Available online 14 August 2023
1876-1070/© 2023 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
research progress
Jothika Jeyabalan
1, Ajithkumar Veluchamy
1, Vishnu Priyan V, Ajit Kumar, Ragavan Chandrasekar, Selvaraju Narayanasamy
*Biochemical and Environmental Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India
A R T I C L E I N F O Keywords:
Immobilization Redox mediator Synergism Enzyme Pollutant Oxidation
A B S T R A C T
Background: Increased industrialization and urbanized civilization lead to the evolution of several persisting hazardous pollutants. In order to degrade these tenacious pollutants in the global environment, bioremediation using microorganisms and enzymes have been exploited. Laccase, a multicopper oxidase enzyme, has been harnessed extensively for the degradation of a broad spectrum of pollutants including endocrine disruptors, microplastics, organic dyes and pharmaceuticals because of its monoelectronic oxidation of substrates and low substrate specificity.
Methods: Though laccase is exploited in various fields like paper & pulp and textile industries, predominantly laccase is utilized in decolourization of innumerable dyes. In this review, properties of laccase based on the source, mechanism of laccase-mediated dye decolourization, factors and operational parameters which affects the dye decolourization efficiency, different supports utilized for the immobilization of laccase, synergistic effect of laccase and its future prospects have been highlighted.
Significant findings: Among the different laccase studied, fungal and bacterial laccase has been harnessed pre- dominantly in decolourization applications while the application of insect laccase is still lacking. Also, recy- clability and elevated operational stability of the laccase can be achieved by trapping or immobilizing the enzyme and synergistic approach has shown promising effect on enhancing the decolourization efficiency.
1. Introduction
Environmental pollution has become a serious problem due to rapid urbanization and industrialization, leading to adverse health effects by incorporating foreign contaminants into the natural ecosystem [1]. It has been reported that 70% of freshwater resources in India are contaminated by anthropogenic activities, leading to water scarcity that augments the necessity of efficient treatment methods [2]. The major pollutants which contaminate the environment are pesticides, pharma- ceutical compounds, microplastics, endocrine disruptors, organic dyes, heavy metals etc., that cause soil, water and air pollution [3]. Various chemical and physical methods have been exploited for pollutant removal like adsorption, advanced oxidation, ion exchange, membrane filtration, precipitation, constructed wetlands, coagulation, and elec- trochemical methods [4]. But these conventional methods possess some pitfalls like exorbitant cost, sludge production, non-specificity and
probability of secondary contamination [5]. Hence, the eco-friendly and efficient bioremediation approach has gained interest which can be defined as any process of utilizing bacteria, fungi, green plants, or their enzymes to restore the polluted environment to its natural state by intracellular accumulation, bio adsorption or enzymatic transformation [6]. The factors which affect the rate of biological degradation are environmental conditions (temperature, pH, aeration), pollutants properties (concentration, chemical structure, hydrophobicity) and soil features (thickness, pollutant ageing etc.,) [7]. Enzymes are highly specific catalysts that play a major role in microbial bioremediation.
Different enzymes such as hydrolases, peroxidases, halogenases, oxi- doreductases, mono or dioxygenases and phosphotriesterase from various species of bacteria, fungi, algae and plants have been utilized in the bioremediation of organic and inorganic substrates [8]. Among all other enzymes, laccase which belongs to the multicopper oxidase family, which are monomeric, dimeric or trimeric glycoproteins contains four
* Corresponding author.
E-mail address: [email protected] (S. Narayanasamy).
1 Equal authorship.
https://doi.org/10.1016/j.jtice.2023.105081
Received 16 June 2023; Received in revised form 31 July 2023; Accepted 9 August 2023
copper atoms (one type I paramagnetic blue copper, one paramagnetic type II non-blue copper, two diamagnetic type III spin coupled Cu-Cu pair) in three redox sites, gained more interest in environmental appli- cations because of its broad substrate range and high redox potential [9]. Laccase is a secondary metabolite produced under nitrogen limited growth limiting conditions [10]. This benzenediol oxidoreductase EC (1.10.3.2) can oxidize various phenolic and non-phenolic compounds including chlorinated phenols, dioxins, herbicides, pesticides, cresols, pharmaceuticals, synthetic dyes, personal care products etc. [11]. Fig. 1 depicts the various application of laccase in degradation of different pollutants. It is also called as a green catalyst because of its ability to oxidize different substrates that require oxygen as a reactant and generate water molecules as a by-product which is completely non-toxic [12]. Laccases are used widely in industrial processes such as bio- leaching in the pulp and paper industry, textile dye bleaching,
biotransformation, clarification of fruit juice and wine, and detection of phenolic pollutants [10] and also in environmental applications such as dye decolourization [13], microplastic degradation [14], antibiotic removal [15], herbicide degradation [16] and emerging pollutants transformation. Among the various pollutants, dyes are the one of the major pollutants found in the environment. Dyes are highly soluble organic compounds with chromophore and auxochrome groups. It has reported that, 10–15% of wastewater is contributed by the textile in- dustry globally [17] and dyes are the primary pollutants of textile in- dustry wastes. Also, 80% of the total discharge of the textile industries is fed into the water stream [18]. So, the development of efficient dye decolourization method is crucial, where laccase stands one.
The present study, highlights the properties of laccase based on the microbial source, significance of laccase on dye decolourization, factors affecting laccase-mediated dye decolourization, decolourization
Fig. 1. Application of laccase in the degradation of various pollutants.
spin-coupled diamagnetic Cu-Cu pair [20]. Fig. 2 represents the struc- ture of laccase. Hence, the general form of laccase is a three-domain structure with protruding cupredoxin loops. T1 copper act as the sub- strate oxidation site because of its high redox potential with trigonal orientation. T2 and T3 copper effectuate the release of water by the reduction of oxygen [21]. The molecular mass of laccase varies from 50 to 140 KDa and the structure of laccase differs from monomeric, homodimeric, and heterodimeric to multimeric forms [10]. The pre- eminent property of the enzyme is stability, which is ensured by the carbohydrate portion of the structure that provides conformational stability, and resistance against proteolysis and radicals [22].These properties of the laccase such as structure, molecular mass, redox po- tential (E◦) and stability vary among the different sources of laccase.
Typically the types of laccase are fungal laccase, bacterial laccase, plant and insect laccase [23].
cases are second to none in the degradation of compounds but the ac- tivity of the enzyme is limited to certain environmental conditions such as acidic pH range (3.5 to 5.5) and certain temperature range (30 to 55 ◦C) [29].
2.2. Plant laccase
Plant laccases are monomeric extracellular proteins with a molecular mass of 60 to 130 kDa with a higher glycosylation pattern that ensures high stability and an isoelectric point of 7 [30]. But the main disad- vantage of plant laccase is its low redox potential which limits its application in bioremediation [31]. However, it has great potential in biofuel production due to its delignification ability and in some cases it is utilized for the phytoremediation of phenolic compounds [32].
Fig. 2. Structure of laccase.
2.3. Bacterial laccase
Bacterial laccase was first detected in Azospirillum lipoferum in the year of 1993 [33]. The molecular mass of bacterial laccase varies from 50 to 70 kDa with the major form of oligomeric intracellular proteins and predominantly intracellular whereas fungal laccase is both intra- cellular and extracellular [34]. Bacterial laccase is highly stable under various environmental conditions which is a thermostable and pH-stable laccase [35]. Hence, bacterial laccase is the superlative choice for bioremediation in natural environment conditions irrespective of its low redox potential (E◦<460 mV) [36].
As far as there is no evidence on utilization of insect laccase for bioremediation purpose. Table 1 enumerate the properties of laccase (Molecular weight, Optimum Temperature and pH) isolated from different sources.
3. Mechanism of laccase-mediated dye decolourization
Laccase, Copper containing oxidase enzymes is produced by different sources such as bacteria, fungi, and plants. Laccase catalyses pollutants such as synthetic dyes, aromatic compounds, phenolic compounds, and aliphatic amines with the help of four copper atoms (Type I, Type II, Type III) that are present at three different sites [37]. The role of the three copper sites based on their environment and spectroscopic char- acteristic plays a significant role in catalysing the pollutant and acti- vating the laccase enzyme [32].
It is often tough to generalise the laccase by its reducing substrate due to broad range of compounds that gets oxidised and substrate varies from one laccase to other. This is due to their high variation in the redox potential (E◦) of different laccase enzymes [38]. The range of substrates which include aromatic amines, methoxy substituted phenols, ortho (o) and para (p) diphenols and polyphenols [39].
Laccase traditional substrate includes aromatic amines and various lignin-derived phenols. Also, Ortho-substituted compounds (e.g. guaia- col, caffeic acid, gallic acid, dihydroxyphenylalanine, pyrogallol, o- phenylenediamine) suits to be better laccase substrates than para- substituted compounds (e.g. p-cresol, p-phenylenediamine), while the
lowest oxidation rate is attained by meta-substituted compounds (e.g.
m-phenylenediamine, orcinol, resorcinol) [40].
The laccase enzyme oxidizes the substrate and withdraws electrons, converting them to free radicals. The laccase then receives these elec- trons and helps to convert molecular oxygen to water [10]. The degra- dation products resulting from enzymatic reactions become smaller fragments and less toxic than parent dyes and it depend solely on the specific dyes and its structure, particular laccase used and reaction condition during the biodegradation process [41]. In certain instance, the degradation product catalysed by laccase may exhibit reduced colour or become colourless. Such products can then be further degraded by other microorganism or enzymes, enabling them to un- dergo complete mineralisation of dye by converting into simpler com- pounds such as CO2, water leading to their removal from the environment.
The Type I Copper site acts as an electron acceptor where substrate oxidation occurs and releases electrons. These electrons are then transported through the trinuclear cluster, which contains Type II and Type III copper and converts molecular oxygen to water [42].
In the case of bulky polymers such as lignin, catalytic sites of laccase cannot interact directly with the functional groups. The non-phenolic part of this lignin has a higher redox potential, which makes it diffi- cult for laccase to oxidize [43]. Therefore, a small molecule is needed that can act as a shuttle between the active site of the laccase and the lignin molecule. These small molecules are called mediators, which are oxidized first by laccase, after which they oxidize the non-phenolic substrates through radical Hydrogen Atom Transfer (HAT) or electron transfer (ET). This mechanism is termed a laccase-mediated system (LMS) [44]. Fig. 3 elucidates the mechanism of laccase mediated sub- strate oxidation in the presence and in the absence of mediator. 2, 2
′
-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), a synthetic molecule, effectively mediates the oxidation of non-phenolic substrates and it is the first chemical substance to be established as a laccase mediator. The oxidation of ABTS by laccase occurs in two stages that generate ABTS+and, in turn, produce ABTS2+ [45] (Supplementary Fig. S1).These laccase mediators are sorted into two kinds, namely natural
Table 1
Properties of laccase isolated from different sources.
S. No Laccase source Molecular
weight Optimum
temperature Optimum
pH Enzyme
activity Remarks References
1 Ganoderma lucidum 57 kDa 40 to 60 ◦C 5 30.55 U/mg Km − 3, 21.36 mM, Vmax − 0.98, 2.77 for ABTS
and guaiacol [55]
2 Trichoderma harzianum 63 kDa 50 ◦C 3 9.19 U/mg Km − 0.10 mM and Vmax − 0.603 μmol/min for
ABTS [57]
3 Coriolopsis trogii 70 kDa 40 ℃ 3 720 U/L Km − 0.09 mM
Vmax − 0.03 μmol/min for ABTS [112]
4 Methylobacterium extorquens 50 kDa 65 ◦C 1–4 515.52 U/
mg Km − 7.65×10−2, 9.18 ×10−2 mM and Fe2+ ions increased the enzyme activity [113]
5 Enterococcus faecium 50.11 kDa 80 ◦C 6 13.75 U/mg Km − 0.68 mM and Vmax − 5.29 μmol/ml min
for ABTS [56]
6 Fomes fomentarius 44 kDa 35–40 ◦C 7 260 ±1.80
U/mL Km − 1 mM and Vmax − 60 mM/min for ABTS [114]
7 LacMeta gene expressed in
Escherichia coli 107.93 kDa 30–40 ◦C 3–5 Not reported Km − 4.22 mM and Vmax − 24.43 μM/min for
ABTS [58]
8 Coriolopis gallica NCULAC F1 57 kDa 60 ◦C 2.5 188.79 U/
mg Fruit peelings and rice straw were used as a low
cost substrate [24]
9 Ganoderma leucocontextum 65 kDa 40 ◦C 5 5776 U/L Km − 1.66 mM and Vmax − 2.45 mM/min [115]
10 pLac gene (Sulfidobacter indolifex)
transformed into E.coli BL21 48.76 kDa 40 ◦C 6 625 mU/mg Km − 3.21 ±0.27 mM and Vmax − 30.97 ±
0.51 μM min−1 for ABTS [76]
11 Kabatiella bupleuri 70–75 kDa 30 to 40 ◦C 3 - 4 215.40 U/L Km − 5.84 ×10−1, 2.8 ×10−2 mM and Vmax −
1.14, 0.60 U/mg for ABTS and syringaldazine [116]
12 Gymnopus luxurians 64 kDa 55 to 65 ◦C 2.2 118.82 U/
mg Km − 539 μM and Vmax − 3.10 μmol/min for
ABTS [77]
13 Bacillus pumilus ARA 58.7 kDa 85 ◦C 3.5 1283 U/mL Km − 0.33 mM;Vmax − 32.40 U/mg for ABTS [117]
14 Aureobasidium melanogenum strain
11–1 62.5 kDa 40 ◦C 3.2 3120 ±170
mU/ml Km − 6.30 ×10−2 mM Vmax − 177.40 M/min
for ABTS [118]
15 Bacillus velezensis 58 kDa 80 ◦C 5.5 188 U/mg Enzyme showed high stability [119]
and synthetic laccase mediators. Some of the natural mediators are acetosyringone [46,47,48], vanillin[49,50], syringaldehyde, [49,51], acetovanillone and the synthetic mediators are N-hydroxyphthalimide (NHPI), [52] 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), [53,54], N-hydroxyacetanilide (NHA), 2,2
′
-azino-bis-(3-ethylbenzothiazoline-6-- sulfonic acid)(ABTS) [55,56,57,58] used for biodegradation. Fig. 4 de- picts the structure of different natural and synthetic laccase mediators.Enzyme-assisted conversion of pollutants has become an attractive replacement for conventional techniques due to their tendency to react with a complex substance, the rapid pace of degradation, and lower toxic by-products [59]. These advantages elevated the scope of the laccase enzyme in the decolourization of dyes in various types of in- dustries. The possible decolourization mechanism of various common dyes was elucidated in Fig. 5.
4. Application of laccase on dye decolourization
Due to their wide application in several industrial fields, including paper and pulp, food, petrochemical and textile industries, the detoxi- fication of effluents is a major concern. Two-thirds of the dyestuffs come from the textile industry, which uses an enormous amount of water and chemicals during the wet processing of textiles and releases the dye effluent that are difficult to decolourize [60]. A survey on textile effluent found that dyes released on the waterbodies have a detrimental effect on the environment, animals and humans and also show signs of cytotox- icity, genotoxicity and Ecotoxicity [61]. Therefore, treatment of this effluent before discharge is crucial. Development of a laccase based treatment approach would be an attractive strategy due to its degrada- tion capacity of various chemical dyes, including synthetic dyes. The application of laccase is rapidly growing and it is not limited to decol- ourising the textile effluent but is also used in the bleaching and dyeing process. Commercial Laccase from genetically engineered Aspergillus, Fig. 3. Mechanism of laccase mediated substrate oxidation in the presence of mediator (a) and in the absence of mediator (b).
contributed by Novozymes (Denmark), can decolourize reactive black 5, reactive blue 114, reactive yellow 239, and reactive red 239 in presence of a mediator, ABTS [53].
Spore laccase of Bacillus licheniformis LS04 isolated from forest soil was able to decolourize synthetic dyes such as reactive blue 19, indigo carmine and reactive black 5 in absence of laccase mediator. However, in presence of a laccase mediator such as acetosyringone, 80% colour removal was observed within 1 h [62]. Similarly, Zhang et al. [63]
isolated Bacillus vallismortis fmb-103 from textile industry disposal sites which efficiently decolourized the triphenylmethane dyes, including aniline blue, malachite green and brilliant green in presence of laccase mediator substrate such as ABTS. Immobilized Laccase of Streptomyces sviceus, a marine actinobacterium, hydrolysed the Congo red-21 dye (synthetic azo dyes) and showed 92% colour removal within 24 h [64].
Thermo-stable and alkali-stable laccase isolated from Bacillus lichen- iformis, was able to rapidly degrade reactive black 5, indigo carmine and reactive blue 19 with 93% decolourisation within 4 h in contiguity of mediators such as acetosyringone [65]. Simultaneous reduction of methyl orange (50 mg/L) and Chromium (VI) (50 mg/L) was observed in anaerobic conditions using a salt-tolerant bacterium, Bacillus circulans BWL1061. During decolourisation, the reduction of Cr (VI) and decol- ourization of methyl orange occurred due to enzymes such as laccase,
NADH-DCIP reductase and azoreductase [66]. Different azo dyes such as orange 3R, T-blue, and yellow GR were able to decolourize using lac- cases from Pseudomonas parafulva and Bacillus cereus [61]. Table 2, lists out some of the recent studies on application of laccase isolated from various sources in dye decolourization and operational parameters.
Furthermore, Laccase has been widely used in the bioremediation of xenobiotics. Xenobiotics often last for a long period of time in the environment and eventually become serious contaminants in soil and water. Numerous environmental bodies may be concerned more about the removal of such xenobiotics. Halophilic laccase from Alkalibacillus salilacus was able to detoxify the anthracene with the help of 1-Hydrox- ybenzotriazole (HBT) [67].
In the dyeing process, Laccase functions as a catalyst, forming chromophore compounds that contribute colour when fixed to the fibre [68]. Oxidoreductase laccases catalyse the formation of polymeric dyes with chromophores from aromatic compounds and can be used for fabric dyeing in textiles [69].
In Food Industry, laccase has also been widely used to treat olive oil mill wastewater, which contains a significant amount of highly toxic phenolic compound. Laccase from fungi such as Pleurotus ostreatus, Lentinula edodes, Pycnoporus coccineus, Rhus vernicifera, lentinula edodes, Cerrena unicolor is used to detoxify the phenolic compounds present in Fig. 4.Structure of different natural and synthetic laccase mediators.
Fig. 5. Possible decolourization mechanism of various dyes by laccase.
Fig. 5. (continued).
the olive-oil mill wastewater [70].
The brewery industry produces wastewater with high concentrations of polyphenol compounds, mainly tannin, which poses a significant
environmental risk [70]. The white-rot fungus Coriolopsis gallica can decolourize the high tannin containing wastewater and also reduce the Chemical oxygen demand [71]. Furthermore, in the presence of the right Fig. 5. (continued).
Table 2
Recent studies on application of laccase isolated from various sources in dye decolourization and optimum operational parameters.
S. No Dye Micro-organism Decolourization efficiency Parameters Reference
1 Malachite green Bacillus aestuarii KSK 89% Temperature − 35 ◦C; pH-7.0; Time -
60 h [120]
2 Methyl Orange, Congo Red, Malachite Green,
Remazol Brilliant Blue R, and Reactive Blue 4 Bacillus sp. NU2 87%, 70%, 65%, 63% and 51%
respectively Temperature-30 ◦C;
Time −1 h [121]
3 Direct blue Trametes versicolor 81% Dye concentration 40 ppm;
temperature 40 ◦C; pH 4; Time - 24 h [122]
4 Orange 3R, yellow GR and T-blue Pseudomonas parafulva 78.98%, 76.63% and 74.54%
respectively Agitation-100 rpm; Temperature- 40 ◦C;
Time- 120 h
[61]
5 bromophenol blue, phenol red, methylene blue Bacillus licheniformis
NS2324 85%,75%,99.28% respectively Time - 4 h,
Temperature- 40 ◦C [123]
6 acid orange, congo red, methyl orange Enterobacter aerogenes
ES014 82.30%,78.20%,
81.50% respectively Temperature − 30 ◦C,
Time - 48 h [124]
7 Anthracene Alkalibacillus salilacus 94% Time - 72 h;
Temperature- 40 ◦C; pH 8 [67]
8 Indigo carmine Bacillus safensis HL3 97% Time- 2 h;
Temperature- 40 ◦C; Agitation- 170 rpm
[125]
9 Reactive Yellow 145 and Remazol Yellow RR Scedosporium
apiospermum 94.80% and 86.90% respectively Temperature- 30 ºC; Agitation-100
rpm;
Time - 204 h
[126]
10 Malachite green Fusarium oxysporum
HUIB02
>90% pH 4.50,
Temperature- 40 ◦C; Time −24 h; Dye concentration-100 mg/L
[127]
11 Malachite green Streptomyces exfoliatus 96% Time −120 h;
Temperature-25 ◦C;
pH, 4; Dye concentration-0.04 g/L;
Inoculum size-12.50%
[128]
12 Methylene blue Bacillus thuringiensis 95% Temperature-30 ◦C; pH - 6;
Agitation-140 rpm;
NaCl-10 g/L;
Inoculum (v/v)- 4%;
Time - 12 h
[98]
13 Indigo carmine and malachite green Bacillus subtilis DS 95.24% and 90% respectively pH - 7.50;
Time - 50 min;
Temperature - 35 ◦C
[129]
14 Acid black 24 and Direct red 81 Bacillus
amyloliquefaciens 71% and 79%
respectively Temperature - 30 ◦C; Agitation - 140 rpm;
Time - 24 h
[130]
15 Congo Red, Malachite Green, Methyl Orange,
Reactive Blue 4 and Remazol Brilliant Blue R Enterobacter sp AI1 78%, 85.70%, 77%, 81%, and
73% respectively Temperature-30 ◦C;
Time - 4 h [131]
16 Malachite green Bacillus subtilis 92.24% Time - 24 h; Temperature- 37 ◦C [132]
17 Congo red-21 Streptomyces sviceus
KN3 92% Time - 24 h;
Temperature − 30 ◦C; Agitation - 100 rpm
[64]
18 Direct Green 28 and Direct Blue 71 Enterobacter aerogenes
PP002 99.00% and 99.23% respectively pH 7;
Temperature-37 ◦C; Agitation-60 rpm;
dye concentration-100 mg/L; Time
− 168 h
[133]
mediator, highly thermostable and pH-stable recombinant laccase (rLAC) of Bacillus pumilus TCCC 11,568 can rapidly decolourize food dyes at high temperatures and throughout a wide pH range [71].
5. Factors affecting laccase activity and its effect on dye decolourization
The activity of the laccase enzyme depends on the biochemical and structural properties of the enzyme like temperature, pH, the effect of inhibitors, surfactants and metal ions [72]. Metal ions attach to the Type1 copper site of the laccase enzyme and inhibit substrate binding by acting as a competitive inhibitor. These properties vary for each or- ganism and are based on the purification mechanism [73]. As textile effluent comprises of heavy metals which are leached out from the dyes and additives (Pb, Cr, Cd, Cu, Ni, Zn, Fe, Hg and Co) [74], the study of effect of metal ions on dye decolourization is significant. In addition to these parameters, substrate concentration, enzyme dose and reaction time also influence the dye decolourization efficiency. [55] studied about the consequences of different factors on the enzyme activity of laccase isolated from white rot fungi Ganoderma lucidum. Among the various metal ions (CoCl2, CuSO4, ZnSO4, FeSO4 and HgCl2) tested, Hg2+ and Fe2+ions showed greatest inhibitory effects. The presence of in- hibitors and ionic and non-ionic surfactants reduced the enzyme activity by nearly 50%. It was also proposed that increasing initial dye concen- tration has a negative effect on the decolourization efficiency of mala- chite green and the presence of mediators accelerates the decolourization and the optimum temperature and pH for malachite green dye decolourization was found to be 50 ◦C and pH 8 with a 72%
decolourization with 1 h. Birge et al. [56] studied the effect of organic solvents, metal ions and surfactants on decolourization efficiency and inferred that the presence of metal ions had no effect on the dye decolourization by Enterococcus faecium laccase and the presence of metal ions (Cr2+, Cu2+) and organic solvents increased the activity. Also, the optimum reaction conditions for textile dye decolourization were 47.50 ◦C, pH 4, and 1 mM redox mediator concentration and the decolourization efficiency increased while increasing the incubation time. Lima et al. [75] studied the effect of decolourization efficiency of the synthesized recombinant laccase under acidic conditions and rela- tively high temperatures and found that up to 60 ◦C, the enzyme retained its structural stability and showed 91.70% decolourization and the addition of cobalt ions increased the decolourization efficiency.
Sharma and Leung [76] analysed the tolerance of recombinant laccase by transforming the Lac gene of Sulfidobacter indolifex against organic solvents and salts. From the results, this recombinant laccase has high tolerance against sodium chloride and it is susceptible to organic sol- vents (methanol, acetonitrile, dimethyl sulfoxide (DMSO), ethanol, and acetone). The reduction in protein stability is due to the disruption of intra-protein hydrogen bonds by alcohol and acetone, the weakening of hydrophobic interactions by acetonitrile and the sequestration of water molecules by DMSO. Also, decolourization efficiency is improved by the amalgamation of the redox mediator. On the other hand, for indigo carmine dye degradation, significant change in the decolourization ef- ficiency is not observed between with and without redox mediator. Sun et al. [77] investigated the decolourization efficiency of Gymnopus lux- urians laccase under different mediators and reaction conditions. Seven different mediators (ABTS, violuric acid (VIA), acetosyringone (AS), 2,2, 6,6-tetramethylpiperidine-1-oxyl (TEMPO), syringaldehyde (SA), vanillin (VAL), and hydroxybenzotriazole (HBT)) were tested against azo dyes, reactive dyes and triphenylmethane dyes and among these AS and SA performed as an optimal mediators. The optimal reaction con- ditions for decolourization were a temperature of 25 ◦C-60 ◦C, a pH of 4, and a mediator concentration of 0.10 mM with a decolourization effi- ciency of 50–90% after 4 h of degradation. Lima et al. [58] inferred that the incorporation of a redox mediator increases the decolourization of methylene blue, trypan blue and, malachite green except for bromo- phenol blue by LacMeta. Additionally, among the different redox
mediators assessed (acetosyringone, syringaldehyde, methyl syringate, and 1-hydroxy benzotriazole), syringaldehyde substantially improved the decolourization efficiency of Remazol Brilliant blue R.
6. Immobilized laccase and their application in dye decolourization
Though laccase is a fruitful approach in bioremediation, its appli- cation in large scale is still limited because of the some bottlenecks associated with it which includes high production cost, poor reusability and less stability. Immobilization is a promising choice to ensure reus- ability that reduces cost and stability in practical applications [78].
Various laccase immobilization methods include, adsorption, ionic binding, covalent binding, entrapment and encapsulation [79]. Fig. 6 illustrate the immobilization methods and various supports harnessed for laccase immobilization.
6.1. Physical immobilization
Entrapment and encapsulation is a physical retention of laccase in a polymer or membrane. It is the easiest immobilization method with no structural alteration of the enzyme. However, mass transfer limitation plays as a major bottleneck in both of these methods [80]. In the study by Mogharabi et al., laccase was entrapped in alginate-gelatin mixed gel for the decolourization of synthetic dyes. The immobilized laccase shown elevated thermal stability with 85% activity retention after five cycles [81].
6.2. Chemical immobilization
In this method, laccase will be immobilized on the support by chemical bonding which includes, adsorption, covalent binding and ionic binding [82]. Adsorption has high commercial potential than other methods because of its simplicity and inexpensive but the interaction between laccase and immobilization support is weak [83]. In some studies, the low-cost biochar material was utilized as the solid support for the immobilization of the laccase enzyme which also helped in the adsorption of recalcitrant toxic pollutants and degradation [84–87].
Whereas, in covalent and ionic bonding, the enzyme binds to the support matrix strongly which prevents the leaking of laccase. Hence, the quantity of the enzyme will be constant which provide promising pathway for continuous dye decolourization applications. But, the main drawback is, possible modification in the active site of the laccase [88].
In various studies, laccase has been immobilized by covalent binding.
Hariri et al. [89] reported that by immobilizing laccase synthesized from Trametes versicolor on magnetic casein @ CoFe2O4 aggregates, the sta- bility of the enzyme was enhanced by 300% in comparison with the free laccase and the enzyme activity was detained even after 24 reusability cycles in the degradation of crystal violet. In this work, the casein as a immobilizing agent acted as a molecular chaperon as well as a solid support for the biocatalyst. Zhu et al. [90] immobilized the laccase on Fe3O4@SiO2 nanoparticles in order to improve the stability of the bio- catalyst during photothermal catalytic decolourization of textile dyes like brilliant green, malachite green, azophloxin, reactive blue 19, alizarin red and procin red MX 5B. This immobilization improved the pH and thermal stability, and tolerance against, inhibitors, metal ions and organic compounds.
Apart from enzyme stability and reusability, in some studies, immobilized laccase shown elevated enzyme activity and dye decolou- rization efficiency than the free laccase. Generally, dye decolourization in immobilized laccase occurs through three principle mechanisms:
adsorption by the carrier support, enzyme catalysis and their synergistic effect [91]. In this study [92], a commercial laccase was tested for the dye degradation efficiency which is 11.52%. whereas, after immobili- zation on amino-functionalized Metal Organic Framework (MOF), drastic shift in degradation efficiency was observed which is 84.63%
because of the synergistic effect between adsorption and laccase catalysis.
Also, in the study [93], improved enzyme activity was observed because of the immobilization of laccase from Trametes versicolor on Cu Zeolitic Imidazole framework-90, where copper acted as the inducer of laccase and ultimately increased the dye degradation efficiency. Also in [94], 1.70 fold improved laccase activity was attained by the immobi- lization on copper nano architectonic. Similarly on various studies [61, 95], copper based immobilization support has shown improved enzyme activity and dye decolourization by increasing the redox potential and loosening the enzyme’s protein structure.
Table 3 lists out some of the recent studies on immobilization of laccase on different supports and its effect on reusability and stability.
7. Synergistic effect of laccase
In recent times, the synergistic effect of the enzyme has emerged as a viable strategy for the efficient decolourization of dyes in an extensive range of industries. This is a result of the enzyme’s ability to work in tandem with the other enzyme or chemical agents to enhance the decolourization efficiency. Generally, simultaneous coupling of process has been found to accelerate the degradation process more effectively
than the sequential coupling [96].
One of the most commonly used strategies for improving the activity of laccase for efficient degradation is to combine it with other enzymes such as lignin peroxidase (LiP) and manganese peroxidase (MnP) [97].
These enzymes break the complex dye molecules into smaller com- pounds that can be more easily broken down by laccase. Wu et al., re- ported 95% of methylene blue dye decolourization by Bacillus thuringiensis F5 and based on its degradation pathway, it has proven that synergistic action of NADH-DCIP reductase, laccase (lac), lignin perox- idase (LiP), and manganese peroxidase (MnP) improved the decolouri- zation [98]. In another study, Guo et al., degraded mixture of azo dyes by a bacterial consortium and reported that high activity of azo reduc- tase, laccase, and lignin peroxidases appeared in the bacterial con- sortium which decolourized the metanil yellow into p-amino diphenylamine and diphenylamine through synergistic effect [99]. In another study by Zhuo et al., synergistic stimulation of aromatic com- pounds (ferulic acid, cinnamic acid, and vanillic acid) and metal ions (Cu2+, Fe2+) improved the performance of Pleurotus ostreatus HAUCC 162 laccase and showed a higher decolourization rate on different synthetic dyes than the individual effect [100].
Additionally, the combination of various physio-chemical and bio- logical process has led to a synergistic approach, resulting in better Fig. 6. Laccase immobilization methods and immobilization supports.
performance in the dye degradation process. Also, enhancing the sta- bility and activity of the enzyme can be achieved through immobilising the laccase on solid support or encapsulating it in nanoparticle which can protect the laccase from denaturation, extends its shelf life and allow for longer degradation periods [101].
One of the advantages of having synergistic with adsorption offers protection of the enzyme from harsh conditions, reactivity and reus- ability. Combining Advanced Oxidation Method with biological treat- ment offers breakdown followed by the removal of various dyes and a higher degree of mineralization [102]. Yang et al. observed the elevated
laccase activity in decolourization of various dyes (i.e., CR, RB and RG) because of the synergistic action of adsorption and enzyme catalysis by the encapsulation of laccase in bimetallic-centred Cu/ Zn Zeolitic Imi- dazolate Frameworks (ZIFs). It was further demonstrated that, copper ions elevated the binding of substrate to the laccase active site which possibly increase the enzyme catalysis. Also, due to the nanoflower structures of the Cu-ZIF-90, more active sites are exposed to the dye which led to the accumulation of dye molecules and shortening of the contact time [93].
Similarly, Li et al. reported that by the synergistic action of Table 3
Recent studies on immobilization of laccase on different supports and its effect on reusability and stability.
S. No Laccase source Pollutant Immobilization material Results Reference
1 Trametes trogii, Trametes
versicolor Malachite green & Orange II Polycaprolactone/
Polyethyleneimine Electrospun fibre
30% improved stability and reusability up to 10 cycles [134]
2 Fungal laccase Safranin O dye 1,1′-carbonyldiimidazole- magnetic cellulose nanofiber (rice straw)
96% relative activity, 86% increased storage stability & 10
reuse cycles [135]
3 Trametes versicolor Reactive deep green,
Reactive deep blue, Acid red 18 dye
Cu Zeolitic Imidazole Framework-
90 High enzyme activity, environmental stability and 5
reusability cycle [93]
4 Xiasheng Biotech Co.,
Ltd, China Congo red Amino functionalized
Cu - MOF Improved stability and decolourization efficiency because of the synergetic effect of degradation and adsorption [92]
5 Aspergillus sp. Methylene blue and
Bromophenol blue Chitosan hydrogel Improved effect of adsorption and dye decolourization [136]
6 Laccase (120 U/g)- Source Leaf Biotechnology Co., Ltd
bisphenol A ZIF-8(PA) high porosity and stable under different conditions
(temperature, pH, reagents, storage) up to 90.28% efficient removal of BPA
[137]
7 CSIR-Indian Institute of Petroleum Dehradun, India
Malachite green Acid
functionalized pine needle biochar Improved thermal and pH stability and reusability up to 6
reaction cycle [86]
8 Trametes versicolor Direct red Copper
Nano architectonics 1.70 fold improved activity, improved pH and temperature
stability, 10 reusability cycle [94]
9 Sunson Co Ltd., China Congo red Co Cu – Metal Organic Framework Improved laccase loading, decolourization by synergic
adsorption and enzyme catalysis [95]
10 Trametes versicolor Malachite green Alkali modified biochar (rice
straw) Maintained 50% removal rate after 10 cycles [138]
11 PersiLac-1 (model
laccase enzyme) Malachite green & Congo red Nano cellulose (NC) from Quinoa
husk Reusability of 18 consecutive runs, performed as an effective cationic dye adsorption because of the negative charge of NC
[139]
12 Trametes versicolor RB-19 and AO-7 Vault nanoparticle (vaults from
Pichia pastoris) Improved stability, superior decolourization ability and
reduced toxicity [140]
13 Trametes versicolor Naphthol green B and Indigo
Carmine Chitosan – poly acrylic acid
microspheres Best rheological properties, improved stability &
operational profile & immobilization efficiency [141]
14 Oudemansiella canarii Remazol Brilliant Blue R Ammonium sulphate-
Glutaraldehyde Less toxic than the free enzyme, improved storage stability
and operation [142]
15 Aspergillus sp. Remazol Brilliant Blue R Corn cob Improved decolourization efficiency than the free enzyme
and green support [143]
16 Trametes versicolor Phenolic dyes Hybrid super structured
nanomaterials 11 reusability cycle and excellent decolourization capacity [144]
17 Trametes versicolor Acid blue 193 Supermagnetic Fe3O4
nanoparticles The solid support was prepared by a green, low carbon footprint plasma polymerization mediated strategy. It offers reusability of 10 cycles with >90% activity and ease of separation
[145]
18 Trametes versicolor Rhodamine B, Reactive Blue
5, Drimaren red, Drimaren turqoise
Poly(vinyl alcohol) – Alginate
beads An eco-friendly and cost effective solid support was
designed with increased storage ability [146]
19 Penicillium expansium Methyl orange, Remazol
Brilliant Blue R,Reactive black-5, Brilliant blue
Thiol modified Fe3O4
nanoparticles using metabolites of Aspergillus niger
The catalytic affinity (Kcat/Km) was increased 10 fold and retained 84.34% of enzyme activity after 10 cycles [147]
20 Trametes villosa Acid blue 277, Acid blue 172 Iron oxide magnetic particles More efficient dye decolourization even at low pollutant concentration due to synergetic effect of adsorption and degradation
[148]
21 Streptomyces sviceus Congo red-21 Calcium alginate beads Maximum of 92% decolourization observed compared to
crude laccase [64]
22 Trametes hirsuta EDN 082 Remazol Brilliant Blue R Light Expanded Clay Aggregate reusability of 6 cycles and wide pH and temperature [149]
23 Trametes versicolor Aniline Calcium alginate beads Repetitive up to 10 cycles. Higher stability of enzyme;
prevent enzyme denaturation [150]
24 Papaya laccase Indigo carmine Chitosan beads Immobilization yield of 98% with no leaching and 3 fold
improved stability [151]
25 Bacillus cereus and
Pseudomonas parafulva Yellow GR,T-blue and orange
3R copper-alginate entrapment Higher enzyme activity and maximum decolourization
observed in immobilisation compared to free laccase [61]
settings.
8. Emerging applications of laccase and its utilization on dye decolourization
The development of laccase-based methods has paved the way for their potential applications in various fields such as biocatalysis, bio- energy and biosensors over the last few years.
Laccase-based biosensors have been developed for detecting a vari- ety of compounds, such as amines, phenolic compounds and endocrine disruptors [104]. Biosensors are devices that detects the presence of chemicals using biological organisms or biological molecules. Currently, laccase based biosensors are being developed for the detection of phenolic compounds which are the primary product of the laccase based dye decolourization. This primary products will be further mineralized into non-toxic products. So the development of laccase based biosensors for the detection of phenolic compounds can be utilized in dye decol- ourization applications in order to analyse the extend of degradation.
Pillai et al. [105] designed a biosensor based on screen-printed carbon electrodes (SPE) to determine the presence of catechol, utilizing nickel oxide/graphite (NiO/G) immobilized with laccase as a bio-nanocomposite coating. Cevher et al. [106] detected the amount of catechol in water samples using an indenoquinoxalinone polymerized on the surface of the electrode and immobilized laccase as a sensing platform. These biosensors demonstrated good stability, selectivity, and high sensitivity towards catechol. Othman and Wollenberger [107] used chemically modified laccase (isolated from Coriolus hirsute) immobilized on functionalized SPE as a biosensor for phenol detection in polluted water. These findings can also pave the way for the development of simple one pot solution for the detection of dye in water bodies.
In another study [108], laccase has been explored in the humifica- tion of pollutants which could also promotes plant growth. By this process, not only environmental pollution will reduce but also, it will helps in enhancing the soil fertility.
It has also reported that laccase has an ability to act as a biocatalyst in biofuel cells for the production of electricity. Biofuel cells are a sub- stitute to conventional fuel cells that convert the chemical energy into electrical energy through bio-electrochemical reactions involving bio- catalysis by enzymes such as laccases, bilirubin oxidases or peroxidases in cathodic reactions. In the study by Chen et al. [109], laccase was immobilized on the cathode of biofuel cells to produce electrical energy, which is a cleaner form of energy. Another study by Ratautas et al. [110]
developed an AuNP-alcohol dehydrogenase bioanode with AuNPs-laccase biocathode and generated a maximum power output of 130 μW cm–2 at 0.50 V and pH 7. Pelosi et al. [111] developed a novel enzymatic biofuel cell (EBC) based on glucose-oxidase (bioanode) and laccase enzyme (biocathode) immobilized on a commercial flat conductive polymer. The developed EBC generated a maximum open circuit voltage of 590 mV and power density of 2.41 µW cm−2 in three weeks. This principle can be utilized in dye decolourization application, by using dye contaminated water in the anode chamber as a substrate solution which leads to simultaneous dye decolourization and electricity generation.
Recent developments in laccase-based methods have concentrated
Increased discharge of pollutants into the natural water bodies due to anthropogenic activities has seriously threatened our environment and significantly improved the need for bioremediation. Enzymes as a bioremediation agent is a pre-eminent sustainable way. Among the various biocatalysts, a laccase is an attractive option in the degradation of chemical pollutants because of its versatility, easy to operate, highly flexible operating conditions, reduced mass transfer limitation and no need of nutrient supply. Among various laccase, fungal and bacterial laccase are the most utilized in bioremediation applications. However, the function and application of plant and insect laccase is not explored much. Untangling the properties of these laccase can augment the application of laccase on dye decolourization. Also, the complex composition of wastewater (pH, heavy metals, high concentration of salts) can be overcome by production of heterologous laccase and engineered laccase using genome engineering, protein engineering and omics tools. However, the application of laccase in real time applications is quite challenging because of its high production cost and variability of enzymatic efficiency in industrial scale. This can be overcome by the reusing the enzyme by immobilization which also improves the stability.
Reduction in enzyme activity due to mass transfer limitations is a problem in loading the enzyme in a carrier through entrapment or encapsulation. Research can be carried out to maintain the activity of the laccase after loading in a carrier. Interestingly, in some cases, copper-based immobilization support has shown elevated activity. Over and above this, the products of the reaction should be less or non-toxic than the target pollutant and appropriate co-factors should be provided during the degradation process if the enzyme is co-factor mediated. Also, the operational and intrinsic catalytic features of the enzyme should be optimized to improve the enzyme catalysis.
Funding
The authors acknowledge the funding support for the work from the IITG, Assam, India (Grant no. BSBESUGIITG01213xSEN001).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
All the listed authors are grateful to Department of Biosciences and Bioengineering, Indian Institute of Technology, Guwahati, India for providing literature facilities.
Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jtice.2023.105081.
