Search Results (176)

A novel optimized bioactive lipid (OBL) rich in long-chain polyunsaturated omega-3 fatty acids (n-3 LCPUFA) was synthesized through enzymatic acidolysis using concentrated belly oil from rainbow trout (Oncorhynchus mykiss) (CB) and tocopherols obtained from cold-pressed maqui seed oil (Aristotelia chilensis (Mol.) Stuntz) (MSO) under supercritical CO₂ conditions. The reaction was catalyzed by Candida antarctica lipase B (CALB) and optimized using a 3² response surface design with 12 experimental runs and three central points, considering pressure (100–300 bar) and temperature (50–80 °C) as independent variables. The response variables included the concentrations of EPA, DHA, α-, β-, γ-, and δ-tocopherols, as well as β- and γ-tocotrienols. MSO contained 10.63, 25.62, and 53.55 g·100 g⁻¹ total fatty acids (TFAs) of α-linolenic, oleic, and linoleic acids, respectively, together with 280.95 mg α-tocopherol·kg⁻¹ and 89.75 mg β-tocotrienol·kg⁻¹. The CB contained 49.57 g EPA + DHA·100 g⁻¹ TFAs. Optimal conditions (72.7 °C and 248.9 bar), experimentally validated at the RSM-predicted point, yielded an OBL containing 41.28 g EPA + DHA·100 g⁻¹ TFAs, 0.39 mg α-tocopherol·kg⁻¹, 3.54 mg β-tocopherol·kg⁻¹, 18.48 mg β-tocotrienol·kg⁻¹, 6.92 mg γ-tocopherol·kg⁻¹, and 16.36 mg γ-tocotrienol·kg⁻¹. Oil quality evaluation using official AOCS methods showed that the OBL exhibited an acceptable oxidative status within international regulatory limits while retaining a measurable phenolic content and intermediate antioxidant capacity derived from MSO. This study demonstrates the successful synthesis of a stable OBL from agro-industrial by-products as a sustainable source of functional ingredients for food, nutraceutical, and cosmetic applications. Full article

Egg quality is a critical economic trait in poultry production, influencing consumer preference and production efficiency. The genetic and epigenetic regulation of egg quality involves complex biological pathways across various traits such as shell quality, albumen composition, and yolk biochemistry. This review synthesizes recent advances in the genetic, molecular, and epigenetic mechanisms that determine poultry egg quality. Specifically, it focuses on external traits such as eggshell strength, color, and thickness, and internal traits including albumen height, yolk composition, and the Haugh unit. Through genome-wide association studies (GWAS), quantitative trait loci (QTL) mapping, whole-genome sequencing (WGS), and multi-omics approaches, key candidate genes such as OC-116, CALB1, CA2 (shell formation), OVAL, SPINK5, SERPINB14 (albumen quality), and FGF9, PIAS1, NOX5 (lipid metabolism) have been identified. These genes play a pivotal role in shell biomineralization, albumen protein regulation, and yolk lipid transport. This review also explores the heritability of these traits, emphasizing the challenges posed by polygenic architecture and the influence of environmental factors. Furthermore, it addresses the dynamic spatiotemporal regulation of egg quality traits, including epigenetic layers such as DNA methylation, histone modifications, RNA methylation, and post-translational protein modifications. This paper highlights the application of these findings to breeding programs via genomic selection, marker-assisted breeding, and epigenetic engineering approaches. Future directions for precision breeding and the development of functional eggs with enhanced quality are also discussed. Full article

Fast pyrolysis of vegetable oils and residues generates bio-oil (BO), a renewable hydrocarbon source with high acidity that limits its direct use in refineries. In this study, BOs were produced from refined soybean oil (RSO) and waste cooking oil (WCO) at 525 °C in a continuous bench-scale pyrolysis at 525 °C, with a 390 ± 8 g h⁻¹ feed rate, under steady-state conditions. The resulting bio-oils exhibited high acidity (acid index of 145 and 127 mg KOH g⁻¹, respectively) and elevated olefinic and oxygen contents, making them corrosive and unsuitable for co-refining with petroleum. To reduce acidity, ethyl esterification was performed using lipase B from Candida antarctica (CALB), using a Box–Behnken 3³ factorial design. Variables included temperature (40–60 °C), bio-oil:ethanol mass ratio (1:1–1:5), and catalyst concentration (3–10% w/w). The acid index was reduced by up to 76%, with optimal conditions (62 °C, 1:1 mass ratio, 11% CALB) yielding a final value of 28 mg KOH g⁻¹. Similar reductions were obtained for waste cooking oil bio-oil, confirming robustness across feedstocks. CALB retained over 70% activity after three cycles, demonstrating stability. This enzymatic esterification process shows strong potential for lowering bio-oil acidity, enabling integration into petroleum refineries, diversifying feedstocks, and advancing renewable fuel production. Full article

Cold-active lipolytic enzymes enable low-temperature biocatalysis, but remain underexplored in Antarctic actinomycetes. Here, we report the discovery and first-step characterization of a CALB-like cold-active lipolytic enzyme (PanLip) from Pseudonocardia antarctica. Sequence and structure analyses revealed a canonical α/β-hydrolase fold with a conserved Ser–Asp–His triad and short helical elements around the pocket reminiscent of CALB’s α5/α10 lid. Mature PanLip was expressed primarily as inclusion bodies in E. coli; an N-terminally truncation (PanLipΔN) improved solubility and PanLipΔN was purified by Ni–NTA. Far-UV CD confirmed a folded α/β architecture. PanLipΔN favored short-chain substrates (p-NPA, k_cat/K_M = 2.4 × 10⁵ M⁻¹·s⁻¹) but also showed measurable hydrolytic activity toward natural triglycerides, consistently with a lipase-family esterase. The enzyme showed an activity optimum near 25 °C and pH 8.0. The enzyme tolerated low salt (maximal at 0.1 M NaCl), mild glycerol, and selected organic solvents (notably n-hexane), but was inhibited by high salt, Triton X-100, and SDS. AlphaFold predicted high local confidence for the catalytic core; DALI placed PanLip closest to fungal lipases (AFLB/CALB). Temperature-series MD and CABS-flex indicated enhanced surface breathing and flexible segments adjacent to the active site—including a region topologically matching CALB α10—supporting a flexibility-assisted access mechanism at low temperature. Structure-based MSAs did not support a cold adaptation role for the reported VDLPGRS motif. Taken together, these findings position PanLip as a promising cold-active catalyst with CALB-like access control and potential for low-temperature biocatalysis. Full article

Ethyl butyrate is a typical flavor ester with pineapple-banana scents, but the poor yield from natural fruits limits its feasibility in food and fragrance industries. In this study, dendritic fibrous nano-silica (DFNS) was hydrophobically modified with octyl groups (DFNS-C₈) to immobilize Candida antarctica lipase B (CALB) for solvent-free esterification of ethyl butyrate. The immobilized lipase CALB@DFNS-C₈, with the enzyme loading of 354.6 mg/g and the enzyme activity of 0.064 U/mg protein, achieved 96.0% ethyl butyrate conversion under the optimum reaction conditions where the molar ratio of butyric acid to ethanol was 1:3, with a reaction temperature and time of 40 °C and 4 h. Under the solvent-free catalytic reactions, CALB@DFNS-C₈ presented the maximum catalytic efficiency of 35.1 mmol/g/h and retained 89% initial activity after ten reuse cycles. In addition, the immobilized lipase can efficiently catalyze the synthesis of various flavor esters (such as butyl acetate, hexyl acetate, butyl butyrate, etc.) and exhibits excellent thermostability and solvent tolerance. A molecular docking simulation reveals that the hydrophobic cavity around the catalytic triad stabilizes the acyl intermediate and ensures the precise orientation of both acid and alcohol substrates. This work provides new insights into the sustainable production of flavor esters using highly active and recyclable immobilized lipases through rational carrier hydrophobization and structural confinement design. Full article

There are 25 crystal structures of Lipase B from Candida antarctica (CalB) that have been previously reported. In this study, we report the first CalB crystal structure that shows the assumed pro-peptide region at the N-terminus (Ala19–Arg25). This 1.45 Å structure shows that this segment of seven amino acids is an extension of the N-terminal loop and that it does not interact with or effect conformational changes in the flexible lid domain, which covers the active site of the enzyme. As such, this region is unlikely to be a classical pro-peptide. Full article

A promising area of emulsion system research is biocatalysis, particularly lipase-catalyzed reactions. Recognizing the potential of emulsions stabilized by both an emulsifier and a polymer, we conducted experimental studies to evaluate the effectiveness of a dual-stabilized system. In this study, we examined the effect of an emulsion system containing an anionic emulsifier (sodium dodecyl sulfate (SDS)) and a gelling agent (carbomer (Carbopol^® Ultrez 10, cross-linked poly(acrylic acid), PAA)) on the catalytic activity of Candida antarctica lipase B (CALB), in both its free and immobilized forms. The results demonstrated that the activity of immobilized CALB in emulsions containing 5.0% SDS and 0.1% carbomer was significantly higher than in emulsions with 5.0% SDS alone (124.44 ± 5.09 vs. 104.44 ± 5.09 U/g of support). At 2.5% SDS, the addition of 0.1% carbomer also enhanced the activity of immobilized CALB (121.11 ± 1.92 vs. 93.33 ± 3.33 U/g of support, p < 0.05). Notably, in emulsions with 2.5% or 5.0% SDS and 0.1% carbomer, hyperactivation of immobilized CALB was observed, with activity exceeding that of the free form by approximately six-fold. These findings highlight the beneficial effect of combining SDS (2.5% or 5.0%) and 0.1% carbomer to enhance the catalytic activity of immobilized CALB in emulsion-based formulations. Full article

The application of lipases in biphasic oil–water emulsions offers an efficient and sustainable alternative to conventional chemical synthesis. However, the natural immiscibility of these phases is a substantial limitation. To address this issue, we proposed a dual-stabilized emulsion system combining a nonionic emulsifier (Kolliphor^® CS 20) and cross-linked polyacrylic acid (Carbopol^® Ultrez 10), exceeding conventional single-stabilized systems. The activity of Candida antarctica lipase B (CALB), both in its free form and immobilized onto an IB-D152 support, was investigated in the prepared emulsion system. The olive oil emulsion stabilized with 10.0% Kolliphor^® CS 20 and 0.1% Carbopol^® Ultrez 10 significantly enhanced the lipolytic activity of immobilized CALB (156.27 ± 3.91 U/g of support), compared to the activity obtained in the emulsion stabilized only with 10.0% Kolliphor^® CS 20 (71.11 ± 3.86 U/g of support). On the other hand, the activity of immobilized CALB in the emulsion containing 5.0% Kolliphor^® CS 20 and 0.1% Carbopol^® Ultrez 10 (62.22 ± 3.85 U/g of support) was lower than in the corresponding system without Carbopol^® Ultrez 10 (72.03 ± 4.63 U/g of support), stabilized with only 5.0% Kolliphor^® CS 20. Furthermore, immobilization onto IB-D152 led to lipase hyperactivation, with activity approximately eight-fold higher than that of free CALB. This dual emulsion stabilization strategy not only improves emulsion stability but also enhances lipase activity, offering new opportunities for scalable, high-performance biocatalysis using emulsions in industrial applications. Full article

The growing need for developing safer and more effective methods for obtaining enantiomers of chiral compounds, particularly those with pharmacological activity, highlights the potential of biocatalysis as an appropriate pharmaceutical research direction. However, low catalytic activity and stability of free enzymes are often among the substantial limitations to the wide application of biocatalysis. Therefore, to overcome these obstacles, new technological procedures are being designed. In this study, we present optimized protocols for the immobilization of Candida antarctica lipase B (CALB) on an octyl- agarose support, ensuring high enantioselectivity in an organic reaction medium. The immobilization procedures (with drying step), including buffers with different pH values and concentrations, as well as the study of the influence of temperature and immobilization time, were presented. It was found that the optimal conditions were provided by citrate buffer with a pH of 4 and a concentration of 300 mM. The immobilized CALB on the octyl-agarose support exhibited high catalytic activity in the kinetic resolution of (R,S)-1-phenylethanol via enantioselective transesterification with isopropenyl acetate in 1,2-dichloropropane (DCP), as a model reaction for lipase activity monitoring on an analytical scale. HPLC analysis demonstrated that the (R)-1-phenylethyl acetate was obtained in an enantiomeric excess of ee_p > 99% at a conversion of approximately 40%, and the enantiomeric ratio was E > 200. Thermal and storage stability studies performed on the immobilized CALB octyl-agarose support confirmed its excellent stability. After 7 days of thermal stability testing at 65 °C in a climatic chamber, the (R)-1-phenylethyl acetate was characterized by enantiomeric excess of ee_p > 99% at a conversion of around 40% (similar values of catalytic parameters to those achieved using a non-stored lipase). The documented high catalytic activity and stability of the developed CALB-octyl-agarose support allow us to consider it as a useful tool for enantioselective transesterification in organic medium. Full article

Hydroxytyrosol (HT), the primary phenolic compound in virgin olive oil, has notable cardiovascular benefits, particularly in preventing low-density lipoprotein (LDL) oxidation. However, its hydrophilicity limits its solubility and integration into lipid-based formulations. This study aimed to enhance its lipophilicity by synthesizing hydroxytyrosyl eicosapentaenoate (HT-EPA), a derivative of HT and eicosapentaenoic acid (EPA), using a one-step enzymatic catalysis with lipase B from Candida antarctica (CALB). The reaction, performed as a suspension of HT in ethyl eicosapentaenoate (Et-EPA) (1:9 molar ratio) under vacuum, achieved higher yields and shorter reaction times than previously reported, with a purity exceeding 98%, confirmed by ¹H-NMR. For the first time, the antioxidant capacity of HT-EPA in comparison with other natural antioxidants was assessed using the FRAP assay, while its oxidative stability in an omega-3-rich oil matrix was evaluated via the Rancimat method. HT-EPA and hydroxytyrosyl acetate (HT-Ac) displayed antioxidant activity comparable to HT but significantly higher than α-tocopherol, a common food antioxidant. Given the scarcity of effective lipid-soluble antioxidants, HT-EPA represents a promising candidate for omega-3 nutraceuticals, offering enhanced stability and potential health benefits. This study provides a simple, efficient, and scalable strategy for developing functional lipid-based formulations with cardioprotective potential by improving HT solubility while preserving its antioxidant properties. Full article

Consumers tend to favor natural dietary supplements to improve their health. However, vendors rarely cite scientific evidence to justify these claims. In the case of poppy oil, it is often mentioned as having a positive effect on Ca metabolism-related disorders, but no proof has been provided. Therefore, the aim of our trial was to test the possible effect of poppy oil on calcium metabolism using Japanese quail layers as a model animal. A total of 120 four-week-old quail were divided into three dietary treatments (four cages per treatment, with ten birds in each cage): a control group (using sunflower oil as the energy source in the diet), 0.5%, and 1% poppy oil supplementation (replacing sunflower oil). Egg production, eggshell thickness, eggshell strength, and egg yolk color were investigated. Ca retention was determined using the acid-insoluble ash method. At the end of the experiment, two birds per cage were sacrificed, and uterus and jejunum samples were collected for gene expression analyses. Poppy oil supplementation improved egg production in terms of intensity, egg weight, and eggmass production. The thickness and eggshell strength decreased when 1% poppy oil was fed to the animals, while Ca retention improved. Poppy oil supplementation increased the expression of Ca transporter genes (CALB1, SLC8A1, and SLC26A9) in the uterus and ITPR1 in the jejunum. Our results indicate a possible effect of poppy oil on Ca metabolism. Further studies are needed to identify the active compound and to understand the mode of action. Full article

Natural compounds with significant bioactive properties can be found in abundance within biomasses. Especially prominent for their anti-inflammatory, neuroprotective, antibacterial, and antioxidant activities are cinnamic acid derivatives (CAs). Ferulic acid (FA), a widely studied phenylpropanoid, exhibits a broad range of therapeutic and nutraceutical applications, demonstrating antidiabetic, anticancer, antimicrobial, and hepato- and neuroprotective activities. This research investigates the green enzymatic synthesis of innovative and potentially bifunctional prodrug derivatives of FA, designed to enhance solubility and stability profiles. Selective esterification was employed to conjugate FA with xylitol, a biobased polyol recognized for its bioactive antioxidant properties and safety profile. Furthermore, by exploiting t-amyl alcohol as a green solvent, the enzymatic synthesis of the derivative was optimized for reaction parameters including temperature, reaction time, enzyme concentration, and molar ratio. The synthesized derivative, xylitol monoferulate (XMF), represents a novel contribution to the literature. The comprehensive characterization of this compound was achieved using advanced spectroscopic methods, including ¹H-NMR, ¹³C-NMR, COSY, HSQC, and HMBC. This study represents a significant advancement in the enzymatic synthesis of high-value biobased derivatives, demonstrating increased biological activities and setting the stage for future applications in green chemistry and the sustainable production of bioactive compounds. Full article

The current paper reports the asymmetric synthesis of a focused library of enantiostructured triacylglycerols (TAGs) constituting a potent drug of the NSAID type (ibuprofen or naproxen) along with a pure bioactive n-3 polyunsaturated fatty acid (PUFA) intended as a novel type of prodrug. In this second category, a TAG prodrug of the terminal sn-1 or sn-3 position of the glycerol skeleton is acylated with a single saturated medium-chain fatty acid (C6, C8, C10, or C12), and another with the drug entity; the PUFA (EPA or DHA) is located in the sn-2 position. This was accomplished by a six-step chemoenzymatic approach, two of which were promoted by a lipase, starting from enantiopure (R)- and (S)-solketals. The highly regioselective immobilized Candida antarctica lipase (CAL-B) played a crucial role in the regiocontrol of the synthesis. The most challenging key step involved the incorporation of the drugs that were activated as oxime esters by the lipase exclusively in the terminal position of glycerol that is protected as a benzyl ether. All combinations, a total of 32 such prodrug TAGs, were prepared, isolated, and fully characterized, along with 24 acylglycerol intermediates, obtained in very-high-to-excellent yields in the majority of cases. Full article

Proteins involved in synaptic transmission in normal hearing, acoustic stimulation, and tinnitus were identified using protein–protein interaction (PPI) networks. The gene list for tinnitus was compiled from the GeneCards database using the keywords “synaptic transmission” AND “inferior colliculus” AND “tinnitus” (Tin). For comparison, two gene lists were built using the keywords “auditory perception” (AP) and “acoustic stimulation” (AS). The STRING and the Cytoscape data analyzer were used to identify the top two high-degree proteins (HDPs) and the corresponding high-score interaction proteins (HSIP). The top1 key proteins of the AP and AS processes are BDNF and the receptor NTRK2; the top2 key proteins in the AP process are PVALB, together with GAD1, CALB1, and CALB2, which are important for the balance of excitation and inhibition. In the AS process, the top2 key proteins are FOS, CREB1, EGR1, and MAPK1, reflecting an activated state. The top1 key proteins of the Tin process are BDNF, NTRK3, and NTF3; these proteins are associated with the proliferation and differentiation of neurons and indicate the remodeling of synaptic transmission in IC. The top2 key proteins are GFAP and S100B, indicating a role for astrocytes in the modulation of synaptic transmission. Full article

Enzymes play an essential role in many different industries; however, their operating conditions are limited due to the loss of enzyme activity in the presence of proteases and at temperatures significantly above physiological conditions. One way to improve the stability of these enzymes against high temperatures and proteases is to encapsulate them in protective shells or virus-like particles. This work presents a streamlined, three-step, cell-free protein synthesis (CFPS) procedure that enables rapid in vitro enzyme production, targeted encapsulation in protective virus-like particles (VLPs), and facile purification using a 6× His-tag fused to the VLP coat protein. This process is performed in under 12 h and overcomes several limitations of enzyme encapsulation, such as the control of packing density, speed, and complexity of the process. Here, we encapsulate the enzyme Candida antarctica lipase B in the VLP from the bacteriophage Qβ, while in the presence of a linking RNA aptamer. The encapsulated enzymes largely retained their activity in comparison to the free enzymes. Additionally, when subjected to 90 °C temperatures or 5 h incubation with proteases, the encapsulated enzymes maintained their activity, whereas the free enzymes lost their activity. In this work, we also demonstrate control over packing density by achieving packing densities of 4.7 and 6.5 enzymes per VLP based off the concentration of enzyme added to the encapsulation step. Full article