Enzymatic Cascade Catalysis in a Nanofiltration Membrane

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Energy, Environmental, and Catalysis Applications

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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05371 • Publication Date (Web): 03 Jun 2019 Downloaded from http://pubs.acs.org on June 5, 2019

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1

Enzymatic Cascade Catalysis in a Nanofiltration

2

Membrane: Engineering the Microenvironment

3

by Synergism of Separation and Reaction

4

Huiru Zhang, †,§ Hao Zhang,†,§ Jianquan Luo,*,†,§ Yinhua Wan†,§

5

†State

Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China

6 7 8

§School

of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, PR China

9 10

ABSTRACT: Microenvironment plays an significant role in enzymatic catalysis,

11

which directly influences enzyme activity and stability. It is important to regulate the

12

enzyme microenvironment, especially for the liquid with unfavored properties (e.g. pH,

13

dissolved oxygen). In this work, we propose a methodology that can regulate pH and

14

substrate concentration for enzymatic catalysis by a biocatalytic membrane, which was

15

is composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) co-

16

immobilized in a polyamide nanofiltration (NF) membrane (i.e. beneath the separation

17

layer). By virtue of the selective separation function of NF membrane and in-situ

18

production of organic acid/electron donor with GOx, a synergism effect of separation

19

and reaction in the liquid/solid interface was manipulated for engineering the

20

microenvironment of HRP to enhance its activity and stability for micro-pollutant

21

removal in water. The outcome of this work not only provides a new methodology to

22

precisely control enzymatic reaction, but also offers a smart membrane system to


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23

efficiently and steadily remove the micro-pollutants in portable water.

24

KEYWORDS:

25

microenvironment engineering

nanofiltration,

cascade

catalysis,

micro-pollutants,

enzymes,

26 27

1. INTRODUCTION

28

Due to human activity, there are increasing micro-pollutants such as

29

pharmaceuticals, endocrine disruptors and polycyclic aromatic hydrocarbons in water,

30

which arouse serious attentions because of their adverse effects on human body

31

Numerous strategies have been applied for micro-pollutants removal such as

32

adsorption3, membrane filtration4-5, advanced oxidation process6, and biodegradation7,

33

among which enzyme-based (e.g. laccase, peroxidase and tyrosinase) methods are more

34

attractive due to their high efficiency, eco-environmentally friendly, mild operation and

35

effective detoxification. However, the trade-off between catalytic efficiency and long-

36

term stability is a big issue that limits the application of enzymatic degradation of

37

micro-pollutants. For example, laccase as a multicopper redox enzyme, can catalyze

38

numerous recalcitrant micro-pollutants such as bisphenol A (BPA), tetracycline,

39

diclofenac8-12, but it was reported that the higher laccase activity resulted in the worse

40

reusability due to more polymerization products depositing close to laccase

41

products with poor solubility may form in the narrow channels connecting T2/T3 sites

42

of laccase that are crucial for electron delivery, which potentially block the further

43

entrance of dissolved oxygen into the channel, eventually causing a decrease of laccase

44

activity with time

14.

13.

1-2.

The

A strategy to break the trade-off between laccase activity and



45

operation stability is to add mediators into reactants. Mediator is a kind of small-size

46

compounds able to generate soluble radicals which won’t cause the enzyme inactivation.

47

It can act as a carrier of electrons between the enzyme and the substrate thereby

48

overcoming the steric hindrances between them15, as well as avoiding the accumulation

49

of the oxidation products around the enzyme active sites and further

50

polymerization/deposition. However, mediators such as 1-hydroxybenzotriazole (HBT)

51

and 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) are toxic, which

52

seem unsuitable for water treatment.

53

Horseradish peroxidase (HRP), an enzyme widely used in the treatment of micro-

54

pollutants16-17 that needs hydrogen peroxide (H2O2) as an electron donor. HRP requires

55

a constant supply of H2O2 to keep its activity, while excess H2O2 triggers the substrate

56

inhibition and the change of enzyme conformation, lowering the activity of HRP18. It’s

57

hard to precisely control a suitable H2O2 concentration in the reactants since the reaction

58

efficiency is changing. Moreover, it is commonly known that potable water is at neutral

59

pH, while HRP has higher activity in acidic pH19. On the other hand, glucose oxidase

60

(GOx) is a promising enzyme which oxidizes the β-D-glucose in the presence of oxygen

61

for in-situ production of gluconic acid and H2O220-21. If HRP coexists with GOx, the

62

produced gluconic acid lowers the pH of the microenvironment for HRP, while the

63

other product (H2O2) can be used as the electron donor22. Therefore, the presence of

64

GOx provides a possibility for the precise engineering of the HRP microenvironment,

65

which is of great importance for achieving high catalytic activity and stability of HRP23.

66

However, for free enzymes, there is a certain spatial distance between the two enzymes,


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67

which weakens the mass transfer between their active centers24, decreasing the

68

efficiency of such micro-environment regulation.

69

Co-immobilization of GOx and HRP in/on a matrix is a practical strategy that shortens

70

the spatial distance between two enzymes and enhances enzyme stability as well as

71

reusability. Polymeric capsules25, nanoparticles26, and supramolecular hydrogel27 are

72

the most frequently-used matrixes for enzymes immobilization. It is well known that

73

enzymes were normally entrapped in the matrixes to increase the loading amount and

74

maintain the activity. Thereinto, concentration gradient diffusion is the major mass

75

transfer mechanism of substrates, intermediates and products around the immobilized

76

enzymes, and the high diffusion resistance inside the immobilization matrix greatly

77

limits the catalysis efficiency28-29. Great efforts have been devoted to enhancing mass

78

transfer efficiency for benign interactions between enzymes and substrates30-32. The

79

porous membranes, served as a support for enzyme immobilization enable enzyme

80

reuse, improve enzyme stability and promote enzymatic efficiency due to the co-effect

81

of diffusion and convection33-34. Therefore, biocatalytic membranes with enzymes

82

immobilized on/in membranes have attracted increasing attention in various areas. In

83

the early stage of biocatalytic membrane development, the most commonly used matrix

84

was microfiltration (MF) membrane, and such membrane was only acted as a carrier of

85

enzyme immobilization35. Gradually, with the wide applications of ultrafiltration (UF)

86

membranes, biocatalytic membranes with both separation (only for macromolecules)

87

and catalysis functions appeared36. Nanofiltration (NF) membrane, the ultrathin skin

88

layer of which adheres onto the supporting base by physical entanglement37, acting as



89

both the support for enzymes and the selective barrier for small molecules, becomes a

90

burgeoning tool to enhance the enzymatic reaction. Biocatalytic membrane based on

91

NF membrane was mainly applied for micro-pollutants removal1, 38. Enzymes could be

92

immobilized on either the skin layer or the supporting layer of the membrane. Normally,

93

NF membrane is designed to fully or partly retain the substrates, and on the one hand,

94

it can realize in-situ product removal which reduces product inhibition for macro-

95

molecule hydrolysis39; on the other hand, it is able to alleviate enzymatic catalysis

96

burden or regulate the substrate concentration involving the reaction if the enzymes are

97

immobilized beneath the skin layer

98

membrane could be used to manipulate the cascade catalysis behavior via controlling

99

the involved substrate concentration.

13, 40.

Inspired by this, we speculated that NF

100

Recently, mussel-inspired chemistry has attracted widespread interest in membrane

101

technology. Dopamine (DA), as a well-known neurotransmitter, can self-

102

polymerization to form a stable polydopamine (PDA) coating layer in alkaline solutions

103

and adhere onto nearly all kinds of substrates by different interactions including

104

electrostatic and hydrophobic absorptions, hydrogen bonds, π-π stacking and

105

chelation41. It was found that the homogeneous polymerization and deposition of DA

106

could be promoted by the addition of polyethyleneimine (PEI)42. The PDA/PEI co-

107

deposition strategy was also widely used in enzyme immobilization, due to its

108

simplicity, stability and biocompatibility. Additionally, PDA layer can also reduce the

109

leakage of enzymes13 and improve the catalysis efficiency by adsorbing and enriching

110

the substrates43.


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111

Inspired by these, we proposed a new, simple and effective method for multi-enzyme

112

immobilization utilizing co-deposition of PDA/PEI in this work. As illustrated in Fig.

113

1, a biocatalytic NF membrane was prepared via co-immobilization of GOx and HRP

114

in a NF membrane, where a cascade catalysis was carried out for degrading micro-

115

pollutant (BPA as an example). In detail, glucose partly passed through the NF

116

membrane and was oxidized with GOx in the membrane, and the produced gluconic

117

acid would decrease the microenvironment pH, which was supposed to improve the

118

HRP-catalyzed oxidation of BPA with the generated H2O2. Here, we attempted to

119

control the H2O2 concentration in the membrane by tuning the glucose permeation

120

through the NF separation layer since H2O2 is very crucial for the activity and stability

121

of the immobilized HRP. Theoretically, a solute retention of NF membrane is mainly

122

determined by feed concentration and operating pressure (i.e. diffusion and

123

convection)44. Thus, by manipulating the diffusion and convection transport of glucose

124

across the membrane (via adjusting glucose concentration in the reactants and operating

125

pressure), a high and stable BPA removal by the prepared biocatalytic membrane may

126

be realized. For the first time, a synergism of separation and reaction accomplished by

127

a biocatalytic NF membrane was proposed for engineering the microenvironment of

128

enzymes, which would not only provide a new methodology to manipulate enzymatic

129

reaction, but also offer a smart membrane system to efficiently remove the micro-

130

pollutants in portable water. In the real application, by simply adding biocompatible

131

glucose which could trigger multi-enzyme reaction, the efficient and stable micro-

132

pollutants removal could be achieved. In addition, a quick-response system will be



133

constructed by regulating the glucose permeance though the NF membrane according

134

to the micro-pollutants concentration in water.

135 136 137

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

138 139

2. EXPERIMENTAL SECTION

140

2.1. Materials. In this research, a dead-end filtration cell (Amicon 8050, Millipore,

141

U.S.A) was utilized and the effective area of which is 13.4 cm2. Polyethyleneimine (PEI)

142

(molecular weight (MW) ~ 600 Da) was supplied by Aladdin. Glucose oxidase (GOx)

143

(EC 1.1.3.4, 160 kDa, 100 U mg-1) from Aspergillus niger and peroxidase from

144

horseradish (HRP) (EC 1.11.1.7, 44 kDa, >300 U mg-1) were obtained from Aladdin

145

(Shanghai, China). NF270 membrane (polyamide, molecular weight cutoff (MWCO)

146

~200-300 Da) was supplied by DOW-Filmtech, which has a dense skin layer to partly

147

retain micropollutant and glucose. Dopamine hydrochloride was obtained from Sigma-

148

Aldrich (Shanghai, China). Bisphenol A (BPA, 96%) was supplied by J&K (Beijing,


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149

China).

D-Glucose

(198.17 Da) was purchased from Xilong Scientific Co., Ltd.

150

(Guangdong, China). Peroxide (H2O2, 30%) was obtained from Beijing Chemical

151

Works (Beijing, China). Fluorescein isothiocyanate (FITC, MW ~389.38 Da) was

152

purchased from MedChemExpress Co., Ltd. (Shanghai, China). Rhodamine B

153

isothiocyanate (RhB, MW ~479.02 Da) was bought from Aladdin (Shanghai, China).

154

All chemicals used in experiments were of analytical grade.

155

2.2. GOx/HRP co-immobilization in NF membrane. In this work, GOx and HRP

156

immobilization was carried out using the reverse filtration method and dopamine/PEI

157

co-deposition technique. The experimental details are presented in supporting

158

information.

159

The enzyme immobilization amount was determined using the mass balance equation

160

(the difference between protein in the original feed and the sum of protein in the

161

remaining and washing solutions), and the protein content was measured via Bradford

162

method 40. The Bradford reagent and the sample were mixed at a ratio of 1:1, and the

163

absorbance was measured at 595 nm. The enzyme detection limit was 20 μgL−1.

164

2.3. Characterization. The fluorescence-labeled enzymes were used to explore the

165

enzymes immobilization distribution in NF270 membrane, and the enzyme distribution

166

was analyzed by confocal laser scanning microscopy (CLSM) on a Leica SP8 STED

167

3X system (Germany). FITC-labeled and RhB-labeled HRP were synthesized by the

168

method reported in literature45 (More details can be found in supporting information).

169

The morphologies of the membranes were examined by a Scanning Electron

170

Microscope (SEM) (SU8020, Hitachi, Japan). Zeta potentials of different membranes



171

were measured by the SurPASS Anton Paar analyzer at a pH range from 4 to 10.

172

Contact angle measurements before and after enzyme immobilization were measured

173

using a water drop shape system (OCA20, Dataphysics, Germany).

174

2.4. BPA removal. The pristine and biocatalytic membranes were respectively placed

175

into the dead-end filtration cell. 40 ml 10 mg·L-1 of BPA solution was poured into the

176

filtration cell for BPA removal under 100 rpm agitation at a certain transmembrane

177

pressure (2, 3, 4 bar). The experimental temperature was controlled at 20 ± 1℃ by

178

placing the cell into water bath. After 10 ml of liquid passed though the membrane,

179

another 10 ml of BPA solution was added to treat a total of 40 ml BPA solution. The

180

BPA content in the permeate and retentate was measured using high-performance liquid

181

chromatography, which is reported in our previous work1, 40.

182

2.5. Synergism of separation and reaction. The cascade catalysis efficiency relied on

183

the suitable H2O2 concentration supplied to HRP, which was directly related to the

184

glucose concentration passing though the membrane. By utilizing the separation

185

function of NF membrane, the glucose permeation through the membrane could be

186

regulated via changing bulk concentration (1, 5, 50 mM) and operating pressure (2, 3,

187

4 bar). In order to ascertain the optimal conditions to maximize the synergism of

188

separation and reaction, the effect of H2O2 concentration (0.02-5 mM) on the stability

189

of the immobilized HRP during BPA removal with reuse cycle was studied. Then, the

190

in-situ generated H2O2 caused by glucose accessible to the immobilized GOx was also

191

detected using the feed solution only containing glucose at different concentrations (1,

192

5, 50 mM) and pressures (2, 3, 4 bar).


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193

2.5.1. Glucose retention. Glucose concentration was measured by using 3,5-

194

dinitrosalicylic (DNS) method46-47. The retention of glucose (Rglu) by the pristine and

195

biocatalytic membranes is calculated by following equation:

196

𝑅𝑔𝑙𝑢 = 1 ― 𝐶𝑓𝑔 × 100%

197

where Cpg and Cfg represented glucose concentrations in the permeate and feed,

198

respectively.

199

2.5.2. Measurement of H2O2 concentration. H2O2 concentrations were determined by

200

visible spectrophotometry after color development with Ti(SO4)248-49. In the presence

201

of H2SO4, the H2O2 reacts with Ti(SO4)2 to form yellow substance which has an

202

adsorption maximum at 410 nm.

(

)

𝐶𝑝𝑔

(1)

Ti(SO4)2 + H2O2 + H2O→H4TiO5 + H2SO4

203

(2)

204

60 µl of H2SO4 (0.3 mol·L-1) and 200 µl of Ti(SO4)2 (0.3 mol·L-1) were added into 1.74

205

ml of samples, which were incubated for 5 min and then measured at 410 nm.

206

2.5.3. Determination of substrate inhibition. The substrate inhibition of H2O2 on

207

HRP was explored by measuring the BPA oxidation rate with various concentrations

208

of H2O2 in 10 ml of BPA solution (containing 1 mgL-1 HRP and 10 mgL-1 BPA). The

209

H2O2 concentration ranges were 0.005-20 mM. The BPA concentration in solution after

210

oxidation was measured by HPLC, and the BPA oxidation rate is calculated by

211

following equation:

212

V=

(𝐶𝑏 ― 𝐶𝑎)

(3)

𝑇



213

where Cb and Ca represented BPA concentrations before and after oxidation in solution,

214

respectively; T is the reaction time.

215

2.6. Reusability and stability of biocatalytic membrane. The biocatalytic membrane

216

reusability and stability were examined by testing the BPA removal for 7 days (20 ml

217

for each day) and continuous 10 h (300 ml BPA solution was treated totally),

218

respectively. The operational conditions: 3 bar, 100 rpm, 21 oC, 10 mg·L-1 BPA and 1

219

mM glucose in deionized water. For the 7 days measurement, the membrane was

220

continuously washed under pressure each time until no BPA was detected in washing

221

solution before stored at 4 ℃. The BPA removal efficiency is calculated as the BPA

222

amount difference between original feed and permeate divided by the BPA amount in

223

the original feed.

224 225

3. RESULTS AND DISCUSSION

226

3.1. Characterization. Distribution of the immobilized GOx and HRP in the NF270

227

membrane was clarified by CLSM. The CLSM images of the biocatalytic membrane

228

obviously indicated that both GOx-FITC (green) and HRP-RhB (red) were well-mixed

229

and mainly anchored in the intermediate layer between skin layer and support layer (Fig.

230

2).


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231 232 233 234

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) crosssection.

235

The contact angles of the pristine and biocatalytic membranes are shown in Fig. 3.

236

It was found that the contact angle of the support layer significantly decreased after

237

enzyme immobilization and PDA/PEI coating while the hydrophilicity of the skin layer

238

did not change. Such hydrophilicity improvement on support layer was attributed to the

239

highly hydrophilic PDA/PEI coating layer, indicating that the coating was successful

240

and this might be beneficial to membrane permeability. SEM images and Zeta potential

241

of the pristine and biocatalytic membranes (Figs. S1 and S2) also revealed that the

242

homogenous coating in the support layer was successfully obtained by co-deposition of

243

PEI and PDA.



244 245

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

246

3.2. Enzymatic cascade catalysis for BPA removal. We first compared BPA removal

247

efficiency by the pristine, PDA/PEI coated, and biocatalytic membranes. As shown in

248

Fig. 4a, the pristine NF membrane can retain more than 70% of BPA in the first cycle,

249

however, BPA molecules would adsorb and dissolve in the polyamide skin layer, and

250

then diffuse through the membrane, resulting in a continuous retention decrease with

251

filtration cycle ( it becomes below 40% in the fourth filtration cycle), which agrees well

252

with the previous results in literature40. Even though the BPA retention by the PDA/PEI

253

coated membrane increased a little thanks to the possible pore narrowing effect induced

254

by the coating layer, the BPA retention decline with reuse cycle was still observed.

255

While for the biocatalytic membrane with two enzymes, the BPA retention almost kept

256

constant (close to 100%) with reuse cycle when a suitable glucose concentration was

257

added. Thus, enzyme catalysis plays a significant role to improve the efficiency and

258

stability of BPA removal. During the biocatalytic membrane preparation, we explored

259

the permeability variation of the membrane (Fig. 4b). The membrane permeability

260

decreased remarkably after enzymes immobilization, which might be derived from the

261

protein fouling layer formed during the reverse filtration, increasing the filtration


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262

resistance of the membrane. After the PDA/PEI coating, the membrane permeability

263

recovered greatly because the hydrophilicity of the membrane support was improved

264

(Fig. 3). Moreover, the membrane permeability further increased after membrane

265

reversal due to the enzyme movement and leakage.

266 267

Figure 4(a) BPA removal efficiency by the pristine, PDA/PEI coated, biocatalytic membranes

268

when 10 gL-1 BPA solution containing 5 mM β-D-glucose was treated with an agitation speed of

269

100 rpm and an operation pressure of 3 bar for 4 cycles (10 ml per cycle). (b) Membrane

270

permeability variations at different enzyme immobilization stages.

271

The enzyme immobilization mechanisms included adsorption, entrapment and

272

covalent bonding as discussed in our previous work, and it was also confirmed that

273

there was almost no enzyme leakage during the storage for 9 days

274

loading in the membrane is about 29.85 µg cm-1 in this work, which was much higher

275

than in literature. On the other hand, under pressure-driven flow-through mode, the as-

276

prepared

277

substrate/intermediates/products around the anchored enzymes, but the contact time

278

between substrates and enzymes became short, thus the enzyme activity had to be high

membrane

would

facilitate

the


mass

13, 40.

The enzyme

transfer

of


Page 16 of 28

279

enough to ensure a high micro-pollutant removal. It was reported that the cascade

280

catalysis by GOx and HRP followed these steps (Eqs. 4-7)50-51. In the first step, the

281

required gluconic acid and H2O2 are in-situ produced by GOx through oxidizing β-D-

282

glucose in the presence of oxygen:

283

β-D-glucose + O2

284

In the next step, HRP is activated by H2O2, leading to the generation of compound I

285

and H2O. Then, compound I can oxidize BPA via oxidoreduction and generate

286

compound II and free radicals. Finally, the produced compound II interacts with other

287

BPA to generate radicals and regenerated HRP.

288

HRP + H2O2

289

compound I +BPA

290

compound II +BPA

GOx

gluconic acid + H2O2

compound I + H2O compound II + BPA radical BPA radical + HRP

(4)

(5) (6) (7)

291

In order to clarify the effect mechanisms of the cascade catalysis on BPA removal

292

by the biocatalytic membrane, we then investigated the effect of pH and glucose

293

concentration on the BPA removal efficiency by free enzymes. The results revealed

294

that the acidic pH caused by gluconic acid could enhance the activity of HRP toward

295

BPA degradation by virtue of H2O2 although gluconic acid itself had a little inhibition

296

effect on HRP compared to hydrochloric acid at the same pH (Fig. S3). It also was

297

confirmed that for the free GOx and HRP, the BPA removal efficiency was closely

298

related with the available glucose concentration, which was eventually attributed to the

299

produced H2O2 concentration since the pH variation was similar at the glucose


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300


concentrations from 5 to 125 mM (Fig. S4a).

301

Though increasing glucose concentration resulted in higher BPA removal by free

302

enzymes for a long reaction time (Fig. S4b), the catalytic behavior in the biocatalytic

303

membrane (a very short reaction time in flow-through mode) may be different, and

304

moreover, the reusability of the biocatalytic membrane may be worse at high glucose

305

concentration because the excessive H2O2 in-situ generated by GOx would inhibit or

306

damage the immobilized HRP. Next we investigated the effect of the glucose

307

concentration in the reactants on the BPA removal by the biocatalytic membrane. As

308

shown in Fig. 5a, in the first flow-through cycle, the BPA removal efficiency reaches

309

around 100% with 5 mM and 50 mM glucose, while it is only 93% with 1 mM glucose,

310

indicating that a higher glucose concentration would promote HRP activity on BPA by

311

decreasing pH and offering more H2O2 for the microenvironment of the immobilized

312

HRP in the membrane (Fig. 5b). However, with increase of reuse cycle, the BPA

313

removal by the biocatalytic membrane declined gradually when the glucose

314

concentration was 5 and 50 mM, on the contrary, with 1 mM glucose, the biocatalytic

315

membrane kept 100% of BPA removal in the next three reuse cycles. This can be

316

explained by the inactivation effect and substrate inhibition of the excessive H2O2 on

317

HRP, and the latter was confirmed by the BPA oxidation rate variation along with H2O2

318

concentration via free HRP-induced catalysis (Fig. 5c). Furthermore, the optimal range

319

of H2O2 concentration was determined by adding H2O2 instead of glucose into BPA

320

solution (Fig. 5d), and the BPA removal efficiency by the biocatalytic membrane could

321

reach nearly 100% in four reuse cycle when the H2O2 concentration ranges from 0.05



322

to 1 mM. It is worth noting that the BPA removal efficiency by the biocatalytic

323

membrane decreased with the reuse cycle when the H2O2 concentration was 0.02 mM,

324

which was attributed to the insufficient H2O2 concentration. In the initial filtration stage,

325

the BPA retention was high due to the adsorption effect (Fig. 4a) and the BPA in the

326

permeate could be fully oxidized under a low H2O2 concentration. However, with the

327

increase of reuse cycles, the BPA concentration in the permeate gradually increased

328

due to “adsorption saturation” of the membrane (Fig. 4a), and such low H2O2

329

concentration was insufficient to oxidize the flowing BPA. Besides the improper H2O2,

330

the undesirable pH (~ 7) would result in severer decline of the BPA removal efficiency

331

with increasing reuse cycle (Fig. S5).

332 333 334 335 336

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.


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337

3.3. Synergism of separation and reaction for engineering microenvironment. It

338

was verified that adding a suitable H2O2 concentration could achieve stable BPA

339

removal, but the pH of the real potable water is unfavored for HRP activity and the

340

H2O2 concentration in the membrane could not be adjusted by NF (H2O2 passes through

341

the membrane freely). Glucose is partly retained by the NF270 membrane, and the

342

glucose accessible to the immobilized enzymes can be regulated by both its bulk

343

concentration and permeate flux, which provides a simple and rapid way to control pH

344

and H2O2 concentration in the reaction zone. First, the biocatalytic membrane had a

345

little higher retention of glucose than the pristine NF270 due to the pore blocking effect

346

by the immobilized enzymes, and the glucose retention increased with permeate flux at

347

higher operating pressure owing to the enhanced convection of solvent. (Fig. S6).

348

Second, the permeate flux at higher glucose concentration was lower because the larger

349

osmotic pressure reduced the effective driving force across the membrane (Fig. 6a).

350

Third, the glucose permeance was affected by both its feed concentration and permeate

351

flux, which became larger at higher feed concentration and lower permeate flux (Fig.

352

6b). The above results regarding the glucose separation behavior could be used to

353

understand the BPA removal efficiency by the biocatalytic membrane at different

354

conditions. As shown in Fig. 6c-e, with 1 mM glucose, the BPA removal is less 95% in

355

the first flow-through cycle due to insufficient glucose passing through the membrane,

356

and although the contact time and glucose permeance are supposed to be higher at lower

357

pressure, the BPA removal at 2 bar is surprisingly lower than the others, implying that

358

the enzymatic reaction in the confined space may be different from in bulk solution. In



359

the second reuse cycle, due to the glucose accumulation in the membrane with filtration

360

time, the BPA removal greatly increased to 100% except for the case at 4 bar possibly

361

because of its higher glucose retention and shorter contact time (Fig. 6c). In the third

362

and fourth cycle, the BPA removal at all operating pressure reached 100%. While with

363

5 mM glucose, the BPA removal efficiency kept at a stable level at 100% under various

364

operating pressure with reuse cycle because such glucose concentration produced a

365

suitable H2O2 concentration in the membrane, which was confirmed by the results in

366

Fig. 6f (0.25-1.0 mM). However, with 50 mM glucose, the BPA removal declined in

367

the second cycle and became worse in the fourth cycle, especially at lower pressure.

368

This was explained by a fact that an excessive H2O2 posed a negative effect on the HRP,

369

and even in the first cycle, the generated H2O2 was much higher than 1.0 mM (Fig. 6f).

370

In the fourth cycle, the BPA removal was a little higher at higher pressure because it

371

resulted in lower glucose permeance and shorter contact time alleviating the inhibition

372

effect of the excessive H2O2. Therefore, by tuning the glucose concentration in water

373

and operating pressure, the BPA removal by the biocatalytic membrane can be

374

manipulated via engineering the microenvironment (pH and H2O2 concentration)

375

around the immobilized enzymes in the membrane. As shown in Fig. 7, a super high

376

and stable BPA removal (>80%) by our biocatalytic membrane (the BPA removal by

377

the pristine NF270 was only 40%) is obtained in a long-term operation (continuous

378

running for 10 h or batch operation for 7 days). To the best of our knowledge, these are

379

the top results on the reusability and stability of BPA removal when compared with

380

other biocatalytic membranes with immobilized laccase reported in literatures (Table


Page 20 of 28

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381

1). In detail, most biocatalytic membranes used for BPA removal were based on laccase

382

due to its high catalysis activity on different micropollutants. However, the laccase is

383

easily deactivated, resulting in its poor stability. In our previous work, the average BPA

384

removal efficiency decline of the laccase-loaded membranes ranged from 4% to 9% in

385

each cycle (10 mg L-1 BPA), while in present work, this number was only 1.36%,

386

showing much higher stability and reusability. Furthermore, the throughput of our

387

biocatalytic membrane was the highest among the results listed in Table 1,

388

demonstrating that this novel biocatalytic membrane could remove much more BPA in

389

a very short filtration time.



390 391 392 393 394 395

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).


Page 22 of 28

Page 23 of 28 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


396 397 398 399 400 401 402 Matrixes

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 1mM 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. a

Enzyme

BPA removal

Initial BPA concentration (mgL-1)

efficiency in the

BPA removal Reuse cycle

first cycle (%)

efficiency in the last cycle (%)

Average decline

Throughput

every cycle (%)

(Lm-2h-1)b

Ref.

CNTs-PVDF (MF)

laccase

4.57

96

4

91

1.25

10.00

52

TiO2 (sol-gel)-PVDF (MF)

laccase

34.24

92

4

84

2.00

20.00

53

TiO2 (NP)-PVDF (MF)

laccase

34.24

95

15

15

5.33

5.00

54

Cu-PVDF (MF)

laccase

10.00

99

5

57

8.68

10.00

55

PDA-NF270 (NF)

laccase

10.00

88

7

34

7.29

5.97

40

CuPDA-NF270 (NF)

laccase

2.00

87

9

77

1.11

2.49

13

PAN-MIL-101 (UF)

laccase

10.00

92

7

62

4.29

6.45

10

PDA/PEI-NF270 (NF)

GOx/HRP

10.00

89

7

80

1.36

21.94

This work

403 404

a

MF: microfiltration, UF: ultrafiltration, NF: nanofiltration;

b

Throughput was calculated by volume of feed

solution/membrane area/processing time.

405 406

4. CONCLUSIONS

407

A biocatalytic membrane for cascade catalysis was simply prepared by co-

408

immobilization of GOx and HRP in a NF membrane via reverse filtration and

409

subsequent co-deposition of PDA/PEI. When glucose in the reactants passed through

410

the biocatalytic membrane, it was catalyzed by GOx to produce gluconic acid and H2O2

411

for engineering microenvironment of the immobilized HRP, where the pH was

412

decreased to enhance the HRP activity, and BPA was degraded by HRP in the presence



413

of H2O2. Such cascade catalysis in a NF membrane toward micro-pollutant removal

414

could be well tuned by changing the bulk glucose concentration and operating pressure.

415

The BPA removal by the biocatalytic membrane in a flow-through cycle achieved 100%

416

with 5 mM glucose and 10 mg·L-1 BPA in the deionized water operated at 2-4 bar,

417

which even kept above 80% in a continuous filtration for 10 h or batch operation for 7

418

days. This biocatalytic NF membrane is promising for efficient and stable micro-

419

pollutant removal in portable water (as domestic water dispensers or tap water filters),

420

and a smart membrane system with a micro-pollutant sensor may be constructed, where

421

the glucose permeance through the NF membrane can be regulated by adjusting glucose

422

concentration and operation pressure according to the micro-pollutant concentration in

423

water, ensuring a suitable microenvironment for the HRP activity and stability.

424 425

ACKNOWLEDGEMENTS

426

The financial supports are supplied by the Beijing Natural Science Foundation

427

(2192053) and Youth Innovation Promotion Association (2017069) of Chinese

428

Academy of Sciences.

429 430

ASSOCIATED CONTENT

431

Supporting Information. SEM images and Zeta potential of pristine and biocatalytic

432

membranes (both sides). Effect of pH and acids on BPA oxidation by free HRP. Effect

433

of glucose concentration on pH of the solution and BPA removal efficiency by cascade

434

catalysis with free enzymes. Effect of pH on BPA oxidation by cascade catalysis in the

435

biocatalytic membrane. Glucose retention as a function of permeate flux by the pristine


Page 24 of 28

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436

and biocatalytic NF membranes at different glucose concentrations. This material is

437

available free of charge via the internet at http://pubs.acs.org.

438 439

AUTHOR INFORMATION

440

* Corresponding author: E-mail: [email protected] (J. Luo)

441 442

NOTES

443

The authors declare no competing financial interest.

444 445

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446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469

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Enzymatic Cascade Catalysis in a Nanofiltration Membrane

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