Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Using low-shear aerated and agitated bioreactor for producing two specific laccases by trametes versicolor cultures induced by 2,5-xylidine: Process development and economic analysis.

Laccase isoforms from basidiomycetes exhibit a superior redox potential compared to commercially available laccases obtained from ascomycete fungi, rendering them more reactive toward mono-substituted phenols and polyphenolic compounds. However, basidiomycetes present limitations for large-scale culture in liquid media, restraining the current availability of laccases from this fungal class. To advance laccase production from basidiomycetes, a newly designed 14-L low-shear aerated and agitated bioreactor provided enzyme titers up to 23.5 IU/mL from Trametes versicolor cultures. Produced enzymes underwent ultrafiltration and LC/MS-MS characterization, revealing the predominant production of only two out of the ten laccases predicted in the T. versicolor genome. Process simulation and economic analysis using SuperPro designer® suggested that T. versicolor laccase could be produced at US$ 3.60/kIU in a 200-L/batch enterprise with attractive economic parameters and a payback period of 1.7 years. The study indicates that new bioreactors with plain design help to produce low-cost enzymes from basidiomycetes.

The production of laccases by white-rot fungi under solid-state fermentation conditions.

Laccases (E.C. 1.10.3.2) produced by white-rot fungi (WRF) can be widely used, but the high cost prevents their use in large-scale industrial processes. Finding a solution to the problem could involve laccase production by solid-state fermentation (SSF) simulating the natural growth conditions for WRF. SSF offers several advantages over conventional submerged fermentation (SmF), such as higher efficiency and productivity of the process and pollution reduction. The aim of this review is therefore to provide an overview of the current state of knowledge about the laccase production by WRF under SSF conditions. The focus is on variations in the up-stream process, fermentation and down-stream process and their impact on laccase activity. The variations of up-stream processing involve inoculum preparation, inoculation of the medium and formulation of the propagation and production media. According to the studies, the production process can be shortened to 5-7 days by the selection of a suitable combination of lignocellulosic material and laccase producer without the need for any additional components of the culture medium. Efficient laccase production was achieved by valorisation of wastes as agro-food, municipal wastes or waste generated from wood processing industries. This leads to a reduction of costs and an increase in competitiveness compared to other commonly used methods and/or procedures. There will be significant challenges and opportunities in the future, where SSF could become more efficient and bring the enzyme production to a higher level, especially in new biorefineries, bioreactors and biomolecular/genetic engineering.

Copper-induced Production of Laccases for Lignin Depolymerisation and Micropollutant Degradation by Laccase-mediator Systems.

Contaminants deriving from human activities represent a constantly growing threat to our environment and have a direct impact on plant and animal health. To alleviate this ecological imbalance, biocatalysis offers a green and sustainable alternative to conventional chemical processes. Due to their broad specificity, laccases are enzymes possessing excellent potential for synthetic biotransformations in various fields as well as for the degradation of organic contaminants. Herein, we produced laccases in submerged cultures of P. ostreatus and T. versicolor in three different media. The fungi/medium combination leading to the highest enzymatic activity was malt extract (2%) + yeast extract (3%) + glucose (0.8%). Laccase production was further increased by supplementing this medium with different concentrations of Cu2+, which also provided a better understanding of the induction effect. Additionally, we disclose preliminary results on the interaction of laccases with mediators (ABTS and violuric acid - VA) for two main applications: lignin depolymerisation with guaiacylglycerol-ß-guaiacyl ether (GBG) as lignin model and micropollutant degradation with Remazol Brilliant Blue (RBB) as enzymatic bioremediation model. Promising results were achieved using VA to increase depolymerization of GBG dimer and to enhance RBB decolorisation.

Enhancement of laccase production from a newly isolated Trichoderma harzianum S7113 using submerged fermentation: Optimization of production medium via central composite design and its application for hydroquinone degradation.

Trichoderma harzianum S7113 as an efficient fungal isolate for laccase production was identified using the 18S rRNA sequencing. T. harzianum S7113 attained its maximal laccase production level on the 14th day of static incubation at 28 °C and pH 5.0 using the inoculum size of 5 discs (14 mm), according to the one factor per time (OFT) method. The most appropriate carbon, organic and inorganic nitrogen sources to promote maximal laccase synthesis were glucose (15 g/L), beef extract (5 g/L), and ammonium chloride (4 g/L), respectively. Results of Response Surface Methodology (RSM) revealed that glucose, meat extract, and ammonium chloride concentrations of 17.54, 7.17, and 4.36 g/L respectively, at a pH value of 6.74 are the favorite conditions for high titer production. The ANOVA analysis highlighted an excellent match between the actual experimental results and the model predicted laccase production levels. The biodegradation of hydroquinone (HQ) by T. harzianum S7113 laccase was most efficient in the pH range of 5.0 to 6.5. The increase in laccase concentration led to a significant increase in the HQ conversion to get a biodegradation rate of 92 ± 2.6% with a laccase concentration of 0.75 U/mL after 3 h of reaction.

Evaluation of Laccase Production by Monokaryotic Strains of Edible Mushrooms.

<b>Background and Objective:</b> Edible mushroom laccases are one of the most attractive enzymes applicable in numerous industrial sectors. The purpose of this research is to construct monokaryotic strains from selected isolates of edible mushrooms and to study the effects of inducers on laccase production under solid-state fermentation. <b>Materials and Methods:</b> Isolation of local commercial strains of edible mushrooms was carried out from the pileus region using standard laboratory techniques. The laccase production was carried out using 40 mM 2,6-Dimethoxyphenol (2,6-DMP) and 40 mM guaiacol as substrate. The generation of monokaryotic strains was performed by mycelium homogenization in sterile water and regrowth in an appropriate medium. Laccase production and study of the effects of inducers on laccase production were then studied. <b>Results:</b> Laccase production of native and monokaryotic strains distinguished these strains into three groups: HIGH-(KK24, KK25), MEDIUM-(KK26, KK1, KK5 and KK23) and LOW (KK13, KK8). Reduced activity was found in almost all isolates after 14 days of inoculation. The effect of pure copper sulfate, copper sulfate with DMP, Tween80 and synthetic melanoidin was studied at 7 and 14 days. KK24 and KK25 showed their positive response to all inducers about 1.5-2.5 folds of activity to their native strains. <b>Conclusion:</b> Eight strains of local and commercial mushrooms were isolated and purified. The corresponding monokaryotic strains were generated from chemical dedikaryotization. Studies of laccase production showed that KK24 and KK25 were high laccase producer's throughout the incubation period. The addition of inducers augmented laccase activity in KK24 and KK25 along with their corresponding monokaryotic strains.

Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae.

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.

Isolation, identification and optimization of enhanced production of laccase from Galactomyces geotrichum under solid-state fermentation.

Laccases are a group of oxidases that catalyze the oxidation of a wide range of electron rich substrates like phenolic compounds, lignin and aromatic amines. They are of interest because of their potential to be used in environmental and industrial applications. In this research, potent laccase producer fungi were screened and isolated from olive mill wastewater (OMW). One of the 23 isolated fungi was identified as Galactomyces geotrichum based on 18S rDNA sequence analysis that detected good laccase activity. Produced laccase had a molecular weight of 55 kDa that was confirmed by zymogram analysis. This is the first report about the optimization of laccase Production by G. geotrichum under solid-state fermentation. The optimization was made by the Taguchi design of experiments (DOE) methodology. An orthogonal array (L25) was designed using Minitab 19 software to study four effective process factors in five levels for laccase production. The optimum condition derived was; moisture content (80%), fermentation time (14 day), CuSO4⋅5H2O as the inducer (300 µM), glucose as a co-substrate (5 g/L). Maximum laccase activity of 52.86 (U/g of dry substrate) was obtained using optimum fermentation condition. This study aimed to better understand the laccase producing microorganisms in OMW and take them to OMW treatment that is rich in phenolic compounds.

Laccase Affects the Rate of Cryptococcus neoformans Nonlytic Exocytosis from Macrophages.

Nonlytic exocytosis is a process in which previously ingested microbes are expelled from host phagocytes with the concomitant survival of both cell types. This process has been observed in the interaction of Cryptococcus spp. and other fungal cells with phagocytes as distant as mammalian, bird, and fish macrophages and ameboid predators. Despite a great amount of research dedicated to unraveling this process, there are still many questions about its regulation and its final benefits for host or fungal cells. During a study to characterize the virulence attributes of Brazilian clinical isolates of C. neoformans, we observed great variability in their rates of nonlytic exocytosis and noted a correlation between this process and fungal melanin production/laccase activity. Flow cytometry experiments using melanized cells, nonmelanized cells, and lac1Δ mutants revealed that laccase has a role in the process of nonlytic exocytosis that seems to be independent of melanin production. These results identify a role for laccase in virulence, independent of its role in pigment production, that represents a new variable in the regulation of nonlytic exocytosis.IMPORTANCECryptococcus neoformans is a yeast that causes severe disease, primarily in immunosuppressed people. It has many attributes that allow it to survive and cause disease, such as a polysaccharide capsule and the dark pigment melanin produced by the laccase enzyme. Upon infection, the yeast is ingested by cells called macrophages, whose function is to kill them. Instead, these fungal cells can exit from macrophages in a process called nonlytic exocytosis. We know that this process is controlled by both host and fungal factors, only some of which are known. As part of an ongoing study, we observed that C. neoformans isolates that produce melanin faster are more-frequent targets of nonlytic exocytosis. Further experiments showed that this is probably due to higher production of laccase, because fungi lacking this enzyme are nonlytically exocytosed less often. This shows that laccase is an important signal/regulator of nonlytic exocytosis of C. neoformans from macrophages.

Lignolytic and hemicellulolytic enzyme cocktail production from Bacillus tequilensis LXM 55 and its application in pulp biobleaching.

Bioprocessing of pulp requires lignolytic as well as hemicellulolytic enzymes. The present study is the first report of a cocktail of laccase (L), xylanase (X), and mannanase (M), from a single bacterium for pulp biobleaching. A novel strain Bacillus tequilensis LXM 55 produced thermo-alkali stable L + X + M. On optimization higher enzyme yield (IUml-1/fold increase) of laccase (396.35/24.16), xylanase (212.95/81.90) and mannanase (153.33/102.90) were achieved in the cocktail. Treatment of pulp with cocktail of enzymes led to 49.35% reduction in kappa number and considerable enhancement in the brightness (11.59%), whiteness (4.11%), and other pulp properties. Most importantly, no mediator system was required for the application of laccase. 40% less chlorine consumption was required to obtain the paper of the same quality as that of pulp treated without enzyme but with 100% chlorine. Therefore, this cocktail of enzymes is highly suitable for pulp biobleaching in the paper mill.

Novel cobiomass degradation of NSAIDs by two wood rot fungi, Ganoderma applanatum and Laetiporus sulphureus: Ligninolytic enzymes induction, isotherm and kinetic studies.

A novel study on biodegradation of 30 mg L-1 of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) mixture (celecoxib, diclofenac and ibuprofen) by two wood-rot fungi; Ganoderma applanatum (GA) and Laetiporus sulphureus (LS) was investigated for 72 h. The removal efficiency of celecoxib, diclofenac and ibuprofen were 98, 96 and 95% by the fungal consortium (GA + LS). Although, both GA and LS exhibited low removal efficiency (61 and 73% respectively) on NSAIDs. However, 99.5% degradation of the drug mixture (NSAIDs) was achieved on the addition of the fungal consortium (GA + LS) to the experimental set-up. Overall, LS exhibited higher degradation efficiency; 92, 87, 79% on celecoxib, diclofenac and ibuprofen than GA with 89, 80 and 66% respectively. Enzyme analyses revealed significant induction of 201, 180 and 135% in laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP) by the fungal consortium during degradation of the NSAIDs respectively. The experimental data showed the best goodness of fit when subjected to Langmuir (R2 = 0.980) and Temkin (R2 = 0.979) isotherm models which suggests monolayer and heterogeneous nature exhibited by the mycelia during interactions with NSAIDs. The degradation mechanism followed pseudo-second-order kinetic model (R2 = 0.987) indicating the strong influence of fungal biomass in the degradation of NSAIDs. Furthermore, Gas Chromatography-Mass Spectrometry (GCMS) and High-Performance Liquid Chromatography (HPLC) analyses confirmed the degraded metabolic states of the NSAIDs after treatment with GA, LS and consortium (GA + LS). Hence, the complete removal of NSAIDs is best achieved in an economical and eco-friendly way with the use of fungi consortium.

Study of Ganoderma lucidum in Laccase Production using Corncob and Paddies Straw Substrates on Submerged Fermentation System.

BACKGROUND AND OBJECTIVE: Ganoderma lucidum a white rot fungi, produce laccase which capable to degrade lignin due to its activity as ligninolytic enzymes. The production of laccase by G. lucidum using various agroindustrial wastes, including corncob and paddies straw, as substrates have been studied. The purpose of this study was to determine substrate that able to produce the highest activity of the laccase from the G. lucidum. MATERIALS AND METHODS: The method used an experimental design followed by descriptive analysis using 4 treatments with duplication including treatment G. lucidum growth into A (control, Potato Dextrose Broth (PDB)), B (PDB+corncob), C (PDB+rice straw) and D (PDB+corncob+rice straw). This study includes; (1) Qualitative assay determination of laccase, (2) Extraction and laccase activity, (3) Cell concentration of G. lucidum measurement and (4) pH measurement. RESULTS: Laccase qualitative assay showed brownish red ring on PDA media that indicated a positive of the laccase enzyme secreted by G. lucidum. Enzyme activity under submerged fermentation condition was achieved by the treatment of adding corncobs with the highest activity accounting 68.75 U mL-1. The fermentation process causes a decrease in pH during the incubation time to pH 4.83. The results of pH measurements showed that the laccase enzyme from G. lucidum worked optimally at pH 4-5 achieved after 5 day of incubation. CONCLUSION: Our results suggest that G. lucidum has potential to produce laccase enzyme by using substrate comprising corncob and rice straw on submerged fermentation.

Refolding, characterization, and dye decolorization ability of a highly thermostable laccase from Geobacillus sp. JS12.

A putative laccase gene (lacG) from Geobacillus sp. JS12 was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli BL21 (DE3) cells, and the protein was primarily found in inclusion bodies. The resulting insoluble proteins were solubilized with 6 M guanidine HCl and refolded using an on-column refolding procedure. Ni-chelation affinity chromatography found the laccase to be a 30 kDa monomeric protein. Spectrophotometry and electron paramagnetic resonance (EPR) analysis indicated LacG as a multi-copper oxidase, with the usual laccase copper sites, Type 1, 2, and 3 Cu(II). The optimum pH for enzymatic activity was 3.0, 6.0, and 6.5 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The recombinant protein displayed high thermostability, with a heat inactivation half-life of approximately 2 h at 95 °C, and an optimum temperature of 80 °C with 2,6-DMP. Catalytic efficiency (kcat/Km) showed that guaiacol and 2,6-DMP were highly oxidized by the enzyme. The enzymatic reaction was significantly enhanced by Co2+ and Mn2+, while activity was strongly inhibited in the presence of Fe2+, Zn2+, and thiol compounds. LacG decolorized 43% of Congo red and 14% of Malachite green, and the addition of ABTS as a redox mediator dramatically increased the dye decolorization efficiency.

Bioprocess optimization of laccase production through solid substrate fermentation using Perenniporia tephropora-L168 and its application in bioremediation of triaryl-methane dye.

Laccases are multi copper oxidases that can oxidize both phenolic and nonphenolic lignin related compounds. Consequently, there has been continuous demand for laccases for the oxidative degradation of phenolic dyes in effluents. In view of this, the present work was focused on laccase production by solid substrate fermentation using a newly isolated fungus Perenniporia tephropora-L168. To intensify the laccase production, the process parameters pH, nitrogen, inducer, and substrate: water ratio were optimized by using statistical model. A set of optimal conditions noted were pH 3, nitrogen 0.001 g/L; inducer 0.5% and substrate: water ratio (1:10), which yielded laccase 1,160 U/g. The crude laccase exhibited noteworthy potential to degrade a triaryl-methane dye especially Malachite green. Also, during bioremediation studies, the statistical process optimization could achieve 81% decolourization within 180 min. The laccase treatment brought chemical transformation in malachite green as evident from UV-Visible spectra, FTIR, HPLC while toxicity against bacteria and fungi was also reduced. During phytotoxicity study, effect of treated and untreated dye on germination of seed was analyzed. Interestingly, the germination index for Vigna aconitifolia and Vigna radiata was increased by two and fourfold, respectively. Overall, this work demonstrates optimized production of laccase using Perenniporia tephropora-L168 and its efficient bioremediation potential for triaryl-methane dye.

Sustainable use of the spent mushroom substrate of Pleurotus florida for production of lignocellulolytic enzymes.

Spent mushroom substrate (SMS), a major byproduct of the mushroom industry, is a lignocellulosic biomass, which contains approximately 57-74.3% of holocellulose fraction. This study was aimed at utilizing SMS of Pleurotus florida for recovery of lignocellulolytic enzymes and sugars and also as a substrate for production of cellulolytic enzymes using different isolates of Trichoderma and Aspergillus under solid-state fermentation (SSF). SMS of P. florida extracts contained significant amounts of laccase (3,015.8 ± 29.5 U/g SMS) and xylanase (1,187.9 ± 12 U/g SMS) activity. Crystallinity pattern and chemical changes in SMS revealed that SMS had a lower crystallinity index (34.2%) as compared with the raw biomass (37.8%), which, in turn, helps in enhancing the accessibility of cellulolytic enzymes to holocellulose. Among the isolates, Trichoderma longibrachiatum A-01 showed maximum activity of endoglucanase (220.4 ± 5.9 U/mg), exoglucanase (78.5 ± 3.2 U/mg) and xylanase (1,550.4 ± 11.6 U/mg) while Aspergillus aculeatus C-08 showed maximum activity of cellobiase (113.9 ± 3.9 U/mg). Extraction with sodium citrate buffer (pH 4.8) showed maximum cellulolytic enzyme activity as compared with other solvents tested. Partial purification of endoglucanase, exoglucanase, xylanase, and cellobiase resulted in 56.3% (1,112.5 U/mg), 48.4% (212.5 U/mg), 44% (4,492.3 U/mg), and 62% (705.0 U/mg) yield with an increase by 5.2-, 4.5-, 4.1-, and 5.0-fold as compared with crude extract. The results reveal that SMS from P. florida could be a potential and cost-effective substrate for production of cellulolytic enzymes from T. longibrachiatum A-01 and A. aculeatus C-08.

Comparative transcriptomics and transcriptional regulation analysis of enhanced laccase production induced by co-culture of Pleurotus eryngii var. ferulae with Rhodotorula mucilaginosa.

The co-culturing of Pleurotus eryngii var. ferulae and Rhodotorula mucilaginosa was confirmed in our previous studies to be an efficient strategy to improve laccase production by submerged fermentation. To determine the possible regulation principles underlying this behaviour, comparative transcriptomic analysis was performed on P. eryngii var. ferulae to investigate the differential expression of genes in co-culture. RNA-seq analysis showed that genes concerning xenobiotic biodegradation and expenditure of energy were upregulated. However, genes related to oxidative stress were downregulated. In addition, the transcription levels of laccase isoenzymes were not consistent in the co-culture system: 3 laccase genes (lacc1, lacc2, lacc12) were upregulated, and 3 laccase genes (lacc4, lacc6, lacc9) were downregulated. The enhancement in laccase activity can be due to upregulation of a laccase heterodimer encoded by the genes lacc2 and ssPOXA3a (or ssPOXA3b), whose expression levels were increased by 459% and 769% (or 585% for ssPOXA3b) compared with those of a control, respectively. ß-Carotene produced by R. mucilaginosa upregulated the transcription of lacc2 only. Combining these results with an analysis of cis-acting responsive elements indicated that four transcription factors (TFs) had potential regulatory effects on the transcription of laccase genes. It was supposed that TFa regulated lacc transcription by binding with methyl jasmonate and heat shock response elements. The expression of TFb, TFc, and TFd was regulated by ß-carotene. However, ß-carotene had no effect on TFa expression. These results provide a possible mechanism for the regulation of laccase gene transcription in the co-culture system and are also beneficial for the future intensification of fungal laccase production.

Trametes versicolor laccase production using agricultural wastes: a comparative study in Erlenmeyer flasks, bioreactor and tray.

Laccases are very interesting biocatalysts of recognized importance for several industrial applications. Its production by Trametes versicolor, a white-rot fungus, was induced by a combination of cotton gin wastes (1%), a lignocellulosic waste, and vinasse (15%), an industrial by-product from sugarcane industry. The use of these agro-industrial wastes are interesting, since it helps in reducing the enzyme production costs, due to their low cost and wide availability, as well as the environmental contamination issues, due to their improper disposal. Thus, laccase production was studied in submerged fermentation of T. versicolor using these agro-industrial wastes (cotton gin waste and vinasse) as carbon source and an additional nitrogen source (0.1% peptone). Three different bioreactors were evaluated for laccase production, such as BioFlo 310 bioreactor, aluminium tray and Erlenmeyer flasks to achieve high levels of laccase production. The highest specific production of laccase was found in BioFlo 310 bioreactor with 12 days of fermentation (55.24 U/mg prot.), which has been shown to be closely related to the oxygen supply to the microorganism through aeration of the fermentation medium. This study brings new insights into green biotechnology regarding vinasse utilization, which is frequently discharged in soils, rivers, and lakes causing adverse effects on agricultural soils and biota, as well as the cotton gin waste recovery.

Purification, Biochemical Characterization and Decolorization Efficiency of Laccases from Peach and Cherry Cultures of Pleutorus eryngii: A Comparative Study.

BACKGROUND: Laccases (Lacs) are used potentially in industrial and biotechnological applications such as decolorization of dyes, degradation of industrial effluents, delignification, etc. thanks to their large varieties of substrate specificities and excellent catalytic efficiencies. The efficient utilizations of Lacs in these applications mostly depend on the identifying their biochemical properties. OBJECTIVE: The goal of this research is to investigate the purification, biochemical characterization and decolorization efficiencies of Lacs. METHODS: Pleurotus eryngii was incubated on peach (PC) and cherry (CC) wastes under optimized solid state fermentation conditions. Then, the enzymes extracts were purified by ammonium sulfate precipitation, anion exchange chromatography, gel filtration, respectively. Lacs fractions were subjected to electrophoretic analyses as well as their structural and kinetic characteristics. Also, the effects of selected chemical agents on purified Lacs activities and determination of decolorization efficiencies were studied. RESULTS: As the results of purification processes of Lacs from both cultures, 3.94-fold purification was obtained for PC, while it was 5.34 for CC. The electrophoretic results of purified Lacs illustrated the single bands of protein (30±1 kDa) in accordance with the results after gel filtration. The Km values of Lacs from PC and CC were respectively detected as 1.1381 and 0.329 mM for ABTS. The selected agents partially/completely inhibited Lac activities. The highest decolorization efficiencies of purified Lacs from PC and CC were separately obtained as 53 and 11.8%. CONCLUSION: The results clearly indicated that the performances of Lacs from both cultures in decolorization application are different from each other depending their activities, biochemical and kinetic characteristics.

Influence of Oxygen Supply on Growth and Laccases Production by Pleurotus sajor-caju PS-2001 in Submerged Process

Abstract (1) Background: Oxygen supply is an important parameter to be considered in submerged cultures. This study evaluated the influence of different conditions for dissolved oxygen (DO) concentration on laccases activities and growth of Pleurotus sajor-caju PS-2001 in submerged process in stirred-tank bioreactor. (2) Methods: Initially, three different conditions were tested: uncontrolled DO and minimum levels of 30% and 80% of saturation, with the pH controlled between 4.5 and 7.0. (3) Results: Best results were observed at 30% DO (26 U mL-1 of laccases at 96 h), whereas higher mycelial biomass was observed at 30% and 80% DO (above 4.5 g L-1). Four different conditions of DO (uncontrolled, 10%, 30% and 50% of saturation) were tested at pH 6.5, with higher laccases activity (80 U mL-1 at 66 h) and lower mycelial growth (1.36 g L-1 at 90 h) being achieved with DO of 30%. In this test, the highest values for volumetric productivity and specific yield factor were determined. Under the different pH conditions tested, the production of laccases is favoured at DO concentration of 30% of saturation, while superior DO levels favours fungal growth. (4) Conclusion: The results indicate that dissolved oxygen concentration is a critical factor for the culture of P. sajor-caju PS-2001 and has important effects not only on laccases production but also on fungal growth.

Structural and biochemical insights into an engineered high-redox potential laccase overproduced in Aspergillus.

Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70 °C = 1.2 h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2 h at 100 °C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding.