Crosslinked on novel nanofibers with thermophilic carbonic anhydrase for carbon dioxide sequestration

Journal Pre-proof Crosslinked on novel nanofibers with thermophilic carbonic anhydrase for carbon dioxide sequestration

Sefli Sri Wahyu Effendi, Chen-Yaw Chiu, Yu-Kaung Chang, ISon Ng PII:

S0141-8130(19)38534-4

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.234

Reference:

BIOMAC 14015

To appear in:

International Journal of Biological Macromolecules

Received date:

22 October 2019

Revised date:

18 November 2019

Accepted date:

29 November 2019

Please cite this article as: S.S.W. Effendi, C.-Y. Chiu, Y.-K. Chang, et al., Crosslinked on novel nanofibers with thermophilic carbonic anhydrase for carbon dioxide sequestration, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/ j.ijbiomac.2019.11.234

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© 2018 Published by Elsevier.

Journal Pre-proof Research article

Crosslinked on novel nanofibers with thermophilic carbonic

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anhydrase for carbon dioxide sequestration

Department of Chemical Engineering, National Cheng Kung University, Tainan 70101,

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Sefli Sri Wahyu Effendi1, Chen-Yaw Chiu2, Yu-Kaung Chang2, I-Son Ng1*

Taiwan, ROC

Graduate School of Biochemical Engineering, Ming Chi University of Technology, New

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Taipei City 24301, Taiwan, ROC

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*Corresponding author: I-Son Ng

Tel: +886-62757575-62648; Fax: +886-62344496 E-mail: [email protected] ORCID: 0000-0003-1659-5814 Co-authors’ email: Sefli Sri Wahyu Effendi ([email protected]) Chen-Yaw Chiu ([email protected]) Yu-Kaung Chang ([email protected])

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Journal Pre-proof Abstract The recombinant Sulfurihydrogenibium yellowstonense carbonic anhydrase (SyCA) was covalently bonded on novel polyacrylonitrile (PAN) and polyethylene terephthalate (PET) nanofibers (PAN-PET-PAN donated as AEA) that was first fabricated by electrospinning. The resulting composite materials further crosslinked by the glutaraldehyde, which significantly increased thermostability up to 89.8% and 18.0% after heating at 60oC for 1 hour for immobilized crude and pure SyCA, respectively. After four repetitive attempts in the

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demonstration of CO2 sequestration, immobilized crude and pure SyCA on AEA also

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effectively improved the total CaCO3 yields to be 5.8 folds and 2.2 folds compared to free

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enzyme. Furthermore, the endurance of immobilized crude was investigated on flue gasses,

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which was retained its activity up to 57% on 50 mM NOx and 61% on 50 mM SOx presence. This is the first report of immobilized thermophilic SyCA on a novel nanofiber at the

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reusability, durability, sequestration of carbon dioxide, tolerant to sulfur oxides (SOx) and

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nitrogen oxides (NOx) toxic gases and to prevent air pollution.

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Keywords: Sulfurihydrogenibium yellowstonense; carbonic anhydrase; nanofiber; immobilization; sequestration

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Journal Pre-proof 1. Introduction Carbonic anhydrase (CA, EC. 4.2.1.1) was an old enzyme since the first discovery in 1932 by Meldrum and Roughton, which were explained the mechanism by hydration reaction of carbon dioxide (CO2) to react with water and converted to bicarbonate ions [1]. The preliminary work has focused on the enzymatic kinetics effects of temperature, salt, and pH of CAs from blood [2]. Afterwards, CAs have become the forefront of scientific interest, from the understanding of mechanism reaction, structural and molecular biology, clinical

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discovery to environmental issues. Recently, CAs have been extensively examined in global

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warming issues [3-4] because ecofriendly in global carbon metabolism and recycle [5],

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biomimetic CO2 sequestration process [4, 6]. On the other hand, CAs are also referred to as

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metalloenzyme which classified into various classes (α, β, γ, δ, ζ, η and θ) hence the implication of zinc ions as its active site [3, 5-8]. The α-CAs are the most protrusive family

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that contain not only in members of mammals, fungi, and bacteria [9] but also represented the

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highest kcat for 106 molecules of CO2 per second [7]. Hence, α-CAs class have been characterized and genetically cloned in model organisms including of CAs from human

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pathogenic bacterium Vibrio cholerae and Neisseria gonorrhoeae [10, 11], halotolerant bacterium Hydrogenovibrio marinus [12], Aeribacillus pallidus and Lactobacillus delbrueckii as alkaline-tolerant [13, 14], and thermophilic bacterium Sulfurihydrogenibium azorense and Sulfurihydrogenibium yellowstonense YO3AOP1 [15, 16]. Our previous study has developed a high-throughput screening platform to explore higher activity CA from S. yellowstonense (SyCA) [17]. The SyCA was presented thermophilic and acidophilic properties that maintained 100% activity at 50oC with optimal pH at 3-5 [18]. Besides, SyCA existed activity at higher temperatures due to immobilizing an anchoring and self-labeling protein tag on it [19]. However, SyCA is still not capable of being intended for use in carbon capture biotechnology because of high costs in the application. Thus, 3

Journal Pre-proof immobilization is one of the solutions that is considered as a brilliant strategy to overcome the problem [3,8,20]. The immobilization of SyCA onto various matrices such as polyurethane foam [5], magnetic nanoparticles [8], and even surface immobilization of enzymes on E. coli outer membrane [17] which have previously been reported. Recently, electrospun nanofibers have been proven for immobilization, since it offers a sizeable surface-to-pores volume ratio, functionalized surfaces, multiple sites for interaction or attachment, low restriction of mass

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transfer [21], higher adsorption capacity and faster than conventional membranes [22].

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Polyacrylonitrile (PAN), as a hydrophobicity electrospun material, has beneficial properties

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such as suitable for immobilization via covalent attachment [23], stable in storage, resistant to

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general solvents, oxidation resistance [24], tolerant to chemical and mechanical strength [22]. In this study, recombinant SyCA in E. coli for protein expression, enzymatic

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characterization, and thermostability of immobilized crude and pure enzyme were performed.

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Then, the eff ect of the glutaraldehyde used in the cross-linking reaction on a novel nanofiber PAN-PET-PAN (donated as AEA) by electrospinning of PAN and polyethylene terephthalate

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(PET) was examined. Finally, the application of SyCA on the nanofiber system is discussed further to accomplish CO2 sequestration and to withstand the presence of toxic chemicals such as NOx and SOx.

2. Material and methods 2.1. Strains, plasmids, and media The E. coli BL21(DE3) harboring pET32a(+) with carbonic anhydrase from S. yellowstonense was used for enzyme production. Polyacrylonitrile (PAN) yarn (Mw 1.2·105 g/mol, containing 93% acrylonitrile and 7% vinyl acetate) was purchased from Fortune 4

Journal Pre-proof Industries Inc. (Taoyuan, Taiwan). Polyethylene terephthalate (PET) spun-bond fabric (basis mass 15 g per m2, thickness 90 μm, fiber diameter 300-500 μm) was supplied by Freudenberg Far Eastern Spunweb Co., Ltd. (Taoyuan, Taiwan). Glutaraldehyde solution of 25 % (wt) was purchased from AppliChem GmbH (Darmstadt, Germany). Other chemicals and solvents were purchased from BioBasic (Toronto, Canada) and Sigma-Aldrich (St. Louis, MO, USA).

2.2. Culture conditions of SyCA

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Recombinant E. coli cells were grown on LB plates (1.5% tryptone, 1.5% NaCl and 0.5%

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yeast extract) with 100 mg/L ampicillin antibiotics at 37°C for 12 h. A single colony was

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inoculated in LB medium with 100 mg/L ampicillin for pre-culture at 37°C for another 12 h

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with shaking at 200 rpm. Then, the cells were diluted by 1:100 in LB medium with antibiotics and cultured at 37°C with 200 rpm agitation. The growth was monitored by measuring the

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biomass or optical density at 600nm (OD600) using the spectrophotometer (Molecular Devices,

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America). As OD600 reached 0.6 ∼ 0.8, the cells were induced by 0.1 mM isopropyl-Dthiogalactopyranoside (IPTG) and 0.5 mM zinc ions (supplied by ZnSO4). Eventually, the

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cells cultured for 16 h were harvested by centrifuging at 10,000 × g for 10 min and washed with deionized water for 2 times.

2.3. Purification and protein analysis Purification of SyCA was carried out by using a His-Trap column chromatography (GE Healthcare, United Kingdom). The crude enzyme of SyCA was filtered by syringe filter 0.22 µm and used for the purification process. The protein separation has used the gradient of imidazole between buffer A (consisted of 20 mM phosphate buffer, 0.5 mM NaCl, 20 mM imidazole) and buffer B (similarly with buffer A except for imidazole concentration which is 500 mM) to elute the target protein. The purification process was performed at 4°C. The total 5

Journal Pre-proof protein concentration was determined using a Bradford assay (Bio-Rad) with bovine serum albumin (BSA) as a protein standard. Finally, whole-cell, crude, and purified samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% separating gel and 4% stacking gel. Proteins were conceived by staining with Coomassie blue R-250 and were scanned using an Image scanner.

2.4. Carbonic anhydrase assay based on CO2 hydration activity

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CA activity was examined by Wilbur-Anderson assay (WAU) with modification [25]. The

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reaction was started by adding 9 mL ice-cold Tris-HCl (20 mM, pH 8.3) buffer with CO2

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saturated solution at 0°C, and 0.2 mL enzyme was mixed and transferred to stirring vials. The

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probe of a standardized pH meter was inserted into test vials with further incubation at 0°C. Next, 6 mL of the substrate was added immediately into vials, and the time required for the

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pH changing from 8.3 to 6.3 was recorded. CA activity was calculated using WAU per

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milliliter of sample. The definition for WAU is (T0–T)/T where T0 and T are measurements taken for the buffer (control) and the buffer containing sample, respectively. In order to

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obtain the kinetic data, diff erent volumes of saturated CO2 solution ranging from 3 to 6 mL were added into 9 mL Tris−HCl (20 mM, pH 8.3) buff er augmented with deionized water to a total volume of 15 mL. The remainder of procedure were the same as described above. A approach involved an R-value of the net enzyme reaction rate was reported in the previous study [25], which can be calculated by Eq. (1), in which A means a mole of converted CO2 during the pH decrease from pH 8.3 to pH 6.3: 𝑅=

𝐴 𝐴 − 𝑇 𝑇𝑂

(1)

All of the data were equipped with the Michaelis-Menten model for calculation of Vmax and Km based on the following Eq. (2). 6

Journal Pre-proof 𝑉 = 𝑉𝑚𝑎𝑥

𝐶 𝐾𝑚 + 𝐶

(2)

In which C and V indicated CO2 concentration and velocity, respectively.

2.5. Preparation of PAN nanofibers and composite AEA The electrospinning process was performed at 298 K, and the operating parameters were

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determined based on the previous study [22]. 15 g solution of PAN was prepared in dissolve in 100 mL of dimethylacetamide (DMA). 10 mL of PAN solution was placed into a syringe

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with a 21-gauge stainless steel nozzle. A syringe attached to a power supply and electrospun

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into nanofiber under the following conditions: voltage: 26.5 kV, syringe rate: 1.0 mL/h, tip to

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collector distance: 15.8 cm, and the rotation rate of the collector: 24.0 cm/s. The nozzle

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moved along with the y-axis (20.0 cm) at a frequency of 12 times per min. The PAN electrospun nanofibrous mats were collected on PET fabric, which was attached to a ground

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steel drum. One PET fiber and two PAN nanofibrous layers, designated as AEA, were

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detached from the collector and processed using thermal pressing at 373 K for 1 h.

2.6. Immobilization of SyCA on AEA nanofibers The procedure of SyCA immobilization consists of three parts: (1) modification functional group of nanofibers (COONa to COOH), (2) immobilized SyCA by covalent bonding, and (3) enzymes crosslinked by glutaraldehyde (GA) treatment. The AEA nanofibers were hydrolyzed by using chemical treatment with 2 mL of 3 M NaOH at 358 K for 20 min. The excess alkaline solution was removed, and nanofibers were washed with water. A 4 mL of 0.1 M HCl was transferred into the hydrolyzed nanofibers (designated as AEA-COOH), followed by enzyme washing and immobilization. The activated AEA-COOH nanofibers were adsorbed with SyCA solution (2 g/L) in 1 mL of deionized water, and the 7

Journal Pre-proof reaction vials were shaken at 85 rpm for 3 h at ambient temperature. The immobilization process was continued at low temperature at 4oC for 6 h, and the unbounded enzymes were removed by washing the nanofibers with 10 mM phosphate buffer (pH 7.0). Finally, the immobilized enzymes were crosslinked to glutaraldehyde (GA) solution consisting of 25 % (wt) GA water solution and deionized water (1%, v/v), and then shaken at 60 rpm for 5 min at room temperature. Unreacted GA was washed excessively with 10 mM phosphate buffer (pH

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7.0). The activity of immobilized CA was further examined using Wilbur-Anderson assay.

2.7. Characterization of immobilized SyCA

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The surface morphology of nanofibers before and after CA immobilization was examined

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by scanning electron microscopy (SEM; SU8100 Hitachi, Japan) operating at a 15 kV

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acceleration voltage. The surface coverage of crude and purified immobilized SyCA was analyzed using Biorad assay to determine protein adsorbed on nanofibers membranes, which

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were calculated as follows:

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parameters can be easily modeled by the Langmuir equation. The protein adsorbed amounts

𝑄=

(𝑞0 𝑥 𝑉0 ) − (𝑞 𝑥 𝑉) 𝑚

(3)

Besides, the calculation of equilibrium immobilization on the polymer surface based on the following equation.

𝜃=

𝑄 𝑞0

(4)

where Q is the protein adsorbed amounts (mg/g) onto the AEA nanofibers, qo is the initial concentration of protein (mg/mL), q is the final concentration (mg/mL), V0 and V are initial volume and final volume, m is the weight of nanofiber mats (g), and θ indicates surface coverage.

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Journal Pre-proof 2.8. Effect of glutaraldehyde (GA) and temperature on the SyCA activities Enzymes including crude, pure, and immobilized samples were placed in a vial and then exposed to glutaraldehyde (GA) solution composed of 25 % (wt) GA solution and deionized water (1%, v/v). The mixture was incubated at various temperatures ranging from 60 to 80°C and room temperature. The residual activity of each temperature was examined by determining the enzyme activity of the original sample (at room temperature) as 100%. After that, the effect of GA was further examined for long-term stability (i.e., 15, 30, 45, and 60

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min) while the residual activity was determined each time by the enzyme activity of sample,

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which was heating 15 min as 100%.

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2.9. Reusability of immobilized SyCA

The reusability of immobilized SyCA was washed with 10 mM sodium phosphate buffer

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solution (pH 7.0) and then added a new CO2 saturated substrate solution to start a new cycle.

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activity as 100%.

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The residual activity of each cycle was examined by determining the first cycle of enzyme

2.10. CO2 sequestration to CaCO3 by SyCA A two-column design was used for CO2 sequestration to form CaCO3 precipitation. CO2 gas was bubbled into the water, which was added with the SyCA crude enzyme or immobilized enzyme at a uniform flow rate for 6 min. Afterward, the mixture reaction was dropped down into the second column consisting of Ca(OH)2 saturated solution to form CaCO3 precipitated and collected by centrifugation. After one cycle use, immobilized SyCA was washed with 10 mM sodium phosphate buffer solution (pH 7.0) and reused in the twocolumn reactor for CO2 sequestration. Each cycle was centrifuged to collect the CaCO3 precipitate solids and weigh the dried. 9

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2.11. Antitoxicity to sulfur oxides (SOx) and nitrogen oxides (NOx) The immobilized sample was incubated in HNO3 and H2SO4 solution, respectively, at concentrations of 25, 50, 75, 100, and 300 mM at room temperature for 15 min. The residual CA activity was measured by WAU, as described previously. All experiments were

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performed in triplicate.

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3. Results and discussion

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3.1. Expression, activity and kinetic parameters of SyCA

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The carbonic anhydrase gene was cloned in E. coli BL21 (DE3) and expressed using pET32a(+) vector, which driven by strong T7/lac promoter, as shown in Fig. 1a. Protein

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expression of SyCA that induced by IPTG was determined by SDS PAGE analysis (Fig. 1b)

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As expected, at approximately 47.5 kDa in the soluble cytoplasmic form due to pET32a(+) contained a fused chaperone (Trx) in the N-terminal to assist the folding of protein [26, 27].

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The recombinant SyCA from the soluble fraction has been purified by using one-step affinity chromatography and eluted the imidazole afterward by ultra-filtration. In this study, the recovery and purification fold of SyCA were 43.6% and 5 times, respectively. The previous study showed a 27% yield and 16 times of purification fold of recombinant CA from Sulfuryhydrogenibium sp. YO3APO1 after purification by affinity chromatography [5]. Another purified β- and γ- CAs from Bacillus sp. SS501 has been found 17 and 23 purification folds, whereas the recovery as 23% and 31%, respectively [28]. It implies SyCA purification by one-step affinity chromatography is a powerful and effective way to obtain higher yield, with the result of 43.6%.

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Journal Pre-proof With respect to CA kinetic data, the mole of Tris-HCl buffer in the Tris part and dissociation rate of saturated CO2 in carbonic acid part were adopted to calculate A value. Tris was used for buffering the pH change, and the reaction could be simplified to the following equation: 𝐵𝐻 + ⇌ 𝐻 + + 𝐵 𝑝𝐾𝑎 = 8.76 at 0oC

(5)

By the thermodynamics property and calculation, the ratio of [B] and [BH+] was obtained as

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-0.46 at pH 8.3. The mole of Tris was calculated as 4.64 x 10-5. Moreover, the dissociation

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rate found as 1.87 × 10−3, which was calculated by solubility (1.337 % mole fraction) as

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0.074 M. Finally, the total treated CO2 by Tris part and carbonic acid part is 2.48 × 10−2 mole as A value. After obtaining A value, the kinetic parameters between crude and purified SyCA

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were determined by Lineweaver Burk plotting (Fig. 1c). The kinetic parameters were shown

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in Table 1, which the purified enzyme was presenting a higher Km value and indicating that it has a higher substrate affinity compared to that in the crude enzyme. Km was achieving half of

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Vm, lower Km may be attributed to diffusion limitations, or cell membrane would inhibit the

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CO2 diffusion [23, 25]. Thus, kcat/Km in terms of catalytic efficiency reported that the catalytic efficiency of the purified enzyme is 2.8 folds higher than that of crude enzyme. Moreover, SyCA has shown higher kcat than hCAI (i.e. 2.0 x 10−5) [29]. The activity crude and pure enzyme activities were 20557.2 U/mg and 35771.6 U/mg, respectively (Fig. 1d). Besides, crude CA from EX-H1 Persephonella marina, which has an extremely high thermostability of 100°C of 4960 U/mg [30], while crude mesophilic CA from Lactobacillus delbrueckii CGMCC 8137 showed 494 U/mg [31]. The purified CA from Black Sea trout (Salmo trutta Labrax Coruhensis) only showed 603.7 U/mg [31]. Therefore, S. yellowstonense was greatly possessed a higher CA activity compared to other thermophilic CAs. 11

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3.2. Characterization of immobilized SyCA on nanofiber The procedure of SyCA immobilization was illustrated schematically in Fig. 2a. The structures of nanofibers before and after immobilizing SyCA were confirmed with SEM micrographs where the surface of the nanofibers became rougher after immobilizing enzymes (Fig. 2b). SEM was used to observe the structure of enzymes that were covalently attached to nanofibers [21]. Actually, the functional modification group of nanofiber was proved by color

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changing from orange turn into yellowish under our observation, which indicated cation

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exchange of –COONa to –COOH was worked out [33]. The nanofibers can be produced in a

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thinner dimension to obtain more surface areas by using electrospun, which offers an

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advantage in the increasing amount of functional groups on the matrix surfaces [34]. Therefore, the size of the nanofiber is an effective factor in determining the attachment and

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stabilization of the enzyme [35, 36]. Herein, the AEA produced by electrospun is a 1 to 2 m

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homogenous fiber, which is similar to amylase immobilized on nanofiber of PVA/PAA [37]. Figure 3 demonstrates protein adsorbed, and surface coverage of SyCA prepared with

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different protein concentrations on the hydrophobic AEA nanofibers. The optimum condition of pure and crude enzyme protein adsorbed on AEA was achieved using a protein concentrations of 0.4 mg/mL and 3 mg/mL, respectively. The maximum protein adsorbed on AEA were obtained to be 7.1 and 130.2 mg protein/g-AEA for pure and crude CA. Whereas, the maximum surface coverage was 85.7% in 0.3 mg/mL and 98.3% in 3 mg/mL. However, further increasing concentration of protein (up to 0.4 and 4 mg/mL) did not affect to enhance the surface coverage. Other studies have been reported the adsorption capacities on PAN nanofiber mats for dye and lipase to be 48.6 mg/g and 21.2 mg/g, respectively [38, 39]. The higher result of the adsorbed SyCA protein has proven that SyCA successfully bonded onto the modification of AEA nanofibers. 12

Journal Pre-proof On the other hand, the corresponding semireciprocal plots (qo/q versus qo) of the experimental data indicated the adsorption homogeneous as Langmuir model since it presented a linear plot [40, 41], which was R2 = 0.9505 for crude and R2 = 0.9775 for pure SyCA. Langmuir model showed not only the homogenous adsorption but also monolayer surface coverage. Monolayer immobilization enzymes offered stable covalent bonds on the support surface carrying compatible chemical groups. It often desired to achieve a controlled covalent binding, which provides a low surface agglomerate and an optimal orientation of

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protein [42]. The orientation of surface coverage, as an independent measurement, presented

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a correlation enzyme immobilization with enzymatic activity [43].

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3.3. Characteristics of glutaraldehyde (GA) effect on SyCA activity

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AEA, as the immobilization matrix, has a high surface area of electrospun fibers, but it accounted for the initial higher release rate of protein from the matrix [34]. Glutaraldehyde

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(GA) plays an essential role in a crosslinking reaction that prevents the leakage of protein

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from the matrix and improves reusability on the immobilization of enzymes [44]. Despite this, sometimes, GA causes inactive proteins to reduce enzyme activity [45]. The effect of 0.1% GA on SyCA activity was studied and showed in Fig. 4. The activity of enzymes declined as GA added, especially immobilized samples. The CA activity of crude/GA, immobilized crude/GA, pure/GA, and immobilized pure/GA samples were decreased to 18615, 1481, 23459, and 6295 U/mg. In order to examine thermostability, the samples were incubated at temperature range 6080oC using the same concentration of GA after heating for 15 mins (Fig. 5a). Interestingly, immobilized crude/GA achieved the most stable residual activity up to more than 150% at all temperatures when compared to other samples. In contrast, immobilized of pure/GA was declined the residual activity to 28% as increasing temperature to 80oC. We speculated that at 13

Journal Pre-proof higher temperatures, the purified samples could easily undergo denaturation, while crude enzymes might be due to the conformation of other rigid proteins. Besides, the secondary covalent bonds would be formed between immobilized crude and GA crosslinking. GA has been reported showing high reactivity towards the amine group of CA and hydroxyl group of nanofibers [46]. In further experiments, the thermal stability of the GA effect was also demonstrated by using crude, pure, and immobilized samples. All samples were inspected using various heating time at 60oC as optimum temperature. As shown in Fig. 5b, crude/GA,

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and its immobilization were presented unique ability with the enhancement of their residual

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activity as heating time increased. Differently, the free of pure samples only retained 27.2%

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activity after 60 min, while immobilized pure enzyme had 118% of its activity after 60 min at

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60oC. The improving thermal stability on immobilized enzyme referred to increasing interactions between the structure of nanofibers in surface hydrophobic and enzyme with the

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reduction of the conformational arrangement of enzyme molecules [44]. Practical

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applications of immobilized SyCA have been described in previous studies, which were used covalent binding between enzyme and the support. Immobilization via covalent bonds is

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suitable for SyCA molecules due to the availability of functional groups between hydroxyl groups and amine groups. As list in Table 2, the long-term stability of immobilized SyCA over 100% after one month has been maintained by using polyurethane foam and magnetic nanoparticles as matrix support [7, 8]. Merlo et al. established an in vivo one-step procedure of enzyme immobilization on the E. coli surface as biological supports. A novel Anchoringand-Self-Labelling-protein-tag (ASLtag) can be a way to increase the thermostability of enzymes [47].

3.4. Application of immobilized SyCA in biomineralization and tolerant to toxicity

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Journal Pre-proof In the commercial point of view, reusability is an important characterization for potential applications of biocatalysts. Figure 6a shows the effect of repeated uses on the residual activity of the immobilized crude and purified SyCA, even though the residual activities of the immobilized CA decayed with the increasing number of recycles. The loss of activity in these steps may be related to the inactivation of the enzyme caused by protein denaturation, and the leakage of protein from the support surfaces upon use [21]. The immobilized purified SyCA non-GA was retained 36.6% activity whereas the best reusability after 5 consecutive

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uses since immobilized pure/GA only retained 8.0% activity. Besides, immobilized crude

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SyCA/GA showed higher activity than non-GA, which was proved crude enzyme has unique

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characteristics on GA crosslinked effects. The potential application of immobilized SyCA

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would be extended by investigating the sequestration of CaCO3 and toxic resistance ability. From other CA species, rBhCA was successfully modified with functional group in the

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original adsorption to increase reusability [48]. Furthermore, BCA has potentially interacting

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enzymes that are covalently attached to support by functional groups in bonding via sidechain amino groups [49] and epoxy groups [50]. Although SyCA did not show more recycle

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uses than other CA species, a novel nanofiber materials has the first successfully reported and demonstrated its reusability.

As given in Fig. 6b and 6c, the CaCO3 yields of immobilized SyCA were higher than the free one. The immobilized samples were prepared by using different conditions and demonstrated up to 4 times repeated uses. Among crude samples: crude, immobilized crude and immobilized crude/GA were obtained 28.1, 163.4, and 110.3 mg of total CaCO3 yields. Surprisingly, purified samples obtained prodigious CaCO3 solids. Pure, immobilized pure and immobilized pure/GA were gained 315.9, 688.1, and 591.9 mg of total CaCO3 yields. However, to our best knowledge, purified SyCA was challenging to prepare, which requires a higher cost of equipment and also is impossible to recycle. 15

Journal Pre-proof Other requirements of CA used in the industrial application is tolerance of toxic chemical species from flue gases (i.e., SOx and NOx). As shown in Fig. 7a and 7b, immobilized crude/GA represented a significant increase of residual enzyme activity up to 57.1% and 61.6% in the presence of 50 mM HNO3 and H2SO4 when compared to free samples, which was retained only 14.8% and 10.2% at the same conditions. The enzymatic tolerance of SOx and NOx is ranking from immobilized with GA > immobilized > free CA. Finally, immobilized crude/GA also lost the activity at higher concentrations solution representative

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of HNO3 and H2SO4. It was assumed that GA has secondary interaction to make substantial

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denaturation of proteins. The effects of SOx and NOx are of interest since the trace amount of

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chemicals is strictly control and inhibits CA activity [51, 52]. However, the potential of co-

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immobilized CA with GA on AEA would eliminate the adverse effect and increased the

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tolerance in this study.

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Journal Pre-proof 4. Conclusions A novel of crosslinked GA effect on enzyme bounded has been explored for the first time. Crosslinked GA has shown unique characteristics on immobilized crude SyCA, which is different from other CAs type, leading to tolerance towards GA effect and the enhanced thermal stability. Furthermore, it could make the process more economically feasible and effectively reduced the cost since it was able to reuse for CO2 biomineralization and the preparation of crude enzyme uncomplicated as the pure enzyme. The findings of the present

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study suggested that surface-functionalized electrospun nanofibers are potential support for

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immobilization and immobilized SyCA as a promising novel for industrial application since

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its durability to NOx and SOx presence.

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Journal Pre-proof Acknowledgements The authors are grateful to the financial support for this study provided by the Ministry of Science and Technology (MOST 108-2621-M-006-015, MOST 108-2221-E-006-004-MY3 and MOST 108-2218-E-006-006) in Taiwan.

Ethics approval and consent to participate

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All the authors have read and agreed the ethics for publishing the manuscript.

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Consent for publication

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The authors approved the consent for publishing the manuscript.

Competing interests

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The authors declare that they have no competing interests.

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Journal Pre-proof References: [1]

N.U. Meldrum, F.J. Roughton, Carbonic anhydrase. Its preparation and properties, J. Physiol. 80 (1933) 113-142.

[2]

C. Geers, G. Gros, Carbon dioxide transport and carbonic anhydrase in blood and muscle, Physiological Reviews 8 (2000) 681-715.

[3]

S.S. Effendi, I.S. Ng, The prospective and potential of carbonic anhydrase for carbon

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dioxide sequestration: a critical review, Process Biochem. (2019)

[4]

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25

Journal Pre-proof Figure legends

Fig. 1. (a) Map of pET32a-SyCA. (b) Protein expression analysis by SDS PAGE. Lane 1: M, lane 2: no isopropyl β-D-1thiogalactopyranosdie (IPTG) induction, lane 3 to 7 are with 0.1 mM IPTG. M, WC, C, UB, P1 and P2 mean marker, whole cell, crude enzyme, purification sample from unbound peak, purification sample containing imidazole, purification sample after 30 kDa ultrafiltration. (c) Lineweaver-Burk plot for inverse of CA activity (CO2 sec

of

mole-1, Y) and 1/P (atm-1, X) of CO2, where Y = 0.7151 X + 4763 with R2 = 0.9946 for crude

ro

(black) and Y = 0.7861 X + 3694 with R2 = 0.9910 for pure (white) CA, respectively. (d)

re

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crude and pure enzymatic activity determined by Wilbur-Anderson assay.

Fig. 2. (a) The schematic illustration of CA immobilization on AEA nanofibers. SEM

lP

analysis of the AEA nanofibers without enzyme at (b) 5 m (c) 50 m, and with the enzyme

na

at (d) 5 m (e) 50 m, respectively.

Jo ur

Fig. 3. Physical equilibrium of immobilization CA on the AEA membrane surface. The relative surface coverage and protein adsorbed of crude (black) and pure SyCA (grey) were analyzed by different protein concentrations.

Fig. 4. Characterization of glutaraldehyde (GA) effect on SyCA activity between crude and pure, crude, or pure with GA, crude or pure immobilized on AEA.

Fig. 5. Temperature effect on SyCA activity between crude and pure, crude or pure with GA, crude or pure immobilized on AEA. (a) 60oC (blue bar), 70oC (red bar), and 80oC (green bar).

26

Journal Pre-proof (b) thermal stability at 60oC heating treatment and for 15 min (purple bar), 30 min (blue bar), 45 min (red bar), and 60 min (green bar).

Fig. 6. (a) Residual relative activity for reusability of immobilized SyCA. Crude (white circle), crude/GA crosslinked (red circle), purified (white triangle), and purified/GA crosslinked (red triangle). (b) Biosequestration of CO2 to CaCO3 between crude and pure, crude or pure with GA, crude or pure immobilized on AEA by 4 recycling uses (1st cycle:

ro

of

gray bar, 2nd cycles: tosca bar, 3rd cycles: red bar and 4th cycles: green bar).

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Fig. 7. (a) NOx and (b) SOx effects on the crude enzyme (circle), immobilized crude

Jo ur

na

lP

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(square), and immobilized crude with GA crosslinked (triangle).

27

Journal Pre-proof Table 1. Comparison of kinetic parameters of crude enzyme and purified of SyCA Specific activity (U/mg)

Protein concentration (mg/mL)

Enzyme

kcat (1/s)

Km (M)

kcat / Km (1/M s)

Crude enzyme

8.818 x 104

0.150

5.879 x 105

20,557

3.616

Purified

2.444 x 105

0.213

1.147 x 106

35,771

1.578

Residual activity (%)

Ref.

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Table 2. Characteristics of different materials used for immobilization of carbonic anhydrase. Immobilization method

Reusability (times) 30* 30*

100 100

[7] [8]

ND

ND

[46]

22

50

[47]

PLA

Adsorption of functional groups Covalent binding

10

43.7

[48]

BCA

GO/PLA

Covalent binding

10

78.9

[48]

BCA

nMOF/PLA

Covalent binding

10

69.3

[48]

BCA

Magnetic microspheres AEA nanofibers

Covalent binding

6

47.6

[49]

Covalent binding

5

36.6

This study

Support material

SspCA SspCA

Covalent binding Covalent binding

SspCA

PU foam magnetic Fe3O4 nanoparticles E. coli surface

ASLtag in vivo

rBhCA

Si-MNPs

BCA

*

re

lP

na

Jo ur

SyCA

-p

CA sources

The long-term stability is for 30 days. ND means not-determined.

28

Journal Pre-proof

Highlight:  Recombinant carbonic anhydrase (CA) was immobilized on a novel nanofiber.  CA was immensely coverage on the nanofiber surface via covalent binding.  Glutaraldehyde aided on improvement thermostability of immobilized CA.  Immobilized CA was successfully reused and augmented CaCO3 yields.

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na

lP

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 Immobilized CA has a remarkable sulfur oxides and nitrogen oxides tolerance.

29

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7