Maximum Laccase Research Articles

Vinasse odyssey: sugarcane vinasse remediation and laccase production by Trametes sp. immobilized in polyurethane foam

Vinasse is a high pollutant liquid residue from bioethanol production. Due to its toxicity, most vinasse is used not disposed of in water bodies but employed for the fertigation of sugarcane crops, potentially leading to soil salinization or heavy metal deposition. The anaerobic digestion of vinasse for energy production is the main alternative to fertigation, but the process cannot eliminate colored compounds such as melanoidins, caramels, or phenolic compounds. The treatment of raw vinasse with white-rot fungi could remove colored and persistent toxic compounds, but is generally considered cost-ineffective. We report the treatment of vinasse by an autochthonous Trametes sp. strain immobilized in polyurethane foam and the concomitant production of high titers of laccase, a high value-added product that could improve the viability of the process. The reuse of the immobilized biomass and the discoloration of raw vinasse, the concentration of phenolic compounds, BOD and COD, and the phytotoxicity of the treated vinasse were measured to assess the viability of the process and the potential use of treated vinasse in fertigation or as a complementary treatment to anaerobic digestion. Under optimal conditions (vinasse 0.25X, 30°C, 21days incubation, 2% glucose added in the implantation stage), immobilized Trametes sp. causes a decrease of 75% in vinasse color and total phenolic compounds, reaching 1082 U L-1 of laccase. The fungi could be used to treat 0.50X vinasse (BOD 44,400mg O2 L-1), causing a 26% decolorization and a 30% removal of phenolic compounds after 21days of treatment with maximum laccase titers of 112 U L-1, while reducing COD and BOD from 103,290 to 42,500mg O2 L-1 (59%) and from 44,440 to 21,230mg O2 L-1 (52%), respectively. The re-utilization of immobilized biomass to treat 0.50X vinasse proved to be successful, leading to the production of 361 U L-1 of laccase with 77% decolorization, 61% degradation of phenolic compounds, and the reduction of COD and BOD by 75% and 80%, respectively. Trametes sp. also reduced vinasse phytotoxicity to Lactuca sativa seedlings. The obtained results show that the aerobic treatment of vinasse by immobilized Trametes sp. is an interesting technology that could be employed as a sole treatment for the bioremediation of vinasse, with the concomitant the production of laccase. Alternatively, the methodology could be used in combination with anaerobic digestion to achieve greater decolorization and reduction of phenolic compounds, melanoidins, and organic load.

Optimization, purification and characterization of laccase from lignocellulolytic fungi Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535

In the present study, lignocellulolytic fungi were isolated by lignocellulolytic waste (Banana waste) soil from the Bhilai-Durg region of Chhattisgarh, India, which was capable to produce laccase enzyme. The laccase producing potent organisms was identified as Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535 using molecular characterization by Internal Transcribed Spacer (ITS). For the better production of laccase enzyme physical (incubation period, temperature and pH) and nutritional (carbon, nitrogen and vitamin source) parameters optimized by one factor-at-a time and response surface methodology approach were used. Laccase enzyme was partially purified by ammonium sulphate precipitation and dialysis. The partially purified laccase was characterized by the effect of substrate concentration, temperature, pH, metal ions and inhibitors. Results showed that out of 5 positive cultures only 2 cultures showed the best laccase production after the quantitative screening. ITS-rDNA gene sequencing and BLAST analysis were used to characterize the isolated fungal species and they were found to be D. funicola NFCCI 4534 and A. padwickii NFCCI 4535. The temperature 24 °C and 32 °C and pH 6.0 and 6.5 were suitable for production of D. funicola and A. padwickii, respectively. It was found that Ferric chloride and ferrous sulphate exhibited maximum laccase activity as compared to other metal ions. However, EDTA and l-arginine inhibited D. funicola and A. padwickii, respectively.

Improved laccase production in Pleurotus djamor RP by atmospheric and room temperature plasma (ARTP) mutagenesis

BackgroundPleurotus djamor is an edible mushroom with medicinal properties, and degrades lignocellulose by producing enzymes that have a variety of industrial applications. The aim of this study was to improve laccase producing potential of P. djamor that may easily convert lignocellulose waste into high-value products. ResultsIn the present study, protoplast of the newly isolated fungus P. djamor RP was mutated by atmospheric pressure and room temperature plasma (ARTP) method. Guaiacol was used as an indicator for the rapid visual appearance of laccase positives in the screening process. From the results of preliminary screening, 10 promising positive mutants with a maximum laccase production were selected for further experimentation. Lac activity by the ABTS method showed that the mutant P. djamor strain 51-4 exhibited the maximum laccase production of 494.44 U/L in shake flask cultivation, which was 86.36% higher than wild strain. Notably, the other mutant P. djamor strain 62-27 exhibits a higher laccase production of 26.06 U/g which was 30.28% higher than wild strain when grown on solid-state fermentation. In addition to a higher yield of laccase, all mutants showed better growth and genetic stability than the wild strain. ConclusionsP. djamor RP mutants with high laccase-producing potential were developed by ARTP Mutagenesis technique, and these isolates could be used as a possible candidate for lignocellulose conversion into high-value products.How to cite: Zhang Z, Shah AM, Mohamed H, et al. Improved laccase production in Pleurotus djamor RP by atmospheric and room temperature plasma (ARTP) mutagenesis. Electron J Biotechnol 2022;58. https://doi.org/10.1016/j.ejbt.2022.03.005

Electricity Production and Phenol Removal of Winery Wastewater by Constructed Wetland – Microbial Fuel Cell Integrated With Ethanol Tolerant Yeast

Winery wastewater is one of the most concerning wastewater because it contains a high concentration of ethanol and phenolic compound that can inhibit the wastewater treatment potential of microorganisms. In this study, the ethanol tolerant yeast with laccase activity was integrated with constructed wetland -microbial fuel cell (CW-MFC) to remove ethanol contamination wastewater treatment limitation and simultaneously generate electrical energy. For the experiment, ethanol tolerant yeast with laccase activity was selected and grown in the CW-MFC filled with winery wastewater. The wastewater treatment (chemical oxygen demand (COD) and phenol removal) potential and electrochemical properties were monitored. The results indicated that the maximal laccase activity of 158.12±0.52 U/mL was gained from the yeast strain ET-KK. The maximal current density and power density of 139.17±1.44 mA/m2 and 38.74±0.80 mW/m2 were generated where the maximal COD and phenol removal of 79.14±0.92% and 85.04±0.07% were obtained. This study used the CW-MFC integrated with laccase-producing ethanol tolerant yeast for winery wastewater treatment and electricity generation.

Laccase-Driven Transformation of High Priority Pesticides Without Redox Mediators: Towards Bioremediation of Contaminated Wastewaters.

In this study, Pleurotus dryinus was grown on municipal biosolids (BS) as the substrate to produce laccase for the removal of pesticides (fungicides, herbicides, and insecticides) from wastewater. Among the various types of BS tested, sterilized biosolids were the most promising substrate for laccase production by P. dryinus with a maximal laccase activity (162.1 ± 21.1 U/g dry substrate), followed by hygenized biosolids (96.7 ± 17.6 U/g dry substrate), unsterilized biosolids (UBS) (31.9 ± 1.2 U/g dry substrate), and alkali-treated biosolids (8.2 ± 0.4 U/g dry substrate). The ultrasound-assisted extraction of this enzyme from fermented UBS was carried out with 0.1 M phosphate buffer at pH 7.0, which increased the enzyme activity of the crude extract by 30%. To test the catalytic potential of the biocatalyst in real matrices, 1 U/ml of recovered crude laccase extract was applied for 24 h for the removal of 29 pesticides (nine fungicides, 10 herbicides, and 10 insecticides) either separately or as a mixture from spiked biologically treated wastewater effluent. When treated with crude enzyme extract, high-priority herbicides metolachlor and atrazine were completely removed, while 93%–97% of the insecticides aldicarb, spinosad, and azinphos-methyl and up to 91% of kresoxim-methyl were removed. Promising results were obtained with BS-derived crude enzyme extract exhibiting improved pesticides removal, which may be due to the mediator effect resulting from the catalytic transformation of other molecules in the cocktail. The results demonstrated a promising integrated bioprocess for the removal of pesticides in wastewater using crude laccase obtained from BS.

Diclofenac biotransformation and toxicity assessment of laccase from Pleurotus floridanus.

Laccase producing fungus Pleurotus floridanus was isolated from Siruvani forest, Tamil Nadu, India. The potential of P. floridanus to produce laccase by using various lignocellulosic substrates was screened under submerged fermentation. Laccase production in the presence of lignocellulosic substrates such as rice, wheat and maize bran as a sole source of carbon as well as an additional supplement was examined. Laccase activity of P. floridanus using varied substrates was observed in the order of rice bran > wheat bran > maize bran. The isolate showed maximum laccase activity of 13.29±0.01 U/mL using rice bran as a carbon source within 11 days. This was 18 fold higher than the control media that lacks lignocellulosic substrates. The diclofenac tolerance was assessed in solid media at various concentrations and the results showed that the mycelia growth is not significantly affected by the drug. Finally, the laccase mediated degradation of diclofenac at a concentration of 10 mg/L showed 98% degradation in 2 h. The phytotoxicity of the crude laccase treated diclofenac was lower than the untreated diclofenac. In conclusion, findings suggested direct application of crude laccase produced from P. floridanus using agro-residues as ideal substrate for environmental applications.

Production of fungal laccase on pineapple waste and application in detoxification of malachite green

The main purpose of this work was to use pineapple crowns as substrate for optimizing laccase production by Trametes versicolor in lab-scale experiments. One-factor-at-the-time analysis and response surface methodology were used to optimize production. A single laccase with molecular weight of 45 kDa was the main protein produced. A maximal laccase activity of 60.73 ± 1.01 U/g was obtained in 7-day cultures, representing a 6.7-fold increase compared to non-optimized conditions. The optimized conditions were temperature: 28 °C; initial moisture: 90%; glucose: 8.38%; yeast extract: 2.86%. Combining activity and stability, the best conditions for using this laccase during the long periods required by large-scale processes are pH 4.0–5.0 and temperature of 40–50 °C. Under these conditions, the crude laccase was efficient in detoxifying the dye malachite green with a KM of 14.33 ± 1.94 µM and a Vmax of 0.482 ± 0.029 µM/min with 0.1 units/mL. It can be concluded that pineapple crown leaves can be effectively used as substrate by T. versicolor for producing laccase under solid-state culture conditions. Laccase is an industrially relevant enzyme and its production with concomitant valorization of pineapple crowns as substrate offers highly interesting perspectives.

Optimization of Laccase Production, and Characterization of Lignin Degradation Products by Fusarium oxysporum JUMAD-053

The objective of this work was to optimize the culture conditions for laccase production in the presence of Kraft lignin by Fusarium oxysporum JUMAD-053, and to evaluate the biodegradation products of lignin. The fungal isolate that presented highest laccase activity had its production optimized by a statistical factorial design 3 3 in 15 experimental runs. F. oxysporum presented the highest constitutive laccase titer (5.37 U/mL). Statistical factorial design demonstrated a maximum laccase titer of 9.8 U/mL when assayed against ABTS under the conditions optimized: 1.125% (w/v) yeast extract, 0.5% (w/v) Kraft lignin and 10 days of cultivation. The maximum laccase titer when assayed on DMP was 8.4 U/mL, following the conditions optimized: 1.125% yeast extract, 0.25% Kraft lignin and 7 days of cultivation. The analysis of cultures led to identification of metabolites; two being aromatic: 2,6-dimethoxy benzoic acid and sesamin; also, fumonisin and long-chain fatty acids. As a result of the study, the maximum laccase activities of 9.8 and 8.4 U/mL measured from ABTS and DMP substrates, respectively. The search shows new sources of fungal laccase for obtaining new metabolites of biodegradation from Kraft lignin in culture medium. DOI: http://dx.doi.org/10.17807/orbital.v13i5.1623

Artificial intelligence modeling and molecular docking to analyze the laccase delignification process of rice straw by Comamonas testosteroni FJ17

The laccase enzymatic characteristics and delignification processes of rice straw by Comamonas testosteroni FJ17 were investigated. Artificial intelligence modeling and molecular docking revealed the specific functional properties involved in the interaction between laccase and lignin compounds with a maximum laccase activity of 2016.7 U L-1 at 24 h. Scanning electron microscopy and X-ray diffractometer analysis confirmed that laccase caused fractures and holes on the surface of rice straw, where crystallinity decrease from 45.3 to 39.9%, and lignin content decreased from 19.0 to 10.3%. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analysis showed that the main delignification process for laccase was via β-o-4 and α-aryl ether cleavage, which generated several small molecular products. The laccase gene was cloned and bioinformatics analysis presented 317 amino acids with a predicted molecular weight of 33.13 kDa. Finally, laccase protein was found to have low binding energies with all lignin compounds tested, and lignin compounds were oxidized by laccase through hydrogen-bonding interactions with the amino acid residues.

The Valorization of Biolignin from Esparto Grass (Stipa tenacissima L.) Produced by Green Process CIMV (Compagnie Industrielle de la Matière Végétale) for Fertilization of Algerian Degraded Soil: Impact on the Physicochemical and Biological Properties

This study proposes a new use for a paper industry waste material, lignin, in agriculture and agronomy as a fertilizer for arid soils, while following a strategy aiming to both increase the amount of organic matter in these soils and decrease the impact of pollution caused by industrial discharges that contain organic and/or inorganic pollutants generated by the paper industry. In fact, this method works to improve soil quality through a new carbon-rich bioorganic fertilizer (biolignin) that results from a green method called CIMV, a targeted depollution objective of the paper industry. Over the course of 180 days, we monitored the physicochemical and biological characteristics of degraded soils treated with three different biolignin treatments of 0 (D0), 2 (D1), and 4 (D2) g/kg. The humification was then evaluated by the equation E4/E6. A remarkable variation of the physicochemical and biological parameters was observed in D1 and D2: temperature 12–38 °C, humidity 9–29%, and pH 7.06–8.73. The C/N ratio decreased from 266 to 49. After 180 days, the improvement in soil carbon content for the three treatments D0, D1, and D2 was 14%, 19%, and 24%, respectively. A maximum bacterial biomass of 152 (CFU/g soil) was observed on the 30th day for D1. Maximum laccase activity for D2 was observed on the 120th day. D1 and D2 recorded a significant degree of humification compared to D0. The best indicator of humification E4/E6 was observed in D1, where the value reached 2.66 at the end of the treatment period. The D2 treatment showed a remarkable effect improving the fertility of the degraded soil, which confirms that biolignin is a good fertilizer.

Degradation of lignin by Bacillus altitudinis SL7 isolated from pulp and paper mill effluent.

Lignin is a major by-product of pulp and paper industries, and is resistant to depolymerization due to its heterogeneous structure. Degradation of lignin can be achieved by the use of potential lignin-degrading bacteria. The current study was designed to evaluate the degradation efficiency of newly isolated Bacillus altitudinis SL7 from pulp and paper mill effluent. The degradation efficiency of B. altitudinis SL7 was determined by color reduction, lignin content, and ligninolytic activity from degradation medium supplemented with alkali lignin (3 g/L). B. altitudinis SL7 reduced color and lignin content by 26 and 44%, respectively, on the 5th day of incubation, as evident from the maximum laccase activity. Optimum degradation was observed at 40 °C and pH 8.0. FT-IR spectroscopy and GC-MS analysis confirmed lignin degradation by emergence of the new peaks and identification of low-molecular-weight compounds in treated samples. The identified compounds such as vanillin, 2-methyoxyhenol, 3-methyl phenol, oxalic acid and ferulic acid suggested the degradation of coniferyl and sinapyl groups of lignin. Degradation efficiency of B. altitudinis SL7 towards high lignin concentration under alkaline pH indicated the potential application of this isolate in biological treatment of the lignin-containing effluents.

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.

Studies on production and evaluation of biopigment and synthetic dye decolorization capacity of laccase produced by A. oryzae cultivated on agro-waste.

The present study investigated the screening of mono and co-culture fungal cultivations for laccase production using extracted lignin as the substrate obtained from cauliflower wastes by two different pretreatment methods. Amongst mono and mixed culture fungal cultivations, monoculture of Aspergillus oryzae exhibited the highest enzymatic activity of 29.7 ± 0.6 U mL-1 under submerged conditions and using alkali extracted lignin as substrate. Under the optimal conditions (pH 4.5, 30°C, 12days, 1% (w/v) lignin and 0.5mM Cu2+ concentration) the maximum laccase activity was estimated to be 41.3 ± 2.8 U mL-1 and production yield of 153.3 ± 2.4mg L-1. Maximum decolorization of pigment extracted from Aspergillus heteromorphus CBS 117.55 cultivated culture media was achieved by administration of 40 U g-1 of crude enzyme concentration. Thermal and pH stability of crude laccase was observed over wide ranges. The dye decolorization efficiency of crude A. oryzae laccase was studied and Congo Red exhibited maximum decolorization percentage (64 ± 1.3%) at 15µM, 50°C and pH 4.5. The kinetic study of different dye (Congo Red) concentrations obtained Vmax and Km values of 0.123 × 10-3M and 0.724mol L-1min-1, respectively.

Laccase activity from Pleurotus ostreatus and Flammulina velutipes strains grown on agro- and forestry residues by solid-state fermentation

Laccase activity from Pleurotus ostreatus and Flammulina velutipes strains was investigated with various agro- and forestry residues by solid-state fermentation. Different species or strains belonging to the same species had the unique capacity of secreting laccase on solid-state fermentation with various agro- and forestry residues. Overall, the capacity of secreting laccase for P. ostreatus strains was superior to F. velutipes strains due to the value of maximum activity on various agro- and forestry residues, except on the stalk of straw. Compared with Populus beijingensis, corncob, and stalk of straw, the presence of cottonseed hull was helpful to improve laccase activity for P. ostreatus strains because the maximum laccase activity from cottonseed hull was higher than that from the other three agro- and forestry residues. The presence of stalk of straw was more helpful to improve laccase activity for F. velutipes strains because of the maximum laccase activity from stalk of straw was higher that from Populus beijingensis, corncob, and cottonseed hull. These results indicated the importance of selecting suitable agro- and forestry residues for fungi producing laccase. These findings contributed to the selection of suitable strains to obtain an integrated application of low-cost laccase in the factory.

Effect of Pretreated Colza Straw on the Growth and Extracellular Ligninolytic Enzymes Production by Lentinula edodes and Ganoderma lucidum

Lentinula edodes 3565 and Ganoderma lucidum 9621 were compared for their ability to produce lignocellulolytic enzymes in submerged (SM) and surface liquid (SL) fermentation of hydrolysed colza straw lignin waste that remained after the production of furfural and bioethanol (CS lignin). Application of cultivated mushrooms to dispose of pretreated colza straw agricultural waste is an approach to decrease the quantity of residual lignin while simultaneously obtaining active substances, e.g., the ligninolytic enzyme complex from mycelium. The effect of adding CS lignin to culture media on the yield of L. edodes and G. lucidum mycelium and extracellular laccase activity was studied. It was revealed that the mycelial growth of G. lucidum on solid media was significantly improved by adding CS lignin. Laccase activity during SL cultivation of L. edodes on medium with CS lignin gradually increased over the experiment starting on day 21 and peaked at 520 U/mL on day 28. G. lucidum expressed the maximum laccase activity, 540 U/mL, during the first 14 days of mycelium SM cultivation. Extracellular laccase activity was enhanced about 35- to 40-fold at cultivation of L. edodes and about 10- to 15-fold in the case of G. lucidum by supplementing liquid culture media with CS lignin.

Galactomannans for Entrapment of Gliomastix murorum Laccase and Their Use in Reactive Blue 2 Decolorization

In the present study, a novel laccase from ascomycete Gliomastix murorum was produced in agro-industrial wastes and entrapped in galactomannan beads for Reactive Blue 2 (Rb-2) decolorization. The maximum laccase production in agave bagasse-based medium occurred at 72 h (1798.6 UL−1). Entrapped laccase decolorized ˃80% of 0.5 mM Rb-2 in 2 h without the addition of redox mediator. Km for Rb-2 substrate was 1.42 mM, with a Vmax of 1.19 µmol min−1 for entrapped laccase. Galactomannan matrices produce stability to acid pH (2–5) and temperatures from 20–70 °C. Reusability assays showed that entrapped laccase could retain efficient Rb-2 decolorization of ˃80% six times. In general, galactomannan used for entrapment of laccase provides economic advantages in large-scale wastewater treatment due to its natural origin and efficient results.

Evaluation of the capacity of laccase secretion of four novel isolated white-rot fungal strains in submerged fermentation with lignocellulosic biomass

The capacity of novel isolated white-rot fungi secreting laccase was evaluated for various kinds of lignocellulosic biomass in submerged fermentation. The laccase secreted by Neofomitella fumosipora Han 386 and Pleurotus pulmonarius Han 527 was significantly faster than that by Coriolopsis trogii Han 751 and Coriolopsis sanguinaria An 282. Maximum laccase from N. fumosipora Han 386 on the four kinds of lignocellulosic biomass tested appeared on the first day. This phenomenon indicated that N. fumosipora Han 386 secreted laccase rapidly compared with other tested strains in this study and showed the superiority in the rate of secreting laccase. Based on the maximum laccase activity, the ability of secreting laccase of C. sanguinaria An 282 was superior to other tested novel isolated strains. On the whole, N. fumosipora Han 386 and P. pulmonarius Han 527 preferred Toona sinensis to produce laccase, C. trogii Han 751 preferred to produce laccase on Populus beijingensis, and C. sanguinaria An 282 grown on Sorghum straw was more suitable for secreting laccase. The results will be helpful for developing bioprocesses using various kinds of lignocellulosic biomass for lignocellulolytic enzyme production and enlarging the number of laccase producing strains for industrial application.

Enrichment of laccase production by Phoma herbarum isolate KU4 under solid-state fermentation by optimizing RSM coefficients using genetic algorithm.

The process parameters were optimized to obtain enhanced enzyme activity from the fungus Phoma herbarum isolate KU4 using rice straw and saw dust as substrate under solid-state fermentation using Response surface methodology (RSM). Genetic algorithm was used to validate the RSM for maximum laccase production. Six variables, viz., pH of the media, initial moisture content, copper sulphate concentration, concentration of tannic acid, inoculum concentration and incubation time were found to be effective and optimized for enhanced production. Maximum laccase production was achieved by RSM at pH 5·0 and 86% of initial moisture content of the culture medium, 150µmoll-1 of CuSO4 , 1·5% tannic acid and 0·128g inoculum g-1 dry substrate inoculum size on the fourth day of fermentation. The highest laccase activity was observed as 79008Ug-1 , which is approximately sixfold enhanced production compared to the unoptimized condition (12085·26Ug-1 ).

Characterization of a Potential Isozyme Laccase from Trametes polyzona MPS1-3 and its Contribution to Palm Oil Mill Effluent Treatment.

The high phenolic content of Palm Oil Mill Effluent (POME) constitutes an environmental concern. In this study, laccase producing microorganisms were isolated from POME samples collected in Côte d'Ivoire for their possible use in POME treatment. Strain showing the highest laccase activity was identified by ITS1-5.8S-ITS2 region sequencing as Trametes polyzona. A maximum laccase production (156.3 U/mL) was obtained after 10days of incubation under shaking condition, at 37°C, pH 4, with starch (1%), tryptone (0.3%) and 10mM of guaiacol. The partially purified laccase of 31kDa exhibited maximum activity at 50°C and pH 4.5 with a Km for guaiacol and Vmax of 0.7mM and 0.04mM/min, respectively. Metals, SDS and EDTA did not inhibit his activity. Used as biotreatment agent, T. polyzona MPS1-3 reduced COD, total suspended solids, total solids and total phenolics by 16.03%, 70.15%, 38.9%, 50.84%, respectively, for sterilized POME and by 13.09%, 58.07%, 36.53%, 42.05% for unsterilized POME. These results showed the promising application of T. polyzona for bioremediation of phenolics compounds in wastewater and it potentially useful in several other biotechnological applications.

Enhancing the expression of recombinant small laccase in Pichia pastoris by a double promoter system and application in antibiotics degradation.

Low-expression levels remain a challenge in the quest to use the small laccase (rSLAC) as a viable catalyst. In this study, a recombinant Pichia pastoris strain (rSLAC-GAP-AOX) producing rSLAC under both AOX and GAP promoters (located in two different plasmids) was generated and cultivated in the presence of methanol and mixed feed (methanol:glycerol). Induction with methanol resulted in a maximum laccase activity of 1200 U/L for rSLAC-GAP-AOX which was approximately 2.4-fold higher than rSLAC-AOX and 5.1-fold higher than rSLAC-GAP. The addition of methanol:glycerol in a stoichiometric ratio of 9:1 consistently improved biomass and led to a 1.5-fold increase in rSLAC production as compared to induction with methanol alone. The rSLAC removed 95% of 5mg/L ciprofloxacin (CIP) and 99% of 100mg/L tetracycline (TC) in the presence of a mediator. Removal of TC resulted in complete elimination of antibacterial activity while up to 48% reduction in antibacterial activity was observed when CIP was removed. Overall, the present study highlights the effectiveness of a double promoter system in enhancing SLAC production.