Moringa oleifera Lam. (Moringaceae) is revered as s ‘miracle tree’ due to its remarkable nutritional, medicinal and industrial uses. In our study, a comparative analysis of the nutritional parameters , phytochemicals , and cytotoxicity of M. oleifera leaf extracts (MLEs) of five selected varieties , was performed. Jaffna variety possessed the highest antioxidant activity (FRAP) followed by other four varieties. The trend observed was: Jaffna  > PKM-1  > PKM-2  > ODC  > Conventional , total soluble proteins (TSP) [0.69 ± 0.01 and 0.94 ± 0.01 µg/mL ], sugar content (TSS) [0.39 ± 0.01 and 0.51 ± 0.01 µg/mL ], chlorophyll content , followed by the other four varieties. The trend observed for cytotoxic activities of ethanolic MLEs on HepG2 cell line, based on the IC50 values, was conventional (1.22 mg/mL) > ODC (0.90 mg/mL) > PKM-2 (0.65 mg/mL) > PKM-1 (0.35 mg/mL) > Jaffna (0.15 mg/mL). The results of HPLC quantification of anticancer compounds  was also in consonance with that of MTT assay. In summary, the trend observed in all the parameters tested was Jaffna > PKM-1 > PKM-2 > ODC > conventional. Thus, Jaffna variety has a better potential to combat malnutrition and cancer and must be recommended for commercial plantations. Moringa oleifera Lam. (Moringaceae) is a ‘miracle tree’ which possesses excellent medicinal and nutritional properties and can combat malnutrition among the young population of the world who are devoid of a nutritious diet , were procured from Tamil Nadu Agriculture University (TAU), India. The wings of the seeds were removed and were soaked in water for 15 min. The seeds were then wrapped in wet paper towels and incubated for sprouting at 25 °C for three days. The seedlings of the aforementioned varieties were transferred into separate plastic pots (3.9 × 3.5 inches) filled with soil and soilrite (soil conditioning mixture) in 1:1 ratio (w/w) and kept in a polyhouse maintained at 30 °C, relative humidity (RH) of 60%, with a 16/8 h light/dark photoperiod.Healthy and uniform seeds of five Fresh leaves of 3 months and 15 months old plants were harvested, shade dried (5 days), powdered, and sieved through a mesh (pore size: 297 microns). Leaf powder (50 g each) of all the varieties was suspended in conical flasks containing 80% ethanol (100 mL) and incubated on a rotary shaker set at 250 rpm, for 6 h. The filtered MLEs were concentrated at 40–45 °C on a rotary evaporator set at 200 rpm. Thereafter, MLEs (~5 mg/mL) were labeled and refrigerated for further use  was added and the reaction mixture was incubated for an hour at RT prior to the addition of the Folin-Ciocalteu reagent (FCR). The absorbance was read at 660 nm spectrophotometrically  plus 10 mL of concentrated sulphuric acid was added to the flasks and incubated overnight. The flasks were placed in the Kjeldahl digestion unit set at 100 °C for 20 min, to digest the mixture followed by adjusting the volume to 100 mL with distilled water. To estimate the mineral contents, 1 g shade-dried leaves of each of the five varieties was powdered separately and the leaf powder was transferred into separate Erlenmeyer flasks (100 mL capacity) and were digested in triacid containing nitric, perchloric and sulphuric acids (9:3:1 3) was released from the distillation of the sample. After distillation, titration of the sample was carried out against 0.01 N H2SO4 until the green color changed into purple. A blank (without plant sample) was also used to assess the contamination.The analysis of nitrogen was done by the Kjeldahl method . An aliqCalcium estimation was conducted by titration with EDTA . A 20 mLM. oleifera was transferred into a 500 mL conical flask, containing K2Cr2O7 , followed by the addition of H2SO4 (20 mL concentrated) to it. The solution was incubated for 1/2 h followed by the addition of orthophosphoric acid (10 mL), and diphenylamine indicator (1 mL). Finally, the titration was performed against (0.05 N) ferrous ammonium sulphate (139 g of ferrous ammonium sulphate and 15 mL of concentrated H2SO4).Carbon estimation was carried out by the protocol of Nelson and Sommers . Leaf poThe Na and K contents were estimated using a flame photometer, as per the protocol of Gloterman et al.  and SahrEstimation of sulphur was performed by the protocol of Mottershead . MLEs (12CO3 (4 N) were added to it until the yellow color disappeared. The pH was adjusted to 4.8 with HCl . Thereafter, 5 mL vanadate-molybdate reagent was added and deionized water was added to make up the volume to 25 mL. Transmittance was read at 470 nm after 30 min incubation (as the color starts to develop). Meanwhile, 5, 10, 15, 20, and 25 mL of 100 ppm of the phosphorous standard was transferred to the 25 mL volumetric flasks and the same procedure was followed as mentioned above. Transmittance was read at 470 nm and the standard curve was drawn.Phosphorous was estimated by the vanadate-molybdate method of Piper . An aliqM. oleifera were suspended in 100 mL of ethanol in respective conical flasks. Thereafter, the flasks were placed on a rotatory shaker (200 rpm), set at 30 °C for 48 h. The MLE was evaporated in the evaporator shaker and was collected in labeled vials for further use. To the different concentrations of MLE (10–40 µg/mL) of each variety, 625 µL of Na2PO4 buffer (pH 7.4) plus 625 µL of potassium ferricyanide were added and the mixture was incubated for 20 min at 50 °C. The mixture was allowed to cool and then 625 µL of trichloroacetic acid (TCA) was added. The mixture was centrifuged at 2000 rpm for 10 min. To the supernatant, 625 µL distilled water plus 125 µL FeCl3 were mixed, and absorbance was recorded spectrophotometrically at 700 nm  at 37 °C. These cells were treated with different concentrations  of MLEs. Thereafter, 50 µL MTT solution [5 mg MTT dye dissolved in 1 mL PBS] was poured into each well and the plate was incubated for 4 h at 37 °C. After incubation, DMSO (15µL) was added into each well. The absorbance was recorded at 570 nm using an ELISA reader. Percentage cytotoxicity was calculated from the absorbance values using the formula given below:For MTT assay, the human liver cell line (HepG2) was procured from NCCS, Pune, India, and culture was maintained at Lovely Professional University, Punjab, India. The cells were grown in DMEM media , supplemented with fetal bovine serum (10%), in a COn (ATCC) ,37. CellM. oleifera varieties was analyzed at the young stage (3-months-old) and mature stage (15-months-old). Maximum sugar content was recorded in Jaffna (0.39 ± 0.04 and 0.51 ± 0.01 µg/mL) followed by PKM-1 (0.25 ± 0.001 and 0.45 ± 0.006 µg/mL), PKM-2 (0.12 ± 0.001 and 0.301 ± 0.004 µg/mL), ODC (0.08 ± 0.003 and 0.16 ± 0.01 µg/mL) and Conventional (0.05 ± 0.002 and 0.08 ± 0.01 µg/mL)  A. Oxidatg sugars . Among all the five varieties, Jaffna showed the highest values of protein content in comparison with the others. The trend observed for the total soluble protein in leaves was: Jaffna (0.695 ± 0.01 and 0.94 ± 0.01 µg/mL) > PKM-1 (0.58 ± 0.01 and 0.88 ± 0.01 µg/mL) > PKM-2 (0.57 ± 0.004 and 0.80 ± 0.01 µg/mL) > ODC (0.44 ± 0.005 and 0.74 ± 0.01µg/mL) > conventional (0.31 ± 0.02 and 0.64 ± 0.01 µg/mL) B. SancheM. oleifera varieties at two different stages of plant growth (young stage: 3-months-old and mature stage: 15-months-old) were assessed to estimate the amount of chlorophyll a, chlorophyll b and total chlorophyll content. Jaffna variety exhibited the highest chlorophyll content at all the stages. The trend observed was: Jaffna  > PKM-1  > PKM-2  > ODC  > conventional   ,41. In o30 mg/g) C. M. oleifera were estimated by using a flame photometer (Na and K), spectrophotometer , Kjeldahl method (N), and titration (Ca and C) as shown in M. oleifera is the most nutrient-rich variety, among the five. It contains macro-essential minerals such as Na, K, Ca, N, S, C, and P which make it a potential source of food and is suitable to combat malnutrition. Our results are in agreement with that of Sodamade et al., Oluwole et al., and El Sohaimy et al. , PKM-2 , ODC , and conventional  ] . M. oleifera contain sufficient amounts of the aforementioned anti-cancer compounds. The obtained results are in consonance with the findings of Fahey  isothiocyanate, 3-caffeoylquinic, and 5-caffeoylquinic acid [M. oleifera is shown in M. oleifera indicates that the Jaffna variety is endowed with a higher content of anti-cancer compounds than the other four. Thus, these results suggest the cultivation and consumption of Jaffna leaves in cancer-affected zones worldwide.The present study showed that the different varieties of of Fahey , who repof Fahey , showed nic acid . A non-eMLEs of all five varieties were also evaluated for their cytotoxic activity against the HepG2 cancer cell line, through the MTT assay. M. oleifera also inhibits the cell viability of liver carcinoma, lymphoblastic, and myeloid leukemia [M. oleifera on the HepG2 cell line. Jaffna variety showed the highest cytotoxic effect on cancer the cell line as compared to the other varieties.Cancer cells possess tremendous potential to divide and therefore anticancer agents are required to stop their growth by targeting them. Interestingly, MLE performs a wide range of biological activities and even targets several proteins and biomolecules to retard cancer progression ,58. It cleukemia . These aleukemia . These tleukemia . The realeukemia . The preM. oleifera) to combat malnutrition. The fact that Moringa is a source of nutrition and medicine has been tested over time and is deeply rooted in Ayurveda, Siddha, Unani, and the traditional Chinese system of medicine (TCM). It is interesting to note that people of all age groups can consume moringa leaf powder and can replenish vitamins and minerals. The present study shows the comparison of biochemical properties of PKM-1, PKM-2, ODC, Jaffna, and conventional varieties of M. oleifera on the basis of different parameters: Frap antioxidant activity, sugar, protein and mineral contents, percentage of phytochemicals (four anticancer compounds), and cytotoxicity against the HepG2 cell line. By assessing these parameters, it was possible to conclude that the Jaffna variety is the most beneficial variety in terms of nutrition, anticancer bioactive compounds, and cytotoxic activity. However, the Jaffna variety needs to be explored further to be involved in reforestation programs so that many people may fully capitalize on its amazing benefits. Thus, meticulous studies on other nutritional and medicinal parameters of Jaffna should be conducted to explore its potential as an alternative and cost-effective source of nutrition and medicine, for better healthcare.The easiest solution for affordable health and nutrition is to shake hands with nature. As mentioned, mother nature has provided mankind with the gift of ‘superfood trees’ (known as


Here, we synthesized a glassy-state CP, [Zn3(H2PO4)6(H2O)3], that exhibited a low melting temperature (114 °C) and a high anhydrous single-ion proton conductivity (8.0 × 10−3 S cm−1 at 120 °C). Converting crystalline CPs to their glassy-state counterparts via melt-quenching not only initiated an isotropic disordered domain that enhanced H+ dynamics, but also generated an immersive interface that was beneficial for solid electrolyte applications. Finally, we demonstrated the first example of a rechargeable all-solid-state H+ battery utilizing the new glassy-state CP, which exhibited a wide operating-temperature range of 25 to 110 °C.Designing solid-state electrolytes for proton batteries at moderate temperatures is challenging as most solid-state proton conductors suffer from poor moldability and thermal stability. Crystal–glass transformation of coordination polymers (CPs) and metal–organic frameworks (MOFs)  + conductivity  and optimal mechanical properties (42.8 Pa s at 120 °C), enables the operation of an all-solid-state proton battery from RT to 110 °C.Melt-quenched coordination polymer glass shows exclusive H Since the H+ charge radius is significantly smaller than that of other ions, faster ion migration and negligible volume changes upon H+ insertion/desertion are expected. Additionally, replacing high-cost Li+ with cheaper and more abundant H+ provides a promising platform for environmentally benign and intrinsically safe energy storage.5–7 Redox-active organic molecules, such as quinone-functionalized conductive polymers,4,8 and metal oxides, including MoO3, WO3, and HxIrO4, are available as H+ electrodes.9–11 Though proton batteries show a smaller specific capacity with a limited number of applications, as compared to their metallic counterparts, diffusion-free charge transport via the Grotthuss mechanism in a defective, Prussian blue analog establishes a high-rate capability (380 A g−1) and extends cycling stability to over 0.5 million charge–discharge cycles, which is a unique advantage of aqueous proton batteries.6,12,13 In spite of various choices of electrodes, electrolytes are mostly limited to aqueous H2SO4 or H3PO4, which dictates the operating-temperature window and selection of usable electrodes.8,13–15The proton  equipment in the oil and gas industries.16 As employing a conventional aqueous electrolyte is not possible at these high temperatures, solid-state H+ batteries with anhydrous solid electrolytes would be more suitable. There are no reports of solid-state H+ batteries working near or above 100 °C due to the difficulties in achieving high anhydrous H+ conductivity, high-temperature stability, and moldability required for H+ conductors.17 Apart from achieving a high H+ conductivity value (near 10−2 S cm−1), high thermal/chemical stability, processing ability, and ion selectivity are also needed to expand the practicality of solid-state electrolytes. Single-ion conductivity in solid-state electrolytes is a core factor that promotes charge-transport efficiency and prevents anion polarization.18,19 Discontinuities along the electrode–electrolyte interfaces and grain boundaries are primary bottlenecks for efficient utilization of solid electrolytes.17,20,21 H+ conductivity at the grain boundary of most crystalline compounds requires a higher migration activation energy than that required by H+ conductivity through the bulk crystal .22–25Safely extending the operating-temperature window to ∼100 °C is essential for H+ conductivity over a wide temperature regime (∼200 °C) represent a new class of solid-state H+ conductors.26–29 Despite their remarkable H+ conductivity, their crystalline nature hinders their processing ability, thus limiting their practicality.30 The glassy state of CP/MOFs is a strong platform to tackle these issues, and there have been increasing numbers of glassy-state CPs recently made from crystalline-state CPs.31–34 Some of these glassy-state CPs show anhydrous H+ conductivity superior to that of their crystalline counterparts by several orders of magnitude.35,36 Moreover, the vitrifying/melting behavior provides these CPs with processing capabilities and forms a grain-boundary free monolith and a flawless heterogeneous interface.31–34,37–41Coordination polymers (CPs) and metal–organic frameworks (MOFs) exhibiting high H+-conductive CP glass suitable for high-temperature anhydrous solid-state H+ batteries. By optimizing the pKa value of the component with 1,2,3-benzotriazole  and the extended hydrogen-bonding network in Zn2+-based CPs, the material demonstrated high anhydrous H+ conductivity (8.0 × 10−3 S cm−1 at 120 °C), relatively low melting point (114 °C), and mechanical softness (42.8 Pa s at 120 °C), which are suitable for electrolytes. The structure and properties were characterized by single-crystal X-ray diffraction (SC-XRD), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), impedance spectroscopy, electromotive force measurements, and solid-state NMR. We also demonstrated a full-cell evaluation of the anhydrous solid-state H+ batteries at 25, 100, and 110 °C.To address this issue, we have developed a new H1a) as a white crystalline powder. SC-XRD analysis of 1a provided its chemical formula, [Zn3(H2PO4)6(H2O)3](BTA), and it was found to exist as a one-dimensional (1D) chain along the a-axis 6(H2O)3](benzimidazole),38 and it is expected that the dynamics of the phosphates bridging the Zn2+ ions (through a single bridging oxygen atom (μ2)) and the non-coordinating BTA could facilitate an anhydrous H+ migration.2,38,42,43Zinc oxide, phosphoric acid, and BTA were subjected to mechanical milling to form the CP  of 1a  and 31P magic-angle spinning (MAS) solid-state NMR  slightly lower than the theoretical ratio (1 : 2) suggested the presence of a small amount of structural defects. All peaks in 31P NMR were located in the range of orthophosphate, suggesting that no condensation occurs during the mechanical synthesis.45–47 TGA of 1a showed a gradual weight loss due to the release of coordinated water at 100 °C . This dehydrated state is henceforth denoted as 1. A reversible structural change between 1a and 1 upon water adsorption and desorption was observed by PXRD  coordination sphere caused the 1D chain structure to deform around the Zn2+ ion. Under ambient air, 1 converted to 1a by capturing atmospheric moisture.38The gram-scale synthesis of a Fig. S1 demonstrR Fig. S2.44 A P tC Fig. S4. The totD Fig. S5.48–50 Th1a by TGA  of 114 °C. Two minor endothermic peaks before that of the Tm were assigned to the dehydration of adsorbed water during the measurement setup.33 The Tm of 1 was 50 °C lower than that of the isostructure, [Zn3(H2PO4)6(H2O)3](benzimidazole), as BTA exhibits lower Tm and pKa values than those of benzimidazole.31,33 Additionally, no significant weight loss was seen at 120 °C after 12 h, confirming a stable liquid state  that demonstrated a glass transition temperature (Tg) of 7.6 °C, exhibiting no Bragg diffraction, and was categorized as melt-quenched glass (MGQ)  Fig. S7.341g further determined its processing ability, where its viscosity (6.6 Pa s) from −30 to 90 °C until it sharply decreased below the working point regime (103 Pa s) above 100 °C. The working point defines the viscosity regime in which the viscosity of a substance is equivalent to that of soda–lime–silica glass above 1100 °C .51 The storage modulus (G′) dominated the loss modulus (G′′) from −30 to 90 °C, verifying the solid character of 1g  impedance under an Ar atmosphere to exclude the influence of water molecules 6](HBim) at 30 °C (1.2 × 10−7 S cm−1) and 60 °C (1.5 × 10−5 S cm−1).38We measured the Holecules  and S9†.(1g) via melt-quenching directly into the electrochemical cell for impedance analysis. Upon the crystalline-to-glassy state transformation, only the bulk impedance response pattern was obtained . Long-term conductivity retention was also evaluated. After 12 h, less than 4% and 10% loss in conductivity was observed at 100 and 120 °C, respectively  measurements.52,54 The transport numbers of most aqueous and ionic liquid electrolytes are lower than 0.6.54–56 The transport number of 1m was elucidated via electromotive force (EMF) measurements, which were conducted for different hydrogen partial pressure (−ln(P1/P2) values of 0.22, 0.51, 0.69, 0.92, and 1.61) at 120 °C ,+ transference number of 1g is 1.0, indicating an ideal single-ion H+ conductivity. The absence of anion mobility suggests that the coordination networks are retained even in the molten state.31,58To highlight the advantage of glass transformation on ionic conductivity, we prepared a monolith d Fig. S9 and it wius plot  shows tw Fig. S10. The con Fig. S11.57 Accor+ conductivity would be dominated by either the phosphate or BTA dynamics; therefore, we utilized variable-temperature 1H MAS solid-state NMR to study their mobilities  electrode was pressed to 1m at 120 °C and quenched to room temperature to provide the electrode–electrolyte interface (CF–1g). Cross-sectional scanning electron microscopy (SEM) images of CF–1g were collected  mapping (CF electrode (intense C) with homogeneously distributed Zn, P, and O signals. To amplify the benefits of melt-quenching glass, we re-examined the morphological alteration of CF–1g after recrystallization. 1g undergoes the recrystallization process upon humidity exposure and transforms back to 1a. As confirmed by PXRD 6]2/3·4H2O (CuFe-TBA) were selected as a model cathode and anode, respectively.10,12 As a reference, we also evaluated a full-cell configuration in 2 M H2SO4 solution at 25 °C. It exhibited a discharge capacity of 35.8 mA h g−1 at 100 mA g−1  in 1m at 120 °C under an Ar atmosphere, where subsequent quenching to room temperature gave the 1g electrolyte. + batteries under an Ar atmosphere utilizing the 1g electrolyte at 25 °C. The highest discharge capacity was 55.4 mA h g−1 at 10 mA g−1. Another advantage the 1g-electrolyte system has over the aqueous system is its large operating-temperature range. The elevated-temperature H+ battery was evaluated at 100 and 110 °C under an Ar atmosphere 3 as elevated temperature promotes a larger structural distortion between Li3V2(PO4)3 and V2(PO4)3 limiting the re-insertion of Li+.63 Additionally, 76% of the original capacity was retained after 1000 cycles of the charge–discharge process at 110 °C  conditions, even though the thickness of this electrolyte was ten times smaller than that of the 1g electrolyte. This emphasizes the importance of interface engineering that endows soft glass materials with high H+ conductivity and moldability.17,20,21Adequately high H Fig. S14. The spemosphere  and the  Fig. S16. We also+ conductive CP, [Zn3(H2PO4)6(H2O)3](BTA), where the dehydrated state (1) integrated promising anhydrous H+ conductivity (1.2 × 10−3 S cm−1 at 100 °C) and relatively low melting point (114 °C). The melt-quenched glass of 1 (1g) enhanced the H+ dynamics of both phosphate and BTA, resulting in a H+ conductivity value of 8.0 × 10−3 S cm−1, a H+ transference number of 1.0, and a viscosity of 42.8 Pa s at 120 °C. The coexistence of high conductivity, transport number, and moldability of 1g, as well as its flawless interface, encouraged us to implement it in solid-state H+ battery applications. A solid-state H+ battery with an operating temperature range above room temperature (25–110 °C) was demonstrated for the first time. The tuning capability of the CP glass H+ conductivity, working temperature, and softness could provide H+ batteries with wider applications.We synthesized a new HS. H. designed the project, and N. M. and A. Y. synthesized the compounds. S. K. collected and analyzed solid-state NMR measurements. N. M. collected and analyzed SC-XRD, PXRD, TGA, SEM, DSC, ICP-ES, DMA, FTIR, conductivity and transport number measurements and battery evaluation. S. H. and N. M. wrote the paper.There are no conflicts to declare.SC-012-D1SC00392E-s001SC-012-D1SC00392E-s002


Chronic heart failure (CHF) is a prevalent health concern with complex pathogenesis. This current study set out to estimate the function of the miR-129-5p/Smurf1/PTEN axis on cardiac function injury in CHF. The model of CHF in rats was established. The cardiac function indexes, myocardial tissue damage, and oxidative stress-related factors in CHF rats were evaluated after the interference of Smurf1/miR-129-5p/PTEN. The targeting relationships between miR-129-5p and Smurf1 and between PTEN and Smurf1 were verified. It was found that that after modeling, cardiac functions were impaired, heart/left ventricular/lung weight and the myocardial structure was destroyed, and the degree of fibrosis of myocardial tissue was increased. After Smurf1 knockdown, the cardiac function, myocardial structure, and oxidative stress were improved, and the fibrosis in myocardial tissue was decreased. Smurf1 was a target of miR-129-5p. miR-129-5p could annul the protective effect of Smurf1 silencing on CHF rats. Smurf1 inhibited PTEN expression by promoting PTEN ubiquitination, while miR-129-5p enhanced PTEN expression by inhibiting Smurf1. Meanwhile, overexpression of PTEN annulled the cardiac dysfunction in CHF rats induced by Smurf1. In conclusion, miR-129-5p targeted Smurf1 and repressed the ubiquitination of PTEN, and promoted PTEN expression, thus improving the cardiac function of CHF rats.  In receHuman Smurf1 (Smad ubiquitin regulatory factor 1) is a tumor promoter by ubiquitination modification and/or degradation of tumor-suppressing proteins, and it is principally expressed in the nervous system, bone, lung, reproductive organs, and other tissues . More imOn a separate note, Smurf1 ubiquitylates and degrades phosphatase and tensin homolog (PTEN) in glioblastoma . Down-remicroRNAs (miRNAs) are small endogenous non-coding RNAs that are essential tools for regulating gene expression and are involved in a variety of biological events, like cell proliferation, differentiation, and apoptosis . Smurf1 2.2.1.Animal experiments conformed to the standards established by the animal experiment committee of Tongji Hospital affiliated to Tongji University and were approved by the ethics committee of Tongji Hospital affiliated to Tongji University.2.2.ad libitum access to food and water. The CHF model was established by ligation of the anterior descending branch of the left coronary artery. The control rats were treated with the same surgical method without arterial ligation. Four weeks after arterial ligation, the rats were allocated to 13 groups with 10 rats per group as follows: control group, CHF group (model establishment), sh-NC group , sh-Smurf1 group , agomiR-NC+oe-NC group (rats were injected with of miR-129-5p agomiR-NC and negative control of Smurf1 overexpression vectors), agomiR-NC + oe-Smurf1 group , miR-129-5p agomiR + oe NC group , miR-129-5p agomiR + oe-Smurf1 group (rats were injected with miR-129-5p agomiR and Smurf1 overexpression vectors), oe-NC group , oe-NC + oe-PTEN group , oe-Smurf1+ oe-NC group , and oe-Smurf1 + oe-PTEN group . Four weeks after injection, transthoracic echocardiography was performed to assess the heart function. After that, the rats were anesthetized using 50 mg/kg pentobarbital sodium to remove the heart and lung tissues.Male Wistar rats  from Hunan SJA Laboratory Animal Co. Ltd  were placed in 12-h light/dark cycles with 2.3.3  −  ESD3)/EDD3]  ×  100; FS (%) =  [(LVEDD − LVESD)/LVEDD]  ×  100. All values were continuously measured for 6 consecutive cardiac cycles in the same rat. The rats were selected blindly.The Vevo 2100 system  was used to display the left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), ejection fraction (EF), and fractional shortening (FS). The calculation formula is as follows: EF (%) =  [ for 5 minutes, differentiated with 1% hydrochloric acid and ethanol for 3 seconds, and stained with 5% eosin solution . After that, the sections were dehydrated, cleared, sealed, and observed [2.5.After dewaxing, sections were sliced, stained with ponceau solution for 2 minutes, immersed in 0.2% glacial acetic acid solution, differentiated in 5% phosphomolybdate solution, and immersed in 0.2% glacial acetic acid solution, and stained with methyl green dye. Finally, the sections were sealed with neutral gum and observed .2.6.Paraffined specimens of each group were collected, sliced at a thickness of 4 μm), dewaxed, and steps were operated according to the conventional immunohistochemical staining method. The primary rabbit anti-Smurf1  was added for incubation. Staining results were determined by randomly selecting 5 lesion areas at high magnification and counting the number of cells with positive staining. Results can be calculated according to the percentage of positive cells. Each experiment was conducted 3 times .2.7.The myocardial tissue of the left ventricular was minced evenly, and 1 g tissue was resuspended in 2 mL RIPA lysis buffer. Then, the activity of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) was measured using enzyme-linked immunosorbent assay (ELISA) kits (Solarbio). Finally, the absorbance was measured using a microplate reader  .2.8.Synthetized Smurf1 3ʹUTR fragments were inserted into the pGL3-reporter  using endonuclease sites XhoI and BamH I to design complementary sequence mutation sites on Smurf1 wild-type seed sequences. The target fragment was inserted into the pGL3-reporter using a T4 DNA ligase after digestion with restriction enzymes. The constructed Smurf1 wild-type (wt) or mutant type (mut) was co-transfected into HEK293T cells with miR-129-5p mimic or mimic-NC. After 48 hours, the cells were lysed and luciferase activity was testified using the dual-luciferase assay kit (Promega) on the Luminometer TD-20/20 detector (Promega Biotech), respectively .2.9.Primary rat cardiomyocytes were provided by ATCC . To produce cells overexpressing the Smurf1 (the Smurf1 group), cells were delivered with the Smurf1 plasmid for 8 hours using 5 µL Lipofectamine 3000 . Next, cells were cotransfected with Smurf1 plasmids and miR-129-5p mimic  to produce the Smurf1 + miR-129-5p group. The proteasome inhibitor MG132 (10 µM) or DMSO (0.1%) was used as solvent control. They were added to the cell culture medium 2 hours prior to mechanical strain application and remained in the culture medium throughout the experiment .2.10.−ΔΔCt method. The internal parameters were U6 and GAPDH, and the primer sequences are shown in Cardiomyocytes were collected and the total RNA content was extracted using the TRIzol reagent (Invitrogen). The first-strand cDNA synthesis kit  was used to synthesize the first strand of cDNA by reverse transcription. The gene expression was tested by qRT-PCR using the SYBR Premix Ex Taq kit (Takara Bio). The PCR was performed using ABI Prism 7500 Fast Real-Time PCR System  under the subsequent conditions: pre-denaturation at 95°C for 10 minutes, and 40 cycles of denaturation at 94°C for 30 seconds, annealing at 59°C for 30 seconds and extension at 72°C for 30 seconds. Gene expression was calculated based on the 22.11.The total protein was extracted using RIPA . An appropriate amount of protein lysate was supplemented, collected in an EP tube, and placed on ice for lysis for 30 minutes. The supernatant was collected and protein concentration was determined using the bicinchoninic acid (BCA) kit . Following protein separation using polyacrylamide gel electrophoresis (PAGE), the protein transferred onto 0.2 μm polyvinylidene fluoride (PVDF) membranes . The antibody was diluted with tris-buffered saline-tween buffer containing 1% skim milk powder, which was used as the primary antibody diluent and added to the membranes and incubated overnight at 4°C. The next day, the horseradish peroxidase-labeled goat anti-rabbit IgG antibody  was diluted with TBST containing 1% skim milk powder, which was used as the secondary antibody diluent. The PVDF membranes were incubated at room temperature for 1 hour and detected using a digital chemiluminescence analyzer  and analyzed using Image J. GAPDH was used as an internal reference, and the ratio of the gray value of the target band to that of GAPDH band was used as the relative level of proteins. The primary antibodies used, including rabbit anti-Smurf1 , PTEN , and GAPDH ab8245; 1:1000), were all from Abcam  [5; 1:10002.12.Cells in each group were lysed in a lysate buffer (a mixture of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerin, 1 mM ethylene diamine tetraacetic acid (EDTA), 0.5% NP-40, and protease inhibitors), and cell debris was removed following centrifugation. After the measurement of lysate concentration using BCA kits, the same amount of proteins was taken from each group and supplemented with cell lysate to the same volume, then 1 μg anti-Smurf1 , PTEN , and 15 μL protein A/G beads  were added and incubated for 2 hours. After washing with cell lysate 3 times, beads were collected by centrifugation, and then an equal volume reductive loading buffer was added. The beads were boiled at 100°C for 5 minutes. Samples were separated by sodium dodecyl sulfate-PAGE, and the proteins were transferred onto PVDF membranes (Millipore), which were then analyzed by immunoblotting .2.13.t-test was used for comparisons between 2 groups. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey’s test. A value of P < 0.05 was regarded statistically significant.All data were processed by SPSS 21.0  and tested for normal distribution and homogeneity of variance. Measurement data complied with the assumption of normal distribution and homogeneity of variance and were displayed as mean ± standard deviation. An unpaired 3.This study was designed to investigate the effect of miR-129-5p on cardiac function of CHF rats by regulating the expression of Smurf1/PTEN. We proved that miR-129-5p may block the ubiquitination of PTEN by targeting Smurf1, thus improving the cardiac function of CHF.3.1.P < 0.05) (P < 0.05) (Previous studies have shown that Smurf1 is overexpressed in tumors  and over < 0.05) ). The ra < 0.05) ). HE sta < 0.05) ). Masson < 0.05) ). Smurf1 < 0.05) ). Meanwh < 0.05) ). Briefl3.2.P < 0.05) (P < 0.05) (P < 0.05) (P < 0.05)  ), indica < 0.05) ). Compar < 0.05) ). HE sta < 0.05) ). Masson < 0.05) ). Oxidat < 0.05) ). Altoge3.3.http://www.sysu.edu.cn/403.html) (P < 0.05). There was no significant difference in the luciferase activity in MUT-Smurf1 3ʹUTR (P > 0.05), indicating that miR-129-5p may specifically bind to Smurf1 (P < 0.05) (P < 0.05)  ). At theo Smurf1 ). Then,  < 0.05) ). Smurf1 < 0.05) . Overall3.4.P < 0.05) (P < 0.05)  ). HE sta < 0.05) ). Masson < 0.05) ). Oxidat < 0.05) ). Altoge3.5.It has been reported that overexpression of Smurf1 promotes PTEN ubiquitination and degradation , and the3.6.To further validate whether PTEN as a downstream of Smurf1 to influence CHF, we divided the CHF rats into the oe-NC group, oe-Smurf1 + oe-NC group, oe-NC + oe-PTEN group, and oe-Smurf1 + OE-PTEN group.P < 0.05) (P < 0.05)  . HE stai < 0.05) ). Masson < 0.05) ). Compar < 0.05) ). Overal4.CHF, now a well-known public health concern, is the chief cause of death worldwide ,14. miRNPrevious research has identified the involvement of Smurf1 in heart failure . But itsSubsequently, we focused our efforts on elaborating the upstream mechanism of Smurf1. The Starbase website predicted the targeted binding sites between miR-129-5p and Smurf1. miR-129-5p is decreased in CHF patients . Dual-luAccording to a previous report, overexpression of Smurf1 promotes the ubiquitination degradation of PTEN . Up-reguRecently, it has been proposed that PTEN can be regulated by post-translational modifications (such as ubiquitination), and the ectopic expression of Smurf1 significantly increases the ubiquitinated PTEN protein level . To explCollectively, our findings indicated that miR-129-5p can improve cardiac function of CHF rats by targeting E3 ubiquitin ligase Smurf1 and promoting the expression of PTEN. However, due to the limited time and funds, the expression patterns and mechanisms of miR-129-5p, Smurf1, and PTEN in the cell models and the clinical setting are not investigated. Therefore, further studies should be conducted on the cell level to find out multiple mechanisms of miR-129-5 on CHF, so as to offer novel insights into CHF management.5.In summary, this study suggested that the protective effect of miR-129-5p overexpression on CHF is mainly reflected in the improvement of cardiac function, anti-myocardial fibrosis, and inhibition of oxidative stress in rats. In addition, the key mechanism of miR-129-5p acting on CHF was revealed, that is, by targeting Smurf1, miR-129-5p prevented the ubiquitination of PTEN and promoted the expression of PTEN, thus improving the cardiac function of CHF. Taken together, miR-129-5p has great potential in the treatment of CHF and is expected to become a new target for the treatment of CHF.


In this article, an anthracene-functionalized semiconducting compound (DPPA) has been designed and synthesized. With irradiation, the compound is able to undergo efficient intersystem crossing (ISC) and non-radioactive decay for photodynamic/photothermal synergistic therapy. In addition, the anthracene module is able to capture and release 1O2 reversibly with or without irradiation. DPPA nanoparticles (NPs) obtained by nanoprecipitation with DSPE-PEG exhibit considerable high phototoxicity on human kidney cancer cells (A498), and the half maximum inhibitory concentration (IC50) is 15.8 μg/ml. Furthermore, an in vivo study demonstrates that complete tumor suppression was observed when the mice were administered DPPA NPs with the help of laser, compared with the control and dark groups. The H&E analysis of the normal tissues  indicates that such NPs cause no side effects, indicating the biosafety of DPPA NPs. The results provide a strategy to design a heavy-atom–free photosensitizer for photothermal and fractionated PDT against kidney tumors.Photodynamic therapy (PDT) utilizes the photogeneration of reactive oxygen species (ROS) with high cytotoxicity to kill cancer cells, holding great promise for cancer treatment. Fractionated delivery of singlet oxygen ( With the increasing cases of cancer worldwide, the development of new therapeutic methods for cancer treatment is of tremendous significance. . Phototh1O2 to regenerate the anthracene modules.  of the as-obtained DPPA is 21.3% in dichloromethane (DCM). DPPA nanoparticles (NPs) obtained by nanoprecipitation exhibit spherical morphology with an average diameter of 52 nm. Such NPs are able to capture singlet oxygen with irradiation and release it in the dark condition. The photothermal conversion efficiency of DPPA NPs is 35.6%. The PDT and PTT synergistic effect may promise the excellent therapeutic efficacy of DPPA NPs.  of DPPA NPs is as low as 15.8 μg/ml in human kidney cells (A498) with laser irradiation. Further fluorescence imaging in vivo suggests that DPPA NPs are able to passively target the tumor by the EPR (enhanced permeability and retention) effect. With the help of a laser, DPPA NPs are capable of inhibiting the tumor growth while exerting little side effects on normal tissues, including the heart, spleen, liver, kidney, and lungs. The results suggest that DPPA NPs have great potential for photothermal and fractionated photodynamic therapy.In this work, we have designed and prepared a heavy-atom-free semiconducting compound 3,6-bis[5-(anthracen-9-yl)furan-2-yl]-2,5-bis(2-octyldodecyl)pyrrolo pyrrole-1,4-dione (denoted as DPPA) by a C-H activation reaction (Scheme). Compared with the standard substance methylene blue (MB), the singlet oxygen quantum yield (PPA NPs. . In vitr1H NMR and 13C NMR spectra were performed on a Bruker DRX NMR spectrometer in CDCl3 (δ = 7.26 ppm) at 298 K as the internal standard. UV-vis and fluorescence spectra were measured on a Shimadzu spectrophotometer, from Japan, (UV-3600) and a HITACHI spectrometer , respectively. TEM of the nanoparticles were measured on equipment (JEOL JEM-2100). Dynamic light scattering (DLS) of DPPA NPs was tested on a particle size analyzer . Fluorescence imaging of DPPA NPs in nude mice was recorded on an IVIS spectrum.2 , and K2CO3  was dissolved in 5 ml DMA . Then, N2 was bubbled to drive off possible oxygen and water in the system. The mixture was heated to 110°C under the protection of N2 gas for 12 h. After cooling to room temperature, the mixture was poured into saturated sodium chloride solution (150 ml) and extracted with dichloromethane (100 ml) three times. The organic layer was washed with brine, followed by drying with anhydrous sodium sulfate. The solvent was removed by rotary evaporation and purified by silica gel column chromatography with dichloromethane and hexane  as the developing solvent. Dark blue solids were obtained . 1HNMR: δ H 8.60-8.56 , 8.55-8.51 , 8.22-8.17 , 8.12-8.01 , 7.76-7.66 , 4.36-4.23 , 2.12-2.02 , 1.33-1.21 , and 0.96-0.76 . 13CNMR: 160.12, 143.83, 128.53, 127.70, 125.67, 124.55, 115.38, 57.13, 45.73, 37.35,30.84, 25.07, 21.63, 17.31, and 13.09. MS: m/z: 1,180.80, found: 1,181.85.A mixture of DPP , 9-bromoanthracence , pivalic acid , Pt (OAc)2. DPPA NPs with different concentrations were co-cultivated with A498 cells in the 96-well plate. For the illumination group, each well was irradiated with a 660 nm laser for 8 min. In contrast, the wells in the control and no illumination groups have not been irradiated. Relative cell viability was determined by recording the absorbance of MTT . MTT in PBS (5 mg/ml) was added to the well (20 μl) and then incubated for 4 h. After that, the mother liquid was discarded, and DMSO (200 μl) was added. The absorbance of each well was recorded on a Bio-Tek microplate reader. Cell viability was then calculated according to the equation:At 37°C, human kidney cancer (A498) cells were cultured with a medium consisting of 12% fetal bovine serum (FBS) in DMEM (Gibico) under the atmosphere of 5% COCell viability (%) = mean absorbance of the group incubated with DPPA NPs/mean absorbance of the group.All the cell experiments were repeated three times.2). The cells were excited at 633 nm, and fluorescence was observed from 650 to 750 nm to investigate the cellular uptake. They were excited with a 488 nm laser, and fluorescence was observed from 490 to 560 nm to show the ROS generation.A498 cells were cultured with DPPA NPs (3 ml) in a confocal dish for 4 h. Then, the medium was discarded, and the cells were washed with PBS , followed by the co-culture with 1 ml polyoxymethylene for 25 min. Then, polyoxymethylene was discarded, and the cells were also washed with PBS three times (1 ml). The cells were further co-cultivated with DCF-DA  for 5 min, followed by washing with PBS (1 ml) three times. A 660 nm laser was then applied to the sample for 3 min . After 12 h, the tumors of the PBS + laser and DPPA + laser groups were irradiated by a 660 nm laser (1 W/cm2) for 8 min, while the mice in the DPPA-only group were not irradiated. These nude mice were then sacrificed for histology analysis.The procedure follows the rules of the National Institutes of Health (NIH). The animal study was approved by Guilin Medical University (SCXK 2007-001). A total of 15 nude mice were purchased and then inoculated with A498 cells. Three mice have been chosen to perform p values of less than 0.05 were considered significant .All numeric data are expressed as mean ± s.d., unless otherwise indicated. The significance between two groups was analyzed by the two-tailed Student’s t-test. Statistical analysis was performed by GraphPad Prism 6.0. 1HNMR, 13CNMR, and mass spectroscopy  promises excellent phototherapeutic efficacy. Therefore, the 1O2 QY of DPPA was calculated by recording the absorbance of 1,3-diphenylisobenzofuran (DPBF) with laser irradiation. With methylene blue (MB ∼Φ = 57%) as the standard substance in DCM . DPPA NPs are able to be uptaken by human kidney cells (A498) after incubation for 6 h . MTT assay shows that DPPA NPs show quite low dark toxicity but high phototoxicity with a low IC50 of 15.8 μg/ml. In vivo photothermal- and fluorescence imaging-guided phototherapy suggest that such NPs are capable of suppressing the tumor growth at a low dose but cause no damage to normal tissues, suggesting the biocompatibility. These results provide some insights to design semiconducting photosensitizers with high phototoxicity, low dark toxicity, and good biocompatibility for photothermal and fractionated photodynamic therapy.In summary, an anthracene-functionalized semiconducting photosensitizer DPPA has been designed and prepared with a high


KM. Here, monomodular GH5_8 mannanases from Eubacterium siraeum (EsGH5_8) and Xanthomonas citri pv. aurantifolii (XcGH5_8) were produced and characterized to clarify if GH5_8 mannanases from Firmicutes and Proteobacteria without CBM(s) possess distinct properties. EsGH5_8 showed a remarkably high temperature optimum of 55 °C, while XcGH5_8 had an optimum at 30 °C. Both enzymes were highly active on carob galactomannan and konjac glucomannan. Notably, EsGH5_8 was equally active on both substrates, whereas XcGH5_8 preferred galactomannan. The KM values were comparable with those of catalytic domains of truncated GH5_8s, while the turn-over numbers (kcat) were in the higher end. Notably, XcGH5_8 bound to but did not degrade insoluble ivory nut mannan. The findings support the hypothesis that GH5_8 mannanases with CBMs target insoluble mannans found in plant cell walls and seeds, while monomodular GH5_8 members have soluble mannans and mannooligosaccharides as primary substrates.Glycoside hydrolase family 5 subfamily 8 (GH5_8) mannanases belong to Firmicutes, Actinomycetia, and Proteobacteria. The presence or absence of carbohydrate-binding modules (CBMs) present a striking difference. While various GH5_8 mannanases need a CBM for binding galactomannans, removal of the CBM did not affect activity of some, whereas it in other cases reduced the catalytic efficiency due to increased  Many carbohydrate-active enzymes acting on polymeric substrates, especially insoluble substrates, contain one or more non-catalytic carbohydrate-binding modules (CBMs) together with the catalytic domain. CBMs can improve catalytic function of glycoside hydrolases (GHs) through targeting the enzymes to the substrate and increasing substrate-enzyme proximity as well as disrupting the crystallinity of insoluble substrates or stabilizing the enzyme ,2,3,4. GA number of endo-β-1,4-mannanases (E.C. 3.2.1.78) have been characterized due to their potential applications in different sectors of industry including detergent, textile, food, animal feed, and bioethanol ..XcGH5_8 B. animalis, which has a CBM10 only capable of binding CGM but not insoluble cellulose or INM [The CBM2 and CBM10 of GH5_8 mannanases are known to facilitate binding to cellulose, despite the enzymes not being active on cellulose ,18,21,26e or INM . The monXanthomonas campestris, Avicel , and unmodified wheat starch were from Sigma-Aldrich .CGM-lv, INM, KGM, and linear β-1,4-mannooligosaccharides were from Megazyme ; CGM-hv (locust bean gum), GG, xanthan from https://itol.embl.de/ accessed on 8 March 2022) [Protein sequences for all GH5_8 proteins in the CAZy database  (778 seqch 2022) .EsGH5_8 and XcGH5_8 were predicted using AlphaFold run in the Colab notebook version [Models of  version .EsGH5_8 (GenBank accession no.: CBK96294) and XcGH5_8 (GenBank accession no.: AMU98328) without predicted signal peptides  were purchased  sub-cloned into pET28a using the NheI and XhoI restriction sites resulting in a cleavable N-terminal His-tag. The plasmids were transformed into E. coli BL21. Starter cultures (10 mL) were made by inoculating LB medium including 50 µg/mL kanamycin with a single colony and incubated at 37 °C overnight. LB medium (750 mL) containing 10 mM glucose and 50 µg/mL kanamycin in shake flask was inoculated with the overnight culture and propagated  to an absorbance at 600 nm of 0.6. The temperature was decreased to 16 °C and expression was induced by a final concentration of 0.1 mM IPTG. Cells were harvested  after approximately 20 h and stored at −20 °C until protein purification.The genes encoding g, 4 °C, 30 min). The supernatant was loaded onto a 5 mL HisTrap HP column  pre-equilibrated with HisTrap equilibration buffer and eluted using HisTrap elution buffer . The eluate was further purified by gel filtration (Superdex 16/60 75). EsGH5_8 was purified using 10 mM Hepes pH 7.4, 150 mM NaCl, while XcGH5_8 was purified using 10 mM Hepes at a pH of 7.0, 150 mM NaCl, and 10% glycerol. Fractions containing pure recombinant protein of correct size as based on SDS-PAGE were pooled and concentrated to 2–3 mg/mL ). XcGH5_8 was stored in 10 mM Hepes pH 7.0, 150 mM NaCl, and 30% glycerol, while EsGH5_8 was stored in the gel filtration buffer. The absorbance at 280 nm was measured, and protein concentration was determined using theoretical extinction coefficients .The proteins were purified in two steps. Cells were resuspended in HisTrap equilibration buffer , lysed using a high-pressure homogenizer at 1 bar, added 3 μL of Benzonase Nuclease (Sigma-Aldrich), and centrifuged . Fluorescence at 330 nm and 350 nm upon excitation at 280 nm were measured using Prometheus NT.48 High Sensitivity capillaries (NanoTemper Technologies) with a protein concentration of 2 µM. The excitation power was determined from a pre-scan of the samples and set to 61%. The unfolding was followed by ramping the temperature from 20 to 95 °C with a temperature increment of 1 °C/min.The melting temperatures (Tm) of g for 10 min. Finally, the absorbance was measured at 540 nm. Mannose was used as standard. One unit of mannanase activity was defined as the amount of enzyme that liberates reducing sugars equivalent to one µmol mannose per minute.Enzymatic activity was determined using a standard reducing end 3,5-dinitrosalicylic acid (DNS) assay essentially as previously described ,19,38: 3EsGH5_8, 7.4 nM; XcGH5_8, 15.3 nM) in a universal buffer  [pH optimum was determined using the standard assay in triplicate using a final concentration of 2.5 mg/mL CGM-lv with enzyme (mM NaCl)  in the rXcGH5_8, 20–45 °C; EsGH5_8, 20–65 °C) in 50 mM sodium phosphate-citrate pH 7.0 including 0.005% Triton X-100, as a buffer.The temperature optimum was determined using the standard assay with 2.5 mg/mL of CGM-lv at different temperatures (EsGH5_8) and 30 °C (XcGH5_8). EsGH5_8 (3.5–28 nM) and XcGH5_8 (5.1–51.1 nM) were incubated with low- and high-viscosity CGMs , KGM , GG , xanthan , or INM  in triplicate.The specific activity was determined using the standard assay with 50 mM sodium phosphate-citrate pH 7.0, 0.005% Triton X-100, as buffer at 37 °C  in 2000 μL and withdrawing 400 μL aliquots at 3, 6, 9, and 12 min. The kinetic assays were done in triplicate. The Michaelis–Menten model was fitted to the initial velocity data using GraphPad Prism 6 .The kinetics on CGM-lv were determined using the same buffer and temperatures as for specific activity determination with a range of substrate concentrations (0.45–9 mg/mL CGM-lv) and enzyme  in 50 mM sodium phosphate-citrate pH 7.0, 0.005% Triton X-100, was incubated at 25 °C (XcGH5_8) or 37 °C (EsGH5_8) for 20 h followed by 10 min at 95 °C to inactivate the enzyme. A sample (2 µL) was applied twice on a silica gel 60 F454 plate (Merck Millipore) and 2 µL of a mixture of 2 mM mannooligosaccharides (mannose to mannohexaose) was applied as standard. The separation was carried out in isopropanol:n-butanol:water (12:4:5) (v/v) as the mobile phase and run twice. Sugars were visualized with a diphenylamine alanine solution  and heat treatment.The product profile from hydrolysis of mannooligosaccharides was analysed using TLC: 50 µL of the reaction mixture, containing 2 mM of mannooligosaccharide (mannobiose to mannohexaose) or 2.5 mg/mL of CGM-lv and enzyme (EsGH5_8 was performed by AGE with 2.5 mg/mL and 5 mg/mL of CGM-lv in 10% acrylamide gels without stacking gels at pH 7.4 (43 mM imidazole and 35 mM Hepes) [Qualitative screening of binding of M Hepes) . ProteinEsGH5_8 and XcGH5_8 to insoluble crystalline INM, microcrystalline cellulose (Avicel), and wheat starch, was performed by a pull-down assay, where the samples were analysed by SDS-PAGE: 10 mg insoluble polysaccharide (prewashed with buffer three times) was mixed with 200 μL of 0.5 mg/mL protein in an assay buffer . Bovine serum albumin  was used as a control. The samples were incubated at 4 °C for 1 h with gentle agitation and then centrifuged . Supernatants were transferred to fresh tubes and centrifuged again, before 4 μL of a supernatant was heat treated in the presence of SDS-loading buffer and subjected to SDS-PAGE. The pellets from the pull-down assay were washed with 250 μL of assay buffer and pelleted as aforementioned, resuspended in 200 μL of assay buffer, added 50 μL of SDS-loading buffer, boiled for 10 min, and applied (5 μL) on the SDS-PAGE. For each protein, a sample of the 0.5 mg/mL protein stock used for the pull-down assay was treated as the supernatant sample and included on the gel.A qualitative screening of the binding of


Tyrosinase is a multifunctional copper-containing oxidase enzyme that initiates melanin synthesis in humans. Excessive accumulation of melanin pigments or the overexpression of tyrosinase may result in skin-related disorders such as aging spots, wrinkles, melasma, freckles, lentigo, ephelides, nevus, browning and melanoma. Nature expresses itself through the plants as a source of phytochemicals with diverse biological properties. Among these bioactive compounds, flavonoids represent a huge natural class with different categories such as flavones, flavonols, isoflavones, flavan-3-ols, flavanones and chalcones that display antioxidant and tyrosinase inhibitor activities with a diversity of mechanistic approaches. In this review, we explore the role of novel or known flavonoids isolated from different plant species and their participation as tyrosinase inhibitors reported in the last five years from 2016 to 2021. We also discuss the mechanistic approaches through the different studies carried out on these compounds, including in vitro, in vivo and in silico computational research. Information was obtained from Google Scholar, PubMed, and Science Direct. We hope that the updated comprehensive data presented in this review will help researchers to develop new safe, efficacious, and effective drug or skin care products for the prevention of and/or protection against skin-aging disorders. Tyrosinase is a multifunctional copper-containing oxidase enzyme, involved in the initial steps of melanin production . MelaninExcessive production of melanin pigments resulted in different dermatological disorders such as skin aging spots, wrinkles, melasma, freckles, lentigo, ephelides, nevus and melanoma . More seAsphodelus microcarpus, Morus nigra L. Limonium tetragonum, Arctostaphylos uva-ursi, Artocarpus lowii, Artemisia aucheri, Cassia tora, Silybum marianum, Humulus lupulus, Rhodiola crenulata, Alpinia officinarum, Mangifera indica, Podocarpus falcatus, Momordica charantia, Cymbopogon citrates, Vitis vinifera and Glycyrrhiza glabra −5′-O-β-d-glucopyranoside (32) [50 = 100 ± 0.5 μM), comparable to kojic acid (6.4 ± 0.04 μM). Quercetin (33), the most abundant flavonol in green tea, apples, berries, onions, and many other fruits, vegetables and grains was proved to possess potent inhibitory activity on the monophenolase and diphenolase activities of tyrosinase. An in silico study by Fan et al. [33) the reversible and competitive inhibition of quercetin on the diphenolase activity of tyrosinase with an IC50 value of (3.08 ± 0.74) × 10−5 mol L−1. The interaction of the 3′, 4′-dihydroxy groups of quercetin (33) with copper ions at the active sites is beyond the inhibitory activity of quercetin (33), as revealed by molecular docking studies. In another study with quercetin (33) isolated from Persicaria senticosa (Polygonaceae), it was reported to inhibit tyrosinase with IC50 values of 14.31 ± 3.93 μM, which is close to the positive control  [50 = 22.71 ± 0.53 μM), comparable to BHA (IC50 = 271.70 ± 19.45 μM).A certain in vitro study was conducted on the aerial parts of ide (32) . This con et al. , demonst4.16 μM) . MoreoveO-α-arabinopyranosyl-(1→6)-β-glucopyranoside (34) inhibited the enzyme with an IC50 value of 46.94 ± 3.09, near that of kojic acid (IC50 = 45.69 ± 1.65 µM). Similarly, the methanol extract of Scrophularia lucida’s aerial parts yielded hesperidin (hesperetin-7-rutinoside) (35) (394 μg/g extract) as a major component [Atraphaxis frutescens and investigated for their inhibitory actions on mushroom tyrosinase [β-d-glucopyranosyloxy-3′,4′,5,5′-tetrahydroxy-7-methoxy-3-α-l-rhamnopyranosyloxyflavone (36) (IC50= 0.90 ±0.02 mM), 3′,4′,5,5′,8-pentahydroxy-7-methoxy-3-α-l-rhamnopyranosyloxyflavone (37) (IC50 =1.20 ± 0.03 mM) and europetin 3-O-α-l-rhamnopyranoside (38) (IC50 =1.10 ± 0.07 mM), compared to kojic acid (IC50 = 0.088 ± 0.002 mM). However, isolated flavonol aglycones did not show any inhibitory properties. Hence, the presence of 3-O-rhamnosyl moiety was suggested to participate in tyrosinase inhibition. Kishore et al. investigated the tyrosine inhibitory activity of myricetin-3-O-α-l-rhamnopyranoside (39), isolated from Myrsine africana methanol extract [50 value of 0.12 ± 0.002 mM. The molecular docking analysis supported its inhibitory activity. The chemical structures of the flavonols with anti-tyrosinase activities are presented in A molecular modeling study evaluated the anti-tyrosinase activity of nine structurally related flavonols, naturally occurring in many plants . Among tomponent . This corosinase . It was  extract . It demo40), an isoflavanoid isolated from Glycyrrhiza glabra roots, was investigated for inhibitory effects and binding mechanisms on tyrosinase [50 value of 0.43 μmol/L through a multiphase kinetic process. Further, glabridin (40) forms stable a glabridin-tyrosinase complex through the strong quenching of intrinsic tyrosinase fluorescence. The molecular docking studies confirmed non-direct binding of glabridin (40) to the tyrosinase active site. Moreover, glabridin (39) did not exert any side effects on melanin synthesis or toxicity in a zebrafish model [41) from the ethanolic extract of the aerial parts of Pueraria 1lobate (Fabaceae) [41) significantly reduced the mRNA and protein expression levels of MITF, TRP-1, and tyrosinase in α-MSH-stimulated B16F10 murine melanoma cells. It potently inhibited tyrosinase activity at a rate of 45.50% at 50 µM, compared to arbutin (12%). Additionally, it effectively reduced melanin content in a reconstructed human 3D skin model, compared to arbutin. Mechanistically, it suppressed the melanin production through modulation of Akt/GSK−3β and MEK/ERK signaling transduction pathways. About twenty-seven flavonoids were identified by Promden et al. [Dalbergia parviflora and tested for tyrosinase inhibition. Among these flavonoids, -3,8-dihydroxy-9-methoxy pterocarpan (42) was the most promising tyrosinase inhibitor by 84.60%, showing an IC50 of 16.70 ± 5.00 μM, similarly to kojic acid (IC50 = 16.80 ± 4.60 μM) at a concentration of 200 μM. Furthermore, this compound (42) effectively suppressed the melanin production by 60% at a concentration of 15 μM without any effect on the cell viability of B16-F10 melanoma cells. Recently, Qu et al. [C-glucoside) (43) (7.66 mg/g), as a major compound in the Puerariae lobatae radix. The isoflavone (43) effectively inhibited the catalytic oxidation process of the tyrosinase enzyme with an IC50 of 0.537 mg/mL in a concentration-dependent manner. In 2018, Wagle et al. [Pueraria lobata (Fabaceae) roots. Among the list of these compounds, calycosin  (44) was characterized as the only potent inhibitor one , superior to the standard, kojic acid . Kim and his team published a report about the isoflavonoids isolated from Apios americana (Fabaceae) tubers and screened them for tyrosinase inhibition [45) and 2′-hydroxygenistein-7-O-gentibioside (46) dose-dependently displayed moderate competitive tyrosinase inhibitors with IC50 values of 39.70 ± 1.5 and 50.00 ± 3.70 µg/mL, respectively, whereas the IC50 of kojic acid was equal 25.20 ± 0.80 µg/mL. The chemical structures of the isoflavones with anti-tyrosinase activities are presented in Glabridin  . This con et al.  in Dalbeu et al.  identifie et al.  describehibition . Of the Silybum marianum) is well-known to be rich in flavonolignans with antioxidant, anti-inflammatory, antiviral and antifibrotic properties [47) was demonstrated to be the most potent one for tyrosinase inhibition, with an IC50 value of 1.70 ± 0.07 µM, compared to kojic acid (IC50 = 15.30 ± 0.50 µM) [47) showed mixed type 1 inhibition of enzyme and a significant binding affinity with Ki value of 0.7 µM. A study published by Chunhakant and Chaicharoenpong reported characterization of phytochemicals from Manilkara zapota (Sapotaceae) bark [48) demonstrated the most potent tyrosinase inhibitory effect with IC50 of 55.41 ± 0.38 μM, comparable to kojic acid (IC50 = 53.43 ± 0.38 μM) and α-arbutin (IC50 = 365.93 ± 0.45 μM). Furthermore, (+)-dihydrokaempferol (48) exhibited the strongest antioxidant activity of isolated compounds through three different assays; 2,2-diphenyl-1-picrylhydrazyl , 2,2′-azino-bis , and ferric reducing antioxidant power , compared with Trolox . Fan et al. [49), a common natural flavonoid, on tyrosinase. This compound is fitted in the pocket of tyrosinase with hydrophobic interactions and hydrogen bonds, resulting in conformational changes of tyrosinase that hinder substrate binding. Consequently, it inhibited tyrosinase activity in a mixed-type manner with an IC50 value of 3.66 ± 0.14 × 10−5 mol/L, compared with kojic acid (IC50 = 4.64 ± 0.37 × 10−5 mol/L). In addition, the combination of dihydromyricetin (49) with vitamin D3 displayed a synergistic effect on the enzyme inhibition. In Turkey, a group of researchers isolated a new catechin, namely (−)-8-chlorocatechin (50), from Quercus coccifera bark [50) expressed tyrosinase inhibition with IC50 value of 4.05 ± 0.30 µg/mL, more potent even than kojic acid (IC50 = 50.75 ± 0.25 µg/mL). Molecular modeling studies revealed the good fitting properties of the compound to the catalytic site of tyrosinase via 4-chromanone moiety with its hydroxyl groups. The chemical structures of the flavan-3-ols with anti-tyrosinase activities are presented in The milk thistle (operties . Among t0.50 µM) . Based oae) bark . Among tn et al.  studied era bark . The isoMorus alba leaves resulted in the isolation of twelve flavonoids which were evaluated for anti-melanogenesis effects in B16-F10 mouse melanoma cells [51) dose-dependently inhibited melanin production via the suppression of intracellular tyrosinase activity, MITF and TRP-1. Moreover, steppogenin (51) effectively modulated CREB and p38 signaling pathways in alpha-melanocyte stimulating hormone (α-MSH)-activated B16-F10 melanoma cells, proceeding to anti-melanogenesis effects. Interestingly, the structure–activity relationship elucidated that the compound possesses a unique structure for tyrosinase inhibition, including hydroxyl groups at C-2′ and C-4′ of the B-ring, at C-5 and C-7 of the A-ring and free C-3. Eriodictyol  (52), a flavanone isolated from Eriodictyon californicum, has been investigated for anti-melanogenic effects in cultured murine melanoma cells (B16-F10) and primary human keratinocytes (PHK) [51) displayed time and concentration-dependent inhibition of tyrosinase in the tested cells. Moreover, it exerted intracellular free radical scavenging potential against hydrogen peroxide (H2O2)-induced oxidative stress at a concentration of 50 μM in B16-F10 and PHK cells. Among the flavanones isolated from the wood of Artocarpus heterophyllous, artocarpanone  (53) demonstrated the most potent inhibitory activity against the tyrosine enzyme with an IC50 of 2.00 ± 0.1 μM [51) and liquiritigenin  (54) demonstrated concentration-dependent inhibition of the enzyme with IC50 values of 7.50 ± 0.50 and 22.00 ± 2.50 μM, respectively. They demonstrated more potent inhibition compared to the positive control, kojic acid, with an IC50 of 44.60 ± 0.40 μM. Pinostrobin (55) was isolated from Egyptian Propolis among other phenolic compounds and was evaluated for its tyrosinase inhibitory activities [55) was the most potent of the isolated compounds of Propolis, demonstrating a moderate inhibitory activity (36.30%) compared with vitamin C, demonstrating 60% inhibition. It was concluded that flavonoids possessing hydroxyl groups at the A and B rings are crucial tyrosinase inhibitors via Cu2+ chelate formation. Investigating the tyrosine inhibitory activity of the isolated flavonoids of Sophora flavescens, kushenol A (56) demonstrated the most potent inhibitory activity, demonstrating an IC50 value of 1.10 ± 0.70 μM [An in depth phytochemical study of 70% ethanolic extract of ma cells . Among tes (PHK) . Steppog± 0.1 μM . Additiotivities . Pinostr 0.70 μM . MoleculLoranthus acutifolius’s aerial parts. These compounds were identified as 2′4′-dihydroxy-6′-methoxy-chalcone (57) (IC50 = 5.70 ± 0.02 μM), comparable with kojic acid (IC50 = 13.90 ± 0.34 μM). Among seventeen phenolic compounds isolated from Angelica keiskei (Umbelliferae) roots, Lee et al. [58), showed a moderate tyrosinase inhibitory property with IC50 values of 15.87 ± 1.21 μM, compared to kojic acid (IC50 = 3.80 ± 0.20 μM). Interestingly, this study used a combinatorial novel approach that includes in vitro tyrosinase inhibition assay coupled with UPLC-MS/MS to screen isolated compounds. Flavokawain A  (59) and flavokawain B  (60), naturally occurring chalcones of Kava (Piper methysticum), were investigated for their melanogenic inhibition in α-MSH-induced B16/F10 cells and zebrafish [Humulus lupulus through tyrosinase activity-guided fractionation of its methanol extract [61), xanthoumol B (dehydrocycloxanthohumol Hydrate) (62) and xanthohumol C (dehydrocycloxanthohumol) (63) demonstrated dose-dependent inhibition of the monophenolase and diphenolase activities of tyrosinase, with IC50 values ranging from from 15.40 to 22.1 μM and from 41.10 to 46.70 μM, respectively. The more potent inhibition was demonstrated with chalcones bearing an isoprenyl group at ring A. Kinetic studies using Dixon and Lineweaver–Burk plots demonstrated the competitive inhibitory activity of the isolated chalcones against tyrosinase.Ticona et al.  identifie et al.  found thebrafish . Both co extract . Among tGreyia radlkoferi and its isolated flavonoids was investigated for their tyrosinase inhibitory effects [50 value of 17.96 μg/mL. Among the isolated flavonoids, 2′,4′,6′-trihydroxydihydrochalcone (64) was characterized as the most potent tyrosinase inhibitor, with an IC50 value of 17.70 μg/mL, comparable to that of kojic acid (IC50 = 3.87 μg/mL). The investigation of the underlying mechanism of inhibition using RT-qPCR was demonstrated to be post-transcriptional. Moreover, molecular docking studies revealed the influence of interaction with Cu2+ ions at the active site on tyrosinase. The chemical structures of the chalcones with anti-tyrosinase activities are presented in The methanolic extract of  effects . The ext65), sanggenon C (66), and sanggenon M (67) with sanggenon O (68) were isolated from the petroleum ether extract of Morus nigra twigs [65) being the most potent, with IC50 values of 0.17 ± 0.01 µM, followed by sanggenon O (68) (IC50 = 1.15 ± 0.03 µM), sanggenon C (66) (IC50 = 1.17 ± 0.03 µM), then sanggenon M (67) (IC50 = 13.06 ± 0.58 µM), compared to kojic acid (IC50 = 32.62 ± 1.24 μM). Two major prenylated flavonoids, namely kuwanon O (69) and sanggenon T (70), were isolated from Morus australis root extract and evaluated for depigmenting effects in different melanocyte systems and artificial skin models [69) and sanggenon T (70), demonstrated an outstanding depigmenting effect in the artificial skin model. Dehydroglyasperin C (71), a major prenylflavonoid of Glycyrrhiza uralensis (Fabaceae), suppressed intracellular tyrosinase activity and related proteins (TYR-1 and TRP-2) at a low concentration (1 μM) in α-MSH-induced B16F1 melanoma cells [Three new isoprenylated flavonoids, namely, kuwanon J , a prenylated form of Broussonetia kazinoki Sieb bark (Moraceae), to evaluate its anti-melanogenic effect on human melanoma cells, normal human melanocytes, and zebrafish [72) potently downregulated MITF by means of the acceleration of AMPK and MAPK protein phosphorylation, which are MITF inhibitors. Moreover, it exhibited anti-melanogenic effects in an vivo model of zebrafish without developmental defects. Cycloheterophyllin (73), a prenylated flavonoid isolated from Artocarpus lowii heartwood and leaves (Moraceae), was characterized as a tyrosinase inhibitor (IC50 = 104.6 μM), compared to kojic acid (IC50 = 219.6 μM) [50 = 102.80 μM), FRAP method (4.70 mM) and ABTS (SC50 = 320.00 μM) [Another study was conducted on kazinol U , a preny19.6 μM) . Furtherophyllin , a prenyArtocarpus pithecogallus (Moraceae) twigs, morachalcone A (74) and 6-prenylapigenin (75) exhibited the most potent tyrosinase effects with IC50 values of 0.77 ± 0.01 μM and 24.29 ± 0.12 μM, respectively, compared to kojic acid (IC50 = 17.32 ± 0.24 μM) [76) and isoxanthohumol (77), from Humulus lupulus methanol extract, among other flavonoids. Both compounds demonstrated significant inhibition of tyrosinase, with IC50 values of 38.1 and 77.4 µM, respectively, for monophenolase and IC50 values of 77.2 and 157.4 µM, respectively, for diphenolase. Kinetic studies using Dixon and Lineweaver–Burk plots demonstrated mixed type inhibition by both flavanones against monophenolase. However, noncompetitive inhibition was demonstrated by both compounds against diphenolase. A study by Kim et al. [Sophora flavescents. Investigating the inhibitory effects of the isolated flavonoids, 8-prenylkaempferol (78) demonstrated potent inhibitory activity (IC50 = 2.40 ± 1.10 μM) against tyrosinase exceeding the chalcones isolated. Molecular docking simulation revealed the competitive inhibition of the prenylated flavonol via its docking to His244 residue of tyrosinase. The chemical structures of the prenylated flavonoids with anti-tyrosinase activities are presented in Upon screening analysis of nine prenylated phenolic compounds isolated from alcone A  and 6-pr0.24 μM)  isolatedm et al.  led to t79) and mesuaferrone B (80) are major bioflavonoids, isolated from the methanolic extract of Mesua ferrea L. stamens with contents of 0.35 ± 0.04% and 0.55 ± 0.06%, respectively [50 values of 10.6 and 10.3 µg/mL, respectively, compared with arbutin (IC50 = 87.20 µg/mL). Furthermore, the dimerization of flavonoid units was found to contribute effectively in potent tyrosinase inhibitory activities, comparable to the original flavonoid monomers. The chemical structures of the biflavonoids with anti-tyrosinase activities are presented in Rhusflavanone  of flavonoids and tyrosinase inhibitory activity. It is worth mentioning that the number and the position of hydroxyl groups especially at ring B drastically affect the activity of different flavonoid classes via CuDespite this extensive research, we observed that the studies addressing the safety profile the promising flavonoids are very scarce. Additionally, some studies are recommended to be extrapolated from in vitro assays to animal and clinical studies to assess the pharmacokinetics, topical permeation, bioavailability, dose issues, lifespan, anatomy and metabolic status. As the conventional whitening market products suffer from severe side effects such as dermatitis and skin cancer in long-term use, we highly recommend well-designed long-term studies in the human subjects to investigate the potential toxic effects, biochemical and molecular mechanisms of the bioactive flavonoids. Interestingly, these studies will potentially push researchers to develop a novel drug for treating skin-aging ailments using medicinal chemistry approaches to synthesize more potent derivatives and study their detailed mechanisms of action.


Bacillus pumilus SK52.001. The BpFAE gene from B. pumilus SK52.001 was speculated and heterogeneously expressed in Bacillus subtilis WB800 for the first time. The enzyme exists as a monomer with 303 amino acids and a molecular mass of 33.6 kDa. Its specific activity was 377.9 ± 10.3 U/(mg protein), using methyl ferulate as a substrate. It displays an optimal alkaline pH of 9.0, an optimal temperature of 50 °C, and half-lives of 1434, 327, 235, and 68 min at 50, 55, 60, and 65 °C, respectively. Moreover, the purified BpFAE released 4.98% FA of the alkali-acidic extractable FA from de-starched wheat bran (DSWB). When the DSWB was enzymatically degraded by the synergistic effect of the BpFAE and commercial xylanase, the FA amount reached 49.47%. It suggested that the alkaline BpFAE from B. pumilus SK52.001, which was heterologously expressed in B. subtilis WB800, possesses great potential for biomass degradation and achieving high-added value FA production from food by-products.Feruloyl esterase  catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl group in an esterified sugar to assist in waste biomass degradation or to release ferulic acid (FA). An FAE-producing strain was isolated from humus soil samples and identified as  Feruloyl esterase FAE; EC 3.1.1.73) is a member of the alpha/beta hydrolase family that cleaves ester or ether linkages between ferulic acid (FA) and sugars in order to facilitate plant cell wall degradation and release FA . FA, a p.1.1.73 iEscherichia coli, a common and highly efficient expression system, is often a poor choice for industrial food production since it produces endotoxins and is not a “Generally Regarded as Safe” (GRAS) host. In recent years, an increasing number of researchers have begun to use Bacillus subtilis as a host to express enzymes, mainly because it is a food-grade microorganism and suitable for the industrial production of FAEs. B. subtilis is also amenable to a simple fermentation process and lacks obvious codon bias [Owing to their utility, strains that secrete FAEs continue to be identified, and some FAEs from fungi have been studied extensively, including their classification , biochemdon bias .B. subtilis system. In this study, the FAE from B. pumilus SK52.001 (BpFAE) was identified, cloned, and heterogeneously expressed in B. subtilis for the first time. The studies were conducted to characterize the biochemical properties and liberate FA from de-starched wheat bran (DSWB) by synergy action of BpFAE and commercial xylanase.To date, there have been few studies on the heterogeneous expression of FAE using the Lysozyme, ampicillin, and kanamycin sulfate were purchased from Sangon Biotech Co., Ltd. . FA (content > 99%) was obtained from J&K Scientific Ltd. . Methyl ferulate (MFA) (content > 99%) was purchased from ThermoFisher Scientific . Commercial xylanase  was obtained from Macklin Co., Ltd. . Wheat bran was obtained from Auchan Supermarket . Other chemicals were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. .3, 1 g/L K2HPO4, 0.5 g/L KCl, 0.5 g/L MgSO4·7H2O, 0.01 g/L FeSO4·7H2O, and 20 g/L ager), with ethyl ferulate (10 g/L) as the sole carbon source at 30 °C for 48 h. The resultant colonies surrounded by halo were purified in a non-selective isolation medium 3 times and inoculated in a fermentation medium  at 30 °C, with shaking at 200 r/min for 26 h. The enzyme activity of FAE was estimated and the strain with the highest activity was identified as the target strain for further study.The soil samples from Wuxi, Jiangsu Province were suspended in sterile saline at a final concentration of 1 g/mL at 30 °C and 200 r/min for 30 min. The strains were isolated using gradient dilution in a non-selective isolation medium . Subsequently, the colonies were picked up from the selective medium  and B. subtilis WB800 (preserved in our laboratory) were hosts for cloning and expression, respectively. The shuttle plasmid pMA5 with NdeI and BamHI sites  was used as the expression vector. Luria-Bertani (LB) broth  was prepared for the routine growth of E. coli. Ampicillin was dissolved in sterile water and used at a final concentration of 100 μg/mL. A super-rich medium (SR) containing 50 μg/mL kanamycin consisted of 3 g/L K2HPO4, 20 g/L yeast extract, 25 g/L tryptone, and 30 g/L glucose for B. subtilis WB800 cultivation.B. pumilus SK52.001 strain was extracted using the Ezup Column Bacteria Genomic DNA Purification Kit . The fae gene fragment was amplified with primers, Bpfae forward: AAAAGGAGCGATTTACATATGATGAACTTACAAGAGCAAATCAAAATCGCTGC and Bpfae reverse: GAGCTCGACTCTAGAGGATCCTTAATGGTGATGGTGATGATGTTCAAATGCCTTT. The FAE sequence fused with a 6 × histidine tag at C-terminus was ligated with the expression vector pMA5 between the NdeI and BamHI sites, resulting in pMA5-fae. The recombinant plasmids were transformed into competent E. coli DH5α cells via heat shock. Positive transformants were cultured overnight in 5 mL LB medium supplemented with ampicillin, and plasmids pMA5-fae were extracted by FastPure Plasmid Mini Kit  and sequenced by Sangon Biotech Co., Ltd. .Genomic DNA from the fae, the resultant plasmids pMA5-fae were introduced into B. subtilis WB800 to obtain the recombinant strains, B. subtilis WB800/pMA5-fae. A colony positive for kanamycin resistance was incubated in LB broth supplemented with 50 μg/mL kanamycin at 37 °C, with shaking at 200 r/min for 12 h as seed. To express the target enzyme, the engineered B. subtilis cells were cultured with 3% inoculation in 50 mL SR medium, with 50 μg/mL kanamycin at 37 °C, with shaking at 200 r/min for 14 h.For the expression of g for 15 min. The cells were resuspended in a sample buffer  containing 100 mmol/L NaCl and disintegrated by biological lysis with 20 mg/mL lysozyme in a water bath at 37 °C for 30 min, with gentle shaking every 10 min. Then, cells were ruptured by ultrasonication for 10 min  and centrifuged at 6000× g for 15 min. To purify the enzyme, the nickel affinity column was washed with deionized water and pre-equilibrated with a binding buffer . The supernatant was loaded into the column, which was then washed with a binding buffer and a washing buffer  to remove unbound or weakly bound proteins. Subsequently, the target enzyme was eluted with an elution buffer  and dialyzed against a sample buffer, with three buffer exchanges to remove imidazole.Following overexpression, all purification steps of the target protein were carried out at 4 °C. Cells expressing BpFAE were harvested by centrifugation at 8000× The subunit and native molecular mass (Mm) of BpFAE were evaluated by sodium dodecyl sulfate (SDS) and native polyacrylamide gel electrophoresis , respectively. The protein was stained with Coomassie brilliant blue R250 and de-stained with a decoloring solution (ethanol:acetic acid:distilled water = 1:2:17).The hydrolytic activity of BpFAE was measured using MFA as substrate. The enzymatic reaction was conducted in a 1 mL mixture containing 990 μL MFA  and 10 μL enzyme solution at 50 °C for 10 min, and terminated by placing this mixture in a boiling water bath for 10 min.v/v) acetic acid in H2Odd (eluent A) and methanol (eluent B). The elution gradient protocol was: 0 min (90% A/10% B); 0–0.23 min (70% A/30% B); 0.23–1.66 min (50% A/50% B); 1.66–4.97 min (100% B); 4.97–5.57 min (85% A/15% B); 5.57–7.52 min (90% A/10% B). A standard curve was generated by linear fitting based on the retention time and FA peak area. One unit of FAE hydrolytic activity was defined as the amount of enzyme required to release 1 μmol FA per minute under standard assay conditions.The concentration of FA was analyzed with a high-performance liquid chromatography (HPLC) system , equipped with an ultraviolet detector at 320 nm and a ZORBAX Eclipse Plus C18 column . The injection volume was 10 μL, and the flow rate and temperature were controlled at 1 mL/min and 30 °C. The mobile phase consisted of 1% , 2–(N–morpholino) ethanesulfonic acid (MES) buffer , sodium phosphate buffer , Tris-HCl , and glycine-NaOH buffer . The relative activity was normalized to the maximum activity set at 100%. In order to study pH stability, the enzyme was pretreated at pH 8.5, 9.0, and 9.5, at 4 °C for 28 h. The activity at the beginning was set at 100%.Tm) were determined. The slope defined as k was obtained according to a linear fitting of the natural logarithm of the relative activity versus time. The half-life was calculated by ln2/k. The molar ellipticity at 222 nm of the enzyme was recorded as a function of variable temperature ranging from 25 to 90 °C on a circular dichroism spectropolarimeter (CD) , with a bandwidth of 1.0 nm, sensitivity of 20 mdeg, heating rate of 0.2 °C/min, and a 0.1 cm quartz cuvette. The Tm was calculated by the acquisition of maximum slope.To investigate the optimal temperature, enzyme activity was assayed at different temperatures ranging from 35 to 90 °C. The highest enzyme activity was set at 100%. For thermal stability, the enzyme was incubated at 50, 55, 60, and 65 °C prior to the residual enzyme activity assayed. The activity of enzyme stored at 4 °C was specified as 100%. The half-life time and melting temperature .The kinetic parameters of the purified BpFAE were measured at different concentrations of MFA (0.1–8 mmol/L) at 50 °C and pH 9.0. The DSWB was incubated with 3 g/L potassium acetate at 95 °C for 1 h with constant stirring, followed by the removal of soluble starch using distilled water. Subsequently, the treated DSWB was dried to a constant weight at 55 °C for later use.g for 10 min. The supernatant was analyzed by HPLC-UV, as described above.The total amount of FA in the DSWB above was chemically extracted by incubation in 2 mol/L NaOH at 50 °C, with shaking at 150 r/min for 4 h, and supplementing 2 mol/L HCl at 85 °C for 1 h. Then, the suspension was centrifuged at 8000× g for 10 min. The analysis of the released FA amount was conducted by HPLC.The FA release from DSWB was carried out by enzymatic degradation. The 75 mg DSWB was suspended in 3 mL of the Tris-HCl buffer , which was enzymatically degraded by adding 0.18 mg of purified BpFAE, 0.31 mg of commercial xylanase, and 0.18 mg of BpFAE along with 0.31 mg xylanase. It was conducted at 50 °C for 4 h, with shaking at 150 r/min. The reaction mixture was terminated by boiling for 10 min and centrifuged at 8000× n = 3). Statistical analyses were performed using OriginPro 9.1 . The data are presented as the mean ± standard deviation  with carboxylesterase A from B. pumilus NCTC10337 (GenBank ID: LT906438.1). In addition, the nucleotide sequence was 96.64% identical with alpha/beta hydrolase from B. pumilus ZB201701 (GenBank ID: CP029464), 90.03% with esterase from B. safensis U14–5 (GenBank ID: CP015607), and 85.54% with alpha/beta hydrolase from B. altitudinis 11–1-1 (GenBank ID: CP054136).PCR sequencing results showed that the open reading frame of B. pumilus SK52.001 is composed of 303 amino acid residues without a signal peptide and has a theoretical isoelectric point (pI) and subunit Mm of 5.22 and 33,633.13 Da, respectively, on the ExPASy website . The grand average of the hydropathicity of BpFAE was −0.286, indicative of a hydrophilic protein. Additionally, BpFAE contains a sole alpha/beta hydrolase fold with a fairly wide range, from residues 67 to 272 as a core domain, which was forecast on the Conserved Domain Database website .The FAE protein from B. altitudinis (GenBank ID: AKC64980). However, BpFAE showed low identity (less than 50%) with FAEs from Pseudonocardia eucalypti , Saccharopolyspora pogona , Paraphaeosphaeria minitans , and Amycolatopsis umgeniensis . Nevertheless, BpFAE shared irrelative homology with FAEs from other Bacillus species, including B. amyloliquefaciens , B. megaterium , B. aryabhattai (GenBank ID: SDE01879), B. timonensis , and B. onubensis (GenBank ID: WP_099362906). BpFAE contains a conserved motif GHSAG in line with the typical GXSXG motif of the alpha/beta hydrolases of the serine proteinase family.The amino acid sequence homology analysis a indicatThe evolutionary relationship between BpFAE and other reported FAEs was evaluated on a phylogenetic tree based on the neighbor-joining algorithm using 1000 bootstrap replicates in MEGA 7 b. From FE. coli [Kluyveromyces marxianus [Pichia pastoris [Trichoderma ressei [B. subtilis expression system of FAE. To explore the feasibility of B. subtilis as an expression system, B. pumilus SK52.001-derived fae was heterogeneously expressed in B. subtilis WB800, a safe and effective food-grade host. Because BpFAE was fused with 6× His-tags at the C-termini before construction, it was purified with a nickel-affinity column. After nickel-affinity column purification, the specific activity of the purified protein was 377.9 ± 10.3 U/(mg protein), which is the highest reported value , and 514.91 1/s, respectively. BpFAE showed a lower Km value for MFA as compared to FAEs from Eupenicillium parvum (6.24 mmol/L) [Km of BpFAE was similar to those of BaFae04 and AnFaeA from B. amyloliquefaciens and A. niger  [Kcat/Km) of MFA was 542.01 mmol/(L·s), reflecting a high substrate conversion rate. Compared to the catalytic efficiency of FAE from B. intestinalis (321.9 mmol/(L·s) [A. terreus (254.9 mmol/(L·min)) [The kinetic parameters were obtained by fitting the Hill function under various substrate concentrations (0.1–8 mmol/L). The parvum 6. mmol/L [ctively) ,28. The ol/(L·s)  from theol/(L·s) , and A. Thermomyces lanuginosus to greatly increase the amount of FA from corn cobs. Combined with the previous results, FAE along with xylanase is a reasonably applicable approach to produce FA from biomass.The enzymes, especially FAE, were used to produce FA from biomass, since FA was linked to lignin or arabinoxylans in the plant cell walls ,32,33. SBacillus pumilus SK52.001 was identified and heterogeneously expressed in B. subtilis WB800 for the first time. The enzyme exhibited an optimal alkaline pH and good thermal stability. Additionally, high specific activity was observed in this study, which was up to 377.9 ± 10.3 U/mg of protein using MFA as a substrate. The production of FA from DSWB was greatly improved to 49.47% using purified BpFAE in the presence of commercial xylanase, compared to 5% of FA production using BpFAE alone. In summary, BpFAE adopting the B. subtilis expression system is an efficient and promising biocatalyst. It proves that FAEs based on the recombinant B. subtilis may be conceivable for waste biomass degradation to promote the production of FA from food by-products, as a response to sustainable development.In this study, an alkaline FAE from