Linguo Zhao

Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose

Backgroundβ-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.ResultsThe 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 Vmax of 64 U/mg and 120 U/mg were found for p-nitrophenyl-β-D-glucopyranoside (Km value of 0.62 mM) and cellobiose (Km value of 7.9 mM), respectively. It displayed high tolerance to glucose and cellobiose. The Kcat 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.ConclusionsThe 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.

Effect of feeding fermented Ginkgo biloba leaves on growth performance, meat quality, and lipid metabolism in broilers

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.

Effects of dietary supplementation with fermented ginkgo leaves on antioxidant capacity, intestinal morphology and microbial ecology in broiler chicks

Abstract The purpose of this study was to evaluate the effects of supplementing diets with three types of fermented Ginkgo-leaves (FGL) on growth, antioxidant capacity, intestinal morphology and microbial ecology in broiler chicks. A total of 300 d-old broilers were randomly allocated to 4 dietary treatments with 6 replications of 10 birds each. Birds were fed on basal diets (Control) or basal diets supplemented with 0.5% FGL with Candida utilis (CF group), Aspergillus niger (AF group) or their combined fermentation (CAF group), respectively, for a 42 d feeding trial. AF and CAF supplementation improved body weight gain (BWG) (22–42 d) and feed conversion ratio (22–42 d and 1–42 d). Concentrations of serum α-tocopherol in CAF group, as well as hepatic α-tocopherol in the three FGL groups were increased, while hepatic reactive oxygen species (ROS) levels were greatly decreased in group AF and CAF. Chickens in AF and CAF groups had decreased hepatic protein carbonyls and malondialdehyde (MDA), as well as jejunal and ileal protein carbonyls. The total superoxide dismutase (T-SOD) activities and glutathione (GSH) of both jejunum and ileum of the CAF group were higher than the other groups. Duodenal and jejunal villous height of birds fed on the AF and CAF diets were increased, while jejunal crypt depth (CD) was decreased. Furthermore, birds fed on AF and CAF supplemented diets had increased ileal lactobacilli populations. Decreased ileal and caecal Escherichia coli and Salmonellas populations was found for the birds fed on CAF supplemented diets. The present study may indicate that the improved feed efficiency and intestinal functions in the group supplemented with AF and CAF are directly connected with the improved antioxidant capacity and intestinal microbial ecology.

Overexpression and characterization of laccase from Trametes versicolor in Pichia pastoris

AbstarctA laccase-encoding gene of Trametes versicolor, lccA, was cloned and expressed in Pichia pastoris X33. The lccA gene consists of a 1560 bp open reading frame encoding 519 amino acids, which was classified into family copper blue oxidase. To improve the expression level of recombinant laccase in P. pastoris, conditions of the fermentation were optimized by the single factor experiments. The optimal fermentation conditions for the laccase production in shake flask cultivation using BMGY medium were obtained: the optimal initial pH 7.0, the presence of 0.5 mM Cu2+, 0.6% methanol added into the culture every 24 h. The laccase activity was up to 11, 972 U/L under optimal conditions after 16 days of induction in a medium with 4% peptone. After 100 h of large scale production in 5 L fermenter the enzyme activity reached 18, 123 U/L. The recombinant laccase was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimum pH and temperature for the laccase were pH 2.0 and 50°C with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. The recombinant laccase was stable over a pH range of 2.0–7.0. The Km and the Vmax value of LccA were 0.43 mM and 82.3 U/mg for ABTS, respectively.

Metabolic Engineering of Escherichia coli for Astragalin Biosynthesis

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

Structures and bioactivities of seven flavonoids from Osmanthus fragrans ‘Jinqiu’ essential oil extraction residues

Abstract 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.

Effect of supplemental fermented Ginkgo biloba leaves at different levels on growth performance, meat quality, and antioxidant status of breast and thigh muscles in broiler chickens

&NA; 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.

Characterization of a novel arabinose-tolerant α-L-arabinofuranosidase with high ginsenoside Rc to ginsenoside Rd bioconversion productivity.

(i) To investigate the enzymatic characterization of α‐l‐arabinofuranosidase from Thermotoga thermarum DSM5069. (ii) To evaluate the performance of its excellent properties on converting ginsenoside Rc to ginsenoside Rd.

Consensus Scoring Model for the Molecular Docking Study of mTOR Kinase Inhibitor

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

Enrichment and Purification of Total Ginkgo Flavonoid O-Glycosides from Ginkgo Biloba Extract with Macroporous Resin and Evaluation of Anti-Inflammation Activities In Vitro

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.