By monitoring these aldehyde–2-piperidinol products, we evaluated the lysyl oxidase–like activity of polyphenols. We also observed that this reaction was mediated by some polyphenols, especially o-diphenolic-type polyphenols, in the presence of copper ions. Interestingly, the natural immunoglobulin M monoclonal antibody recognized these aldehyde–2-piperidinol products as an innate epitope. These findings establish the existence of a dynamic equilibrium of oxidized lysine and provide important insights into the chemopreventive function of dietary polyphenols for chronic diseases.Polyphenols, especially catechol-type polyphenols, exhibit lysyl oxidase–like activity and mediate oxidative deamination of lysine residues in proteins. Previous studies have shown that polyphenol-mediated oxidative deamination of lysine residues can be associated with altered electrical properties of proteins and increased crossreactivity with natural immunoglobulin M antibodies. This interaction suggested that oxidized proteins could act as innate antigens and elicit an innate immune response. However, the structural basis for oxidatively deaminated lysine residues remains unclear. In the present study, to establish the chemistry of lysine oxidation, we characterized oxidation products obtained  A)  , 4. LysyA) , 6. OtheA) , 8, 9.Fio-quinone derivatives that can catalyze oxidative deamination of primary amines , is known to be an efficient mediator of the oxidation of primary amines such as ε-amino group of lysine residues in elastin and collagen  , the most abundant polyphenol in green tea, to proteins, where EGCG oxidizes specific lysine residues in the protein (C)  . In our  protein . In addi protein C 17). M. Mo-quintein (C) , 17. DesN-biotinyl-5-aminopentylamine (Bt-APA), was used as the substrate for oxidation by eggshell membranes (ESMs) containing lysyl oxidase  was incubated with hen's ESM containing lysyl oxidase in 0.1 M sodium phosphate buffer for 48 h, and the reaction mixtures were analyzed by LC–MS. As shown in B and C, incubation with ESM gave three supposedly oxidized Bt-APAs; two major products (I and II) and one minor product (III). Incubation of the lysine analog with polyphenols and o-quinone compounds, such as PQQ, also generated the same products (data not shown).To gain structural insights into the polyphenol-mediated oxidation of lysine residues, the lysine analog (Bt-APA) A was inc+ at m/z 328 , suggesting that the product might be identical to the oxidatively deaminated aldehyde species, N-biotinyl-5-aminopentanal . This predicted structure was confirmed by the detection of the signal (9.52 ppm), corresponding to the aldehyde group, in the NMR spectra. The other major product II gave an intense M + H+ at m/z 310 on LC–MS analysis , suggesting further dehydration of product I. Based on MS data, it was initially predicted that II might have a tetrahydropyridine structure . Kijewska et al. , corresponding to either a Schiff base–type (imine) linkage (m/z 638) between Bt-APA and aldehyde product or aldol condensation (m/z 637) of two aldehyde products . The product was finally identified as an aldol condensation product based on NMR detection at two proton signals (6.67 and 9.12 ppm), corresponding to the α,β-unsaturated aldehyde.Molecular-ion MS information about the products was collected using LC–MS. The mass spectrum of I showed an M + Ha et al.  recentlyA). Similarly, the evaporation of isolated I resulted in both I and II (B), suggesting that both products are in equilibrium in solution. The isolated sample that appeared to be a mixture of I and II was then characterized by NMR. Compared with the 13H-NMR spectrum between the original lysine analog and the sample . Curiously, the characteristic signals of the olefinic protons and carbons of tetrahydropyridine were not detected in the NMR analysis of the product. Instead, 1H-NMR of the product detected two signals at 5.55 and 5.90 ppm and 13C-NMR detected two signals at 71.92 and 76.507 ppm  and observations (m/z = 310) can be explained by the dehydration of the product in the positive ionization mode of electrospray ionization (ESI)–MS. In fact, the negative ionization mode of the ESI–MS showed an M − H+ signal at m/z 326 (data not shown).To elucidate product II, the lysine analog was treated with ESM and the product was isolated by HPLC on a reverse-phase column. After evaporation, however, the isolated sample (II) gave I in addition to II A. SimilaI and II B, suggese sample , a new s.507 ppm . These s4), and their reactivity for reduction reaction was examined. As shown in Figure 6A, the reduction profile was dependent on the concentration of NaBH4. When the sample was treated with the lowest concentration (0.5 mM) of the reducing agent, the product I was selectively lost along with the appearance of a new product at 3.47 min, which was determined to be a reduced form of the aldehyde species, N-biotinyl-5-aminopentanol (IV), by the MS analyses (B and C). Treatment with 5 mM NaBH4 resulted in complete and partial loss of I and II, respectively, and only a new product (N-biotinyl-5-aminopentanol) was detected (A). At the highest concentration (50 mM) of NaBH4, both I and II were converted to N-biotinyl-5-aminopentanol (A). In addition, methylation of II with methanol/trifluoroacetic acid (TFA) gave a product (retention time of 4.20 min) with M + H+ at m/z 342 (D and E), corresponding to N-biotinyl-2-methoxypiperidine (F). Based on all these data, the product equilibrated with the aldehydic species putatively presumed to be the tetrahydropyridine product (F) was finally determined to be the 2-piperidinol product (G).On the other hand, the hemiaminal ring opens and reforms, giving products with different configurations at the anomeric center. To identify the putative hemiaminal product, the isolated sample comprising ring-open and ring-closed structures was treated with sodium borohydride , 2-piperidinol (GK-2), and aldol condensation (GK-3). When a mixture of the putative aldehyde and the 2-piperidinol product separated by HPLC was subjected to 1H-NMR, the characteristic signals of the aldehyde (9.53 ppm) and hemiacetal methine region (5.55 and 5.90 ppm) were indeed detected in the oxidized peptide . Then, we tested the reaction of 2-piperidinol (II) with biotin thioacid and detected an intense M + H+ at m/z 570.8, corresponding to the 2-piperidine thioester derivative, by LC–MS (A and B). The underlying reaction mechanism of derivatization may include dehydration of 2-piperidinol to a tetrahydropyridine product, followed by the formation of a thioester derivative by the addition of thioacid (C). Using this derivatization reaction, we attempted to detect the 2-piperidinol product in proteins by Western blotting with streptavidin-horseradish peroxidase (HRP). Treatment of bovine serum albumin (1 mg/ml) with PQQ in the presence of Cu2+ showed a slight expansion of the protein and a shift in mobility on native PAGE analysis . Western blot analysis of oxidized proteins gave a strong signal because of the binding of biotin thioacid to the protein. In addition, the treatment of the oxidized protein with NaBH4 prior to the derivatization with biotin thioacid resulted in partial loss of the signals .To detect 2-piperidinol product in proteins, we utilized an hioacids . In our by LC–MS , A and Bthioacid C. Using A). In the absence of copper ions, the potential source of aldehyde–piperidinol products includes piceatannol and baicalein, both of which were much more efficient than EGCG. Copper ions drastically promoted the formation of oxidatively deaminated products. Piceatannol showed the greatest activity, which was followed by catechin, epicatechin, baicalein, quercetin, and catechol. Because these polyphenols contain an o-diphenolic structure in common (B), it is reasonable to estimate that polyphenol-mediated oxidative deamination may proceed via a copper/catechol-driven mechanism. This speculation can be supported by the previous findings that polyphenols, o-diphenolic-type polyphenols in particular, gain lysyl oxidase–like activity via oxidation to the corresponding o-quinone derivatives  was incubated with polyphenols (2.5 mM) in the presence and absence of copper ions (0.5 mM) in 0.1 M sodium phosphate buffer (pH 7.4) for 24 h, and the products comprising both aldehyde and 2-piperidinol products were analyzed by LC–ESI–MS/MS in the positive ion mode using multiple reaction monitoring. Of interest, not all the polyphenols exhibited an aldehyde/piperidinol–producing activity upon incubation with the lysine analog A. In then common B, it is ivatives , 13.FiguA). We then tested the immunoreactivity of the isolated sample  and found that the product was indeed recognized by IgM mAb SBM3 .We have shown that polyphenols with lysyl oxidase–like activity can be an endogenous source of innate epitopes recognized by natural IgM antibodies and suggested a mechanism, in which the changes in the electronegative potential of the polyphenol-treated proteins might be involved, at least in part, in the recognition by the antibodies . HoweverFig. 10o-diphenolic-type polyphenols in the presence of copper ions. These data provide the first experimental evidence for the existence of the ring-closed intermediates generated during oxidative deamination of lysine residues. More notably, the aldehyde–2-piperidinol product also functioned as an innate epitope recognized by the natural IgM antibodies. The data provide the basis for the hypothesis that the specificity of the natural IgM for polyphenol-modified proteins may be due, at least in part, to aldehyde–piperidinol products. Furthermore, these findings may indicate that the dynamic equilibrium of oxidatively deaminated lysine between the ring-open and ring-closed intermediates in proteins is associated with the potential beneficial effects of polyphenols.In the present study, to gain more insight into the oxidative deamination of lysine residues, we characterized the oxidation products generated by incubating the lysine analog (Bt-APA) with ESM and established a unique equilibrium between aldehyde and 2-piperidinol products. In addition, based on the presence of such equilibrium, we assessed the lysyl oxidase–like activity of polyphenols by monitoring both products and observed that the reaction was specifically mediated by several m/z 227, throughout this study. It was expected that this characteristic common fragment ion might allow selective analysis of products derived from the lysine analog and facilitate product identification by LC–MS. When Bt-APA was incubated with ESM under physiological conditions, reverse-phase HPLC analysis of the reaction mixture yielded mainly two major products (I and II) and one minor product (III). Incubation of the lysine analog with PQQ in the presence of Cu2+ also generated the same products. The data suggest that these products could commonly be generated by the action of polyphenols with lysyl oxidase–like activity. LC–MS data suggested that product I might represent an aldehydic product, N-biotinyl-5-aminopentanal, and this hypothesis was confirmed by experiments with NaBH4. III was identified as a cross-linking product generated via the aldol condensation reaction of two aldehyde species known to be the source of further crosslinks, such as desmosine and its isomer isodesmosine, in elastin and collagen  detected two signals at 5.55 and 5.90 ppm corresponding to the methine proton of the hemiaminal group. In addition, two proton signals, 3.60 and 4.06 ppm, corresponding to the methylene group adjacent to the hemiaminal methine group were also detected. The 13C-NMR spectrum also showed two signals, 71.92 and 76.50 ppm, corresponding to the methine carbon of hemiaminal. Therefore, in agreement with theory, it was shown that 2-piperidinol products are composed of two configurations. Considering the steric effect of the hydroxy group, it was speculated that the equatorial position is preferable to the axial position of the hydroxyl group. However, the signal intensity of the methine proton in the hemiaminal group revealed that the two stereoisomers might be equally present.Since the 2-piperidinol product has a chiral carbon center (C-2) and stereogenic nitrogen, it is thought to be composed of two diastereomers because of its equilibrium with the 5-aminopentanal derivative. It was previously reported that the formation of 2-piperidinol product was predicted as an intermediate in the process of conversion of aldehyde intermediate to tetrahydropyridine products . In the 2+ accelerated the formation of oxidation products suggests that the reaction can proceed via a metal-catalyzed mechanism. Polyphenols are generally very sensitive to Cu2+-catalyzed oxidation. Suzuki et al.  was obtained from Thermo Fisher Scientific. All the other reagents used in this study were of analytical grade and obtained from commercial sources.N-biotinyl-5-aminopentanal [I] and N-biotinyl-5-aminopentanal dimer [III]) and 30 eV (for N-biotinyl-2-piperidinol [II]); desolvation gas flow rate is 800 l/h.ESM was used for the lysyl oxidase–mediated reactions . BrieflyNα-benzoylglycyl-lysine, the peptide (3 mM) was incubated with 10 mg/ml ESM in 0.1 M sodium phosphate buffer (pH 7.4) for 48 h at 37 °C. After incubation, the reaction mixtures were analyzed by LC–ESI–MS analysis using an SQD single quadrupole mass spectrometer (Waters) equipped with an ACQUITY UPLC system (Waters). The sample injection volumes of 5 μl each were separated on Develosil HB C30-UG  at the flow rate of 0.3 ml/min. A gradient of solvent A (water) with solvent B (acetonitrile) was used as follows: 5% B at 0 min, 95% B at 8 min, and 95% B at 13 min. An MS scan was performed in the positive ion mode using nitrogen as the nebulizing gas. The experimental conditions were as follows: ion source temperature, 120 °C; desolvation temperature, 350 °C; cone voltage, 30 eV; and desolvation gas flow rate, 800 l/h.For the lysyl oxidase–mediated oxidation of 2O). The 1H NMR spectra (500 MHz) and 13C NMR spectra (125 MHz) were recorded at 21 °C by a JNM-ECA500 II (JEOL).The reaction mixture was separated by a reverse-phase HPLC as already mentioned. After evaporation, the resulting precipitate was dissolved in heavy water : δ1.22 to 1.47 , 2.10 , 2.64 , 2.72 , 2.85 , 3.04 , 3.19 , 4.28 , 4.46 . 13C NMR : δ23.16, 25.21, 27.70, 27.89, 27.99, 28.01, 35.52, 39.03, 39.72, 55.45, 60.28, 62.13, 165.38, and 176.71. The spectra of a mixture composed of I and II: 1H NMR : δ1.24 to 1.76 , 2.11 , 2.29 , 2.46 , 2.65 , 2.83 , 3.06 , 3.19 , 3.60 , 4.06 , 4.28 , 4.46 , 5.55 , 5.90 , 9.52 . 13C NMR : δ17.32, 18.72, 21.48, 24.29, 24.64, 24.89, 25.21, 27.68, 30.03, 30.81, 35.52, 36.61, 36.95, 38.89, 39.15, 39.70, 41.20, 42.74, 55.38, 60.28, 62.10, 71.92, 76.5, 91.01, 165.40, 175.28, 176.71, 208.59. The spectra of III: 1H NMR : δ 1.26 to 1.61 , 2.11 , 2.29 , 2.61 , 2.82 , 3.01 , 3.06 , 3.16 , 4.25 , 4.44 , 6.67 , and 9.12 .The spectra of Bt-APA: 1H NMR : δ1.33 to 2.47 , 4.04 , 4.33 , 5.62 , 5.97 , 7.43 , 7.53 , 7.71 , and 9.53 . The spectra of GK-3: 1H NMR : δ1.28 to 2.57 , 3.93 to 4.04 , 6.56 , 7.38 , 7.48 , 7.67 , 9.03 , 3.16 , 4.25 , 4.44 , 6.67 , and 9.12 .The spectra of a mixture of GK-1 and GK-2: N-biotinyl-5-aminopentanal (I) and N-biotinyl-2-piperidinol (II) dissolved in 0.1 M sodium phosphate buffer (pH 7.4) was treated with 10 μl of NaBH4 in methanol at the indicated final concentrations for 1 h on ice. Polyphenol-modified protein (1 mg/ml) in 0.1 M sodium phosphate buffer (pH 7.4) was treated with 50 mM NaBH4. The reduced product was characterized by LC–ESI–MS, 1H NMR, and 13C NMR. 1H NMR : δ1.19 to 1.58 , 2.10 , 2.64 , 2.85 , 3.04 , 3.19 , 3.45 , 4.28 , and 4.46 . 13C NMR : δ22.45, 25.21, 27.67, 27.84, 28.11, 30.94, 35.53, 39.20, 39.70, 55.40, 60.27, 61.64, 62.10, 165.38, and 176.68.The mixture (10 μg/ml) of N-biotinyl-5-aminopentanal (I) and N-biotinyl-2-piperidinol (II) was dissolved in 1 ml of methanol containing 0.1% TFA. After incubation for 12 h at room temperature, the reaction mixture was evaporated and dissolved in 1 ml of water for the LC–ESI–MS analysis.The mixture (∼1 mg) composed of 1H NMR : δ1.35 , 1.46 , 1.61 , 2.63 , 2.84 , 3.09 , 4.13 , and 4.30 .Biotin (200 μmol) was dissolved in dimethylformamide. To this solution, diisopropylethylamine (400 μmol) was added. This reaction mixture was then cooled to 4 °C prior to addition of acetic anhydride (200 μmol) and stirred for another 30 min. The reaction mixture was treated with sodium hydrosulfide (220 μmol) and stirred for another 4 h. After drying the reaction mixture, the dried mixture was dissolved in a 3:1 mixture of 0.1% TFA and dimethyl sulfoxide (DMSO) and subjected to a fractionation by HPLC on a ChromaNik Sunniest RP-AQUA column  at a flow rate of 10 ml/min. A gradient of solvent A (water containing 0.1% TFA) with solvent B (acetonitrile containing 0.1% TFA) was used as follows: 20% B at 0 to 5 min, 50% B at 20 min, 95% B at 22 min, and 95% B at 25 min. The elution profiles were monitored by absorbance at 220 nm. Fractions corresponding to the peak of biotin thioacid were dried and stored at −80 °C. The spectra of a mixture of biotin thioacid: 2CO3. After incubation for 2 h on ice, the protein samples were run on nondenaturing native PAGE. After electrophoresis, the gel was transblotted onto a polyvinylidene difluoride membrane , incubated with blocking one , washed, and then incubated with streptavidin-HRP (1:5000) for 1 h at room temperature. This procedure was followed by the addition of SuperSignal West Pico PLUS Chemiluminescent Substrate . The blots were visualized by ImageQuant LAS4000 (Cytiva).Biotin labeling of the polyphenol-modified protein was performed using the reaction mixtures containing 1.0 mg/ml protein with 3 mM biotin thioacid and 50 mM Na4 in 0.1 M sodium phosphate buffer (pH 7.4) containing 10% DMSO for 24 h at 37 °C. The samples were subjected to an LC–ESI/MS/MS analysis using a TQD triple-stage quadrupole mass spectrometer (Waters) equipped with an ACQUITY UPLC system (Waters). The sample injection volumes of 5 μl each were separated on Develosil HB C30-UG  at the flow rate of 0.3 ml/min. A gradient of solvent A (water containing 0.1% formic acid) with solvent B (acetonitrile containing 0.1% formic acid) was used as follows: 5% B at 0 min, 95% B at 8 min, and 95% B at 13 min. Multiple reaction monitoring was performed in the positive ion mode using nitrogen as the nebulizing gas. The experimental conditions were as follows: ion source temperature, 120 °C; desolvation temperature, 350 °C; cone voltage, 30 eV; collision energy, 20 eV; desolvation gas flow rate, 800 l/h; cone gas flow rate, 50 l/h; and collision gas, argon. The strategy was designed to detect the product ion (m/z 227.2) from the positively ionized N-biotinyl-5-aminopentanal (m/z 328.2) and N-biotinyl-2-piperidinol (m/z 328.2) by monitoring the samples transmitting their [M-H2O + H]+ >227.2  and [M-H2O + H]+ >227.2 (for N-biotinyl-2-piperidinol) transitions. Since N-biotinyl-5-aminopentanal and N-biotinyl-2-piperidinol were easily dehydrated, we fragmented both dehydrated forms. These two molecules were distinguished by their retention times .Bt-APA (1 mM) was incubated with 2.5 mM polyphenols in the presence or absence of 0.5 mM CuSON-biotinyl-5-aminopentanal (I) and N-biotinyl-2-piperidinol (II) in PBST was added to the wells. After a 1-h incubation, streptavidin-HRP in PBST was added, incubated for 1 h, and then developed using the TMB Ultra substrate (Thermo Scientific). The reaction was terminated by the addition of 2 M sulfuric acid, and the binding to IgM was quantified by measuring the absorbance at 450 nm.Either the isotype control IgM  or monoclonal IgM SBM3 (1 μg/ml) showing specificity toward the EGCG-modified proteins  was immoAll data are included within this article.The authors declare that they have no conflicts of interest with the contents of this article.


N-phenacyldibromobenzimidazoles against C. albicans and C. neoformans. Firstly, we selected the non-cytotoxic most active benzimidazoles based on the structure–activity relationships showing that the group of 5,6-dibromobenzimidazole derivatives are less active against C. albicans vs. 4,6-dibromobenzimidazole analogues (5e–f and 5h). The substitution of chlorine atoms to the benzene ring of the N-phenacyl substituent extended the anti-C. albicans action . The excellent results for N-phenacyldibromobenzimidazole 5h against the C. albicans reference and clinical isolate showed IC50 = 8 µg/mL and %I = 100 ± 3, respectively. Compound 5h was fungicidal against the C. neoformans isolate. Compound 5h at 160–4 µg/mL caused irreversible damage of the fungal cell membrane and accidental cell death (ACD). We reported on chitinolytic activity of 5h, in accordance with the patterns observed for the following substrates: 4-nitrophenyl-N-acetyl-β-d-glucosaminide and 4-nitrophenyl-β-d-N,N′,N″-triacetylchitothiose. Derivative 5h at 16 µg/mL: (1) it affected cell wall by inducing β-d-glucanase, (2) it caused morphological distortions and (3) osmotic instability in the C. albicans biofilm-treated. Compound 5h exerted Candida-dependent inhibition of virulence factors.Our study aimed to characterise the action mode of  Candida spp. and Cryptococcus spp. ; 5h or chitinase [mL] (here 0.004 mL).Test was preformed using Chitinase Assay Kit . Procedure was based on technical bulletin obtained from producer . Four grts, USA) . ChitinoC. albicans SC5314 ref. strain’s, C. albicans SPZ176 clinical strains and C. neoformans SPZ173 clinical strain’s culture were prepared as previously described. Test samples were prepared by adding of 100 μL of 105-fold diluted cells suspensions to 900 μL YNB medium with 5h at conc. of 160, 16 or 4 μg/mL. Control was obtained by adding 105-fold diluted cells suspensions to 900 μL YNB without 5h. All samples were incubated at 30 °C with shaking at 120 rpm for 18 h. Suspensions were then centrifuged at 9500 rpm for 2 min and cells were washed with PBS. Following, 100 μL of the washed cells were added to 900 μL of PBS with glucose (5 mM) and rhodamine B (7.18 mg/mL) . After 30 min of incubation at 37 °C, suspensions were centrifuged at 9500 rpm for 2 min and the cells were washed with PBS. Then, the cells were resuspended in PBS with glucose (1 mM) and incubated at 37 °C with shaking at 120 rpm for 18 h. Then, the post growth medium was separated from the cells by centrifugation at 9500 rpm for 2 min and 20 μL of supernatant was added to microtiter plate. To prepare 10-fold diluted samples, 180 μL of sterile water was added to each well. Fluorescence was measured with excitation at 521 nm and emission at 627 nm using Synergy H4 Hybrid Reader . Concentration of Rho123 was calculated using formula: C = (E − Blank − 1151.2) × 10/556.91; where: E—emission; Blank—emission of PBS/glucose medium; 1151.2 and 556,91—coefficients of rhodamine standard curve; 10—dilution coefficient. Decrease of the Rho123 content was determined using the formula: ΔC% = [C(Test) − C(Control)]/C(Control) × 100; where: C(Test)—concentration of rhodamine in tested samples; C(Control)—concentration of rhodamine in control samples [ samples .C. albicans SC5314, C. albicans SPZ 176 clinical isolate and C. neoformans SPZ 173 clinical isolate were prepared as previously described. Test samples were prepared as described in Rho123 assay  . C. albissay see . Positiv5h, flow cytometry analysis was performed using the protoplasts and C. albicans SPZ176 and C. neoformans SPZ173 cells. Protoplasts were obtained according to the method previously described [5h at 30 °C with shaking at 120 rpm for 24 h. Compound-free growth controls were also prepared. After harvesting by centrifugation at 3000 rpm at 4 °C for 5 min; cells were washed and resuspended with sterile water. Determination of the cell death type was conducted by staining using annexin V and propidium iodide  [To determine the type of cell death induced by the action of escribed . Cells aMA, USA) . SuspensC. albicans SPZ 176 and clinical C. neoformans SPZ 173 cultures were prepared, as previously described [5h solution in PBS  was added (test sample), to the control one 500 μL PBS was added. Plate was incubated at 37 °C for 18 h. Biofilms were then washed twice with PBS and then 495 μL of PBS and 5 μL of staining solution was added. The following staining solutions were used: CR  at stock conc. of 200 µg/mL; CFW  at stock conc. of 250 µg/mL; AO  at stock conc. of 100 µg/mL; EB  at stock conc. of 100 µg/mL [escribed . Suspens00 µg/mL . Final s


Bacillus aryabhattai GEL-09 and evaluated the hydrolytic performance of this enzyme. Firstly, the lacZBa-encoding gene was cloned and overexpressed in Escherichia coli BL21(DE3). Phylogenetic analyses revealed that lacZBa belonged to the glycoside hydrolase family 42. Using SDS-PAGE, we determined that the molecular weight of lacZBa was ~75 kDa. Purified lacZBa exhibited a maximum activity at 45 °C, pH 6.0, and could be activated following incubation at 45 °C for several minutes. The half-life of lacZBa at 45 °C and 50 °C was 264 h and 36 h, respectively. While Co2+, Mn2+, Zn2+, Fe2+, Mg2+, and Ca2+ enhanced enzymatic activity, Cu2+ and ethylenediaminetetraacetic acid inhibited enzymatic activity. Moreover, lacZBa could hydrolyze lactose and oNPG with Km values of 85.09 and 14.38 mM. Molecular docking results revealed that lacZBa efficiently recognized and catalyzed lactose. Additionally, the hydrolysis of lactose by lacZBa was studied in lactose solution and commercial milk. Lactose was completely hydrolyzed within 4 h with 8 U/mL of lacZBa at 45 °C. These results suggested that lacZBa identified in this study has potential applications in the dairy industry.β-Galactosidase has been greatly used in the dairy industry. This study investigated a novel thermostable β-galactosidase (lacZBa) from  It can be used to yield dairy products and lactose-free milk. Lactose-free milk is suitable for people with lactose intolerance, which is prevalent in more than 70% of the world’s adult population, and more than 90% among East Asians [.23 naturLactobacillus [Bifidobacterium [Escherichia coli [Bacillus sp. [Aspergillus oryzae [Aspergillus niger [Kluyveromyces lactis [Kluyveromyces fragilis [Geobacillus stearothermophilus is thermostable, and its optimum temperature is 70 °C [Arthrobacter sp. ON14, isolated from Antarctica, exhibits high catalytic activity at 15 °C [Exiguobacterium acetylicum can maintain 80% residual enzyme activity at 40 °C for 200 h and has good thermostability [β-Galactosidases are ubiquitous enzymes produced by animals, plants, and microorganisms. In animals, β-galactosidases exist in the intestine  and milkbacillus ,5, Bifidacterium , Escherihia coli , Bacillullus sp. ,9,10. Ths oryzae , Aspergius niger ,13, and s lactis , Kluyverfragilis , etc. Bafragilis . All theis 70 °C . Moreoveat 15 °C . β-Galactability . FurtherAspergillus oryzae, Aspergillus niger or Lactobacillus. However, most of them have the disadvantage of poor thermostability [β-Galactosidases have been isolated from different sources, such as microorganisms, animals, and plants. However, the most widely used enzymes are microbial β-galactosidases . The reltability .Bacillus aryabhattai Gel-09 (CCTCC M2017320), a newly isolated raw starch-digesting amylase-producing strain, was screened and identified in our laboratory [B. aryabhattai Gel-09, we found that a protein encoded in the genome of this strain may be a new β-galactosidase (lacZBa). We expect that the new protein will have different properties from the existing β-galactosidases, which will provide value for future scientific research or commercial production. In this study, the β-galactosidase-encoding gene of B. aryabhattai GEL-09 was cloned and heterologously expressed in Escherichia coli BL21(DE3), and the recombinant enzyme was characterized in detail. Furthermore, the hydrolysis of lactose by lacZBa was studied in lactose solution and commercial milk.boratory . After fBacillus aryabhattai GEL-09, newly isolated by our laboratory, has been deposited in the China General Microbiological Culture Collection Center  under accession No. M2017320 [E. coli JM109 was used as host for gene cloning, and E. coli BL21(DE3) was used for the production of recombinant protein. The pET24a was used as the expression vector.M2017320 . E. coliEcoRI), rTaq, DNA standards, DNA fragment recovery kit, genomic DNA isolation kit, and plasmid extraction kit were purchased from Sangong Biotech . PCR primers were synthesized by Takara Bio Inc. . 2-nitrophenyl-β-D-galactopyranoside (oNPG) was obtained from J & K Scientific . All other chemicals were purchased from Nanjing Chemical Reagent Co., Ltd.  unless otherwise indicated.Restriction enzymes (B. aryabhattai GEL-09 genomic DNA was extracted by using the genomic DNA isolation kit (Sangong Biotech). Two primers were designed (lacZBa-Forword: 5′-GAAGGAGATATACATATGTACATCGGCGTCGATT-3′ and lacZBa-Reverse: 5′-TTGTCGACGGAGCTCCTACATGTTTTCTACAACTCGACT-3′) for amplification. The lacZBa gene was amplified by PCR, and the PCR products were ligated into the pET24a vector digested by EcoR I. The ligation mixture was transformed E. coli JM109, followed by DNA sequencing of recombinant plasmid. Then, the confirmed plasmid (pET24a-lacZBa) was transformed E. coli BL21(DE3) for expression.The E. coli BL21(DE3) single-colony cells, the cells were cultivated for 8 h at 37 °C. The cells were then cultured for 36 h at 30 °C in TB medium to express lacZBa. The bacteria were collected, suspended in pH 6.0 phosphate buffer, and broken by ultrasonic cell crusher. The supernatant and sediment of recombinant lacZBa were separated under centrifugal conditions for 20 min at 8000 rpm, and the supernatant fraction of recombinant lacZBa was collected and purified using a prepacked DEAE-μSphere ion-exchange column . The purity of recombinant lacZBa was appraised by 12% sodium dodecyl sulfate polyacrylamide gel [LB broth, which contained kanamycin (30 μg/mL), was used to culture transformed mide gel .2CO3 solution was used to stop the reaction, and absorbance was determined at 420 nm. One unit is the amount of β-galactosidase that catalysis the hydrolysis of 1 μM of oNPG per minute.Typically, the measurement of β-galactosidase activity using oNPG as the substrates . FirstlyThe optimal pH of lacZBa was measured over a pH range of 4.5 to 8.0 at 45 °C using sodium phosphate buffer (100 mM), and the pH stability was determined by incubation in the same pH ranges at 4 °C for 24 h. The optimal temperature for β-galactosidase was determined in 100 mM sodium phosphate buffer (pH 6.0) from 30 °C to 55 °C. To study the thermostability of lacZBa, the purified lacZBa was incubated at 45 °C and 50 °C, and the residual lacZBa activity was measured. The initial lacZBa activity was considered as 100% .2+, Zn2+, Cu2+, Co2+, Fe2+, Mg2+, and Ca2+) and disodium EDTA at final concentrations of 1 mM and 5 mM were added, respectively, and then reacted at 45 °C for 10 min [Seven metallic divalent cations  were calculated using Graphpad Prism8 . [Different concentrations (1–40 mM) and (10–2000 mM) of oNPG and lactose were used as substrates to measure the kinetic parameters of lacZBa, respectively. The reactions were performed in 100 mM sodium acetate buffer (pH 6.0) at 45 °C for 10 min. The kinetic parameters . .v/v) reagents was used as mobile phase. The gel plate was stained by spraying with aniline-diphenylamine phosphate reagent, and maintained at 85 °C for 10 min.Briefly, 1 mL of purified lacZBa (8 U/mL) was added into lactose solution (containing 50 g/L lactose substrate), and incubated at 45 °C for various times . Then, thin-layer chromatography analysis on a silica gel plate  was used to analyze the reaction mixture. The mixture n-butanol/HAc/water/ methanol  and commercial skim milk containing 5 g/100 mL of lactose in incubation at 45 °C for different times . The reaction was stopped by incubating the reaction mixture in boiling water bath for 5 min. After deproteinization with trichloroacetic acid (10%) at 4 °C for 3 h, the supernatant was collected by centrifugation at 12,000 rpm for 15 min .The lactose in commercial milk and its hydrolysis products  was analyzed by TLC, and quantified by HPLC. HPLC analysis was performed on Waters alliance 2695 separation module connected with 2414 refractive index detector using NH2P-504E column  eluted with 75% acetonitrile (1 mL/min). The concentration of glucose in the hydrolysis products was quantified by using an SBA-40D biological sensing analyzer, a kind of glucose biosensor  .t-test. p values of <0.01 were considered statistically significant.All assays were performed at least three times, and the results were expressed as the mean ± standard deviation of these replicates. Statistical analysis was performed with Student’s Multiple sequence alignment was carried out by using Clustal Omega, and displayed by ESPript 3.0. The phylogenetic tree was constructed in the MEGA 7 package using the maximum likelihood (ML) method. ProtParam tool of ExPASy was used to predict the Mw (molecular weight) and theoretical pI (isoelectric point) of the lacZBa .https://swissmodel.expasy.org/) (accessed in 2 April 2020) was used to develop a homology model for lacZBa. The crystal structure of β-galactosidase (PDB 3tts.1) from Bacillus circulans sp. alkalophilus was used as the template. The structure with the highest GMQE score  was chosen for further structure refinement. The model structure cleaning, and minimization was carried out by using Yasara software . The 3D structure of lactose (ligand) was drawn in ChemDraw 18.0. After the ligand and the receptor preparation, docking runs was performed using Yasara software, and the docking parameters is shown in + V2.2 , and BIOVIA Discovery Studio Visualizer 2020  [The SWISS-MODEL server  .B. aryabhattai Gel-09. Following genome data analyses, we found a hypothetical β-galactosidase protein, lacZBa. For a more in-depth study, we cloned and overexpressed the encoding gene lacZBa in E. coli. The length of the lacZBa open reading frame was 1950 bp, and the lacZBa protein consisted of 649 amino acids (AAs). The MW (molecular weight) and theoretical pI (isoelectric point) of lacZBa were 75.42 kDa and 5.38, respectively.We isolated the novel bacterium Bacillus sp. ME75. To verify the phylogenetic position of lacZBa among the characterized β-galactosidases, we used the maximum likelihood method to construct a phylogenetic tree of lacZBa and 24 β-galactosidases, which have been reported from different glycoside hydrolases (GH) families  with β-galactosidase (WP_182028680.1) of families . The resfamilies , whereasch other . In addiechanism .Bacillus circulans sp. alkalophilus was used as the template to simulate the lacZBa structure. Moreover, the ramachandran plots of the structure was also analyzed 8 TIM barrel structure [The crystal structure of β-galactosidase (PDB 3tts.1) from analyzed . The pref lacZBa , which wa RUK-10  and T. ttructure .E. coli BL21(DE3). Cells were cultured in LB medium for 6–8 h, and seeded into TB medium at 5% (v/v) inoculum. The cells were induced by adding 0.2 mmol/L IPTG after 3 h of incubation in TB medium, and continued to be cultured for 36 h. After fermentation, the optical density (OD600) reached 22.14. Additionally, there was significant lacZBa activity in the recombinant cell lysates. Because lacZBa is an intracellular enzyme, the recombinant cells were disrupted by ultrasonic treatment, and the soluble protein fractions of the lysed cells were collected as crude enzyme. The crude enzyme was purified by ammonium sulfate precipitation and dialysis following DEAE-μSphere anion-exchange chromatography  contains 649 AA residues. Members of GH-2 contain more AA residues than those of GH-42, including β-galactosidase from Saccharopolyspora rectivirgula [Alteromonas sp. ML52 [Enterobacter cloacae GAO [Bacillus circulans (GH-35) contains 586 AA residues, while β-galactosidase from Leptotrichia buccalis ATCC 14201 (GH-1) [To investigate its biochemical properties, we expressed lacZBa in tography . SDS-PAGtography . By compMB 41171  β-galactivirgula  (1251 AAsp. ML52  (1059 AAArthrobacter sp. 32c [Rahnella sp. R3 [Planococcus isolate [B. coagulans NL01 (50–65 °C) [Thermotoga naphthophila RUK-10 (70–90 °C) [These results reveal that the optimal temperature of lacZBa is 45 °C, with 97.1% and 96.3% relative activities at 40 °C and 50 °C, respectively C. Howeve sp. 32c  and 4–35 isolate  contains0–65 °C)  and Ther0–90 °C)  exhibit Rahnella sp. R3 loses ~70% of its activity following incubation at 45 °C for 15 min [Planococcus sp. L4 [Based on its sequence homology, lacZBa belongs to GH-42. Several GH-42 β-galactosidases are derived from extremophiles such as thermophilic, cryophilic, and halophilic microbes, and most GH-42 β-galactosidases have good thermostability. When evaluating the thermostability of lacZBa, we found that lacZBa could be activated following incubation at 45 °C for several minutes. Moreover, the activity of the purified enzyme was ~117% following storage at 45 °C for 24 h and 97% following storage at 45 °C after 100 h. Moreover, the half-life of lacZBa was 264 h at 45 °C and 36 h at 50 °C D. Therefr 15 min  and is fs sp. L4  is fully2+, Mg2+, and Fe2+ moderately improved enzymatic activity (109–127%). Mn2+, Zn2+, Co2+, and higher concentrations (5 mM) of Ca2+, Mg2+, Fe2+ significantly enhanced enzyme activity (150%). In contrast, enzymatic activity was inhibited by Cu2+. Therefore, the most common divalent cations are unable to inhibit the activity of lacZBa, and lacZBa is suitable in dairy products where the mineral concentration is relatively high and the pH of milk ranges between 6 and 7. Additionally, we have completed with the effects of anionic groups including 1 mM and 5 mM Cl−, 2 mM and 10 mM Cl−, 1 mM and 5 mM SO4 2− on lacZBa activity. The results showed that anionic groups did not significantly promote or inhibit on lacZBa activity.Different concentrations of EDTA significantly inhibited lacZBa activity, indicating that the catalytic action of lacZBa required the presence of divalent cations . Further2+ was beneficial for the activity of most β-galactosidases [Alteromonas sp. ML117 [Paracoccus sp. 32d [Planococcus sp. L4 [2+. Conversely, Cu2+ inhibits the activity of most β-galactosidases including β-galactosidases from Arthrobacter sp. ON14 [Arthrobacter sp. 20B [B. aryabhattai GEL-09 (this study). Unlike lacZBa, some β-galactosidases like YesZ from Bacillus subtilis [Akkermansia muciniphila [2+, which has is greatly beneficial for its use in the dairy industry. However, Ca2+ had no influence on β-galactosidase  from Arthrobacter sp. ON14 [Previous studies have shown that Mgosidases , regardlp. ML117 , Paracoc sp. 32d , and Plas sp. L4  are actisp. ON14 , Arthrob sp. 20B , and B. subtilis  and Am08iniphila  do not rsp. ON14 .Km) was 14.38 mM for oNPG and 85.09 mM for lactose  was 1.75 and 14.66 U/mg when lactose and oNPG were used as substrate, respectively. Similar to lacZBa, β-galactosidase  from Bacillus circulans sp. alkalophilus has higher affinity for oNPG [Rahnella sp. R3, has a higher substrate affinity towards lactose than oNPG. Its Km is three to 10 times higher for oNPG than for lactose [Kinetic analysis showed that the Michaelis constant ( lactose . At 45 °for oNPG . However lactose .B. circulans (PDB: 3tts.1) and lactose.Lactose was docked into the substrate binding pocket of lacZBa to generate the binding mode. The docking results revealed that lactose binds to the active site pocket of lacZBa A. To revIn addition, molecular docking was used to analyze the interaction between lactose and catalytic residues (Glu142 and Glu304). The carboxyl groups of Glu142 and Glu304 located on the hydrophilic face of the glucose residue and hydrophobic face of the galactose residue, respectively. Based on multiple sequence alignment  and moleLactose is a unique carbohydrate present in milk. Human milk contains ~7.4% lactose, which is the primary source of energy to newborns. The lactose content of commercially available cow milk is ~4.8%. In East Asia, more than 90% of people are unable to digest lactose, and may develop symptoms of lactose intolerance. β-Galactosidases, which have been widely used to hydrolyze lactose in dairy products, are beneficial for lactose-intolerant people.To determine the lactose hydrolysis ability of lacZBa, 8 U/mL lacZBa was used to hydrolyze a 50 g/L lactose solution. The hydrolytic products of lacZBa were analyzed by TLC. The results showed that lactose could be completely hydrolyzed by lacZBa within 4 h A. To furSaccharomyces cerevisiae and Aspergillus oryzae [Even though several β-galactosidases have been heterologously expressed and identified, only a few are available for commercial applications . Currents oryzae , both ofs oryzae , experims oryzae . Additios oryzae . TherefoB. aryabhattai Gel-09. Based on protein sequence alignment and phylogenetic analyses, lacZBa was determined to be a member of GH-42. The activity of the enzyme could be thermally activated and exhibited excellent thermal stability at 45–50 °C. Furthermore, molecular docking and hydrolysis experiments showed that lacZBa could efficiently recognize and catalyze lactose. Notably, lactose in milk was completely hydrolyzed within 4 h following addition of 8 U/mL of lacZBa at 45 °C, suggesting that it is a promising candidate for further use and research in the dairy industry. However, lacZBa has limited activity toward lactose, and the affinity of lacZBa for lactose as a substrate is not as good as that of oNPG. In future study, we are going to design a series of mutants of lacZBa, which can be better applied to the production of lactose-free milk by screening mutants of lacZBa with high hydrolysis performance. Moreover, we will try to heterologously express the protein in a generally recognized as safe host and optimize its enzyme activity for a better application in food industry.In this study, we expressed and characterized a novel β-galactosidase lacZBa from


Arabidopsis thaliana in the Pichia pastoris KM71H (MutS) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/Lmedium/h was achieved, which was 102.5-fold higher compared to flask cultivations.Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from  Myrosinase EC 3.2.1.147), a β-thioglucosidase, is a part of the plant defence system. Depending on the reaction conditions, it catalyses the hydrolysis of glucosinolates into a range of products  [1]. One.147, a βArabidopsis thaliana possesses six different myrosinase genes, while Brassica napus was reported to have at least 20 myrosinase isoenzymes, which are divided into three subclasses based on enzyme activity, solubility, and molecular weight  significantly enhanced the long-term stability of recombinant myrosinase. After 12 months of storage, the supernatant stored at −20 °C in 50% (v/v) glycerol had 90% of its original activity. No significant changes in myrosinase activity were observed when storing the supernatant at −80 °C. After 12 months, myrosinase exhibited 99% residual activity without glycerol and 108% residual activity with added glycerol. Hence, myrosinase can be stored at −80 °C without loss of its activity even without the need for use of any cryo-preservative agent. Arsiccio et al.  in 30% (v/v) glycerol and E. coli in LB  in 50% (v/v) glycerol. Microorganisms were also cultivated on plates containing 2% (w/v) agar and 50 mg/L ZeocinTM .w/v) YNB (Yeast Nitrogen Base), 4 × 10−5% (w/v) biotin, 1% (w/v) glucose, and 200 mM potassium phosphate buffer, (pH 6)], BMM2  YNB, 4.10−5% (w/v) biotin, 1% (v/v) methanol, 200 mM potassium phosphate buffer (pH 6)), and BMM10  YNB, 4.10−5% (w/v) biotin, 5% (v/v) methanol, and 200 mM potassium phosphate buffer (pH 6)) media according to Weis et al. [Cultivations in 96-deep well plates were performed in BMD [Buffered Minimal Dextrose medium; 1.34%  yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB, 4 × 10−5% (w/v) biotin, 1% (v/v) glycerol, and 100 mM potassium phosphate (pH 6)) and BMMH  YNB, 4 × 10−5% (w/v) biotin, 0.5% (v/v) methanol, and 100 mM potassium phosphate (pH 6)) media were used.For flask experiments, BMGY  supplemented with 4.35 mL PTM1  per litre of BSM medium. PTM1 solution was also added to the methanol feed in high-cell density fermentations (12 mL PTM1 per litre of methanol).Fermentation experiments were performed in BSM medium  was purchased from Generay Biotech Co., Ltd. . Plasmid pPICZαA-myr was transformed to E. coli DH5α cells. Plasmid DNA was extracted using the Genopure Plasmid Midi Kit  and linearised with SacI . Approximately 5 μg of linearised plasmid was transformed to P. pastoris KM71H competent cells by electroporation. Competent P. pastoris cells were prepared by a condensed protocol according to [TM concentrations (100–250 mg/L) and cultivated at 30 °C for 48 h.Plasmid pPICZαA containing the codon-optimised TGG1 gene for myrosinase from rding to . TransfoP. pastoris transformants were screened for myrosinase activity in a 96-deep well plate according to the protocol used by Markošová et al. [g, 10 °C, 10 min), and the supernatants were screened for myrosinase activity.á et al. . Each we® Flash microplate reader . The reaction mixture (100 μL), containing 50 μL of cell-free supernatant, 0.3 mM sinigrin , 0.5 mM ascorbic acid, and 0.1 M sodium phosphate buffer (pH 6.5), was incubated in a microplate reader at 30 °C and shaken at 240 spm (8 mm diameter). After 20 h, the released glucose was determined by adding 50 μL of solution containing 1.5 mM NAD+, 0.347 mg/mL glucose dehydrogenase , and 0.1 M potassium phosphate buffer (pH 7). The reaction was performed at 30 °C, shaken at 240 spm (8 mm diameter), and measured at 340 nm for 20 min. Maximal velocity was calculated by Varioskan® Flash software .The myrosinase activity assay for best transformant determination was performed in a 96-well microplate  using a VarioskanP. pastoris colony and cultivated at 30 °C and 200 rpm for 18 h. Then, the cells were harvested by centrifugation  and resuspended in 100 mL of BMMH medium. Pure methanol (100 μL) was added to BMMH medium 3 times a day. Cultivation was terminated after 90 h (a 72-h induction with methanol). Supernatants from the cultivation of the untransformed P. pastoris KM71H strain and a P. pastoris KM71H strain expressing a different gene were used as controls.Flask cultivation was performed in 500 mL shake-flasks. First, 100 mL of BMGY medium was inoculated with a single ® Mini Reactor Systems, Eppendorf, Germany). The cultivation procedure was described previously [v/v) inoculum, OD600: 14–16) 150 mL BSM medium supplemented with 0.653 mL PTM1. A 20% dissolved oxygen (DO) saturation was maintained by an agitation cascade (50–2000 rpm). After glycerol depletion (approx. 22 h), two methanol pulses to 3 g/L were performed, and after the second methanol depletion (approximately 45 h), continuous methanol feeding was started. Agitation was fixed at 1000 rpm, the DO control was turned off, and a third methanol pulse was performed. Methanol feeding was controlled by an automated program that switched the pump on when DO saturation was between 30–40%, maintaining the level of methanol below 3 g/L. Agitation parameters were altered from the original protocol [TM BioFlo® 115, Eppendorf, Germany) with 1.5 L working volume. Temperature and pH were set to different values based on each experiment.Fermentations were performed in 350 mL mini fermenters  was added directly to the BSM medium before sterilisation (200 μL of antifoam per 1.5 L of BSM medium). No additional antifoam was needed throughout the fermentation.g, 10 °C, 15 min), the biomass was discarded, and the supernatant was either filtered through 0.45 μm mixed cellulose ester membrane filter  or subjected to tangential membrane filtration on a 0.44 μm membrane  using ÄKTA flux . The cell-free supernatant was concentrated and desalted with Amicon® Ultra Centrifugal Filters with a 30 kDa cut-off  or using a 30 kDa cut-off membrane  with 50 mM sodium phosphate buffer (pH 6.5).After fermentation, the cultivation medium was centrifuged  for the myrosinase activity assay contained 100 μL cell-free supernatant, 0.6 mM sinigrin, 1 mM ascorbic acid, and 50 mM sodium phosphate buffer (pH 6.5). The reaction was performed in a thermomixer  at 30 °C and 600 rpm. Samples were taken at different times, and the reaction was terminated by boiling the reaction mixture for 5 min. The sinigrin concentration was then analysed by HPLC according to Tsao et al. . One uniReaction conditions were changed for temperature and pH profile determination and ascorbic acid activation measurement. For pH profile determination, 50 mM sodium phosphate-citrate (pH 3.5–5.5), 50 mM sodium phosphate (pH 5.5–7.5), and 50 mM Tris-HCl (pH 7.5–8.8) were used. In temperature profile measurements, a temperature range of 20–60 °C was tested. Myrosinase activation was tested with 0, 0.25, 0.5, 0.75, 1, 1.5, 3, 5, and 10 mM ascorbic acid.Operational stability was determined during one-hour incubation of myrosinase at 30 °C, 40 °C, and 50 °C. Samples were taken every 15 min. Then, a standard reaction was used for myrosinase activity determination.600) measurement at 600 nm . A calibration curve was then used to calculate the dry cell weight (DCW): DCW (g/L) = 0.2001 × OD600 + 0.1075 [Biomass growth was controlled by optical density  using WATREX Polymer IEX H form 8 μm (250 × 8 mm column) and WATREX Polymer IEX H form 8 μm (40 × 8 mm guard column). A flow rate of 0.8 mL/min of 9 mM sulfuric acid at 45 °C was used.® NX 5μm C18 110Å (150 × 4.6 mm) was used at 30 °C. Sinigrin was purchased from Sigma-Aldrich .The sinigrin concentration was determined by HPLC  according to Tsao et al. . A PhenoThe protein concentration in cell-free supernatants was determined by the Bradford method . The BraP. pastoris KM71H (MutS) strain expressing TGG1 myrosinase from A. thaliana was performed for the first time. It is the first report of this type of strain used for myrosinase expression. The importance of producing myrosinase in fermenters was demonstrated since the productivity increased 102.5-fold compared to flask cultivation. Optimisation of cultivation temperature and pH led to a threefold increase in fermentation productivity. The produced myrosinase can be stored at −80 °C without a significant loss of activity for 12 months.High-cell density cultivation of the


The chromatographic profiles of the hydroethanolic extract and a flavone-rich fraction (ACFF) from A. chica were determined by high-performance liquid chromatography coupled with a diode-array UV-Vis detector (HPLC-DAD-UV) and electrospray ionization mass spectrometry in tandem (LC-ESI-MS-MS). The flavones luteolin (1) and apigenin (2), isolated from chromatographic techniques and identified by Nuclear Magnetic Resonance of 1H and 13C, were also quantified in ACFF, showing 190.7 mg/g and apigenin 12.4 mg/g, respectively. The other flavones were identified by comparing their spectroscopic data with those of the literature. The in vitro activity was assayed against promastigotes and intramacrophagic amastigote forms of L. amazonensis. Cytotoxicity tests were performed with peritoneal macrophages of BALB/c mice. Nitrite quantification was performed with Griess reagent. Ultrastructural investigations were obtained by transmission electron microscopy. Anti-Leishmania assays indicated that the IC50 values for ACFF, apigenin, and luteolin were obtained at 40.42 ± 0.10 and 31.51 ± 1.13 μg/mL against promastigotes, respectively. ACFF and luteolin have concentration-dependent cytotoxicity. ACFF and luteolin also inhibited the intra-macrophagic parasite , with a selectivity index of 11.44 for ACFF. Promastigotes exposed to ACFF and luteolin exhibited ultrastructural changes, such as intense cytoplasm vacuolization and mitochondrial swelling. These findings data evidence the antileishmanial action of flavone-rich fractions of A. chica against L. amazonensis, encouraging further studies.Acknowledging the need of identifying new compounds for the treatment of leishmaniasis, this study aimed to evaluate, from  Flavonoids are an important class of secondary metabolites with a low molecular weight polyphenolic structure, widely distributed in the plant kingdom among subgroups that include chalcones, flavones, flavonols, and isoflavones . This meLeishmania infection  B. Verlot, syn. Bignonia chica, belongs to the family Bignoniaceae. It comprises about 120 genera and 860 species  (morphotype IV) were cultivated and collected in March 2016 at Fiocruz Atlantic Forest Campus, municipality of Rio de Janeiro, Rio de Janeiro State, Brazil (S22.9406 W43.4046). Plant material was identified by Dr. Marcus Felipe Oliveira da Silva at the Botanical Collection of Medicinal Plants of Farmanguinhos/FIOCRUZ, where a voucher specimen was deposited and registered under the number CBPM666.Leaves of Arrabidaea chica leaves were dried in a forced circulation oven at a temperature of 45°C for 4 days. After drying, the plant material was powered using a knife-mill affording 0.85 mm particles that were stored in an amber flask. The hydroethanolic extract was prepared by exhaustive maceration of dried and powdered leaves (1.5 kg) in 70% ethanol solution (v/v) with three changes of solvent, once every 48 h for 7 days at room temperature. After filtration, the solvent was evaporated under reduced pressure to yield 226.19 g of a red extract (ACCE) corresponding to yielding of 15.07%. The crude hydroethanolic extract (60 g) was then submitted to liquid-liquid partition with n-hexane (3 × 200 mL), dichloromethane (3 × 200 mL), ethyl acetate (3 × 200 mL) and n-butanol (3 × 200 mL). The final aqueous residue was discarded. Dichloromethane fraction was successively chromatographed by column chromatography on Sephadex LH-20 , using methanol as eluent to produce a purified flavone-rich fraction determined by thin layer chromatoghapy (TLC) analysis, and named ACFF. Successive chromatographic fractionation steps of ACFF on Sephadex LH-20 led to the isolation of two flavonoids . Isolated compounds and a flavone-rich fraction were analyzed by TLC  using acetone: chloroform: formic acid (75:16:0.8 v/v/v) as eluent and, subsequently, sprayed with 1% NP/PEG reagent , as well as 1H and 13C NMR spectrometry. Comparison with literature records allowed the identification of (1) luteolin and (2) apigenin (apigenin .(1) Luteolin 1H NMR (400 MHz-methanol-d6) d: 6.47 ; 6.13 ; 6.35 ; 7.35 ; 6.88 ; 7.35 . 13C NMR (400 MHz-methanol-d6) d: 166.05 (C-2); 103.41(C-3); 183.49 (C-4); 163.05 (C-5); 95.91(C-6); 166.05 (C-7); 101.31 (C-8); 159.72 (C-9); 104.27 (C-10); 123.43 (C-1′); 113.89 (C-2′); 147.31 (C-3′); 151.65 (C-4′); 116.86 (C-5′); 120.23 (C-6′).(2) Apigenin 1H NMR  d: 6.55 ; 6.16 ; 6.39 ; 7.35 ; 6.88 ; 13C NMR (400 MHz-Methanol-d6) d: 170.32 (C-2); 103.60 (C-3); 183.69 (C-4); 163.15 (C-5); 100.93 (C-6); 166.08 (C-7); 95.65 (C-8); 163.93 (C-9); 104.65 (C-10); 123.25 (C-1′); 129.42 (C-2′ and C-6′); 117.14 (C-3′ and C-5′); 159.65 (C-4′).® liquid chromatographer coupled to a Shimadzu UV detector with diode array SPDM20A, equipped with a CBM20A controller, DGU20A degasser, LC20AD binary pump, CTO20A oven, and SILA20A auto-injector. A Shimadzu LabSolutions Software Version 5.3  was used to analyze chromatograms. Combinations of acidified ultrapure water  (A) and acetonitrile  (B) were used as the mobile phase . HPLC column was silica-based C18 . The oven was set at 50°C and the injection volume was 10 μL for all analyses.Chromatographic analyses were performed on HPLC-DAD-UV using a Shimadzu Nexera XRA total of 1,000 µL of acetonitrile: methanol  mixture  was added to 5 mg of ACCE and ACFF, previously weighed in a 4 mL vial. The vial was sealed and the sample was sonicated for 10 min with occasional swirling. Posteriorly, the sample was vortexed to mix thoroughly, followed by filtering through a 0.45 µm PTFE filter  before further analyses into an HPLC vial.Stock solutions of analytical standards luteolin and apigenin  were prepared at 1,000 mg/mL in MeOH  in volumetric flasks. Six concentration of work solutions  were done on the day for calibration curves of each compound. The solutions were filtered in a 0.45 μm PTFE filter  before analyses by HPLC-DAD-UV. Injections of 20 μL were performed in triplicate to obtain the calibration curves from the areas corresponding to the peaks of luteolin and apigenin. The analytical curve (1–20 μg/mL) of the standards was plotted based on the UV-Vis signal at 254 nm: luteolin content (μg/mL) = (Abs (mAu) + 21,030)/83,557; R2 = 0.9995 and apigenin content (μg/mL) = (Abs (mAu) + 27,059)/77,296; R2 = 0.996. Flavones amounts were calculated in mg/g of dry extract. The following dilution factors were used for luteolin and apigenin quantitative analysis: 147.06 and 11.47, respectively.Liquid chromatography coupled to electrospray ionization mass spectrometry in tandem (LC-ESI-MS-MS) was performed with an LC Shimadzu Nexera Ultra-Fast Liquid Chromatography (UFLC) coupled to an ion trap Bruker amaZon MS. Analyses were performed at ambient temperature in a silica-based C18 column . The mobile phase consisted of ultrapure water obtained from the Milli-Q Millipore purification system, acidified at pH 3.0 with anhydrous acetic acid  (A) and acetonitrile  (B). The gradient of B was as follows: in 54.86 min from 5 to 95%; from 54.86 to 55.54 min returns to 5% B, remaining like this until 62.0 min to column re-equilibration. The flow rate was set at 0.5 mL/min and the injection volume was 1 μL. ESI-MS/MS were recorded in a Bruker Ion trap amazon SL mass spectrometer in the positive ionization mode (ESI+). The operating conditions were 1 μL/min infusion, 3.0–4.0 kV capillary voltage, 100°C temperature source, and cone voltage of 20–40 V. Mass spectra were recorded and interpreted by Bruker Compass Data Analysis 4.2 .All procedures performed with 4–6-weeks old female BALB/c mice were in accordance with the National Council for Control of Animal Experimentation (CONCEA). These animals were obtained from the Institute of Science and Technology in Biomodels of Oswaldo Cruz Institute and the experiments were approved by the local Ethics Committee on Animal Care and Utilization (CEUA-IOC L53/2016).Leishmania amazonensis H21 (MHOM/BR/76/MA-76) was maintained in the laboratory by successive passages in BALB/c mice. Parasites were isolated from a non-ulcerated nodular lesion in the footpad and amastigote viability was checked by light microscopy. 106 amastigote forms were transferred to the NNN medium  and maintained for seven days to differentiate into promastigote forms. Then, these forms were cultured at 26°C in Schneider’s Insect medium , supplemented with 10% fetal bovine serum , 100 IU/mL of penicillin, and 100 μg/mL of streptomycin in a maximum of ten in vitro passages , penicillin (100 U/mL) and (100 μg/mL) streptomycin, at 37°C and 5% COvernight .A. chica on the promastigote forms of L. amazonensis, viable promastigotes were counted in a Neubauer chamber according to the method described by 6 cells/mL) harvested from the logarithmic growth phase were added to 96-well flat-bottomed microtiter plates. Then, 50 µL of the ACCE , ACFF (12.5–200 μg/mL) or isolated flavonoids (3.125–100 μg/mL) were added to each well and incubated at 26 ± 1°C for 72 h. Wells with parasites and DMSO 1% only were used as untreated control and amphotericin B (0.03125–1.0 μg/mL) was used as a reference drug. After the incubation, viable promastigotes were counted in a Neubauer chamber. The experiments were conducted in triplicate. Percentage of growth inhibition was calculated from the count of viable parasites relative to the untreated control, and 50% inhibitory concentration (IC50) values were determined.To evaluate the anti-promastigote effects of the ACCE, ACFF, and isolated flavonoids from Peritoneal Macrophage Obtaining and Cell Culture) were seeded at 5 × 105 cells per milliliter in a 96-well plate and allowed to adhere overnight at 37°C and 5% CO2. Subsequently, the cells were treated with different concentrations of the ACCE, ACFF , flavonoids (1.95–500 μg/mL) or amphotericin B (0.19–25 μg/mL), in a final volume of 100 μL/well, incubated for 72 h at the same conditions. Wells without cells were used as blank and wells with cells and 1% DMSO were used as controls. The cytotoxicity was determined with the MTT  assay. Half-maximal cytotoxic concentration (CC50) was calculated according to Peritoneal macrophages collected as previously described (5 cells/well), containing round coverslips and incubated at 37°C in 5% CO2 overnight. The cells were then infected with promastigote forms of L. amazonensis, in the parasite/cell ratio of 10:1, for 6 h followed by washing with PBS to remove free parasites. Infected cells were treated with different concentrations of ACFF (0.15–2.5 μg/mL) or luteolin (1.56–25 μg/mL), and the plates were incubated in similar conditions for 24 h. The macrophages containing amastigotes without compounds treatment and those treated with amphotericin B (2.5–0.15 μg/mL) were considered the negative and positive controls, respectively. Finally, the coverslips of the infected and treated cells were fixed in Bouin's solution, stained by Giemsa and observed under a light microscope. The activity anti-intramacrophage amastigotes of the compounds was evaluated by counting the number of amastigotes in each macrophage by examining 200 macrophages in comparison with the untreated control. The percentage of infected cells was obtained from the number of infected cells divided by two. The mean number of amastigotes per cell was obtained from the number of intracellular amastigotes in 200 cells divided by the number of infected cells . Selectivity index (SI), which was calculated using the equation of CC50 for murine macrophage/IC50 for the intracellular amastigote forms of L. amazonensis, was used to compare the toxicity and activity of the compounds.Peritoneal macrophages of BALB/c mice were cultured in 24-well plates (5 × 10ed cells . The per6 cells/mL) by the Griess reaction for nitrite according to the method described by L. amazonensis (3 × 107 parasites/mL), and added in 96-well plates. Then, were added to supernatants 50 µL of Griess reagent (25 µL of sulfanilamide 1% in 2.5% H3PO4 solution and 25 µL of N-(1-naphthyl ethylenediamine 0.1% solution). After 10 min, the plates were read at 570 nm on the spectrophotometer and the nitrite values were obtained from the standard sodium nitrite curve (1.5–100 μM).Nitric oxide (NO) release was indirectly measured in the supernatants of macrophage culture  in 0.1 M sodium cacodylate buffer, pH 7.2, overnight. Then, parasites were washed three times with 0.1 M sodium cacodylate buffer and post-fixed in a solution containing 1% osmium tetroxide, 0.8% potassium ferrocyanide, and 5 mM calcium chloride, washed in 0.1 M sodium cacodylate buffer, dehydrated in graded acetone, and embedded in EPON 812 resin . Ultrathin sections were obtained from 100 nm cuts in Sorvall MT 2-B (Porter Blum) ultramicrotome  stained with 5% uranyl acetate aqueous solution and lead citrate (1.33% lead nitrate and 1.76% sodium citrate), and examined in a transmission electron microscope JEM-1011  operating at 80 kV  and Mann-Whitney test.The numerical results were expressed as mean ± standard deviation and the statistical analyses were conducted through the statistical software GraphPad PrismA. chica ((1) and apigenin (2), which had already been reported in the literature, and had their structures confirmed by 1H and 13C NMR  also resulted in a chromatogram with six peaks . Mass spA. chica, as shown in The results of HPLC-DAD-UV and LC-ESI-MS-MS allowed the identification of the six component flavones of the rich fraction in flavonoids of r2) for linear regression equations of both standards were obtained: 0.9995 for luteolin and 0.9960 for apigenin. Luteolin and apigenin contents were calculated using the equations Absorbance (mAu) = 83,557 (concentration)—21,030 and Absorbance (mAu) = 77,296 (concentration)—27,059, respectively. The content of luteolin and apigenin  and UV spectra of the corresponding peaks in the flavone-rich fraction with authentic standards apigenin and luteolin. Flavones quantification was carried out through calibration curves obtained by triplicate injections. Calibration curves showed to be linear in the ranges of 1–20 μg/mL for luteolin and apigenin. Besides that, good regression coefficients   .From the results described in p = 0.0010, p = 0.0043, p = 0.0002, p = 0.0011, p = 0.0049, p = 0.0005, p = 0.0002, p = 0.0002, L. amazonensis and treated with amphotericin B, ACFF and luteolin corroborate with the results of The parameters of infection of untreated cells were used as comparative control for the treatment with the different compounds at different concentrations. Infected and untreated macrophages presented 783.1 ± 80.43 amastigotes per 200 cells, 87.74 ± 7.95% of infected cells, and mean of amastigotes per infected cell of 5.135 ± 0.53 . The treA. chica compounds on nitrite production in the supernatant of BALB/c peritoneal macrophages is shown in 2, p = 0.0857) and luteolin , when compared to untreated cells (4.34 ± 1.12 μM NaNO2). Low nitrite levels were also observed in cells stimulated with L. amazonensis and treated with ACFF  and luteolin , when compared to stimulated and untreated cells (5.83 ± 1.69 μM NaNO2), although the difference was not statistically significant. In the test, macrophages when stimulated with lipopolysaccharide (LPS) produced high levels of nitrite compared to cultures not stimulated with LPS.The effect of A. chica promoted morphological and structural changes, analysis by transmission electron microscopy (TEM) was performed. Parasites were treated or not with ACFF and luteolin IC50 for 24 h. Promastigote forms showed the cellular morphology with an elongated body and all its intact organelles  and 256 and 283 nm (band II). Band I is associated with absorption due to the B ring of cinnamoyl system and when registered in the range of 304–350 nm it is characteristic of flavones . Band II(3), scutellarein (4), carajuflavone (5) and chrysoeriol (6).The flavone-rich fraction (ACFF) was also analyzed by LC-ESI-MS-MS, whose results compared with literature data  allowed m/z 153 and 169 are characteristic of compounds containing di-hydroxylated and tri-hydroxylated A ring, respectively. Retro-Diels-Alder fragmentation also produces information about B ring. Fragments at m/z 119 and 135 are related to mono- and di-hydroxylated B ring, respectively. Positive LC-ESI-MS-MS data analyses were performed to confirm structures in addition to data already obtained in HPLC-DAD-UV and Rt of flavones from ACFF. Both luteolin (1) and apigenin (2) have been identified by 1H NMR and 13C NMR spectrometric techniques, UV, elution order, and Rt, and were also evaluated by fragmentation pattern. These compounds showed the Retro-Diels-Alder fragmentation pattern confirming the di-hydroxylation of A ring . Luteolin (1) showed experimental pseudo-molecular ion (M + [H]+) at m/z 287.0496, which is compatible to the molecular formula C15H10O6 as well as the mass fragment at m/z 241.0447 [(M-H2O-CO)]+. Apigenin (2) was confirmed by the pseudo-molecular ion at m/z 271.0548, corresponding to the molecular formula C15H10O5, as well as the fragments at m/z 243 characterized by C=O loss. Retro-Diels-Alder fragments correspondent to a tri-hydroxylated A ring at m/z 169.0103 and 169.0105 were observed to 6-hydroxy-luteolin (3) and scutellarein (4), respectively. The compound 6-hydroxy-luteolin or 5,6,7,3′,4′-penta-hydroxy flavone (3) also showed the experimental pseudo-molecular ion (M + [H]+) at m/z 303.0449, compatible to the molecular formula C15H11O7, as well as the mass fragment at m/z 257.0389 [(M-H2O-CO)]+, confirming their identification. The structure of scutellarein (4) was also confirmed by the experimental pseudo-molecular ion at m/z 287.0496, which is compatible with the molecular formula C15H10O6. Carajuflavone or 6,7,3′,4′-tetrahydroxy-5-methoxy flavone (5) showed a pseudo-molecular ion at m/z 317.0595 corresponding to the molecular formula C16H13O7. The fragment at m/z 168.0017 indicated that in Retro-Diels-Alder fragmentation, one of the three hydroxyl groups of A ring was substituted. It was confirmed by their fragmentation pattern that produced ions at m/z 302.0360 confirming the loss of a methyl group (M—[CH3]+). Chrysoeriol (6) showed experimental pseudo-molecular ion at m/z 301.0646 compatible with the molecular formula C16H13O6. Chrysoeriol fragmentation pattern produced ions at m/z 286.0414 [(M)-CH3]+ and at m/z 258.0467 [(M)-CH3-CO]+. These pieces of evidence confirm compound (6) as chrysoeriol , luteolin (1), apigenin (2), and scutellarein (4)—were identified in ACFF. Carajuflavone (5), another 6-hydroxylated compound from A. chica had already been described by (3) has been reported in the Bignoniaceae family, and 6-hydroxylation is a common structural feature of the Bignoniaceae flavonoids having particular chemotaxonomy relevance for this reason. Besides, 6-hydroxylation is also characteristic of the structures of the red pigments anthocyanidins carajurin and carajurone found in A. chica (All identified flavones in ACFF have been previously described for  extract . In anotA. chica .(1) as the most representative flavone of ACFF ((2) represented only 12.43 mg/g dry extract. The results obtained in the present study are already better than other publications on concentrations and contents of luteolin and apigenin in extracts obtained from A. chica leaves  of the promastigote forms of L. amazonensis determined at 155.9 μg/mL. Phytochemical screening of that study showed an extract containing flavonoids, phenolic compounds, tannins, anthocyanidins and chalcones. In addition, the crude extract of the leaves of A. chica had a cytotoxic effect at a concentration of 189.9 μg/mL. Factors that can explain the difference in phytochemical and biological results are probably due to the climatic differences between the two locations where the plant was collected, as well as the way of obtaining the extract  . Such characteristics are observed in apigenin and luteolin. Thus, it is possible to infer that the different leishmanicidal activity of these two flavones might be due to the replacement pattern in the B-ring, since luteolin has a catechol portion  while apigenin has only one hydroxyl in 4'  . The antyl in 4' .L. amazonesis intracelular amastigote. In the search for new drugs against Leishmania spp., intracellular amastigote is the stage of the parasite considered as the most relevant target for the primary screening of new compounds . In addition, when the infected macrophages were treated with increasing concentrations of apigenin, there was a decrease in the number of infected cells  in activated macrophages . TherefoA. chica flavonoids against intracellular amastigote.In a study with apigenin and luteolin, both inhibited NO production, considering that a C-2,3 double bond may be important, and that the patterns of substitution of flavonoid molecules can determine the potency of the inhibition in NO production . In addiA. chica compounds directly on the parasite, ultrastructural evaluation of the promastigote forms of L. amazonensis was performed by transmission electron microscopy. The treatment with ACFF promoted ultrastructural alterations such as the vacuolization process of the cytoplasm, lipid and multivesicular bodies, swelling of the kinetoplast and mitochondria with the breaking of the mitochondrial ridges. Ultrastructural changes were also observed in the study by L. infantum promastigotes treated with fraction B2 (1:1 n-hexane/ethyl acetate) obtained from the crude hexane extract of A. chica. In this study, mitochondrial edema with loss of matrix content and the presence of vesicles within this organelle were observed.Trying to understand the leishmanicidal effect of L. amazonensis promastigotes. It was possible to observe vacuolization of the cytoplasm, change in nuclear chromatin, lipid bodies, with kinetoplast and fully degenerated mitochondria, and vacuoles similar to autophagosomes. Studies performed by Leishmania, an inhibition similar to the well-known anti-Leishmania drug, pentamidine. Another study elucidates the mechanism of action of luteolin by analyzing mitochondrial and cytosolic changes associated with death similar to L. donovani cell apoptosis (Leishmania cells.Luteolin, the metabolite with the highest concentration in ACFF composition, was evaluated for ultrastructural alterations induced in poptosis . In thisA. chica. However, based on the findings obtained, further studies should be carried out to elucidate the mechanisms of action, as well as in vivo studies are essential to assess the leishmanicidal activity of the ACFF against Leishmania.Our results provide additional evidence on the antileishmanial activity induced by A. chica. The flavones luteolin and apigenin were isolated from the ACFF using chromatographic techniques and identified by NMR spectrometry techniques. ACCE, ACFF and flavones showed leishmanicidal activity against the promastigotes of L. amazonensis. The antiparasitic effect of ACFF and luteolin was confirmed by the ultrastructural changes with induction of mitochondrial damage. ACFF also showed low cytotoxicity in host cells if compared with the isolated flavones. ACFF and luteolin showed leishmanicidal activity against the intracellular amastigotes, however, this activity is not related to the production of NO by host cells. Thus, ACFF is a suitable candidate for further in vivo investigations against L. amazonensis.Chromatographic techniques and analysis of the mass spectra obtained from the ACCE allowed the identification of compounds of the ACFF derived from


Panax ginseng C.A. Mey (ginseng) is a classic medicinal plant which is well known for enhancing immune capacity. Polysaccharides are one of the main active components of ginseng. We isolated water-soluble ginseng polysaccharides (WGP) and analyzed the physicochemical properties of WGP including molecular weight, monosaccharide composition, and structural characteristics. WGP had minimal effect on the growth of hepatocytes. Interestingly, WGP significantly increased the mRNA and protein levels of complement component 4 (C4), one of the core components of the complement system. Promoter reporter gene assays revealed that WGP significantly enhanced activity of the C4 gene promoter. Deletion analyses determined that the E-box1 and Sp1 regions play key roles in WGP-induced C4 transcription. Taken together, our results suggest that WGP promotes C4 biosynthesis through upregulation of transcription. These results provide new explanation for the intrinsic mechanism by which ginseng boosts human immune capacity. Panax ginseng C.A. Mey (ginseng), a perennial plant belonging to genus Panax, is one of the most appreciated medicinal plants  have been proved playing an important role in the modulation of immunity . Human iC4 gene product, fourth component of human complement (C4), is an important component of the complement system  was boiled in deionized water (4 L) for 3 h. After filtration, the solid phase was boiled in deionized water at the same ratio (g/ml) two more times. The filtrate was combined, centrifuged, and concentrated. The concentrated solution was mixed with anhydrous ethanol at a volume ratio of 1:3 and left overnight at 4°C. Discard the supernatant by centrifugation and the precipitate was successively washed with 75% ethanol, 95% ethanol, anhydrous ethanol, and ether. The crude ginseng polysaccharides were obtained after drying and subsequently redissolved in deionized water. After removal of proteins using a Sevage reagent, a 3 KD molecular sieve was used to remove small molecules to obtain WGP. The content of sugar was tracked and monitored by the phenol sulfuric acid method .via 0.45 μm microfiltration membrane. The filtered sample (20 μL) was loaded into a TSK-Gel G4000PWXL column  controlled by LC-10Avp system . High performance gel permeation chromatography (HPGPC) was performed using 0.2 M NaCl as mobile phase at flow rate of 0.5 ml/min. T-series Dextran standards were used for reference standards.WGP (5 mg/ml) was filtered via 0.22 μm organic membrane. Conversion of monosaccharides with PMP were detected via high-performance liquid chromatography (HPLC). The sample (10 μL) was injected into a 4.6 mm × 250 mm COSMOSIL 5C18-PAQ column  controlled by an LC-20AT system . HPLC was performed using a mobile phase composed of 19.5% Acetonitrile and 80.5% 0.1 M PBS (pH 7.0) at flow rate of 1 ml/min. The absorbance values at wavelength 245 nm were compared with those of monosaccharide standards including arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose, and xylose to determine the monosaccharide composition of WGP.Monosaccharide composition analysis was performed as previously described . Brieflyvia Tenor 27 spectrophotometer . WGP was ground with KBr powder at a mass ratio of 3:1 and compressed into a pellet. The FT-IR spectra were recorded in range of 400–4,000 cm−1.Fourier transform-infrared spectroscopy (FT-IR) spectra of WGP was acquired 2O. The 1H and 13C nuclear magnetic resonance (NMR) spectrum were performed on AV-500, 600, and 800 instruments  using tetramethylsilane as the internal standard.The lyophilized WGP was dissolved in Dmycoplasma monthly by the PCR method described by Uphoff and Drexler . Cells were cultured using DMEM media containing 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine  and 10% (V/V) fetal bovine serum  and incubated in HERAcell150i incubator (Thermo Fisher Scientific) set to 5%CO2/95% air and 37°C. Cells were tested for  Drexler .via a microplate reader.The L-O2 cells were treated with a series of equal ratio gradient concentrations of WGP for 72 h in a 96-well plate. MTT   was added at the final concentration of 0.5 mg/ml and incubated for 4 h in incubator. Then, the cells were lysed in 10% SDS containing 10 mM HCl overnight. The absorbance values at wavelength 590 nm were measured via ultrasonic cell disruption and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred onto PVDF (polyvinylidene difluoride) membrane (Thermo Fisher Scientific), and subsequently immunoblotted utilizing anti-β-actin  and -C4  antibodies. Visualization of immunoreactive proteins was conducted using the Odyssey Infrared Imaging System . Densitometry measurements were performed using Odyssey V3.0 software (LiCor).Whole cell lysates were obtained via reverse transcription PCR Kit (Thermo Fisher Scientific), as described previously .Total RNA was extracted by TRIZOL method and used to make cDNAs eviously . The Lig changes . C4 tranvia full length-forward and reverse primers (C4 promoter was identified and digested by XhoI and HindIII (Promega). The C4 promoter was then subcloned into reporter gene vector pGL4.19 basic (Promega) at the XhoI and HindIII restriction sites to generate pC4-1,007/+44. To generate the 5’-deletion constructs, pC4-119/+44, pC4-102/+44, pC4-92/+44, pC4-72/+44, and pC4-48/+44, genomic DNA fragments were PCR amplified from the pGL4.19 plasmid using forward (segment 1 to segment 5) and reverse primers . Differences were compared via hot-water extraction and alcohol precipitation from ginseng powder. The yield of WGP was 0.85%. Carbohydrate content of WGP was over 98% and protein impurity was less than 0.1%. HPGPC was used for molecular weight analysis of WGP. We obtained two overlapping peaks and a single symmetric peak indicating that the molecular weight range of the WGP was from 1 kD to 79.4 kD  was used to confirm the effect of WGP on C4 transcription. Transient transfection of pC4-1,007/+44 into L-O2 cells was performed first, then the cells were treated with WGP for 72 h. The p-C4-1,007/+44 construct showed significant reporter activity compared to pGL4.19 basic in the absence or presence of WGP. Interestingly, treatment of the cells with WGP for 72 h significantly enhanced the C4 promoter reporter gene activity , one NF1 binding site (positions −110 to −97), and one Sp1 (positions −57 to −49) binding element  site at −110 to −97, an Sp1 site at −57 to −49, and three basic helix-loop-helix -like transcription factor sites at −137 to −132, −98 to −93, and −78 to −73, respectively referred to as E-box1, E-box2, and E-box3. These regions appear to be critical for the transcript activity of C4 gene (C4 gene promoter. We found that E-box1 and Sp1 elements play key roles in WGP-regulated C4 transcription. A previous study has shown that the E-box motif of C4 promoter can be recognized by one of the many basic helix-loop-helix (bHLH) or basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor family members  class III area on the sixth human chromosome.  closely . C4 promerase II . C4 prom C4 gene . Thus, w members . Moreove8 to −73 . Thus, aIn conclusion, our results demonstrate that polysaccharides derived from ginseng can significantly increase production of C4 through transcriptional mechanisms. These results provide a molecular explanation for the immunity enhancing function of ginseng extracts.


The PDFs were stored at 50 °C for 0, 1, 2, 4, 13, and 26 weeks. The total GDP concentration of single-chamber PDFs did not change considerably during storage , but individual GDPs were affected differently. 3-DG (− 82.6 µM) and 3-DGal (− 71.3 µM) were degraded, whereas 5-HMF (+ 161.7 µM), glyoxal (+ 32.2 µM), and formaldehyde (+ 12.4 µM) accumulated between 0 and 26 weeks. Acetaldehyde, glucosone, MGO, and 3,4-DGE showed time-dependent formation and degradation. The GDP concentrations in double-chamber fluids were generally lower and differently affected by storage. In conclusion, the changes of GDP concentrations during storage should be considered for the evaluation of clinical effects of PDFs.Reactive glucose degradation products (GDPs) are formed during heat sterilization of glucose-containing peritoneal dialysis fluids (PDFs) and may induce adverse clinical effects. Long periods of storage and/or transport of PDFs before use may lead to de novo formation or degradation of GDPs. Therefore, the present study quantified the GDP profiles of single- and double-chamber PDFs during storage. Glucosone, 3-deoxyglucosone (3-DG), 3-deoxygalactosone , 3,4-dideoxyglucosone-3-ene , glyoxal, methylglyoxal (MGO), acetaldehyde, formaldehyde, and 5-hydroxymethylfurfural (5-HMF) were quantified by two validated UHPLC-DAD methods after derivatization with  During the heat sterilization of PDFs, monocarbonyls like acetaldehyde, formaldehyde, furfural, and 5-hydroxymethylfurfural (5-HMF) as well as α-dicarbonyl compounds such as glucosone, 3-deoxyglucosone (3-DG), 3-deoxygalactosone , 3,4-dideoxyglucosone-3-ene , glyoxal, and methylglyoxal (MGO) are formed3. There is strong evidence that GDPs impair the biocompatibility of PDFs and that the formation of advanced glycation end-products from GDPs, for example, limits the long-term treatment with peritoneal dialysis5. Moreover, some GDPs can induce inflammatory mechanisms, reduced cellular stress response, or apoptosis, which leads to a loss of cell viability8. Among the GDPs, 3,4-DGE shows the highest cytotoxicity and is responsible for the most pronounced enzyme inactivation10.Peritoneal dialysis is a renal replacement therapy utilizing peritoneal dialysis fluids (PDFs), which contain an osmotic agent such as glucose, lactate buffer, and electrolytes. For microbial safety, PDFs are usually heat-sterilized, but the process leads to the formation of clinically relevant glucose degradation products (GDPs)12. Immediately prior to use, the glucose solution is mixed with a buffer from the second compartment yielding a physiological pH of about 7.4.As an alternative to the widely used single-chamber PDFs, double-chamber-bag products have been developed. Sterilizing the glucose-containing solution separately from lactate at a lower pH of about 3 reduces the GDP load14. Previously, Zimmeck et al. observed that the concentration of 3-DG decreased during six months of storage at 25, 30, and 40 °C, whereas the concentration of 5-HMF increased13. Erixon et al. detected an increasing amount of 3,4-DGE and 5-HMF in a conventional PDF after incubation for 21 days at 25, 40, or 60 °C, respectively, while the concentration of 3-DG decreased during the incubation experiments with the exception of the PDFs stored at 25 °C14. Another study monitored the UV absorbance at 228 nm  and 284 nm (absorption maximum of 5-HMF) of a heat-sterilized fifty percent glucose solution during storage15. All three studies revealed that the changes in GDP concentrations are more pronounced at higher temperatures15. To date, however, no comprehensive quantitative screening of GDP profiles (including up to ten mono- and dicarbonyls) has been performed in stored PDFs. Additionally, the existing data is not sufficient to compare the behaviors of GDPs in single-chamber and double-chamber-bag PDFs during storage.It is well established that GDPs are mainly formed during heat sterilization, but their contents in PDFs may change when PDFs are exposed to elevated temperatures during storage and transport. Thus, the GDP composition of PDFs used for the treatment of patients may vary considerably from the contents in the freshly produced solution. To our knowledge, only the behaviors of a few selected GDPs in PDFs have been investigated yet10 so that their concentration levels in the PDFs that are administered to the patients can be of clinical relevance. Therefore, the present study aimed to investigate storage-induced changes of the major mono- and α-dicarbonyl GDPs in single- and double-chamber PDFs. The present test conditions of 50 °C and 45% relative humidity simulated possible storage or transport conditions during the summer season, in hot climate zones, or during overseas transport in non-tempered cargo containers. The GDPs were quantified in the PDFs by ultrahigh-performance liquid chromatography (UHPLC) coupled with diode array detection (DAD) after derivatizing the six major α-dicarbonyls with o-phenylenediamine (OPD) and the three major monocarbonyls with 2,4-dinitrophenylhydrazine (DNPH).A comprehensive analysis of the entire GDP profiles in single- and double-chamber PDFs is, however, of high relevance, because it can be expected that different GDP structures react differently during long-term exposure to storage temperatures. To date, it is not sufficiently clear how the concentrations of glucosone, 3-DGal, glyoxal, MGO, formaldehyde, and acetaldehyde change during storage or transport. Individual GDPs have specific physiological effectsThe present study investigated the influence of storage at 50 °C and 45% relative humidity on the GDP profiles of single- and double-chamber PDFs for up to 26 weeks.3. The monocarbonyl GDP 5-HMF also reacts with OPD yielding a benzimidazole derivative, which gives a signal at 316 nm and can coelute with the quinoxaline derivatives of 3-DGal or glyoxal16, thus limiting their accurate quantification. In order to achieve baseline separation of 5-HMF and all major α-dicarbonyls, the pH of the eluent A was set to 3.3 . Therefore, we applied weighted linear regression models  and the apex of the furfural signal was observed in heated PDFs, but neither in standard solutions nor in spiked unheated PDFs . Therefore, an unknown compound was assumed to coelute with furfural in heat-sterilized glucose-containing PDFs, which may lead to an overestimation of the furfural content. Furfural was the least quantifiable GDP in the PDFs  and dropped to 3.1 µM during further incubation , followed by 5-HMF (22.5 µM), and 3-DGal (19.7 µM). The compounds 3,4-DGE (2.8 µM), acetaldehyde (2.2 µM), and glucosone (1.6 µM) were present at much lower concentrations. Formaldehyde was below the limit of quantification  and glyoxal below the limit of detection  at week 0. MGO was not detectable in any double-chamber PDF , the transport routes (long/short), or the ambient temperatures (summer/winter). In particular, temperatures can fluctuate remarkably and may reach almost 60 °CThe current analysis investigated products ready for dispatch with the consequence that the PDFs had been pre-stored at room temperature for two and five months, respectively. Even though this random selection represented realistic conditions, the pre-storage may be a limitation of the study, because changes of the GDP profile between production and dispatch cannot be excluded.2. In both types of PDF, the concentrations of 3-DG and 3-DGal decreased during prolonged storage. This result is in line with previous studies that also observed a decrease of 3-DG during storage over 21 days at 40 and 60°C14, or six months at 25, 30, and 40 °C, respectively13. 3-DG and 3-DGal undergo reactions leading, for example, to the formation of 3,4-DGE19 and further to 5-HMF, which seems to be a stable end-product in PDFs2  and 3-DGal (− 71.2 µM). Interestingly, the concentration of 3,4-DGE peaked after one week of incubation. Erixon et al. also reported increased 3,4-DGE concentrations, especially at incubation temperatures of 40 °C and 60 °C, during one week of incubation and observed that the 3,4-DGE content decreased again until the end of the 21-day storage period at 60°C21. Recently, it was shown that traces of redox-active metal ions, such as iron(II), can promote the formation of the oxidized GDP glucosone and, to a lesser extent, also glyoxal and MGO22. The concentrations of 3-DG, 3-DGal, and 3,4-DGE, which are formed by non-oxidative mechanisms2 were not affected by metal ions22. Thus, traces of metal ions from raw materials or production equipment may be responsible for the formation of glucosone during the storage of double-chamber PDFs.The concentrations of glucosone and MGO decreased in single-chamber PDFs over time, but the glucosone content increased slightly in double-chamber solutions. MGO was below LOD in the double-chamber-bag fluids at any time point. Glucosone is formed by the oxidation of glucose13. Single-chamber bags contain lactate and glucose, which mediate the formation of acetaldehyde13. In double-chamber bags, glucose and lactate are stored separately so that the reaction is inhibited. Thus, double-chamber bags contain considerably less acetaldehyde compared to single-chamber solutions. Previous studies reported concentrations below LOD or LOQ in double-chamber PDFs, which ranged from 2 to 18 µM, respectively24. The present method is more sensitive, so that acetaldehyde contents between 1.5 and 2.2 µM could be quantified in the PDFs. These results indicate that very minor concentrations of acetaldehyde may be formed from glucose either independently from lactate, during/after mixing the solutions of both compartments, or directly from lactate.The acetaldehyde content peaked in single-chamber PDFs at week 13, while it decreased in double-chamber bags from the beginning of storage. Acetaldehyde is formed from lactate but is considered as GDP, because its formation is glucose-dependent21. Alternatively, Yaylayan and Keyhani proposed a mechanism based on the loss of two water molecules from glucose and subsequent retro-aldol reaction25. In addition, glyoxal can be formed via oxidation of glycolaldehyde after retro-aldol cleavage of glucose20. The reaction mechanism leading to the formation of formaldehyde has yet to be elucidated.The concentrations of glyoxal and formaldehyde increased over the storage period in both types of PDFs, which may result from the degradation of long-chain GDPs such as glucosone. Glyoxal can be formed from glucosone via retro-aldol cleavageThe contents of 3-DG and 3-DGal decreased in both PDF types during 26 weeks of storage. However, 5-HMF, glyoxal, and formaldehyde were formed during the incubation time in both PDF systems. The acetaldehyde content decreased in double-chamber PDFs, while the concentration peaked in single-chamber fluids after 13 weeks. The concentration of 3,4-DGE reached its maximum in single-chamber PDFs after one week of incubation and decreased afterwards. After 26 weeks of storage, however, the 3,4-DGE concentration was still slightly higher than in week 0. In double-chamber bags, a continuous decline of 3,4-DGE was observed. The GDP profiles of single- and double-chamber bags underwent different changes. The PD solutions in both systems vary in pH as well as in glucose, buffer, and electrolyte concentrations. These factors influence the formation and degradation of the GDPs during heat sterilization and subsequent storage. At the beginning, and even at the end of the study, double-chamber PDFs contained less GDPs than single-chamber solutions  unless indicated otherwise. Liquid chromatography/mass spectrometry-grade solvents  and formic acid  were used in all experiments. 3-DG (purity > 95%) was obtained from Chemos . Glucosoned-lactate (3.9 g/L), calcium chloride dihydrate (0.26 g/L), sodium chloride (5.8 g/L), and magnesium chloride hexahydrate (0.10 g/L). The ready-to-use double-chamber PDFs contained 4.25% glucose (w/w), sodium d-lactate (3.9 g/L), calcium chloride dihydrate (0.26 g/L), sodium chloride (5.6 g/L), and magnesium chloride hexahydrate (0.10 g/L). Because the glucose solution and the buffer compartment of double-chamber systems must be mixed prior to sampling, the samples were always drawn from originally sealed bags. Directly after production, the PDFs were kept at room temperature before the present storage experiments started five months (single-chamber PDFs) or, respectively, two months (double-chamber PDFs) after production. For the storage experiments, the PDFs were kept at 50 °C and 45% relative humidity. Samples were drawn after 0, 1, 2, 4, 13, and 26 weeks from three new bags each, except for the single-chamber PDF at week 13, which was only available in duplicate. The study design followed the European Medicines Agency ICH Topic Q 1 A guideline using accelerated testing conditions plus 10 °C to cover extreme temperature scenarios37.The commercial single-chamber PDFs contained 4.25% (w/w) glucose, sodium An Ultimate 3000RS system  was used with a Waters ACQUITY UPLC® Phenyl column  equipped with a corresponding guard column. System control, data acquisition, and processing were performed by Chromeleon 6.8 software.3. Briefly, α-dicarbonyl compounds were converted into their corresponding quinoxaline derivatives by derivatization with OPD. For this purpose, 80 µL of the sample was mixed with 10 µL of derivatizing reagent (4% OPD in 1 M 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic (HEPES) buffer, pH 7) and 10 µL of internal standard . The samples were incubated in the dark between 2 and 16 h. Afterwards, the solutions were analyzed by UHPLC-DAD using ammonium formate buffer  and methanol (eluent B) at 55 °C with a flow rate of 0.4 mL/min. The eluent composition was [time (minutes)/percent B]: − 3.0/10, 0.0/10, 8.5/25, 10/50, 10.1/80, and 12.0/80. DAD spectra for peak verification were recorded from 190 to 490 nm. 3,4-DGE was quantified at a wavelength of 335 nm, all other analytes at 316 nm by external calibration. The UHPLC-DAD analysis was performed in duplicates.α-Dicarbonyl GDPs were quantified as previously reported with minor modifications24 with some modifications. DNPH (10 mg) was dissolved in 4 mL of acetonitrile and 100 µL of 60% sulfuric acid. Aliquots of 500 µL of each PDF were mixed with 200 µL of the DNPH solution, filtered into amber glass vials, and allowed to stand light-protected at room temperature for one (minimum) to 8 h (maximum). The analytes were eluted at 50 °C with a flow rate of 0.4 mL/min using ammonium formate buffer  and acetonitrile (eluent B). The eluent composition was [time (minutes)/percent B]: 0.0/20, 1.0/20, 2.5/31, 12.0/31, 13.0/37, 18.0/37, 22.0/55, 22.2/90, 25.0/90, 25.1/20, 30.0/20. The injection volume was 10 µL. DAD spectra for peak verification were recorded from 190 to 600 nm. Except for furfural, no discrepancies between the expected and the recorded UV/Vis spectra were observed in any of the standard solutions or PDFs. Thus, it can be assumed that recovery experiments for 5-HMF, formaldehyde, and acetaldehyde are not negatively influenced in heated PDFs. The analysis of monocarbonyl compounds in PDFs was performed in duplicates.For the profiling of monocarbonyl GDPs, all samples were reacted with DNPH according to Tauer et al.To test whether the derivatization procedure yielded stable hydrazone derivatives in lactate-buffered PDFs, a commercial single-chamber PDF was treated as described above. UHPLC-DAD analysis was performed after incubation at room temperature for different periods of time up to 12.5 h. Each sample was analyzed in triplicate and the mean values of the peak areas were plotted against the derivatization time.2) of 0.990, a relative error of < 5%, and homogeneity of variances across the concentration range .Formaldehyde and acetaldehyde were quantified at 356 nm and furfural and 5-HMF at 390 nm by external calibration. Since the derivatization reagent contained traces of formaldehyde and acetaldehyde, solvent blanks were analyzed in the same way and the peak areas of formaldehyde and acetaldehyde in the samples were corrected for these background signals. A ten-point calibration curve from 0.6 to 320.0 µM was recorded for 5-HMF and a nine-point calibration curve from 1.3 to 320.0 µM for acetaldehyde. Seven-point calibration curves were obtained for formaldehyde and furfural ranging from 0.8 to 64.0 µM  and 0.2 to 16.0 µM . Each calibration level was analyzed in duplicate. The linearity of the calibration curves was evaluated by linear regression analysis with a minimally acceptable coefficient of determination , 5-HMF , formaldehyde , or acetaldehyde . Spiked and unspiked PDFs were analyzed as described above. The mean recovery of six experiments for each concentration level was determined and expressed as /added concentration × 100%. Precision was expressed as standard deviation and coefficient of variations.38 and were as follows (LOD/LOQ): formaldehyde 0.1/0.6 µM, acetaldehyde 0.05/0.2 µM, 5-HMF 0.02/0.1 µM, furfural 0.04/0.2 µM.The LOD and LOQ were determined using the calibration method according to German technical standard DIN 32645:2008-11This article does not contain any studies with human participants or animals performed by any of the authors.Supplementary Information.