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1.
Int J Biol Macromol ; 93(Pt A): 837-842, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27642125

RESUMO

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.


Assuntos
Agaricales/enzimologia , Corantes/química , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Lacase/química , Lacase/isolamento & purificação , Estabilidade Enzimática , Concentração de Íons de Hidrogênio
2.
Int J Biol Macromol ; 81: 785-93, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26361865

RESUMO

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.


Assuntos
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
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