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Table 1 Application of some interesting fungal laccases that degrade different compounds and may be useful in water treatement

From: Laccases: structure, function, and potential application in water bioremediation

Laccase source

Applied enzyme form

Type of culture, ingredients and enzyme form

Application

Reaction parameters

Results obtained

Main putative mechanisms involved

References

Pharmaceutical compounds

 Pycnoporus sanguineus CCT-4518

C

CI

The fungus was grown in PDA solid medium for 7 days at 28 °C. Laccase extract was produced in 50 mL of liquid media, at 28 °C for 72 h and supported

Laccase removal of 17-alpha-ethynilestradiol (EE2)

Free and immobilized laccase extract (100 U/L) were mixed with 10 mL of EE2 at 10 mg/L, 10 mL of acetate buffer, pH 4 or 5 or 10 mL of distilled water, all of this at 28 °C

80% of removal of EE2 after 24 h by the free and immobilized laccase extract at pH 4 and 5. The immobilized form had three cycles of reusability with high transformations

The laccase is able form dimers of the EE2 by polymerization of it

[49]

 Pycnoporus sanguineus

C

The Theobroma grandiflorum AW was used as Pycnoporus sanguineus laccase (Lac) inducer, cultivated for 7 days at 28 ± 2 °C and 150 rpm

Degradation of estrogens tested

100 U/L of laccases, with 17-α-ethinylestradiol at 10 μg/mL, and 1% of inducer by 24 h

Removal 96% of estrogens after 8 h of reaction

They suggest the degradation product, with hydroxylation of estrogens

[56]

 Trametes versicolor

F

Commercial laccase powder from T. versicolor (activity ≥ 0.5 U/mg) from Sigma-Aldrich

Degradation of PhAC: diclofenac, trimethoprim, carbamazepine, and sulfamethoxazole

Selected PhAC concentrations were added to the enzyme solution in individual beakers. The beakers were incubated on a rotary shaker for 48 h at 80 rpm and 25  °C

The results of this study revealed that laccase can effectively degrade diclofenac (100%), trimethoprim (95%), carbamazepine (85%), and sulfamethoxazole (56%)

Not reported

[50]

 Trametes hirsuta

C

It was grown on PDA medium for 5 days at 28 °C and then on petri plates, pH 5 in static condition for 10 days, on Kirk’s medium. The supernatant was used

Degradation of chloramphenicol (CAP)

Different mediators like syringaldehyde, naphthol, vanillin and ABTS were added at 0.25, 0.50, 1, 3, 5 and 10 mM, to the reaction with 100 U of laccase enzyme and 10 mg/L of CAP in 0.1 M acetate buffer pH 5, by 48 h

The laccase enzyme degraded 0.5 mg/L CAP within 7 days without mediators and was efficiently degraded in the presence of laccase mediator system (syringaldehyde, vanillin, ABTS and α-naphthol)

Dehalogenation and oxidation of CAP by laccase to form chloramphenicol aldehyde which was non-toxic to the microorganisms studied

[53]

 Trametes versicolor

C

SF (500 mL) with 20 g of dried apple pomace, Tween 80 (0.1%) and moisture of 75% (w/w), inoculated with mycelia by 14 days, 30 °C with 200 mL. Enzyme from supernatant extract

Chlortetracycline (CTC) degradation

CTC at 2 mg/L, laccase dose at 0.5 IU, pH 4.5 or 6.0, and ultrasonication

60% of CTC, considered as a recalcitrant pollutant, was removed in 2 h by ultrasonication and assisted laccase at pH 6.0. While at pH 4.5, 80% of CTC was degraded, resulting non estrogenic by products

Oxidation of C–C and C–O bonds

[51]

 Pleurotus ostreatus

FP

PDA medium at 25 °C, and added ciprofloxacin (CIP: at 100, 200, 300, 400 and 500 ppm). The enzyme was secreted

Degradation of ciprofloxacin (CIP)

Fungi growth by 14 days with 100, 200, 300, 400 and 500 ppm of CIP

Antibiotic degradation of about 68.8, 94.25 and 91.34% was estimated after 14 days of incubation at 500 ppm CIP

Not reported

[52]

 Pycnoporus sanguineus CS43f

F

STR of 10 L with 36.8% tomato juice medium, by 15 days, induced with CuSO4 and soybean oil at 48 h. LacI and LacII were purified

Degradation of endocrine disrupting chemicals (EDCs): nonylphenol and triclosan (a biocide)

EDC at 10 ppm final concentration were prepared in pH 5 McIlvaine buffer with 100 U/L laccase. Samples were tested every 30 min for 8 h at 25 °C

More than 95% removal after 8 h of treatment with 100 U/L at pH 5

Enzyme-driven oxidation

[41]

Plastics, personal care and herbicide compounds

 Pycnoporus sanguineus (CS43)

CI

11-days cultures in 10-L STR in complex liquid medium at 28 °C. Crude extract enzyme immobilized

Degradation of emerging endocrine disruptor (bisphenol A)

800 μL McIlvaine buffer (pH 3), 100 µL of ABTS (5 mM, 1.0% w/v) and 100 µL of laccase extract of P. sanguineus (CS43)

100% degradation of bisphenol A (20 mg/L) was achieved in less than 24 h

Probably degradation ends in the formation of 4-isopropenylphenol

[42]

 Trametes versicolor BAFC 2234

MI

7-days cultures in 30-L STR with complex liquid medium (50% tomato juice). Purified enzymes

In vitro oxidation of phenol

The reaction mixture in 1.5-mL contained dissolved phenol (0.5 mM), 50 mM sodium citrate pH 4.5 and 0.1 U/mL laccase

84% phenol removal in 4 h. Dark colored products partly precipitated were found

Oxidative coupling of phenoxy radicals as major pathway of phenol conversion

[43]

 Recombinant laccase from Trametes sanguineus in  Trichoderma atroviride

F

Cultures grown in 50 mL, incubated for 4 days at 28 °C/150 rpm. Purified laccase

Degradation of xenobiotic compounds (phenanthrene and benzo[α]pyrene)

Phenanthrene and benzo[α]pyrene were added into supernatants up to at 10 ppm, incubated at 28 °C and shaken at 150 rpm for 24 h

57.5 U/L of laccase in supernatant removed phenanthrene and benzo[α]pyrene (97 and 99% respectively) present in wastewater from a biofuel industry plant

Not reported

[67]

 Nicotiana tabacum expressing a laccase from Pleurotus ostreatus

C

Plants were grown for 16 days in a growing chamber at 24 °C under a photoperiod of 16:8 h (light:darkness). Enzyme secreted into rhizosphere

Phytoremediation of phenol content from olive mill wastewaters

Laccase activity of transgenic root exudates was evaluated by oxidation of 2 mM ABTS at 420 nm in 0.1 M citrate buffer pH 3.0 at 25 °C

Transgenic tobacco plants cultivated in a hydroponic solution with olive mill wastewaters were able to reduce the total phenol content up to 70%

Not reported

[92]

 Anthracophyllum discolor

MI

It was grown in Kirk liquid medium with Tween 80 or soil supplemented with Tween 80 and wheat grains. Whole cultures

Degradation of polycyclic aromatic hydrocarbons (PAH)

Cultures and 50 mg/L of PAH at 30 °C by 28 days. 10 g soil and 0.5 g wheat grains in 30 mL tubes contaminated with a 50 mg/kg of PAHs at 30 °C by 60 days

54 up to 75% removal of phenanthrene, anthracene. fluoranthene, pyrene and benzo (a)pyrene in soil with A. discolor

Products of degradations were anthraquinone, phthalic acid, 4-hydroxy-9-fluorenone, 9-fluorenone and 4,5-dihydropyrene

[39]

 Trametes pubescens CBS 696.94

C

1L SF with synthetic liquid medium supplemented with dry coffee husk. 23 days static incubation at 30 °C. Crude extracts filtered

Biodegradation of a mixture of 2-chlorophenol (CP), 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TCP), pentachlorophenol (PCP)

Degradation of CPs during 8 h at 40 °C, 200 rpm in flasks containing 100 mL of a CP mixture, with 15 mg/L of each CP in 50 mM phosphate buffer, pH 6.0. Enzymatic extract (5 mL) and 10 U/L

Biodegradation of 100%, 99%, 82.1% and 41.1% of CP, DCP, TCP and PCP, respectively, after 4 h. The reduction in chlorophenols, allowed 90% reduction toxicity

Not reported

[44]

 Neosartorya fischeri

C

50 mL SF with modified Czapek medium and 20 mg of asphaltenes as carbon source, at 37 °C 100 rpm, 4 weeks. Whole cultures

Metabolization and mineralization of asphaltenes (recalcitrant petroleum fraction)

Asphaltene mineralization was quantified by measuring CO2 production. Cell-free extracellular medium was solvent extracted and analyzed by GC–MS

After 11 weeks of growth, the fungus metabolize 15.5% of the asphaltenic carbon, including 13.2% transformed to CO2

Generation of oxidized metabolites such as hydroxypyrenedione and hydroxyphenylacetic acid

[40]

 Coriolopsis rigida LPSC 232

C

15-days liquid cultures in modified Czapek Dox medium (0.5% peptone and 0.15 mM Cu2+)

Detoxification of water soluble fraction from ‘‘alpeorujo” (WSFA)

Reaction mixtures containing WSFA 20% (v/v) and 20 U laccase were incubated 24 h at 28 °C and 150 rpm

Reduction of free phenols from the WSFA

Oxidation of free phenols, resulting in radical formation, leading to polymerization as well as detoxification

[208]

 Trametes villosa

C

F

Extracted and purified enzyme (Novozymes)

Bisphenol A (BPA) degradation

2.2 mM BPA incubated for 1 h with 1.0 unit/mL of laccase. The reaction mixture: 0.5 mM ABTS, 0.1 M sodium acetate, pH 5.0, and an enzyme in a total volume of 1.0 ml was incubated at 37 °C

BPA was degraded by a laccase, which was extracted and purified from DeniLite, a Novozymes’ product. Transforming and important endocrine-disturbing compound

BPA was metabolized to two compounds: one with high molecular weight due to oxidative condensation, and another identified as 4-isopro-penylphenol

[54]

Dye-based pollutants

 Pleurotus ostreatus URM 4809

C

250-mL SF with 50 mL of effluent with 0.05 g/L Remazol Brilliant Blue R and 107 spores/mL and incubated

Decolorization dyes used in the textile industry

Cell in a microbial fuel cell with continuous laccase synthesis; and 0.05 g/L of anthraquinone remazol brilliant blue R dye

Laccase promoted decolorization by 86% of the anthraquinone dye remazol brilliant blue R (used in the textile industry)

Not mentioned, but phytotoxicity results showed that the process did not generate detectable toxic products

[46]

 Ganoderma lucidum E47 strain

C

Solid-state fermentation in MYSA medium, pH 5.5, kept in darkness for 7 days at 25 °C. Supernatants was used as enzyme preparation. 5 compounds were tested in 0.5 L minireactor simulating an effluent

Decolorizing xanthene, azo and triarylmethane dyes

0.1 mM of organic dyes: Bengal rose; blue black naphthol; congo red; methyl orange; bromocresol green; bromocresol purple; bromophenol blue; and phenol red, 550 nm; 100 mM potassium acetate buffer pH 4.8, 5% butyl acetate, 25 °C

The best activity-stability reached in pH 4.8 at 37 °C, decolorizing xanthene, azo and triarylmethane dyes, with selectivity on bromocresol green and bromocresol purple. Activity on effluent biotreatment

Not reported

[47]

 Oudemansiella canarii

C

SF, mycelial from petri dishes were incubated without agitation under air at 28 °C and in the absence of light by 14 days. Extract was dialyzed and partially purified

Decolorization of congo red

50 mM acetate buffer (pH 5.5) in 250-mL

140 SF with 50 mL and containing 50 mg/L of Congo red and native 141 laccase (5 U). The mixtures were incubated at 30 °C in the dark in a rotary shaker at 100 rpm

5 U were able to decolorize 80% of 50 mg/L Congo red within 24 h at 30 °C and pH 5.5

Laccase acts not only on the dye chromophore group, but also that it cleaves different covalent bonds, causing an effective fragmentation of the molecule

[48]

 P. pastoris or A. thaliana expressing Lcc9 from Laccaria bicolor

F

SF in BMGY medium at 28 °C, the cells were suspended in of BMMY. Methanol was added to 1% every 24 h

Decolorization of triphenylmethane dyes, employed in industrial dyeing processes

The reaction mixture for the decolorization assay contained 0.1 mM of crystal violet, McIlvaine buffer and 50 μL of the enzyme in a total of 200 μL. ABTS, as the mediator, was added if necessary. Incubated in dark for 24 h

In the presence of ABTS, the decolorization rates of Crystal violet by laccases in P. pastoris or A. thaliana reached 90.7% and 83.6%, respectively

Not reported

[68]

 Recombinant laccase (Lcc IIIb) from Trametes versicolor expressed in Yarrowia lipolytica

C

Cultures grown in optimized PPB medium pH 7.0 at 2 L STR

Decolorization of pollutant dyes: bromocresol purple, safranin, malachite green, kristal violet, bromothymol blue, nigrosine and phenol red

Reaction mixture was composed of 10 µL of supernatant and 90 µL of a buffer prepared by dissolving 0.1 mg of each dye in 1 mL of citrate buffer at pH 3. Dye decolorization was followed spectrophotometrically

The dye decolorization rates after the first hour were 43%, 54%, 55%, 49%, 56%, 53% and 37% for bromocresol purple, safranin, malachite green, kristal violet, Bromothymol blue, nigrosine and phenol red, respectively

Not reported

[71]

 Recombinant LCC3 from Trametes trogii BAFC 463 in Pichia pastoris

C

4-days liquid cultures induced with methanol

Synthetic dye decolorization

50 µM of dye, citrate–phosphate buffer pH 4.5 at 30 °C, 1–10 U/mL laccase. Mediators used ρ-coumaric acid, HBT, violuric acid (200 µM) acetosyringone (10–200 µM)

50–100% decolorizing ability of azoic, indigoid, triarylmethane, and anthraquinonic with acetosyringone within 2 h incubation at pH 6, 70 °C

Decolorization effectiveness depended on the chemical characteristics of redox mediators and dyes, and the ratio

[70]

 Trametes trogii BAFC 463

C

F

22-days static liquid cultures in glucose (20 g/L), asparagine (3 g/L) medium with 1 mM Cu2+

Decolorization of synthetic dyes

19.5 U laccase per reaction, in test tubes at 30 °C with sodium acetate buffer (10 mM, pH 4.5) in a total volume of 3 mL. The effect of different salts, heavy metals, reaction temperature, pH and redox was analyzed

Laccase decolorized 85% of indigo carmine, xylidine, malachite green, gentian violet, bromophenol blue, 65% of fast blue RR and 30% of Azure B and Methylene Blue in 24 h

Direct oxidation of certain dyes and/or by the LMS

[58]

 Trametes versicolor

FP

F

The fungus was maintained on 2% malt agar slants at 25 °C

Commercial purified enzyme (Fluka)

Biodegradation of triphenylmethane dyes

Reactions in SF with 100 mL dye solution (150 mg/L) buffered with 1.6 mM 2,2-dimethyl succinate, pH 4.5, at 25 °C (laccase 1225 U/L), HBT (10−3 M)

Degradation dye brilliant green1 and acid green 16. resulting benzoic acid and diethylamine and 5,7-disulfo-2-naphtoic acid respectively

Oxidation of the methyl carbon of dye structure, giving stable products

[57]

 Aspergilus expressing a laccase from Myceliophthora thermophila

I

Submerged fermentation of a recombinant Aspergillus sp. A commercial formulation, DeniLite II S, from novozymes A/S covalently immobilized

Decolorization of synthetic dyes

20 U/mL of immobilized laccase or 0.5 g in a FBR. Several dyes at 0.02% (w/v) 30 °C 0.1 M sodium acetate buffer (pH 4.5) 90 rpm

The anthraquinonic dyes acid blue 25 and acid green 27 were decolorized. The RBBR and the diazo RB-5 were only decolorized with laccase/HBT, 31 and 60%, respectively, after 24 h

Direct oxidation of certain dyes and/or by the LMS

[45]

 Recombinant lcc1 gene from Trametes trogii in Pichia pastoris

F

SF at 30 °C and STR 2 L at 25 °C cultures in phosphate buffered minimal methanol (BMM), supplemented with yeast extract or casaminoacids

Decolorization dyes (amaranth, carmoisine, cochineal red, sunset yellow, patented blue, blue indigo and alizarin red S

1 mL (0.05 mg/mL of dye in 0.1 M sodium phosphate buffer, pH 5.0) and 1 IU of laccase with or without 1 mM redox mediator 1-hydroxybenzotriazole or violuric acid at 25 °C, plus dyes

All the dyes were decolorized up to 60% percent after 2 h with containing 1 U of Lcc1 and the redox mediator violuric acid 1 mM

Generation of a phenoxy radical resulting in the cleavage of azo linkages with nitrogen release

[62]

  1. F free purified enzyme, I immobilized purified enzyme, FP fungal pellets, C crude extract or culture supernatant, CI crude extract immobilized, MI mycelium immobilized, STR stirred-tank reactor, FBR fixed-bed bioreactor, PhAC pharmaceutically active compound, SF shake flask, PDA potato dextrose agar, LMS laccase-mediator system, HBT hydroxybenzotriazole, EE2 17-alpha-ethynilestradiol, CAP chloramphenicol, CTC chlortetracycline, CIP ciprofloxacin, SFMZ sulfamethoxazole, EDCs endocrine disrupting chemicals, AzBTS-(NH4)2 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, ABTS diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate), PAHs polycyclic aromatic hydrocarbons, CP 2-chlorophenol, DCP 2,4-dichlorophenol, TCP 2,4,6-trichlorophenol, PCP pentachlorophenol, BPA bisphenol A, WSFA water soluble fraction from ‘‘alpeorujo”, BMGY buffered glycerol-complex medium