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The root of Panax notoginseng (Burk.) F.H. is one of the most famous natural medicines and functional foods for numerous diseases. The saponin constituents present in Panax notoginseng leaves (PNGL) demonstrate an elevated level of glycosylation in comparison to those in the roots, resulting in a significantly diminished bioavailability. In this study, we systematically screened nine recombinant thermostable glycosidases and identified a synergistic combination, namely Tpexy3 and Tpebgl3, to catalyze PNGL. At 70 °C, 2000 U of both glycosidases were added before and after 1 g of PNGL, yielding 650 mg of new fractions, of which ginsenosides Rg3, F2, CK, and PPD were identified as the major products. The enzymatic PNGL demonstrated a lipid droplet-lowering effect on the 3 T3-L1 lipid deposition model. Furthermore, the enzymatic PNGL exhibited significant inhibitory effects on weight gain, hyperlipidemia, hyperglycemia, and inflammation in high-fat diet (HFD) mice. Importantly, the enzymatic PNGL restored the HFD-induced increase in the Firmicutes/Bacteroidetes (F/B) ratio and promoted the growth of beneficial gut microbes with anti-inflammatory properties, improved lipid metabolism, and short-chain fatty acid production. These results strongly indicate that enzyme-catalyzed pretreatment of PNGL can effectively enhance its bioavailability and anti-obesity effects. Biological enzyme catalysis not only provides technical support for the development of specific functional products but also promotes the efficient and high-value utilization of natural product resources.

Abstract Industrial application of icariin and baohuoside I has been hindered by the short supply to a great extent. In this work, a novel GH78 α- l-rhamnosidase AmRha catalyzed the bioconversion of low-value epimedin C in crude Epimedium Folium flavonoids (EFs) to icariin and baohuoside I was developed. Firstly, the high-level expression of AmRha in Komagataella phaffii GS115 attained an enzyme activity of 571.04 U/mL. The purified recombinant AmRha could hydrolyze α-1,2-rhamnoside bond between two rhamnoses (α-Rha(2 → 1)α-Rha) in epimedin C to produce icariin with a molar conversion rate of 92.3%, in vitro. Furtherly, the biotransformation of epimedin C to icariin by the recombinant Komagataella phaffii GS115 cells was also investigated, which elevated the EFs concentration by fivefold. In addition, biotransformation of epimedins A-C and icariin in the raw EFs to baohuoside I was fulfilled by a collaboration of AmRha and β-glucosidase/β-xylosidase Dth3. The results obtained here provide a new insight into the preparation of high-value products icariin and baohuoside I from cheap raw EFs.

It is generally believed that there is a great relationship between hangover and the age of Baijiu. However, what factors make Baijiu (stored for a long time) feel better after drinking has not been well explained. In this study, ethanol metabolism, oxidation stress, inflammation and release of inhibitory neurotransmitter were selected as the indicator of hangover. The results showed that the longer the age of Baijiu, the higher the antioxidant and anti-inflammatory levels, the less damage to the liver of mice. In addition, we also found that the longer the age of Baijiu, the faster the ethanol metabolism rate, the smaller the impact on the brain. A correlation analysis on Baijiu ingredients and hangover related indicators was conducted. These results showed that ethyl acetate, n-butanol, n-hexanol, butyl acetate, ethyl octanoate, isovaleric acid, 2-hydroxypropionic acid had a great correlation with all hangover related indicators.

ABSTRACTObjective: Hydroxytyrosol (HT) is a polyphenol with a wide range of biological activities. Excessive drinking can lead to oxidative stress and inflammation in the liver, which usually develop into alcohol liver disease (ALD). At present, there is no specific drug to treat ALD. In this paper, the protection effect of HT on ALD and the underline mechanism were studied.Methods: HepG2 cells were exposed to ethanol in vitro and C57BL/6J mice were fed with a Lieber-DeCarli ethanol liquid diet in vivo.Results: triglyceride (TG) level in serum and the expression of fatty acid synthase (FASN) were reduced significantly by the treatment with HT The acetaldehyde dehydrogenase (ALDH) activity was increased, the serum level of malondialdehyde (MDA) was decreased, catalase (CAT) and glutathione (GSH) were increased, suggesting that HT may reduce its oxidative damage to the body by promoting alcohol metabolism. Furthermore, according to the mRNA levels of tnf-α, il-6 and il-1β, HT inhibited ethanol-induced inflammation significantly. The anti-inflammatory mechanism of HT may be related to suppress the STAT3/iNOS pathway.Dissussion: Our study showed that HT could ameliorate ethanol-induced hepatic steatosis, oxidative stress and inflammation and provide a new candidate for the prevention and treatment of ALD.

Abstract Background C-glycosylated flavonoids are a main type of structural modification and can endow flavonoids with greater stability, bioactivity, and bioavailability. Although some C-glycosylated flavonoids have been biosynthesized in vivo or vitro, only a few C-glycosylflavonols have been prepared by these methods. Results In this study, several uridine 5’-diphosphate (UDP)-glucose biosynthesis pathways and Escherichia coli hosts were screened to reconstruct recombinant strains for producing the novel C-glycosylflavonols kaempferol 8-C-glucoside and quercetin 8-C-glucoside. To increase C-glycosylflavonol production, the timing of flavonol addition was adjusted, and glycerol was added to avoid degradation of C-glycosylflavonols. By using resting cell bioconversion, the highest kaempferol 8-C-glucoside and quercetin 8-C-glucoside production reached 16.6 g/L and 12.5 g/L, respectively. Then, ultrasound-assisted adsorption/desorption was used to prepare C-glycosylflavonols by using macroporous resins. Through screening macroporous resins and optimizing the adsorption/desorption conditions, the highest adsorption capacity and desorption capacity for kaempferol 8-C-glucoside on HPD100 reached 28.57 mg/g and 24.15 mg/g, respectively. Finally, kaempferol 8-C-glucoside (15.4 g) with a yield of 93% and quercetin 8-C-glucoside (11.3 g) with a yield of 91% were obtained from 1 L of fermentation broth. Conclusions Kaempferol 8-C-glucoside and quercetin 8-C-glucoside are novel C-glycosylflavonols, which have not been extracted from plants. This study provides an efficient method for the preparation and biocatalytic synthesis of kaempferol 8-C-glucoside and quercetin 8-C-glucoside by metabolic engineering of Escherichia coli. Graphical Abstract

There are some differences in the anti-inflammatory activities of four typical components in EGB (extracts of ginkgo biloba leaves), and there is also a synergistic relationship. The order of inhibiting the NO-release ability of single functional components is OA > GF > OPC > G. Ginkgolide (G), proanthocyanidins (OPC), and organic acids (OA) all have synergistic effects on ginkgo flavonoids (GF). GF:OA (1:9) is the lowest interaction index among all complexes, showing the strongest synergy. The anti-inflammatory mechanism of the compound affects the expression of p-JNK, p-P38, and p-ERK1/2 proteins by inhibiting the expression of iNOS and COX2 genes on NFKB and MAPK pathways. This also provides a research basis for the development of anti-inflammatory deep-processing products of EGB.

It has been reported that Celtis sinensis Pers. is employed as a folk medicine for the treatment of inflammatory diseases. But the mechanism supporting its use as anti-inflammatory remains unclear. To investigate the anti-inflammatory of Celtis sinensis Pers. ICR mice were provided Celtis sinensis Pers. leaf extract (CLE) at 100, 200 mg/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, 20 mg/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 inhibited 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.

As is well-known, endo-1,4-β-xylanase and β-xylosidase are the rate-limiting enzymes in the degradation of xylan (the major hemicellulosic component), main functions of which are cleavaging xylan to release xylooligosaccharides (XOS) and xylose that these two compounds have important application value in fuel, food, and other industries. This study focuses on enzymatic hydrolysis of poplar sawdust xylan for production of XOS and xylose by a GH11 endo-1,4-β-xylanase MxynB-8 and a GH39 β-xylosidase Xln-DT. MxynB-8 showed excellent ability to hydrolyze hemicellulose of broadleaf plants, such as poplar. Under optimized conditions (50°C, pH 6.0, dosage of 500 U/g, substrate concentration of 2 mg/mL), the final XOS yield was 85.5%, and the content of XOS2−3 reached 93.9% after 18 h. The enzymatic efficiency by MxynB-8 based on the poplar sawdust xylan in the raw material was 30.5%. Xln-DT showed excellent xylose/glucose/arabinose tolerance, which is applied as a candidate to apply in degradation of hemicellulose. In addition, the process and enzymatic mode of poplar sawdust xylan with MxynB-8 and Xln-DT were investigated. The results showed that the enzymatic hydrolysis yield of poplar sawdust xylan was improved by adding Xln-DT, and a xylose-rich hydrolysate could be obtained at high purity, with the xylose yield of 89.9%. The enzymatic hydrolysis yield was higher (32.2%) by using MxynB-8 and Xln-DT together. This study provides a deep understanding of double-enzyme synergetic enzymolysis of wood polysaccharides to valuable products.

Curcumin is a polyphenolic natural product that has promising anticancer properties. However, its clinical utility is limited by its chemical instability and poor metabolic properties. In this paper, a series of new curcumin analogs were synthesized and found to be potent antiproliferative agents against the HepG2 cell line by MTT assay. In general, Group B with single ketone and group C with chalcone were markedly more cytotoxic than group A with diketone. Compound B5 was found as the most potent analog (IC50 = 11.33 μM) compared to curcumin (IC50 = 32.83 μM) and the mechanism of its cytotoxicity was investigated. The result of the wound healing assay indicated B5 strong potential to suppress HepG2 cell migration in a dose- and time-dependent manner. Subsequent assays (including JC-1 staining, Bcl-2, and caspase 3 protein levels by Western blotting) confirmed that B5 exposure induced apoptosis in HepG2 cells. Curcumin-induced comprehensive transcriptomes profile, Western blotting, molecular docking, and molecular dynamics analysis showed that the mechanism may relate to the regulation of cellular metabolic process and the expression of AKT protein. Taken together, we could conclude that curcumin and its analogs induced HepG2 cell proliferation, migration, and apoptosis via AKT signaling pathway and the mitochondrial death pathway. This study could lay the foundation for optimizing curcumin and provide valuable information for finding novel anti-HCC drugs.

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.

The husks of Xanthoceras sorbifolia Bunge (X. sorbifolia), as by-products of industrial production, have brought a severe burden to the environment and caused an enormous waste of resources. Bioactive triterpenoid saponins are rich in the husks. To reuse the husks and gain high-quality saponin products, saponin-oriented deep eutectic solvents (DESs), as an efficient and selective extraction strategy from X. sorbifolia husks, were designed for the first time. The enhancement of the extraction rate was investigated by screening solvents from acidic DESs and response surface methodology (RSM) optimization. As a result, the tetrapropylammonium bromide-lactic acid (TPMBr-La) was the most efficient DESs, with an extraction efficiency of up to 135% higher than 70% ethanol. A maximum extraction rate of 72.11 ± 0.61 mg Re/g dw was obtained under the optimized parameters. Scanning electron microscope graphs revealed that damage to the microstructure caused by DESs enhanced the extraction efficiency. Moreover, the recovery of total saponins with D101 macroporous resin was consistent with the pseudo-second-order kinetic model. Seven saponins were also identified by HPLC-MS analysis. Finally, TPMBr-La extracts exhibited 92.30 ± 1.10% DPPH radical scavenging rate at 100 μg/mL, and 92.20 ± 0.30% ABTS radical scavenging rate at 1200 μg/mL. Our current research proposes a selective and high-efficiency substitute for the extraction of saponins and might contribute to further DESs application in the recycling of by-products.

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.

Abstract 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.

Abstract 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 K m and V max 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.

The predicted anti-oxidation is related to apoptosis, proliferation, lipid metabolism, cell differentiation, and immune response. There are some differences in the antioxidant capacity of the four typical components of ginkgo biloba extract (EGb) including ginkgo flavone (GF), ginkgolide (G), procyanidins (OPC), and organic acids (OA), and any two members of them can exhibit apparent synergistic effects. The order of DPPH scavenging ability was: OPC > GF > OA > G. The scavenging ability of procyanidins was close to that of VC; the scavenging capacity of ABTS was GF > OPC > OA > G. The GF:OPC (1:9) showed the best synergism in scavenging DPPH and ABTS radicals. The 193 kinds of small molecules reported in EGb were obtained by analyzing the properties of EGb. In order to construct a corresponding biological activity target set, molecular docking and the network pharmacology method were employed to build the molecular action mechanism network of a compound target, and the main biological functions and signaling pathways involved with their antioxidant activities were predicted. The results displayed that the top ten compounds which belonged to the two broad categories, ginkgo flavonoids and proanthocyanidins, could interact closely with several important target proteins (CASP3, SOD2, MAPK1, HSPA4, and NQO1). This would be expected to lay a theoretical foundation for the deep development of Ginkgo biloba extract.

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.

Ginkgo biloba extract (EGB) has many pharmacological activities. In the quality standard of EGB, the main quality control indexes are total flavone (content ≥ 24%) and total lactone (content ≥ 6%). There are no specific limits for nearly 70% of “other components”. 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’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’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’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.

Kaempferol (kae) and its glycosides are widely distributed in nature and show multiple bioactivities, yet few reports have compared them. In this paper, we report the antitumor, antioxidant and anti-inflammatory activity differences of kae, kae-7-O-glucoside (kae-7-O-glu), kae-3-O-rhamnoside (kae-3-O-rha) and kae-3-O-rutinoside (kae-3-O-rut). Kae showed the highest antiproliferation effect on the human hepatoma cell line HepG2, mouse colon cancer cell line CT26 and mouse melanoma cell line B16F1. Kae also significantly inhibited AKT phosphorylation and cleaved caspase-9, caspase-7, caspase-3 and PARP in HepG2 cells. A kae-induced increase in DPPH and ABTS radical scavenging activity, inhibition of concanavalin A (Con A)-induced activation of T cell proliferation and NO or ROS production in LPS-induced RAW 264.7 macrophage cells were also seen. Kae glycosides were used to produce kae via environment-friendly enzymatic hydrolysis. Kae-7-O-glu and kae-3-O-rut were hydrolyzed to kae by β-glucosidase and/or α-L-rhamnosidase. This paper demonstrates the application of enzymatic catalysis to obtain highly biologically active kae. This work provides a novel and efficient preparation of high-value flavone-related products.

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 °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 °C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 μM, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 °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 β-glucosidase (β-G) and α-rhamnosidase (α-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 β-G and α-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.

To further improve the expression level of recombinant xylanase in Pichia pastoris, the xynB gene, encoding the mature peptide from Aspergillus niger NL-1, was designed and synthesized based on the synonymous condon bias of P. pastoris and optimized G+C content. 155 nucleotides were changed, and the GC content decreased from 57.7% to 43.6%. The synthetic xynB was inserted into the pPICZaA and then integrated into P. pastoris GS115. The activity of the recombinant xylanase reached 1414.7 U/mL, induced with 0.8% methanol after 14-day cultivation at a temperature of 28oC in shake flasks, which was 267% higher than that of the native gene. Furthermore, the maximum xylanase activity of 20424.2 U/mL was obtained by high-density fermentation in a 5-L fermenter, which was the highest xylanase expression in P. pastoris yet reported. The recombinant xylanase had its optimal activity at a pH of 5.0 and temperature of 50oC. The recombinant xylanase was stable over a pH range of 4.5 to 8.0. Thus, this report provides an industrial means to produce the recombinant xylanase in P. pastoris.