Laccase Production Research Articles

Overproduction of Laccase by Trametes versicolor and Pycnoporus sanguineus in Farnesol-Pineapple Waste Solid Fermentation

The effect of farnesol, a sesquiterpene alcohol, on the production of laccases by Trametes versicolor and Pycnoporus sanguineus in pineapple waste solid-state fermentation was evaluated. Extracellular laccase production reached a maximum of 77.88 ± 5.62 U/g (236% above control) in farnesol-induced cultures of T. versicolor on the 17th day, whereas in a similar P. sanguineus culture, a maximal laccase activity of 130.95 ± 2.20 U/g (159% increase) was obtained on the 17th day. A single 45 KDa laccase was produced by both fungi under the influence of farnesol. These and other data allow us to conclude that farnesol acted as an inducer of the same form of laccase in both fungi. Farnesol disfavored fungal growth by increasing the lag phase, but it also clearly improved the oxidative state of the cultures. Contrary to the results obtained previously in submerged cultures, farnesol did not promote hyperbranching in the fungal mycelia. This is the first demonstration that farnesol is an excellent inducer of laccases in T. versicolor and P. sanguineus in solid-state cultivation. In quantitative terms, the results can be regarded as an excellent starting point for developing industrial or at least pre-industrial procedures to produce laccases using T. versicolor and P sanguineus under the stimulus of farnesol.

Laccase Production from Local Biomass Using Solid State Fermentation

The large family of enzymes, known as polyphenols oxidases, includes laccase. Due to the inclusion of a copper atom in their catalytic core, laccases are frequently referred to as multi-copper oxidases. Laccases are versatile enzymes that can catalyze the oxidation of a wide range of phenolic and non-phenolic substances. In the current study, a local strain of Aspergillus niger was used for solid-state fermentation to produce fungal laccase, as well as purify and optimize laccase. The enzyme profile, which was acquired using guaiacol to measure enzyme activity, showed that after five days of incubation, wheat straw provided the highest level of laccase activity, or 2.551 U/mL. A technique called response surface methodology (RSM) was used to examine the effects of various conditions on the production of enzymes. The RSM results demonstrated that after five days of incubation, the enzyme activity was at its highest at 45 °C, pH 5.5, and 30% moisture level, inoculated with 2 mL mycelium. Through ammonium sulphate precipitation and dialysis, the enzyme was purified. Additionally, column chromatography was used to further purify laccase. The next step was enzyme characterization to evaluate how temperature and pH affected enzyme activity. At 45 °C and pH 5.5, the isolated enzyme produced its highest level of activity. The findings of the current study showed that A. niger is capable of producing laccase in an economical and environmentally friendly way. Due to its unique oxidative and catalytic features, this enzyme has received a lot of attention recently.

Utilization potential of agro-industrial by-products and waste sources: Laccase production in bioreactor with Pichia pastoris

Valorization of waste materials has been an attractive approach due to ensuring circular bioeconomy model in bioprocesses depending on annual billion metric tons production of agro-industrial by-products and wastes. With being an influential platform of Pichia pastoris fermentation to produce value-added bioproducts, especially for recombinant proteins, the studies have been gaining speed to apply the circular bioeconomy model to these processes by enhancing production efficiency. In this study, solid and liquid dextrose from a local corn processing industry and watermelon peel from a local market were evaluated for production of Madurella mycetomatis laccase from P. pastoris X33 in a bioreactor. When compared to fermentation including alternative sources and glycerol/methanol which was performed as a control study, almost 2-fold higher laccase activity with 41 g DCW/L and 1.30-fold increase in laccase activity with 50 g DCW/L were determined, respectively for fermentation strategies including solid dextrose and liquid dextrose. Maximum laccase activity was obtained from watermelon peel hydrolysate utilization although lowest cell density was obtained in the growth stage. One-way ANOVA analysis confirmed the effect of carbon sources encouraging heterologous protein production in P. pastoris expression system. These results proved suitability of bioprocesses which were designed for the valorization of solid/liquid dextrose and watermelon peel hydrolysate as alternative carbon sources in P. pastoris fermentation for future scale-up.

A review of microbial laccase production and activity toward different biotechnological applications

A review of microbial laccase production and activity toward different biotechnological applications

Depolymerization of lignin using laccase from Bacillus sp. PCH94 for production of valuable chemicals: A sustainable approach for lignin valorization

Lignin is the most abundant aromatic polymer in nature, and its depolymerization offers excellent opportunities to develop renewable aromatic chemicals. In the present study, Bacillus sp. PCH94 was investigated for laccase production and lignin depolymerization. Maximum production of laccase enzyme was achieved within 6.0 h at 50 °C on a natural lignocellulosic substrate. Furthermore, Bacillus sp. PCH94 was used to bioconvert lignin dimeric and polymeric substrates, validated using FT-IR, NMR (1H, 13C), and LCMS. Genome mining of Bacillus sp. PCH94 revealed laccase gene (lacBl) as multicopper oxidase (spore coat CotA). Further, lacBl from Bacillus sp. PCH94 was cloned, expressed, and kinetically characterized. LacBl enzyme showed activity for substrates ABTS (40.64 IU/mg), guaiacol (5.43 IU/mg), and DMP (11.93 IU/mg). The LacBl was active in higher temperatures (10 to 100 °C) and showed a half-life of 36 and 27 h at 50 and 60 °C, respectively. The purified LacBl was able to depolymerize kraft lignin into valuable products (ferulic acid and acetovanillone), which have applications in the pharmaceutical and food industries. Overall, the current study demonstrated the role of bacterial laccase in the depolymerization of lignin and opened a promising prospect for the green production of valuable compounds from recalcitrant lignin.

Production of a novel laccase from Ceratorhiza hydrophila and assessing its potential in natural dye fixation and cytotoxicity against tumor cells

BackgroundFlavonoid natural dyes have gained attention because they are nontoxic and eco-friendly. However, they do not work effectively with artificial fibers and require the use of mordants, which are considered as hazardous chemicals. Laccase enzyme catalyzes the oxidation of phenols, forming phenoxyl radicals that undergo a further polymerization process. So, laccase can oxidize flavonoid dyes, and it can be used instead of harmful mordants in flavonoid dye fixation on cotton fabrics. Laccases also are involved in a variety of metabolic processes, and they have anti-proliferative effects toward HepG2 and MCF-7 tumor cells. ResultsAmong fifteen fungal isolates, the fungus Ceratorhiza hydrophila isolated from the submerged plant Myriophyllum spicatum was selected as the most potent laccase producer. Optimization of the production medium resulted in a 9.9-fold increase in laccase productivity. The partially purified Ceratorhiza hydrophila laccase could successfully improve the affinity of cotton fabrics toward quercetin (flavonoid) dye with excellent color fastness properties. The partially purified laccase also showed anti-proliferative activity against HepG2 and MCF-7 tumor cells. However, high laccase concentration is required to estimate IC50. ConclusionsCeratorhiza hydrophila MK387081 is an excellent laccase producer. The partially purified laccase from Ceratorhiza hydrophila can be used in textile dyeing and printing processes as a safer alternative to the conventional hazardous mordants. Also, it can be used in preparation of cancer treatment drugs. However, further studies are needed to investigate IC50 for both cell types at higher laccase concentrations.

Differential Laccase Production among Diverse Fungal Endophytes in Aquatic Plants of Hulimavu Lake in Bangalore, India

The ability of plants to acclimatise and thrive in stressed environments can be attributed, in part, to the reserve of endophytic fungi that they harbour, that help enhance physiological and immunological defence and tolerance to various biotic and abiotic stressors. The present work has focussed on screening laccase producing endophytic fungi residing in different aquatic plants isolated from Hulimavu Lake, Bengaluru. This lake is well known for its water pollution contributed by anthropogenic factors. Survival of plants in this lake can hence be associated with their rich repertoire of endophytic fungi that enhance host plant defence towards stressors. Upon isolation and culturing of endophytic fungi, qualitative laccase detection using laccase specific growth media and quantitative laccase estimation using ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) substrate were performed. Differential production rates were observed for the laccase enzyme by different endophytic fungi; production rates also varied between fungi isolated from different parts like node, stem, root and leaf of the same plant species too. Phylogenetic analysis of fungal isolates with highest laccase production was performed and the species was found to be Cladosporium tenuissimum. Even the crude extract of this strain displayed laccase production of 42.16U/L, as revealed by ABTS assay. Hence this strain is a promising candidate for optimization studies for utilisation in the domain of bioremediation and industrial applications.

PRODUCTION OF LACCASE FROM GANODERMA SP. BY USING AGRO-RESIDUAL WASTES

Solid-state fermentation (SSF) has emerged as a potential technology for the production of microbial products such asfeed, fuel, food, industrial chemicals and pharmaceutical products. Its application in bioprocesses such as bioleaching, biobeneficiation, bioremediation, biopulping, etc. has offered several advantages. Utilisation of agro-industrial residues assubstrates in SSF processes provides an alternative avenue and value-addition to these otherwise under- or non-utilisedresidues. The aim of this study was to determine the most promising fungus, the best solid substrate and the optimalconditions for the production of laccase. A laccase producing fungus Ganoderma sp. was isolated and produces laccase byutilizing the agro-wastes. The study revealed that the rice bran was found to be the best supported lignocellulosicsubstrate for extracellular laccase production under SSF and optimum condition for highest lacacse activity of pH andtemperature were observed at 6.0 and 45°C respectively. The incubation periods for laccase maximum activity was at 8thday. Glucose and Peptone were the best supported carbon and nitrogen source for the maximum laccase activity.

Enhanced laccase separation from fermentation medium using cryogel columns

The laccase enzyme family belongs to the oxidoreductase enzyme class and is one of the most commercially valuable enzymes that catalyzes the oxidation of one electron of a wide range of phenolic compounds. Separation and purification of laccases are crucial for industry since they play an important role in dye decolorization, biodegradation and food processing. Therefore, developing effective, high yielding and cost-effective methods for laccase production is vital. In this study, it was aimed to prepare cryogel columns for laccase purification following the bioproduction of laccase via Aspergillus niger. 2-hydroxyethyl methacrylate based cryogels were synthesized in the presence of 1-vinylimidazole as the affinity ligand and characterized by swelling tests, Brunauer–Emmett–Teller surface area measurement and scanning electron microscopy analysis. Surface area and water uptake ratio of cryogel columns were 35 m2/g and 93 %, respectively. The effect of pH, equilibrium laccase concentration, flow rate, interaction time and temperature on laccase adsorption were examined. The purification factor was calculated as 10.53 under optimum conditions and the enzyme recovery was found to be 86.7 % from fermentation medium. Current study revealed that laccase purification using cryogels following filtration of fermentation medium could be a promising candidate for industrial applications with eliminating the need for complex chromatographic steps.

Enhancing the production of a heterologous Trametes laccase (LacA) by replacement of the major cellulase CBH1 in Trichoderma reesei.

The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei.

Laccase Production Under Optimized Parameters by Aspergillus oryzae, an Endophytic Fungus and their Application to Waste Water Treatment

Objective: To isolate an endophytic fungal laccase producer from the Ziziphus mauritiana plant leaves in the surroundings of paper mill effluents and to study its role in the decolorization of synthetic dyes, removal of COD and phenol from industrial effluents. Methods: Laccase-producing endophytic fungi were isolated and screened by using an agar plate method. The positive isolates were identified, and laccase activity was determined by using spectrophotometric methods to monitor the oxidation of guaiacol and optimizing various parameters affecting laccase production. The molecular mass of the purified laccase enzyme was determined by using 12% SDSgel electrophoresis. Industrial effluents were treated with laccase to remove phenol, decolorize dyes, and reduce chemical oxygen demand. For the analysis, a spectrophotometric method was employed. Findings: One of the most effective endophytic fungal isolates, Aspergillus oryzae, was screened as a maximum laccase producer. The optimal pH of 6, temperature of 35 oC, inoculation period of 8 days, and the inoculum number of 3 discs/100 ml of Czapek Dox Broth in submerged culture were determined for the maximum laccase production. Sucrose and sodium nitrate, as carbon and nitrogen sources, considerably assisted laccase production. The molecular weight of the isolated laccase from A. oryzae was 66 kDa. The greatest activity was determined to be 64.2 U/mL, which is two times more than under unoptimized conditions. After the fifth day of exposure, the A. oryzae laccase decolorizes the synthetic dyes Bromophenol blue, Congo red, Methyl orange, and Phenol red.Chemical oxygen demand and phenolic pollutants’ clearance rates were 38–43% and 60% from coal and textile effluents during their exposure times, respectively. Novelty: A. oryzae was discovered to be a potent natural laccase producer endophytic fungus from paper mill effluents, which may be used for decolorizing non-textile dyes, treatment of various industrial effluents, and other industrial purposes. Keywords: Endophytic Fungi; Aspergillus oryzae; Laccase; Chemical oxygen demand; Synthetic dyes

Characterization of the biosurfactant production and enzymatic potential of bacteria isolated from an oil-contaminated saline soil.

Biosurfactants are amphiphilic compounds with extensive applications in oily contaminated environments to remove hydrocarbons. Moreover, enzymes such as laccase and manganese peroxidase are responsible for the oxidation of a variety of phenolic compounds and aromatic amines. Therefore, in the present study, bacteria with the potential to produce biosurfactants and enzymes (namely, laccase, manganese peroxidase, and endoglucanase carboxymethyl cellulose (CMCase)) were isolated from petroleum oil-contaminated soil. From 15 isolated bacteria, three isolates were selected as the best producers of biosurfactants according to the related tests, such as tests for surface tension reduction. These three bacteria indicated tolerance to a salinity test and were classified as resistant and very resistant. The isolates 3, 12, 13, and 14 showed positive results for the degradation of guaiacol, phenol red, and carboxymethylcellulose, as well as the decoloration of methylene blue by the creation of a clear halo around the bacterial colony. Upon the quantitation of the laccase and manganese peroxidase activities, 22.58U/L and 21.81U/L, respectively, were measured by isolate 13. Furthermore, CMCase activity was recorded with 0.057436U/ml belonging to isolate 14. Bacterial strains with appreciable laccase, peroxidase, CMCase activity, and biosurfactant production potentials were identified through 16S rDNA sequence analysis as Bacillus sp. (isolate 3), Bacillus toyonensis (isolate 12), Bacillus cereus (isolate 13), and Bacillus tropicus (isolate 14), and their nucleotide sequences were deposited in the GenBank. The potentials for the industrial applicability of the biosurfactants and enzymes abound, and production needs to be optimized by the selected bacterial strains.

Optimization of laccase production by Pleurotus ostreatus Florida and evaluation of metabolites generated during Kraft lignin biotransformation

Optimization of laccase production by Pleurotus ostreatus Florida and evaluation of metabolites generated during Kraft lignin biotransformation

Biodecolourization of Textile Effluents using Ligninolytic Enzymes Produced from Selected Bacterial and Fungal Isolated From Soil Contaminated Textile Waste

ABSTRACT Textile effluent contains enormous chemicals with detrimental environmental effects on both fauna and flora due to its chemical compositions. In this study, the effect of lignolytic enzymes produced by microorganisms for the treatment of textile effluent was examined using standard microbiological techniques. The potential of the isolates to produce laccase (L), lignin peroxidases (LiP) and manganase peroxidase (MnP) was investigated using streak plate method and assay methods. Finally, the L, LiP and MnP enzymes produced with the optimal processing parameter were used to decolorize textile effluent singly and in combination for ten (10) days. Fourteen (14) microbial isolates which include eight (8) bacterial and four (4) fungi were isolated from soil contaminated with textile effluents. Aspergillus terreus and Aspergillus niger showed higher production of laccase with 8.0 mm diameter zone of inhibition. Bacillus licheniformis and Bacillus subtilis had the widest zone of inhibition (12.0 and 8.0 mm) respectively. Only Aspergillus flavus however had the potential to produce lignin peroxidase (with 10 mm zones of clearance) of all the fungi isolated in this study. Laccase caused the highest decolourization of 72.5% comparable to 71.1% observed for the three enzyme combination while LiP has 57.0%. This finding established the potential use of bacterial ligninocellulolytic enzymes for the decolourization of textile effluent.

Solation, Screening and Optimization for Laccase production by Fusarium spp. under submerged fermentation

Laccases are oxidases with broad substrate specificity and ability to oxidize various phenolic and nonphenolic compounds. This study was carried out to isolate and characterizes laccase producing fungi from the environmental samples. Soil samples were collected from different locations. Suspensions of the samples (1g in 10 ml sterile physiological saline) were serially diluted, inoculated onto Czapek Dox Agar (CZA) containing antibiotic chloramphenicol and incubated for 7 days at 30 o C. The fungal isolates were characterized macroscopically and microscopically. Screening was done using Czapek-Dox agar was supplemented with 0.1% tannic acid, 0.5% Bromophenol blue, 0.05 ml in 25 ml of Methanol Guaiacol the pure culture of the single colonies from the spread plate method were inoculated on the media and incubated at room temperature for 5-6 days. After 5-6 days the plates were checked for clear transparent zone.

Yeni İzole Edilen Trametes versicolor'un Kesikli, Tekrarlı-Kesikli ve Katı-Faz Fermentasyon Süreçlerinde Lakkaz Üretimi

In this study, the laccase production ability of the newly isolated Trametes versicolor strain was investigated in three different fermentation processes. In all three fermentation processes, the fungus was able to produce the laccase enzyme. During the solid-state fermentation process 13.21 U/mL laccase activity was detected on the 20th day in the 10 mM copper-containing medium, while this value reached to 27.30 U/mL in the medium containing 0.5 mM ABTS+10 mM copper. During the liquid batch fermentation process, laccase activity was significantly induced in the medium containing 1 mM copper and the laccase activities reached 2.25, 19.83 and 24.57 U/mL compared to the medium without copper on the 3rd, 6th, and 9th days, respectively. ABTS and xylidine induced the laccase production of this strain at a much lower level than copper. The liquid repeated-batch process also significantly induced the laccase production. While low level of enzyme activities were detected in a copper-free medium, laccase activities were induced in the copper-containing medium and the activity increased from 0.66 U/mL to 9.87 U/mL at the 6th use of the pellets. Copper was detected as an effective inducer for laccase production in all fermentation processes and activity staining after native polyacrylamide gel electrophoresis clearly showed the active laccase bands. The results revealed that this strain is a good laccase producer and the laccase production yield varies depending on the fermentation process, production time, and inducer used.

A comparative screening of laccase-mediator systems by white-rot fungi laccases for biocatalytic benzyl alcohol oxidation

Production of value-added compounds from waste materials is of utmost importance for the development of a sustainable society especially regarding their use as catalysts in industrially relevant synthetic reactions. Herein, we show the production of laccases from four white-rot fungi, which were grown on agricultural residues, specifically Trametes versicolor 11269, Pleurotus ostreatus 1020, Panus tigrinus 707 and Lentinula edodes SC-495. The produced laccases were tested on a laccase-mediator system (LMS) for the biocatalytic oxidation of the model substrate benzyl alcohol into benzaldehyde. The LMS was carried out in the presence both of tetrahydrofuran as co-solvent and of the mediator 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) due to its high redox potential and its ability to perform the oxidation. Tolerance studies showed that the dialyzed solutions were able to tolerate 1% (99:1 v/v) of co-solvent, whereas a concentration of 10% v/v had a detrimental activity. Performances in the biocatalytic oxidation of laccase solutions from different purification steps were compared. Similar conversion was observed for laccase in dialysis (raw) and gel filtration (GF) product versus commercial T. versicolor laccase. The latter oxidized almost 99% of substrate while the other laccase solutions were able to reach a conversion from 91% for the laccase solution from P. tigrinus 707 after dialysis, to 50% for the laccase solution from P. ostreatus 1020 after gel filtration. This work highlights the potential of unpurified laccase solutions to be used as catalysts in synthetic reactions.

Yellow laccase produced by Trametes versicolor K1 on tomato waste: A comparative study with the blue one produced on semi-synthetic medium

Laccase production by fungal growth on agrifood waste is still poorly studied. Trametes versicolor K1 isolated from palm bark produced a yellow non glycosylated laccase from tomato waste based medium (TMT) and a blue glycosylated laccase on glucose medium (GLU). Lignocellulosic biomass, such as pinecones (PIN), palm leaves (PLM), olive pomace (OLV), and alfa stems (ALF) have also been used as growth medium for T. versicolor K1. In these conditions, very low or no laccase production was observed. When peptone was supplied in TMT medium, the laccase activity increased from 4170 U/L to 8618 U/L. By increasing the culture volume up to 1 L, laccase production on TMT was 9929 U/L. The yellow laccase (TmtLac) was purified from the supernatant TMT medium and has shown similar characteristics with the blue laccase (GluLac) purified from the GLU medium. Their apparent protein size was 63 kDa. Catalytic activities of the yellow form were not very different from those of the blue form, but specific activity of the purified yellow laccase produced on tomato waste was much higher. The Km and Vm values for four substrates, ABTS, DMP, guaiacol, and pyrogallol were almost similar for both isoenzymes. The optimum pH and temperature were respectively 4.0 and 50 °C. Although the level of glycosylation is clearly different, the thermostability of TmtLac and GluLac are quite similar. TmtLac is even slightly more tolerant at 60 °C for 24 h than GluLac. Moreover TmtLac showed greater stability at alkaline pH after 24 h compared to that of GluLac.We demonstrate that activity of the yellow TmtLac is not significantly affected compared to the blue laccase and that tomato waste is a simple and interesting lignocellulosic substrate to the laccase producer Trametes sp.

Characterisation of a laccase isolated from Trametes hirsuta and its application in the oligomerisation of phenolic compounds

Phenolic compounds are widely distributed in nature and industrial environment, and their detoxification or bioactive enhancement is of great value to environmental protection and industrial development. Laccases are multicopper oxidases that catalyse the oligo- or polymerisation of phenolic compounds. Identifying new laccase producers and investigating their application potential are of great importance. In this study, a white-rot fungus, Trametes hirsuta EZ1, with significantly high laccase productivity was isolated. The optimum conditions were studied for the maximum fermentation of extracellular laccase, which was achieved at 150 U/mL with a medium containing 10% strain EZ1, 7% maltodextrin, 1.5% peptone, and 0.5 mM Cu2+, and incubation at initial pH 6.0, 32 °C, and 180 rpm for nine days. Subsequently, a 70-kDa laccase was purified that showed activity over a wide range of temperature and pH, sensitivity to many metal ions and sodium dodecyl sulphate, and high tolerance to organic solvents. Purified laccase showed a significant unreported effect by catalysing catechol or ferulic acid into dimers, trimers, and tetramers or caffeic acid into dimers, trimers, tetramers, and pentamers. The oligomeric mixtures exhibited increased antioxidative capacity compared to that of each parent monomer, except for caffeic acid derivatives. Our study offers a novel strain source for laccase production and broadens its application in the enhancement of bioactive compounds.

Laccase production from Bacillus aestuarii KSK using Borassus flabellifer empty fruit bunch waste as a substrate and assessing their malachite green dye degradation.

The lignocellulosic waste, Borassus flabellifer empty fruit bunch waste (BFEFBW), was employed to produce laccase using Bacillus aestuarii KSK under solid-state fermentation (SSF) conditions and to assess the efficiency of malachite green (MG) dye decolourization. Abiotic factors such as pH (5.0-9.0), temperature (25-45°C) and incubation time (24-96 h) were optimized using Response surface methodology-Box-Behenan Design (RSM-BBD) to exploit the laccase production. The anticipated model revealed that the highest laccase activity of 437 U/ml shows after 60 h of incubation at 35°C at pH7.0. The bacterial laccase was used to remove 89% of the MG dye in less time. The laccase from B. aestuarii KSK decolorizes the MG and thereby making it a suitable choice for wastewater treatment from industrial effluents. This study is the first report on the production of laccase from B. flabellifer empty fruit bunch waste as a substrate. Bacillus aestuarii KSK was isolated from the soil sample and used to produce laccase under SSF conditions. The bacterial laccase has the potential for industrial application in textile waste dye treatment.