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Appl Microbiol Biotechnol (2014) 98:65256542

DOI 10.1007/s00253-014-5810-8

MINI-REVIEW

Laccase applications in biofuels production: current status


and future prospects
Tukayi Kudanga & Marilize Le Roes-Hill

Received: 21 February 2014 / Revised: 30 April 2014 / Accepted: 1 May 2014 / Published online: 20 May 2014
# Springer-Verlag Berlin Heidelberg 2014

Abstract The desire to reduce dependence on the ever


diminishing fossil fuel reserves coupled with the impetus
towards green energy has seen increased research in biofuels
as alternative sources of energy. Lignocellulose materials are
one of the most promising feedstocks for advanced biofuels
production. However, their utilisation is dependent on the
efficient hydrolysis of polysaccharides, which in part is dependent on cost-effective and benign pretreatment of biomass
to remove or modify lignin and release or expose sugars to
hydrolytic enzymes. Laccase is one of the enzymes that are
being investigated not only for potential use as pretreatment
agents in biofuel production, mainly as a delignifying enzyme,
but also as a biotechnological tool for removal of inhibitors
(mainly phenolic) of subsequent enzymatic processes. The
current review discusses the major advances in the application
of laccase as a potential pretreatment strategy, the underlying
principles as well as directions for future research in the search
for better enzyme-based technologies for biofuel production.
Future perspectives could include synergy between enzymes
that may be required for optimal results and the adoption of
the biorefinery concept in line with the move towards the
global implementation of the bioeconomy strategy.

Keywords Laccase . Lignocellulose . Pretreatment . Biofuels

T. Kudanga (*) : M. Le Roes-Hill


Biocatalysis and Technical Biology Research Group, Cape Peninsula
University of Technology, PO Box 1906, Bellville 7535, South
Africa
e-mail: kudangat@cput.ac.za
T. Kudanga
e-mail: tikudanga@yahoo.co.uk

Introduction
Physical and chemical processes have been, and continue to
be, an integral aspect of modern life. This is mainly due to
their utility in the conversion of raw materials into valueadded products widely available on the market. However, in
recent years, there has been a growing interest in the use of
bio-based processes, which is attributable to the increasing
evidence of their advantages as alternatives to physicochemical processes and in some cases, the fact that they may
be the only viable option. Unlike physico-chemical processes,
the bio-based processes are considered more efficient, costeffective, environmentally friendly and sustainable; mostly
because they are not overly reliant on non-renewable resources such as fossil fuels. A combination of punitive action
and legislations introduced to improve protection of human
health and the environment, pressure from environmental
protection lobbyists and conscious proactive efforts, have seen
an upsurge in the search for alternative biotechnological processes. Consequently, biotechnology, and in particular biocatalysis, is now established as an important option in many
applications. The main success factors for biocatalytic routes
include the selectivity of the catalyst, the efficiency of the
process (Burton et al. 2002), potential reusability of the enzymes and their biodegradability (once no longer needed), and
general environmental considerations (Pollard and Woodley
2007). Thus, many industries are currently pursuing enzymatic approaches for the development of green chemistry technologies. Laccases are among the enzymes which are being
intensively investigated for various biotechnological
applications.
Laccase (benzenediol: oxygen oxidoreductase, EC
1.10.3.2) is a multi-copper-containing enzyme which performs one-electron oxidation of various substrates such as
diphenols, methoxy-substituted monophenols, as well as aromatic and aliphatic amines to form radicals and at the same

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Appl Microbiol Biotechnol (2014) 98:65256542

time reduces molecular oxygen to water (Kudanga et al.


2011a). Laccase-generated radicals undergo a number of enzymatic or non-enzymatic reactions, which have implications
in many industries. Coupling of the radicals to form oligomers
has been applied in organic synthesis, whereas polymerisation
to form insoluble compounds (which can be easily removed
by filtration or sedimentation) has been utilised in bioremediation. The radicals can also participate in the degradation of
complex polymers by cleavage of covalent bonds especially
alkylaryl bonds (sometimes in the presence of mediators),
releasing monomers (Breen and Singleton 1999) and in the
ring cleavage of aromatic compounds (Claus et al. 2002;
Durn and Esposito 2000; Kawai et al. 1988b). These processes have potential application in the degradation of lignin
and xenobiotic compounds and therefore have implication in
the paper and pulp industry, textile industry, biofuel industry
and in bioremediation, among others. The broad substrate
range of laccase, which is further widened by the use of a
laccase-mediator system (LMS), allows the enzyme to be
applied in many industries. Currently, the main applications
are in the pulp and paper industry, textile industry and in
bioremediation while application potential has been demonstrated for the food industry, pharmaceutical industry, organic
synthesis, lignocellulose modification, biofuels production

and diagnostic purposes (Burton and Le Roes-Hill 2008).


While most of these applications have been reviewed
(Table 1), the potential of the enzyme in biofuel production
has not been comprehensively reviewed in recent articles.
Recent reviews on the pretreatment strategies have focused
mainly on physico-chemical processes (Mood et al. 2013;
Bensah and Mensah 2013; Talebnia et al. 2010; Alvira et al.
2010; Hendriks and Zeeman 2009; Mosier et al. 2005) while
biotechnological processes have been confined to detoxification processes in the broad sense of application of all potentially relevant enzymes and whole cells (Parawira and Tekere
2011). The main advantages of using enzymes over whole
cells include the possibility of using higher temperatures
which are not necessarily optimal for the growth of the microorganisms, high catalytic efficiencies of the enzymes and
low utility costs associated with the use of mild conditions.
However, there are some disadvantages which include
prolonged incubation periods which may be required, high
enzyme production costs despite the possibility of recombinant production of laccases (Parawira and Tekere 2011) and
missed opportunity of using a combination of enzymes available in whole cells. In nature, various enzymes (laccases and
peroxidases) produced by whole cells are involved in the
degradation of lignin, cumulatively generating unstable

Table 1 Reviewed applications of laccases


Application

Main reactions

Pulp and paper industry

Oxidative delignification (mainly using LMS)

Relevant reviews

Arora and Sharma (2010), Bourbonnais et al. (2001), Breen


and Singleton (1999), Caas and Camarero (2010),
Kunamneni et al. (2008b), Mayer and Staples (2002),
Nyanhongo et al. (2007), Rodrguez-Couto and TocaHerrera (2006), Thurston (1994) and Widsten and
Kandelbauer (2008)
Textile industry
Oxidative delignification (mainly using LMS) and Arora and Sharma (2010), Kudanga et al. (2011b), Kunamneni
oxidative degradation
et al. (2008b) and Rodrguez-Couto and Toca-Herrera (2006)
Food industry
Coupling/crosslinking reactions
Brijwani et al. (2010), Kudanga et al. (2011b), Kunamneni
et al. (2008b), Minussi et al. (2002) and Osma et al. (2010)
Wood products and modification Coupling and grafting and co-polymerisation
Kudanga et al. (2011b) and Widsten and Kandelbauer (2008)
of lignocellulose materials
Bioremediation
Oxidative degradation (usually in the presence
Arora and Sharma (2010), Caas and Camarero (2010), Durn
of mediators); coupling coupled with
and Esposito (2000), Durn et al. (2002), Kudanga et al.
polymerisation, demethylation or
(2011a, b), Kunamneni et al. (2008b), Majeau et al. (2010),
dechlorination; and oxidative cleavage
Mayer and Staples (2002) Nyanhongo et al. (2007),
Rodrguez-Couto and Toca-Herrera (2006) and Strong and
Claus (2011)
Organic synthesis
Oxidative oligomerisation
Durn et al. (2002), Kunamneni et al. (2008a, b), Mikolasch
and Schauer (2009); Riva (2006), Rodrguez-Couto and
Toca-Herrera (2006) and Witayakran and Ragauskas (2009)
Pharmaceutical industry
Oxidative oligomerisation
Arora and Sharma (2010), Kudanga et al. (2011b), Kunamneni
et al. (2008b), Mikolasch and Schauer (2009) and
Nyanhongo et al. (2007)
Biosensors and other
Oxidation
Arora and Sharma (2010), Durn et al. (2002), Kunamneni
bio-analytical purposes
et al. (2008a), Mayer and Staples (2002), Nyanhongo et al.
(2007) and Rodrguez-Couto and Toca-Herrera (2006)

Appl Microbiol Biotechnol (2014) 98:65256542

radicals that can attack the lignin component in a process often


referred to as enzymatic combustion (Prez et al. 2002). While
other oxidase enzymes such as lignin peroxidases, manganese
peroxidases and versatile peroxidases can also play a key role
in lignin biodegradation and therefore biofuels production,
these enzymes do, however, require the presence of cofactors,
e.g. H2O2 and Mn(II) to catalyse the reaction, which makes
the process expensive and environmentally unfriendly. On the
contrary, laccases are essentially green catalysts; they require
only air (oxygen) as co-substrate, producing water as the only
by-product. Therefore the main aim of this article is to provide
a focused comprehensive review of the application potential
of laccases in the production of biofuels, mainly as a
delignifying enzyme to facilitate access of hydrolytic enzymes
to carbohydrate moieties and as a detoxifying enzyme for
removal of inhibitory phenolic compounds from lignocellulose hydrolysates, as well as providing the mode of action of
the enzyme in each case. In addition, the review provides
directions for future research as a way of highlighting future
prospects of the enzyme in biofuels production.

Laccase: sources, structure and function


Laccase was first isolated from sap of the Japanese lacquer
tree Rhus vernicifera (Yoshida 1883) and is now widely
regarded as a ubiquitous enzyme which has been isolated from
higher plants (Dwivedi et al. 2011), insects (Kramer et al.
2001), invertebrates, specifically oysters, mussels and other
bivalves (Luna-Acosta et al. 2010), archaea (Uthandi et al.
2010), prokaryotes (Claus 2003) and is widespread in fungi
(Baldrian 2006).
Structurally, laccase is a glycosylated monomer or homodimer and generally laccase of fungal or bacterial origin is less
glycosylated (1025 %) than those of plant origin. The carbohydrate component is believed to enhance stability of the
enzyme, and its main building units are glucose, hexoamines, fucose, mannose, arabinose and galactose. Each functional laccase monomeric unit consists of four copper atoms
namely type 1 copper (T1), type 2 copper (T2) and two type 3
copper (T3) atoms. The T1 copper is the site where oneelectron oxidation of substrates takes place and therefore acts
as the primary electron acceptor. The two T3 copper atoms
and T2 copper form a trinulear site where molecular oxygen is
reduced to water. It is widely believed that the axial ligand in
the T1 copper influences its redox potential. Recently, a second group of laccases with two copper atoms per molecule
which work in concert with pyrolloquinoline quinone (PQQ)
factor has been reported (Rogalski and Leonowicz 2004).
These laccases show high N-terminus amino acid sequence
homology with blue laccases but do not show the typical
spectroscopic and paramagnetic properties of blue laccases
(Leontievsky et al. 1997a). They are yellow-brown in colour

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and are now frequently referred to as yellow laccases. The


oxidation state of the copper in their active site appears to be
different from that of blue laccases and there have been reports
that, unlike blue laccases, they can oxidise non-phenolic lignin models and veratryl alcohol in the absence of mediators
(Leontievsky et al. 1997b; Giardina et al. 2010). They are
therefore interesting candidates in the search for laccases for
use in lignin degradation with potential for biofuel production.
A third type of laccase, the white laccase, has been identified
in various fungal strains (Giardina et al. 2010). The white
laccase from Trametes hirsuta, as in the case of the yellow
laccases, is able to oxidise complex dyes in the absence of
mediators and therefore also show potential for application in
biofuel production.
The three-dimensional structure of fungal laccases has been
elucidated by a number of researchers and has been reviewed
by Morozova et al. (2007a). Generally, the structure is
organised as monomeric units consisting of three sequentially
arranged domains of a barrel-type structure. Recently, it was
confirmed that fungal laccases show no evidence of higherorder oligomeric assemblies in the crystal lattice and essentially exist as monomers (Ge et al. 2010). The T1 copper site
of laccases is located in the third domain, and the tri-nuclear
cluster T2/T3 is located between the first and third domains
while the amino acid residues of the second and third domains
are involved in formation of the substrate-binding pocket
(Morozova et al. 2007a). Although laccases are typically
three-domain monomeric enzymes, a small four-copper
laccase was identified from Streptomyces coelicolor A3(2)
(Machczynski et al. 2004) and its three-dimensional structure
shows that it consists of only two domains, which are similar
to domains 1 and 3 of the classical fungal and plant laccases
(Sklov et al. 2009). Unlike typical laccases, this two domain
laccase named small laccase, because of its size, is active in
the oligomeric form as dimers (Machczynski et al. 2004) or
more frequently trimers (Sklov et al. 2009). The trimeric
organisation of the enzyme and relative proximity of the type
1 copper ions bring the three substrate binding sites close in
space effectively forming a shallow trimeric substrate binding
site (Sklov et al. 2009). This organisation highlights the
importance of a stable trimeric structure since the active sites
are placed between the individual protein chains.
Laccase-catalysed oxidation proceeds through the direct
interaction with the substrate or indirectly via the oxidation
of a substrate by a laccase oxidised mediator. With direct
oxidation the substrate is oxidised by one electron oxidation
at the T1 copper atom to form an unstable reactive radical.
After four cycles of one-electron oxidation, the electrons are
transferred to the trinuclear centre where they reduce one
oxygen molecule to two molecules of water (Kudanga et al.
2011b; Riva 2006). However, some potential substrates cannot be oxidised directly because they are either too large to
enter the laccase active site or they have a higher redox

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potential than the enzyme. Such substrates can be


oxidised using a LMS in which a small, low redox
potential substrate is first oxidised by laccase and then
its radical can be utilised for the oxidation of complex
or high redox potential substrates. The LMS could be
particularly important in the use of blue laccases in
biofuel production due to high redox potential of substrates in particular the non-phenolic moieties, and inaccessibility of active site by complex lignocellulosic
substrates. However, many laccase mediators are either
toxic or expensive, which has necessitated the search for
natural mediators.

Search for natural mediators


As mentioned previously, the group of laccases often referred
to as the blue laccases, typically require the presence of small
compounds (mediators) to oxidise compounds that are considered to be atypical substrates for laccases, e.g. complex
biopolymers such as lignin (Desai and Nityanand 2011;
Kunamneni et al. 2008b). The molecule 2,2-azino-bis-(3ethyl-benzothizoline-6-sulphonic acid) (ABTS) was the first
mediator described and was used in the application of laccases
in the bio-bleaching of wood pulps (Madhavi and Lele 2009).
With the development of the LMS, laccases have been successfully applied in the oxidation of aromatic methyl groups,
benzyl alcohols, as well as in bioremediation such as the
removal of pesticides, polycyclic aromatic hydrocarbons
(PAH), explosives and chemical warfare agents, and in the
bleaching of various textile dyes (Madhavi and Lele 2009;
Torres-Duarte et al. 2009; Trovaslet-Leroy et al. 2010). It is
therefore conceivable that the LMS have huge potential in
biofuels production.
There are two main categories of mediators: synthetic and
natural (Fig. 1a, b) (Caas and Camarero 2010). To date, more
than 100 synthetic mediators have been identified and their
effects on laccase activity described. Among these, ABTS, 1hydroxybenzotriazole (HBT), violuric acid (VIO), Nhydroxyphthalimide (HPI), N-hydroxyacetanilide (NHA),
2,2,6,6,-tetramethyl-1-piperidinyloxy-free radical (TEMPO),
phenothiazines and other heterocycles have been extensively
studied (Kunamneni et al. 2008b). As pointed out earlier, these
synthetic mediators have two major drawbacks: (1) They are
expensive and therefore not feasible for application at industrial scale, and (2) They can cause the formation of toxic
derivatives that may result in the inactivation of laccases.
There has therefore been an increased interest in the identification and utilisation of cheaper, eco-friendly natural mediators (Kunamneni et al. 2008b), with the main focus of identifying natural mediators from renewable sources (such as
industrial by-products) and from plants (Johannes and
Majcherczyk 2000; Moldes et al. 2008; Vila et al. 2011).

Appl Microbiol Biotechnol (2014) 98:65256542

Natural mediators include 4-hydroxybenzoic acid, 4hydroxybenzyl alcohol, aniline, phenol and phenolic compounds derived from lignin degradation such as
acetosyringone, acetovanillone, p-coumaric acid, ferulic acid,
syringaldehyde and vanillin (Kunamneni et al. 2008b). It has
been determined that natural mediators act very similar to
synthetic mediators and sometimes have a greater effect
(Fillat et al. 2010). Lignin-derived phenolic mediators have
successfully been applied to fungal laccase reactions involving dye decolourisation, delignification of paper pulp from
wood and non-wood fibres, delignification of flax pulp and
kenaf pulp, delignification of Eucalyptus wood chips, removal
of lipophilic extractives and the oxidation of PAH (Andreu
and Vidal 2011; Fillat et al. 2010; Rico et al. 2014). However,
one potential drawback is that in some instances, an opposite
effect can be achieved when using natural mediators vs synthetic mediators, e.g. Vila et al. (2011) reported on a laccase
that acted as a polymerising agent in the presence of natural
mediators, while acting as a delignifying agent in the presence
of a synthetic mediator. Aracri et al. (2009) reported a
similar result in their study on the bleaching of pulp
and effluents obtained from sisal pulp. In this study,
natural mediators resulted in laccase-mediated coupling
reactions rather than bleaching observed in the presence
of synthetic mediators. This could compromise the use
of the mediators in biofuel production. Such mediators
may be useful in laccase-assisted biografting applications (Aracri et al. 2009). Indeed, we have observed
that lignin model compounds guaiacylglycerol guaiacyl ether (erol) and syringylglycerol -guaiacyl
ether acted as mediators during laccase assisted coupling
of fluorophenols to the models (Kudanga et al. 2010c).

Lignin: the target component in laccase-assisted biofuel


production
Lignin is one of the three major components of lignocellose
materials comprising (1835 %); the other ones being cellulose (4050 %) and hemicellulose (1525 %). It is thus the
second most abundant organic polymer on Earth, representing
a huge reserve of non-fossil carbon which at the moment is
heavily underutilised. Structurally, lignin is a heterogeneous,
optically inactive, three-dimensional polymer of hydroxylated
and methoxylated phenylpropane units, linked in an irregular
manner through oxidative coupling to form ether and C-C
bonds. It is the binding agent which holds cells together,
giving rigidity to the cell. Its complex and hydrophobic nature
and ability to act as an encrusting agent on and around the
carbohydrate fraction, limits enzymatic hydrolysis of the carbohydrate fraction hence the need for pretreatment when
converting biomass to fuels such as bioethanol (Asgher et al.
2014) (Fig. 2). Apart from lignin content, other prominent

Appl Microbiol Biotechnol (2014) 98:65256542


Fig. 1 Examples of natural (a)
and synthetic (b) laccase
mediators. HPI Nhydroxyphthalimide, TEMPO
2,2,6,6-tetramethyl-piperidineN-oxyl, SPP-m 1-(3sulphophenyl)-3-methylpyrazolone-5, HBT 1hydroxybenzotriazole,
ABTS 2,2-azino-bis-(3-ethylbenzothiazoline-6 sulphonic
acid), NHA N-hydroxyacetanilide

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a
COCH 3

MeO

OMe

HO

OH

CH 2OH

OH

OH

OH

Vanillyl alcohol

OMe

OMe

OH

OH

HO

MeO

OMe

OH

Gallic acid

Acetosyringone

COOH

Syringic acid

Vanillin

COCH 3

OH

OH

OH

NH 2

OH

MeO

OMe

OMe
OH

OH

p-coumaric acid

OH

3-hydroxyanthranilic acid Catechol

Syringaldehyde Acetovanillone
CHO

OMe

OH

MeO
MeO

OMe
OH

MeO

OH

OH

2,6-dimethylphenol

HO

OCH 2CH 3

OMe

MeO

2,4,6-trimethylphenol Ethyl vanillin

Sinapic acid
OH

OH
O

O
HO

MeO

OH

OH

HO
MeO

4-hydroxybenzoic acid

Ferulic acid

Cinnamic acid

OH

Coniferyl alcohol

H3C

O
N OH

H3C

CH3
N

H3C

OH

CH3

OH

SO3H

TEMPO

HPI

SPP-m

Violuric acid
OH

H 3C

N
N

OH

OH

OH

HBT

NHA

S
O
O

HO
S

N
N

OH
S

Phenol red

O
N

H3C

CH3

ABTS

lignin-related factors that impact biomass conversion may


include lignin composition, its chemical structures and
lig nin-ca rb ohydrate co mplex (LCC) linkage s in

lignocellulosic biomass (Pu et al. 2013). There are three


monolignol monomers; p-coumaryl alcohol, coniferyl
alcohol, and sinapyl alcohol (Fig. 3ac), which are connected

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Fig. 2 Lignocellulose model structure, delignification strategies, and subsequent steps in the production of bioethanol (Adapted from Du et al. 2013b;
Asgher et al. 2014 with permission from ACS Publications and Elsevier)

through radical coupling to form inter-unit linkages such as O-4, -5, 5-5, -, 5-O-4 and the more recently discovered
dibenzodioxocin 5-5-O-4 (Karhunen et al. 1995a, b) (Fig. 3d
k).
The first comprehensive model of softwood lignin structure
was developed by Sakakibara (1980) based upon the degradation products formed from lignin samples as a result of
hydrolysis in aqueous dioxane and catalytic hydrogenolysis.
According to the Sakakibara model, the most prevalent interunit linkages were thought to be the -O-4 alkyl aryl ether, 5-5
biphenyl, -5 and 1 alkyl arene and -O-4 benzyl aryl ether
linkages. However, recent developments have shown that
there are very low levels, if any of -1 and non-cyclic -O-4
linkages (Kilpelinen et al. 1994; Ede and Kilpelinen 1995)

and that many of the 5-5-biphenyl inter-unit linkages are


etherified with phenylpropanoid units to form 5-5-O-4
dibenzodioxocin structures (Karhunen et al. 1995a, b).
Consequently, a new improved model, which took into account the new knowledge and the proposed structures involved in the formation of lignin-carbohydrate complexes,
was developed (Chen and Sarkanen 2003, 2010) (Fig. 4).
Lignin carbohydrate complexes are formed when lignin is
covalently linked to hemicellulose. Xylan, glucan and
glucomannan residues appear to be the major interface between lignin and the carbohydrate components (Du 2013). For
example spruce wood was found to consist of 49.5 % glucanlignin, 30.9 % glucomannanlignin and 12.0 % xylanlignin
(Du 2013) while benzyl ether, -ester and phenyl glycoside

Appl Microbiol Biotechnol (2014) 98:65256542

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Fig. 3 Lignin precursor


molecules (ac) and common
linkages in lignin (dk)

LCC bonds have been reported as the predominant


linkages between lignin and carbohydrates (Du 2013;
Balakshin et al. 2011). An interesting observation on
gymnosperm lignin is that the lignin residues that are
bound primarily to xylans are composed almost exclusively of -O-4 alkyl aryl ether substructures while
those which are predominantly attached to
glucomannans, are thought to encompass all the other
substructures with a lower proportion of -O-4 ethers
(Chen and Sarkanen 2010) (Fig. 4). However, recent
research on spruce wood has shown that lignin in
glucomannan lignin has a higher content of -O-4 ether
substructures (62 %) than in xylanlignin complexes
(53 %) (Du et al. 2014). It has been suggested that it
is not just the lignin content that effects recalcitrance of
lignocellulosic materials, but rather and possibly more
importantly, the integration of lignin and polysaccharides within the cell wall, and their associations with
one another and with other wall components (DeMartini
et al. 2011). Therefore the cleavage of the linkages
between lignin and carbohydrates in LCCs should be
an important consideration in the pretreatment of biomass for biofuel production.

Application of laccases in biofuel production


The use of second-generation biofuels such as biogas and
bioethanol is generally regarded as greener because they
are produced from sustainable feedstocks. Biogas is produced
in an anaerobic process in which organic matter is converted
into methane and carbon dioxide. The process involves four
main phases namely pretreatment, hydrolysis, acidogenesis
and acetogenesis, and methane fermentation. In the first
phase, pretreatment is performed to improve the hydrolysis
yield and consequently total methane yield. The hydrolysis
step, which is carried out by obligate anaerobes such as
Bacteroides spp., Clostridium spp. and facultative bacteria
such as Streptococcus spp., involves enzymatic breakdown
of insoluble organic material and higher molecular mass compounds such as lipids, polysaccharides, proteins, fats and
nucleic acids into soluble organic materials such as monosaccharides, amino acids and other simple organic compounds. In
the third step, primary fermenting bacteria (obligate and facultative anaerobes) convert the products of hydrolysis into
volatile fatty acids, hydrogen and alcohols (acidogenesis).
Volatile fatty acids longer than two carbon atoms and alcohols
longer than one carbon atom cannot be utilised by

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Appl Microbiol Biotechnol (2014) 98:65256542

Fig. 4 A model of the structural features of gymnosperm lignin and its association with hemicellulose components to form lignin-carbohydrate
complexes (Chen and Sarkanen 2010). Reprinted with permission from Elsevier

methanogenic bacteria and must be converted to acetic acid,


hydrogen and carbon dioxide (acetogenesis) by obligate
hydrogen-producing secondary fermenting bacteria (Bruni
2010). In the final phase, acetic acid, hydrogen, carbon dioxide and other one-carbon compounds such as formate and
methanol are converted into methane and carbon dioxide
(biogas) by obligate anaerobic methanogenic bacteria (acetate
utilisers such as Methanosarcina spp. and Methanothrix spp.
and hydrogen and formate utilising species of genera such as
Methanobacterium and Methanococcus). Bioethanol production involves sequential steps of pretreatment (for the same
reasons as in biogas production), enzymatic hydrolysis, fermentation of monomeric sugars to ethanol and product separation by distillation (not necessary in biogas production since
the methane escapes from the water phase) (Bruni 2010)
(Fig. 2).

Currently, biogas and bioethanol only contribute a minor


fraction to fuel products from the energy industry. In recent
years the demand for biogas has been increasing as the world
seeks ways to reduce consumption of fossil fuels and
substituting firewood in rural households. This is driven by
the need to respond to climate change and to shift the prime
resource base from non-renewable to renewable feedstocks,
an aspect clearly highlighted in the global bio-economy strategy. Consequently, research efforts to increase output of biogas are being intensified in the hope that it may one day be a
leading energy source. A number of factors that limit the
hydrolysis of lignocellulose materials and therefore reduce
biofuel yields have been identified (Alvira et al. 2010;
Hendriks and Zeeman 2009). One of the factors is the lignin
component which limits the rate and extent of enzymatic
hydrolysis by shielding the digestible parts of the substrate

Appl Microbiol Biotechnol (2014) 98:65256542

from enzymes (Ding et al. 2012; Jung et al. 2000; Chang and
Holtzapple 2000) or by binding cellulolytic enzymes nonproductively (Kumar et al. 2012; Palonen et al. 2004;
Esteghlalian et al. 2001). A number of physical and chemical
pretreatment processes are being considered to minimise the
effect of lignin for example removal or breakdown of the
lignin component in order to expose the carbohydrate component to hydrolytic enzymes or hydrolysis of hemicellulose to
release monomeric sugars and soluble oligomers from the cell
wall matrix into the hydrolysate, leaving the lignin with the
residual substrate for subsequent removal after the hydrolysis
of cellulose or even after distillation. These include mechanical treatment, thermal treatment, acid treatment, alkali treatment, oxidative treatment with hydrogen peroxide or peracetic
acid, ammonia treatment, carbon dioxide treatment and using
various combinations of these physico-chemical processes.
However, these processes are in most cases unattractive as
they fail to meet the basic pretreatment requirements such as
avoiding the degradation or loss of carbohydrates, avoiding
the formation of by-products which are inhibitory to the
subsequent hydrolysis and fermentation processes, and being
cost-effective (Sun and Cheng 2002; Talebnia et al. 2010). For
example, acid treatment causes enzyme inhibition, and condensation and precipitation of solubilised lignin components
which decreases digestibility (Hendriks and Zeeman 2009). In
addition, acid treatment has a risk in the formation of volatile
degradation products and this carbon is lost during conversion
to ethanol (not a major issue in methane production, as volatile
products can be converted to methane). Steam pretreatment
causes production of furfural, hydroxy-methylfurfural and
soluble phenolic compounds which inhibit ethanol or biogas
production. Alkali treatment can cause solubilisation, redistribution and condensation of lignin and modifications in the
crystalline state of the cellulose which counteract the positive
effects of lignin removal and cellulose swelling (Gregg and
Saddler 1996). Oxidative pretreatment causes solubilisation of
lignin which inhibits conversion of the cellulose to ethanol or
methane, and also causes loss of sugars due to non-selective
oxidation. Ammonia treatment can only be carried out at
ambient temperatures for several days while carbon dioxide
treatment is carried out at high temperatures of up to 200 C.
In general, these physico-chemical processes are energy intensive, uneconomical and environmentally unfriendly.
In light of this, enzymatic approaches, using lignin
oxidising or delignifying enzymes are being considered for
the pretreatment of lignocellulosic materials either alone or in
combination with other pretreatment methods. Laccase is one
enzyme which is being intensively investigated for the pretreatment of lignocellulosic materials in bioethanol or biogas
production since it is one of the most common ligninolytic
enzymes. Table 2 summarises some of the research output in
the application of laccases in biogas and bioethanol production. As shown in Table 2, a number of substrates have been

6533

used as feedstock for second generation biofuels production.


Composition of lignocellulosic materials of the starting materials varies depending on the plant species, the age and growth
conditions of the plant material and how the material has been
processed, e.g. lignin content is higher in soft wood than in
hard wood, wheat straw and switchgrass. Access to the sugarrich cellulose and hemicellulose components would therefore
require varying degrees of treatment. In nature, synergism
between different sets of enzymes (oxidative and hydrolytic)
is required for the effective breakdown of lignocellulose.
There are, however, certain factors that limit the enzymatic
hydrolysis of lignocellulose, for example, type of raw material, pretreatment of the raw material (e.g. drying could collapse
cell walls and pores thereby limiting the binding ability of the
enzymes), end-product inhibition, depletion of accessible degradable parts, enzyme inactivation and unproductive binding
or entrapment of cellulases. Laccase increase fermentability of
lignocellulosic materials mainly through lignin degradation
(Tabka et al. 2006). Delignification by laccases is often carried
out in combination with mediators due to the low redox
potential of laccases and the complexity and size of lignocellulose materials. Laccase alone can oxidise only the easily
oxidisable phenolic units at the surface of the substrate which
often constitute less than 10 % of the polymer. LMS generate
radicals which are able to extensively cleave covalent bonds in
lignin as they can oxidise both phenolic and non-phenolic
lignin components. Recent research efforts have been dedicated to understanding the mechanism of oxidative cleavage of
non-phenolic lignin and LCCs using LMS. Building on earlier
findings (Fabbrini et al. 2002; Kawai et al. 1988a, 2002;
Camarero et al. 1997), the mechanism of enzymatic cleavage
of LCCs using Pycnoporus cinnabarinus laccase-HBT system
was elucidated (Du et al. 2013b) (Fig. 2). The LMS is able to
oxidise the C carbon to form oxidised G and S structures
(structure I in Fig. 2). This can be followed by a nonenzymatic reaction in which O2 attacks the carbon-centered
radical intermediates, forming unstable structures IIa which
undergo a number of reactions such as C oxidation (to
produce structure III), CC cleavage, and aromatic ring
cleavage. An alternative route involves structure IIa
abstracting a proton to form IIb before undergoing CC
cleavage to form structures IV and V. Recently, the mechanism of LMS-catalysed delignification using the natural mediator methyl syringate was elucidated through the use of
lignin markers which showed shortened side chains and increased syringyl-to-guaiacyl ratio which suggests preferential
removal of guaiacyl units over syringyl units (Rico et al.
2014). However, the most noticeable modification observed
was the formation of C-oxidised syringyl lignin units during
the enzymatic treatment which indicates a C-oxidation
mechanism of degradation. Mechanisms of oxidation of lignin
using LMS-based on other mediators have been reviewed
(Morozova et al. 2007b). However, some of the mechanisms

Lignin peroxidase; manganese


peroxidase; -glucosidase
(whole cells used)
Whole cells used (other enzymes
such as lignin peroxidase,
xylanase, cellulose,
-glucosidase present)
Steam explosion; no mediator

Alone or with HBT

P. cinnabarinus

P. cinnabarinus

P. cinnabarinus

Modified strains of
Cerrena unicolor

Trametes versicolor
immobilised

Pycnoporus sp.
SYBC-L3

Streptomyces
griseorubens ssr38

P. cinnabarinus and
Trametes villosa

T. villosa

Trametes hirsuta

Ganoderma lucidum
Yarrowia lipolytica

Wheat straw slurries

Wheat straw

Wheat straw

Cotton gin trash

Wheat straw

Switchgrass

Paddy straw

Wheat straw

Eucalypt (Eucalyptus
globulus) and
elephant grass
(Pennisetum
purpureum)

Perennial weed
Parthenium sp.

Sugar cane bagasse


Rice straw
(Oryza sativa L.)

Steam explosion
Alone or with mediators; mild
acid/steam explosion

Whole cells use (other lignolytic


enzymes present)

With mediator 3,5-dimethoxy-4hydroxybenzonitrile alone or


combined with ultrasonication,
hot water treatment
Ethanol organosolv process

Steam explosion

Steam explosion; no mediator

Steam explosion; no mediator

Laccase alone or with methyl


syringate as mediator

Myceliophthora
thermophila

Eucalyptus

Combination treatment/mediator

Source of laccase

Substrate

Table 2 Applications of laccases in the production of bioethanol and biogas

Delignification
Removal of phenolic compounds

Delignification

Delignification

Polymerisation and removal


of phenolics

Delignification

Adsorption; polymerisation;
oligomerisation and removal
of phenolics
Delignification; modification
of biomass (increased porosity)

Delignification and modification


of cellulose structure

Polymerisation and removal


of phenolics
Removal of phenolic
compounds

Polymerisation and reduction


of phenolic content

Delignification

Mode of action

Reduction of phenolic content by up to 94 %;


shortening of lag phase of fermentation
yeast Kluyveromyces marxianus;
enhanced ethanol concentration
(increase of up to ~13X)
Up to 48 % lignin reduction with HBT;
5 % reduction without mediator; increase
in ethanol production (over 4 g L1 in
17 h in eucalypt and 2 g L1 in 17 h
in elephant grass); no significant
increase without mediator
Improved rate of sugar release
(up to 18 times); 5 fold higher
saccharification efficiency; 52.65 higher
availability of holocellulosea
~75 % increase in glucose equivalents
Up to 75 % removal of phenolic
compounds (with HBT as mediator);
Increased saccharification yield
(90.4 % with mediator; 68 %
laccase only; 52.5 %)

30 % reduction in lignin content after


36 days; 50 % improvement in
enzymatic hydrolysisa
25 % of depolymerised recovered; high
saccharification efficiency of 97.8 %a

Up to 50 % lignin removal accompanied by


approximately 40 % increase in glucose
and xylose yields after enzymatic hydrolysis.
Laccase alone removed up to 20 % of lignin
Prolonged hydrolysis time from 48 to 144 h;
reduced lag phase and increased viability of
Saccharomyces cerevisiae F12; boosted f
ermentation (yield, 22 g/L ethanol) without
adding extra nitrogen
Increased substrate loading up to 12 % (w/v);
increased ethanol concentration (16.7 g/L)
Up to 73 % removal of phenolic compounds;
increased fermentation yeast cell viability
and ethanol yield
10 % decrease in lignin and up to 23 %
cellulose conversion and 31.6 % ethanol
yield after combination treatment of
ultrasonication, hot water and LMS
Improvement of ethanol productivity from
0.13 to 0.17 g L1 h1

Positive effects observed

Sitarz et al. (2013)


Lee et al. (2012)

Rana et al. (2013)

Gutirrez et al. (2012)

Moreno et al. (2012)

Saritha et al. (2013)

Liu et al. (2013)

Ludwig et al. (2013)

Plcido et al. (2013)

Moreno et al. (2013b)

Moreno et al. (2013a)

Moreno et al. (2013c)

Rico et al. (2014)

Reference

6534
Appl Microbiol Biotechnol (2014) 98:65256542

Sclerotium sp.

Trametes sp.

T. versicolor

Trametes sp. AH28-2


heterologously expressed
in Trichoderma reesei
C. unicolor and T. hirsuta

Wheat straw

Newspaper

Corn stover

Corncob residue

Commercial Novozyme
51003
Phellinus robustus, Pleurotus
eryngii, Irpex lacteus and
Poria subvermispora
Pleurotus ostreatus

Manure

None

Ethanol and hot water extraction;


violuric acid as mediator

Trametes hirsuta

Cyathus stercoreus

Trametes versicolor

Pycnoporus sanguineus
H275

Steam-pretreated
softwood

Sugarcane bagasse

Willow pretreated
with steam and
SO2
Wheat straw and
corn stover

N-hydroxy-N-phenylacetamide
(NHA) and N-acetoxy-Nphenylacetamide
Acid hydrolysis

Steam explosion

Coriolopsis rigida and


T. villosa

Wheat straw

Wheat bran as mediator

Delignification

Polymerisation and removal


of phenolic compounds
Detoxification through removal
of phenolic compounds

Delignification (partial)

Polymerisation of phenolic
compounds

Delignification

Delignification

Ultrasonic and H2O2

Ceriporiopsis subvermispora
ATCC 90467, CZ-3 and
CBS 347.63

Delignification

Delignification

Delignification

Lignin modification

Delignification (probable;
not investigated)

Delignification

Loosening of lignin-carbohydrate
complex
Delignification/lignin
modification; de-inking

Removal of phenolic compounds

Mode of action

Mild alkali treatment

None

Substrate crushed to powder

Impregnated with SO2 gas and


steam pretreated (spruce);
steam treated (giant reed)

1-HBT; cellulase and


hemicellulase mixture
Cellulase

None

Japanese cedar
wood

Rice hull

Wheat straw

Pleurotus sp.

Ricinus communis

Spruce (Picea abies)


and giant reed
(Arundo donax)

Steam explosion

Coltricia perennis
Commercial
Novozym 51003

Rice straw
(O. sativa L.)

Steam explosion

Combination treatment/mediator

Source of laccase

Substrate

Table 2 (continued)

97.8 % klason lignin removed from 1 g


wheat straw and 19.98 % increase in
Somogyi carbohydrate (fermentable sugar)

77.5 % reduction in total phenolics;


1.7-fold increase in ethanol yield
Rapid consumption of glucose and
increased ethanol productivity

44 % lignin degraded, net yield of total


sugars increased (~1236.7 %); glucose
content increase (~1038.9 %)
7476 % decrease of -O-4 aryl ether
linkages; methane yield increased from
6 to 9 % in absence of mediator to
25 % of theoretical yield with mediator
Reduction of the toxic effect of phenolic
compounds, improved fermentability
of hydrolysates
13 % (using laccase alone) or 21 %
(using LMS) increase in sugar yield

Improved the hydrolysis yield of spruce


by 12 %; reduced hydrolysis yield
of giant reed by 17 %; reduced binding
of enzymes on modified spruce lignin
(opposite effect on giant reed)
Maximum delignification 85.69 %;
reducing sugar yield increased 2.68-fold
Increased the methane yield by
(19.80.4 m3 CH4 (t WW)1)
Ethanol yield increased by 62 %

Increase in ethanol concentration (0.28 %


(v/v) in laccase treated vs 0 % in
absence of laccase)
Downstream cellulose hydrolysis
was improved 7 %
Increase in reducing sugar yields by
up to 71.6 %

Removal of phenolic compound (76 %


with C. perennis; 52 % with
commercial laccase); Increased
saccharification yield by 48 and 28 %,
respectively
16.8 % increase in cellulose conversion

Positive effects observed

Lu et al. (2010)

Jnsson et al. (1998)

Chandel et al. (2007)

Palonen and Viikari


(2004)

Jurado et al. (2009)

Amirta et al. (2006)

Yu et al. (2009)

Salvacha et al. (2011)

Mukhopadhyay
et al. (2011)
Bruni et al. (2010)

Moilanen et al. (2011)

Zhang et al. (2012)

Chen et al. (2012)

Nakanishi et al. (2012)

Qiu and Chen (2012)

Kalyani et al. (2012)

Reference

Appl Microbiol Biotechnol (2014) 98:65256542


6535

Not stated
Pleurotus sp.

Myceliopthera thermophilae
(NS51003 Novozymes A/S)

Basidiomycete Euc-1

Saccharomyces cerevisiae
transformants
(with laccase gene)
T. versicolor, Bjerkandera
adusta, Ganoderma
applanatum and
Phlebia rufa

Cotton stalk
Ricinus communis

Wheat straw

Wheat straw

Spruce hydrolysate
supplemented with
coniferyl aldehyde
Wheat straw

Rhus vernificera

Rice straw

Delignification

None

Laccase activity was not determined

Result includes effect of other enzymes since whole cells were used

Polymerisation and
detoxification

Delignification

Lignin oxidation

Not specified (possible


combination of
delignification and
detoxification)
Polymerisation of lignin residues
Delignification

None

None

None

Alkali treatment
None

None

None

Lentinus edodesb

Mushroom logs

Delignification

Detoxification

None

Ureibacillus
thermosphaericus

Waste house wood

Mode of action

Combination treatment/mediator

Source of laccase

Substrate

Table 2 (continued)

43 % decrease in lignin content

Glucose yield increased by 5.45 % after 72 h


85.69 % delignification, 2.68-fold increase
in yield of reducing sugars
Increase in O2 consumption ascribed to lignin
oxidation and therefore chemical alteration
of lignin
Percentage of Klason lignin present decreased
from 20 to 13 %; 4-fold increase in substrate
saccharification; ratio of cellulose/lignin
increased from 2.7 to 5.9
Removal of coniferyl aldehyde; ethanol produced
only by transformants

Reduced sugar loss when compared


with overliming, removal of toxic
compounds
Decrease in lignin content from 21.07 %
to 18.78 %; higher total sugar and
ethanol yield
Improved monosaccharide production
by 29.13 %

Positive effects observed

Dinis et al. (2009)

Larsson et al. (2001)

Dias et al. (2010)

Haykir (2009)
Mukhopadhyay
et al. (2011)
Kaparaju and
Felby (2010)

Chang et al. (2011)

Lee et al. (2008)

Okuda et al. (2008)b

Reference

6536
Appl Microbiol Biotechnol (2014) 98:65256542

Appl Microbiol Biotechnol (2014) 98:65256542

were not well known at the time and were therefore presented
as suggestions or possible mechanisms. Besides
delignification, laccase or LMS can improve biofuels production through changes in the structure of the lignocellulose
microfiber which modify properties such as porosity, surface
area, and hydrophobicity resulting in the reduction of unproductive binding of cellulases (Moilanen et al. 2011). For
example, it has been observed that laccase-treatment of steam
pretreated spruce resulted in an increase in acidic groups
which would indicate a decrease in lignin hydrophobicity
and an increase in negative surface charge (Palonen and
Viikari 2004). This could result in electrostatic repulsion
toward cellulases which subsequently decreases the unproductive binding of cellulases. It is worth mentioning, however,
that lignin degradation using LMS can also be accompanied
by the release of inhibitory mono-aromatic phenolic compounds in the medium that decreases activity of cellulases
and other hydrolases (Gamble et al. 2000). Similarly, thermochemical pretreatment steps such as acid and steam explosion
typically lead to the production of furans, phenols and weak
acids which can inhibit the activity of cellulases and sugar
fermenting organisms. Treating the steam exploded material is
costly, requires special separation equipment (filtration) and
generates large amounts of wastewater. Subsequently, research efforts are now also being directed towards detoxification of hydrolysates through laccase-catalysed removal of
phenolic compounds and other inhibitory compounds such
as furans and weak acids (Moreno et al. 2013a, c, 2012;
Kolb et al. 2012; Parawira and Tekere 2011; Jnsson et al.
1998). The process involves laccase-catalysed oxidation of
the compounds to unstable radicals which couple to form
oligomeric and polymeric compounds which are less toxic to
enzymes required for subsequent processes than the corresponding monomeric units. The reaction mechanism is well
characterised. For example, we have shown recently that
laccase-catalysed oxidation of the phenolic molecule ferulic
acid led to coupling of the radicals through -5 and -
coupling to form dimers (Adelakun et al. 2012) and the
reaction also produces oligomers and polymers.
Polymerisation of phenolic compounds is so typical of
laccases that we have observed this phenomenon in virtually
all the coupling reactions we carried out with simple phenolic
compounds and oligomeric lignin models (Kudanga et al.
2009, 2010a, b, c; Widsten et al. 2010). Although physicochemical methods are also efficient at removing phenolics,
they also remove compounds such as acetic acid (Chandel
et al. 2007) which are important in biogas production. Some
oxidised mediators such as oxidised N-hydroxy-Nphenylacetamide can also inhibit the cellulases required for
subsequent hydrolysis (Palonen and Viikari 2004). The effect
of such inhibitors can be reduced by using excess substrate
(probably by offering sites for the radical attacks) or by using
acetylated mediators which are slowly released by a lipase in

6537

situ (Palonen and Viikari 2004). Recently a system that uses


immobilised laccase for detoxifying lignocelluloses hydrolysates was developed (Ludwig et al. 2013). By immobilising
laccase onto a hydrophobic carrier, compounds that could not
be removed by free laccase (such as furfural) could be fully
removed while incorporation of an anion exchanger as a subsequent step led to the reduction of lignocellulose degradation
products such as HMF, formic acid, levulinic acid and acetic
acid by 90, 62, 39 and 20 %, respectively before fermentation
for the production of ethanol (Ludwig et al. 2013).
Combining laccase treatment with other treatment methods
usually results in higher yields than the individual methods
alone (Bruni et al. 2010). Laccase treatment can be accomplished using free purified laccase or whole cells which produce the enzyme. However, where whole cells are used it is
usually not easy to find a direct correlation among enzyme
production, lignin degradation, and sugar yield (Salvacha
et al. 2011; Yamagishi et al. 2011) because oxidative lignin
degradation is a nonspecific process where other extracellular
ligninolytic enzymes and low molecular-weight extracellular
oxidant compounds (such as Mn3+ and oxygen free radicals)
generated during the process, participate (Guilln et al. 2000;
Hammel et al. 2002). It is also worth noting the work of Zhang
et al. (2012) in which Trametes sp. AH28-2 laccase gene lacA
fused to cellobiohydrolase I signal peptide coding sequence
was heterologously expressed in Trichoderma reesei resulting
in a huge improvement in yield of reducing sugars. It was
proposed that the improvement in cellulolytic activity could
be due to the modification of the lignin surface which decreased the unproductive binding of cellulases to lignin and/or
partial breakdown of lignin structure by laccase which increased access of cellullase to cellulose and/or possible synergistic effects in which depolymerisation of cellulose and of
lignin could accelerate each other (Zhang et al. 2012;
Westermark and Eriksson 1974).
Generally, there have been few research outputs on laccasemediated delignification for the direct purpose of bioethanol
or biogas production. However, most delignification processes catalysed by laccase and LMS either for research purposes
or for the purposes of biopulping as recently reviewed (Caas
and Camarero 2010; Martnez et al. 2009) and reported
(Eugenio et al. 2010a, b; Fillat et al. 2010; Crestini et al.
2011; Martn-Sampedro et al. 2011a, b; Moldes et al. 2008,
2010; Du et al. 2013a, b), could be adapted for biogas or
bioethanol production. In related uses, laccases have been
used for de-inking newspapers which were subsequently used
for bioethanol production (Kuhad et al. 2010; Nakanishi et al.
2012). Although there was no ethanol production using newspapers which were not treated with laccase, Nakanishi et al.
(2012) did not observe significant evidence of delignification
in laccase-treated newspapers. They proposed that laccasemediated de-inking and change in the structure of biomass
exposed the cellulose in newspaper to cellulolytic enzymes.

6538

Conclusion and directions for future research


Across the globe, countries have implemented, defined or are
working towards the development of a bio-economy strategy.
The need for feasible and sustainable bio-based processes
underpins a large part of the bio-economy approach. The
application of laccases in biofuel production is currently based
on our understanding of the role of laccase and laccasemediator systems in lignocellulose degradation or modification. Designing bio-based processes around what happens in
nature, e.g. understanding the synergy between all the enzymes involved in the breakdown of lignocellulosics, and
the application of this knowledge in the design of new biobased processes will greatly contribute towards more efficient
processes. For example it may be better to target the LCC with
a cocktail of enzymes (e.g. laccases and hemicellulases) that
degrade the lignin and hemicellulose components and possibly the benzyl ether, -ester and phenyl glycoside linkages
between lignin and carbohydrates. The success recorded by
Chen et al. (2012) emphasises the need for more research in
this area. A different approach would be the heterologous
expression of laccase in for example a host that has
hemicellulase capacity in a similar way to experiments carried
out by Zhang et al. (2012) in which they successfully
expressed Trametes sp. AH28-2 laccase in the cellulolytic
fungus T. reesei.
There is also a continued need for the discovery of natural
mediators that can be applied in a LMS for the cleavage of
lignin linkages, as well as a need for the discovery of novel
enzymes with new and interesting activities (e.g. the activities
observed for the yellow and white laccases). Even though
there may be many advantages to the LMS (e.g. they can be
used for woody and non-woody lignocellulose materials
where they can have direct action on lignin), there are certain
disadvantages, e.g. the generation of by-products that can lead
to the inactivation of laccases and other enzymes such as
xylanases when using HBT as mediator. However, another
synthetic mediator NHA is much less toxic as mediator
(Woolridge 2014) while the bulk of natural mediators produce
much better results in mixed enzyme systems. However, in
general, mediators add cost when compared with using
laccase alone and a costbenefit analysis should be considered
before choosing a laccase only or a LMS. The application of
metagenomic techniques in mining enzymes from microbial
communities (Xing et al. 2012) which has become more
feasible due to advances in sequencer technology and
metagenomic sequencing should also be considered in the
search for novel laccases. Other related approaches include
site-directed mutagenesis to improve activity of laccases and
application of new bioinformatics tools such as the Laccase
Engineering Database (LccED) (Sirim et al. 2011), that, for
example, have allowed researchers to design better laccases.
Such technologies can be adapted in the search for better

Appl Microbiol Biotechnol (2014) 98:65256542

pretreatment enzyme systems that can be applied in biobased processes for the production of biofuels.
Acknowledgments Financial support from the National Research
Foundation (NRF)South Africa and the Cape Peninsula University of
Technology Research Funding (URF) is gratefully acknowledged. Any
opinion, findings and conclusions or recommendations expressed in this
material are those of the author(s) and therefore the NRF does not accept
any liability in regard thereto.

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