Application Of Laccase Research Articles

Application of laccase immobilized rice straw biochar for anthracene degradation.

The present study explores the immobilization of ligninolytic enzyme-laccase on the surface of rice straw biochar and evaluates its application for anthracene biodegradation. The rice straw biochar was acid-treated to generate carboxyl functionality on its surface, followed by detailed morphological and chemical characterization. The surface area of functionalized biochar displayed a two-fold increase compared to the untreated biochar. Laccase was immobilized on functionalized biochar, and an immobilization yield of 66% was obtained. The immobilized enzyme demonstrated operational stability up to six cycles while retaining 40% of the initial activity. Laccase immobilization was further investigated by performing adsorption and kinetic studies, which revealed the highest immobilization concentration of 500 U g-1 at 25°C. The adsorption followed the Langmuir isotherm model at equilibrium, and the kinetic study confirmed pseudo-second-order kinetics. The equilibrium rate constant (K2) at 25°C and 4°C were 3.6×10-3g U-1 min-1 and 4×10-3g U-1 min-1 respectively for 100 U g-1 of enzyme loading. This immobilized system was applied for anthracene degradation in the aqueous batch mode, which resulted in complete degradation of 50mgL-1 anthracene within 24h of interaction exposure.

PRODUCTION OF LACCASE BY FUNGI ISOLATED FROM SOIL VIA SUBMERGED FERMENTATION USING CORN COB AS SUBSTRATE

One of the limitations of large scale application of laccase (EC 1.10.3.2) is the inability to produce them in large quantity at an affordable cost. This study was carried out to screen indigenous fungi for their ability to produce laccase using the locally available substrate. Five soil samples were collected and diluted serially, 0.1 mL of the 10-5 and 10-6 dilutions were inoculated onto Potato dextrose agar (PDA) plates. The fungal isolates were identified based on their macroscopic and microscopic characteristics. The isolates were then screened for laccase production by growing them on PDA containing tannic acid as an indicator compound. The laccase producing isolates were further screened for their ability to utilize corn cob as a substrate for laccase production. Ten fungal species were isolated and identified as Trichoderma viridae (3), Trichoderma harzianum (3), Aspergillus niger (2), Fusarium sp. (1) and Penicillium sp. (1). Only two of the isolates namely T. viridae and T. harzianum were found to be laccase producers. Both laccase producing fungal species were able to utilize corn cob as substrate for laccase production. T. viridae had higher enzyme activity (2.228 U/mL) than T. harzianum (2.1583 U/mL) after 9 days of incubation. Laccase producing fungi were isolated in this study and they were able to use corn cob as substrate for laccase production.

Insight into Depolymerization Mechanism of Bacterial Laccase for Lignin

Laccase is essential for the biodepolymerization of lignin, but the challenge is that the reaction mechanism has not been fully elucidated. The laccase (Lacc) inactivated mutant of Bacillus ligniniphilus L1 had a sharp decline in its ability to degrade lignin, which proved its indispensable role in lignin depolymerization. The purified Lacc from recombinant Escherichia coli BL21 and its mediator system (LMS) displayed significant lignin degradation capacities as well as remarkable thermotolerance and solvent resistance. The chemical oxygen demand removal rates of LMS for alkaline and milled wood lignin have reached 67.0% and 80.9%, respectively. Comprehensive analyses, including Fourier-transform infrared spectrometry, gas chromatography–mass spectrometry, 2D-HSQC-NMR, and time-of-flight secondary ion mass spectrometry, unveiled that Lacc- and LMS-oxidized lignin include at least 10 or more catalytic reactions. Lacc can effectively degrade G-lignin even without a mediator, and the removal rate of G-lignin is higher than that of S-lignin. In addition, the supplementation of the mediator increased the removal rate of H-lignin by Lacc and the cleavage of interunit linkages such as β-O-4, β-5, β-β, 4-O-5, and 5-5. Moreover, we found that Lacc cannot polymerize some aromatic monomers into dimers or polymers, which is different from fungal and plant laccases. It is by far the most detailed study describing the reaction mechanism of lignin oxidation by bacterial laccase. These results provide new insights into the catalytic mechanism of bacterial laccase and lay the foundation for the application of laccase in lignin valorization.

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

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

An insight into the production strategies and applications of the ligninolytic enzyme laccase from bacteria and fungi

The ligninolytic multi-copper enzyme laccase, is widely produced by bacteria and fungi. It has been extensively used in industries for its versatile applications due to its catalytic properties of oxidation of both phenolic and non-phenolic lignin based compounds. For this purpose, the enzyme needs to be produced in large quantities. Though there have been multiple reviews focused on the biochemical aspects, molecular properties and wide applications of laccases, none of them have elaborately discussed on their production, which is the prerequisite for its use. Through this review a novel attempt has been made to discuss the production strategies of laccases from mesophilic and extremophilic microorganisms using different media ingredients, agro-industrial wastes and culture conditions. These enzymes from various sources possess unique properties, yet sharing some common structural and catalytic features which can be improved using enzyme immobilization techniques. The review also outlines various applications of laccases in different industries including food, textile, paper and pulp. The enzyme has also been used for green synthesis of nanoparticles. Further, its use in biosensors and polymer syntheses have also been elaborated.

Treasure from dross: Application of agroindustrial wastes-derived thermo-halotolerant laccases in the simultaneous bioscouring of denim fabric and decolorization of dye bath effluents

In this research paper, thermo-halotolerant laccases from microbial fermentation of agroindustrial residues by Achromobacter xylosoxidans HWN16 (Hb9c) and Citrobacter freundii LLJ16 (Ie1c) were evaluated for their ability to decolorize synthetic dyes and also wash indigo from denim fabric. Denim bioscouring and dye decolorization were monitored at different incubation times and uniquely high dye concentrations. The application of the free laccases in denim bleaching had no empirical effect, when applied unaided. However, their homogenization with an oxidized mediator (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) yielded experiential effects after 6 h of incubation under orbital conditions, and improved discoloration of fabrics was observed with the aid of a dissection microscope. Furthermore, the dye baths were decolorized by the homogenates, after scouring of the fabrics, thereby producing colorless effluents. The results presented revealed the possibility of replacing conventional caustic and environmentally toxic approaches with cleaner sustainable methods for washing of denims, and dye decolorization.

Biomedical and Pharmaceutical-Related Applications of Laccases.

The oxidation of a vast range of phenolic and non-phenolic substrates has been catalyzed by laccases. Given a wide range of substrates, laccases can be applied in different biotechnological applications. The present review was conducted to provide a broad context in pharmaceutical- and biomedical- related applications of laccases for academic and industrial researchers. First, an overview of biological roles of laccases was presented. Furthermore, laccase-mediated strategies for imparting antimicrobial and antioxidant properties to different surfaces were discussed. In this review, laccase-mediated mechanisms for endowing antimicrobial properties were divided into laccase-mediated bio-grafting of phenolic compounds on lignocellulosic fiber, chitosan and catheters, and laccase-catalyzed iodination. Accordingly, a special emphasis was placed on laccase-mediated functionalization for creating antimicrobials, particularly chitosan-based wound dressings. Additionally, oxidative bio-grafting and oxidative polymerization were described as the two main laccase-catalyzed reactions for imparting antioxidant properties. Recent laccase-related studies were also summarized regarding the synthesis of antibacterial and antiproliferative agents and the degradation of pharmaceuticals and personal care products.

Environmentally Friendly and Recyclable Natural-Mediator-Modified Magnetic Nanoparticles for Laccase-Catalyzed Decolorization

The high cost, potential toxicity, and possible enzyme inhibition ability of artificial mediators have limited the large-scale application of laccase (Lac)/mediator systems. Here, sinapic acid (SA), a natural mediator, was covalently attached to amino-functionalized magnetic nanoparticles (MNPs) via amide bond formation. The as-prepared SA@MNPs were characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy, cyclic voltammetry, and thermogravimetric analysis. The SA@MNPs were then applied to evaluate the activity of the immobilized mediator for Lac-catalyzed dye decolorization using indigo carmine (IC) as a model dye. When SA and SA@MNPs were used as Lac mediators, IC decolorization yields of ∼93% and 96%, respectively, were obtained after 60 min. Moreover, SA@MNPs exhibited an IC decolorization yield of ∼90% after being reused for 8 cycles. The Lac/SA@MNP system was shown to degrade IC by breaking down the chromophoric group. The easy recyclability, good reusability, nontoxicity, and relatively low cost of SA@MNPs make this immobilized natural mediator a promising tool for dye treatment.

Applications of Microbial Laccases: Patent Review of the Past Decade (2009–2019)

There is a high number of well characterized, commercially available laccases with different redox potentials and low substrate specificity, which in turn makes them attractive for a vast array of biotechnological applications. Laccases operate as batteries, storing electrons from individual substrate oxidation reactions to reduce molecular oxygen, releasing water as the only by-product. Due to society’s increasing environmental awareness and the global intensification of bio-based economies, the biotechnological industry is also expanding. Enzymes such as laccases are seen as a better alternative for use in the wood, paper, textile, and food industries, and they are being applied as biocatalysts, biosensors, and biofuel cells. Almost 140 years from the first description of laccase, industrial implementations of these enzymes still remain scarce in comparison to their potential, which is mostly due to high production costs and the limited control of the enzymatic reaction side product(s). This review summarizes the laccase applications in the last decade, focusing on the published patents during this period.

A novel method for improving laccase activity by immobilization onto copper ferrite nanoparticles for lignin degradation

In this paper, a novel method for simultaneous enhancement of catalytic activity and reusability of laccase was carried out to overcome the limitations on industrial application of laccase. The immobilization of laccase onto copper ferrite magnetic nanoparticles (CuMNPs) and ferrite magnetic nanoparticles (MNPs) were optimized at 50 mM glutaraldehyde concentration and 1:5 enzyme:nanoparticles (NPs) ratio for 9 h of cross-linking time, yielding a maximum activity recovery of 94.68 ± 0.92% and 89.78 ± 1.24%, respectively. The laccase immobilized NPs were characterized using physico-chemical methods such as SEM-EDAX, FTIR, XRD, TGA and VSM and the laccase immobilized CuMNPs showed 18% higher activity as compared to free enzyme. The prepared CuMNPs and MNPs showed superior thermal stability (50–70 °C) with t1/2 increased by 5.7 and 4.1 folds, respectively, as compared to free laccase. The laccase in immobilized forms exhibited higher kinetic potential and stable at wide temperature and pH range. In addition, laccase immobilized NPs retained more than 70% residual activity during reuse up to 6 cycles and storing for 20 days at 4 °C. The laccase immobilized CuMNPs showed higher delignification (43.28 ± 1.46%) on Ipomoea carnea than laccase immobilized MNPs.

Laccases as versatile enzymes: from industrial uses to novel applications

AbstractThe application of enzymes offers an enormous potential in the improvement of existing industrial procedures and in the establishment of new processes for obtaining high‐added value products. Enzymes provide cleaner and more efficient industrial processes and contribute to the sustainability concept. In this sense, laccases are very versatile biocatalysts currently used in food, textile and pulp and paper sectors among others. During the last years, scientific efforts have been diverted to the exploitation of such interesting enzymes in novel fields like lignocellulosic biorefineries, biosensors or enzymatic biofuel cells. This review provides a general vision of the use of laccase enzymes describing their main characteristics and mode of action. Furthermore, their current uses in industrial processes are summarized and the most novel potential application of laccases are revealed. The increasing interest on laccases is also demonstrated by the research efforts on enzyme engineering as it is detailed in this review. © 2019 Society of Chemical Industry

High-level expression of Bacillus amyloliquefaciens laccase and construction of its chimeric variant with improved stability by domain substitution.

Large-scale application of bacterial laccases is usually limited by their low production, and their recombinant expression in Escherichia coli is prone to form inactive aggregates in the cytoplasm. In this work, we optimized the expression conditions of Bacillus amyloliquefaciens laccase (LacA) in E. coli, and obtained high yield for the extracellular production of LacA. The final activity reached 20,255 U/L for LacA, which is among one of the highest activities for recombinant bacterial laccases. Moreover, a chimeric enzyme (Lac3A/S) was designed based on LacA by domain substitution with a stable laccase from B. subtilis. The hybrid laccase could also be secreted into the culture medium with high expression level, and had higher thermal and alkaline stabilities than those of LacA. It was fully active after 10-day incubation at pH 9.0, and retained 47% of its initial activity after incubation at 70°C for 5h. Homology analysis of protein structure indicated Lac3A/S had a more packed structure in the copper-binding sites than LacA, which might lead to an enhancement in stability under harsh conditions.

Short-term and Residual Effects of Laccase Application on Creeping Bentgrass Thatch Layer

Organic layer formation in the form of thatch is a major problem in managed turfgrass systems. Biweekly application of laccase enzyme has been well-documented to facilitate the degradation of thatch and reduce the accumulation rate of organic matter in ‘Crenshaw’ creeping bentgrass (Agrostis stolonifera L.). A field experiment involving creeping bentgrass was conducted to evaluate the residual effects on thatch accumulation after ceasing laccase applications. A significant reduction in thatch layer thickness was observed at 6, 12, and 18 months after treatment initiation when laccase was applied at different rates and frequencies. Residual effects of laccase application were observed for thatch layer thickness, but no additional accumulation of thatch was observed 6 months after treatment cessation. At 18 months after treatment initiation, a significant increase in the thatch layer was observed where treatments had been ceased for 12 months, but no thatch accumulation was observed for laccase treatment for a second 6-month period during the second year. This information is critical to turf practitioners when developing laccase application protocols. Limiting laccase applications for a period of 6 months during 1 year was shown to be effective for thatch control.

Enhancing laccase production by white-rot fungus trametes hirsuta SSM-3 in co-culture with yeast sporidiobolus pararoseus SSM-8

Due to wide application of laccase, many researchers have shown great interest in over production of white-rot fungi laccase by co-culture. In this study, a white-rot fungus Trametes hirsuta SSM-3, and a yeast Sporidiobolus pararoseus SSM-8 were isolated and identified from Mulberry fruit. The capacity of S. pararoseus to enhance laccase production was remarkable in T. hirsuta, yielding 31777 ± 742 U/L, about 9.9 times higher than the result from the monoculture. The stimulatory factor in the S. pararoseus cells might be temperature-sensitive. The laccase production was enhanced by oil-extract of S. pararoseus and β-carotene induction. The amylase activity was decreased rapidly when strain S. pararoseus SSM-8 was inoculated. The glucose deprivation was occurred both in the mono-culture and co-culture process, and S. pararoseus propagated slowly in co-culture all the time. Native-PAGE revealed an increase of laccase-1(lac-1) level and a laccase-3 (lac-3) in the co-culture. Therefore, it was concluded that competition for resources between the co-cultured microbes leaded to amylase decreasing and the enhanced production of laccase. This conclusion was helpful for the development of laccase fermentation industry because it provided an effective, simple and economic method to improve the yield of laccase.

Synthesizing Polyaniline With Laccase/O2 as Catalyst.

The polymerization of aniline to polyaniline (PANI) can be achieved chemically, electrochemically or enzymatically. In all cases, the products obtained are mixtures of molecules which are constituted by aniline units. Depending on the synthesis conditions there are variations (i) in the way the aniline molecules are connected, (ii) in the average number of aniline units per molecule, (iii) in the oxidation state, and (iv) in the degree of protonation. For many possible applications, the synthesis of electroconductive PANI with para-N-C-coupled aniline units in their half-oxidized and protonated state is of interest. This is the emeraldine salt form of PANI, abbreviated as PANI-ES. The enzymatic synthesis of PANI-ES is an environmentally friendly alternative to conventional chemical or electrochemical methods. Although many studies have been devoted to the in vitro synthesis of PANI-ES by using heme peroxidases with added hydrogen peroxide (H2O2) as the oxidant, the application of laccases is of particular interest since the oxidant for these multicopper enzymes is molecular oxygen (O2) from air, which is beneficial from environmental and economic points of view. In vivo, laccases participate in the synthesis and degradation of lignin. Various attempts of synthesizing PANI-ES with laccase/O2 in slightly acidic aqueous media from aniline or the linear aniline dimer PADPA (p-aminodiphenylamine) are summarized. Advances in the understanding of the positive effects of soft dynamic templates, as chemical structure guiding additives (anionic polyelectrolytes, micelles, or vesicles), for obtaining PANI-ES-rich products are highlighted. Conceptually, some of these template effects appear to be related to the effect “dirigent proteins” exert in the biosynthesis of lignin. In both cases intermediate radicals are formed enzymatically which then must react in a controlled way in follow-up reactions for obtaining the desired products. These follow-up reactions are controlled to some extent by the templates or specific proteins.

Simultaneous purification and immobilization of laccase on magnetic zeolitic imidazolate frameworks: Recyclable biocatalysts with enhanced stability for dye decolorization

Laccases are versatile oxidases showing strong ability to eliminate hazardous pollutants from wastewater. Magnetic zeolitic imidazolate framework-8 (Fe3O4@ZIF-8) nanoparticles were synthesized by a facile method and used for single-step purification and immobilization of recombinant laccase from Escherichia coli culture supernatant. An activity recovery of 75.7% was achieved for the immobilized biocatalyst. The storage and thermal stability of laccase was remarkably enhanced after immobilization. The immobilized laccase exhibited optimum temperature at 80°C. It retained about 46.0% of initial activity after 6 h incubation at 80°C, while the free laccase was totally inactivated by this treatment. In addition, the immobilized enzyme maintained 87.1% activity after 10 days of storage at 30°C. Finally, excellent reusability was also observed for the immobilized laccase, which was able to completely decolorize indigo carmine after 5 consecutive cycles. Therefore, the immobilization of laccase on Fe3O4@ZIF-8 nanoparticles offers a facile and cost-effective way for environmental application of bacterial laccase.

Immobilized fungal laccase as "green catalyst" for the decolourization process – State of the art

Abstract The omnipresence of enzyme decides on their essential role in the infinite number of catalytic transformations taking place in nature as well as on their undisputed physiological significance in a series of metabolic changes. They comprise an extremely valuable tool as “green catalysts” in many technological processes in various industries. Removal of dyes from industrial wastewater constitutes a significant problem for highly developed countries resulting from the continuous economic progress and the increased production of coloured impurities. This generates the need to search for new, effective and ecological technologies for removal of toxic dyes. The enzymes, immobilised on a carrier to increase and facilitate their recovery from the post-process mixture, more and more often find application in various industries. The use of enzymes, including laccase, is a promising technology for the removal of coloured impurities from wastewater. This article reviews the application of laccase as the “green catalyst” in the decolourization process. Research results published so far indicate that the immobilization positively affects the entire protein structure of the enzyme and strengthening, which translates into an increased resistance of the enzyme to unfavourable process conditions. Selected immobilization methods and the carriers most commonly used for this purpose are also presented.

From agro-waste to tool: biotechnological characterization and application of Ganoderma lucidum E47 laccase in dye decolorization.

The culture of fungal species from agro-waste allows for the sustainable preparation of valuable biotechnological products and contributes to establish the Circular Economy concept. The Ganoderma lucidum species is well known as producer of laccases (EC 1.10.3.2), which serves as a tool to oxidize chemicals. When producing G. lucidum E47 basidiomes with edible purposes out of rice crop residues, its laccase remains as by-product. In this work, we report the biotechnological characterization and application of the laccase recovered from spent cultures of the G. lucidum E47 strain. We detected at least one polypeptide (ca. 59kDa) which displays attractive activity and stability values when used in the range of 18-45°C in mildly acidic environment (pH 4.8-5.8). These parameters can be enhanced in the presence of organic cosolvents such as butyl acetate and methyl iso-butyl ketone, but the opposite effect is observed with solvents of lower log P. The best activity-stability performance is reached when the biocatalyst is used in pH 4.8 buffer with 5% (v/v) butyl acetate at 37°C. The laccase was capable of decolorizing xanthene, azo and triarylmethane dyes, exhibiting excellent selectivity on bromocresol green and bromocresol purple. Furthermore, the biocatalyst displayed an attractive activity when assessed for the decolorization of bromocresol green in a proof-of-concept effluent biotreatment.

Laccase from Aspergillus nidulans TTF6 Showing Pb Activation for Smaller Substrates and Dyes Remediation in All Climates

Prompted by multiple applications of laccases, the fungi yielding enzyme with wider geographical applicability and other novel features was explored from Jaisalmer desert (India). One of the isolates TTF6 yielded laccase exhibiting novel many-fold amplification in activity in the presence of lead (Pb) and tolerance to Na. The peroxide-free enzyme showed apparent molecular mass 40 kDa, activity from 10 to 80 °C, temperature optima at 60 °C and pH optima at 5.0. It was highly thermostable losing only 22 and 44% of its maximal activity in 24 and 48 h, respectively, with a half-life of 60 h at 60 °C. It was found resistant to studied metals including Cu and Zn and showed tolerance to many organic solvents. The enzyme was produced best in Olga medium and induced by ethanol and copper sulfate but not by guaiacol, though guaiacol could be utilized as substrate. The partially purified enzyme decolorized selected synthetic dyes at 20–40 °C, the decolorization rate, however, was not affected by Pb. The fungus was identified on the basis of morphological and ITS sequence characteristics as A. nidulans. This is the first report of a Pb-activated laccase and the sixth phenol oxidase from A. nidulans reported so far.

Mode of Action, Properties, Production, and Application of Laccase: A Review.

Background and Source: Laccase belongs to the blue multi-copper oxidases, which are widely distributed in fungi and higher plants. It is present in Ascomycetes, Deuteromycetes, and Basidiomycetes and found abundantly in white-rot fungi. Applications: Laccase enzymes because of their potential have acquired more importance and application in the area of textile, pulp and paper, and food industry. Recently, it is being used in developing biosensors for detection and removal of toxic pollutants, designing of biofuel cells and medical diagnostics tool. Laccase is also being used as a bioremediation agent as they have been found potent enough in cleaning up herbicides pesticides and certain explosives in soil. Because of having the ability to oxidize phenolic, non-phenolic lignin-related compounds and highly fractious environmental pollutants, laccases have drawn the attention of researchers in the last few decades. Commercially, laccases have been used to determine the difference between codeine and morphine, produce ethanol and are also being employed in de-lignify woody tissues. We have revised patents related to applicability of laccases. We have revised all the patents related to its wide applicability. Conclusion: For fulfillment of these wide applications, one of the major concerns is to develop a system for efficient production of these enzymes at a broad scale. Research in the field of laccases has been accelerated because of its wide diversity, utility, and enzymology. This paper deals with recent trends in implementation of the laccases in all practical possibilities with the help of optimizing various parameters and techniques which are responsible for mass production of the enzyme in industries.