Concanavalin A Coated Activated Carbon for High Performance

Subscriber access provided by GAZI UNIV

Letter

Concanavalin A Coated Activated Carbon for High Performance Enzymatic Catalysis Weina Xu, You Yong, Zheyu Wang, Guoqiang Jiang, Jianzhong Wu, and Zheng Liu ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Sustainable Chemistry & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Concanavalin A Coated Activated Carbon for High Performance Enzymatic Catalysis †

†

†

†

‡

Weina Xu, You Yong, Zheyu Wang, Guoqiang Jiang, Jianzhong Wu and Zheng Liu*

†

†Key Lab for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China. ‡ Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, USA. Corresponding author * Email: [email protected] KEYWORDS: Enzyme immobilization, Concanavalin A, activated carbon, phenol removal, laccase ， horseradish peroxidase ABSTRACT: A facile method of immobilizing enzymes on activated carbon (AC) is proposed, in which the first step is to coat AC with Concanavalin A (ConA), followed by the adsorption of enzymes. Two model glycoenzymes, horseradish peroxidase (HRP) and laccase, were immobilized on the ConA adsorbed AC through the tightly specific recognition of ConA to their glycosidic moieties, as confirmed by laser confocal microscopy. The coating layer of ConA

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 24

reduced the deactivation of enzymes and prevented the leakage of enzymes, as indicated by the significantly improved yield of enzymatic activity. The immobilized enzymes on ConA coated AC appeared an improved stability against pH and temperature, offering an expanded operation “window” for growing applications of the enzymatic catalysis. Finally, the integration of the adsorption capacity of AC and the chemical degradation of laccase promises the efficient removal of aqueous phenol. The improved recovery of enzyme activity, enhanced stability as well as the combination of substrate adsorption and enzymatic reaction make ConA coated AC appealing for various enzymatic catalysis.

INTRODUCTION Activated carbon (AC) is a common porous material with proven advantages in terms of high adsorption capacity, excellent mechanical property, and ease of functionalization with numerous chemical or physical methods.1-2 In practice, AC is widely used for liquid and gas treatment,3-4 supercapacitors,5 fuel cells,6 and various heterogeneous catalysis.7 Also noteworthy is that AC can be made from diversified renewable resources such as wood,8 coconut shell,9 bamboo,10 making AC attractive in sustainable chemical engineering. Immobilized enzymes are a preferable choice for industrial practice since immobilization simplifies the enzyme recovery, stabilizes multiple protein complexes, and enables a wider “operation window” of enzymatic catalysis as compared with their native counterparts in solution form.11-16 In fact, the earliest report of immobilizing enzyme on AC can be found in 1910s.17 While physical adsorption is seemingly straightforward for enzyme immobilization, the surface force may lead to undesirable conformational changes and thus deactivation of the enzymes.18-19 Moreover, the leakage of adsorbed enzymes prevents the practice of this idea. Chemical approaches such as crosslinking with glutaraldehyde, as described by Stoner and co-workers,20 and covalent binding with


2

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


chemically modified AC, as described by Baily21 and co-workers, are effective in preventing enzyme leakage, the low activity caused by chemical treatment and harsh reaction conditions remain a major challenge hindering their practical applications.1, 22-23 Concanavalin A (ConA) is a type of lectin known for its capability to agglutinate glycoproteins through molecular recognition of carbohydrate containing non- reducing terminal α-D-mannosyl or α-D-glucosyl moieties.24 ConA has been used as a molecule linker in enzyme and cell systems. Mislovicova25 and co-workers examined the effect of bioaffinity immobilization by precipitation of glycoenzyme with lectin. Chen26 and co-workers applied ConA modified nanoparticles to evaluating N-glycan expression on cell surface. Moreover, Mallardi27 and co-workers found that the leakage of enzymes inside calcium alginate beads could be avoided by forming large protein aggregates with ConA. On the other hand, Huang and coworkers28 proposed a ConA enabled self-assembly procedure to immobilize glycoenzymes on single-walled carbon nanotube (SWNT), in which the first step is to coat SWNT with n-dodecyl β-D-maltoside through hydrophobic adsorption, followed by assembly of ConA and glycoenzymes on the surface of SWNT upon the specific affinity between ConA and glycosyl groups. In addition to a higher enzyme loading, a higher activity is obtained in comparison to that obtained by direct adsorption. This can be attributed to the presence of ConA that stabilizes the tertiary structure of glycoenzymes by shielding the direct contact with hydrophobic surface of SWNT. While ConA has been widely used for the immobilization of glycoenzymes,29-31 its application as a molecule linker to immobilize enzymes on AC has not been reported before. We thus directed our efforts to explore the potential of ConA for enzyme immobilization on AC. We hope that, in addition to inhibit the leakage of enzymes by forming enzyme aggregate, ConA


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 24

may help to prevent the deactivation of enzymes at the AC surface as shown in physical adsorption and covalent binding. Here we report a two-step adsorption procedure to immobilize enzyme on AC, which yields both a higher activity and a higher reusability, as compared to above-mentioned methods. As shown in Scheme 1, the first step is to adsorb ConA on AC, and the second step is to attach enzyme on the ConA- coated AC surface through affinitive adsorption. In the present study, we demonstrate the feasibility of this method using laccase and horseradish peroxidase (HRP), two frequently-used glycoenzymes, and examine the mechanism of the improved catalytic performance. We use lipase as a negative control to illustrate the molecular recognition of ConA and its impact on the enzyme immobilization. In addition to showing its effectiveness in preventing enzyme leakage, we find that such a two-step procedure also improves the enzyme stability against pH and temperature. Finally, we show that the immobilized laccase has a high efficiency for the removal of aqueous phenol, a toxic and carcinogenic pollutant.32 The improved efficiency may be attributed to integration of the phenol enrichment by the AC and the efficiently chemical degradation by laccase.

Scheme 1. Enzyme immobilization on the ConA coated AC. RESULTS AND DISCUSSION


4

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


First we examined the colocalization of ConA and enzyme. In a typical run, 0.1 g AC was added to 5 mL of phosphate buffer (PBS, 50 mM, pH 7) containing ConA of given concentration. The mixture was placed in a shaker with a rotation speed of 170 rpm and the adsorption was conducted at room temperature for 12 h to ensure that the adsorption reaches saturation (AC/ConA, Figure S1a). After 3 cycles of washing and centrifugation, 5 mL PBS solution containing enzymes was added. Here two glycoenzyme, laccase and HRP were applied to examine the versatility of this two-step adsorption method. The concentration of laccase and HRP was 4 mg mL-1 laccase and 1 mg mL-1 HRP respectively according to their activity. The adsorption of enzymes on ConA coated AC (AC/ConA/enzyme) was equilibrated for 12 h in the same conditions described above (Figure S1b). The enzyme loading was determined by size exclusion chromatography and bicinchoninic acid (BCA) protein assay.33 Figure 1a shows the scanning electron microscope (SEM) images of laccase immobilized on the AC, while the SEM of AC, AC/ConA and HRP immobilized on the AC are given in Figure S2 and S3a. Here the AC particle retains its porous structure after the adsorption of ConA and laccase (Figure 1a and Figure S4), with a 15.7 % decrease in surface area (Table S1). The insets show protein aggregates of 25 nm in diameter appeared at the pore surface of the AC. Figure 1b presents the confocal microscopy image of ConA (labelled with fluorescein isothiocyanate, FITC) and laccase (labelled with rhodamine B, RhB) inside the AC, respectively. It shows abundant contact points of ConA with a whole grain of AC. The yellow color indicates the colocalization of labelled ConA and laccase (HRP in Figure S3b). Figure 1c shows the Fourier transform infrared (FITR) spectra of the AC as well as the AC coated with various protein and enzymes. Here the signal around 1718 cm-1 corresponding to the stretching of a carbonyl bond34 confirms the existence of protein species such as ConA, HRP and laccase. Figure 1d shows the


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dynamic light scattering (DLS) analysis of ConA agglutinated

Page 6 of 24

enzyme aggregate

(ConA/enzyme) in the PBS solution. Here the measurement was taken after 1 h of shaking. The DLS results indicate that both ConA agglutinated laccase and HRP have a size of 400 nm in diameter (Figure S3c), confirming the biological recognition of ConA to glycoenzymes.

Figure 1. Characterization of laccase immobilized on the ConA-coated AC. (a) SEM (inset: high-resolution SEM); (b) laser confocal microscopy, in which laccase was labelled with RhB (red) and ConA was labelled with FITC (green), respectively; (c) FTIR (d) DLS of ConA (1mg mL-1), laccase (4mg mL-1) and ConA/laccase (the mass ratio is 1:4) in the PBS solution. Then we studied the effect of ConA on enzyme immobilization. Firstly, we examined the stability of ConA adsorption on AC by elution experiment, in which only 10 % percent of adsorbed ConA was released after shaking ConA coated AC in 5 mL PBS for 10 hours alone. Figure 2 presents the mass yield and apparent activity of laccase at different yields of ConA on 0.1 g AC. The mass yields were calculated from the laccase concentration in the supernatant after immobilization. It is shown that the coating of AC with ConA increases the apparent


6

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


laccase activity from 13.5 to 31.7 U g-1AC35 (1 U g-1 AC is defined as 1 unit of enzyme activity on 1 g of AC). Similar increase was also found for HRP, from 16.7 to 40.2 U g-1 AC (Figure S5). The enhanced performance may be attributed to ConA agglutination and stabilization of the agglutinated protein.36 Further increase in ConA concentration leads to a slight increase in both the mass yield and apparent activity. According to the results shown in Figure S6, we can achieve a better reusability of immobilized enzyme on AC in mass ratio 50. Considering both activity and reusability, mass ratio 50 was chosen for the following experiments for enzyme immobilization on AC.

Figure 2. Effect of ConA on laccase immobilized on AC. We have also examined specifically the effect of ConA on inhibiting both the leakage of immobilized enzyme from AC and the deactivation of enzyme at the surface of AC. In a typical run, 10 mg immobilized enzyme particles were incubated with 10 mL substrate solution for 1 min at room temperature with a shaking speed of 170 rpm. Then the immobilized enzyme was filtrated from the suspension and subjected to activity assay. As shown by Figure 3a, laccase immobilized on the ConA-coated AC remains 98 % of its initial activity after 10 cycles and 80 % after 25 cycles. By contrast, the one without ConA (AC/laccase) lost all laccase activity after 22 cycles. Similarly, HRP immobilized on ConA-coated AC remained 50 % of its initial activity


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 24

after 25 cycles, while its counterpart prepared without ConA (AC/HRP) lost all activity after 12 cycles (Figure S7a). Finally, we compared above mentioned two-step adsorption procedure against the crosslinking with glutaraldehyde, which has been extensively used for enzyme immobilization.20, 37-38

The crosslinked one shows a reusability around 90 % after 10 cycles, which is comparable

to 98 % of laccase immobilized on ConA-coated AC (Figure 3b). However, the crosslinking step led to a loss of laccase activity up to 55 %. Similar results were obtained in the case of HRP, as shown in Figure S7b, in which the glutaraldehyde crosslinked HRP retains 65 % of its initial activity after 10 cycles, similar to that for ConA-coated AC. The crosslinking of HRP adsorbed on AC with glutaraldehyde led to a 45 % loss of activity while gave no significant improvement in extending the reusability. To understand the mechanism underlying the significantly improved activity and reusability by ConA coating, we carried out similar experiments for enzyme immobilization using bovine serum albumin (BSA), an extensively used protein stabilizer, to coat the AC prior to enzyme adsorption (AC/BSA/enzyme). Pretreatment with BSA leads to an increased apparent activity by 0.8-fold for laccase and 1.5-fold for HRP (The increased apparent activity = (activity of AC/ConA/enzyme - activity of AC/enzyme) / activity of AC/enzyme). The aqueous contact angles (Table S2) show that the adsorption of ConA and BSA changes the AC surface from hydrophobic to hydrophilic. This is favorable for shielding undesirable hydrophobic interactions with the AC, preventing conformational transition of the immobilized enzyme and, consequently, inhibiting enzyme deactivation.28 In other words, the surface coating prevents conformational transition of the immobilized enzyme and, consequently, inhibits enzyme deactivation. However, unlike ConA, BSA fails to stop the leakage and the deactivation of adsorbed enzyme, as


8

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


indicated by the consecutive reduction of the apparent activity as shown in Figure 3a. Notwithstanding, a non-glycoenzyme, lipase from Candida Rugosa (CRL), was adopted to immobilized on ConA- and BSA-coated ACs, in which the results show no significant difference in reusability between ConA and BSA coated AC (Figure 3c). We also examined the effect of pore size (Figure S8) and particle diameter of the AC on enzyme immobilization. As shown in Figure 3d and Figure S7c, in all cases, the coating of AC with ConA results in a significant enhancement in apparent activity. Considering both the enzyme loading and uptake of substrate, 40 mesh AC was used as supports for the following experiments.


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 24

Figure 3. Reusability of (a) laccase immobilized on ConA-coated AC, on BSA-coated AC, and on bare AC (20-40 mesh), (b) cross-linked laccase immobilized on ConA-coated AC and on bare AC, (c) CRL immobilized on ConA-coated AC, on BSA-coated AC, and on bare AC, and (d) laccase immobilized on ConA coated-AC in 8-16 mesh with large pore size distribution and 100 mesh with small pore size distribution (Figure S8). One major objective of enzyme immobilization is to improve enzyme stability and expand its applications to broader operational conditions.39-40 Here we examined the stability of the immobilized enzyme at different temperature and pH. As shown Figure 4a, the immobilized laccase is able to retain 30 % of its activity at 65 oC after 50 min, while the free laccase has been fully deactivated. Figure 4a shows that we can obtain a 4-fold and 1-fold increase in half-life for the immobilized laccase at 60 oC and 65 oC, respectively. Figure 4b indicates that the immobilized laccase maintains higher activity at the temperature range from 30 to 70 oC compared with its native counterpart in solution form. Figure 4c shows that the activity of immobilized laccase falls monotonically in response to pH increase from 3 to 7, which, however, is 10-20 % higher than that of the free laccase. Similar improvement was observed in thermal stability and catalytic performance in the case of HRP immobilized on ConA-coated AC (Figure S9).


10

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (a)-(c) Stability of AC/ConA/laccase and free laccase (4 mg mL-1) as measured by residual activity: (a) thermostability in 60 oC and 65 oC; (b) effect of temperature on the apparent activity; (c) effect of pH on the apparent activity. (d) Phenol removal with laccase, bare AC, laccase immobilized on bare AC, and laccase immobilized on ConA-coated AC. Whereas incorporation of solid matrix generates extra mass transport resistance,41 it may promote the affinitive uptake of substrate thereby facilitating the enzymatic reactions.37, 42 To study the effect of the solid matrix on mass transport, we compared the Michaelis–Menten kinetics of immobilized enzymes and free enzymes. As shown in Table 1, the Km values for laccase and HRP immobilized on ConA-coated AC are almost the same as those for free laccase and HRP, indicating that immobilized enzymes appeared a nearly identical substrate affinity as their native counterparts. These, we believe, can be attributed to the adsorptive effect of AC to


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 24

the substrate. However, immobilized enzymes have shown a significant reduction of kcat from 0.76 s-1 to 0.11 s-1). This may be attributed to the hindered transport of substrate from bulk liquid phase to the interior region of AC saturated with ConA agglutinated enzyme, as can be interpreted from Figure S4 where a significant reduction of pore size of AC occurs after protein adsorption. Table 1. Catalysis kinetics of AC/ConA/enzymes and free enzymes Km

kcat

(mM)

(s-1)

AC/ConA/laccase

0.06

0.11

ConA/laccase

0.07

0.67

free laccase

0.04

0.76

AC/ConA/HRP

0.92

6

ConA/HRP

0.94

142

free HRP

0.88

148

kcat = the mole number of reacted substrate / (the mole number of enzyme * time (s)) Finally, we tested the potential of immobilized enzyme on ConA-coated AC by its application to the removal of phenol. During the test run, 70.2 µL 2 mg mL-1 free laccase, 10 mg laccase immobilized on bare or ConA-coated AC was applied to 2 mM phenol solution. The residual phenol concentration was determined by colorimetric method with potassium ferricyanide(III) and 4-aminoantipyrine (4-APP).39 As shown in Figure 4d, the immobilized laccase gives the highest removal rate at all stages. A rapid adsorption of phenol by AC was also observed, which not only benefit the high removal rate, but also, and more importantly, the completeness of the removal, accounting for the 90 % removal of phenol, which was significantly higher than 64 % and 78 % reported elsewhere.39, 43 Such an active uptake by AC, as a universal adsorbent, brings an additional benefit to intensify the enzymatic reactions, in particularly the complete conversion of a dilute substrate.


12

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CONCLUSION We presented a two-step adsorption procedure to immobilize enzyme on AC with the application of ConA. The distinct advantage of this new procedure, as compared to either direct adsorption or covalent binding, is that it dramatically reduces both enzyme leakage and enzyme deactivation. This significant change is attributed to the presence of ConA which immobilizes glycoproteins via molecular recognition of their glycosidic residues and, in the meantime, shields the unfavorable interactions between enzymes and the hydrophobic AC surface and thus prevents enzyme deactivation.28 Using laccase and HRP as model enzymes, we demonstrated the effectiveness of this new immobilization procedure. In both cases, the immobilized enzymes greatly extend reusability and the “operation window” in terms of both temperature and pH. Moreover, the AC intensifies the enzymatic process through enhanced substrate uptake, as shown in the case of phenol removal, promising for diverse future applications such as conversion of volatile or diluted substrates. EXPERIMENTAL SECTION Preparation of AC/ConA/enzymes In a typical run, 0.1 g AC, washed with deionized water and dried, was dispersed in 5 mL phosphate buffer solution (PBS, 50 mM, pH 7) containing ConA with different mass ratio to AC, followed by shaking at 170 rpm and room temperature for 12 h to form AC/ConA. After washing and centrifugation, 5 mL 4 mg mL-1 laccase (1 mg mL-1 HRP and 1 mg mL-1 CRL) in PBS was added, shaking for another 12 h in the same condition to form AC/ConA/laccase (AC/ConA/HRP and AC/ConA/CRL). Finally, AC/ConA/enzymes were obtained by centrifugation and washing with PBS to remove the unadsorbed proteins.


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 24

Following above-mentioned procedure, AC/BSA/enzymes and AC/enzymes were prepared. In the preparation of AC/BSA/enzymes, 5 mL 1 mg mL-1 BSA in PBS solution was applied instead of ConA. As for AC/enzymes, AC was directly dispersed in enzyme solution of given concentration, followed by shaking and treatment in the same condition. Cross-linking of enzymes immobilized on AC To prepare cross-linked enzymes on AC, glutaraldehyde (0.2 % w/v) was applied to AC/ConA/enzyme and AC/enzyme suspension. After 2 h shaking at room temperature, NaBH4 (5 % weight of protein) was added, followed by 4 h shaking to reduce the Schiff base. Enzyme loading determination The enzyme loading on AC is obtained by subtracting the enzyme content in the supernatant after immobilization from the initial enzyme content. In the first run, the size exclusion chromatography on a TSK-GEL G2000SWXL column was applied to differentiate enzyme and ConA in the supernatant after 12-hour adsorption of enzyme on AC/ConA. Since no ConA leaks in the second step (see Figure S10), BCA protein assay with BSA as standard protein was applied to determine the enzyme concentration in the following experiments. Phenol removal During a run, 10 mg AC/ConA/laccase, 10 mg AC and 70.2 µL 2 mg mL-1 free laccase were shaking with 10 mL 2 mM phenol at 170 rpm and room temperature, respectively. The removal of phenol was determined by measuring the residual phenol in supernatants with colorimetric method. At set intervals, 100 µL supernatant was added into a mixture of 200 µL 83.4 mM potassium ferricyanide(III) in Na2CO3-NaHCO3 buffer (0.25 mM, pH 10), 200 µL 20.8 mM 4-APP in Na2CO3-NaHCO3 buffer (0.25 mM, pH 10) and 1.5 mL Na2CO3-NaHCO3 buffer


14

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(0.25 mM, pH 10) to determine the removal of phenol. The mixture was incubated for 30 min (after complete reaction) before the detection of absorbance in 505 nm. ASSOCIATED CONTENT Supporting Information. Materials and methods, enzyme activity assays, synthesis of labelled proteins, figures for the adsorption kinetic curves of proteins to AC, SEM images of AC/ConA and AC, structure characterizations of AC/ConA/HRP, pore size distribution of AC before and after immobilization, effect of ConA on HRP immobilized on AC, effect of ConA on the reusability of immobilized enzyme, reusability of AC/ConA/HRP and AC/BSA/HRP, SEM images of AC in different size, stability of AC/ConA/HRP and free HRP, tables for the surface area and contact angle of AC before and after protein adsorption. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China under grant No. 21520102008. J.W. is grateful to the financial support by the U. S. National Science Foundation (NSF-CBET-474 0852353). The authors are grateful for the supports from Dr. Diannan Lu, Dr. Jun Ge and Dr. Yifei Zhang. AUTHOR INFORMATION * E-mails: [email protected] Notes The authors declare no competing financial interest. REFERENCES (1) Jain, A.; Ong, V.; Jayaraman, S.; Balasubramanian, R.; Srinivasan, M. P., Supercritical fluid immobilization of horseradish peroxidase on high surface area mesoporous activated carbon. J.


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 24

Supercrit. Fluids 2016, 107, 513-518. (2) Kandasamy, R.; Kennedy, L. J.; Vidya, C.; Boopathy, R.; Sekaran, G., Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil. J. of Mol. Catal. B: Enzym. 2010, 62 (1), 59-66. (3) Zeng, H. C.; Jin, F.; Guo, J., Removal of elemental mercury from coal combustion flue gas by chloride-impregnated activated carbon. Fuel 2004, 83 (1), 143-146. (4) Mohan, D.; Singh, K. P., Single- and multi-component adsorption of cadmium and zinc using activated carbon derived from bagasse - an agricultural waste. Water Res. 2002, 36 (9), 23042318. (5) Hulicova-Jurcakova, D.; Seredych, M.; Lu, G. Q.; Bandosz, T. J., Combined Effect of Nitrogen- and Oxygen-Containing Functional Groups of Microporous Activated Carbon on its Electrochemical Performance in Supercapacitors. Adv. Funct. Mater. 2009, 19 (3), 438-447. (6) Dong, H.; Yu, H.; Wang, X.; Zhou, Q.; Feng, J., A novel structure of scalable air-cathode without Nafion and Pt by rolling activated carbon and PTFE as catalyst layer in microbial fuel cells. Water Res. 2012, 46 (17), 5777-5787. (7) Ghaedi, M.; Heidarpour, S.; Kokhdan, S. N.; Sahraie, R.; Daneshfar, A.; Brazesh, B., Comparison of silver and palladium nanoparticles loaded on activated carbon for efficient removal of Methylene blue: Kinetic and isotherm study of removal process. Powder Technol. 2012, 228, 18-25. (8) Wang, T.; Tan, S.; Liang, C., Preparation and characterization of activated carbon from wood via microwave-induced ZnCl2 activation. Carbon 2009, 47 (7), 1880-1883. (9) Daud, W.; Ali, W. S. W., Comparison on pore development of activated carbon produced from palm shell and coconut shell. Bioresour. Technol. 2004, 93 (1), 63-69.


16

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(10) Hameed, B. H.; Din, A. T. M.; Ahmad, A. L., Adsorption of methylene blue onto bamboobased activated carbon: Kinetics and equilibrium studies. J. Hazard. Mater. 2007, 141 (3), 819825. (11) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O., Enzyme immobilization in a biomimetic silica support. Nat. Biotechnol. 2004, 22 (2), 211-213. (12) Wu, X.; Yang, C.; Ge, J.; Liu, Z., Polydopamine tethered enzyme/metal-organic framework composites with high stability and reusability. Nanoscale 2015, 7 (45), 18883-18886. (13) Ruedaa, N.; dos Santos, C. S.; Daniela Rodriguez, M.; Albuquerque, T. L.; Barbosa, O.; Torres, R.; Ortiz, C.; Fernandez-Lafuente, R., Reversible immobilization of lipases on octylglutamic agarose beads: A mixed adsorption that reinforces enzyme immobilization. J. of Mol. Catal. B: Enzym. 2016, 128, 10-18. (14) Hanefeld, U.; Gardossi, L.; Magner, E., Understanding enzyme immobilisation. Chem. Soc. Rev. 2009, 38 (2), 453-468. (15) Sheldon, R. A.; van Pelt, S., Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev. 2013, 42 (15), 6223-6235. (16) Kuchler, A.; Yoshimoto, M.; Luginbuhl, S.; Mavelli, F.; Walde, P., Enzymatic reactions in confined environments. Nature Nanotech. 2016, 11 (5), 409-420. (17) Nelson, J. M.; Griffin, E. G., Adsorption of invertase. J. Am. Chem. Soc. 1916, 38, 11091115. (18) Sadana, A., PROTEIN ADSORPTION AND INACTIVATION ON SURFACES INFLUENCE OF HETEROGENEITIES. Chem. Rev. 1992, 92 (8), 1799-1818. (19) Di Risio, S.; Yan, N., Adsorption and inactivation behavior of horseradish peroxidase on cellulosic fiber surfaces. J. Colloid Interface Sci. 2009, 338 (2), 410-419.


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

(20) Stoner, G. E.; Gileadi, E.; Ludlow, J. C.; Kirwan, D. J., IMMOBILIZATION OF TRYPSIN ON CARBON. Biotechnol. Bioeng. 1975, 17 (3), 455-456. (21) Cho, Y. K.; Bailey, J. E., ENZYME IMMOBILIZATION ON ACTIVATED CARBON ALLEVIATION OF ENZYME DEACTIVATION BY HYDROGEN-PEROXIDE. Biotechnol. Bioeng. 1977, 19 (5), 769-775. (22) Liu, C. C.; Lahoda, E. J.; Galasco, R. T.; Wingard, L. B., IMMOBILIZATION OF LACTASE ON CARBON. Biotechnol. Bioeng. 1975, 17 (11), 1695-1696. (23) Ramani, K.; Karthikeyan, S.; Boopathy, R.; Kennedy, L. J.; Mandal, A. B.; Sekaran, G., Surface functionalized mesoporous activated carbon for the immobilization of acidic lipase and their application to hydrolysis of waste cooked oil: Isotherm and kinetic studies. Process Biochem. 2012, 47 (3), 435-445. (24) Gray, R. D.; Glew, R. H., KINETICS OF CARBOHYDRATE BINDING TO CONCANAVALIN-A. J. Biol. Chem. 1973, 248 (21), 7547-7551. (25) Mislovicova, D.; Gemeiner, P.; Sandula, J.; Masarova, J.; Vikartovska, A.; Docolomansky, P., Examination of bioaffinity immobilization by precipitation of mannan and mannan-containing enzymes with legume lectins. Biotechnol. Appl. Biochem. 2000, 31, 153-159. (26) Chen, Z.; Liu, Y.; Wang, Y.; Zhao, X.; Li, J., Dynamic Evaluation of Cell Surface N-Glycan Expression via an Electrogenerated Chemiluminescence Biosensor Based on Concanavalin AIntegrating Gold-Nanoparticle-Modified Ru(bpy)(3)(2+)-Doped Silica Nanoprobe. Anal. Chem. 2013, 85 (9), 4431-4438. (27) Mallardi, A.; Angarano, V.; Magliulo, M.; Torsi, L.; Palazzo, G., General Approach to the Immobilization of Glycoenzyme Chains Inside Calcium Alginate Beads for Bioassay. Anal. Chem. 2015, 87 (22), 11337-11344.


18

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(28) Li, Y.; Huang, X.; Qu, Y., A strategy for efficient immobilization of laccase and horseradish peroxidase on singleSwalled carbon nanotubes. J. Chem. Technol. Biotechnol. 2013, 88 (12), 2227-2232. (29) Saleemuddin, M.; Husain, Q., CONCANAVALIN-A - A USEFUL LIGAND FOR GLYCOENZYME IMMOBILIZATION - A REVIEW. Enzyme Microb. Technol. 1991, 13 (4), 290-295. (30) Yavuz, H.; Akgol, S.; Arica, Y.; Denizli, A., Concanavalin a immobilized affinity adsorbents for reversible use in yeast invertase adsorption. Macromol. Biosci. 2004, 4 (7), 674-679. (31) Datta, S.; Christena, L. R.; Rajaram, Y. R. S., Enzyme immobilization: an overview on techniques and support materials. 3 Biotech 2013, 3 (1), 1-9. (32) Xu, F., Oxidation of phenols, anilines, and benzenethiols by fungal laccases: Correlation between activity and redox potentials as well as halide inhibition. Biochemistry 1996, 35 (23), 7608-7614. (33) Smith, P. K.; Krohn, R. I.; Hermanson, G.; Mallia, A.; Gartner, F.; Provenzano, M.; Fujimoto, E.; Goeke, N.; Olson, B.; Klenk, D., Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150 (1), 76-85. (34) El Hajjouji, H.; Fakharedine, N.; Baddi, G. A.; Winterton, P.; Bailly, J. R.; Revel, J. C.; Hafidi, M., Treatment of olive mill waste-water by aerobic biodegradation: An analytical study using gel permeation chromatography, ultraviolet-visible and Fourier transform infrared spectroscopy. Bioresour. Technol. 2007, 98 (18), 3513-3520. (35) Hublik, G.; Schinner, F., Characterization and immobilization of the laccase from Pleurotus ostreatus and its use for the continuous elimination of phenolic pollutants. Enzyme Microb. Technol. 2000, 27 (3-5), 330-336.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 24

(36) Haider, T.; Husain, Q., Calcium alginate entrapped preparations of Aspergillus oryzae beta galactosidase: Its stability and applications in the hydrolysis of lactose. Int. J. Biol. Macromol. 2007, 41 (1), 72-80. (37) Zhang, Y.; Yong, Y.; Ge, J.; Liu, Z., Lectin Agglutinated Multienzyme Catalyst with Enhanced Substrate Affinity and Activity. ACS Catal. 2016, 6 (6), 3789-3795. (38) Talekar, S.; Ghodake, V.; Ghotage, T.; Rathod, P.; Deshmukh, P.; Nadar, S.; Mulla, M.; Ladole, M., Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase. Bioresour. Technol. 2012, 123, 542-547. (39) Zhang, F.; Zheng, B.; Zhang, J. L.; Huang, X. L.; Liu, H.; Guo, S. W.; Zhang, J. Y., Horseradish Peroxidase Immobilized on Graphene Oxide: Physical Properties and Applications in Phenolic Compound Removal. J. Phys. Chem. C 2010, 114 (18), 8469-8473. (40) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R., Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40 (6), 1451-1463. (41) Cho, Y. K.; Bailey, J. E., IMMOBILIZATION OF ENZYMES ON ACTIVATED CARBON - PROPERTIES OF IMMOBILIZED GLUCOAMYLASE, GLUCOSE OXIDASE, AND GLUCONOLACTONASE. Biotechnol. Bioeng. 1978, 20 (10), 1651-1665. (42) Zhang, Y.; Ge, J.; Liu, Z., Enhanced Activity of Immobilized or Chemically Modified Enzymes. ACS Catal. 2015, 5 (8), 4503-4513. (43) Liu, Y. Y.; Zeng, Z. T.; Zeng, G. M.; Tang, L.; Pang, Y.; Li, Z.; Liu, C.; Lei, X. X.; Wu, M. S.; Ren, P. Y.; Liu, Z. F.; Chen, M.; Xie, G. X., Immobilization of laccase on magnetic bimodal mesoporous carbon and the application in the removal of phenolic compounds. Bioresour. Technol. 2012, 115, 21-26.


20

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For table of content use only:

Concanavalin A Coated Activated Carbon for High Performance Enzymatic Catalysis Weina Xu, † You Yong, † Zheyu Wang, † Guoqiang Jiang, †Jianzhong Wu‡ and Zheng Liu*†


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

Synopsis: Enzymes immobilized on Concanavalin A coated activated carbon appeared improved activity and reusability with high efficiency in phenol removal.

Abstract Graphic：：


22

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For table of content use only:

Concanavalin A Coated Activated Carbon for High Performance Enzymatic Catalysis Weina Xu, † You Yong, † Zheyu Wang, † Guoqiang Jiang, †Jianzhong Wu‡ and Zheng Liu*†

Synopsis: Enzymes immobilized on Concanavalin A coated activated carbon appeared improved activity and reusability with high efficiency in phenol removal.


1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 24

Abstract Graphic：


2

Concanavalin A Coated Activated Carbon for High Performance

Recommend Documents