Supplementary data

A New Polymer-based Laccase for Decolorization of AO7: Long-term Storage

and Mediator Reuse

Xiaolin Zhang, Bing Wu, Bingcai Pan*, Lv Lu, Weiming Zhang

State Key Laboratory of Pollution Control and Resource Reuse, School of the

Environment, Nanjing University, Nanjing 210023, P.R. China

*To whom correspondence should be addressed

E-mail: bcpan@nju.edu.cn (B. Pan)

Tel: +86-25-8968-0390

The Supporting Information Available contains 16 pages, including Scheme S1-S2;

Fig. S1-S7; and Tables S1-S4.

1

2

3

4

5

6

7

8

9

10

111213141516

17

1819

1

Scheme S1. Schematic illustration of fabrication of polymer-support laccase (N-PS-

Lac)

Scheme S2. Illustration of two-step decolorization, traditional LMS and control

methods

Fig. S1 TEM photographs of N-PS-3 (a) and N-PS-Lac-3 (b)

Fig. S2. The comparison of N-PS-3 and N-PS-Lac-3 FT-IR spectrum in the amide I

region. (a) IR spectra of N-PS-3 and N-PS-Lac-3, (b) subtracted spectra of N-PS-Lac

(N-PS subtracted from N-PS-Lac) and laccase IR spectrum

Fig. S3. FT-IR spectra of laccase and N-PS-Lac-3 in the amide I region and their

Gaussian curve-fitting before and after 2-year storage

Fig. S4. HPLC results of Solution-1 and Solution-2 after seven-cyclic assay as

compared to Solution-3. Solution-1, 2 and 3 were illustrated in scheme S2

Fig. S5. The HPLC results of Hobt, AO7, solution-1’ and solution-2’. Hobt: 1 mM

Hobt solution; AO7: 100 mg/L AO7 solution; solution-1’: solution in step 1 in control

method; solution-2’: solution in step 2 in control method.

Fig. S6. UV-vis adsorption spectrum of solution containing AO7, Hobt and laccase at

initial and after decolorization

Fig. S7. Phytotoxicity study of two-step decolorization assay on rice seed

germination. (a) Photos of rice seeds after seed germination (n=10), (b) length of rice

seedling, (c) length of rice root. (*: P<0.05, **: p<0.01) Blank: deionized water, AO7:

100 mg/L AO7 solution; HOBT: 1 mM HOBT solution; Solution-1 and 3 were

illustrated in scheme S2

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

2

Scheme S1. Schematic illustration of fabrication of polymer-support laccase (N-PS-

Lac)

42

43

44

3

Scheme S2. Illustration of two-step decolorization process, traditional LMS and

control methods

45

46

47

4

Fig. S1 TEM photographs of N-PS-3 (a) and N-PS-Lac-3 (b).

48

49

50

5

Fig. S2 The comparison of N-PS-3 and N-PS-Lac-3 FT-IR spectrum in the amide I

region. (a) IR spectra of N-PS-3 and N-PS-Lac-3, (b) subtracted spectra of N-PS-Lac

(N-PS subtracted from N-PS-Lac) and laccase IR spectrum

51

52

53

54

55

56

6

Fig. S3 FT-IR spectra of laccase and N-PS-Lac-3 in the amide I region and their

Gaussian curve-fitting before and after 2-year storage

57

58

59

60

7

Fig. S4 HPLC results of Solution-1 and Solution-2 after seven-cyclic assay as

compared to Solution-3. Solution-1, 2 and 3 were illustrated in scheme S2

61

62

63

64

8

Fig. S5 The HPLC results of Hobt, AO7, solution-1’ and solution-2’. Hobt: 1 mM

Hobt solution; AO7: 100 mg/L AO7 solution; solution-1’: solution in step 1 in control

method; solution-2’: solution in step 2 in control method.

6566

67

68

9

Fig. S6 UV-vis adsorption spectrum of solution containing AO7, Hobt and laccase at

initial and after decolorization

6970

71

72

10

Fig. S7 Phytotoxicity study of two-step decolorization assay on rice seed germination.

(a) Photos of rice seeds after seed germination (n=10), (b) length of rice seedling, (c)

length of rice root. (*: P<0.05, **: p<0.01) Blank: deionized water, AO7: 100 mg/L

AO7 solution; HOBT: 1 mM HOBT solution; Solution-1 and 3 were illustrated in

scheme S2

73

74

75

76

77

78

79

11

Table S1. The laccase loading amount and activity of N-PS-Lac fabricated

Catalyst Functional

Groups

(mmol/g)

Laccase Loading Amount

(nkat/g)

Activity

(nkat/g)

Activity

Retention (%)

N-PS-Lac-1 2.09 201.23 4.10 2.04

N-PS-Lac-2 2.72 399.27 21.17 5.30

N-PS-Lac-3 3.24 466.56 27.90 5.98

80

81

82

83

12

Table S2. Infrared band positions and band assignments of laccase and N-PS-Lac-3

SampleInitial After 2-year storage Assignment

aPeak Position Area Peak Position Area

Laccase

1692.57 1.44% 1694.48 0.99% β-Sheet

1680.98 11.36% 1689.11 1.99% β-Turn

1668.35 16.65% 1680.14 13.10% β-Turn

1660.08 11.84% 1666.57 21.55% β-Turn b

1651.86 9.40% 1653.01 23.95% α-Helix

1645.13 10.06% 1642.61 14.89% Unordered

1635.62 28.11% 1632.19 17.08% β-Sheet

1619.78 11.13% 1619.10 6.45% β-Sheet

N-PS-Lac-

3

1695.42 0.62% 1693.64 0.39% β-Sheet

1690.67 1.37% 1688.54 1.48% β-Sheet

1679.20 13.04% 1673.13 16.82% β-Turn

1665.13 18.56% 1661.16 9.92% β-Turn

1655.56 12.10% 1652.48 12.91% α-Helix

1646.06 18.88% 1646.20 5.75% Unordered

1636.14 13.28% 1640.32 15.50% β-Sheet

1630.49 24.88% β-Sheet

1625.67 22.15% 1624.38 12.35% β-Sheet

a. The peaks are assigned according to references De Rosa, 2006; Griebenow and

Klibanov, 1995; 1996; Zhai et al., 2013 . b. Uncertain

Table S3. The P-value of the root length between any two groups based on ANOVA

analysis

AO7 solution-1 solution-3 HobtBlank 2.90E-03 2.20E-01 5.68E-11 1.92E-09AO7 5.21E-04 1.28E-08 5.02E-07solution-1 2.63E-10 3.41E-09solution-3 3.99E-01

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

13

Table S4. The P-value of the seedling length between any two groups based on

ANOVA analysis

AO7 solution-1 solution-3 Hobt

Blank 1.86E-03 2.51E-01 3.86E-05 2.43E-06AO7 9.60E-03 5.68E-02 4.35E-03

solution-1 1.03E-04 3.14E-06

solution-3 3.55E-01

99

100

101

102

103104

14

References

De Rosa, G., Larobina, D., La Rotonda, M.I., Musto, P., Quaglia, F. and Ungaro, F.

(2005) How cyclodextrin incorporation affects the properties of protein-loaded

PLGA-based microspheres: the case of insulin/hydroxypropyl-beta-cyclodextrin

system. Journal of Controlled Release 102(1), 71-83.

Griebenow, K. and Klibanov, A.M. (1995) Lyophilization-induced reversible changes

in the secondary structure of proteins. Proceedings of the National Academy of

Sciences of the United States of America 92(24), 10969-10976.

Griebenow, K. and Klibanov, A.M. (1996) On protein denaturation in aqueous-

organic mixtures but not in pure organic solvents. Journal of the American

Chemical Society 118(47), 11695-11700.

Zhai, J.L., Day, L., Aguilar, M.I. and Wooster, T.J. (2013) Protein folding at emulsion

oil/water interfaces. Current Opinion in Colloid & Interface Science 18(4), 257-

271.

105

106

107

108

109

110

111

112

113

114

115

116

117

118

15