Improvement of laccase activity by silencing PacC in Ganoderma lucidum

Abstract Ganoderma lucidum is a representative white-rot fungus that has great potential to degrade lignocellulose biomass. Laccase is recognized as a class of the most important lignin-degrading enzymes in G. lucidum. However, the comprehensive regulatory mechanisms of laccase are still lacking. Based on the genome sequence of G. lucidum, 15 laccase genes were identified and their encoding proteins were analyzed in this study. All of the laccase proteins are predicted to be multicopper oxidases with conserved copper-binding domains. Most laccase proteins were secreted enzymes in addition to Lac14 in which the signal peptide could not be predicted. The activity of all laccases showed the highest level at pH 3.0 or pH 7.0, with total laccase activity of approximately 200 U/mg protein. Silencing PacC resulted in a 5.2 fold increase in laccase activity compared with WT. Five laccase genes (lac1, lac6, lac9, lac10 and lac14) showed an increased transcription levels (approximately 1.5-5.6 fold) in the PacC-silenced strains versus that in WT, while other laccase genes were downregulated or unchanged. The extracellular pH value was about 3.1, which was more acidic in the PacC-silenced strains than in the WT (pH 3.5). Moreover, maintaining the fermentation pH resulted in a downregulation of laccase activity which is induced by silencing PacC Our findings indicate that in addition to its function in acidification of environmental pH, PacC plays an important role in regulating laccase activity in fungi.

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Cerrena unicolor Laccases, Genes Expression and Regulation of Activity

Biomolecules ◽

10.3390/biom11030468 ◽

2021 ◽

Vol 11 (3) ◽

pp. 468

Author(s):

Anna Pawlik ◽

Beata Ciołek ◽

Justyna Sulej ◽

Andrzej Mazur ◽

Przemysław Grela ◽

...

Keyword(s):

Laccase Activity ◽

Environmental Changes ◽

Specific Activity ◽

Electrochemical Analysis ◽

White Rot ◽

White Rot Fungus ◽

Cerrena Unicolor ◽

Genes Expression ◽

Regulation Of Activity ◽

Laccase Genes

A white rot fungus Cerrena unicolor has been identified as an important source of laccase, unfortunately regulation of this enzyme genes expression is poorly understood. Using 1D and 2D PAGE and LC-MS/MS, laccase isoenzymes were investigated in the liquid filtrate of C. unicolor culture. The level of expression of laccase genes was measured using qPCR. The elevated concentrations of copper and manganese in the medium caused greatest change in genes expression and three laccase transcripts were significantly affected after culture temperature was decreased from 28 to 4 °C or increased to 40 °C. The small differences in the PAGE band intensities of individual laccase proteins were also observed, indicating that given compound affect particular laccase’s transcript. Analyses of laccase-specific activity, at all tested conditions, showed the increased activities as compared to the control, suggesting that enzyme is regulated at the post-translational stage. We observed that the aspartic protease purified from C. unicolor, significantly stimulate laccase activity. Moreover, electrochemical analysis of protease-treated laccase sample had 5 times higher redox peaks. The obtained results indicate that laccases released by C. unicolor are regulated at transcriptional, translational, and at the post-translational steps of gene expression helping fungus adapt to the environmental changes.

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Lignin-Modifying Enzymes of the White Rot Basidiomycete Ganoderma lucidum

Applied and Environmental Microbiology ◽

10.1128/aem.65.12.5307-5313.1999 ◽

1999 ◽

Vol 65 (12) ◽

pp. 5307-5313 ◽

Cited By ~ 116

Author(s):

Trevor M. D’Souza ◽

Carlos S. Merritt ◽

C. Adinarayana Reddy

Keyword(s):

Ganoderma Lucidum ◽

Laccase Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Mesh Size ◽

Syringic Acid ◽

White Rot ◽

White Rot Fungus ◽

Laccase Production ◽

Malt Extract

ABSTRACT Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (M r of 40,000 and 66,000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity (∼100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence oflip-like sequences in G. lucidum.

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Introducing a Thermo-Alkali-Stable, Metallic Ion-Tolerant Laccase Purified From White Rot Fungus Trametes hirsuta

Frontiers in Microbiology ◽

10.3389/fmicb.2021.670163 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jing Si ◽

Hongfei Ma ◽

Yongjia Cao ◽

Baokai Cui ◽

Yucheng Dai

Keyword(s):

Specific Activity ◽

Endocrine Disrupting Chemicals ◽

Environmental Pollutants ◽

Fold Increase ◽

Industrial Applications ◽

White Rot ◽

White Rot Fungus ◽

Metallic Ions ◽

Trametes Hirsuta ◽

Ph Range

This study introduces a valuable laccase, designated ThLacc-S, purified from white rot fungus Trametes hirsuta. ThLacc-S is a monomeric protein in nature with a molecular weight of 57.0 kDa and can efficiently metabolize endocrine disrupting chemicals. The enzyme was successfully purified to homogeneity via three consecutive steps consisting of salt precipitation and column chromatography, resulting in a 20.76-fold increase in purity and 46.79% yield, with specific activity of 22.111 U/mg protein. ThLacc-S was deciphered as a novel member of the laccase family and is a rare metalloenzyme that contains cysteine, serine, histidine, and tyrosine residues in its catalytic site, and follows Michaelis-Menten kinetic behavior with a Km and a kcat/Km of 87.466 μM and 1.479 s–1μM–1, respectively. ThLacc-S exerted excellent thermo-alkali stability, since it was markedly active after a 2-h incubation at temperatures ranging from 20 to 70°C and retained more than 50% of its activity after incubation for 72 h in a broad pH range of 5.0–10.0. Enzymatic activities of ThLacc-S were enhanced and preserved when exposed to metallic ions, surfactants, and organic solvents, rendering this novel enzyme of interest as a green catalyst for versatile biotechnological and industrial applications that require these singularities of laccases, particularly biodegradation and bioremediation of environmental pollutants.

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Enhanced Formation of Extracellular Laccase Activity by the White-Rot Fungus Trametes multicolor

Biotechnology for Fuels and Chemicals ◽

10.1007/978-1-4612-0119-9_18 ◽

2002 ◽

pp. 229-241 ◽

Cited By ~ 2

Author(s):

Johann Hess ◽

Christian Leitner ◽

Christiane Galhaup ◽

Klaus D. Kulbe ◽

Barbara Hinterstoisser ◽

...

Keyword(s):

Laccase Activity ◽

White Rot ◽

White Rot Fungus ◽

Extracellular Laccase ◽

Trametes Multicolor

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Protective effect of vanilloids against chemical stress on the white-rot fungus Ganoderma lucidum

Journal of Environmental Management ◽

10.1016/j.jenvman.2013.03.040 ◽

2013 ◽

Vol 124 ◽

pp. 1-7 ◽

Cited By ~ 5

Author(s):

Francisco Kuhar ◽

Leandro Papinutti

Keyword(s):

Protective Effect ◽

Ganoderma Lucidum ◽

White Rot ◽

White Rot Fungus ◽

Chemical Stress

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Enhancement of lignin degradation and laccase activity in Pleurotus ostreatus by cotton stalk extract

Canadian Journal of Microbiology ◽

10.1139/w98-054 ◽

1998 ◽

Vol 44 (7) ◽

pp. 676-680 ◽

Cited By ~ 17

Author(s):

Orly Ardon ◽

Zohar Kerem ◽

Yitzhak Hadar

Keyword(s):

Oxygen Consumption ◽

Pleurotus Ostreatus ◽

Lignin Degradation ◽

Laccase Activity ◽

White Rot ◽

White Rot Fungus ◽

Lignin Biodegradation ◽

Cotton Stalk ◽

Fermentation System ◽

Uv Visible

The white rot fungus Pleurotus ostreatus was grown in a chemically defined solid state fermentation system amended with cotton stalk extract (CSE).Treated cultures exhibited increased laccase activity as well as enhanced lignin mineralization. Mineralization of [14C]lignin initialized 4 days earlier in CSE-supplemented cultures than in control cultures. Total mineralization in the first 16 days was 15% in the CSE-treated cultures, compared with only 7% in the controls. Cotton stalk extract also contained compounds that serve as substrates for laccase purified from P. ostreatus as shown by oxygen consumption, as well as changes in the UVvisible spectrum.Key words: cotton, Pleurotusostreatus, white rot, laccase, lignin biodegradation.

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Application of ligninolytic potentials of a white-rot fungus Ganoderma lucidum for degradation of lindane

Environmental Monitoring and Assessment ◽

10.1007/s10661-016-5606-7 ◽

2016 ◽

Vol 188 (10) ◽

Cited By ~ 19

Author(s):

Harsimran Kaur ◽

Shammi Kapoor ◽

Gaganjyot Kaur

Keyword(s):

Ganoderma Lucidum ◽

White Rot ◽

White Rot Fungus

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Characteristics of Compost from Balinese Cattle Dung (CD) and Rice Straw (RS) using White Rot Fungus (Wrf) (Ganoderma sp) As Bioactivators

Jurnal Ilmu dan Teknologi Hasil Ternak ◽

10.21776/ub.jitek.2020.015.03.7 ◽

2020 ◽

Vol 15 (3) ◽

pp. 194-204

Author(s):

Muhammad Irfan Said ◽

Jamila Mustabi ◽

Siti Amelia Putri Syamsuddin

Keyword(s):

Rice Straw ◽

Ph Value ◽

White Rot ◽

White Rot Fungus ◽

Cattle Dung ◽

Randomized Design ◽

Test Parameters ◽

The Difference ◽

The Republic ◽

Range Of Values

To maximize the performance of the fermentation process, there is a need for a biactivator. The purpose of this study was to evaluate the characteristics of compost-based waste from cattle dung (CD) combined with rice straw (RS) by applying white rot fungus (Wrf) (Ganoderma sp) as a bioactivator. In this study, 6 types of treatments have been applied, namely T1(CD:RS)(25%:75%)+5% of Wrf; T2(CD:RS)(50%:50%)+5% of Wrf; T3(CD:RS)(75%:25%)+5% of Wrf; T4 (CD:RS)(25%:25%)+0% of Wrf; T5(CD:RS)(50%:50%)+0% of Wrf; T6 (CD:RS)(75%:25%)+0% of Wrf. The study was designed experimentally using a completely randomized design (CRD), 6 treatments with 3 replications. Based on the research results, the difference in the ratio of CD to RS and the use of Wrf bioactivator has a significant effect (p<0.05) in increasing the pH, temperature, C-organic and C/N ratio in compost. However, this difference had no significant effect (p>0.05) on the C-organic value. The pH value of compost was obtained with variations of 5.50±0.43-6.73±0.06. Temperature of compost is in the range of values 24.00±0.00-25.67 ±0.58 ºC. The C-organic value is in the range of 24.38±0.48-31.32±0.80%, while the N-organic value varies at 1.34±0.11-2.10±0.15%. The C/N ratio is in the range of 15.00± 0.11-19.33±1.15. Based on the interaction, the results showed that there was a significant interaction (p>0.05) between the use of Wrf bioactivator with the ratio of CD to RS on the parameters of pH, N-organic and C/ Nratio, while, C-organic and temperature had no significant effect. The results of the study was concluded that several test parameters produced compost characteristics that were in accordance with Standar Nasional Indonesia (SNI) and the Regulation of the Minister of Agriculture of the Republic of Indonesia. Compost production using T3 treatment ((CD:RS)(75%:25%) + 5% of Wrf) produces the best characteristics compared to other treatments.

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A New Laccase of Lac 2 from the White Rot Fungus Cerrena unicolor 6884 and Lac 2-Mediated Degradation of Aflatoxin B1

Toxins ◽

10.3390/toxins12080476 ◽

2020 ◽

Vol 12 (8) ◽

pp. 476 ◽

Cited By ~ 1

Author(s):

Zhimin Zhou ◽

Renkuan Li ◽

Tzi Bun Ng ◽

Yunyun Lai ◽

Jie Yang ◽

...

Keyword(s):

Aflatoxin B1 ◽

Environmental Pollutants ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Mass Spectrometry Data ◽

White Rot ◽

White Rot Fungus ◽

Multicopper Oxidases ◽

Cerrena Unicolor

Aflatoxin B1 (AFB1) is a known toxic human carcinogen and can be detoxified by laccases, which are multicopper oxidases that convert several environmental pollutants and toxins. In this study, a new laccase that could catalyze AFB1 degradation was purified and identified from the white-rot fungus Cerrena unicolor 6884. The laccase was purified using (NH4)2SO4 precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis of C. unicolor 6884. Six putative laccase protein sequences were obtained via functional annotation. The lac 2 cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB1 detoxification. AFB1 degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 °C for 24 h. The half-life of AFB1 degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and [2,2′ -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB1 degradation by Lac 2. The product of AFB1 degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q1 (AFQ1) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB1 present in food and feeds.

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