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1.
Appl Biochem Biotechnol ; 190(3): 1035-1048, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31659712

RESUMO

Laccases are a group of enzymes with a critical activity in the degradation process of both phenolic and non-phenolic compounds. These enzymes present in a diverse array of species, including fungi and bacteria. Since this enzyme is in the market for different usages from industry to medicine, having a better knowledge of its structures and properties from diverse sources will be useful to select the most appropriate candidate for different purposes. In the current study, sequence- and structure-based characteristics of these enzymes from fungi and bacteria, including pseudo amino acid composition (PseAAC), physicochemical characteristics, and their secondary structures, are being compared and classified. Autodock 4 software was used for docking analysis between these laccases and some phenolic and non-phenolic compounds. The results indicated that features including molecular weight, aliphatic, extinction coefficient, and random coil percentage of these protein groups present high degrees of diversity in most cases. Categorization of these enzymes by the notion of PseAAC, showed over 96% accuracy. The binding free energy between fungal laccases and their substrates showed to be considerably higher than those of bacterial ones. According to the outcomes of the current study, data mining methods by using different machine learning algorithms, especially neural networks, could provide valuable information for a fair comparison between fungal and bacterial laccases. These results also suggested an association between efficacy and physicochemical features of laccase enzymes from different sources.


Assuntos
Aminoácidos/análise , Bactérias/enzimologia , Fungos/enzimologia , Lacase/química , Lacase/isolamento & purificação , Modelos Químicos , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína , Reprodutibilidade dos Testes
2.
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
3.
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
4.
Bioprocess Biosyst Eng ; 42(10): 1635-1645, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31203448

RESUMO

Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Lacase/química , Lacase/isolamento & purificação , Polyporales/enzimologia
5.
Carbohydr Res ; 474: 57-66, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30738956

RESUMO

The N-glycans in Toxicodendron vernicifluum (Rhus vernicifera) lacquer laccase was elucidated for the first time through a combination of enzymatic digestion and subsequent mass spectrometry measurements using LC-MS/MS and MALDI-TOF MS. Lacquer laccase was isolated from a Japanese lacquer acetone powder from consecutive Sephadex C-50 and DEAE A-50 column chromatography. Trypsin and chymotrypsin digestions of the lacquer laccase resulted in a mixture of peptides and N-glycopeptides, which were treated with peptide-N-glycosidases and then Nα-(aminooxyacetyl)tryptophanylarginine methyl ester (aoWR) to give the aoWR-labelled N-glycans. The MS measurements revealed that GlcNAc4Hex5Fuc3Xyl1 N-glycan was attached at 12 N-glycosylation sites (Asn 5, 14, 180, 194, 233, 274, 284, 347, 364, 381, 398, and 519), GlcNAc3Hex4Fuc2Xyl1 N-glycan at two sites (Asn 124 and 454), and GlcNAc3Hex6Fuc1Xyl1 N-glycan at one site (Asn 28). A database search (Mascot search) of the peptides also suggested the presence of N-glycans at the 15 potential N-glycosylation sites (Asn-X-Ser/Thr).


Assuntos
Glicopeptídeos/análise , Lacase/química , Proteínas de Plantas/química , Polissacarídeos/química , Toxicodendron/química , Sequência de Aminoácidos , Sequência de Carboidratos , Cromatografia em Gel , Quimotripsina/química , Glicopeptídeos/química , Glicosídeo Hidrolases/química , Glicosilação , Lacase/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/química
6.
Bioconjug Chem ; 30(3): 679-697, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30620558

RESUMO

Trametes versicolor can degrade bark as a source for carbon necessity. Therefore, it secretes lignin peroxidase, mangan peroxidase, and laccase. The laccase enzyme was produced in high yield at pH of 5 and glucose concentration of 10 g L-1. In optimized medium, the enzyme activity was between 200 and 250 U L-1 when an inducer was absent. It was seen that the activity reached 400 U L-1 when phenol was used as an inducer. The molecular weight of purified laccase was found to be 80 kDa with SDS-PAGE, and kinetic constant Km and Vmax values for 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonate were determined to be 3.66 × 10-4 µM and 1652 U L-1, respectively. Because of these properties, these enzymes are widely used, free or immobilized, in industrial areas. Laccase enzyme decolorization of six different dyes was carried out. A decolorization capacity of 50-99% was achieved by cultivation for 20 days using a beginning dye concentration of 20 ppm. The removal of color with an active enzyme was obtained around 90%. Also, the laccase enzyme was conjugated, amine-functionalized, low-symmetry phthalocyanine. This conjugate was examined by both photodynamic therapy and chemosensor application. This conjugate fluorescence had a quantum yield of 0.32 (lifetime 3.59 ns) and efficiently generated singlet oxygen (quantum yield 0.4). The conjugate successfully displayed photodamage in HeLa and HuH-7 cells in the photodynamic therapy application. These results indicate that the conjugate represents an interesting agent with potential applications in photodynamic therapy. In addition, the chemosensor behavior of this compound to different metal ions has been studied, and this conjugate is displayed as a fluorescence chemosensor for the determination of Fe3+ions.


Assuntos
Cobre/química , Indóis/química , Peroxidases/química , Linhagem Celular , Precipitação Química , Cromatografia por Troca Iônica , Cor , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Humanos , Lacase/química , Lacase/isolamento & purificação , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Trametes/enzimologia
7.
Int J Biol Macromol ; 128: 132-139, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30684571

RESUMO

5-Formyl-2-furancarboxylic acid (FFCA) is increasingly important building blocks, which has promising applications in fuels, chemical intermediates, drugs, etc. However, highly selective oxidation of 5-hydroxymethylfurfural (5-HMF) to FFCA by enzyme catalysis is a new challenging problem due to the absence of specific enzymes. Recently we identified a kind of laccase (CotA-TJ102) from Bacillus subtilis TJ-102 could convert selectively 5-HMF to FFCA, and no further oxidation production (such as FDCA). The initial conditions resulted in low 5-HMF conversion ratio at 23.5% and low FFCA yield at 19.3% after 96 h, but high FFCA selectivity at 82.4%. Then the proposed mechanism in detail for the selective oxidation of HMF to FFCA by CotA-TJ102 was deduced and discussed. After optimization of reaction parameters such as pH, TEMPO concentration and temperature, FFCA could be obtained in a high yield of 98.55% with a 5-HMF conversion ratio of nearly 100% after a short reaction time of 12 h. Finally, after immobilization of CotA-TJ102 on magnetic nanoparticles, the FFCA yield remained 83.28% for 10 of recycling times based on the high FFCA selectivity (>96%), which reflected high stability and good reusability. Therefore, this enzymatic approach constitutes a promising method for the green production of FFCA.


Assuntos
Furaldeído/análogos & derivados , Furanos/metabolismo , Lacase/metabolismo , Oxirredução , Vias Biossintéticas , Biotransformação , Catálise , Ativação Enzimática , Estabilidade Enzimática , Enzimas Imobilizadas , Furaldeído/metabolismo , Expressão Gênica , Lacase/química , Lacase/genética , Lacase/isolamento & purificação , Nanopartículas de Magnetita , Relação Estrutura-Atividade
8.
Int J Biol Macromol ; 124: 530-536, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30500499

RESUMO

Laccase is one of the widely used enzymes for biotechnological processes. Immobilization of enzymes is a universally accepted approach to increase their reusability and stability. In this study, laccase enzyme from Trametes versicolor was encapsulated for the first time in a chitosan-nanobiochar matrix. The chitosan-tripolyphosphate gel formation technique was employed to produce homogeneous biocatalyst nanoparticles, with 35% effective binding efficiency and 3.5 Units/g apparent activity under the best configuration. The reusability of the encapsulated laccase was demonstrated towards the oxidation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) for several consecutive cycles, exhibiting 30% of the initial activity after 5 cycles. The encapsulated laccase showed a moderate increase in enzyme stability against pH and temperature variation compared to the free enzyme. Moreover, the storage stability of laccase at both 4 °C and 25 °C was increased after immobilization. Only 2% of laccase was leaked during a 5-day period from biocatalyst. Laccase in its free form showed no antibacterial activity against Gram positive and Gram-negative model microorganisms, while encapsulated laccase showed antibacterial activity towards Gram-positive ones. Thus, the encapsulation of the laccase is an efficient method to keep the enzyme active and stable for different applications.


Assuntos
Carvão Vegetal/química , Quitosana/análogos & derivados , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Lacase/química , Nanocompostos/química , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Benzotiazóis/química , Biocatálise , Quitosana/química , Composição de Medicamentos/métodos , Estabilidade Enzimática , Enzimas Imobilizadas/isolamento & purificação , Enzimas Imobilizadas/farmacologia , Reutilização de Equipamento , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Lacase/isolamento & purificação , Lacase/farmacologia , Testes de Sensibilidade Microbiana , Oxirredução , Ácidos Sulfônicos/química , Temperatura , Trametes/química
9.
Int J Biol Macromol ; 123: 1052-1061, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30465829

RESUMO

A bacterial laccase having potential to work in industry without mediator is of special interest. In this work, gene (1.83kb) encoding a novel laccase from Rheinheimera sp., having potential to deink waste paper without mediator, was cloned, over-expressed and the induced 69kDa protein (RhLacc) was purified and characterized. rhlacc gene was mutated by error prone PCR and mutants were sequenced. One mutant showed protein truncation resulting in the absence of domain 3 that contains T1 copper center. It is known that redox potential of T1 is the key parameter for substrate oxidation. Overexpression of this mutant gene showed induced 41.1kDa protein (∆RhLacc) that exhibited laccase activity but in the presence of added copper, compared to RhLacc which showed activity without added copper ions. Optimum temperature for both was 55°C. However, optimum pH varied with substrates. Kinetic studies showed ∆RhLacc had lower affinity for substrates except for guaiacol and reduced kcat in comparison to RhLacc. Both were able to deink old newspaper and degrade indigo carmine without mediator. The study suggests that the novel property to deink waste paper without mediator may not depend on the redox potential of T1 but other mechanisms using domains 1 and 2 may be involved.


Assuntos
Cobre/metabolismo , Lacase/química , Lacase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biocatálise , Simulação por Computador , Estabilidade Enzimática , Genoma Bacteriano , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Tinta , Íons , Cinética , Lacase/genética , Lacase/isolamento & purificação , Mutagênese/genética , Fenóis/análise , Domínios Proteicos , Alinhamento de Sequência , Solventes , Açúcares/análise , Temperatura
10.
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
11.
Int J Biol Macromol ; 125: 1042-1055, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30562519

RESUMO

The white laccase was produced from Myrothecium verrucaria ITCC-8447 under submerged fermentation. The media components were optimized by response surface methodology (CCD-RSM). The nutritional components (glucose and peptone) and physical parameters (pH and temperature) were optimized by response surface methodology for enhanced laccase production by Myrothecium verrucaria ITCC-8447. The enzyme activity under optimum condition exhibited 1.45 fold increases in laccase activity. The white laccase was subjected to ion exchange chromatography with 6 fold purification. The molecular weight of white laccase was ~63-75kDa as estimated by SDS-PAGE followed by the activity staining with ABTS where green bands confirmed the presence of laccase. The enzyme was stable over an alkaline pH range of 7-9 and the temperature range of 30-40°C. The characterization of white laccase was done by CD spectra, UV-visible absorption, FTIR and XRD. The Km and Vmax values of the purified laccase were 2.5mM and1818.2µmol/min/L. The delignification capability of the white laccase was determined by reduction in Kappa number (58.8%) and Klason lignin (64.7%) of wheat straw after 12h of incubation. Further the delignification was confirmed FTIR and XRD.


Assuntos
Proteínas Fúngicas/química , Hypocreales/enzimologia , Lacase/química , Lignina/química , Triticum/química , Estabilidade Enzimática , Análise Fatorial , Fermentação , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Hypocreales/química , Hypocreales/efeitos dos fármacos , Cinética , Lacase/isolamento & purificação , Lacase/metabolismo , Peso Molecular , Peptonas/metabolismo , Peptonas/farmacologia , Caules de Planta/química , Caules de Planta/metabolismo , Especificidade por Substrato , Temperatura , Triticum/metabolismo
12.
Int J Biol Macromol ; 120(Pt B): 1744-1751, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30268749

RESUMO

A laccase-producing ascomycete was isolated from arid soil in Tunisia. This fungus was identified as Thielavia sp. using the phylogenetic analysis of rDNA internal transcribed spacers. The extracellular laccase produced by the fungus was purified to electrophoretic homogeneity, showing a molecular mass around 70 kDa. The enzyme had an optimum pH of 5.0 and 6.0 for ABTS and 2,6­DMP, respectively and it showed remarkable high thermal stability, showing its optimal temperature at 70 °C (against 2,6­DMP). It presented slight inhibiting effect by EDTA, SDS and l­cyst although this effect was more marked by sodium azide (0.1 mM). On the other hand, it showed tolerance to up to 300 mM NaCl, retaining around 50% of its activity at 900 mM. Among the metal ions tested on TaLac1, Mn2+ showed an activating effect. Their kinetic parameters Km and kcat were 23.7 µM and 4.14 s-1 for ABTS, and 24.3 µM and 3.46 s-1 towards 2,6­DMP. The purified enzyme displayed greater efficiency in Remazol Brilliant Blue R decolorization (90%) in absence of redox mediator, an important property for biotechnological applications.


Assuntos
Ascomicetos/enzimologia , Corantes/química , Corantes/metabolismo , Lacase/isolamento & purificação , Lacase/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Cor , Poluentes Ambientais/química , Poluentes Ambientais/metabolismo , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Cloreto de Sódio/farmacologia , Temperatura
13.
PLoS One ; 13(8): e0202440, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30138464

RESUMO

The white-rot fungus Cerrena unicolor BBP6 produced up to 243.4 U mL-1 laccase. A novel laccase isoform LacA was purified; LacA is a homodimer with an apparent molecular mass of 55 kDa and an isoelectric point of 4.7. Its optimal pH was 2.5, 4.0, and 5.5 when 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP) were used as the substrates, respectively. The optimal temperature was 60°C for ABTS and 80°C for both guaiacol and 2, 6-DMP. LacA retained 82-92% activity when pH was greater than 4 and 42%-92% activity at or below 50°C. LacA was completely inhibited by 0.1 mM L-cysteine, 1 mM Dithiothreitol, and 10 mM metal ions, Ca2+, Mg2+ and Co2+. LacA had good affinity for ABTS, with a Km of 49.1 µM and a kcat of 3078.9 s-1. It decolorized synthetic dyes at 32.3-87.1%. In the presence of 1-hydroxybenzotriazole (HBT), LacA decolorized recalcitrant dyes such as Safranine (97.1%), Methylene Blue (98.9%), Azure Blue (96.6%) and simulated textile effluent (84.6%). With supplemented manganese peroxidase (MnP), Mn2+ and HBT, the purified LacA and BBP6 fermentation broth showed great potential in denim bleaching, with an up to 5-fold increase in reflectance values.


Assuntos
Corantes/química , Proteínas Fúngicas , Lacase , Polyporales/enzimologia , Têxteis , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Isoenzimas/química , Isoenzimas/isolamento & purificação , Lacase/química , Lacase/isolamento & purificação
14.
Fungal Biol ; 122(5): 302-309, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29665956

RESUMO

Laccase production in saline conditions is still poorly studied. The aim of the present study was to investigate the production of laccase in two different types of bioreactors by the marine-derived basidiomycete Peniophora sp. CBMAI 1063. The highest laccase activity and productivity were obtained in the Stirred Tank (ST) bioreactor, while the highest biomass concentration in Air-lift (AL) bioreactor. The main laccase produced was purified by ion exchange and size exclusion chromatography and appeared to be monomeric with molecular weight of approximately 55 kDa. The optimum oxidation activity was obtained at pH 5.0. The thermal stability of the enzyme ranged from 30 to 50 °C (120 min). The Far-UV Circular Dichroism revealed the presence of high ß-sheet and low α-helical conformation in the protein structure. Additional experiments carried out in flask scale showed that the marine-derived fungus was able to produce laccase only in the presence of artificial seawater and copper sulfate. Results from the present study confirmed the fungal adaptation to marine conditions and its potential for being used in saline environments and/or processes.


Assuntos
Organismos Aquáticos/metabolismo , Basidiomycota/metabolismo , Reatores Biológicos/microbiologia , Meios de Cultura/química , Lacase/metabolismo , Solução Salina/metabolismo , Organismos Aquáticos/crescimento & desenvolvimento , Basidiomycota/crescimento & desenvolvimento , Cromatografia em Gel , Cromatografia por Troca Iônica , Dicroísmo Circular , Sulfato de Cobre/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Lacase/química , Lacase/isolamento & purificação , Peso Molecular , Oxirredução , Estrutura Secundária de Proteína , Temperatura
15.
Int J Biol Macromol ; 113: 1142-1148, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29545062

RESUMO

Agaricus bisporus CU13 laccase was purified using ammonium sulfate precipitation (40-80%), Sephadex G100, and DEAE Sephadex A50 anion exchange column chromatography, respectively. Two laccase isoenzymes (Lacc1 & Lacc2) with purification folds of 1.40 and 5.81 respectively, were obtained from DEAE Sephadex A50 column. Optimal temperature and pH were recorded at 55 °C and pH 5.0 for both laccase isoenzymes using ABTS as substrate. Lacc1 was more thermostable than Lacc2 with residual activity of 95, 80 and 6%, while Lacc2 only retained 72, 25 and 0.4% of its activity after incubation for 90 min. at 50, 60 and 70 °C, respectively. Lacc2 retained about 93 and 86% of the initial activity at pH 9.0 and 7.0, whereas Lacc1 was stable at pH 7.0 and 5.0 followed by pH 9.0 and retained about 87, 76, and 36% of its activity respectively, after 4 h of incubation. Lacc1 was activated by 40% in the presence of Cu2+ (10 mM). Km and Vmax values found to be 0.394 and 0.158 µM, and 0.1351 and 0.4755 µmol min-1 for Lacc1 and Lacc2, respectively. The efficiency of both isoenzymes to decolorize Acid blue dye, make the enzyme seems to be a prospective for further biotechnological applications.


Assuntos
Agaricus/enzimologia , Corantes/metabolismo , Lacase/isolamento & purificação , Lacase/metabolismo , Agaricus/citologia , Biocatálise , Cor , Estabilidade Enzimática , Espaço Extracelular/enzimologia , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Lacase/química , Metais/farmacologia , Oxirredução , Temperatura
16.
J Appl Microbiol ; 124(6): 1454-1468, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29405500

RESUMO

AIMS: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. METHODS AND RESULTS: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat /Km values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. CONCLUSIONS: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. SIGNIFICANCE AND IMPACT OF THE STUDY: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.


Assuntos
Proteínas Fúngicas/genética , Lacase/genética , Lignina/metabolismo , Polyporales/enzimologia , Clonagem Molecular , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Expressão Gênica , Cinética , Lacase/química , Lacase/isolamento & purificação , Lacase/metabolismo , Pichia/genética , Pichia/metabolismo , Reação em Cadeia da Polimerase , Polyporales/química , Polyporales/genética , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
Int J Biol Macromol ; 112: 775-779, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29425870

RESUMO

Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60min at 80°C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants.


Assuntos
Biocatálise , Lacase/metabolismo , Sequência de Aminoácidos , Bacillus/enzimologia , Dicroísmo Circular , Estabilidade Enzimática , Cinética , Lacase/química , Lacase/isolamento & purificação , Proteínas Mutantes/química , Oxirredução
18.
Appl Microbiol Biotechnol ; 102(5): 2425-2439, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29380032

RESUMO

Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes. Integrity of laccase genes in anaerobes has been questioned, since laccases oxidize a variety of compounds using molecular oxygen as the electron acceptor. The genome of the anaerobe Geobacter metallireducens GS-15 contains five genes for laccase-like multicopper oxidases. In order to show whether one of the predicted genes encodes a functional laccase, the protein encoded by GMET_RS10855 was heterologously expressed in Escherichia coli cells. The His6-tagged enzyme (named GeoLacc) was purified to a large extent in the apoprotein, inactive form: incubation with CuSO4 allowed a 43-fold increase of the specific activity yielding a metallo-enzyme. The purified enzyme oxidized some of the typical laccase substrates, including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (2,6-DMP), along with pyrogallol and K4[Fe(CN)6]. Temperature optimum was 75 °C and pH optimum for ABTS and 2,6-DMP oxidation was ~ 6.0. As observed for other laccases, the enzyme was inhibited by halide anions and was sensitive to increasing concentrations of dimethyl sulfoxide and Tween-80. Notably, GeoLacc possesses a very high affinity for dioxygen: a similar activity was measured performing the reaction at air-saturated or microaerophilic conditions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Expressão Gênica , Geobacter/enzimologia , Lacase/química , Lacase/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Geobacter/química , Geobacter/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Lacase/genética , Lacase/metabolismo , Especificidade por Substrato
19.
J Biosci Bioeng ; 125(4): 371-376, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29331528

RESUMO

The laccase gene from Pycnoporus sanguineus was cloned and inserted between the strong Pcbh1 promoter and the Tcbh1 terminator from Trichoderma reesei to form the recombinant plasmid pCH-lac. Using Agrobacterium-mediated technique, the pCH-lac was integrated into the chromosomes of T. reesei. Twenty positive transformants were obtained by employing hygromycin B as a selective agent. PCR was used to confirm that the laccase gene was integrated into the chromosomal DNA of T. reesei. Laccase production by recombinant transformants was performed in shaking flasks, and the activity of laccase reached 8.8 IU/mL after 96-h fermentation under a batch process, and 17.7 IU/mL after 144-h fermentation using a fed-batch process. SDS-PAGE analysis of the fermentation broth showed that the molecular mass of the protein was about 68 kDa, almost the same as that of the laccase produced by P. sanguineus, which indicated that laccase was successfully expressed in T. reesei and secreted out of the cells. The laccase produced by the recombinant T. reesei showed good thermal stability, and could degrade the toxic phenolic material bisphenol A efficiently, after 1-h reaction with 0.06 IU/mL laccase and 0.5 mmol/L ABTS as the mediator at 60 °C and pH 4.5, the degradation rate reached 95%, which demonstrated that it had great potential value in treating the household garbage and wastewater containing the bisphenol A.


Assuntos
Compostos Benzidrílicos/metabolismo , Lacase/metabolismo , Fenóis/metabolismo , Pycnoporus/enzimologia , Trichoderma/genética , Trichoderma/metabolismo , Agrobacterium/genética , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Fermentação , Concentração de Íons de Hidrogênio , Lacase/química , Lacase/isolamento & purificação , Pycnoporus/genética
20.
Int J Biol Macromol ; 108: 642-649, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29203348

RESUMO

This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene (rSLAC) produced high titres of extracellular laccase (500 ±â€¯10 U/l), which were further increased seven fold by pre-incubation at 80 °C for 30 min. The enzyme (∼38 kDa) had an optimum activity at 80 °C, but optimum pH varied with substrate used. Km values for ABTS, SGZ and 2,6-DMP were 142.85 µM, 10 µM and 54.55 µM and the corresponding kcat values were 60.6 s-1, 25.36 s-1 and 27.84 s-1, respectively. The t1/2 values of the rSLAC at 60 °C, 70 °C, 80 °C were 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (Ed) was 117.275 kJ/mol while ΔG, ΔH and ΔS for thermal inactivation of the rSLAC were all positive. The rSLAC decolourised more than 90% of Brilliant Blue G and Trypan Blue dye in 6 h without the addition of a mediator. High titres of SLAC expressed in P. pastoris enhance its potential for various industrial applications.


Assuntos
Expressão Gênica , Lacase/genética , Lacase/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genética , Clonagem Molecular , Ativação Enzimática , Concentração de Íons de Hidrogênio , Lacase/química , Lacase/isolamento & purificação , Peso Molecular , Especificidade por Substrato , Termodinâmica
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