González K.González K.11, Arias E., Arias E.22, Carnicero A., Carnicero A.11, Perestelo F., Perestelo F.11and and Falcón M. A.Falcón M. A.11

1. Department of Microbiology and Cellular Biology. University of La 1. Department of Microbiology and Cellular Biology. University of La Laguna. Tenerife. SpainLaguna. Tenerife. Spain

2. Department of Microbiology and Parasitology. University of Alcalá. 2. Department of Microbiology and Parasitology. University of Alcalá. Alcalá de Henares. SpainAlcalá de Henares. Spain

“Improvements in an industrial lignin (Indulin AT) with laccase and laccase-

mediator systems evaluated by spectroscopic and chromatographic

techniques”

Laccase properties of Laccase properties of Fusarium Fusarium proliferatumproliferatum

0

25

50

75

100

0 30 60 90 120

Act

ivity

(%)

Time (min)

―― pH 2.2 pH 2.2 ―― pH 5pH 5

―― pH 3 pH 3 ―― pH 9pH 9

―― pH 4, 6, 7, 8pH 4, 6, 7, 8

0

25

50

75

100

0 30 60 90 120

Act

ivity

(%)

Time (min)

―― pH 2.2 pH 2.2 ―― pH 5pH 5

―― pH 3 pH 3 ―― pH 9pH 9

―― pH 4, 6, 7, 8pH 4, 6, 7, 8

0

25

50

75

100

0 30 60 90 120

Act

ivity

(%)

Time (min)

―― 2020--3030ººCC ―― 6060ººCC

―― 4040ººCC ―― 7070ººCC

―― 5050ººCC

0

25

50

75

100

0 30 60 90 120

Act

ivity

(%)

Time (min)

0

25

50

75

100

0 30 60 90 120

Act

ivity

(%)

Time (min)

―― 2020--3030ººCC ―― 6060ººCC

―― 4040ººCC ―― 7070ººCC

―― 5050ººCC

StabilitypH Temperature

OptimalTemperature: 60ºC

pH: 3.5

Kinetics properties

Characteristics of industrial lignin Characteristics of industrial lignin (Indulin AT)(Indulin AT)

0

1

2

3

210 260 310 360 410 460

Ab

so

rba

nce

Wavelength (nm)

220 nm

245 nm

280 nm

340 nm

UVUV--Vis SpectrumVis Spectrum

0

1

2

3

210 260 310 360 410 460

Ab

so

rba

nce

Wavelength (nm)

220 nm

245 nm

280 nm

340 nm

UVUV--Vis SpectrumVis Spectrum

0

1

2

3

210 260 310 360 410 460

Ab

so

rba

nce

Wavelength (nm)

220 nm

245 nm

280 nm

340 nm

UVUV--Vis SpectrumVis Spectrum

0

1

2

3

210 260 310 360 410 460

Ab

so

rba

nce

Wavelength (nm)

220 nm

245 nm

280 nm

340 nm

UVUV--Vis SpectrumVis Spectrum

0

0,05

0,1

0,15

0,2

0 10 20 30 40 50 60 70A

bs

orb

an

ce 2

80

nm

Time (min)

87 kDa 135 Da

Molecular mass Molecular mass profileprofile

0

0,05

0,1

0,15

0,2

0 10 20 30 40 50 60 70A

bs

orb

an

ce 2

80

nm

Time (min)

87 kDa 135 Da

Molecular mass Molecular mass profileprofile

0

0,05

0,1

0,15

0,2

0 10 20 30 40 50 60 70A

bs

orb

an

ce 2

80

nm

Time (min)

87 kDa 135 Da

Molecular mass Molecular mass profileprofile

0

0,05

0,1

0,15

0,2

0 10 20 30 40 50 60 70A

bs

orb

an

ce 2

80

nm

Time (min)

87 kDa 135 Da

Molecular mass Molecular mass profileprofile

Klason lignin: 78% Reducing sugars: 0.12%

UV-Vis Spectrum of treated UV-Vis Spectrum of treated industrial ligninindustrial lignin

Outset of Outset of incubation incubation

with laccasewith laccase

ControlControl Treated sampleTreated sampleOH

CH2OH

OCH3

CH

CH

OH

CH2OH

OCH3

CH

CH

OH

CH3

OCH3

C

CH2

O

OH

CH3

OCH3

C

CH2

O

OH

CH3

OCH3

CH

CH

OH

CH3

OCH3

CH

CH

Coniferyl Coniferyl alcoholalcohol

IsoeuglenolIsoeuglenol

AcetovanilloneAcetovanilloneQuinoneQuinone

AbsorbanceAbsorbance

0

0,2

0,4

0,6

220 270 320 370

Ab

so

rba

nc

e

Wavelength (nm)

Min 27

―― Treated sampleTreated sample

―― controlcontrol

0

0,2

0,4

0,6

220 270 320 370

Ab

so

rba

nc

e

Wavelength (nm)

Min 27

―― Treated sampleTreated sample

―― controlcontrol

O

OCH3

R

CH

O

OCH3

R

CH

Molecular mass distribution of Molecular mass distribution of industrial ligninindustrial lignin

Time (min)

0

0,04

0,08

0,12

0,16

0 10 20 30 40 50 60 70

Ab

so

rba

nc

e 2

80

nm

a b c d

― 0 h0 h

― controlcontrol

Molecular weight

fractions (%) 0 h

Control

400 - 87 kDa (a) 7.41 6.14

87 – 5.67 kDa (b)

36.66 30.33

5.67 - 0.8 kDa (c)

31.54 32.82

< 0.8 kDa (d)

24.39 30.71

%Polymerization 7.6 -

Outset of Outset of incubation incubation

with laccasewith laccase

7.6% 7.6% PolymerizationPolymerization

Molecular mass distribution after 48 hMolecular mass distribution after 48 h

Molecular weight

fractions (%) 48 h

Control

400 - 87 kDa (a) 16.37 6.14

87 – 5.67 kDa (b)

41.05 30.33

5.67 - 0.8 kDa (c)

24.04 32.82

< 0.8 kDa (d)

18.54 30.71

%Polymerization 20.95 -

21 % 21 % PolimerizationPolimerization

Time (min)

0

0,04

0,08

0,12

0 10 20 30 40 50 60 70

Ab

so

rba

nc

e 2

80

nm

a b c d

― 48 h48 h

― controlcontrol

Ab

so

rba

nc

e

Wavelength (nm)

0

0,02

0,04

0,06

215 265 315 365

Min 30

UV-Vis spectra

Laccase-ABTS systemLaccase-ABTS system

― Treated ligninTreated lignin

― controlcontrol

― Treated ligninTreated lignin

― controlcontrol

0

0,5

1

1,5

2

210 260 310 360

Ab

sorb

ance

Wavelength (nm)

Ab

so

rba

nc

e

Wavelength (nm)

0

0,2

0,4

0,6

0,8

210 260 310 360

Min 50.8

Min 66.1

Time (min)

Ab

so

rba

nc

e 2

80

nm

0

0,04

0,08

0,12

0,16

0 10 20 30 40 50 60 70

60.5 kDa 400 kDa 135 Da

Molecular mass distribution

UV-Vis spectra

22% depolymerization

In summary:In summary: We can conclude that at the very beginning of the We can conclude that at the very beginning of the

incubation the recovered Indulin AT showed an incubation the recovered Indulin AT showed an intense functionalization accompanied by a slight intense functionalization accompanied by a slight polymerization, which reached its polymerization, which reached its maximum maximum after 48 after 48 h of incubation. When ABTS was used as mediator a h of incubation. When ABTS was used as mediator a strong depolymerization and a new peak at 400 kDa strong depolymerization and a new peak at 400 kDa were registered in the lignin elution profile. In were registered in the lignin elution profile. In addition, a strong oxidation was detected in the addition, a strong oxidation was detected in the spectra below 50 min. Other mediators like HBT or spectra below 50 min. Other mediators like HBT or violuric acid did not produce such changes.violuric acid did not produce such changes.