Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer

A monokaryotic strain of the white-rot fungus Pycnoporus cinnabarinus was shown to produce, in a 2-L bioreactor culture, 100 mg·L-1 benzaldehyde (bitter almond aroma) from L-phenylalanine with a productivity of 33 mg·L-1·day-1. The addition of HP20 resin, a styrene divinylbenzene copolymer highly selective for benzaldehyde, enabled an eightfold increase in the production of benzaldehyde and a twofold increase in productivity. In the presence of HP20 resin, the production of 790 mg·L-1 benzaldehyde was concomitant with the synthesis of cinnamic acid derivatives of high organoleptic notes such as cinnamaldehyde, cinnamyl alcohol, and methyl cinnamate.Key words : benzaldehyde, L-phenylalanine, Pycnoporus cinnabarinus, adsorbents.

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Highly Efficient Production of Laccase by the Basidiomycete Pycnoporus cinnabarinus

Applied and Environmental Microbiology ◽

10.1128/aem.70.11.6379-6384.2004 ◽

2004 ◽

Vol 70 (11) ◽

pp. 6379-6384 ◽

Cited By ~ 68

Author(s):

Alexandra M. C. R. Alves ◽

Eric Record ◽

Anne Lomascolo ◽

Karin Scholtmeijer ◽

Marcel Asther ◽

...

Keyword(s):

Gene Expression ◽

Laccase Activity ◽

Expression System ◽

Schizophyllum Commune ◽

Laccase Production ◽

Efficient Production ◽

Laccase Gene ◽

Mrna Accumulation ◽

Pycnoporus Cinnabarinus ◽

Monokaryotic Strain

ABSTRACT An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml−1 in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter−1 was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml−1. These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter−1 when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml−1 (i.e., 360 mg liter−1) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter−1. In this case, maximal activities were 3,900 and 4,660 nkat ml−1, respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.

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DEGRADAÇÃO DA MADEIRA DE EUCALYPTUS SP. POR BASIDIOMICETOS DE PODRIDÃO BRANCA

Arquivos do Instituto Biológico ◽

10.1590/1808-1657v74p3212007 ◽

2007 ◽

Vol 74 (4) ◽

pp. 321-328

Author(s):

L.D. Abreu ◽

R.H. Marino ◽

J.B. Mesquita ◽

G.T. Ribeiro

Keyword(s):

Pleurotus Ostreatus ◽

Schizophyllum Commune ◽

Pycnoporus Cinnabarinus

RESUMO Avaliou-se a degradação de Eucalyptus sp. pelos basidiomicetos Pleurotus ostreatus, Pycnoporus cinnabarinus e Schizophyllum commune, in vitro e em condições de campo. Para tanto, na degradação in vitro foram utilizados discos de Eucalyptus sp. submetidos aos seguintes tratamentos: T1 controle; T2 – 5 mL de água; T3 – 10 mL de água; T4 meio de cultura batata-dextrose-ágar. O parâmetro analisado foi a perda de massa (em %), após 60 e 120 dias de incubação. Em condições de campo foram utilizados corpos de prova deEucalyptus sp. inoculados com substrato “spawn” dePleurotusostreatus, Pycnoporus cinnabarinus e Schizophyllum commune. Foram realizados os seguintes tratamentos: T1 – controle (sem água e sem inóculo); T2 – corpos de prova submersos por 24h em água e T3 – corpos de prova não submersos por 24h em água e avaliada a perda de massa (%), após 60 e 120 dias de incubação. A degradação dos discos de eucalipto in vitro e em condições de campo foi influenciada pelos isolados. Os discos de eucalipto, in vitro, inoculados com Pycnoporus cinnabarinus apresentaram, em média, 25,33% de perda de massa e o micélio foi mais vigoroso em relação ao isolado Pleurotus ostreatus e ao Schizophyllum commune. Os tratamentos empregados e o período de incubação, in vitro, não influenciaram a perda de massa dos discos de eucalipto. Em condições de campo, a perda de massa dos corpos de prova de eucalipto, inoculados com Pycnoporus cinnabarinus, foi de 15,79%, já com Pleurotus ostreatus foi de 12,45% e Schizophyllum commune 12,95%.

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Identification of an Oligosaccharide Dehydrogenase from Pycnoporus Cinnabarinus Provides Insights into Fungal Breakdown of Lignocellulose

10.21203/rs.3.rs-237113/v1 ◽

2021 ◽

Author(s):

Gabriele Cerutti ◽

Elena Gugole ◽

Linda Celeste Montemiglio ◽

Annick Turbé-Doan ◽

Dehbia Chena ◽

...

Keyword(s):

Function Analysis ◽

Sequence Similarity ◽

Functional Characterization ◽

Physiological Role ◽

Free Form ◽

Lignocellulose Degradation ◽

Carbohydrate Active Enzymes ◽

Recognition Mechanism ◽

Pycnoporus Cinnabarinus ◽

Enzymatic Function

Abstract Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin.Results: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a b(1à3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-p interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario.Conclusions: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

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Nueva variedad chilena de pycnoporus cinnabarinus (jacq. Ex.fr.) Karst. Estudio de sus características morfológicas y fisiológicas.

Boletín Micológico ◽

10.22370/bolmicol.1988.3.4.1551 ◽

2019 ◽

Vol 3 (4) ◽

Author(s):

Julio A. Burgos ◽

Sylvia E. Ortiz

Keyword(s):

Pycnoporus Cinnabarinus

Se describe la variedad ligninolítica Pycnoporus cinnabarinus (Jacq.ex Fr.) Kant. var. osorninus Burgos var. nov. basándonos en que sus características morfológicas difieren claramente de P. cinnabarinus. Además esta variedad tiene una tasa de crecimiento superior a la de P. cinnabarinus. El medio liquido con sulfato de amonio como fuente de nitrógeno estimula la formación de clamidosporas fructificacines en agar-extracto de malta o medios con aserrín de pino forman tubos con 4 poros/mm.

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Pretreatment of lignocellulosic material with fungi capable of higher lignin degradation and lower carbohydrate degradation improves substrate acid hydrolysis and the eventual conversion to ethanol

Canadian Journal of Microbiology ◽

10.1139/w08-003 ◽

2008 ◽

Vol 54 (4) ◽

pp. 305-313 ◽

Cited By ~ 75

Author(s):

Sarika Kuhar ◽

Lavanya M. Nair ◽

Ramesh Chander Kuhad

Keyword(s):

Phanerochaete Chrysosporium ◽

Lignin Degradation ◽

Ethanol Yield ◽

Fungal Isolate ◽

Fermentation Inhibitors ◽

Pycnoporus Cinnabarinus ◽

Acid Hydrolysate ◽

Carbohydrate Degradation ◽

Fungal Pretreatment ◽

Acid Load

Phanerochaete chrysosporium , Pycnoporus cinnabarinus ,and fungal isolates RCK-1 and RCK-3 were tested for their lignin degradation abilities when grown on wheat straw (WS) and Prosopis juliflora (PJ) under solid-state cultivation conditions. Fungal isolate RCK-1 degraded more lignin in WS (12.26% and 22.64%) and PJ (19.30% and 21.97%) and less holocellulose in WS (6.27% and 9.39%) and PJ (3.01% and 4.58%) after 10 and 20 days, respectively, than other fungi tested. Phanerochaete chrysosporium caused higher substrate mass loss and degraded more of holocellulosic content (WS: 55.67%; PJ: 48.89%) than lignin (WS: 18.89%; PJ: 20.20%) after 20 days. The fungal pretreatment of WS and PJ with a high-lignin-degrading and low-holocellulose-degrading fungus (fungal isolate RCK-1) for 10 days resulted in (i) reduction in acid load for hydrolysis of structural polysaccharides (from 3.5% to 2.5% in WS and from 4.5% to 2.5% in PJ), (ii) an increase in the release of fermentable sugars (from 30.27 to 40.82 g·L–1in WS and from 18.18 to 26.00 g·L–1in PJ), and (iii) a reduction in fermentation inhibitors (total phenolics) in acid hydrolysate of WS (from 1.31 to 0.63 g·L–1) and PJ (from 2.05 to 0.80 g·L–1). Ethanol yield and volumetric productivity from RCK-1-treated WS (0.48 g·g–1and 0.54 g·L–1·h–1, respectively) and PJ (0.46 g·g–1and 0.33 g·L–1·h–1, respectively) were higher than untreated WS (0.36 g·g–1and 0.30 g·L–1·h–1, respectively) and untreated PJ (0.42 g·g–1and 0.21 g·L–1·h–1, respectively).

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Enzymatic Synthesis of Galactooligosaccharides by the Condensation Action of Thermostable .ALPHA.-Galactosidase from Pycnoporus cinnabarinus.

NIPPON SHOKUHIN KOGYO GAKKAISHI ◽

10.3136/nskkk1962.38.722 ◽

1991 ◽

Vol 38 (8) ◽

pp. 722-728 ◽

Cited By ~ 5

Author(s):

Masaru MITSUTOMI ◽

Junya HONDA ◽

Akira OHTAKARA

Keyword(s):

Enzymatic Synthesis ◽

Pycnoporus Cinnabarinus ◽

Alpha Galactosidase

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Bioinformatics Analysis of Laccase (Lac1) in Pycnoporus cinnabarinus

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.121-122.502 ◽

2010 ◽

Vol 121-122 ◽

pp. 502-506

Author(s):

He Li ◽

Guo Ying Zhou ◽

Huai Yun Zhang ◽

Liang Guo

Keyword(s):

Amino Acid ◽

Manganese Peroxidase ◽

Bioinformatics Analysis ◽

Instability Index ◽

Pycnoporus Cinnabarinus ◽

Grand Average ◽

Glycosylation Sites ◽

The World ◽

Aliphatic Index

Pycnoporus cinnabarinus is a plant pathogen. It is common in many areas and is widely distributed throughout the world. Laccases of are some of the few oxidoreductases commercialized as industrial catalysts. In the present study, some characters of the amino acid sequence of P.cinnabarinus laccase (Lac1) were predicted and analyzed with the tools of bioinformatics. These results showed that the protein was composed of 20 kinds of amino acid; the theoretical pI of manganese peroxidase was 4.81 and the theoretical molecular weight of manganese peroxidase was 56292.0 Da; total number of atoms was 7806; the extinction coefficient was 58120 (280 nm). The N-terminal of the sequence considered was M (Met) and the estimated half-life was 30 hours (mammalian reticulocytes, in vitro). The instability index (II) was computed to be 34.50; this classifies the protein as stable. Aliphatic index was 82.64. Grand average of hydropathicity (GRAVY) was -0.063. There were 8 glycosylation sites, a signal peptide and conserved domains.

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Induction of Extracellular Hydroxyl Radical Production by White-Rot Fungi through Quinone Redox Cycling

Applied and Environmental Microbiology ◽

10.1128/aem.02137-08 ◽

2009 ◽

Vol 75 (12) ◽

pp. 3944-3953 ◽

Cited By ~ 99

Author(s):

Víctor Gómez-Toribio ◽

Ana B. García-Martín ◽

María J. Martínez ◽

Ángel T. Martínez ◽

Francisco Guillén

Keyword(s):

Hydroxyl Radical ◽

Fenton Reaction ◽

White Rot Fungi ◽

Ligninolytic Enzymes ◽

White Rot ◽

Oh Radicals ◽

Production Rates ◽

Pycnoporus Cinnabarinus ◽

Simple Strategy ◽

The One

ABSTRACT A simple strategy for the induction of extracellular hydroxyl radical (OH) production by white-rot fungi is presented. It involves the incubation of mycelium with quinones and Fe3+-EDTA. Succinctly, it is based on the establishment of a quinone redox cycle catalyzed by cell-bound dehydrogenase activities and the ligninolytic enzymes (laccase and peroxidases). The semiquinone intermediate produced by the ligninolytic enzymes drives OH production by a Fenton reaction (H2O2 + Fe2+ → OH + OH− + Fe3+). H2O2 production, Fe3+ reduction, and OH generation were initially demonstrated with two Pleurotus eryngii mycelia (one producing laccase and versatile peroxidase and the other producing just laccase) and four quinones, 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and 2-methyl-1,4-naphthoquinone (menadione [MD]). In all cases, OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H2O2 levels and Fe3+ reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.

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