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.

Binding interaction of laccases from Bacillus Subtilis after industrial dyes exposure: Molecular docking and molecular dynamics simulation studies

Dyes are becoming more widely used around the world wide, but there is no effective bioremediation approach for removing them completely from the environment. Several dyes are mentioned to be degraded through bacteria; however, it's still unknown how the particular enzymes act throughout the dye degradation. The behavior and function of these enzymes in the biodegradation of azo dyes (Textile dyes) had been investigated experimentally by the numbers of the researchers, however, the molecular mechanisms remain unclear. Therefore, the interaction mechanisms of textile dye (methyl orange) with laccase from B. subtilis were explored through molecular docking and molecular dynamics simulations, the three selected dyes (methyl orange, malachite green, and acid blue 62) that interact positively with laccase on the basis of their maximum binding energy, molecular docking results indicate that one of the three dyes is more stable as a target for degradation through Bacillus subtilis laccase. Therefore, subsequent research focused solely on one substrate: methyl orange. Molecular Dynamics simulation study was applied after the molecular docking to determine the interaction between laccases and methyl orange dyes. The trajectory was proved with root mean square deviation and root mean square fluctuation analysis. According to the molecular dynamics simulation results, laccase-methyl orange complexes remain stable during the catalytic reaction. So, this study demonstrates how laccase is involved in methyl orange bioremediation.

A comparative study of free laccase and laccase immobilized in copper alginate

Enzyme immobilization has gained considerable attention due to the incredible properties exhibited by the enzymes in immobilized condition. Therefore, in the present study, a comparative analysis of free and immobilized laccase is reported using various substrates. The ideal substrate for immobilization was found to be copper alginate with an immobilization yield of 91.078%. The optimum pH was 4 and 5, respectively, for free and immobilized enzymes while the optimum temperature was found to be 50 and 60?C, respectively. The kinetic parameters Vmax and Km were found from the Line weaver-Burk plot and were 48.076 U/mL and 0.480 mM for free enzyme while 55.55 U/mL and 1.277 mM for the immobilized enzyme, respectively. The catalytic efficiency Kcat was found as 100.01s?1 for free enzyme and 43.5s?1 for its immobilized counterpart. Out of the various metal ions used, Co2+ was found to enhance the activity of an immobilized enzymes. The storage stability of the enzyme was studied and was found that only 32.44% of initial activity was retained by free enzyme whereas, 70.21% of activity was retained by immobilized enzyme upon storage for four weeks at 4?C. The thermal stability studies shows that the immobilized enzyme retained 32.60% of its initial activity and the free enzyme retained 1.14% of initial activity on exposure to 60?C for 3 h. Finally, the reusability of immobilized laccase beads was evaluated by decolorization of methyl orange for five repeated cycles and a percentage decolorization of 32.04% could be retained at the fifth cycle. This study therefore, suggests copper alginate-immobilized beads to be an effective option for various applications

Identification of laccase gene family members in peach and its relationship with chilling induced browning / 生物工程学报

The laccase (PpLAC) gene family members in peach fruit were identified and the relationship between their expression pattern and chilling induced browning were investigated. The study was performed using two varieties of peaches with different chilling tolerance, treated with or without exogenous γ-aminobutyric acid (GABA) during cold storage. Twenty-six genes were screened from the peach fruit genome. These genes were distributed on 6 chromosomes and each contained 5-7 exons. The PpLAC gene family members shared relatively similar gene structure and conserved motifs, and they were classified into 7 subgroups based on the cluster analysis. Transcriptome sequencing revealed that the expression levels of PpLAC7 and PpLAC9 exhibited an increasing pattern under low temperature storage, and displayed a similar trend with the browning index of peach fruit. Notably, GABA treatment reduced the degree of browning and inhibited the expression of PpLAC7 and PpLAC9. These results suggested that PpLAC7 and PpLAC9 might be involved in the browning of peach fruit during cold storage.

Isolation, identification, and optimization of laccase from Alternaria alternata

Laccase, lignin peroxidase, and manganese peroxidase have a synergistic effect on a wide range of recalcitrantcompounds. Among them, laccase is polyphenol oxidase widely available in fungi, plant species, and insects.Laccase has an important role in effluent decoloration, detoxification of pulp bleaching, and bioremediationprocess. Screening was carried out to find new fungal isolate for the presence of laccase activity with guaiacolas indicator compound. Sixteen fungal isolates were obtained from biodeteriorated agro waste and the woodsamples were collected from North Gujarat region of India. Among these isolates, one of the fungal isolateswas observed with good laccase activity and identified as Alternaria alternata. Laccase activity was determinedusing 2,2’-azinobis-(3-ethylbenzethiazoline-6-sulfonate) as substrate. Various production parameters such aspH, temperature, various carbon sources, nitrogen source, inducers, and cations were used to select the optimumcondition for further increase in the production of this enzyme. Maximum laccase activity was obtained withglucose and sucrose as carbon source, 0.2% ammonium sulfate as nitrogen source, and 0.06% Cu+2 with 1.5 mMveratryl alcohol as inducer under optimized condition.

Evolutionary divergence of function and expression of laccase genes in plants

Laccases (LACs) are versatile enzymes that catalyze oxidation of a wide range of substrates, thereby functioning in regulation of plant developmental processes and stress responses. However, with a few exceptions, the function of most LACs remains unclear in plants. In this study, we newly identified 4, 12, 22, 26, 27, 28 and 49 LAC genes for Physcomitrella patens, Amborella trichopoda, Zea mays, Ricinus communis, Vitis vinifera, Triticum aestivum and Glycine max, on the basis of exhaustive homologous sequence searches. In these plants, LACs differ greatly in sequence length and physical properties, such as molecular weight and theoretical isoelectric point (pI), but majority of them contain a signal peptide at their N-terminus. The originality of LACs could be traced back to as early as the emergence of moss. Plant LACs are clearly divided into seven distinct classes, where six ancient LACs should be present prior to the divergence of gymnosperms and angiosperms. Functional divergence analysis reveal that functional differentiation should occur among different groups of LACs because of altered selective constraints working on some critical amino acid sites (CAASs) within conserved laccase domains during evolution. Soybean and maize LACs have significantly different exon frequency (6.08 vs 4.82), and they are unevenly distributed and tend to form gene clusters on some chromosomes. Further analysis shows that the expansion of LAC gene family would be due to extensive tandem and chromosomal segmental duplications in the two plant species. Interestingly, *81.6% and 36.4% of soybean and maize LACs are potential targets of miRNAs, such as miR397a/b, miR408d, or miR528a/b etc. Both soybean and maize LACs are tissuespecifically and developmental-specifically expressed, and are in response to different external abiotic and biotic stressors. These results suggest a diversity of functions of plant LAC genes, which will broaden our understanding and lay solid foundation for further investigating their biological functions in plants.

Agro-food waste employed to design and develop culture media for fungal growth

Aim: Waste from the agricultural and food industries could be used as a source of nutrients in the culture media for fungal growth and thus reduce environmental pollution and the cost of bioprocesses. In this work, it was determined if some agro-food waste can improve the mycelial growth of fungi and laccase production.Methodology: Culture media with agar and vermicompost, vermiwash, calcined pork bones or fish waste for mycelial growth of fungi were elaborated. The laccase activity of each strain grown in all culture media was also estimated. Results: Pleurotus eryngii, Lentinula edodes, Pleurotus ostreatus, Flammulina velutipes, Pleurotus citrinopileatus and Ganoderma lucidum grew in all culture media, except Lentinula edodes, obtaining in general higher biomass and radial growth rate than those observed on potato-dextrose agar. The maximum laccase activity was found in Pleurotus citrinopileatus cultivated in fish waste medium. The C: N ratio, ashes and pH were important factors for the growth of the strains. Interpretation: There were significant differences in biomass concentration and laccase activity in the designed culture media. These results provide an interesting alternative for the use of wastes and metabolites production of industrial interest.

Structural-functional analyses of textile dye degrading azoreductase, laccase and peroxidase: a comparative in silico study

Background: Textile industry not only plays a vital role in our daily life but also a prominent factor in improving global economy. One of the environmental concern is it releases huge quantities of toxic dyes in the water leading to severe environmental pollution. Bacterial laccase and azoreductase successfully oxidize complex chemical structure of nitrogen group-containing azo dyes. Additionally, the presence of textile dye infuriates bacterial peroxidase to act as a dye degrading enzyme. Our present study deals with three textile dye degrading enzymes laccase, azoreductase, and peroxidase through analyzing their structural and functional properties using standard computational tools. Result: According to the comparative analysis of physicochemical characteristics, it was clear that laccase was mostly made up of basic amino acids whereas azoreductase and peroxidase both comprised of acidic amino acids. Higher aliphatic index ascertained the thermostability of all these three enzymes. Negative GRAVY value of the enzymes confirmed better water interaction of the enzymes. Instability index depicted that compared to laccase and preoxidase, azoreductase was more stable in nature. It was also observed that the three model proteins had more than 90% of total amino acids in the favored region of Ramachandran plot. Functional analysis revealed laccase as multicopper oxidase type enzyme and azoreductase as FMN dependent enzyme, while peroxidase consisted of α-ß barrel with additional haem group. Conclusion: Present study aims to provide knowledge on industrial dye degrading enzymes, choosing the suitable enzyme for industrial set up and to help in understanding the experimental laboratory requirements as well.

Effectiveness of application of lignolytic fungal strains, Cladosporium uredinicola GRDBF21 and Bipolaris maydis GRDBF23 in the treatment of tannery effluent

Aim: This study aims to investigate the ability of laccase producing fungal strains Cladosporium uredinicola GRDBF21 and Bipolaris maydis GRDBF23 isolated from decaying wood bark in decolouration and detoxification of tannery effluent. Methodology: Fungal strains from decaying wood bark samples were isolated by serial dilution technique followed by single spore isolation method. The selected fungal isolates were investigated for their laccase enzyme production. Their effect on physio-chemical properties of tannery effluent collected from final effluent drainage of a leather-tanning factory in Chrompet, Chennai, Tamil Nadu, India was analysed. Toxicity of treated and untreated tannery effluent was analysed by seed germination test. Results: The lignolytic and constitutive producers of laccase enzyme, C. uredinicola GRDBF21 and B. maydis GRDBF23 exhibited a tolerance index of 1.2 and 1.5, respectively, at 60% effluent concentration. The isolates were able to increase pH and reduce colour, turbidity, total suspended solids and electrical conductivity of the effluent. Besides observing a decrease in the BOD and COD levels, there was also a reduction in the sodium and hexavalent chromium content. C. uredinicola GRDBF21 and B. maydis GRDBF23 treated effluent showed a seed germination percentage of 66.6% and 76.6%, respectively. The untreated effluent completely inhibited the seed germination. Interpretation: The study confirms that the fungal species C. uredinicola GRDBF21 and B. maydis GRDBF23 could be effectively used in decolouration and detoxification of tannery effluent.

Screening, Production and Partial Characterization of a Thermostable Laccase from Trametes sp. Isolate B7 with Biotechnological Potentials

The search for efficient and green oxidation technologies has increased interest in utilization of laccases in non conventional methods. Laccases catalyze a wide range of substrates due to low substrate specificity and strong oxidative potentials. Challenges to large-scale enzyme utilization include, low enzyme activity and instability which restrict use in many areas of biotechnology. In the study, 59 fungi comprising Aspergillus niger (40%), Trichoderma harzianum (31%), Aspergillus flavus (9.0%), Trichoderma viride (5.0%), Fusarium oxysporum (5.0%), Rhizopus stolonifer (5.0%), Trametes sp. (3.0%) and Aspergillus nidulans (2.0%) were isolated and screened for laccase production. Plate screening test showed 57.5%, 34.0% and 8.5% of fungi were laccase-positive on ABTS, Guaiacol, and α-naphthol agar respectively. Isolates were further screened in liquid cultures, and the highest laccase producer identified molecularly. Trametes sp isolate B7 was selected for solid state fermentation (SSF). Laccase production in SSF was highest at pH 5.0 (2356 U/mL). The purified laccase showed high activity (pH 3.0 - 6.0) and stability (pH 3.0 - 8.5) using ABTS. It was active (20 - 80°C) and thermostable (30 - 80°C) with optimum stability at 70°C (100% for 1 hour). The percentage decolourization of Phenol red were 28% and 36% using 1000 U/mL and 2000 U/mL crude laccases respectively. Similarly, RBBR (100%), Congo red (75%) and Malachite green (62%), and 77.4%, 64% and 28% were decolourized using 1000 U/mL and 2000 U/mL crude laccases respectively. ABTS agar was very reliable in large-scale screening for laccase which possessed thermostable property and degraded synthetic dyes without use of enzyme mediators. These attribute made the enzyme suitable for application in industry and biotechnology.

Laccase production by Myrothecium gramineum and its optimization under solid state fermentation using cowpea pod as substrate

The present study highlights the utilization of wastes such as cowpea outer pod generated from agro industries forlaccase production using Myrothecium gramineum LCJ177 under solid-state fermentation. Conventional methodswere used to optimize the process parameters. The classical one-factor-at-a-time method showed that the optimalstarch concentration was 1 g/L, peptone concentration was 0.5 g/L, copper sulfate concentration was 0.6 mM, andpyrogallol concentration was 0.8 mM. Likewise, the suitable physical conditions were an initial pH of five of theculture medium, the temperature of 30°C and moisture content of 60%. Utilization of dried cowpea outer pod as asubstrate reduces the pollution levels by converting agro-wastes as useful by-products.

Degradação do repelente DEET pelas lacases do Pleurotus ostreatus / Degradation of repellent DEET by Pleurotus ostreatus laccases

Objetivo: Avaliar a capacidade de remediação do DEET em meio líquido, pelo fungo de decomposição branca Pleurotus ostreatus usando como indutor enzimático os resíduos sólidos do cacau e realizar bioensaios de toxicidade com as amostras pós-tratamento, para aplicações em tratamentos de águas. Método: Foi realizada a produção enzimática com resíduos do Cacau. A biorremediação com o caldo enzimático foi realizada em erlenmeyers de 250mL, contendo a solução do composto, tampão acetato de sódio pH 5 e o caldo batata, incubados à 28°C, com rotação de 120 rpm, por 48 horas. Já com o fungo ativo, o mesmo foi incubado a 28 ºC e teve em seu meio a adição do composto. As amostras foram quantificados em Cromatografia líquida de alta performance (CLAE). O teste de adsorção foi feito com o fungo autoclavado e analisado após 14 dias. Resultado: O composto se apresentou possivelmente tóxico e a remediação mostrou uma tendência linear de degradação com o fungo de 39%. Conclusão: Pleurotus ostreatus é um candidato promissor para o tratamento de contimanações geradas por DEET.

Objective: We evaluated the remediation capacity of DEET in liquid medium by the white decomposition fungus Pleurotus ostreatus using the solid residues of cocoa as an enzymatic inducer and performed toxicity bioassays with the post-treatment samples, for water treatment applications. Method: Enzymatic production with cocoa residues was performed. Bioremediation with the enzyme broth was performed in a 250mL erlenmeyer flasks, containing the solution of the compound, sodium acetate buffer pH 5 and the potato broth, incubated at 28 °C, with rotation of 120 rpm, for 48 hours. With the active fungus, the same was incubated at 28 ºC and had in its culture medium the addition of the compound. The samples were quantified in high performance liquid chromatography (HPLC). The adsorption test was performed with the autoclaved fungus and analyzed after 14 days. Results: The compound was possibly toxic and the remediation showed a linear tendency of degradation of 39% with the fungus. Conclusion: Pleurotus ostreatus is a promising candidate for the treatment of contaminants generated by DEET.

Progress in pitch control in pulp by enzymes / 生物工程学报

Pitch deposits have negative effects on product quality, machine performance and production line profitability during pulp and paper manufacture. As traditional pitch control technology cannot provide satisfactory solutions in the pitch deposits, the enzymatic treatment has been rapidly developed for its high efficiency and pollution-free property. In this review, the chemical composition and present form of the pitch in pulp is first introduced, followed by a description of the pitch control enzymes. The emphasis is on the current research on enzymatic solutions to pitch problems, including the reaction mechanism, technology, and the present main problems of lipase, sterol esterases, laccase and lipoxygenase. Finally, the technology prospects in this field are proposed.

Opuntia Extractable and Non-extractable (NEP) Residues as Co-carbon Sources for Improved Extracellular Laccase Production: Partial Purification and Characterization

Aim: The study of laccase production by Trametes pubescens cultured on cladode extractive residues alone, and as co-carbon source with wheat, using submerged fermentation condition. Place and Duration of Study: Biotechnology Laboratory, Durban University of Technology, Durban, South Africa. Methodology: Plant extraction, submerged fermentation, enzyme activity and characterization techniques were utilized. Results: The highest laccase activity was observed at 60-80% wheat: residue ratio combinations under submerged conditions with copper and xylidine supplementation. Partially purified enzyme fractions showed similar characteristics at different temperatures and pHs. There was good retention of relative enzyme activity at temperatures near 60°C, and pH stability from pH 4.0-6.0. At optimal culture conditions laccase activity was highest at 49.01±0.21 U/ml (80% wheat: 20% residue ratio), and lower at 10.02±0.51 U/ml (80% wheat:20% NEP residue). The optimum temperature for laccase fractions was 25°C and pH optimum at 5. Highest specific activity was 3.55 U/mg of protein for the 80% wheat: 20% residue ratio laccase extract. Conclusion: These results show the potential of cladode extractable phenol residues as potential economic growth medium for laccase production, offering a new alternative use for this agro-industrial and/or laboratory by-product.

Decolorization of Cibacron Blue 3G-A Dye by Agaricus bisporus CU13 Laccase - Mediator System: A Statistical Study for Optimization via Response Surface Methodology

Aim: the present study aims to optimize Cibacron Blue 3G-A decolorization as a model dye through laccase ‎enzymatic biocatalysis presenting the role of HBT as a redox mediator via ‎RSM approach.‎ Study Design: RSM using Central Composite Design (CCD) was used in order to determine the most effective variables levels in Cibacron Blue 3G-A decolorization and to investigate their interactions. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Research Division, National Research Centre (NRC), Cairo, Egypt, between August 2017 and January 2018. Methodology: The evaluation of Cibacron Blue 3G-A decolorization by A. bisporus CU13 crude laccase was conducted through different trials using a 1.5 mL reaction mixture containing different concentration of crude laccase, Cibacron Blue 3G-A, and HBT in 0.1 M sodium citrate buffer (pH 4.5) at room temperature for different incubation periods. Results: Hydroxybenzotriazole (HBT) as a mediator enhanced Cibacron Blue 3G-A decolorization levels significantly, where decolorization percentage caused by laccase enzyme alone were ‎11.92 and ‎23.78%, ‎whereas that caused by laccase HBT mediator system under the same conditions were 43.43 and ‎76.34% after 1 and 22 h of incubation, respectively. HBT concentration, dye concentration, enzyme activity, and incubation time were chosen as study variables to optimize Cibacron Blue 3G-A dye decolorization through RSM approach via central composite design (CCD). The optimum conditions for Cibacron Blue 3G-A decolorization were found to be under using 0.50 U/mL of Agaricus bisporus CU13 laccase, 92.19 ppm of Cibacron Blue 3G-A, and 1 mM of HBT in order to get decolorization percentage of 29.29% in 35 min. Conclusion: Agaricus bisporus CU13 crude laccase was used as a biocatalyst to decolorize Cibacron Blue 3G-A in presence of HBT as a mediator through utilizing the response surface methodology approach. HBT concentration, dye concentration, enzyme activity, ‎and incubation time affects the decolorization levels considerably.

Laccase Activity and Azo Dye Decolorization Potential of Podoscypha elegans

Azo dyes containing effluents from different industries pose threats to the environment. Though there are physico-chemical methods to treat such effluents, bioremediation is considered to be the best eco-compatible technique. In this communication, we discuss the decolorization potentiality of five azo dyes by Podoscypha elegans (G. Mey.) Pat., a macro-fungus, found growing on the leaf-litter layer of Bethuadahari Wildlife Sanctuary in West Bengal, India. The fungus exhibited high laccase and very low manganese peroxidase activities under different culture conditions. Decolorization of five high-molecular weight azo dyes, viz., Orange G, Congo Red, Direct Blue 15, Rose Bengal and Direct Yellow 27 by the fungus was found to be positive in all cases. Maximum and minimum mean decolorization percentages were recorded in Rose Bengal (70.41%) and Direct Blue 15 (24.8%), respectively. This is the first record of lignolytic study and dye decolorization by P. elegans.

Nucleus-Selective Expression of Laccase Genes in the Dikaryotic Strain of Lentinula edodes

In mating of Lentinula edodes, dikaryotic strains generated from certain monokaryotic strains such as the B2 used in this study tend to show better quality of fruiting bodies regardless of the mated monokaryotic strains. Unlike B2, dikaryotic strains generated from B16 generally show low yields, with deformed or underdeveloped fruiting bodies. This indicates that the two nuclei in the cytoplasm do not contribute equally to the physiology of dikaryotic L. edodes, suggesting an expression bias in the allelic genes of the two nuclei. To understand the role of each nucleus in dikaryotic strains, we investigated single nucleotide polymorphisms (SNPs) in laccase genes of monokaryotic strains to reveal nuclear origin of the expressed mRNAs in dikaryotic strain. We performed reverse transcription PCR (RT-PCR) analysis using total RNAs extracted from dikaryotic strains (A5B2, A18B2, and A2B16) as well as from compatible monokaryotic strains (A5, A18, and B2 for A5B2 and A18B2; A2 and B16 for A2B16). RT-PCR results revealed that Lcc1, Lcc2, Lcc4, Lcc7, and Lcc10 were the mainly expressed laccase genes in the L. edodes genome. To determine the nuclear origin of these laccase genes, the genomic DNA sequences in monokaryotic strains were analyzed, thereby revealing five SNPs in Lcc4 and two in Lcc7. Subsequent sequence analysis of laccase mRNAs expressed in dikaryotic strains revealed that these were almost exclusively expressed from B2-originated nuclei in A5B2 and A18B2 whereas B16 nucleus did not contribute to laccase expression in A2B16 strain. This suggests that B2 nucleus dominates the expression of allelic genes, thereby governing the physiology of dikaryons.

Bioinformatics analysis of laccases involved in active lignans′ biosynthesis in Isatis indigotica / 药学实践杂志

Objective Identifying laccases, as one of the key synthetases in the lariciresinol biosynthetic pathway, by analyzing the transcriptome sequencing results in Isatis indigotica would provide a dependable foundation for later functional study of Isatis indigotica′s laccases. Methods Bioinformatical softwares and kinds of analytical methods online were used to find out the characteristics of the laccases from I. indigotica, including physical and chemical properties, homology, and the properties after induction of MeJA. Results The transcriptional results showed that Iilac3 and Iilac5 from I. indigotica were corresponded ith the accumulation of the effective metabolites, making them the potential functional genes participated in lariciresinol synthesis. Conclusion Through the detailed bioinformatical analysis of Iilacs,which laid a solid foundation for the further study of the physiological and biochemical mechanisms and structural characteristics of the functional proteins.

Bioinformatic analysis and characterization of myxobacteria laccase-like multicopper oxidases / 生物工程学报

Laccase is a widely-used environment-friendly copper-containing oxidase found in many plants, insects and fungi. Recently, more and more laccases are also found in bacteria. Myxobacteria are an important bacteria resource. However, myxobacteria are much more difficult to isolate and purify than other bacteria. We used bioinformatic approach to screen myxobacteria proteomes available in NCBI. Based on conserved sequences of four copper binding sites in multicopper oxidase, 30 potential laccase sequences were obtained. Among them, nine genes were synthesized and expressed in Escherichia coli BL21 (DE3). Seven proteins showed laccase activity when tested with traditional laccase substrates. One protein, named rSC-2, was chosen for further research because it exhibited the highest activity towards 2,6-dimethyl phenol (DMP). The molecular weight of rSC-2 was 57 kDa. Its specific activity to DMP was 0.27 U/mg. The optimal temperature and the optimal pH were 60 ℃ and 7.0, respectively. About 50% of the original activity was retained after incubation at 60 ℃ and pH 7.0-8.0 for 1 h. Metals showed different effects on rSC-2. rSC-2 activity was enhanced by several metalsat concentration of 1 mmol/L, such as Ca²⁺ and Mn²⁺. With a higher concentration of 5 mmol/L, the activity of rSC-2 was apparently inhibited. This is the first report of bioinformatics screening myxobacteria laccases in combination with expression in E. coli.