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LC-MS analysis of β-estradiol degradation by recombinant laccases. A) Chromatogram of β-estradiol by NSP4-Lac1326 and NSP4-tvel5 Laccase from cell lysates and cell-free medium extraction, with pET-11a as control B) LC-MS analysis of β-estradiol degradation by different laccases. Protein expression from cell lysed and cell free medium (Ni: Nickle Pulldown Assay) was from E. coli induced under the condition of OD 600 = 0.4, IPTG = 0.4 mM and 25 • C.
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Endocrine Disrupting Chemicals (EDCs) are a group of molecules that can influence hormonal balance, causing disturbance of the reproductive system and other health problems. Despite the efforts to eliminate EDC in the environment, all current approaches are inefficient and expensive. In previous research, studies revealed that laccase-producing mic...
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Effluent discharged from textile industries, particularly the denim dyeing units containing indigo carmine/crystal violet/highly dimethyl-aminated crystal violet, is a huge threat to flora and fauna in that area. These effluents are highly toxic, mutagenic, and linked to prevalence of cancer in these areas. Since no single enzyme is capable of brea...
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... Recent research has revealed that laccase heterologous expression and secretion may be accomplished using modified E. coli. When expressing extracellular enzymes, however, E. coli easily forms inclusion bodies and preserves part of the produced enzyme in these inclusions, heterologous laccase expression in E. coli offers a possible strategy for PE biodegradation 87,88 . Microalgae as phototrophic microorganisms are so advantageous for use as microbial cell factories because of their high growth rates, scalability, easy and low-cost cultivation, and potentially suitable for genetic manipulation. ...
Microplastic contamination is an increasing environmental concern worldwide, with microplastic particles found in various ecosystems, including soil. Because microplastics are non-biodegradable, they pose severe removal and disposal issues. The employment of biological organisms to break down or degrade toxins, known as bioremediation, has emerged as a remedy for microplastic pollution. Microbial enzymes play an important role in bioremediation by facilitating the breakdown and degradation of microplastics, as many plastic-degrading enzymes have been discovered and purified over the last decades. Thanks to the high-throughput newly investigated "omics" techniques and also efforts on enzyme engineering, this exploration has now been speeded up, but despite these advances, plastic and microplastic pollution issues are still among the unsolved environmental concerns. This chapter emphasizes the importance of microbial enzymes in the bioremediation of microplastics by exploring their diversity, methods of action, and prospective uses in tackling microplastic pollution and how far are we from using this approach to solving this problem. This understanding will pave the way for the creation of efficient and environmentally friendly ways to tackle the growing menace of microplastics.
... Laccase-based biosensors have been developed to detect p-coumaric acid [11] and epinephrine [12]. Furthermore, toxic substances can be degraded by laccases [13], and various investigators have used laccases for construction of biofuel cells [14,15]. Nevertheless, new laccases with properties suitable for specific applications are needed. ...
... Application of laccase on large scale are limited by the cost of production and efficiency of the enzyme [33]. Production of large amounts of laccase can be achieved at lower costs with the use of recombinant protein expression technology [13,34]. Enzyme activity and stability can be improved by protein engineering [35,36]. ...
Laccases are enzymes that catalyze oxidation of a wide range of substrates. The enzyme shows promising applications in various fields such as toxic substance degradation, organic synthesis, and biosensors. In this study, we purified and characterized laccase from Ganoderma sp. Three different Ganoderma sp. found in the northeast Thailand were studied for laccase activity. When cultured in media containing rice bran and rice husk, Ganoderma sp. 03 showed high laccase activity. The laccase produced by this strain was purified using Phenyl Sepharose fast flow (FF) chromatography and quaternary amine (Q) Sepharose chromatography. Estimated molecular weight of the purified laccase was 39.81 kDa as determined by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified laccase had an optimal potential hydrogen (pH) of 3.5, showed high stability at pH 3.0-5.0 using 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate, and exhibited an optimal temperature of 40.0-55.0C. The best temperature stability was observed at 30C. Effect of different substances on laccase activity showed that p-coumaric acid and dithiothreitol (DTT) strongly inhibited laccase activity. Especially, DTT could inhibit 100% of laccase activity, even at a concentration as low as 0.1 mM. All the tested metal ions, except ZnSO4, decreased laccase activity. This information may be useful for further studies and applications of this enzyme.
... As shown in Table 3, the microorganisms that are capable of secreting polyolefin biodegrading enzymes are not model organisms, which means that they are difficult to genetically engineer. Two model organisms, E. coli and Y. lipolytica, have been applied to the expression and secretion of polyolefin-biodegrading enzymes to date [12,78,79]. The heterologous expression of polyolefin-degrading enzymes in model organisms can efficiently increase the expression level of polyolefin-degrading enzymes through genetic engineering in model organisms. ...
... However, a potential issue with laccase expression in E. coli is that it is easy for E. coli to form inclusion bodies when expressing extracellular enzymes. Mo et al. [79] expressed three laccases from three different organisms, namely Lac1326 from marine sediment samples, fungal tvel5 laccase from Trametes versicolor and bacterial BPUL laccases from Bacillus pumilus for the purpose of degrading β-estradiol. The result of Western blot analysis indicates that laccase was detected both in vivo and in vitro in E. coli, which means that some laccase stayed in inclusion bodies instead of being secreted in vitro. ...
... Moreover, the effect of polyolefin degradation by artificial microbial consortia can be more complete than that of pure culture. Finally, the construction of artificial microbial consortia is a time-saving and efficient method relative to other metabolic engineering techniques [79]. Therefore, constructing artificial microbial consortia is regarded as a promising means of polyolefin degradation. ...
Polyolefins, including polyethylene (PE), polypropylene (PP) and polystyrene (PS), are widely used plastics in our daily life. The excessive use of plastics and improper handling methods cause considerable pollution in the environment, as well as waste of energy. The biodegradation of polyolefins seems to be an environmentally friendly and low-energy consumption method for plastics degradation. Many strains that could degrade polyolefins have been isolated from the environment. Some enzymes have also been identified with the function of polyolefin degradation. With the development of synthetic biology and metabolic engineering strategies, engineered strains could be used to degrade plastics. This review summarizes the current advances in polyolefin degradation, including isolated and engineered strains, enzymes and related pathways. Furthermore, a novel strategy for polyolefin degradation by artificial microbial consortia is proposed, which would be helpful for the efficient degradation of polyolefin.
... Conversely, Steel and co-workers aimed their efforts to tackle the degradation of perfluorinated compounds (PFC) via bio-remediation, as enabled by the identification and engineering of PFC-degrading enzymes [16]. Similarly, Mo et al. worked on bio-engineering microorganisms to express and secrete recombinant laccases with confirmed degrading activity [17], thus helping in the degradation of Endocrine Disrupting chemicals from the environment. ...
Polyethylene (PE), a widely used recalcitrant synthetic polymer, is a major global pollutant. PE has very low biodegradability due to its rigid C-C backbone and high hydrophobicity. Although microorganisms have been suggested to possess PE-degrading enzymes, our understanding of the PE biodegradation process and its overall applicability is still lacking. In the present study, we used an artificial bacterial consortium for PE biodegradation to compensate for the enzyme availability and metabolic capabilities of individual bacterial strains. Consortium members were selected based on available literature and preliminary screening for PE-degrading enzymes, including laccases, lipases, esterases, and alkane hydroxylases. PE pellets were incubated with the consortium for 200 days. A next-generation sequencing analysis of the consortium community of the culture broth and on the PE pellet identified Rhodococcus as the dominant bacteria. Among the Rhodococcus strains in the consortium, Rhodococcus erythropolis was predominant. Scanning electron microscopy (SEM) revealed multilayered biofilms with bacteria embedded on the PE surface. SEM micrographs of PE pellets after biofilm removal showed bacterial pitting and surface deterioration. Multicellular biofilm structures and surface biodeterioration were observed in an incubation of PE pellets with R. erythropolis alone. The present study demonstrated that PE may be biodegraded by an artificially constructed bacterial consortium, in which R. erythropolis has emerged as an important player. The results showing the robust colonization of hydrophobic PE by R. erythropolis and that it naturally possesses and extracellularly expresses several target enzymes suggest its potential as a host for further improved PE biodeterioration by genetic engineering technology using a well-studied host-vector system.
Plastic pollution has emerged to be the biggest global concern in the modern day. The production of large volumes and different varieties of plastics has led to their huge accumulation and contamination in environmental matrices. Along with plastics, there are additional problems posed by microplastics and related chemicals like phthalates that leach into surrounding resources, thereby polluting them and causing health hazards to living organisms. Thus, their degradation becomes an immediate concern. Although some strategies like photodegradation have been suggested to tackle plastic menace, microbial degradation has been found to be the most effective one. Several microbes have been isolated, characterized, and screened for plastic degradation. Various screening procedures provide more potent microorganisms that break down a variety of plastics by producing several specific and non-specific enzymes. These microbes constitute bacteria, fungi, and other classes like diatoms and microalgae. In-depth studies linked to potent microbes like Ideonella sakaiensis and establishment of databases like PlasticDB make the process more convenient. Further, advancements in molecular techniques like metagenomics and genetic engineering have opened doors for prospecting newer tactics against this global issue. This chapter deals with the microbial cornucopia that are capable of metabolizing various types of plastics and their allied aspects.
Mushroom laccases play a crucial role in lignin depolymerization, one of the most critical challenges in lignin utilization. Importantly, laccases can utilize a wide range of substrates, such as toxicants and antibiotics. This study isolated a novel laccase, named HeLac4c, from endophytic white-rot fungi Hericium erinaceus mushrooms. The cDNAs for this enzyme were 1569 bp in length and encoded a protein of 523 amino acids, including a 20 amino-acid signal peptide. Active extracellular production of glycosylated laccases from Saccharomyces cerevisiae was successfully achieved by selecting an optimal translational fusion partner. We observed that 5 and 10 mM Ca2+, Zn2+, and K+ increased laccase activity, whereas 5 mM Fe2+ and Al3+ inhibited laccase activity. The laccase activity was inhibited by the addition of low concentrations of sodium azide and L-cysteine. The optimal pH for the 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was 4.4. Guaiacylglycerol-β-guaiacyl ether, a lignin model compound, was polymerized by the HeLac4c enzyme. These results indicated that HeLac4c is a novel oxidase biocatalyst for the bioconversion of lignin into value-added products for environmental biotechnological applications. .
Multicopper oxidases (MCOs) share a common catalytic mechanism of activation by oxygen and cupredoxin-like folding, along with some common structural determinants. Laccases constitute the largest group of MCOs, with fungal laccases having the greatest biotechnological applicability due to their superior ability to oxidize a wide range of aromatic compounds and lignin, which is enhanced in the presence of redox mediators. The adaptation of these versatile enzymes to specific application processes can be achieved through the directed evolution of the recombinant enzymes. On the other hand, their substrate versatility and the low sequence homology among laccases make their exact classification difficult. Many of the ever-increasing amounts of MCO entries from fungal genomes are automatically (and often wrongly) annotated as laccases. In a recent comparative genomic study of 52 basidiomycete fungi, MCO classification was revised based on their phylogeny. The enzymes clustered according to common structural motifs and theoretical activities, revealing three novel groups of laccase-like enzymes. This review provides an overview of the structure, catalytic activity, and oxidative mechanism of fungal laccases and how their biotechnological potential as biocatalysts in industry can be greatly enhanced by protein engineering. Finally, recent information on newly identified MCOs with laccase-like activity is included.
The laccase-mediator system (LMS) is an important solution to eliminate veterinary residues. In this study, a novel laccase from Lysinibacillus fusiformis (Lyfu-Lac) showed promising removal efficiency towards sulfonamides and tetracyclines residues in the presence of syringic acid (SA) and 2'-Azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS). The optimum temperature of Lyfu-Lac against ABTS was 80 °C, which is much higher than that of most laccases. At 40 °C, more than 85% of sulfamethazine (SMZ), sulfamethoxazole (SMX), and sulfadiazine (SDZ) were removed by the Lyfu-Lac-SA system after 12 h. Meanwhile, tetracycline (TC), and oxytetracycline (OTC) were significantly reduced to 0% and 16.9% by the Lyfu-Lac-ABTS system after 6 h. Based on the analysis of transformation products (TPs), a two-step reaction was reasonably proposed in the removal of sulfonamides: (i) the oxidation of mediators by Lyfu-Lac to radicals, (ii) the adductive reaction between radicals and sulfonamides. Moreover, the removal mechanism of tetracyclines was proposed, including the cleavage of the N-N bond after the adductive reaction between ABTS radicals and tetracyclines.
