Enhancing antioxidant performance of lignin by enzymatic treatment

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Enhancing antioxidant performance of lignin by enzymatic treatment with laccase Zhili Li, Jiubing Zhang, Li Qin, and Yuanyuan Ge ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04070 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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ACS Sustainable Chemistry & Engineering

1

Enhancing Antioxidant Performance of Lignin by Enzymatic

2

Treatment with Laccase

3

Zhili Li*, Jiubing Zhang, Li Qin, Yuanyuan Ge*

4

School of Chemistry and Chemical Engineering, Guangxi University, Nanning, 530004, China

5

*E-mail: [email protected] (Z Li); [email protected] (Y Ge)

6

ABSTRACT

7

The utilization of lignin is of high interest since it represents the most abundant

8

polyphenol worldwide. For the application of lignin as a natural antioxidant more

9

phenolic hydroxyl groups (Ph-OH) and lower molecular weight (Mw) are essential. Here,

10

we proposed a green method based on enzymatic treatment of two lignins (alkaline

11

lignin: AL, and hydrolysis lignin: HL) with laccase, by which the structure, especially

12

the Ph-OH contents and Mw of lignin could effectively controlled. The antioxidant

13

activity of the lignin was evaluated in vitro by DPPH• (1,1-Diphenyl-2-Picrylhydrazyl)

14

radicals scavenging ability. The mechanism of antioxidant activity improvement was

15

also elucidated. As expected, the enzymatic treated lignin (HL-L1.0) with the highest

16

Ph-OH content and lower Mw exhibited the strongest antioxidant activity (IC50=28.8

17

µg/mL), which was even stronger than the commercial antioxidants BHT

18

(3,5-di-tert-butyl-4-hydroxytoluene,

19

(3-tert-butyl-4-hydroxyanisole, IC50=56.3 µg/mL). This study suggested that the

20

enzymatic treatment was a green and effective way to obtain lignin derivatives that

21

showed great potential for the antioxidant application.

IC50=38.2

1

ACS Paragon Plus Environment

µg/mL)

and

BHA

ACS Sustainable Chemistry & Engineering 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

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KEYWORDS: Lignin; Antioxidant; Free radical; Enzyme; Laccase

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INTRODUCTION

25

Nowadays, natural antioxidants due to their abundance, biocompatibility and ability

26

to capture and neutralize free radicals to reduce the damage to the body 1, and prevent

27

the oxidation of oils and fats, and avoid nutrition of food fading 2, are more and more

28

favored in food, medicine and chemical industry 3. Lignin, one of the main constituents

29

of lignocellulosic biomass, is the most abundant aromatic biopolymer on the Earth 4-5. It

30

is a three-dimensional polymer composed by three phenyl propane units including

31

syringyl alcohol (S), guaiacyl alcohol (G) and p-hydroxyphenol alcohol (H)

32

natural abundant biopolymer with polyphenol structure, lignin shows potential

33

antioxidant properties

34

lignin including the molecular weight and the diversity of functional groups, results in

35

the inhomogeneous structures and properties of lignin which also covers the antioxidant

36

activities 4.

6-7

. As a

8-9

. Previous studies have indicated that the heterogeneity of

37

One strategy that has proved to be highly successful assisting in the conversion of

38

lignin into valuable products is enzymatic treatment 10. Using enzymes to modify lignin

39

provides environmental and economic advantages over chemical or thermal processes,

40

such as mild reaction conditions, the use of renewable and inexpensive biocatalysts, and

41

reduction in the use of toxic chemicals. Laccase is a class of enzyme that has received

42

much use in the field of lignocellulose modification to create products with increased

43

strength, hydrophobicity, and other properties

44

effect of enzymatic treatment of lignin with laccase on its structure and antioxidant

11

. However, to date, the study about

3



45 46

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activity has yet to be seen. In this study, alkaline lignin (AL) and hydrolysis lignin (HL), the most dominant 12

47

by-products from pulp & paper industry (about 70 million tons of AL per year)

48

bio-ethanol industry (about 60 million tons of HL annually)

49

enzymatically treated with laccase. The influences of laccase dosages on the chemical

50

structures and properties of the lignin derived fractions, especially their molecular

51

weights (Mw) and phenolic hydroxyl (Ph-OH) contents, were comprehensively studied

52

to evaluate the possibility of improving the antioxidant activity of lignin. For

53

characterization of antioxidant activity of naturally occurring phenolic compounds, the

54

method using 2, 2-diphenyl-1-picrylhydrazyl free radical (DPPH•) as a reactive free

55

radical is now recognized as a good one that gives an opportunity to search for radical

56

scavenging ability 9. Thus, the in vitro DPPH• scavenging assay was used to evaluate

57

the antioxidant activities of the lignin fractions

58

antioxidant activity and the structure of lignin was discussed. The antioxidant activities

59

of the lignin fractions, some reported natural-occurring and commercial antioxidants

60

were also compared. The objective of this study is not only to investigate the effect of

61

enzymatic treatment with laccase on lignin’s antioxidant activity but also to obtain

62

lignin derivatives with strong antioxidant activity through the green enzymatic

63

treatment method.

64

MATERIALS AND METHODS

65

and

10

, respectively, were

13

. The relationship between the

Materials and reagents. Alkaline lignin (AL) was obtained from Nanpu Pulp Mill

4


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66

(Nanning, China) and HL was purchased from Long Li Biological Technology Co.

67

(Shandong, China), which were purified by an acidulation precipitation method 8.

68

Guaiacol was purchased from Damao Chemical (Tianjin, China); Laccase (activity: 10

69

units per mg) from Aspergillus oryzae was purchased from Xiacheng Co. (Ningxia,

70

China); DPPH• and dimethyl sulfoxide-d6 (DMSO-d6) were purchased from

71

Sigma-Aldrich

72

3-methoxybenzaldehyde, sodium acetate (NaAc), imidazole, phenolphthalein, pyridine,

73

3,5-di-tert-butyl-4-hydroxytoluene (BHT) and 3-tert-butyl-4-hydroxyanisole (BHA)

74

were purchased from Macklin Biochemical Co. (Shanghai, China).

(Shanghai,

China).

Folin

Cioulteau

(FC)

reagent,

75

Enzymatic treatment of lignin with laccase. 1.0 g of AL or HL was dissolved in a

76

NaAc buffer solution in a round-bottomed flask at 25 ℃ with continuous stirring, then

77

laccase (0.8~1.6 mL) was added for the treatment of lignin under air for 6 h. After the

78

reaction completed, diluted sulfuric acid was added into the mixture and stewed

79

overnight. The brown precipitate was formed and collected by filtration, washing and

80

dried under vacuum at 60 ℃ for 24 h. The enzymatic treated lignin fractions could be

81

obtained after finely milled, which were named as, AL fractions: AL-L0.8, AL-L1.2,

82

AL-L1.6, and HL fractions: HL-L0.8, HL-L1.0 and HL-L1.2, respectively, in which the

83

numbers indicated the dosage of laccase used for treating lignin.

84

Characterizations. FTIR (Fourier-transform infrared spectroscopy) spectra of the

85

lignins were analyzed by Thermo Scientific Nicolet iS50, United States in the range of

86

500-4000 cm-1 using a KBr disk containing 1% finely ground samples. The

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87

number-average (Mn), weight-average (Mw) molecular weights, and polydispersity

88

(Mw/Mn) of the lignin fractions were measured with an Agilent 1100 GPC (gel

89

permeation chromatography). HPLC-grade tetrahydrofuran was used as solvent and

90

eluent. Sodium polystyrene sulfonate was used as a standard sample (Sigma-Aldrich).

91

The 1H NMR (nuclear magnetic resonance) analysis was carried out on a Bruker

92

AVANCE III 600 MHz spectrometer (Switzerland) at 600 MHz. Lignin was acetylated

93

before test: 2.0 g of lignin was dissolved in 40 mL of pyridine: acetic anhydride (1:1,

94

v/v) solution and stirring for 24 h at room temperature. Then 15 mL of 1.5% HCl was

95

added at 0 ℃, and the resulting precipitate was filtered and washed with deionized water

96

to neutral. The acetylated lignin was finally dried in a vacuum oven at 50 ℃ overnight.

97

For 1H NMR analysis, 20 mg sample was dissolved in DMSO-d6 (0.6 mL, containing

98

0.03% Tetramethylsilane (TMS), v/v).

99

Measurement of Ph-OH groups’ content. The main functional Ph-OH groups’

100

contents in lignin fractions were measured with FC method using Guaiacol as a standard

101

according to literature 14. 1 mL of lignin solution (0.5 mg/mL) was mixed with 2 mL of

102

the FC reagent (1N) and incubated for 5 min at ambient temperature. Subsequently, 45

103

mL of deionized water and 2 mL of a 20% (w/v) sodium carbonate solution was added.

104

After

105

spectrophotometrically at 760 nm (Uv-vis TU-1880, China). All measurements were

106

carried out in triplicate with the relative deviations of AL-L0.8 & AL, and HL-L1.0 >HL-L1.2 >HL-L0.8 >HL,

193

respectively, which were in agreement with the increasing order of Ph-OH content and

194

decreasing order of Mw of these lignins (Table 1). The earlier reports had revealed that

195

the Ph-OH content and Mw of lignin were closely correlated to the antioxidant activity 24,

196

and this result confirmed the hypothesis as well. Furthermore, by comparing AL

197

fractions with HL fractions, the HL fractions showed lower IC50 values, i.e. higher

198

antioxidant activity, than the AL fractions, suggesting the Ph-OH groups played a more

199

important role in the antioxidant activity of lignins than the Mw did, since HL fractions

200

had higher Mw than AL fractions. It has been believed that the free radical scavenging

201

ability of lignin is mainly from its hydrogen donating ability and the stability of the free

202

radical intermediate formed during the scavenging reaction

203

ability of lignin is primarily from the Ph-OH groups that is why the Ph-OH content

204

accounts for the antioxidant activity of lignin. The possible pathways for lignin

205

polyphenol reacting with DPPH• were exhibited in SI Figure S3. Lignin polyphenol

206

firstly donated a hydrogen from Ph-OH to scavenge one DPPH• molecule (Figure S3a).

207

Then the aryl could stabilize the phenoxyl radical (Figure S3b). Next, one DPPH•

208

molecule might combine with one aryl radical to form electron pair as indicated in

209

Figure S3c. The other possible mechanism involved the stabilization of the phenoxyl

210

radical through extended double bonds in the side chain of lignin unit structure (Figure

211

S3d, e) 25.

212

25

. The hydrogen donating

Table 2 also quantitatively listed the IC50 values of the commercial BHT and BHA,

11



213

and some reported natural-occurring antioxidant for inhibiting DPPH•. It was noted that

214

the DPPH• scavenging ability of AL-L1.2 (IC50= 55.6 µg/mL) was lower than the

215

commercial antioxidant BHT (IC50=38.2 µg/mL), whereas close to the BHA (IC50=56.3

216

µg/mL). More importantly, the antioxidant activities of all the treated HL fractions, for

217

example, HL-L1.0 (IC50= 28.8 µg/mL), were higher than that of both BHA and BHT,

218

and other reported antioxidants extracted from biomass. The fact indicated that the

219

lignins after treating with laccase could be acted as natural occurring antioxidants in

220

medicine, food oils and fats, preventing or delaying the oxidation of oils and fats, and

221

avoiding nutrition of food damaging and fading. Moreover, the enzymatic treated lignin

222

provided a natural, non-toxic and abundant source for antioxidants as compared with the

223

costs and the relatively poor biocompatibility of synthetic antioxidants (including BHA,

224

BHT) 26.

225

In summary, the enzymatic treatment with laccase proposed in this work successfully

226

realized the modification of alkaline lignin and hydrolysis lignin and six lignin fractions

227

with different molecular weights and Ph-OH contents were obtained. The further

228

analysis of chemical structure demonstrated the aromatic skeleton of the lignins was

229

maintained well, with increasing dosage of laccase, the Ph-OH content of lignin

230

increased distinctly while the molecular weight decreased. Due to the effects of Ph-OH

231

and molecular weight on the antioxidant activity of lignin, the HL-L1.0 showed

232

outstanding DPPH• scavenging ability stronger than both of commercial BHT and BHA,

233

which indicated its promising application as natural occurring antioxidants.

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Consequently, this research suggested that enzymatic treatment of lignin could achieve a

235

remarkable improvement in antioxidant performance and further provide the potential

236

for value-added applications of lignin biopolymers in antioxidants.

237

Supporting Information

238

The Supporting Information is available free of charge on the ACS publications website.

239

Corresponding author

240

*E-mail: [email protected] (Z Li); [email protected] (Y Ge).

241

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No.

242 243

21464002).

244

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328 329

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330

Figure captions

331

Figure 1. FTIR spectra of the enzymatic treated lignin fractions.

332

Figure 2. 1H NMR spectra of the enzymatic treated lignin fractions.

333

Figure 3. Proposed interaction mechanism of lignin with laccase.

334

Figure 4. DPPH• free radical scavenging ability of the lignin fractions and BHA, BHT

335

in terms of Inhibition Percentage (IP, %).

336

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337

Table 1. Molecular weight and functional groups contents of lignin fractions. Lignin fraction

Mw (Da)

Mn (Da)

Mw/Mn

Ph-OH (mmol/g)

AL

1400

1000

1.4

0.51±0.02

AL-L0.8

1300

1000

1.3

1.28±0.06

AL-L1.2

1300

900

1.4

1.39±0.05

AL-L1.6

1100

900

1.2

1.33±0.07

HL

2400

1400

1.7

1.81±0.08

HL-L0.8

1900

1200

1.6

1.93±0.09

HL-L1.0

1500

1200

1.3

1.96±0.08

HL-L1.2

1400

1100

1.3

1.95±0.09

338 339

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340

Table 2. Comparison of IC50 values of the lignin fractions with BHA, BHT and some

341

reported antioxidants. Samples

IC50 (µg/mL)

Ref

AL

--

This work

AL-L1.2

55.6

This work

AL-L1.6

58.9

This work

HL

59.9

This work

HL-L0.8

34.3

This work

HL-L1.0

28.8

This work

HL-L1.2

31.6

This work

BHA

56.3

This work

BHT

38.2

This work

Tocopherol

380.0

2

Astragalus honey

123.5

27

Clover honey

98.2

27

G. lucidum polysaccharide

100.0

28

Kiwifruit extract

83.4

29

342 343

18


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344 345

Figure 1.

346

19



347 348

Figure 2.

349

20


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350 351

Figure 3.

352

21



353

80

AL AL-L1.2 BHT

AL-L0.8 AL-L1.6 BHA

60 IP (%)

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

40 20 0

354 355

20

40 60 80 Concentration (µg/mL)

100

Figure 4.

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TOC – For Table of Contents Use Only

D

P P H

·

358 359

Synopsis

360

The enzyme treatment of lignin with laccase provides a strategy to enhance its

361

antioxidant activity as a natural-occurring antioxidant.

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Enhancing antioxidant performance of lignin by enzymatic treatment

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