β-Galactosidases (β-d-galactohydrolases, EC 3.2.1.23) are valuable enzymes for industrial applications ranging from the production of lactose-free products to prebiotics, low-calorie sweeteners, and surfactants. Specifically, the β-galactosidase from Aspergillus oryzae (GalAo) is recognized as more suitable for synthetic applications owing to its superior lactose transgalactosylation-to-hydrolysis ratio. However, the limited stability of GalAo under the harsh conditions dictated by synthetic reactions remains a significant constraint. We produced GalAo as His-tagged recombinant protein in the Kluyveromyces lactis heterologous system to comparatively study its properties with respect to the commercial enzyme. Recombinant WT GalAo is slightly more stable (+1.4 °C Tm value) compared to the commercial protein. Either enzyme showed comparable immobilization yields (~80%), but the recombinant enzyme displayed about 2-fold higher activity, with resulting better recovery (33% vs 18%), which was maintained even after 5 months. Three engineered variants were produced by targeting surface residues that were mutated to Lys to promote the formation of imine linkages with the carrier (glyoxyl Sepabeads™) in a controlled rather than random orientation. All variants could be immobilized, though with lower activity compared to WT (0.73–1.25 U/g). Although the recombinant expression system has still low productivity for biocatalytic applications, it represents a starting point for further optimization.
Biological CO₂ fixation is sustainable, but Chloroflexus aurantiacus performance across diverse cultivation regimes remains poorly characterized and understood. This study assessed CO₂ fixation potential and pigment accumulation in C. aurantiacus Y-400-fl (DSM 637) as the dominant carbon source in a single culture shifted from organic carbon (heterotrophic) to inorganic carbon. A duplicate 672-h batch culture (50 °C, 331.2 ± 35.8 µmol m⁻² s⁻¹) passed through three stages: photoheterotrophic growth on organic carbon, an inorganic-carbon (IC)/sulfide shock, and sodium-acetate supplementation (photomixotrophic). CO₂ fixation potential was inferred from IC removal efficiency, and carotenoids and bacteriochlorophylls (BChl) a and c were tracked. Growth on organic carbon gave the highest biomass (0.48 ± 0.03 g VSS L⁻¹) and productivity (46.45 ± 1.94 mg L⁻¹ d⁻¹), which fell to 18.32 ± 0.30 and 10.54 ± 1.57 mg L⁻¹ d⁻¹ in the two inorganic-carbon stages. Cumulative IC removal relative to the post-shock IC was near zero or negative until 579 h and positive only at 672 h (33.84 ± 34.65%; bottles 58.3% and 9.3%); because total nitrogen fell similarly at that point, this decline is uncertain and indicates a possible fixation capacity. Per litre, carotenoids rose by about half after the shift to IC and sulfide (1.8–2.6–2.8 mg L⁻¹) and persisted under acetate. In contrast, bacteriochlorophylls a and c, unchanged until 336 h, fell by more than half by 672 h. C. aurantiacus has potential for combined CO₂ fixation and pigment production, but performance is modest and variable; staged cultivation merits testing.
Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria perform poorly under multiple environmental stresses (low temperature, high salinity, extreme pH, and high ammonia nitrogen), restricting their large-scale engineering applications. Existing reviews on HN-AD stress tolerance mostly summarize strain phenotypic traits and single‑omics findings, lacking systematic interpretation of the molecular mechanisms of stress adaptation from an integrated multi-omics perspective. This narrative review distinguishes between simple parallel omics analysis and authentic cross-omics integration, and summarizes four types of experimental results of the HN-AD stress mechanism. From published multi-omics studies, molecular adaptation features are classified into bacterial universal stress-response modules, putative stress-nitrogen metabolism coupling modules specific to HN-AD bacteria, and strain-specific response features. These frequently reported candidate regulatory targets still lack sufficient functional validation. Most of the existing results are derived from omics-level correlation analysis. Cross-genus genetic verification and in-situ multi-omics studies in reactors are still scarce. Based on available literature findings, four core biological questions are distilled to build links between laboratory mechanistic insights and reactor engineering performance. This review provides multi-omics molecular comparisons to address limitations of phenotype-centered reviews, and offers insights for the study of biological denitrification of complex industrial wastewater.
Urokinase is an important thrombolytic enzyme for the treatment of thromboembolic disorders, but its clinical application is limited by poor stability and lack of reusability. In this study, urokinase was covalently immobilized onto Fe₃O₄ magnetic nanoparticles using glutaraldehyde as a crosslinker to obtain a magnetically recoverable biocatalyst. Successful immobilization was confirmed by FT-IR, FE-SEM/EDX, XRD, and VSM analyses, demonstrating that the structural integrity and magnetic properties of the nanoparticles were preserved. The immobilization conditions were optimized, and the highest immobilization efficiency (79.1%) was achieved using 1000 IU urokinase and 0.5% glutaraldehyde. Compared with the free enzyme, immobilized urokinase retained comparable catalytic activity while exhibiting higher specific activity. Kinetic analysis showed only a slight increase in Km with no significant change in Vmax. The immobilized enzyme also displayed enhanced pH and thermal stability, retained approximately 65% of its initial activity after six reuse cycles, and showed improved storage stability and greater tolerance to several metal ions and organic solvents. Furthermore, it maintained substantial fibrinolytic activity, achieving 51.55% clot lysis compared with 60.41% for the free enzyme. These findings demonstrate that Fe₃O₄-immobilized urokinase is a stable, reusable, and magnetically recoverable biocatalyst with promising potential for thrombolytic applications.
