To date, enzymatic systems capable of phosphorylating phenolic compounds are rare, and phenolic phosphate synthetase from Bacillus subtilis (BsPPS) is distinguished by its ability to regioselectively phosphorylate a broad range of structurally diverse natural polyphenols, a transformation valuable for improving the aqueous solubility and bioavailability of phytochemicals. However, the lack of an efficient high-throughput screening (HTS) platform has limited its engineering and application. Here, we developed a fluorescence-based HTS assay for rapid estimation of BsPPS activity using umbelliferone as a fluorogenic probe. Phosphorylation of umbelliferone causes pronounced fluorescence quenching, providing an inverse correlation between fluorescence intensity and catalytic activity. The assay was systematically optimized and validated, demonstrating high accuracy, precision, and reproducibility. Application to an error-prone PCR library identified BsPPS variants with improved thermostability while maintaining catalytic activity. Overall, this assay fills a critical methodological gap and provides a practical foundation for future exploration of related phenolic compound-phosphorylating enzymes.

Despite therapeutic advances, atherosclerosis remains a major global health challenge. Most current treatments target systemic risk factors rather than the diseased vascular wall. Our previous work identified genistein, a soy isoflavone, as a cannabinoid receptor 1 (CB1) antagonist capable of suppressing CB1-mediated vascular inflammation and atherosclerosis. However, its poor water solubility and low oral bioavailability limit clinical application. We aimed to develop water-soluble, orally bioavailable CB1 antagonists for atherosclerosis and to investigate the role of endothelial CB1 in hemodynamic regulation. RNA-sequencing datasets from the NCBI GEO repository were analyzed to assess CB1 expression in atherosclerotic patients. Apolipoprotein E-deficient (Apoe−/−) mice with or without partial carotid artery ligation (PCAL) were used to model acute and chronic atherosclerosis. A cone-and-plate viscometer was employed to simulate disturbed flow. A ligand-based high-throughput virtual screening approach combined with SWEETLEAD chemical database analysis was used to discover new CB1 antagonists. A biotransformation-based strategy was used to generate isoflavone monophosphate prodrugs. We found CB1 was upregulated in atherosclerotic lesions from patients and mice, and in endothelial cells exposed to disturbed flow. Mechanistically, this was driven by ZNF610 and Spi1 binding and KLF4 dissociation at the CB1 promoter. Daidzein, a soy isoflavone structurally similar to genistein, was identified as a novel CB1 antagonist. To enhance solubility and bioavailability, we developed genistein 7-O-phosphate (G7P) and daidzein 7-O-phosphate (D7P). Pharmacological treatment with these isoflavone monophosphates or genetic CB1 ablation reversed disturbed flow-induced endothelial dysfunction and endothelial-to-mesenchymal transition (EndMT). Oral administration of G7P and D7P significantly reduced atherosclerotic plaque formation in mice. This is the first study to identify transcriptional regulators that drive endothelial CB1 upregulation in response to disturbed flow. We further demonstrated that isoflavone monophosphates ameliorate disturbed flow-induced endothelial dysfunction and EndMT via CB1 inhibition, offering promising oral therapeutics for atherosclerosis.

Flavonols (3-hydroxy flavones) have been studied for their beneficial bioactivities for human health. Recently, we reported that a flavonoid phosphate synthetase (BsFPS) from Bacillus subtilis BCRC 80517 can transform several flavonoids into their phosphate conjugates, which become more water-soluble and thus increase the oral bioavailability. However, the in vivo metabolism of different flavonols has yet to be determined. Here, we investigated biotransformation of three flavonols (quercetin, kaempferol and fisetin) by B. subtilis BCRC 80517. C-ring cleavage products of quercetin and kaempferol, i.e., 2-protocatechuoyl-phloroglucinol carboxylic acid (2-PCPGCA), were produced, whereas two phosphate derivatives of fisetin (fisetin 4 & PRIME;-O-phosphate and fisetin 3 & PRIME;-O- phosphate) were generated by cultivation with B. subtilis BCRC 80517. Our results indicated that there are structure-specific metabolic pathways in B. subtilis toward different flavonols, where the 5-hydroxy group de-termines metabolic priority. Our findings provide new insights for developing bioproduction platform to produce flavonol phosphate derivatives for nutraceutical applications.

Flavonoids, with therapeutic potential, often encounter challenges due to poor aqueous solubility, which limits their oral bioavailability and efficacy. Phosphorylation presents a promising solution, yet current chemical methods require protective agents to mitigate side product formation. Here, we developed a coupled bienzymatic system using an ATP-dependent dikinase, flavonoid phosphate synthetase (BsFPS), Bs FPS), for selective synthesis of flavonoid monophosphates. This system integrates a class III polyphosphate kinase 2 (PPK2-III) for ATP regeneration from AMP, thereby reducing costly ATP consumption and enhancing economic viability. Through PPK2III screening, Er PPK was identified as a compatible candidate, facilitating a streamlined one-pot system. Structural analysis revealed that the residue at the equivalent site of Arg75ErPPK Er PPK influences the binding pocket entry distance and flavonoid derivative inhibition propensity on PPK2-III, confirming the compatibility of Er PPK and providing structural insights for identifying PPK2-III with reduced inhibition likelihood. Further optimization strategies, including balancing Mg2+ 2 + and polyphosphate ratios, incorporating Tween surfactants for enhanced substrate solubilization without compromising enzyme stability, and increasing the rate-limiting enzyme amount, significantly elevated the conversion rate. Notably, pH control was crucial due to pH decline caused by Bs FPS catalysis. pH-stat conversion achieved a 99.0 % conversion of luteolin, the flavonoid target, within 8 h, yielding up to 21.5 mM (7.9 g/L) luteolin monophosphates with up to 95 % reduction in ATP requirement. This enzymatic phosphorylation approach shows promise for advancing polyphenolic monophosphate bioproduction and transforming their functional applications.

Luteolin (Lut) and apigenin (Apn), flavones present in various edible plants, exhibit diverse antioxidant and pharmacological activities but have limited in vivo efficacy due to low water solubility and poor bioavailability. Here, we generated luteolin and apigenin monophosphate derivatives (LutPs and ApnPs) individually via microbial biotransformation. We then characterized their physicochemical properties and evaluated their in vitro and in vivo pharmacokinetics and bioavailability. Both LutPs and ApnPs showed enhanced solubility and dissolution and remained stable in simulated gastrointestinal conditions. Additionally, they efficiently reverted to parental forms via alkaline phosphatase in Caco-2 cells. Following oral administration in rats, LutPs and ApnPs exhibited higher plasma exposure to both aglycone and conjugated forms compared to Lut and Apn. Notably, the in vivo biotransformation of Apn to Lut was observed in all apigenin-related groups. Our study suggests that flavone monophosphates are effective alternatives with enhanced bioavailability, providing insights for the potential application of emerging bioactive nutraceuticals.

Flavonoids are associated with health benefits, but most of them have poor oral bioavailability due to their extremely low aqueous solubility. Flavonoid O-phosphorylation suggests a potent modification to solve the problems. Here, we isolated, identified and characterized an unprecedented phosphotransferase, flavonoid phosphate synthetase (BsFPS), from B. subtilis. The enzyme catalyzes the ATP-dependent phosphorylation of flavonoid to generate flavonoid monophosphates, AMP and orthophosphate. BsFPS is a promiscuous phospho-transferase that efficiently catalyzes structurally-diverse flavonoids, including isoflavones, flavones, flavonols, flavanones and flavonolignans. Based on MS and NMR analysis, the phosphorylation mainly occurs on the hy-droxyl group at C-7 of A-ring or C-4 ' of B-ring in flavonoid skeleton. Notably, BsFPS is regioselective for the ortho-3 ',4 '-dihydroxy moiety of catechol-containing structures, such as luteolin and quercetin, to produce phosphate conjugates at C-4 ' or C-3 ' of B-ring. Our findings highlight the potential for developing biosynthetic platform to obtain new phosphorylated flavonoids for pharmaceutical and nutraceutical applications.

Luteolin (LUT), a plant-derived flavone, exhibits various bioactivities; however, the poor aqueous solubility hampers its applications. Here, we revealed bioconversion of LUT by Bacillus subtilis BCRC 80517, yielding three water-soluble phosphate conjugates. These derivatives were identified as luteolin 4'-O-phosphate (L4'P), luteolin 3'-O-phosphate (L3'P), and luteolin 7-O- phosphate (L7P) by LC-ESI-MS/MS and NMR. Besides, we found that Bacillus subtilis BCRC 80517 was able to convert different levels of LUT but showed a limited conversion rate. By observing bacterial morphology with transmission electron microscopy and confocal fluorescence microscopy, we found that LUT disrupted the bacterial membrane integrity, which explained the incomplete conversion. Additionally, we revealed a spontaneous intramolecular transesterification of L4'P to L3'P, the thermodynamically more stable form, under acidic conditions and proposed the possible mechanism involving a cyclic phosphate as the intermediate. This study provides insight into development of a potent structural modification strategy to enhance the solubility of LUT through biophosphorylation.

The flavanoid hesperidin (Hsd) is one of the major polyphenols in citrus fruits. Hsd and its aglycone hesperetin (Hst) have a broad array of bioactivities; however, their low aqueous solubility and low intestinal permeability lead to their limited oral bioavailability. In the present study, we generated two water-soluble derivatives of Hst, namely, Hst 7-O-phosphate and Hst3'-O-phosphate, by a unique bioconversion process of Bacillus subtilis var. natto BCRC80517. The phosphorylated products showed superior aqueous solubility and distinct physicochemical properties compared with the original Hst. The Hst phosphate derivatives (HstPs) remained stable in simulated gastric and intestinal fluids for 240 min and could revert to the original Hst form by alkaline phosphatase treatment in Caco-2 cells, showing enhanced intestinal permeability in vitro. After oral administration in rats, HstPs greatly elevated plasma exposure to Hst and showed better bioavailability than did Hsd. HstPs may be a potential and efficient alternative to Hst.

The study on alleviating blood pressure was carried on a series of essential-lemon oil products.The pure essential-lemon oils were obtained by solution extraction distillation, and were prepared for the assessment of acidity and saponification values.The composed oils from pure essential-lemon oils with black sesame oils and olive grape seed blending oils were prepared from oil and soap smells.The receptors of stimulated nanovapors from the essential oils and soaps were compared.The assist of bathing with water could be better for alleviating blood pressure.These results are for the research only.

Growing interest in the health benefits of soy isoflavones has led to research in the isolation of individual isoflavone species for further application. Herein, we develop a new strategy to isolate daidzein, genistein, daidzin and genistin in soybean. We investigated the impact of solvents used and the extraction time on the extracted isoflavone contents from soybean. A 30-min extraction with 65% aqueous methanol gave a total isoflavone yield of 345 mg/100 g soybean, the highest value among tested conditions. Further, we proposed a two-stage adsorption/desorption chromatography comprising macroporous resin and aluminium oxide to isolate isoflavone. First, HP-20 resin was used to separate the glucosidic and aglyconic forms of isoflavone, then individual species of isoflavone could be isolated using aluminium oxide by specific retention of 5-hydroxy isoflavone. This process achieved overall high recovery (82-97%) and purity (92-95%) of the four isoflavones, which confirms a high separating efficiency for isoflavones from soybean.

Genistein 7-O-phosphate (G7P), generated by a bioconversion process of Bacillus subtilis var. natto with soy isoflavone genistein, is practically water-soluble and more bioavailable than aglyconic genistein. In this study, we assessed the ability of G7P to prevent bone loss in rats with ovariectomy. Twelve-week-old female rats underwent ovariectomy, and genistein or G7P was administered to rats 2 weeks after the surgery by daily oral gavage for another 12 weeks. After sacrifice at the 12th week, the femurs were analyzed by micro computed tomography, histology and biomechanical study. G7P administration prevented the loss of bone mineral density and the reduced Young’s modulus caused by ovariectomy and improved bone architecture observed in histology and tomography. Additionally, G7P treatment resulted in a higher plasma level and inhibition of post-ovariectomy bone loss than did genistein at the same dosage. Therefore, G7P may be potent functional food agent for dealing with postmenopausal osteoporosis.

This paper proposes a novel battery with zinc oxalate (ZnC2O4) electrode exhibiting superior charging and rechargeable characteristics compared to an original zinc-carbon battery.ZnC2O4 electrode provides homogeneous anodic Zn +2 and catholic C2O4 -2 ions.The effect of adding different percentages of ZnC2O4 on the charging voltage and current are investigated.The possibility of achieving charging and discharging recycles in the cell electrolyte are studied.The electrode ZnC2O4 was prepared by the sol-gel method.Based on the voltage, current, and energy calculations, it concludes that adding different percentages of ZnC2O4 to the battery electrolyte have different effects on the internal resistance of the battery.

Antimelanogenic agents from natural sources have been widely investigated. Urolithin A (UA) and B (UB), the main gut microflora metabolites of dietary ellagic acid derivatives, have various bioactivities such as anti-inflammatory and antiaging effects. In this study, the metabolites were found to possess depigmentation efficacy by suppressing tyrosinase activity. Both UA and UB could attenuate melanogenesis in B16 melanoma cells to 55.1 ± 3.8 and 76.4 ± 17.4% of control at noncytotoxic dosage, 10 μM, respectively. UA showed comparable efficacy to positive control, 5 μM of kojic acid treatment (51.2 ± 7.8). RT-PCR results revealed that UA and UB inhibited melanin formation by affecting the catalytic activity of tyrosinase rather than its mRNA expression. Kinetics for UA and UB on tyrosinase activity revealed that their inhibition behavior toward cellular tyrosinase involved competitive inhibition. UA and UB may be potent tyrosinase inhibitors and they possess significant antimelanogenesis ability as novel skin-whitening ingredients.

The soy isoflavones daidzein (DAI) and genistein (GEN) have beneficial effects on human health. However, their oral bioavailability is hampered by their low aqueous solubility. Our previous study revealed two water-soluble phosphorylated conjugates of isoflavones, daidzein 7-O-phosphate and genistein 7-O-phosphate, generated via biotransformation by Bacillus subtilis BCRC80517 cultivated with isoflavones. In this study, two novel derivatives of isoflavones, daidzein 4'-O-phosphate and genistein 4'-O-phosphate, were identified by HPLC-ESI-MS/MS and 1H, 13C, and 31P NMR, and their biotransformation roadmaps were proposed. Primarily, isoflavone glucosides were deglycosylated and then phosphorylated predominantly into 7-O-phosphate conjugates with traces of 4'-O-phosphate conjugates. Inevitably, trace quantities of glucosides were converted into 6″-O-succinyl glucosides. GEN was more efficiently phosphorylated than DAI. Nevertheless, the presence of GEN prolonged the time until the exponential phase of cell growth, whereas the other isoflavones showed little effect on cell growth. Our findings provide new insights into the novel microbial phosphorylation of isoflavones involved in xenobiotic metabolism.

Research has indicated the health beneficial effects of soyfoods. Genistein, one of the primary bioactive agents in soybeans, has a number of pharmacological and biological activities; however, its low water solubility and low intestinal permeability may lead to the depletion of genistein absorption from dietary intake and contribute to its poor oral bioavailability. Previous study revealed a water-soluble phosphate conjugate of genistein, genistein 7-O-phosphate (G7P), generated by biotransformation of Bacillus subtilis var. natto BCRC80517 with genistein. This study aimed to investigate the dissolution profile, intestinal permeability and oral bioavailability of genistein and G7P. G7P improved water solubility and also enhanced intestinal permeability in vitro and in situ, and greatly increased plasma exposure to genistein after oral administration in rats. The aqueous solubility of genistein is the absorption barrier to its oral bioavailability. G7P may be a promising and efficient alternative to genistein.

Daidzein and genistein have many benefits for human health; however, their applications may be restricted because of their low aqueous solubility. In this work, we generated two water soluble isoflavone derivatives, daidzein 7-O-phosphate and genistein 7-O-phosphate, by incubating Bacillus substilis var. natto BCRC 80517 with daidzein and genistein. These two isoflavone derivatives were characterized by HPLC-ESI–MS/MS, 13C NMR and 31P NMR. Genistein was phosphorylated more rapidly than daidzein. In addition, this bacterial strain could transform glucosidic isoflavones via aglucones into the corresponding 7-O-phosphate conjugates. However, despite the production of agluconic daidzein and genistein, cells could not transform malonyl glucosidic isoflavones into the 7-O-phosphate conjugates. Furthermore, alkaline phosphatase activities from human colon carcinoma (Caco-2) cells were found effective in the dephosphorylation of daidzein 7-O-phosphate and genistein 7-O-phosphate into the corresponding aglyconic isoflavones.

Sinter-hardening alloy steels have been widely applied in press-and-sinter products. However, research on these cost-effective materials for metal injection molding (MIM) has been limited. This study identified a new MIM alloy steel (Fe-6 w/o Ni-0.8 w/o Cr-0.8 w/o Mo-0.4 w/o C) with attractive properties. To achieve high sintered density and homogeneous alloying, fine powders and high-temperature sintering were used. To obtain enhanced combinations of strength and ductility in the sinter-hardened alloy steel, a high nickel content was used, along with chromium and molybdenum, which enhanced homogenization of the nickel and increased hardenability. With a cooling rate of <30 degrees C/min, and no quenching treatment, the sintered-and-tempered alloy steel attained an ultrahigh tensile strength of 1,900 MPa (275,000 psi) with an elongation of 7.6% and a toughness of 55 J (40 ft..lbf) surpassing the properties of quenched-and- tempered MIM-4140 and MIM4605. The improved mechanical properties of the new alloy steel are understood in terms of the attendant transformation kinetics and microstructure.

The optical responses of Au nanoparticle arrays dispersed within porous anodic alumina (PAA) have been investigated. The 2D Au/PAA structures were well preparing by immersing various pore sizes of PAAs into the mixing solution of HAuCl4, cetyl-trimethyl ammonium bromide, and NaBH4 under Au phase transfer processes. With the dispersion of Au nanoparticles in those nanopores in PAA, the intensity of photoluminescence light and cathodoluminescence light spectra largely decreases. The dissipated emission could be interpreted by the partly sealing of the light emission oxygen defect centers in PAA with the dispersion of Au nanoparticles. The major absorption comes from the interface plasmon resonance of Au nanoparticles surrounding with PAAs. The excited Au nanoparticles act as a Bragg grating for wave propagation scattering into the alumina matrix.