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Table 2 Current overview on laccases characteristics, immobilization techniques and related applications

From: Laccases as green and versatile biocatalysts: from lab to enzyme market—an overview

Immobilization methods Laccase source Immobilization supports Characteristics of the native enzyme Results after immobilization Results after application of the immobilized enzyme Reference
Optimum pH Optimum temperature (°C) Molecular weight (kDa)
Entrapment- Physical retention of the enzyme in a network, usually an insoluble sol–gel porous matrix, followed by cross-linking Cyberlindnera fabianii Calcium (Ca-AIL) and copper alginate beads (Cu-AIL) 5.0 40 52 Increased storage stability after 21 days at 4 °C;
Increased thermostability;
After 8 reuses the residual activities were 36 and 40%, using ABTS as substrate
Degradation of 42.7% (Ca-AIL) and 39.1% (Cu-AIL) of bisphenol A (100 μM BPA) after 24 h Olajuyigbe et al. (2019)
Trametes pubescens Calcium alginate beads and Crosslinking prior to entrapment in calcium alginate beads 3.0 40 Thermal and pH stability was improved;
40.3% of activity retention after 24 h at pH 9,0;
Increased storage stability after 35 days at 4 °C;
70% of activity retention after 10 successive cycles of reuse
Degradation was greater than 99% of bisphenol A (20 mg/L) after 2 h Lassouane et al. (2019)
Bacillus subtilis MTCC 2414 Copper alginate beads 9.0 35 37 Thermal stability increase 81.72% degradation of Yellow GR dye (0.1% w/v) after 120 h Narayanan et al. (2015)
Brevibacterium halotolerans N11 (KY883983) Alginate-gelatin 5.0 35 55 86.7% recovered activity;
Increased pH and temperature stability;
65% of retention activity after 7 reuses using guaiacol as a substrate
Efficiency in decolorization of different classes of synthetic dyes Reda et al. (2018)
Trichoderma harzianum (HZN10) Sol–gel matrix 6.0 50 56 93% immobilization efficiency;
Increase in thermal, pH, and operational stability
82% of activity retention after 6 cycles of reuse using ABTS as a substrate
Dye decolorization: 100% of malachite green, 90% of methylene blue and 60% degradation of congo red (200 mg/L each) in the presence of 1-hydroxybenzotriazole (HBT) mediator Bagewadi et al. (2017)
Cyathus bulleri Poly Vinyl Alcohol-based polymers crosslinked either by nitrate (PVA-nitrate) or boric acid (PVA-boric) High immobilization yield, with 65 and 90% for PVA-boric and PVA-nitrate, respectively;
- High resistance when exposed to high temperatures;
80% of activity retention after four months of storage at 4 °C
Batch decolorization of 95% of Basic Green 4 dye up to 20 cycles and 90% of Acid Red 27 up to 10 cycles (100 µM each)
Continuous decolorization of 90% Acid Red 27 with a mediator (ABTS)
Chhabra et al. (2015)
Pseudomonas putida Nanofibers and carbon nanotubes (SWNT) 8.5 10 45 Increase in thermal, stability;
After 5 cycles of reuse, 75% and 95% of the initial activity were maintained at 80 °C and 4 °C, respectively;
Laccase activity was retained over 10 cycles of random freeze–thaw treatment
- Mukhopadhyay et al. (2015)
Encapsulation- The enzyme is retained in spheres, such as semipermeable membranes thus preventing direct contact with the external environment Bacillus safensis sp. strain S31 Alginate beads 5.0 30 Removed 95% of reactive black (10 mg/L) after 1 h Siroosi et al. (2018)
Adsorption- Simple adsorption of the enzyme on support by bonds such as hydrophobic interactions, Van der Waals forces, hydrogen bonds, and ionic bonds Aspergillus oryzae Granular activated carbon 7.0 30 56 Thermal and pH stability was improved;
55% of activity retention after 20 cycles of reuse
Removal of more than 80% of sulfamethoxazole, carbamazepine, diclofenac
and bisphenol A (each at 2.5 mg/L)
Nguyen et al. (2016)
Coprinus comatus Biochar 3.0 Immobilization yield of 64.2%;
Thermal stability increase;
34% of activity retention after 7 cycles of reuses
71.4% removal of chlorinated biphenyl (0.04 g/L) Li et al. (2018b)
Pleurotus ostreatus MANAE-agarose 5.0 50 60 Thermal stability increase;
70% of activity retention after 170 days of storage at 4 °C;
In reuse, more than 90% of Bisphenol-A was degraded in the 15th consecutive cycle
Degradation of 90%Bisphenol-A (100 mg/L) Brugnari et al. (2018)
Pleurotus ostreatus Porous acrylic carrier with octadecyl groups (C18) 94% of activity retention after 21 days (4 °C);
Increase of thermal stability at 40 °C and 60 °C;
63% of activity retention, after four transformations for the synthesis of the dye in a continuous system
Production of orange dye (N15) through the transformation of 2-amino-3-methoxybenzoic acid Wlizło et al. (2020)
Polyporus durus Nanoporous Zeolite-X 4.0 70 Immobilization yield of 83%;
Thermal and pH stability was improved;
After 7 cycles of decolorization, 100% of laccase activity was maintained
Decolorization of 100% of dyes Acid Blue 225 e Reactive Blue 19 (100 mg/L each) after 15 and 45 min, respectively Wehaidy et al. (2019)
Rhus vernicifera Sepiolite (A); Sepiolite modified with chitosan (B); Sepiolite plus Cu(II) (C); Sepiolite modified with both chitosan and Cu(II) (D) Improvement in enzymatic activity;
Low desorption (< 10%) in all samples
Olshansky et al. (2018)
Trametes versicolor Metal-chelated chitosan-based copolymer nanoparticles 5.5 30 Thermal and pH stability was improved;
50% of activity retention after 8 cycles of reuse
Phenol degradation (20 mg/L) about 82% after 4 h, without a mediator; The addition of mediator ABTS improved phenol degradation (100% with 1 mM ABTS) Alver and Metin (2017)
Chelation—The enzyme binds to the support by coordinated bonds, where the charged and polar amino acids such as histidine residues bind to metal ions Escherichia coli (recombinant laccase) Magnetic zeolitic imidazolate nanoparticles 70 65 75.7% recovered activity;
Increased thermal and storage stability;
The immobilized laccase retained 46.0% of the initial activity after 6 h at 80 °C;
87.1% of activity retention after 10 days of storage at 30 °C;
Greater affinity (Km) to ABTS than the free enzyme
Complete decolorization of indigo carmine (25 mg/L) after five consecutive cycles  
Covalent bonds- Covalent binding of the enzyme to the support Aspergillus sp. Graphene oxide nanosheets 5.0 Immobilization yield of 64.6%;
Decolorization of more than 75% of the dyes evaluated after 6 consecutive cycles
High efficiency of biodegradation of azo dyes in different concentrations Kashefi et al. (2019)
Trametes versicolor Polyacrylonitrile-biochar composite nanofibrous membrane 4.5 30–40 Thermal, pH, and storage stability were improved;
71% of activity retention after one month of storage at 4 °C;
50% of activity retention after seven cycles of oxidation with ABTS
The biodegradation in the continuous mode of Chlortetracycline (200 ppb) exhibited 58.3% removal efficiency at the flux rate of 1 mL/h·cm2 Taheran et al. (2017)
Trametes versicolor Polyaniline electrodeposited onto a glassy carbon electrode/ 3.0 Laccase biosensor has the highest current, demonstrating the highest catalytic ability for catechol oxidation than those without the enzyme Biosensor for the detection of phenolic compounds (catechol) Nazari et al. (2015)
Trametes versicolor Graphene oxide/CuFe2O4 nanocomposite 6.0 35 88% of activity recovery;
Increased thermal and pH stability;
83% of activity retention after 30 days of storage at 4 °C
High efficiency in the synthesis of arylsulfonyl benzenediols (up to 91%);
Conversion capacity of about 80% after 10 cycles of reuse
Rouhani et al. (2018)
Trametes versicolor Copper ferrite magnetic nanoparticles (CuMNPs) and ferrite magnetic nanoparticles (MNPs) 5.0 40 Activity recovery of 94.68 ± 0.92% and 89.78 ± 1.24% for CuMNPs and MNPs, respectively;
Increased thermal and pH stability;
At 70 °C, CuMNPs and MNPs showed relative activity of more than 70 and 60%, respectively;
MNPs and CuMNPs retained more than 70% of its initial residual activity after 20 days (4 °C);
After 6 cycles, the immobilized exhibited more than 70% of the initial activity
Delignification of plant biomass:
CuMNPs: 43.28 ± 1.46% of lignin removal (160.6 mg lignin/g biomass);
MNPs: 40.10 ± 1.35% (169.5 mg lignin/g biomass)
Muthuvelu et al. (2020)
Trametes versicolor Immobilized on the electrospun
zein fiber (ceZL)
    Relative activity of 92.76 ± 3.65% after immobilization;
After 10 days of storage (4 °C), the ceZL remained higher than 81% of residual activity;
The ceZL exhibited high relative activity between 4 and 40 °C
Optical biosensor to indicate the shelf life of food based on temperature (time–temperature indicator, TTI) Jhuang et al. (2020)
  Bacillus atrophaeus Magnetic-nanoparticles 5.5 35 31 Immobilization yield of 50%;
The immobilized exhibited 60% of residual activity after 10 consecutive cycles with ABTS as a substrate;
Greater stability at temperatures above 40 °C
Juice clarification:
Reduction of total phenolic compounds (41–58%);
Reduction of color and turbidity of 49–59% and 50–59%, respectively
Narnoliya et al. (2019)
Protein-inorganic hybrid nanoflower—This immobilization consists of a complex of enzymes cross-linked with metal ions with nanoflower (NF) morphology Trametes versicolor Cross-linked of laccase-Cu3(PO4)2∙3H2O hybrid NF 3.0 40 Yield and activity recovery of 78.1 and 204%, respectively;
Improved catalytic efficiency, storage stability and greater solvent tolerance;
CL-NF maintained 91.5% of the initial activity after 60 days of incubation at 4 °C;
Residual activity of 92.3% after 10 reuse cycles
Decolorization of synthetic dyes (120 µg/mL each): bromophenol blue (41.2%), CBBR-250 (73.2%) and xylene cyanol (73.0%) after 48 h without mediators; the mediator (ABTS) increased the efficiency of decolorization Patel et al. (2018)
CLEAs- In this method, there is no need for support. The immobilization occurs through cross-links between aggregated enzymes using bifunctional or multifunctional reagents Fomes fomentarius 2.6 30 At pH 4.6 the immobilized enzyme retained twice the value of relative activity (60%) than the free one;
Increased thermal and storage stability;
74% of activity retention after 70 days at 4 °C;
After six reuse cycles, residual activity was about 50%
Decolorization around 90% of dyes malachite green (7 mg/L), bromothymol blue (50 mg/L) and methyl red (100 mg/L) after 10 h Vršanská et al. (2018)
E-CLEAs- The soluble enzyme is aggregated by cross-linking and then entrapped within supports for the obtention of a better operational stability and reusability Trametes versicolor Entrapped cross-linked enzyme aggregate in mesoporous silica 4.5 Increased thermal, pH and storage stability;
Retention of activity in solvents;
79% of activity retention after 20 cycles of reuse
Removal total of phenol (0.4 mM) in 40 min Fathali et al. (2019)
M-CLEAs—Magnetic particles are bonded with CLEAs to provide enhanced mechanical stability. These biocatalysts are the easy separation from the reaction mixture and recycled by using a simple magnetic field Trametes versicolor Magnetically activated chitosan CLEAs (MAC-CLEAs) 4.0 40 The activity recovery of MAC-CLEAs reached 62.2%;
–54% activity retention at pH 7.0;
67% of the activity at 60 °C;
32% of activity retention after 35 cycles of reuse
–Elimination of 13 pharmaceuticals (100 µg/L each):
–After 6 h MAC-CLEAs effectively removed mefenamic acid (99%), acetaminophen (85%) and diclofenac (85%) and other compounds were partially removed, in some cases, ABTS was used as a mediator
Kumar and Cabana (2016)