Biotechnology Journal最新文献

Microfluidic brain-on-a-chip and angiogenesis-on-a-chip models have been employed by leading research groups around the world. These models have great potential, but they have not yet been used in the context of personalized medicine and diagnostics, nor have they been widely adopted for testing drug candidates. The reproduction of a physiologically relevant in vitro brain-on-a-chip model with essential similarity to in vivo structural and functional characteristics, including integrity and complexity of the brain tissue, limited and controlled permeability of barriers, and plastic changes induced by brain stimulation or injury, is a highly challenging task that requires a comprehensive approach and a tour de force in advanced biomedical engineering and living soft matter physics. This approach necessitates not only the combination of well-established chip production technologies and cell biology protocols but also the integration of knowledge from neuroscience, (bio)physics, hydrodynamics, material science, (bio)chemistry, electronics, and soft matter physics. This review covers current understandings of the establishment of microvascular network formation in the active brain in vivo, as well as analysis of novel approaches to reconstruct the basic mechanisms of cerebral angiogenesis and barrier genesis in a physiologically relevant brain-on-a-chip model. The novel concept, which employs the application of an external electric field to stimulate vasculogenesis/angiogenesis on a chip, demonstrates how a synergistic approach may help to solve this non-trivial problem and to establish the vascularized brain tissue sensitive to various stimuli and suitable for the high-precision analysis of its functional activity and plasticity.

One of the main obstacles to preparing oligoxanthan through the enzymatic degradation of xanthan gum (XG) is its dense structure. To achieve the large-scale production of oligoxanthan, it is necessary to perturbate the conformation of XG and increase enzyme accessibility. This research developed an efficient system for the enzymatic degradation of XG assisted by low concentrations of ionic liquids (ILs). Structural characterization results indicated that a low concentration of [EMIM]DEP (1%, v/v) not only loosens the structure of XG but also alters the secondary structure of enzyme, thereby dramatically increasing its hydrolytic activity and eventually enhancing the production of oligoxanthan. The results were further elucidated by molecular dynamics (MD) simulation. Under the hydrolysis conditions of 1.5 mg/mL Aspergillus niger cellulase (AnCel), 0.2 mg/mL XG, and 1% (v/v) [EMIM]DEP at 40°C and pH 5.0 for 20 min, XG can be hydrolyzed into a mixture with a glucose equivalent of 32.25%. The degree of polymerization (DP) of XG hydrolysates was 3–10. Moreover, the xanthan hydrolysates obtained using this system exhibit excellent antioxidant activity. This study proposed an effective method for the preparation of functional oligosaccharides, which is of significant importance for the valorization of XG.

Methylene-tetrahydrofolate reductase (MTHFR) is an important enzyme for acetogenic carbon fixation, but the redox mechanism driving this reaction is not clearly understood. Previous enzymology work and energetic accounting on species such as Clostridium autoethanogenum has led to confounding results when placed in the context of in vivo experiments. In this work, we create multiple C. autoethanogenum strains harboring alternative MTHFR enzyme complexes as well as genome-scale metabolic models to better understand how these organisms conserve energy on gas substrates. The inclusion of a Type-III MTHFR unexpectedly allows for higher growth than expected and suggests the possibility of an additional redox balancing cycle employed during autotrophic growth.

The SpyTag/SpyCatcher system is a modular protein assembly tool. Its core mechanism is the formation of isopeptide bonds, which achieves protein assembly through covalent coupling. This system is characterized by mild reaction conditions, rapid connection and no need for additional reagents, and shows good application potential in fields such as enzyme engineering. Although the system has made progress in application, it still faces challenges such as industrial scale and clinical immunogenicity. This paper systematically reviews the principle of the SpyTag/SpyCatcher system and its development progress. It also analyzes the deficiencies of the system in industrial applications, focuses on elaborating its specific application examples in enzyme engineering, discusses existing challenges, and looks forward to future research directions. Overall, this review aims to provide references and new ideas for research in related fields.

Biocatalytic esterification in water is a green alternative to chemical synthesis but often faces challenges such as low enzyme efficiency, poor substrate solubility, and expensive cofactors. Here, we present a streamlined aqueous esterification system utilizing the adenylation domain of carboxylic acid reductase (A-domain_CAR), a minimal catalyst that efficiently activates carboxylic acids. A-domain_CAR exhibited superior catalytic performance over full-length CARs, achieving up to 96% yield of methyl cinnamate under optimized aqueous conditions. To improve cost-efficiency and scalability, the system was coupled with a Class III polyphosphate kinase 2 (Class III PPK2) from Deinococcus proteolyticus for in situ ATP regeneration using AMP and polyphosphate. This two-enzyme platform enabled high-yield esterification across a broad range of cinnamic and benzoic acid derivatives and various alcohols. Incorporating micellar media further enhanced the conversion of poorly soluble aromatic alcohols such as benzyl and phenethyl alcohol. Preparative-scale esterification of methyl caffeate, a bioactive antioxidant ester, was successfully demonstrated with a 66% yield in a 500 mL aqueous reaction. This study highlights A-domain_CAR as a modular, efficient, and scalable biocatalyst, advancing sustainable ester synthesis for applications in pharmaceuticals, fine chemicals, and bio-based materials.

Recombinant antibody production in mammalian cells is essential for therapeutic, diagnostic, and research applications. Single-expression plasmid systems that co-express both heavy and light chains under balanced promoters offer advantages in improving expression consistency. However, the relationship between intracellular antibody expression levels and secretion efficiency following clonal selection remains unclear. In this study, a single-expression plasmid encoding a chimeric anti-CD99 antibody (ChAbMT99/1) was constructed and used to establish stably expressing HEK293T mini-pool populations through zeocin selection. Intracellular ChAbMT99/1 expression levels in five selected mini-pools were assessed using intracellular immunofluorescence staining and flow cytometry. Antibody secretion into the culture supernatants was quantified by indirect enzyme-linked immunosorbent assay (ELISA) and further validated using indirect immunofluorescence assay and flow cytometry with native CD99-expressing cells. The results revealed a strong positive correlation between intracellular antibody expression and secretion levels. Mini-pools with higher intracellular fluorescence intensities produced greater antibody yields, indicating that intracellular antibody expression levels, within the context of single-plasmid expression systems, can reliably predict secretion efficiency. This finding facilitates the efficient selection of high-producing mini-pools for large-scale recombinant antibody production.

Aldo-keto reductase (AKR) is an important biocatalyst for the synthesis of chiral alcohols; however, its inability to catalyze bulky substrates severely limits its industrial applications. Previously, the T23V/Q213A mutant of AKR from Kluyveromyces marxianus (KmAKR) exhibited an extended substrate scope, but it still showed poor catalytic activity toward some valuable aliphatic and aromatic ketones. Here, we developed a computer-assisted strategy to virtually screen a mutation library constructed from residues 23 and 213. Guided by the binding free energy calculated from high-throughput molecular dynamics simulations, the top ten mutants with the lowest binding energies in each group were selected for testing against the corresponding substrates. It was found that most selected mutants exhibited enhanced catalytic activity, yielding the corresponding pharmaceutically important alcohols with high enantioselectivities. Structural and dynamic analyses indicated that residues 23 and 213 functioned as molecular switches to control the dynamics of the loop regions that constitute the substrate-binding pocket, thereby influencing the substrate specificity of KmAKR.