Engineering in Life Sciences最新文献

Posttreatment of the effluents from wastewater treatment plants is becoming increasingly important, as the conventional treatment cannot completely remove organic trace contaminants. Promising techniques like chemical oxidation methods, including ozonation, face the challenge of potentially generating more toxic transformation products than their parent substances due to incomplete oxidation. In this work, the laccase from Trametes versicolor was immobilized on a polyester textile to create a biocatalytic textile filter for the posttreatment of organic trace contaminants and their ozonation by-products. Different filter designs for reactive filtration with biocatalytic textiles were implemented on the laboratory scale and tested for their effectiveness in degrading the dye Remazol Brilliant Blue, the pharmaceutical diclofenac, and its ozonation products. The plate module, inspired by lamellar clarifiers and featuring the textile with covalently immobilized enzyme on the lamella surfaces, exhibited the best performance characteristics. Employing this module, a continuous process of diclofenac ozonation and subsequent posttreatment with the biocatalytic filter was conducted. This not only demonstrated the feasibility of continuous biocatalytic wastewater filtration but also highlighted improved degradation efficiencies of ozonation products compared to the batch process using laccase in solution.

Establishing manufacturing processes for cell culture-based pharmaceutical products involves managing multiple parameters that can affect yield and efficiency, as well as process robustness and product quality. Implementing Quality by Design (QbD) principles can support process optimization, while streamlining the chemistry, manufacturing, and control aspects for regulatory approval. In this study, we mimic a QbD approach based on an influenza A virus production process using two clonal suspension Madin-Darby canine kidney (MDCK) cell lines with distinct characteristics. We performed a quantitative risk assessment including biological and technical parameters to identify the Critical Process Parameters (CPPs). To comprehensively study the effects and interactions of four CPPs, we used an Ambr 15 scale-down system following a Design of Experiments (DoE) approach. After data analysis and modeling, we obtained design spaces characterized by high robustness with a less than 1% risk of failure and even some indications for virus titer and yield improvement, while keeping process-related impurities such as DNA and total protein concentration low. These findings were subsequently verified at a more than 100-fold higher working volume. Taken together, our approach may stimulate ideas for the implementation of streamlined process development and regulatory approval in the field of viral vaccine production.

To increase their throughput, reduce laboratory work and improve reproducibility, automation of bioprocesses is gaining in importance nowadays. This applies in particular to microbioreactors (MBRs), which can be easily integrated in highly parallelized and automated platforms and, therefore, be applied for screenings, cell-based assays, and bioprocess development. One promising pharmaceutical application for MBRs is the performance of phage sensitivity tests called phagograms in phage therapy. However, there is no automated and parallelized platform available so far that fulfills the requirements of phagograms. Therefore, a novel highly parallelizable capillary-wave microbioreactor (cwMBR) with a volume of 7 µL, which has already been successfully applied for phagograms, was extended by an in-house built platform for automated fluid addition in the single-digit nanoliter range. The cwMBR has a phage-repellent hydrophilic glass surface. Furthermore, a custom-made highly parallelizable device for biomass measurement in the lower microliter scale was developed and validated in the cwMBR. To prove the applicability of the platform for the generation of phagograms, a phagogram using Escherichia coli and automated phage addition was performed. The results indicate a clear lysis of the bacteria by the phages and thus confirm the applicability of performing automated phagograms in the highly parallelizable cwMBR platform.

Melt Electrowriting (MEW) is a powerful technique in tissue engineering, enabling the precise fabrication of scaffolds with complex geometries. One of the most important parameters of MEW is collector speed, which has been extensively studied in relation to critical translation speed. However, its influence on crystallinity was overlooked. Crystallinity is crucial for the mechanical properties and degradation behavior of the scaffolds. Therefore, in this study, we present how printing affects the crystallinity of fibers and the resulting mechanical properties of MEW scaffolds. In systematic analysis, we observed a significant reduction in scaffold crystallinity with increased speed, as evidenced by wide-angle X-ray scattering. This decrease in crystallinity was attributed to differences in cooling rates, impacting the polycaprolactone molecular orientation within the fibers. By using tensile testing, we observed the decrease in scaffold Young's modulus with increasing collector speed. Given the relation between crystallinity and mechanical properties of the material, we developed a finite element analysis model that accounts for changes in crystallinity by employing distinct bulk Young's modulus values to help characterize scaffold mechanical behavior under tensile loading. The model reveals insights into scaffold stiffness variation with different architectural designs. These insights offer valuable guidance for optimizing 3D printing to obtain scaffolds with desired mechanical properties.

Ferulic acid is a high-value chemical synthesized in plants. The ferulic acid biosynthesis is still affected by the insufficient methylation activity of caffeic acid O-methyltransferase (COMT). In this study, we engineered COMT from Arabidopsis thaliana to match caffeic acid, and the mutant COMT^{N129V-H313A-F174L} showed 4.19-fold enhanced catalytic efficiency for degrading caffeic acid. Then, we constructed the de novo synthesis pathway of ferulic acid by introducing tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), 4-hydroxyphenylacetate 3-hydroxylase from Escherichia coli (EcHpaBC), and mutant COMT^{N129V-H313A-F174L}, and further increased tyrosine synthesis. Furthermore, we overexpressed two copies of COMT^{N129V-H313A-F174L} and enhanced the supply of S-adenosyl-L-methionine (SAM) by expressed S-ribosylhomocysteine lyase (luxS) and 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (mtn) to increase the production of ferulic acid. Finally, the production of ferulic acid reached 1260.37 mg/L in the shake-flask fermentation and 4377 mg/L using a 50 L bioreactor by the engineered FA-11. In conclusion, COMT enzyme engineering combined with global metabolic engineering effectively improved the production of ferulic acid and successfully obtained a fairly high level of ferulic acid production.

Trypanosoma brucei phospholipase A₂ (TbPLA₂) is a validated drug target but the difficulty in expressing its soluble recombinant protein has limited its exploitation for drug and vaccine development for African and American trypanosomiases. We utilized recombinant deoxyribonucleic acid (DNA) technology approaches to express soluble TbPLA₂ in Escherichia coli and Pichia pastoris and biochemically characterize the purified enzyme. Full-length TbPLA₂ was insoluble and deposited as inclusion bodies when expressed in E. coli. However, soluble and active forms were obtained when both the full-length and truncated TbPLA₂ were expressed in fusion with N-terminal FLAG tag and C-terminal eGFP in P. pastoris, and the truncated protein in fusion with N-terminal FLAG tag and C-terminal mClover in E. coli. Truncated TbPLA₂ lacking the signal peptide and transmembrane domain was finally expressed in Rosetta 2 cells and purified to homogeneity. Its migration on sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed its size to be 39 kDa. Kinetic studies revealed that the enzyme has a specific activity of 107.14 µmol/min/mg, a V_max of 25.1 µmol/min, and a K_M of 1.58 mM. This is the first report on the successful expression of soluble and active recombinant TbPLA₂, which will facilitate the discovery of its specific inhibitors for the development of therapeutics for trypanosomiasis.

Deep eutectic solvents (DESs) hold the potential to serve as a sustainable and environmentally friendly substitute for supercritical fluids, ionic liquids, and organic solvents. Moreover, DESs have been demonstrated to assist in stabilizing the structure of enzyme. The enzymatic synthesis of epoxy vegetable oil in a DES-system was developed in this study, and the influence of DESs viscosity on the epoxidation system was investigated for the first time. The results demonstrated that the epoxy value reached 8.97, and the double bond conversion rate was 82.48%. The viscosity of the reaction system decreased from 209.32 to 91.35 (mPa·s). The application of DES in epoxidation was confirmed through structural characterization, indicating that eutectic solvents could serve as substitutes for toxic and volatile organic solvents in synthesizing high-epoxide vegetable oils using an enzymatic method, thus facilitating the production of environmentally friendly plasticizers.