An extracellular yellow laccase from white rot fungus Trametes sp. F1635 and its mediator systems for dye decolorization.

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.

Purification, characterization, and cloning of an extracellular laccase with potent dye decolorizing ability from white rot fungus Cerrena unicolor GSM-01.

A novel laccase was purified from fermentation broth of the white rot fungus Cerrena unicolor strain GSM-01 following three ion-exchange chromatography steps and one gel-filtration step. The purified enzyme was determined to be a monomeric protein of 63.2kDa and demonstrated high oxidation activity of 2.05×104U/mg towards ABTS. Its cDNA, gene, and amino acid sequences were obtained. It possessed high sequence similarity with that of other laccases but different enzymatic properties. It manifested optimal pH and temperature of 2.6 and 45°C, respectively. Fe3+ and Fe2+ were the most efficient inhibitors towards Cerrena unicolor laccase (CUL), while Mn2+ can slightly enhance the laccase activity of 3.8-10.5%. The Km and Vmax of CUL were estimated to 302.7µM and 13.6µMm-1, respectively. CUL was effective in the decolorization of bromothymol blue, evans blue, methyl orange, and malachite green with decolorization efficiencies of 50%-85%. It possesses potential application in textile and environmental industries.

An extracellular yellow laccase with potent dye decolorizing ability from the fungus Leucoagaricus naucinus LAC-04.

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.

An extracellular laccase with potent dye decolorizing ability from white rot fungus Trametes sp. LAC-01.

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.

Identification of potential biomarkers for clear cell renal cell carcinoma based on microRNA-mRNA pathway relationships.

BACKGROUND: MicroRNAs (miRNAs) play important roles in tumor genesis. miRNA dysregulation has been widely studied and demonstrated in clear cell renal cell carcinoma (ccRCC). MATERIALS AND METHODS: We applied a newly proposed method for selecting miRNAs that discriminate between healthy controls and cancers. We initially extracted different miRNAs and mRNAs and then selected miRNA-mRNA dysregulation pairs. The pathways that involved mRNAs were acquired according to the functional enrichment. We integrated the miRNAs, mRNAs, and pathways and constructed the miRNA-mRNA pathway relationships based on the derived significant miRNAs. RESULTS: We acquired 566 antiregulated miRNA-mRNA pairs including 56 miRNAs and 485 mRNAs. Three significant pathways related to ccRCC, namely, arginine and proline metabolism, aldosterone-regulated sodium reabsorption, and oxidative phosphorylation, were observed. Based on the miRNA-mRNA pathway relationships, five significant miRNAs were identified as potential biomarkers: hsa-miR-425, hsa-miR-136, hsa-miR-335, hsa-miR-340, and hsa-miR-320d. CONCLUSION: This integrative network approach revealed important miRNAs in the ccRCC that can identify specific disease biomarkers, which can be used as targets for cancer treatment.