Applied Biochemistry and Biotechnology最新文献

Developing thermally stable, reusable enzymes is key to eco-friendly biodiesel production. Here, we report the first immobilization of a sequence-reconstructed ancestral lipase from family 1.3 lipase, last universal common ancestor (LUCA) onto Seplite LX120, a cross-linked styrene-divinylbenzene resin, via a simple physical adsorption strategy. The immobilized LUCA exhibited broad operational stability, maintaining over 80% activity from 20-100 °C and above 50% across pH 4-9, with optimal performance at 70 °C (565.48 U/g) and pH 9 (534.52 U/g). Enhanced solvent and storage stability were observed compared to the free enzyme, with over 50% residual activity after 12 weeks at 4 °C and excellent tolerance in polar solvents. Notably, the biocatalyst retained 95.35% activity after 10 reuse cycles. Characterization by Scanning Electron Microscopy (SEM) revealed enzyme coverage on the Seplite LX120 surface, while Brunauer-Emmett-Teller (BET) analysis confirmed suitable surface area and porosity for effective immobilization. Both free and immobilized LUCA catalyzed efficient transesterification of waste cooking oil (WCO), yielding 95% and 100% biodiesel, respectively, within 3 h under optimized conditions: methanol-to-oil molar ratio of 6:1, shaking at 150 rpm, and 70 °C. These findings highlight the promise of ancestral enzyme immobilization as a novel route to high-performance, sustainable biocatalysts for biodiesel synthesis from low-cost feedstocks.

The 5-methylcytosine (5mC) epigenetic modification is a prominent pattern of DNA methylation. Nonetheless, its function and relationship with the immune microenvironment (IME) in heart failure (HF) are unclarified. Firstly, seven microarrays and two high-throughput sequencing datasets were downloaded from GEO for this research. Secondly, random forests, LASSO logistic regression, and SVM-RFE were leveraged for screening hub genes. Quantitative PCR, Western blot, and immunofluorescence staining were conducted in a male rat model of HF after myocardial infarction for validation. Thirdly, 5mC-related HF samples were allocated into two distinct categories using consensus clustering algorithms. Finally, single-sample gene-set enrichment analysis and CIBERSORT deconvolution algorithm were utilized for further exploring the IME in HF, including infiltrating immune cell abundance score, human leukocyte antigen (HLA), and immune checkpoints (ICPs). Among these 5mC regulators, four hub genes were uncovered, and the diagnostic model exhibited an outstanding ability to distinguish HF and healthy samples (0.969, 95%CI, 0.953-0.985). DNMT3B was identified as greatly influencing cardiac function. DNMT3B and MBD2 were finally identified as hub genes in the HF model. Two 5mC subtypes manifested different modification patterns, infiltrating immunocytes, ICPs, and HLA gene expression. Furthermore, 305 DEGs were found in 5mC subtypes, and many functions were associated with important pathophysiological mechanisms of HF. The HF diagnostic model was established using 5mC robust core biomarkers. 5mC methylation regulators may offer novel perspectives for understanding the mechanisms, accurate diagnosis, and effective interventions for HF.

Enzyme-protein interactions (EPIs) are critical targets in both pharmaceutical development and industrial biocatalysis, but their effective modulation has been challenged by high false-positive rates (20-30%) in conventional screening methods and limited accuracy in computational predictions. To address these issues, we have developed an innovative hybrid platform that integrates computational modeling with experimental validation. Our approach combines (1) advanced simulation techniques, including molecular docking, MD simulations, and QM/MM calculations; (2) machine learning-driven candidate prioritization; and (3) high-precision validation methods such as FRET/BRET and SPR. This integrated framework achieved exceptional predictive accuracy, with computational binding energies (ΔG = -8 to -10 kcal/mol) strongly correlating with experimental measurements (K_D = 100-500 nM), while enhancing target specificity by 40% and reducing off-target effects by 30% (p < 0.01). The platform's therapeutic potential was demonstrated through the identification of BRAF V600E inhibitors (predicted ΔG = -9.5 kcal/mol, experimental EC50 = 11 nM), which induced 45% tumor regression in vivo. In industrial applications, our structure-guided engineering approach boosted biofuel and L-lysine production yields by 35% and 28%, respectively, with successful scale-up to 10,000-50,000 L bioreactors. Implemented using open-source computational tools (GROMACS, AutoDock Vina), the platform reduced screening false positives to < 5%, shortened development timelines by 20%, and reduced production costs by 18.6%. The demonstrated success in both biomedical and biotechnological applications establishes our framework as a versatile solution for precision EPI modulation, offering transformative potential for drug discovery and sustainable manufacturing.

The production and consumption of plant-based foods are increasing due to health, ethical, and environmental concerns. However, plants do not synthesize vitamin B₁₂, and the fortification of plant-based foods with this vitamin is highly recommended to prevent its deficiency. This work aimed to optimize vitamin B₁₂ production by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 growing on stirred tank bioreactors using the liquid acid protein residue of soybean, an agroindustrial waste, as medium culture. The influence of medium supplementation, presaccharification of sugars, and aeration strategies was investigated. The pH and temperature controls were optimized by applying the design of experiments (doe) approach, based on a central composite design. Vitamin B₁₂ production more than tripled (from ~ 1.5 mg • L^-1 to 5 mg • L^-1) after the optimization process. Cultures of P. freudenreichii produced high concentrations of biomass (> 6 g • L^-1), increasing by threefold the specific biomass yield of vitamin B₁₂ (> 0.8 mg • g^-1 dry cell). This bioprocess, based on the use of cheap and readily available soybean agroindustrial waste as microbial medium, and using a GRAS microorganism, could become an efficient alternative source of vitamin B₁₂ production to fortify plant-based products.

This study aims to evaluate the potential of green-synthesized titanium dioxide (TiO₂) nanoparticles (NPs) derived from herbal extracts-namely Camellia sinensis (Green Tea), Eclipta prostrata (Bhringraj), Glycyrrhiza glabra (Licorice), and Cinnamomum zeylanicum (Cinnamon)-for enhancing the Sun Protection Factor (SPF) in sunscreen formulations. Addressing the environmental and toxicity concerns associated with conventionally synthesized TiO₂ NPs, this eco-friendly approach offers a sustainable and effective alternative for cosmetic applications without compromising efficacy. The synthesized nanoparticles were characterized using UV-Vis spectroscopy, X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR), and then incorporated into oil-in-water emulsions containing 5% homosalate. In vitro SPF was measured using the Labsphere UV-2000S system, while formulation stability was assessed via pH monitoring, rheological behavior, and accelerated centrifugation using the LUMiSizer. Statistical analysis, including one-way ANOVA and effect size metrics (η², Cohen's d), confirmed significant SPF enhancement, particularly in formulations using Green Tea and Cinnamon-derived TiO₂ NPs, which showed 3.12-fold and 3.11-fold improvements, respectively. Notably, smaller crystallite size (30.2 nm in Green Tea-derived TiO₂) correlated with higher SPF (14.97 ± 0.31). All formulations exhibited excellent physical and thermal stability. These findings underscore the promise of plant-mediated TiO₂ NPs as a viable, sustainable solution for next-generation sunscreen products, with further investigation warranted for scale-up and in vivo validation.

Melanoma, an aggressive skin cancer, leads to over 10,000 deaths annually, necessitating novel therapeutic approaches. Lagerstroemia floribunda (LF), a plant rich in phenolics and flavonoids, has been traditionally used in East Asia for its anticancer properties. This study investigates the biochemical and anti-melanoma effects of LF methanol extracts in B16-F10 melanoma cells. Antioxidant activity was evaluated through total phenolic and flavonoid content, SOD scavenging (IC₅₀ = 181.9 µg/mL), and ORAC assay (15.034 ± 4.405 µmol TE/g). The extract inhibited melanoma cell proliferation by 35.1% at 200 µg/mL, reduced colony formation (30%), and suppressed migration (21.32%). Mechanistically, LF extract disrupted MAPK signaling, decreasing phosphorylation of JNK (30%), ERK1/2 (23%), and p38 (14%). Cell cycle analysis revealed G2/M arrest, while apoptosis induction (98.5% at 100 µg/mL) was mediated by Bax upregulation (2.5-fold) and caspase-3/-9 activation (2-fold and 2.3-fold, respectively). Molecular docking identified quercetin, a major LF compound, as a potent binder to JNK (- 7.505 kcal/mol), p38 (- 7.903 kcal/mol), and Bax (- 5.753 kcal/mol), supporting its role in MAPK inhibition and apoptosis. These findings highlight LF's ability to modulate key oncogenic pathways and induce programmed cell death in melanoma cells. Given its traditional use and demonstrated bioactivity, LF presents a promising source of bioactive compounds for melanoma treatment, aligning with biotechnological applications in natural product-based drug discovery.

Background: Diabetic retinopathy (DR) affects vision and can even cause blindness. Kruppel-like factor 7 (KLF7) takes part in high-glucose (HG)-prompted retinal pigment epithelial cell (RPE) apoptosis in vitro, the molecular mechanisms of KLF7-mediated DR pathogenesis are poorly studied.

Methods: HG-challenged RPEs were used as a model for DR in vitro. Cell viability, proliferation, apoptosis, and inflammation were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide, 5-ethynyl-2'-deoxyuridine, flow cytometry, and TUNEL assays. Oxidative stress damage was determined by detection of ROS and MDA. The interaction between KLF7 and forkhead box protein O4 (FOXO4) was estimated by chromatin immunoprecipitation (ChIP)-qPCR and dual-luciferase reporter assays. The toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway was assessed by western blot and the NF-κB inhibitor BAY 11-7085.

Results: HG induced up-regulation of KLF7 in RPEs, and KLF7 silencing weakened HG-induced RPE apoptosis, inflammation, and oxidative stress damage. FOXO4 activated the transcription of KLF7, and FOXO4 silencing demonstrated the same function as KLF7 knockdown in HG-challenged RPEs. Moreover, KLF7 down-regulation reversed FOXO4 overexpression-mediated promoting effect on HG-induced RPE injury. Interestingly, FOXO4 activated the TLR4/MyD88/NF-κB pathway by KLF7, and BAY 11-7085 overturned KLF7 elevation-mediated effects on HG-induced RPE injury.

Conclusion: FOXO4 participated in HG-induced RPE injury via activation of the TLR4/MYD88/NF-κB pathway by enhancing the transcription of KLF7, supporting that FOXO4 and KLF7 as potential targets for DR treatment.

B-cell lymphoma 9 (BCL9) is a regulatory protein that plays a key role within the Wnt/β-catenin signaling pathway. In recent years, it has garnered significant attention across the domains of tissue repair and tumour biology. The Wnt/β-catenin signaling pathway is of great importance in regulating bone tissue formation and osteoblast differentiation. As a co-activator of β-catenin, BCL9 is capable of governing bone development and regeneration by augmenting the transcriptional activity of the Wnt signaling. In addition, BCL9 is also closely related to diverse biological processes, such as angiogenesis, muscle regeneration and tumour progression. Research findings have indicated potential correlations between BCL9 and the treatment of osteoporosis, fracture healing, as well as other bone-related ailments, thereby positioning it as a crucial target in the exploration of bone regeneration. In oncology research, BCL9 affects tumour cell proliferation and metastasis by regulating the Wnt/β-catenin pathway, thus emerging as a promising target for anticancer therapy. In this paper, we conducted a comprehensive review of the discovery, structure, and mechanism of action of BCL9 within the Wnt/β-catenin signaling pathway, along with its potential applications in bone development, tissue regeneration, and cancer therapy, with the intention of furnishing novel perspectives and a theoretical foundation for future investigations.