Search

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The present study was conducted to investigate the effect of 3 types of fermented Ginkgo-leaves (FGL) on layer performance, egg quality, lipid metabolism, microbial populations, and egg-yolk fatty acids composition in laying hens. A total of 432 Lohmann Brown laying hens, 49 weeks of age, were randomly allocated to 4 dietary treatments with 6 replications of 18 birds each. Layers were fed basal diets (Control) or basal diets supplemented with 0.5% Ginkgo-leaves fermented with Candida utilis (CF group), Aspergillus niger (AF group), or their combined fermentation (CAF group), respectively, for an 8-week feeding trial. Compared with the control group, AF and CAF supplementation improved (P

This paper presents a prefermentation treatment method involving fungi to improve flavonoid extraction from the leaves of Ginkgo biloba . The fungi employed for this treatment were screened from the soil present under an ancient ginkgo tree. Seventy-six strains belonging to 23 genera were isolated and identified by a molecular identification method employing 18S rDNA sequences. Thirty-three strains grew well using ginkgo leaves as the growth medium. One strain, Gyx086, with higher extracted yield of flavonoids and more similar to the control, was finally selected for prefermentation processing. The major fermentation factors were optimized by response surface methodology. The optimal conditions for the highest total falvonoid yield were 27.8 °C for temperature, 64.2% for moisture content, and 61 h for fermentation time. Under the optimal condition, a actual total flavonoid yield of 27.59 ± 0.52 mg/g dry weight culture sample was obtained, which was about 70% higher than that of unfermented gingko leaf samples.

The recombinant strain P. pastoris GS115-lccC was used to produce laccase with high activity. Factors influencing laccase expression, such as pH, methanol concentration, copper concentration, peptone concentration, shaker rotate speed, and medium volume were investigated. Under the optimal conditions, laccase activity reached 12,344 U/L on day 15. The recombinant enzyme was purified by precipitating and dialyzing to electrophoretic homogeneity, and was estimated to have a molecular mass of about 58 kDa. When guaiacol was the substrate, the laccase showed the highest activity at pH 5.0 and was stable when the pH was 4.5~6.0. The optimal temperature for the laccase to oxidize guaiacol was 60°C, but it was not stable at high temperature. The enzyme could remain stable at 30°C for 5 days. The recombinant laccase was used to degrade chlorpyrifos in several laccase/mediator systems. Among three synthetic mediators (ABTS, HBT, VA) and three natural mediators (vanillin, 2,6-DMP, and guaiacol), vanillin showed the most enhancement on degradation of chlorpyrifos. Both laccase and vanillin were responsible for the degradation of chlorpyrifos. A higher dosage of vanillin may promote a higher level of degradation of chlorpyrifos, and the 2-step addition of vanillin led to 98% chlorpyrifos degradation. The degradation of chlorpyrifos was faster in the L/V system (kobs = 0.151) than that in the buffer solution (kobs = 0.028).

A β-glucosidase gene bglI from Aspergillus niger NL-1 was cloned and expressed in Pichia pastoris. The bglI gene consists of a 2583 bp open reading frame encoding 861 amino acids; the enzyme was classified into glycoside hydrolases 3. To improve the expression level of recombinant BGL in P. pastoris, fermentation conditions were optimized by the single-factor experiments. The optimal fermentation conditions were obtained: initial pH 5.0, methanol concentration 0.5% added into the culture every 24 h, and initial cell density (OD600) of 10 for induction. The activity of BGL was increased from 4 U/mL to 45 U/mL in optimal conditions. The BGL was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS-PAGE. The optimal activity was at pH 4.0 and 60°C. The recombinant enzyme was stable over a pH range of 3.0–7.0 and retained more than 85% activity after incubation at 60°C for 30 min. The kinetic experiments revealed Km and Vmax for p-nitrophenyl-β-D-glucoside of 0.64 mM and 370 U/mg, for cellobiose 8.59 mM and 1480 U/mg. The activity of BGL was not or only a little affected by many metal ions and EDTA and was enhanced by methanol or n-butyl alcohol. The BGL had a Ki of 48 mM for glucose and retained 76% activity in the presence of 50 mM glucose. The favorable properties of BGL offer the potential for industrial application.

Aspergillus niger-fermented-Ginkgo biloba leaves (FG) and the comparative effect with nonfermented (NF)-Ginkgo leaves were investigated on morphology, absorption, and immunity of small intestine after lipopolysaccharide (LPS)-challenge in chicks at an early age. Broiler chicks (180 d of age) were divided into 3 treatment groups and were fed 1 of 3 diets: basal diet or basal diet supplemented with 0.5% NF or FG (control, NF, and FG group, respectively). Half of the birds from each treatment group were injected intraperitoneally with LPS (500 μg/kg of BW) or 0.9% NaCl solution (the sham control groups) at 10, 12, 15, 17, 19, and 21 d of age. The results indicated that when LPS-challenged birds were pretreated with FG, the decrease of ADG, ADFI, duodenal and jejunal relative weights, villus height, crypt depth, alkaline phosphatase activity, and plasma d-xylose were dramatically attenuated (P < 0.05 or P < 0.01). Meanwhile, a significant decrease (P < 0.05 or P < 0.01) of duodenal and jejunal interferon-γ, interleukin (IL)-4, IL-13, IL-18, inducible nitric oxide synthase, and duodenal sodium glucose co-transporter 1 mRNA expressional levels were found in LPS-challenged birds pretreated with FG. In conclusion, FG-supplemented diets minimized the deleterious effects of LPS and improved intestinal development, absorption, and immunity in immune-stressed chickens.

The present study was conducted to investigate the influence of dietary supplementation with different levels of fermented Ginkgo biloba leaves (FGBL) on growth performance, slaughter performance, meat quality, antioxidant enzyme capacity, and free radical scavenging activities of muscles in broiler chickens. A total of 648 one-d-old broiler chickens were randomly allocated into six dietary treatments, including control group (CON 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). Body weight gain and feed intake were recorded at 1, 21, and 42 d. At 42 d, 2 birds from each replicate were slaughtered. The results indicated that 3.5 g/kg FGBL diet significantly increased (P 0.05) ADFI and ADG in 1 to 42 d and ADFI in 22 to 42 d compared with the CON group. In 1 to 21 d, 4.5 g/kg FGBL diet improved (P 0.05) ADFI and ADG. With dietary FGBL increasing, the feed: gain ratio (F/G) in 1 to 21 d was significantly decreased (P 0.05). However, birds fed with 5.5 g/kg FGBL had a higher (P 0.05) F/G compared with other groups in 22 to 42 d and 1 to 42 d. In addition, FGBL3 and FGBL4 showed lower (P 0.05) L* value in breast muscle, cooking loss in thigh muscle and lower 24 h and 48 h drip loss in both breast and thigh muscles than those of other groups. Furthermore, birds in the FGBL3 and FGBL4 groups increased (P 0.05) the activity of total superoxide dismutase and total antioxidant capability in muscles, and the scavenging activities of 2,2΄-azino-bis (3-ethylbenzothiazoline-6-sulfonic) acid radical, OH•, and O2•- in thigh muscle, decreased (P 0.05) malondialdehyde concentration in thigh muscle, as compared to the CON group. In conclusion, FGBL had the potential to improve the growth performance, meat quality and antioxidant status of broiler chickens. The optimal dose in the present study of FGBL in broiler diets was from 3.5 to 4.5 g/kg.

Aspergillus niger-fermented Ginkgo biloba leaves (FR) and its comparative effect with non-fermented G. biloba leaves (NF) were investigated on growth performance, small intestinal structure and function of broiler chicks. A total of 300 d-old Arbor Acress broilers were randomly allocated into 5 dietary treatments, which were then denoted as the control group (basal diet), and the NF group (containing 0.35% and 0.7% NF in the starter and grower diets, respectively), FR1, FR2 and FR3 groups containing 0.2%, 0.35% and 0.5% FR in the starter diets and 0.4%, 0.7% and 1.0% FR in the grower diets, respectively. The results showed that, compared with the control and NF groups, reduced (P

Aspergillus niger-fermented Ginkgo biloba leaves (FR) and its comparative effect with vitamin E (VE) and nonfermented (NF) Ginkgo leaves on growth, lipid metabolism, antioxidant capacity, and meat quality of broiler chicks were investigated. In total, 360 one-day-old broiler chicks were randomly allocated into 6 dietary treatments, which were then denoted as control group (basal diet), VE group (containing respectively 15 and 30 IU/kg of all-rac-α-tocopherol acetate in the starter and grower phase), NF group (containing respectively 0.35% and 0.7% NF in the starter and grower phase), and FR1, FR2, and FR3 groups containing respectively 0.2, 0.35, and 0.5% FR in the starter and 0.4, 0.7, and 1.0% FR in the grower phase. The results on performance showed that a significant (P < 0.05) reduction of feed:gain ratio of birds in the FR2 group (22-42 d and 1-42 d) was observed when compared with that of the control and NF groups. With dietary FR increasing, the serum α-tocopherol concentration increased linearly (P = 0.001). Compared with the control, broilers had higher (P < 0.05) serum high-density lipoprotein concentration, total superoxide dismutase activities, and total antioxidant capacity when they were provided with the FR2 and FR3 diet. Whereas the low-density lipoprotein and triglyceride concentrations were lower (P < 0.05 or P < 0.01) in broilers from FR2 or FR3 groups. As the dietary FR increased, abdominal fat (P = 0.002) and muscle malondialdehyde (P = 0.001) concentrations decreased. Furthermore, 24-h pH, 24-h drip loss, and cooking loss were greatly improved (P < 0.05) as the levels of FR increased. Birds fed with FR had a lower (P < 0.05) C16:0 and C18:0 concentrations but a greater (P = 0.001) concentration of C18:2, C18:3, and C20:4 than that of the control. In conclusion, FR can improve the growth performance and lipid metabolism of broilers with decreased abdominal fat deposition. Also, the antioxidant capacity and meat quality improving effects observed in broilers fed FR products might result from the increased retention of α-tocopherol and reduction in lipid peroxidation, as evidenced by the decrease in malondialdehyde and the increase in total superoxide dismutase activities.

The genus Carpinus of Betulaceae is the most widely distributed in the European landscape. This study reports a comparative study based on the pheophorbide a and flavonoid content from the two main species of the genus Carpinus, Carpinus betulus and Carpinus turczaninowii, respectively, in Nanjing, China. The pheophorbide a and flavonoid content depends on the organ, species, and season. HPLC analysis showed that the pheophorbide a and flavonoid levels were the highest in May and June, respectively, from the leaves of C. betulus 'Fastigiata.' In contrast, the content of pheophorbide a and flavonoid in the stems of C. betulus 'Fastigiata' or in other species was low. The immunosuppressive effects of the ethyl acetate extracts and methanol extracts from the two Carpinus species were also evaluated. The ethyl acetate extracts of C. betulus 'Fastigiata' in May and the methanol extracts of C. betulus 'Fastigiata' in June showed better immunosuppressive activity than in other seasons, which coincided with the content of pheophorbide a and flavonoid, respectively. Our findings indicated that C. betulus 'Fastigiata' can serve as a medicinal plant against inflammation because of its pheophorbide a and flavonoid content.

This study reports the combination of Ginkgo flavonoid (GF) andCoriolus versicolorpolysaccharide (CVP) in the prevention and treatment of a mouse model of Alzheimer’s disease (AD). GF is a traditional health product, and CVP is the main active ingredient of the medicinal fungusCoriolus versicolor. The Morris water maze test, the Y maze, and the step-through test showed that the combinational use of CVP and GF synergistically improved memory in a mouse model of AD. Based on H&E staining analysis, the combination of CVP and GF decreased the severity of the pathological findings in the brain. Given that the expression of IL-1β, IL-6, and TNF-αwas downregulated, the inflammation response in AD mice was considered to be inhibited. The downregulation of GFAP further demonstrated that inflammation was reduced in the brain of AD mice following treatment. Moreover, the expression levels of superoxide dismutase (SOD) and catalase (CAT) were elevated in the brains of treated mice, indicating that oxidation levels were reduced upon the combination treatment. Our results provide new insights into the efficient utilization of traditional medicine for preventing dementia.

This study was conducted to investigate the effect of Candida utilis and Aspergillus niger, combined with fermented Ginkgo biloba leaves (FGB) on immunity, antioxidant capability, lipid metabolism, and disease resistance against Aeromonas hydrophila infection in juvenile blunt snout bream (Megalobrama amblycephala). Fish were fed basal diets supplemented with fermented Ginkgo biloba leaves FGB1(0.125%), FGB2(0.25%), FGB3(0.5%), FGB4(1.0%), or Control (0.00%) respectively. At the end of the 60-day feeding trial, fish were challenged by A. hydrophila and mortality rate was recorded for the next 7 days. Results showed that, compared with the control, serum myeloperoxidase (MPO) activity of groups FGB1 and FGB2, alkaline phosphatase (AKP) and lysozyme (LZM) activity of group FGB2 increased significantly (P0.05). After A. hydrophila challenge, the relative mortality of group FGB1 and FGB2 were significantly lower than the control (P

In an attempt to shift the optimal pH of the xylanase B (XynB) from Aspergillus niger towards alkalinity, target mutation sites were selected by alignment between Aspergillus niger xylanase B and other xylanases that have alkalophilic pH optima that highlight charged residues in the eight-residues-longer loop in the alkalophilic xylanase. Multiple engineered XynB mutants were created by site-directed mutagenesis with substitutions Q164K and Q164K+D117N. The variant XynB-117 had the highest optimum pH (at 5.5), which corresponded to a basic 0.5 pH unit shift when compared with the wild-type enzyme. However, the optimal pH of the XynB- 164 mutation was not changed, similar to the wild type. These results suggest that the residues at positions 164 and 117 in the eight-residues-longer loop and the cleft's edge are important in determining the pH optima of XynB from Aspergillus niger.

The complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. But endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation. The glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum β-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in V max /K m in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188. The hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the operational cost of the hydrolysis process.

Background β-Glucosidase is an important component of the cellulase enzyme system. It does not only participate in cellulose degradation, it also plays an important role in hydrolyzing cellulose to fermentable glucose by relieving the inhibition of exoglucanase and endoglucanase from cellobiose. Therefore, the glucose-tolerant β-glucosidase with high specific activity for cellobiose might be a potent candidate for industrial applications. Results The β-glucosidase gene bgl that encodes a 443-amino-acid protein was cloned and over-expressed from Thermoanaerobacterium thermosaccharolyticum DSM 571 in Escherichia coli. The phylogenetic trees of β-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the BGL was a novel β-glucosidase. By replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of bgl was increased from 6.6 to 11.2 U/mg in LB medium. Recombinant BGL was purified by heat treatment followed by Ni-NTA affinity. The optimal activity was at pH 6.4 and 70°C. The purified enzyme was stable over pH range of 5.2–7.6 and had a 1 h half life at 68°C. The activity of BGL was significantly enhanced by Fe2+ and Mn2+. The V max of 64 U/mg and 120 U/mg were found for p-nitrophenyl-β-D-glucopyranoside (K m value of 0.62 mM) and cellobiose (K m value of 7.9 mM), respectively. It displayed high tolerance to glucose and cellobiose. The K cat for cellobiose was 67.7 s-1 at 60°C and pH 6.4, when the concentration of cellobiose was 290 mM. It was activated by glucose at concentrations lower that 200 mM. With glucose further increasing, the enzyme activity of BGL was gradually inhibited, but remained 50% of the original value in even as high as 600 mM glucose. Conclusions The article provides a useful novel β-glucosidase which displayed favorable properties: high glucose and cellobiose tolerance, independence of metal ions, and high hydrolysis activity on cellobiose.

The fungal degradation of toxic alkaloids, papaverine and harmaline, which present in some wild grass, were firstly investigated by using the lignin-degrading basidiomycete Phanerochaete chrysosporium. The fungi could quickly degrade the papaverine and harmaline, especially in the case of P. chrysosporium in Mn-including medium. The nutrient nitrogen concentration had a great effect on the degradation rate of substrates. The papaverine was degraded more effectively under low concentration of nitrogen (LN) condition. But the harmaline could be greatly degraded under both low and high nitrogen concentration. These results strongly suggest that the lignin-degrading basidiomycete Phanerochaete chrysosporium have great potential in degradation of toxic alkaloids of wild grass.

Problems such as agricultural safety and environmental pollution by using chlorpyrifos and other pesticides had become major issues which needed to be solved. The white rot fungi had a unique degradation ability and mechanism to a variety of xenobiotic, which had a good prospect in the bioremediation of the environment. Optimizing the conditions of degrading pesticides chlorpyrifos by using phanerochaete chrysosporium were studied in this work. The optimal culture medium and initial pH value were HCHN and 5.5 respectively. The initial concentration of chlorpyrifos added into the culture could be reach 200mg/L with a highest degradation rate at the range of experimental concentration. In addition, veratryl alcohol and Mn2+ can effectively promote the degradation. The degradation rate of chlorpyrifos could arrive to 90% in the 15 day under optimal conditions. The present works lay the foundation of study on reducing the residual chlorpyrifos in agricultural and forest products and soil bioremediation in the next step.