rst washed with ultrapure water (Milli-Q) at room according to Bradford assay28) or 5 mg of support (with FDH temperature for 10 minutes. Next, a dealumination process was immobilized) was added to a solution of 2.3 mL of 0.1 M performed with HCl 1 M at 100 °C for 4 h. This treatment was phosphate buffer pH 7.5, 100 mL of cofactor NAD+ 50 mM and repeated 4 times to remove the aluminum in excess, which is 500 mL of sodium formate 0.3 M. Similarly, 100 mL of GlyDH reported to be detrimental to the mesostructuring process. Aer solution (0.05 mg mL−1 according to Bradford assay28) or 5 mg the dealumination process, an alkaline desilication was carried of support (with GlyDH immobilized) was added to 1.850 mL of out with NaOH 0.2 M at 100 °C for 30 minutes, followed by glycerol solution 100 mM (in 0.1 M phosphate buffer pH 7) and a milder acid wash with HCl 0.5 M at the same temperature for 50 mL of NAD+ 100 mM. The increase in absorbance at 340 nm, 4 h, to remove the aluminum on the surface. Aer each deal- corresponding to the peak of formation of NADH, was recorded umination and desilication treatment, the zeolites were cen- for two minutes and correlated to the specic activity of the free trifugated, washed, and dried overnight at 60 °C. The sample of enzyme (AFE, in UI mgprot−1) and immobilized (AIE, in UI gsup−1). mesostructured sodium-clinoptilolite (M-Na-Clino) was ob- The activity is expressed in IU (international unit) and corre- tained by replacing the washing with Milli-Q water with an ion sponds to the amount of enzyme necessary to produce one mmol exchange with NaCl 2 M at 50 °C for 3 h, to have sodium ions on per minute of NADH at the dened conditions of pH and the framework of the clinoptilolite instead of the native cations temperature. of the framework. The other steps are the same described above. 2.2.4 Immobilization and co-immobilization of FDH and 2.2.2 Supports functionalization. All the zeolites (B-Clino, GlyDH on supports. The immobilization was carried out in two M-Clino, and M-Na-Clino) were hetero-functionalized with different ways. In the rst case, the two enzymes were immo- glyoxyl and amino groups, using respectively GPTMS and bilized separately, while in the second case a co-immobilization APTES, by adapting the method reported in the literature from was carried out by immobilizing the two enzymes at the same Bernal et al.,31 Pietricola et al.16 and Cesar et al.8 The formation time. In this way, 1 g of support was put in contact with 2 mg of of glyoxyl groups went through a series of three reactions: rstly, FDH or 2 mg of GlyDH. The process was adapted from Pietricola the generation of epoxy groups on the surface of the support, et al.16 and Rocha-Martin et al.29 First, FDH or GlyDH was 31144 | RSC Adv., 2022, 12, 31142–31155 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances dissolved in 20 mL of 25 mM phosphate buffer pH 7, and this 2000). Sample morphology was analyzed through FE-SEM mixture was maintained at a temperature of 4 °C in an ice bath, microscopy (Zeiss MERLIN, Gemini-II column) and HR-TEM with gentle stirring. Aer adding the support, the immobiliza- microscopy (JEM-3100F, FE-LaB6 gun). Infrared measurements tion took place, and the time of immobilization was estimated were carried out on the three functionalized supports B-Clino, by collecting samples of the supernatant and monitoring the M-Clino, and M-Na-Clino. IR spectra were acquired on Bruker activity of the enzyme still in solution. At the same time, enzyme INVENIO equipped with liquid nitrogen cooled MCT detector. activity under the same conditions, but not in contact with the The Fourier transform IR (FT-IR) spectra were collected, aer support, was evaluated and used as a blank. When the activity of a pretreatment of 1 hour at 300 °C in standard vacuum condi- the supernatant in the immobilization solution dropped to zero tions, at room temperature, and atmospheric pressure in the or remained constant for two consecutive measurements, the range of 4000–400 cm−1 with 64 scans and a resolution of 4 mixture was ltered under vacuum in a Buchner funnel. cm−1. The analyses were performed on pellets, obtained by Subsequently, the solid was resuspended in 20 mL of 100 mM pressing the sample powder at 150 bar, and the intensity was carbonate buffer pH 10 for 30 minutes. Aerward, 10 mg of normalized by dividing it by the surface density of the pellet. sodium borohydride was added to the mixture with 5 mL of 2.2.6 Temperature and pH prole of biocatalyst. The glycerol, to reduce the Schiff's bases formed between the glyoxyl temperature and pH proles of the biocatalysts were evaluated groups of the functionalized support and the amino groups of by calculating the activity, as expressed in Section 2.2.5, varying the enzyme. Aer 10 minutes, the solid was ltered under only the pH or temperature. Specically, to evaluate the pH vacuum, washed with abundant water and 25 mM phosphate prole, the temperature was kept xed at 30 °C and the pH of buffer pH 7, and dried in a desiccator at the temperature of 4 °C. the solution varied from 6 to 10. A 0.1 M phosphate buffer was Next, Bradford assay28 was carried out to determine the used for pH 6, 7, and 8 solutions while a 0.1 M carbonate buffer concentration of protein at the beginning (Cprot,t0) and at the was used for pH 9 and 10 solutions. To evaluate the temperature end (Cprot,tf) of the immobilization process. Finally, the immo- prole, activity tests at a temperature ranging from 30 to 60 °C bilization yield (IY) was evaluated and expressed in eqn (2) as: were carried out (at pH 7.5 for FDH enzyme and 7 for GlyDH Cprot;t0  Cprot;tf enzyme). IY ð%Þ ¼  100 (2) 2.2.7 Thermal stability of free and immobilized enzymes. Cprot;t0 Both free and immobilized enzymes were incubated at 50 °C. To evaluate the residual activity over time, an activity test was The activity of the immobilized enzyme can be expressed in carried out as described previously, at 30 °C (at pH 7.5 for FDH the form of the retained activity (Ract) shown in eqn (3) as: enzyme and 7 for GlyDH enzyme). The residual activity (A) was AIE AIE expressed as a percentage of the initial activity (A0). To describe Ract ð%Þ ¼  100 ¼  100 (3) the deactivation of the free enzyme, a rst-order deactivation q  AFE;t0 Cact model with no residual activity was used, expressed in eqn (4) where AIE is the specic activity of the immobilized enzyme (in as: UI gsup−1), q is the enzymatic load provided in mgprot/gsup, AFE,t0 is the specic activity of the free enzyme at the beginning of the A = A0 × e−kDt (4) immobilization (in UI mgprot−1), calculated as described in Section 2.2.5. Cact represents the contact activity, namely the where kD represents the deactivation constant (h−1) and t is the maximum specic activity of the support if all the protein in time (h). Conversely, immobilized enzymes were more likely to contact was immobilized and fully active. follow an asymptotic deactivation model, which can be 2.2.5 Characterization of materials. The specic surface described by eqn (5): area (SBET) of the samples of zeolite involved in this work was evaluated by nitrogen physisorption at −196 °C, with a Micro- A = A0[(1 − a)e−kDt + a] (5) meritics TriStar II 3020 instrument and then by processing the where a is the residual activity at innite time. From eqn (4) and results with the Braunauer–Emmet–Teller method (BET). (5) it was possible to obtain the half-life (t1/2) and nally the However, for zeolites, the BET method usually provides quite stabilization factor (SF), expressed in eqn (6): underestimated values of the surface area, hence it was also calculated with the Langmuir method SLAN. Furthermore, the t1=2;imm: enz: SF ¼ (6) micropores volume (Vmicro) were evaluated by analyzing the t1=2; sol: enz: desorption stage with the Barrett–Joyner–Halenda (BJH) method. Before testing, all the samples were subjected to an 2.2.8 Co-immobilization of uorescence-labeled enzymes. outgassing pre-treatment at a high temperature (400 °C) for 2 h FDH and GlyDH were labeled with ATTO 488 and ATTO 550, to remove impurities on the surface of the support. The crys- respectively, using a protein labeling kit and separation talline structure of materials was determined with X-ray columns (from Sigma Aldrich) according to the procedure diffractometry (XRD) by using an X'Pert Philips PW3040 indicated by the manufacturer, a similar procedure is also re- diffractometer using Cu Ka radiation (2q range = 5°–40°; step = ported in the literature by Rocha-Martı́n et al.33 Each enzyme 0.05° 2q; time per step = 0.2 s). The diffraction peaks were was incubated in soluble form with the respective dye for 2 h at compared according to the Powder Data Files database (PDF © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31142–31155 | 31145 RSC Advances Paper pH 9.5 (carbonate buffer 0.1 M) and puried into a separation Table 1 Results of nitrogen physisorption tests and mesostructuring column. Immobilization and co-immobilization of the labeled process enzymes were carried out in the dark, following a similar B-Clino M-Clino M-Na-Clino procedure to the non-labeled enzymes, previously described. B- Clino was chosen as a support because it did not show any SBET (m2 g−1)a 37 195 216 uorescence emittance in the considered wavelength range, SLang (m2 g−1)b 39 282 311 unlike the mesostructured supports. The sample was deposited Vp (cm3 g−1)c 0.14 0.22 0.25 Vmicro (cm3 g−1)d 0.005 0.064 0.066 on a glass slide and covered with a cover-glass slip, then a observed with a Nikon Eclipse Ti-e uorescence optical inverted Specic surface area obtained through the BET method. b Specic surface area obtained through the Langmuir method. c Total pore microscope, at 100× magnication. The microscope is equip- volume, calculated with the BJH method. d Volume of micropores ped with a super bright wide-spectrum source (Shutter Lambda calculated applying the t-plot method. XL), a high-resolution camera (Zyla 4.2 Plus, 4098 × 3264 pixels, Andor Technology) and an immersion oil 100× objective (Nikon). Fluorescence lters were set to an excitation centered by 427% for M-Clino and 454% for M-Na-Clino while SLang at 488 nm (obtaining a green light emission) and an excitation increased by 662% for M-Clino and 697% for M-Na-Clino. This centered at 550 nm (obtaining a red light emission). The outcome is also observable from the change of shape and collected images were analyzed with the NIS-Element soware position of the isotherms (Fig. 1). The pore size distribution of (NIS-Element AR 4.5, Nikon). A merge of brighteld, green and both mesostructured samples showed multi-modal behavior, red emission images was performed to obtain co-localization with peaks in the meso-macroscopic range between 30 and images where the two uorescence signals collected result to 200 nm (Fig. S1†). Isotherms of the nitrogen physisorption test, be superimposed. alongside pore size distribution diagrams, were reported to 2.2.9 Reduction of CO2 into formic acid. The reduction of show the entity of Slit-like porosity, typically generated by the CO2 to formic acid was conducted in a sealed glass bottle, at 30 ° aky morphology of clinoptilolite particles, which was further C under stirring, 1 atm, and pH between 6.5 and 7.5, blowing conrmed by the shape of the hysteresis. Furthermore, the CO2 in solution during all the tests. For each reaction, 500 mg of values of the volume of micropores of M-Clino and M-Na-Clino biocatalyst were suspended into 30 mL of phosphate buffer were increased due to acid/alkaline attacks, (total pore volume 100 mM pH 7.5 and, to ensure a solution constantly saturated of the modied clinoptilolites was increased too but to a lesser with carbon dioxide, 100 mL min−1 of CO2 was bubbled from extent compared to Vmicro values). the bottom, causing the pH to decrease. Firstly, only FDH was The mesostructuring process (Fig. S1†). In addition, in our supported over M-Clino, and 10 mM of NADH was dissolved previous work, it was reported that the functionalization into the initial solution, to assess the single reaction. Secondly, procedures with APTES and GPTMS do not affect the meso- FDH and GlyDH were co-immobilized over M-Clino, and the porosity of material.16 initial feed was 10 mM of NAD+ and equimolar glycerol. In this 3.1.2 XRD. The XRD patterns of the studied samples are way, the chain reaction of conversion of cofactor and reduction reported in Fig. 2. The clinoptilolite diffractogram (B-Clino) of CO2 was studied. The variation of formic acid concentration exhibits a high crystallinity pattern with its well-known peaks was observed in a Shimadzu 20A prominence device, equipped at 2q = 9.75, 22.34, and 29.98° which are indexed with the with an RDA detector at 210 nm, on a Kinetex Phenomenex C18 (4.6 × 250 mm, 10 mm) column kept at 30 °C. The mobile phase used was 5 mM sulphuric acid in Milli-Q water, the ow rate was 0.4 mL min−1, and the retention time is 3.1 minutes. 3 Results and discussion 3.1 Support characterization 3.1.1 Nitrogen physisorption at 77 K. To create a meso- porous structure inside the clinoptilolite that is more suitable for enzyme immobilization, the natural clinoptilolite was sub- jected to two different treatments. The structure of the resulting three materials, untreated clinoptilolite (B-Clino), mesoporous clinoptilolite (M-Clino), and ion exchange mesostructured cli- noptilolite (M-Na-Clino) were characterized. Table 1 summarizes the results obtained from nitrogen physisorption tests (BET surface area SBET, Langmuir surface area SLang, pore volume Vp, and micropore volume Vmicro). The specic surface area of clinoptilolite (B-Clino) is relatively low, Fig. 1 Isotherms from nitrogen physisorption tests for the base (B- this can be ascribed to the fact that it is a natural zeolite. Due to Clino) and the mesostructured (M-Clino, M-Na-Clino) clinoptilolite the mesostructuring process, the SBET of clinoptilolite increased samples. 31146 | RSC Adv., 2022, 12, 31142–31155 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances possible to observe a new additional peak at about 22°. This new peak can be ascribed to the presence of Na in the structure of M- and M-Na-Clino. The reference pdf number is ascribed to Na- clinoptilolite (reference nr. 96-900-1393). The presence of Na in M-Na-Clino is clearly explained due to the ion-exchange treat- ment to obtain this element inside the clinoptilolite structure. On the other hand, for M-Clino the presence of Na could be explained by the desilication treatment with NaOH. When the B-Clino reacts in NaOH solution, the desilication process occurs because the Si inside clinoptilolite was exchanged with Na+ from NaOH. Thus, in M-Clino, there is a small amount of Na. 3.1.3 Electron microscopy. With electron microscopy analysis, the morphology of the support at different levels of magnication was dened. Fig. 3 compares FE-SEM images of B-Clino (a, b) M-Clino (c, d), and M-Na-Clino (e, f) at 1k× and 10k× magnication. Likewise, Fig. 4 displays B-Clino, M-Clino, and M-Na-Clino subjected to HR-TEM analysis at different magnications. As previously mentioned, Fig. 3 demonstrates the aky morphology of clinoptilolite, which was substantially Fig. 2 XRD diagram comparison between B-Clino, M-Clino, and M- maintained during the acid/alkaline attacks. From a macro- Na-Clino. scopic point of view, the particles of M-Clino appear to be slightly larger, as if some sort of aggregation took place (Fig. 3c). On the contrary, M-Na-Clino particles seem to be less aggregate clinoptilolite reference code: 00-039-1383. Furthermore, the than B-Clino and M-Clino (Fig. 3e). At higher magnication, the modied M-Clino and M-Na-Clino preserve the same charac- scales of M-Clino are ner and more spaced than the ones of B- teristics peaks of B-Clino. However, it is possible to observe Clino, while M-Na-Clino akes are more similar to the ones of B- a decrease in the peak intensities in these two materials. The Clino (Fig. 3b, d, and f). In light of this, the mesopores and reason could be due to the acid/alkaline treatment used for the macropores probably coincide with these distances. HR-TEM mesostructuring process which allows new cavities and pores, images of the supports reveal that the acid/alkaline attacks but irreversibly changes the B-Clino crystal structure. It is maintained the aky morphology of clinoptilolite (Fig.4a) but modied the aspect of the akes. The M-Clino (Fig. 4b) seems to Fig. 3 FE-SEM images for B-Clino (a, b), M-Clino (c, d), and M-Na-Clino (e, f) at magnification 1k× and 10k×. © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31142–31155 | 31147 RSC Advances Paper Fig. 4 HR-TEM images of (a) B-Clino (b) M-Clino and (c) M-Na-Clino samples. have been eroded by acid/alkaline attacks; in fact, the edges The spectra conrm the correct functionalization of the look smoother, and the surface of the akes appears rougher. supports because all the samples present bands related to the These changes to akes contributed to increase the surface vibration of ammino groups and to the organosilanes (CH2) area, as conrmed by the N2 physisorption analysis. On the used for functionalizing the supports. Moreover, the supports other hand, the M-Na-Clino (Fig. 4c) exhibits sharper edges than exhibit also peaks that can be correlated to the structure of the B-Clino and M-Clino. Moreover, it presents small particles zeolite. A summary of the vibration bands is reported in Table 2. partially incorporated into the structure (one of them is high- All samples present a band in the range 3100–3750 cm−1, this lighted in the gure). These particles are different from the can correspond to the tensile vibrations of –NH, provided by the surrounding material and exhibit a more organized structure, it functionalization process, and –OH still present in the material is possible to recognize parallel rows. despite the pre-treatment, due to the high specic surface area 3.1.4 FT-IR spectroscopy. FT-IR measurements were con- of the material. The spectrum of B-Clino presents an absorption ducted on the three different supports used for the immobili- band centered at 487 cm−1, that can correspond to the vibra- zation of FDH and GlyDH. The IR spectra are reported in Fig. 5. tions of the bonding T–O (where T is Si or Al of the zeolitic structure). The absence of this peak, in the two modied supports, can be due to the acid and alkaline attacks that have slightly changed the structure of the clinoptilolite as has been highlighted by the XRD analysis. In addition, it is possible assuming that the M-Clino is the sample for which the process of functionalization has been the most effective: it has the highest ratio between the propylamine vibrational peaks, provided by APTES and GPTMS, and the stretching vibration peak O–T^–O. The FT-IR spectra of the support alone, the functionalized support, and the enzymes individually and co-immobilized, for all the materials used are reported in Fig. S2.† 3.2 Immobilization and co-immobilization of FDH and GlyDH FDH and GlyDH were individually immobilized and co- immobilized, following a two-step protocol, as previously described, on the three different zeolites. The hetero-functional supports are used to rapidly immobilize enzymes through a multipoint covalent attachment. This technique can be used to immobilize different proteins.25 The rst step conducted at Fig. 5 FT-IR spectra in absorbance of the three functionalized pH 7 enables the formation of ionic interactions between the supports: B-Clino, M-Clino, M-Na-Clino. enzymes and the amine groups provided by APTES; while the 31148 | RSC Adv., 2022, 12, 31142–31155 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances Table 2 Absorption bands for B-Clino, M-Clino, and M-Na-Clino of its units and their desorption from the support.43 M-Clino functionalized with APTES and GPTMS support allows to obtain the best Ract of co-immobilized FDH, and although the Ract of co-immobilized GlyDH is slightly lower, Vibrational features Wavenumber (cm−1) Reference it is still sufficiently high to perform the reaction. The yields of Vibration T–O 487 34 immobilization and retained activities of GlyDH are compa- Stretching O–T ^–O 795 34 and 35 rable with those obtained by Rocha-Martin and colleagues on Deformation Si–CH2 1406 36 glyoxyl agarose,29 with immobilization yields in the 70–100% Bending C–N and bending N–H 1511 37 Water associated with Na and Ca 1637 37 and 38 range and expressed activities in the 5–40% range. The specic in the zeolite channels activity of the co-immobilized FDH resulted to be approximately Symmetric vibration CH2 2879 36 2.5 times the one of GlyDH presents on the same support. For Asymmetric vibration CH2 2933 35 and 36 this reason, a further co-immobilization with the ratio of 1 : 4 by Tensile vibration –OH and –NH 3750–3100 26 and 35 weight between FDH and GlyDH was performed, to increase the specic activity of the biocatalyst. Also, the IY of this last co- immobilization resulted to be 100%. The specic activity (AIE) calculated for the co-immobilized FDH (1 : 4 ratio by weight) is second step at pH 10 promotes the creation of Schiff's bases 2.34 UI g−1 while the one of GlyDH is 1.35 UI g−1. Using a 1 : 4 between Lysine residues and the glyoxyl groups introduced in loading ratio of the two enzymes, the specic activity gap is the support. The last step of the immobilization involves the use reduced, in this case, the AIE of FDH resulted to be approxi- of sodium borohydride to reduce the Schiff's bases and nally mately 1.7 times the one of GlyDH. obtain covalent bonds. This technique enables the use of hydrophobic support and high ionic strength solutions to be 3.3 Characterization of biocatalysts avoided. For example, with epoxy hydrophobic support 1 M buffer phosphate solutions are usually used.25 The immobili- 3.3.1 Distribution of the enzymes. To understand the zation yields (IY) are 100% for all the immobilizations per- inuence of the structural changes on the zeolite, a study of the formed. The other immobilization metrics (retained activity, distribution of the enzymes was carried out. For that purpose, Ract; and specic activity, AIE) of GlyDH and FDH separately the enzymes were labeled with uorescence dyes, FDH and immobilized and co-immobilized on the supports are reported GlyDH with ATTO 488 dye and ATTO 550, respectively. The in Table 3. Both enzymes immobilized on the mesostructured images collected with the uorescence microscope depict materials displayed an increased retained activity if compared a grain of the clinoptilolite in brighteld (Fig. 6a). to the ones on B-Clino. The retained activities obtained FDH is reported in the green emission channel (Fig. 6c), and immobilizing FDH and GlyDH on the mesostructured zeolites GlyDH, thus is expected to visualize in the red emission channel are very similar: the Ract of GlyDH separately immobilized is (Fig. 6d). Besides, unloaded B-Clino was observed with the same better on M-Clino, Ract of FDH co-immobilized with GlyDH is parameters to check that the support itself has no uorescence slightly higher using M-Clino as support, on the contrary, the emission, as reported in the ESI (Fig. S3†). On the contrary, Ract of co-immobilized GlyDH is a bit lower on M-Clino. The mesostructured supports (M-Clino and M-Na-Clino) themselves retained activity obtained on the three supports is comparable have uorescence emission preventing the observation of the with other works where the expressed activity ranged from 5% enzyme, probably due to the reagent used for the meso- to 90% with FDH from Candida boidinii immobilized on glyoxyl structuring and functionalization process. For this reason, the agarose.39 In our previous works, FDH on glyoxyl silica showed images of FDH and GlyDH immobilized on M-Clino and M-Na- a retained activity of 10–14%, while with FDH on glyoxyl or Clino are not reported. From the merged image (Fig. 6b), the amino natural zeolite a retained activity from 4 to 40% was two enzymes appear not to be equally distributed over the obtained.15,16 Approximating the shape of the two enzymes with zeolite particle. In particular, the signal related to ATTO 488 a sphere,40 a diameter of 5.54 nm and 8.99 nm is obtained, (green), corresponding to FDH, seems to be more uniformly starting with molecular weights of 74 kDa,41 and 31.6 kDa,42 spread, while the one by ATTO 550 (red), labeled GlyDH, is more respectively for FDH and GlyDH. Moreover, GlyDH is a multi- likely conned on the edges. This can be attributed to the meric enzyme composed of 8 subunits,42 therefore the loss of different sizes of the two enzymes, which causes GlyDH to activity during immobilization is likely due to the dissociation remain at the edges of the particle. This heterogeneous distri- bution opens opportunities for the development of positional Table 3 Retained activity (Ract) and specific activity (AIE) of biocatalysts FDH FDH co-imm. GlyDH GlyDH co-imm. AIE (IU g−1) Ract (%) AIE (IU g−1) Ract (%) AIE (IU g−1) Ract (%) AIE (IU g−1) Ract (%) B-Clino 0.64 18 0.05 3 0.29 16 0.09 9 M-Na-Clino 3.22 89 1.45 80 0.40 22 0.77 83 M-Clino 3.17 88 1.68 93 0.65 35 0.69 74 © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31142–31155 | 31149 RSC Advances Paper 3.3.2 Effect of temperature and pH on biocatalysts activity. To determine the optimal pH and temperature of the two enzymes in different operational conditions, the enzymatic activity was measured by changing the pH or temperature concerning the conditions described in Section 2.2.5. For the sake of brevity, the tests were performed to have a comparison between the free enzymes, the enzymes separately immobilized (FDH/MClino or GlyDH/MClino), and co-immobilized (FDHco- imm/MClino or GlyDH co-imm/MClino) on mesostructured clinoptilolite (M-Clino), the material used as support. The results for the optimal temperature are reported in Fig. 7, while the corresponding values of optimal pH are in Fig. 8. Both free FDH and GlyDH showed their maximum activity at 50 °C. FDH immobilized over M-Clino was most active at 60 °C, with a shi of 10 °C in optimal temperature. Regarding the GlyDH enzyme on M-Clino, the trend is very similar to that of the free enzyme. Being supported over M-Clino seems slightly unfavorable for both enzymes and the two curves appear to be narrower. Also in Fig. 6 Optical fluorescence microscopy images of clinoptilolite (a) our previous works, with FDH on glyoxyl or amino mordenite with ATTO-488 labeled FDH enzyme in the green channel (c) and and glyoxyl silica, an increase in optimal temperature was not ATTO-550 labeled GlyDH enzyme in the red channel (d) co-immo- bilized over B-Clino. Image (b) shows the two enzymes in the red and obtained.15,16 Concerning the behavior of co-immobilized green channels. The image (a) is in brightfield. enzymes, the co-presence of FDH and GlyDH on the same support seems to increase the activity at a lower temperature, FDH is most active at 60 °C while GlyDH is at 50 °C, and the co-immobilization on zeolites.44,45 Furthermore, it raises the optimal temperature is the same obtained for the enzymes question of how the distribution of the enzymes is in the mes- separately immobilized. As far as pH is concerned, the two free ostructured zeolites, but unfortunately as mentioned before the enzymes are characterized by signicantly different optimal autouorescence of the material did not allow to observe the values, around 7 for FDH and up to 10 for GlyDH. This becomes enzyme distribution. In any case, the uorescence microscopy a problem in the context of a chain reaction that involves both. also shows the co-presence of the two enzymes in the same sites On the other hand, the optimal pH of GlyDH supported over M- of the support particles: this is a conrmation of having effec- Clino is shied towards 8, closing the gap with FDH pH. On the tively co-immobilized FDH and GlyDH on Clinoptilolite contrary, GlyDH co-immobilized with FDH over M-Clino is particles. qualied by an optimal pH of around 9. Nevertheless, co- immobilized FDH presents generally higher stability than the Fig. 7 Optimal temperature results for FDH (a) and GlyDH (b). The tests were performed several times. 31150 | RSC Adv., 2022, 12, 31142–31155 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances Fig. 8 Optimal pH results for FDH (a) and GlyDH (b). The tests were performed many times. free or individually immobilized enzyme, which is most active for the models used to describe the thermal deactivation of the at pH 8. So, the gap between FDH and GlyDH remains constant. different biocatalysts, namely deactivation constant KD, residual 3.3.3 Thermal stability of free and immobilized enzymes. activity Ract, and half-life t1/2. The stability of FDH was visibly The kinetic of thermal deactivation was represented as the enhanced by the immobilization over mesostructured zeolite, percentage of the initial activity, the results are shown in Fig. 9. obtaining an increase in terms of half-life. Furthermore, the The tests were carried out only on the biocatalysts obtained biocatalyst had a residual activity of 40%, while the residual immobilizing the enzymes on M-Clino. Free FDH displayed activity of the free enzyme was under 10% aer 72 hours. The exponential deactivation kinetics, described by eqn (4). While stabilization factor of FDH/M-Clino (around 2.7) is comparable the loss of activity of immobilized enzymes and free GlyDH was to the results present in the literature. For example, with described using eqn (5) since at the end of the test a residual Candida methylica FDH immobilized on aldehyde immobead activity was observed. Table 4 summarizes the main parameters 150, a stabilization factor of 3.6 was obtained.46 In our previous Fig. 9Thermal deactivation of FDH (a) and GlyDH (b): thermal deactivation models for the soluble enzyme (black dot line) and the enzyme immobilized on M-Clino separately (FDH green dot line, GlyDH yellow dot line), and co-immobilized (FDH orange dot line, GlyDH red dot line). The points had been replicated. © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31142–31155 | 31151 RSC Advances Paper Table 4 Thermal deactivation kinetic parameters for FDH and GlyDH derivates at 50 °C Free FDH FDH/M-Clino FDH co-imm/M-Clino Free GlyDH GlyDH/M-Clino GlyDH co-imm/M-Clino Decay constant KD (h−1) 0.034 0.027 0.037 0.020 0.050 0.020 Residual activity a 0 0.35 0.40 0.25 0.40 0.34 Half-life time t1/2 (h) 20 54 48 55 36 71 works, an FS of 3.7 and 18.9 was obtained, respectively with formic acid (the concentration of DHA in the solution was not Candida boidinii FDH immobilized respectively on glyoxyl measured). MCF0.75 and amino mordenite.15,16 Samples of the reacting mixture were collected periodically to With glyoxyl agarose, a stabilization factor of 150 by Bolivar evaluate formic acid concentration, for the different reactions. and colleagues was obtained.39 To enhance the biocatalyst The rst reaction is carried out only with FDH supported over M- stability, the immobilization rate could be optimized, as Clino using NADH 10 mM as a reducing agent, and the results are observed by Mateo and co-workers.47 When FDH is co- shown in Fig. S4a.† The second reaction is carried out using FDH immobilized with GlyDH similar results are obtained. and GlyDH co-immobilized on M-Clino (a scheme of the two Regarding the stability of GlyDH, no signicant differences reactions catalyzed by FDH and GlyDH is reported in Fig. 10a), in were observed between its immobilized and soluble forms. this case starting with NAD+ 10 mM, this reaction is conducted in The structural complexity of GlyDH, its large size, and the two different cases varying the FDH : GlyDH w/w weight ratio composition of 8 subunits, make its stabilization very complex. during the immobilization procedure (FDH : GlyDH 1 : 1 ratio and Increased stability of this enzyme has only been reported when its FDH : GlyDH 1 : 4 ratio in Fig. S4b and c† respectively). In these quaternary structure has been stabilized by crosslinking with tests, the oxidized form of the nicotinamide cofactor was added polymers.29 Considering that both enzymes have similar lifetimes, (NAD+), with glycerol in the solution. If formic acid is observed, the use of this type of modication with polymers was discarded. the regeneration of the reduced form of the cofactor (NADH) carried out by the GlyDH enzyme is conrmed. Fig. 10b shows the 3.4 CO2 reduction maximum concentration of formic acid obtained in the solution for the three reaction conditions. To carry out the CO2 reduction a 0.1 M buffer phosphate pH 7 The reduction of CO2, performed by FDH over M-Clino, was used to avoid the deactivation of the cofactor, which proceeds successfully (Fig. S4a†), providing a peak of 5.5 mM happens at a more basic or acidic pH, during the reaction.48 of formic acid aer 2 hours of reaction. This means that the Furthermore, even if the highest activity was observed at 50 °C, mass production of formic acid, related to the activity of FDH, is the reaction was carried out at room temperature and atmo- 2.65 mg IUFDH−1. At equilibrium, the conversion settles down at spheric pressure. Higher temperatures have the drawback of around 50%. A similar result was obtained in our previous work, substantially reducing the CO2 solubility in aqueous media and where a concentration of about 4 mM of formic acid was it causes a faster deactivation of cofactor.48,49 The results of the observed with FDH on glyoxyl and amino mordenite.16 reactions were evaluated by monitoring the production of Fig. 10 (a) Scheme of the reactions of CO2 conversion into formic acid in the presence of NADH catalyzed by FDH(i) CO2 conversion and oxidation of NAD+ to obtain and regenerate NADH, catalyzed respectively by FDH and GlyDH co-immobilized on M-Clinoptilolite(ii). (b) Maximum concentration of formic produced after 2 hours of reaction due to the reduction of CO2 by FDH (red) FDH : GlyDH w/w ratio 1 : 1 (blue) FDH : GlyDH w/w ratio 1 : 4 (yellow). 31152 | RSC Adv., 2022, 12, 31142–31155 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances The reaction catalyzed by FDH, with cofactor regenerated by the GlyDH enzyme from Geobacillus stearothermophilus, capable GlyDH, with the same amount in weight of the two enzymes, of carrying out a regeneration of the NADH cofactor. instead, produced around 100 mM of formic acid (Fig. S4b†). In particular, the inuence of the surface area and the This indicates a mass production of formic acid, related to the support morphology was investigated. A mesostructuring activity of FDH, of about 0.05 mg IUFDH−1. These performances process was carried out on the support using an acid/base may be due to the rst reaction of the chain, catalyzed by treatment. Through physisorption with N2 at −196 °C, GlyDH, which is slower than the reaction catalyzed by FDH, a noticeable increase in surface area and an enlargement of the producing a “bottleneck” phenomenon. In this case, the formic pores were observed. To carry out a covalent immobilization, acid produced is very little, so the concentration of NADH a process of hetero-functionalization using APTES and GPTMS regenerated by GlyDH is not enough to permit a reduction of was performed. In this way, amino and glyoxyl groups were CO2 catalyzed by FDH. In light of this, to improve the reaction, generated on the supports and the immobilization was carried the loading ratio (in weight) of FDH/GlyDH was raised to 1 : 4. out on both the pristine support and the mesostructured ones. By increasing the amount of GlyDH, NADH was produced in The co-immobilization and co-localization of both enzymes a greater amount by oxidizing glycerol to 1,3-dihydroxyacetone, were conrmed by uorescence microscopy. improving the reaction condition, compared to the previous An increase in the activity of the two enzymes was observed case. Therefore, the regenerated NADH is used to reduce CO2 to when they are immobilized on mesostructured clinoptilolite formic acid. It is possible to see the reaction mechanism in compared to when untreated clinoptilolite is used as support. Fig. 10a-II. Aer 2 h a maximum of 3 mM of formic acid is Both the free and immobilized enzymes were characterized for reached. This implies that the mass production of formic acid, optimal pH and T. In addition, a thermal stability test at 50 °C related to the activity of FDH, is 1.44 mg IUFDH−1. In this way, it was performed, demonstrating a signicant increase in half-life is also possible to conrm the regeneration of the NADH for the FDH enzyme. The GlyDH enzyme, on the other hand, is cofactor. Subsequently, a part of formic acid is converted back more stable in free than immobilized form. to CO2, in fact aer 4 hours the concentration of formic acid in Finally, the two enzymes were used to carry out the reduction the solution is about 1.5 mM (Fig. S4c†). As the reaction goes of CO2 to formic acid. FDH alone produced a concentration of ahead, GlyDH is progressively subjected to product inhibition, 5 mM, resulting in a conversion of approximately 50% because DHA is not removed from the reacting medium.50 As compared to the initially provided NADH cofactor. The reduc- a result, the rate of NADH regeneration becomes slower until it tion reaction was also possible using the two co-immobilized reaches zero. This decrease in the regeneration rate leads to an enzymes and starting from the cofactor in an oxidized form. accumulation of NAD+ in the media, which shis the direction The GlyDH enzyme, using glycerol, generated NADH starting of the reaction to the consumption of formic acid. Finally, aer from NAD+ which was then used by the FDH enzyme to carry out 6 hours a concentration of 2 mM of formic acid was observed, the reduction reaction. In this case, at the end of the test, but it does not achieve the maximum reached aer 2 hours. a concentration of about 2 mM of formic acid was obtained. On Rocha-Martin and colleagues reported that the GlyDH, as the other hand, to increase the performance, it would be a free enzyme, has a half-maximal inhibitory concentration possible to extract the two enzymes from different microor- (IC50) of DHA, produced by the oxidation of glycerol, equal to ganisms that are more efficient in carrying out these two 0.43 mM.29 Furthermore, its acid sites are the most commonly reactions. used to carry out the immobilization on hetero-functionalized The strategy of mesostructuring the porous materials and support. These sites mainly correspond to the active sites the subsequent immobilization seems very promising and where the non-competitive inhibition by DHA takes place, as paves the way for the future development of processes applied demonstrated by Rocha-Martin et al. using a 3D structure,29 to a wider array of enzymes. increasing the half-maximal inhibitory concentration. To avoid this inhibition and recover the DHA product, alternatives such Author contributions as in situ product removal have been proposed.51 This could be a strategy to enhance the regeneration of the cofactor and Conceptualization: M. P., C. O.; methodology: M. P., C. O., T. T.; facilitate the purication of the products of the reaction. investigation: G. P., C. C., I. Z., M. D., O. R., V. C.; funding The immobilization of FDH and GlyDH on hetero- acquisition: C. O., O. R.; resources: M. P., C. O., T. T., V. C., D. F., functionalized support, even if it does not allow an increase writing – original dra: I. Z., G. P., C. C.; writing – review and in thermal stability, it could be used to carry out CO2 reduction editing: O. R., T. T. C. C.; supervision: M. P., C. O. with a biocatalyst that can be recovered and potentially re-used. Conflicts of interest 4 Conclusions There are no conicts to declare. The conversion of CO2 to formic acid was evaluated using the Acknowledgements enzyme FDH from Candida boidinii, covalently immobilized on the hetero-functionalized clinoptilolite. For the rst time, the Politecnico di Torino for the funding support required to FDH enzyme was co-immobilized on porous support along with publish this work in gold open access. O. R. acknowledges the © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31142–31155 | 31153 RSC Advances Paper support of Generalitat de Catalunya AGAUR for a Beatriu de 20 S. Mitchell and J. Pérez-Ramı́rez, Catal. Today, 2011, 168, 28– Pinós H2020 MSCA-Cofund (2019-BP-00180). The funding 37. support provided by ANID-Chile through the project FONDE- 21 M. Hartmann and D. Jung, J. Mater. Chem., 2010, 20, 844– CYT 11180967 is acknowledged. 857. 22 N. Ramı́rez, M. Serey, A. Illanes, M. Piumetti and C. Ottone, J. Photochem. Photobiol., B, 2021, 215, 112113. Notes and references 23 A. 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