Laccases have been shown in Bacillus sp., Streptomyces

Laccases (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) are a
ubiquitous in nature enzyme having the capability to oxidize a wide range
of recalcitrant phenolic and nonphenolic compounds by converting oxygen
molecule to reactive radicals and water on concomitant four-electron reduction
(Hakulinen and Rouvinen 2015). These free electrons catalyze the oxidation of various
aromatic and non-aromatic compounds as well as phenolic ring-containing amines
substituted with various functional groups such as methoxy, amino, diamino and
hydroxyindols and few other metal compounds Mo(CN)84-, Fe(CN)64- and
Os(CN)64- (Chandra and Chowdhary 2015; Rezaei et al. 2017). Laccase was first
reported from Japanese laquer tree Rhus vernicifera (Yoshida., 1883). Laccases
are the members of multi-copper oxidases and contain histidine-rich copper
binding domains participate in (1) cross-linking of monomers, (2) degradation
of polymers, and (3) ring cleavage of aromatic compounds (Kawai et al. 1988). Laccases
are found glycoproteins, ranging from various fungi, bacteria, higher plants
and some insects. It is mainly produced from fungi, especially white rot, and
has been widely exploited for the application in industrial and biotechnology,
for instance, in the detoxification of chemicals in wastewater, degradation of
lignin in pulp and paper, degradation of inorganic compounds to soil organic
matter and the decolorization of dyes in textile due to their high redox
potential (Niladevi and Prema, 2008). However, the majority of the industrial processes are
carried out in extreme conditions, i.e., higher temperature and pH, and high
salt concentration, and fungal laccase generally fails to work in these extreme
environments (Du et al. 2015; Wang and Zhao 2016). In recent years, application
of bacterial laccases are growing rapidly due to their many remarkable features
in comparison to fungal laccase from the industrial point of view, such as work
in a broad range of temperature and pH with enormous stability against various
inhibitory agents (Guan et al. 2015).

        Bacterial
laccase was first reported in Azospirillum lipoferum (Givaudan et al.,
1993); it plays a role in cell pigmentation, oxidation of organic compounds
(Faure et al., 1994, 1995) and/or electron transport (Alexandre et al., 1999).
Bacterial protein sequence studies have indicated that the laccases are
represented by high G+C Gram positive bacteria and ?-, ?- and ?–proteobacteria
(Alexandre and Zhulin, 2000); as have been shown in Bacillus sp., Streptomyces
sp., and a ? proteobacterium (Bains et al., 2003; Sharma et al., 2007).
Most bacterial laccases are intracellular enzymes or periplasmic proteins as
shown in A. lipoferum and B. subtilis. Laccase like activity has
also been found in bacteria, for example, the copper efflux protein CueO from Escherichia
coli and the copper resistance protein CopA from Pseudomonas syringae
and Xanthomonas campestris. These were due to the dependence of the
2,6-dimethoxyphenol oxidation on Cu2+ addition (Solano et al.,
2001). Besides,
bacterial laccases have some additional advantages because of their practically
feasible, cost-effective
used in various industrial applications, which include broad substrate
specificity, enzyme production in a short time, and easiness to clone and
express in the host with suitable manipulation (Fernandes et al. 2014; Prins et
al. 2015). Multiple
sequence alignments of more than 100 laccases resulted in identification of
four ungapped sequence regions, L1–L4, as the overall signature of laccases,
distinguishing them within the broader class of multi-copper oxidases (Kumar et
al., 2003). The 12 amino acid residues in the enzymes serving as the copper
ligands are housed within these conserved active site. The amino acid ligands
of the trinuclear cluster are the eight histines, which occur in a highly
conserved pattern of four helix-X-helix motifs. In one of this motifs, X is the
cysteine bound to the T1 copper while each of the histines is bound to one of
the two type 3 coppers. Intraprotein homologies between signatures L1 and L3
and between L2 and L4 suggest the occurrence of duplication events. In the
proposed review, comprehensive information of laccase-producing bacteria for
degradation organic pollutants release from pulp and paper industry.

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Laccase structure and mechanisms

Bacterial
laccases often occur as isoenzymes that oligomerize to form multimeric
complexes. Structurally,
laccases contain 15–30% carbohydrate and have molecular masses of 60–90 kDa with
acidic isoelectric points around pH 4.0, which shows high enzymatic stability
(Chandra et al., 2015). More recently the spore coat protein cotA of Bacillus
subtilis has been identified as a laccase (Hullo et
al., 2001; Martins et al., 2002) and the crystal structure has been
presented (Enguita et al., 2003). For the catalytic activity
a minimum of four copper atoms and three types of copper active protein unit is
needed:

Type 1: copper
has a trigonal coordination, with two histines and a cysteine as conserved
equatorial ligands and one position usually variable. This axial ligand is
methionine in the bacterial (CotA) and leucine or phenylalanine in fungal
laccases. It has been widely argued that this axial position ligand strongly
influences the oxidation potential of the enzyme, possibly providing the
mechanism for regulating its activity. Copper (T1Cu) is
substrate binding site responsible for the oxidation of the substrate and also for
the blue color Cu site, characterized by an intense SCys ? Cu(II) charge
transfer transition and high redox potential of ca. +790 mV, having strong electronic
absorbance caused by the covalent copper–cysteine bond around 610 nm and detectable
electro-paramagnetic resonance (EPR).

Type 2: Copper (T2Cu)
is inducer/ inhibitor and oxygen binding site. It is colorless no absorption in
the visible spectrum and also detectable electro-paramagnetic resonance (EPR). It
is stategically positioned close to the type 3 copper.

Type 3: Copper (T3Cu)
is a binuclear center also binds
inducer/ inhibitor and oxygen diamagnetic spin-coupled copper-copper pairs gives
a weak absorbance near the UV spectrum 330 nm but it is not detectable electro-paramagnetic
resonance (EPR).

Laccase

Type 2 and type 3 copper form a
trinuclear cluster, where reduction of molecular oxygen and release of water
takes place. Type 1 copper coordinate two histidines, one methionine and one
cysteine, Type 2 copper is coordinated by two and type 3 copper atoms by six
histidines. The strong anti-ferromagnetical coupling between the two type 3
copper atoms, is maintained by a hydroxyl bridge. For laccase and
Ceruloplasmin, the substrate is a metal ion which binds tightly to a substrate
binding site. As shown in figure 1, these substrate binding sites are located
near the His ligands of the T1 Cu center. The electron from substrate is first
transferred to the T1 and then over >13Å through a Cys-His pathway to a trinuclear
Cu cluster where O2 is reduced to water (figure 1A) (Solomon et al.,
2010).         The catalytic cycles of laccase have increased
significantly; it plays an important role in the degradation of aromatic organic
compounds which leads to radicals generation. The reaction of the fully reduced enzyme with O2 occurs in two 2
electron steps, with the second being fast, so it is effectively a four
electron process (Solomon et al. 2008; Chauhan et al., 2017).). The abstracted electron moves from the T1 center to the
trinuclear cluster via a cysteine– histidine pathway that is highly conserved
among multicopper oxidases. This so-called super exchange pathway is built by
overlapping redox active molecule orbitals of T1 coordinating cysteine,
backbone atoms and T3 copper coordinating histidine residues (Solomon et al.
2008). The trinuclear center which plays an important role in the catalytic
mechanism is made up of Type 2 and 3 copper. The catalytic process begins after
oxygen molecules attach to the trinuclear cluster and inhibit further entry of
any other molecule (Chauhan et al., 2017). The T2Cu site react with two
molecules of histidine and one molecule of water, whereas T3Cu react with three
histidines and hydroxide molecules. In the final step, the oxygen molecule is
converted to water by laccase in two steps (Chauhan et al., 2017). In the first
step, first electron is reduced by T2Cu and T3Cu, however reduction of the
second step two electron
reduction of peroxide mediator combined with the triangular topology of the
trinuclear centre for T2Cu site and T1Cu linked to T3Cu by covalent Cys–His
bonds (Madhavi and Lele 2009; Chandra and Chowdhary 2015). Importantly, native intermediate is a fully oxidized
all bridged structure and is the catalytically relevant fully oxidized form of
the enzyme (Solomon et al. 2008) (figure 1B).Laccase-mediator systems

However, this limitation has been
overcome through mimicking nature, by using redox mediators in the so-called
laccase-mediator systems. The presence of certain small-molecular weight
compounds, that act as redox mediators, expand the catalytic activity of
laccase towards more recalcitrant compounds such as non-phenolic lignin units
(Barreca et al., 2003). Mediators act as electrons shuttles, providing the
oxidation of complex substrates (such as lignin polymers) that do not enter the
active site due to steric hindrances. Once oxidized by the enzyme and stabilized
in more or less stable radicals, mediators diffuse far away from the enzymatic
pocket and, by mechanisms different from the enzymatic one, enable the oxidation
of target compounds that in principle are not substrates of laccase because of
their high size or high redox potential (Bourbonnais and Paice, 1990;
Bourbonnais et al., 1997b; Kawai et al., 1989). The ideal redox mediator would
be a small-size compound, able to generate stable radicals (in its oxidized
form) that do not inactivate the enzyme, and which reactivity would allow its
recycling without degeneration. In addition, from the point of view of their
industrial and environmental application, laccase mediators should be
environmental- friendly and available at low cost.Natural
mediators and synthetic mediators

Laccases can oxidize a wide range of molecules; more than
a hundred different types of compounds have been identified as substrates for
laccases and there are various natural and synthetic, chemical mediators also
referred to as ”enhancers” used for laccase assays. All of the substrates
cannot be directly oxidized by laccases, either because of their large sizes,
which inhibit their penetration into the enzyme active site or because of their
particularly high redox potentials. To overcome this hindrance, suitable
chemical mediators are used that are oxidized by the laccase, and their
oxidized forms are then able to interact with the high redox potential
substrate targets65. The first synthetic mediator to be used in the
laccase-mediator system (LMS) for pulp delignification was ABTS, which was
introduced in 1990.35 More than 100 other compounds have been tested since then
for their ability to oxidize lignin or lignin containing models compounds through
the selective oxidation of theirbenzylic/ hydroxyl groups. The most effective
mediators for lignin degradation proved to be the N-heterocyclic bearing NAOH
groups including violuric acid (VIO), N-hydroxyl-N-phenylacetamide (NHA) and
HBT.5Several issues still remain to be addressed before the industrial
implementation of synthetic mediators. Whereas the cost of synthetic mediator
such as HBT could be feasible as compared to the present consumption of
chemical additives in the pulp industry,36 an effort towards finding a more cost
effective and more environment friendly alternative would help for economic application.
Moreover, a concern exists about the possible toxicity of some of the most
powerful laccase mediators, such as the AN(OH)A compounds or their reaction
products.10 Natural mediators prove to be ecofriendly alternative to synthetic
mediators (Figure 5). It is notable to point out the high availability of
natural mediators such as syringaldehyde (SA) and acetosyringone (AS), which
are readily available in the black liquors made from the kraft pulping of
eucalyptus pulp.37 Hence, they are cost effective, less toxic, and contribute
to the reusability of enzyme.Organic pollutants

Paper-mill effluents are
characterized by the presence of bad smell, color and suspended solids, high
concentration of nutrients and Levels of different families of organic
compounds containing effluents that contribute to the toxicity of paper-mill
waters and effluents, their levels, toxicological characterization and the
methodologies used for their analysis. To decrease the problems associated
with microbial, bacterial, fungal and algal growth, it is common to dose
biocides for wood preservation and during paper-making (Lacorte et al., 2003). Nevertheless
wood extractives include lipophilic (fatty and resin acids, sterols, steryl
esters and triglycerides) hydrophilic (cellulose, lignans, lignin-like
substances and hemicelluloses) compounds that dissolve in white waters during
paper production. Surfactants, such as linear alkylbenzene sulfonates and
alkylphenol ethoxylates, are present in white waters because of their use as
cleaning agents or as additives in antifoamers, deinkers, dispersants, etc. In
paper-making, chlorine and chlorinated compounds are also sources of dioxins
and furans, which have been detected in sediments in the vicinity of a pulp and
paper-mill 38 and in effluents, along with polychlorinated
dibenzothiophenes39. Different fauna and flora living close to paper-mill
discharges have manifested some effects, such as skin and physiological
diseases in fish, decreases in the number of juveniles, changes in communities
and population structure, changes in growth rates, and delayed sexual
maturation and reproduction, among others 1,44,45. In addition, oxygen depletion is common in
such effluents, that cause eutrophication of receiving waters, and high
toxicity overall aquatic ecosystem.Potential
industry and biotechnological applications of laccase enzyme

Laccases
are able to depolymerize lignin and lignin containing compounds, delignify wood
pulps, kraft pulp fibers and chlorine-free in the biopolpation process
(Bourbonnais et al. 1997; Lund and Ragauskas, 2001; Chandra and Ragauskas,
2002; Camarero et al. 2004; Rodríguez and Toca, 2006; Vikineswary et al. 2006).
Laccases-mediator system finds potential application in enzymatic modification of
bleaching of kraft pulp, textile, dyes industries and the efficiency of which
has been proven in mill-scale trials (Strebotnik and Hammel, 2000). Therefore,
the development of processes based on laccases seems an atractive solution due
their potential in degrading various type recalcitrant organic and inorganic, xenobiotic
compounds and polycyclic aromatic hydrocarbons. Most currently existing
processes to treat waste and wastewater are ineffective and cost effective. This
ability could be used in the future to attach chemically versatile compounds in
the fiber surfaces and let recycled pulp for new use (Rodriguez and Toca, 2006;
Mocchiutti et al. 2005; Saparrat et al. 2008; Widsten and Kandelbauer, 2008).

          Laccases can be useful to certain
processes that enhance or modify the color appearance of food or beverage for
the removal of undesirable phenolics, responsible for the browning, haze
formation and turbidity in clear fruit juice, beer, wine (Rodriguez and Toca,
2006) and also employed to ascorbic acid resolve, sugar beet pectin gelation,
baking and in the treatment of olive mill wastewater (Ghindilis, 2000; Minussi
et al. 2002; Rodriguez and Toca, 2006; Selinheimo et al. 2006; Minussi et al.
2007). Recently, laccases have been employed for several applications a new
biocatalyst in organic synthesis as the oxidation of functional groups, the
coupling of steroids, aromatic amines and phenols, medical agents (antibiotics
and sedatives, anti-inflammatory, anesthetics), the making of carbon-nitrogen
bonds and in synthesis of complex natural products in cosmetics industries and
are considered among some of the most promising enzymes for future industrial applications
(Mikolasch and Schauer, 2009; Baminger et al. 2001; Fabbrini et al. 2001;
D’Acunzo et al. 2002; Mikolasch et al. 2002; Baiocco et al. 2003; Barilli et
al. 2004; Nicotra et al. 2004; Xu, 2005; Rodriguez and Toca, 2006; Ponzoni et
al. 2007). A new enzymatic method based on laccase was developed in medicinal
area to distinguish simultaneously codeine and morphine in drug samples
injected into a flow detection system (Bauer et al. 1999). Laccases also can be
applied as biosensors or bioreporters, biolinkers and immunoassay (Bauer et al.
1999; Xu, 1999; Duran and Esposito, 2000; Ghindilis, 2000; D’Souza, 2001;
Kuznetsov et al. 2001; Kunamneni et al. 2008; Szamocki, et al. 2009). Laccases
still could be immobilized on the cathode of biofuel cells that could provide
for small transmitter systems (Ghindilis, 2000) and laccase-based miniature
biological fuel cell is of particular interest for many medical applications
calling for a power source implanted in a human body (Rodriguez and Toca, 2006;
Heller, 2004). Immobilized commercial laccase have been reported to degrade meta-,
ortho- and para-substituted methoxyphenols, chlorophenols and
cresols, and also some refractory compounds because when the enzyme is
immobilized, it becomes more durable, vigorous and resistant towards
alterations in the environment, allowing for easy recovery and multiple reuses
of these enzymes (Chandra et al., 2015).Conclusion and future recommendations

The first effect of greatest paper
industry wastewater organic discharge is depletion of oxygen in the receiving
waters, caused by oxygen-consuming microbial degradation of readily biodegradable
organic matter being discharged. This has led to a rapid interest in developing
new methods for in-mill treatment of whitewater to remove organic matter. A
review of treatment of pulp and paper-mill effluents indicates that there are
processes to minimize the discharge of wastewater to the environment. Biological
treatment removes the bulk of organic matter, but the fraction remaining, often
dominated by lignin, makes further biotreatment difficult. This gives a
significant increase in color of the treated water and unacceptable coloring of
the product in paper of qualities for which color is important. Laccase enzymes
play important roles in the biodegradation and biotransformation of the complex
recalcitrant compounds, in economical treatment of wastewater containing
phenolic compounds, PHAs, chemical pesticides, synthetic dyes and various
emerging pollutants. This enzyme is highly versatile in nature and they find
application in a wide variety of industries such as wastewater detoxification, textile
dye transformation, in food technology, personal and medical care applications,
organic synthesis, biolinkers, biosensors and analytical applications. The
biotechnological significance of these enzymes has led to a drastic increase in
the demand for these enzymes in the recent day. 

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