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Prenyl groups increase the lipophilicity of flavonoids, endowing them with a special activity, selectivity, and pharmacological properties by prenylation. Herein, a novel prenyltransferase (ShFPT) gene from Streptomyces sp. NT11 was expressed in Escherichia coli, and its biochemical characteristics were determined. ShFPT exhibited high selectivity to prenylate naringenin at C-6 to generate 6-prenylnaringenin. The optimal activity was observed at pH 6.0 and 55 °C. The Kcat and Km for naringenin were 0.0095 s-1 and 0.20 mM, respectively. Several promiscuous kinase and isopentenyl phosphate kinase genes were screened to develop the most efficient dimethylallyl diphosphate (DMAPP) synthesis pathway for 6-prenylnaringenin synthesis in E. coli. The 6-prenylnaringenin production was improved by changing the induction strategies and optimizing the bioconversion conditions. Finally, 6-prenylnaringenin production reached the highest yield of 69.9 mg/L with average productivity of 4.0 mg/L/h after 16 h incubation, which is the highest yield for any prenylated flavonoid reported to date in E. coli. Therefore, this study provides an efficient method for 6-prenylnaringenin production and reveals the DMAPP synthesis pathway.

The response surface methodology was used for the optimization of different submerged fermentation conditions for the production of recombinant laccase Lcc1 in Pichia pastoris KM71H. The initial screening of production parameters was performed using a Plackett–Burman design, and the variables with significant effects on laccase production were identified as follows: medium initial pH, methanol additive amount and liquid volume. These variables were selected for further optimization studies using a Box-Behnken design. The results indicated that the optimum fermentation conditions were as follows: medium optimal initial pH value of 7.01, methanol additive amount of 0.63% (v/v) per 24 h and liquid volume of 19.50% (v/v). These conditions provided the highest laccase enzyme activity of 12,491 U/L, resulting in a 3-fold increase in the production of recombinant laccase. Using industrial basic salt as culture medium, the maximum enzyme activity of Lcc1 was 22,594 U/L after high-density fermentation, which was 1.8-fold increase by shake flask optimization. Furthermore, partially purified laccase was used for the degradation of the octyl phenol and 4-tert-octylphenol, which displayed excellent degradation capacity. After 24 h, the degradation rate of 200 mg/L of 4-tert-octylphenol at 40 °C, pH 4.5 using 50 mM tartaric acid buffer with 1500 U/L purified laccase enzyme in L/A (laccase/ABTS) system was 97.2%. For octyl phenol, the degradation rate was 93.1%. All of the results suggested that the laccase Lcc1 significantly reduced or eliminated the toxicity of octyl phenol and 4-tert-octylphenol, which may be suitable for typical phenolic pollutants in the environment.

The thermostable GH78 family α-L-rhamnosidase DthRha from Dictyoglomus thermophilum DSM3960 was found to have the ability to remove the α-L-rhamnose moiety that was directly linked to the aglycone by an α-1-rhamnosidic bond at the C-3 position of epimedium flavonoids. It exhibited high specific activity on p-nitrophenyl-α-L-rhamnopyranoside (pNPR) (281.06 U/mg), followed by epimedin C (30.42 U/mg), icariin (9.74 U/mg), baohuoside I (1.02 U/mg) and 2’-O-rhamonosylicariside II (3.74 U/mg). In addition, the thermostable glucosidase Dth3 from Dictyoglomus thermophilum DSM3960, which exhibits β-xylosidase activity and β-glucosidase activity, was purified. The main enzymatic properties of the two thermostable glycosidases were compared, and these glycosidases were successfully utilized in the preparation of pharmacologically active icaritin from the total flavonoid extract of epimedium. Finally, 5 g/L of the total flavonoid extract of epimedium was completely transformed into 207.95 mg/L icaritin at pH 5.5 and 80 °C for a total of 4 h. This is the first report on the efficient biotransformation of the total flavonoid extract of epimedium into icaritin by two thermostable enzymes with high productivity. The cost of icaritin preparation will be substantially reduced by converting all the major ingredients in the total flavonoid extract of epimedium.

In recent years, with the global environmental deterioration and air pollution, the incidence of bronchitis has increased year by year, and the sales of anti-bronchitis drugs are growing rapidly, mainly due to the long treatment cycle and the difficulty of curing. Developing available traditional Chinese medicines with significant curative effect against bronchitis would be a promising strategy; for instance, Ginkgo seeds, as the fruit of natural plant ginkgo, has been used in ancient times to cure coughs. However, the detailed mechanism of curing cough has not been shown yet. Investigate the mechanism of Ginkgo seeds in the treatment of bronchitis by establishing a series of molecular networks including active ingredients-targets, proteins interactions, biological functions, pathway, and biological processes of targets. In this study, the main active ingredients of Ginkgo seeds and the potential targets related to bronchitis could be obtained by retrieving corresponding database. The molecular docking study between active molecules and protein targets was performed by Glide 6.6. Subsequently, a total of forty potential targets were manually selected. Based on this, the ingredients-target network was constructed using Cytoscape software, as well as proteins interactions network combing with the String database. Finally, the molecular biological function, metabolic pathway, and biological processes of these forty targets were analyzed by Clue GO plug-in. The results indicated that these protein targets were closely related to lipid transport, positive regulation of DNA replication, cAMP metabolic pathway, and other processes, which played a vital role in the treatment of bronchitis by mediating interleukin 17, fluid shear stress and atherosclerosis, asthma, renin secretion, p53, and other signaling pathways. Among these targets, the two protein ALB (Albumin) and DHRS2 (Dehydrogenase 2) can interact with compounds more frequently, and the top three compounds ranked by the docking scores were amentoflavone, (+)-catechin-5-O-glucoside, and liquiritin, implying that these compounds might be used for the treatment of bronchitis. It is obvious that the pharmacological effect of Ginkgo seeds on bronchitis displayed a characteristic of multi-components, multi-targets, and multi-pathways. Nevertheless, the two protein targets and three compounds derived from Ginkgo seeds could be further used for the explanation for Ginkgo seeds in curing bronchitis. This research can provide a scientific basis for studying on the anti-bronchitis mechanism of Ginkgo seeds.

To efficiently recycle the GH78 thermostable rhamnosidase (TpeRha) and easily separate it from reaction mixture and furtherly improve the enzyme properties, the magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNcZ8) was prepared through modifying Fe3O4-NH2 with tetraethyl silicate (TEOS), microcrystalline cellulose and zinc nitrate hexahydrate. The FSNcZ8 displayed better magnetic stability and higher-temperature stability than unmodified Fe3O4-NH2 (FN), and it was used to adsorb and immobilize the GH78 thermostable rhamnosidase (TpeRha) from the Thermotoga petrophilea 13995. The properties of FSNcZ8-TpeRha were as follows: The optimal reaction temperature and pH were 90°C and 5.0; the highest activity of FSNcZ8-TpeRha approached 714 U/g; In addition, FSNcZ8-TpeRha had better higher-temperature stability than FN, after incubation at 80°C for 3 h, the residual enzyme activities of FSNcZ8-TpeRha, FN-TpeRha and free enzyme were 93.5%, 63.32% and 62.77%, respectively. The organic solvent tolerance and the monosaccharides tolerance of FSNcZ8-TpeRha, compared with free TpeRha, were greatly improved. Using Naringin (1 mmol/L) as the substrate, the optimal converting conditions were as follows: FSNcZ8-TpeRha concentration is 6 U/mL; induction temperature is 80°C; the pH is 5.5; induction time is 30 min, and the yield of products was same as free enzymes. After 10 times of repeated reaction, the conversion of Naringin remained above 80%, which showed a great improvement of the catalytic efficiency and repeated utilization at the immobilized α-L-rhamnosidase.

A thermostable and organic solvent-tolerant bacterial laccase from Bacillus pumilus ARA has been expressed heterologously and characterized, which shows potential decolorization capacity to various types of industrial synthetic dyes. The optimal temperature and pH were 85 °C and 3.5, respectively, while the purified recombinant laccase B.P.Lacc was stable under 55-75 °C and pH 5.0-8.0 conditions. The apparent kinetic parameters Km and Vmax of B.P.Lacc for ABTS as the substrate were 0.33 mM and 32.4 U/mg, respectively. Ethanol (1%, v/v) and methanol (2%, v/v) could stimulate the enzyme activity. The recombinant laccase retained over 95% of its initial activity in 10% (v/v) methanol. The optimal expression conditions for the laccase production of B.P.Lacc in LB medium were obtained: induction temperature of 25 °C, 0.4 mM Cu2+, and 1.0 mM IPTG added into the culture. After 5 h, the final laccase production was 1283 U/mL. Moreover, the laccase activity increased to 4822 U/mL after follow-up 2 h stationary cultivation, with about a 3.76-fold increase. The purified B.P.Lacc was able to efficiently decolorize synthetic dyes combined with mediators. Adding 1.0 mM ABTS, more than 90% of BRRB was decolorized by the enzyme, whether at pH 4.0 or pH 7.9. The outstanding enzymatic properties suggested that B.P.Lacc may be suitable for a wide application in future biodegradation fields.

The thermostable GH3 family β-glucosidase DthBgl3 and thermostable GH78 family α-l-rhamnosidase DthRha from Dictyoglomus thermophilum DSM3960 were successfully immobilized by industrial amino resin 1000NH. The optimal reaction temperature and pH of 1000NH-DthBgl3 was 85 °C and pH 5.0. Over 65% residual enzyme activity maintained for 1000NH-DthBgl3 after a 3-h incubation under 85 °C, while about 20% residual enzyme activity maintained for free DthBgl3. The optimal reaction temperature and pH of 1000NH-DthRha was 95 °C and pH 6.5. Over 90% residual enzyme activity maintained for 1000NH-DthRha after a 3-h incubation under 90 °C, while about 22% residual enzyme activity maintained for free DthRha. Meanwhile, immobilized 1000NH-DthBgl3 and 1000NH-DthRha were successfully and could completely transform all major ingredient of 10 g/L total flavonoids extract from Epimedium (TFEE) into icaritin. After 15 cycles (45 h) of repeated use at 85 °C, 3 U 1000NH-DthBgl3 showed a molar conversion rate of 73.12%, initial activity of 30.09% and productivity of 124 mg/L/h, while 15 U 1000NH-DthRha showed a molar conversion rate of 88.50%, initial activity of 85.31% and productivity of 75 mg/L/h after ten cycles (60 h) of repeated use at 85 °C. The cooperation of two immobilized enzymes showed a molar conversion rate of 87.21% and productivity of 141 mg/L/h after 15 cycles of repeated use at 85 °C. This is the first report on immobilization of two thermostable glycosidases from thermophilic bacteria and their cooperated biocatalysis of all major ingredients of TFEE into icaritin with high productivity under a high substrate concentration.

In recent years, deep eutectic solvent (DES) has attracted comprehensive attention on the extraction of natural products, and is regarded as an alternative to traditional organic solvents for the environmental advantages. Twenty-six DESs were compared for their extraction yield of total flavonoids and isovitexin (the main flavonoid in Celtis sinensis) from Celtis sinensis. The results show that the extraction yields of total flavonoids by betaine/glycolic acid (DES8), ethylamine hydrochloride/1,2-propanediol (DES12) and tetrapropylammonium bromide/lactic acid (DES17) are the highest, while the extraction yields of isovitexin by ethylene glycol/malonic acid (DES23), ethylene glycol/glycolic acid (DES24) and 1,2-propanediol/glycolic acid (DES26) are the highest. The extraction conditions using the above six DESs were further optimized systematically. Under optimum conditions, the extraction rates of total flavonoids and isovitexin can be increased up to 95.39 and 10.58 mg g−1, respectively, which were significantly higher than that of methanol extraction. In order to exclude the effect of DESs on the bioactivity of Celtis sinensis extract, the macroporous resin D-101 was used to purify the total flavonoids from DESs extract, and the recovery rates of flavonoids from the above six kinds of DESs were all over 80%. Next, the anti-inflammatory activity of DES extracts was compared using a lymphocyte transformation experiment. The result showed that the inhibition rate of the DES24 extract on the proliferation of Con A-activated T cells was up to 72% with an IC50 value of 124.8 μg mL−1. None of the DESs extracted exhibited cytotoxicity on normal T cells. The mechanism of the anti-inflammatory activity against Con A-activated T cells may be that DES24 flavonoids extract induced the apoptosis of inflammatory T cells, and activated the expression of pro-apoptotic protein. Taken together, DES has showed significant advantages on the extraction of natural products for the relatively mild extraction condition, high yield and environmental-friendliness.

Hyperoside is used as the standard to assess the quality of Hyperin perforatum L and exhibits many biological properties. To improve the yield of hyperoside in Escherichia coli, four UDP-galactose synthesis pathways were screened and a recombinant strain was reconstructed by using an efficient UDP-galactose generation system coupled with Petunia hybrida glycosyltransferase to biosynthesis hyperoside from quercetin. By adopting the resting cell transformation, maximal hyperoside production of 4574 mg/L and average productivity of 63.5 mg/L/h were achieved after 72 h of biotranformation. Subsequently through recycling of resting cells, average productivity within 72 h reached 126 mg/L/h, which was 100% higher than that in the batch fermentation. Hyperoside production reached equivalent to 18,000 mg/L by recycling seven times of resting cell in 168 h, which was 393% of that in the batch fermentation and the first to reach 10 g per liter scale in E. coli. Therefore, this study provides an efficient method for construction of UDP-galactose synthesis pathway and hyperoside production in E. coli.

The development of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is an ongoing and challenging research field. However, the dynamic motion of the binding site of EGFR has not been accurately depicted, hindering the improvement of EGFR TKI. For this reason, about 33 protein complexes (32 EGFR proteins plus 1 ErbB4 protein) were carefully curated and subsequently studied for dynamic movements of their binding sites by molecular dynamics simulations in this study. The analysis of root mean square deviation (RMSD) revealed that T790M mutation can make an impact on dynamic motion of binding sites; the RMSD value of the EGFR binding site was unrelated to inhibitory activity. The analysis of the radius of gyration (Rg) revealed that T790M can slightly shrink the value of Rg, thereby influencing the shape of the EGFR binding site. More interestingly, the Rg value can exhibit weak correlation with inhibitory activity of most inhibitors. The relationship between Rg and biological activity deserve our serious interest since the best scoring function, Xscore, cannot distinguish highly active EGFR inhibitors. The root mean square fluctuation (RMSF) analysis of key residues derived from binding sites indicated that the most flexible residue was ASP800 with a large RMSF value against the steady residue ALA743 with a small RMSF value, and two other residues (MET793 and LEU844) were supposed to be involved with molecular recognition. In short, the obtained results would be more effective for guiding the development of a novel EGFR kinase inhibitor.

Synthesis of β-ionone in microbial cell factories is limited by the efficiency of carotenoid cleavage dioxygenases (CCDs). To obtain genes responsible for specific cleavage of carotenoids generating β-ionone, a novel carotenoid cleavage dioxygenase 1 from Morus notabilis was cloned and overexpressed in Escherichia coli . The MnCCD1 protein was able to cleave a variety of carotenoids at the positions 9, 10 (9', 10') to produce β-ionone, 3-hydroxy-4-oxo-β-ionone, 3-hydroxy-β-ionone, and 3-hydroxy-α-ionone in vitro . MnCCD1 could also cleave lycopene and β-carotene at the 9, 10 (9', 10') bind bond to produce pseudoionone and β-ionone respectively in E.coli accumulating carotenoids. The enzyme activity of MnCCD1 was reached 2.98 U/mL at optimized conditions (temperature 28 ℃, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of MnCCD1 revealed the optimal activities were at pH 8.4 and 35 ℃. The addition of 10% ethanol could increase enzyme activity at above 15%. However, an obvious decline was observed on enzyme activity as the concentration of Fe 2+ increased (0-1 mM). The V max for β-apo-8'-carotenal was 72.5 U/mg, while the K m was 0.83 mM. The results provide a foundation for developing the application of carotenoid cleavage dioxygenases as biocatalysis and synthetic biology platforms to produce volatile aroma components from carotenoids.

As a fast-growing tree, poplar is widely planted and typically used for wood processing in China. During poplar wood processing, a large amount of poplar sawdust (PS) and poplar leaves (PL) are produced and abandoned. To make full use of poplar resources and clarify the use of poplar as a feed additive, the active ingredients in PS and PL were extracted and isolated, and the anti-inflammatory effects of the extracts on mice with dextran sulfate sodium (DSS)-induced colitis were investigated. In vitro anti-inflammatory experiments showed that the ethyl acetate extract of PS and PL (PSE and PLE, respectively) could significantly inhibit the proliferation of concanavalin A (Con A)-activated lymphocytes. Salicortin, tremulacin and salireposide were identified in both PSE and PLE. Oral administration of PSE and PLE rescued DSS-induced colonic shortening, repaired tissue damage, and decreased the disease activity index (DAI). The antioxidant capacity, including the increased activities of glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD and catalase (CAT) and decreased activity of myeloperoxidase (MPO), in the colons of mice with colitis was enhanced through the activation of ERK after treatment with PSE and PLE. The ratio of Th1 to Th17 cells, which can lead to inflammation in the spleen, was significantly decreased by the administration of PSE and PLE, while the phosphorylation of related transcription factors (p65, STAT1, and STAT3) was inhibited. Furthermore, PSE and PLE could induce apoptosis in Con A-activated lymphocytes, which may be associated with the increase in p-TBK1, as the molecular docking results also indicated that salireposide in PSE and PLE could interact with the TBK1 protein. Overall, our study provides a promising feed additive for improving intestinal inflammation in animals and a method for the full utilization of poplar resources.

In this study, a GH78 α- L -rhamnosidase AtRha from Aspergillus terreus CCF3059 was screened and expressed in Pichia pastoris KM71H. The maximum enzyme activity of AtRha was 1000 U/mL after 12 days. AtRha was most active at 65 °C and pH 6.5, displaying excellent thermal stability and pH stability. The kinetic parameters Km, Vmax, kcat and kcat/Km values for pNPR were 0.481 mM, 659 μmol/min·mg, 1065 s-1 and 2214 s-1 mM-1, respectively. AtRha could be inhibited by Fe2+, Hg2+ and Cu2+. Moreover, it displayed good tolerance to organic reagents with 52.6% activity in 15%(w/v) methanol. AtRha can hydrolyze icariin containing the α-1 rhamnoside linkage. Furthermore, AtRha and β-glucosidase TthBg3 showed excellent selectivity to cleave the rhamnose at the 3rd position and the glucosyl at the C-7 group of icariin, which established an effective and green method to produce the more pharmacological active icaritin. In addition, the optimal enzyme addition schemes and the reaction conditions were screened and optimized. After a two-stage transformation under optimized conditions, 0.5 g/L of icariin was transformed into 0.25 g/L of icaritin, with a corresponding molar conversion rate of 91.2%. Our findings provide a new, specific and cost-effective method for the production of icaritin in the industry.

β-Glucosidases and β-xylosidases are two categories of enzymes that could cleave out nonreducing, terminal β-D-glucosyl and β-D-xylosyl residues with release of D-glucose and Dxylose, respectively. In this paper, two functional β-glucosidase Dth3 and β-xylosidase Xln-DT from Dictyoglomus thermophilum were heterologously expressed in E.coli BL21 (DE3). Dth3 and Xln-DT were relatively stable at 75°C and were tolerant or even stimulated by glucose and xylose. Dth3 was highly tolerant to glucose with a Ki value of approximately 3 M. Meanwhile, it was not affected by xylose in high concentration. The activity of Xln-DT was stimulated 2.13- fold by 1 M glucose and 1.29-fold by 0.3 M xylose, respectively. Furthermore, the β- glucosidase Dth3 and β-xylosidase Xln-DT showed excellent selectivity to cleave the outer C-6 and C-3 sugar moieties of ASI, which established an effective and green method to produce the more pharmacologically active CAG, an exclusive telomerase activator. We measured temperature, pH and dosage of enzyme using a single-factor experiment in ASI biotransformation. After optimization, the optimal reaction conditions were as follows: 75°C, pH 5.5, 1 U of Dth3 and 0.2 U of Xln-DT, respectively. Under the optimized conditions, 1 g/l ASI was transformed into 0.63 g/l CAG with a corresponding molar conversion of 94.5% within 3 h. This is the first report to use the purified thermostable and sugar-tolerant enzymes from Dictyoglomus thermophilum to hydrolyze ASI synergistically, which provides a specific, environment-friendly and cost-effective way to produce CAG.

Aims The aim of this work was to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3 by three thermostable glycosidases. Methods and results The GH1 thermostable beta-glucosidase Tpebgl1 from Thermotoga petrophlia was found to have the ability to convert ginsenosides Rb1 and Rb2. Its properties concerning ginsenoside conversion were systematically investigated. It had high specific activity on pNPG (162·20 U mg-1 ) and pNPArp (22·14 U mg-1 ). The Km and Vmax of Tpebgl1 for pNPG were 0·28 mmol l-1 and 470·2 U mg-1 and for pNPArp were 17·30 mmol l-1 and 74·28 U mg-1 . Therefore, it could successfully convert ginsenosides Rb1 and Rb2 into ginsenoside Rd, which has been proven by experiments in this paper then. Tpebgl1 also had good tolerance to glucose and some organic solvents. These made Tpebgl1 a good catalyst candidate for industrial application. Finally, it was applied to convert ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, combined with thermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase Tt-Afs and ginsenoside Rd converting β-glucosidase Tpebgl3. A quantity of 10 g l-1 of ginsenoside extract was transformed into 3·93 g l-1 of Rg3 at 90°C, pH 5·0 for 3 h, with a corresponding molar conversion of 98·19%. Conclusion The thermostable enzyme Tpebgl1 was found to be a ginsenoside-converting enzyme and successfully applied in the preparation of ginsenoside 20(S)-Rg3 from ginsenoside extract. The three-step cooperate transformation system of ginsenoside extract was established by using Tpebgl1, Tt-Afs (a thermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase) and Tpebgl3 (a thermostable ginsenoside Rb1 converting β-glucosidase). Significance and impact of the study Converting all the major ginsenosides into protopanaxadiol-type ginsenoside extract would greatly reduce the cost of ginsenoside Rg3 preparation. Enzymes from thermophilic bacteria can meet the requirement of higher reaction temperatures in industrial reactions for substrate solubility promotion and bacterial contamination prevention.

Isoorientin and isovitexin, kinds of flavone C-glycosides, exhibit a number of biological properties. In this work, The C-glucosyltransferase (Gt6CGT) gene from Gentiana triflora was cloned and expressed in Escherichia coli BL21(DE3). The optimal activity of Gt6CGT was at pH 7.5 and 50° C. The enzyme was stable over pH range of 6.5–9.0, and had a 1-h half-life at 50° C. The Vmax for luteolin and apigenin was 21.1 nmol/min/mg and 31.7 nmol/min/mg, while the Km was 0.21 mM and 0.22 mM, respectively. Then, we developed an environmentally safe and efficient method for isoorientin and isovitexin production using the coupled catalysis of Gt6CGT and Glycine max sucrose synthase (GmSUS). By optimizing coupled reaction conditions, the titer of isoorientin and isovitexin reached 3820 mg/L with a corresponding molar conversion of 94.7% and 3772 mg/L with a corresponding molar conversion of 97.1%, respectively. The maximum number of UDP-glucose regeneration cycles (RCmax) reached 28.4 for isoorientin and 29.1 for isovitexin. The coupled catalysis reported herein represents a promising method to meet industrial requirements for large-scale isoorientin and isovitexin production in the future.

Isoquercitrin exhibits a number of biological properties and has a higher water solubility than quercetin. In this work, an Arabidopsis glycosyltransferase with high activity on quercetin (AtUGT78D2) was coupled with an efficient quercetin-tolerant Glycine max sucrose synthase (GmSUS) to synthesize isoquercitrin by regenerating UDP-glucose from UDP. The important factors for optimal synergistic catalysis were determined. Through the use of a fed-batch operation, the final titer of isoquercitrin increased to 3830 mg/L with a corresponding molar conversion of 94.3% and a maximum number of UDP-glucose regeneration cycles (RCmax) of 33.

The current study was carried out to determine the influence of supplemental fermented Ginkgo biloba leaves (FGBL) at different levels on serum biochemistry and antioxidant capacity in broilers. A total of 648 one-d-old broiler chickens were randomly allocated into six dietary treatments, including control group (basal diet), FGBL1, FGBL2, FGBL3, FGBL4 and FGBL5 groups (basal diet containing 1.5, 2.5, 3.5, 4.5 and 5.5 g/kg FGBL, respectively). The experiment lasted for 6 weeks. At 42 d, 2 birds with nearly equal body weight from each replicate were slaughtered. The results indicated that gain to feed ratio had linear and quadratic influences by FGBL addition. The activity of glutathione peroxidase (GSH-PX) in serum, total antioxidant capability and total superoxide dismutase activities in liver and nuclear hepatic transcription factor erythroid 2-related factor mRNA expression followed the same fashion. The supplementation of FGBL linearly increased serum albumin concentration and hepatic GSH-PX mRNA expression, linearly decreased serum triglycerides concentration, malondialdehyde concentration in both serum and liver. There were quadratic influences on body weight and serum total protein concentration as supplemental FGBL level increased. Finally, it can be summarized that addition of FGBL affected growth performance, serum biochemistry and antioxidant capacity of broiler chickens. The optimal dose in the present study of FGBL in broiler diets was from 3.5 to 4.5 g/kg.

It has been reported that Celtis sinensis Pers. is employed as a folk medicine for the treatment of inflammation diseases. But the mechanism supporting its use as anti-inflammatory remain unclear. To investigate the anti-inflammatory of Celtis sinensis Pers. ICR mice were provided Celtis sinensis Pers. leaf extract (CLE) at 100, 200mg/kg after ginkgolic acids (GA) sensitization. Our data showed that CLE and the main flavonoid isovitexin in CLE could ameliorate GA-induced contact dermatitis in mice. Ear swelling, inflammatory cell infiltration and splenomegaly were inhibited significantly by isovitexin, while the weight loss of mice in the isovitexin-treated group was much better than that in the dexamethasone-treated group (positive control drug). It has been reported in previous research that GA-induced inflammation is closely related to the T cell response. Therefore, T cells were the focus of the anti-inflammatory effect of isovitexin in this paper. The in vivo results showed that isovitexin (10, 20mg/kg) inhibited the expression of proinflammatory cytokines (TNF-α, IFN-γ, IL-2 and IL-17A) in lymph nodes, inhibited the secretion of cytokines into the serum from mice with contact dermatitis and promoted the expression of apoptosis-related proteins. In vitro, isovitexin also induced apoptosis and proinflammatory cytokine expression in Con A-activated T cells. Further study showed that the MAPK and STAT signaling pathways and the phosphorylation of SHP2 were inhibited by isovitexin. Both molecular docking and biological experiments indicated that SHP2 may be an anti-inflammatory target of isovitexin in T cells. Taken together, isovitexin can serve as a potential natural agent for the treatment or prevention of GA-induced inflammatory problems.

Orientin and vitexin, important components of bamboo-leaf extracts, are C-glycosylflavones which exhibit a number of interesting biological properties. In this work, we developed an efficient biocatalytic cascade for orientin and vitexin production consisting of Trollius chinensis C-glycosyltransferase (TcCGT) and Glycine max sucrose synthase (GmSUS). In order to relieve the bottleneck of the biocatalytic cascade, the biocatalytic efficiency, reaction condition compatibilities and the ratio of the enzymes were determined. We found that the specific activity of TcCGT was significantly influenced by enzyme dose and Triton X-100 or Tween 20 (0.2%). Co-culture of BL21-TcCGT-Co and BL21-GmSUS-Co affected the catalytic efficiency of TcCGT and GmSUS, and the maximum orientin production rate reached 47 μM/min at the inoculation ratio of 9:1. The optimal pH and temperature for the biocatalytic cascade were pH 7.5 and 30 °C, respectively. Moreover, the high dose of the enzymes can improve the tolerance of biocatalytic cascade to substrate inhibition in the one-pot reaction. By using a fed-batch strategy, maximal titers of orientin and vitexin reached 7090 mg/L with a corresponding molar conversion of 98.7% and 5050 mg/L with a corresponding molar conversion of 97.3%, respectively, which is the highest titer reported to date. Therefore, the method described herein for efficient production of orientin and vitexin by modulating catalytic efficiencies of enzymes can be widely used for the C-glycosylation of flavonoids.

Ginkgo acids (GAs) in ginkgo products usually lead to allergies or liver toxicity. In this study, the GA-induced toxicity was attenuated and Con A-induced T-lymphocyte proliferation was inhibited by extracts of Celtis sinensis leaves (ECSL). So, the active ingredients in ECSL were studied to solve the problems caused by GAs. First, the eight components of MeOH extracts were determined by HPLC-DAD/LC-MS. Then, the 12 active ingredients were separated based on the anti-inflammatory activity. Lymphocyte conversion showed that the inhibition rates of apigenin, quercetin, and isovitexin at 100 μM on Con A-activated proliferation of T cells were up to 82.46%, 62.86% and 42.76%, respectively. The inhibition rate on the LPS-induced NO release in RAW 264.7 cells of quercetin, apigenin, isovitexin, and vitexin were exceeding 80% at 100 μM. Taken together, the material foundation for the screen of GAs toxicity-attenuated ingredients were provided here. PRACTICAL APPLICATIONS: Ginkgo biloba extracts (EGBs) have been conducted to develop functional food which could increase blood circulation and enhance memory. Nevertheless, people in consumption of ginkgo products, often caused severe allergic reactions due to the potential allergens identified ginkgolic acids (GAs) of ginkgo products. We first find that the extracts of Celtis sinensis leaves can reduce GAs-induced damage on HepG2 liver cells. Then, the bioactive compounds in C. sinensis leaves were separated and purified based on anti-inflammatory activities against T cells. Quercetin, apigenin, and isovitexin showed well anti-inflammatory activities against Con A-activated T-lymphocytes and LPS activated RAW 264.7 macrophages. However, quercetin and apigenin are flavones O-glycosides which are rich in Ginkgo biloba. To solve the problems in Ginkgo biloba products caused by GAs, flavone C-glycoside (isovitexin) may be used for the further study in GAs toxicity-reduction.

Ginkgo biloba extract (EGB) has many pharmacological activities. In the quality standard of EGB, the main quality control indexes are total flavone (content &ge, 24%) and total lactone (content &ge, 6%). There are no specific limits for nearly 70% of &ldquo, other components&rdquo, In recent years, in order to pursue the production of a high-ketone ester, some enterprises removed the unwanted components, including some organic acids. Protocatechuic acid (PCA), as an important organic acid, has been reported to have a variety of biological activities. It is necessary to explore whether it can promote the biological activities of the main functional components of EGB. In this study, PCA was selected to be combined with Ginkgolide B (GB) for the treatment of Parkinson&rsquo, s disease. In vitro, rotenone (rot) was used to induce PC12 cells. The survival rate was tested by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dimethyltetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) and antioxidase were detected to analyze the effects of drugs on oxidative stress. The apoptosis was tested via Western blot. The results show that the cell viability was increased, morphology was improved, the oxidative stress level decreased, and the apoptosis was inhibited after the combination treatment of GB and PCA, and the effect was better than GB or PCA alone. In vivo, MPTP (30 mg/kg) was used to induce Parkinson&rsquo, s disease (PD) in male C57BL/6 mice. The motor ability of the mice was measured by pole-climbing and the suspension. The injury of nerve cells was indicated by HE staining. Oxidative stress levels were tested via antioxidant enzyme activity. The number of dopaminergic neurons was reflected by TH staining. Results show that the combination treatment of GB and PCA could significantly restore the motor ability of PD mice, reduce the injury of nerve cells, improve the activity of the antioxidant enzyme in the brain tissue, and increase the expression of TH in the substantia nigra of midbrain. Our study shows that PCA increases the efficacy of GB (the main functional ingredient of EGB) in the treatment of Parkinson&rsquo, s disease, which provides a new idea for the treatment of nervous system diseases and a new concept for the efficient utilization of active components in Ginkgo biloba leaves.

Failure of cancer chemotherapy is mostly due to multidrug resistance (MDR). Overcoming MDR mediated by overexpression of ATP binding cassette (ABC) transporters in cancer cells remains a big challenge. In this study, we explore whether NVP-TAE684, a novel ALK inhibitor which has the potential to inhibit the function of ABC transport, could reverse ABC transporter-mediated MDR. MTT assay was carried out to determine cell viability and reversal effect of NVP-TAE684 in parental and drug resistant cells. Drug accumulation and efflux assay was performed to examine the effect of NVP-TAE684 on the cellular accumulation and efflux of chemotherapeutic drugs. The ATPase activity of ABCG2 transporter in the presence or absence of NVP-TAE684 was conducted to determine the impact of NVP-TAE684 on ATP hydrolysis. Western blot analysis and immunofluorescence assay were used to investigate protein molecules related to MDR. In addition, the interaction between NVP-TAE684 and ABCG2 transporter was investigated via in silico analysis. MTT assay showed that NVP-TAE684 significantly decreased MDR caused byABCG2-, but not ABCC1-transporter. Drug accumulation and efflux tests indicated that the effect of NVP-TAE684 in decreasing MDR was due to the inhibition of efflux function of ABCG2 transporter. However, NVP-TAE684 did not alter the expression or change the subcellular localization of ABCG2 protein. Furthermore, ATPase activity analysis indicated that NVP-TAE684 could stimulate ABCG2 ATPase activity. Molecular in silico analysis showed that NVP-TAE684 interacts with the substrate binding sites of the ABCG2 transporter. Taken together, our study indicates that NVP-TAE684 could reduce the resistance of MDR cells to chemotherapeutic agents, which provides a promising strategy to overcome MDR.

UDP-rhamnose is synthesized by UDP-rhamnose synthase, but the reaction requires two kinds of cofactors, NAD+ and NADPH. In this work, a cofactor self-sufficient UDP-rhamnose regeneration system was described herein. The UDP-rhamnose synthase (VvRHM) gene from Vitis vinifera was cloned and expressed in Escherichia coli and characterizations of VvRHM were determined. The N-terminal region of VvRHM was fused with the bifunctional UDP-4-keto-6-deoxy- d -glucose 3,5-epimerase/UDP-4-keto-rhamnose 4-keto-reductase (NRS/ER) from Arabidopsis thaliana to obtain the fusion enzyme (VvRHM-NRS), which was NADPH-independent and could convert UDP-glucose to UDP-rhamnose with NADH self-sufficient. The optimal activity of VvRHM-NRS was at pH 7.5 and 30 °C. Apparent Km and Vmax of VvRHM-NRS for UDP-glucose were 88 ± 9 μM and 12.7 ± 0.6 nmol/min/mg, and for NAD+ were 69 ± 7 μM and 11.9 ± 0.5 nmol/min/mg. Then, a novel process for the synthesis of UDP-rhamnose from sucrose, NAD+, and UDP was developed by using VvRHM-NRS coupled with Glycine max sucrose synthase (GmSUS). By optimizing coupled reaction conditions, UDP-rhamnose production reached 0.57 mM. Finally, UDP-rhamnose regeneration system was tested to synthesize quercitrin with A. thaliana glycosyltransferase (AtUGT78D1). Therefore, VvRHM-NRS coupled with GmSUS presented a green chemistry approach for the UDP-rhamnose regeneration in glycosylation of flavonoids and other bioactive substances.

In an attempt to evaluate the effects of thermal treatment on wood cell walls (CWs), Masson pine (Pinus massoniana Lamb.) wood was thermally modified (TM) at 150, 170 and 190°C for 2, 4 and 6 h, respectively. The chemical properties, cellulose crystallinity (C rI) and micromechanics of the control and thermally modified wood (TMW) were analyzed by wet chemical analysis, X-ray diffraction and nanoindentation. The relative lignin content and C rI increased after the TM partly degraded the amorphous wood polymers. The relative lignin content was higher in TMW and the equilibrium moisture content decreased. Moreover, the elastic modulus (E r) and hardness (H) of TMW were lowered along with the creep ratio decrement (C IT) of CWs. However, a severe treatment (e.g. 190°C/6 h) may negatively affect the mechanical properties of CWs caused by the partial degradation of hemicelluloses and also cellulose.

In this article, the methods for detection of enzyme activity and protein concentration are described. The data of the calibration curves can be used for a further understanding on the assays of enzyme activity measured with p-nitrophenyl-β-D-glucopyranoside (pNPG) or cellobiose as the substrate. In addition, the data presented provides an analytic method for measuring protein concentration in mixed samples.

Background Dihydro-β-ionone is a principal aroma compound and has received considerable attention by flavor and fragrance industry. The traditional method of preparing dihydro-β-ionone has many drawbacks, which has restricted its industrial application. Therefore, it is necessary to find a biotechnological method to produce dihydro-β-ionone. Results In this study, the enoate reductase with high conversion efficiency of β-ionone to dihydro-β-ionone, DBR1, was obtained by screening four genetically engineered bacteria. The product, dihydro-β-ionone, was analyzed by GC and GC-MS. The highest dihydro-β-ionone production with 308.3 mg/L was detected in the recombinant strain expressing DBR1 which was later on expressed and purified. Its optimal temperature and pH were 45 °C and 6.5, respectively. The greatest activity of the purified enzyme was 356.39 U/mg using β-ionone as substrate. In the enzymatic conversion system, 1 mM of β-ionone was transformed into 91.08 mg/L of dihydro-β-ionone with 93.80% of molar conversion. Conclusion DBR1 had high selectivity to hydrogenated the 10,11-unsaturated double bond of β-ionone as well as high catalytic efficiency for the conversion of β-ionone to dihydro-β-ionone. It is the first report on the bioconversion of β-ionone to dihydro-β-ionone by using enoate reductase. Electronic supplementary material The online version of this article (10.1186/s12896-018-0438-x) contains supplementary material, which is available to authorized users.

Background β-D-xylosidase is a vital exoglycosidase with the ability to hydrolyze xylooligosaccharides to xylose and to biotransform some saponins by cleaving outer β-xylose. β-D-xylosidase is widely used as one of the xylanolytic enzymes in a diverse range of applications, such as fuel, food and the pharmaceutical industry; therefore, more and more studies have focused on the thermostable and xylose-tolerant β-D-xylosidases. Results A thermostable β-xylosidase gene (xln-DT) of 1509 bp was cloned from Dictyoglomus thermophilum and expressed in E.coli BL21. According to the amino acid and phylogeny analyses, the β-xylosidase Xln-DT is a novel β-xylosidase of the GH family 39. The recombinant β-xylosidase was purified, showing unique bands on SDS-PAGE, and had a protein molecular weight of 58.7 kDa. The β-xylosidase Xln-DT showed an optimal activity at pH 6.0 and 75 °C, with p-nitrophenyl-β-D-xylopyranoside (pNPX) as a substrate. Xln-DT displayed stability over a pH range of 4.0-7.5 for 24 h and displayed thermotolerance below 85 °C. The values of the kinetic parameters Km and Vmax for pNPX were 1.66 mM and 78.46 U/mg, respectively. In particular, Xln-DT displayed high tolerance to xylose, with 60% activity in the presence of 3 M xylose. Xln-DT showed significant effects on the hydrolyzation of xylobiose. After 3 h, all the xylobiose tested was degraded into xylose. Moreover, β-xylosidase Xln-DT had a high selectivity for cleaving the outer xylose moieties of natural saponins, such as notoginsenoside R1 and astragaloside IV, which produced the ginsenoside Rg1 with stronger anti-fatigue activity and produced cycloastragenol with stronger anti-aging activity, respectively. Conclusion This study provides a novel GH 39 β-xylosidase displaying extraordinary properties of highly catalytic activity at temperatures above 75 °C, remarkable hydrolyzing activity of xylooligosaccharides and rare saponins producing ability in the pharmaceutical and commercial industries. Electronic supplementary material The online version of this article (10.1186/s12896-018-0440-3) contains supplementary material, which is available to authorized users.

Aim and Objective: EGb761, a standardized and well-defined product extract of Ginkgo biloba leaves, has beneficial role in the treatment of multiple diseases, particularly Alzheimer's disease (AD). Identification of natural acetylcholinesterase (AChE) inhibitors from EGb761 would provide a novel therapeutic approach against the Alzheimer's disease. Material and Method: A series of 21 kinds of promising EGb761 compounds were selected, and subsequently evaluated for their potential ability to bind AChE enzyme by molecular docking and a deep analysis of protein surface pocket features. Results: Docking results indicated that these compounds can bind tightly with the active site of human AChE, with favorable distinct interactions around several important residues Asp74, Leu289, Phe295, Ser293, Tyr341, Trp286 and Val294 in the active pocket. Most EGB761 compounds could form the hydrogen bond interactions with the negatively charged Asp74 and Phe295 residues. Among these compounds, diosmetin is the one with the best-predicted docking score while three key hydrogen bonds can be formed between small molecule and corresponding residues of the binding site. Besides, other three compounds luteolin, apigenin, and isorhamnetin have better predicted docking scores towards AChE than other serine proteases, i.e Elastase, Tryptase, Factor XA, exhibiting specificity for AChE inhibition. The RMSD and MM-GBSA results from molecular dymamic simulations indicated that the docking pose of diosmetin-AChE complex displayed highly stable, which can be used for validating the accuracy of molecular docking study. Subsequently, the AChE inhibitory activities of these compounds were evaluated by the Ellman's colorimetric method. Conclusion: The obtained results revealed that all the four compounds exhibited modest AChE inhibitory activity, among which Diosmetin manifested remarkable anti-AChE activity, comparable with the reference compound, Physostigmine. It can be deduced that these EGB761 compounds can be regarded as a promising starting point for developing AChE inhibitors against AD.

To improve the quality of Ginkgo biloba leaves as biological feed additives, twelve Aspergillus niger strains were evaluated for their growth in the moisture ginkgo leaf meal media through solid-state fermentation. The results relating to flavor, flavonoids, enzymes, crude protein, and reducing sugars showed A. niger Gyx086 strain was capable of efficiently fermenting ginkgo leaves. The optimal cultural conditions were three loops of spores inoculation to every 75 g medium containing 60 % water, grew at 28˚C for 48 h. The Gyx086 grew well in the medium. The fermented leaves generated a strong sweet-smelling odor, could be identified by electronic nose equipment using a cluster analysis, other than the original offensive smell from non-fermented ginkgo leaves. Each gram dried culture with Gyx086 showed 2.83 × 109 CFU of A. niger; 3.19 ± 0.37 FPU of acid-resistant filter paper activity. Its total contents of flavonoids, reducing sugars, and crude proteins were 19.95 ± 0.23 mg, 24.28 ± 2.35 mg, and 162.81 ± 3.46 mg in each gram of leaves, 26.03 %, 62.73 %, and 14.58 % higher than the controls, respectively. The essential amino acids and total amino acids contents were 96.41 % and 16.49 % higher than the controls.

The discovery of mammalian target of rapamycin (mTOR) kinase inhibitors has always been a research hotspot of antitumor drugs. Consensus scoring used in the docking study of mTOR kinase inhibitors usually improves hit rate of virtual screening. Herein, we attempt to build a series of consensus scoring models based on a set of the common scoring functions. In this paper, twenty-five kinds of mTOR inhibitors (16 clinical candidate compounds and 9 promising preclinical compounds) are carefully collected, and selected for the molecular docking study used by the Glide docking programs within the standard precise (SP) mode. The predicted poses of these ligands are saved, and revaluated by twenty-six available scoring functions, respectively. Subsequently, consensus scoring models are trained based on the obtained rescoring results by the partial least squares (PLS) method, and validated by Leave-one-out (LOO) method. In addition, three kinds of ligand efficiency indices (BEI, SEI, and LLE) instead of pIC50 as the activity could greatly improve the statistical quality of build models. Two best calculated models 10 and 22 using the same BEI indice have following statistical parameters, respectively: for model 10, training set R2 = 0.767, Q2 = 0.647, RMSE = 0.024, and for test set R2 = 0.932, RMSE = 0.026; for model 22, raining set R2 = 0.790, Q2 = 0.627, RMSE = 0.023, and for test set R2 = 0.955, RMSE = 0.020. These two consensus scoring model would be used for the docking virtual screening of novel mTOR inhibitors.

Glycosylation and methylation of flavonoids are the main types of structural modifications and can endow flavonoids with greater stability, bioactivity, and bioavailability. In this study, five types of O-methyltransferases were screened for producing O-methylated luteolin, and the biosynthesis strategy of 3’-O-methylisoorientin from luteolin was determined. To improve the production of 3’-O-methylluteolin, the S-adenosyl- l -methionine synthesis pathway was reconstructed in the recombinant strain by introducing S-adenosyl- l -methionine synthetase genes. After optimizing the conversion conditions, maximal 3’-O-methylluteolin production reached 641 ± 25 mg/L with a corresponding molar conversion of 76.5 %, which was the highest titer of methylated flavonoids reported to date in Escherichia coli. 3’-O-Methylluteolin (127 mg) was prepared from 250 mL of the broth by silica gel column chromatography and preparative HPLC with a yield of 79.4 %. Subsequently, we used the biocatalytic cascade of Gentiana triflora C-glycosyltransferase (Gt6CGT) and Glycine max sucrose synthase (GmSUS) to biosynthesize 3’-O-methylisoorientin from 3’-O-methylluteolin in vitro. By optimizing the coupled reaction conditions and using the fed-batch operation, maximal 3’-O-methylisoorientin production reached 226 ± 8 mg/L with a corresponding molar conversion of 98 %. Therefore, this study provides an efficient method for the production of novel 3’-O-methylisoorientin and the biosynthesis strategy for methylated C-glycosylation flavonoids by selective O-methylation/C-glycosylation motif on flavonoids.

Summary A novel α-L-rhamnosidase was isolated and purified from Aspergillus oryzae NL-1. The enzyme was purified 13.2-fold by ultrafiltration, ion exchange and gel filtration chromatography with an overall recovery of 6.4% and specific activity of 224.4 U/mg, and the molecular mass of its subunit was approximately 75 kDa. Its optimal temperature and pH were 65 °C and 4.5, respectively. The enzyme was stable in the pH range 3.5–7.0, and it showed good thermostability at higher temperatures. The KM, kcat and kcat/KM values were 5.2 mm, 1624 s−1 and 312 s−1 mm−1 using pNPR as substrates, respectively. Moreover, the enzyme exhibited transglycosylating activity, which could synthesise rhamnosyl mannitol through the reactions of transglycosylation with inexpensive rhamnose as the glycosyl donor. Our findings indicate that the enzyme has potential value for glycoside synthesis in the food industry.

Hyperoside exhibits many biological properties and is more soluble in water than quercetin. A uridine 5′-diphosphate (UDP) galactose regeneration system and one-pot synthesis of hyperoside was described herein. Glycine max sucrose synthase (GmSUS) was coupled with Escherichia coli UDP-galactose 4-epimerase (GalE) to regenerate UDP-galactose from sucrose and UDP. Petunia hybrida glycosyltransferase (PhUGT) with high activity toward quercetin was used to synthesize hyperoside via the UDP-galactose regeneration system. The important factors for optimal synergistic catalysis were determined. Through the use of a fed-batch operation, the final titer of hyperoside increased to 2134 mg/L, with a corresponding molar conversion of 92% and maximum number of UDP-galactose regeneration cycles (RCmax) of 18.4 under optimal conditions. Therefore, the method described herein for the regeneration of UDP-galactose from UDP and sucrose can be widely used for the glycosylation of flavonoids and other bioactive substances.

Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal activity was at pH 7.0 and 45 &deg, C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 &deg, C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 &mu, M, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 &deg, C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids.

Ginkgo tea is a kind of health food produced from Ginkgo biloba leaves. The market of Ginkgo tea encountered many difficulties because of its bad palatability and vague function statement. In this study, two kinds of glycosidase were used to improve the flavor of Ginkgo tea, and three kinds of bioactivities were selected to investigate the health care function of the tea infusion. The aroma components extracted by headspace absorb (HSA) method during the making of Ginkgo tea were analyzed by GC-MS. The flavonoids and ginkgolides released into the tea infusion were studied by HPLC. A combination of &beta, glucosidase (&beta, G) and &alpha, rhamnosidase (&alpha, R) was applied during the making of the tea. The contents of characteristic aroma components and the release of total flavonoids and ginkgolides were increased significantly by adding &beta, G and &alpha, R. The composition of flavone glycosides was changed greatly. The free radical scavenging, inhibition of inflammatory cell activation, and tumor cytotoxicity activities of the tea were demonstrably improved. According to the release of active components, Ginkgo tea can be brewed repeatedly for at least three times. The enzymes used here show potential application prospects in the making of Ginkgo tea or tea drink to get higher contents of flavonoids, ginkgolides, and aroma components.

A novel β-xylosidase Dt-2286 from Dictyoglomus turgidum was cloned and overexpressed in Escherichia coli BL21 (DE3). Dt-2286 belonging to glycoside hydrolase (GH) family 3 encodes a polypeptide with 762 amino acid residues with a molecular weight of 85.1 kDa. By optimization of the growth and induction conditions, the activity of β-xylosidase reached 273 U/mL, which is the highest yield reported to date from E. coli in a shake-flask. The optimal activities of the purified Dt-2286 were found at pH 5.0 and 98 °C. It also shows excellent thermostable/haloduric/organic solvent-tolerance. Dt-2286 was revealed to be a multifunctional enzyme with β-xylosidase, α-arabinofuranoside, α-arabinopyranoside and β-glucosidase activities, and Kcat/Km was 5245.316 mM−1 s−1, 2077.353 mM−1 s−1, 1626.454 mM−1 s−1, and 470.432 mM−1 s−1 respectively. Dt-2286 showed significant synergistic effects on the degradation of xylans, releasing more reduced sugars (up to 15.08 fold) by simultaneous addition with endoxylanase. Moreover, this enzyme has good activity in the hydrolysis of epimedium B, demonstrating its versatility in practical applications.

In this study, a recombinant carotenoid cleavage dioxygenase 4 was produced from Osmanthus fragrans by E . coli under different bacterial growth conditions and used to develop a biotechnological method for preparation of natural β-ionone from β-carotene. β-ionone was analyzed by HPLC and OfCCD4 activity was measured based on concentration change of β-ionone. At pH 8.0 and 35 °C, the greatest activity of the purified recombinant protein was 14.3 U/mg and the maximum concentration of β-ionone was 71.186 mg/L within 1 h. Both the enzyme activity and the concentration of β-ionone could increase by nearly 6 times with addition of 9% Triton X-100 and 2% liquid paraffin.

Astragalin (kaempferol 3-O-glucoside) is used as a standard to assess the quality of Radix astragali and has exhibited a number of biological properties. In this work, we screened several UDP-dependent glycosyltransferases (UGT) for their potential as efficient biocatalysts for astragalin synthesis. The highest astragalin production with 285 mg/L was detected in the recombinant strain expressing UGT from Arabidopis thaliana (AtUGT78D2). To further improve astragalin production, an efficient UDP-glucose synthesis pathway was reconstructed in the recombinant strain by introducing sucrose permease, sucrose phosphorylase, and uridylyltransferase. On the basis of those results, a recombinant strain, BL21-II, was constructed to produce astragalin. By optimizing conversion conditions, astragalin production was increased from 570 to 1708 mg/L. The production was scaled up using a fed-batch fermentation, and maximal astragalin production was 3600 mg/L, with a specific productivity of 150 mg/L/h after 24 h incubation and a corresponding molar conversion of 91.9%, the highest yield reported to date.

Although ginkgolic acids (GAs) are identified as allergenic components in the leaves and seed coat of ginkgo biloba, it is widely revealed that GAs can exhibit various promising biological activities such as anti-tumor activity and antibacterial activity. Thus, investigating molecular targets underlying biological activities of GAs can contribute to the further development and utilization of GAs. In this paper, five kinds of common GAs were prepared for the large-scale reverse docking experiment. The results obtained by network analysis displayed that GAs can directly target at least four important druggable proteins: peroxisome proliferator-activated receptor (PPARA, PPARD, and PPARG), and dihydroorotate dehydrogenase (DHODH). Subsequently, molecular dynamics (MD) simulations and MM/GBSA calculations demonstrated that all the GAs can bind tightly with DHODH thermodynamically, implying that DHODH was probably considered as one of potential protein targets of GAs. Given the clinical significance of DHODH, these bioactive molecules (GAs) could serve as promising therapeutic agents against acute myeloid leukemia (AML) or the starting scaffold for the development of natural DHODH inhibitors.

The overexpressing ABCB1 transporter is one of the key factors leading to multidrug resistance (MDR). Thus, many ABCB1 inhibitors have been found to be able to overcome ABCB1-mediated MDR. However, some inhibitors also work as a substrate of ABCB1, which indicates that in order to achieve an effective reversal dosage, a higher concentration is needed to overcome the pumped function of ABCB1, which may concurrently increase the toxicity. WYE-354 is an effective and specific mTOR (mammalian target of rapamycin) inhibitor, which recently has been reported to reverse ABCB1-mediated MDR. In the current study, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to determine the cell viability and reversal effect of WYE-354 in parental and drug-resistant cells. Drug accumulation was performed to examine the effect of WYE-354 on the cellular accumulation of chemotherapeutic drugs. The ATPase (adenosine triphosphatase) activity of the ABCB1 transporter in the presence or absence of WYE-354 was conducted in order to determine the impact of WYE-354 on ATP hydrolysis. Western blot analysis and immunofluorescence assay were used to investigate the protein molecules related to MDR. In addition, the interaction between the WYE-354 and ABCB1 transporter was investigated via in silico analysis. We demonstrated that WYE-354 is a substrate of ABCB1, that the overexpression of the ABCB1 transporter decreases the efficacy of WYE-354, and that the resistant WYE-354 can be reversed by an ABCB1 inhibitor at a pharmacological achievable concentration. Furthermore, WYE-354 increased the intracellular accumulation of paclitaxel in the ABCB1-mediated MDR cell line, without affecting the corresponding parental cell line, which indicated that WYE-354 could compete with other chemotherapeutic drugs for the ABCB1 transporter substrate binding site. In addition, WYE-354 received a high score in the docking analysis, indicating a strong interaction between WYE-354 and the ABCB1 transporter. The results of the ATPase analysis showed that WYE-354 could stimulate ABCB1 ATPase activity. Treatment with WYE-354 did not affect the protein expression or subcellular localization of the ABCB1. This study provides evidence that WYE-354 is a substrate of the ABCB1 transporter, implicating that WYE-354 should be avoided for use in ABCB1-mediated MDR cancer.

In the present study, the performance and separation characteristics of six macroporous resins for the enrichment and purification of total ginkgo flavonoid O-glycosides (TGFs) (quercetin (I), kaempferol (II), isorhamnetin (III)) from Ginkgo Biloba extracts (EGB) are evaluated. The adsorption and desorption properties of TGFs are studied on macroporous resins, including D101, D201, AB-8, HPD400, D301, and D311. Along with the results, AB-8 resin exhibits the best adsorption and desorption capacity for these three ginkgo flavonoid O-glycosides among the six resins. Adsorption isotherms are created on AB-8 resin and fit well to the Langmuir (R2 > 0.96) and Freundlich (R2 > 0.92, 0.3 < 1/n < 0.7) models. After the treatment with gradient elution on AB-8 resin packed chromatography column, the contents of the three main ginkgo flavonoid O-glycosides (I, II, and III) increase from 8.93%, 9.88%, and 6.11% in the extracts to 30.12%, 35.21%, and 14.14%, respectively, in the product. The recoveries of compounds I, II, and III are 88.76%, 93.78%, and 60.90%, respectively. Additionally, the anti-inflammatory effects of TGFs are evaluated in LPS-treated RAW 264.7 macrophages, and the result demonstrates that TGFs could significantly inhibit LPS-induced NO release in vitro in a dose-dependent manner compared with the control group. These findings suggest that TGFs could potentially be natural antioxidants and anti-inflammatory ingredients that could be used in pharmaceutical products and functional food additives.

To comprehend the molecular mechanisms that control the differences in the composition of Osmanthus essential oils, the RNA-seq data and differentially expressed genes in different cultivar Osmanthus were studied. cDNA libraries of "jinqiugui," "baijie," and "rixianggui" were sequenced using Illumina HiSeq TM 2000. All of the enzymes involved in ionone synthesis were verified. DEGs were revealed and their enriched pathways were analyzed. A total of 20 DEGsencoding four enzymes that were potential candidates involved in ionone biosynthesis, as well as ispH, GPPS, ZDS, and CCD. It provided a way for Osmanthus oil monomer material to be synthesized in vitro.

Chlorogenic acid (CGA) has been reported to exhibit potent anti-inflammatory activity. However, the development of anti-inflammatory agent based on CGA has not been investigated. In this paper, a series of caffeoyl salicylate compounds derived from CGA were designed, synthesized, and evaluated by LPS-induced nitric oxide synthase inhibition and QRT-PCR technique. Most compounds showed modest activity to inhibit production of nitric oxide (NO) in RAW 264.7 cells induced by lipopolysaccharides (LPS). Among these compounds, QRT-PCR and western blotting results indicated that compounds 6b, 6c, 6f, 6g and D104 that possess 5-member ring or 6-member ring caused a significant inhibition against expression of the iNOS2 in LPS-induced macrophages. In addition, cytotoxic assay displayed most derivatives have good safety in vitro. This new promising scaffold could be further exploited for the development of anti-inflammatory agent in the future.

The α-L-Rhamnosidase is an important enzyme with applications in the food and pharmaceutical industries because it can release terminal L-rhamnose residues from various natural products. In this study, the B-factor-saturation mutagenesis strategy was used to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus. The D594Q and G827K/D594Q mutant enzymes were obtained by screening a series of mutants; they prolonged the half-life of the enzyme at 70 °C by 2.1-fold and 2.3-fold, respectively. Analysis of the 3D structure showed that in the thermostable variants the number of hydrogen bonds and salt bridges was increased, explaining the enhanced thermostability. Kinetic studies showed that the KM values for the D594Q and G827K/D594Q mutant enzymes decreased by 4.0% and 3.8%, respectively. Additionally, the kcat/KM values for the D594Q and G827K/D594Q mutant enzymes increased by 15.5% and 9.2%, respectively. Moreover, the D594Q and G827K/D594Q mutant enzymes exhibited markedly improved isoquercitrin yield at 70 °C that increased by 13.5% and 11.0%, respectively. Therefore, the D594Q and G827K/D594Q mutant enzymes are more suitable for the industrial processes of isoquercitrin preparation.

Osmanthus fragrans are well-known for their fragrance, but it is wasteful if to discard O. fragrans flower after extracting their essential oils. In this paper, we found that O. fragrans flower residues were rich in flavonoids. Six flavonoids and one phenylethanoid glycoside were isolated from the ethanol extract of O. fragrans flower residues, identified as quercetin (1), rutin (2), verbascoside (3), genistin (4), kaempferol (5), isorhamnetin (6) and naringin (7). In bioactivity study, kaempferol (IC50 = 1.43 μg/mL) showed the best anti-inflammatory activity. Isorhamnetin, quercetin, kaempferol, verbascoside and rutin (the values of IC50 were 18.30, 11.05, 16.88, 20.21 and 22.76 μg/mL, respectively) showed excellent DPPH free radical scavenging activity. Verbascoside performed relatively well at inhibiting the growth of both CT26 colonic carcinoma cells (IC50 = 46.87 μg/mL) and HepG2 hepatocarcinoma cells (IC50 = 30.58 μg/mL). In addition, quercetin and kaempferol showed strong anti-proliferation activity against HepG2 cells.

In the fermentation progress, fermentation parameters including the feed rate, induction temperature, and induction pH evidently regulate the accumulation of acetic acid generated by recombinant E. coli in the medium. The production of thermostable β-glucosidase (Tpebgl3) was increased by optimizing the parameters mentioned step by step. The optimal conditions were obtained with the highest enzyme expression (560.4 U/mL) and the maximum DCW (65 g/L) at the pre-induction specific growth rate of 0.2 h−1 followed by a post-induction specific growth rate (0.18 h−1); induction temperature is 39 °C; the pH is 7.2; the concentration of acetic acid was maintained all along below 0.9 g/L. Results show it is necessary for the synthesis of Tpebgl3 to regulate the accumulation of acetic acid at the premise of feeding to meet the normal growth of E. coli. The production of Tpebgl3 by recombinant E. coli is the highest reported to date.

Background Thermally stable α-L-rhamnosidase with cleaving terminal α-L-rhamnose activity has great potential in industrial application. Therefore, it is necessary to find a proper method to improve the thermal stability of α-L-rhamnosidase. Results In this study, addition of sorbitol has been found to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus at temperatures ranging from 65 °C to 80 °C. Half-life and activation free energy with addition of 2.0 M sorbitol at 70 °C were increased by 17.2-fold, 8.2 kJ/mol, respectively. The analyses of the results of fluorescence spectroscopy and CD have indicated that sorbitol helped to protect the tertiary and secondary structure of α-L-rhamnosidase. Moreover, the isoquercitrin yield increased from 60.01 to 96.43% with the addition of 1.5 M of sorbitol at 70 °C. Conclusion Our findings provide an effective approach for enhancing the thermostability of α-L-rhamnosidase from Aspergillus terreus, which makes it a good candidate for industrial processes of isoquercitrin preparation. Electronic supplementary material The online version of this article (doi:10.1186/s12896-017-0342-9) contains supplementary material, which is available to authorized users.

It has been previously demonstrated that laccases exhibit great potential for use in several industrial and environmental applications. In this paper, two laccase isoenzyme genes, lccB and lccC, were cloned and expressed in Pichia pastoris GS115. The sequence analysis indicated that the lccB and lccC genes consisted of 1,563 and 1,584 bp, and their open reading frames encoded 520 and 527 amino acids, respectively. They had 72.7% degree of identity in nucleotides and 86.7% in amino acids. The expression levels of LccB and LccC were up to 32,479 and 34,231 U/l, respectively. The recombinant laccases were purified by ultrafiltration and (NH4)2SO4 precipitation, showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimal pH and temperature for LccB were 2.0 and 55°C with 2,2'-azino-bis-[3-ethylbenzthiazolinesulfonic acid (ABTS) as a substrate, whereas LccC exhibited optimal pH and temperature at 3.0 and 60°C. The apparent kinetic parameters of LccB were 0.43 mM for ABTS with a Vmax value of 51.28 U/mg, and the Km and Vmax values for LccC were 0.29 mM and 62.89 U/mg. The recombinant laccases were able to decolorize five types of dyes. Acid Violet 43 (100 g/ml) was completely decolorized by LccB or LccC (2 U/ml), and the decolorization of Reactive Blue KN-R (100 g/ml) was 91.6% by LccC (2 U/ml). Thus, the study characterizes useful laccase isoenzymes from T. versicolor that have the capability of being incorporated into the treatment of similar azo and anthraquinone dyes from dyeing industries.

SUMMARY In this study, our objective was to compare the effects of sulfated Agrocybe chaxingu polysaccharide (saCP) and nonsulfated aCP (naCP) on lymphocyte proliferation in vitro and immune-enhancing activity in broiler chicks. In the in vitro test, 3 saCP with different degrees of sulfation—saCP0.83, saCP1.32, and saCP 1.79—were used to study the peripheral lymphocyte proliferation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay with naCP as the control. On the basis of this test, saCP 1.32 and saCP 1.79 were selected for the in vivo test. In the in vivo test, 300 one-day-old chicks were randomly divided into 5 groups (negative control, vaccine control, naCP, saCP 1.32, and saCP 1.79). Chicks in the vaccine control and aCP groups were vaccinated with newcastle disease (nD) vaccine at 10 d of age, with a booster dose at 21 d of age. at the time of the first vaccination, the chicks in the 3 aCP groups were injected with naCP, saCP 1.32, and saCP 1.79, whereas those in the vaccine control group were injected with 0.5 ml of physiological saline once a day for 3 successive days. In comparison to naCP, saCP 1.32 and saCP 1.79 significantly increased the relative weight of the thymus and spleen at 22 d of age, enhanced serum nD antibody titers at 16, 22, 28, 34, and 42 d of age, and enhanced splenic t- and B-lymphocyte proliferation at 22 and 28 d of age. Furthermore, saCP 1.32 and saCP 1.79 could induce significant augmented levels of interferon-γ and Il-6. Sulfation could enhance both cellular and humoral immunity in broiler chicks on a degrees of sulfation basis. the saCP 1.79 exerted the best efficacy and would be expected to be immunopotentiator molecule during nD vaccination in broilers.