Laccase Enzyme Research Articles

Biotransformation of Pollutants by Pycnoporus spp. in Submerged and Solid-State Fermentation: Mechanisms, Achievements, and Perspectives

Substantial amounts of organo-pollutants, often persistent and toxic, are generated globally each year, posing a threat to soil, water, groundwater, and air. The pollutants encompass a wide range of substances from various sources, which include solid as well as liquid ones, such as landfill leachates and wastewaters. The compounds include paper and pulp mill byproducts, pharmaceuticals, diverse types of plastics, hydrocarbons, pigments, and dyes, as well as pesticides and insecticides. Fungal bioremediation stands out as a promising technology that uses the metabolic potential of fungi to eliminate or mitigate the impact of pollutants. Notably, species of the genus Pycnoporus exhibit significant capabilities for degrading a broad spectrum of toxic molecules. This degradation is facilitated by released ligninolytic enzymes, especially laccase, and cellular enzymes pertaining to the cytochrome P450 monooxygenase system. The laccase, which is overproduced by the genus Pycnoporus, is quite remarkable for its high redox potential. The objective of this review is to highlight the proficiency of the Pycnoporus genus in the degradation of pollutants in submerged and solid-state fermentation. Recent studies conducted over the past decade consistently highlight the Pycnoporus genus as a robust contender in the realm of white biotechnology.

Nanoparticles based on polyferylic and polygentisic acids as new carriers of anticancer drugs

Lignin polymers and their derivatives are actively used in various fields of biomedicine to create biocompatible materials, as medications, and to form nanoparticles. However, natural polymeric compounds derived from plant materials or monomers are defined as a mixture of compounds having a high heterogeneity in chemical structure, which greatly complicates the determination of their biological activity. This paper describes a new method of controlled synthesis using the enzyme laccase, which can be applied to obtain polymers with a specific structure. Based on enzymatically synthesized lignin-like polymers from ferulic and gentisiс phenolic monomers, nanoparticles with stable properties under physiological conditions were formed. The nanoparticles can differ in morphology from globular to fibrillar structures, depending on monomers used in the enzymatic reaction and the method of their formation. Nanoparticles obtained from lignin-like polymers of ferulic and gentisic acids can be loaded with low molecular weight hydrophobic compounds, including the anticancer drug doxorubicin. It has been shown that polyferulic nanoparticles are actively penetrate in tumor cells growing both in a monolayer culture and as part of spheroids, and, compared with a free compound, doxorubicin in the composition of nanoparticles has a greater cytotoxic effect on breast cancer cells. These data indicate the possibility of effective use of these carriers as passive targeted drug delivery in the treatment of tumors.

Periodate oxidation of nanofibrillated cellulose films for active packaging applications

An alternative packaging material based on cellulose that possesses excellent barrier properties and is potentially useful for active packaging has been developed. Cellulose nanofibril was efficiently and selectively oxidized with sodium periodate generating reactive aldehyde groups. These groups formed hemiacetal and hemialdal bonds during film formation and, consequently, highly transparent, elastic and strong films were created even under moisture saturation conditions. The periodate oxidation treatment additionally decreased the polarity of the films and considerably enhanced their water barrier properties. Thus, the water contact angle of films treated for 3 and 6 h was 97° and 102°, their water drop test value was higher than in untreated film (viz., 138 and 141 min with 3 and 6 h of treatment) and their water vapour transmission rate was substantially better (3.31 and 0.78 g m−2 day−1 with 3 and 6 h, respectively). The presence of aldehyde groups facilitated immobilization of the enzyme laccase, which efficiently captures oxygen and prevents food decay as a result. Laccase-containing films oxidized 80 % of Methylene Blue colorant and retained their enzymatic activity after storage for 1 month and 12 reuse cycles, opening the door to the possible creation of a reusable packaging to replace the single-use packaging.

Degradation of textile polyazodyes by Polyporus ciliatus laccase produced in peanut shell solid medium: purification and characterization

This study describes decolorization of reactive sulfonated azoic dye Direct Blue 71, one of the recalcitrant and highly used industrial dyes, by Polyporus ciliatus derived laccase. Agro-industrial wastes are increasingly used as substrates for laccase production from white rot fungi through solid-state fermentation (SSF). Peanut shell (PS) is an attractive substrate for laccase production, and SSF is an adequate strategy for that purpose. It has been employed as a novel support-substrate for laccase production by Polyporus ciliatus under solid-state fermentation. The highest laccase yield of 1260 U/ml was attained using PS after 13 days of incubation at 30°C. The purified laccase (55 kDa) showed maximum activity at pH 3 and 60°C. The extracellular laccase enzyme was purified 67.6-fold to a specific activity of 1466 U/mg by a Mono-Q anion-exchange column. The purified enzyme Km value was found to be 24 µM, while the Vmax value was observed to be 0.96 µM s-1 with ABTS as a substrate. It had high thermotolerance and pH stability and was resistant to several metal ions including copper, cadmium, and iron. P. ciliatus pure laccase showed high potential for the decolorization and detoxification of the reactive sulfonated azoic dye Direct Blue 71, which suggested that this enzyme could be used for textile effluent treatment.

Potential of White Rot Fungi from Berbak-Sembilang National Park, Indonesia for Decolorization and Detoxification Commercial Direct Dyes

Commercial direct dyes are frequently employed in the dyeing of Jumputan, a traditional craft originating from South Sumatra, Indonesia. The introduction of synthetic dyes into the environment can have a detrimental impact on ecosystem stability, necessitating the implementation of remedial measures. There has been a growing interest in the utilization of White Rot Fungi (WRF) as a viable biological agent for the purpose of decolorizing and detoxifying synthetic colors. However, it is imperative to investigate the isolated WRF from Berbak-Sembilang National Park (TNBS) due to the varying capacities of decolorization and detoxification exhibited by each WRF. The study involved 5 WRF from TNBS screened in solid media, and grown in a liquid medium containing commercial direct dyes (direct turquoise, direct orange and direct yellow). WRF grown on liquid medium contains a single dye, mixture dyes and temperature characterization. It measured decolorization, biomass and enzyme activities. Toxicity is measured by the BSLT method. The findings indicated that 3 specific fungi, namely Phellinus noxius (BRB 11), Lentinus sajor-caju (BRB 12) and Leotrametes menziesii (BRB 73), exhibited a significant capacity to remove the color from both individual and mixture of direct dyes through the production of laccase and MnP enzymes. The optimal temperature for decolorization of the mixture of direct dyes was 35 °C for BRB 11 (61.4 %) and BRB 73 (60.7 %), whereas BRB 12 (47.4 %) exhibited optimal temperature at 30 °C. The toxicity assay conducted using A. salina showed a progressive rise in the LC50 value, from 15.37 mgL−1 in the control group to a range of 21.63 - 35.84 mgL−1 in the treated group, indicating a detoxification process. However, the most toxic isolate was BRB 12. This study proposes the potential of 2 isolates, Phellinus noxius (BRB 11) and Leotrametes menziesii BRB 73, from TNBS for degradation of single and mixture dye wastewater in the environment. HIGHLIGHTS The discovery of 3 White Rot Fungi from TNBS (Indonesia) has the ability to remove color and reduce the level of toxicity of commercial direct dyes, both individual and mixture of commercial direct dyes. Uncovering one of White Rot Fungi has not been widely published for decolorization and detoxification commercial direct dyes, namely Leotrametes menziesii (BRB 73). The optimal temperature was 35 °C for decolorization of commercial direct dyes by Phellinus noxius (BRB 11) and Leotrametes menziesii (BRB 73) GRAPHICAL ABSTRACT

Evaluating mechanism of banana pseudo-stem retting using seawater: A cost-effective surface pre-treatment approach

Retting has been employed to extract natural fibers from agricultural wastes as a biological and cost-effective approach for centuries. With its global abundance, banana pseudo-stem is a promising agro-waste for lignocellulosic fiber extraction. In this study, fibers were extracted from the pseudo-stems after being pre-treated under four conditions using seawater at room temperature for up to 35 d Bacterial isolation from the fresh seawater sample and screening for ligninolytic ability were conducted. Bacterial load as well as laccase and manganese peroxidase enzyme activity profile assay during the retting duration were analyzed. Fourier transform infrared (FT-IR) and X-day diffraction (XRD) analyses were also examined for both pre-treated and untreated extracted fibers. The results shows that six out of the eight bacterial isolates had the ability to degrade lignin. The treatments (Raw stem + Raw seawater) and (Autoclaved stem + Raw seawater) recorded the highest viable bacterial load of 9.24 × 102 and 4.46 × 102 CFU, respectively, on the 14th day of the retting process. Additionally, the highest laccase and manganese peroxidase enzymes activity was recorded for (Raw stem + Raw seawater) and (Autoclaved stem + Raw seawater) treatments in the second to the third week. The FT-IR spectra of the pre-treated fibers revealed relative reductions in peaks attributed to polysaccharides and other amorphous substances for all retting conditions. The XRD diffractogram revealed that the crystallinity index (CI) of pre-treated fibers increased in all seawater retting treatment conditions. However, the CI for fibers pre-treated under enzymatic conditions were enhanced even after five weeks. Sequence analysis for selected bacterial isolates showed homology to sequences of Bacillus velezensis, Shewanella sp. L8–5, and Citrobacter amalonaticus and Bacillus subtilis j8 strain. From these findings, it was suggested that physical, biological, and chemical actions were collectively involved in the seawater retting process of banana pseudo-stems.

Wastewater Nutrient Recovery via Fungal and Nitrifying Bacteria Treatment

In efforts to reduce the consumption of fossil fuels and promote recycling biowaste, there is an interest in the production of post-hydrothermal liquefaction wastewater (HTL-AP) from the hydrothermal liquefaction (HTL) process that converts wet biomass into biocrude oil. This study explores ways of transforming potentially toxic HTL-AP into a fertilizer source for hydroponic cropping systems. This study specifically investigates the integration of the white-rot fungus Trametes versicolor with nitrifying bacteria (Nitrosomonas and Nitrobacter) to convert the organic nitrogen compounds into inorganic nitrogen while also producing the enzyme laccase, which has been shown to remove toxic compounds. This study aims to increase the concentration of nitrate-N to valorize wastewater as a suitable fertilizer by measuring several parameters, including laccase activity, pH, nitrate-N, and ammonia/ammonium-N concentrations, and analyzes interactions to optimize the conversion process. The data support the claim that the simultaneous inoculation of T. versicolor and nitrifying bacteria significantly increases nitrate-N concentrations in HTL-AP, as it increased by 17 times, or an increase of 32.69 mg/L. In addition, HTL-AP treated with T. versicolor and nitrifying bacteria reduced the treatment time by 120 h, highlighting a reduction in personnel time and energy consumption. Therefore, this research accentuates sustainability through fungal and bacterial treatments to develop eco-friendly hydroponic fertilizers. Future research should explore the potential of utilizing the combination of T. versicolor and nitrifying bacteria for the treatment of other industrial wastewaters.

Enzymatic Bioregeneration of Activated Carbon by Laccase

Activated carbon is widely used in combination with biological treatment systems for the treatment of organic compounds, which are refractory or toxic in conventional biological treatment systems. In these systems, compounds adsorbed on activated carbon may desorb within time due to a concentration gradient between adsorbent and the bulk liquid caused by the biodegradation of substrates in the liquid phase by microorganisms. The desorbed compounds are further biodegraded by microorganisms. This mechanism is called bioregeneration of activated carbon. Previous studies showed that bioregeneration percentages could be higher than the concentration gradient-driven desorbability. This was attributed to exoenzymatic bioregeneration occurring due to the activity of extracellular enzymes secreted by microorganisms in these systems. These extracellular enzymes can diffuse into the activated carbon pores where they can react with the previously adsorbed compounds resulting in their desorption from the carbon surface and degradation. However, the effect of extracellular enzymes on bioregeneration was not conclusively proven in any of the literature studies on bioregeneration because extracellular enzymes were not directly used for the purpose of bioregeneration. In this study, enzymatic bioregeneration of activated carbon was investigated by directly using an extracellular enzyme, laccase, which is known from the literature to catalyze the oxidation reactions of phenolic substances and is commercially available in its pure form. Therefore phenol, 2-nitrophenol, and bisphenol-A were used as the target compounds. For this purpose, batch adsorption, abiotic desorption, enzymatic degradation and enzymatic bioregeneration experiments were performed using two different activated carbon types; thermally and chemically activated ones. The results showed that there was a significant difference between the total enzymatic bioregeneration efficiencies and abiotic desorption efficiencies for each phenolic compound depending on the activated carbon type. Thereby, exoenzymatic bioregeneration has been quantitatively shown for the first time in the literature.

Enzymatic coloration of polyamide fabrics and its comparison with conventional dyeing

AbstractAmong the textile wet processes, the colouring process is the process step that causes the most environmental pollution. New approaches that support sustainable production instead of environmentally harmful processes are extensively researched in the literature. When considering substitution of chemical processes with environmentally friendly methods, one of the issues that comes to mind is enzymatic processes. It is noteworthy that in recent years, interesting and promising studies on the use of laccase enzymes in textile dyeing have been published in the literature. However, studies on enzymatic colouring are still very limited and sufficient success has not been achieved especially on polyamide. The aim of this study is to develop an environmentally friendly enzymatic dyeing method, which can be an alternative to dyeing polyamide knitted fabrics with 1:2 metal complex dyes according to the exhaustion method. For this purpose, various phenol and amine compounds, and their mixtures were used as precursor, and the colours that can be obtained by enzymatic colouring have been determined. In addition, optimisation of enzymatic colouring conditions (enzyme concentration, temperature and time) was carried out for the precursors that gave the best results in terms of colour and fastness. Furthermore, enzymatic dyeing was compared with conventional 1:2 metal complex dyeing in terms of technical and economic aspects. The reaction pathways in enzymatic coloration with laccase enzyme by using various precursors were also explained by Fourier‐transform infrared (FTIR) analysis.

Microbial degradation of low-density polyethylene, polyethylene terephthalate, and polystyrene by novel isolates from plastic-polluted environment.

Biodegradation is an eco-friendly measure to address plastic pollution. This study screened four bacterial isolates that were capable of degrading recalcitrant polymers, i.e., low-density polyethylene, polyethylene terephthalate, and polystyrene. The unique bacterial isolates were obtained from plastic polluted environment. Dermacoccus sp. MR5 (accession no. OP592184) and Corynebacterium sp. MR10 (accession no. OP536169) from Malaysian mangroves and Bacillus sp. BS5 (accession no. OP536168) and Priestia sp. TL1 (accession no. OP536170) from a sanitary landfill. The four isolates showed a gradual increase in the microbial count and the production of laccase and esterase enzymes after 4weeks of incubation with the polymers (independent experiment set). Bacillus sp. BS5 produced the highest laccase 15.35 ± 0.19 U/mL and showed the highest weight loss i.e., 4.84 ± 0.6% for PS. Fourier transform infrared spectroscopy analysis confirmed the formation of carbonyl and hydroxyl groups as a result of oxidation reactions by enzymes. Liquid chromatography-mass spectrometry analysis showed the oxidation of the polymers to small molecules (alcohol, ethers, and acids) assimilated by the microbes during the degradation. Field emission scanning electron microscopy showed bacterial colonization, biofilm formation, and surface erosion on the polymer surface. The result provided significant insight into enzyme activities and the potential of isolates to target more than one type of polymer for degradation.

Кислородный биокатод на основе laccase Pleurotus ostreatus HK-35 для биотопливного элемента

The main reasons that make mankind look for new sources of energy are decreasing dependence on fossil fuels and reducing pollution. Wastewater treatment with the help of microbial fuel cells is an area where these two goals can be combined. Microbial fuel cells, in which microorganisms catalyze the oxidation of organic substances, represent a new and promising alternative for electricity generation. The creation of an efficient cathode in such systems is the important problem. It is evident that in these applications an oxygen (air) electrode is a promising cathode. In this study, the oxygen biocathode based on the laccase enzyme Pleurotus ostreatus HK-35 was developed and its electrochemical properties were studied depending on the immobilization method of the enzyme on the surface of a carbon-graphite electrode and the type of the electrolyte. It was experimentally established that laccase injection using a sol-gel matrix was the effective method for immobilizing laccase on the surface of a carbon-graphite electrode. It was shown that the more efficient operation of the laccase-based biocathode was observed in a phosphate-citrate (pH 4.0) buffer solution, i.e., in an acidic environment.

Bacillus licheniformis O12'den Yeni Bir Termostabil Lakkazın Tek Adım Afinite Kromatografisi Kullanılarak Saflaştırılması, Karakterizasyonu ve Renk Giderme Potansiyeli

Laccases are copper-containing enzymes that can oxidize a wide variety of substrates. Thanks to this feature of laccase, some dyes that cause environmental pollution can be decolorized. Some bacteria, such as Bacillus licheniformis, naturally produce the enzyme laccase. A new affinity column was tested in this study. For this purpose, the extracellular laccase sepharose 4B-L-tyrosine-ρ-aminobenzoic acid produced by bacteria grown in suitable media was isolated by affinity chromatography method. Its purity was checked by SDS-PAGE method. The decolorization effect of some dyestable in textile wastewater of laccase isolated from B. lichenisformis O12 by affinity column was investigated. No mediator was used in this procedure. .As a result, laccase was purified 4.82-fold purification with a yield of 38.3% respectively, The molecular weight of the purified enzyme was determined as ~70 kDa by the SDS-PAGE method. The enzyme showed optimum activity at pH 4.0 and temperature 92°C. The enzyme was found to retain 100% activity even after 12 hours of incubation at 60°C and 92°C. The kinetic parameters were determined with laccase substrates such as ABTS, 2,6-DMP, and guaiacol. The purified laccase was decolorized with varied efficiencies such as 35% of Reactive black, 31% of Acid black 1, 28% of Methylene blue, and 15% of Acid red 27 without the use of any redox mediators. These properties of B. licheniformis O12 laccase enzyme make it a potential candidate enzyme for use in various biotechnological and industrial applications.

A smartphone-based colorimetric assay using Cu-tannic acid nanosheets (Cu-TA NShs) as a laccase-mimicking nanozyme for visual detection of quercetin in vegetables.

Theinteraction of Cu-tannic acid nanosheets (Cu-TA NShs) as nanozyme in a surfactant solution of CTAB under relatively acidic conditions is shown toexhibit a catalytic effect on quercetin (Qur). This catalytic property of Cu-TA NShs, which mimics laccase enzyme with many advantages, has been applied todeveloping a selective colorimetric sensor for the determination of trace amounts of Qur in vegetable samples. This strategy presents a desirable linear relationship between the absorbance signal intensity and the concentrations of Qur from 0.350 to 32.09 µM with a detection limit (LOD) of 0.064 µM (S/N = 3). The feasibility of the proposed portable colorimetric sensor for in situ analysis of the real samples has been validated with the high-performance liquid chromatography (HPLC) method as reference method, and two-tailed test (t test) statistical analysis certifies good agreement between the results. This enzyme-free and sensitive naked-eye sensor with the smartphone-based color map is promising to provide technical support for the rapid and visual detection of Qur in vegetables.

Single-bath-single-stage enzymatic treatment of denim

Denim has been a popular choice of apparel worldwide, prized for its versatility and long-lasting qualities. Being a core part of fashion trends for ages, a variety of techniques are being introduced in the denim industry to create a diverse and aged look. This research investigates the application of a combination of amylase, cellulase, and laccase at different temperatures on denim to achieve desired aged or faded appearance. The samples from 9 different washing recipes were analyzed properly for tensile strength, seam strength, color changes, back staining, stiffness, and chemical oxygen demand (COD). The results led to the finding of an optimal washing condition. An aged look on denim fabric can be varied by varying washing temperatures and amounts of laccase enzyme. The highest fading of denim garment was obtained when washed at a temperature of 65 °C for 45 min using 1.5 g/L of amylase, 1.5 g/L of cellulase, and 2 g/L of laccase enzyme. This was confirmed by the lowest whiteness index of the pocketing fabric (70.1) with an improved fabric feel as evident from the lowest bending length of 2.254 cm. The enzymatic process was found to be significantly environment-friendly as generated wastewater required almost 4 times less oxygen to degrade than the wastewater from the conventional process. Notably, this temperature selection did not negatively impact the fabric's tensile strength and abrasion resistance, emphasizing the efficiency of enzymatic processes. It further strengthened the position of single-bath-single-stage enzymatic treatment of denim as an alternative to conventional denim washing.

Efficient immobilization of highly stable Bacillus amyloliquefaciens spore laccase for biodecolorization of textile dyes in water

Globally, millions of cubic meters of dye-contaminated wastewater are generated annually from the textile industry. Discharging the dye-contaminated wastewater into natural water streams is detrimental to the ecosystem and human health. Wastewater bioremediation is considered a practical solution for converting wastewater into value-added water for safe reuse especially in irrigation. The present study investigated the immobilization of highly stable spore laccase enzyme of Bacillus amyloliquefaciens A1 by entrapment and adsorption techniques. Based on the results, agarose gel was the most efficient support with 94% immobilization yield. Optimum temperature of immobilized enzyme was at 60 ℃ with complete thermal stability for 120 min. The maximum activity of immobilized enzyme was at pH 6–7 and retained 91% of its activity after 24 h incubation. It showed 100% storage stability after 8 weeks which was higher than that of un-immobilized enzyme (4 weeks). The enzyme activity was nearly stable after 8 consecutive cycles of reuse. Decolorization of 8 textile dyes was studied using the immobilized enzyme. Crystal violet, direct red 81 and acid black 24 were efficiently decolorized (94.5, 77.4, and 68.8%, respectively) within 24 h in the first cycle. Reusability of immobilized enzyme showed 74.6, 53.5, and 51.8% decolorization of the same dyes in the same order after the second cycle. Immobilized spore laccase-treated dye water was used to irrigate lentil seeds and showed germination index about 73% compared to 10.8% for untreated one indicating very low toxicity of the treated dye water.

BIODELIGNIFICATION OF AGRO-INDUSTRIAL WASTE AS THE SUBSTRATE TO PRODUCE LACCASE ENZYME FROM TRAMETES VERSICOLOR

Agro-industrial waste in Indonesia is a significant problem for the environment. Optimization of agro-industrial waste utilization is an exciting thing to be researched further. This study aims to utilize agro-industrial waste to optimize laccase enzyme production. The agro-industrial waste used is in the form of bagasse, rice straw, and corn stalks. The waste is used as biomass and treated to extract cellulose through bio-delignification. Cellulose serves as a substrate substance in the production of laccase enzymes. Laccase enzymes can be applied as a biocatalyst in the environmental, pharmaceutical, and chemical industries. Cellulose from agroindustry waste was synthesized by heat treatment using steam explosion with the operation conditions 200°C and 10 bar for 10 minutes. The production of laccase enzymes was done using the green technology submerged fermentation method for 32 hours at 25°C. The optimal result in producing laccase enzyme is using substrate from corn stalks and adding nutrients with an activity value of 25.46 U/mL. The reaction kinetics of laccase enzyme and commercial enzyme against 0.1 M substrate were modelled using the Euler method, and the k value was 2x10-4 min-1 (crude laccase) and 1.9x10-2 min-1 (commercial laccase).

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Identification and Validation of Magnolol Biosynthesis Genes in Magnolia officinalis.

Bacterial infections pose a significant risk to human health. Magnolol, derived from Magnolia officinalis, exhibits potent antibacterial properties. Synthetic biology offers a promising approach to manufacture such natural compounds. However, the plant-based biosynthesis of magnolol remains obscure, and the lack of identification of critical genes hampers its synthetic production. In this study, we have proposed a one-step conversion of magnolol from chavicol using laccase. After leveraging 20 transcriptomes from diverse parts of M. officinalis, transcripts were assembled, enriching genome annotation. Upon integrating this dataset with current genomic information, we could identify 30 laccase enzymes. From two potential gene clusters associated with magnolol production, highly expressed genes were subjected to functional analysis. In vitro experiments confirmed MoLAC14 as a pivotal enzyme in magnolol synthesis. Improvements in the thermal stability of MoLAC14 were achieved through selective mutations, where E345P, G377P, H347F, E346C, and E346F notably enhanced stability. By conducting alanine scanning, the essential residues in MoLAC14 were identified, and the L532A mutation further boosted magnolol production to an unprecedented level of 148.83 mg/L. Our findings not only elucidated the key enzymes for chavicol to magnolol conversion, but also laid the groundwork for synthetic biology-driven magnolol production, thereby providing valuable insights into M. officinalis biology and comparative plant science.

Investigation of Bioremediation Potential of Laccase Enzyme Immobilized on Silver-Coated Magnetic Nanoparticles

Investigation of Bioremediation Potential of Laccase Enzyme Immobilized on Silver-Coated Magnetic Nanoparticles

Chiral CuxCoyS-CuzS Nanoflowers with Bioinspired Enantioselective Catalytic Performances.

Nanomaterials with biomimetic catalytic abilities have attracted significant attention. However, the stereoselectivity of natural enzymes determined by their unique configurations is difficult to imitate. In this work, a kind of chiral CuxCoyS-CuzS nanoflowers (L/D-Pen-NFs) is developed, using porous CuxCoyS nanoparticles (NPs) as stamens, CuzS sheets as petals, and chiral penicillamine as surface stabilizers. Compared to the natural laccase enzyme, L/D-Pen-NFs exhibit significant advantages in catalytic efficiency, stability against harsh environments, recyclability, and convenience in construction. Most importantly, they display high enantioselectivity toward chiral neurotransmitters, which is proved by L- and D-Pen-NFs' different catalytic efficiencies toward chiral enantiomers. L-Pen-NFs are more efficient in catalyzing the oxidation of L-epinephrine and L-dopamine compared with D-Pen-NFs. However, their catalytic efficiency in oxidizing L-norepinephrine and L-DOPA is lower than that of D-Pen-NFs. The reason for the difference in catalytic efficiency is the distinct binding affinities between CuxCoyS-CuzS nano-enantiomers and chiral molecules. This work can spur the development of chiral nanostructures with biomimetic functions.