Laccase Isozymes Research Articles

Effects of Orange Peel Extract on Laccase Activity and Gene Expression in Trametes versicolor.

The genome of Trametes versicolor encodes multiple laccase isozymes, the expression of which is responsive to various conditions. Here, we set out to investigate the potential of orange peel extract as an inducer of laccase production in this white-rot fungus, in comparison to the previously identified inducing chemical compound, veratryl alcohol. For four geographically distinct T. versicolor strains, a positive correlation has been observed between their oxidative activity and incubation time in liquid cultures. The addition of 20% orange peel extract or 5 mM veratryl alcohol caused a rapid increase in the oxidative potential of T. versicolor M99 after 24 h, with a more pronounced effect observed for the orange peel extract. To elucidate the underlying molecular mechanisms of the induced laccase activity, a transcriptional gene expression analysis was performed for the seven individual laccase genes in T. versicolor, revealing the upregulation of several laccase genes in response to the addition of each inducer. Notably, the gene encoding TvLac5 demonstrated a substantial upregulation in response to the addition of 20% orange peel extract, likely contributing to the observed increase in its oxidative potential. In conclusion, our results demonstrate that orange peels are a promising agro-industrial side stream for implementation as inducing agents in large-scale laccase production with T. versicolor.

Functional Characterization of Laccase Isozyme (PoLcc1) from the Edible Mushroom Pleurotus ostreatus Involved in Lignin Degradation in Cotton Straw.

Fungal laccases play important roles in the degradation of lignocellulose. In this study, the laccase producing cotton straw medium for Pleurotus ostreatus was optimized by single-factor and orthogonal experiments, and to investigate the role of Lacc1 gene, one of the laccase-encoding genes, in the degradation of cotton straw lignin, an overexpression strain of Lacc1 gene was constructed, which was analyzed for the characteristics of lignin degradation. The results demonstrated that the culture conditions with the highest lignin degradation efficiency of the P. ostreatus were the cotton straw particle size of 0.75 mm, a solid-liquid ratio of 1:3 and containing 0.25 g/L of Tween in the medium, as well as an incubation temperature of 26 °C. Two overexpression strains (OE L1-1 and OE L1-4) of Lacc1 gene were obtained, and the gene expression increased 12.08- and 33.04-fold, respectively. The results of 1H-NMR and FTIR analyses of significant changes in lignin structure revealed that Lacc1 gene accelerated the degradation of lignin G-units and involved in the cleavage of β-O-4 linkages and the demethylation of lignin units. These findings will help to improve the efficiency of biodelignification and expand our understanding of its mechanism.

Fungal oxidoreductases and CAZymes effectively degrade lignocellulosic component of switchgrass for bioethanol production

• Fungal oxidoreductases and CAZymes were produced in a bioreactor. • Laccase isozymes removed lignin up to 33.9 % from switchgrass. • Microscopic and spectroscopic analysis clearly indicated lignin degradation. • Laccase treatment improved 22.47 fold sugar hydrolysis over untreated biomass. • Fermentation studies showed high ethanol yield (78.32 % of theoretical yield). Ganoderma lucidum , a basidiomycetous fungus, has enormous potential in the co-production of diverse extracellular oxidoreductases and CAZymes. In the present study, G. lucidum MDU-7 produced 510 U/ml of laccases, xylanase (3.0 IU/ml) and cellulase (3.1 FPU/ml) in a lab-scale bioreactor. Laccase pretreatment of switchgrass in a laboratory-designed bioreactor resulted in better phenol degradation (64.27 %), lignin removal (33.9 %), and increase in cellulose crystallinity (12.66 %). The pretreatment efficacy was validated using various microscopic and spectroscopic techniques (SEM, FTIR, and XRD). The hydrolysis of laccase pretreated biomass using diverse cellulases from Aspergillus oryzae MDU-4, Aspergillus flavus MDU-5, Trichoderma citrinoviride MDU-1, and Trichoderma longibrachiatum MDU-6 resulted in 162.7 mg/g, 120.4 mg/g, 157.3 mg/g, and 176.25 mg/g release of sugars, respectively. The enzymatic hydrolysates containing 3.37–5.11 g/L sugars were fermented by Saccharomyces cerevisiae NCIM-3640 strain which resulted into 1.32–1.96 g/L ethanol, with high production yield (31.42–39.16 %). Our findings suggest that laccase pretreatment technology may provide a promising and environmentally benign alternative for the sustainable development of lignocellulosic biorefinery.

Comparative Analysis of Peniophora lycii and Trametes hirsuta Exoproteomes Demonstrates "Shades of Gray" in the Concept of White-Rotting Fungi.

White-rot basidiomycete fungi are a unique group of organisms that evolved an unprecedented arsenal of extracellular enzymes for an efficient degradation of all components of wood such as cellulose, hemicelluloses and lignin. The exoproteomes of white-rot fungi represent a natural enzymatic toolbox for white biotechnology. Currently, only exoproteomes of a narrow taxonomic group of white-rot fungi—fungi belonging to the Polyporales order—are extensively studied. In this article, two white-rot fungi, Peniophora lycii LE-BIN 2142 from the Russulales order and Trametes hirsuta LE-BIN 072 from the Polyporales order, were compared and contrasted in terms of their enzymatic machinery used for degradation of different types of wood substrates—alder, birch and pine sawdust. Our findings suggested that the studied fungi use extremely different enzymatic systems for the degradation of carbohydrates and lignin. While T. hirsuta LE-BIN 072 behaved as a typical white-rot fungus, P. lycii LE-BIN 2142 demonstrated substantial peculiarities. Instead of using cellulolytic and hemicellulolytic hydrolytic enzymes, P. lycii LE-BIN 2142 primarily relies on oxidative polysaccharide-degrading enzymes such as LPMO and GMC oxidoreductase. Moreover, exoproteomes of P. lycii LE-BIN 2142 completely lacked ligninolytic peroxidases, a well-known marker of white-rot fungi, but instead contained several laccase isozymes and previously uncharacterized FAD-binding domain-containing proteins.

Production and properties of a laccase isozyme (FvLcc3) from the edible mushroom Flammulina velutipes, which is undetectable under the culture condition for fruiting body formation.

Extracellular laccase isozyme (FvLcc3) from the edible mushroom Flammulina velutipes was found to be undetectable under the culture condition for fruiting body formation. FvLcc3 was purified and determined to be an approximately 53-kDa monomeric protein. FvLcc3 showed the highest catalytic efficiency (kcat/Km) toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) followed by 2,6-dimethoxyphenol and guaiacol and did not oxidize 3,4-dihydroxy-l-phenylalanine and l-tyrosine.

Biodegradation of Endocrine Disrupting Chemicals with Laccase Isozymes from Recombinant Pichia pastori

Biodegradation of Endocrine Disrupting Chemicals with Laccase Isozymes from Recombinant Pichia pastori

Deciphering the Amendment-Induced Secretion of Ligninolytic Enzymes by Pleurotus pulmonarius

Pleurotus pulmonarius belonging to the white-rot fungal basidiomycetes group secretes extracellular ligninolytic enzymes for the degradation of agroresidues. The present study was carried out to evaluate the efficacy of different agro-residues for the enhanced production of ligninolytic enzymes and to authenticate their ability by protein analysis. The morphological and molecular sequences of white-rot fungi were characterised. Besides, the efficacy of organic and inorganic amendments in the secretion of ligninolytic enzymes by P. pulmonarius was characterised using SDS-PAGE and native PAGE analysis. The characterised strain of P. pulmonarius secreted enhanced laccase enzyme levels in the liquid medium through supplementation with organic and inorganic amendments. Wheat bran and groundnut cake each @5% enhanced secretions of Laccase, LiP and MnP. Copper sulphate at 150 μM enhanced the laccase enzyme and at 100 μM enhanced the LiP enzyme level by P. pulmonarius. Similarly, supplementation with manganese sulphate at 150 μM enhanced laccase, LiP and MnP enzyme levels compared to control. SDS-PAGE results showed protein banding patterns in the range of 50–85 kDa for the Lac enzyme in samples drawn from wheat bran and groundnut cake-supplemented substrates. Native PAGE results of laccase enzymes also showed that wheat bran (5%) + groundnut cake (5%) + CuSO4 (150 M) + MnSO4 (150 M) induced four laccase isozymes. Supplementing organic and inorganic amendments to the substrates would enhance the secretion of laccase enzyme that would aid in better breakdown of lignin.

Elucidating fungal Rigidoporus species FMD21 lignin-modifying enzyme genes and 2,3,7,8-tetrachlorodibenzo-p-dioxin degradation by laccase isozymes

White-rot fungus Rigidoporus sp. FMD21 is a lignin-modifying enzyme producing fungus that can degrade dioxin. Extracellular enzymes from FMD21 include laccase and manganese peroxidase which are promising enzymes for myco-remediation because of their wide substrate specificity and mild catalysis conditions. The FMD21 genome was sequenced using Ion Torrent technology and consists of 38.98 Mbps with a GC content of 47.4 %. Gene prediction using Augustus with Basidiomycota reference setting resulted in 8245 genes. Functional gene annotations were carried out by using several programs and databases. We focused on laccase and ligninolytic peroxidase genes, which are most likely involved in the degradation of aromatic pollutants. The genome of FMD21 contains 12 predicted laccase genes (10 out of 12 predicted as full length) and 13 putative ligninolytic peroxidases which were annotated as MnP or versatile peroxidases. Four predicted laccases showed a higher than 65 % binding chance to 2,3,7,8-TCDD with the highest at 72 % in in silico docking analysis. Heterologous expressed laccases showed activity towards three tested substrates included ABTS, guaiacol and 2,6-DMP. ABTS displayed two-stage oxidation which differed from natural FMD21 laccases. 2,3,7,8-TCDD was degraded by 50 % after two weeks of enzymatic treatment by three out of five laccase isozymes which were natural laccases secreted by FMD21. In this study, we provide direct evidence for the 2,3,7,8-TCDD biodegradation capability of fungal laccases.

Purification and Characterization of Two Novel Laccases from Peniophora lycii.

Although, currently, more than 100 laccases have been purified from basidiomycete fungi, the majority of these laccases were obtained from fungi of the Polyporales order, and only scarce data are available about the laccases from other fungi. In this article, laccase production by the white-rot basidiomycete fungus Peniophora lycii, belonging to the Russulales order, was investigated. It was shown that, under copper induction, this fungus secreted three different laccase isozymes. Two laccase isozymes—Lac5 and LacA—were purified and their corresponding nucleotide sequences were determined. Both purified laccases were relatively thermostable with periods of half-life at 70 °C of 10 and 8 min for Lac5 and LacA, respectively. The laccases demonstrated the highest activity toward ABTS (97 U·mg−1 for Lac5 and 121 U·mg−1 for LacA at pH 4.5); Lac5 demonstrated the lowest activity toward 2,6-DMP (2.5 U·mg−1 at pH 4.5), while LacA demonstrated this towards gallic acid (1.4 U·mg−1 at pH 4.5). Both Lac5 and LacA were able to efficiently decolorize such dyes as RBBR and Bromcresol Green. Additionally, phylogenetic relationships among laccases of Peniophora spp. were reconstructed, and groups of orthologous genes were determined. Based on these groups, all currently available data about laccases of Peniophora spp. were systematized.

Stabilization–destabilization and redox properties of laccases from medicinal mushroom Ganoderma lucidum and human pathogen Yersinia enterocolitica

Laccases or benzenediol oxygen oxidoreductases (EC 1.10.3.2) are polyphenol multicopper oxidases that are known for their structural and functional diversity in various life forms. In the present study, the molecular and physico-chemical properties (redox-potential and secondary structures) of fungal laccase isozymes (FLIs) isolated from a medicinal mushroom Ganoderma lucidum were analyzed and compared with those of the recombinant bacterial laccases (rLac) obtained from different Yersinia enterocolitica strains. It was revealed that the FLIs contained His-Cys-His as the most conserved residue in its domain I Cu site, while the fourth and fifth residues were variable (Ile, Leu, or Phe). Evidently, the cyclic voltammetric measurements of Glac L2 at Type 1 Cu site revealed greater E° for ABTS/ABTS+ (0.312 V) and ABTS+/ABTS2+ (0.773 V) compared to the E° of rLac. Furthermore, circular dichroism-based conformational analysis revealed structural stability of the FLIs at acidic pH (3.0) and low temperature (<30 °C), while the isozymes were destabilized at neutral pH (7.0) and high-temperature conditions (>70 °C). The zymographic studies further confirmed the functional inactivation of FLIs at high temperatures (≥70 °C), predominantly due to domain unfolding. These findings provide novel insight into the evolution of the catalytic efficiency and redox properties of the FLIs, contributing to the existing knowledge regarding stress responses, metabolite production, and the biotechnological utilization of metabolites.

Metabolic coupling in the co-cultured fungal-yeast suite of Trametes ljubarskyi and Rhodotorula mucilaginosa leads to hypersecretion of laccase isozymes

Trametes ljubarskyi produces multiple laccase isozymes under various physicochemical conditions. During co-cultivation condition Rhodotorula mucilaginosa showed inter-specific interactions with T. ljubarskyi and hypersecretion of laccases; however, the underlying molecular mechanism is less-known. The analysis of proteomics data of co-cultivated cultures revealed the mechanism of metabolic coupling during fungal-yeast interactions. The results suggested high score GO terms related to stimulus-response, protein binding, membrane components, transport channels, oxidoreductases, and antioxidants. The SEM studies confirmed the cellular communication and their inter-specific interactions. This study allows us to deepen and refine our understanding of fungal-yeast symbiotic interaction; further, it also establishes a mutual relation by metabolic coupling for 10-fold higher laccase isozyme secretion (6532 U/ml). The purified laccase isozymes showed acidic pH optima (pH 3–4), higher thermo-stability (60 °C), and broad enzyme kinetics (Km) values. Our study also provides an in-depth understanding of laccase isozymes and their potential to degrade synthetic dyes, which may help the fungi to survive in an adverse environment.

Molecular cloning of cDNA encoding a major laccase isozyme from Grifola frondosa and its expression in Pichia pastoris

Molecular cloning of cDNA encoding a major laccase isozyme from Grifola frondosa and its expression in Pichia pastoris

Development of an eco-friendly deinking process for the production of bioethanol using diverse hazardous paper wastes

Bioethanol production using paper wastes seems a promising approach towards sustainable energy, but mainly hindered by hazardous ink. Therefore, in our experimental plan various methods were applied for the deinking of waste newspapers, laser printed papers and examination papers. A newly designed paper pulper reduced the pulping time from 6 h to 2 h. Cellulase (15 FPU/g) from Aspergillus oryzae MDU-4 was found effective for the deinking of newspapers, whereas laccase isozymes (150 U/g) from Ganoderma lucidum MDU-7 along with 2 mM HOBt was preferred for the ink removal and degradation from the examination papers. Ozonation in the presence of Tween-80 was found to be efficient in the removal of toxic toners used in laser printing papers. The biologically and physically deinked papers, studied with the help of SEM, TEM, FTIR, and XRD analysis revealed significant changes in the chemical and surface structure. Moreover, the saccharification of deinked papers with the help of an enzymatic consortium of Trichoderma citrinoviride MDU-1 resulted in 305 mg/g, 377 mg/g, and 409 mg/g release of sugars from the newspaper, examination paper, and laser printed paper, respectively. Finally, the enzymatic hydrolysates fermented with Saccharomyces cerevisiae NCIM-3640 produced 3.35 g/L ethanol, with 40.85% ethanol yield.

Heterologous expression of Stlac2, a laccase isozyme of Setosphearia turcica, and the ability of decolorization of malachite green

The active laccases of ascomycetous fungus Setosphaeria turcica were identified by Native-PAGE and ESI-MS/MS, and one of these isozymes Stlac2 was heterologous expressed to investigate the decolorization of malachite green. Setosphaeria turcica produced three active laccase isozymes: Stlac1, Stlac2, and Stlac6. Stlac2 was heterologously expressed in both eukaryotic and prokaryotic expression systems. The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive, and also showed a higher molecular weight than predicted because of glycosylation. The depression of laccase activity was attributable to the incorrect glycosylation at Asn97. Stlac2 expressed in Escherichia coli and the recombinant Stlac2 exhibited activity of 28.23 U/mg with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. The highest activity was observed at pH of 4.5 and the temperature of 60 °C. The activity of recombinant Stlac2 was inhibited by 10 mM Na+, Mg2+, Ca2+, Mn2+, and increased by 10 mM of Fe3+ with a relatively activity of 315% compared with no addition. Cu2+ did not affect enzyme activity. Recombinant Stlac2 was capable of decolorizing 67.08% of 20 mg/L malachite green in 15 min without any mediators. ConclusionsGenerally, recombinant protein of fungal laccase Stlac2 was active without glycosylation and decolorize malachite green efficiently, which has potential industrial applications.

The Minor Recombinant Laccase Isozymes of Trametes hirsuta 072: Preparation and Properties

Laccases (EC 1.10.3.2) are multicopper polyphenol oxidases found in various organisms, in particular, in fungi. Fungal laccases are encoded by multigene families, which can contain up to 17 genes. However, not all isozymes can be obtained from native producers. Previous studies have shown that the filamentous fungus Penicillium canescens is a promising object for the heterologous expression of various laccase isozymes of Trametes hirsuta 072. In this work, the cultivation conditions of P. canescens strains, recombinant producers of T. hirsuta heterologous minor laccase, are optimized. The optimization of the rLacD and rLacF purification method increased their yield and specific activity of rLacD. In addition, the melting points of minor laccase isoenzymes are measured and the glycosylation of isoenzymes is studied.

Evolutionary Relationships Between the Laccase Genes of Polyporales: Orthology-Based Classification of Laccase Isozymes and Functional Insight From Trametes hirsuta.

Laccase is one of the oldest known and intensively studied fungal enzymes capable of oxidizing recalcitrant lignin-resembling phenolic compounds. It is currently well established that fungal genomes almost always contain several non-allelic copies of laccase genes (laccase multigene families); nevertheless, many aspects of laccase multigenicity, for example, their precise biological functions or evolutionary relationships, are mostly unknown. Here, we present a detailed evolutionary analysis of the sensu stricto laccase genes (CAZy – AA1_1) from fungi of the Polyporales order. The conducted analysis provides a better understanding of the Polyporales laccase multigenicity and allows for the systemization of the individual features of different laccase isozymes. In addition, we provide a comparison of the biochemical and catalytic properties of the four laccase isozymes from Trametes hirsuta and suggest their functional diversification within the multigene family.

Heterologous expression of two minor laccase isozyme cDNAs from the edible mushroom Grifola frondosa.

Two cDNAs encoding the minor laccase isozymes (Lac2 and Lac3) of Grifola frondosa were cloned, characterized, and expressed in Pichia pastoris. The recombinant Lac2 (rLac2) was stable at pH 6.0, whereas the recombinant Lac3 (rLac3) was stable in a broad pH range (pH 4.0-8.0). In addition, rLac2 and rLac3 showed the highest catalytic efficiency (kcat/Km) for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid).

Properties of two laccases from the Trametes hirsuta 072 multigene family: Twins with different faces

Utilization of laccases in biotechnology and bioremediation has created a strong demand for the characterization of new enzymes and an increase in production of known laccases. Thus, additional research into these enzymes is critically needed.In this study, we report a comparative study of the biochemical and transcriptional properties of two different laccase isozymes from Trametes hirsuta 072 – the constitutive and inducible forms. A recombinant LacC enzyme was expressed in Penicillium canescens to characterize its properties. LacC is single-purpose enzyme, unlike LacA, which can operate efficiently under a wide range of temperatures and pHs (55–70 °C and pH 3–5, respectively). LacC has a lower RedOx potential than LacA and does not oxidize substrates containing amine groups. Expression of the lacC gene was selective compared to that of the lacA gene and increased significantly in the presence of complex synthetic compounds such as dyes and xenobiotics.This study shows that laccases from the multigene families of basidiomycetes differ significantly in their properties, thus providing a complementary effect during lignin degradation.

Characterization of a thermostable mutant of Agaricus brasiliensis laccase created by phylogeny-based design

Laccases are enzymes that oxidize various aromatic compounds, and therefore they have attracted much attention from the standpoints of medical and industrial applications. We previously isolated the cDNA that codes for a laccase isozyme (Lac2a) from the medicinal mushroom Agaricus brasiliensis (Matsumoto-Akanuma etal., Int. J. Med. Mushrooms, 16, 375-393, 2014). In this study, we first attempted heterologous expression of the wild-type laccase using a Pichia pastoris secretory expression system. However, the trial was unsuccessful most likely because the enzyme was too unstable and degraded immediately after production. Therefore, we improved the stability of the laccase by using a phylogeny-based design method. We created a mutant laccase in which sixteen original residues were replaced with those found in the phylogenetically inferred ancestral sequence. The resulting mutant protein was successfully produced using the P.pastoris secretory expression system and then purified. The designed laccase showed catalytic properties similar to those of other fungal laccases. Moreover, the laccase is highly thermally stable at acidic and neutral pH and is also stable at alkaline pH at moderate temperatures. We expect that the laccase will serve as a useful tool for enzymatic polymerization of di-phenolic compounds.

Diversity of Ligninolytic Enzymes and Their Genes in Strains of the Genus Ganoderma: Applicable for Biodegradation of Xenobiotic Compounds?

White-rot fungi (WRF) and their ligninolytic enzymes (laccases and peroxidases) are considered promising biotechnological tools to remove lignin related Persistent Organic Pollutants from industrial wastewaters and contaminated ecosystems. A high diversity of the genus Ganoderma has been reported in Cuba; in spite of this, the diversity of ligninolytic enzymes and their genes remained unexplored. In this study, 13 native WRF strains were isolated from decayed wood in urban ecosystems in Havana (Cuba). All strains were identified as Ganoderma sp. using a multiplex polymerase chain reaction (PCR)-method based on ITS sequences. All Ganoderma sp. strains produced laccase enzymes at higher levels than non-specific peroxidases. Native-PAGE of extracellular enzymatic extracts revealed a high diversity of laccase isozymes patterns between the strains, suggesting the presence of different amino acid sequences in the laccase enzymes produced by these Ganoderma strains. We determined the diversity of genes encoding laccases and peroxidases using a PCR and cloning approach with basidiomycete-specific primers. Between two and five laccase genes were detected in each strain. In contrast, only one gene encoding manganese peroxidase or versatile peroxidase was detected in each strain. The translated laccases and peroxidases amino acid sequences have not been described before. Extracellular crude enzymatic extracts produced by the Ganoderma UH strains, were able to degrade model chromophoric compounds such as anthraquinone and azo dyes. These findings hold promises for the development of a practical application for the treatment of textile industry wastewaters and also for bioremediation of polluted ecosystems by well-adapted native WRF strains.