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Journal Article•DOI•

A commercial laccase-mediator system to delignify and improve saccharification of the fast-growing Paulownia fortunei (Seem.) Hemsl.

Jorge Rencoret1, Antonio Pereira González1, Gisela Marques1, José Carlos del Río Andrade1, Ángel T. Martínez1, Ana Suárez1 •
Spanish National Research Council1
19 Dec 2018-Holzforschung (Walter de Gruyter GmbH)-Vol. 73, Iss: 1, pp 45-54
TL;DR: The presence of oxidized lignin compounds in the filtrates of the enzymatic treatments – such as vanillin, vanillic acid, syringaldehyde and syringic acid – conclusively demonstrates the ability of L/MeS treatment to oxidize and depolymerize the lign in paulownia wood.
Abstract: 10 paginas.-- 5 figuras.-- 3 tablas.-- 41 referencias.-- El archivo adjunto no es la ultima version postprint

Summary (2 min read)

Jump to: [Introduction] – [Material and methods] – [Result and discussion] and [Conclusions]

Introduction

  • Its properties such a lightweight, strength, insulation, fast drying, high ignition point and rot resistance makes it popular for house construction and furniture making.
  • Paulownia also has a potential use as energy crop for the production of bioethanol (Ye et al. 2016; Domínguez et al. 2017) also by enzymatic hydrolysis (Chandra et al. 2007) and subsequent fermentation by yeasts (Park et al. 2013) or bacteria (Ng et al. 1981) to ethanol.
  • Different physical and chemical pretreatments have been proposed for a better saccharification of paulownia wood, such as dilute acid, alkali, ultrasonic-assisted alkali treatments (Ye et al. 2015, 2016), autohydrolysis (Domínguez et al. 2017), and steam explosion (Radeva et al. 2012).
  • Methyl syringate (MeS) will serve as mediator, which is obtained from syringic acid present in pulp and paper side-streams (Rosado et al. 2012).

Material and methods

  • Paulownia wood, enzymes and mediator: 3-year-old P. fortunei trees were provided by the University of Huelva .
  • At the 95% confidence level, the enzymatic experiments are smaller than the differences found between the control, laccase alone, and L/MeS treatments.
  • The 2D HSQC NMR experiments were performed at the gel state, which is an in situ analysis of the whole cell-wall (Kim et al. 2008, Rencoret et al. 2009b).
  • The 1H-13C correlation signals from the aromatic region of the spectrum were used to estimate the content of lignin (relative to the content of amorphous carbohydrates, estimated from the anomeric xylose and glucose signals), and the lignin composition in terms of G, S and oxidized S (S′) and G (G′) units.
  • The extraction process was repeated three times, using fresh chloroform each time and the supernatants were combined.

Result and discussion

  • Delignification of paulownia wood by the enzymatic pretreatment The Klason lignin content of the paulownia control, processed as the full enzymatic treatment but without laccase and mediator, was only slightly reduced from 23.8 to 22.0% with respect to the initial paulownia lignin content (Table 1).
  • This is the effect of alkaline peroxide extractions.
  • The treatment with laccase alone only resulted in a 5% lignin reduction compared to the control sample.
  • This low efficiency of laccase alone is well known (Lai, 1992).
  • The pretreatment with L/MeS, however, resulted in a lignin decrement up to 24%, with respect to the control, but this delignification is degree is lower than reported about eucalypt wood Page 7 of 23 https://mc.manuscriptcentral.com/holz.

Conclusions

  • The lignin in paulownia wood was modified and partially removed by pretreatment with recombinant laccase of M. thermophila in the presence of MeS as phenolic mediator, in spite of its high G-unit content.
  • The L/MeS system acts selectively on the lignin polymer, leaving the carbohydrate signals in the HSQC spectra practically unaffected.
  • The utilization of The commercially available L/MeS system has a high application potential as pretreatment for the saccharification of paulownia wood.
  • The authors thank Dr. Manuel Angulo (CITIUS, University of Seville) for technical assistance with the NMR experiments.

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1
1
Short title: Enzymatic pretreatment of P. fortunei
A commercial laccase-mediator system to delignify and improve
saccharification of the fast-growing Paulownia fortunei
Jorge Rencoret
1,*
, Antonio Pereira
1
, Gisela Marques
1
, José Carlos del Río
1
, Ángel T.
Martínez
2
and Ana Gutiérrez
1
1
Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, E-41012-Seville, Spain
2
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040-Madrid, Spain
* Corresponding author: (Tel: +34 954624711; E-mail: jrencoret@irnase.csic.es)
Abstract: It was demonstrated for the first time that a laccase-based enzymatic pretreatment
is able to delignify fast-growing paulownia species. The treatment was performed with a
commercial low-redox potential laccase isolated from Myceliophthora thermophila and
methyl syringate (MeS) as a natural phenolic mediator. Up to 24% lignin removal was
attained by the laccase-mediator treatment (L/MeS), followed by alkaline peroxide extraction
in a multistage sequence. The reduction in lignin content was accompanied by a significant
improvement in the subsequent enzymatic saccharification ending up in 38% glucose and
34% xylose yields. The structural modifications of the lignin were analyzed in situ by 2D-
NMR spectroscopy. A considerable removal of guaiacyl and syringyl lignin units with respect
to the carbohydrate signals was visible as well as the cleavage of β-O-4
ʹ, β
-5
ʹ and β
-β
ʹ
linkages leading to elevated amounts of Cα-oxidized guaiacyl and syringyl units. The
presence of oxidized lignin compounds in the filtrates of the enzymatic treatments ─ such as
vanillin, vanillic acid, syringaldehyde and syringic acid ─ conclusively demonstrates the
ability of L/MeS treatment to depolymerize the lignin in paulownia wood.
Keywords: pretreatment; paulownia; lignin; 2D-NMR; laccase; saccharification
Introduction
Paulownia genus comprises of nine species and a few hybrids native to China and East Asian
(Zhu et al. 1986), which are grown commercially for the production of hardwood timber
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Holzforschung - International Journal of the Biology, Chemistry, Physics and Technology of Wood
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Postprint of: Holzforschung (2018) Article in Press
For Review Only
2
2
(Bergmann 1998), while Paulownia is a fast-growing tree with a high biomass production
(Jiménez et al. 2005). Its properties such a lightweight, strength, insulation, fast drying, high
ignition point and rot resistance makes it popular for house construction and furniture making.
It was also investigated as a raw material for the production of chemical pulp (Jiménez et al.
2005; Caparrós et al. 2007, 2008). Paulownia fortunei is the most suitable species to this
purpose (Rai et al. 2000). P. fortunei shows extraordinary high growth rates under suitable
conditions (Ede et al. 1997), reaching up to 15-20 m high in only 5-7 years, and annual
productions as high as 150 t ha
-1
y
-1
(Jiménez et al. 2005).
Paulownia also has a potential use as energy crop for the production of bioethanol (Ye
et al. 2016; Domínguez et al. 2017) also by enzymatic hydrolysis (Chandra et al. 2007) and
subsequent fermentation by yeasts (Park et al. 2013) or bacteria (Ng et al. 1981) to ethanol.
To this purpose, the cross-linked a macro-molecular assembly of the cell wall must be
submitted to a pretreatment (Chen 2014). Especially lignins limits the enzymatic hydrolysis
by steric hindrance of the enzymes access to the polysaccharides and their inactivation
(Kumar and Wyman 2009; Rahikainen et al. 2013).
Paulownia wood contains ~24% lignin, which is composed of guaiacyl (G) and
syringyl (S) units with an S/G ratio of 0.66 (Rencoret et al. 2009a). Different physical and
chemical pretreatments have been proposed for a better saccharification of paulownia wood,
such as dilute acid, alkali, ultrasonic-assisted alkali treatments (Ye et al. 2015, 2016),
autohydrolysis (Domínguez et al. 2017), and steam explosion (Radeva et al. 2012). Biological
pretreatments are also possible via ligninolytic fungi or their enzymes, but these methods have
not yet been investigated on paulownia.
The aim of the present study is to evaluate the pretreatment of paulownia wood by
commercially available laccase-mediator (L/M) system to improve the subsequent
saccharification. The thermostable laccase from the fungus M. thermophila will be applied
from Novozymes (Bagsvaerd, Denmark) that has been cloned and expressed in Aspergillus
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oryzae (Xu et al. 1996; Berka et al. 1997). Methyl syringate (MeS) will serve as mediator,
which is obtained from syringic acid present in pulp and paper side-streams (Rosado et al.
2012). MeS is cheaper and less toxic than synthetic mediators such as HBT, violuric acid or
ABTS. The L/MeS treatment was proved to be highly effective on eucalypt wood (
Rico et al.
2014), which contains a syringyl-rich lignin (Rencoret et al. 2008) that is easier to degrade
under alkaline conditions (González-Vila et al. 1999; Shimizu et al. 2017). The modification
of the structure of the lignin polymer in the pretreated paulownia will be monitored by 2D-
NMR spectroscopy of the whole sample at the gel state (Kim et al. 2008; Rencoret et al.
2009b), and the effect of the L/MeS treatment on the saccharification yield will be reported.
Material and methods
Paulownia wood, enzymes and mediator: 3-year-old P. fortunei trees were provided by the
University of Huelva (Spain). The wood was manually debarked, chipped, air-dried and
ground in a knife mill IKA MF1O to pass 1 mm screen, and then finely milled in a planetary
mill Retsch PM100 at 400 rpm for 2 h (including pause times to prevent sample heating),
using a 500 mL agate jar and agate balls (20 × 20 mm). The commercial (recombinant) fungal
laccase from M. thermophila (Novozym 51003) was supplied by Novozymes. The enzymatic
activity was measured as initial velocity during oxidation of 5 mM ABTS from Roche to its
cation radical (ε
436
29300 M
-1
cm
-1
) in 0.1 M sodium acetate (pH 5) at 24°C. The laccase
activity was 1000 U mL
-1
. One activity unit (U) is defined as the amount of enzyme
transforming 1 µmol of ABTS per min. MeS (methyl 4-hydroxy-3,5-dimethoxybenzoate from
Alfa Aesar (Karlsruhe, Germany) served as the redox mediator.
L/MeS treatments: A sequence of four L/MeS treatments was applied, each one followed by
an alkaline peroxide extraction step. Laccase doses of 50 U g
-1
were assayed, together with
3% MeS (% is b.o. dry wood). Paulownia samples (4 g) at 6% consistency (w:w) in 50 mM
NaH
2
PO
4
(pH 6.5) were placed into 200 mL pressurized bioreactors (Labomat, Mathis,
Formatted: Font color: Auto
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Switzerland) and treated under O
2
atmosphere (2 bar), in a thermostatic shaker (adjusted at
50°C and 190 rpm), for 24 h. Then, samples were filtered through a Büchner funnel and
washed with 1 L of water. Subsequently, samples at 6% consistency (w:w) were submitted to
an alkaline peroxide extraction wit 1% (w:w) NaOH and 3% (w:w) H
2
O
2
at 80°C for 90 min
in a thermostatic shaker at 140 rpm, followed by water washing. Treatments with laccase
alone (50 U g
-1
), without mediator, and controls without laccase and mediator were also
performed. A control treatment with mediator alone was not included in view of the negative
results of previous studies. The Klason lignin contents (TAPPI Method T222 om-88, 2004)
were corrected for ash.
Saccharification of pretreated samples: After L/MeS treatment, the samples were
hydrolyzed in an enzyme cocktail of commercial enzymes (Novozymes) with cellulase
(Celluclast 1.5 L; 2 FPU g
-1
) and β-glucosidase (Novozym 188; 6 U g
-1
) at 1% solid loading
in 3 mL of 100 mM sodium citrate (pH 5) for 72 h at 45°C, in a shaker bath at 140 rpm. The
released monosaccharides were determined as alditol acetate derivatives by GC (Selvendran
et al. 1979) on an HP 5890 instrument (Hewlett-Packard, Hoofddorp, The Netherlands), as
previously described (Rencoret et al. 2017). Chromatographic peaks were quantified by area,
and different standards (including glucose and xylose, among others) were used to elaborate
calibration curves. Duplicate experiments were performed in terms of of L/MeS treatment
(including control, laccase and laccase-MeS) and glucose and xylose release, and Klason
lignin contents were analysed in triplicate measurements. At the 95% confidence level, the
enzymatic experiments are smaller than the differences found between the control, laccase
alone, and L/MeS treatments. Moreover, ANOVA experiments between subjects were
performed. Post hoc pairwise comparisons, using the Tukey
HSD test, were also performed.
The data from both enzymatic and technical replicates were averaged.
2D-NMR analyses: The 2D HSQC NMR experiments were performed at the gel state, which
is an in situ analysis of the whole cell-wall (Kim et al. 2008, Rencoret et al. 2009b). This
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approach does not require a previous lignin isolation and avoids possible alterations and
material losses during the isolation process. Theseis experiments provided also structural
information on the hemicelluloses in the cell wall. 70 mg of ball-milled samples (and filtrate
samples) were transferred into 5-mm NMR tubes, and swelled in 1 mL of DMSO-d
6
, forming
a gel inside the tube (Kim et al. 2008; Rencoret et al. 2009b). Heteronuclear single quantum
coherence (HSQC) 2D-NMR spectra were recorded at 300 K on a Bruker AVANCE III 500
MHz spectrometer equipped with a 5 mm TCI gradient cryoprobe with inverse geometry. An
adiabatic HSQC pulse sequence (Bruker standard ‘hsqcetgpsisp.2’), which enabled a
semiquantitative analysis of the different
1
H-
13
C correlation signals (Kupče and Freeman,
2007), was utilized. HSQC spectra were acquired from 10 to 0 ppm (5000 Hz) in F2 (
1
H)
using 1000 data points for an acquisition time (AQ) of 100 ms, an interscan delay (D1) of 1 s,
and from 200 to 0 ppm (25,168) in F1 (
13
C) using 256 increments of 32 scan, for a total
acquisition time of 2 h 34 min. The
1
J
CH
used was 145 Hz. Processing used typical matched
Gaussian apodization in
1
H and a squared cosine bell in
13
C. The central solvent peak was
used as an internal reference (δ
C
/δ
H
39.5/2.49). Lignin and carbohydrate correlation signals in
the HSQC spectra were assigned by comparison with the literature (Rencoret et al. 2009a;
2009b; Kim et al. 2014). The
1
H-
13
C correlation signals from the aromatic region of the
spectrum were used to estimate the content of lignin (relative to the content of amorphous
carbohydrates, estimated from the anomeric xylose and glucose signals), and the lignin
composition in terms of G, S and oxidized S (S′) and G (G′) units. The C
α
-H
α
correlation
signals in the aliphatic-oxygenated region were used to estimate the abundance of the various
lignin inter-unit linkages whereas the C
γ
-H
γ
correlation signals were used to estimate the
abundance of the cinnamyl alcohol end-units. Likewise, S
2,6
(and S′
2,6
) and G
2
(and G′
2
)
signals were used to estimate the relative abundances of the aromatic units – as signals S
2,6
and S′
2,6
involve two proton-carbon pairs, their volume integrals were halved. The content of
Cα-oxidized S-lignin units in the HSQC spectrum of paulownia treated with laccase and
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Citations
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Journal Article•DOI•
Utility of laccase in pulp and paper industry: A progressive step towards the green technology.

[...]

Gursharan Singh1, Shailendra Kumar Arya2•
Lovely Professional University1, Panjab University, Chandigarh2
01 Aug 2019-International Journal of Biological Macromolecules
TL;DR: This review is providing the proper updated information on the state of the art of different applications of laccase in paper industry and confer the interpretation to the readers about the areas of extensively studied and the field where there is still much left to be done.
Abstract: Laccase has the enormous potential to be implemented as the multitasking biocatalyst in whole process of paper making. The enzyme can utilize effectively for pulping, delignification of pulps as alone or in the combination with other bleaching enzymes. Laccase has been evaluated for the biografting of pulp fibers, decolorize and stabilize the effluent of the paper mills, biotransformation of high molecular weight (HMW) lignins to lower molecular weight (LMW) aromatic compounds. Further this enzyme has the huge possibilities to apply for deinking of old newsprint (ONP) and pitch removal from varieties of the different pulps. There is no doubt on versatility mode of action of laccases in paper industry, but still there is limited commercialization of this enzyme has been possible, because of laccases has the less redox potential (E0) and needs mediators for the oxidation of non phenolic substrates, production of enzyme is cost intensive at large scale. This review is providing the proper updated information on the state of the art of different applications of laccase in paper industry. It also confer the interpretation to the readers about the areas of extensively studied and the field where there is still much left to be done.

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Journal Article•DOI•
Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

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Davinia Salvachúa1, Davinia Salvachúa2, Allison Z. Werner1, Allison Z. Werner2, Isabel Pardo1, Martyna Michalska3, Brenna A. Black1, Bryon S. Donohoe1, Stefan J. Haugen1, Rui Katahira1, Sandra Notonier2, Sandra Notonier1, Kelsey J. Ramirez1, Antonella Amore1, Samuel O. Purvine4, Erika M. Zink4, Paul E. Abraham2, Richard J. Giannone2, Suresh Poudel2, Philip D. Laible3, Robert L. Hettich2, Gregg T. Beckham1, Gregg T. Beckham2 •
National Renewable Energy Laboratory1, Oak Ridge National Laboratory2, Argonne National Laboratory3, Environmental Molecular Sciences Laboratory4
03 Apr 2020-Proceedings of the National Academy of Sciences of the United States of America
TL;DR: This work proposes a mechanism for extracellular nutrient acquisition from aromatic compounds by soil bacteria, which holds promise for improving the efficiency of microbial lignin conversion and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.
Abstract: Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

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Cites methods from "A commercial laccase-mediator syste..."

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Journal Article•DOI•
Process optimization, purification and characterization of alkaline stable white laccase from Myrothecium verrucaria ITCC-8447 and its application in delignification of agroresidues.

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Komal Agrawal1, Nisha Bhardwaj1, Bikash Kumar1, Venkatesh Chaturvedi, Pradeep Verma1 •
Central University of Rajasthan1
15 Mar 2019-International Journal of Biological Macromolecules
TL;DR: The white laccase was produced from Myrothecium verrucaria ITCC-8447 under submerged fermentation by response surface methodology and the enzyme activity under optimum condition exhibited 1.45 fold increases in lAccase activity.
Abstract: The white laccase was produced from Myrothecium verrucaria ITCC-8447 under submerged fermentation. The media components were optimized by response surface methodology (CCD-RSM). The nutritional components (glucose and peptone) and physical parameters (pH and temperature) were optimized by response surface methodology for enhanced laccase production by Myrothecium verrucaria ITCC-8447. The enzyme activity under optimum condition exhibited 1.45 fold increases in laccase activity. The white laccase was subjected to ion exchange chromatography with 6 fold purification. The molecular weight of white laccase was ~63-75kDa as estimated by SDS-PAGE followed by the activity staining with ABTS where green bands confirmed the presence of laccase. The enzyme was stable over an alkaline pH range of 7-9 and the temperature range of 30-40°C. The characterization of white laccase was done by CD spectra, UV-visible absorption, FTIR and XRD. The Km and Vmax values of the purified laccase were 2.5mM and1818.2μmol/min/L. The delignification capability of the white laccase was determined by reduction in Kappa number (58.8%) and Klason lignin (64.7%) of wheat straw after 12h of incubation. Further the delignification was confirmed FTIR and XRD.

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Journal Article•DOI•
Lignin degradation and detoxification of eucalyptus wastes by on-site manufacturing fungal enzymes to enhance second-generation ethanol yield

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Willian Daniel Hahn Schneider1, Roselei Claudete Fontana1, Henrique Macedo Baudel, Félix Gonçalves de Siqueira2, Jorge Rencoret3, Ana Gutiérrez3, Laura I. de Eugenio3, Alicia Prieto3, María Jesús Martínez3, Ángel T. Martínez3, Aldo José Pinheiro Dillon1, Marli Camassola1 •
University of Caxias do Sul1, Empresa Brasileira de Pesquisa Agropecuária2, Spanish National Research Council3
15 Mar 2020-Applied Energy
TL;DR: An important finding of the present work is the successful wood delignification in the absence of laccase mediators, making M. palmivorus laccases a potential candidate not only for the production of biofuels but also for the generation of lignin-derived aromatic compounds for different applications in the biotechnology industry.
Abstract: Novel laccases have promising and valuable applications in biorefineries. This investigation documents, for the first time, the potential of depolymerising and repolymerising lignin by the secretome, rich in laccases, from a newly isolated white-rot basidiomycete Marasmiellus palmivorus VE111, for further saccharification and ethanolic fermentation steps. Proteomic analyses of the secretome of M. palmivorus show that laccases are the most predominant enzyme released by this fungus. The whole crude enzymatic broth is used for the delignification of lignin in Eucalyptus globulus wood, with the aim of enhancing the saccharification by cellulolytic and xylanolytic enzymes from Penicillium echinulatum S1M29. In addition, two different strategies, namely, laccase treatment before and after enzymatic hydrolysis, are employed to detoxify steam-exploded E. globulus wood. The objective is to increase the fermentative performance by removing substances formed during the feedstock pretreatment that can inhibit microbial fermentation. The E. globulus wood delignification results in a 31% decrease in the lignin content and a 10% increase in the glucose yield after hydrolysis. An important finding of the present work is the successful wood delignification in the absence of laccase mediators. This laccase-rich preparation also demonstrates its potential in removing the phenolic inhibitors present in steam-exploded E. globulus wood, increasing the ethanol yield by an additional 10%. Furthermore, it is important to highlight that these findings are achieved in the absence of commercial enzymes, making M. palmivorus laccases a potential candidate not only for the production of biofuels but also for the generation of lignin-derived aromatic compounds for different applications in the biotechnology industry.

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Laccase-mediated delignification and detoxification of lignocellulosic biomass: removing obstacles in energy generation.

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Indian Institute of Petroleum1
12 Mar 2021-Environmental Science and Pollution Research
TL;DR: In this article, a review of recent studies in bioenergy generation using laccase biocatalyst in the pretreatment processes is presented, which summarizes the advantages, associated challenges, and potential prospects to overcome the limitations.
Abstract: The rising global population and worldwide industrialization have led to unprecedented energy demand that is causing fast depletion of fossil reserves. This has led to search for alternative energy sources that are renewable and environment friendly. Use of lignocellulosic biomass for energy generation is considered a promising approach as it does not compete with food supply. However, the lignin component of the biomass acts as a natural barrier that prevents its efficient utilization. In order to remove the lignin and increase the amount of fermentable sugars, the lignocellulosic biomass is pretreated using physical and chemical methods which are costly and hazardous for environment. Moreover, during the traditional pretreatment process, numerous inhibitory compounds are generated that adversely affect the growth of fermentative microbes. Alternatively, biological methods that use microbes and their enzymes disrupt lignin polymers and increase the accessibility of the carbohydrates for the sugar generation. Microbial laccases have been considered as an efficient biocatalyst for delignification and detoxification offering a green initiative for energy generation process. The present review aims to bring together recent studies in bioenergy generation using laccase biocatalyst in the pretreatment processes. The work provides an overview of the sustainable and eco-friendly approach of biological delignification and detoxification through whole-cell and enzymatic methods, use of laccase-mediator system, and immobilized laccases for this purpose. It also summarizes the advantages, associated challenges, and potential prospects to overcome the limitations.

27 citations

References
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Journal Article•DOI•
Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation.

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Robert Bourbonnais1, Michael G. Paice1•
St. John's University1
02 Jul 1990-FEBS Letters
TL;DR: Laccase is capable of oxidizing both phenolic and non‐phenolic moieties of lignin but that the latter is dependent on the co‐presence of primary laccase substrates.
Abstract: In the presence of substrates such as Remazol Blue and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate) (ABTS), laccases Coriolus (Trametes) versicolor can also oxidize non-phenolic lignin model compounds. Veratryl alcohol (I) and 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-propane-1,3-diol (III) were oxidized by laccase and mediator to give the alpha-carbonyl derivatives. The beta-1 lignin model dimer, 1-(3,4-dimethoxyphenyl)-2-phenoxy-ethane-1,2-diol (II) was cleaved by laccase in the presence of ABTS to give veratraldehyde and benzaldehyde. On the basis of these observations, we propose that laccase is capable of oxidizing both phenolic and non-phenolic moieties of lignin but that the latter is dependent on the co-presence of primary laccase substrates.

1,296 citations

Journal Article•DOI•
Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5

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Hoon Kim1, John Ralph1•
Great Lakes Bioenergy Research Center1
20 Jan 2010-Organic and Biomolecular Chemistry
TL;DR: This gel-state NMR fingerprinting of the components of finely divided plant cell walls swelled in DMSO may provide the basis for an attractive approach to providing a secondary screen for selecting biomass lines and for optimizing biomass processing and conversion efficiencies.
Abstract: NMR fingerprinting of the components of finely divided plant cell walls swelled in DMSO has been recently described. Cell wall gels, produced directly in the NMR tube with perdeutero-dimethylsulfoxide, allowed the acquisition of well resolved/dispersed 2D 13C–1H correlated solution-state NMR spectra of the entire array of wall polymers, without the need for component fractionation. That is, without actual solubilization, and without apparent structural modification beyond that inflicted by the ball milling and ultrasonication steps, satisfactorily interpretable spectra can be acquired that reveal compositional and structural details regarding the polysaccharide and lignin components in the wall. Here, the profiling method has been improved by using a mixture of perdeuterated DMSO and pyridine (4 : 1, v/v). Adding pyridine provided not only easier sample handling because of the better mobility compared to the DMSO-d6-only system but also considerably elevated intensities and improved resolution of the NMR spectra due to the enhanced swelling of the cell walls. This modification therefore provides a more rapid method for comparative structural evaluation of plant cell walls than is currently available. We examined loblolly pine (Pinus taeda, a gymnosperm), aspen (Populus tremuloides, an angiosperm), kenaf (Hibiscus cannabinus, an herbaceous plant), and corn (Zea mays L., a grass, i.e., from the Poaceae family). In principle, lignin composition (notably, the syringyl : guaiacyl : p-hydroxyphenyl ratio) can be quantified without the need for lignin isolation. Correlations for p-coumarate units in the corn sample are readily seen, and a variety of the ferulate correlations are also well resolved; ferulates are important components responsible for cell wall cross-linking in grasses. Polysaccharide anomeric correlations were tentatively assigned for each plant sample based on standard samples and various literature data. With the new potential for chemometric analysis using the 2D NMR fingerprint, this gel-state method may provide the basis for an attractive approach to providing a secondary screen for selecting biomass lines and for optimizing biomass processing and conversion efficiencies.

566 citations

"A commercial laccase-mediator syste..." refers methods in this paper

  • ...Seventy milligrams of ball-milled samples (and filtrate samples) were transferred into 5-mm NMR tubes and swelled in 1 ml of DMSO-d6, forming a gel inside the tube (Kim et al. 2008; Rencoret et al. 2009b)....

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  • ...2D-NMR analyses: The 2D heteronuclear single quantum coherence (HSQC) NMR experiments were performed at the gel state, which is an in situ analysis of the whole cell wall (Kim et al. 2008; Rencoret et al. 2009b)....

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  • ...The modification of the structure of the lignin polymer in the pretreated paulownia was monitored by two dimensional-nuclear magnetic resonance (2D-NMR) spectroscopy of the whole sample at the gel state (Kim et al. 2008; Rencoret et al. 2009b), and the effect of the L/MeS treatment on the saccharification yield was reported....

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Journal Article•DOI•
A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability

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Feng Xu1, Woonsup Shin2, Stephen H. Brown1, Jill Angela Wahleithner1, Uma M. Sundaram2, Edward I. Solomon2 •
Novo Nordisk1, Stanford University2
08 Feb 1996-Biochimica et Biophysica Acta
TL;DR: It is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.
Abstract: A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7-0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity. By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series. At the optimal pH, the two substrates studied, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging form 10 to 120 and from 1 to 45 microM; and kcat values ranging from 50 to 16 000 and 200 to 3000 per min, respectively. The enzymes are more stable in the neutral-alkaline pH range. The thermal stability is in the order of bilirubin oxidase equivalent to Myceliophthora laccase equivalent to Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase. Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.

536 citations

Journal Article•DOI•
Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound

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Kaichang Li1, Feng Xu2, Karl-Erik Eriksson1•
University of Georgia1, Novo Nordisk2
01 Jun 1999-Applied and Environmental Microbiology
TL;DR: Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methOxyphenoxy)propan-1,3-diol (I), and a Phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid.
Abstract: Conventional pulp-bleaching techniques with chlorine or chlorine-based chemicals can, under certain conditions, generate chlorinated organic compounds that are toxic to the environment. The pulp and paper industry is facing an increasing pressure from environmentally concerned organizations to replace the conventional bleaching techniques with environmentally benign ones. Enzymatic bleaching methods have recently drawn much attention as being environmentally friendly. In addition to xylanase, laccase has been the most actively investigated enzyme for biobleaching of kraft pulp because laccase can be produced in large amounts at a reasonable price and use cheap oxygen as an electron acceptor. However, expensive redox mediators are still a hurdle in the implementation of laccase in pulp bleaching. Laccase (EC 1.10.3.1) belongs to a family of multi-copper oxidases that are widespread in numerous fungi, in various plant species (18), in the bacterium Azospirillum lipoferum (10), and in a dozen of studied insects (25). Laccase has various functions, including participation in lignin biosynthesis (21), plant pathogenicity (22), the degradation of plant cell walls (12, 17), insect sclerotization (3), bacterial melanization (10), and melanin-related virulence for humans (26). Chemically, all of these functions of laccases are related to oxidation of a range of aromatic substances. However, the net effect of such oxidations could be very different and even work in opposite directions. Plant laccases, for example, oxidize monolignols to form polymeric lignins, whereas laccases from white-rot fungi degrade and depolymerize lignins. In the degradation of lignin by white-rot fungi, the redox potential of the lignin-degrading enzymes has long been believed to play a crucial role because nonphenolic subunits, the most predominant lignin substructures in wood, have high redox potentials. The well-studied lignin peroxidase is able to oxidize nonphenolic aromatic compounds with very high ionization potentials such as 1,2-dimethoxybenzene (E1/2 = 1,500 mV) and veratryl alcohol (14, 20). Lignin peroxidase was thus once believed to be a key enzyme for fungal degradation of lignin, whereas laccase was believed to be less important because it could not oxidize veratryl alcohol (a typical model compound for nonphenolic lignin). The highest redox potential of a laccase reported so far does not exceed 800 mV, which is believed not to be high enough to oxidize a nonphenolic lignin structure. However, it has been demonstrated that laccase is able to oxidize some compounds (redox mediators) with a higher redox potential than laccase itself, although the mechanism by which this happens is not known (2, 7). In the presence of such redox mediators, laccase is also able to oxidize nonphenolic lignin model compounds and decrease pulp kappa number to a great extent (5, 8). Several effective redox mediators have been reported so far (2, 5, 6, 8, 13). The importance of the redox potential of laccases in the oxidation of lignin model compounds by laccase/mediator systems will be addressed here. While much effort has been devoted to search for more effective redox mediators, the laccase parameters governing lignin degradation and pulp bleaching are still not fully elucidated. In an effort to determine these parameters, we compared the ability of different laccases for the oxidation of lignin model compounds in a laccase-mediator system. More specifically, four laccases from different fungal species were purified and used to oxidize the β-O-4 dimer I (the most predominant lignin substructure) and phenol red (a phenolic lignin model compound). Laccases from the different sources were found to oxidize dimer I and phenol red at different rates. Criteria for a better laccase and more effective laccase-mediator systems for pulp bleaching have been suggested.

434 citations

Journal Article•DOI•
Application of quantitative 31P NMR in biomass lignin and biofuel precursors characterization

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Yunqiao Pu1, Shilin Cao1, Arthur J. Ragauskas1•
Georgia Institute of Technology1
26 Aug 2011-Energy and Environmental Science
TL;DR: Using lignin isolated from native and transgenic plants as well as from pretreatment and biological/thermal deconstruction processes, researchers have demonstrated that this technique has unique characterization capabilities which have broad applicability in the biofuels research community.
Abstract: The last decade has seen tremendous growth and interest in renewable energy and fuels aimed primarily at addressing issues of climate change, energy security, and rising energy costs. These efforts coupled with the demand for efficient utilization of biomass place a premium on the detailed analysis of the fundamental chemical structures of biomass, especially in light of the ever-increasing efforts to generate transgenic plants with reduced recalcitrance and altered lignin structure. This review examines the growing application of phosphitylation followed by 31P NMR to quantitatively analyze biomass lignin structures including guaiacyl, syringyl, guaiacyl with carbon substituents at the C5 position, catechol, p–hydroxyphenyl, aliphatic and carboxylic hydroxyl groups. The application of this methodology to provide a rapid analytical tool for lignin/biomass derived bio-oils and biodiesel precursors is also discussed. Utilizing lignin isolated from native and transgenic plants as well as from pretreatment and biological/thermal deconstruction processes, researchers have demonstrated that this technique has unique characterization capabilities which have broad applicability in the biofuels research community.

432 citations

Related Papers (3)
Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin and Improves Saccharification

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12 May 2016-Bioenergy Research
Jorge Rencoret, Antonio Pereira, José C. del Río, Ángel T. Martínez, Ana Gutiérrez 
Delignification and Saccharification Enhancement of Sugarcane Byproducts by a Laccase-Based Pretreatment

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19 Jul 2017-ACS Sustainable Chemistry & Engineering
Jorge Rencoret, Antonio Pereira, José C. del Río, Ángel T. Martínez, Ana Gutiérrez 
Mechanisms of Lignin Degradation with Three Species of Enzymes(Systems)

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01 Jan 2008
YE Han-ling
Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "Short title: enzymatic pretreatment of p. fortunei a commercial laccase-mediator system to delignify and improve saccharification of the fast-growing paulownia fortunei" ?

Up to 24 % lignin removal was attained by the laccase-mediator treatment ( L/MeS ), followed by alkaline peroxide extraction in a multistage sequence.