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A highly thermotolerant laccase produced by Cerrena unicolor strain CGMCC 5.1011 for complete and stable malachite green decolorization.

ABSTRACT: Laccases are a class of multi-copper oxidases with important industrial values. A thermotolerant laccase produced by a basidiomycete fungal strain Cerrena unicolor CGMCC 5.1011 was studied. With glycerin and peptone as the carbon and nitrogen sources, respectively, a maximal laccase activity of 121.7 U/mL was attained after cultivation in the shaking flask for 15 days. Transcriptomics analysis revealed an expressed laccase gene family of 12 members in C. unicolor strain CGMCC 5.1011, and the gene and cDNA sequences were cloned. A glycosylated laccase was purified from the fermentation broth of Cerrena unicolor CGMCC 5.1011 and corresponded to Lac2 based on MALDI-TOF MS/MS identification. Lac2 was stable at pH 5.0 and above, and was resistant to organic solvents. Lac2 displayed remarkable thermostability, with half-life time of 1.67 h at 70 ºC. Consistently, Lac2 was able to completely decolorize malachite green (MG) at high temperatures, whereas Lac7 from Cerrena sp. HYB07 resulted in accumulation of colored MG transformation intermediates. Molecular dynamics simulation of Lac2 was conducted, and possible mechanisms underlying Lac2 thermostability were discussed. The robustness of C. unicolor CGMCC 5.1011 laccase would not only be useful for industrial applications, but also provide a template for future work to develop thermostable laccases.

SUBMITTER: Yao Y

PROVIDER: S-EPMC7532254 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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A highly thermotolerant laccase produced by Cerrena unicolor strain CGMCC 5.1011 for complete and stable malachite green decolorization.

Yao Yanhua Y Zhou Guimei G Lin Yonghui Y Xu Xinqi X Yang Jie J

AMB Express 20201002 1

Laccases are a class of multi-copper oxidases with important industrial values. A thermotolerant laccase produced by a basidiomycete fungal strain Cerrena unicolor CGMCC 5.1011 was studied. With glycerin and peptone as the carbon and nitrogen sources, respectively, a maximal laccase activity of 121.7 U/mL was attained after cultivation in the shaking flask for 15 days. Transcriptomics analysis revealed an expressed laccase gene family of 12 members in C. unicolor strain CGMCC 5.1011, and the gen ...[more]

PMID: 33006679

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Project description:Chronic lymphocytic leukemia (CLL) is the most commonly observed adult hematological malignancy in Western countries. Despite the fact that recent improvements in CLL treatment have led to an increased percentage of complete remissions, CLL remains an incurable disease. Cerrena unicolor is a novel fungal source of highly active extracellular laccase (ex-LAC) that is currently used in industry. However, to the best of our knowledge, no reports regarding its anti-leukemic activity have been published thus far. In the present study, it was hypothesized that C. unicolor ex-LAC may possess cytotoxic activity against leukemic cell lines and CLL primary cells. C. unicolor ex-LAC was separated using anion exchange chromatography on diethylaminoethyl cellulose-Sepharose and Sephadex G-50 columns. The cytotoxic effects of ex-LAC upon 24- and 48-h treatment on HL-60, Jurkat, RPMI 8226 and K562 cell lines, as well as CLL primary cells of nine patients with CLL, were evaluated using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Annexin V/propidium iodide staining of Jurkat cells treated with ex-LAC was used to investigate apoptosis via flow cytometry. Ex-LAC induced changes in Jurkat and RPMI 8226 cells, as visualized by fluorescence and scanning electron microscopy (SEM). The XTT assay revealed high cytotoxic rates following treatment with various concentrations of ex-LAC on all the cell lines and CLL primary cells analyzed, with a half maximal inhibitory concentration ranging from 0.4 to 1.1 µg/ml. Fluorescence microscopy and SEM observations additionally revealed apoptotic changes in Jurkat and RPMI 8226 cells treated with ex-LAC, compared with control cells. These results were in agreement with the apoptosis analysis of Jurkat cells on flow cytometry. In conclusion, C. unicolor ex-LAC was able to significantly induce cell apoptosis, and may represent a novel therapeutic agent for the treatment of various hematological neoplasms.

Project description:BackgroundLaccases are green biocatalysts with wide industrial applications. The study of efficient and specific laccase producers remains a priority. Cerrena species have been shown to be promising basidiomycete candidates for laccase production. Although two sets of Cerrena genome data have been publicly published, no comprehensive bioinformatics study of laccase gene family in C. unicolor has been reported, particularly concerning the analysis of their three-dimensional (3D) structures and molecular docking to substrates, like ABTS and aflatoxin B1 (AFB1).ResultsIn this study, we conducted a comprehensive genome-wide analysis of laccase gene family in C. unicolor 87613. We identified eighteen laccase genes (CuLacs) and classified them into three clades using phylogenetic analysis. We characterized these laccases, including their location in contig 5,6,9,12,15,19,26,27, gene structures of different exon-intron arrangements, molecular weight ranging from 47.89 to 141.41 kDa, acidic pI value, 5-15 conserved protein motifs, signaling peptide of extracellular secretion (harbored by 13 CuLacs) and others. In addition, the analysis of cis-acting element in laccase promoters indicated that the transcription response of CuLac gene family was regulatable and complex under different environmental cues. Furthermore, analysis of transcription pattern revealed that CuLac8, 12 and CuLac2, 13 were the predominant laccases in response to copper ions or oxidative stress, respectively. Finally, we focused on the 3D structure analysis of CuLac proteins. Seven laccases with extra transmembrane domains or special sequences were particularly interesting. Predicted structures of each CuLac protein with or without these extra sequences showed altered interacting amino acid residues and binding sites, leading to varied affinities to both ABTS and AFB1. As far as we know, it is the first time to discuss the influence of the extra sequence on laccase's affinity to substrates.ConclusionsOur findings provide robust genetic data for a better understanding of the laccase gene family in C. unicolor 87613, and create a foundation for the molecular redesign of CuLac proteins to enhance their industrial applications.

Project description:The white rot fungus Cerrena unicolor 87613 has been previously shown to be a promising resource in laccase production, an enzyme with significant biotechnological applications. Conventional methods face technical challenges in improving laccase activity. Attempts are still being made to develop novel approaches for further enhancing laccase activity. This study aimed to understand the regulation of laccase activity in C. unicolor 87613 for a better exploration of the novel approach. Transcriptomic and metabolomic analyses were performed to identify key genes and metabolites involved in extracellular laccase activity. The findings indicated a strong correlation between the glutathione metabolism pathway and laccase activity. Subsequently, experimental verifications were conducted by manipulating the pathway using chemical approaches. The additive reduced glutathione (GSH) dose-dependently repressed laccase activity, while the GSH inhibitors (APR-246) and reactive oxygen species (ROS) inducer (H2O2) enhanced laccase activity. Changes in GSH levels could determine the intracellular redox homeostasis in interaction with ROS and partially affect the expression level of laccase genes in C. unicolor 87613 in turn. In addition, GSH synthetase was found to mediate GSH abundance in a feedback loop. This study suggests that laccase activity is negatively influenced by GSH metabolism and provides a theoretical basis for a novel strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.IMPORTANCEThe production of laccase activity is limited by various conventional approaches, such as heterologous expression, strain screening, and optimization of incubation conditions. There is an urgent need for a new strategy to meet industrial requirements more effectively. In this study, we conducted a comprehensive analysis of the transcriptome and metabolome of Cerrena unicolor 87613. For the first time, we discovered a negative role played by reduced glutathione (GSH) and its metabolic pathway in influencing extracellular laccase activity. Furthermore, we identified a feedback loop involving GSH, GSH synthetase gene, and GSH synthetase within this metabolic pathway. These deductions were confirmed through experimental investigations. These findings not only advanced our understanding of laccase activity regulation in its natural producer but also provide a theoretical foundation for a strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.

Dataset Information

A highly thermotolerant laccase produced by Cerrena unicolor strain CGMCC 5.1011 for complete and stable malachite green decolorization.

Publications

A highly thermotolerant laccase produced by Cerrena unicolor strain CGMCC 5.1011 for complete and stable malachite green decolorization.

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