Cell Wall Bound Anionic Peroxidases from Asparagus Byproducts

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Cell wall bound anionic peroxidases from asparagus by-products Sara Jaramillo-Carmona, Sergio Lopez, Sara Vazquez-Castilla, Ana Jimenez-Araujo, Rocio Rodríguez-Arcos, and Rafael Guillén-Bejarano J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf502560k • Publication Date (Web): 08 Sep 2014 Downloaded from http://pubs.acs.org on September 15, 2014

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Journal of Agricultural and Food Chemistry

1

Cell wall bound anionic peroxidases from asparagus

2

by-products

3

4

Sara Jaramillo-Carmona1, Sergio López2, Sara Vazquez-Castilla1, Ana

5

Jimenez-Araujo1, Rocio Rodriguez-Arcos1, Rafael Guillen-Bejarano1,*

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1

8

Seville, Spain

9

2

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Phytochemicals and Food Quality Group, Instituto de la Grasa (CSIC), 41014

Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC),

41012 Seville, Spain

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12

*Telephone number 954611550; Fax number 954616790; E-mail:

13

[email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

15

Asparagus byproducts are a good source of cationic soluble peroxidases (CAP)

16

useful for the bioremediation of phenol contaminated wastewaters. In this study,

17

we have purified and characterized ionically and covalently cell wall bound

18

peroxidases (POD) from the same byproducts. The covalent forms of POD

19

represent more than 90% of the total cell wall bound POD. Isoelectric focusing

20

showed that while the covalent fraction is constituted primarily by anionic

21

isoenzymes , the ionic fraction is a mixture of anionic, neutral, and cationic

22

isoenzymes. Covalently bound peroxidases were purified by means of ion

23

exchange chromatography and affinity chromatography. In vitro detoxification

24

studies showed that while CAP is more effective for the removal of 4-CP and

25

2,4-DCP, anionic asparagus peroxidase (AAP) is better option for the removal

26

of hidroxytirosol (HT), the main phenol present in olive mill wastewaters.

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KEYWORDS: cell wall bound peroxidase, anionic isoperoxidase, asparagus by-

29

product, phenols, bioremediation.


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INTRODUCTION

31

The development of sustainable technology for the treatment of industrial

32

wastewaters provides a link between sustainable industry and environmental

33

balance. Phenols and their derivatives are the major pollutants in wastewaters

34

from the chemical and pharmaceutical industries.1 The common treatments of

35

these effluents to remove their phenol contamination

36

electrocoagulation,2 photodecomposition,3 adsorption,4 and enzymatic and

37

microbiological methods.5 There is a wide range of enzymes and genetically

38

modified microorganisms that are used to oxidize

39

peroxidase (POD) for bioremediation is based on the oxidation of aromatic

40

compounds by hydrogen peroxide and was first reported by Klibanov and Morris

41

in 1981.6 The efficiency of horseradish POD for phenol removal was reported to

42

be around 90%.7 Several factors should be considered when the industry

43

choose the use of POD for the elimination of phenols, such as the high-

44

quantity,high efficiency, and low-cost of the enzyme. Agricultural by-products

45

are promising alternative raw materials to horseradish (Armoracia rusticana)

46

root for the extraction of POD.

47

Asparagus is a high-value, labor-intensive, perennial vegetable and a

48

continuously growing crop. Only 50% of the spear is used for human

49

consumption and the rest is considered a by-product that has been traditionally

50

used for animal feeding and low-value products. Previous studies revealed that

51

these by-products are rich in many of the same phytochemicals that can be

52

found in the edible part of the spears, mainly phenols (flavonoids and

53

hydroxycinnamic acids) and saponins. We have recently optimized a method for

54

the extraction of these phytochemicals from asparagus by-products and their

55

use as functional ingredients.8 In addition, we have demonstrated that

are diverse:

phenols. The use of



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asparagus by-products are a major source of a highly efficient soluble cationic

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POD. These CAP (cationic soluble peroxidases) have a significant effectiveness

58

for the bioremediation of the phenols present in different industrial effluents,

59

such us 4-chlorophenol, 2,4-dichlorophenol and hydroxytyrosol.9

60

PODs are differentially expressed in various plant tissues in multiple molecular

61

forms, soluble, ionic, and covalently bound to the cell walls.10 All of these

62

isoenzymes contain identical heme groups but differ in the precise composition

63

of the glycoprotein and widely varying in their isoelectric point and with different

64

thermal stability, substrate specificity and physiological roles in plant tissues.11

65

The isoenzymes can be classified in two groups, one having the isoelectric

66

point in an acidic range, which is generally associated with lignin biosynthesis

67

and the other, in a basic range, associated with indole-3-acetic acid

68

degradation.12 Different isoelectric patterns are reported for different vegetables

69

tissues13,14,15 and the interaction with phenolics compounds and hidrolityc

70

enzymes may be responsible for the formation of POD with different

71

electrophoretic mobilities.16 However, the physiological function of individual

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isoenzymes is only partially understood and is complicated by the presence of

73

multiple POD isoenzymes. Such isoforms are often rather difficult to be isolated

74

and purified.

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Asparagus, as many other plants, contains a complete set of isoperoxidases,

76

associated with the soluble and bound fractions of the same tissue. The aim of

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this study was to develop a simple and rapid method for the purification and

78

characterization of one cell wall bound POD of asparagus tissues and to study

79

their potential use for the bioremediation of different industrial effluents.

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MATERIALS AND METHODS

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Chemicals and enzymatic preparations

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All the solvents used for chromatography were HPLC grade. 4-Chlorophenol, 2,

84

4-dichlorophenol were from Sigma Aldrich (Madrid, Spain) and hydroxytyrosol

85

were from Extrasynthese (Genay Cedex, France). The commercial enzymes,

86

Olivex, Novoferm 31, Celluclast, Pectinex AR were kindly provided by

87

Novozymes A/S (Madrid, Spain).

88 89

Plant material

90

White asparagus by-products were obtained from local producers (Seville,

91

Spain) and were frozen with liquid nitrogen until further use.

92 93

Extraction of cell wall bound peroxidases

94

The residue from the extraction of the soluble POD9 was re-extracted as

95

described below. The residue (500 g) was homogenized in a Janke & Kunkel

96

Ultraturrax T-25 (IKA®-Labortechnik, Staufen, Germany) at top speed for 3

97

minutes with 500 mL of 50 mM Tris/HCl buffer, pH 7.0 containing 1 g/L ascorbic

98

acid and 1 M of potassium chloride.Afterwards, the solid residue was recovered

99

by filtration through nylon. This procedure was repeated twice to maximize the

100

extraction yield. The filtrates were pooled to get the ionic POD extract. The solid

101

residue was washed three times with water prior to the extraction of the

102

covalent peroxidases. Then it was re-suspended in 50 mM of a sodium acetate

103

buffer, pH 5.0, with 5.0% (v/v) of a commercial enzymatic mixture “Olivex”

104

(Novozymes A/S, Madrid, Spain) and subjected to agitation for 12 h at 25 °C

105

followed by centrifugation at 21,214g for 30 min. The supernatant was saved

106

and aliquoted for further analysis. 5 ACS Paragon Plus Environment


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Assay for the POD Activity

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POD activity was determined with ABTS as the reducing substrate in a total

110

volume of 0.2 mL. The assay mixture contained 20 µL of a 1 M acetate buffer

111

with a pH of 5.0, 20 µL of 1 mM ABTS, and 20 µL of 0.3% H2O2 and variable

112

amounts of H2O and enzyme preparations to reach the final assay volume. The

113

oxidation of ABTS was followed by monitoring the increase in absorbance at

114

415 nm and using ε415 nm = 31.1 mM−1 cm−1 for ABTS cation radical formation.17

115

One unit of activity (UA) was defined as the amount of enzyme required to

116

oxidize 1 µmol of ABTS per minute at pH 5.0 and 25 °C. A Bio-Rad iMark

117

microplate reader was used for the spectroscopic measurements.

118 119

Protein Determination

120

Protein was quantified on the basis of the dye-binding method of Bradford using

121

bovine serum albumin (BSA) as standard.18

122 123

Isoelectric Focusing

124

Preparative isoelectric focusing was carried out using a Rotofor preparative IEF

125

cell (Bio-Rad). The protein samples were dialyzed against deionized water

126

overnight, supplemented with 2% (v/v) Biolytes (Bio-Rad) of 3.0−10.0 pH range,

127

and then loaded into the Rotofor cell. The isoelectric focusing, without pre-

128

running, was done according to the manufacturer’s instructions. The power

129

supply was set at a constant power of 12 W. Once focusing was complete, the

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electro-focusing cell was fractionated into 20 aliquots ranging from acidic to

131

basic isoelectric point (pI) proteins. POD activity and protein contents were

132

determined for each aliquot. 6 ACS Paragon Plus Environment

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133 134

Gel Electrophoresis

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SDS−PAGE of the crude extract and purified fraction was performed on a 12%

136

acrylamide minigel in a Bio-Rad protein II electrophoresis cell set at 150 V for

137

120 min according to the method of Laemmli.19 Protein band detection was

138

conducted by silver staining as previously described by Rabilloud.20 Molecular

139

weights of the bands were calculated using Labimage software v2.7.1 (Kapelan

140

Bio-Imaging).

141 142

Purification of anionic POD

143

The different samples were dialyzed against deionized water, centrifuged at

144

21,214g for 2 h at 4 °C, and filtered prior to purification. The chromatography

145

purification steps were carried out at room temperature.

146 147

Affinity chromatography

148

The low pI fractions from the IEF showing POD activity were combined and

149

loaded into a column Concanavaline A-Sepharose (Amersham Pharmacia

150

Biotech, Sweden) affinity column pre-equilibrated with 20 mM Tris/HCl buffer at

151

pH 7.0 containing NaCl 0.5M at a flow rate of 0.3 mL/min. Unbound proteins

152

were removed by repeated washings with concanavalin A loading buffer at 1.0

153

mL/min. Anionic POD were eluted from the resin with two successive washes

154

with five column volumes of loading buffer containing 0.5 M methyl-α-D-

155

glucopyranoside. The resin was left in contact with the buffer for 1 h for the first

156

elution and overnight for the second elution. Afterwards, the column was

157

washed with 1.0 M methyl-α-D- glucopyranoside to elute the proteins strongly



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bound to the column. Fractions of 6 mL were collected during all the

159

chromatography steps and the POD activity and protein content were

160

measured.

161 162

Effect of pH on POD

163

POD activity as a function of pH was established by incubating the purified POD

164

in 1 M sodium acetate buffer (pH 3.0−5.0), 1 M sodium phosphate buffer (pH

165

5.0−7.0), and 0.05 M Tris-HCl buffer (pH 7.0−9.0). Analysis conditions were the

166

same as those described for the POD activity assay.

167 168

Determination of Optimum Temperature and Thermal Stability

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The optimal temperature and thermal stability of POD were assayed using a

170

water bath (Precis-Term P-Selecta) with the optimal pH at temperatures ranging

171

from 15 to 80 °C, for 10 min prior to substrate addition. After heating, the

172

solutions were rapidly cooled in ice water, and the POD activity was

173

immediately determined. The percent residual activity was plotted against

174

different

175

temperatures in the range of 50−85 °C with exposure times ranging from 1 to 30

176

min.

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Kinetic constants for ABTS and H2O2

178

Kinetic parameters were calculated from Lineweaver-Burk plots for the two-

179

substrate mechanisms followed by POD15 using H2O2 and ABTS concentrations

180

from 0.1-10 mM, respectively. Temperature and pH were kept constant in the

181

optimum values determined previously. Taking into account the reciprocal of

temperatures.

Thermal

stability

was

also

tested

at

varying


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both sides of the Michaelis-Menten equation leads to the Lineweaver-Burk

183

relationship:

184

1/ν0 = Ka / A0Vmax + Kb / B0Vmax + 1/ Vmax

185 186 187

where ν0 is the initial reaction velocity, Vmax is the maximum reaction velocity, Ka

188

and Kb are Km values, the Michaelis-Menten constant for substrate A (H2O2) and

189

B (ABTS), respectively, and A0 and B0 are substrate A and B concentrations.

190

The ν0 were determined as a function of both substrate concentrations. A plot of

191

1 / ν0 vs 1 / [B0] for varying values of [A0] will give a series of parallel lines, each

192

of slope Kb / Vmax. A plot of intercept vs 1 / [A0] will give a line of slope Ka / Vmax

193

and intercept 1 / Vmax. Hence Ka, Kb and Vmax may be determined.

194 195

Removal of Phenolic Compounds

196

We evaluated the catalytic activity of the purified POD, using different model

197

compounds such as monoaromatic phenolic type 4-chlorophenol (4-CP), 2,4-

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dichlorophenol (2,4-DCP) as a model of the disubstituted substrate, and 2-(3,4-

199

dihydroxyphenyl)ethanol [hydroxytyrosol (HT)], a polyphenol present in olive mill

200

wastewater. The assays were performed in a reactor at room temperature. All

201

substances were dissolved in 1 M acetate buffer at pH 4.0. The optimal ratio of

202

AAP and H2O2 concentration for phenol removal was investigated using

203

H2O2concentrations between 1 and 20 mM. In order to study the influence of

204

AAP,

205

different incubation conditions: (a) a control solution that consisted of 2 mM

206

phenol, (b) 2 mM phenol and H2O2, (c) 2 mM phenol and CAP, and (d) 2 mM

H2O2, and their combination on the removal of phenols, we followed



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phenol, H2O2, and CAP. The course of the reaction was monitored by taking 1

208

mL of sample, and the concentration of the phenol was analyzed until the value

209

was constant.

210 211

Phenol Quantification

212

The removal of each phenol was monitored by high-performance liquid

213

chromatography (HPLC) and an ultraviolet detector. The aliquots were taken

214

from model wastewaters at the indicated times. The reaction was stopped by

215

the addition of 10 µL of 20% trichloroacetic acid (TCA). After centrifugation, 20

216

µL were injected into a MediterraneaSea C18 (Teknokroma), 5 µm, 25 × 0.6

217

reversed phase HPLC column. The initial mobile phase consisted of 70:30 (v/v)

218

water:acetonitrile with 0.1% formic acid, which was brought to 0:100 (v/v) in a

219

linear gradient lasting for 20 min, held for another 10 min, and then 5 min of

220

30% acetonitrile. Phenol concentrations were determined from a straight-line

221

standard calibration obtained using known concentrations (standards) of each

222

compound. The results are expressed as removal efficiency, which is defined as

223

the percentage of phenols removed from the solution under the established

224

experimental conditions.

225 226

Statistical Analysis

227

The mean ± SD of three replicates was calculated. All data were analyzed using

228

multivariate analysis of variance (MANOVA) followed by the Fisher−LSD multi-

229

comparison test. The level of significance was p < 0.05.

230 231


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RESULTS AND DISCUSSION

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Extraction of cell wall bound POD

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It is known that a great part of the apoplastic POD is associated to cell walls

235

either by ionic interactions or by covalent bonds. In this study, we have

236

developed a sequential method to extract the different forms of peroxidases that

237

were ionically and covalently bound to the cell wall of the asparagus tissues.

238

The wall-bound POD can partially be released by treatment with a high molarity

239

solution of NaCl or other salts. In our method, after the extraction of the soluble

240

fraction,9 the cell walls still contained a considerable amount of POD activity.

241

We performed a washing procedure to determine if POD activities were loosely

242

bound to the cell wall or entrapped inside the intracellular vesicles. The residue

243

obtained after soluble POD extraction was suspended in the same buffer used

244

but added 1.0 M KCl. Then we followed the extraction procedure for ionic

245

isoenzymes as described in the materials and methods section. The crude

246

extract contained 0.89 UA of POD/g fresh material. This value represents a

247

small quantity of ionically bound POD (IBP) compared to soluble POD (5.31

248

UA/g fresh material).9 The highly soluble to IBP ratio has also been reported for

249

other plant foods, such as peaches (5:1)21, mustard (2:1).22 and artichoke hearts

250

(6:1)29.

251

After extraction with high ionic strength, the cell walls were used as substrate to

252

test the further extractability of POD by the enzymatic method in order to get the

253

covalently bound POD (CBP). Treatment of the cell walls for 12 hours with 50

254

mM acetate buffer, pH 5.0 and various commercial enzymatic preparations with

255

different hydrolytic activities was assayed (Table 1). The POD activity was

256

measured after the enzymatic hydrolysis of the cell walls. As shown in Figure 1,



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Novoferm 31 extracted the same peroxidase amounts as those detected in the

258

control. After the hydrolysis by Celluclast, the POD activity was higher than that

259

detected in the control sample but Clluclast did no efficiently extract the CBP.

260

The treatments of cell wall with Pectinex AR or Olivex resulted in the most

261

efficient

262

polygalacturonase and pectin esterase activities (Table 1) but Olivex possesses

263

higher cellulase activity. In order to determine if the different efficiency of these

264

two commercial preparations was due to their different cellulase activity, a

265

mixture of Pectinex AR plus Celluclast was also tested. A similar treatment on

266

oat cell wall led to maximum recovery of CBP.23 However, in asparagus by-

267

product cell walls, the CBP released after the treatment with the combination

268

enzymatic mixture was the same as that obtained with Olivex alone. On the

269

other hand, it is noteworthy that Novoferm 31, despite

270

enzymatic composition similar to Olivex and Pectinex AR, is not effective for the

271

release of peroxidase tightly bound to the cell wall. This fact could be due to the

272

inactivation of the enzymatic mixture or to the presence of some inhibitors.

273

These findings suggest that the concerted action of polygalacturonase, pectin

274

esterase and cellulase is required to efficiently release CBP. In fact, with this

275

method we found that the cell walls from the asparagus by-product possess a

276

high CBP (4.11 UA/g fresh material). This value was similar to the previously

277

reported cytoplasmatic POD (5.31 UA/g).9 In total, the POD activity in the crude

278

extract of asparagus by-products was 10,31 UA/g of fresh material. This value

279

is higher than values reported for typical sources of peroxidase such as turnip

280

extract (6,5 UA/g),24 and broccoli (3,5 UA/g).25 Ionic forms of POD represent

281

less than 10% of the activity detected (0,89 UA/g) thereby the CBP is a good

extractions.

These

enzymatic

mixtures

contain

similar

possessing an


Page 13 of 37


282

source for peroxidase, while in other plants this fraction is considered too low to

283

be assayed.13

284 285

Isoelectric Focusing of cell wall bound POD

286

The distinct forms of the peroxidases from the cell walls were characterized by

287

IEF in a pH range of 1 to 12. Different patterns of activity were obtained for the

288

ionic and covalent fractions (Figure 2). IBP included a broad range of acid (pI ca

289

4-5), as neutral (pI ca 6-7) and basic (pI ca 8-12) isoforms (Figure 2A). A similar

290

IEF profile has been described in flax fibers.26 However, in green asparagus the

291

IEF profile for ionic POD was similar to the soluble POD.9 These isoenzymes

292

are mainly neutral isoforms and the activities of the isoenzymes with pI between

293

5-7 were weak.27

294

The IEF profiles obtained for CBP showed a group of anionic isoforms (pI, ca.

295

5-6) while the extreme cationic isoforms present in the soluble and ionic

296

fractions were absent or negligible in the covalent fraction (Figure 2B). A Similar

297

IEF pattern has been described for the covalent fraction of proteins from pea

298

root.28

299

Consequently while CBP is constituted mainly of anionic isoenzymes, the IBP

300

showed a very broad isoenzymatic profile and is a mixture of cationic neutral

301

and anionic isoforms.

302 303

Purification of anionic POD

304

The POD activity in the crude IBP was lower (0.89 UA/g of fresh material) than

305

the activity measured in the CBP (4.11 UA/g of fresh material) which was similar

306

to the POD activity in the soluble fraction.9 Previously we had developed a



Page 14 of 37

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method for the purification of cationic POD9 and here we present a new method

308

to purify the anionic asparagus POD present in the cell wall of asparagus by-

309

products (AAP). The specific activity and purification factor for the CBP extract

310

are shown in Table 2. The CBP was purified

311

isoelectric focusing and affinity chromatography on concanavalin A. The specific

312

activity of the crude extract was low due to a high content of proteins from the

313

commercial enzymatic mixture and the proteins extracted.

314

The IEF profile of the CBP showed several bands with pI values between 4.3

315

and 5.8 (Figure 2B). These fractions were pooled and separated from the basic

316

isoenzymes, which improved the purity of the enzymes (21-fold, Table 2) and

317

reduced the volume of the crude sample by 32% before following the

318

chromatography step. The pooled acidic fractions, representing 68% of the total

319

activity of the crude extract, were dyalized and transferred onto the

320

Concanavaline A-Sepharose column as described in the materials and methods

321

section. Concanavaline A is a lectin which binds to the carbohydrate molecules

322

containing α-D-mannopyranosyl, α-D-glucopyranosyl and sterically analogous

323

residues. Figure 3 indicates that POD activity was mainly distributed as a single

324

peak separated from the protein fraction. This purification step allowed us to

325

obtain pure anionic asparagus POD (AAP) with high specific activity from

326

asparagus by-products (Table 2). Overall the enzyme was purified about 236

327

times and showed a high yield for recovery of the activity.

328

Partial characterization of AAP

329

SDS-electrophoresis was monitored through protein band analysis to determine

330

the molecular weight of the proteins of the isolated fractions (Figure 4). After the

331

isoelectric focusing purification step, the ionic and covalent proteins showed

in two consecutives steps:


Page 15 of 37


332

different protein bands after SDS-electrophoresis. The ionic fraction contained

333

two main bands of 25 and 28 kDa, and three other faint bands of 23, 32, and

334

40 kDa. Conversely, the IEF fraction contained two main bands of 40 and 53

335

kDa, and four other faint bands of 20, 65, 90, and 130 kDa. These faint bands

336

were not present after Concanavalin A column purification and the band of 53

337

kDa was greatly diminished, suggesting that most of the anionic proteins of this

338

molecular weight lack glycosidic residues. Interestingly, small proteins seemed

339

to be ionically bound to the cell walls while medium and large proteins are

340

mainly bound to the cell wall covalently. A previous study on artichokes also

341

found that the molecular weight of POD isoforms in the ionic fraction were

342

smaller than that of covalently bound proteins in the leaf and the heart.29 In pea

343

root, a covalent POD of 70 kDa with nine isoenzimes was described.28 A

344

relationship between the size, the ionic strength of the POD, and the number of

345

glycosylated residues of amino acids for cell wall anchoring may exist. As we

346

have detailed by IEF, the pI of the ionically bound POD is lower than the

347

covalently bound POD. Moreover, basic POD from asparagus have a pI far

348

from 7.0 and their molecular weights have been described to be of 23, 27, and

349

43 kDa.9

350 351

Optimal pH of AAP

352

The optimal activity of AAP was tested against different pH by using ABTS as

353

substrate. To rule out any buffer effect on the peroxidase activity we checked

354

that at pH 5 with acetate buffer and with phosphate buffer the POD activity was

355

the same and that this was also true at pH 7 with phosphate buffer and Tris

356

buffer respectively. AAP displayed a typical bell-shaped pH profile (Figure 5A),



Page 16 of 37

357

which exhibited stronger activity in a pH range of 3.0–5.0, with an optimal pH of

358

3.5. At pH 5.0 the AAP activity was around 50% of the maximum activity. In

359

asparagus, the optimal pH for the activity of AAP was lower than those purified

360

from other vegetable sources such as red cabbage (pH 4.0),12 red algae (pH

361

5.0)30 or sweet potato tubers (pH 4.5).31

362 363

Thermal stability of AAP

364

The optimal temperature for AAP was between 35-40 ºC (Figure 5B). These

365

values agree with those obtained for the covalent POD of green asparagus.27

366

The Tm value for AAP was around 100 ºC, which highlights the thermal stability

367

of the enzyme compared to CAP.9 In order to characterize the biocatalytic

368

properties of the AAP, the percent remaining activity was plotted against time

369

(Figure 6). The exposure of the AAP for 30 minutes at 60 ºC did not decrease

370

its activity, while the exposure of the AAP at 70 ºC for 30 minutes reduced the

371

POD activity by up to 50%. These results corresponded with the monophasic

372

behavior described for green asparagus extracts treated between 50-60 ºC.32

373

Other authors reported that several POD forms had a higher thermal stability as

374

no reduction in the POD activity was found below 80 ºC.32 From a technological

375

point of view, this is an important outcome as treatment at high temperatures

376

(80-90 ºC) provoked a drastic loss in the AAP activity in 5 minutes. Similar to

377

CAP,9 Figure 6 showed that the remaining activity gradually decreased as the

378

temperature increased, yielding nonlinear curves. Our results agreed with those

379

that Ganthavorn33 reported on partially purified raw POD extracts from green

380

asparagus. Their data closely corresponded to a monophasic behavior at 50

381

and 60 °C, and a biphasic behavior at 70 °C.


Page 17 of 37


382

Kinetic Constants for ABTS and H2O2

383

.The Michaelis-Menten equation is used to distinguish among the ordered,

384

random, and ping-pong mechanisms and to obtain the Km values for both

385

substrates (ABTS and H2O2). The purified AAP showed typical Michaelis–

386

Menten kinetics for both ABTS and H2O2. The substrate saturation curve was

387

obtained by interpolating the substrate concentrations against activity values.

388

Michaelis–Menten constants (Km) were determined from Lineweaver–Burk

389

plots. The double reciprocal plots of the AAP kinetics are shown in Figure 7A,

390

where the parallel lines indicate that AAP follows a ping-pong mechanism. The

391

calculated y-intercepts were plotted against 1/A0 and resulted in a straight line

392

(Figure 7B) from which we achieved the Vmax = 0.695 mM ABTS min−1. The Km

393

value for H2O2 was found to be 0.53 mM. This value was lower than those

394

reported for turnip POD (0.8 mM).34 The ABTS Km value was 0.21 mM, which is

395

60 times lower than the value reported for red algae POD (13mM)30 or turnip

396

POD (0.71 mM).34This indicated that AAP may be suitable for applications

397

where a high sensitivity for ABTS is required.

398 399

Phenolic compound removal by AAP

400

We have previously characterized the activity of cationic POD from asparagus

401

by-products for removing phenolic compounds such as HT (which are found in

402

olive oil wastewaters at high concentrations), 4-CP, and 2,4-DCP9. We now

403

extend this knowledge to the AAP from the same source. The relationship

404

between phenols removed as a function of hydrogen peroxide for 1 h in a stirred

405

batch reaction is shown in Figure 8A. All of the experiments were carried out at

406

the optimal pH and temperature for AAP (as shown in Figure 5). At 2 mM H2O2,



Page 18 of 37

407

59% of the total HT was already removed while only 14.6% and 4% were

408

removed for 2,4-CP and 4-C, respectively. The optimal H2O2 concentration for

409

HT and 2,4-CP removal was 4 mM while for 4-CP an 8 mM H2O2 was needed.

410

Within 1 h, the preferred substrate for AAP was HT>2,4-DCP>4-CP. The results

411

clearly show that the optimal relationship of AAP/H2O2 for the removal of 2,4

412

DCF and HT was 5 UA AAP mL−1 mmol−1 H2O2 while for 4-CP it was 10 UA

413

AAP mL−1 mmol−1 H2O2. The incubation of AAP with phenolic compounds

414

assayed without H2O2 did not produce a significant decreased in the

415

concentration of the initial compounds (data not shown), suggesting that

416

phenolic oxidation is due to POD activity and not to other enzymatic activities

417

that could be present in the extract.

418

Finally, we studied the time courses of the changes in the phenolic compound

419

concentrations (Figure 8B). The rates of degradation of the three phenols

420

assayed differed. AAP was most active against HT, more than 70% of the total

421

HT disappeared within the first 1 h compared to 2,4-CP and 4-CP, which were

422

removed in 42% and 25% of the initial concentration, respectively. In the same

423

conditions CAP was able to remove only 25% of the HT.9 Longer times of

424

exposure of the HT and 4-CP to the AAP did not reduce the amount of phenol

425

concentrations. However, AAP was still active after 2 h in the presence of 2,4-

426

DCP, which was removed by up to 50% for its initial concentration (Figure 8B).

427

Intriguingly most of the POD from vegetables are reported to be more active for

428

monophenols such as 4-CP35 than for ortho phenols such as HT. So, the

429

botanical source of POD plays an important role in the efficiency of

430

bioremediation. Taking into account our previous studies,9 it can be concluded

431

that in the POD from asparagus by-products, CAP isoforms have higher


Page 19 of 37


432

efficiency in removing chorophenols while AAP have higher efficiency in

433

removing

434

bioremediation of mono and di- chorophenols exists36 while little information can

435

be found in relation to the removal of the phenols present in olive oil

436

wastewater.

437

In conclusion, large differences were found between acidic and basic wall

438

isoperoxidases in relation to their efficacy in the oxidation of the phenolic

439

compounds assayed. While CAP was found to be a good alternative for the

440

removal of chlorinated compounds, the AAP is a better option for the removal of

441

the main phenol present in olive mill wastewaters. Both isoenzymes might exert

442

their activities in an acidic environment and AAP showed a very high

443

thermostability and resistance to inhibitors action. All these features make AAP

444

a valuable enzyme for the removal of highly polluted wastewater produced by

445

the agro-food industries.

ortophenols

such

as

HT.

Extensive

knowledge

about

the

446 447

Abbreviations used

448

A, activity; ABTS, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid); BSA,

449

bovine serum albumin; AAP, anionic asparagus peroxidase; CAP, cationic

450

asparagus peroxidase; CE, crude extract; CBP, covalently bound peroxidase; 4-

451

CP, 4-chlorophenol; 2,4-DCP, 2,4-dichlorophenol; E, enzyme; HPLC, high-

452

performance liquid chromatography; HT, hydroxytyrosol; IBP, ionically bound

453

peroxidase; IEF, isoelectric focusing; pI, isoelectric point; POD, peroxidase(s);

454

Tm, midpoint inactivation temperature; TCA, trichloroacetic acid; Tris, 2-Amino-

455

2-hydroxymethyl-propane-1,3-diol; UA, units of activity.

456



457

AUTHOR INFORMATION

458

Corresponding Author

459

*Phone: +34 954611550. Fax: +34 954616790. E-mail: [email protected]

460

Notes

461

The authors declare no competing financial interest

462

ACKNOWLEDGEMENTS

463

S. Jaramillo-Carmona and S Lopez acknowledge financial support from the

464

Spanish CSIC (JAE-doc Program), a contract co-funded by the ESF. S

465

Vazquez-Castilla wishes to thank the Spanish CSIC for her contract (JAE Pre

466

Program).

Page 20 of 37

467 468

REFERENCES

469

(1) Botalova, O.; Schwarzbauer, J.; al Sandouk, N. Identification and chemical

470

characterization of specific organic indicators in the effluents from chemical

471

production sites. Water Res. 2011, 45, 3653−3664.

472 473

(2) Rajkumar, D; Palanivelu, K. Electrochemical treatment of industrial wastewater. J. Hazard. Mater. 2004, 113, 123-129.

474

(3) Du, Y. X; Fu, Q. S.; Li, Y; Su, Y. L. Photodecomposition of 4-chlorophenol

475

by reactive oxygen species in UV/air system. J. Hazard. Mater. 2011, 186,

476

491-496.

477

(4) Gholizadeh, A.; Kermani, M.; Gholami, M.; Farzadkia, M; Yaghmaeian, K .

478

Removal Efficiency, Adsorption Kinetics and Isotherms of Phenolic

479

Compounds from Aqueous Solution Using Rice Bran Ash. Asian J. Chem.

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2013, 25, 3871-3878.


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(5) Maza-Marquez, P.; Martinez-Toledo, M. V.; Gonzalez-Lopez, J.; Rodelas,

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B.; Juarez-Jimenez, B.; Fenice, M. Biodegradation of olive washing

483

wastewater pollutants by highly efficient phenol-degrading strains selected

484

from adapted bacterial community. Int. Biodeterior. Biodegrad. 2013, 82, 192-

485

198.

486

(6) Klibanov, A. M.; Morris, E. D. Horseradish peroxidase for the removal of

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carcinogenic aromatic amines from water. Enzyme Microb. Technol. 1981,

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3, 119−122.

489

(7) Wagner, M.; Nicell, J. A. Detoxification of phenolic solutions with

490

horseradish peroxidase and hydrogen peroxide. Water Res. 2002, 36,

491

4041–4052.

492

(8) Fuentes-Alventosa, JM; Jaramillo-Carmona, S; Rodriguez-Gutierrez, G;

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Guillen-Bejarano, R; Jimenez-Araujo, A; Fernandez-Bolanos, J; Rodriguez-

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Arcos, R. Preparation of bioactive extracts from asparagus by-product.

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Food Bioprod. Process., 2013, 91, 74-82.

496

(9) Jaramillo-Carmona, S.; Lopez, S.; Vazquez-Castilla, S.; Rodriguez-Arcos,

497

R.; Jimenez-Araujo, A.; Guillen-Bejarano, R. Asparagus by-products as a

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new source of peroxidases. J. Agric. Food Chem. 2013; 61, 6167-6174.

499

(10) Mader, M.. Compartimentation of peroxidase isoenzymes in plant cells. In

500

Plant

501

Biochemical and Physiological Aspects. Ed. Penel, C.; Gaspar, Th.;

502

Greppin, H. University of Geneva, Italy, 1992.

503

(11)

Peroxidases,

Topics

and Detailed

Literature

on

Molecular

Sergio L., Cardinali A., De Paola A., Di Venere D., Biochemical

504

properties of soluble and bound peroxidase from artichoke heads and

505

leaves. Food Technol. Biotechnol., 2009, 47, 32-38.



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506

(12) Boeuf, G.; Bauw, G.; Legrand, B.; Rambour, S. Purification and

507

characterization of a basic peroxidase from the medium of cell suspension

508

cultures of chicory. Plant Physiol. Biochem. 2000, 38, 217-224.

509

(13) Monerri, C.; Guardiola, J. L. Peroxidase activity and isoenzymes profile in

510

buds and leaves in relation to flowering in Satsuma mandarin (Citrus

511

unshiu). Sci. Hortic. 2001, 90, 43-56.

512

(14) Martinez-Pastur, G.; Zappacosta, D.; Arena, M.; Curvetto, N. Changes in

513

isoperoxidase patterns during the in vitro rooting of nothofagus Antarctica.

514

Bulg. J. Plant Physiol. 2001, 27, 43–53.

515

(15) Lepeduš, H.; Cesar, V.; Krsnik-Raso, M. Guaiacol Peroxidases in Carrot

516

(Daucus carota L.) Root. Food Technol. Biotechnol. 2004, 42, 33–36

517

(16) Hamed, R. R.; Maharem, T. M.; Fatah, M. M. A.; Ataya, F. S. Purification

518

of peroxidase isoenzymes from turnip roots. Phytochemistry. 1998, 48,

519

1291-1294.

520

(17) Fortea, M. I.; Pellicer, J. A.; Serrano-Martínez, A.; López-Miranda, S.;

521

Lucas-Abellán, C.; Núñez-Delicado, E. Red cabbage (Brassica oleracea)

522

as a new source of high-thermostable peroxidase. J. Agric. Food. Chem.

523

2012, 60, 10641−10648.

524

(18) Bradford, M. M. A rapid and sensitive method for the quantitation of

525

microgram quantities of protein utilizing the principle of protein-dye

526

binding. Anal. Biochem. 1976, 72, 248−254.

527 528 529 530

(19) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227, 680−685. (20) Rabilloud, T. Mechanisms of protein silver staining in polyacrylamide gels: A 10-year synthesis. Electrophoresis. 1990, 11, 785−794.


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531 532

(21) Neves, V. A. Ionically bound peroxidase from peach fruit. Braz. Arch. Biol. Technol. 2002, 45, 7-16.

533

(22) Saroop, S; Chanda, S. V.; Singh, Y. D. Changes in soluble and ionically

534

bound peroxidase activities during Brassica Juncea seed development.

535

Bulg. J. Plant Physiol. 2002, 28, 26–34.

536

(23) González, L. F.; Rojas, M .C.; Perez, F. J. Diferulate and lignin formation is

537

related

to

biochemical

differences

538

Phytochemistry. 1999, 50, 711-717.

of

wall-bound

peroxidases.

539

(24) Duarte-Vázquez, M. A.; García-Almendárez, B.; Regalado, C.; Whitaker, J.

540

R. Purification and partial characterization of three turnip (Brassica napus

541

L. var esculenta D.C.) peroxidases. J. Agric. Food. Chem. 2000, 48,

542

1574−1579.

543

(25) Duarte-Vázquez, M. A.; García-Padilla, S.; García-Almendárez, B. E.;

544

Whitaker, J. R.; Regalado, C. Broccoli processing wastes as a source of

545

peroxidase. J. Agric. Food. Chem. 2007, 55, 10396−10404.

546 547 548 549

(26) McDougall, G. J. Changes in cell wall-associated peroxidases during the

lignification of flax fibers. Phytochemistry. 1992, 31, 3385-3389. (27) Wang, Z.; Luh, B. S. Characterization of soluble and bound peroxidases in green asparagus. J. Food Sci. 1983, 48, 1412-1421.

550

(28) Kukavica, B. M.; Veljovic-Jovanovic, S. D.; Menckhoff, L.; Luthje, S. Cell wall-

551

bound cationic and anionic class III isoperoxidases of pea root: biochemical

552

characterization and function in root growth. J. Exp. Bot. 2012, 63, 4631-4645.

553

(29) Sergio, L.; Pieralice, M.; Di Venere, D.; Cardinali, A. Thermostability of

554

Soluble and Bound Peroxidases from Artichoke and a Mathematical Model

555

of Its Inactivation Kinetics. Food Technol. Biotechnol. 2007, 45, 367–373.



Page 24 of 37

556

(30) Fortea, M. I.; Lopez-Miranda, S.; Serrano-Martinez, A.; Hernandez-

557

Sanchez, P.; Zafrilla, M. P.; Martinez-Cacha, A.; Nunez-Delicado, E.

558

Kinetic characterisation and thermal inactivation study of red alga

559

(Mastocarpus stellatus) peroxidase. Food Chem. 2011, 127, 676

560

1091−1096.

561

(31) Castillo-Leon, J.; Alpeeva, I. S.; Chubar, T. A.; Galaev, I. Y.; Csoregi, E.;

562

Sakharov, I. Y. Purification and substrate specificity of peroxidase from

563

sweet potato tubers. Plant Sci. 2002, 163, 1011-1019.

564

(32) Morales-Blancas, E. F.; Chandia, V. E.; Cisneros-Zevallos, L. Thermal

565

inactivation kinetics of peroxidase and lipoxygenase from broccoli, green

566

asparagus and carrots. J. Food Sci. 2002, 67 , 146-154.

567

(33) Ganthavorn, C.; Nagel, C. W.; Powers, J. R. Thermal inactivation of

568

asparagus lipoxygenase and peroxidase. J. Food Sci. 1991, 56, 47−49

569

(34) Agostini, E.; Hernández-Ruiz, J.; Arnao, M. B.; Milrad, S. R.; Tigier, H. A.;

570

Acosta, M. A peroxidase isoenzyme secreted by turnip (Brassica napus)

571

hairy-root cultures: Inactivation by hydrogen peroxide and application in

572

diagnostic kits. Biotechnol. Appl. Biochem. 2002, 35, 1−7.

573

(35) Lopez-Molina, D.; Hiner, A. N. P.; Tudela, J.; Garcia-Canovas, F.;

574

Rodriguez-Lopez, J. N. Enzymatic removal of phenols from aqueous

575

solution by artichoke (Cynara scolymus L.) extracts. Enzyme Microb.

576

Technol. 2003, 33, 738−742.

577

(36) Praveen, K.; Usha, K. Y.; Viswanath, B.; Reddy, B. R. Kinetic Properties of

578

Manganese Peroxidase from the Mushroom Stereum ostrea and its Ability

579

to Decolorize Dyes. J. Microbiol. Biotechnol. 2012, 22 , 1540-1548.

580


Page 25 of 37


581

Figure legends.

582

Figure 1: Covalently bound peroxidase activity released by different enzymatic

583

mixtures.

584

Figure 2. Isoelectric profile for ionically bound (A) and covalently bound (B) to

585

cell wall POD from asparagus by-products.

586

Figure 3. Typical elution profile for affinity chromatography of the anionic

587

isoperoxidase fraction.

588

Figure 4. SDS-PAGE electrophoresis after 5 minutes of exposure to the

589

developer (Na2CO3) of the crude extract, the A1 fraction, and the Con A-purified

590

AAP. Molecular weights (MW) are for 250, 150, 100, 75, 50, 37, 25, 20, 15, 10

591

KDa.

592

Figure 5. Optimal pH (A) and temperature (B) for the enzymatic activity of

593

purified AAP using ABTS as substrate. Values are the means of triplicate ± SD.

594

Figure 6. Thermal inactivation of purified AAP at 60 ºC, 70 ºC, 80 ºC and 90 ºC.

595

Values are means of triplicate ± SD.

596

Figure 7. Kinetic behavior of the two substrate reactions for purified AAP: (A)

597

plot of the 1/[substrate] versus 1/ velocity according to Michaelis-Menten

598

equation. (B) plot of the y- intercepts the lines of part A versus 1/[H2O2]

599

Figure 8. Phenols removed as a function of AAP and peroxide concentrations

600

in batch treatment. (A) Effect of H2O2 concentration on phenol reduction.

601

Conditions: 2 mM phenol; 20 UA ml-1 of CAP after 1 hour. (B) Course of the

602

oxidation with time of 4-chlorophenol, 2,4-dichlorophenol and hydroxytyrosol



Page 26 of 37

603

(2mM each) at pH 4.0, 5 UA CAP ml-1 and either 4 mM H2O2 (2,4 DCF and HT)

604

or 8 mM H2O2 (4.-CP).


Page 27 of 37


TABLES Table 1. Relation of enzymatic commercial mixtures used for covalently bound to the cell wall peroxidase extraction and enzymatic activitya

Celluclast

Pectinex AR

Olivex

Novoferm 31

Polygalacturonase

0.0

12.3

15.8

17.3

Pectin esterase

0.3

3.6

3.3

2.3

Cellulase

8.6

0.1

1.5

3.8

Glucosidase

0.1

0

0

0

a

values corresponding to activity measured as µkat mL-1



Page 28 of 37

Table 2. Summary of the purification of AAP from asparagus by-products

Purification step

Total

Total

Specific

Recovery

Purification

activity

Protein

activity

activity

factor

(UA)

(mg)

(UA/mg)

(%)

Crude extract

491 ± 7

923 ± 8

0.53

100 ± 1

1

IEF

334 ± 9

30 ± 4

11.08

68 ± 4

21

Dialyzed extract

323 ± 7

27 ± 5

11.96

66 ± 6

22

Con-A

75± 4

0.6 ±0.03

125.0

25 ± 3

236


Page 29 of 37


1000

a b

600 400

c

c

c

200

Pe ct in

ex

AR

N

t

+

ov

C el lu cl as

of er m

31

ex liv O

ex in ct Pe

C el

on

lu cl

as

AR

t

tro l

0

C

POD activity (U)

800

Figure 1 ACS Paragon Plus Environment


8

Page 30 of 37

Proteins (g)

POD activity

500 400

6

300 4 200 2

100

1,1 2,3 4,1 4,8 5,3 5,9 6,4 6,6 6,9 7,1 7,5 7,7 8,0 8,2 8,5 9,0 9,3 10,3 12,1 12,8

0

Proteins (g)

POD activity (U)

A)

0

pI

Proteins (g)

POD activity

250

1400

B)

1200 1000

150

800

100

600 400

50 0

2,6 3,2 3,7 4,3 4,9 5,3 5,6 5,8 6,3 6,8 7,0 7,3 7,5 7,6 8,1 8,4 8,7 9,1 9,6 9,9

200

pI


0

Proteins (g)

POD activity (U)

200

Page 31 of 37


POD activity

70

Proteins (g) 140

Me-Gluc 0.5M

50

Me-Gluc 120 1M 100

40

80

30

60

20

40

10

20

0

0

5

10 15 20 Fraction number


25

0 30

Proteins (g)

POD activity (U)

60


MW

CE

IEF

AAPc

250 kDa 150 kDa 100 kDa 75 kDa

MW AAPi 250 kDa 150 kDa

50 kDa

100 kDa 75 kDa

37 kDa

50 kDa 37 kDa

25 kDa 20 kDa

25 kDa 20 kDa 15 kDa 15 kDa

10 kDa


Page 32 of 37

Page 33 of 37


POD activity (U)

140 Remaining POD activity (%)

A)

120 100 80 60 40 20 0

0

2

4

6

8

10

100

120

pH

POD activity (U)


B)

100 80 60 40 20 0

0

20

40 60 80 Temperature (ºC)




70ºC

60ºC

80 oC

Page 34 of 37

90oC

100 80 60 40 20 0

0

20

40

60 80 Time (min)


100

120

Page 35 of 37

1/v0 (min(mM-1))

A)


0.012

1.0 mM H2O2

0.5 mM H2O2

0.010

0.25 mM H2O2

0.12 mM H2O2

0.008 0.006 0.004 0.002 0.000

0

2

B)

4 6 1/[ABTS](mM-1)

8

10

9 y = 0.7593x + 1.4378 R2 = 0.9867

Intercept (min(mM -1))

8 7 6 5 4 3 2 1 0 0

2

4

6

1/[H2O2](mM-1)


8

10


A)

2,4-DCP

HT

Page 36 of 37

4-CP

Remaining efficiency (%)

100

75

50

25

0

0

2

B)

4

6

2,4-DCP

8 10 mM H2O2

HT

12

14

16

4-CP

Remaining Phenol (%)

100 80 60 40 20 0

0

20

40

60 Time (min)

80


100

120

Page 37 of 37


TOC Graphic

ACS Paragon Plus Environment

Cell Wall Bound Anionic Peroxidases from Asparagus Byproducts

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