Effect of lysozyme combined with hydrothermal pretreatment on excess sludge and anaerobic digestion.

Anaerobic digestion (AD) is widely employed for sludge stabilization and waste reduction. However, the slow hydrolysis process hinders methane production and leads to prolonged sludge issues. In this study, an efficient and eco-friendly lysozyme pre-treatment method was utilized to address these challenges. By optimizing lysozyme dosage, hydrolysis and cell lysis were maximized. Furthermore, lysozyme combined with hydrothermal pretreatment enhanced overall efficiency. Results indicate that: (1) When lysozyme dosage reached 90 mg/g TS after 240 min of pretreatment, SCOD, soluble polysaccharides, and protein content reached their maxima at 855.00, 44.09, and 204.86 mg/L, respectively. This represented an increase of 85.87%, 365.58%, and 259.21% compared to the untreated sludge. Three-dimensional fluorescence spectroscopy revealed the highest fluorescence intensity in the IV region (soluble microbial product), promoting microbial metabolic activity. (2) Lysozyme combined with hydrothermal pretreatment significantly increased SCOD, soluble proteins, and polysaccharide release from sludge, reducing SCOD release time. Orthogonal experiments identified Group 3 as the most effective for SCOD and soluble polysaccharide release, while Group 9 released the most soluble proteins. The significance order of factors influencing SCOD, soluble proteins, and polysaccharide release is hydrothermal temperature > hydrothermal time > enzymatic digestion time.(3) The lysozyme-assisted hydrothermal pretreatment group exhibited the fastest release and the highest SCOD concentration of 8,135.00 mg/L during anaerobic digestion. Maximum SCOD consumption and cumulative gas production increased by 95.89% and 130.58%, respectively, compared to the control group, allowing gas production to conclude 3 days earlier.

Indicator displacement-based colorimetric assay for dibutyl phthalate in pharmaceutical products with titanium(IV)-pyridylazo resorcinol (PAR).

Taking advantage of the competitive binding affinity towards Ti(IV) between 4-(2-pyridylazo) resorcinol (PAR) and phthalate, a simple indicator displacement (ID)-based colorimetric assay was designed for indirect determination of a well-known phthalic acid ester, dibutyl phthalate (DBP). The indicator PAR and Ti(IV) formed a purplish-red-colored Ti(IV)-PAR complex (λmax = 540 nm) at a 1:1 ratio. In the presence of pre-hydrolyzed DBP, colorless complex formation of phthalate ion (emerging from alkaline hydrolysis of DBP) with Ti(IV) resulted in a hypsochromic shift in absorbance maximum, accompanying a color change from purplish-red to yellowish-orange (λmax = 390 nm) by the release of PAR from Ti(IV)-PAR system. Based on this mechanism, the linear response range of the system for DBP was found to lie between 0.16 and 0.37 mmol L-1 with an experimental detection limit of 11.6 µmol L-1. The recommended Ti(IV)-PAR system was successfully applied to DBP-containing pharmaceutical products (as real sample) after a simple clean-up process for removing possible water-soluble interferents. The analytical results obtained from the recommended method (by applying the standard addition approach) and the reference liquid chromatography-tandem mass spectrometric (LC-MS/MS) method were statistically compared using DBP-extract of the drug samples. Consequently, a simple and selective colorimetric ID strategy was proposed for the analysis of DBP in pharmaceuticals for the first time.

Improvement of Bacillus subtilis PI agarase production, hydrolysate scavenging capability assessment, and saccharification of algal biomass for green ethanol generation.

The goal of the current work was to optimize the growth parameters needed to manufacture agarase enzyme from a non-marine PI strain of Bacillus subtilis on an agar-based medium. Using Plackett-Burman design (PBD), nine process parameters were evaluated, and agar, peptone, and yeast-extract were identified as the most significant independent factors influencing agarase production with confidence levels more than 90%. To evaluate the optimal concentrations of the indicated process parameters on agarase production, the Box-Behnken design (BBD) was applied. After optimization, B. subtilis strain PI produced 119.8 U/ml of agarase, representing a 1.36-fold increase. In addition the agar hydrolysate fermented products contain the liberated oligosaccharide acts as strong antioxidant which has 62.4% scavenging activity. Also, the agarase yields increased (1141.12, 1350.253, 1684.854 and 1921.863 U/ml) after substitution the agar with algal biomass of Carolina officinalis at different concentrations (2, 5, 10 and 15%), respectively. After completing the saccharification process, the resulted hydrolysate was used to produce ethanol through fermentation with Pichia pastoris yeast strain as an economical method giving yields (6.68317, 7.09748, 7.75648 and 8.22332 mg/ml), that are higher than using yeast extract peptone dextrose (YPD) medium (4.461 mg/ml).

Real-Time Monitoring of Hydrolysis Reactions of Pyrophosphates with Dissolution Dynamic Nuclear Polarization.

Dissolution dynamic nuclear polarization (d-DNP) has enabled applications such as the real-time monitoring of chemical reactions. Such applications are mainly for 13C and 15N spins with long spin-lattice relaxation times in the molecules of interest. However, the only applications for phosphorus using d-DNP are pH imaging and nucleation during crystallization due to the short relaxation times. Here we show that it is possible to observe enzyme reactions using d-DNP with phosphorus. Hyperpolarized 31P spins in pyrophosphate were obtained using bullet-DNP, which requires less dilution of highly polarized solid samples. Real-time monitoring of the hydrolysis reaction of pyrophosphate by inorganic pyrophosphatase from baker's yeast at physiological pH and was successfully achieved and the reaction rate was determined. This is an important reaction for a wide range of applications related to medicine, agriculture, and quantum life science.

Sources and control of impurity during one-pot enzymatic production of dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) has a promising market due to its capacity to regulate human hormone levels as well as preventing and treating various diseases. We have established a chemical esterification coupled biocatalytic-based scheme by lipase-catalyzed 4-androstene-3,17-dione (4-AD) hydrolysis to obtain the intermediate product 5-androstene-3,17-dione (5-AD), which was then asymmetrically reduced by a ketoreductase from Sphingomonas wittichii (SwiKR). Co-enzyme required for KR is regenerated by a glucose dehydrogenase (GDH) from Bacillus subtilis. This scheme is more environmentally friendly and more efficient than the current DHEA synthesis pathway. However, a significant amount of 4-AD as by-product was detected during the catalytic process. Focused on the control of by-products, we investigated the source of 4-AD and identified that it is mainly derived from the isomerization activity of SwiKR and GDH. Increasing the proportion of glucose in the catalytic system as well as optimizing the catalytic conditions drastically reduced 4-AD from 24.7 to 6.5% of total substrate amount, and the final yield of DHEA achieved 40.1 g/L. Furthermore, this is the first time that both SwiKR and GDH have been proved to be promiscuous enzymes with dehydrogenase and ketosteroid isomerase (KSI) activities, expanding knowledge of the substrate diversity of the short-chain dehydrogenase family enzymes. KEY POINTS: • A strategy of coupling lipase, ketoreductase, and glucose dehydrogenase in producing DHEA from 4-AD • Both SwiKR and GDH are identified with ketosteroid isomerase activity. • Development of catalytic strategy to control by-product and achieve highly selective DHEA production.

Analysis of Polypeptides by Amino Acid Analysis.

Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.

Angiotensin-Converting Enzyme and Renin-Inhibitory Activities of Protein Hydrolysates Produced by Alcalase Hydrolysis of Peanut Protein.

Hypertension is a major controllable risk factor associated with cardiovascular disease (CVD) and overall mortality worldwide. Most people with hypertension must take medications that are effective in blood pressure management but cause many side effects. Thus, it is important to explore safer antihypertensive alternatives to regulate blood pressure. In this study, peanut protein concentrate (PPC) was hydrolyzed with 3-5% Alcalase for 3-10 h. The in vitro angiotensin-converting enzyme (ACE) and renin-inhibitory activities of the resulting peanut protein hydrolysate (PPH) samples and their fractions of different molecular weight ranges were determined as two measures of their antihypertensive potentials. The results show that the crude PPH produced at 4% Alcalase for 6 h of hydrolysis had the highest ACE-inhibitory activity with IC50 being 5.45 mg/mL. The PPH samples produced with 3-5% Alcalase hydrolysis for 6-8 h also displayed substantial renin-inhibitory activities, which is a great advantage over the animal protein-derived bioactive peptides or hydrolysate. Remarkably higher ACE- and renin-inhibitory activities were observed in fractions smaller than 5 kDa with IC50 being 0.85 and 1.78 mg/mL. Hence, the PPH and its small molecular fraction produced under proper Alcalase hydrolysis conditions have great potential to serve as a cost-effective anti-hypertensive ingredient for blood pressure management.

In Vitro Multi-Bioactive Potential of Enzymatic Hydrolysis of a Non-Toxic Jatropha curcas Cake Protein Isolate.

The Jatropha curcas cake, a protein-rich by-product of biofuel production, was the subject of our study. We identified and quantified the ACE inhibitory, antioxidant, and antidiabetic activities of bioactive peptides from a Jatropha curcas L. var Sevangel protein isolate. The protein isolate (20.44% recovered dry matter, 38.75% protein content, and 34.98% protein yield) was subjected to two enzyme systems for hydrolysis: alcalase (PEJA) and flavourzyme (PEJF), recording every 2 h until 8 h had passed. The highest proteolytic capacity in PEJA was reached at 2 h (4041.38 ± 50.89), while in PEJF, it was reached at 6 h (3435.16 ± 59.31). Gel electrophoresis of the PEJA and PEJF samples showed bands corresponding to peptides smaller than 10 kDa in both systems studied. The highest values for the antioxidant capacity (DPPH) were obtained at 4 h for PEJA (56.17 ± 1.14), while they were obtained at 6 h for PEJF (26.64 ± 0.52). The highest values for the antihypertensive capacity were recorded at 6 h (86.46 ± 1.85) in PEJF. The highest antidiabetic capacity obtained for PEJA and PEJF was observed at 6 h, 68.86 ± 8.27 and 52.75 ± 2.23, respectively. This is the first report of their antidiabetic activity. Notably, alcalase hydrolysate outperformed flavourzyme hydrolysate and the cereals reported in other studies, confirming its better multi-bioactivity.

The Discovery, Molecular Cloning, and Characterization of Dextransucrase LmDexA and Its Active Truncated Mutant from Leuconostoc mesenteroides NN710.

Dextransucrases play a crucial role in the production of dextran from economical sucrose; therefore, there is a pressing demand to explore novel dextransucrases with better performance. This study characterized a dextransucrase enzyme, LmDexA, which was identified from the Leuconostoc mesenteroides NN710. This bacterium was isolated from the soil of growing dragon fruit in Guangxi province, China. We successfully constructed six different N-terminal truncated variants through sequential analysis. Additionally, a truncated variant, ΔN190LmDexA, was constructed by removing the 190 amino acids fragment from the N-terminal. This truncated variant was then successfully expressed heterologously in Escherichia coli and purified. The purified ΔN190LmDexA demonstrated optimal hydrolysis activity at a pH of 5.6 and a temperature of 30 °C. Its maximum specific activity was measured to be 126.13 U/mg, with a Km of 13.7 mM. Results demonstrated a significant improvement in the heterologous expression level and total enzyme activity of ΔN190LmDexA. ΔN190LmDexA exhibited both hydrolytic and transsaccharolytic enzymatic activities. When sucrose was used as the substrate, it primarily produced high-molecular-weight dextran (>400 kDa). However, upon the addition of maltose as a receptor, it resulted in the production of a significant amount of oligosaccharides. Our results can provide valuable information for enhancing the characteristics of recombinant dextransucrase and potentially converting sucrose into high-value-added dextran and oligosaccharides.

Enhanced anaerobic digestion of waste-activated sludge by thermal-alkali pretreatment: a pilot-scale study.

The composition of waste-activated sludge (WAS) is complex, containing a large amount of harmful substances, which pose a threat to the environment and human health. The reduction and resource utilization of sludge has become a development demand in sludge treatment and disposal. Based on the technical bottlenecks in the practical application of direct anaerobic digestion technology, this study adopted two different thermal and thermal-alkali hydrolysis technologies to pretreat sludge. A pilot-scale experiment was conducted to investigate the experimental conditions, parameters, and effects of two hydrolysis technologies. This study showed that the optimal hydrolysis temperature was 70 °C, the hydrolysis effect and pH can reach equilibrium with the hydrolysis retention time was 4-8 h, and the optimal alkali concentration range was 0.0125-0.015 kg NaOH/kg dry-sludge. Thermal-alkali combination treatment greatly improved the performance of methane production, the addition of NaOH increased methane yield by 31.2% than that of 70 °C thermal hydrolysis. The average energy consumption is 75 kWh/m3 80% water-content sludge during the experiment. This study provides a better pretreatment strategy for exploring efficient anaerobic digestion treatment technologies suitable for southern characteristic sewage sludge.

Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile.

Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC-PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.

Sequential Enzymatic Hydrolysis and Ultrasound Pretreatment of Pork Liver for the Generation of Bioactive and Taste-Related Hydrolyzates.

In the study of protein-rich byproducts, enzymatic hydrolysis stands as a prominent technique, generating bioactive peptides. Combining exo- and endopeptidases could enhance both biological and sensory properties. Ultrasound pretreatment is one of the most promising techniques for the optimization of enzymatic hydrolysis. This research aimed to create tasteful and biologically active pork liver hydrolyzates by using sequential hydrolysis with two types of enzymes and two types of ultrasound pretreatments. Sequential hydrolyzates exhibited a higher degree of hydrolysis than single ones. Protana Prime hydrolyzates yielded the largest amount of taste-related amino acids, enhancing sweet, bittersweet, and umami amino acids according to the Taste Activity Value (TAV). These hydrolyzates also displayed significantly higher antioxidant activity. Among sequential hydrolyzates, Flavourzyme and Protana Prime hydrolyzates pretreated with ultrasound showed the highest ferrous ion chelating activity. Overall, employing both Alcalase and Protana Prime on porcine livers pretreated with ultrasound proved to be highly effective in obtaining potentially tasteful and biologically active hydrolyzates.

Three-Step Enzymatic Remodeling of Chitin into Bioactive Chitooligomers.

Here we describe a complex enzymatic approach to the efficient transformation of abundant waste chitin, a byproduct of the food industry, into valuable chitooligomers with a degree of polymerization (DP) ranging from 6 to 11. This method involves a three-step process: initial hydrolysis of chitin using engineered variants of a novel fungal chitinase from Talaromyces flavus to generate low-DP chitooligomers, followed by an extension to the desired DP using the high-yielding Y445N variant of ß-N-acetylhexosaminidase from Aspergillus oryzae, achieving yields of up to 57%. Subsequently, enzymatic deacetylation of chitooligomers with DP 6 and 7 was accomplished using peptidoglycan deacetylase from Bacillus subtilis BsPdaC. The innovative enzymatic procedure demonstrates a sustainable and feasible route for converting waste chitin into unavailable bioactive chitooligomers potentially applicable as natural pesticides in ecological and sustainable agriculture.

Suspensions of antibiotics in self-emulsifying oils as a novel approach to formulate eye drops with substances which undergo hydrolysis in aqueous environment.

The aim of this study was to develop eye-drops with cefuroxime (CEF) sodium or vancomycin (VAN) hydrochloride, antibiotics that are instable in water. Anhydrous self-emulsifying oils (SEO) are proposed as a carrier and antibiotics are suspended. In the contact with tear fluid, the formulation should transform into emulsion, with fast dissolution of an antibiotic. CEF or VAN (5% w/w) was suspended in SEO carriers prepared by dissolving surfactants (Tween 20 or Span 80 5% w/w) in Miglyol, castor oil, or olive oil. Formulations with or without sodium citrate (2% w/w) were compared. Six-months or 1-year stability tests were carried out at 40 °C. The content of CEF and VAN was evaluated using HPLC and the potency of the antibiotic was assessed with agar diffusion method. In contact with water, drug particles suspended in SEO dissolved rapidly and o/w emulsion was formed. After 1-year at 40 °C, the content of degradation products was at most 0.5% in CEF and 4.0% in VAN formulations. The agar diffusion assay has shown that CEF and VAN loaded into SEO retained its potency against the sensitive microorganisms comparable to an aqueous solution. Therefore, SEO can be used as a novel carrier for the active substances which may not require improved solubility or absorption but need to be protected from moisture. This is a formulation that can be produced on industrial scale, with no limitation of stability or drug concentration.

Engineered polyethylene terephthalate hydrolases: perspectives and limits.

Polyethylene terephthalate (PET) is a major component of plastic waste. Enzymatic PET hydrolysis is the most ecofriendly recycling technology. The biorecycling of PET waste requires the complete depolymerization of PET to terephthalate and ethylene glycol. The history of enzymatic PET depolymerization has revealed two critical issues for the industrial depolymerization of PET: industrially available PET hydrolases and pretreatment of PET waste to make it susceptible to full enzymatic hydrolysis. As none of the wild-type enzymes can satisfy the requirements for industrialization, various mutational improvements have been performed, through classical technology to state-of-the-art computational/machine-learning technology. Recent engineering studies on PET hydrolases have brought a new insight that flexibility of the substrate-binding groove may improve the efficiency of PET hydrolysis while maintaining sufficient thermostability, although the previous studies focused only on enzymatic thermostability above the glass transition temperature of PET. Industrial biorecycling of PET waste is scheduled to be implemented, using micronized amorphous PET. Next stage must be the development of PET hydrolases that can efficiently degrade crystalline parts of PET and expansion of target PET materials, not only bottles but also textiles, packages, and microplastics. This review discusses the current status of PET hydrolases, their potential applications, and their profespectal goals. KEY POINTS: • PET hydrolases must be thermophilic, but their operation must be below 70 °C • Classical and state-of-the-art engineering approaches are useful for PET hydrolases • Enzyme activity on crystalline PET is most expected for future PET biorecycling.

Small molecule substrates for the rapid quantification of acyl transfer activity of nylon hydrolase NylCA.

The widespread use of polyamides such as nylons has led to the accumulation of nylon waste, which is particularly resistant to decomposition due to the intrinsic stability of the amide bond. New methods are required for the true recycling of these waste materials by depolymerization. Enzymes that are capable of hydrolyzing polyamides have been proposed as biocatalysts that may be suitable for this application. NylC is an enzyme that can mediate the hydrolysis of aminohexanoic acid oligomers, and to some extent, bulk polymers. However, current assays to characterize the activity of this enzyme require long reaction times and/or rely on secondary reactions to quantify hydrolysis. Herein, we have designed structurally-optimized small molecule chromogenic esters that serve as substrate analogues for monitoring NylC acyltransferase activity in a continuous manner. This assay can be performed in minutes at room temperature, and the substrate N-acetyl-GABA-pNP ester (kcat = 0.37 s-1, KM = 256 µM) shows selectivity for NylC in complex biological media. We also demonstrate that activity towards this substrate analogue correlates with amide hydrolysis, which is the primary activity of this enzyme. Furthermore, our screening of substrate analogues provides insight into the substrate specificity of NylC, which is relevant to biocatalytic applications.

Probing the Electrostatic Effects of H-Ras Tyrosine 32 Mutations on Intrinsic GTP Hydrolysis Using Vibrational Stark Effect Spectroscopy of a Thiocyanate Probe.

The wildtype H-Ras protein functions as a molecular switch in a variety of cell signaling pathways, and mutations to key residues result in a constitutively active oncoprotein. However, there is some debate regarding the mechanism of the intrinsic GTPase activity of H-Ras. It has been hypothesized that ordered water molecules are coordinated at the active site by Q61, a highly transforming amino acid site, and Y32, a position that has not previously been investigated. Here, we examine the electrostatic contribution of the Y32 position to GTP hydrolysis by comparing the rate of GTP hydrolysis of Y32X mutants to the vibrational energy shift of each mutation measured by a nearby thiocyanate vibrational probe to estimate changes in the electrostatic environment caused by changes at the Y32 position. We further compared vibrational energy shifts for each mutation to the hydration potential of the respective side chain and demonstrated that Y32 is less critical for recruiting water molecules into the active site to promote hydrolysis than Q61. Our results show a clear interplay between a steric contribution from Y32 and an electrostatic contribution from Q61 that are both critical for intrinsic GTP hydrolysis.

Improvement of a low-cost protocol for a simultaneous comparative evaluation of hydrolytic activity between sessile and planktonic cells: Candida albicans as a study model.

Candida albicans is often implicated in nosocomial infections with fatal consequences. Its virulence is contributed to hydrolytic enzymes and biofilm formation. Previous research focused on studying these virulence factors individually. Therefore, this study aimed to investigate the impact of biofilm formation on the hydrolytic activity using an adapted low-cost method. Eleven strains of C. albicans were used. The biofilms were formed on pre-treated silicone discs using 24-well plates and then deposited on the appropriate agar to test each enzyme, while the planktonic cells were conventionally seeded. Biofilms were analysed using Raman spectroscopy, fluorescent and scanning electron microscopy. The adapted method provided an evaluation of hydrolytic enzymes activity in C. albicans biofilm and showed that sessile cells had a higher phospholipase and proteinase activities compared with planktonic cells. These findings were supported by spectroscopic and microscopic analyses, which provided valuable insights into the virulence mechanisms of C. albicans during biofilm formation.

PEGylation renders carnosine resistant to hydrolysis by serum carnosinase and increases renal carnosine levels.

Carnosine's protective effect in rodent models of glycoxidative stress have provided a rational for translation of these findings in therapeutic concepts in patient with diabetic kidney disease. In contrast to rodents however, carnosine is rapidly degraded by the carnosinase-1 enzyme. To overcome this hurdle, we sought to protect hydrolysis of carnosine by conjugation to Methoxypolyethylene glycol amine (mPEG-NH2). PEGylated carnosine (PEG-car) was used to study the hydrolysis of carnosine by human serum as well as to compare the pharmacokinetics of PEG-car and L-carnosine in mice after intravenous (IV) injection. While L-carnosine was rapidly hydrolyzed in human serum, PEG-car was highly resistant to hydrolysis. Addition of unconjugated PEG to carnosine or PEG-car did not influence hydrolysis of carnosine in serum. In mice PEG-car and L-carnosine exhibited similar pharmacokinetics in serum but differed in half-life time (t1/2) in kidney, with PEG-car showing a significantly higher t1/2 compared to L-carnosine. Hence, PEGylation of carnosine is an effective approach to prevent carnosine degradations and to achieve higher renal carnosine levels. However, further studies are warranted to test if the protective properties of carnosine are preserved after PEGylation.

Exploring the Anticonvulsant Properties of a Celecoxib-Phospholipid Conjugate: Synthesis, Activation, and Evaluation of Cytotoxicity.

BACKGROUND: Epilepsy poses a significant global health challenge, particularly in regions with limited financial resources hindering access to treatment. Recent research highlights neuroinflammation, particularly involving cyclooxygenase-2 (COX-2) pathways, as a promising avenue for epilepsy management. METHODS: This study aimed to develop a Cyclooxygenase-2 inhibitor with potential anticonvulsant properties. A promising drug candidate was identified and chemically linked with phospholipids through docking analyses. The activation of this prodrug was assessed using phospholipase A2 (PLA2)-mediated hydrolysis studies. The conjugate's confirmation and cytotoxicity were evaluated using Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), and Sulphoramide B (SRB) assays. RESULTS: Docking studies revealed that the Celecoxib-Phospholipid conjugate exhibited a superior affinity for PLA2 compared to other drug-phospholipid conjugates. FT-IR spectroscopy confirmed the successful synthesis of the conjugate, while DSC analysis confirmed its purity and formation. PLA2-mediated hydrolysis experiments demonstrated selective activation of the prodrug depending on PLA2 concentration. SRB experiments indicated dose-dependent cytotoxic effects of Celecoxib, phospholipid non-toxicity, and efficient celecoxib-phospholipid conjugation. CONCLUSION: This study successfully developed a Celecoxib-phospholipid conjugate with potential anticonvulsant properties. The prodrug's specific activation and cytotoxicity profile makes it a promising therapeutic candidate. Further investigation into underlying mechanisms and in vivo studies is necessary to assess its translational potential fully.