Immobilization of laccase on magnetic PEGDA-CS inverse opal hydrogel for enhancement of bisphenol A degradation in aqueous solution.

Endocrine disruptors such as bisphenol A (BPA) adversely affect the environment and human health. Laccases are used for the efficient biodegradation of various persistent organic pollutants in an environmentally safe manner. However, the direct application of free laccases is generally hindered by short enzyme lifetimes, non-reusability, and the high cost of a single use. In this study, laccases were immobilized on a novel magnetic three-dimensional poly(ethylene glycol) diacrylate (PEGDA)-chitosan (CS) inverse opal hydrogel (LAC@MPEGDA@CS@IOH). The immobilized laccase showed significant improvement in the BPA degradation performance and superior storage stability compared with the free laccase. 91.1% of 100 mg/L BPA was removed by the LAC@MPEGDA@CS@IOH in 3 hr, whereas only 50.6% of BPA was removed by the same amount of the free laccase. Compared with the laccase, the outstanding BPA degradation efficiency of the LAC@MPEGDA@CS@IOH was maintained over a wider range of pH values and temperatures. Moreover, its relative activity of was maintained at 70.4% after 10 cycles, and the system performed well in actual water matrices. This efficient method for preparing immobilized laccases is simple and green, and it can be used to further develop ecofriendly biocatalysts to remove organic pollutants from wastewater.

Cu,Ce-containing phosphotungstates as laccase-like nanozyme for colorimetric detection of Cr(VI) and Fe(â ¢).

Herein, a nanocomposite of Cu,Ce-containing phosphotungstates (Cu,Ce-PTs) with outstanding laccase-like activity was fabricated via a one-pot microwave-assisted hydrothermal method. Notably, it was discovered that both Fe3+ and Cr6+ could significantly enhance the electron transfer rates of Ce3+ and Ce4+, along with generous Cu2+ with high catalytic activity, thereby promoting the laccase-like activity of Cu,Ce-PTs. The proposed system can be used for the detection of Fe3+ and Cr6+ in a range of 0.667-333.33 µg/mL and 0.033-33.33 µg/mL with a low detection limit of 0.135 µg/mL and 0.0288 µg/mL, respectively. The proposed assay exhibits excellent reusability and selectivity and can be used in traditional Chinese medicine samples analysis.

Designing Artificial Laccase Catalysts by Introducing Substrate Oxidation Metals into Oxygen-Reducing Metal-Organic Frameworks: Cu-doped ZIF-67.

Laccase, a multi-copper oxidase, is limited by its optimal temperature range and isolation costs. To overcome these challenges, we synthesized copper-doped zeolitic imidazolate framework-67 (Cu-ZIF-67) with 16 mol% Cu as an artificial laccase catalyst. The introduced Cu site acts as the phenol oxidation site, and Co-based ZIF-67 is the four-electron oxygen reduction site. Laccase also employs this division of oxidation and reduction sites. Cu-doped ZIF-67 demonstrated significant catalytic activity, superior to natural laccase, especially at elevated temperatures, and maintained stability across multiple reaction cycles. These findings suggest that Cu-doped ZIF-67 is a robust, reusable alternative for industrial applications requiring high thermal stability and efficient catalysis.

Identification of Grape Laccase Genes and Their Potential Role in Secondary Metabolite Synthesis.

Laccase, a copper-containing oxidoreductase, has close links with secondary metabolite biosynthesis in plants. Its activity can affect the synthesis and accumulation of secondary metabolites, thereby influencing plant growth, development, and stress resistance. This study aims to identify the grape laccases (VviLAC) gene family members in grape (Vitis vinifera L.) and explore the transcriptional regulatory network in berry development. Here, 115 VviLACs were identified and divided into seven (Type I-VII) classes. These were distributed on 17 chromosomes and out of 47 VviLACs on chromosome 18, 34 (72.34%) were involved in tandem duplication events. VviLAC1, VviLAC2, VviLAC3, and VviLAC62 were highly expressed before fruit color development, while VviLAC4, VviLAC12, VviLAC16, VviLAC18, VviLAC20, VviLAC53, VviLAC60 and VviLAC105 were highly expressed after fruit color transformation. Notably, VviLAC105 showed a significant positive correlation with important metabolites including resveratrol, resveratrol dimer, and peonidin-3-glucoside. Analysis of the transcriptional regulatory network predicted that the 12 different transcription factors target VviLACs genes. Specifically, WRKY and ERF were identified as potential transcriptional regulatory factors for VviLAC105, while Dof and MYB were identified as potential transcriptional regulatory factors for VviLAC51. This study identifies and provides basic information on the grape LAC gene family members and, in combination with transcriptome and metabolome data, predicts the upstream transcriptional regulatory network of VviLACs.

Detoxification of aflatoxin B1 by a Bacillus subtilis spore coat protein through formation of the main metabolites AFQ1 and epi-AFQ1.

A variety of important agricultural crops host fungi from the Aspergillus genus can produce cancerogenic secondary metabolites such as aflatoxins. Consequently, novel strategies for detoxification and their removal from food and feed chains are required. Here, detoxification of Aflatoxin B1 (AFB1) by the Bacillus subtilis multi-copper oxidase CotA (BsCotA) was investigated. This laccase was recombinantly produced in E. coli while codon optimization led to duplication of the amount of active protein obtained. CuCl2 was added to the cultivation medium leading to a 25-fold increase of V max corresponding to improved incorporation of Cu2+ into the enzyme protein which is essential for the catalytic reaction. To avoid potential cytotoxicity of Cu2+, cultivation was performed at microaerobic conditions indeed leading to 100x more functional protein when compared to standard aerobic conditions. This was indicated by an increase of V max from 0.30 ± 0.02 to 33.56 ± 2.02 U/mg. Degradation kinetics of AFB1 using HPLC with fluorescence detection (HPLC-FLD) analysis indicated a theoretical substrate saturation above solubility in water. At a relatively high concentration of 500 µg/L, AFB1 was decomposed at 10.75 µg/Lh (0.17 nmol*min-1*mg-1) at a dosage of 0.2 µM BsCotA. AFQ1 and epi-AFQ1 were identified as the initial oxidation products according to mass spectrometry (i.e., HPLC-MS, HPLC-QTOF). None of these molecules were substrates for laccase but both decomposed in buffer. However, decomposition does not seem to be due to hydration of the vinyl ether in the terminal furan ring. Genotoxicity of the formed AFB1 was assessed in several dilutions based on the de-repression of the bacterial SOS response to DNA damage indicating about 80-times reduction in toxicity when compared to AFQ1. The results of this study indicate that BsCotA has high potential for the biological detoxification of aflatoxin B1.

Characterization and Degradation of Triphenylmethane Dyes and Their Leuco-Derivatives by Heterologously Expressed Laccase From Coprinus cinerea.

Laccase is a copper-containing polyphenol oxidase that can oxidize phenolic and non-phenolic organic substrates. In the past decades, laccases had received considerable attention because of the ability to degrade various organic substances. Based on the codon preference of the Pichia pastoris expression system, this study optimized the gene structure of the laccase gene Lcc1 from Coprius cinerea through synthetic biology methods. A new gene Lcc1I was synthesized and heterologously expressed in P. pastoris. After 3 days of cultivation in a shake flask at 30°C, the transformants produced at a yield of 890 mg L-1protein. The highest production level of the recombinant laccase was 2760 U L-1. The molecular mass of the recombinant laccase was estimated at 60 kDa. The enzyme showed highest activity at pH 3.4 and 45°C. It possessed better stability at higher pH and lower temperature condition. Using 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as the substrate, the Km and Vmax values were 0.136 mM and 9778 µM min-1 mg-1, respectively. The recombinant laccase could directly oxidize some triphenylmethane dyes like leuco-crystal violet (LCV) and leuco-malachite green (LMG). With the help of ABTS mediator, it could oxidize and degrade 77.7% crystal violet (CV) and 79.2% malachite green (MG) within 1 h. Our results indicate that optimization of the laccase gene achieves good expression results in the host system. The dye degradation model constructed in this study may also be applied to the degradation of other organic pollutants and toxic substances, providing new solutions for environmental remediation against the increasingly severe environmental pollution.

Bacillus CotA laccase improved the intestinal health, amino acid metabolism and hepatic metabolic capacity of Pekin ducks fed naturally contaminated AFB1 diet.

BACKGROUND: Aflatoxin B1 (AFB1) is a prevalent contaminant in agricultural products, presenting significant risks to animal health. CotA laccase from Bacillus licheniformis has shown significant efficacy in degrading mycotoxins in vitro test. The efficacy of Bacillus CotA laccase in animals, however, remains to be confirmed. A 2 × 2 factorial design was used to investigate the effects of Bacillus CotA laccase level (0 or 1 U/kg), AFB1 challenge (challenged or unchallenged) and their interactions on ducks. The purpose of this study was to evaluate the efficacy of Bacillus CotA laccase in alleviating AFB1 toxicosis of ducks. RESULTS: Bacillus CotA laccase alleviated AFB1-induced declines in growth performance of ducks accompanied by improved average daily gain (ADG) and lower feed/gain ratio (F/G). Bacillus CotA laccase ameliorated AFB1-induced gut barrier dysfunctions and inflammation testified by increasing the jejunal villi height/crypt depth ratio (VH/CD) and the mRNA expression of tight junction protein 1 (TJP1) and zonula occluden-1 (ZO-1) as well as decreasing the expression of inflammation-related genes in the jejunum of ducks. Amino acid metabolome showed that Bacillus CotA laccase ameliorated AFB1-induced amino acid metabolism disorders evidenced by increasing the level of glutamic acid in serum and upregulating the expression of amino acid transport related genes in jejunum of ducks. Bacillus CotA laccase ameliorated AFB1-induced liver injury testified by suppressing oxidative stress, inhibiting apoptosis, and downregulating the expression of hepatic metabolic enzyme related genes of ducks. Moreover, Bacillus CotA laccase degraded AFB1 in digestive tract of ducks, resulting in the reduced absorption level of AFB1 across intestinal epithelium testified by the decreased level of AFB1-DNA adduct in the liver, and the reduced content of AFB1 residues in liver and feces of ducks. CONCLUSIONS: Bacillus CotA laccase effectively improved the growth performance, intestinal health, amino acid metabolism and hepatic aflatoxin metabolism of ducks fed AFB1 diets, highlighting its potential as an efficient and safe feed enzyme for AFB1 degradation in animal production.

The effects of applying cellulase and laccase on fermentation quality and microbial community in mixed silage containing corn stover and wet brewer's grains.

Objective: The purpose of this experiment was to explore the effect of adding cellulase and laccase on fermentation quality and microbial community in mixed silage of corn stover and wet brewer's grains. Try to a new approach for the proper preservation and utilization of the agro-industrial by-products (corn stover and wet brewer's grains). Methods: The experiment was divided into four groups: CK (control), C (cellulase, 120 U/g fresh matter [FM]), L (laccase, 50 U/g FM), CL (cellulase 120 U/g FW and laccase 50 U/g FM), and the chemical composition, fermentation quality, microbial population and microbial community in mixed silage of corn stover and wet brewer's grains after 30 day's fermentation were determined. Results: Compared to control, the addition of cellulase significantly increased crude protein (CP), water-soluble carbohydrate (WSC), lactic acid bacteria (LAB) counts, while significantly decreased the neutral detergent fiber (NDF), acid detergent fiber (ADF) content (P < 0.05). Adding laccase significantly decreased the acid detergent lignin (ADL) content (P < 0.05). Combined application of cellulase and laccase significantly increased the CP, WSC content and LAB counts, while significantly decreased pH value, NDF, ADF and ADL content (P < 0.05), thereby improving fermentation quality. In addition, the application of cellulase and laccase increased the abundance of Firmicutes and LAB genera, and decreased microbial diversity level of the sample. Conclusion: The combined application of cellulase and laccase further improved fermentation quality and microbial community in mixed silage of corn stover and wet brewer's grains.

Competitive antagonistic action of laccase between Trichoderma species and the newly identified wood pathogenic Ganoderma camelum.

Ganoderma, a well-known genus in the Ganodermataceae family, has caused the extinction of several tree species due to its pathogenicity. This study explored the pathogenic effect of a newly identified Ganoderma species on trees and its competitive efficiency against Trichoderma species. Ganoderma camelum sp. nov. is characterized by small sessile basidiomata and a velvety, soft, camel-brown pileus. Phylogenetic analysis and ITS rDNA sequences indicated that the species were Trichoderma and Ganoderma camelum. Both fungal species competed antagonistically by secreting laccase. The laccase activity of G. camelum, with a value of 8.3 ± 4.0 U/mL, demonstrated the highest competitive activity against Trichoderma species. The laccase produced by T. atroviride (2.62 U/mL) was most effective in countering the pathogenic action of the novel G. camelum. The molecular weights of laccase were determined using SDS-PAGE (62.0 kDa for G. camelum and 57.0 kDa for T. atroviride). Due to the white rot induced by this Ganoderma species in the host tree, G. camelum showed the highest percentage inhibition of radial growth (76.3%) compared to T. atroviride (28.7%). This study aimed to evaluate the competitive antagonistic activity of Ganoderma and Trichoderma on malt extract agar media in the context of white rot disease in the host tree. This study concluded that the laccase from G. camelum caused weight loss in rubber wood blocks through laccase action, indicating tissue injury in the host species. Therefore, it was also concluded that G. camelum was more effective in pathogenic action of the host and resisted the biological action of T. atroviride. In principal components analysis (PCA), all the species associated with laccase exhibited a very strong influence on the variability of the system. The PIRG rate (percentage inhibition of radial growth) was strongly and positively correlated with laccase activity.

Machine learning-assisted laccase-like activity nanozyme for intelligently onsite real-time and dynamic analysis of pyrethroid pesticides.

The intelligently efficient, reliable, economical and portable onsite assay toward pyrethroid pesticides (PPs) residues is critical for food safety analysis and environmental pollution traceability. Here, a fluorescent nanozyme Cu-ATP@ [Ru(bpy)3]2+ with laccase-like activity was designed to develop a versatile machine learning-assisted colorimetric and fluorescence dual-modal assay for efficient onsite intelligent decision recognition and quantification of PPs residues. In the presence of alkaline phosphatase (ALP), the laccase-like activity of Cu-ATP@ [Ru(bpy)3]2+ was enhanced to oxidize colorless o-phenylenediamine (OPD) into dark-yellow 2,3-diaminophenazine (DAP) via electron transfer, appearing a new yellow fluorescence at 550 nm. Meanwhile, the red fluorescence of Cu-ATP@ [Ru(bpy)3]2+ at 600 nm was quenched due to the internal filter effect (IFE) of DAP towards Cu-ATP@ [Ru(bpy)3]2+. However, the selective inhibition of PPs toward ALP activity enabled to observe a dual-modal response of PPs concentration-dependent decrease in colorimetric signal and enhancement in the fluorescence intensity ratio of F600 nm/F550 nm. On this basis, both the colorimetric and fluorescence images were captured and processed with a home-made WeChat applet-installed smartphone to extract the corresponding image color information, thus achieving machine learning-assisted onsite real-time and dynamic intelligent decision recognition and quantification of PPs residues in real samples, which shows a promising potential in safeguarding food safety and environmental health.

Integration of Fe-MOF-laccase-magnetic biochar: From Rational Designing of a biocatalyst to aflatoxin B1 decontamination of peanut oil.

Enzymatic degradation of aflatoxins in food commodities has gained significant attention. However, enzyme denaturation in organic media discourages their direct use in oils to remove aflatoxins. For that, enzymes are immobilized or encapsulated for improved stability and reusability under unfavorable conditions. We sandwiched the laccase between a carrier and an outer protective layer. We used spent-mushroom-substrate (SMS) derived porous magnetic biochar as the laccase carrier and coated it with an iron MOF to create a biocomposite, Fe-BTC@Lac@FB. The immobilized laccase demonstrated enhanced chemical, thermal, and storage stability and proficient reusability. Fe-BTC@Lac@FB exhibited 11 times enhanced aflatoxin B1 (AFB1) degradation compared to free laccase (FL). In addition, thermally inactivated Fe-BTC@Lac@FB could adsorb 11.2 mg/g of AFB1 from peanut oil. Multi-aflatoxin removal also proved promising, while Fe-BTC@Lac@FB could retain > 85 % of AFB1 removal efficacy after five reusability cycles. Fe-BTC@Lac@FB treatment did not affect peanut oil quality as indicated by different oil quality parameters and proved essentially non-cytotoxic. All these aspects helped recognize Fe-BTC@Lac@FB as an excellent laccase-carrying material with exceptionally higher stability, activity, and reusability.

High-Valence Mn MOF Inspired by Laccase Mediators Enables Versatile Nature-Mimicking Catalysis.

In nature, active Mn3+ -ligand complexes produced by laccase catalyzed oxidation can act as the low-molecular mass, diffusible redox mediators to oxidize the phenolic substrates overcoming the limitations of natural enzymes. Learning from the metal-ligand coordination of natural functional units, high-valence Mn metal-organic framework (Mn MOF) is constructed to simulate the catalysis in natural mediator system. Benefiting from the characteristics of nanoscale size, rich metal coordination unsaturated sites, and mixed valence state dominated by Mn(III), Nano Mn(III)-TP exhibits superior laccase-mimicking activity, whose Vmax (maximal reaction rate) is much higher than that of natural laccase. Referring to natural systems, relevant free radical experiments prove that the material induces the production of active oxygen species with the assistance of carboxylic acid, and active oxygen species further oxidize phenolic substrates. Based on its robust performances, the primary oxidative degradation of an emerging pollutant triclosan (TCS) is creatively applied, an important antiasthmatic medicine terbutaline sulfate (TBT) detection, and the synthesis of non-toxic and black near-natural dyes for dyeing. By simulating the essential mediators of natural enzymatic catalysis, an Mn MOF-based material that demonstrates multiple novel applications is successfully developed, which introduces a new reliable strategy for achieving versatile nature-mimicking catalysis.

Expression of laccase and ascorbate oxidase affects lignin composition in Arabidopsis thaliana stems.

Lignin is a phenolic polymer that is a major source of biomass. Oxidative enzymes, such as laccase and peroxidase, are required for lignin polymerisation. Laccase is a member of the multicopper oxidase family and has a high amino acid sequence similarity with ascorbate oxidase. However, the process of functional differentiation between the two enzymes remains poorly understood. In this study, the common ancestry sequence of laccase and ascorbate oxidase (AncMCO) was predicted via phylogenetic reconstruction, and its in vivo effect on lignin biosynthesis in Arabidopsis thaliana was assessed. The estimated AncMCO sequence conserved key residues that coordinate with copper ions, implying that the electron transfer system is likely to be conserved in AncMCO. However, multiple insertions/deletions corresponding to protein surface structures have been found between laccase, ascorbate oxidase, and AncMCO. The overexpression of canonical laccase (AtLAC4) and ascorbate oxidase (AtAAO1) in A. thaliana resulted in notable increases of syringyl/guaiacyl lignin unit ratio in stems, whereas, in contrast, the overexpression of AncMCO did not show any detectable change in lignin deposition. Transcriptomic analysis revealed that the AtAAO1-overexpressing line exhibited significant changes in the expression of a wide range of cell wall biosynthesis genes. These results highlight the importance of the molecular evolution of multicopper oxidase, which drives lignin biosynthesis during plant evolution.

Laccase: A Green Biocatalyst Offers Immense Potential for Food Industrial and Biotechnological Applications.

Laccase, a multipurpose biocatalyst, is widely distributed across all kingdoms of life and plays a key role in essential biological processes such as lignin synthesis, degradation, and pigment formation. These functions are critical for fungal growth, plant-pathogen interactions, and maintenance of soil health. Due to its broad substrate specificity, multifunctional nature, and environmentally friendly characteristics, laccase is widely employed as a catalyst in various green chemistry initiatives. With its ability to oxidize a diverse range of phenolic and nonphenolic compounds, laccase has also been found to be useful as a food additive and for assessing food quality parameters. Ongoing advancements in research and technology are continually expanding the recognition of laccase's potential to address global environmental, health, and energy challenges. This review aims to provide critical insights into the applications of laccases in the biotechnology and food industry.

Optimization, purification and characterization of laccase from a new endophytic Trichoderma harzianum AUMC14897 isolated from Opuntia ficus-indica and its applications in dye decolorization and wastewater treatment.

BACKGROUND: Hazardous synthetic dye wastes have become a growing threat to the environment and public health. Fungal enzymes are eco-friendly, compatible and cost-effective approach for diversity of applications. Therefore, this study aimed to screen, optimize fermentation conditions, and characterize laccase from fungal endophyte with elucidating its ability to decolorize several wastewater dyes. RESULTS: A new fungal endophyte capable of laccase-producing was firstly isolated from cladodes of Opuntia ficus-indica and identified as T. harzianum AUMC14897 using ITS-rRNA sequencing analysis. Furthermore, the response surface methodology (RSM) was utilized to optimize several fermentation parameters that increase laccase production. The isolated laccase was purified to 13.79-fold. GFC, SDS-PAGE revealed laccase molecular weight at 72 kDa and zymogram analysis elucidated a single band without any isozymes. The peak activity of the pure laccase was detected at 50 °C, pH 4.5, with thermal stability up to 50 °C and half life span for 4 h even after 24 h retained 30% of its activity. The Km and Vmax values were 0.1 mM, 22.22 µmol/min and activation energy (Ea) equal to 5.71 kcal/mol. Furthermore, the purified laccase effectively decolorized various synthetic and real wastewater dyes. CONCLUSION: Subsequently, the new endophytic strain produces high laccase activity that possesses a unique characteristic, it could be an appealing candidate for both environmental and industrial applications.

Enhanced biocatalytic laccase production using agricultural waste in solid-state fermentation by Aspergillus oryzae for p-chlorophenol degradation.

Agricultural residues are one of the most cost-effective and readily accessible carbon resources for producing commercially significant enzymes. Several enzymes have been used in different industries like pharmaceuticals, foods, textiles, and dyes that can be generated by various species of microbes found in waste from agriculture. The current research investigated laccase production by Aspergillus oryzae utilizing agricultural wastes. Physical and chemical properties, including pH, temperature, sucrose, yeast extract, and copper sulfate levels, were optimized. The utilization of the response surface methodology along with the centralized composite design method, which assesses multiple media parameters and utilizes a two-level experimental approach, aids in determining the variable and its significance in increasing production quality. The centralized composite design enhancement showed that the optimal conditions for highest laccase activity (623.16 U/mL) were pH 7.0, temperature 25 °C, corn cobs as substrate, sucrose (2.0 %), yeast extract (1.0 %), and copper sulfate (0.1 mM) level. The laccase enzyme was optimized using various pH, temperature, metal ions, and inhibitors combinations. The extracted laccase enzyme maximum activity was attained at pH 6.0 and 40 °C. The inclusion of divalent ions can enhance laccase activity, while the use of various inhibitors decreases laccase activity. Under various pH circumstances, the Aspergillus oryzae laccase enzyme can successfully degrade p-chlorophenol. The present study describes statistically validated optimal methodologies for enhancing laccase synthesis, leading to a laccase production technique that is simultaneously highly efficient and economically profitable, with possible use of p-chlorophenol degradation.

Fabrication, characterization, and application of laccase-immobilized membranes for acetamiprid and diuron degradation.

Water and wastewater pollution by acetamiprid and diuron is considered a serious environmental problem. In this study, chitosan (CHS), a naturally occurring bioadsorbent considered ecologically harmless to remove these micropollutants, was developed as a possible carrier to immobilize laccase (Lac) from Trametes trogii. Polyethylene glycol methyl ether (PEGME) was chosen for blending CHS, so a hybrid biocatalyst-based Lac/CHS-PEGME membrane was prepared. The prepared CHS-PEGME and Lac/CHS-PEGME membranes were characterized by Fourier-transformed-infrared (FTIR) spectroscopy, scanning-electron-microscopy (SEM), and X-ray-diffraction (XRD). Pesticide degradation tests with Lac/CHS-PEGME were performed at different contact times and initial concentrations. Acetamiprid degradation was most effective (84 %) at the 12th hour, at an initial concentration of 0.1 mg/L, while diuron degradation was most effective (65 %) at an initial concentration of 6 mg/L and a contact time of 16th hour. Under optimum conditions, the reusability of Lac/CHS-PEGME was found to be 8 cycles for acetamiprid and 5 cycles for diuron. From these results, it is understood that acetamiprid is degraded more quickly and effectively than diuron. Adsorption process data were well fitted to the Langmuir isotherm model and the pseudo-first-order kinetic model. These findings showed that using Lac/CHS-PEGME was a practical and environmentally friendly method for acetamiprid and diuron degradation.

Laccase immobilized on amino modified magnetic biochar as a recyclable biocatalyst for efficient degradation of trichloroethylene.

Bioremediation of trichloroethylene (TCE) contaminated groundwater has recently attracted considerable attention. In this study, laccase was immobilized on amino modified magnetic pine biochar (MBC-NH2) by adsorption-crosslinking-covalent binding method, and its application in the degradation of TCE was evaluated. MBC-NH2 was obtained from pine sawdust by calcination, magnetic modification and amino modification. MBC-NH2 had high specific surface area (71.3 m2/g), rich surface functional groups and good magnetism. Under the conditions of 25 °C, pH = 4, glutaraldehyde (GA) concentration of 7 %, crosslinking time of 1 h, laccase concentration of 0.75 mg/mL, and immobilization time of 7 h, the loading capacity of laccase on MBC-NH2 carrier was as high as 782 mg/g. Compared with free laccase, immobilized laccase showed higher pH stability and thermal stability, and its activity remained 48.5 % after being reused for 10 times, and 80.8 % after being stored at 4 °C for 30 days. The immobilized laccase exhibited a good degradation effect on TCE. At 25 °C, pH = 4, immobilized laccase concentration of 0.35 g/L, and initial TCE concentration of 10 mg/L, the degradation efficiency of TCE by immobilized laccase was as high as 92.1 % within 48 h. In addition, the degradation products of TCE were analyzed, and the results showed that immobilized laccase could degrade TCE into non-toxic products through epoxidation, hydroxylation, and dechlorination. The immobilized laccase biocatalyst prepared in this study can achieve efficient degradation of TCE, which provides a feasible solution for chlorinated pollution of water resources. These research results are of great significance for the synthesis of biocatalysts for the efficient degradation of chlorinated hydrocarbons.

Synergistic bioremediation of petroleum-contaminated soil using immobilized consortium of Rhodococcus rhodochrous and Bacillus subtilis laccase.

Petroleum-contaminated soil represents a significant environmental and public health challenge on a global scale. Microbial bioremediation has shown potential, yet the role of enzymes in enhancing petroleum degradation remains underexplored. In this study, the synergistic effects of Rhodococcus rhodochrous (R.rh) and Bacillus subtilis-derived laccase (BsLac) was investigated in the remediation of petroleum-contaminated soil. Immobilized R.rh (PSIMRH) and BsLac (ADIMLac) exhibited higher petroleum degradation rates than their free state, achieving 78.3% and 56.3% degradation in liquid systems, respectively. The combined treatment of PSIMRH and ADIMLac demonstrated a synergistic effect on petroleum degradation, achieving 43.6% with a maximum degradation constant of 0.0335 d-1, representing a 202.7% improvement over untreated soil. PSIMRH enhanced petroleum degradation through microbial metabolism, while ADIMLac accelerated the initial breakdown of complex hydrocarbons into simpler, more bioavailable ones via enzymatic oxidation, providing growth substrates for microbes and significantly improving petroleum degradation rates. The microbial analysis revealed an increase abundance of known petroleum-degrading bacterial genera, including Rhodococcus, Lysobacter, Micromonospora, and Streptomyces. However, the presence of BsLac appeared to reduce the competitive advantage of Rhodococcus, promoting the proliferation of indigenous strains like Lysobacter and Streptomyces. These results suggest that enzyme-microbe synergy can enhance the bioremediation process by altering microbial community dynamics and accelerating petroleum degradation. This study attempts to remediate petroleum-contaminated pollution with the combined use of strains and enzymes, providing a new approach for the remediation of other pollution problems.

Sustainable pretreatment method of lignocellulosic depolymerization for enhanced ruminant productivity using laccase protein immobilized agarose beads.

Laccase, the selectively lignin degrader, vital to the initiation of lignocellulosic deconstruction was immobilized onto activated agarose beads to increase its reuse potential. Laccase cross-linked beads (~ 3.42 mm) recorded a specific activity of 23 Umg- 1, retaining about 80.43% enzyme activity after 45 days of storage. The immobilization yield and efficiency were 89% and 97% respectively. The equilibrium data fitted the Freundlich equation (R2 = 0.9987) demonstrating multilayer adsorption and the presence of Cu, Fe, and S in the elemental analysis of immobilized beads established effective binding between activated agarose beads and the laccase protein. Characterization studies of the immobilized laccase-treated crop residues revealed significant differences in the lignin polymer after each treatment cycle. An increase in digestibility of 26.21% and 7.62% was observed in paddy and finger millet-treated straws respectively, over the controls corroborating efficient lignin depolymerization. The propitious performance of laccase beads authenticated in the batch enzymatic reactor to treat crop residues paves headway as a sustainable green technology in the deconstruction of crop residues for use as ruminant feed, augmenting productivity.