Enhanced degradation of phototreated recycled and unused low-density polyethylene films by Pleurotus ostreatus.

Polyethylene, one of the most used petroleum-derived polymers, causes serious environmental pollution. The ability of Pleurotus ostreatus to degrade UV-treated and untreated recycled and unused (new) low-density polyethylene (LDPE) films was studied. We determined the fungal biomass production, enzyme production, and enzyme yield. Changes in the chemical structure and surface morphology of the LDPE after fungal growth were analyzed using FTIR spectroscopy and SEM. Functional group indices and contact angles were also evaluated. In general, the highest Lac (6013 U/L), LiP (2432 U/L), MnP (995 U/L) and UP (6671 U/L) activities were observed in irradiated recycled LDPE (IrRPE). The contact angle of all samples was negatively correlated with fermentation time; the smaller the contact angle, the longer the fermentation time, indicating effective biodegradation. The IrRPE samples exhibited the smallest contact angle (49°) at 4 weeks, and the samples were fragmented (into two pieces) at 5 weeks. This fungus could degrade unused (new) LDPE significantly within 6 weeks. The biodegradation of LDPE proceeded faster in recycled than in unused samples, which can be enhanced by exposing LDPE to UV radiation. Enzymatic production during fungal growth suggest that LDPE degradation is initiated by laccase (Lac) followed by lignin peroxidase (LiP), whereas manganese peroxidase (MnP) and unspecific peroxygenase (UP) are involved in the final degradation process. This is the first experimental study on the fungal growth and its main enzymes involved in LDPE biodegradation. This fungus has great promise as a safe, efficient, and environmentally friendly organism capable of degrading LDPE.

Bioprospecting of endophytic fungi from semidesert candelilla (Euphorbia antisyphilitica Zucc): Potential for extracellular enzyme production.

Endophytic microbial communities colonize plants growing under various abiotic stress conditions. Candelilla (Euphorbia antisyphilitica Zucc.) is a shrub that develops functionally in arid and semi-arid zones of Mexico; these conditions generate an association between the plant and the microorganisms, contributing to the production of enzymes as a defense mechanism for resistance to abiotic stress. The objective of this research was to isolate and identify endophyte fungi of candelilla and bioprospection of these endophytic fungi for enzyme production using candelilla by-products. Fungi were isolated and identified using ITS1/ITS4 sequencing. Their potency index (PI) was evaluated in producing endoglucanase, xylanase, amylase, and laccase. Fermentation was carried out at 30°C for 8 days at 200 rpm, with measurements every 2 days, using candelilla by-products as substrate. All fungi exhibited higher cellulase, amylase, and laccase activities on the 2nd, 6th, and 8th day of fermentation, respectively, of fermentation. The fungus Aspergillus niger ITD-IN4.1 showed the highest amylase activity (246.84 U/mg), the genus Neurospora showed the highest cellulase activity, reaching up to 13.45 FPU/mg, and the strain Neurospora sp. ITD-IN5.2 showed the highest laccase activity (3.46 U/mg). This work provides the first report on the endophytic diversity of E. antisyphilitica and its potential role in enzyme production.

Optimization studies on laccase activity of Proteus mirabilis isolated from treatment sludge of textile industry factories : Optimization of laccase activity of Proteus mirabilis.

Laccase is an exothermic enzyme with copper in its structure and has an important role in biodegradation by providing oxidation of phenolic compounds and aromatic amines and decomposing lignin. The aim of this study is to reach maximum laccase enzyme activity with minimum cost and energy through optimization studies of Proteusmirabilis isolated from treatment sludge of a textile factory. In order to increase the laccase enzyme activities of the isolates, medium and culture conditions were optimized with the study of carbon (Glucose, Fructose, Sodium Acetate, Carboxymethylcellulose, Xylose) and nitrogen sources (Potassium nitrate, Yeast Extract, Peptone From Soybean, Bacteriological Peptone), incubation time, pH, temperature and Copper(II) sulfate concentration then according to the results obtained. Response Surface Method (RSM) was performed on six different variables with three level. According to the data obtained from the RSM, the maximum laccase enzyme activity is reached at pH 7.77, temperature 30.03oC, 0.5 g/L CuSO4, 0.5 g/L fructose and 0.082 g/L yeast extract conditions. After all, the laccase activity increased 2.7 times. As a result, laccase activity of P. mirabilis can be increased by optimization studies. The information obtained as a result of the literature studies is that the laccase enzymes produced in laboratory and industrial scale are costly and their amounts are low. This study is important in terms of obtaining more laccase activity from P.mirabilis with less cost and energy.

Decisional tool development and application for techno-economic analysis of fungal laccase production.

Textile and medical effluents causing bioaccumulation and biomagnification have been successfully biodegraded by fungal laccases. Here, a decision-making tool was developed and applied to evaluate 45 different laccase production strategies which determined the best potential source from a techno-economical perspective. Laccase production cost was calculated with a fixed output of 109 enzymatic units per batch (USD$per109U) and a sensitivity analysis was performed. Results indicate that optimization of enzymatic kinetics for each organism is essential to avoid exceeding the fermentation time point at which production titer reaches its peak and, therefore, higher production costs. Overall, the most cost-effective laccase-producing strategy was obtained when using Pseudolagarobasidium acaciicola with base production cost of USD $42.46 per 109 U. This works serves as platform for decision-making to find the optimal laccase production strategy based on techno-economic parameters.

Lignin degradation by a novel thermophilic and alkaline yellow laccase from Chitinophaga sp.

Laccases (EC 1.10.3.2) are oxidoreductases that belong to the multicopper oxidase subfamily and are classified as yellow/white or blue according to their absorption spectrum. Yellow laccases are more useful for industrial processes since they oxidize nonphenolic compounds in the absence of a redox mediator and stand out for being more stable and functional under extreme conditions. This study aimed to characterize a new laccase that was predicted to be present in the genome of Chitinophaga sp. CB10 - Lac_CB10. Lac_CB10, with a molecular mass of 100.06 kDa, was purified and characterized via biochemical assays using guaiacol as a substrate. The enzyme demonstrated extremophilic characteristics, exhibiting relative activity under alkaline conditions (CAPS buffer pH 10.5) and thermophilic conditions (80-90°C), as well as maintaining its activity above 50% for 5 h at 80°C and 90°C. Furthermore, Lac_CB10 presented a spectral profile typical of yellow laccases, exhibiting only one absorbance peak at 300 nm (at the T2/T3 site) and no peak at 600 nm (at the T1 site). When lignin was degraded using copper as an inducer, 52.27% of the material was degraded within 32 h. These results highlight the potential of this enzyme, which is a novel yellow laccase with thermophilic and alkaline activity and the ability to act on lignin. This enzyme could be a valuable addition to the biorefinery process. In addition, this approach has high potential for industrial application and in the bioremediation of contaminated environments since these processes often occur at extreme temperatures and pH values. IMPORTANCE: The characterization of the novel yellow laccase, Lac_CB10, derived from Chitinophaga sp. CB10, represents a significant advancement with broad implications. This enzyme displays exceptional stability and functionality under extreme conditions, operating effectively under both alkaline (pH 10.5) and thermophilic (80-90°C) environments. Its capability to maintain considerable activity over extended periods, even at high temperatures, showcases its potential for various industrial applications. Moreover, its distinctive ability to efficiently degrade lignin-demonstrated by a significant 52.27% degradation within 32 h-signifies a promising avenue for biorefinery processes. This newfound laccase's characteristics position it as a crucial asset in the realm of bioremediation, particularly in scenarios involving contamination at extreme pH and temperature levels. The study's findings highlight the enzyme's capacity to address challenges in industrial processes and environmental cleanup, signifying its vital role in advancing biotechnological solutions.

Evaluation of Antibiotic Biodegradation by a Versatile and Highly Active Recombinant Laccase from the Thermoalkaliphilic Bacterium Bacillus sp. FNT.

Laccases are industrially relevant enzymes that have gained great biotechnological importance. To date, most are of fungal and mesophilic origin; however, enzymes from extremophiles possess an even greater potential to withstand industrial conditions. In this study, we evaluate the potential of a recombinant spore-coat laccase from the thermoalkaliphilic bacterium Bacillus sp. FNT (FNTL) to biodegrade antibiotics from the tetracycline, ß-lactams, and fluoroquinolone families. This extremozyme was previously characterized as being thermostable and highly active in a wide range of temperatures (20-90 °C) and very versatile towards several structurally different substrates, including recalcitrant environmental pollutants such as PAHs and synthetic dyes. First, molecular docking analyses were employed for initial ligand affinity screening in the modeled active site of FNTL. Then, the in silico findings were experimentally tested with four highly consumed antibiotics, representatives of each family: tetracycline, oxytetracycline, amoxicillin, and ciprofloxacin. HPLC results indicate that FNTL with help of the natural redox mediator acetosyringone, can efficiently biodegrade 91, 90, and 82% of tetracycline (0.5 mg mL-1) in 24 h at 40, 30, and 20 °C, respectively, with no apparent ecotoxicity of the products on E. coli and B. subtilis. These results complement our previous studies, highlighting the potential of this extremozyme for application in wastewater bioremediation.

Caatinga, Amazon and Atlantic Forest as natural sources for microbial lignocellulolytic enzymes.

Brazilian biomes are important sources for environmental microorganisms, including efficient metabolic machineries, like actinomycetes. These bacteria are known for their abilities to produce many bioactive compounds, including enzymes with multiple industrial applications. The present work aimed to evaluate lignocellulolytic abilities of actinomycetes isolated from soil and rhizosphere samples collected at Caatinga, Atlantic and Amazon Forest. Laccase (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP) and cellulase were evaluated for their efficiency. These enzymes have an essential role in lignin decomposition, through oxidation of phenolic and non-phenolic compounds, as well as enzymatic hydrolysis of vegetal biomass. In this sense, a total of 173 actinomycetes were investigated. Eleven (11) of them were selected by their enzymatic performance. The actinomycete AC166 displayed some activity in all analysed scenarios in terms of Lac, MnP and LiP activity, while AC171 was selected as the most promising strain, showing the following activities: 29.7 U.L-1 for Lac; 2.5 U.L-1 for LiP and 23 U.L-1 for MnP. Cellulolytic activities were evaluated at two pH conditions, 4.8 and 7.4, obtaining the following results: 25 U.L-1 and 71 U.L-1, respectively. Thermostability (4, 30 and 60 o C) and salinity concentrations (0 to 4 M) and pH variation (2.0 to 9.0) stabilities of the obtained LiP and Lac enzymatic extracts were also verified. The actinomycete strain AC171 displayed an adaptable response in distinct pH and salt profiles, indicating that bacterial LiP was some halophilic type. Additionally, the strain AC149 produced an alkali and extreme halophilic lignin peroxidase, which are promising profiles for their future application under lignocellulosic biomass at bioethanol biorefineries.

Laccase-based catalytic microreactor for BPA biotransformation.

A laccase-based catalytic reactor was developed into a polydimethylsiloxane (PDMS) microfluidic device, allowing the degradation of different concentrations of the emergent pollutant, Bisphenol-A (BPA), at a rate similar to free enzyme. Among the immobilizing agents used, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was capable of immobilizing a more significant amount of the laccase enzyme in comparison to glutaraldehyde (GA), and the passive method (2989, 1537, and 1905 U/mL, respectively). The immobilized enzyme inside the microfluidic device could degrade 55 ppm of BPA at a reaction rate of 0.5309 U/mL*min with a contaminant initial concentration of 100 ppm at room temperature. In conclusion, the design of a microfluidic device and the immobilization of the laccase enzyme successfully allowed a high capacity of BPA degradation.

Copper increases laccase gene transcription and extracellular laccase activity in Pleurotus eryngii KS004.

The white-rot fungus Pleurotus eryngii secretes various laccases involved in the degradation of a wide range of chemical compounds. Since the laccase production is relatively low in fungi, many efforts have been focused on finding ways to increase it, so in this study, we investigated the effect of copper on the transcription of the pel3 laccase gene and extracellular laccase activity. The results indicate that adding 0.5 to 2 mM copper to liquid cultures of P. eryngii KS004 increased both pel3 gene transcription and extracellular laccase activity in a concentration-dependent manner. The most significant increase in enzyme activity occurred at 1 mM Cu2+, where the peak activity was 4.6 times higher than in control flasks. Copper also induced the transcription of the laccase gene pel3. The addition of 1.5 and 2 mM Cu2+ to fungal culture media elevated pel3 transcript levels to more than 13-fold, although the rate of induction slowed down at Cu2+ concentrations higher than 1.5 mM. Our findings suggest that copper acts as an inducer in the regulation of laccase gene expression in P. eryngii KS004. Despite its inhibitory effect on fungal growth, supplementing cultures with copper can lead to an increased extracellular laccase production in P. eryngii.

Oxidation of chlortetracycline and its isomers by Botrytis aclada laccase in the absence of mediators: pH dependence and identification of transformation products by LC-MS.

Tetracyclines are antibiotics considered emerging pollutants and currently, wastewater treatment plants are not able to remove them efficiently. Laccases are promising enzymes for bioremediation because they can oxidize a wide variety of substrates. The aim of this study was to evaluate the Botrytis aclada laccase for the oxidation of chlortetracycline and its isomers in the absence of a mediator molecule, at a pH range between 3.0 to 7.0, and to characterize the transformation products by LC-MS. Chlortetracycline and three isomers were detected in both, controls and reaction mixtures at 0 h and in controls after 48 h of incubation but in different proportions depending on pH. An additional isomer was also detected, but only in the presence of BaLac. Based on the transformation products identified in the enzymatic reactions and information from literature, we assembled a network of transformation pathways starting from chlortetracycline and its isomers. The spectrometric analysis of the products indicated the probable occurrence of oxygen insertion, dehydrogenation, demethylation and deamination reactions. Four new products were identified, and we also described a novel transformation product without the chloro group. We observed that increasing pH led to higher diversity of main products. This is the first study using the laccase from fungi Botrytis aclada to oxidate chlortetracycline and its isomers and it can be considered as an ecological alternative to be used in bioremediation processes such as wastewater.

Thermosensitive polymer-assisted extraction and purification of fungal laccase from citrus pulp wash effluent.

BACKGROUND: This study explores the use of liquid-liquid extraction with thermosensitive polymers for producing laccase (Lac) from Pleurotus sajor-caju. This process leverages liquid waste from the citrus industry, specifically pulp wash. The research delves into extractive fermentation and thermoseparation, both processes being facilitated by a polymer exhibiting a lower critical solution temperature transition. RESULTS: Key factors considered include the choice of polymer, its concentration, pH, separation temperature, and the behavior of the polymer-rich phase post-extractive fermentation concerning the lower critical solution temperature. Notably, under conditions of 45% by weight of Pluronic L-61 and pH 5.0 at 25 °C, the Lac resulted in an enhancement in the purification factor of 28.4-fold, compared with the Lac obtained directly from the fermentation process on the eighth day. There was an 83.6% recovery of the Lac enzyme in the bottom phase of the system. Additionally, the unique properties of Pluronic L-61, which can induce phase separation and also allow for thermoseparation, led to a secondary fraction (aqueous solution) of Lac with purification factor of 2.1 ± 0.1-fold (at 32 ± 0.9 °C and 30 ± 0.3 min without stirring) from the polymeric phase (top phase). Fourier-transform infrared analysis validated the separation data, particularly highlighting the α-helix content in the amide I region (1600-1700 cm-1 ). CONCLUSION: In summary, the insights from this study pave the way for broader industrial applications of these techniques, underscoring benefits like streamlined process integration, heightened selectivity, and superior separation efficacy. © 2023 Society of Chemical Industry.

Control of selectivity in the oxidation of 5-hydroxymethylfurfural to 5- formyl-2-furancarboxylic acid catalyzed by laccase in a multiphasic gas-liquid microbioreactor.

The selectivity of 5-formyl-2-furancarboxylic acid (FFCA) was studied in a batch bioreactor and microbioreactors with different internal diameters (ID). Using microbioreactors, the effect of the flow rate of the liquid and gas phase on the yield, space time yield (STYFFCA), and gas-liquid mixture velocity (UM) of the reaction was evaluated. The biooxidation in flow microbioreactors, a selectivity of 100 % for FFCA was achieved, while with the batch bioreactor at the same substrate concentration a selectivity of 6.7 % was obtained. The highest yield (30 %) with 15 mM of 5-hydroxymethylfurfural (HMF) was reached at a gas-liquid flow rate of 0.5 µL/min and the highest STYFFCA (0.07 mol m-3 min-1) was achieved at a gas-liquid flow rate of 1.5 µL/min with the microbioreactor with an ID of 0.5 mm. The UM values (0.5 to 1.6 cm min1) indicated that the reaction takes place under a kinetic regime without mass transfer limitations.

Immobilizing white-rot fungi laccase: Toward bio-derived supports as a circular economy approach in organochlorine removal.

Despite their high persistence in the environment, organochlorines (OC) are widely used in the pharmaceutical industry, in plastics, and in the manufacture of pesticides, among other applications. These compounds and the byproducts of their decomposition deserve attention and efficient proposals for their treatment. Among sustainable alternatives, the use of ligninolytic enzymes (LEs) from fungi stands out, as these molecules can catalyze the transformation of a wide range of pollutants. Among LEs, laccases (Lac) are known for their efficiency as biocatalysts in the conversion of organic pollutants. Their application in biotechnological processes is possible, but the enzymes are often unstable and difficult to recover after use, driving up costs. Immobilization of enzymes on a matrix (support or solid carrier) allows recovery and stabilization of this catalytic capacity. Agricultural residual biomass is a passive environmental asset. Although underestimated and still treated as an undesirable component, residual biomass can be used as a low-cost adsorbent and as a support for the immobilization of enzymes. In this review, the adsorption capacity and immobilization of fungal Lac on supports made from residual biomass, including compounds such as biochar, for the removal of OC compounds are analyzed and compared with the use of synthetic supports. A qualitative and quantitative comparison of the reported results was made. In this context, the use of peanut shells is highlighted in view of the increasing peanut production worldwide. The linkage of methods with circular economy approaches that can be applied in practice is discussed.

Testing for the ability to modify antibiotics of Panus tigrinus 8/18 Lentinus strigosus 1566 laccase / Teste de capacidade para modificar antibióticos a partir da lacase de Panus tigrinus 8/18 e Lentinus strigosus 1566

In advanced biotechnology, the utilization of enzymes to achieve new or modified compounds with antibacterial, fungicidal, and anti-cancer specifications is crucial. Mushroom lactases are a hopeful biocatalyst for the synthesis and modification of different compounds. They are an accessible and inexpensive enzyme for the preparation of reaction objects and have recently received attention. Laccase purification was performed from basidiomycete Lentinus strigosus (LS) in several stages: Stage 1. On ion-exchange chromatography on TEAE Servacell 23 (400 ml), two distinctly separated laccase activity peaks were observed, eluted from the carrier at 0.21 and 0.27 M NaCl. In order to reduce the loss of enzymes, all fractions with laccase activity were collected, concentrated, and desalted using an ultrafiltration cell (Amicon, United States) with a UM-10 membrane. Stage 2. The resulting preparation with laccase activity was applied to a Q-Sepharose column (60 ml). Two well-separated peaks with laccase activity were obtained during the elution: laccase I (0.12 M NaCl) and laccase II (0.2 M NaCl). Stage 3. In the course of further purification of both enzymes, carried out on anion-exchange carrier Resource Q (6 ml), a broken gradient was used: 0 - 10%, 10 - 20%, and 20 - 100% with 1M NaCl. Stage 4. Both laccase I and laccase II, obtained after Resource Q, were desalted, concentrated to 1 ml each, and applied to a Superdex 75 gel filtration column. As a result, two laccases were obtained in a homogeneous form.

Na biotecnologia moderna, o uso de enzimas para obter compostos novos ou modificados com propriedades antibacterianas, antifúngicas e anticancerígenas é crucial. Lactases de cogumelos são biocatalisadores promissores para síntese e modificação de diferentes compostos, por serem enzimas baratas e disponíveis para a preparação de componentes de reação, e vem recebendo a devida atenção recentemente. A purificação da lacase foi realizada a partir do basidiomiceto Lentinus strigosus em vários estágios: Etapa 1 - na cromatografia de troca iônica em TEAE Servacell 23 (400 ml), foram observados dois picos de atividade da lacase distintamente separados, com eluição do transportador a 0,21 e 0,27 M de NaCl. Para reduzir a perda de enzimas, todas as frações com atividade de lacase foram coletadas, concentradas e dessalinizadas em uma célula de ultrafiltração (Amicon, Estados Unidos) com membrana UM-10; Etapa 2 - a preparação resultante com atividade de lacase foi aplicada a uma coluna Q-Sepharose (60 ml). Durante a eluição, foram obtidos dois picos bem separados com atividade de lacase: lacase I (NaCl 0,12 M) e lacase II (NaCl 0,2 M); Etapa 3 - no decurso da purificação adicional de ambas as enzimas, realizada no Recurso Q de transportador de troca aniônica (6 ml), um gradiente quebrado foi usado: 0-10%, 10-20% e 20-100% com NaCl 1M; Etapa 4 - tanto a lacase I como a lacase II, obtidas após o Recurso Q, foram dessalinizadas e concentradas para 1 ml cada e aplicadas a uma coluna de filtração em gel Superdex 75. Como resultado, duas lacases foram obtidas de forma homogênea.

Testing for the ability to modify antibiotics of Panus tigrinus 8/18 Lentinus strigosus 1566 laccase

Abstract In advanced biotechnology, the utilization of enzymes to achieve new or modified compounds with antibacterial, fungicidal, and anti-cancer specifications is crucial. Mushroom lactases are a hopeful biocatalyst for the synthesis and modification of different compounds. They are an accessible and inexpensive enzyme for the preparation of reaction objects and have recently received attention. Laccase purification was performed from basidiomycete Lentinus strigosus (LS) in several stages: Stage 1. On ion-exchange chromatography on TEAE Servacell 23 (400 ml), two distinctly separated laccase activity peaks were observed, eluted from the carrier at 0.21 and 0.27 M NaCl. In order to reduce the loss of enzymes, all fractions with laccase activity were collected, concentrated, and desalted using an ultrafiltration cell (Amicon, United States) with a UM-10 membrane. Stage 2. The resulting preparation with laccase activity was applied to a Q-Sepharose column (60 ml). Two well-separated peaks with laccase activity were obtained during the elution: laccase I (0.12 M NaCl) and laccase II (0.2 M NaCl). Stage 3. In the course of further purification of both enzymes, carried out on anion-exchange carrier Resource Q (6 ml), a broken gradient was used: 0 - 10%, 10 - 20%, and 20 - 100% with 1M NaCl. Stage 4. Both laccase I and laccase II, obtained after Resource Q, were desalted, concentrated to 1 ml each, and applied to a Superdex 75 gel filtration column. As a result, two laccases were obtained in a homogeneous form.

Resumo Na biotecnologia moderna, o uso de enzimas para obter compostos novos ou modificados com propriedades antibacterianas, antifúngicas e anticancerígenas é crucial. Lactases de cogumelos são biocatalisadores promissores para síntese e modificação de diferentes compostos, por serem enzimas baratas e disponíveis para a preparação de componentes de reação, e vem recebendo a devida atenção recentemente. A purificação da lacase foi realizada a partir do basidiomiceto Lentinus strigosus em vários estágios: Etapa 1 - na cromatografia de troca iônica em TEAE Servacell 23 (400 ml), foram observados dois picos de atividade da lacase distintamente separados, com eluição do transportador a 0,21 e 0,27 M de NaCl. Para reduzir a perda de enzimas, todas as frações com atividade de lacase foram coletadas, concentradas e dessalinizadas em uma célula de ultrafiltração (Amicon, Estados Unidos) com membrana UM-10; Etapa 2 - a preparação resultante com atividade de lacase foi aplicada a uma coluna Q-Sepharose (60 ml). Durante a eluição, foram obtidos dois picos bem separados com atividade de lacase: lacase I (NaCl 0,12 M) e lacase II (NaCl 0,2 M); Etapa 3 - no decurso da purificação adicional de ambas as enzimas, realizada no Recurso Q de transportador de troca aniônica (6 ml), um gradiente quebrado foi usado: 0-10%, 10-20% e 20-100% com NaCl 1M; Etapa 4 - tanto a lacase I como a lacase II, obtidas após o Recurso Q, foram dessalinizadas e concentradas para 1 ml cada e aplicadas a uma coluna de filtração em gel Superdex 75. Como resultado, duas lacases foram obtidas de forma homogênea.

Systematic screening strategy for fungal laccase activity of endophytes from Otoba gracilipes with bioremediation potential.

Fungal laccases are promising for biotechnological applications, including bioremediation and dye biotransformation, due to their high redox potential and broad substrate specificity. However, current bioprospecting methods for identifying laccase-producing fungi can be challenging and time-consuming. For early detection, it was developed a three-step, multi-criteria weighting system that evaluates fungal strains based on: First, the biotransformation capacity of three dyes (i.e., Congo red, brilliant blue G-250, and malachite green), at three different pH values, and with a relative weighting supported for the redox potential of each colorant. The relative decolorization coefficient (RDC), used as th2e first classification criterion, expressed their potential performance. Second, under the same conditions, laccase activity was estimated by observing the different degrees of oxidation of a given substrate. The selection criterion was the relative oxidation coefficient (ROC). Finally, laccase activity was quantified in submerged fermentations using three inducers (i.e., loofah sponge, Tween 80, and veratyl alcohol). This multicriteria screening strategy evaluated sixteen isolated endophytic fungal strains from Otoba gracilipes. The system identified Beltraniopsis sp. ET-17 (at pH values of 5.00 and 5.50) as a promising strain for dye biotransformation, and Phlebia floridensis as the best laccase producer, achieving a high activity of 116 µmol min-1 L-1 with loofah sponge as an inducer. In-vitro testing confirmed the efficacy of P. floridensis, with 53.61 % decolorization of a dye mixture (brilliant blue-Congo red. ratio 1:1) after 15 days of incubation. Thus, with the proposed screening strategy it was possible to highlight two species of interest at an early bioprospecting stage on a Colombian native tree poorly explored.

Enhanced laccase activity in Trametes versicolor (L.: Fr.) Pilát by host substrate and copper.

Laccases are appealing biocatalysts for various industrial utilizations. The fungus Trametes versicolor (L.: Fr.) Pilát causes white rot in wood and has been identified as an important fungal laccase producer. To investigate laccase production and activity in T. versicolor, the native isolate was collected from the host (Quercus castaneifolia) in the forests of Guilan province, northern Iran, and then purified and identified using the molecular marker. Its ability to produce laccase enzyme in the presence of different plant substrates including sawdust and wood chips of oak, poplar, and pine was evaluated. Also, the effect of copper as an enzyme inducer was investigated in vitro. The results showed that adding the wood to the culture medium increased laccase production, and among these, oak sawdust had the greatest effect, a 1.7-fold increase from that in the control (4.8 u/l vs. 2.8 u/l). Also, the enzyme extraction time effect on the optimal recovery yield showed that the 5-h enzyme extraction cycle resulted in the highest yield of the enzyme (18.97 u/l). Moreover, adding different concentrations of copper to the fungal culture medium increased the production of laccase, and the highest amount of enzyme (92.04 u/l) was obtained with 3.5 mM of CuSO4 along with oak sawdust. Based on the results, the addition of host wood sawdust ("oak" in this work) and copper particles together stimulates the fungal growth and the laccase production during submerged cultivation of T. versicolor. Therefore, it would be a safe and cheap strategy for the commercial production of laccase by filamentous fungi.

Purification and characterization of an alkali-organic solvent-stable laccase with dye decolorization capacity from newly isolated Lysinibacillus fusiformis W11.

A new Lysinibacillus fusiformis strain with abundant laccase activity was isolated from soil under forest rotted leaf and identified as L. fusiformis W11 based on its 16S rRNA gene sequence and physiological characteristics. The laccase LfuLac was purified and characterized. The optimum temperature and pH of LfuLac on guaiacol were 45 °C and pH 9, respectively. LfuLac kept 78%, 88%, 92%, 74%, and 47% of activity at pH 7-11, respectively, suggesting the alkali resistance of the enzyme. The effects of various metal ions on LfuLac showed that Cu2+, Mg2+, and Na+ were beneficial to laccase activity and 10 mM Cu2+ increased the activity of LfuLac to 216%. LfuLac showed about 90% activity at 5% organic solvents and more than 60% activity at 20%, indicating its resistance to organic solvents. In addition, LfuLac decolorized different kinds of dyes. This study enriched our knowledge about laccase from L. fusiformis W11 and its potential industrial applications.

Lignin-Degrading Bacteria in Paper Mill Sludge.

The effluents generated in the paper industry, such as black liquor, have a high content of lignin and other toxic components; however, they represent a source of lignin-degrading bacteria with biotechnological potential. Therefore, the present study aimed to isolate and identify lignin-degrading bacteria species in paper mill sludge. A primary isolation was carried out from samples of sludge present in environments around a paper company located in the province of Ascope (Peru). Bacteria selection was made by the degradation of Lignin Kraft as the only carbon source in a solid medium. Finally, the laccase activity (Um-L-1) of each selected bacteria was determined by oxidation of 2,2'-azinobis-(3-etilbencenotiazolina-6-sulfonate) (ABTS). Bacterial species with laccase activity were identified by molecular biology techniques. Seven species of bacteria with laccase activity and the ability to degrade lignin were identified. The bacteria Agrobacterium tumefasciens (2), Klebsiella grimontii (1), and Beijeinckia fluminensis (1) were reported for first time. K. grimowntii and B. fluminensis presented the highest laccase activity, with values of 0.319 ± 0.005 UmL-1 and 0.329 ± 0.004 UmL-1, respectively. In conclusion, paper mill sludge may represent a source of lignin-degrading bacteria with laccase activity, and they could have potential biotechnological applications.

Improvement of Laccase Production by Thielavia terrestris Co3Bag1. Enhancing the Bio-Catalytic Performance of the Native Thermophilic TtLacA via Immobilization in Copper Alginate Gel Beads.

A 32-fold increase in laccase activity production by the thermophilic biomass-degrading fungus T. terrestris Co3Bag1 was achieved when the microorganism was grown on a modified medium containing fructose, sodium nitrate, and copper. A 70 kDa laccase (TtLacA), produced under the above conditions, was purified, immobilized in copper alginate gel beads, and characterized. TtLacA, both free and immobilized enzymes, exhibited optimal activity at pH 3.0, at a temperature of 65 and 70 °C, respectively, although both displayed 70% of activity from 40 to 70 °C. Free and immobilized enzymes retained at least 80% of relative activity in the pH range from 3 to 4.6. Immobilized TtLacA manifested a 2.3-fold higher thermal stability than the free form of the enzyme at 60 and 70 °C. Immobilized TtLacA retained 95% initial activity for six consecutive reuse cycles at 60 °C, and also retained 86% of initial activity after 12 days of storage at 4 °C. Based on the biochemical features, thermophilic TtLacA may be an efficient enzyme for dye decolorization and other industrial applications at high temperatures or acidic conditions. This work represents the first report about the immobilization and biochemical characterization of a thermophilic laccase from a member of the genus Thielavia.