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Cellulose nanofibers for magnetically-separable and highly loaded enzyme immobilization
Accepted Manuscript Cellulose Nanofibers for Magnetically-Separable and Highly Loaded Enzyme Immobilization Hwa Heon Je, Sora Noh, Sung-Gil Hong, Youngjun Ju, Jungbae Kim, Dong Soo Hwang PII: DOI: Reference:
S1385-8947(17)30653-8 http://dx.doi.org/10.1016/j.cej.2017.04.110 CEJ 16864
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
29 November 2016 8 April 2017 25 April 2017
Please cite this article as: H.H. Je, S. Noh, S-G. Hong, Y. Ju, J. Kim, D.S. Hwang, Cellulose Nanofibers for Magnetically-Separable and Highly Loaded Enzyme Immobilization, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.04.110
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Cellulose Nanofibers for Magnetically-Separable and Highly Loaded Enzyme Immobilization
Hwa Heon Je1,†, Sora Noh2,†, Sung-Gil Hong2, Youngjun Ju2, Jungbae Kim2,3,*, Dong Soo Hwang1,4,*
1
Division of Integrative Bioscience and Biotechnology, Pohang University of Science and
Technology (POSTECH), Pohang 37673, Republic of Korea 2
Department of Chemical and Biological Engineering, Korea University, Seoul 02841,
Republic of Korea 3
Green School, Korea University, Seoul 02841, Republic of Korea
4
School of Environmental Science and Engineering, Pohang University of Science and
Technology (POSTECH), Pohang 37673, Republic of Korea
†These authors contributed equally to this work *Corresponding author. E-mail: [email protected] or [email protected]
1
Abstract Cellulose nanofibers (CNFs) are one of attractive supporting materials for enzyme immobilization due to their unique properties such as high surface area, high porosity and surface carboxyl groups for chemical bonding. In this study, CNFs were prepared via TEMPO-mediated oxidation and physical grinding of cellulose, and further used for the immobilization of α-chymotrypsin (CT) enzyme via four different approaches such as covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically-separable EPC (Mag-EPC). EPC approach consists of three steps: covalent enzyme attachment, enzyme precipitation and crosslinking, while EC represents a control without the step of enzyme precipitation. Amine-functionalized magnetic nanoparticles were added during the enzyme precipitation and crosslinking steps to produce magneticallyseparable EPC (Mag-EPC). The activities of CA, EC, EPC and Mag-EPC were 0.067, 0.14, 1.3 and 2.6 units per mg CNFs, respectively, representing that the activity of Mag-EPC was 38-, 19- and 2-times higher than those of CA, EC and EPC, respectively. After incubation under shaking (200 rpm) for 30 days, CA, EC, EPC and Mag-EPC maintained 12%, 46%, 77% and 50% of their initial activities, respectively, while free CT showed only 0.2% of its initial activity even after 8 days. Because CT is a tricky enzyme to stabilize due to its inactivation mechanism via autolysis, the present results of stable EPC and Mag-EPC on CNFs have demonstrated the great potential of CNFs as an environmentally-friendly and economical carrier of enzyme immobilization, which allows for magnetic separation as well as high enzyme activity/loading and stability.
2
Keywords Cellulose nanofibers; TEMPO-mediated oxidation; alpha-Chymotrypsin; Enzyme precipitate coating, Enzyme immobilization; Enzyme stabilization;
3
1. Introduction
Cellulose materials have gathered growing attention in various fields due to their natural abundance, low cost, and intriguing mechanical properties [1-5]. In particular, they are environmentally-friendly sustainable biomaterials with light weight, low thermal expansion, and non-toxicity [6]. Despite of these advantages, cellulose itself is a matrix with inherent problems, such as serious diffusional limitation, low surface area and low functional group density for chemical modification, mainly due to its highly crystalline hierarchical structure based on high density of intramolecular hydrogen bonding between cellulose molecules [7-9]. Many efforts have been made in order to break the intramolecular structure of cellulose by using ionic liquids or acid hydrolysis [10, 11], the high crystalline structure of cellulose is still limiting the versatile applications of cellulose in various fields. As one of the approaches to generate the cellulose derivatives, 2,2,6,6tetramethylpiperidine-1-oxyl
(TEMPO)
mediated
oxidation
of
cellulose
was
performed to prepare cellulose nanofibers (CNFs) with low energy cost [12]. The action of co-catalysts such as NaBr and NaClO allows for regioselective oxidation of C6 primary hydroxyls, producing electrostatically repulsive C6 carboxyl groups on cellulose microfibers in aqueous solution [13, 14]. Mechanical disintegration using a grinder after TEMPO oxidation enables the production of CNFs in a mild condition [15, 16]. The resulting CNFs are well dispersed in aqueous solution and contain larger surface area than cellulose before the treatment [12]. CNFs have been used as a carrier for the immobilization of biomolecules such as antibodies, DNAs, RNAs, and enzymes [17, 18]. In particular, enzymes such as lipase [19], laccase [20], and glucose oxidase [21] have been immobilized on CNFs due to 4
their promising properties as an environmentally-friendly biocatalysts working under mild reaction [22-24]. However, the approaches of enzyme immobilization such as physical adsorption [19] and covalent attachment [21] on CNFs resulted in low enzyme loading and poor enzyme stability, which has hampered their successful applications. To improve both loading and stability of enzymes, a three-step approach of enzyme precipitate coating (EPC), consisting of covalent enzyme attachment, enzyme precipitation and crosslinking, was proposed and has been successful in achieving both high enzyme loading and stability of glucose oxidase and carbonic anhydrase on nanostructured materials such as carbon nanotubes (CNTs) [25] and carboxylated polyaniline nanofibers (cPANFs) [26], respectively. In the present work, CNFs were prepared via TEMPO mediated oxidation and mechanical grinding, and used for the immobilization of model enzyme, αchymotrypsin (CT), via the EPC approach. Covalent attachment (CA) and enzyme coating (EC) with no enzyme precipitation step were also prepared as the control samples of EPC. CT is selected as a model enzyme, because CT is a tricky enzyme to stabilize due to its inactivation mechanism via autolysis [27] while it can be used for a variety of hydrolytic and synthetic reactions [28, 29]. We also mixed aminefunctionalized magnetic nanoparticles with enzymes during the precipitation and crosslinking steps to produce magnetically-separable EPC (Mag-EPC) allowing for facile magnetic separation. We investigated the morphology, activities and stabilities of CA, EC, EPC and Mag-EPC, and compared them from the standpoints of enzyme loading and stability, which are important for the practical successes of enzymes on environmentally friendly and economical CNFs in various enzyme applications.
2. Experimental 5
2.1. Materials
α-Chymotrypsin (CT) (EC.232-671-2) from bovine pancreas, glutaraldehyde (GA), ammonium sulfate (AS), N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP), N, Ndimethylformamide (DMF), ethyl(dimethylaminopropyl) carbodiimide (EDC), Nhydroxysuccinimide (NHS), ethylenediamine, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), sodium hypochlorite (NaClO) solution (4.5 v/v%), 2-(N-morpholino) ethanesulfonic acid (MES), sodium phosphate and Tris-HCl were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amine-functionalized magnetic nanoparticles (MNPs, 50 nm) were purchased from Chemicell (Berlin, Germany). A commercial bleached coffee filters from Thomas & Green Pte Ltd. (Singapore) were used as a source for native cellulose. Sodium bromide (>99.5%) was purchased from Samchun chemical (Pyeongtaek, South Korea).
2.2. Cellulose nanofiber preparation
Cellulose (20 g) from coffee filter was cut down into small pieces and dispersed in deionized water (DW) (2 L) containing TEMPO (0.312 g, 1 mM) and sodium bromide (2.058 g, 10 mM). TEMPO-mediated oxidation was initiated by adding NaClO solution (4.5%, 250 mL) to oxidize cellulose, followed by the stirring (100 rpm) at room temperature. The pH of cellulose slurry was maintained at 10 by adding NaOH solution (0.1 M) [12, 30-32]. After 12 hours of oxidation, TEMPO-oxidized cellulose was excessively washed with DW by filtration. Then, the grinding of oxidized cellulose was carried out by electric grinder (MKCA6-2, Masuko Sangyo, Kawaguchi, 6
Japan) at 15,000 rpm. Finally, cellulose nanofibers (CNFs) in the form of hydrogel were stored at room temperature until further usage.
2.3. Electric conductivity titration
The content of carboxyl group on CNFs was estimated by the electric conductivity titration method [31, 33, 34]. Freeze-dried CNF sample (230 mg) was added to DW (55 mL) with NaCl (2.922 mg), and the solution was stirred to obtain well-dispersed suspension. The pH of the suspension was set to 3.0 by adding 0.1 M HCl. After 3 hours of reaction, the pH was increased up to 11 by continuous addition of 0.04 M NaOH (0.1 mL/min). Conductivity was measured using the conductivity meter (Starter 3000c, Ohaus, Parsippany, NJ, USA), and the content of carboxyl groups was estimated by the titration curve of conductivity.
2.4. Enzyme immobilization on cellulose nanofibers
Carboxyl groups of CNFs were modified by EDC/NHS reaction for covalent attachment of α-chymotrypsin (CT). EDC (10 mg/mL) and NHS (50 mg/mL) in MES buffer (100 mM, pH 6.0) were added to CNFs, and the mixture was shaken at 200 rpm for 1 h. Then, EDC-treated CNFs were washed two times by distilled water. CA, EC, EPC and Mag-EPC were prepared in the following ways. To prepare the CA sample, 1 mL of CT solution containing 2 or 10 mg CT was mixed with EDC-treated CNFs, and the mixture was incubated under shaking (200 rpm) for 2 h in order to form covalent bonds between enzyme molecules and CNFs. For the preparation of EC sample, GA solution was added to the CA sample in the final GA concentration of 0.5% w/v, and 7
the mixture was incubated under shaking (200 rpm) for 30 min. The EPC sample was prepared by adding ammonium sulfate for the final concentration of 40% w/v, and the mixture was incubated under shaking (200 rpm) for 30 min. Then, the same GA treatment was performed as that for the preparation of EC sample. To make the MagEPC sample, amine-functionalized magnetic nanoparticles (MNP) and ammonium sulfate were added to the CA sample, and the mixture was incubated under shaking (200 rpm) for 30 min. Then, the same GA treatment was performed as those of EC and EPC samples. All samples were incubated at 4 °C under shaking (50 rpm) for 14 h. After rigorous washing by 100 mM PB (pH 7.8) three times, the mixtures were incubated under shaking (200 rpm) in the presence of ethylenediamine (100 mM) to cap the unreacted aldehyde groups for 30 min. Then, all the samples were washed with 100 mM PB (pH 7.8) under shaking (200 rpm) three times, and recovered by centrifugation at 12,000 rpm (CA, EC, and EPC) or by magnetic capture for 5 min (Mag-EPC). The CA, EC, EPC and Mag-EPC samples were stored in 100 mM PB (pH 7.8) at 4 °C until use.
2.5. Activity and stabilities measurements of immobilized CT samples on CNFs
The activities of CT samples were measured by the hydrolysis of N-Succinyl-AlaAla-Pro-Phe p-nitroanilide (TP) in 100mM PB (pH 7.8). 100 μL of CT samples were mixed with 1.9 mL of 100mM PB (pH 7.8) containing 20 μL of the TP solution (20 mg/mL in DMF), and the mixture was shaken at 200 rpm. Aliquots were taken timedependently for spectrophotometric measurement of product (p-nitroanilide) at the wavelength of 410 nm, and the activity was calculated from the initial slope of absorbance change with time. One unit of CT is defined by the CT amount to generate 8
1 μmole of p-nitroanilide from the CT-catalysed hydrolysis of TP within 1 min. The enzyme loadings of EC, EPC and Mag-EPC were estimated by directly measuring the nitrogen amounts in the final immobilizations via elemental analysis (EA 1112/2000, Thermo Fisher Scientific, Waltham, MA, USA). Elemental analysis was performed by preparing samples without the capping of unreacted aldehyde groups to prevent the addition of nitrogen. By this way, the nitrogen amount in each sample could be used to calculate the enzyme amount because the enzyme would be the only source for the elemental nitrogen. In order to estimate the enzyme loading of CA, the BCA assay (Thermo Fisher Scientific, Waltham, MA, USA) was performed by measuring the enzyme amount in solution before and after immobilization. To estimate the stability of immobilized CT samples, the activities of CA, EC, EPC and Mag-EPC were incubated in 100 mM PB (pH 7.8) at room temperature under rigorous shaking (200 rpm). At each time point, the residual activity of each sample was measured by using an aliquot as described above. The relative activity was calculated from the ratio of residual activity at each time point to the initial activity of each sample.
2.6. Transmission electron microscope (TEM) and field emission-scanning electron microscope (FE-SEM) analyses
The samples of CNFs, CA, EC, EPC and Mag-EPC were analyzed by using a high resolution-transmission electron microscope (HR-TEM, JEM-1011, JEOL, Tokyo, Japan). For HR-TEM analysis, a droplet of 0.05 wt% CNF hydrogel and enzyme immobilized samples were mounted on formvar/carbon coated copper grid. One drop of 2% uranyl acetate was added on each sample before drying for negative staining, and the samples were dried at room temperature. 9
For FE-SEM analysis, the samples of CA, EC, EPC and Mag-EPC were excessively washed with deionized water and freeze-dried for 24 h. After platinum coating, the samples were imaged using a field emission-scanning electron microscope (FE-SEM, Quanta 250 FEG, FEI, Hillsboro, OR, USA) at an accelerating voltage of 15 kV.
2.7. Cryogenic Transmission electron microscope (cryo-TEM) analysis
1 μL of the CNF hydrogel was transferred to a B-type aluminum planchette (Technotrade International, Manchester, NH, USA), and rapidly frozen in a Bal-Tec HPM 010 high-pressure freezer (Boeckeler Instruments, Tusson, AZ, USA). After freeze substitution for 5 days at −80 °C in from water to anhydrous acetone containing 2% OsO4, the samples were warmed to room temperature over 2 days (24 h from −80 to −20 °C, 20 h from −20 to 4 °C, 4 h from 4 to 20 °C). The structure of the CNF hydrogel was fixed with osmium during the freeze substitution. After 3 times washes with anhydrous acetone, the fixed CNF hydrogel was embedded in a graduated Epon resin (Ted Pella, Redding, CA, USA) dilution in acetone (5, 15, 25, 50, 75, and 100% (v/v)) over 3 days. After polymerization in a 60 °C oven for 24 h, the sample was microtomed into 200 nm-sections using a EM UC7 microtome (Leica, Munchen, Germany) and applied onto copper grids for TEM analysis.
2.8. Physical properties of the CNF hydrogel
Diameter of the cellulose nanofibers, average area of the pore in the CNF hydrogel were estimate from cryo-TEM image of the microtomed the CNF hydrogel with Image J software [35]. Porosity was calculated from the followed equation [36]. 10
VHydrogel = VCNF +Vpore = Wdry weight of CNF/ρCNF + Vpore. Thermogravimetric analysis (TGA, TA Instrument Q600, PH407, New Castle, DE, USA) was used to calculate the mass changes in the hydrogel and CNF as the temperature increases.
3. Results and discussion
3.1.Preparation and characterization of CNFs
To improve both loading and stability of enzymes for the successful applications of CNFs in practical enzyme reaction process, we synthesized cellulose nanofibers (CNFs) for enzyme immobilization by treating cellulose via TEMPO-mediated oxidation and follow-up grinding. Fig. 1 shows the production of CNF hydrogel from cellulose with the descriptions of structural and chemical changes during the conversion of cellulose to CNFs. The first step is the TEMPO oxidation to loosen up the tight cellulose structure, and the second step is the grinding in water to prepare the final 2 L of CNF hydrogels (Fig. 1a), which is massive production compare to previous studies [12, 13]. The FTIR spectra of cellulose and CNFs show the absorbance increase of peaks at 1620 and 1415 cm-1 with CNFs (Fig. 2a), which correspond to the C=O stretching frequency of free and salt forms of carboxylic acid, respectively. According to the electric conductivity titration, the amount of carboxyl groups per unit weight of CNFs was 1.41 mmol COOH/g CNFs. (Fig. S1 in Supporting Information) This result is within the previously reported range of carboxylate contents for effective disintegration of CNFs (0.7 to 1.52 mmol/g) [37]. It should be noted that longer TEMPO oxidation to increase carboxylate content in the 11
cellulose induce the depolymerization of glycoside bonds by β-elimination, which leads to lower CNFs yield [31, 37]. The TEMPO oxidation selectively oxidizes the 6OH groups at the glucose units on the surface of cellulose to deprotonated negatively charged carboxyl groups (COO-) upon the TEMPO oxidation [38]. In other words, the TEMPO oxidation changes the attractive hydrogen bonding force of 6-OH group to repulsive long-range electrostatic force of 6-COOH (Fig. 1b) [38]. As a result, many hydrogen bonds between cellulose fibers were broken upon TEMPO-oxidation, and the oxidized cellulose fibers with negatively charged carboxyl groups on their surface were further grinded in distilled water for the physical disintegration of cellulose into the hydrogel of CNFs. The grinding process led to the exposure of carboxyl and hydroxyl groups on the surface of CNF. Attractive hydrogen bonds between CNF can make crosslinks in the gel, and electrostatic repulsions due to the carboxyl groups can generate pores in the gel [39]. Indeed, these intermolecular interactions produce the cellulose nanofiber hydrogel (Fig. 1b), which has a nanofiber matrix with porous structure as observed in TEM images (Figs. 2b and 2c). According to analysis of cryoTEM image, the average diameter of the cellulose nanofibers in the hydrogel was uniform (~ 10 nm), the average surface area of pores in the CNF hydrogel, estimated from Cryo-TEM image, was 2.55 ± 0.78 μm2. The porosity of the CNF hydrogel was 99.3%. Thermogravimetric analysis (TGA) on the hydrogel was also conducted to check the thermos-stability of the CNF. No detectable mass change below 200 °C (Fig. S2 in Supporting Information) suggests the CNF is stable up to 200 °C. CNFs of high aspect ratios and fairly uniform diameters with large porosity can be used as the carrier for enzyme immobilization, starting with covalent enzyme attachment using the carboxyl groups on their surface. In addition, 1.41 mmol COOH/g CNFs from the titration measurement is approximately corresponding to one carboxyl group per four 12
monomeric units, which is sufficient for immobilizing enzymes with high density. The massive production of CNF hydrogel is efficient for making a large amount of enzyme immobilized substrate.
3.2. Immobilization of CTs on CNFs
Fig. 3a shows the immobilization procedures of CTs on the surface of CNFs based on approaches of covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically separable EPC (Mag-EPC). Covalent bonds were formed between enzymes and CNFs by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) linker for CA sample, while glutaraldehyde (GA) treatment was additionally carried out for the cross-linking of enzyme molecules to prepare EC sample. EPC was prepared via three steps of covalent attachment, enzyme precipitation by ammonium sulfate, and enzyme cross-linking with GA, while aminefunctionalized magnetic nanoparticles were added to enzyme solution in the step of enzyme precipitation to prepare Mag-EPC sample. To find an optimum condition of CNF concentration for the immobilization of CTs on CNFs, we checked the fluidity of CNFs hydrogels in various concentrations of CNFs from 1 mg/ml to 10 mg/ml. (Fig. S3 in Supporting Information). When the vial was upside down, 10 mg/ml of CNFs was too dense for the CNF hydrogel to flow down, while 1, 2, and 5 mg/ml of CNFs flew down under gravity. Preliminary experiments were done by using 2 mg/ml and 10 mg/ml of CNFs for the immobilization of CTs (Fig. S4 in Supporting Information). EPC and Mag-EPC showed apparent colour change due to the enzyme crosslinking upon GA treatment. Interestingly, EPC and Mag-EPC samples with 10 mg/ml CNFs show serious 13
aggregation of final immobilizations, while the use of 2 mg/ml CNFs did not show this kind of aggregation at all. This suggests that the dense CNF hydrogel with 10 mg/ml of CNFs (as shown in Fig. S3) prevents the efficient contact between CNFs and enzyme molecules, leading to inefficient immobilization of CTs on the surface of CNFs and aggregation of CNFs. On the other hand, the use of less dense CNF hydrogel with 2 mg/ml of CNFs resulted in an efficient enzyme immobilization on the CNF surface based on good contact between CNFs and enzyme molecules, which inhibits the aggregation of CNFs due to the shielding of CNF surface by the immobilized enzyme molecules. The activity of Mag-EPC with 2 mg/ml CNFs was about 8.1 times higher than that of Mag-EPC with 10 mg/ml CNFs (Fig. S5 in Supporting Information). If the enzyme molecules are immobilized on CNFs in the same efficiency of enzyme immobilization, the activity of Mag-EPC with 2 mg/ml CNFs should be five times higher than that of Mag-EPC with 10 mg/ml CNFs. The present result of better enzyme activity with 2 mg/ml CNFs, 8.1 times rather than 5 times, can be explained by the better contact between enzyme molecules and CNFs, which allow for more efficient enzyme immobilization. Mag-EPC with 2 mg/ml of CNFs could be captured by using a magnet within 30 seconds (Fig. 3b), and 2 mg/ml of CNFs was chosen for further studies of enzyme immobilization and their characterizations.
3.3. Activities and stabilities of CT/CNFs
The activities of immobilized CTs on CNFs via CA, EC, EPC and Mag-EPC were measured by the hydrolysis of N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP) in 100 mM PB (pH 7.8). Fig. 4a shows the activities of CA, EC, EPC, and Mag-EPC, which 14
are 0.067±0.010, 0.14±0.03, 1.3±0.3, and 2.6±0.2 units per milligram of CNFs, respectively (Fig. 4a). The activity of Mag-EPC was 38, 19, and 2.0 times higher than those of CA, EC and EPC, respectively. Higher activities of EPC and Mag-EPC than CA and EC reveals that ammonium sulfate precipitation and GA cross-linking improved the amount of enzyme loading on CNFs. The enzyme loadings of CA, EC, EPC and Mag-EPC were estimated by using BCA assay and elemental analysis. Conventionally, the enzyme loading of immobilized enzyme systems is indirectly estimated by measuring the enzyme amount in solution before/after immobilization and then calculating the disappeared enzyme amount from the solution upon enzyme immobilization. However, the enzyme loadings of EC, EPC and Mag-EPC are difficult to obtain due to the formation of insoluble crosslinked enzymes, which interferes with the correct measurement of enzyme amount via conventional protein assays because they are based on the soluble form of enzymes. As a bypass, elemental analysis was performed by using samples prepared without capping unreacted aldehyde groups by ethylenediamine. By this way, the nitrogen amount in each sample can be used for the calculation of the enzyme amount because the enzyme would be the only source for the elemental nitrogen. In case of CA, the BCA assay was used for estimation of its enzyme loading. The enzyme loadings of CA, EC, EPC and Mag-EPC were estimated to be 0.15, 1.7, 3.0 and 3.5 mg CT/mg CNF, respectively. Higher enzyme loadings of EPC and Mag-EPC were also observed in the SEM and TEM images (Figs. 5a and 5b). Interestingly, Mag-EPC showed two times higher activity than EPC. Due to a lot more amine groups on the surface of magnetic nanoparticles, the efficiency of cross-linking between enzymes and magnetic nanoparticles was improved, resulting in increased enzyme loading of Mag-EPC than EPC, as reflected in the activity data. The specific enzyme activities of free CT, CA, EC, EPC and Mag-EPC were 5.6, 0.45, 0.08, 0.43 15
and 0.74 units/mg CT, respectively. The low specific activity of immobilized CT on CNFs can be explained by the enzyme denaturation under shaking, the mass transfer limitation, and the enzyme deformation upon crosslinking. Interestingly, the specific activity of EC was 5.6 times lower than that of CA, revealing that the enzyme molecules would be inactivated upon chemical crosslinking without being precipitated. CNFs are mechanically stable (stiffness ~120 GPa) in both dry and wet conditions, and they do not dissolve in most aqueous and organic solvents [1]. In addition, TGA result (Fig. S2 in Supporting Information) shows that CNFs has high thermo-stability. These mechanical, chemical, and thermos- stabilities of CNF enable the long-term stability test (~30 days) of the immobilized CTs on CNFs under rigorous shaking (200 rpm). Fig. 4b shows the stabilities of CA, EC, EPC, and Mag-EPC, and the relative activity is defined as the ratio of residual activity at each time point to the initial activity of each sample. After incubation under rigorous shaking for 30 days, CA, EC, EPC and Mag-EPC maintained 12%, 46%, 77% and 50% of their initial activities, respectively, while free CT showed only 0.2% of its initial activity even after incubation in the same condition for 8 days. High stability of EPC can be explained by ammonium sulfate precipitation and cross-linking of CTs. Enzyme precipitation by ammonium sulfate allows enzyme molecules to be closely packed based on ‘salting out’ effect [40]. In other words, the addition of ammonium sulfate increases the ionic strength of enzyme solution, resulting in the aggregation and precipitation of enzyme molecules. The close-packing of enzyme molecules as a result of enzyme precipitation would allow for an efficient formation of multi-point chemical cross-linkages among enzyme molecules on CNFs upon GA treatment. Multi-point cross-linkages can effectively prevent both CT denaturation and detachment of CTs from CNFs [41, 42]. Interestingly, the activity of Mag-EPC was lower than that of EPC after 30 days, even 16
though Mag-EPC was prepared based on the same precipitation and cross-linking procedure. Some of large aggregates of CTs and magnetic nanoparticles, as reflected on the highest activity of Mag-EPC, could be detached from CNFs more easily than EPC, and detached CTs would be more vulnerable to denaturation under rigorous shaking (200 rpm).
3.4. Electron microscopic analyses of CT/CNFs
CA, EC, EPC and Mag-EPC samples were analyzed by using FE-SEM and FE-TEM. According to the FE-SEM images (Fig. 5a), CA and EC showed negligible changes in the morphology of CNFs when compared to pristine CNFs, and immobilized CT enzyme molecules could not be observed. On the other hand, the images of EPC and Mag-EPC showed that CNFs were covered by the large quantity of enzyme aggregates. This suggests that a lot more amount of CTs were immobilized on the CNFs by the approach of EPC and Mag-EPC than CA and EC approaches. These observations well correlate with the higher activities of EPC and Mag-EPC than CA and EC. In FE-TEM images (Fig. 5b), CNF and CA showed no difference between two of them. Mainly negative stained CNFs (white rod-shaped) were found. EC showed certain amount of stained enzymes (dark part around CNFs) around CNFs. In the case of EPC, a substantial amount of enzyme aggregates was visualized due to high enzyme density. Mag-EPC showed a vividly different image by barely showing the CNF image when compared to the other samples due to a lot more electron-dense magnetic inorganic nanoparticles than organic CNFs. However, we can still observe the rod shaped structure CNFs through the massive aggregation of enzymes and magnetic nanoparticles. Based on FE-SEM and FE-TEM analyses, it is confirmed that 17
the steps for ammonium sulfate precipitation and GA crosslinking have a major role in improving enzyme loadings of EPC and Mag-EPC.
4. Conclusions
This study has opened up the potential of cellulose nanofibers (CNFs) as a support material for stable, efficient, and recyclable enzyme immobilizations. Chymotrypsin (CT), a tricky enzyme to stabilize due to its inactivation via autolysis, could be successfully immobilized on CNFs via EPC and Mag-EPC methods. EPC and MagEPC significantly improved both enzyme loading and stability because ammonium sulfate mediated enzyme precipitation facilitates the stable form of CT aggregates on CNFs. In addition, magnetically-separable Mag-EPC allows for the recycling of stabilized and immobilized enzymes via a facile magnet capture. The present results of EPC and Mag-EPC have demonstrated the great potential of CNFs as a carrier of massive enzyme immobilization, which allows for magnetic separation as well as high enzyme loading and stability for the practical application of CNFs. Taken together, our comprehensive data suggest that other enzymes with various functions can also be massively immobilized and stabilized on CNFs for their practical successes in various enzyme applications.
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Acknowledgements This
research
was
supported
by
Global
Research
Laboratory
Program
(2014K1A1A2043032), the ‘2016, University-Institute Cooperation Program’, and the Basic Core Technology Development Program for the Oceans and the Polar Regions (2016M1A5A1027594) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning. This research was also supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (No. 20142020200980). This research was also supported by the Marine Biotechnology program (Marine BioMaterials Research Center) funded by the Ministry of Oceans and Fisheries, Korea (D11013214H480000110).
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[23] J. Kim, J.W. Grate, P. Wang, Nanobiocatalysis and Its Potential Applications, Trends Biotechnol. 26 (2008) 639-646. [24] K.M. Koeller, C.H. Wong, Enzymes for Chemical Synthesis, Nature 409 (2001) 232-240. [25] B.C. Kim, X. Zhao, H.-K. Ahn, J.H. Kim, H.-J. Lee, K.W. Kim, S. Nair, E. Hsiao, H. Jia, M.-K. Oh, B.I. Sang, B.-S. Kim, S.H. Kim, Y. Kwon, S. Ha, M.B. Gu, P. Wang, J. Kim, Highly Stable Enzyme Precipitate Coatings and Their Electrochemical Applications, Biosens. Bioelectron. 26 (2011) 1980-1986. [26] S.-G. Hong, H. Jeon, H.S. Kim, S.-H. Jun, E. Jin, J. Kim, One-Pot Enzymatic Conversion of Carbon Dioxide and Utilization for Improved Microbial Growth, Environ. Sci. Tehcnol. 49 (2015) 4466-4472. [27] S. Kumar, G.E. Hein, Concerning Mechanism of Autolysis of Alpha-Chymotrypsin, Biochemistry 9 (1970) 291-&. [28] M.E. Kimball, D.S.T. Hsieh, C.K. Rha, Chymotrypsin Hydrolysis of Soybean Protein, J. AGR. FOOD. CHEM. 29 (1981) 872-874. [29] D. Zhou, Z.X. Liu, D.H. Zhang, Y. Xu, W.B. Tan, L. Ma, Y. Sun, B. Shen, C.L. Zhu, Chymotrypsin Both Directly Modulates Bacterial Growth and Asserts Ampicillin DegradationMediated Protective Effect on Bacteria, Ann. Microbiol. 63 (2013) 623-631. [30] A.E.J. Denooy, A.C. Besemer, H. Vanbekkum, Highly Selective Nitroxyl Radical-Mediated Oxidation of Primary Alcohol Groups in Water-Soluble Glucans, Carbohyd. Res. 269 (1995) 89-98. [31] T. Saito, A. Isogai, TEMPO-Mediated Oxidation of Native Cellulose. The Effect of Oxidation Conditions on Chemical and Crystal Structures of The Water-Insoluble Fractions, Biomacromolecules 5 (2004) 1983-1989. [32] C. Tahiri, M.R. Vignon, TEMPO-Oxidation of Cellulose: Synthesis and Characterisation of Polyglucuronans, Cellulose 7 (2000) 177-188. [33] J. Araki, M. Wada, S. Kuga, Steric Stabilization of A Cellulose Microcrystal Suspension by Poly(Ethylene Glycol) Grafting, Langmuir 17 (2001) 21-27.
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Figure Captions
Fig. 1. (a) Schematic illustration for the preparation of cellulose nanofibers from cellulose via TEMPO-mediated oxidation and follow-up grinding. (b) Regioselective oxidation of C6 primary hydroxyls of cellulose to C6 carboxylate groups by TEMPO-mediated oxidation. Fig. 2. (a) FTIR spectra of cellulose source (A) and cellulose nanofibers (B) after TEMPO oxidation and grinding. (b) TEM and (c) cryo-TEM images of cellulose nanofibers. Fig. 3. (a) Schematics for enzyme immobilization methods: covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically separable EPC (Mag-EPC). (b) Magnetic separation of Mag-EPC. Fig. 4. (a) Activities of CA, EC, EPC and Mag-EPC. The CT activity was measured by the hydrolysis of N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP) in 100 mM PB (pH 7.8). One unit is defined by the enzyme amount to generate one μmole of p-nitroanilide within one minute from the CT-catalysed hydrolysis of TP. (b) Stabilities of CA, EC, EPC and MagEPC in aqueous buffer (100 mM PB, pH 7.8) under shaking (200 rpm). The relative activity is defined as the ratio of residual activity at each time point to the initial activity of each sample. Fig. 5. (a) FE-SEM and (b) HR-TEM images of CNFs, CA, EC, EPC and Mag-EPC.
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Figures
Fig. 1.
25
Fig. 2.
26
Fig. 3.
27
Fig. 4.
28
Fig. 5.
29
Highlights
Cellulose nanofibers (CNFs) were used as a carrier of chymotrypsin (CT) immobilization. Four different CT immobilization approaches on CNFs were performed. CT, a tricky enzyme to stabilize, was stably and massively immobilized on CNFs. Magnetic separation of enzyme precipitate coating on CNFs was effective. Potential of CNFs for stable and recyclable enzyme immobilization was demonstrated.
30
S1385-8947(17)30653-8 http://dx.doi.org/10.1016/j.cej.2017.04.110 CEJ 16864
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
29 November 2016 8 April 2017 25 April 2017
Please cite this article as: H.H. Je, S. Noh, S-G. Hong, Y. Ju, J. Kim, D.S. Hwang, Cellulose Nanofibers for Magnetically-Separable and Highly Loaded Enzyme Immobilization, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.04.110
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Cellulose Nanofibers for Magnetically-Separable and Highly Loaded Enzyme Immobilization
Hwa Heon Je1,†, Sora Noh2,†, Sung-Gil Hong2, Youngjun Ju2, Jungbae Kim2,3,*, Dong Soo Hwang1,4,*
1
Division of Integrative Bioscience and Biotechnology, Pohang University of Science and
Technology (POSTECH), Pohang 37673, Republic of Korea 2
Department of Chemical and Biological Engineering, Korea University, Seoul 02841,
Republic of Korea 3
Green School, Korea University, Seoul 02841, Republic of Korea
4
School of Environmental Science and Engineering, Pohang University of Science and
Technology (POSTECH), Pohang 37673, Republic of Korea
†These authors contributed equally to this work *Corresponding author. E-mail: [email protected] or [email protected]
1
Abstract Cellulose nanofibers (CNFs) are one of attractive supporting materials for enzyme immobilization due to their unique properties such as high surface area, high porosity and surface carboxyl groups for chemical bonding. In this study, CNFs were prepared via TEMPO-mediated oxidation and physical grinding of cellulose, and further used for the immobilization of α-chymotrypsin (CT) enzyme via four different approaches such as covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically-separable EPC (Mag-EPC). EPC approach consists of three steps: covalent enzyme attachment, enzyme precipitation and crosslinking, while EC represents a control without the step of enzyme precipitation. Amine-functionalized magnetic nanoparticles were added during the enzyme precipitation and crosslinking steps to produce magneticallyseparable EPC (Mag-EPC). The activities of CA, EC, EPC and Mag-EPC were 0.067, 0.14, 1.3 and 2.6 units per mg CNFs, respectively, representing that the activity of Mag-EPC was 38-, 19- and 2-times higher than those of CA, EC and EPC, respectively. After incubation under shaking (200 rpm) for 30 days, CA, EC, EPC and Mag-EPC maintained 12%, 46%, 77% and 50% of their initial activities, respectively, while free CT showed only 0.2% of its initial activity even after 8 days. Because CT is a tricky enzyme to stabilize due to its inactivation mechanism via autolysis, the present results of stable EPC and Mag-EPC on CNFs have demonstrated the great potential of CNFs as an environmentally-friendly and economical carrier of enzyme immobilization, which allows for magnetic separation as well as high enzyme activity/loading and stability.
2
Keywords Cellulose nanofibers; TEMPO-mediated oxidation; alpha-Chymotrypsin; Enzyme precipitate coating, Enzyme immobilization; Enzyme stabilization;
3
1. Introduction
Cellulose materials have gathered growing attention in various fields due to their natural abundance, low cost, and intriguing mechanical properties [1-5]. In particular, they are environmentally-friendly sustainable biomaterials with light weight, low thermal expansion, and non-toxicity [6]. Despite of these advantages, cellulose itself is a matrix with inherent problems, such as serious diffusional limitation, low surface area and low functional group density for chemical modification, mainly due to its highly crystalline hierarchical structure based on high density of intramolecular hydrogen bonding between cellulose molecules [7-9]. Many efforts have been made in order to break the intramolecular structure of cellulose by using ionic liquids or acid hydrolysis [10, 11], the high crystalline structure of cellulose is still limiting the versatile applications of cellulose in various fields. As one of the approaches to generate the cellulose derivatives, 2,2,6,6tetramethylpiperidine-1-oxyl
(TEMPO)
mediated
oxidation
of
cellulose
was
performed to prepare cellulose nanofibers (CNFs) with low energy cost [12]. The action of co-catalysts such as NaBr and NaClO allows for regioselective oxidation of C6 primary hydroxyls, producing electrostatically repulsive C6 carboxyl groups on cellulose microfibers in aqueous solution [13, 14]. Mechanical disintegration using a grinder after TEMPO oxidation enables the production of CNFs in a mild condition [15, 16]. The resulting CNFs are well dispersed in aqueous solution and contain larger surface area than cellulose before the treatment [12]. CNFs have been used as a carrier for the immobilization of biomolecules such as antibodies, DNAs, RNAs, and enzymes [17, 18]. In particular, enzymes such as lipase [19], laccase [20], and glucose oxidase [21] have been immobilized on CNFs due to 4
their promising properties as an environmentally-friendly biocatalysts working under mild reaction [22-24]. However, the approaches of enzyme immobilization such as physical adsorption [19] and covalent attachment [21] on CNFs resulted in low enzyme loading and poor enzyme stability, which has hampered their successful applications. To improve both loading and stability of enzymes, a three-step approach of enzyme precipitate coating (EPC), consisting of covalent enzyme attachment, enzyme precipitation and crosslinking, was proposed and has been successful in achieving both high enzyme loading and stability of glucose oxidase and carbonic anhydrase on nanostructured materials such as carbon nanotubes (CNTs) [25] and carboxylated polyaniline nanofibers (cPANFs) [26], respectively. In the present work, CNFs were prepared via TEMPO mediated oxidation and mechanical grinding, and used for the immobilization of model enzyme, αchymotrypsin (CT), via the EPC approach. Covalent attachment (CA) and enzyme coating (EC) with no enzyme precipitation step were also prepared as the control samples of EPC. CT is selected as a model enzyme, because CT is a tricky enzyme to stabilize due to its inactivation mechanism via autolysis [27] while it can be used for a variety of hydrolytic and synthetic reactions [28, 29]. We also mixed aminefunctionalized magnetic nanoparticles with enzymes during the precipitation and crosslinking steps to produce magnetically-separable EPC (Mag-EPC) allowing for facile magnetic separation. We investigated the morphology, activities and stabilities of CA, EC, EPC and Mag-EPC, and compared them from the standpoints of enzyme loading and stability, which are important for the practical successes of enzymes on environmentally friendly and economical CNFs in various enzyme applications.
2. Experimental 5
2.1. Materials
α-Chymotrypsin (CT) (EC.232-671-2) from bovine pancreas, glutaraldehyde (GA), ammonium sulfate (AS), N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP), N, Ndimethylformamide (DMF), ethyl(dimethylaminopropyl) carbodiimide (EDC), Nhydroxysuccinimide (NHS), ethylenediamine, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), sodium hypochlorite (NaClO) solution (4.5 v/v%), 2-(N-morpholino) ethanesulfonic acid (MES), sodium phosphate and Tris-HCl were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amine-functionalized magnetic nanoparticles (MNPs, 50 nm) were purchased from Chemicell (Berlin, Germany). A commercial bleached coffee filters from Thomas & Green Pte Ltd. (Singapore) were used as a source for native cellulose. Sodium bromide (>99.5%) was purchased from Samchun chemical (Pyeongtaek, South Korea).
2.2. Cellulose nanofiber preparation
Cellulose (20 g) from coffee filter was cut down into small pieces and dispersed in deionized water (DW) (2 L) containing TEMPO (0.312 g, 1 mM) and sodium bromide (2.058 g, 10 mM). TEMPO-mediated oxidation was initiated by adding NaClO solution (4.5%, 250 mL) to oxidize cellulose, followed by the stirring (100 rpm) at room temperature. The pH of cellulose slurry was maintained at 10 by adding NaOH solution (0.1 M) [12, 30-32]. After 12 hours of oxidation, TEMPO-oxidized cellulose was excessively washed with DW by filtration. Then, the grinding of oxidized cellulose was carried out by electric grinder (MKCA6-2, Masuko Sangyo, Kawaguchi, 6
Japan) at 15,000 rpm. Finally, cellulose nanofibers (CNFs) in the form of hydrogel were stored at room temperature until further usage.
2.3. Electric conductivity titration
The content of carboxyl group on CNFs was estimated by the electric conductivity titration method [31, 33, 34]. Freeze-dried CNF sample (230 mg) was added to DW (55 mL) with NaCl (2.922 mg), and the solution was stirred to obtain well-dispersed suspension. The pH of the suspension was set to 3.0 by adding 0.1 M HCl. After 3 hours of reaction, the pH was increased up to 11 by continuous addition of 0.04 M NaOH (0.1 mL/min). Conductivity was measured using the conductivity meter (Starter 3000c, Ohaus, Parsippany, NJ, USA), and the content of carboxyl groups was estimated by the titration curve of conductivity.
2.4. Enzyme immobilization on cellulose nanofibers
Carboxyl groups of CNFs were modified by EDC/NHS reaction for covalent attachment of α-chymotrypsin (CT). EDC (10 mg/mL) and NHS (50 mg/mL) in MES buffer (100 mM, pH 6.0) were added to CNFs, and the mixture was shaken at 200 rpm for 1 h. Then, EDC-treated CNFs were washed two times by distilled water. CA, EC, EPC and Mag-EPC were prepared in the following ways. To prepare the CA sample, 1 mL of CT solution containing 2 or 10 mg CT was mixed with EDC-treated CNFs, and the mixture was incubated under shaking (200 rpm) for 2 h in order to form covalent bonds between enzyme molecules and CNFs. For the preparation of EC sample, GA solution was added to the CA sample in the final GA concentration of 0.5% w/v, and 7
the mixture was incubated under shaking (200 rpm) for 30 min. The EPC sample was prepared by adding ammonium sulfate for the final concentration of 40% w/v, and the mixture was incubated under shaking (200 rpm) for 30 min. Then, the same GA treatment was performed as that for the preparation of EC sample. To make the MagEPC sample, amine-functionalized magnetic nanoparticles (MNP) and ammonium sulfate were added to the CA sample, and the mixture was incubated under shaking (200 rpm) for 30 min. Then, the same GA treatment was performed as those of EC and EPC samples. All samples were incubated at 4 °C under shaking (50 rpm) for 14 h. After rigorous washing by 100 mM PB (pH 7.8) three times, the mixtures were incubated under shaking (200 rpm) in the presence of ethylenediamine (100 mM) to cap the unreacted aldehyde groups for 30 min. Then, all the samples were washed with 100 mM PB (pH 7.8) under shaking (200 rpm) three times, and recovered by centrifugation at 12,000 rpm (CA, EC, and EPC) or by magnetic capture for 5 min (Mag-EPC). The CA, EC, EPC and Mag-EPC samples were stored in 100 mM PB (pH 7.8) at 4 °C until use.
2.5. Activity and stabilities measurements of immobilized CT samples on CNFs
The activities of CT samples were measured by the hydrolysis of N-Succinyl-AlaAla-Pro-Phe p-nitroanilide (TP) in 100mM PB (pH 7.8). 100 μL of CT samples were mixed with 1.9 mL of 100mM PB (pH 7.8) containing 20 μL of the TP solution (20 mg/mL in DMF), and the mixture was shaken at 200 rpm. Aliquots were taken timedependently for spectrophotometric measurement of product (p-nitroanilide) at the wavelength of 410 nm, and the activity was calculated from the initial slope of absorbance change with time. One unit of CT is defined by the CT amount to generate 8
1 μmole of p-nitroanilide from the CT-catalysed hydrolysis of TP within 1 min. The enzyme loadings of EC, EPC and Mag-EPC were estimated by directly measuring the nitrogen amounts in the final immobilizations via elemental analysis (EA 1112/2000, Thermo Fisher Scientific, Waltham, MA, USA). Elemental analysis was performed by preparing samples without the capping of unreacted aldehyde groups to prevent the addition of nitrogen. By this way, the nitrogen amount in each sample could be used to calculate the enzyme amount because the enzyme would be the only source for the elemental nitrogen. In order to estimate the enzyme loading of CA, the BCA assay (Thermo Fisher Scientific, Waltham, MA, USA) was performed by measuring the enzyme amount in solution before and after immobilization. To estimate the stability of immobilized CT samples, the activities of CA, EC, EPC and Mag-EPC were incubated in 100 mM PB (pH 7.8) at room temperature under rigorous shaking (200 rpm). At each time point, the residual activity of each sample was measured by using an aliquot as described above. The relative activity was calculated from the ratio of residual activity at each time point to the initial activity of each sample.
2.6. Transmission electron microscope (TEM) and field emission-scanning electron microscope (FE-SEM) analyses
The samples of CNFs, CA, EC, EPC and Mag-EPC were analyzed by using a high resolution-transmission electron microscope (HR-TEM, JEM-1011, JEOL, Tokyo, Japan). For HR-TEM analysis, a droplet of 0.05 wt% CNF hydrogel and enzyme immobilized samples were mounted on formvar/carbon coated copper grid. One drop of 2% uranyl acetate was added on each sample before drying for negative staining, and the samples were dried at room temperature. 9
For FE-SEM analysis, the samples of CA, EC, EPC and Mag-EPC were excessively washed with deionized water and freeze-dried for 24 h. After platinum coating, the samples were imaged using a field emission-scanning electron microscope (FE-SEM, Quanta 250 FEG, FEI, Hillsboro, OR, USA) at an accelerating voltage of 15 kV.
2.7. Cryogenic Transmission electron microscope (cryo-TEM) analysis
1 μL of the CNF hydrogel was transferred to a B-type aluminum planchette (Technotrade International, Manchester, NH, USA), and rapidly frozen in a Bal-Tec HPM 010 high-pressure freezer (Boeckeler Instruments, Tusson, AZ, USA). After freeze substitution for 5 days at −80 °C in from water to anhydrous acetone containing 2% OsO4, the samples were warmed to room temperature over 2 days (24 h from −80 to −20 °C, 20 h from −20 to 4 °C, 4 h from 4 to 20 °C). The structure of the CNF hydrogel was fixed with osmium during the freeze substitution. After 3 times washes with anhydrous acetone, the fixed CNF hydrogel was embedded in a graduated Epon resin (Ted Pella, Redding, CA, USA) dilution in acetone (5, 15, 25, 50, 75, and 100% (v/v)) over 3 days. After polymerization in a 60 °C oven for 24 h, the sample was microtomed into 200 nm-sections using a EM UC7 microtome (Leica, Munchen, Germany) and applied onto copper grids for TEM analysis.
2.8. Physical properties of the CNF hydrogel
Diameter of the cellulose nanofibers, average area of the pore in the CNF hydrogel were estimate from cryo-TEM image of the microtomed the CNF hydrogel with Image J software [35]. Porosity was calculated from the followed equation [36]. 10
VHydrogel = VCNF +Vpore = Wdry weight of CNF/ρCNF + Vpore. Thermogravimetric analysis (TGA, TA Instrument Q600, PH407, New Castle, DE, USA) was used to calculate the mass changes in the hydrogel and CNF as the temperature increases.
3. Results and discussion
3.1.Preparation and characterization of CNFs
To improve both loading and stability of enzymes for the successful applications of CNFs in practical enzyme reaction process, we synthesized cellulose nanofibers (CNFs) for enzyme immobilization by treating cellulose via TEMPO-mediated oxidation and follow-up grinding. Fig. 1 shows the production of CNF hydrogel from cellulose with the descriptions of structural and chemical changes during the conversion of cellulose to CNFs. The first step is the TEMPO oxidation to loosen up the tight cellulose structure, and the second step is the grinding in water to prepare the final 2 L of CNF hydrogels (Fig. 1a), which is massive production compare to previous studies [12, 13]. The FTIR spectra of cellulose and CNFs show the absorbance increase of peaks at 1620 and 1415 cm-1 with CNFs (Fig. 2a), which correspond to the C=O stretching frequency of free and salt forms of carboxylic acid, respectively. According to the electric conductivity titration, the amount of carboxyl groups per unit weight of CNFs was 1.41 mmol COOH/g CNFs. (Fig. S1 in Supporting Information) This result is within the previously reported range of carboxylate contents for effective disintegration of CNFs (0.7 to 1.52 mmol/g) [37]. It should be noted that longer TEMPO oxidation to increase carboxylate content in the 11
cellulose induce the depolymerization of glycoside bonds by β-elimination, which leads to lower CNFs yield [31, 37]. The TEMPO oxidation selectively oxidizes the 6OH groups at the glucose units on the surface of cellulose to deprotonated negatively charged carboxyl groups (COO-) upon the TEMPO oxidation [38]. In other words, the TEMPO oxidation changes the attractive hydrogen bonding force of 6-OH group to repulsive long-range electrostatic force of 6-COOH (Fig. 1b) [38]. As a result, many hydrogen bonds between cellulose fibers were broken upon TEMPO-oxidation, and the oxidized cellulose fibers with negatively charged carboxyl groups on their surface were further grinded in distilled water for the physical disintegration of cellulose into the hydrogel of CNFs. The grinding process led to the exposure of carboxyl and hydroxyl groups on the surface of CNF. Attractive hydrogen bonds between CNF can make crosslinks in the gel, and electrostatic repulsions due to the carboxyl groups can generate pores in the gel [39]. Indeed, these intermolecular interactions produce the cellulose nanofiber hydrogel (Fig. 1b), which has a nanofiber matrix with porous structure as observed in TEM images (Figs. 2b and 2c). According to analysis of cryoTEM image, the average diameter of the cellulose nanofibers in the hydrogel was uniform (~ 10 nm), the average surface area of pores in the CNF hydrogel, estimated from Cryo-TEM image, was 2.55 ± 0.78 μm2. The porosity of the CNF hydrogel was 99.3%. Thermogravimetric analysis (TGA) on the hydrogel was also conducted to check the thermos-stability of the CNF. No detectable mass change below 200 °C (Fig. S2 in Supporting Information) suggests the CNF is stable up to 200 °C. CNFs of high aspect ratios and fairly uniform diameters with large porosity can be used as the carrier for enzyme immobilization, starting with covalent enzyme attachment using the carboxyl groups on their surface. In addition, 1.41 mmol COOH/g CNFs from the titration measurement is approximately corresponding to one carboxyl group per four 12
monomeric units, which is sufficient for immobilizing enzymes with high density. The massive production of CNF hydrogel is efficient for making a large amount of enzyme immobilized substrate.
3.2. Immobilization of CTs on CNFs
Fig. 3a shows the immobilization procedures of CTs on the surface of CNFs based on approaches of covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically separable EPC (Mag-EPC). Covalent bonds were formed between enzymes and CNFs by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) linker for CA sample, while glutaraldehyde (GA) treatment was additionally carried out for the cross-linking of enzyme molecules to prepare EC sample. EPC was prepared via three steps of covalent attachment, enzyme precipitation by ammonium sulfate, and enzyme cross-linking with GA, while aminefunctionalized magnetic nanoparticles were added to enzyme solution in the step of enzyme precipitation to prepare Mag-EPC sample. To find an optimum condition of CNF concentration for the immobilization of CTs on CNFs, we checked the fluidity of CNFs hydrogels in various concentrations of CNFs from 1 mg/ml to 10 mg/ml. (Fig. S3 in Supporting Information). When the vial was upside down, 10 mg/ml of CNFs was too dense for the CNF hydrogel to flow down, while 1, 2, and 5 mg/ml of CNFs flew down under gravity. Preliminary experiments were done by using 2 mg/ml and 10 mg/ml of CNFs for the immobilization of CTs (Fig. S4 in Supporting Information). EPC and Mag-EPC showed apparent colour change due to the enzyme crosslinking upon GA treatment. Interestingly, EPC and Mag-EPC samples with 10 mg/ml CNFs show serious 13
aggregation of final immobilizations, while the use of 2 mg/ml CNFs did not show this kind of aggregation at all. This suggests that the dense CNF hydrogel with 10 mg/ml of CNFs (as shown in Fig. S3) prevents the efficient contact between CNFs and enzyme molecules, leading to inefficient immobilization of CTs on the surface of CNFs and aggregation of CNFs. On the other hand, the use of less dense CNF hydrogel with 2 mg/ml of CNFs resulted in an efficient enzyme immobilization on the CNF surface based on good contact between CNFs and enzyme molecules, which inhibits the aggregation of CNFs due to the shielding of CNF surface by the immobilized enzyme molecules. The activity of Mag-EPC with 2 mg/ml CNFs was about 8.1 times higher than that of Mag-EPC with 10 mg/ml CNFs (Fig. S5 in Supporting Information). If the enzyme molecules are immobilized on CNFs in the same efficiency of enzyme immobilization, the activity of Mag-EPC with 2 mg/ml CNFs should be five times higher than that of Mag-EPC with 10 mg/ml CNFs. The present result of better enzyme activity with 2 mg/ml CNFs, 8.1 times rather than 5 times, can be explained by the better contact between enzyme molecules and CNFs, which allow for more efficient enzyme immobilization. Mag-EPC with 2 mg/ml of CNFs could be captured by using a magnet within 30 seconds (Fig. 3b), and 2 mg/ml of CNFs was chosen for further studies of enzyme immobilization and their characterizations.
3.3. Activities and stabilities of CT/CNFs
The activities of immobilized CTs on CNFs via CA, EC, EPC and Mag-EPC were measured by the hydrolysis of N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP) in 100 mM PB (pH 7.8). Fig. 4a shows the activities of CA, EC, EPC, and Mag-EPC, which 14
are 0.067±0.010, 0.14±0.03, 1.3±0.3, and 2.6±0.2 units per milligram of CNFs, respectively (Fig. 4a). The activity of Mag-EPC was 38, 19, and 2.0 times higher than those of CA, EC and EPC, respectively. Higher activities of EPC and Mag-EPC than CA and EC reveals that ammonium sulfate precipitation and GA cross-linking improved the amount of enzyme loading on CNFs. The enzyme loadings of CA, EC, EPC and Mag-EPC were estimated by using BCA assay and elemental analysis. Conventionally, the enzyme loading of immobilized enzyme systems is indirectly estimated by measuring the enzyme amount in solution before/after immobilization and then calculating the disappeared enzyme amount from the solution upon enzyme immobilization. However, the enzyme loadings of EC, EPC and Mag-EPC are difficult to obtain due to the formation of insoluble crosslinked enzymes, which interferes with the correct measurement of enzyme amount via conventional protein assays because they are based on the soluble form of enzymes. As a bypass, elemental analysis was performed by using samples prepared without capping unreacted aldehyde groups by ethylenediamine. By this way, the nitrogen amount in each sample can be used for the calculation of the enzyme amount because the enzyme would be the only source for the elemental nitrogen. In case of CA, the BCA assay was used for estimation of its enzyme loading. The enzyme loadings of CA, EC, EPC and Mag-EPC were estimated to be 0.15, 1.7, 3.0 and 3.5 mg CT/mg CNF, respectively. Higher enzyme loadings of EPC and Mag-EPC were also observed in the SEM and TEM images (Figs. 5a and 5b). Interestingly, Mag-EPC showed two times higher activity than EPC. Due to a lot more amine groups on the surface of magnetic nanoparticles, the efficiency of cross-linking between enzymes and magnetic nanoparticles was improved, resulting in increased enzyme loading of Mag-EPC than EPC, as reflected in the activity data. The specific enzyme activities of free CT, CA, EC, EPC and Mag-EPC were 5.6, 0.45, 0.08, 0.43 15
and 0.74 units/mg CT, respectively. The low specific activity of immobilized CT on CNFs can be explained by the enzyme denaturation under shaking, the mass transfer limitation, and the enzyme deformation upon crosslinking. Interestingly, the specific activity of EC was 5.6 times lower than that of CA, revealing that the enzyme molecules would be inactivated upon chemical crosslinking without being precipitated. CNFs are mechanically stable (stiffness ~120 GPa) in both dry and wet conditions, and they do not dissolve in most aqueous and organic solvents [1]. In addition, TGA result (Fig. S2 in Supporting Information) shows that CNFs has high thermo-stability. These mechanical, chemical, and thermos- stabilities of CNF enable the long-term stability test (~30 days) of the immobilized CTs on CNFs under rigorous shaking (200 rpm). Fig. 4b shows the stabilities of CA, EC, EPC, and Mag-EPC, and the relative activity is defined as the ratio of residual activity at each time point to the initial activity of each sample. After incubation under rigorous shaking for 30 days, CA, EC, EPC and Mag-EPC maintained 12%, 46%, 77% and 50% of their initial activities, respectively, while free CT showed only 0.2% of its initial activity even after incubation in the same condition for 8 days. High stability of EPC can be explained by ammonium sulfate precipitation and cross-linking of CTs. Enzyme precipitation by ammonium sulfate allows enzyme molecules to be closely packed based on ‘salting out’ effect [40]. In other words, the addition of ammonium sulfate increases the ionic strength of enzyme solution, resulting in the aggregation and precipitation of enzyme molecules. The close-packing of enzyme molecules as a result of enzyme precipitation would allow for an efficient formation of multi-point chemical cross-linkages among enzyme molecules on CNFs upon GA treatment. Multi-point cross-linkages can effectively prevent both CT denaturation and detachment of CTs from CNFs [41, 42]. Interestingly, the activity of Mag-EPC was lower than that of EPC after 30 days, even 16
though Mag-EPC was prepared based on the same precipitation and cross-linking procedure. Some of large aggregates of CTs and magnetic nanoparticles, as reflected on the highest activity of Mag-EPC, could be detached from CNFs more easily than EPC, and detached CTs would be more vulnerable to denaturation under rigorous shaking (200 rpm).
3.4. Electron microscopic analyses of CT/CNFs
CA, EC, EPC and Mag-EPC samples were analyzed by using FE-SEM and FE-TEM. According to the FE-SEM images (Fig. 5a), CA and EC showed negligible changes in the morphology of CNFs when compared to pristine CNFs, and immobilized CT enzyme molecules could not be observed. On the other hand, the images of EPC and Mag-EPC showed that CNFs were covered by the large quantity of enzyme aggregates. This suggests that a lot more amount of CTs were immobilized on the CNFs by the approach of EPC and Mag-EPC than CA and EC approaches. These observations well correlate with the higher activities of EPC and Mag-EPC than CA and EC. In FE-TEM images (Fig. 5b), CNF and CA showed no difference between two of them. Mainly negative stained CNFs (white rod-shaped) were found. EC showed certain amount of stained enzymes (dark part around CNFs) around CNFs. In the case of EPC, a substantial amount of enzyme aggregates was visualized due to high enzyme density. Mag-EPC showed a vividly different image by barely showing the CNF image when compared to the other samples due to a lot more electron-dense magnetic inorganic nanoparticles than organic CNFs. However, we can still observe the rod shaped structure CNFs through the massive aggregation of enzymes and magnetic nanoparticles. Based on FE-SEM and FE-TEM analyses, it is confirmed that 17
the steps for ammonium sulfate precipitation and GA crosslinking have a major role in improving enzyme loadings of EPC and Mag-EPC.
4. Conclusions
This study has opened up the potential of cellulose nanofibers (CNFs) as a support material for stable, efficient, and recyclable enzyme immobilizations. Chymotrypsin (CT), a tricky enzyme to stabilize due to its inactivation via autolysis, could be successfully immobilized on CNFs via EPC and Mag-EPC methods. EPC and MagEPC significantly improved both enzyme loading and stability because ammonium sulfate mediated enzyme precipitation facilitates the stable form of CT aggregates on CNFs. In addition, magnetically-separable Mag-EPC allows for the recycling of stabilized and immobilized enzymes via a facile magnet capture. The present results of EPC and Mag-EPC have demonstrated the great potential of CNFs as a carrier of massive enzyme immobilization, which allows for magnetic separation as well as high enzyme loading and stability for the practical application of CNFs. Taken together, our comprehensive data suggest that other enzymes with various functions can also be massively immobilized and stabilized on CNFs for their practical successes in various enzyme applications.
18
Acknowledgements This
research
was
supported
by
Global
Research
Laboratory
Program
(2014K1A1A2043032), the ‘2016, University-Institute Cooperation Program’, and the Basic Core Technology Development Program for the Oceans and the Polar Regions (2016M1A5A1027594) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning. This research was also supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (No. 20142020200980). This research was also supported by the Marine Biotechnology program (Marine BioMaterials Research Center) funded by the Ministry of Oceans and Fisheries, Korea (D11013214H480000110).
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Figure Captions
Fig. 1. (a) Schematic illustration for the preparation of cellulose nanofibers from cellulose via TEMPO-mediated oxidation and follow-up grinding. (b) Regioselective oxidation of C6 primary hydroxyls of cellulose to C6 carboxylate groups by TEMPO-mediated oxidation. Fig. 2. (a) FTIR spectra of cellulose source (A) and cellulose nanofibers (B) after TEMPO oxidation and grinding. (b) TEM and (c) cryo-TEM images of cellulose nanofibers. Fig. 3. (a) Schematics for enzyme immobilization methods: covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically separable EPC (Mag-EPC). (b) Magnetic separation of Mag-EPC. Fig. 4. (a) Activities of CA, EC, EPC and Mag-EPC. The CT activity was measured by the hydrolysis of N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP) in 100 mM PB (pH 7.8). One unit is defined by the enzyme amount to generate one μmole of p-nitroanilide within one minute from the CT-catalysed hydrolysis of TP. (b) Stabilities of CA, EC, EPC and MagEPC in aqueous buffer (100 mM PB, pH 7.8) under shaking (200 rpm). The relative activity is defined as the ratio of residual activity at each time point to the initial activity of each sample. Fig. 5. (a) FE-SEM and (b) HR-TEM images of CNFs, CA, EC, EPC and Mag-EPC.
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Figures
Fig. 1.
25
Fig. 2.
26
Fig. 3.
27
Fig. 4.
28
Fig. 5.
29
Highlights
Cellulose nanofibers (CNFs) were used as a carrier of chymotrypsin (CT) immobilization. Four different CT immobilization approaches on CNFs were performed. CT, a tricky enzyme to stabilize, was stably and massively immobilized on CNFs. Magnetic separation of enzyme precipitate coating on CNFs was effective. Potential of CNFs for stable and recyclable enzyme immobilization was demonstrated.
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