Process Biochemistry最新文献

Biotin is essential for metabolizing the three major nutrients and is vital for animal growth and development. Industrially, biotin production has relied on chemical synthesis, a method fraught with disadvantages including complex processes, environmental risks, and high costs. Consequently, researchers have increasingly recognized the benefits of biosynthesizing biotin. Native transcriptional regulation and metabolic bottlenecks restrict biotin production. Replacing the native promoter and bioO with the constitutive P₄₃ promoter, along with swapping the native birA for a non-regulatory variant from Mycobacterium tuberculosis, significantly upregulated biotin biosynthetic gene expression, effectively bypassing native transcriptional regulation. Subsequent strategies to bypass the inefficient native BioW included knocking out ydbM in the β-oxidation pathway—a potential pimeloyl-CoA degrader, expressing heterologous bioW genes, and introducing alternative pimeloyl-ACP biosynthetic pathways, collectively increasing biotin titers to 6.91 mg/L. This study demonstrates a multi-faceted approach to overcoming metabolic and regulatory barriers, providing a template for enhancing biotin production in B. subtilis, with implications for the broader field of microbial biotechnology.

There has been recent interest in using the β-galactosidase of Bacillus circulans to produce prebiotic oligosaccharides, including galactooligosaccharides (GOS), lactulose and lactosucrose. The types and amounts of the transgalactosylation products produced depend on the selectivities of the enzyme for the various transgalactosylation and hydrolysis reactions that occur. To date, the selectivities of the β-galactosidase of B. circulans for these reactions have not been adequately characterized. In the current work, we undertake four case studies using literature data for systems in which different product mixtures are produced (i) GOS; (ii) GOS and disaccharides; (iii) GOS and lactulose; and (iv) GOS and lactosucrose. We analyze this data to obtain quantitative estimates of the relevant selectivities. We show that, in the production of GOS, the isoform β-Gal-A is significantly less selective against hydrolysis reactions than the other β-galactosidase isoforms. In the production of lactulose, the β-galactosidase of B. circulans gives high lactulose yields, despite having a 19- to 33-fold preference for producing GOS over lactulose. In the production of lactosucrose, the β-galactosidase of B. circulans also prefers to produce GOS.

In this study, alkali-soluble polysaccharide from Manilkara zapota (MZAP) was extracted and polysaccharide-iron complexes MZAP-Fe (III) was synthesized. Structural characterization, antioxidant and digestive properties were used to evaluate the feasibility of MZAP-Fe (III) as an iron supplement. The results showed that when the reaction temperature was 70 ℃, the pH was 9 and the addition of sodium citrate was 0.025 g, MZAP- Fe (Ⅲ) had the highest iron content (14.68 %) and its average molecular weight increased compared to MZAP. The UV and FT-IR results indicated that MZAP- Fe (Ⅲ) was formed by the carboxyl and hydroxyl groups in MZAP with the iron group to form Fe-O bonds. MZAP-Fe (III) maintained high thermal stability over the temperature range of 20°C to 450℃. When the concentration of MZAP- Fe (Ⅲ) was 1.0 mg/mL, hydroxyl radical-scavenging activity and DPPH radical scavenging activity reached 57.03 % and 82.21 %, respectively, and both were significantly higher than the antioxidant activity of MZAP. Moreover, in the simulating human digestion, MZAP- Fe (Ⅲ) was more stable than FeSO₄ and less likely to be destroyed by stomach acid. The present study demonstrated the potential of MZAP-Fe (III) as a novel iron supplement.

Plasmodium falciparum is the pathogen responsible for 90 % of all malaria cases and is associated with severe complications and the highest fatality rate. Plasmodium falciparum lactate dehydrogenase (PfLDH) is a useful biomarker for the treatment and diagnosis of malaria; however, its use is limited by low production yield and functional activity. In this study, using Escherichia coli Rosetta(DE3) strain specialized for eukaryotic protein expression, we successfully expressed and purified PfLDH without a codon optimization process, which has previously been considered essential for protein expression in E. coli strains. The induction temperature and time were optimized for the overexpression of PfLDH using the transformed strain, and 31.3 mg/L of PfLDH protein was successfully purified using immobilized metal affinity chromatography. The physical properties of the purified PfLDH were assessed by verifying tetramer formation, and the functional properties of PfLDH were assessed with a colorimetric assay using a substrate that reacts with PfLDH. Subsequently, the binding of PfLDH with a DNA aptamer, which is known to specifically bind to PfLDH, was verified. These results are expected to provide important suggestions for research on obtaining large amounts of PfLDH from bacteria, thereby facilitating the treatment and diagnosis of malaria.

The cyclic lipopeptide bacillomycin D exhibits potent antifungal activity against a wide range of fungi, particularly filamentous fungi, thereby exhibiting significant potential for application in food preservation. However, the limited yield obtained from wild strains hinders the practical application of bacillomycin D. In this study, we used different strategies to modify the original strain Bacillus amyloliquefaciens fmbJ, including promoter replacement, competitive synthetic gene knockout, and overexpression of the lipopeptide transporter gene. Subsequently, we analyzed changes in the production and expression of bacillomycin D genes in the engineered strain. The results demonstrated that P_bacA exhibits the highest promoter activity, leading to a significant increase (2.05-fold) in bacillomycin D production compared with control group. Additionally, deletion of the epsA-O gene cluster also significantly enhances the synthesis of bacillomycin D. However, when amyloid gene cluster tasA-sipW-yqxM was deleted,bacillomycin D accumulation time was shortened, resulting in no significant increase in bacillomycin D production. In addition, lipopeptide transporters SwrC, KrsE and YcxA did not promote bacillomycin D production. Finally, by replacing the promoter P_bacA, we successfully disrupted the epsA-O gene cluster, and bacillomycin D production increased by 2.55 times compared to the initial bacillomycin D strain. In conclusion, this study indicated that genetic engineering of regulatory genes was an effective strategy to improve the yields of bacillomycin D and provided promising strains for industrial production of bacillomycin D.

Aromase™ H2 is an enzymatic cocktail marketed to intensify the aroma, promote clarity and overall flavor of tea by releasing aromatic compounds and antioxidants that are naturally glycosylated. The hydrolysis of a wide range of glycoconjugates is the key to its action, which can arise from the substrate promiscuity of the main diglycosidase or other enzymes that are in the mixture. The characterization focusing on the activity toward flavonoids at different pH values allowed to increase the cocktail ratio of diglycosidase to α-rhamnosidase activity. In that way, the free disaccharide rutinose was selectively obtained in a laboratory scale reactor with 90 % conversion after 3.5 h reaction at pH 8. Purification was carried out by liquid-liquid extraction, recovering the aglycone hesperetin. Subsequent extraction with activated carbon allowed a recovery of 84 % of the rutinose. This work demonstrates the practical application of a commercial cocktail for the selective hydrolysis of rutinosylated compounds.

Large-scale application of MFCs is limited by the high cost of electrode and low power density. In this study, the electrochemical structure and power output performance of three novel straw-derived biochar are discussed and characterized by scanning electron microscope, specific surface area, cyclic voltammetry, Raman spectrum, electrochemical impedance spectroscopy etc. Compared with the common carbon felt electrode, the optimal electricity generation ability and electrochemical affinity are obtained in corn straw biochar electrode: the rough surface with macroporous structure and high degree of graphitization facilitates the attachment of electroactive bacteria; the maximum output voltage, open circuit voltage, power density and coulombic efficiency reach 662.64±4.03 mV, 798.24±3.65 mV, 1.54±0.18 W m⁻² and 50.39±1.68 %, respectively; the maximum COD removal efficiency of 77.45±1.69 % is achieved; exchange current density (4.6142×10⁻⁴ A cm⁻²) in corn straw electrode presents the better electrochemical activity than that of carbon felt electrode. These implies that corn straw-derived biochar is a competitive raw material for MFC anode.

The peel of Citrus limon is a colossal organic byproduct of the juice industry containing a significant quantity of bioactive polyphenols with diverse therapeutic potentials. As they are often underutilized the present investigation aims to valorize and promote the utilization of these polyphenols through encapsulation in a hydrocolloid-based hydrogel matrix for prolonged shelf-life and sustainable delivery. The hydrogels were prepared by blending sodium alginate and pectin through ionotropic gelation which achieved ∼86.72 % polyphenol encapsulation efficiency. The polyphenol-encapsulated hydrogels had maintained the shelf life and effectively preserved >95 % of polyphenols at 30 °C with no discernible variation for >30 days. Multiple physicochemical investigations were performed on the prepared formulation. Additionally, the release kinetics of polyphenols from beads were examined in a simulated gastrointestinal environment. The result showed that ∼87.54 % of entrapped polyphenol was released after 9 h and the pattern was found controlled release type and pH-responsive. The degree of fluctuation of a few specific polyphenolic compounds was noted and evaluated using principal component analysis. Furthermore, the encapsulating procedure protected a considerable amount of antioxidant activity (>60 %) while demonstrating good hemocompatibility. Therefore, the produced formulation may be used by the juice industries to create a valorization step using the byproduct.

A novel endo-polygalacturonase (PGC-AN64) from Aspergillus niger HO32 was produced (88.22 U/mL) on orange peel by-product (OPP), purified, biochemically characterized, and its utility in clarification of orange juice was elucidated. The enzyme was purified by ammonium sulfate fractionation (80 %) and gel filtration chromatography on Sephacryl® S-200 HR column. Its pH and temperature optima were 6.5 and 70 °C, respectively, and was stable over a pH range of 5–7 and temperature 60–80 °C. The 26-residue NH₂-terminal sequence of PGC-AN64 showed high homology with those of Aspergillus pectinases. Metal ions (Na⁺, Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺) positively affect PGC-AN64 activity, while Ba²⁺, Cd²⁺, and Cr³⁺ negatively affect its original activity. Interestingly, PGC-AN64 exhibited a considerable substrate specificity and catalytic efficiency compared to the commercial enzymes PECLYVE V (P1) and PECLYVE CP (P2). More interestingly and compared to P1 and P2, PGC-AN64 showed that the clarification process had a significant effect on the transmittance percentages with 84.68 ± 2.08, 88.05 ± 1.79, and 93.13 ± 3.58 % for PGC-AN64, P1, and P2, respectively. Furthermore, color parameters after the clarification process increase in the L* and b* values, and led to a decrease in the a* value. The obtained results suggest that PGC-AN64 revealed its potential in orange juice clarification.

Developing novel nanomedicine formulations that successfully penetrate the blood-brain barrier (BBB) to treat and diagnose glioma effectively is still a significant obstacle. This study presents the development of polymer nanogels camouflaged with macrophage membranes. The nanogels are loaded with manganese dioxide (MnO₂) and carboplatin. They are designed for the treatment of glioma using a combination of chemotherapy and chemodynamic therapy (CDT). The precipitation polymerization process was used to create redox-responsive poly(caprolactone) (PCL) nanogels (NGs). These NGs were then loaded with MnO₂ and physically encapsulated with carboplatin. The resulting nanogels had an average diameter of 102.52 nm and were covered with macrophage membranes to provide excellent stability. The developed NGs exhibit redox and pH responsiveness, releasing carboplatin and Mn(II) in a controlled manner. NGs may effectively deplete glutathione (GSH) in the tumour milieu. The Mn(II) generated in this process enhances the effectiveness of the loaded carboplatin by exerting its chemotherapeutic action and promoting the formation of reactive oxygen species to improve the efficiency of CDT. The results of our study show a very efficient and promising nanomedicine platform for the treatment and diagnosis of glioma, a type of cancer. This platform is self-adaptive, cooperative, and based on NG technology. Furthermore, this platform can potentially be used for other challenging types of cancer.