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 | ||
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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) |