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1.
Int J Biol Macromol ; 141: 855-867, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505206

RESUMO

Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70 °C = 1.2 h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2 h at 100 °C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding.


Assuntos
Aspergillus/enzimologia , Lacase/química , Modelos Moleculares , Conformação Proteica , Sequência de Aminoácidos , Catálise , Fenômenos Químicos , Estabilidade Enzimática , Glicosilação , Concentração de Íons de Hidrogênio , Lacase/biossíntese , Lacase/isolamento & purificação , Peso Molecular , Oxirredução , Relação Estrutura-Atividade , Especificidade por Substrato , Difração de Raios X
2.
J Basic Microbiol ; 59(8): 784-791, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31259434

RESUMO

Laccases are multicopper oxidases with high potential for industrial applications. Several basidiomycete fungi are natural producers of this enzyme; however, the optimization of production and selection of inducers for increased productivity coupled with low costs is necessary. Lignocellulosic residues are important lignin sources and potential inducers for laccase production. Pinus taeda, a dominant source of wood-based products, has not been investigated for this purpose yet. The aim of this study was to evaluate the production of laccase by the basidiomycete fungus Ganoderma lucidum in the presence of different inducers in submerged and solid-state fermentation. The results of submerged fermentation in presence of 5 µM CuSO 4 , 2 mM ferulic acid, 0.1 g/L P. taeda sawdust, or 0.05 g/L Kraft lignin indicated that although all the tested inducers promoted increase in laccase activity in specific periods of time, the presence of 2 mM ferulic acid resulted in the highest value of laccase activity (49 U/L). Considering the submerged fermentation, experimental design following the Plackett-Burman method showed that the concentrations of ferulic acid and P. taeda sawdust had a significant influence on the laccase activity. The highest value of 785 U/L of laccase activity on submerged fermentation was obtained on the seventh day of cultivation. Finally, solid-state fermentation cultures in P. taeda using ferulic acid or CuSO 4 as inducers resulted in enzymatic activities of 144.62 and 149.89 U/g, respectively, confirming the potential of this approach for laccase production by G. lucidum.


Assuntos
Fermentação , Lacase/biossíntese , Reishi/metabolismo , Sulfato de Cobre/metabolismo , Ácidos Cumáricos/metabolismo , Meios de Cultura/metabolismo , Lacase/metabolismo , Lignina/metabolismo , Pinus/metabolismo , Reishi/enzimologia , Fatores de Tempo
3.
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


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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
4.
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
5.
Int Microbiol ; 22(2): 217-225, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30810987

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 α- 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.


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
6.
Bioresour Technol ; 281: 99-106, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30807996

RESUMO

In this study, Plackett-Burman and central composite designs were applied to improve polyhydroxybutyrate (PHB) production from alkaline pretreatment liquor (APL) by Cupriavidus necator DSM 545 using a supplement system consisting of oxidative enzymes (laccase, aryl alcohol oxidase (AAO)), mediators (ABTS, HOBT), DMSO, silica nanoparticle Aerosol R816 and surfactant Tween 80. First, screening experiments under Plackett-Burman design showed R816, ABTS and Tween 80 could significantly enhance PHB production. Additional experiments showed that HOBT and DMSO could be removed, and laccase and AAO were needed to remain in the system. Second, a central composite design was applied to obtain the optimum supplemental levels of R816, ABTS and Tween 80. Under optimum conditions, theoretical maximum PHB production (1.9 g/L) was close to experimental PHB production (2.1 g/L). With the supplement system, a 10-fold increase was achieved compared to PHB production (0.2 g/L) without any supplements.


Assuntos
Hidroxibutiratos/metabolismo , Polissorbatos/farmacologia , Tensoativos/farmacologia , Oxirredutases do Álcool/metabolismo , Cupriavidus necator/enzimologia , Lacase/biossíntese , Oxirredução
7.
PLoS One ; 14(2): e0210892, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30753186

RESUMO

The content and size of stone cell clusters affects the quality of pear fruit, and monolignol polymerization and deposition in the cell walls constitute a required step for stone cell formation. Laccase (LAC) is the key enzyme responsible for the polymerization of monolignols. However, there are no reports on the LAC family in pear (Pyrus bretschneideri), and the identity of the members responsible for lignin synthesis has not been clarified. Here, 41 LACs were identified in the whole genome of pear. All Pyrus bretschneideri LACs (PbLACs) were distributed on 13 chromosomes and divided into four phylogenetic groups (I-IV). In addition, 16 segmental duplication events were found, implying that segmental duplication was a primary reason for the expansion of the PbLAC family. LACs from the genomes of three Rosaceae species (Prunus mummer, Prunus persica, and Fragaria vesca) were also identified, and an interspecies collinearity analysis was performed. The phylogenetic analysis, sequence alignments and spatiotemporal expression pattern analysis suggested that PbLAC1, 5, 6, 29, 36 and 38 were likely associated with lignin synthesis and stone cell formation in fruit. The two target genes of Pyr-miR1890 (a microRNA identified from pear fruit that is associated with lignin and stone cell accumulation), PbLAC1 and PbLAC14, were selected for genetic transformation. Interfamily transfer of PbLAC1 into Arabidopsis resulted in a significant increase (approximately 17%) in the lignin content and thicker cell walls in interfascicular fibre and xylem cells, which demonstrated that PbLAC1 is involved in lignin biosynthesis and cell wall development. However, the lignin content and cell wall thickness were not changed significantly in the PbLAC14-overexpressing transgenic Arabidopsis plants. This study revealed the function of PbLAC1 in lignin synthesis and provides important insights into the characteristics and evolution of the PbLAC family.


Assuntos
Frutas , Genoma de Planta , Lacase , Lignina , Proteínas de Plantas , Pyrus , Frutas/enzimologia , Frutas/genética , Estudo de Associação Genômica Ampla , Lacase/biossíntese , Lacase/genética , Lignina/biossíntese , Lignina/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Pyrus/enzimologia , Pyrus/genética
8.
Bioprocess Biosyst Eng ; 42(4): 567-574, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30652220

RESUMO

Chemical composition and physical structure of solid substrate have significantly impacts on fermentation performance. The aqueous ammonia was used to pretreat rice straw. Furthermore, the feasibility of pretreatment to improve laccase production was also evaluated in terms of the enzymatic digestibility, chemical structure, physical structure, and laccase production. The results showed that aqueous ammonia pretreatment could modify chemical compositions, destroy rigid structure of the lignocellulosic substrate, increase enzymatic digestibility and change water state, which were beneficial to facilitate the fungus growth and nutrition utilization. Pretreatment of lignocellulosic substrate with aqueous ammonia at 80 °C gave the best effect on laccase production, yielding 172.74 U/g laccase at 14 days, which was 3.4 times higher than that of the control. The aqueous ammonia pretreatment could alternate the physicochemical characteristics of lignocellulosic substrate, resulting in the improved laccase production, which was a promising method that might be explored in solid-state fermentation.


Assuntos
Basidiomycota/crescimento & desenvolvimento , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Lignina , Oryza/química , Fermentação , Hidrólise , Lignina/química , Lignina/metabolismo
9.
N Biotechnol ; 50: 44-51, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30668986

RESUMO

The high-molecular weight fraction of olive mill wastewater (HMW-OMW), a byproduct of olive oil biorefinery, was used at the reactor level as the basal medium for production of laccase and Mn-dependent peroxidase (MnP) by Trametes ochracea. Three reactor systems, namely stirred tank reactors equipped with either Rushton turbines or marine impeller and draft tube (STR and STR-MD, respectively) and an air-lift reactor (ALR) were compared for this purpose. Although inocula were supplied as intact pellets, in both STR-based systems fungal growth evolved rapidly into a dispersed form while the ALR enabled the maintenance of the pellet growth mode. STR was deemed to be the most promising system since it best supported the production MnP activity on the HMW-OMW-based medium and its performance in laccase production did not differ from that observed with the STR-MD. Among the stirring regimes considered (250, 400, 500 and 600 rpm), the best production in the STR was observed at 500 rpm and 1.0 vvm for both laccase (8850 ± 270 IU L-1 on day 15) and MnP (17,027.4 ± 87.2 IU L-1 on day 13). When the inocula were supplied to the STR in homogenized form, the MnP production peak (16,856 ± 1070 IU L-1) was attained 8 days earlier than the previous condition and that of laccase was nearly doubled (14,967 ± 907 IU L-1). When compared with literature data, T. ochracea MnP production and productivity on the HMW-OMW-based medium were the highest reported for a wild-type fungal strain.


Assuntos
Lacase/biossíntese , Lignina/metabolismo , Azeite de Oliva/metabolismo , Peroxidases/biossíntese , Trametes/metabolismo , Eliminação de Resíduos Líquidos , Águas Residuárias/química , Peso Molecular , Azeite de Oliva/química , Trametes/enzimologia
10.
Bioprocess Biosyst Eng ; 42(3): 367-377, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30470907

RESUMO

Production of laccase from Ganoderma lucidum RCK 2011 under solid-state fermentation (SSF) conditions was optimized using response surface methodology, resulting in an approximate eightfold increase compared to that in the unoptimized media. Further, the enzyme produced under SSF as whole fermented substrate (in situ SSF laccase) was found to be more stable than the in vitro enzyme (harvested by downstreaming processing of fermented wheat bran). Interestingly, the biobleaching potentials of both in situ and in vitro SSF laccases were comparable, saving 25% chlorine dioxide for achieving similar pulp brightness as obtained in the pulp treated chemically. The reduction in the demand of chlorine dioxide in the pulp bleaching sequence subsequently decreased the levels of adsorbable organic halogen (AOX) in the resulting effluents of the process by 20% compared to the effluents obtained from chemical bleaching sequence. Therefore, direct application of in situ SSF laccase in pulp biobleaching will be environmentally friendly as well as economical and viable for implementation in paper mills.


Assuntos
Proteínas Fúngicas , Lacase , Papel , Reishi/enzimologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Lacase/biossíntese , Lacase/química
11.
Appl Microbiol Biotechnol ; 103(2): 747-760, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30362077

RESUMO

Bacterial laccases have received considerable attention because of several advantages associated with the higher environmental stability of these enzymes compared with fungal laccases. In this study, a laccase-like gene from Burkholderia cepacia BNS was successfully cloned. This gene was found to encode a mature protein of 279 amino acids that exhibited laccase activity in dimer form. The mature protein was found to contain approximately 4 mol of copper per monomer, and the metal ion-binding sites were predicted. BC_lacL gene transcription levels were analyzed by qRT-PCR to study expression patterns in the presence of different putative inducers (copper ions, guaiacol, veratryl alcohol, vanillin, coniferaldehyde, p-coumaric acid, sinapic acid, and ferulic acid). Copper ions had a positive effect on both transcription levels and intracellular laccase activity. Interestingly, upon induction with sinapic acid, BC_lacL gene transcription was lower than in the presence of copper ions, but laccase activity was highest under these conditions. The BC_lacL protein expressed in Escherichia coli exhibited a specific activity of 7.81 U/mg with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate and 12.3 U/mg with 2,6-dimethoxyphenol (2,6-DMP) as the substrate after purification through Ni-affinity chromatography. The optimal activity and kinetic parameters of the recombinant BC_lacL protein were observed (kcat/Km = 3.96 s-1 µM-1) at a pH of 4.0 at 55 °C for ABTS oxidization and (kcat/Km = 11.6 s-1 µM-1) at a pH of 10.0 at 75 °C for 2,6-DMP oxidization. The protein exhibited high stability in an alkaline environment, with a half-life of more than 12 h. The same results were obtained via decolorization of eight dyes. Hence, this laccase-like enzyme may have potential industrial applications.


Assuntos
Burkholderia cepacia/enzimologia , Burkholderia cepacia/genética , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Expressão Gênica , Lacase/biossíntese , Lacase/genética , Sítios de Ligação , Clonagem Molecular , Coenzimas/análise , Cobre/análise , Ativadores de Enzimas/análise , Escherichia coli/genética , Cinética , Lacase/química , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Reação em Cadeia da Polimerase em Tempo Real , Temperatura
12.
Protein Expr Purif ; 154: 16-24, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30248451

RESUMO

The fungal laccase Lcc9 from Coprinopsis cinerea is a promising candidate for biotechnological applications due to its distinct biochemical properties. In the present work, Lcc9 cDNA was cloned from C. cinerea using reverse transcription polymerase chain reaction and heterologously expressed in Pichia pastoris GS115. The recombinant laccase was found to be a heavily hyperglycoprotein, with the molecular weight of 60.2 kDa as determined by MALDI-TOF. Laccase activity in the culture supernatant was 1750 ±â€¯83 U/L and reached 3138 ±â€¯62 U/L after expression condition optimization using orthogonal experiment. The biochemical property of the purified recombinant Lcc9 (rLcc9) was compared to that of wild-type Lcc9. rLcc9 shows a higher specific activity (315.3 U/mg) than Lcc9 (92.9 U/mg) when using ABTS (2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate)) as the substrate. Although rLcc9 and Lcc9 showed comparable optimal pH (6.5) and temperature (70 °C) toward syringaldazine, rLcc9 displayed higher activity and stability in the pH range of 6.5-8.5. rLcc9 showed improved ability to oxidize indigo carmine and 5 azo dyes when methyl syringate was used as the mediator, with the decolorization rate range from 71.9 ±â€¯3.2% to 99.1 ±â€¯1.6% for different dyes in a wide pH (4.5-9.0) and temperature (4-70 °C) ranges. In comparison, Lcc9 decolorized 50.3 ±â€¯2.1% to 98.2 ±â€¯2.0% of the dyes used. The improved activity and stability in alkaline pH of rLcc9 relative to Lcc9, and improved dye decolorization ability towards 6 dyes suggested greater application potential of rLcc9 in biotechnologies such as wastewater treatment.


Assuntos
Agaricales , Proteínas Fúngicas , Expressão Gênica , Lacase , Agaricales/enzimologia , Agaricales/genética , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Lacase/biossíntese , Lacase/química , Lacase/genética , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
13.
Folia Microbiol (Praha) ; 64(1): 91-99, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30084087

RESUMO

To obtain enzymatic preparations with higher laccase activity levels from Funalia floccosa LPSC 232, available for use in several applications, co-cultures with six filamentous microfungi were tested. A laccase non-producing soil fungus, identified as Penicillium commune GHAIE86, showed an outstanding ability to increase laccase activity (3-fold as compared to that for monoculture) when inoculated in 6-day-old F. floccosa cultures. Maximum laccase production with the F. floccosa and P. commune co-culture reached 60 U/mL, or twice that induced by chemical treatments alone. Our study demonstrated that co-culture with soil fungi might be a promising method for improving laccase production in F. floccosa. Although the enhancement of laccase activity was a function of P. commune inoculation time, two laccase isoenzymes produced by F. floccosa remained unchanged when strains were co-cultured. These data are compatible with the potential of F. floccosa in agricultural applications in soil, whose enzyme machinery could be activated by soil fungi such as P. commune.


Assuntos
Lacase/biossíntese , Interações Microbianas , Penicillium/fisiologia , Polyporaceae/enzimologia , Técnicas de Cocultura , Contagem de Colônia Microbiana , Lacase/química , Lacase/metabolismo , Penicillium/genética , Penicillium/crescimento & desenvolvimento , Polyporaceae/crescimento & desenvolvimento , Polyporales , Microbiologia do Solo , Fatores de Tempo
14.
Mar Biotechnol (NY) ; 21(1): 76-87, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30456695

RESUMO

Aureobasidium melanogenum strain 11-1 with a high laccase activity was isolated from a mangrove ecosystem. Under the optimal conditions, the 11-1 strain yielded the highest laccase activity up to 3120.0 ± 170 mU/ml (1.2 U/mg protein) within 5 days. A laccase gene (LAC1) of the yeast strain 11-1 contained two introns and encoded a protein with 570 amino acids and four conserved copper-binding domains typical of the fungal laccase. Expression of the LAC1 gene in the yeast strain 11-1 made a recombinant yeast strain produce the laccase activity of 6005 ± 140 mU/ml. The molecular weight of the recombinant laccase after removing the sugar was about 62.5 kDa. The optimal temperature and pH of the recombinant laccase were 40 °C and 3.2, respectively, and it was stable at a temperature less than 25 °C. The laccase was inhibited in the presence of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), and DL-dithiothreitol (DTT). The Km and Vmax values of the laccase for 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was 6.3 × 10-2 mM and 177.4 M/min, respectively. Many synthetic dyes were greatly decolored by the laccase.


Assuntos
Organismos Aquáticos , Proteínas Fúngicas/genética , Lacase/genética , Plasmídeos/metabolismo , Saccharomycetales/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Lacase/biossíntese , Lacase/isolamento & purificação , Peso Molecular , Filogenia , Plasmídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/classificação , Saccharomycetales/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Temperatura
15.
J Gen Appl Microbiol ; 65(1): 26-33, 2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-29952346

RESUMO

Laccases are unable to oxidize the non-phenolic components of complex lignin polymer due to their less redox potential (E0). Catalytic efficiency of laccases relies on the mediators that potentiates their oxidative strength; for breaking the recalcitrant lignin. Laccase from Bacillus sp. SS4 was evaluated for its compatibility with natural and synthetic mediators. (2 mM). It was found that acetosyringone, vanillin, orcinol and veratraldehyde have no adverse effect on the laccase activity up to 3 h. Syringaldehyde, p-coumaric acid, ferulic acid and hydroquinone reduced the enzyme activity ≥50% after 1.0 h, but laccase activity remained 100 to ~120% in the presence of synthetic mediators HBT (1-Hydroxylbenzotrizole) and ABTS. (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) after 3 h. MgSO4 and MnSO4 (40 mM) increased the enzyme activity 3.5 fold and the enzyme possessed ≥70% activity at a very high concentration. (2 M) of NaCl. The enzyme retained 40-110% activity in the presence of 10% DMSO (dimethylsulfoxide), acetone, methanol and ethyl acetate. On the other hand, CuSO4 (100 µM) induced the laccase production 8.5 fold without increasing the growth of bacterial cells. Laccase from SS4 appropriately decolorized the indigo carmine (50 µM) completely in the presence of acetosyringone (100 µM) within 10 min and 25% decolorization was observed after 4 h without any mediator.


Assuntos
Bacillus/enzimologia , Microbiologia Industrial , Lacase/fisiologia , Estresse Fisiológico/fisiologia , Sulfato de Cobre/farmacologia , Ativadores de Enzimas/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Índigo Carmim/metabolismo , Lacase/biossíntese , Lacase/metabolismo , Metais , Compostos Orgânicos , Oxirredução , Biossíntese de Proteínas/efeitos dos fármacos , Temperatura
16.
Bioprocess Biosyst Eng ; 42(3): 499-512, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30536123

RESUMO

Repeated batch semi-solid fermentation (sSF) process using wheat straw substrate and fungal growth of Ganoderma lucidum on solid substrate was studied for production of laccase. pH showed significant effect on laccase production. Highest laccase activity with pH controlled to 5.0 in batch sSF was 15257.2 ± 353.4 U L- 1 on 9th day. In repeated batch process at pH 5.0, insoluble biomass substrate and fungal growth were reused after liquid part of medium was replaced with glucose, ammonium phosphate (best nitrogen source) and combined glucose and ammonium phosphate solution separately. Refilled to 80% w v- 1 of initial soluble sugar of first batch resulted in highest laccase production with peak activity after 4 days from replacement. Production of enzyme increased from 15257.2 U L- 1 in first batch to cumulative 90164.4 U L- 1 in 29 days after six repeated batches, productivity increased from 1680.2 to 3110.3 U L- 1 day- 1 (∼ 1.9 times) due to reductions in inhibitory effects and time required for fungal growth. Utilization of wheat straw in repeated batch sSF was supported by composition analysis and morphological changes (scanning electron microscopy) of substrate. Economic production of laccase using agricultural residues in repeated batch sSF could be possible.


Assuntos
Biomassa , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Reishi/crescimento & desenvolvimento , Triticum/química , Técnicas de Cultura Celular por Lotes
17.
J Microbiol ; 57(2): 127-137, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30552631

RESUMO

Interspecific mycelial interactions between white rot fungi are always accompanied by an increased production of laccase. In this study, the potential of the white rot fungus Dichomitus squalens to enhance laccase production during interactions with two other white rot fungi, Trametes versicolor or Pleurotus ostreatus, was assessed. To probe the mechanism of laccase induction and the role that laccase plays during combative interaction, we analyzed the differential gene expression profile of the laccase induction response to stressful conditions during fungal interaction. We further confirmed the expression patterns of 16 selected genes by qRT-PCR analysis. We noted that many differentially expressed genes (DEGs) encoded proteins that were involved in xenobiotic detoxification and reactive oxygen species (ROS) generation or reduction, including aldo/keto reductase, glutathione S-transferases, cytochrome P450 enzymes, alcohol oxidases and dehydrogenase, manganese peroxidase and laccase. Furthermore, many DEG-encoded proteins were involved in antagonistic mechanisms of nutrient acquisition and antifungal properties, including glycoside hydrolase, glucanase, chitinase and terpenoid synthases. DEG analyses effectively revealed that laccase induction was likely caused by protective responses to oxidative stress and nutrient competition during interspecific fungal interactions.


Assuntos
Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Lacase/biossíntese , Lacase/genética , Interações Microbianas/fisiologia , Polyporaceae/enzimologia , Polyporaceae/genética , Técnicas de Cocultura , Genes Fúngicos/genética , Micélio/enzimologia , Micélio/genética , Micélio/fisiologia , Nutrientes , Estresse Oxidativo , Pleurotus/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA , Trametes/fisiologia , Transcriptoma
18.
J Biosci Bioeng ; 127(6): 672-678, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30573384

RESUMO

In this study, a novel thermophilic bacterial strain was isolated from Tattapani hot spring located in the Chhattisgarh state of India. The laccase was produced via submerged fermentation and purified by ammonium sulfate precipitation and anion exchange chromatography up to 13.7 fold. The 16S rRNA gene sequence and biochemical analysis revealed that the bacterial isolate is Bacillus sp. strain PC-3. The activity of extracellular crude laccase was determined to be 11.2 U/mL using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate. The SDS-PAGE revealed that the enzyme consists of single subunit with molecular size of 36 kDa. The laccase exhibited the maximum enzyme activity at temperature of 60°C and pH 7. Moreover, the laccase retained 99.1% of its original activity for 180 min and exhibited half-life of 3.75 h at 60°C. Similarly, the laccase retained 95% activity at pH 7 for 240 min and displayed significant activity at wider pH range. In addition, the laccase was used for functionalization of chitosan film and characterized for antioxidant and antimicrobial activity. Interestingly, the functionalized chitosan film showed the improved antioxidant and antimicrobial activity.


Assuntos
Bacillus/metabolismo , Quitosana/metabolismo , Lacase/metabolismo , Temperatura , Bacillus/enzimologia , Bacillus/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Lacase/biossíntese , Lacase/química , RNA Ribossômico 16S/genética , Especificidade por Substrato
19.
Pol J Microbiol ; 67(4): 417-430, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30550228

RESUMO

We isolated three laccase-producing fungus strains from Taxus rhizosphere. Myrotheium verrucaria strain DJTU-sh7 had the highest laccase activity of 216.2 U/ml, which was increased to above 300 U/ml after optimization. DJTU-sh7 had the best decolorizing effect for three classes of reactive dyes. The DJTU-sh7-containing fungal consortium displayed the robust decolorizing ability. Both color removal efficiency and chemical oxygen demand were increased in the consortium mediated biotransformation. Transcriptome changes of M. verrucaria elicited by azo dye and phenolic were quantified by the high throughput transcriptome sequencing, and the activities of the selected oxidases and reductases were determined. The possible involvement of oxidases and reductases, especially laccase, aryl alcohol oxidase, and ferric reductase in the biotransformation of dye and phenolic compounds was revealed at both transcriptomic and phenotypic levels. Revealing the transcriptomic mechanisms of fungi in dealing with organic pollutants facilitates the fine-tuned manipulation of strains in developing novel bioremediation and biodegradation strategies.We isolated three laccase-producing fungus strains from Taxus rhizosphere. Myrotheium verrucaria strain DJTU-sh7 had the highest laccase activity of 216.2 U/ml, which was increased to above 300 U/ml after optimization. DJTU-sh7 had the best decolorizing effect for three classes of reactive dyes. The DJTU-sh7-containing fungal consortium displayed the robust decolorizing ability. Both color removal efficiency and chemical oxygen demand were increased in the consortium mediated biotransformation. Transcriptome changes of M. verrucaria elicited by azo dye and phenolic were quantified by the high throughput transcriptome sequencing, and the activities of the selected oxidases and reductases were determined. The possible involvement of oxidases and reductases, especially laccase, aryl alcohol oxidase, and ferric reductase in the biotransformation of dye and phenolic compounds was revealed at both transcriptomic and phenotypic levels. Revealing the transcriptomic mechanisms of fungi in dealing with organic pollutants facilitates the fine-tuned manipulation of strains in developing novel bioremediation and biodegradation strategies.


Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Corantes/metabolismo , Rizosfera , Taxus/microbiologia , Transcriptoma , Compostos Azo , Biodegradação Ambiental , Biotransformação , Sequenciamento de Nucleotídeos em Larga Escala , Lacase/biossíntese , Fenóis/metabolismo , Fenótipo , Microbiologia do Solo
20.
World J Microbiol Biotechnol ; 34(11): 160, 2018 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341455

RESUMO

Pleurotus tuoliensis is a valuable, rare and edible mushroom that is been commercially cultivated and is rapidly developing in China markets. Low temperatures are required to induces primordia initiation for the successful production of fruiting bodies (basidiomes) during commercial cultivation. In this work, we investigated the enzymatic activities and performed transcription profiling analysis of enzymatic genes under different low temperature conditions. The results suggest that the enzymatic activities and transcription levels decrease or increase significantly at 4 and 13 °C. Lacc10 and mnp6 seems to play a dominant role during nutrition growth. Furthermore, the expression of laccase and peroxidase genes was highly correlated to the detected extracellular enzymatic activity. Cold stress genes expression profiles were upregulated under 4 °C/13 °C (3 days), while only the Hsp70 gene was downregulated (at the stage of fruiting bodies production) at 13 °C (12 days). Our results showed that the transcriptional regulation of laccase and ligninolytic peroxidase genes plays an important role in the fruiting bodies of Bailinggu under low temperature induction (4 °C). Induction at low temperatures was a highly important cultivation condition in Bailinggu.


Assuntos
Temperatura Baixa , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Pleurotus/enzimologia , Pleurotus/genética , Catalase/biossíntese , Catalase/genética , Catecol Oxidase/biossíntese , Catecol Oxidase/genética , China , Ensaios Enzimáticos , Perfilação da Expressão Gênica , Lacase/biossíntese , Lacase/genética , Peroxidase/biossíntese , Peroxidase/genética , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Transcriptoma
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