Applied Biochemistry and Biotechnology最新文献

This work aims to provide a basis for the enhancement of fucoxanthin (FCX) and eicosapentaenoic acid (EPA) biosynthesis in the microalga Phaeodactylum tricornutum using metabolomics and computational biology. To achieve this, both targeted (UHPLC and GC-FID) and untargeted (FTIR and NMR) analyses were conducted throughout various stages of cell cultivation. Targeted analyses revealed that EPA concentrations peaked at the end of the logarithmic growth phase, while fucoxanthin levels remained consistent from the onset of this phase through to the stationary phase. Untargeted analyses provided metabolic profiles by correlating FTIR absorbance bands with functional groups. When combined with cultivation strategies designed to improve EPA and FCX content, the optimal time for harvesting cells was identified as the end of the logarithmic phase. NMR further highlighted potentially key metabolic pathways for optimizing EPA and FCX production in Phaeodactylum tricornutum, particularly those involved in glyoxylate and dicarboxylate metabolism.

Mushrooms, being a source of therapeutically active compounds, are of great interest to researchers due to their historical usage in traditional therapies and the significant role that natural products have played in the development of contemporary medications. Lentinus polychrous is one underutilized mushroom species collected from the laterites of West Bengal, India. Our study aims toward its taxonomic validation, deciphering the secondary metabolic fingerprint, and testing its efficiency in countering many clinical issues, including oxidative stress, growing microbial drug resistance, and cancer. In vitro investigations have shown that the methanolic extract of the mushroom has a broad spectrum of antioxidant activities with effective concentration (EC₅₀) ranging from 403.6 ± 3.8 to 841.2 ± 10.7 µg/mL depending on the type of free radicals and is effective in combating human pathogenic bacterial strains where MIC₅₀ varies from as low as 302.2 ± 3.8 to 570.6 ± 1.8 µg/mL, mediated likely through inducing the breakdown of the outer coat and inducing increased porosity. The fraction was also shown to possess anticancer properties against A549 cells (LD₅₀ 120.9 ± 1.83 µg/mL) by triggering apoptosis. The modulation of Bcl-2 family gene expression was found to be the primary factor responsible for the induction of apoptosis in A549 cells during the experimental approaches. The findings revealed that the mushroom exhibits significant antioxidant, antibacterial, and particular cytotoxic effects on lung cancer cells, indicating its potential medical importance. These results provide essential insights into possibilities for the development of new therapeutic medicines derived from this mushroom.

Lipases from the basidiomycete fungus Ustilago maydis are promising but underexplored biocatalysts due to their high homology with Candida antarctica lipases. This study provides a comprehensive characterization of a recombinant CALB-like lipase from U. maydis, expressed in Pichia pastoris (rUMLB), and compares its properties with those of the well-studied recombinant lipase B from C. antarctica (rCALB). Biochemical analyses included evaluations of optimal pH, temperature, triglyceride (TG) preference for short- and medium-chain acyl groups, phospholipase and amidase activities, enantiopreference, thermostability, stability in organic solvents, and response to NaCl concentrations. rUMLB, a glycosylated enzyme with a molecular weight of 38.6 kDa, exhibited cold-active behavior at 0 °C and preferred hydrolysis of partially soluble short-chain fatty acid TGs, like rCALB. In addition, rUMLB was also capable of hydrolyzing insoluble long-chain triglycerides like rCALB. The half-life at 50 °C for rCALB was approximately 1.6 times greater than that of UMLB, which has fewer surface-exposed proline residues. Both enzymes displayed strong (R)-enantiopreference on (R)-glycidyl butyrate, (R)-ethyl hydroxy butyrate, and (R)-methyl hydroxy valerate enantiomers with increased activity in non-polar solvents. However, rUMLB was more sensitive to polar solvents. Notably, rUMLB was activated at high NaCl concentrations, as previously reported for rCALB. rUMLB showed amidase activity on capsaicinoids similar to rCALB; however, rUMLB uniquely demonstrated significant phospholipase activity toward natural phospholipids, a feature not observed in rCALB. The analysis of the cavity adjacent to the active site in the UMLB model and CALB structure revealed slightly larger area, volume, and hydrophobicity values for UMLB. These comparative insights highlight the functional diversity within the CALB-type lipase family, underscoring the potential of UMLB as a versatile biocatalyst and providing valuable information for biotechnological applications and for understanding enzyme structure-function relationships within the CALB superfamily.

Lipases have catalytic capacity in various processes such as hydrolysis. Those derived from plant sources, such as linseed, offer an economical alternative. The immobilization process facilitates the recovery and reuse of lipase, providing advantages such as resistance to high temperatures and difficulties in recovering and reusing free lipases, which makes product separation difficult. This study presents the immobilization of lipases extracted from flax seeds on octylfunctional hydrophobic supports. Additionally, the thermal stability of the derived products was evaluated in comparison with freely soluble lipase. The lipase exhibited a strong affinity for the evaluated heterofunctional hydrophobic supports, with DVS-activated octylagarose emerging as the most efficient method for immobilization, thus increasing catalytic activity upon resuspension. Furthermore, the octylagarose derivative demonstrated a notable increase in thermal stability. The main results of the study include that the soluble enzyme showed greater activity after 24 h, regardless of the temperature evaluated. The benzamide extract showed greater thermal stability, and all supports evaluated showed greater activity than the soluble enzyme after immobilization. Notably, lipase immobilized on octyl glyoxyl agarose showed the highest activity, demonstrated stability for 840 h at 60 °C, and had a half-life of 1242 h. Furthermore, the lipase immobilized in octyl glyoxyl agarose showed a stabilization factor approximately nine times greater than the free enzyme. These results suggest that immobilization, probably achieved through interfacial activation and multipoint covalent bonds, prevented the release of the enzyme into the medium with increasing temperature. This study thus highlights the significant potential of immobilizing flaxseed-derived lipase.

The relationship between circular RNAs (circRNAs) and tumor growth and metastasis is increasingly well-established. In this study, we sought to shed light on circ-NMNAT1's potential molecular mechanisms in bladder cancer (BCa). circ-NMNAT1, miR-370-3p, and ATXN2L expression profiles were explored using RT-qPCR and/or Western blot techniques. Cell proliferation was detected by MTT and colony formation assay. Transwell assay was used to detect the migration and invasion ability of cells. Western Blot was used to detect the protein expression level of ATXN2L. The targeting relationship between miR-370-3p and circ-NMNAT1 or ATXN2L was confirmed by dual luciferase reporter gene and RIP assay. A xenograft tumor model was created to investigate circ-NMNAT1's function in BCa in vivo. The high expression of circ-NMNAT1 was measured in BCa. circ-NMNAT1 bound competitively to miR-370-3p and downregulated miR-370-3p expression. After knocking down circ-NMNAT1, the proliferation ability of EJ cells was significantly inhibited, and the number of cell colonies was (80.00 ± 7.10). The number of migrated and invaded cells was significantly reduced by (35.49 ± 0.05)% and (59.00 ± 0.04)%, respectively, after silencing circ-NMNAT1. In addition, downregulation of circ-NMNAT1 also significantly increased the apoptosis rate of EJ cells by (23.55 ± 2.95)%. Knockdown of miR-370-3p or overexpression of ATXN2L reduced the effect of circ-NMNAT1 silencing on BCa cells. The promoting effect of circ-NMNAT1 on BCa progression was further validated in vivo tumor models. The weight and volume of the tumor were significantly inhibited after circ-NMNAT1 knockdown, which were (87.50 ± 20.40) mg and (238.90 ± 21.38) mm³, respectively. Circ-NMNAT1 is highly expressed in BCa and promotes the proliferation, migration, and invasion of BCa cells by regulating the miR-370-3p/ATXN2L axis, thereby accelerating the progression of BCa. Our results suggest that circ-NMNAT1 may be a new therapeutic target for BCa.

The primary objective of this study was to identify bioactive compounds from four medicinal plants with multi-targeting activity against hepatocellular carcinoma (HCC). A comprehensive analysis led to the identification of a subset of compounds possessing favorable drug-likeness, pharmacokinetics, and absence of toxicity profiles. Target analysis for 42 phytochemicals revealed 210 potential targets associated with HCC. Protein-protein interaction (PPI) analysis of these targets uncovered five critical hub genes, STAT3, SRC, AKT1, MAPK3, and EGFR, in our study. Correlation analysis of these hub genes indicated a strong positive correlation between EGFR, MAPK3, and SRC expression highlighting their interconnected roles in HCC. Survival analysis underscored the significant prognostic role of these hub genes in HCC underscoring their potential as biomarkers. The co-expression analysis unveiled an intricate network of interactions among the hub genes, while the enrichment analysis demonstrated their enrichment in diverse biological and signaling pathways related to HCC. Molecular docking analysis between the seven phytochemicals and five identified targets revealed that bauerenol exhibited good affinity towards all the targets. Subsequent molecular dynamics (MD) simulations demonstrated that bauerenol formed stable complexes with STAT3, AKT1, EGFR, and MAPK3, suggesting its potential as a multi-targeted inhibitor. Our research suggests that bauerenol shows promise as an inhibitor for HCC targets and stands out as a notable lead compound. However, further experimental studies are necessary to confirm its activity and to evaluate its potential as a therapeutic agent for HCC.

Polyvinyl chloride (PVC) plastics are widespread around the globe, and each year, thousands of tons of PVC end up in the environment in the form of micro-/nanoplastics. Literature has reported extensively on the biodegradation of its PVC additives/plasticizers; however, bio-based treatment approaches for its polymers have been scanty. The current review has discussed elaborately all possible PVC degradation processes and the toxicity challenges faced during its mitigation. This review has also delineated and assessed all physical, chemical, and biological approaches reported for PVC treatments. All the biodeterioration, biocatalysis, and biodegradation mechanisms reported for PVC have been comprehensively discussed. Recent advances have also been highlighted like the direct application of invertebrate species and selective enzymes like peroxidases, alkane monooxygenase, and laccase during PVC treatment. Insights of functional genomes/genes and OMICS have been recommended, which might help predict and address any future issues during the mitigation of PVC pollution in the environment.

Objective: A growing body of evidence suggests the involvement of long noncoding ribose nucleic acids (lncRNAs) in acute kidney injury (AKI). This study focused on the mechanistic role of lncRNA small nucleolar RNA host gene 12 (SNHG12) in ischemia/reperfusion (I/R)-mediated AKI. A model of hypoxia/reoxygenation (H/R) was created using human kidney cells (HK-2). Expression levels of SNHG12 and miR-129-1-3p mRNAs, and USP25 protein were determined through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting analyses, respectively. Furthermore, the relationship between SNHG12 and miR-129-1-3p, as well as miR-129-1-3p and Ubiquitin Specific Peptidase 25 (USP25), was investigated using dual-luciferase reporter gene, RNA pull-down, and immunoprecipitation assays. To further evaluate the role of SNHG12 in AKI, a mouse model was established to study the pathological changes in kidney tissues after SNHG12 knockdown. SNHG12 was upregulated in H/R-induced HK-2 cells and I/R-induced AKI mouse model. Conversely, the expression of miR-129-1-3p showed a significant downregulation. Through dual-luciferase assay and RNA pull-down analysis, it was demonstrated that SNHG12 interacted with miR-129-1-3p, and miR-129-1-3p acted as a negative regulator of USP25. Silencing SNHG12 attenuated the detrimental effect of H/R on HK-2 cells, which was counteracted by miR-129-1-3p antagomir. USP25 overexpression also reversed the effect of miR-129-1-3p on H/R-induced HK-2 cells. SNHG12 knockdown was further found to ameliorate I/R-induced renal injury, apoptosis, oxidative stress, and inflammation in AKI mouse model. SNHG12 was upregulated in I/R-induced AKI and its knockdown ameliorated AKI through the miR-129-1-3p/USP25 axis. SNHG12/miR-129-1-3p/USP25 axis serves as a potential therapeutic target for I/R-related renal injury.

Epidemiologic study suggests that nicotine reduces the risk of Parkinson's disease (PD) and thus could serve as a potential treatment. In this study, we aimed to investigate the effect of nicotine on the behavioral phenotypes and pathological characteristics of mice induced by human alpha-synuclein preformed fibers (α-syn-PFF). Mice were injected with 5 µg of human α-syn-PFF in the hippocampus while administering nicotine-containing drinking water (200 µg/mL). After 1 month, the motor ability, mood, spatial learning, and memory ability of the PD phenotype-like model mice were detected using open field, rotarod, Y maze, and O maze tests. The expression of pathological α-syn and apoptotic proteins, as well as the number of glial and neural stem cells in the hippocampus of mice, was detected using western blot and immunofluorescence. The results demonstrated that nicotine significantly reduced pathological α-syn accumulation, α-syn serine 129 phosphorylation, and apoptosis induced by α-syn-PFF injection in the hippocampus of mice. Nicotine also inhibited the increase in the number of glia, microglia, and neuronal apoptotic cells, and it decreased the expression of PI3K and Akt while also exhibiting significant memory impairment, motor deficits, and anxiety-like behavior. In conclusion, our findings suggest that nicotine ameliorates behavioral deficits and pathological changes in mice by inhibiting human α-syn-PFF-induced neuroinflammation and apoptosis.

Cyanobacteria are advantageous hosts for industrial applications toward achieving sustainable society due to their unique and superior properties such as atmospheric CO₂ fixation via photosynthesis. However, cyanobacterial productivities tend to be weak compared to heterotrophic microbes. To enhance them, it is necessary to understand the fundamental metabolic mechanisms unique to cyanobacteria. In cyanobacteria, NADPH and ATP regenerated by linear and cyclic electron transfers using light energy are consumed by CO₂ fixation in a central metabolic pathway. The previous study demonstrated that the strain deleted a part of respiratory chain complex (ΔndhF1) perturbed NADPH levels and photosynthetic activity in Synechocystis sp. PCC 6803. It is expected that disruption of ndhF1 would result in a decrease in the function of cyclic electron transfer, which controls the ATP/NAD(P)H production ratio properly. In this study, we evaluated the effects of ndhF1 deletion on central metabolism and photosynthesis by ¹³C-metabolic flux analysis. As results of culturing the control and ΔndhF1 strains in a medium containing [1,2-¹³C] glucose and estimating the flux distribution, CO₂ fixation rate by RuBisCO was decreased to be less than half in the ΔndhF1 strain. In addition, the regeneration rate of NAD(P)H and ATP by the photosystem, which can be estimated from the flux distribution, also decreased to be less than half in the ΔndhF1 strain, whereas no significant difference was observed in ATP/NAD(P)H production ratio between the control and the ΔndhF1 strains. Our result suggests that the ratio of utilization of cyclic electron transfer is not reduced in the ΔndhF1 strain unexpectedly.