Process Biochemistry最新文献

Aspergillus terreus is the most important strain for the industrial production of itaconic acid. Few works have reported the critical metabolic nodes in the synthesis of itaconic acid in A. terreus. Herein a high-throughput screening method was established to screen an efficient mutant A. terreus 6–12#, which could produce 28.6 g/L itaconic acid with a productivity of 0.36 g/g glucose, about 66 % and 57 % higher than that of the parent strain. On this basis, the expression of the key enzymes was analyzed centering on the glycolytic pathway, the precursor synthesis pathway, the itaconic acid synthesis and degradation pathways, and the membrane transport system. It was proven that the expressions of enzymes favoring itaconic acid synthesis in each of these modules were significantly elevated in the mutant. But the enzymes CAD, MTT, MFS in the itaconic acid synthesis pathway and the membrane transport system displayed low elevation levels, while the degradation pathway was activated with the improvement of itaconic acid, which was considered the possible limited step in itaconic acid synthesis in A. terreus. These findings provided potential metabolic modification targets for further acquisition of excellent A. terreus cell factories and helped to facilitate the industrial production of itaconic acid.

Methyl thiophanate (MT) is a widely used fungicide in agriculture; however, it presents substantial health risks to humans. Exposure to MT can damage the human nervous and reproductive systems and inhibit pepsin, a crucial enzyme for digestion and metabolism. This research aimed to investigate the interaction between MT and pepsin using different spectroscopic techniques and dynamic simulations. The UV-Vis results further validated the Trp delocalization findings through molecular docking. At various temperature levels, the effective quenching constants (kq) were found to be 8.28×10³, 5.36×10³ and 2.43×10³ L mol⁻¹, respectively, indicating static quenching with a kq greater than 2 × 10¹⁰. Thermodynamic analysis revealed ${\Delta H}^0$= −56.56 kJ ${\mathrm{mol}}^{-1}$and${∆S}^0$ = −102 J ${\mathrm{mol}}^{-1}{K}^{-1}$, highlighting the significance of hydrogen bonds and Van der Waals interactions in the complex, consistent with docking analyses. By studying the interaction between pepsin and MT, valuable insights can be gained into its impact on digestive system enzymes, leading to a better understanding of MT's toxicological and inhibitory effects on digestion. This interaction disrupts protein digestion, which has broader implications for nutrient absorption and overall digestive health.

The recalcitrant nature of biomass hampers the enzymes' access to polysaccharides. Ionic liquids (ILs) and deep eutectic solvents (DESs) are eco-friendly solubilizing agents since these facilitate effective delignification and enhance carbohydrate's accessibility to enzymes for proficient bioconversion. This study focuses on the pretreatment of sugarcane bagasse with a series of choline-based ILs and DESs to enhance biomass hydrolysis and lignin extraction. Initially, a screening of the green solvents in the dissolution process was performed, where Choline chloride/acetic acid DES showed the best results for xylan (61.7 %) and Choline lactate IL for cellulose (53.4 %) and both with complete dissolution (100 %) for lignin. The results suggested that increased temperature and biomass/solvent ratio lead to higher dissolution yields, which achieved 80 % for most solvents studied. In contrast, the opposite is seen for water content. Pretreatment with the green solvents allowed for greater hydrolysis of the non-dissolved materials, with a 2–4-fold increase compared to untreated bagasse. Enzymatic hydrolysis with Cellic CTec2 showed high yields (above 85 %). Furthermore, remarkable results were obtained, where more than 80 % of the non-dissolved material pretreated with DES was hydrolyzed. The lower concentrations of enzymes led to better hydrolysis results for the material pretreated with DES (100 %) and the untreated material (60.8 %). A wide optimum range was observed for temperature and pH with temperatures from 30 to 60 °C and pH between 3 and 6. Overall, the results of this study provide an effective and facile pretreatment method for lignocellulosic biomass waste with green solvents to enhance enzymatic hydrolysis.

Glucagon-like peptide-1 (GLP-1) analogues have demonstrated greater efficacy and safety in treating type 2 diabetes mellitus (T2DM) due to their strong ability to regulate hypoglycemia. The short plasma half-life of native GLP-1 has prompted the development of novel strategies, such as the conjugation of a C-16 fatty acid to lysine in the GLP-1 analogue sequence, to enhance its longevity. With the escalating burden of T2DM, there is a pressing need for the development of innovative GLP-1 analogues with enhanced efficacy and increased production capacities. In this study, we engineered three recombinant DNA-based clones by incorporating distinct upstream fusion tags, enzyme cleavage sites, and charge-based additional amino acid sequences. These constructed plasmids were subsequently transformed into E. coli BL21 DE3 hosts to enhance solubility and streamline downstream protein purification and were evaluated for yield, purity, and biological effectiveness. A high-yield clone was selected, and the peptide was purified using column chromatography, yielding 150–180 mg per Liter of fermentation culture. The purified C-16 fatty acid-conjugated GLP-1 analogue peptide was analysed for its biological activity, particularly cAMP generation in pancreatic β-cells. Results revealed a concentration of 10.58 pmol/mL of generated cAMP and an average 1.5-fold up-regulation of genes in the cAMP downstream pathway compared to the innovator standard. Based on these findings, we conclude that the developed clone, featuring the enterokinase cleavage site with a sequence of modified TrxA tag with five additional hydrophobic amino acids, not only enhances yield but also preserves the biological efficacy of the final product.

The innate immune system, present in various species, functions as the primary barrier against pathogens, preventing infection and maintaining homeostasis. It consists of mechanical, chemical, and cellular elements across eukaryotic organisms, including physical barriers, physiological processes, pattern recognition molecules, proteins, and cytokines. Despite significant taxonomic differences, both bacteria and complex multicellular organisms exhibit strikingly analogous immune system attributes. Specifically, conserved features in their anti-phage defense mechanisms include the regulation of cellular responses to cytosolic double-stranded DNA and the presence of functional nucleases for viral nucleic acid degradation. Both bacteria and eukaryotes also employ pattern recognition receptors (PRRs) with central nucleoside triphosphatases domains for phage identification and targeting, alongside shared strategies such as gasdermins and pore-forming proteins that confer protection against phages and induce cellular apoptosis. These evolutionary parallels suggest that innate immunity has deep, ancient roots and may reflect the progression of complex life forms from simpler ancestors. Understanding these similarities could inspire new therapeutic strategies and provide deeper insight into the diverse evolutionary pathways of immunity across species. The current article examines these evolutionary connections by comparing innate immune systems in bacteria and eukaryotes, focusing on shared components and molecular insights. Future research should aim to identify and bridge knowledge gaps, further elucidating the evolutionary origins and functional diversity of innate immunity across species.

The COVID-19 pandemic has brought global attention towards the readiness of biotechnology platforms for rapid development of immunogens to be used as vaccine antigens, in diagnostics and in fundamental research. In the present study we provide a bioprocess that is both robust and industry-compatible for high level expression, efficient purification, and scale-up of IMT-CVAX, a prefusion-stabilized trimeric spike protein of SARS-CoV-2. The recombinant IMT-CVAX protein was produced using stable cell pool of Chinese Hamster Ovary (CHO-S) cells that were banked in compliance with cGMP (Current Good Manufacturing Practices) regulations. High quantity expression of IMT-CVAX was achieved through optimization of the shake flask-based process with regards to media, nutrient feeding regimen, incubation temperature, viable cell density and cell viability. We employed refined expression procedures to scale up IMT-CVAX using bioreactor, resulting in a significantly higher yield of around 500 mg/L. Subsequently, a straightforward and simple purification process was developed to produce high-quality IMT-CVAX protein. This procedure consisted of centrifugation, tangential flow filtration (TFF), and either one- or two-step liquid chromatography. A robust anti-spike IgG response was observed in mice after immunization with purified adjuvanted IMT-CVAX. In pseudoviral neutralization assay, mice-generated anti-IMT-CVAX sera could neutralize various SARS-CoV-2 variants. The human convalescent sera from COVID-19-recovered patients recognized IMT-CVAX effectively, which confirms the conformational integrity of IMT-CVAX epitopes. The bioprocess demonstrated here is able to produce sufficient quantities of biopharmaceutical-quality spike protein, which can be used as immunogen against emerging SARS-CoV-2 variants, and can aid in the rapid response to any other pandemic-potential pathogens.

Lignocellulosic biomasses are promising source of fermentative sugars for the production of succinic acid. The lignocellulosic matrix must be pretreated to make the sugars available for the fermentation, but the most tested operative conditions can generate inhibitors as acetic acid, furans, phenolic compounds. Inhibitors remained an obstacle for the implementation of succinic acid production starting from recalcitrant biomasses as wheat straw. Batch tests were performed at two starting concentrations of strain, sugars (glucose, glucose and xylose) and inhibitors (acetic acid and furfural) by comparing the fermentation in standard broth medium and hydrolysate. Notwithstanding the presence of acetic acid (52.5 mg/L) and furfural (15 mg/L), succinic acid was obtained at 9*10⁻² ± 7*10⁻³ g/L by starting from wheat straw hydrolysate that contained glucose (1.1 g/L), xylose (0.4 g/L) and without additional nitrogen source. Therefore, the study highlighted that a more concentrated inoculum was able to reduce the synergistic effect of inhibitors at their highest concentrations. The results obtained may contribute to improve succinic acid production from the biomasses that have been under-exploited but abundantly available, as wheat straw, for which solutions must be found to solve the problem of inhibitors production or to mitigate its effect on the fermentation process.

Lipase inhibitors are used in the treatment of candidiasis, obesity, and acne problems. Therefore, the investigation of new natural lipase inhibitors has generated great interest. Juniperus phoenicea is a small Mediterranean tree considered an important medicinal plant. Through this work, we investigated the composition and inhibition effect of J. phoenicea extract on Candida rugosa and human lipases; and we analyzed the interactions between detected compounds and lipases using molecular docking, ADME-T, and dynamic studies. We extracted the secondary metabolites from the plant aerial parts and tested their activities against lipase. We examined major components of J. phoenicea, using Autodock vina for molecular docking, Desmond from Schrodinger suite 2021–1 for dynamics, and evaluating its drug-likeness and toxicity properties using ADMET webserver. We found that the IC₅₀ values of ethyl acetate and methanol extracts are 1.33±0.10 and 1.731 mg/mL. Using HPLC quantitative analysis we identified in ethyl acetate extract the following phenolic compounds gallic acid, rutin, apigenin-7-O-glucoside, cinnamic acid, and quercetin. The complex apigenin-7-O-glucoside-lipase is stabilized by four hydrogen bonds and hydrophobic interactions, and the binding energy equals -10 Kcal/mol, which is better than the interactions saved for orlistat. Dynamic Simulation demonstrated better stability in the ligand-protein complex apigenin-7-O-glucoside-1lpb within 100 ns than the orlistat-1lpb complex. This study is described in this work for the first time. The results indicated that apigenin-7-O-glucoside from J. phoenicea L could be a good choice to develop a new drug candidate against candidiasis, obesity, and other lipase-related diseases.

Isocorydine is an aporphine alkaloid that is mainly produced via chemical synthesis and extracted from plants. Its hydrochloride has been authorized to be a prescription drug for clinical use. This study aims to discover potential alkaloidal metabolites from endophytic fungi associated with the plant. Endophytic fungi were subjected to solid-state fermentation (SSF) to investigate the production of isocorydine. An aporphine alkaloid was obtained from the metabolite of SSF with Aspergillus sydowii G12. Its chemical structure was deduced to be isocorydine according to the spectroscopic data of ESI-MS, ¹H and ¹³C NMR spectroscopy. The fermentation conditions including substrate, fermentation temperature, and time were optimized. As a result, SSF with A. sydowii G12 using potato as substrate at 20 °C for 15 d could produce isocorydine with a high yield of 2.51 ± 0.09 mg/g. This paper is the first to report an approach to producing isocorydine from microorganisms instead of plants.

As a precursor, caproate is widely used in food and medicine for production of various valuable products. From an environmental point of view, caproate production by anaerobic fermentation is a sustainable way. The cost of caproate could be reduced if the wasted biomass is used as substrate. In this study, the caproate production from corn straw and food waste was investigated. Results showed that the sulfuric acid – enzyme method was the optimum approach for hydrolysis of corn straw, and the corresponding glucose concentration was 24.3 g/L. At the optimum condition, the 5-hydroxymethyl furfural (HMF) was found to be the main inhibitor of the hydrolysate, which exhibited inhibitory effect to ethanol and caproate fermentation. Further analysis showed that the inhibition threshold of HMF to Clostridium kluyveri was 1.2 g/L. The formation of biofilm in the fermentation system was found to be an effective way for improving the robustness of cells to inhibitors. The caproate production of 13.5 g/L was obtained when the ethanol and acetate derived from corn straw and food waste was used as substrates. This study cast an insight that it was a promising way for caproate production from the corn straw and food waste.