Laccase from the white-rot fungus Trametes trogii

The goal of this work was to investigate the decomposition of azo dyes by oxidative methods, such as laccase and ultrasound treatments. Each of these methods has strong and feeble sides. The laccase treatment showed high decolorization rates but cannot degrade all investigated dyes (reactive dyes), and high anionic strength led to enzyme deactivation. Ultrasound treatment can decolorize all tested dyes after 3 h at a high energy input, and prolonged sonication leads to nontoxic ionic species, which was demonstrated by ion chromatography and toxicity assays. For the first time, it was shown that a combination of laccase and ultrasound treatments can have synergistic effects, which was shown by higher degradation rates. Bulk light absorption and ion-pairing high-performance liquid chromatography (IP-HPLC) were used for process monitoring, while with reversedphase HPLC, a lower number of intermediates than expected by IP-HPLC was found. Liquid chromatographymass spectrometry indicated that both acid orange dyes lead to a common end product due to laccase treatment. Acid Orange 52 is demethylated by laccase and ultrasound treatment. Further results confirmed that the main effect of ultrasound is based on˙OH attack on the dye molecules.

Section: Discussionmentioning

confidence: 99%

“…The enzyme laccase generally is very sensitive to halide ions and small anions (18,53), which can bind on the T2/T3 trinuclear copper cluster site inhibiting the internal electron transfer.…”

Section: Discussionmentioning

confidence: 99%

Degradation of Azo Dyes by Laccase and Ultrasound Treatment

Tauber

Guebitz

Rehorek

2005

“…Laccase activity was assessed by measuring the enzymatic oxidation of 0.02 M 2,2Ј-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma) in McIlvine buffer (pH 3.4) at 30°C (9). The buffer solution was saturated with air by bubbling prior to the assay.…”

Section: Methodsmentioning

confidence: 99%

“…They are multicopper enzymes, highly glycosylated, employed by fungal organisms to colonize the environment, especially roots or, in general, the tree's surface. These enzymes are very attractive for biotechnological applications, mainly in the paper and textile industries, and the interest in their production is strong (9). Strain CBS2359/152F of K. lactis was transformed with the centromeric vector pLC12, carrying lcc1, a laccase gene from T. trogii, and subjected to the usual induction procedure.…”

Section: Klpdc1 Hypoxic Expressionmentioning

confidence: 99%

Induction by Hypoxia of Heterologous-Protein Production with the Kl PDC1 Promoter in Yeasts

Camattari

Bianchi

Branduardi

et al. 2007

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1␤ was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get ␤-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.

“…For instance, groups in the ortho or para position with respect to the hydroxyl groups of phenolic compounds, hydroxindoles, and aromatic compounds favor their oxidation by laccases. However, the presence of various groups, depending on their position and size, may produce the opposite effect due to steric inhibition of the enzyme (6,12,29). Specific receptors for phenolic compounds on the fungal hyphae surface of Heterobasidion annosum have been reported (19).…”

Section: Resultsmentioning

confidence: 99%

Use of Multiplex Reverse Transcription-PCR To Study the Expression of a Laccase Gene Family in a Basidiomycetous Fungus

González

Terrón

Zapico

et al. 2003

Laccases produced by white rot fungi are involved in the degradation of lignin and a broad diversity of other natural and synthetic molecules, having a great potential for biotechnological applications. They are frequently encoded by gene families, as in the basidiomycete Trametes sp. strain I-62, from which the lcc1, lcc2, and lcc3 laccase genes have been cloned and sequenced. A multiplex reverse transcription-PCR method to simultaneously study the expression of these genes was developed in this study. The assay proved to be quick, simple, highly sensitive, and reproducible and is particularly valuable when numerous samples are to be analyzed and/or if the amount of initial mRNA is limited. It was used to analyze the effect of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) and two of its isomers (2,5-dimethoxybenzyl alcohol and 3,5-dimethoxybenzyl alcohol) on differential laccase gene expression in Trametes sp. strain I-62. These aromatic compounds produced different induction patterns despite their chemical similarity. We found 2,5-dimethoxybenzyl alcohol to be the best inducer of laccase activity while also producing the highest increase in gene expression; 3,5-dimethoxybenzyl alcohol was the next best inducer. Transcript amounts of each gene fluctuated dramatically in the presence of these three inducers, while the total amounts of laccase mRNAs seemed to be modulated by a coordinated regulation of the different genes.