Methyl syringate: An efficient phenolic mediator for bacterial and fungal laccases

Rosado, Tânia; Bernardo, Pedro; Kočí, Kamila; Coelho, Ana Varela; Robalo, M. Paula; Martins, Lı́gia O.

doi:10.1016/j.biortech.2012.08.023

Cited by 60 publications

(48 citation statements)

References 35 publications

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“…Syringaldehyde oxidation finally rendered a strong absorption maximum at 284 nm with a smaller peak at 370 nm, in concordance with the yellow product 2,6-dimethoxy p -benzoquinone. The latter has been reported as end product from the enzymatic oxidation of syringaldehyde, acetosyringone, syringic acid or sinapic acid, depending on the reaction conditions [21,29,30]. …”

Section: Resultsmentioning

confidence: 99%

New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

et al. 2013

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BackgroundFungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies.ResultsFirstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast.ConclusionsThe high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.

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Section: Resultsmentioning

confidence: 99%

New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

et al. 2013

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“…Slight variations of the optimal pH (< 3 to 5) were observed for the phenolic substrates, which are assumed to be related to the protonation/deprotonation state of the compound (Rosado et al 2012). The pH range in which significant activity was measured was wider for L Tv than for L Sc , with L Tv showing higher activity in the pH range 5.5 to 7.…”

Section: Discussionmentioning

confidence: 99%

“…L Sc was much less active on the phenolic substrates than on ABTS compared to L Tv . These differences in reactivity are reported to be related to differences in shape and chemical composition of the substrate binding site of the enzymes (Rosado et al 2012; Xu et al 1996). Differences in the phenol substitution seemed also to influence the activity.…”

Section: Discussionmentioning

confidence: 99%

Bacterial versus fungal laccase: potential for micropollutant degradation

et al. 2013

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Relatively high concentrations of micropollutants in municipal wastewater treatment plant (WWTP) effluents underscore the necessity to develop additional treatment steps prior to discharge of treated wastewater. Microorganisms that produce unspecific oxidative enzymes such as laccases are a potential means to improve biodegradation of these compounds. Four strains of the bacterial genus Streptomyces (S. cyaneus, S. ipomoea, S. griseus and S. psammoticus) and the white-rot fungus Trametes versicolor were studied for their ability to produce active extracellular laccase in biologically treated wastewater with different carbon sources. Among the Streptomyces strains evaluated, only S. cyaneus produced extracellular laccase with sufficient activity to envisage its potential use in WWTPs. Laccase activity produced by T. versicolor was more than 20 times greater, the highest activity being observed with ash branches as the sole carbon source. The laccase preparation of S. cyaneus (abbreviated LSc) and commercial laccase from T. versicolor (LTv) were further compared in terms of their activity at different pH and temperatures, their stability, their substrate range, and their micropollutant oxidation efficiency. LSc and LTv showed highest activities under acidic conditions (around pH 3 to 5), but LTv was active over wider pH and temperature ranges than LSc, especially at near-neutral pH and between 10 and 25°C (typical conditions found in WWTPs). LTv was also less affected by pH inactivation. Both laccase preparations oxidized the three micropollutants tested, bisphenol A, diclofenac and mefenamic acid, with faster degradation kinetics observed for LTv. Overall, T. versicolor appeared to be the better candidate to remove micropollutants from wastewater in a dedicated post-treatment step.

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“…In native plants, in-situ oxido-reductase catalysis activity initiates the polymerization through the cross-linking of phenoxy radicals, and the same technique can be applied in the in-vitro bonding of lignocellulosic materials (Cesarino et al 2012;Koch and Schmitt 2013). The laccase activity during delignification can be improved by adding a redox mediator, which increases the range of substrates from phenolic to non-phenolic compounds (Gutiérrez et al 2012;Rosado et al 2012).…”

Section: Lignin Polymerizationmentioning

confidence: 99%

Laccase, an Emerging Tool to Fabricate Green Composites: A Review

Mahmood¹,

Hashim²,

Sulaiman³

et al. 2015

BioResources

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In the last two decades, laccases have received much attention from researchers because of their specific ability to oxidize lignin. This function of laccase is very useful for applications in several biotechnological processes, including delignification in the pulp and paper industry and the detoxification of industrial effluents from the textile and petrochemical industries. This review focuses on laccase-mediated fiberboard synthesis. Growing concerns regarding the emission of formaldehyde from wood composites has prompted industrialists to consider the fabrication of green composites. Laccase-mediated fiber treatments oxidize the lignin component without affecting the cellulose structure. As a result, free radicals are generated on the fiber surface, and these can act as potential reactive sites for further cross-linking reactions in board manufacturing. Binderless fiberboards prepared using such methods can be considered as green composites because the manufacturing process involves no additional resin.

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Methyl syringate: An efficient phenolic mediator for bacterial and fungal laccases

Cited by 60 publications

References 35 publications

New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

Bacterial versus fungal laccase: potential for micropollutant degradation

Laccase, an Emerging Tool to Fabricate Green Composites: A Review

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