Applied Biochemistry and Biotechnology最新文献

In this study, a thermostable phytase enzyme was produced from Mucor indicus through the optimization of media components, followed by immobilization, with the aim of enhancing the nutritional value of broiler and layer feed via dephytinization process. Various agricultural by-products, including wheat bran, rice bran, chickpea husk, and black gram husk were evaluated for their efficacy in phytase production. Among these, black gram husk demonstrated the highest enzyme activity, achieving 92.10 U/ml. Optimization of media components and physical parameters, including a temperature of 50 °C, a pH of 5.5, an inoculum age of 72 h, an inoculum size of 1.5%, glucose as the carbon source, and peptone as the nitrogen source, resulted in a significant enhancement in enzyme activity, reaching 184.03 U/ml. The catalytic efficiency of the free enzyme was determined to be 5.68 ± 0.28 mM/s, whereas the immobilized enzyme exhibited a substantially higher catalytic efficiency of 17.26 ± 0.24 mM/s. Application of the immobilized enzyme for the dephytinization of broiler and layer feed led to phosphorus liberation of 35.45 mg/g and 58.46 mg/g, respectively, after 24 h of incubation. These results demonstrate the potential of immobilized thermostable phytase for improving the nutritional quality of animal feed by utilizing sustainable agricultural by-products.

Cell-based biosensors are evolving as versatile tools for biological research, drug development, and environmental monitoring. Living cells are used to detect elements in these biosensors, which offer significant advantages over standard transducers. The purpose of this review article is to provide an in-depth overview of cell-based biosensors, emphasizing their working principles, fabrication processes, and applications. The potential of living cells to respond to particular analytes or stimuli supports the design and operation of cell-based biosensors. Real-time and label-free identification can be accomplished by combining these cells with transducers like microelectrodes or optical sensors. Genetically engineered cells or changed microenvironments can be used in cell-based biosensors to improve performance by optimizing cell types for increased dynamic range, sensitivity, and selectivity. Cell-based biosensors are developed by meticulously cultivating and immobilizing cells on transducer surfaces while retaining their vitality and performance. Cell-based biosensors have a wide range of applications, including monitoring the environment, healthcare, and pharmaceutical research. These biosensors have been used to detect diseases, toxic substances, pollutants, and therapeutic drug screening. Cell-based biosensors are cutting-edge technology that brings together the capabilities of live cells and transducers to detect analytes in a sensitive and specific manner. These biosensors illustrate the tremendous potential for upcoming uses in healthcare and monitoring environmental conditions with further developments in fabrication methods and the inclusion of artificial intelligence.

Triple-negative breast cancer (TNBC) poses significant challenges as it lacks specific treatment approaches. In this study, we synthesized niobium-nitrogen-doped titanium dioxide (Nb-NTO) nanoparticles (NPs) functionalized with Mentha arvensis ethanolic and Mucuna pruriens methanolic extracts and evaluated their anti-cancer potential against MDA-MB-231 TNBC cells. The functionalization of doped Nb-NTO NPs with Mentha arvensis and Mucuna pruriens extract exhibited significant synergistic effects, reducing cell viability in a dose-dependent manner with enhanced cytotoxicity at lower concentrations compared to individual treatments. Microscopic analysis revealed morphological changes indicative of apoptosis and necrosis, while flow cytometry demonstrated increased apoptotic and necrotic cell populations in the combination-treated groups. These treatments also significantly reduced the secretion of pro-inflammatory cytokines IL-6 and IL-8, suggesting modulation of the inflammatory tumor microenvironment. Furthermore, the functionalized Nb-NTO NPs effectively targeted cancer stem cell (CSC) properties, inhibiting mammosphere formation and clonogenic survival and downregulating critical CSC markers, including c-Myc, OCT4, and NANOG. This study highlights the potential of Nb-NTO NPs functionalized with Mentha arvensis and Mucuna pruriens as a novel therapeutic strategy for TNBC, addressing key hallmarks of cancer, including apoptosis, inflammation, and CSC targeting. While these findings demonstrate promising in vitro anti-cancer efficacy, further in vivo validation and mechanistic studies are necessary to advance these treatments toward clinical anti-cancer applications.

Aphanothece is a genus of colonial cyanobacteria with a global distribution that is found in various aquatic and terrestrial environments. It has garnered interest because of its high content of amino acids, carbohydrates, fatty acids, and pigments, which possess bioactive and biotechnological properties. This review analyzes articles highlighting Aphanothece species in biotechnological contexts and describes their biochemical composition. Among its primary metabolites are glutamic acid, alanine, palmitic acid, chlorophyll a, echinenone, and β-carotene. The biotechnological potential of Aphanothece spans the fields of biofuel, health, agro-industry, and bioremediation. The notable bioactivities of species such us A. sacrum, A. pallida, and A. bullosa include photoprotective, immunostimulant, antimicrobial, anticancer, and biostimulant activities due to secondary metabolites such as mycosporine-like amino acids, peptides, betaines, and glycerophospholipids. The high production of hydrogen and lipids by A. halophytica supports its use in biofuels. Species such as A. microscopica are effective at treating agro-industrial and domestic effluents and water polluted by metals and hydrocarbons, alongside simultaneous CO₂ capture. This review provides information that can guide the sustainable use of Aphanothece species and identifies gaps in current knowledge, particularly in the development of commercial products. Continuous exploration of this genus can significantly promote environmental sustainability and biotechnological innovation.

Diabetic retinopathy (DR) is a microvascular complication of diabetes. Insulin-like growth factor 1 receptor (IGF1R) has been implicated in the pathogenesis of DR; however, the underlying mechanism remains unclear. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess IGF1R mRNA expression. Western blotting assays were performed to analyze the protein expression of IGF1R, ubiquitin-specific peptidase 14 (USP14), and BRCA1-associated protein 1 (BAP1). Cell viability, apoptosis, interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels were analyzed using cell counting kit-8 assay, flow cytometry, and enzyme-linked immunosorbent assays, respectively. Fluorescent microscopy and flow cytometry were performed for reactive oxygen species (ROS) level assessment, and colorimetric assays for iron (Fe²⁺) and glutathione (GSH) levels. Co-immunoprecipitation assays and/or colocalization techniques were employed to validate the association of IGF1R with USP14 and BAP1. Treatment with high glucose (HG) increased the protein expression of IGF1R, USP14, and BAP1 in ARPE-19 cells. Silencing of IGF1R mitigated HG-induced apoptosis, inflammatory response, and ferroptosis in ARPE-19 cells. USP14 was found to stabilize IGF1R protein expression through deubiquitination. Overexpression of USP14 exacerbated HG-induced cellular injury, whereas silencing of USP14 protected ARPE-19 cells by reducing IGF1R expression. Interaction between IGF1R and BAP1 was confirmed in ARPE-19 cells and IGF1R silencing protected cells from HG-induced injury by regulating BAP1 expression. Thus, USP14-dependent regulation of IGF1R expression and its interaction with BAP1 play a crucial role in the pathogenesis of high glucose-induced diabetic retinopathy.

We investigated the mechanisms of polymer-lipase interactions that govern the catalytic activity of lipases in the presence of polymers. Using a combination of fluorescence correlation spectroscopy (FCS), activity analysis, fluorescence spectroscopy, and computational surface analysis, three model lipases-Thermomyces lanuginosus lipase (TLL), Candida antarctica lipase B (CalB), and Bacillus subtilis lipase A (BSLA), with different degrees of hydrophobic active site exposure were studied. Low-molecular-weight poly(methyl methacrylate) (PMMA), synthesized via ARGET ATRP, was employed to study the effect of unstructured polymers in dispersed solution on lipase activity. PMMA significantly enhanced TLL and BSLA hydrolytic activity, while no CalB activation was observed. FCS analysis indicated that this activation was facilitated by polymer lipase binding, a phenomenon observed with TLL and BSLA but not with CalB. Computational analysis further revealed that the surface properties of the lipases were critical for the lipases' susceptibility to activation by PMMA. Although CalB exhibited the largest total hydrophobic surface area, its homogeneous distribution prevented activation, whereas strong, localized hydrophobic interactions allowed PMMA to bind and activate TLL and BSLA. Supported by the quantitative correlation between elevated 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence in the presence of PMMA and lipase activity, the activation was attributed to locally increased hydrophobicity of the lipases upon polymer binding. These findings provide critical insights into the role of polymer interactions in lipase activation and stabilization, highlighting the potential for designing tailored polymer carriers to optimize enzyme performance in industrial and biotechnological applications.

Microbial transformation has enabled phytosterols as readily available and bio-renewable starting materials for the industrial synthesis of steroidal active pharmaceutical ingredients (APIs). Editing the phytosterol side chain would create various steroidal compounds with a specific C17-side chain, which will greatly facilitate the synthesis of steroidal APIs. Precise cleavage of the phytosterol side chain requires identification of the key enzymes and the reaction pathways of phytosterol side chain metabolism. In this study, a hydratase EchA19 was identified in Mycolicibacterium neoaurum NRRL B-3805, a strain which was engineered by traditional mutation and screening or genetic manipulation, generating recombinant strains for the industrial-scale production of androstenedione (AD), androstadienedione (ADD), and 9α-hydroxy-androstenedione (9α-OH-AD) from phytosterols. It was found that EchA19 is the key hydratase affecting the first β-oxidation pathway of phytosterol side chain metabolism. The previously proposed carboxylation at the C28 position might occur after the cleavage of the C24 branched alkyl side chain, rather than after the dehydrogenation reaction. This study has provided us with new insights and a deeper understanding of the metabolic pathways of phytosterol side chain, and laid a foundation for synthesizing valuable steroid drug intermediates from phytosterols through metabolic regulation by precisely editing the side chain.

Anaerobic digestate food waste effluent (ADFE) contains high nutrient loads and causes water pollution and waste of resources if discharged without treatment. Microalgae provides a promising strategy for nutrients recovery, biomass production, and CO₂ capture. However, due to the characteristics of high ammonia nitrogen and low C/N ratio, it is hard for the original algae to remove N and P in ADFE. Hence, this study investigated the gradient domestication of C. vulgaris and S. quadricauda to enhance their tolerance to high concentrations of ADFE and evaluated their growth and metabolic responses under ADFE concentrations ranging from 20 to 60%. The results revealed that S. quadricauda exhibited better tolerance and growth performance compared to C. vulgaris, with a 42.31% biomass increase in the 40% BG11-diluted ADFE group after domestication. Additionally, extracellular polymeric substances (EPS) secretion increased by 27.66% to help shield algal cells from pollutants. The final removal efficiencies of total nitrogen, total phosphorus, and soluble chemical oxygen demand by S. quadricauda in BG11 dilution group were 37.13%, 40.64%, and 75.45%, respectively, which indicated that domestication alleviated oxidative stress to algal cells. This work demonstrates the simultaneous ADFE treatment and cost-effective microalgal biomass production, providing new insights into microalgal cultivation for the efficient treatment and valorization of nutrient-rich ADFE for sustainability.

Geobacillus sp. represents an important source of thermophilic esterases, yet studies on the rational design and industrial application of these enzymes remain limited. In our previous research, we identified the esterase Gju768 from Geobacillus jurassicus DSMZ 15726. In the present study, we employed a novel computer-aided rational design approach, ACDP (AutoDock, Consurf, Discovery Studio, PoPMuSiC), to enhance the enzyme's thermal stability. Through molecular docking and conservation analysis, three hotspots were identified. Virtual saturation mutagenesis was subsequently performed, yielding two selected mutations, Q78I and Q78L, from the resulting library. Notably, mutants Q78I and Q78L exhibited significant improvements in thermal stability and enzyme activity compared to the wild type (WT). Compared to WT, mutants Q78I and Q78L exhibited a 65.27% and 38.38% increase in half-life at 65 °C, along with a 14.48% and 1.60% improvement in specific activity at their respective optimal temperatures. Furthermore, under optimized conditions for cinnamyl acetate production, mutant Q78I demonstrated a yield of 68%, compared to only 31% for WT. This study underscored the potential of protein engineering strategies to enhance enzyme performance in industrial applications, particularly for the synthesis of value-added compounds such as cinnamyl acetate.