Applied Biochemistry and Biotechnology最新文献

In this study, the liquid lipase Yarrowia lipolytica lipase 2 (YLLip2) expressed by Pichia pastoris GS115 was used to produce biodiesel from waste oil. Four signal peptides were compared to express YLLip2 in P. pastoris, among which SP23 exhibited greater secretion performance. In a 1.3-L bioreactor with FM22 medium for 7 days incubation, the maximum YLLip2 activity and total protein content reached 895.44 ± 27.31 U/mL and 3.83 ± 0.31 g/L, respectively. Under the optimal reaction conditions of 30 °C, 20% moisture content, 50 U/g oil of enzyme dosage, and distributed methanol addition, the reaction yield reached 80.99% after 12 h. To further improve the biodiesel yield, some additives were used to assist with YLLip2. The results showed that adding G50 (approximately 1/20 of YLLip2) increased the yield by approximately 90% after 6 h cultivation without changing the enzyme dosage. Compared with previous studies, the reaction time for biodiesel production from waste oil in this study was significantly shortened. This study provides a workable method for converting low-quality feedstocks containing high free fatty acids into biodiesel using a liquid lipase as the catalyst.

The drug development process is a critical challenge in the pharmaceutical industry due to its time-consuming nature and the need to discover new drug potentials to address various ailments. The initial step in drug development, drug target identification, often consumes considerable time. While valid, traditional methods such as in vivo and in vitro approaches are limited in their ability to analyze vast amounts of data efficiently, leading to wasteful outcomes. To expedite and streamline drug development, an increasing reliance on computer-aided drug design (CADD) approaches has merged. These sophisticated in silico methods offer a promising avenue for efficiently identifying viable drug candidates, thus providing pharmaceutical firms with significant opportunities to uncover new prospective drug targets. The main goal of this work is to review in silico methods used in the drug development process with a focus on identifying therapeutic targets linked to specific diseases at the genetic or protein level. This article thoroughly discusses A-to-Z in silico techniques, which are essential for identifying the targets of bioactive compounds and their potential therapeutic effects. This review intends to improve drug discovery processes by illuminating the state of these cutting-edge approaches, thereby maximizing the effectiveness and duration of clinical trials for novel drug target investigation.

The viability of probiotic cells decreases during passage through the gastrointestinal tract. The process of probiotics encapsulation with sodium alginate and chitosan polymers was carried out to protect the Lactobacillus plantarum in adverse conditions. Lactobacillus plantarum was entrapped in sodium alginate/chitosan (SA/BChi) and sodium alginate/nano-chitosan (SA/NChi) wall materials. Encapsulating L. plantarum with SA/BChi and SA/NChi resulted in a high encapsulation efficiency % of ~ 86.41 to 91.09%. In addition, coating bacteria cells in encapsulants improved the survivability of the cells under the simulated gastrointestinal fluids by ~ 52.61% in SA/Chi and 58.04% in SA/NChi compared to 29% for unencapsulated forms. Probiotic beads under field emission-scanning electron microscopy (FE-SEM) were morphologically compact with a cracked appearance of SA/NChi beads. The Fourier transform-infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC) showed vigorous electrostatic interaction between polymers, as well as the high melting points, which corroborate the previous investigations in the field for using SA/BChi or SA/NChi as a promising encapsulating agent for ameliorating the survivability of probiotics under harsh conditions. The distinctive properties possessed by the two coatings make them excellent candidates for use as polymeric carriers in probiotic delivery systems.

Taxifolin, also known as dihydroquercetin (DHQ), is a flavonoid recognized for its potent antioxidant properties and a wide range of biological activities, including anti-tumor, antiviral, and immunomodulatory effects. Conventional extraction and chemical synthesis methods for taxifolin are often limited by low yields and associated environmental concerns. In this study, we investigated the heterologous biosynthesis of taxifolin in Yarrowia lipolytica through a combination of metabolic engineering and genome-scale metabolic modeling (GSM), complemented by flux balance analysis (FBA). We engineered Yarrowia lipolytica by introducing key biosynthetic genes and successfully synthesized taxifolin using naringenin (NAR) as a substrate, chosen for its low cost. Fermentation experiments demonstrated an optimal taxifolin yield of 10% at a substrate concentration of 200 mg/L naringenin, with a maximum yield of 26.4 mg/L taxifolin at 1 g/L naringenin. To further enhance production, we applied a marker-free Cre-loxP-based gene integration method, allowing stable genomic integration of key genes, which increased taxifolin yield to 34.9 mg/L at 1 g/L naringenin. Additionally, intermediate metabolites eriodictyol (ERI) and dihydrokaempferol (DHK) accumulated to concentrations of 89.2 mg/L and 21.7 mg/L, respectively. Furthermore, we integrated metabolic data into a GSM and applied FBA to optimize the taxifolin biosynthetic pathway. Through Pareto frontier analysis, sensitivity analysis, flux variability analysis, and single gene deletion simulations, we identified key genetic modifications that significantly enhanced taxifolin yield. Overexpression of GND1 and IDP2 increased yields by 94% and 155%, respectively, while knockout of LIP2 led to a 46% increase. Using tri-baffled shake flasks to improve oxygen supply resulted in a 120% yield increase, whereas YPG medium decreased yield by 59%, validating our model's accuracy. To ensure stable and efficient gene expression, we integrated multi-copy constructs into the ribosomal DNA (rDNA) locus of Yarrowia lipolytica, doubling taxifolin production. These results demonstrate the effectiveness of GSM and FBA in addressing bottlenecks in microbial taxifolin biosynthesis and provide a basis for future optimization and large-scale production.

Large quantities of by-products are generated after processing of rose snapper (Lutjanus guttatus), such as viscera, head, tail, skin, and bones, which are considered a potential source of valuable molecules. Therefore, the aim of the present study was the biochemical characterization of alkaline proteases isolated from the intestines of L. guttatus and the evaluation of their stability against different chemical denaturants (salts, surfactants/reducing agents, organic solvents, and commercial detergent formulations). In addition, the efficiency to hydrolyze proteins from rose snapper wastes (head, tail, skin, and muscle trimmings) by Alcalase® and alkaline protease extract (APE) isolated from Lutjanus guttatus intestine was evaluated. The APE exhibited a maximum activity at pH 12 and 45 °C and high stability at pH and temperature ranges from 9 to 12 and 10 to 40 °C, respectively. Assays with specific protease inhibitors indicated that trypsin and chymotrypsin are the main types of proteases in APE. An 80% of the proteolytic activity was retained in the presence of 25% NaCl and was stable in the presence of the reducing agent DTT; however, it lost around 70% of proteolytic activity in the presence of 2-mercaptoethanol. The enzymatic activity of APE was maintained above 60% in methanol, ethanol, and propanol as well as in liquid commercial detergents. Alkaline proteases from rose snapper exhibited higher hydrolytic efficiency, compared to the microbial enzyme Alcalase when protein from L. guttatus wastes were hydrolyzed. According to these results, the integral exploitation of rose snapper could be reached by proper usage of its by-products, creating a baseline to promote circular economy.

Nanozymes are a class of nanomaterials that are capable of mimicking the activities of natural enzymes. They are currently receiving considerable attention due to their advantageous properties. The objective of this study is to provide a comprehensive analysis of the advancements and trends in nanozymes for microbial theranostics research over the past decade through a detailed bibliometric approach. For this purpose, an effective search query was formulated, and relevant publications from 2013 to 2023 were collected from the Web of Science Core Collection database. Subsequently, the following softwares were employed for analysis: VOSviewer, the Bibliometrix R package, and GraphPad Prism 8.0.2. The findings revealed a statistically significant positive correlation (r = 0.993; p < 0.0001) between publications and citations, in addition to an important growth rate of scientific output of approximately 28.90%. China, India, and the USA were the most productive countries, whereas progress in low- and middle-income countries remained constrained. The Chinese Academy of Sciences was the most productive institution, and remarkably almost the top 10 productive authors were from China. Regarding keywords analysis, current research hotspots are primarily concentrated on the application of nanozymes in anti-biofilm-related research, antibacterial activity and therapy, the development of biosensors for microbial detection and control, and the advancement of wound disinfection and therapy.

Zinc oxide nanoparticles (ZnO-NPs) can enhance zinc bioavailability in plants, improving crop nutritional quality and addressing global zinc deficiency. This study aimed to investigate the effects of zinc oxide nanoparticles (ZnO-NPs), obtained by a green synthesis method, on the growth, yield parameters, and zinc content of rice plants. In the study, two different application strategies of ZnO-NPs on rice plants were evaluated, i.e., foliar spray and seed priming. To compare the effects of these application strategies, rice plants were treated with ZnO-NPs at two different concentrations, 25 mg/L and 50 mg/L. Effects of ZnO-NPs on plant growth (shoot and root length, fresh and dry mass), chlorophyll and carotenoid content, grain yield and zinc content were investigated. The results showed that both ZnO-NP application methods increased rice growth and yield, especially the combined method (seed priming + foliar spray) provided the highest efficiency. It was observed that seed zinc content was increased up to 10% by seed priming method and foliar spray application increased the zinc content up to 23% while the combination of seed priming and foliar spray increased zinc content up to 122%. In general, applications at 50 mg/L concentration increased more than 25 mg/L. These results indicate that ZnO-NPs can be an effective tool for zinc deficiency management in agricultural practices and can improve the yield and nutritional content of staple foods such as rice.

Chitin, a natural organic compound with content slightly lower than cellulose, is also known for chitosan, a substance derived from chitin through deacetylation. In this experiment, preliminary screening was conducted using the plate discoloration circle method, leading to the selection of a high-yield CDA-producing strain from 28 candidates through rescreening. Morphological characteristics and 16S rDNA sequence analysis revealed 99.93% homology with Enterobacter sichuanensis strain N24, thus naming this strain Enterobacter strain ZCDA27. Initial fermentation of the strain yielded CDA activity of 9.29 U/mL. Single-factor optimization was then performed, followed by a PB test to screen for significant factors affecting enzyme production. The response surface method was used to further optimize the fermentation conditions. The optimal fermentation conditions for the carbon source, nitrogen source, metal ion, fermentation temperature, time, liquid volume, and initial pH were explored. Significant factors affecting enzyme production, including MgSO₄, initial medium pH, and fructose levels, were identified using the PB test. Finally, the fermentation conditions of ZCDA27 were optimized using the Box-Behnken design combined with RSM, which comprised fructose at 1.020%, magnesium sulfate at 0.016%, and peptone at 1%. The fermentation conditions included a temperature of 37, initial pH of 7.1, rotation speed of 140 × g, fermentation time of 28 h, inoculation amount of 2%, and liquid volume of 40%. Under these conditions, the enzyme activity of ZCDA27 reached 14.52 U/mL, a 1.6-fold increase from the pre-optimization levels. In summary, this study provides an experimental foundation for further development and application of Enterobacter spp. ZCDA27 CDA.

Recessive shrunken2 (sh2)-based sweet corn is preferred worldwide as it possesses higher sugar and extended shelf life. However, traditional sh2-based sweet corn is poor in vitamin A and vitamin E. Here, parental lines of two sh2-based sweet corn hybrids, viz. PSSC-2 and ASKH-2, were targeted for introgression of β-carotene hydroxylase 1 (crtRB1) and γ-tocopherol methyltransferase (vte4) genes through marker-assisted backcross breeding. Seeds with sh2sh2sh2 genotype in the endosperm were selected based on the shrunken phenotype in BC₁F₁, BC₂F₁ and BC₂F₂ generations. Gene-based markers, viz. 3'-TE-InDel and 118-InDel specific for crtRB1 and vte4, respectively, were successfully utilized for foreground selection in BC₁F₁, BC₂F₁ and BC₂F₂. Reconstituted hybrids showed high provitamin A (proA: 19.52 ± 0.52 µg/g) with a maximum of 7.8-fold increase over original hybrids (ASKH-2 and PSSC-2: 3.33 ± 0.28 µg/g). High α-tocopherol (20.75 ± 0.44 µg/g) and α/γ-tocopherol ratio (0.55 ± 0.02) with an average enhancement of 2.3- and 1.7-fold, respectively, was recorded among reconstituted hybrids over original versions (α-tocopherol: 9.21 ± 0.33 µg/g, α/γ-tocopherol ratio: 0.31 ± 0.01). The average yield of reconstituted hybrids (11.40 ± 0.22 t/ha) was at par with the original sweetcorn hybrids (11.60 ± 0.20 t/ha). This is the first report of stacking sh2, crtRB1 and vte4 genes to improve nutritional quality in sweet corn. These biofortified sweet corn hybrids hold immense significance to alleviate micronutrient malnutrition.

Gentiana kurroo Royle, a critically endangered Himalayan herb, is valued in treating leucoderma, syphilis, bronchial asthma, hepatitis, etc. The current investigation performed quantitative and qualitative phytochemical analysis of G. kurroo root extracts prepared in chloroform, methanol, and ethyl acetate. The phenolic and flavonoid contents were the highest in methanol and chloroform extract, respectively. Several pharmacologically important compounds were identified through gas chromatography-mass spectrometry. Antioxidant analysis revealed methanolic extract to be the most efficient scavenger of 2,2-diphenyl-1-picrylhydrazyl (IC₅₀ = 114 µg mL^-1), hydrogen peroxide (IC₅₀ = 109.9 µg mL^-1), and superoxide (IC₅₀ = 74.63 µg mL^-1) radicals. Gentiopicroside was isolated from the methanolic root extract through silica-gel column-chromatography, and the characterization of concentrated fractions was achieved employing various analytical techniques. Pertaining to silver nanoparticle (GkAgNPs) synthesis, different physicochemical parameters were optimized and it was observed that root extract treated with silver-nitrate (0.5 mM) at 60 °C and incubated in dark for at least 120 min after initial color change, yielded GkAgNPs optimally. GkAgNPs were anisotropic and polydisperse and exhibited characteristic surface plasmon resonance (424 nm), crystalline face-centered cubic geometry, size (50-300 nm), and zeta-potential (- 16.3 mV). FT-IR spectra indicated the involvement of phenols and flavonoids in AgNPs synthesis. GkAgNPs were evidenced as strongly cytotoxic (IC₅₀ = 1.964 µg mL^-1) against HeLa cells and also showed deformed cellular morphology, a significant reduction in viable cell counts and colony-forming efficiency (4.08%). The findings suggest potential applications in drug development for treating serious human diseases. To the best of our knowledge, this study represents the first report on the isolation of gentiopicroside, the bio-fabrication of GkAgNPs using G.kurroo root extract, and their strong bioefficacy against HeLa cells.