Catalase immobilization: Current knowledge, key insights, applications, and future prospects - A review.

Catalase (CAT), a ubiquitous enzyme in all oxygen-exposed organisms, effectively decomposes hydrogen peroxide (H2O2), a harmful by-product, into water and oxygen, mitigating oxidative stress and cellular damage, safeguarding cellular organelles and tissues. Therefore, CAT plays a crucial role in maintaining cellular homeostasis and function. Owing to its pivotal role, CAT has garnered considerable interest. However, many challenges arise when used, especially in multiple practical processes. "Immobilization", a widely-used technique, can help improve enzyme properties. CAT immobilization offers numerous advantages, including enhanced stability, reusability, and facilitated downstream processing. This review presents a comprehensive overview of CAT immobilization. It starts with discussing various immobilization mechanisms, support materials, advantages, drawbacks, and factors influencing the performance of immobilized CAT. Moreover, the review explores the application of the immobilized CAT in various industries and its prospects, highlighting its essential role in diverse fields and stimulating further research and investigation. Furthermore, the review highlights some of the world's leading companies in the field of the CAT industry and their substantial potential for economic contribution. This review aims to serve as a discerning, source of information for researchers seeking a comprehensive cutting-edge overview of this rapidly evolving field and have been overwhelmed by the size of publications.

Shallow water seeding cultivation enhances cold tolerance in tobacco seedlings.

Cold stress can impact plant biology at both the molecular and morphological levels. We cultivated two different types of tobacco seedlings using distinct seeding methods, observing significant differences in their cold tolerance at 4 °C. After 12 h cold stress, shallow water seeding cultivation treatment demonstrates a relatively good growth state with slight wilting of the leaves. Tobacco grown using the float system exhibited short, thick roots, while those cultivated through shallow water seeding had elongated roots with more tips and forks. After cold stress, the shallow water seeding cultivation treatment demonstrated higher antioxidant enzyme activity, and lower malondialdehyde (MDA) content.Transcriptome analysis was performed on the leaves of these tobacco seedlings at three stages of cold treatment (before cold stress, after cold stress, and after 3 days of recovery). Upon analyzing the raw data, we found that the shallow water seeding cultivation treatment was associated with significant functional enrichment of nicotinamide adenine dinucleotide (NAD) biosynthesis and NAD metabolism before cold stress, enrichment of functions related to the maintenance of cellular structure after cold stress, and substantial functional enrichment related to photosynthesis during the recovery period. Weighted gene co-expression network analysis (WGCNA) was conducted, identifying several hub genes that may contribute to the differences in cold tolerance between the two tobacco seedlings. Hub genes related to energy conversion were predominantly identified in shallow water seeding cultivation treatment during our analysis, surpassing findings in other areas. These include the AS gene, which controls the synthesis of NAD precursors, the PED1 gene, closely associated with fatty acid ß-oxidation, and the RROP1 gene, related to ATP production.Overall, our study provides a valuable theoretical basis for exploring improved methods of cultivating tobacco seedlings. Through transcriptome sequencing technology, we have elucidated the differences in gene expression in different tobacco seedlings at three time points, identifying key genes affecting cold tolerance in tobacco and providing possibilities for future gene editing.

The intratumoral microbiota biomarkers for predicting survival and efficacy of immunotherapy in patients with ovarian serous cystadenocarcinoma.

BACKGROUND: Ovarian serous cystadenocarcinoma, accounting for about 90% of ovarian cancers, is frequently diagnosed at advanced stages, leading to suboptimal treatment outcomes. Given the malignant nature of the disease, effective biomarkers for accurate prediction and personalized treatment remain an urgent clinical need. METHODS: In this study, we analyzed the microbial contents of 453 ovarian serous cystadenocarcinoma and 68 adjacent non-cancerous samples. A univariate Cox regression model was used to identify microorganisms significantly associated with survival and a prognostic risk score model constructed using LASSO Cox regression analysis. Patients were subsequently categorized into high-risk and low-risk groups based on their risk scores. RESULTS: Survival analysis revealed that patients in the low-risk group had a higher overall survival rate. A nomogram was constructed for easy visualization of the prognostic model. Analysis of immune cell infiltration and immune checkpoint gene expression in both groups showed that both parameters were positively correlated with the risk level, indicating an increased immune response in higher risk groups. CONCLUSION: Our findings suggest that microbial profiles in ovarian serous cystadenocarcinoma may serve as viable clinical prognostic indicators. This study provides novel insights into the potential impact of intratumoral microbial communities on disease prognosis and opens avenues for future therapeutic interventions targeting these microorganisms.

Enhanced immune reconstitution with albuvirtide in HIV-infected immunological non-responders.

Background: Incomplete immune recovery in people living with HIV/AIDS (PLWHA) remains an important clinical challenge with the lack of an effective strategy currently available to restore their T-cell immune response. This study aimed to evaluate the effect of Albuvirtide (ABT) on immune recovery in immunological non-responders (INRs) and attempted to explore potential mechanisms of ABT on the functionality of immune cells. Methods: In this prospective, open-label, controlled clinical study, participants with incomplete immune reconstitution (continuous ART over 5 years and CD4+T lymphocyte absolute count of <500 cells/µl or ART for 2-5 years and CD4+T cell count of <200 cells/µl with undetectable viral load) were received intensive treatment with ABT or maintained on the original ART regimen at a ratio of 1:1. Immune response and safety were examined within 24 weeks. In the cytological study, T subsets, cell apoptosis and cell autophagy were analyzed using immunofluorescence staining and flow cytometry from 25 blood specimens. Results: Both groups (n=25 each) were comparable in age, gender, and ART duration. At week 12, CD4+T cell count increased significantly in the intensive ABT group compared with control group (the change from baseline in CD4+T cell count: 45 vs. -5 cells/µL, p<0.001). After ABT discontinuation, CD4+T cell counts remained significantly higher in the intensive ABT group at week 24 (55 vs. -5 cells/µL, p=0.012). In laboratory analysis, naïve CD4+ T cell amounts were lowest among participants with unsatisfactory immune response (uIR) to ABT (p=0.001). The proportion of caspase 3+CD45RA+CD31+CD4+ T cells was significantly lower in participants with satisfactory immune response (sIR) to ABT (p<0.05). Conclusion: Significant CD4+T cell count increase suggests ABT enhances immune function in INRs which may be attributed to its antiviral properties as well as its ability to increase thymic cell output and decrease cell apoptosis.

AI-based digital pathology provides newer insights into lifestyle intervention-induced fibrosis regression in MASLD: An exploratory study.

BACKGROUND AND AIMS: Lifestyle intervention is the mainstay of therapy for metabolic dysfunction-associated steatohepatitis (MASH), and liver fibrosis is a key consequence of MASH that predicts adverse clinical outcomes. The placebo response plays a pivotal role in the outcome of MASH clinical trials. Second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) microscopy with artificial intelligence analyses can provide an automated quantitative assessment of fibrosis features on a continuous scale called qFibrosis. In this exploratory study, we used this approach to gain insight into the effect of lifestyle intervention-induced fibrosis changes in MASH. METHODS: We examined unstained sections from paired liver biopsies (baseline and end-of-intervention) from MASH individuals who had received either routine lifestyle intervention (RLI) (n = 35) or strengthened lifestyle intervention (SLI) (n = 17). We quantified liver fibrosis with qFibrosis in the portal tract, periportal, transitional, pericentral, and central vein regions. RESULTS: About 20% (7/35) and 65% (11/17) of patients had fibrosis regression in the RLI and SLI groups, respectively. Liver fibrosis tended towards no change or regression after each lifestyle intervention, and this phenomenon was more prominent in the SLI group. SLI-induced liver fibrosis regression was concentrated in the periportal region. CONCLUSION: Using digital pathology, we could detect a more pronounced fibrosis regression with SLI, mainly in the periportal region. With changes in fibrosis area in the periportal region, we could differentiate RLI and SLI patients in the placebo group in the MASH clinical trial. Digital pathology provides new insight into lifestyle-induced fibrosis regression and placebo responses, which is not captured by conventional histological staging.

CRISPR-Cpf1 system and its applications in animal genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein (Cas) system is a gene editing technology guided by RNA endonuclease. The CRISPR-Cas12a (also known as CRISPR-Cpf1) system is extensively utilized in genome editing research due to its accuracy and high efficiency. In this paper, we primarily focus on the application of CRISPR-Cpf1 technology in the construction of disease models and gene therapy. Firstly, the structure and mechanism of the CRISPR-Cas system are introduced. Secondly, the similarities and differences between CRISPR-Cpf1 and CRISPR-Cas9 technologies are compared. Thirdly, the main focus is on the application of the CRISPR-Cpf1 system in cell and animal genome editing. Finally, the challenges faced by CRISPR-Cpf1 technology and corresponding strategies are analyzed. Although CRISPR-Cpf1 technology has certain off-target effects, it can effectively and accurately edit cell and animal genomes, and has significant advantages in the preclinical research.

Bacterial outer membrane vesicle nanorobot.

Autonomous nanorobots represent an advanced tool for precision therapy to improve therapeutic efficacy. However, current nanorobotic designs primarily rely on inorganic materials with compromised biocompatibility and limited biological functions. Here, we introduce enzyme-powered bacterial outer membrane vesicle (OMV) nanorobots. The immobilized urease on the OMV membrane catalyzes the decomposition of bioavailable urea, generating effective propulsion for nanorobots. This OMV nanorobot preserves the unique features of OMVs, including intrinsic biocompatibility, immunogenicity, versatile surface bioengineering for desired biofunctionalities, capability of cargo loading and protection. We present OMV-based nanorobots designed for effective tumor therapy by leveraging the membrane properties of OMVs. These involve surface bioengineering of robotic body with cell-penetrating peptide for tumor targeting and penetration, which is further enhanced by active propulsion of nanorobots. Additionally, OMV nanorobots can effectively safeguard the loaded gene silencing tool, small interfering RNA (siRNA), from enzymatic degradation. Through systematic in vitro and in vivo studies using a rodent model, we demonstrate that these OMV nanorobots substantially enhanced siRNA delivery and immune stimulation, resulting in the utmost effectiveness in tumor suppression when juxtaposed with static groups, particularly evident in the orthotopic bladder tumor model. This OMV nanorobot opens an inspiring avenue to design advanced medical robots with expanded versatility and adaptability, broadening their operation scope in practical biomedical domains.

Thallium spill shifts the structural and functional characteristics of viral communities with different lifestyles in river sediments.

Thallium (Tl), a highly toxic heavy metal, can affect microbial community, while little is known about its effect on viral community. The present study investigated the variation of viral communities, as well as their interactions with microbial hosts under Tl stress. Tl in sediments significantly altered the composition and diversity of the viral communities, but showed no significant links with the prokaryotic communities, which may reveal a potential discrepancy in the sensitivity of the viral and prokaryotic communities to heavy metal stress. Auxiliary metabolic genes (AMGs) involved in denitrification, methane oxidation and organic sulfur transformation were enriched at T1-contaminated sites, while the abundance of AMGs related to methanogenesis and sulfate reduction were higher at pristine sites. Specially, the enrichment of AMGs involved in assimilatory sulfate reduction in Tl-contaminated sites could possibly reduce Tl bioavailability by enhancing the microbially-driven sulfur cycling to generate sulfides that could be complexed with Tl. Moreover, there was a significantly positive correlation between virus-carrying metal resistant genes and the sedimentary Tl concentration, implying that Tl contamination might enhance the metal resistant potential of the viruses. Serving as the functional gene reservoir, the response of viral AMGs to Tl stress could represent a potential pathway for microorganisms to be adapted to the metal-polluted environments. Our study provided novel insights into the impact of Tl spill on viral communities, shedding light on functional characteristics and the links of virus-host interaction with Tl level.

Degradation and mechanism analysis of protein macromolecules by functional bacteria in tobacco leaves.

Tobacco, a crop of significant economic importance, was greatly influenced in leaf quality by protein content. However, current processing parameters fail to adequately meet the requirements for protein degradation. Microorganisms possess potential advantages for degrading proteins and enhancing the quality of tobacco leaves, and hold substantial potential in the process of curing. To effectively reduce the protein content in tobacco leaves, thereby improving the quality and safety of the tobacco leaves. In this study, tobacco leaf were used as experimental material. From these, the BSP1 strain capable of effectively degrading proteins was isolated and identified as Bacillus subtilis by 16S rDNA analysis. Furthermore, the mechanisms were analyzed by integrating microbiome, transcriptome, and metabolome. Before curing, BSP1 was applied to the surface of tobacco leaves. The results indicated that BSP1 effectively improves the activity of key enzymes and the content of related substances, thereby enhancing protein degradation. Additionally, protein degradation was achieved by regulating the diversity of the microbial community on the surface of the tobacco leaves and the ubiquitin-proteasome pathway. This study provided new strategies for extracting and utilizing functional strains from tobacco leaves, opening new avenues for enhancing the quality of tobacco leaves.

Development and characterization of wax-bovine bone protein-grapeseed oil composite oleogels: Experimental and molecular simulation studies.

Three new types of composite oleogel formulations were designed. Specifically, oleogels were prepared using 90% grapeseed oil as the oil phase and carnauba wax (CW)/beeswax/rice bran wax-bovine bone protein (BBP) as gelators. All samples were solid and had an oil-binding capacity of >90%. BBP addition considerably improved the waxy texture of the oleogel and had an important effect on the crystalline network. X-ray diffractometry indicated that BBP increased the ß'-crystal content. All samples showed sol-gel thermodynamic behavior under temperature scanning. Fourier-transform infrared spectroscopy and molecular docking confirmed the formation of noncovalent interactions dominated by van der Waals forces during the development of the oleogel. The optimal components of the three oleogels exhibited an excellent effect of slowing down the release of free fatty acids. This study could serve as a reference for the development and application of wax-protein as a new binary gelator in the food industry.

Sub-pixel target fine spatial feature extraction method based on aperture coding and micro-scanning imaging mechanism.

The small imaging size of targets over long distances results in the loss of geometry and spatial features. Current methods are subject to sampling limitations and cannot accurately capture the spatial features of sub-pixel targets. This paper proposes a method to accurately locate and extract the fine spatial features of sub-pixel targets through aperture coding and micro-scanning imaging. First, the formation mechanism of imaging features for sub-pixel targets is analyzed. Second, the optical aperture is anisotropically coded in different directions to modulate the spreading spots of the target. The primary spreading direction and the center of the anisotropic spreading spots are extracted. The contour and the location of the target are determined from the spreading length and the intersections of the primary spreading directions. Then, the target is sampled by different detector units through various micro-scanning offsets. The pixel units containing different sub-pixel components of the target after offset are determined based on the location results. The fine spatial distribution of the sub-pixel target is reconstructed based on the intensity variations in the pixel units containing the target. Finally, the accuracy of the sub-pixel target fine spatial feature extraction method is validated. The results show a sub-pixel localization error of less than 0.02 and an effective improvement of the sub-pixel target spatial resolution. This paper provides significant potential for improving the ability to capture spatial features of targets over long distances.

Coulomb-induced emission time shifts in high-order harmonic generation from H2.

Accurate emission times of high-order harmonic generation (HHG) are vital for high-precision ultrafast detection in attosecond science, but a quantitative analysis of Coulomb effects on this time is absent in the molecular HHG. Here, we investigate the Coulomb-induced emission-time shift in HHG of H2+ with two different internuclear distances R, where the times obtained via the Gabor transform of numerical data from solving the time-dependent Schrödinger equation are used as simulation experiment results. Based on the molecular strong-field approximation, we develop a trajectory-resolved classical model that takes into account the molecular two-center structure. By selecting appropriate electron trajectories and including Coulomb interactions, the classical trajectory method can reproduce Gabor emission times well. This consistence reveals that Coulomb tails cause an emission-time shift of ∼35 as at the R = 2.0 a.u. case and of ∼40-60 as at the R = 2.6 a.u. case under the present laser parameters when compared to the Coulomb-free quantum-orbit model. Our results are of significance to probe the attosecond dynamics via two-center interference.

Enantioselective Synthesis of ß-Aminoboronic Acids via Borylalkylation of Enamides.

Aminoboronic acids represent a class of significant compounds that have attracted significant attention in the fields of drug discovery and organic synthesis. Despite notable progress in their synthesis, the efficient construction of chiral ß-aminoboronic acids with alkyl side chains remains a challenging endeavor. Here, we introduce an unprecedented nickel-catalyzed asymmetric borylalkylation of enamides, employing a simple chiral diamine ligand, readily available B2pin2, and alkyl halides as coupling partners. This reaction serves as an efficient platform for assembling a diverse range of ß-aminoboronic acid derivatives with flexible alkyl side chains, displaying exceptional regio-, stereo-, and enantioselectivities. Moreover, this transformation exhibits a broad substrate scope and remarkable tolerance toward various functional groups. Theoretical calculations demonstrate that the benzyl group on the ligand is the key to the high enantiocontrol in this transformation. Additionally, we exemplify the practical application of this strategy through the concise synthesis of complex bioactive molecules.

GWAS combined with QTL mapping reveals the genetic loci of leaf morphological characters in Nicotiana tabacum.

BACKGROUND: Leaf morphology plays a crucial role in photosynthetic efficiency and yield potential in crops. Cigar tobacco plants, which are derived from common tobacco (Nicotiana tabacum L.), possess special leaf characteristics including thin and delicate leaves with few visible veins, making it a good system for studying the genetic basis of leaf morphological characters. RESULTS: In this study, GWAS and QTL mapping were simultaneously performed using a natural population containing 185 accessions collected worldwide and an F2 population consisting of 240 individuals, respectively. A total of 26 QTLs related to leaf morphological traits were mapped in the F2 population at three different developmental stages, and some QTL intervals were repeatedly detected for different traits and at different developmental stages. Among the 206 significant SNPs identified in the natural population using GWAS, several associated with the leaf thickness phenotype were co-mapped via QTL mapping. By analyzing linkage disequilibrium and transcriptome data from different tissues combined with gene functional annotations, 7 candidate genes from the co-mapped region were identified as the potential causative genes associated with leaf thickness. CONCLUSIONS: These results presented a valuable cigar tobacco resource showing the genetic diversity regarding its leaf morphological traits at different developmental stages. It also provides valuable information for novel genes and molecular markers that will be useful for further functional verification and for molecular breeding of leaf morphological traits in crops in the future.

Companion cell mediates wound-stimulated leaf-to-leaf electrical signaling.

Leaf wounding triggers rapid long-range electrical signaling that initiates systemic defense responses to protect the plants from further attack. In Arabidopsis, this process largely depends on clade three GLUTAMATE RECEPTOR-LIKE (GLR) genes GLR3.3 and GLR3.6. In the cellular context, phloem sieve elements and xylem contact cells where GLRs were mostly present are implicated in the signaling events. In spite of that, the spatial requirements of different leaf cell types for leaf-to-leaf signaling remain poorly investigated. In this study, we dissected cell-type-specific long-distance wound signaling mediated by GLR3s and showed that phloem companion cells are critical in shaping the functions of GLR3.3 and GLR3.6 in the signaling pathway. GLR3.3-mediated response is phloem-specific, during which, GLR3.3 has to be renewed from companion cells to allow its function in sieve elements. GLR3.6 functions dually in ectopic phloem companion cells, in addition to xylem contact cells. Furthermore, the action of GLR3.6 in phloem is independent of its paralog GLR3.3 and probably requires synthesis of GLR3.6 from xylem contact cells. Overall, our work highlights that the phloem companion cell is crucial for both GLRs in controlling leaf-to-leaf electrical signaling.

Polyoxometalate functionalized magnetic metal-organic framework with multi-affinity sites for efficient enrichment of phosphopeptides.

The reasonable design of metal-organic framework (MOF)-derived nanomaterial has important meaning in increasing the enrichment efficiency in the study of protein phosphorylation. In this work, a polyoxometalate (POM) functionalized magnetic MOF nanomaterial (Fe3O4@MIL-125-POM) was designed and fabricated. The nanomaterial with multi-affinity sites (unsaturated metal sites and metal oxide clusters) was used for the enrichment of phosphopeptides. Fe3O4@MIL-125-POM had high-efficient enrichment performance towards phosphopeptides (selectivity, a mass ratio of bovine serum albumin/α-casein/ß-casein at 5000:1:1; sensitivity, 0.1 fmol; satisfactory repeatability, ten times). Furthermore, Fe3O4@MIL-125-POM was employed to enrich phosphopeptides from non-fat milk digests, saliva, serum, and A549 cell lysate. The enrichment results illustrated the great potential of Fe3O4@MIL-125-POM for efficient identification of low-abundance phosphopeptides.

PxBLAT: an efficient python binding library for BLAT.

BACKGROUND: With the surge in genomic data driven by advancements in sequencing technologies, the demand for efficient bioinformatics tools for sequence analysis has become paramount. BLAST-like alignment tool (BLAT), a sequence alignment tool, faces limitations in performance efficiency and integration with modern programming environments, particularly Python. This study introduces PxBLAT, a Python-based framework designed to enhance the capabilities of BLAT, focusing on usability, computational efficiency, and seamless integration within the Python ecosystem. RESULTS: PxBLAT demonstrates significant improvements over BLAT in execution speed and data handling, as evidenced by comprehensive benchmarks conducted across various sample groups ranging from 50 to 600 samples. These experiments highlight a notable speedup, reducing execution time compared to BLAT. The framework also introduces user-friendly features such as improved server management, data conversion utilities, and shell completion, enhancing the overall user experience. Additionally, the provision of extensive documentation and comprehensive testing supports community engagement and facilitates the adoption of PxBLAT. CONCLUSIONS: PxBLAT stands out as a robust alternative to BLAT, offering performance and user interaction enhancements. Its development underscores the potential for modern programming languages to improve bioinformatics tools, aligning with the needs of contemporary genomic research. By providing a more efficient, user-friendly tool, PxBLAT has the potential to impact genomic data analysis workflows, supporting faster and more accurate sequence analysis in a Python environment.

The TCR ß chain CDR3 insights of bovine liver immune repertoire under heat stress.

Objective: The liver plays a dual role in regulating temperature and immune responses. Examining the influence of Heat stress (HS) on liver T cells contributes significantly to understanding the intricate interplay between the immune system and hepatic tissues under thermal stress. This study focused on investigating the characteristics of the T-cell receptor (TCR) ß chain CDR3 repertoire in bovine liver samples under both HS and pair-fed (PF) environmental conditions. Methods: Sequencing data from six samples sourced from the GEO database underwent annotation. Utilizing immunarch and VDJtool software, the study conducted comprehensive analyses encompassing basic evaluation, clonality assessment, immune repertoire comparison, diversity estimation, gene usage profiling, VJ gene segment pairing scrutiny, clonal tracking, and Kmers analysis. Results: All four TCR chains, namely α, ß, γ, and δ, were detected, with the α chains exhibiting the highest detection frequency, followed closely by the ß chains. The prevalence of αß TCRs in bovine liver samples underscored their crucial role in governing hepatic tissue's physiological functions. The TCR ß CDR3 repertoire showcased substantial inter-individual variability, featuring diverse clonotypes exhibiting distinct amino acid lengths. Intriguingly, HS cattle displayed heightened diversity and clonality, suggesting potential peripheral T cell migration into the liver under environmental conditions. Notably, differential VJ gene pairings were observed in HS cattle compared to the PF, despite individual variations in V and J gene utilization. Additionally, while most high-frequency amino acid 5-mers remained consistent between the HS and PF, GELHF and YDYHF were notably prevalent in the HS group. Across all samples, a prevalent trend of high-frequency 5mers skewed towards polar and hydrophobic amino acids was evident. Conclusion: This study elucidates the characteristics of liver TCR ß chain CDR3 repertoire under HS conditions, enhancing our understanding of HS implications.

A smartphone-adaptable fluorescent probe for visual monitoring of fish freshness and its application in fluorescent dyes.

Due to increasing food safety issues, developing intelligent, on-site, and visual methods for detecting fish freshness has attracted significant attention. Here, we have prepared a benzo[h]chromene derivative BCN that can visually detect 12 biogenic amines (BAs) with high sensitivity. The mechanism for recognizing cadaverine (Cad) is that the probe reacts with Cad to produce a Schiff base derivative, which alters the charge distribution within the molecule, resulting in significant colorimetric and fluorescence changes. The sensing label BCN/FPS was prepared by loading the probe BCN on filter paper, and a visual detection platform was constructed by combining it with a smartphone. By monitoring the correspondence between label color and TVB-N content, a working curve of (R + B)/(R + B + G) with TVB-N content was obtained, enabling visual evaluation of salmon freshness using only a mobile phone. In addition, based on the good solubility and processability of BCN, its application in fluorescent dyes including impregnating dyes, printing inks, coatings, and flexible films has been explored, which opens up new directions for the application of BCN. Therefore, BCN has the potential for real-time monitoring of meat freshness and preparation of fluorescent materials.

Ancestral sequence reconstruction of the prokaryotic three-domain laccases for efficiently degrading polyethylene.

Biodegradation of polyethylene (PE) plastics is environmentally friendly. To obtain the laccases that can efficiently degrade PE plastics, we generated 9 ancestral laccases from 23 bacterial three-domain laccases through ancestral sequence reconstruction. The optimal temperatures of the ancestral laccases were between 60 °C-80 °C, while their optimal pHs were at 3.0 or 4.0. Without substrate pretreatment and mediator addition, all the ancestral laccases can degrade low-density polyethylene (LDPE) films at pH 7.0 and 60 °C. Among them, Anc52, which shared low sequence identity (18 %-41.7 %) with the reported PE-degrading laccases, was the most effective for LDPE degradation. After the catalytic reactions at 90 °C for 14 h, Anc52 (0.2 mg/mL) induced clear wrinkles and deep pits on the PE film surface detected by scanning electron microscope, and its carbonyl and hydroxyl indices reached 2.08 and 2.42, respectively. Then, we identified the residues 203 and 288 critical for PE degradation through site-directed mutation on Anc52. Moreover, Anc52 be activated by heat treatment (60 °C and 90 °C) at pH 7.0, which gave it a high catalytic efficiency (kcat/Km= 191.73 mM-1·s-1) and thermal stability (half-life at 70 °C = 13.70 h). The ancestral laccases obtained here could be good candidates for PE biodegradation.