Preparative Biochemistry & Biotechnology最新文献

This study demonstrates a circular economy approach by utilizing glycerol pitch, biodiesel co-product as a carbon source to produce biodegradable terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] using Cupriavidus malaysiensis USMAA1020. Various carbon sources, including oleic acid, 1,4-butanediol, and 1-pentanol, were combined with glycerol pitch as main substrates. The experiments were conducted in a mineral salt medium (MSM) with a high concentration of carbon source and limited nitrogen, which promotes both bacterial growth and PHA accumulation. Preliminary studies indicated, the composition of the polymer was triggered by varying the concentration of 1,4-butanediol, oleic acid, and glycerol pitch while keeping the amount of 1-pentanol constant. After optimization, the PHA content in dry biomass increased from 60 ± 12 to 88 ± 5 wt%, while the residual dry cell weight (RDCW) slightly decreased from 1.77 ± 0.53 to 1.11 ± 0.41 g/L. The combination of glycerol pitch in the biosynthesis of the terpolymer proved effective for the cost-efficient and sustainable production of P(3HB-co-3HV-co-4HB). This process could drive innovation while reducing waste, by harnessing glycerol pitch's properties, sustainable co-product of biodiesel production into a valuable biodegradable polymer. The characterization of the terpolymer revealed a diverse range of properties, making it suitable for various applications.

Crustacean residues are potential sources of carotenoids, bioactive molecules with antioxidant and anti-inflammatory activities. The lipid extract from shrimp consists mainly of astaxanthin and unsaturated fatty acids with high nutraceutical potential. However, the chemical instability of astaxanthin represents a drawback that needs to be overcome to enable the application in food products. Therefore, this work aimed to obtain microcapsules from the lipid extract of L. vannamei cephalothorax in a calcium alginate matrix. The extraction method showed relevant technical and economic viability, according to the results of yield and astaxanthin content. The lipid extract showed excellent antioxidant potential, given the predominant concentration of astaxanthin and oleic acid in the chemical constitution of the extract. The microencapsulation method used resulted in microcapsules with morphology and size suitable for use in food matrices.

Microgreens are young, immature plants widely used in salads and dishes for their nutritional value, color, texture, and flavor. Their compatibility with indoor and small-scale cultivation makes them a unique crop of modern controlled environment agriculture, addressing the growing demand of urban populations. Beyond their culinary appeal, microgreens are gaining recognition as nutrient-dense functional foods due to their high concentrations of vitamins, minerals, antioxidants, and bioactive compounds, including flavonoids, carotenoids, and phenolics, compared to mature greens and sprouts. Unlike sprouts, which are harvested after 2-7 days, microgreens are harvested between 7 and 21 days after the development of young true leaves, offering enhanced microbial safety. Among them, herb microgreens such as fenugreek, basil, mustard, fennel, coriander, and moringa stand out for their rich bioactive composition and potential therapeutic benefits. Their health-promoting properties include anti-hyperglycemic, anti-carcinogenic, and anti-inflammatory effects, and roles in immune modulation, cardiovascular health, and managing age-related diseases. As a result, their demand is increasing in both the food and pharmaceutical industries. This review explores the nutritional profile, therapeutic significance, human health perspectives, and culinary applications of various herb microgreens. Additionally, this review highlights the key research gaps and future directions to optimize their functional food potential for human health.

Tomatoes, rich in phenolics and antioxidants, offer a rich source of dietary antioxidants with potential health-promoting properties. Owing to the involvement of toxic solvents, high energy consumption, and hazardous waste, a safer and more sustainable extraction method has been preferred in the food and pharmaceutical industries. This study systematically investigated three advanced extraction techniques-microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), and accelerated solvent extraction (ASE)-to maximize phenolic yield and antioxidant capacity while concurrently minimizing the use of hazardous solvents, focusing Baccarat tomato variant. Among the tested methods, ASE demonstrated superior DPPH radical scavenging activity (87.480 ± 0.856%) with an IC50 of 4.65 mg/mL, while UAE achieved the highest total phenolic content (TPC, 9.060 ± 0.028 mg GAE/g DW). A strong correlation between TPC and antioxidant activities (DPPH and FRAP) was observed, emphasizing interdependence between phenolic content and bioactivity. Hierarchical cluster analysis (HCA) revealed 66.66% of ASE samples grouped into a cluster based on enhanced antioxidant properties. While UAE is superior for maximizing phenolic yield, ASE is the optimal method for producing extracts with the highest antioxidant potency, highlighting the critical role of technology selection in developing targeted ingredients for functional foods and therapeutic agents toward good health and well-being.

The synthesis of optically active alcohols is vital for the pharmaceutical and fine chemical industries, yet traditional chemical methods generally lack efficiency and stereoselectivity. Although ketoreductases are a more environmentally friendly option, many of them need to have their catalytic performance improved to satisfy industrial demands. This study focused on engineering Komagataella phaffii ketoreductase (KpKR), the first characterized ketoreductase from this yeast, by applying an innovative combination of error-prone PCR and ribosome display for rapid directed evolution. Through the use of kinetic analysis, molecular docking, homology modeling, and substrate specificity assays, five evolved variants (M1-M5) were identified and characterized. Compared to a range of microbial ketoreductases, such as Bacillus sp. ECU0013 and yeast-derived enzymes, the KpKR variants demonstrated efficient catalytic performance; notably, M5 showed enhanced catalytic efficiency (k_cat/k_m = 199.58 s^-1mM^-1) toward ketoesters, while M1 demonstrated remarkable activity for halogenated substrates, outperforming Bacillus enzymes by thousands-fold. Structural investigation identified key C-terminal changes that likely contributed to improved active site accessibility. Furthermore, sequence analysis confirmed KpKR as a member of the short-chain dehydrogenase/reductase (SDR) family. These results underscore the innovative utility of ribosome display in enzyme engineering and establish the evolved KpKR variants as powerful, highly efficient biocatalysts suitable for challenging pharmaceutical synthesis.

Polyhydroxybutyrate (PHB) is an environmentally friendly polymer that is synthesized by bacteria and stored as a carbon source. One of the main problems in PHB production is the price of materials for production. This study aims to optimize environmentally friendly and affordable materials such as molasses, tofu wastewater, and coconut water to increase PHB yield. Statistical optimization with central composite design (CCD) was used to optimize materials in Bacillus cereus B52 (B. cereus B52) for PHB production. The concentration of molasses, tofu wastewater and coconut water were used as optimization parameters to increase the PHB yield. The optimization results resulted in the production of PHB yield of 1.45 ± 0.14 g/L. Chemical analysis was used to determine the biopolymer validation of PHB. In conclusion, production optimization using a combination of materials from molasses, tofu wastewater, and coconut water can increase PHB production in B. cereus B52.

The optimization for the extraction of bioactive compounds from Garcinia cambogia (GC) and Myristica fragrans (nutmeg rind (NM)) using Peleg's kinetic model with Response Surface Methodology (RSM) was observed. The extraction efficiency was assessed through Total Phenolic Content (TPC), Total Flavonoids (TF), and Antioxidant activity. The effect of particle size, temperature, and solvent concentration on extraction performance was facilitated by RSM. The kinetics of both initial rapid solute release and eventual equilibrium phase were carried out using Peleg's model. The model demonstrated high predictive accuracy of TPC values with R² = 0.985 and Root Mean Square Error (RSME) = 0.90 μg Gallic Acid Equivalence/mL (μg GAE/mL) and TF values with R² = 0.907 and RSME = 0.752 μg Quercetin Equivalence/mL (μg QE/mL) and R² = 0.961 and RSME = 0.316% for antioxidant activity for G. cambogia. Similarly, a satisfactory fit was observed for M. fragrans (R² = 0.971; RMSE = 11.19 μg GAE/mL), (R² = 0.0.571 and RSME = 0.266 μg QE/mL), and (R² = 0.923 and RSME = 0.511%), respectively. The close agreement between observed and predicted values supports the robustness of Peleg's model and RSM in optimizing extraction parameters.

This study investigates the immobilization of peroxidase (POD) concentrated from celery root onto multi-walled carbon nanotubes (MWCNTs), focusing on its effects on enzymatic activity, stability, and reusability. POD was extracted using phosphate buffer, followed by ammonium sulfate precipitation and dialysis. Immobilization conditions were optimized based on contact time and support amount. The immobilized enzyme showed maximum activity after 300 minutes, whereas increasing MWCNT content led to reduced activity due to diffusion limitations. Kinetic analysis revealed that immobilized POD retained a similar V_max compared to the free enzyme, but exhibited significantly higher K_M values. Comprehensive characterization using BET, FTIR, SEM/EDX, TGA, and TEM confirmed successful immobilization and enzyme-nanotube interactions. BET analysis showed a decrease in surface area from 275 to 197 m²/g. FTIR spectra confirmed the appearance of protein-specific bands post-immobilization, and EDX data revealed increased nitrogen and oxygen levels, along with Fe as a cofactor marker. Thermal degradation profiles also changed, while SEM and TEM images demonstrated morphological alterations on the nanotube surfaces. Immobilized POD preserved activity at pH 4.0-6.0 and optimum temperature (30 °C), and retained functionality over multiple cycles and storage periods. These findings highlight the potential of MWCNT-supported POD systems in environmentally relevant and industrial biocatalytic applications.

Affinity chromatography-based methods for immunoglobulin G quantification present an attractive alternative to widely used nephelometry due to their simplicity, speed, and compatibility with various sample types. This study validates the efficient analytical use of commercially available POROS CaptureSelect FcXP affinity resin, a stationary phase optimized for fast recombinant human IgG purification. The analytical method was validated in a simple bind-elute mode with a cycle time of 5 minutes. Linearity was confirmed in a range of 4 µg (lower limit of quantification) to 260 µg, allowing direct quantification of IgG from human plasma without further dilution. The method demonstrated excellent precision, with %RSD values ranging from 5% to 1% and satisfactory recovery 99% on average, respectively. By decreasing the residence time from 0.2 minutes to 0.1 minutes, a cycle time of 2.5 minutes was achieved, demonstrating a powerful chromatographic method for fast and reliable IgG quantification. This method offers significant advantages over nephelometry, including reduced analysis time and enhanced compatibility with high-throughput workflows.

A polypropylene (PP) degrading bacterium derived from plastic waste site was identified as Microbacterium hydrocarbonoxydans KRS13 following 16S rDNA sequence analysis. Biodegradation assay for 90 days based on gravimetric method demonstrated a measurable weight reduction of 2.1 ± 0.10% in un-pretreated polypropylene films. The protein content and viability of KRS13 in terms of CFU/cm² was examined along with hydrophobicity and biosurfactant characterization. The polypropylene degradation was assessed through analytical techniques such as FTIR, EDX, FE-SEM and GC-MS. Robust growth of KRS13 as indicated by CFU (10⁸ CFU mL^-1) and protein content (4.71 µg mL^-1) demonstrated efficient biofilm formation and polypropylene degradation along with a removal rate of 0.00024 day^-1 (t₁/₂ = 2887.5 days). FE-SEM analysis revealed holes, cracks, and roughening on plastic surface, whereas increased oxygen (13.9%) and decreased carbon (86.1%) content noticed in EDX analysis. FTIR detected carbonyl and oxygenated groups, supported by increased keto, ester carbonyl and vinyl bond indices. Pretreatment of PP films on biodegradation also assessed by gravimetric method that displayed highest degradation (3.2 ± 0.4%) among UV treated films. Collectively, these findings highlight the significant potential of our strain M. hydrocarbonoxydans strain KRS13 as an effective PP degrader, supporting sustainable microbial-mediated biodegradation of plastic waste for PP waste management.