González K. 1, Arias E. 2, Carnicero A. 1, Perestelo F. 1 and Falcón M. A. 1 1. Department of...
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Transcript of González K. 1, Arias E. 2, Carnicero A. 1, Perestelo F. 1 and Falcón M. A. 1 1. Department of...
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.