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Biochimie ; 148: 46-54, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29501482


A novel extracellular laccase was purified from fermentation broth of the white rot fungus Trametes sp. F1635 by a three-step protocol including two consecutive ion-exchange chromatography steps on DEAE-Sepharose and SP-Sepharose, and a final gel-filtration on Superdex 75. The purified laccase (TsL) was a monomeric protein with the molecular mass of 64.8 kDa. It demonstrated high oxidation activity of 4.00 × 104 U/mg towards ABTS. Its N-terminal amino acid sequence was AIGPVADLTIINNAV which was unique and sharing high similarity of other fungal laccases. TsL was a yellow laccase based on absorption spectrum analysis. It demonstrated an acidic pH optimum of 2.6 and temperature optimum of 50 °C towards ABTS. The Km and Vmax values towards ABTS were estimated to 18.58 µM and 1.35 µmol/min, respectively. TsL manifested effective decolorization activity towards eriochrome black T (EBT), remazol brilliant blue R (RBBR), malachite green (MG), and eriochrome black T (EBT) (over 60%). Violuric acid (VA) and acetosyringone (AS) were the optimal mediators for the laccase in dye decolorization. Results suggest that TsL demonstrates great potential for dye decolorization and water treatment.

Corantes/metabolismo , Espaço Extracelular/enzimologia , Lacase/metabolismo , Trametes/citologia , Sequência de Aminoácidos , Cor , Fermentação , Concentração de Íons de Hidrogênio , Cinética , Lacase/química , Peso Molecular , Temperatura , Trametes/enzimologia
Int J Biol Macromol ; 93(Pt A): 837-842, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27642125


A novel laccase was isolated from fermentation broth of the mycorrhizal fungus Leucoagaricus naucinus LAC-04 by using a protocol that comprising ion-exchange chromatography steps on DEAE-cellulose, SP-Sepharose, and Q-Sepharose, and finally gel filtration by fast protein liquid chromatography on Superdex 75. The laccase (LNL) was purified with a purification fold of 21.19 and a recovery rate of 19.8%. It is a monomeric protein with a molecular mass of 56kDa. LNL lacks absorption around 600nm, which indicates that the purified laccase is a yellow laccases. LNL demonstrates an optimal pH of 2.2 and an optimal temperature range of 30-60°C using ABTS as the substrate. It is inhibited in the presence of EDTA and metal ions including Cd2+, Co2+, Cu2+. The Km of the laccase towards ABTS is estimated to 50.12µM at pH 2.2 and 30°C. Moreover, the purified laccase manifests effective decolorizing activity towards azo, heterocyclic, and aromatic dyes including Bromothymol Blue, Eriochrome Black T, Evans Bue, Fuchsin Basic, and Remazol Brilliant Blue R.

Agaricales/enzimologia , Corantes/química , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Lacase/química , Lacase/isolamento & purificação , Estabilidade Enzimática , Concentração de Íons de Hidrogênio
Int J Biol Macromol ; 81: 785-93, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26361865


A novel laccase was purified from fermentation broth of white rot fungus Trametes sp. LAC-01 using an isolation procedure involving three ion-exchange chromatography steps on DEAE-cellulose, SP-Sepharose, and Q-Sepharose, and one gel-filtration step. The purified enzyme (TSL) was proved as a monomeric protein with a Mr of 59kDa based on SDS-PAGE and FPLC. Partial amino acid sequences were obtained by LC-MS/MS sharing considerably high sequence similarity with that of other laccases. It possessed optimal pH of 2.6 and temperature of 60°C using ABTS as the substrate. The Km of the laccase toward ABTS was estimated to 30.28µM at pH 2.6 and 40°C. TSL manifested considerably high oxidizing activity toward ABTS, but was avoid of degradative activity toward benzidine, caftaric acid, etc. It was effective in the decolorization of phenolic dyes - Bromothymol Blue and Malachite Green with decolorization rate higher than 60% after 24h of incubation. Adjunction of Cu(2+) with the final concentration of 2.0mmol/L significantly activated laccase production with a steady high level of 275.8-282.2U/mL in 96-144h. The high yield and short production period makes Trametes sp. LAC-01 and TSL potentially useful for industrial and environmental application and commercialization.

Corantes/química , Fungos/enzimologia , Lacase/química , Cromatografia Líquida , Cobre/química , DNA Intergênico , Ativação Enzimática , Estabilidade Enzimática , Fermentação , Fungos/classificação , Fungos/genética , Concentração de Íons de Hidrogênio , Cinética , Lacase/biossíntese , Lacase/isolamento & purificação , Peso Molecular , Filogenia , Especificidade por Substrato , Espectrometria de Massas em Tandem , Temperatura , Trametes/classificação , Trametes/enzimologia , Trametes/genética
Biotechnol Appl Biochem ; 60(4): 393-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24033593


A 36-kDa protein, with an N-terminal sequence highly homologous to polygalacturonase (PG) inhibiting proteins, was isolated from small brown-eyed cowpea seeds. The protein was unadsorbed on diethylaminoethyl cellulose but adsorbed on both Affi-gel blue gel and SP-sepharose. It inhibited mycelial growth in the fungus Mycosphaerella arachidicola with an half-maximal (50%) inhibitory concentration (IC50 ) of 3.3 µM. It reduced [methyl-(3) H] thymidine incorporation into MBL2 lymphoma and L1210 leukemia cells with an IC50 of 7.4 and 5.4 µM, respectively. It inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with an IC50 of 12.9 µM. However, it did not inhibit PG. The potent antifungal and antitumor activities of the protein suggest that it can be developed into an antifungal agent for combating M. arachidicola invasion in crops and an agent for cancer therapy in humans.

Antifúngicos/isolamento & purificação , Fabaceae/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Inibidores da Transcriptase Reversa/isolamento & purificação , Sementes/química , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fungos/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Proteínas de Plantas/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia
J Biomed Biotechnol ; 2012: 736472, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23093860


A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7'-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, K(m) values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC(50) of 1.8 µM, 1.7 µM, and 1.25 µM, respectively, signifying that it is an antipathogenic protein.

Agaricus/enzimologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Lacase/administração & dosagem , Lacase/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Inibidores da Transcriptase Reversa/química , Proliferação de Células/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Células Hep G2 , Humanos , Células MCF-7 , Neoplasias Experimentais/patologia