Enzymatic Cascade Catalysis in a Nanofiltration Membrane

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 22419−22428

www.acsami.org

Enzymatic Cascade Catalysis in a Nanofiltration Membrane: Engineering the Microenvironment by Synergism of Separation and Reaction Huiru Zhang,†,‡ Hao Zhang,†,‡ Jianquan Luo,*,†,‡ and Yinhua Wan†,‡ †

Downloaded via UNIV OF SOUTHERN INDIANA on July 23, 2019 at 01:12:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China ‡ School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, PR China S Supporting Information *

ABSTRACT: Microenvironment plays a significant role in enzymatic catalysis, which directly influences enzyme activity and stability. It is important to regulate the enzyme microenvironment, especially for the liquid with unfavored properties (e.g., pH and dissolved oxygen). In this work, we propose a methodology that can regulate pH and substrate concentration for enzymatic catalysis by a biocatalytic membrane, which is composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) co-immobilized in a polyamide nanofiltration (NF) membrane (i.e., beneath the separation layer). By virtue of the selective separation function of NF membrane and in situ production of organic acid/electron donor with GOx, a synergism effect of separation and reaction in the liquid/solid interface was manipulated for engineering the microenvironment of HRP to enhance its activity and stability for micropollutant removal in water. The outcome of this work not only provides a new methodology to precisely control enzymatic reaction but also offers a smart membrane system to efficiently and steadily remove the micropollutants in portable water. KEYWORDS: nanofiltration, cascade catalysis, micropollutants, enzymes, microenvironment engineering decrease of laccase activity with time.14 A strategy to break the trade-off between laccase activity and operation stability is to add mediators into reactants. Mediator is a kind of smallsize compounds able to generate soluble radicals which will not cause enzyme inactivation. It can act as a carrier of electrons between the enzyme and the substrate, thereby overcoming the steric hindrances between them,15 as well as avoiding the accumulation of the oxidation products around the enzyme active sites and further polymerization/deposition. However, mediators such as 1-hydroxybenzotriazole (HBT) and 2,2′azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) are toxic, which seem unsuitable for water treatment. Horseradish peroxidase (HRP), an enzyme widely used in the treatment of micropollutants,16,17 needs hydrogen peroxide (H2O2) as an electron donor. HRP requires a constant supply of H2O2 to maintain its activity, while excess H2O2 triggers the substrate inhibition and the change of enzyme conformation, lowering the activity of HRP.18 It is hard to precisely control a suitable H2O2 concentration in the reactants because the

1. INTRODUCTION Because of human activity, there are increasing micropollutants such as pharmaceuticals, endocrine disruptors, and polycyclic aromatic hydrocarbons in water, which arouse serious attention because of their adverse effects on the human body.1,2 Numerous strategies have been applied for micropollutants removal such as adsorption,3 membrane filtration,4,5 advanced oxidation process,6 and biodegradation,7 among which enzyme-based (e.g., laccase, peroxidase, and tyrosinase) methods are more attractive because of their high efficiency, eco-friendliness, mild operation, and effective detoxification. However, the trade-off between catalytic efficiency and longterm stability is a big issue that limits the application of enzymatic degradation of micropollutants. For example, laccase as a multicopper redox enzyme can catalyze numerous recalcitrant micropollutants such as bisphenol A (BPA), tetracycline, and diclofenac,8−12 but it has been reported that the higher laccase activity resulted in worse reusability because of more polymerization products depositing close to laccase.13 The products with poor solubility may form in the narrow channels, connecting T2/T3 sites of laccase that are crucial for electron delivery, which potentially block further entrance of dissolved oxygen into the channel, eventually causing a © 2019 American Chemical Society

Received: March 26, 2019 Accepted: June 3, 2019 Published: June 3, 2019 22419

DOI: 10.1021/acsami.9b05371 ACS Appl. Mater. Interfaces 2019, 11, 22419−22428

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic of biocatalytic membrane preparation and cascade catalysis in the NF membrane for engineering microenvironment and removing micropollutants.

the coeffect of diffusion and convection.33,34 Therefore, biocatalytic membranes with enzymes immobilized on/in membranes have attracted increasing attention in various areas. In the early stage of biocatalytic membrane development, the most commonly used matrix was microfiltration (MF) membrane, and such membrane acted only as a carrier of enzyme immobilization.35 Gradually, with the wide applications of ultrafiltration (UF) membranes, biocatalytic membranes with both separation (only for macromolecules) and catalysis functions appeared.36 Nanofiltration (NF) membrane, the ultrathin skin layer of which adheres onto the supporting base by physical entanglement,37 acting as both the support for enzymes and the selective barrier for small molecules, becomes a burgeoning tool to enhance the enzymatic reaction. Biocatalytic membrane based on NF membrane was mainly applied for micropollutants removal.1,38 Enzymes could be immobilized on either the skin layer or the supporting layer of the membrane. Normally, NF membrane is designed to fully or partly retain the substrates, and on the one hand, it can realize in situ product removal which reduces product inhibition for macro-molecule hydrolysis;39 on the other hand, it is able to alleviate enzymatic catalysis burden or regulate the substrate concentration involving the reaction if the enzymes are immobilized beneath the skin layer.13,40 Inspired by this, we speculated that NF membrane could be used to manipulate the cascade catalysis behavior via controlling the involved substrate concentration. Recently, mussel-inspired chemistry has attracted widespread interest in membrane technology. Dopamine (DA), as a well-known neurotransmitter, can self-polymerize to form a stable polydopamine (PDA) coating layer in alkaline solutions and adhere onto nearly all kinds of substrates by different interactions including electrostatic and hydrophobic absorp-

reaction efficiency is changing. Moreover, it is commonly known that potable water is at neutral pH, while HRP has higher activity in acidic pH.19 On the other hand, glucose oxidase (GOx) is a promising enzyme that oxidizes the β-Dglucose in the presence of oxygen for in situ production of gluconic acid and H2O2.20,21 If HRP coexists with GOx, the produced gluconic acid lowers the pH of the microenvironment for HRP, while the other product (H2O2) can be used as the electron donor.22 Therefore, the presence of GOx provides a possibility for the precise engineering of the HRP microenvironment, which is of great importance for achieving high catalytic activity and stability of HRP.23 However, for free enzymes, there is a certain spatial distance between the two enzymes, which weakens the mass transfer between their active centers,24 decreasing the efficiency of such microenvironment regulation. Co-immobilization of GOx and HRP in/on a matrix is a practical strategy that shortens the spatial distance between two enzymes and enhances enzyme stability as well as reusability. Polymeric capsules,25 nanoparticles,26 and supramolecular hydrogel27 are the most frequently used matrixes for enzymes immobilization. It is well-known that enzymes were normally entrapped in the matrixes to increase the loading amount and maintain the activity. Thereinto, concentration gradient diffusion is the major mass-transfer mechanism of substrates, intermediates, and products around the immobilized enzymes, and the high diffusion resistance inside the immobilization matrix greatly limits the catalysis efficiency.28,29 Great efforts have been devoted to enhancing mass-transfer efficiency for benign interactions between enzymes and substrates.30−32 The porous membranes, serving as a support for enzyme immobilization, enable enzyme reuse, improve enzyme stability, and promote enzymatic efficiency because of 22420


Research Article


filtration method and dopamine/PEI codeposition technique. The experimental details are presented in Supporting Information. The enzyme immobilization amount was determined using the mass balance equation (the difference between protein in the original feed and the sum of protein in the remaining and washing solutions), and the protein content was measured via Bradford method.40 The Bradford reagent and the sample were mixed at a ratio of 1:1, and the absorbance was measured at 595 nm. The enzyme detection limit was 20 μg L−1. 2.3. Characterization. The fluorescence-labeled enzymes were used to explore the enzymes immobilization distribution in NF270 membrane, and the enzyme distribution was analyzed by confocal laser scanning microscopy (CLSM) on a Leica SP8 STED 3X system (Germany). FITC-labeled and RhB-labeled HRP were synthesized by the method reported in the literature.45 (More details can be found in Supporting Information.) The morphologies of the membranes were examined by a scanning electron microscope (SEM) (SU8020, Hitachi, Japan). Zeta potentials of different membranes were measured by the SurPASS Anton Paar analyzer at a pH range from 4 to 10. Contact angle measurements before and after enzyme immobilization were measured using a water drop shape system (OCA20, Dataphysics, Germany). 2.4. BPA Removal. The pristine and biocatalytic membranes were respectively placed into the dead-end filtration cell. BPA solution (40 mL, 10 mg·L−1) was poured into the filtration cell for BPA removal under 100 rpm agitation at a certain transmembrane pressure (2, 3, and 4 bar). The experimental temperature was controlled at 20 ± 1 °C by placing the cell into a water bath. After 10 mL of liquid passed though the membrane, another 10 mL of BPA solution was added to treat a total of 40 mL of BPA solution. The BPA content in the permeate and retentate was measured using high-performance liquid chromatography, which is reported in our previous work.1,40 2.5. Synergism of Separation and Reaction. The cascade catalysis efficiency relied on the suitable H2O2 concentration supplied to HRP, which was directly related to the glucose concentration passing though the membrane. By utilization of the separation function of NF membrane, the glucose permeation through the membrane could be regulated via changing bulk concentration (1, 5, and 50 mM) and operating pressure (2, 3, and 4 bar). To ascertain the optimal conditions to maximize the synergism of separation and reaction, the effect of H2O2 concentration (0.02−5 mM) on the stability of the immobilized HRP during BPA removal with reuse cycle was studied. Then the in situ generated H2O2 caused by glucose accessible to the immobilized GOx was also detected using the feed solution containing only glucose at different concentrations (1, 5, and 50 mM) and pressures (2, 3, and 4 bar). 2.5.1. Glucose Retention. Glucose concentration was measured by using 3,5-dinitrosalicylic (DNS) method.46,47 The retention of glucose (Rglu) by the pristine and biocatalytic membranes is calculated by the following equation:

tions, hydrogen bonds, π−π stacking, and chelation.41 It was found that the homogeneous polymerization and deposition of DA could be promoted by the addition of polyethylenimine (PEI).42 The PDA/PEI codeposition strategy was also widely used in enzyme immobilization because of its simplicity, stability, and biocompatibility. Additionally, PDA layer can also reduce the leakage of enzymes13 and improve the catalysis efficiency by adsorbing and enriching the substrates.43 Inspired by these, we proposed a new, simple, and effective method for multienzyme immobilization utilizing codeposition of PDA/PEI in this work. As illustrated in Figure 1, a biocatalytic NF membrane was prepared via co-immobilization of GOx and HRP in a NF membrane, where a cascade catalysis was carried out for degrading micropollutant (BPA as an example). In detail, glucose partly passed through the NF membrane and was oxidized with GOx in the membrane, and the produced gluconic acid would decrease the microenvironment pH, which was supposed to improve the HRP-catalyzed oxidation of BPA with the generated H2O2. Here, we attempted to control the H2O2 concentration in the membrane by tuning the glucose permeation through the NF separation layer because H2O2 is very crucial for the activity and stability of the immobilized HRP. Theoretically, a solute retention of NF membrane is mainly determined by feed concentration and operating pressure (i.e., diffusion and convection).44 Thus, by manipulating the diffusion and convection transport of glucose across the membrane (via adjusting glucose concentration in the reactants and operating pressure), a high and stable BPA removal by the prepared biocatalytic membrane may be realized. For the first time, a synergism of separation and reaction accomplished by a biocatalytic NF membrane was proposed for engineering the microenvironment of enzymes, which would not only provide a new methodology to manipulate enzymatic reaction but also offer a smart membrane system to efficiently remove the micropollutants in portable water. In the real application, by simply addition of biocompatible glucose which could trigger multienzyme reaction, the efficient and stable micropollutants removal could be achieved. In addition, a quick-response system will be constructed by regulating the glucose permeance though the NF membrane according to the micropollutants concentration in water.

2. EXPERIMENTAL SECTION 2.1. Materials. In this research, a dead-end filtration cell (Amicon 8050, Millipore, U.S.A.) was utilized and the effective area of which is 13.4 cm2. Polyethylenimine (PEI) (molecular weight (MW) ∼ 600 Da) was supplied by Aladdin. Glucose oxidase (GOx) (EC 1.1.3.4, 160 kDa, 100 U mg−1) from Aspergillus niger and peroxidase from horseradish (HRP) (EC 1.11.1.7, 44 kDa, >300 U mg−1) were obtained from Aladdin (Shanghai, China). NF270 membrane (polyamide, molecular weight cutoff (MWCO) ∼200−300 Da) was supplied by DOW-Filmtech, which has a dense skin layer to partly retain micropollutant and glucose. Dopamine hydrochloride was obtained from Sigma-Aldrich (Shanghai, China). Bisphenol A (BPA, 96%) was supplied by J&K (Beijing). D-Glucose (198.17 Da) was purchased from Xilong Scientific Co., Ltd. (Guangdong, China). Peroxide (H2O2, 30%) was obtained from Beijing Chemical Works (Beijing). Fluorescein isothiocyanate (FITC, MW ∼389.38 Da) was purchased from MedChemExpress Co., Ltd. (Shanghai, China). Rhodamine B isothiocyanate (RhB, MW ∼479.02 Da) was bought from Aladdin (Shanghai, China). All chemicals used in experiments were of analytical grade. 2.2. GOx/HRP Co-immobilization in NF Membrane. In this work, GOx and HRP immobilization was carried out using the reverse

ij C pg yzz zz × 100% R glu = jjjj1 − j Cfg zz k {

(1)

where Cpg and Cfg represented glucose concentrations in the permeate and feed, respectively. 2.5.2. Measurement of H2O2 Concentration. H2O2 concentrations were determined by visible spectrophotometry after color development with Ti(SO4)2.48,49 In the presence of H2SO4, the H2O2 reacts with Ti(SO4)2 to form a yellow substance which has an adsorption maximum at 410 nm.

Ti(SO4 )2 + H 2O2 + H 2O → H4TiO5 + H 2SO4

(2)

−1

Sixty microliters of H2SO4 (0.3 mol·L ) and 200 μL of Ti(SO4)2 (0.3 mol·L−1) were added into 1.74 mL of samples, which were incubated for 5 min and then measured at 410 nm. 2.5.3. Determination of Substrate Inhibition. The substrate inhibition of H2O2 on HRP was explored by measuring the BPA oxidation rate with various concentrations of H2O2 in 10 mL of BPA 22421


Research Article


Figure 2. Confocal laser scanning microscopy images of the biocatalytic membrane with immobilized GOx and HRP: (a) support layer; (b) skin layer; (c) 3D structure; and (d) cross section. solution (containing 1 mg L−1 HRP and 10 mg L−1 BPA). The H2O2 concentration ranges were 0.005−20 mM. The BPA concentration in solution after oxidation was measured by HPLC, and the BPA oxidation rate is calculated by the following equation: V=

(C b − Ca) T

3. RESULTS AND DISCUSSION 3.1. Characterization. Distribution of the immobilized GOx and HRP in the NF270 membrane was clarified by CLSM. The CLSM images of the biocatalytic membrane obviously indicated that both GOx-FITC (green) and HRPRhB (red) were well-mixed and mainly anchored in the intermediate layer between skin layer and support layer (Figure 2). The contact angles of the pristine and biocatalytic membranes are shown in Figure 3. It was found that the contact angle of the support layer significantly decreased after enzyme immobilization and PDA/PEI coating while the hydrophilicity of the skin layer did not change. Such hydrophilicity improvement on support layer was attributed to the highly hydrophilic PDA/PEI coating layer, indicating that the coating was successful and this might be beneficial to membrane permeability. SEM images and zeta potential of the pristine and biocatalytic membranes (Figures S1 and S2, Supporting Information) also revealed that the homogeneous coating in the support layer was successfully obtained by codeposition of PEI and PDA.

(3)

where Cb and Ca represented BPA concentrations before and after oxidation in solution, respectively, and T is the reaction time. 2.6. Reusability and Stability of Biocatalytic Membrane. The biocatalytic membrane reusability and stability were examined by testing the BPA removal for 7 days (20 mL for each day) and continuous 10 h (300 mL of BPA solution was treated totally), respectively. The operational conditions were as follows: 3 bar, 100 rpm, 21 °C, 10 mg·L−1 BPA, and 1 mM glucose in deionized water. For the 7 days measurement, the membrane was continuously washed under pressure each time until no BPA was detected in washing solution before being stored at 4 °C. The BPA removal efficiency is calculated as the BPA amount difference between original feed and permeate divided by the BPA amount in the original feed. 22422


Research Article


ability further increased after membrane reversal because of the enzyme movement and leakage. The enzyme immobilization mechanisms included adsorption, entrapment, and covalent bonding as discussed in our previous work, and it was also confirmed that there was almost no enzyme leakage during the storage for 9 days.13,40 The enzyme loading in the membrane is about 29.85 μg cm−1 in this work, which was much higher than those found in the literature. On the other hand, under pressure-driven flowthrough mode, the as-prepared membrane would facilitate the mass transfer of substrate/intermediates/products around the anchored enzymes, but the contact time between substrates and enzymes became short; thus, the enzyme activity had to be high enough to ensure a high micropollutant removal. It was reported that the cascade catalysis by GOx and HRP followed these steps (eqs 4−7).50,51 In the first step, the required gluconic acid and H2O2 are in situ produced by GOx through oxidizing β-D-glucose in the presence of oxygen:

Figure 3. Contact angle of pristine and biocatalytic membranes’ skin and support layers.

3.2. Enzymatic Cascade Catalysis for BPA Removal. We first compared BPA removal efficiency by the pristine, PDA/PEI-coated, and biocatalytic membranes. As shown in Figure 4a, the pristine NF membrane can retain more than 70% of BPA in the first cycle; however, BPA molecules would adsorb and dissolve in the polyamide skin layer, and then diffuse through the membrane, resulting in a continuous retention decrease with filtration cycle (it becomes below 40% in the fourth filtration cycle), which agrees well with the previous results in the literature.40 Even though the BPA retention by the PDA/PEI-coated membrane increased a little thanks to the possible pore narrowing effect induced by the coating layer, the BPA retention decline with reuse cycle was still observed, whereas for the biocatalytic membrane with two enzymes, the BPA retention almost remained constant (close to 100%) with reuse cycle when a suitable glucose concentration was added. Thus, enzyme catalysis plays a significant role to improve the efficiency and stability of BPA removal. During the biocatalytic membrane preparation, we explored the permeability variation of the membrane (Figure 4b). The membrane permeability decreased remarkably after enzymes immobilization, which might be derived from the protein fouling layer formed during the reverse filtration, increasing the filtration resistance of the membrane. After the PDA/PEI coating, the membrane permeability recovered greatly because the hydrophilicity of the membrane support was improved (Figure 3). Moreover, the membrane perme-

GOx

β‐D‐glucose + O2 ⎯⎯⎯→ gluconic acid + H 2O2

(4)

In the next step, HRP is activated by H2O2, leading to the generation of compound I and H2O. Then compound I can oxidize BPA via oxidoreduction and generate compound II and free radicals. Finally, the produced compound II interacts with other BPA to generate radicals and regenerated HRP. HRP + H 2O2 → compound I + H 2O

(5)

compound I + BPA → compound II + BPA radical

(6)

compound II + BPA → BPA radical + HRP

(7)

To clarify the effect mechanisms of the cascade catalysis on BPA removal by the biocatalytic membrane, we then investigated the effect of pH and glucose concentration on the BPA removal efficiency by free enzymes. The results revealed that the acidic pH caused by gluconic acid could enhance the activity of HRP toward BPA degradation by virtue of H2O2, although gluconic acid itself had a little inhibition effect on HRP compared to hydrochloric acid at the same pH (Figure S3). It also was confirmed that, for the free GOx and HRP, the BPA removal efficiency was closely related to the available glucose concentration, which was eventually attributed to the produced H2O2 concentration because the

Figure 4. (a) BPA removal efficiency by the pristine, PDA/PEI-coated, and biocatalytic membranes when 10 g L−1 BPA solution containing 5 mM β-D-glucose was treated with an agitation speed of 100 rpm and an operation pressure of 3 bar for four cycles (10 mL/cycle). (b) Membrane permeability variations at different enzyme immobilization stages. 22423


Research Article


Figure 5. Effect of glucose concentration on (a) BPA oxidation by cascade catalysis in membrane and (b) the permeate pH with reuse cycle. Effect of H2O2 concentration on (c) BPA oxidation rate catalyzed by free HRP and (d) BPA oxidation by immobilized HRP in the membrane with reuse cycle.

mM. It is worth noting that the BPA removal efficiency by the biocatalytic membrane decreased with the reuse cycle when the H2O2 concentration was 0.02 mM, which was attributed to the insufficient H2O2 concentration. In the initial filtration stage, the BPA retention was high because of the adsorption effect (Figure 4a) and the BPA in the permeate could be fully oxidized under a low H2O2 concentration. However, with the increase of reuse cycles, the BPA concentration in the permeate gradually increased because of “adsorption saturation” of the membrane (Figure 4a), and such low H2O2 concentration was insufficient to oxidize the flowing BPA. Besides the improper H2O2, the undesirable pH (∼ 7) would result in a more severe decline of the BPA removal efficiency with increasing reuse cycle (Figure S5). 3.3. Synergism of Separation and Reaction for Engineering Microenvironment. It was verified that adding a suitable H2O2 concentration could achieve stable BPA removal, but the pH of the real potable water is unfavored for HRP activity and the H2O2 concentration in the membrane could not be adjusted by NF (H2O2 passes through the membrane freely). Glucose is partly retained by the NF270 membrane, and the glucose accessible to the immobilized enzymes can be regulated by both its bulk concentration and permeate flux, which provides a simple and rapid way to control pH and H2O2 concentration in the reaction zone. First, the biocatalytic membrane had a little higher retention of glucose than the pristine NF270 because of the pore-blocking effect by the immobilized enzymes, and the glucose retention increased with permeate flux at higher operating pressure because of the enhanced convection of solvent. (Figure S6). Second, the permeate flux at higher glucose concentration was lower because the larger osmotic pressure reduced the effective

pH variation was similar at the glucose concentrations from 5 to 125 mM (Figure S4a). Though increasing glucose concentration resulted in higher BPA removal by free enzymes for a long reaction time (Figure S4b), the catalytic behavior in the biocatalytic membrane (a very short reaction time in flow-through mode) may be different, and moreover, the reusability of the biocatalytic membrane may be worse at high glucose concentration because the excessive H2O2 in situ generated by GOx would inhibit or damage the immobilized HRP. Next, we investigated the effect of the glucose concentration in the reactants on the BPA removal by the biocatalytic membrane. As shown in Figure 5a, in the first flow-through cycle, the BPA removal efficiency reaches around 100% with 5 mM and 50 mM glucose, while it is only 93% with 1 mM glucose, indicating that a higher glucose concentration would promote HRP activity on BPA by decreasing pH and offering more H2O2 for the microenvironment of the immobilized HRP in the membrane (Figure 5b). However, with an increase of reuse cycle, the BPA removal by the biocatalytic membrane declined gradually when the glucose concentration was 5 and 50 mM; in contrast, with 1 mM glucose, the biocatalytic membrane kept 100% of BPA removal in the next three reuse cycles. This can be explained by the inactivation effect and substrate inhibition of the excessive H2O2 on HRP, and the latter was confirmed by the BPA oxidation rate variation along with H2O2 concentration via free HRP-induced catalysis (Figure 5c). Furthermore, the optimal range of H2 O 2 concentration was determined by adding H2O2 instead of glucose into BPA solution (Figure 5d), and the BPA removal efficiency by the biocatalytic membrane could reach nearly 100% in four reuse cycles when the H2O2 concentration ranges from 0.05 to 1 22424


Research Article


Figure 6. (a, b) Effect of operating pressure on (a) permeate flux and (b) glucose permeance at different glucose concentrations. (c−e) Effect of operating pressure on cascade catalysis in the membrane at different glucose concentrations of (c) 1 mM, (d) 5 mM, and (e) 50 mM with reuse cycle. (f) H2O2 concentration produced by immobilized GOx in the membrane at different glucose concentrations with reuse cycle (operating pressure = 3 bar).

driving force across the membrane (Figure 6a). Third, the glucose permeance was affected by both its feed concentration and permeate flux, which became larger at higher feed concentration and lower permeate flux (Figure 6b). The above results regarding the glucose separation behavior could be used to understand the BPA removal efficiency by the biocatalytic membrane at different conditions. As shown in Figure 6c−e, with 1 mM glucose, the BPA removal is less than 95% in the first flow-through cycle because of insufficient glucose passing through the membrane, and although the contact time and glucose permeance are supposed to be higher at lower pressure, the BPA removal at 2 bar is surprisingly lower than the others, implying that the enzymatic reaction in the confined space may be different from that in bulk solution. In the second reuse cycle, because of the glucose accumulation in the membrane with filtration time, the BPA removal greatly increased to 100%, except for the case at 4 bar possibly because of its higher glucose retention and shorter contact time (Figure

6c). In the third and fourth cycle, the BPA removal at all operating pressures reached 100%, whereas with 5 mM glucose, the BPA removal efficiency remained at a stable level at 100% under various operating pressures with reuse cycle because such glucose concentration produced a suitable H2O2 concentration in the membrane, which was confirmed by the results in Figure 6f (0.25−1.0 mM). However, with 50 mM glucose, the BPA removal declined in the second cycle and became worse in the fourth cycle, especially at lower pressure. This was explained by the fact that excessive H2O2 posed a negative effect on the HRP, and even in the first cycle, the generated H2O2 was much higher than 1.0 mM (Figure 6f). In the fourth cycle, the BPA removal was a little higher at higher pressure because it resulted in lower glucose permeance and shorter contact time, alleviating the inhibition effect of the excessive H2O 2. Therefore, by tuning of the glucose concentration in water and operation of the pressure, the BPA removal by the biocatalytic membrane can be 22425


Research Article


Figure 7. BPA removal performance during (a) continuous filtration for 10 h and (b) long-term batch operation for 7 days. Experimental conditions: operating pressure = 3 bar, feed solution = 10 mg·L−1 BPA and 1 mM glucose, the total treated volume was 300 mL for continuous filtration, the treated volume was 20 mL for each batch.

Table 1. Comparison of Reusability of Biocatalytic Membrane in BPA Removal matrixes

a

CNTs-PVDF (MF) TiO2 (sol−gel)-PVDF (MF) TiO2 (NP)-PVDF (MF) Cu-PVDF (MF) PDA-NF270 (NF) CuPDA-NF270 (NF) PAN-MIL-101(UF) PDA/PEI-NF270(NF)

initial BPA concentration (mg L−1)

BPA removal efficiency in the first cycle (%)

reuse cycle

BPA removal efficiency in the last cycle (%)

average decline every cycle (%)

throughputb (L m−2 h−1)

laccase laccase

4.57 34.24

96 92

4 4

91 84

1.25 2.00

10.00 20.00

52 53

laccase laccase laccase laccase laccase GOx/HRP

34.24 10.00 10.00 2.00 10.00 10.00

95 99 88 87 92 89

15 5 7 9 7 7

15 57 34 77 62 80

5.33 8.68 7.29 1.11 4.29 1.36

5.00 10.00 5.97 2.49 6.45 21.94

54 55 40 13 10 this work

enzyme

ref

a

MF, microfiltration; UF, ultrafiltration; NF, nanofiltration. bThroughput was calculated by volume of feed solution/membrane area/processing time.

the immobilized HRP, where the pH was decreased to enhance the HRP activity and BPA was degraded by HRP in the presence of H2O2. Such cascade catalysis in a NF membrane toward micropollutant removal could be well-tuned by changing the bulk glucose concentration and operating pressure. The BPA removal by the biocatalytic membrane in a flow-through cycle achieved 100% with 5 mM glucose and 10 mg·L−1 BPA in the deionized water operated at 2−4 bar, which even remained above 80% in a continuous filtration for 10 h or batch operation for 7 days. This biocatalytic NF membrane is promising for efficient and stable micropollutant removal in portable water (as domestic water dispensers or tap water filters), and a smart membrane system with a micropollutant sensor may be constructed, where the glucose permeance through the NF membrane can be regulated by adjusting glucose concentration and operation pressure according to the micropollutant concentration in water, ensuring a suitable microenvironment for the HRP activity and stability.

manipulated via engineering the microenvironment (pH and H2O2 concentration) around the immobilized enzymes in the membrane. As shown in Figure 7, a super high and stable BPA removal (>80%) by our biocatalytic membrane (the BPA removal by the pristine NF270 was only 40%) is obtained in a long-term operation (continuous running for 10 h or batch operation for 7 days). To the best of our knowledge, these are the top results on the reusability and stability of BPA removal when compared with other biocatalytic membranes with immobilized laccase reported in the literature (Table 1). In detail, most biocatalytic membranes used for BPA removal were based on laccase because of its high catalysis activity on different micropollutants. However, the laccase is easily deactivated, resulting in its poor stability. In our previous work, the average BPA removal efficiency decline of the laccase-loaded membranes ranged from 4% to 9% in each cycle (10 mg L−1 BPA), while in the present work, this number was only 1.36%, showing much higher stability and reusability. Furthermore, the throughput of our biocatalytic membrane was the highest among the results listed in Table 1, demonstrating that this novel biocatalytic membrane could remove much more BPA in a very short filtration time.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b05371.

4. CONCLUSIONS A biocatalytic membrane for cascade catalysis was simply prepared by co-immobilization of GOx and HRP in a NF membrane via reverse filtration and subsequent co-deposition of PDA/PEI. When glucose in the reactants passed through the biocatalytic membrane, it was catalyzed by GOx to produce gluconic acid and H2O2 for engineering microenvironment of

SEM images and zeta potential of pristine and biocatalytic membranes (both sides); effect of pH and acids on BPA oxidation by free HRP; effect of glucose concentration on pH of the solution and BPA removal efficiency by cascade catalysis with free enzymes; effect of pH on BPA oxidation by cascade catalysis in the 22426


Research Article


■

(13) Cao, X.; Luo, J.; Woodley, J. M.; Wan, Y. Mussel-Inspired CoDeposition to Enhance Bisphenol a Removal in a Bifacial Enzymatic Membrane Reactor. Chem. Eng. J. 2018, 336, 315−324. (14) Ji, C.; Hou, J. W.; Wang, K.; Ng, Y. H.; Chen, V. Single-Enzyme Biofuel Cells. Angew. Chem., Int. Ed. 2017, 56 (33), 9762−9766. (15) Canas, A. I.; Camarero, S. Laccases and Their Natural Mediators: Biotechnological Tools for Sustainable Eco-Friendly Processes. Biotechnol. Adv. 2010, 28 (6), 694−705. (16) Jiang, Y. J.; Cui, C. C.; Zhou, L. Y.; He, Y.; Gao, J. Preparation and Characterization of Porous Horseradish Peroxidase Microspheres for the Removal of Phenolic Compound and Dye. Ind. Eng. Chem. Res. 2014, 53 (18), 7591−7597. (17) Escalona, I.; de Grooth, J.; Font, J.; Nijmeijer, K. Removal of Bpa by Enzyme Polymerization Using Nf Membranes. J. Membr. Sci. 2014, 468, 192−201. (18) Rocha-Martin, J.; Velasco-Lozano, S.; Guisan, J. M.; LopezGallego, F. Oxidation of Phenolic Compounds Catalyzed by Immobilized Multi-Enzyme Systems with Integrated Hydrogen Peroxide Production. Green Chem. 2014, 16 (1), 303−311. (19) Xu, J.; Tang, T.; Zhang, K.; Ai, S.; Du, H. Electroenzymatic Catalyzed Oxidation of Bisphenol-a Using Hrp Immobilized on Magnetic Silk Fibroin Nanoparticles. Process Biochem. 2011, 46 (5), 1160−1165. (20) Sun, J. Y.; Ge, J. C.; Liu, W. M.; Lan, M. H.; Zhang, H. Y.; Wang, P. F.; Wang, Y. M.; Niu, Z. W. Multi-Enzyme Co-Embedded Organic-Inorganic Hybrid Nanoflowers: Synthesis and Application as a Colorimetric Sensor. Nanoscale 2014, 6 (1), 255−262. (21) Farrugia, T.; Perriman, A. W.; Sharma, K. P.; Mann, S. MultiEnzyme Cascade Reactions Using Protein-Polymer Surfactant SelfStanding Films. Chem. Commun. 2017, 53 (13), 2094−2097. (22) Elouarzaki, K.; Bourourou, M.; Holzinger, M.; Le Goff, A.; Marks, R. S.; Cosnier, S. Freestanding Hrp-Gox Redox Buckypaper as an Oxygen-Reducing Biocathode for Biofuel Cell Applications. Energy Environ. Sci. 2015, 8 (7), 2069−2074. (23) Shi, J. F.; Wu, Y. Z.; Zhang, S. H.; Tian, Y.; Yang, D.; Jiang, Z. Y. Bioinspired Construction of Multi-Enzyme Catalytic Systems. Chem. Soc. Rev. 2018, 47 (12), 4295−4313. (24) Li, Z. X.; Zhang, Y. F.; Su, Y. C.; Ouyang, P. K.; Ge, J.; Liu, Z. Spatial Co-Localization of Multi-Enzymes by Inorganic NanocrystalProtein Complexes. Chem. Commun. 2014, 50 (83), 12465−12468. (25) Vriezema, D. M.; Garcia, P. M. L.; Sancho Oltra, N.; Hatzakis, N. S.; Kuiper, S. M.; Nolte, R. J. M.; Rowan, A. E.; van Hest, J. C. M. Positional Assembly of Enzymes in Polymersome Nanoreactors for Cascade Reactions. Angew. Chem. 2007, 119 (39), 7522−7526. (26) Li, Z.; Zhang, Y.; Su, Y.; Ouyang, P.; Ge, J.; Liu, Z. Spatial CoLocalization of Multi-Enzymes by Inorganic Nanocrystal−Protein Complexes. Chem. Commun. 2014, 50 (83), 12465−12468. (27) Qu, R.; Shen, L. L.; Qu, A. T.; Wang, R. L.; An, Y. L.; Shi, L. Q. Artificial Peroxidase/Oxidase Multiple Enzyme System Based on Supramolecular Hydrogel and Its Application as a Biocatalyst for Cascade Reactions. ACS Appl. Mater. Interfaces 2015, 7 (30), 16694− 16705. (28) Liese, A.; Hilterhaus, L. Evaluation of Immobilized Enzymes for Industrial Applications. Chem. Soc. Rev. 2013, 42 (15), 6236−6249. (29) Sheldon, R. A.; van Pelt, S. Enzyme Immobilisation in Biocatalysis: Why, What and How. Chem. Soc. Rev. 2013, 42 (15), 6223−6235. (30) Shi, J. F.; Zhang, S. H.; Wang, X. L.; Jiang, Z. Y. Open-Mouthed Hybrid Microcapsules with Elevated Enzyme Loading and Enhanced Catalytic Activity. Chem. Commun. 2014, 50 (83), 12500−12503. (31) Yang, X. W.; Cai, Z. W.; Ye, Z. M.; Chen, S.; Yang, Y.; Wang, H. F.; Liu, Y. F.; Cao, A. N. In Situ Synthesis of Porous Silica Nanoparticles for Covalent Immobilization of Enzymes. Nanoscale 2012, 4 (2), 414−416. (32) Shi, J. F.; Jiang, Z. Y. An Efficient and Recyclable Enzyme Catalytic System Constructed through the Synergy between Biomimetic Mineralization and Polyamine-Salt Aggregate Assembly. J. Mater. Chem. B 2014, 2 (28), 4435−4441.

biocatalytic membrane; glucose retention as a function of permeate flux by the pristine and biocatalytic NF membranes at different glucose concentrations (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.L.). ORCID

Jianquan Luo: 0000-0002-9949-7779 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Financial support was supplied by the Beijing Natural Science Foundation (2192053) and Youth Innovation Promotion Association (2017069) of Chinese Academy of Sciences.

■

REFERENCES

(1) Li, S.; Luo, J.; Wan, Y. Regenerable Biocatalytic Nanofiltration Membrane for Aquatic Micropollutants Removal. J. Membr. Sci. 2018, 549, 120−128. (2) Im, J.; Löffler, F. E. Fate of Bisphenol a in Terrestrial and Aquatic Environments. Environ. Sci. Technol. 2016, 50 (16), 8403− 8416. (3) Zambianchi, M.; Durso, M.; Liscio, A.; Treossi, E.; Bettini, C.; Capobianco, M. L.; Aluigi, A.; Kovtun, A.; Ruani, G.; Corticelli, F.; Brucale, M.; Palermo, V.; Navacchia, M. L.; Melucci, M. Graphene Oxide Doped Polysulfone Membrane Adsorbers for the Removal of Organic Contaminants from Water. Chem. Eng. J. 2017, 326, 130− 140. (4) Cui, Y.; Liu, X. Y.; Chung, T. S.; Weber, M.; Staudt, C.; Maletzko, C. Removal of Organic Micro-Pollutants (Phenol, Aniline and Nitrobenzene) Via Forward Osmosis (Fo) Process: Evaluation of Fo as an Alternative Method to Reverse Osmosis (Ro). Water Res. 2016, 91, 104−114. (5) Yang, X. B.; Sun, H. G.; Pal, A.; Bai, Y. P.; Shao, L. Biomimetic Silicification on Membrane Surface for Highly Efficient Treatments of Both Oil-in-Water Emulsion and Protein Wastewater. ACS Appl. Mater. Interfaces 2018, 10 (35), 29982−29991. (6) Aleksic, M.; Kusic, H.; Koprivanac, N.; Leszczynska, D.; Bozic, A. L. Heterogeneous Fenton Type Processes for the Degradation of Organic Dye Pollutant in Water - the Application of Zeolite Assisted Aops. Desalination 2010, 257 (1−3), 22−29. (7) Balest, L.; Mascolo, G.; Di Iaconi, C.; Lopez, A. Removal of Endocrine Disrupter Compounds from Municipal Wastewater by an Innovative Biological Technology. Water Sci. Technol. 2008, 58 (4), 953−956. (8) de Cazes, M.; Belleville, M. P.; Mougel, M.; Kellner, H.; Sanchez-Marcano, J. Characterization of Laccase-Grafted Ceramic Membranes for Pharmaceuticals Degradation. J. Membr. Sci. 2015, 476, 384−393. (9) Abejon, R.; De Cazes, M.; Belleville, M. P.; Sanchez-Marcano, J. Large-Scale Enzymatic Membrane Reactors for Tetracycline Degradation in Wwtp Effluents. Water Res. 2015, 73, 118−131. (10) Ren, Z.; Luo, J.; Wan, Y. Highly Permeable Biocatalytic Membrane Prepared by 3d Modification: Metal-Organic Frameworks Ameliorate Its Stability for Micropollutants Removal. Chem. Eng. J. 2018, 348, 389−398. (11) Xu, R.; Tang, R. Z.; Zhou, Q. J.; Li, F. T.; Zhang, B. R. Enhancement of Catalytic Activity of Immobilized Laccase for Diclofenac Biodegradation by Carbon Nanotubes. Chem. Eng. J. 2015, 262, 88−95. (12) Xu, R.; Zhou, Q. J.; Li, F. T.; Zhang, B. R. Laccase Immobilization on Chitosan/Poly(Vinyl Alcohol) Composite Nanofibrous Membranes for 2,4-Dichlorophenol Removal. Chem. Eng. J. 2013, 222, 321−329. 22427


Research Article


Performance, Stability, and Regeneration. J. Membr. Sci. 2016, 520, 869−880. (53) Hou, J. W.; Dong, G. X.; Ye, Y.; Chen, V. Enzymatic Degradation of Bisphenol-a with Immobilized Laccase on Tio2 SolGel Coated Pvdf Membrane. J. Membr. Sci. 2014, 469, 19−30. (54) Hou, J. W.; Dong, G. X.; Luu, B.; Sengpiel, R. G.; Ye, Y.; Wessling, M.; Chen, V. Hybrid Membrane with Tio2 Based BioCatalytic Nanoparticle Suspension System for the Degradation of Bisphenol-A. Bioresour. Technol. 2014, 169, 475−483. (55) Fan, J. X.; Luo, J. Q.; Wan, Y. Aquatic Micro-Pollutants Removal with a Biocatalytic Membrane Prepared by Metal Chelating Affinity Membrane Chromatography. Chem. Eng. J. 2017, 327, 1011− 1020.

(33) Luo, J.; Meyer, A. S.; Mateiu, R. V.; Pinelo, M. Cascade Catalysis in Membranes with Enzyme Immobilization for MultiEnzymatic Conversion of Co2 to Methanol. New Biotechnol. 2015, 32 (3), 319−327. (34) Bae, J. S.; Jeon, E.; Moon, S. Y.; Oh, W.; Han, S. Y.; Lee, J. H.; Yang, S. Y.; Kim, D. M.; Park, J. W. Bicontinuous Nanoporous Frameworks: Caged Longevity for Enzymes. Angew. Chem., Int. Ed. 2016, 55 (38), 11495−11498. (35) Smuleac, V.; Butterfield, D. A.; Bhattacharyya, D. Layer-byLayer-Assembled Microfiltration Membranes for Biomolecule Immobilization and Enzymatic Catalysis. Langmuir 2006, 22 (24), 10118−10124. (36) Vanangamudi, A.; Saeki, D.; Dumée, L. F.; Duke, M.; Vasiljevic, T.; Matsuyama, H.; Yang, X. Surface-Engineered Biocatalytic Composite Membranes for Reduced Protein Fouling and SelfCleaning. ACS Appl. Mater. Interfaces 2018, 10 (32), 27477−27487. (37) Shan, L.; Gu, J.; Fan, H.; Ji, S.; Zhang, G. Microphase Diffusion-Controlled Interfacial Polymerization for an Ultrahigh Permeability Nanofiltration Membrane. ACS Appl. Mater. Interfaces 2017, 9 (51), 44820−44827. (38) Li, S.; Luo, J.; Hang, X.; Zhao, S.; Wan, Y. Removal of Polycyclic Aromatic Hydrocarbons by Nanofiltration Membranes: Rejection and Fouling Mechanisms. J. Membr. Sci. 2019, 582, 264− 273. (39) Su, Z.; Luo, J.; Pinelo, M.; Wan, Y. Directing Filtration to Narrow Molecular Weight Distribution of Oligodextran in an Enzymatic Membrane Reactor. J. Membr. Sci. 2018, 555, 268−279. (40) Cao, X.; Luo, J.; Woodley, J. M.; Wan, Y. Bioinspired Multifunctional Membrane for Aquatic Micropollutants Removal. ACS Appl. Mater. Interfaces 2016, 8 (44), 30511−30522. (41) Yang, H.-C.; Waldman, R. Z.; Wu, M.-B.; Hou, J.; Chen, L.; Darling, S. B.; Xu, Z.-K. Dopamine: Just the Right Medicine for Membranes. Adv. Funct. Mater. 2018, 28 (8), 1705327. (42) Yang, H.-C.; Wu, M.-B.; Li, Y.-J.; Chen, Y.-F.; Wan, L.-S.; Xu, Z.-K. Effects of Polyethyleneimine Molecular Weight and Proportion on the Membrane Hydrophilization by Codepositing with Dopamine. J. Appl. Polym. Sci. 2016, 133 (32), 43792. (43) Ma, A.; Xie, Y.; Xu, J.; Zeng, H.; Xu, H. The Significant Impact of Polydopamine on the Catalytic Performance of the Carried Au Nanoparticles. Chem. Commun. (Cambridge, U. K.) 2015, 51 (8), 1469−71. (44) Luo, J. Q.; Wan, Y. H. Effects of Ph and Salt on Nanofiltrationa Critical Review. J. Membr. Sci. 2013, 438, 18−28. (45) Yang, Y.; Zhang, R. Q.; Zhou, B. N.; Song, J. Y.; Su, P.; Yang, Y. High Activity and Convenient Ratio Control: DNA-Directed Coimmobilization of Multiple Enzymes on Multifunctionalized Magnetic Nanoparticles. ACS Appl. Mater. Interfaces 2017, 9 (42), 37254−37263. (46) Zhang, H.; Luo, J.; Li, S.; Wei, Y.; Wan, Y. Biocatalytic Membrane Based on Polydopamine Coating: A Platform for Studying Immobilization Mechanisms. Langmuir 2018, 34 (8), 2585−2594. (47) Soni, A.; Jha, S. K. A Paper Strip Based Non-Invasive Glucose Biosensor for Salivary Analysis. Biosens. Bioelectron. 2015, 67, 763− 768. (48) Ahmad, M.; Simon, M. A.; Sherrin, A.; Tuccillo, M. E.; Ullman, J. L.; Teel, A. L.; Watts, R. J. Treatment of Polychlorinated Biphenyls in Two Surface Soils Using Catalyzed H2o2 Propagations. Chemosphere 2011, 84 (7), 855−862. (49) Schumb, W. C.; Satterfield, C. N.; Wentworth, R. L. Hydrogen Peroxide; Reinhold Pub. Corp.: New York, 1955. (50) Yu, J.; Taylor, K. E.; Zou, H.; Biswas, N.; Bewtra, J. K. Phenol Conversion and Dimeric Intermediates in Horseradish PeroxidaseCatalyzed Phenol Removal from Water. Environ. Sci. Technol. 1994, 28 (12), 2154−60. (51) Wang, Q.; Zhang, X.; Huang, L.; Zhang, Z.; Dong, S. Gox@Zif8(Nipd) Nanoflower: An Artificial Enzyme System for Tandem Catalysis. Angew. Chem., Int. Ed. 2017, 56 (50), 16082−16085. (52) Ji, C.; Hou, J. W.; Chen, V. Cross-Linked Carbon NanotubesBased Biocatalytic Membranes for Micro-Pollutants Degradation: 22428


Enzymatic Cascade Catalysis in a Nanofiltration Membrane

Recommend Documents