Laccases (EC 1.10.3.2) are multi–copper oxidases, which catalyze one electron oxidation of a
wide range of inorganic and organic substances, coupled with one four-electron reduction of
oxygen to water (Xu 1996). Laccases not only catalyze the removal of a hydrogen atom from
the hydroxyl group of methoxy-substituted monophenols, ortho- and para-diphenols, but also
can oxidize other substrates such as aromatic amines, syringaldazine, and non-phenolic
compounds, to form free radicals (Bourbonnais et al. 1997, Li et al. 1999, Robles et al. 2000;
Durán and Esposito 2000). It is known that laccases can catalyze the polymerization of
various phenols and halogen, alkyl- and alkoxy-substituted anilines (Hoff et al. 1985,
Kobayashi et al. 2001, Kobayashi and Higashimura 2003). Only recently has positively been
demonstrated that plant laccases are able to polymerize monolignols within the plant cell wall
matrix, in the complete absence of peroxidase (Sterjiades et al. 1992, Liu et al.1994,
Richardson et al. 2000) and to break down non-phenolic ligno-cellulose by certain phenolic
compounds acting as mediators (Bourbonnais et al. 1997). These studies show that laccases
are involved only in the early stages of lignification, while peroxidases are involved later
(Bao et al. 1993, Wallace & Fry 1999, Boudet 2000). Thus, the oxidation of lignosulfonates
with laccase to binding fiber, particle and paper boards could be an important achievement to
overcome the environmental limitation of the phenol–formaldehyde resins. The objective of
this preliminary work is to characterize four different lignosulfonates after laccase oxidation in order to understand their potentiality toward industrial application as cross-linker for paper
and lignocellulosic substrates.
The characterization of the four lignosulfonates has shown similar results in term of color and
chemical structure as confirmed by the Uv-Vis and FT-IR analysis. However, the particle size
of the DP399 showed higher values then DP398 (6.7 and 9.5 nm respectively) indicating that
slight changes in the chemical structure can have visible changes in the organization behavior
of the lignosulfonates in solution. The lignosulfonate DP400 and DP401 have shown different
chemical structure both in Uv-vis and FT-IR spectra and a different particle size range (7.1
and 5.1 nm).
As the highest concentration of enzyme was the responsible for the maximum change in the
absorbance spectrum the 20 U/mL was the activity selected to perform the assays. The laccase
oxidation of the four lignosulfonate showed dramatic changes in the chemical structure of all the samples that could be attributed to the conversion of the oxidized phenolic hydroxyl
groups. For all the samples the UV peaks at 250 and 280 nm disappeared and the peaks at 320
and 360 nm are significantly decreased and the visible absorbance showed a general increase
in all spectra. In the visible spectra the laccase oxidized lignosulfonates DP398 and DP399
showed similar behavior with the formation of a large shoulder around 400-450 nm.
However, the lignosulfonates DP400 and DP401 showed different behavior with the
formation of a narrow peak at 650 nm for DP400 and at 450 nm for DP401. The comparison
of the FT-IR spectra of the oxidized and raw lignosulfonates showed a similar behavior in all
the samples. The growing in the band of the typical aromatic skeletal vibration bands (1600
and 1510 cm−1) and the decreasing in the bands of the ether group and of the C-O, C-H and
C=O vibration (between 1300 and 1000 cm−1) indicate a breakdowns of the lignosulfonate
structure and a reassembling of the aromatic skeletal in a different polymer structure. The
particle size results confirmed this conclusion indicating a growing in the oxidized
lignosulfonates size, in special for the DP401 (102.6%), and a decreasing in the
polydispersity, in special for DP400 (48.1%), that indicate the decrease of the heterogeneity of the compounds. The K/S values of the lignocellulosic fibers have shown that the oxidized
lignosulfonate compounds catalyzed by laccase have poor affinity toward fibers. However,
the oxidized lignosulfonate showed significant differences in the UV-vis spectra. The
differences between the flax and sisal could be attributed to the different percentage of lignin
in the two substrates. In conclusion, the results showed that a huge oxidation of the aromatic
structure of the lignosulfonates was achieved and a reorganization of the oxidized
lignosulfonate by crosslinking reactions leads to the formation of a new polymer structure
