Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 649
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Gene ; 742: 144586, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32179171

RESUMO

Pycnoporus sanguineus, an edible mushroom, produces antimicrobial and antitumor bioactive compounds and pH- and thermo- stable laccases that have multiple potential biotechnological applications. Here we reported the complete genome of the species Pycnoporus sanguineus ACCC 51,180 by using the combination of Illumina HiSeq X Ten and the PacBio sequencing technology. The represented genome is 36.6 Mb composed of 59 scaffolds with 12,086 functionally annotated protein-coding genes. The genome of Pycnoporus sanguineus encodes at least 19 biosynthetic gene clusters for secondary metabolites, including a terpene cluster for biosynthesis of the antitumor clavaric acid. Seven laccases were identified, while 22 genes were found to be involved in the kynurenine pathway in which the intermediate metabolite 3-hydroxyanthranilic acid were catalyzed by laccases into cinnabarinic acid. This study represented the third genome of the genus Pycnoporus, and wound facilitate the exploration of useful sources from Pycnoporus sanguineus for future industrial applications.


Assuntos
Proteínas Fúngicas/genética , Genoma Fúngico/genética , Microbiologia Industrial/métodos , Lacase/genética , Pycnoporus/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Cinurenina/metabolismo , Lacase/metabolismo , Engenharia Metabólica , Oxazinas/metabolismo , Estabilidade Proteica , Pycnoporus/enzimologia , Metabolismo Secundário/genética
2.
Ecotoxicol Environ Saf ; 193: 110335, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32088549

RESUMO

In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s-1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.


Assuntos
Corantes/metabolismo , Lacase/metabolismo , Corantes de Rosanilina/metabolismo , Corantes/toxicidade , Escherichia coli/genética , Escherichia coli/metabolismo , Lacase/genética , Mutação , Corantes de Rosanilina/toxicidade
3.
BMC Plant Biol ; 19(1): 552, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830911

RESUMO

BACKGROUND: Understanding lignin biosynthesis and composition is of central importance for sustainable bioenergy and biomaterials production. Species of the genus Miscanthus have emerged as promising bioenergy crop due to their rapid growth and modest nutrient requirements. However, lignin polymerization in Miscanthus is poorly understood. It was previously shown that plant laccases are phenol oxidases that have multiple functions in plant, one of which is the polymerization of monolignols. Herein, we link a newly discovered Miscanthus laccase, MsLAC1, to cell wall lignification. Characterization of recombinant MsLAC1 and Arabidopsis transgenic plants expressing MsLAC1 were carried out to understand the function of MsLAC1 both in vitro and in vivo. RESULTS: Using a comprehensive suite of molecular, biochemical and histochemical analyses, we show that MsLAC1 localizes to cell walls and identify Miscanthus transcription factors capable of regulating MsLAC1 expression. In addition, MsLAC1 complements the Arabidopsis lac4-2 lac17 mutant and recombinant MsLAC1 is able to oxidize monolignol in vitro. Transgenic Arabidopsis plants over-expressing MsLAC1 show higher G-lignin content, although recombinant MsLAC1 seemed to prefer sinapyl alcohol as substrate. CONCLUSIONS: In summary, our results suggest that MsLAC1 is regulated by secondary cell wall MYB transcription factors and is involved in lignification of xylem fibers. This report identifies MsLAC1 as a promising breeding target in Miscanthus for biofuel and biomaterial applications.


Assuntos
Lacase/genética , Lignina/química , Proteínas de Plantas/genética , Poaceae/fisiologia , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/fisiologia , Lacase/metabolismo , Lignina/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Poaceae/química , Poaceae/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
4.
Molecules ; 24(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671660

RESUMO

Many dyes and pigments are used in textile and printing industries, and their wastewater has been classed as a top source of pollution. Biodegradation of dyes by fungal laccase has great potential. In this work, the influence of reaction time, pH, temperature, dye concentration, metal ions, and mediators on laccase-catalyzed Remazol Brilliant Blue R dye (RBBR) decolorization were investigated in vitro using crude laccase from the white-rot fungus Ganoderma lucidum. The optimal decolorization percentage (50.3%) was achieved at 35 °C, pH 4.0, and 200 ppm RBBR in 30 min. The mediator effects from syringaldehyde, 1-hydroxybenzotriazole, and vanillin were compared, and 0.1 mM vanillin was found to obviously increase the decolorization percentage of RBBR to 98.7%. Laccase-mediated decolorization percentages significantly increased in the presence of 5 mM Na+ and Cu2+, and decolorization percentages reached 62.4% and 62.2%, respectively. Real-time fluorescence-quantitative PCR (RT-PCR) and protein mass spectrometry results showed that among the 15 laccase isoenzyme genes, Glac1 was the main laccase-contributing gene, contributing the most to the laccase enzyme activity and decolorization process. These results also indicate that under optimal conditions, G. lucidum laccases, especially Glac1, have a strong potential to remove RBBR from reactive dye effluent.


Assuntos
Antraquinonas/metabolismo , Corantes/metabolismo , Lacase/genética , Reishi/enzimologia , Biodegradação Ambiental , Cor , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Lacase/química , Lacase/metabolismo , Metais/farmacologia , Reishi/genética , Temperatura , Fatores de Tempo , Transcrição Genética
5.
Genes (Basel) ; 10(9)2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514462

RESUMO

Laccase is a widely used industrial oxidase for food processing, dye synthesis, paper making, and pollution remediation. At present, laccases used by industries come mainly from fungi. Plants contain numerous genes encoding laccase enzymes that show properties which are distinct from that of the fungal laccases. These plant-specific laccases may have better potential for industrial purposes. The aim of this work was to conduct a genome-wide search for the soybean laccase genes and analyze their characteristics and specific functions. A total of 93 putative laccase genes (GmLac) were identified from the soybean genome. All 93 GmLac enzymes contain three typical Cu-oxidase domains, and they were classified into five groups based on phylogenetic analysis. Although adjacent members on the tree showed highly similar exon/intron organization and motif composition, there were differences among the members within a class for both conserved and differentiated functions. Based on the expression patterns, some members of laccase were expressed in specific tissues/organs, while some exhibited a constitutive expression pattern. Analysis of the transcriptome revealed that some laccase genes might be involved in providing resistance to oomycetes. Analysis of the selective pressures acting on the laccase gene family in the process of soybean domestication revealed that 10 genes could have been under artificial selection during the domestication process. Four of these genes may have contributed to the transition of the soft and thin stem of wild soybean species into strong, thick, and erect stems of the cultivated soybean species. Our study provides a foundation for future functional studies of the soybean laccase gene family.


Assuntos
Evolução Molecular , Lacase/genética , Proteínas de Plantas/genética , Caules de Planta/genética , Seleção Genética , Soja/genética , Resistência à Doença , Lacase/química , Lacase/metabolismo , Família Multigênica , Melhoramento Vegetal/métodos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Caules de Planta/fisiologia , Soja/enzimologia , Soja/microbiologia
6.
J Microbiol Biotechnol ; 29(9): 1383-1390, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31434174

RESUMO

In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE, cotG, and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a His6 tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be 50°C. Upon treatment with known laccase inhibitors, including EDTA, SDS, and NaN3, the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.


Assuntos
Antraquinonas/metabolismo , Bacillus subtilis/enzimologia , Corantes/metabolismo , Lacase/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Inibidores Enzimáticos/química , Expressão Gênica , Cinética , Lacase/antagonistas & inibidores , Lacase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Esporos Bacterianos/enzimologia , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Temperatura
7.
Int J Biol Macromol ; 137: 232-237, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260768

RESUMO

A highly thermostable laccase from Geobacillus sp. strain WSUCF1 was cloned into Escherichia coli (E. coli) using pRham N-His SUMO expression system. The thermostable laccase with a molecular weight ~30 kDa had a t1/2 (pH 6.0) of 120 h at 50 °C. The homology modelling for laccase structure showed the presence of Cu active centers with His and Cys residues involved in the active site and ligand binding activity of the enzyme, respectively. The Km, Vmax, Kcat and Kcat/Km values of the purified enzyme with ABTS were found to be 0.146 mM, 1.52 U/mg, 1037 s-1 and 7102.7 s-1 mM-1, respectively. The doping of recombinant WSUCF1 laccase to commercial enzyme cocktails Accellerase® 1500 and Cellic CTec2 improved the hydrolysis of untreated, alkali and acid treated corn stover by 1.31-2.28 times and bagasse by 1.32-2.02 times. Further, in-house enzyme cocktails with laccase hydrolyzed untreated, alkali and acid treated bagasse and gave 1.44, 1.1, and 0.92 folds higher sugar, respectively, when compared with Accellerase 1500. The results suggested that thermostable laccase can aid in the improved hydrolysis of lignocellulosic biomass.


Assuntos
Biomassa , Lacase/química , Lignina/química , Ativação Enzimática , Estabilidade Enzimática , Hidrólise , Íons/química , Lacase/genética , Lacase/isolamento & purificação , Metais/química , Proteínas Recombinantes , Termodinâmica
8.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261802

RESUMO

Laccases (EC 1.10.3.2) are multicopper oxidoreductases acting on diphenols and related substances. Laccases are highly important for biotechnology and environmental remediation. These enzymes contain mononuclear one T2 copper ion and two T3 copper ions (Cu3α and Cu3ß), which form the so-called trinuclear center (TNC). Along with the typical three-domain laccases Bacteria produce two-domain (2D) enzymes, which are active at neutral and basic pH, thermostable, and resistant to inhibitors. In this work we present the comparative analysis of crystal structures and catalytic properties of recombinant 2D laccase from Streptomyces griseoflavus Ac-993 (SgfSL) and its four mutant forms with replacements of two amino acid residues, located at the narrowing of the presumable T3-solvent tunnels. We obtained inactive enzymes with substitutions of His165, with Phe, and Ile170 with Ala or Phe. His165Ala variant was more active than the wild type. We suggest that His165 is a "gateway" at the O2-tunnel leading from solvent to the Cu3ß of the enzyme. The side chain of Ile170 could be indirectly involved in the coordination of copper ions at the T3 center by maintaining the position of the imidazole ring of His157 that belongs to the first coordination sphere of Cu3α.


Assuntos
Proteínas de Bactérias/química , Cobre/metabolismo , Lacase/química , Simulação de Acoplamento Molecular , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Estabilidade Enzimática , Lacase/genética , Lacase/metabolismo , Ligação Proteica , Streptomyces/enzimologia
9.
Int J Biol Macromol ; 138: 21-28, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31301394

RESUMO

The active laccases of ascomycetous fungus Setosphaeria turcica were identified by Native-PAGE and ESI-MS/MS, and one of these isozymes Stlac2 was heterologous expressed to investigate the decolorization of malachite green. Setosphaeria turcica produced three active laccase isozymes: Stlac1, Stlac2, and Stlac6. Stlac2 was heterologously expressed in both eukaryotic and prokaryotic expression systems. The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive, and also showed a higher molecular weight than predicted because of glycosylation. The depression of laccase activity was attributable to the incorrect glycosylation at Asn97. Stlac2 expressed in Escherichia coli and the recombinant Stlac2 exhibited activity of 28.23 U/mg with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. The highest activity was observed at pH of 4.5 and the temperature of 60 °C. The activity of recombinant Stlac2 was inhibited by 10 mM Na+, Mg2+, Ca2+, Mn2+, and increased by 10 mM of Fe3+ with a relatively activity of 315% compared with no addition. Cu2+ did not affect enzyme activity. Recombinant Stlac2 was capable of decolorizing 67.08% of 20 mg/L malachite green in 15 min without any mediators. CONCLUSIONS: Generally, recombinant protein of fungal laccase Stlac2 was active without glycosylation and decolorize malachite green efficiently, which has potential industrial applications.


Assuntos
Ascomicetos/enzimologia , Lacase/genética , Lacase/metabolismo , Corantes de Rosanilina/metabolismo , Sequência de Aminoácidos , Cor , Expressão Gênica , Glicosilação , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Lacase/química , Modelos Moleculares , Pichia/genética , Conformação Proteica , Temperatura
10.
Chemosphere ; 234: 733-745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31234090

RESUMO

Fungal laccases have shown great potential in industrial and environmental applications. They are generally produced as laccase isoenzymes. Thus, to further study the properties of different laccase isoenzymes and their performance in bio-remediation is essential for a deep understanding of laccase function and application. In this study, three Pleurotus ostreatus HAUCC 162 laccase isoenzymes were heterologously expressed, and the effects of different inhibitors, metal ions, and organic solvents on the activity of recombinant laccases were evaluated. In the dye decolorization test, LACC6 showed the highest ability to remove Malachite green (MG), Remazol Brilliant Blue R (RBBR), Bromophenol blue (BB), and Methyl orange (MO) among the three recombinant laccases. Removal rates within 24 h were 91.5%, 84.9%, 79.1%, and 73.1% for MG (100 mg/L), RBBR (100 mg/L), BB (100 mg/L), and MO (100 mg/L), respectively. The MG and RBBR transformation pathways were proposed by using High Performance Liquid Chromatography-Mass Spectrometry (LC-MS) analysis. Based on the results of this work, the production of recombinant LACC6 or improving the portion of LACC6 in the crude extracellular laccase may advance synthetic dye removal.


Assuntos
Corantes/isolamento & purificação , Lacase/metabolismo , Pleurotus/enzimologia , Antraquinonas/química , Antraquinonas/isolamento & purificação , Biodegradação Ambiental , Cor , Corantes/química , Isoenzimas/metabolismo , Lacase/genética , Proteínas Recombinantes/metabolismo , Corantes de Rosanilina/química , Corantes de Rosanilina/isolamento & purificação
11.
J Biosci Bioeng ; 128(5): 518-524, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31196789

RESUMO

Laccase Lcc9 from Coprinopsis cinerea heterologously expressed in Pichia pastoris (rLcc9) displayed different molecular weight and specific activity from the native laccase (nLcc9). Glycosylation may play a role in regulating the Lcc9 specific activity. To elucidate this hypothesis, in this study, firstly we demonstrated that rLcc9 and nLcc9 were glycoproteins, and then enzymatically deglycosylated them. The obtained drLcc9 and dnLcc9 showed an apparent decrease in their specific activities. Three putative N-glycosylation sites (N293, N313, and N454) were then predicted in Lcc9 and substituted to evaluate their roles in its specific activity. Molecular weight analysis on those mutants suggested that glycosylation should have occurred on N313 and N454 whereas not on N293 in rLcc9. Comparison of catalytic properties of those mutants revealed that glycosylation at N313 and N454 in rLcc9 could affect the binding affinity to substrates and the catalytic rate, respectively. In addition, the glycosylation could also affect the thermal stability of rLcc9 and nLcc9 since deglycosylation of those Lcc9s resulted in decreases in their thermal stability to some extent. These results will help us to understand the effect of glycosylation on biochemical characteristics of fungal laccases, and provide us support for the improvement of fungal laccase activity based on N-linked glycosylation modification.


Assuntos
Lacase/metabolismo , Pichia/metabolismo , Biocatálise , Glicosilação , Lacase/química , Lacase/genética , Pichia/genética , Proteínas Recombinantes/metabolismo
12.
Int. microbiol ; 22(2): 217-225, jun. 2019. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-184828

RESUMO

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) using Pleurotus ostreatus was investigated in the current study along with the expression levels of laccase genes involved in biodegradation under variable conditions. Biodegradation of PAHs (naphthalene, anthracene, and 1,10-phenanthroline) was detected spectrophotometrically. Recorded data revealed that biodegradation of the tested PAHs was time dependent. Elevated level of naphthalene biodegradation (86.47%) was observed compared to anthracene (27.87%) and 1,10-phenanthroline (24.51%) within 3 days post incubation. Naphthalene was completely degraded within 5 days. Further incubation enhanced the biodegradation of both anthracene and 1,10-phenanthroline until reaches 93.69% and 92.00% biodegradation of the initial concentration within an incubation period of 11 and 14 days, respectively. Naphthalene was selected as a PAH model. HPLC and thin layer chromatography of naphthalene biodegradation products at time intervals proposed that naphthalene was first degraded to alpha- and ß-naphthol which was further metabolized to salicylic and benzoic acid. The metabolic pathway of naphthalene degradation by this fungus was elucidated based on the detected metabolites. The expression profile of six laccase isomers was evaluated using real-time PCR. The transcriptome of the fungal laccase isomers recorded higher levels of transcription under optimized fermentation conditions especially in presence of both naphthalene and Tween 80. The accumulation of such useful metabolites from the biodegradation of PAH pollutants recommended white rot fungus as a potential candidate for production of platform chemicals from PAH wastes


No disponible


Assuntos
Perfilação da Expressão Gênica , Lacase/biossíntese , Naftalenos/metabolismo , Pleurotus/enzimologia , Pleurotus/metabolismo , Biotransformação , Lacase/genética , Redes e Vias Metabólicas/genética , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Espectrofotometria , Fatores de Tempo
13.
Bioengineered ; 10(1): 182-189, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31142180

RESUMO

Bacterial CotA-laccases exhibit higher activity in alkaline pH and salt concentration conditions compared to laccases from white-rot fungi. They are considered as green catalysts in decolorizing of industrial dyes. However, CotA-laccases are limited due to the low yield and catalytic efficiency as the spore-bound nature of CotA. A DNA shuffling strategy was applied to generate a random mutation library. To improve laccase activities, a mutant (T232P/Q367R 5E29) with two amino acid substitutions was identified. The catalytic efficiency of mutant 5E29 was 1.21 fold higher compared with that of the wild-type. The Km and kcat values of 5E29 for SGZ were of 20.3 ± 1.3 µM and 7.6 ± 2.7 s-1. The thermal stability was a slight enhancement. Indigo Carmine and Congo red were efficiently decolorized by using this mutant at pH 9.0. These results provide that 5E29 CotA-laccase is a good candidate for biotechnology applications under alkaline condition, with an effective decolorization capability.


Assuntos
Embaralhamento de DNA/métodos , Lacase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Concentração de Íons de Hidrogênio , Lacase/genética
14.
Pol J Microbiol ; 68(1): 71-81, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050255

RESUMO

Few publications report the occurrence of bacteria associated with fungal cells. The presence of bacteria associated with one strain of Pleurotus ostreatus (Fr.) P. Kumm. was described in the literature. We describe the biodiversity of bacteria associated with eight oyster mushroom strains from Japan, Poland, and the USA. The presence of microorganisms associated with all tested P. ostreatus strains was confirmed using fluorescent microscopy. Among 307 sequences, 233 of clones representing 34 genera and 74 sequences were identified as Bacteria. Most of the bacteria associated with the strain PUSAS were related to E. coli and two clones were related to Cupriavidus genus. The biodiversity of clones isolated from fungal strains originating from Japan and Poland ranged from 15 to 32 different bacterial clones. The most often the bacteria related to genus Curvibacter, Pseudomonas, Bacillus, Cupriavidus, Pelomonas, and Propionibacterium were associated with the strains of fungi mentioned above. Laccase-like (LMCO) genes were identified in whole bacterial DNA isolated from the associated bacteria but ß-glucosidase and ß-xylanase genes were not detected.Few publications report the occurrence of bacteria associated with fungal cells. The presence of bacteria associated with one strain of Pleurotus ostreatus (Fr.) P. Kumm. was described in the literature. We describe the biodiversity of bacteria associated with eight oyster mushroom strains from Japan, Poland, and the USA. The presence of microorganisms associated with all tested P. ostreatus strains was confirmed using fluorescent microscopy. Among 307 sequences, 233 of clones representing 34 genera and 74 sequences were identified as Bacteria. Most of the bacteria associated with the strain PUSAS were related to E. coli and two clones were related to Cupriavidus genus. The biodiversity of clones isolated from fungal strains originating from Japan and Poland ranged from 15 to 32 different bacterial clones. The most often the bacteria related to genus Curvibacter, Pseudomonas, Bacillus, Cupriavidus, Pelomonas, and Propionibacterium were associated with the strains of fungi mentioned above. Laccase-like (LMCO) genes were identified in whole bacterial DNA isolated from the associated bacteria but ß-glucosidase and ß-xylanase genes were not detected.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Interações Microbianas/fisiologia , Pleurotus/classificação , Bactérias/genética , Biodiversidade , DNA Bacteriano/genética , Endo-1,4-beta-Xilanases/genética , Japão , Lacase/genética , Microscopia de Fluorescência , Polônia , RNA Ribossômico 16S/genética , Estados Unidos , beta-Glucosidase/genética
15.
Molecules ; 24(10)2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31126120

RESUMO

Lignin is an important biological polymer in plants that is necessary for plant secondary cell wall ontogenesis. The laccase (LAC) gene family catalyzes lignification and has been suggested to play a vital role in the plant kingdom. In this study, we identified 45 LAC genes from the Brassica napus genome (BnLACs), 25 LAC genes from the Brassica rapa genome (BrLACs) and 8 LAC genes from the Brassica oleracea genome (BoLACs). These LAC genes could be divided into five groups in a cladogram and members in same group had similar structures and conserved motifs. All BnLACs contained hormone- and stress- related elements determined by cis-element analysis. The expression of BnLACs was relatively higher in the root, seed coat and stem than in other tissues. Furthermore, BnLAC4 and its predicted downstream genes showed earlier expression in the silique pericarps of short silique lines than long silique lines. Three miRNAs (miR397a, miR397b and miR6034) target 11 BnLACs were also predicted. The expression changes of BnLACs under series of stresses were further investigated by RNA sequencing (RNA-seq) and quantitative real-time polymerase chain reaction (qRT-PCR). The study will give a deeper understanding of the LAC gene family evolution and functions in B. napus.


Assuntos
Brassica napus/fisiologia , Lacase/genética , Estresse Fisiológico , Sequenciamento Completo do Genoma/métodos , Motivos de Aminoácidos , Brassica napus/enzimologia , Brassica napus/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Lacase/química , MicroRNAs/genética , Família Multigênica , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , RNA de Plantas/genética , Análise de Sequência de RNA
16.
Int J Mol Sci ; 20(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991752

RESUMO

: Two laccase-encoding genes from the marine-derived fungus Pestalotiopsis sp. have been cloned in Aspergillus niger for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the presence of sea salt. The optimal pH and temperature of PsLac1 and PsLac2 differed in relation to the substrates tested, and both enzymes were shown to be extremely stable at temperatures up to 50 °C, retaining 100% activity after 3 h at 50 °C. Both enzymes were stable between pH 4-6. Different substrate specificities were exhibited, and the lowest Km and highest catalytic efficiency values were obtained against syringaldazine and 2,6-dimethoxyphenol (DMP) for PsLac1 and PsLac2, respectively. The industrially important dyes-Acid Yellow, Bromo Cresol Purple, Nitrosulfonazo III, and Reactive Black 5-were more efficiently decolorized by PsLac1 in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). Activities were compared in saline conditions, and PsLac2 seemed more adapted to the presence of sea salt than PsLac1. The overall surface charges of the predicted PsLac three-dimensional models showed large negatively charged surfaces for PsLac2, as found in proteins for marine organisms, and more balanced solvent exposed charges for PsLac1, as seen in proteins from terrestrial organisms.


Assuntos
Corantes/metabolismo , Fungos/enzimologia , Lacase/metabolismo , Sequência de Aminoácidos , Aspergillus niger/genética , Clonagem Molecular/métodos , Corantes/isolamento & purificação , Estabilidade Enzimática , Fungos/genética , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Lacase/química , Lacase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salinidade , Especificidade por Substrato , Temperatura
17.
J Agric Food Chem ; 67(19): 5486-5495, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31012315

RESUMO

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.


Assuntos
Cannabis/microbiologia , Proteínas Fúngicas/metabolismo , Pleurotus/enzimologia , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Galactosidases/química , Galactosidases/genética , Galactosidases/metabolismo , Glicosilação , Lacase/química , Lacase/genética , Lacase/metabolismo , Lignina/metabolismo , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Peroxidases/química , Peroxidases/genética , Peroxidases/metabolismo , Caules de Planta/microbiologia , Pleurotus/classificação , Pleurotus/genética , Pleurotus/crescimento & desenvolvimento , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Transporte Proteico
18.
Environ Sci Pollut Res Int ; 26(15): 14943-14950, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30919176

RESUMO

Polychlorinated biphenyls (PCBs) represent a large group of recalcitrant environmental pollutants. Up to now, many studies have focused on bioremediation of PCBs by fungal strains; however, the mechanisms of adaptation of these strains towards PCBs remain unknown despite their importance in developing effective bioremediation processes. We studied five species, each consisting of two strains isolated either from PCB-polluted or PCB-unpolluted substrates (control strains). We investigated their responses to PCB contamination by studying their tolerance to PCBs, their ability to reduce these pollutants, and their expression level of Laccase genes. In Thermothelomyces thermophila, Thermothelomyces heterothallica, Thermoascus crustaceus, and Fusarium solani, all the studied strains showed a similar tolerance and PCB degradation regardless of their origin. In Schizophyllum commune, while both strains showed similar resistance to PCBs, i.e., PCBs and their degradation products presented no toxicity for these strains, the rate of PCB degradation of the strain from a PCB-polluted environment was significantly slightly higher. The PCB degradation did not correlate with the expression level of genes encoding Laccases. These results demonstrate that the tolerance and PCB degradation by the fungal strains, which did not involve Laccase genes, required different adaptation systems which seem to be constitutive or rapidly inducible by PCB according to the fungal species.


Assuntos
Ascomicetos/efeitos dos fármacos , Basidiomycota/efeitos dos fármacos , Lacase/genética , Bifenilos Policlorados/toxicidade , Adaptação Biológica , Ascomicetos/genética , Ascomicetos/metabolismo , Basidiomycota/genética , Basidiomycota/metabolismo , Biodegradação Ambiental , Poluentes Ambientais/metabolismo , Poluentes Ambientais/toxicidade , Regulação Fúngica da Expressão Gênica , Lacase/metabolismo , Bifenilos Policlorados/metabolismo , Microbiologia do Solo
19.
ACS Synth Biol ; 8(4): 833-843, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30897903

RESUMO

Fungal laccases are biotechnologically relevant enzymes that are capable of oxidizing a wide array of compounds, using oxygen from the air and releasing water as the only byproduct. The laccase structure is comprised of three cupredoxin domains sheltering two copper centers-the T1Cu site and the T2/T3 trinuclear Cu cluster-connected to each other through a highly conserved internal electron transfer pathway. As such, the generation of laccase chimeras with high sequence diversity from different orthologs is difficult to achieve without compromising protein functionality. Here, we have obtained a diverse family of functional chimeras showing increased thermostability from three fungal laccase orthologs with ∼70% protein sequence identity. Assisted by the high frequency of homologous DNA recombination in Saccharomyces cerevisiae, computationally selected SCHEMA-RASPP blocks were spliced and cloned in a one-pot transformation. As a result of this in vivo assembly, an enriched library of laccase chimeras was rapidly generated, with multiple recombination events simultaneously occurring between and within the SCHEMA blocks. The resulting library was screened at high temperature, identifying a collection of thermostable chimeras with considerable sequence diversity, which varied from their closest parent homologue by 46 amino acids on average. The most thermostable variant increased its half-life of thermal inactivation at 70 °C 5-fold (up to 108 min), whereas several chimeras also displayed improved stability at acidic pH. The two catalytic copper sites spanned different SCHEMA blocks, shedding light on the recognition of specific residues involved in substrate oxidation. In summary, this case-study, through comparison with previous laccase engineering studies, highlights the benefits of bringing together computationally guided recombination and in vivo shuffling as an invaluable strategy for laccase evolution, which can be translated to other enzyme systems.


Assuntos
Quimera/genética , Proteínas Fúngicas/genética , Lacase/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Aminoácidos/genética , Domínio Catalítico/genética , Temperatura Alta , Engenharia de Proteínas/métodos
20.
Appl Microbiol Biotechnol ; 103(7): 3061-3071, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30783720

RESUMO

A simple and stable immobilization of a laccase from Pleurotus ostreatus was obtained through genetic fusion with a self-assembling and adhesive class I hydrophobin. The chimera protein was expressed in Pichia pastoris and secreted into the culture medium. The crude culture supernatant was directly used for coatings of polystyrene multi-well plates without additional treatments, a procedure that resulted in a less time-consuming and chemicals reduction. Furthermore, the gene fusion yielded a positive effect with respect to the wild-type recombinant enzyme in terms of both immobilization and stability. The multi-well plate with the immobilized chimera was used to develop an optical biosensor to monitor two phenolic compounds: L-DOPA ((S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic acid) and caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid); the estimation of which is a matter of interest in the pharmaceutics and food field. The method was based on the use of the analytes as competing inhibitors of the laccase-mediated ABTS oxidation. The main advantages of the developed biosensor are the ease of preparation, the use of small sample volumes, and the simultaneous analysis of multiple samples on a single platform.


Assuntos
Técnicas Biossensoriais , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Pleurotus/enzimologia , Ácidos Cafeicos/metabolismo , Clonagem Molecular , Meios de Cultura/química , Enzimas Imobilizadas/biossíntese , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Lacase/genética , Levodopa/metabolismo , Oxirredução , Pichia/genética , Poliestirenos , Proteínas Recombinantes de Fusão/biossíntese
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA