Dyes and Pigments 184 (2021) 108749

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Dyes and Pigments

journal homepage: http://www.elsevier.com/locate/dyepig

Review

Discoloration of indigo dyes by eco-friendly biocatalysts

Kwon-Young Choi *
Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, Gyeonggi-do, Republic of Korea
Department of Environmental and Safety Engineering, College of Engineering, Ajou University, Suwon, Gyeonggi-do, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Indigo is one of the most commonly used dyes worldwide. However, indigo dyeing technology and physico­
Indigo chemical treatment processes have generated potentially hazardous by-products and sludge, causing serious
Indigo carmine environmental pollution. Among indigo-treating technologies, biological treatments using a “green” catalysts of
Discoloration
laccase and oxidase enzymes have been spotlighted. This review comprehensively assessed the whole cell-based
Laccase
discoloration and in vitro enzyme treatment of indigo (carmine) and process engineering, including enzyme
Enzyme immobilization
immobilization and aggregation, for indigo degradation. Although both have various advantages, effective
process design to obtain desirable biomass and enzyme production strategy with cost-effective manner should be
considered for indigoids treatment. In addition, the supply of electron mediators and the removal of unexpected
byproducts by the laccase should also be considered throughout the whole process. Therefore, further researches
on enzyme and process engineering such as chemo-enzymatic process to obtain the desired performances at the
industrial level were suggested.

1. Introduction
Indigo is one of the oldest-used dyes in human history and it is still
the main compound used to dye denim due to its outstanding coloration
[1,2]. However, indigo has a very low solubility in water owing to its
chemical structure, such that additional, repeated reduction–oxidation
(redox) processes are required in denim dyeing [3,4]. The dyeing pro­
cess with natural indigo derived from plants includes biotransformation
of a highly soluble glycoside indican (indolyl-β-D-glucopyranoside) by
β-glucosidase and air oxidation [5]. Meanwhile, dyeing with synthetic
indigo includes the reduction of indigo to a highly water-soluble leu­
coindigo using a strong reducing, sodium dithionite (Na2S2O4), followed
by air-oxidation [6]. In this process, the eluted indigo and strong
reducing agents for the treatment of unsolved indigo has a high risk of
being exposed to the aqueous environment [3,7]. To overcome this
problem, several attempts have been made to use either reducing sugars
as a “green” (i.e., eco-friendly) reductase during the reduction process or
glycosylation protection/deglycosylation deprotection using a glyco­
syltransferase enzyme [5,8,9]. Nevertheless, these methods all have the
disadvantage of being accompanied by losses in sugar feedstock. Alter­
natively, a water-soluble indigo derivative could be used by introducing
a functional group, such as carboxylic acid, into indigo, but this has also
shown a disadvantage in the use of an indole-derivative substrate
through chemical synthesis [10,11]. To-date, indigo dyeing still uses
reducing agents, both of which are released into the environment during
the dying process. After dyeing, indigo may also be released during
washing and rubbing due to its dye fastness [3,12–14].
In addition to indigo dyes, various synthetic dyes are used at the
industrial scale, which are released as water in the dyeing process, as
well as after use in dyeing products, such as paper, plastics, fabrics, and
cosmetics [12,14–16]. This threatens aqueous ecosystems and can cause
serious environmental pollution if not properly treated; thus, the process
of decomposing or decoloring dyes is critical for mitigating environ­
mental hazards. During decomposition, indigoids dyes are removed via
physicochemical treatments, such as a chemical catalyst, adsorbent,
photocatalyst, electro-Fenton, and photoelectro-Fenton treatments [12,
17–19]. Microbial methods using enzymes, such as laccase and peroxi­
dase, are also applied [20,21]. The dye can be specifically removed in
the activated sludge during the water treatment process, as the activated
sludge includes various aerobic/anaerobic microorganismic oxidation
processes [22–24].
In addition to eco-friendly dye production technologies, consolidated
eco-friendly bioprocesses leading to environmentally friendly dye
decomposition have been spotlighted. Moreover, recently strengthened
environmental regulations and changes in environmental awareness
have spurred additional research into eco-friendly dying and treatment

* Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, Gyeonggi-do, Republic of Korea.
E-mail address: kychoi@ajou.ac.kr.

https://doi.org/10.1016/j.dyepig.2020.108749
Received 28 May 2020; Received in revised form 28 July 2020; Accepted 30 July 2020
Available online 8 August 2020
0143-7208/© 2020 Elsevier Ltd. All rights reserved.
K.-Y. Choi Dyes and Pigments 184 (2021) 108749

methods using biological processes. As a result, many enzymes involved
in dye degradation have been identified through deoxyribonucleic acid
(DNA) sequencing and the identification of microbes involved in dye
degradation across various biotechnological advances [20,25]. The
chemical structures of intermediates in dye decomposition processes
have also been well established [26,27]. In this review, engineered
bio-indigo degradation and discoloration via eco-friendly, in vitro and in
vivo enzymatic and microbial processes are discussed, with consider­
ation of their limitations in future research. It is expected that this
synthesis will provide useful insights for the treatment of (indigoid) dyes
in the future.
2. Microbial degradation and discoloration of indigo
The overall decolorization of textile dyes, with respect to microbial
and wastewater treatments, were extensively reviewed by Robinson
et al. and Fu et al. [28–30]. The review of Fu et al. [29] especially
highlighted the fungi-dependent decolorization of industrial textile dyes
in wastewater. According to their review, fungi can degrade or decol­
orize through biosorption or enzymes secreted by their host cells. The
screening and identification of microorganisms involved in indigo
degradation and detoxification is quite meaningful and applicable to the
biological wastewater treatment of activated sludge processes [31].
Moreover, indigo-degrading microorganisms can be used as safe pro­
biotics. For example, Fdhila et al. utilized an indigo-degrading Bacillus
consortium as a living probiotic on Crassostrea gigas reared in seawater
contaminated with indigo dye, and found that they greatly decolorized
the indigo and decreased the abundance of hepatocytes [32]. Indigo
degradation and discoloration are also possible through the use of mi­
crobial biotransformation and purified enzymes.
The discoloration of indigo using microbes is carried out in parallel
with removal by biosorption by cells plays an important role in the in­
digo dye discoloration through electrostatic interactions between the
carboxy-, amino-, and phosphate functional groups on cell surfaces and
Congo Red dye [33,34]. According to Manai et al. [35], the decolor­
ization of indigo in batch cultures of Chaetomium globosum could be
achieved with 8% adsorption and 92% biodegradation. However, bio­
sorption is inappropriate for removing dyes due to the need for addi­
tional recovery processes, including cellular recovery, and the superior
efficiency of synthetic sorbents. Instead, the decomposition of dyes by
the various enzymes produced by cells is more efficient, and the
whole-cell biotransformation of indigo carmine by bacteria, cyanobac­
teria, fungi, and yeast cells have extensively studied (Table 1).
2.1. Indigo discoloration by filamentous fungi
Among them, filamentous fungi have been reported to degrade dyes
more effectively than other microorganisms due to the presence of lig­
ninolytic enzymes, such as laccase and peroxidase, involved in dye
degradation [36]. Fungal systems are one of the most efficient biological
treatments of not only undesired industrial wastes, but also
dye-containing effluents [37]. For example, the efficacies of
enzyme-producing fungi, such as Trametes hirsute, Aspergillus alliaceus
strain 121C, Trametes trogii, Trametes villosa, Coriolus versicolor, Chaeto­
mium globosum, Pleurotus ostreatus, Ganoderma applanatum, Lentinula
edodes, Phanerochaete chrysosporium, and Curvularia lunata have been

Table 1
Whole-cell biodegradation of indigoids and metabolites.
Cells/sources Substrate Concentration Reaction engineering Discoloration Metabolites Ref.
(g/L) efficiency (%)

Burkholderia sp. IDO3 Indole 0.1 pH 4–9, 25–35 ◦ C, 0–250 rpm 100% Isatin, anthranilate [38]
Cupriavidus sp. SHE Indole 0.1 24 h, 100-mL flask, 30 ◦ C 100% Isatin, unidentified [39]
C15H8N2O3 (m/z 265.0)
Exiguobacterium sp. PMA 4- 0.075 32 h, 100-mL flask, 30 ◦ C 100% Indole, isatin, anthranilate, [40]
chloroindole salicylic acid
Bacillus sp. MZS10 Indigo 0.1 5-L stirred-tank fermenter, 30 C
◦
87.31% Isatin, isatin sulfonic acid, [41]
carmine indoline sulfonic acid
Bacillus aryabhattai DC100 Indigo 0.18 72 h, 250-mL flask 98.31% – [42]
carmine
Aeromonas hydrophila Indigo 0.1 7 d, 30 ◦ C 85% – [43]
Activated sludge Indigo 0.72 ga activated sludge process (ASP), 98%, KSindb 20.01 g/ – [31]
2.5-L batch reactors m3
Anabaena flos-aquae UTCC64 Indigo 0.01 14 d, 25 ◦ C, 50-mL flask 71.92% Isatin, anthranilate [44]
Phormidium autumnale UTEX1580 Indigo 0.01 14 d, 50-mL flask 91.22% Isatin, anthranilate [44]
Synechococcus sp. PCC7942 Indigo 0.01 14 d, 50-mL flask 0% Isatin, anthranilate [44]
Diutina rugosa KR262715 Indigo 0.01 5 d, 35 ◦ C, 2-g cell biomass 99.97% 1, 2-dihydro-3H-indol-3-one, [45]
cyclopentanone
Chaetomium globosum IMA1 Indigo 0.7 10 d, 6.22-g cell biomass 99.8%c, 88.4% COD – [35]
KJ472923 removal
Pleurotus ostreatus, Ganoderma Indigo 0.1 10–22 d, plate assay 100% – [46]
applanatum, Lentinula edodes
Curvularia lunata URM, Phanerochaete Textile 0.167 (BOD), 10 d, 5-L aerated bioreactor, 28 98% – [47]
chrysosporium URM 6181 effluent 0.354 (COD) ◦
C 93%
Aspergillus alliaceus strain 121C Indigo 0.15 9 d, 30 ◦ C 98.6% – [48]
Trametes hirsuta Indigo 0.07 3 d, 1-L bioreactor, immobilized 100% – [49]
on stainless steel sponge
Trametes trogii, Trametes villosa Indigo 0.0234 In vitro assay, 30 min, 27% – [50]
Coriolus versicolor carmine asparagine as nitrogen source 80%
88%
Trametes trogii Indigo 0.0234 In vitro assay 30 min, glutamic 94% – [50]
carmine acid as nitrogen source
Paenibacillus larvae Indigo 0.1 8 h, 0.5-L flask 100% (30 ◦ C), 92% Isatin sulfonic acid, [51]
carmine (40 ◦ C) anthranilic acid

Abbreviations: ADH, alcohol dehydrogenase; BOD, biological oxygen demand; COD, chemical oxygen demand; DTT, dithiothreitol, Fre, flavin reductase; VSS, volatile
suspended solid.
a
CODS/g VSS.
b
Half-saturation coefficient of heterotrophs for indigo dye.
c
8% by fungal adsorption, 92% by enzymatic degradation.

2
K.-Y. Choi
reported (Table 1). In the case of indigo or indigo carmine (5,5
ind-indigodisulphonic acid salt) in the oxidized state, due to their low
solubilities, studies on discoloration by these fungi have been conducted
at concentrations of <0.1 g/L. The indigo discoloration efficiency in
terms of conversion % by fungi is relatively high, but it takes a relatively
long fermentation cycle, which suggested the improvement of process
efficiency through engineering is necessary.
Most fungal species have shown 100% indigo discoloration activities
after more than 5 days of incubation. While the bio-discoloration of
indigo dyes by fungi are the most effective, the activities and perfor­
mances of fungal species were greatly affected by several factors,
including the presence of metal ions and their concentrations, temper­
ature, pH, and process design, the latter of which needs to be optimized
to obtain the desired discoloration performance. Khelifi et al. [48] used
A. alliaceus strain to discolor indigo carmine. They achieved a 98.6%
discoloration activity against 0.15 g/L of indigo within 9 days. However,
the activity was greatly affected by the nitrogen and carbon sources used
for the A. alliaceus culture, suggesting that the subtle optimization of
fungal cultures could affect indigo treatments. Similarly, Levin et al.
[50] demonstrated the nitrogen source-dependency on indigo decolor­
ization by white-rot fungi. The indigo carmine treatment by culture
filtrates of three different fungi, T. trogii, T. villosa, and C. versicolor var.
antarcticus, revealed that L-glutamate was the best nitrogen source for
laccase and manganese peroxidase enzyme production, allowing for the
highest laccase (188.3 U/ml) and manganese peroxidase (4.5 U/ml)
activities to be obtained. Crude filtrates exhibited 94% indigo carmine
degradation activity within 30 min.
Papadopoulou et al. [46] demonstrated the optimization of several
factors, including the Cu2+ ion concentration for Pleurotus, Ganoderma,
and Lentinula mushroom genera-dependent indigo discoloration via
Box-Behnken design. The lag time of P. pulmonarius varied depending on
Cu2+ and nitrogen concentrations, which affected further mycelium
extension and decolorization rates, and subsequent laccase activity.
Another issue of interest in the process design of large-scale treatments
of textile effluents, including indigo, was noted by Miranda et al. [47],
who designed a 5-L aerated bioreactor for effluent discoloration. The
treated effluent contained indigo dye with a high chemical oxygen de­
mand (COD) of 0.354 g/L, which is commonly observed in textile
effluent. They cultured two strains of C. lunata and P. chrysosporium
independently in a 5-L working volume reactor and achieved higher
enzyme production activities and discoloration efficiencies of >93%
with ten days of incubation. Another process engineering approach to
obtain higher laccase activity was developed by Rodrı ́guez Couto et al.,
who utilized a stainless steel sponge as an extracellular laccase enzyme
carrier [49]. T. hirsuta was used for laccase production in a 1-L biore­
actor, wherein laccase enzymes were immobilized on the stainless steel
sponge. The highest laccase activity (2200 U/L) was obtained and 0.07
g/L of indigo was completely decolorized within 3 days. Yeast has also
been used for indigo degradation. Bankole et al. [45] isolated a strain of
Diutina rugosa that could decolorize 0.01 g/L of indigo dye completely
within 5 days of incubation. In yeast systems, lignin peroxidase and
NADH-DCIP reductase were identified to play major roles in indigo
degradation, and the removal effect by partial adsorption was also
verified.
The primary advantage of fungi-dependent treatments of indigoids is
the use of various extracellular enzymes produced by host cells. Indi­
goids, as well as various toxicant by-products, can be treated by highly
active laccase, manganese peroxidase, and lignin peroxidase, and
applied to various purposes. In particular, ligninolytic extracellular
enzyme treatments involve simple centrifugation, and higher enzyme
activity achieved through the filtration of crude extracts is a substantial
security advantage. However, the use of fungi for indigo degradation
requires more engineering for process applications. The treatment of dye
in mixed cultures, such as biological water treatments, requires the
consideration of various factors, such as the low growth rate of fungi and
laccase, complex incubation conditions needed for peroxidase
3
Dyes and Pigments 184 (2021) 108749
production, and the loss of nutrients by fast growing bacteria. Addi­
tionally, the economics of additional processes should also be accounted
for when using crude extracts via filtration.
2.2. Indigo discoloration by bacteria
In terms of discoloration and degradation activity, fungi are reported
to be superior to other microorganisms. However, the disadvantages of
the delicate culture conditions, such as pH, nutrient load, and long lag
time, as well as the generation of toxic by-products have limited their
large-scale application. Besides fungal strains, several bacteria, cyano­
bacteria, and actinomycetes have also been reported to play a key role in
indigo degradation, and securing active bacterial strains via screening is
regarded as important. For example, Chen et al. [43] screened several
bacterial strains with the capability of degrading indigo from sludge and
mud lakes. Among them, Aeromonas hydrophila was screened and
exhibited an 85% indigo discoloration activity in 7 days. Interestingly,
the bacterial discoloration activity depended on the nitrogen source of
the yeast extract, whereas the carbon source of glucose inversely
inhibited the discoloration. These findings suggest to us that it is
essential to optimize the nitrogen source by balancing its performance
with economic efficiency during process design in order to scale-up to
the industrial level.
Fast-growing bacteria with high degradation potentials have
attracted great attention due to their high efficiency and cost savings.
Among them, Bacillus species are excellent candidates for dye degra­
dation and have been reported to degrade several commercial dyes with
excellent performances. For example, Paz et al. [42] reported on the
ligninolytic enzyme-producing B. arybhattai strain DC100, which could
degrade dyestuffs from the textile industry. At the laboratory scale,
B. arybhattai could degrade 0.18 g/L of indigo carmine by nearly 100%
within 3 days and achieve high reductions of COD. This was due to the
rapid growth rate exhibited by Bacillus strains, which significantly
reduce decomposition times, as compared to fungi. Some peroxidase
and/or laccase enzymes were estimated to be responsible for degrada­
tion in synthetic dyes. Li et al. [41,52] also reported on a similar strain of
Bacillus sp., MZS10, which was able to degrade Azure B dye via quinone
dehydrogenase. This strain could decolorize 0.1 g/L of indigo carmine
by 87.31% in a 5-L fermentor within 15 h. Thus, it can be seen that the
rate of discoloration increases considerably with the use of bacterial
strains when compared to fungi, which take more than a week to achieve
similar levels of decoloration activity. Interestingly, Bacillus sp. MZS10
generated unique metabolites of indoline sulfonic acid, along with
oxidative metabolites of isatin, isatin sulfonic acid, in contrast to other
indigo carmine metabolites. The indoline sulfonic acid was the reductive
metabolite of indigo carmine, suggesting the existence of dehydroge­
nase- or reductase-dependent degradation pathways. In addition to the
laccase- and peroxidase-dependent oxidation pathways (Fig. 1).
Microbes that could degrade the biosynthetic indigo precursor,
indole, have also been assessed. Although indole is not a colorant dye, it
is the key precursor in indigo dye biosynthesis. Indigo can be prepared
by a reversible tryptophanase enzyme reaction, or synthesized in the
plant through the deglycoxylation reaction of indoxyl β-D-glucoside.
The indole is converted into indoxyl (3-hydroxyindole) with a C3-
specific hydroxyation reaction via the oxygenase enzyme and into in­
digo through continuous oxidation [53]. Indole has been used as a type
of dye precursor in the form of indole derivatives and is found in the
effluent of the dyestuff processing industry. Indole is not only a key
precursor of indigo biosynthesis, but also a bottleneck intermediate in its
degradation. Due to its chemotoxicity and deleterious environmental
effects, it has been subjected to decomposition by several treatments. In
biological treatments, several microorganisms, such as Alcaligenes sp.,
Burkholderia sp., Pseudomonas sp., P. aeruginosa Gs, Aspergillus niger, and
P. ostreatus are reported to have the ability to degrade indole anaero­
bically or aerobically [54–60]. For example, Qu et al. [39] isolated the
SHE strain of Cupriavidus sp., which could anaerobically degrade 0.1 g/L
K.-Y. Choi
Fig. 1. Degradation and discoloration of indole, indigo, and indigo carmine by cells and enzymes.
of indole by 100% in 24 h, using indole as the sole carbon source.
Similarly, Ma et al. [38] isolated Burkholderia sp. strain IDO3 from
sludges, which showed an excellent degradation ability of 100% for 0.1
g/L of indole within 14 h. Moreover, they identified the key enzymes of
indole oxygenase (IifC) and reductase (IifD) responsible for indole
degradation that supported indole degradation pathways and generated
intermediates.
2.3. Indigo discoloration by cyanobacteria
As the effluents of dye waste processing contain a variety of chem­
icals, and dye chemicals are very recalcitrant, tertiary treatment
methods, such as algal (cyanobacteria) treatments, are also important in
wastewater treatment. Until now, several cyanobacteria, such as
Chlorella sp., Oscillatoria sp., Westiellopsis sp., and Lynghya sp., have been
reported to have the ability to degrade toxic chemicals and dyes
[61–64]. Recently, Dellamatrice et al. demonstrated the successful
degradation and detoxification of indigo dye using three different cya­
nobacteria: Anabaena flos-aquae UTCC64, Phormidium autumnale
UTEX1580, and Synechococcus sp. PCC7942 [44]. Among them, Syn­
echococcus did not show any indigo degradation activity, while the other
two displayed discoloration activities of 71.92% and 91.22% after 14
days of incubation. However, the intermediates of isatin and anthranilic
acid generated during incubation were not completely degraded.
Further engineering seems to be required to achieve the high perfor­
mance required at the industrial level due to the long degradation time
compared to bacterial treatments and low activity for dye-containing
sludge.
2.4. Identification of indigo degradation products
Recently, Ma et al. [20] extensively reviewed the microbes involved
in indole degradation and indigo production, and several enzymes
involved in indole hydroxylation were also summarized. The represen­
tative intermediates in the indole degradation pathway were identified
as isatin and anthranilate, although all of the intermediates were not
identified and they varied depending upon the microbes present. In
general, indole – as a degradation product of indigo – is a key inter­
mediate, and is further oxidized by oxygenase and converted into
indoxyl, followed by oxidation, resulting in the formation of 2,3-dihy­
droxyindole, isatin, formylanthranilic acid, antihylic acid, salicylic
acid, and catecholoid. Campos et al. proposed a possible mechanism for
4
Dyes and Pigments 184 (2021) 108749
laccase catalyzed degradation of indigo dye (Fig. 2) [24]. The degra­
dation metabolites of gentisate, under anaerobic conditions, and isatin
sulfonic acid and indole sulfonic acid, during reductive degradation,
have also been reported [41]. Interestingly, Qu et al. [39] analyzed the
indole metabolites of a newly isolated Cupriavidus sp. strain SHE with a
mass-to-charge ratio (m/z) of 265 and postulated that they were
dimerization or indole ring-opening products.
3. Enzymatic degradation pathways of indigo
3.1. Fungal enzymes involved in indigo degradation
Ligninolytic enzymes are utilized in indigo degradation, among
which laccase has been widely utilized due to its high activity and sta­
bility. Laccase enzymes (E.C 1.10.3.2), which are copper-containing
polyphenol oxidases, produce four free electrons that react to a wide
range of phenolic and non-phenolic molecules by reducing oxygen
molecules into water molecules. In general, white rot fungi-derived
laccase enzymes have been widely utilized to degrade various dye­
stuffs due to their high activity and redox potential. The laccase enzymes
secreted extracellularly by fungi are used in both their crude extract and
purified forms.
Several fungal laccase enzymes have been reported to be able to
degrade dyestuffs through the production of the intracellular and
extracellular ligninolytic enzymes of laccase, manganese peroxidase,
and versatile oxidases (Table 2). The fungal strains of T. trogii, Funalia
trogii ATCC 200800, T. versicolor ATCC 200801, white rot fungi (WRF-1),
T. trogii BAFC 463, T. orientalis, Cerrena sp., Ceriporiopsis subvermispora
CZ-3, Scytalidium thermophilum, Panus rudi, C. cinerea, L. edodes,
P. ostreatus, and Anoderma sp. En3 were identified to have the capability
of indigo or indigo carmine discoloration [24,65–79,94,95]. In general,
fungal laccases works better at low temperatures and under acidic
conditions. Most fungal laccases show their highest activity at pH values
of 2.5–4.5, with >95% of high discoloration activity against 0.1 g/L of
indigo carmine. The kinetic values of fungal laccase enzymes deter­
mined by 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)
substrate are Km values ranging from 10 μM to 0.62 mM and a kcat range
of 10–400 s− 1. According to Campos et al. [24], T. trogii BAFC 463
laccase showed an excellent kinetic activity of 0.25 mM Km and 399 s− 1
kcat for ABTS, and 46% showed 0.023-g/L indigo carmine decomposition
activity during a 2-h period [94]. Against indigo carmine, Daâssi et al.
[69] reported the kinetic values of T. trogii laccase enzymes as 47.94
K.-Y. Choi Dyes and Pigments 184 (2021) 108749

Fig. 2. Proposed pathway of laccase-dependent indigo degradation [24].

mg/L (0.1 mM) of Km and 10.43 mgL− 1min− 1 (22.3 μM/min) of Vmax. indigo carmine discoloration with an activity of 92% [83]. Fungal lac­
Furthermore, Ben Younes and Sayadi [68] determined the Km and kcat case and peroxidase enzymes are clearly very powerful biocatalysts for
values of Scytalidium thermophilum laccase enzyme to be 7.44 mM and discoloring indigo; however, fungal-derived laccase enzymes are limited
10.9 s− 1, respectively, against indigo carmine. More specifically, they in their industrial application, as it takes a long time to produce enzymes
demonstrated indigo carmine degradation using laccase enzyme isolated with the desired production yields, which also vary depending upon
and purified from S. thermophilum, and transformed the highest con­ culture nutrients due to their recalcitrant growth and low working pH.
centration of 5 g/L of indigo carmine efficiently up to 50% within 6 h Therefore, studies on high-active laccase enzymes isolated from new
using 0.1 U ml/L of purified laccase without any mediators for the first fungal species or evolved enzymes through genetic engineering to obtain
time [68]. high yields and activities are on-going.
The most critical issues regarding fungal (including mushrooms)
laccase enzymes, in spite of their high activity and robustness, are long
3.2. Bacterial enzymes involved in indigo degradation
fermentation cycle in the fungal host system [96]. To overcome these
limitations, several researchers have attempted to generate heterolo­
In addition to fungal laccase and peroxidase enzymes, bacterium-
gous expressions of fungal laccase enzymes in yeast systems. For
derived laccase enzymes are also known to exhibit various dyestuff
example, Xu et al. [75] demonstrated the expression of a thermophilic
decomposition abilities. Unlike fungal laccase, bacterial laccase has the
fungal laccase, Lcc9, from C. cinerea in Pichia pastoris, and obtained
advantage of displaying a relatively high stability under high tempera­
3138 U/L of high-activity recombinant laccase. Additionally, the re­
ture and pH conditions. Above all, it has the great advantage of being a
combinant laccase exhibited improved stability and activity under
recombinant system capable of securing high laccase yields. To date,
alkaline (up to pH 9) conditions. Indeed, several fungal laccases have
various bacterial laccase enzymes have been isolated and produced for
been heterologously expressed in P. pastoris to obtain higher production
extensive application. Among them, several bacteria, such as B. subtilis,
yields and rates [66,77,94,95].
B. amyloliquefaciens, B. licheniformis, B. licheniformis LS04, Bacillus sp.
Recombinant laccase production in yeast systems is clearly a
CF96, γ-proteobacterium JB, A. faecalis XF1, and Thermus thermophilus,
powerful way to obtain desirable laccase enzymes (Table 3). However,
have been reported to have the ability to degrade and decolorize indigo
other issues, such as glycosylation modification and low expression, may
dye [84,85,87–92]. Most bacterial laccase enzymes display optimized
also limit the production of recombinant laccase. Another strategy to
activity at 40–60 ◦ C and broad pH stability at pH 4–8. Most studies of
overcome such problems is to utilize coculture-induced fungal laccase
indigo carmine decolorization activity, from 25 mg/L to 0.1 g/L, have
production in native host systems. For example, Pan et al. [76]
shown activities of >90% for in vitro experiments. Among the most
demonstrated an enhanced Lcc9 laccase production in C. cinerea
interesting laccase enzymes, the B. subtilis CotA laccase enzyme exhibits
Okayama 7 via coculturing with Gongronella sp. w5. In their study,
a 95% discoloration activity against 25 mg/L indigo carmine (10 min,
laccase activity was greatly increased (by up to 1800 U/L) in the
100 ◦ C, and pH 10) [87]. Most bacterial laccase enzymes exhibit sub­
coculture, which was 900 times higher than in the single culture of
strate affinities of several tens to hundreds of micrometers for ABTS, and
C. cineria.
kcat activities on the order of 10− 1 to 103 s− 1.
Besides laccase, fungal oxidase enzymes have also been reported to
In order to utilize a large amount of bacterial laccase enzyme, it is
degrade indigo. For example, fungal manganese peroxidase from Tra­
critical to express the laccase gene in an external host. In general, E. coli,
metes sp. 48424 and P. chrysosporium were used for indigo carmine
P. pastoris, and B. subtilis have been used as host systems, with E. coli
degradation, and showed excellent discoloration activities of 94.6% and
being the most commonly used to produce bacterial enzymes; laccase
90.18%, respectively [80,81]. Also, peroxidase from plant source of
enzymes from various bacteria have been produced in E. coli. To over­
horseradish has been employed for degradation and discoloration of
come low enzyme yields and inhibition during cell lysis, Koschorreck
indigo carmine. Terres et al. reported kinetics of decolorization of indigo
et al. [89] reported the heterologous expression of the Tth laccase
carmine by horseradish peroxidase, which revealed that HRP has rate
enzyme, which was isolated from T. thermophiles in E. coli via purifica­
constant of 5.5 × 1012 min− 1 (kobs) with 12.59 min of t1/2 [82]. Bilirubin
tion through thermolysis, thereby increasing enzyme yields up to 51.33
oxidase from Myrothecium verrucaria 3.2190 has also been utilized for
U/mg. The purified laccase also exhibited a 100% indigo carmine

5
K.-Y. Choi Dyes and Pigments 184 (2021) 108749

Table 2
Enzymatic degradation and discoloration of indigoids.
Enzymes Origin Expression Reaction Substrate (g/L) Discoloration/degradation activity Ref.
(substrate)

Laccase Trametes orientalis Extracellular, 1 U/mL, 48 h, pH 4, 50 ◦ C Indigo (0.05) 42.74% [65]

precipitation Km 0.333 mM, kcat 21.81 s− 1 (for ABTS)
Laccase Trametes trogii BAFC Expressed in Pichia 2 h, pH 6, 70 ◦ C, IC (0.023) 46%, [24]
463 pastoris, purified acetosyringone mediator Km 0.25 mM, kcat 399 s− 1 (for ABTS)
LacI laccase Cerrena sp. Expressed in P. pastoris, 24 h, pH 3.5, 55 ◦ C IC (0.05) 97.5%, [66]
purified Km 0.028 mM, kcat 332.4 s− 1 (for ABTS)
Laccase Ceriporiopsis Extracellular, purified 24 h, pH 2.5, 30 ◦ C IC (0.08) 95.28%, [67]
subvermispora CZ-3 Km 0.222 mM, kcat 109.59 s− 1 (for ABTS)
Laccase Scytalidium Extracellular, purified 0.1 U/mL, 6 h, pH 5, 60 C ◦
IC (5) 50% [68]
thermophilum Km 7.44 mM, kcat 10.9 s− 1 (for indigo
carmine)
Laccase Trametes trogii Extracellular 0.5 U/mL, 2.5 h, pH 4.5, 45 IC (0.06) 99.76%, Km 47.94 mg/L, Vmax 10.43 mg/L [69]
◦
C, HBT mediator (for indigo carmine)
Laccase White rot fungi (WRF- Extracellular, purified 12 d, pH 5, 30 ◦ C IC (0.2) ~100% (by cells), isatin sulfonic acid and 4- [70]
1) amino-3-methylbenzenesulphonic acid
Laccase Trametes trogii Extracellular, purified 6.5 U/mL, 24 h, pH 4.5, 30 IC (0.023, 50 ~90% [71]
◦
C, HBT mediator μM)
Laccase Panus rudis Extracellular, purified 5 ng/mL, 0.5 h, pH 4, 45 ◦ C, IC (0.041) 81%, [72]
ABTS mediator
Laccase Funalia trogii ATCC Culture filtrate 72 h, 30 ◦ C IC 87%, 40.29 U/mL [73]
200800 88%, 12.09 U/mL
Trametes versicolor
ATCC 200801
Laccase T. versicolor Extracellular, purified pH 4.5, 35 ◦ C – Km 0.62 mM, Vmax 8264 U/L (for ABTS) [74]
TH1 Trametes hirsuta, Extracellular, purified 24 h, pH 4.5, 30 ◦ C, Indigo (0.001) 31%, 30.5%, 27% [24]
TH2 Sclerotium rolfsii acetosyringone mediator
SRL1 laccase
Laccase Coprinopsis cinerea Expressed in P. pastoris pH 6.5, 70 ◦ C, methyl IC 98.2%, [75]
syringate mediator 3138 U/L (for ABTS)
Lcc9 laccase Coprinopsis cinerea Gongronella sp. w5 1 h, pH 6.5, 60 ◦ C, Indigo (0.029) 75% [76]
coculture, purified ABTS mediator Km 0.0109 mM, kcat 215.31 s− 1 (for ABTS)
Lcc1B laccase Lentinula edodes Expressed in P. pastoris, 5 μg laccase, 1 h, pH 2, 40 IC >80% [77]
purified ◦
C, TEMPO mediator Km 0.0425 mM, kcat, 67.5 s− 1 (for ABTS)
Laccase Pleurotus ostreatus Three phase partitioning 10 U/mL, 1 h, pH 4.5, 30 ◦ C, IC (0.0001) 97% [78]
purification acetosyringone mediator
Laccase Ganoderma sp.En3 Expressed in P. pastoris, 0.1 U, 36 h, pH 5, 30 ◦ C, Waste indigo 83.72% [79]
purified kojic acid mediator effluent (80%)
Manganese Trametes sp. 48424 Expressed in P. pastoris 100 U/L, 18 h, pH 4.5 IC (0.1) 94.6% [80]
peroxidase
Manganese Phanerochaete Solid state fermentation 6h IC 90.18% [81]
peroxidase chrysosporium
Horseradish Horseradish (TOYOBO) Purified 0.003% H2O2, 60 ◦ C, pH 4-6 IC (25 μM) 100%, kobs 5.5 × 1012min− 1, t1/212.59 min [82]
peroxidase
Bilirubin Myrothecium verrucaria Extracellular, purified 1 U/L, 40 min, pH 7.5, 50 C ◦
IC (0.1) 92%, [83]
oxidase 3.2190 Km 21 μM, kcat 21.8 s− 1 (for bilirubin)
Km 0.126 mM, kcat 19.8 s− 1 (for ABTS)
Laccase Alcaligenes faecalis XF1 Extracellular, purified 1 U/mL, 3 h, pH 8, 50 ◦ C IC (0.1) 70%, [84]
Km 0.5 mM, kcat 1242.75 S-1 (for ABTS)
Laccase Bacillus sp. CF96 Extracellular, purified pH 8, 60 ◦ C Indigo 90% [85]
Km 0.737 mM, Vmax 100.5 U/mg (for
syringaldazine)
Laccase D500G Bacillus licheniformis Expressed in P. pastoris 1 h, pH 4, 50 ◦ C, IC (0.025) ~100% [86]
LS04 acetosyringone mediator Km 0.058 mM, kcat 6.7 s− 1 (for ABTS)
CotA laccase Bacillus subtilis Expressed in P. pastoris, 10 min, pH 10, 100 ◦ C IC (0.025) 95%, [87]
purified Km 0.146 mM, kcat 14.4 s− 1 (for ABTS)
Laccase Bacillus Expressed in P. pastoris, 0.5 h, pH 6.6, 60 ◦ C, IC (0.025) 99% kcat/Km0.16 s− 1 μM− 1 (for ABTS) [88]
amyloliquefaciens purified acetosyringone mediator
2+
Tth Thermus thermophilus Expressed in Escherichia pH 7.5, 40 ◦ C, IC (0.023) 100% (1 h, w/Cu ), 51.33 U/mg [89]
CotA Baccilus subtilis coli, thermolysis acetosyringone mediator 100% (1 h, w/o Cu2+), 7.07 U/mg
Ssl1 laccase Streptomyces sviceus 100% (4 h, w/o Cu2+), 0.81 U/mg
Laccase Bacillus licheniformis Expressed in P. pastoris, 1 h, pH 9, 40 ◦ C, IC (0.025) ~100% [26]
purified acetosyringone mediator Km 0.044 mM, kcat, 5.6 s− 1 (for ABTS)
Laccase Bacillus licheniformis Expressed in P. pastoris, 1 h, pH 9, 40 ◦ C, IC (0.025) ~100% [26]
purified acetosyringone mediator Km 0.044 mM, kcat, 5.6 s− 1 (for ABTS)
Laccase Bacillus licheniformis Spore suspensions lysate 1 h, pH 9, 40 ◦ C, IC (0.025) 91.99% [90]
LS04 acetosyringone mediator
CotA laccase Bacillus subtilis B. subtilis spores display, 2 h, pH 8, 60 ◦ C, HBT IC (0.0446, 0.1 44.6 μg indigo carmine/g spores, [91]
mediator mM) Km 0.443 mM, Vmax 150 U/mg kcat s− 1 (for
ABTS)
Laccase γ-Proteobacterium JB column chromatography 2 h, pH 9, 55 ◦ C, IC (0.004, 10 98.3%, [92]
purification syringaldehyde mediator μM) Km 9.6 μM, Vmax 96.4 mM/min/mg (for
indigo carmine)
Laccase Streptomyces sp. C1 column chromatography 2.0 U mL− 1, 2 h, pH 8, 40 ◦ C, IC (0.016) 83.7% [93]
purification syringate mediator Km 0.43 mM, kcat, 8.45 s− 1 (for ABTS)

6
K.-Y. Choi Dyes and Pigments 184 (2021) 108749

*E. coli whole cell reaction, **conversion.

Abbreviations: IC, indigo carmine; HBT, 1-hydroxybenzotriazole; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl.

Table 3
Enzyme fixation for enhanced indigo carmine degradation.
Enzymes Immobilization template and linkers Reaction Loading Remarks Ref.
capacity

Commercial laccase PVA-co-PE membranes template, laccase- 3 h, IC 0.02 g/L 2 g/L 99.5% discoloration, 14 cycles [97]
Cu2(PO4)3⋅3H2O hybrid nanoflowers (98.52)a
Laccase expressed in Fe3O4@ZIF-8 nanoparticles 6 h, 80 ◦ C – Thermal stability (46% for 6 h at [98]
E. coli 80 ◦ C) 5 cycles
Peroxidase MNPs-TA-starch-CLEAs-peroxidase 3 h, IC 0.02 g/L – 68% discoloration [99]
Glucose oxidase tannin coated NiFe2O4/T/GOx magnetic 90 min – 98.6% discoloration [100]
nanoparticles
Fungal laccase (Tplac) chitosan beads grafted cross-linker genipin 96 h, 55 ◦ C 44.06 g/L 56.25% discoloration, [101]
11 cycles (>55%)
Pycnoporus sanguineus Anode: microporous activated carbon fiber felt- 0.01 g/L, 1 h, 30 ◦ C, 10 V – 83.60% discoloration, bio- [102]
laccase laccase, cathode: Pt electrochemical remediation
Novozym® 51003 EDTA-Cu (II) chelating Fe3O4@SiO2 magnetic 4 h, 40 C pH 3.5, 5 mM IC
◦
– 97% discoloration, Km 37.5 μM, [103]
nanoparticles Vmax 6.12 μM/min, 5 cycles (73%)
Armoracia rusticana Cross-linking with ethylene glycol-bis [succinic – – 84.35% degradation [104]
Horseradish acid N-hydroxysuccinimide, (EG-NHS) packed bed
peroxidase reactor
Laccase and glucose Laccase/MnFe2O4/calcium alginate and glucose pH 7 and pH 5 – 25.09% and 44.03% degradation [105]
oxidase oxidase/MnFe2O4/calcium alginate
nanocomposites
Papaya laccase Chitosan beads entrapments 8 h, pH 10, 37 ◦ C, 0.05 g/L IC, 98% IY and 100% discoloration, 4 cycles [106]
100% LE (<20%)
CotA multicopper Fe3O4 Magnetic Ganoderma lucidum spore 1 h, 25 ◦ C, 10 mg microspheres, 75 mg/g 99% discoloration, 81% recovery, [107]
oxidase microspheres 0.05 g/L IC 10 cycles (75%)
Funalia trogii Laccase Cu-impregnated apricot stone-based activated 5 min, 0.025 g/L IC – 62% discoloration, 29.23 U/mL [108]
carbon (for ABTS), 3 cycles (92.5%)
Cerrena unicolor laccase Adsorption and covalent bonds formation on silica- 256 U, 18 d, 25 C, 0.005 g/L IC
◦
0.75 mmol/g 100% discoloration [109]
gel carriers, continuous process
Trametes versicolor CPC-silica beads 31.5 ± 4.3 U laccase/g, 3 h, pH – 85.2% discoloration, 4.87 μM/U/h [110]
laccase 5, 25 ◦ C, Acid blue 74 0.036 mM initial rate
Cerrena unicolor laccase Covalent-binding on the mesostructured siliceous 5 h, pH 5.3, 25 ◦ C, 0.05 g/L IC 1–1.5 mmol kcat/Km 0.88 min− 1 μM− 1 (for [111]
cellular foams functionalized by glutaraldehyde g− 1 ABTS), 1 month storage (55–75%)

Abbreviations: CLEA, cross-linked enzyme aggregates; CPC, controlled porosity carrier; IC, indigo carmine; IY, immobilization yields; LE, loading efficiency; MNP,
magnetic nanoparticle.
a
Cycles and blanket indicate the reusability and recovered activity.

discoloration activity in the presence of Cu2+ ions. However, in many
cases, recombinant laccase enzymes in E. coli host systems form insol­
uble aggregates. Recently, the production of high-yield laccase enzymes
by expressing Bacillus-derived laccase enzymes in P. pastoris have been
reported [26,86–88]. The methylotrophic P. pastoris is another widely
used expression host for heterologous laccase production, and provides
the advantages of high cell density and protein secretion. One important
process in laccase production through genetic recombination is protein
separation through cell disruption, as protein separation and purifica­
tion processes are additionally required unless secretion is induced. In
this regard, the P. pastoris host is a very efficient system for laccase
enzyme production. Laccase enzymes from B. licheniformis, B. subtilis,
and B. amyloliquefaciens have been expressed in P. pastoris and purified
for the discoloration of indigo carmine [26,86–88].
Recombinant enzyme production enables increased enzymatic ac­
tivity and improved stability through enzyme engineering [112]. Wang
et al. [86] generated B. licheniformis laccase D500G mutants through
site-directed mutagenesis to obtain increased production yields in a
P. pastoris host system. The activity of mutated D500G enzymes was
greatly increased, up to 347 U/L, which was ~9.3 times higher than that
of wild-type laccase. Many laccase enzymes derived from Bacillus strains
have been reported, and the production of laccase enzymes in Bacillus
host systems has also been frequently described. The advantage of
Bacillus-derived laccase enzymes is that when using a Bacillus host, the
laccase enzyme can be utilized with cell incubation through the simul­
taneous expression of CotA proteins involved in spore coats and dis­
plays. For example, Lu et al. [90] employed a spore suspension lysate of
B. licheniformis laccase for indigo carmine discoloration. Their lysate
showed a 91.99% indigo carmine discoloration activity within 1 h of
reaction. Similarly, Cho et al. [91] used B. subtilis CotA laccase enzymes
for indigo carmine discoloration. The self-immobilized CotA laccase
enzyme, which was associated with the surface of B. subtilis spores,
showed a discoloration activity of 44.6 μg indigo carmine/g spores. The
CotA laccase enzyme has been demonstrated to serve as an environ­
mental whole-cell biocatalyst with the advantage of Bacillus strains
which have been generally recognized as safe (GRAS) microbial sources.
Multiple laccases derived from bacteria, particularly those originating
from actinomycetes, have also been reported for indigo carmine
discoloration. Lu et al. [93] reported on a laccase enzyme isolated from
Strepotomyces sp C1 in agricultural waste compost for the same purpose,
which belonged to a laccase-like multicopper oxidase (LMCO) respon­
sible for the decolorization of azo dyes. The Streptomyces LMCO showed
an 83.7% decolorization activity for indigo carmine after 2 h of incu­
bation at 40 ◦ C and pH 8.0. Laccase enzymes originating from γ-pro­
teobacterium have also been used for indigo carmine degradation. Singh
et al. [92] purified laccase enzymes from γ-proteobacterium JB, which
was isolated from an alkali-tolerant soil environment. In the presence of
a syringaldehyde mediator, this laccase could decolorize 10 μM of indigo
carmine with an efficiency of 98.3% in 2 h of reaction at 55 ◦ C. Inter­
estingly, the purified laccase enzyme was highly stable over a pH range
of 4–10, even after 60 days of incubation at 4 ◦ C.
Another issue regarding the laccase-dependent degradation of indigo
carmine that should be considered is the requirement of electron me­
diators for laccase enzyme activation [37]. If the substrate of the laccase
is large or if accessing the active site is difficult due to the inhibition of
diffusion, enzymatic reaction through a mediator is required. Mediators,

7
K.-Y. Choi
such as N-heterocycles bearing N–OH (e.g., violuric acid), N-hydrox­
y-N-phenyl acetamide, and N-hydroxybenzotriazole, have been reported
to be highly effective [113]. In general, indigo discoloration activity is
measurably higher in the presence of mediators, such as acetosyringone,
syringaldehyde, and 1-hydroxybenzotriazole. Although it is possible to
secure high enzyme activities, electron mediators impose limitations on
the terms of use for another substrate and the production of by-products
in industrial color discoloration. It is therefore necessary to select an
optimal mediator depending upon the chemical structure of the target
discoloration substrate and to optimize the mediator concentration
depending upon the substrate concentration employed.
3.3. Enzyme immobilization for indigo degradation and process
engineering
Enzymes involved in the discoloration of indigo, such as laccase and
peroxidase, generally have high activities and stabilities at high tem­
peratures and over a broad range of pH conditions. However, when
applying them at the large-scale industrial level, the demand for enzyme
recovery and reuse in a cost-effective manner is quite high. It is possible
to increase enzyme production yields through strain and enzyme engi­
neering, but it is also feasible to more effectively overcome indigo
discoloration through enzyme immobilization (Fig. 3). Various enzyme
carriers and supports have been used, and engineering studies have re­
ported on improvements made to the recyclability and stability of en­
zymes [114,115].
In general, silica and activated carbon are the commonly used
matrices for immobilizing enzymes. Due to its high biocompatibility and
excellent pH stability, silica-based glass beads are widely used in various
applications. For dye discoloration, several silica matrices, such as
imidazole-modified silica gel, silanized alumina particles, and glutaral­
dehyde cross-linked glass beads, have been examined [116–118]. For
example, Champagne and Ramsay [110] used controlled-porosity car­
rier (CPC) silica beads for T. versicolor laccase immobilization, which
showed an activity of 31.5 U laccase/g. Similarly, Rekuć et al. [109,111]
immobilized C. unicolor laccase into silica-gel carriers, including meso­
structured siliceous cellular foams functionalized by glutaraldehyde,
which showed an ~1 mmol/g loading capacity with 100% discoloration
activity for indigo carmine dye. Immobilized laccase enzymes in
particular showed 55–75% residual activities even after 1 month of
Fig. 3. Immobilization of the indigo-degradating enzymes laccase, peroxidase, and oxidase onto various beads with cross-linkers and nanoparticles.
8
Dyes and Pigments 184 (2021) 108749
storage.
In addition to glass beads, chitosan beads have recently been used for
stable enzyme immobilization due to their excellent biocompatibility
and low toxicity. Furthermore, they have also been applied to various
enzyme immobilization matrices due to their favorable physical prop­
erties, including their high porosity, sufficient adhesion area, and lower
resistance to mass transfer. By using a cross-linking reagent, such as
glutaraldehyde or genipin, it has been possible to stably maintain
enzyme activity across various ranges of pH. Ma et al. [101] utilized a
chitosan-genipin cross-linked entrapping matrix for fungal laccase
(Tplac) immobilization. Compared to free laccase enzymes, the immo­
bilized system showed improved pH, thermal, and storage stabilities,
with a residual activity of more than 55% after 11 cycles of use. The
highest (44.06 g-1) loading capacity (LC) and 96.85% immobilization
efficiency (IE) were achieved by using 0.1% (m/v) of genipin as the
cross-linker. Similarly, Jaiswal et al. [106] used chitosan for the
entrapment of purified papaya laccase, thereby leading to improved
catalytic efficiency (10-fold of kcat/Km) and thermal/pH-stability against
indigo carmine decolorization. The chitosan matrix exhibited an
immobilization yield (IY) of 98% and a 100% loading efficiency (LE).
Besides synthetic matrix surfaces, the direct use of cell surfaces could
be effective for the immobilization of laccase enzymes. Fan et al. [107]
generated hollow microspheres obtained from Ganoderma lucidum
spores by loading Fe3O4 nanoparticles, thereby immobilizing CotA
multicopper oxidase on the magnetic spore microspheres. The immo­
bilized CotA enzymes with 10 mg microspheres could decolorize 99% of
the indigo carmine present within 1 h of incubation, and could retain
75% of their residual activity after 10 consecutive cycles. The most
powerful advantage of magnetic spores is their rapid and easy recovery
by an external magnetic field. Recently, enzyme immobilization in
synthetic nanoparticles has attracted great attention due to the devel­
opment of nanoparticle manufacturing. Several nanoparticles, such as
tannin-coated NiFe2O4/T/GOx magnetic nanoparticles, Fe3O4@ZIF-8
nanoparticles, and Cu2(PO4)3⋅3H2O hybrid nanoflowers, have been
utilized for indigo carmine decolorization. Shojaat et al. immobilized
laccase and glucose oxidase on MNFe2O4 nanoparticles, which were
synthesized via coprecipitation with calcium alginate [105]. Each
immobilized laccase and glucose oxidase enzyme showed an activity of
44.03% and 25.09%, respectively.
The fabrication of the hierarchically porous nanofibrous membranes
K.-Y. Choi
has recently been utilized to immobilize laccase enzymes in order to
degrade indigo carmine. Ge et al. [119] reported on inorganic–organic
hybrid nanoflowers, which formed a flower-like structure via the com­
bination of metal ions and amino groups on enzymes with extraordinary
catalytic performance. These nanoflowers have subsequently been
widely used to immobilize several enzymes [120]. For example, Rong
et al. [121] produced laccase-Cu3(PO4)2 hybrid nanoflowers on the
surfaces of Cu8(PO3OH)2(PO4)4⋅7H2O nanoflowers that exhibited highly
efficient and stable Congo red dye degradation. Meanwhile, Luo et al.
[97] immobilized commercial laccase-Cu2(PO4) 3•3H2O hybrid nano­
flowers on hierarchically porous nanofibrous poly(vinyl)
alcohol-co-ethylene (PVA-co-PE) membranes. This immobilization sys­
tem showed excellent catalytic performance for indigo carmine degra­
dation, with an efficiency of 99.5%, and could maintain its activity for
least 14 cycles, suggesting that it may serve as a robust and eco-friendly
catalyst for wastewater treatment.
Cross-linked enzyme aggregate (CLEA) technology has been used for
carrier free methods of enzyme immobilization. Compared to using a
carrier matrix, such methods offer great advantages of simplicity, cost
efficacy, and feasibility to crude enzyme extracts. Mehde [99] generated
a peroxidase CLEA complex using magnetic nanoparticle (MNPs)-TA-s­
tarch aggregation agents, and could decolorize indigo carmine with 68%
activity in a 3-h reaction. The immobilization of enzymes with aggre­
gates during indigo degradation could also be enhanced in combination
with reaction process design. Bilal et al. [104] generated Armoracia
rusticana horseradish peroxidase aggregates with ethylene glycol-bis
[succinic acid N-hydroxysuccinimide] (HRP-CLEAs), which showed an
84.35% degradation activity against indigo. It is worth noting that they
designed a packed-bed reactor system using HRP-CLEAs, and this system
could retain nearly 60% of its residual activity after seven consecutive
cycles.
The high stability and reusability of immobilized enzymes enables
the electro-oxidation process during indigo dye degradation. For
example, Garcia et al. [102] designed an electro-oxidation reaction
process using a microporous activated carbon fiber felt (ACFF)-based
anode onto which laccase enzymes isolated from Pycnoporus sanguineus
were immobilized. The ACFF exhibited a high surface area and excellent
electric conductivity, which are useful in the bioreactor processes of
electro-oxidation reactions. The designed electro-oxidation reaction
using this ACFF-anode (10 V) showed an 83.6% discoloration activity of
indigo carmine for 1 h in tap water. Another interesting engineering
development for dye degradation involved the use of ionic liquid-based
surfactants to improve the efficiency of laccase enzymes. Bento et al.
[122] used an ionic liquid of decyltrimethylammonium bromide,
[N10111]Br, and 1-decyl-3-methylimidazolium chloride [C10mim]Cl
with surfactant characteristics to improve the degradation of hydro­
phobic textile dyes of indigo carmine. The use of this ionic liquid showed
the remarkable degradation activity for indigo carmine of 82% in just
0.5 h. However, further engineering improvements are needed to ach­
ieve higher reaction activities and reusability among immobilized cat­
alysts. Additionally, the identification of inexpensive enzyme
immobilization scaffolds, optimal conditions, and reaction process de­
signs during the immobilization process remain necessary to enhance
the application of the enzymatic degradation of indigo dyes in immo­
bilization systems.
4. Conclusions and future perspectives
Indigo is one of the most commonly used dyes, both globally and
throughout human history, and it has been widely exposed to the
environment, presenting a threat to natural ecosystems due to its
physicochemical properties. Various physical and chemical removal
processes, such as precipitation, filtration, and advanced oxidation, are
possible; however, problems arising due to operational costs and the
generation of chemical sludge currently limit their use. In the interest of
overcoming these deficiencies, biological treatment processes have
9
Dyes and Pigments 184 (2021) 108749
gained increasing attention; among them, studies on the discoloration
and degradation of indigo using laccase enzymes have been conducted
continuously. Laccase enzymes have been used to degrade various dyes,
as well as in ligninolytic processes and indigo degradation. These en­
zymes have the great advantage of being applicable at the industrial
level due to their high activity and stability. However, the critical lim­
itation to the large-scale application of laccase enzymes is the need for
high-yield enzyme production and separation while maintaining a suf­
ficient level of enzyme activity and stability.
In this review, indigo discoloration and degradation studies in which
the production of various biological laccase enzymes originating from
fungi, bacteria, and plants in order to overcome these limitations were
comprehensively assessed. Indigo degradation and discoloration by
microbial whole cells or enzymatic conversion have their pros and cons.
Although the whole cell system can be directly applied on a large scale to
a water treatment environment such as activated sludge, the process
needs to be optimized for cell culture and intracellular enzyme pro­
duction. In particular, fungi need long fermentation cycle to obtain
desirable cell mass and enzyme yield. In addition, secondary metabolites
in the cell and byproducts produced by intra- and extracellular enzy­
matic reactions also require treatment as laccase can oxidize a variety of
phenolic molecules which lead to the generation of randomly cross-
linked or oxidized byproducts; otherwise, it may inhibit the whole cell
reaction leading to low degradation efficiency. In contrast, in vitro indigo
degradation by enzymes derived from fungi or bacteria can achieve high
decomposition efficiency in a short time, and can be recycled with high
stability through immobilization. One of the most remarkable results in
laccase utilization was the heterologous production of laccase enzymes
and engineering for efficient performance via enzyme fixation and
modification. However, it also requires further efforts in enzyme pro­
duction and purification. In particular, soluble expression and optimi­
zation of fungi-derived enzymes in heterologous host are a big challenge
to be overcome. Eventually, the emphasis should be addressed on aug­
menting the efficiency and yields along with the process costs, which
constitute a key criterion in scalable and sustainable process of indigo
degradation and discoloration.
Further research on enzyme and process engineering are necessary to
obtain the desired performances needed for the widespread application
of enzymatic indigo degradation at the industrial level. Due to the
advent of microbial identification and DNA sequencing technologies, it
is expected that indigo bioprocessing technologies will be further
developed through systematic optimization. For example, microbiome
research, metagenomics sequencing and metatranscriptomics technol­
ogies based on meta-omics can be applied to identify highly active
indigenous degraders and enzymes, which are superior to previously
known. In addition, transcriptomic understandings of indigenous
degrader strains can up-regulate the genes involved in indigo degrada­
tion and lead high yield production of indigo degrading enzymes. Be­
sides, microbes-based catalytic reactions such as Fe(III)-reducing
microbial-driven Fenton reaction can be further applicable for indigo
degradation and discoloration by generating hydroxyl radicals. This
would also require the supply of electron mediator. Currently, low-cost
chemical synthetic electron mediators such as HBT, ABTS and TEMPO
have been utilized, but research on eco-friendly mediators with high
efficiency will also be necessary. Considering the recent progresses in
indigo degradation, the paradigm seems to be shifting from a chemical
process back to a greener and synergistic one. It would be possible, for
example, to apply laccase-immobilized biocatalysts to a synergistic
chemo-enzymatic process such as catalytic treatment, adsorptive
removal, and advanced oxidation process. Meanwhile, the demand for
enzyme recovery and reuse in a cost-effective manner would be quite
high when applying them at synergistic chemo-enzymatic process.
Therefore, it would also be necessary to increase biocatalyst stability
and reusability through enzyme engineering and enzyme immobiliza­
tion technology for further applications.
K.-Y. Choi Dyes and Pigments 184 (2021) 108749

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