Discovery of two novel laccase-like multicopper oxidases from Pleurotus citrinopileatus and their application in phenolic oligomer synthesis

Abstract

Background Laccases and laccase-like multicopper oxidases (LMCOs) oxidize a vast array of phenolic compounds and amines, releasing water as a byproduct. Their low substrate specificity is responsible for their tremendous biotechnological interest, since they have been used for numerous applications. However, the laccases characterized so far correspond to only a small fraction of the laccase genes identified in fungal genomes. Therefore, the knowledge regarding the biochemistry and physiological role of minor laccase-like isoforms is still limited. Results In the present work, we describe the isolation, purification and characterization of two novel LMCOs, PcLac1 and PcLac2, from Pleurotus citrinopileatus. Both LMCOs were purified with ion-exchange chromatographic methods. PcLac2 was found to oxidize a broader substrate range than PcLac1, but both LMCOs showed similar formal potentials, lower than those reported previously for laccases from white-rot fungi. Proteomic analysis of both proteins revealed their similarity with other well-characterized laccases from Pleurotus strains. Both LMCOs were applied to the oxidation of ferulic and sinapic acid, yielding oligomers with possible antioxidant activity. Conclusions Overall, the findings of the present work can offer new insights regarding the biochemistry and variability of low-redox potential laccases of fungal origin. Low-redox potential biocatalysts could offer higher substrate selectivity than their high-redox counterparts, and thus, they could be of applied value in the field of biocatalysis.

Efficient Degradation of Aflatoxin B1 and Zearalenone by Laccase-like Multicopper Oxidase from Streptomyces thermocarboxydus in the Presence of Mediators

“…Multicopper oxidases, particularly laccase, were able to catalyze reactions involving a broad range of substrates, such as the model substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), lignin-related aromatic compounds, metal ions, and so on [32]. The purified recombinant StMCO was capable of oxidizing various substrates, including the model substrate ABTS, phenolic compound 2,6-dimethylphenol (DMP), and azo dye reactive black 5 (RB5), but it could not oxidize the phenolic compound guaiacol (GUA), non-phenolic compound veratryl alcohol (VA), and anthraquinone dye reactive blue 19 (RB19), which was similar to the substrate specificity of most other reported bacterial laccases [33,34]. However, the accurate classification of multicopper oxidase, assigned as laccase, still remained unclear [33], thus StMCO was termed a laccase-like multicopper oxidase.…”

The Laccase of Myrothecium roridum VKM F‐3565: A New Look at Fungal Laccase Tolerance to Neutral and Alkaline Conditions**

“…During polymerization of ferulic acid by laccase from basidiomycete Trametes pubescens the formation of several polymeric products was observed: two different dimeric compounds with [ M +H] + 387, a trimeric product with [ M +H] + 579, and tetrameric product with [ M +H] + 769 [40] . Dimeric ([ M +H] + 297, [ M +H] + 341, [ M +H] + 385) and trimeric ([ M +H] + 533, [ M +H] + 565, [ M +H] + 577) products were also found during oxidation of ferulic acid catalyzed by the laccases of the basidiomycete Pleurotus citrinopileatus [41] …”

“…) products were also found during oxidation of ferulic acid catalyzed by the laccases of the basidiomycete Pleurotus citrinopileatus. [41] The product of condensation of three molecules of coniferyl alcohol (R f 0.0) with successive elimination of the carbonyl group (CO) and ketene (CH 2 =C=O) or elimination of a fragment with m/z 71 ([M + H] + 577 (70 %), 549 (25 %), 506 (100 %), 464 (15 %), 393 (25 %)) was found in a neutral extract of the reaction mixture containing the laccase of M. roridum VKM F-3565. The trimeric polymers with a molecular ion mass [M + H] + 433 and [MH] + 447 were also found during coniferyl acid transformation catalyzed by the laccase of the basidiomycete L. strigosus 1566.…”

Laccase-Mediated Oxygen Reduction in Liquid Flow Fuel Cells for Efficient Oxidation of Biomass-Derived Aldehydes with Co-Generation of Electricity

“…19 Therefore, its substrate spectrum is relatively broad, including monophenols, polyphenols, methoxy-substituted phenols, aromatic compounds, amines, etc. 20 However, for macromolecular substrates such as lignin and its oligomers that cannot enter the substrate binding pocket of laccase, it is necessary to use low-molecularweight mediators to achieve electron transfer between substrate and enzyme to accelerate the electron transfer efficiency and further improve laccase oxidation activity. 21 Common laccase mediators include ABTS, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), HBT, ect.…”

“…Laccase (EC 1.10.3.2) is a structurally stable multicopper oxidase widely derived from fungi, plants, and bacteria, which can oxidize phenolic and nonphenolic compounds with O 2 as an electron acceptor and generate water. , Its active center consists of four copper atoms, and the substrate loses electrons at the mononuclear copper atom at the T1 position, then the electrons transfer through the histidine–cysteine–histidine tripeptide motif to the T2 position and two T3 position copper atoms, and finally to O 2 through the copper atom at the T2 position. , Fungal laccase is also a highly oxidizing biological enzyme with a high redox potential ( E θ ) between 500 and 800 mV vs SHE (standard hydrogen electrode) . Therefore, its substrate spectrum is relatively broad, including monophenols, polyphenols, methoxy-substituted phenols, aromatic compounds, amines, etc . However, for macromolecular substrates such as lignin and its oligomers that cannot enter the substrate binding pocket of laccase, it is necessary to use low-molecular-weight mediators to achieve electron transfer between substrate and enzyme to accelerate the electron transfer efficiency and further improve laccase oxidation activity .…”