Applied Biochemistry and Biotechnology最新文献

Kappaphycus alvarezii is one of the economically important red alga, harvested primarily for the production of hydrocolloids. This study aimed to identify an effective extraction method for the isolation of phycobiliproteins (PBPs) from K. alvarezii and evaluate its biofunctional properties. Organic solvents, inorganic acids, organic acids, phosphate buffer saline (PBS), distilled water, and freeze-thawing were used to identify an effective extraction method. The identification of PBPs was carried out by UV-Vis spectrophotometry, FTIR spectroscopy, and SDS-PAGE analysis. The antioxidant potentials of PBPs was examined by DPPH and ABTS radical scavenging assays. The metal chelating efficiency was determined using divalent, monovalent, and trivalent metal ions. The WST-1 assay and DAPI staining were performed to examine HepG2 cell viability and apoptosis, respectively. Among the extraction methods, higher content of phycoeryhrin was recorded in phosphate buffer saline. The results showed that extraction using PBS exhibits highest content of phycoerythrin. The PBPs fraction contains phycoerythrin as a major pigment molecule. The isolated PBPs exhibited increased DPPH and ABTS radical scavenging activity in a concentration-dependent manner. It has possible quenching ability of Co²⁺ and Cu²⁺. The isolated PBPs inhibited HepG2 cell growth in a dose-dependent manner and induced nuclear fragmentation. The data emphasize that K. alvarezii could be utilized for the extraction of phycoerythrin which has antioxidant, metal chelation, and anticancer properties.

α-Ketoglutaric acid (α-KG) is an important intermediate in the tricarboxylic acid cycle and is widely used in organic synthesis, nutritional fortification agents, and other fields. In this study, we co-expressed L-glutamate oxidase (LGOX) from Streptomyces ghanaensis and catalase (CAT) from Escherichia coli and optimized the expression intensity of LGOX and CAT as well as the whole-cell catalytic conditions, which achieved 87.97% conversion of monosodium glutamate and 96.77 g/L yield of α-KG in the whole-cell catalytic process in 12 h. Further, for the LGOX and CAT dual enzyme system, five immobilization methods were designed, and ten batches of ZIF-8-GA immobilized cells with catalytic efficiency of more than 80% were prepared, and the immobilized cells were catalyzed for 12 h, with the conversion rate of monosodium glutamate of 88.46% and the yield of α-KG of 79.61 g/L. The use of a dual-enzyme system with a single microorganism for α-KG production overcomes the limitations of existing methods relying on exogenous catalase (CAT) addition. By employing a novel immobilization technique, this approach enables continuous, batch-based, low-cost production, offering a new solution for the green and efficient biomanufacturing of α-ketoglutaric acid.

The limited natural occurrence of stilbenes, a valuable compound, in sources such as peanuts restricts their full exploitation for health benefits. To address this challenge, we explored the novel use of Sargassum vulgare liquid extract (SLE) as a biostimulant to enhance stilbene production in peanut plants shoots. While seaweed extracts have been previously studied for general crop improvement, this work uniquely demonstrates a genotype-specific and concentration-dependent enhancement of piceatannol levels. Three peanut cultivars: 'Trabelsia' (AraT), 'Chanfakhi' (AraC), and 'American' (AraA) treated with six foliar SLE concentrations (0%, 0.5%, 1%, 2%, 4%, 8%) weekly for 7 weeks, starting 21 days after germination. Significant improvements (p < 0.05) in plant growth, pigment content, and yield were observed, with the highest biomass in AraT at 8% SLE and the highest pod production in AraC and AraA at 1-2%. Polyphenol and flavonoid content peaked under SLE at 8% AraC and 1%, respectively. Most notably, piceatannol content in AraA increased threefold (30.11 ± 2.6 µg/g DW) with 8% SLE, while resveratrol and piceid levels showed limited changes across treatments. These findings highlight the innovative use of S. vulgare extract as a sustainable elicitor for targeted piceatannol enrichment in peanut byproducts, opening avenues for their valorisation in nutraceutical or pharmaceutical applications.

Dementia is a brain disorder that impairs the cognitive abilities like memory, thinking, reasoning, and judgement, thereby restricts an individual's capacity to carry out daily activities. Alzheimer's disease (AD) is a prominent example of one such condition, representing approximately 60-70% of dementia cases and is characterized as an irreversible multifaceted neurodegenerative disorder. The enzyme Acetylcholinesterase (AChE) is a significant contributor to dementia and other neurodegenerative disorder, where this enzyme hydrolyses acetylcholine, a crucial neurotransmitter, thereby disrupting neurotransmission. AChE inhibitors (AChEi) can help delay or mitigate this degradation process. Black rice (purple rice, forbidden rice), a glutinous pigmented rice variety rich in bioavailable phytonutrients like phenols, has shown the potential in alleviating several biological disorders. It has also been reported to possess anti-Alzheimer's properties. This study aims to investigate the metabolomic changes in an indigenous variety of black rice throughout its different germination stages, as well as the impact of metabolite dynamics on the inhibition of acetylcholinesterase. The germination stages exhibited significant variation in terms of their metabolomic constituents. The G2 stage showed the highest AChE inhibition potential among the germination stages, with an IC₅₀ value of 0.217 ± 0.009 mg mL^- 1. Among the compounds identified in the black rice extract, benzene-1,2,4-triol, pyrogallol, hydroquinone, and phloroglucinol* (reported for the first time) exhibited superior activity than the standard drug galantamine. Furthermore, the combination of these authentic compounds with the standard drug (galantamine) showed promising results in reducing the complications associated with the synthetic drug in both in vitro and in silico studies.

As promising biocatalysts, cytochrome P450 enzymes (CYP450s) participate in various reactions involving complex organic compounds. However, their broader application potential is constrained by the dependence on reductase domains. In this study, we engineered a self-sufficient P450 chimeric enzyme by fusing 11β-hydroxylase CYP11B1 with distinct reductase domains from P450_BM3, P450_RhF, and CYP116B3 through peptide linkers. The results demonstrated that the catalytic efficiency of the chimeric CYP11B1-BMR exceeded that of "natural" electron transfer systems CYP11B1-AdR/Adx by approximately 25%. Under optimized expression conditions, the activity of CYP11B1-BMR using 7-ethoxycoumarin and cytochrome c as substrates increased by 30% and 25%, respectively. Among the various linkers tested for rigidity and length, a flexible linker comprising 15 amino acids yielded the highest reduction activity for CYP11B1-BMR, which was 26.6% greater than that observed for CYP11B1-AdR/Adx. This study not only successfully developed a self-sufficient chimeric enzyme but also enhanced its catalytic performance, thereby providing a practical approach for constructing more efficient biocatalytic systems.

Antimicrobial peptides offer significant potential in addressing antimicrobial resistance, as pathogens develop low or no resistance against them. This study aimed to isolate and screen AMP-producing bacteria from environmental samples with broad-spectrum antibacterial activity. Preliminary screening yielded 185 AMP-producing bacterial isolates and subsequently, the most promising isolate with broad-spectrum antibacterial activity was identified as Bacillus mobilis by 16S rRNA sequencing. The AMP from B. mobilis was named mobilisin and purified by hydrophobic adsorption chromatography using Diaion HP-20, followed by RP-HPLC. The molecular weight and probable sequence of mobilisin were observed to be 861.16 Da and KNMFPPK, respectively, using LC-MS/MS and PEAKS Studio 11 analysis. The purified mobilisin showed a notable antibacterial spectrum against both Gram-negative and Gram-positive bacteria with MICs of 31.25 and 62.5 µg/ml, respectively. The antibacterial potential of mobilisin was further confirmed with the synthesised peptide (KNMFPPK), which showed MIC in a similar range. The purified mobilisin exhibited thermal stability at a wide temperature range (4 °C to 80 °C) and maintained its integrity over a broad pH (2.0-11.0) range. Mobilisin also exhibited stability in the presence of various surfactants and organic solvents. The bactericidal potential of mobilisin was confirmed in vitro by scanning electron microscopy (SEM), where it displayed high efficacy by destroying the cell membrane integrity of both Gram-positive and Gram-negative bacteria. The purified mobilisin further demonstrated its applicability in fresh produce preservation by inhibiting the growth of Gram-negative and Gram-positive bacteria on strawberry fruits. Given its broad-spectrum antibacterial properties and biochemical resilience, mobilisin holds the potential for diverse applications in the pharmaceutical and food sectors.

Achieving soluble expression of disulfide bond-containing recombinant proteins in Escherichia coli is challenging. While strategies such as low post-induction temperature, fusion tags, and engineered strains have been employed to achieve soluble protein expression, their specific effects on E. coli metabolism and, crucially, how these metabolic shifts relate to the challenge of achieving soluble protein expression remain unclear. Here, we performed untargeted metabolomics to study the key metabolic changes associated with co-expression of fusion tags in E. coli strains at low and high cultivation temperatures. Using a mass spectrometry-based approach, we identified 121 differentially abundant metabolites. The metabolomes of BL21 (DE3) and SHuffle strains exhibited distinct intracellular pools of amino acids and redox regulators. We further studied the expression of platelet-derived growth factor (PDGF) as a model disulfide-rich protein that generally tends to aggregate when expressed in E. coli. A lower induction temperature and the addition of a thioredoxin tag were observed to be crucial for enhancing soluble expression of PDGF in both strains. However, SHuffle showed heightened metabolic stress during PDGF production compared to BL21. Soluble PDGF expression was associated with higher levels of peptides, nucleotides, and glycolysis and TCA cycle intermediates, while PDGF expression as inclusion bodies was associated with higher levels of amino acids, nucleobases, and pentose phosphate pathway intermediates. Our findings highlight the potential of LCMS-based metabolic profiling in understanding the capacities of different host strains for protein processing and guiding metabolic engineering for improved recombinant protein folding and expression in E. coli.

Microbial infections are a major healthcare challenge, exacerbated by rising antibiotic resistance. This study aims to synthesize and characterize silver-based nanoparticles (Ag₂O and Ag NPs) via conventional and green routes using Corchorus olitorius leaf extract. Structural, optical, and antibacterial properties were analyzed using XRD, FTIR, and UV-Vis spectroscopy. Antibacterial efficacy was evaluated through disc diffusion, MIC, MBC, and biofilm inhibition assays. Statistical analysis was performed using two-way ANOVA to ensure result reliability. The XRD analysis confirmed that S1 (conventional synthesis) consists of cubic Ag₂O, while S2 (uncalcined green-synthesized NPs) and S3 (calcined green-synthesized NPs) exhibit cubic metallic Ag. The crystallite size increased from S2 (16.11 nm) to S3 (31.73 nm), with improved crystallinity (S3: 93.58%). SEM images revealed that green-synthesized nanoparticles (S2, S3) were more uniform and well-dispersed compared to S1. TG analysis indicated that calcination effectively removed organic residues, enhancing nanoparticle stability. Antibacterial tests demonstrated strong activity against E. coli and B. cereus, with S1 showing the highest inhibition. MIC and MBC values confirmed the bacteriostatic, and bactericidal nature of all samples, with S2 exhibiting the strongest effect on B. cereus. Antibiofilm results showed that all samples inhibited biofilm formation, particularly at high concentrations. Overall, green synthesis produced highly crystalline Ag NPs with enhanced stability and antimicrobial efficacy. Calcination further improved crystallinity and reduced defects, making S3 the most stable. These findings highlight the potential of Ag NPs for biomedical and environmental applications, with synthesis conditions significantly influencing their structural and biological properties.

Pentatropis capensis is traditionally used to treat several ailments, yet its potential as a bioresource for green nanotechnology remains unexplored. Selenium nanoparticles (Se NPs) synthesized via plant-based methods are gaining attention for their biomedical and environmental applications. In this study the synthesized Se NPs were characterized using DLS, TEM, SEM with EDAX, XRD, FTIR and UV-visible spectroscopy. UV analysis revealed a characteristic absorbance at 266 nm, SEM and TEM imaging demonstrated both spherical and rod-shaped nanoparticles, averaging 72 nm and 12 nm in size, respectively. EDAX confirmed elemental composition of Selenium (96%) and oxygen (4%). The PDI was recorded as 0.237 in DLS analysis. Antibacterial assays revealed a 16 mm inhibition zone against S. aureus at 100 µg/mL. Antioxidant potential was demonstrated with IC₅₀ values of 75.71 µg/mL (ABTS) and 72.85 µg/mL (DPPH), while TAC results highest OD value at 100 µg/mL is 0.51. Anti-inflammatory activities showed IC₅₀ values of 25.19 µg/mL (HRBC) and 55.55 µg/mL (albumin denaturation). Cytotoxicity testing on A431 epidermoid carcinoma cells revealed an IC₅₀ of 56.69 µg/mL through MTT assay. Zebrafish embryo assays revealed developmental toxicity at concentrations ≥ 1 µg/mL. Photocatalytic degradation achieved 91% methylene blue removal within 70 min. These findings highlight their potential applications in nanomedicine and environmental remediation, with careful consideration of dose-dependent toxicity.