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

Toxins ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 476 ◽  
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.

scholarly journals Effect of Physisporinus vitreus on wood properties of Norway spruce. Part 2: Aspects of microtensile strength and chemical changes

Holzforschung ◽  
2011 ◽  
Vol 65 (5) ◽  
Author(s):  
Christian Lehringer ◽  
Bodo Saake ◽  
Vjekoslav Živković ◽  
Klaus Richter ◽  
Holger Militz
Keyword(s):  
Norway Spruce ◽  
Lignin Content ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
White Rot ◽  
White Rot Fungus ◽  
Wall Structure ◽  
Fungal Activity ◽  
Selective Delignification ◽  
Simultaneous Degradation

AbstractThe biotechnological application of the white rot fungusPhysisporinus vitreusnamed “bioincising” is currently being investigated for permeability improvement of Norway spruce (Picea abies(L.) Karst.) wood. During short-term (<9 weeks) incubation, fungal activity induces degradation of pit membranes and a simultaneous alteration of the tracheid cell wall structure. In Part 1 of this article series, the occurrence of selective delignification and simultaneous degradation was shown by UV-microspectrophotometry (UMSP). Moreover, significant reduction of Brinell hardness was recorded after 7 and 9 weeks incubation. For a better understanding of the chemical alterations in the wood constituents and the corresponding changes of mechanical properties due to fungal activity, we applied microtensile tests on thin strips that were prepared from the surface of incubated Norway spruce wood. Indications for the occurrence of selective delignification and simultaneous degradation were evident. Determination of lignin content and carbohydrate analysis by borate anion exchange chromatography confirmed the results. The present study verifies the findings from Part 1 of this article series and from previously conducted microscopic investigations. Now, the degradation characteristics ofP. vitreusare established and the bioincising process can be further optimized with higher reliability.

Isolation of a new laccase isoform from the white-rot fungiPycnoporus cinnabarinusstrain ss3

2000 ◽  
Vol 46 (8) ◽  
pp. 759-763 ◽  
Author(s):  
Ludovic Otterbein ◽  
Eric Record ◽  
David Chereau ◽  
Isabelle Herpoël ◽  
Marcel Asther ◽  
...  
Keyword(s):  
First Generation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
White Rot ◽  
White Rot Fungus ◽  
Pycnoporus Cinnabarinus ◽  
Ammonium Sulphate Precipitation ◽  
Liquid Culture Medium

Two extracellular laccase isoforms (Lac I and Lac II) produced by the white-rot fungus Pycnoporus cinnabarinus from the monokaryotic strain ss3 were purified from ferulic-acid-induced liquid culture medium using ammonium sulphate precipitation, followed by anion-exchange chromatography on a Mono Q column. Strain ss3 is the first generation of the parental strain P. cinnabarinus I-937. The new isolated isoform, Lac II, consists of an 86 000 molecular weight protein as determined by SDS polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of both isoforms were determined, and compared to known laccase protein sequences of other organisms.Key words: oxydo-reductase, filamentous fungi, purification.

scholarly journals Cerrena unicolor Laccases, Genes Expression and Regulation of Activity

Biomolecules ◽  
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.

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

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.

scholarly journals Pyrene and Chrysene Tolerance and Biodegradation Capability of Pleurotus Sajor-Caju

2018 ◽  
Vol 12 (1) ◽  
pp. 24-35 ◽  
Author(s):  
Giuliano Saiu ◽  
Stefania Tronci ◽  
Massimiliano Grosso ◽  
Enzo Cadoni ◽  
Nicoletta Curreli
Keyword(s):  
Liquid Medium ◽  
Copper Sulphate ◽  
Negative Impact ◽  
Fungal Growth ◽  
Tween 80 ◽  
White Rot ◽  
White Rot Fungus ◽  
Polycyclic Aromatic ◽  
Pleurotus Sajor Caju

Introduction: The present work focused on the biodegradation capability of a white-rot fungus, the Pleurotus sajor-caju, when exposed to polycyclic aromatic hydrocarbons. Methods: The research was carried out by using in vitro systems developed on Petri dishes, to evaluate the fungal tolerance to pyrene and chrysene, followed by experiments in liquid medium. The first experimental campaign was necessary to evaluate the conditions promoting fungal growth and tolerance (presence of surfactants, peptone, copper sulphate and lecithin) and it was designed and analysed using statistical techniques. Results: It was found that the fungal population growth is strongly inhibited by chrysene presence. On the other hand, pyrene had a mild negative impact on the mycelia growth, which seemed to be positively influenced by the presence of Tween 80 and copper sulphate. Starting from these results, the behaviour of Pleurotus sajor-caju in presence of pyrene was investigated in liquid medium. Results showed that the depletion of pyrene was evident during a period of 20 days, and removal efficiency was greater than 90%.

Ca2+-binding proteins of cilia and infraciliary lattice ofParamecium tetraurelia: their phosphorylation by purified endogenous Ca2+-dependent protein kinases

2002 ◽  
Vol 115 (9) ◽  
pp. 1973-1984
Author(s):  
Kwanghee Kim ◽  
Min Son ◽  
Joan B. Peterson ◽  
David L. Nelson
Keyword(s):  
Protein Kinases ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Soluble Fraction ◽  
Biological Activities ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Paramecium Tetraurelia ◽  
Dependent Protein

We purified two small, acidic calcium-binding proteins(ParameciumCa2+-binding proteins, PCBP-25α and PCBP-25β) from Paramecium tetraurelia by Ca2+-dependent chromatography on phenyl-Sepharose and by anion-exchange chromatography. The proteins were immunologically distinct. Monoclonal antibodies against PCBP-25β did not react with PCBP-25α, and antibodies against centrin from Chlamydomonas reacted with PCBP-25α but not with PCBP-25β. Like the centrins described previously, both PCBPs were associated with the infraciliary lattice (ICL), a fibrillar cytoskeletal element in Paramecium. Both were also present in isolated cilia, from which they could be released (with dynein) by a high-salt wash, and both PCBPs cosedimented with dynein in a sucrose gradient. PCBP-25β was especially prominent in cilia and in the deciliation supernatant, a soluble fraction released during the process of deciliation. The results of immunoreactivity and localization experiments suggest that PCBP-25α is a Paramecium centrin and that PCBP-25β is a distinct Ca2+-binding protein that confers Ca2+ sensitivity on some component of the cilium, ciliary basal body or ICL.We characterized these proteins and Paramecium calmodulin as substrates for two Ca2+-dependent protein kinases purified from Paramecium. PCBP-25α and calmodulin were in vitro substrates for one of the two Ca2+-dependent protein kinases (CaPK-2), but only PCBP-25α was phosphorylated by CaPK-1. These results raise the possibility that the biological activities of PCBP-25α and calmodulin are regulated by phosphorylation.

scholarly journals Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 942-952 ◽  
Author(s):  
L Zhang ◽  
A Jhingan ◽  
FJ Castellino
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Complete Disappearance ◽  
Carboxyglutamic Acid

Abstract To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

scholarly journals Lighting Conditions Influence the Dynamics of Protease Synthesis and Proteasomal Activity in the White Rot Fungus Cerrena unicolor

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1322
Author(s):  
Anna Pawlik ◽  
Magdalena Jaszek ◽  
Anita Swatek ◽  
Marta Ruminowicz-Stefaniuk ◽  
Beata Ciołek ◽  
...  
Keyword(s):  
Expression Profile ◽  
Hydrolytic Degradation ◽  
Culture Fluid ◽  
Global Gene Expression ◽  
26S Proteasome ◽  
Stress Factors ◽  
White Rot ◽  
White Rot Fungus ◽  
Lighting Conditions ◽  
Cerrena Unicolor

Recent transcriptomic and biochemical studies have revealed that light influences the global gene expression profile and metabolism of the white-rot fungus Cerrena unicolor. Here, we aimed to reveal the involvement of proteases and ubiquitin-mediated proteolysis by the 26S proteasome in the response of this fungus to white, red, blue and green lighting conditions and darkness. The changes in the expression profile of C. unicolor genes putatively engaged in proteolysis were found to be unique and specific to the applied wavelength of light. It was also demonstrated that the activity of proteases in the culture fluid and mycelium measured using natural and synthetic substrates was regulated by light and was substrate-dependent. A clear influence of light on protein turnover and the qualitative and quantitative changes in the hydrolytic degradation of proteins catalyzed by various types of proteases was shown. The analysis of activity associated with the 26S proteasome showed a key role of ATP-dependent proteolysis in the initial stages of adaptation of fungal cells to the stress factors. It was suggested that the light-sensing pathways in C. unicolor are cross-linked with stress signaling and secretion of proteases presumably serving as regulatory molecules.

scholarly journals White-Rot Fungi Control on Populus spp. Wood by Pressure Treatments with Silver Nanoparticles, Chitosan Oligomers and Propolis

Forests ◽  
2019 ◽  
Vol 10 (10) ◽  
pp. 885 ◽  
Author(s):  
Casado-Sanz ◽  
Silva-Castro ◽  
Ponce-Herrero ◽  
Martín-Ramos ◽  
Martín-Gil ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
White Rot Fungi ◽  
Ethanolic Extract ◽  
White Rot ◽  
Wood Preservation ◽  
White Rot Fungus ◽  
Limiting Factor ◽  
Populus Spp ◽  
Over Time

There is growing interest in the development of non-toxic, natural wood preservation agents to replace conventional chemicals. In this paper, the antifungal activities of silver nanoparticles, chitosan oligomers, and propolis ethanolic extract were evaluated against white-rot fungus Trametes versicolor (L.) Lloyd, with a view to protecting Populus spp. wood. In order to create a more realistic in-service type environment, the biocidal products were assessed according to EN:113 European standard, instead of using routine in vitro antimicrobial susceptibility testing methods. Wood blocks were impregnated with the aforementioned antifungal agents by the vacuum-pressure method in an autoclave, and their biodeterioration was monitored over 16 weeks. The results showed that treatments based on silver nanoparticles, at concentrations ranging from 5 to 20 ppm, presented high antifungal activity, protecting the wood from fungal attack over time, with weight losses in the range of 8.49% to 8.94% after 16 weeks, versus 24.79% weight loss in the control (untreated) samples. This was confirmed by SEM and optical microscopy images, which showed a noticeably higher cell wall degradation in control samples than in samples treated with silver nanoparticles. On the other hand, the efficacy of the treatments based on chitosan oligomers and propolis gradually decreased over time, which would be a limiting factor for their application as wood preservatives. The nanometal-based approach is thus posed as the preferred choice for the industrial treatment of poplar wood aimed at wood-based engineering products (plywood, laminated veneer lumber, cross-laminated timber, etc.).

scholarly journals Prebiotic Activity of Poly- and Oligosaccharides Obtained from Plantago major L. Leaves

2020 ◽  
Vol 10 (8) ◽  
pp. 2648 ◽  
Author(s):  
Paolina Lukova ◽  
Mariana Nikolova ◽  
Emmanuel Petit ◽  
Redouan Elboutachfaiti ◽  
Tonka Vasileva ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Galacturonic Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Plantago Major ◽  
Prebiotic Activity ◽  
Molecular Fraction ◽  
Exclusion Chromatography

The aim of the present study was to evaluate the prebiotic potential of Plantago major L. leaves water-extractable polysaccharide (PWPs) and its lower molecular fractions. The structure of PWPs was investigated by high pressure anion exchange chromatography (HPAEC), size exclusion chromatography coupled with multi-angle laser light scattering detector (SEC-MALLS) and Fourier-transform infrared (FTIR) spectroscopy. The chemical composition and monosaccharide analyses showed that galacturonic acid was the main monosaccharide of PWPs followed by glucose, arabinose, galactose, rhamnose and xylose. FTIR study indicated a strong characteristic absorption peak at 1550 cm−1 corresponding to the vibration of COO− group of galacturonic acid. The PWPs was subjected to hydrolysis using commercial enzymes to obtain P. major low molecular fraction (PLM) which was successively separated by size exclusion chromatography on Biogel P2. PWPs and PLM were examined for in vitro prebiotic activity using various assays. Results gave evidence for changes in optical density of the bacteria cells and pH of the growth medium. A heterofermentative process with a lactate/acetate ratio ranged from 1:1 to 1:5 was observed. The ability of PLM to stimulate the production of certain probiotic bacteria glycohydrolases and to be fermented by Lactobacillus sp. strains was successfully proved.

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