Purification and Characterization of β-Glucosidase from a Newly Isolated Strain Tolypocladium cylindrosporum Syzx4

A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer(ITS) rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27...     

Aggregation Pathways of the Amyloid β(1-42) Peptide Depend on Its Colloidal Stability and Ordered β-Sheet Stacking

Amyloid β (Aβ) fibrils are present as a major component in senile plaques, the hallmark of Alzheimer's disease (AD). Diffuse plaques (nonfibrous, loosely packed Aβ aggregates) containing amorphous Aβ aggregates are also formed in brain. This work examines the influence of Cu(2+) complexation by Aβ on the aggregation process in the context of charge and structural variations. Changes in the surface charges of Aβ molecules due to Cu(2+) binding, measured with a ζ-potential measurement device, were correlated with the aggregate morphologies examined by atomic force microscopy. As a result of the charge variation, the "colloid-like" stability of the aggregation intermediates, which is essential to the fibrillation process, is affected. Consequently, Cu(2+) enhances the amorphous aggregate formation. By monitoring variations in the secondary structures with circular dichroism spectroscopy, a direct transformation from the unstructured conformation to the β-sheet structure was observed for all types of aggregates observed (oligomers, fibrils, and/or amorphous aggregates). Compared to the Aβ aggregation pathway in the absence of Cu(2+) and taking other factors affecting Aβ aggregation (i.e., pH and temperature) into account, our investigation indicates that formations of amorphous and fibrous aggregates diverge from the same β-sheet-containing partially folded intermediate. This study suggests that the hydrophilic domain of Aβ also plays a role in the Aβ aggregation process. A kinetic model was proposed to account for the effects of the Cu(2+) binding on these two aggregation pathways in terms of charge and structural variations.     

ZrCl4-Catalyzed Synthesis of β-Aminosulfides from Aziridines and Thiols

A simple and efficient synthesis of β-aminosulfides has been introduced by ring opening of aziridine rings with aromatic thiols in the presence of zirconium(IV) chloride under solvent-free conditions at room temperature.     

Molecular Cloning and Expression of a Novel β-Glucosidase Gene from Phialophora sp. G5

A novel β-glucosidase gene, bgl1G5, was cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the gene consists of a 1,431-bp open reading frame encoding a protein of 476 amino acids. The deduced amino acid sequence of bgl1G5 showed a high identity of 85 % with a characterized β-glucosidase from Humicola grisea of glycoside hydrolase family 1. Compared with other fungal counterparts, Bgl1G5 showed similar optimal activity at pH 6.0 and 50 °C and was stable at pH 5.0–9.0. Moreover, Bgl1G5 exhibited good thermostability at 50 °C (6 h half-life) and higher specific activity (54.9 U mg^–1). The K _m and V _max values towards p-nitrophenyl β-d-glucopyranoside (pNPG) were 0.33 mM and 103.1 μmol?min^–1?mg^–1, respectively. The substrate specificity assay showed that Bgl1G5 was highly active against pNPG, weak on p-nitrophenyl β-d-cellobioside (pNPC) and p-nitrophenyl-β-d-galactopyranoside (ONPG), and had no activity on cellobiose. This result indicated Bgl1G5 was a typical aryl β-glucosidase.     

Use of alumina for elimination of sulfinic acid from β-aryl- and β-alkylsulfonyl carbonyl compounds.

Title compounds give α-unsaturated carbonyl compounds by treatment with basic alumina in dichloromethane or diethylether. α-Enones are thus obtained in high yield at room temperature.     

Gene Cloning and Characterization of a Novel Salt-Tolerant and Glucose-Enhanced β-Glucosidase from a Marine Streptomycete

The gene BglNH encoding a β-glucosidase was cloned from a marine streptomycete. Sequence analysis revealed that BglNH encoded a 456-aa peptide with a calculated mass of 51 kDa. The deduced amino acid sequence of BglNH showed the highest identities of 61 % with known β-glucosidases and contained a catalytic domain which belonged to the glycoside hydrolase family 1. The gene BglNH was expressed in Escherichia coli and the recombinant enzyme (r-BglNH) was purified. The optimum pH and temperature of r-BglNH were pH?6.0 and 45 °C, respectively. The r-BglNH displayed the typical salt-tolerant and glucose-enhanced characteristics. Its activity was remarkably enhanced in the presence of 0.5 M NaCl (rose more than 1.6-fold) and 0.1 M glucose (rose more than 1.4-fold). Moreover, r-BglNH displayed good pH stability and metal tolerance. It remained stable after incubating with buffers from pH?4.0 to 10.0, and most metal ions had no significant inhibition on its activity. These properties indicate that r-BglNH is an ideal candidate for further research and industrial applications.     

Synthesis of β-d-glucopyranosyl- and β-d-galactopyranosylamines from 4-bromo-3-methylaniline and 2-amino-5-bromopyridine

The possibility of coupling of d-glucose and d-galactose with 4-bromo-3-methylaniline, 2,4,6-tribromoaniline, and 2-amino-5-bromopyridine was studied. The substituent in the aromatic ring was found to influence the conditions and possibility of the reaction. The yields of β-d-glucopyranosyl- and β-d-galactopyranosylamines from 4-bromo-3-methylaniline and 2-amino-5-bromopyridine were 50–65%; 2,4,6-tribromoaniline did not react at all.     

Expression and Characterization of a GH39 β-Xylosidase II from Caulobacter crescentus

In the present work, the gene xynB2, encoding a ??-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5?%. CcXynB2 appeared as a single band of 60?kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 ??-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55?°C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5?C7.5 for 24?h and thermotolerance up to 50?°C. The K _M and V _Max values were 9.3?±?0.45?mM and 402?±?19???mol?min^?1 for ??-nitrophenyl-??-d-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family ??-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.     

Structure of β-SrRh2O4 from X-ray and neutron powder diffraction

β-SrRh₂O₄, a novel high temperature phase, is prepared as black powder by solid state reaction of SrCO₃ with Rh in air. The compound crystallises in the space group P63c, Z = 1, with a = 3.0626(2) Å and c = 11.3996(7) Å. The structure consists of Cd1₂-type layers of edge sharing RhO₆ octahedra with an AABB oxygen layer sequence. Strontium is found in partial occupation of both available interlayer sites, in trigonal prismatic coordination.     

NMR and Monte Carlo Studies on the Solution Conformation of a Linear Capsular Polysaccharide from a Soybean‐Nodulating Bacterium (Strain B33)

The conformational behavior of the capsular polysaccharide obtained from a fast‐growing soybean‐nodulating rhizobia (strain B33) isolated from Xinjiang Autonomous Region (Eastern China) has been analyzed by NMR and molecular mechanics simulations. This polysaccharide has the repeating unit →6)‐4‐O‐Me‐α‐d‐Glcp‐(1→4)‐3‐O‐Me‐β‐d‐GlcpA‐(1→. The NMR results indicate that the α‐(1→4) linkage may adopt a variety of conformations, and that at least two of the resulting minima must exist in solution. NOE data agree with an 85:10:5 ratio for the lowest‐energy conformations. In the case of the β‐(1→6) linkage, NMR indicates that the rotamer gg is highly populated. Experimental and calculated NOE intensities match well when the global energy minimum conformation for this linkage has exclusively the gg orientation. The influence of the adjacent methyl group on the glycosyloxymethyl population has been evaluated by simulation of a disaccharide without this group. A relative destabilization of gt rotamer has been found. Long chains have been simulated using a Metropolis algorithm at different ratios of the gg and gt rotamers in the glucose moiety. It was observed that the increase in population of the gt rotamer yielded more close contacts in the chain.     

Identification and Functional Characterization of an α-Amylase with Broad Temperature and pH Stability from Paenibacillus sp.

Amylases are important industrial enzymes that have been applied widely in the food, detergent, and pulp industries and fermentation processes. In the present study, a gene encoding an alpha-amylase from the genomic DNA library of Paenibacillus sp. was identified and characterized. The amylase gene designated amy1 was shown to consist of 1,980 bp and shared sequence identity towards α-amylase genes from other Bacillus sp. The deduced amino acid sequence for Amy1 indicated 80 % sequence identity with other Bacillus strains. Heterologous expression of recombinant Amy1 in Escherichia coli BL21(DE3) facilitated the recovery of this protein in soluble form. Enzyme kinetic data revealed Amy1 to have a K _m of 23.83 mg/mL and K _cat of 48.74 min^?1 and K _cat /K _m of 2 min^?1 mg^?1 mL^?1 for starch. The activity of this protein was found to be enhanced by Mn²⁺, and furthermore, Amy1 remained active at a broad pH range (4–10) and temperature (30–90 °C). The ability of Amy1 to act on food waste under broad temperature and pH conditions, together with its ability to produce simple sugars, shows many advantages for further application in the food industry.     

Extraction and Determination of β-Sitosterol from Salicornia herbacea L. Using Monolithic Cartridge

A short ionic liquids-based monolithic cartridge was prepared and used as the selective extraction sorbent. Characteristic and evaluation are investigated by field emission scanning electron microscopy (FE-SEM), and a new approach was developed for the extraction and determination of β-sitosterol from Salicornia herbacea L. using the ionic liquids-based monolithic cartridge. Chromatographic analysis was conducted on a C₁₈ column with UV detection at 210 nm, and an eluting solution consisting of acetonitrile–water (60/40, v/v) was used as the mobile phase at a flow rate of 0.8 mL min^?1. The linearity was confirmed in the concentration range of 0.50–100.00 μg mL^?1, with RSDs within 4.20%, and a recovery of β-sitosterol ranging from 97.20 to 102.93%. This method effectively removed the impurities without any tedious pretreatment, and it provided a fast, economic and effective way to assay trace drugs from natural plants.     

Isolation and Characterization of a Bioactive Polysaccharide from Panax Quinquefolium L.

ＩｓｏｌａｔｉｏｎａｎｄＣｈａｒａｃｔｅｒｉｚａｔｉｏｎｏｆａＢｉｏａｃｔｉｖｅＰｏｌｙｓａｃｃｈａｒｉｄｅｆｒｏｍＰａｎａｘＱｕｉｎｑｕｅｆｏｌｉｕｍＬ．ＭＡＸｉｕ－ｌｉ＊,ＨＡＯＣｈｕｎ－ｙａｎ,ＬＵＳｈｉ－ｘｉａｎｇ,ＳＵＮＹｕｎ－ｘｉｕ,Ｌ...     

Purification and characterization of an intracellular β-glucosidase from a Candida sake strain isolated from fruit juices

A yeast strain isolated in the laboratory from fruit juices was studied and classified as Candida sake. The strain produces an intracellular beta-glucosidase when grown with cellobiose as the carbon source. The enzyme was purified by ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular beta-glucosidase, estimated by gel filtration, was 240 kDa. The tetrameric structure of the beta-glucosidase was determined following treatment of the purified enzyme with sodium dodecyl sulfate. The enzyme exhibited optimum activity at 52 degrees C and pH 4.25 with citrate-phosphate buffer. The enzyme was active against soluble glycosides with the (1-->4)-beta configuration, and from Lineweaver Burk plots, a Km value of 6.9 mmol/L was found for p-nitrophenyl-beta-D-glucopyranoside. The beta-glucosidase was found to be tolerant to glucose inhibition with a Ki value of 0.2 mol/L.     

Isolation and characterization of four novel β-Sitosteryl esters from Salvadora persica Linn.

Salvadora persica is virtuous to have a variety of phytoconstituents responsible for many biological activities some of them identified particularly while some are still to be acknowledged. A number of steroidal, glycosidic, terpenoids, saponins and functional esters are reported till date. The present study deals with extraction, isolation, and characterisation of four novel steroidal esters by systematic cold extraction of S. persica. The extracted phytoconstituents were characterised by sophisticated spectral UV, IR, NMR and MS, techniques. The reported four new β-Sitosteryl esters SP-2, 3, 5 and 6 were extracted and reported for the first time.     

Total synthesis of (−)-jaspine B and its 4-epi-analogue from d-xylose

The total synthesis of the HCl salts of (−)-jaspine B ent-1 and its 4-epi-congener ent-4 was accomplished starting from the common template 13 derived from d-xylose. The cornerstone of our synthesis was [3,3]-heterosigmatropic rearrangements, which effectively provided scaffolds with a chiral amino group. A subsequent Wittig olefination installed a C₁₄ alkyl side chain and acid-mediated ring-closing reaction established the tetrahydrofuran core.     

New norditerpenoids and a diterpenoid from a sponge that inhibit the lyase activity of DNA polymerase β

Using an assay to detect inhibitors of the lyase activity of DNA polymerase β, bioassay-directed fractionation of a CHCl₃ extract of an unidentified sponge of the family Demospongiae resulted in the isolation of the new diterpenoid 1, the new bis-norditerpenoids 2-3, and the two known compounds spongia-13(16),14-dien-19-oic acid (4), and methylspongia-13(16),14-dien-19-oate (5). The structures of the new compounds were established on the basis of extensive 1D and 2D NMR spectroscopic interpretation. All five compounds inhibited the lyase activity of DNA polymerase β.     

One-Pot Oxidation and Nitrosation of β-Alkanolamines with KMnNO4-NaNO2. A Convenient Preparation of β-Ketonitrosamines

The oxidation and nitrosation of β-alkanolamines to the corresponding β-ketonitrosamines is reported. The oxidation is carried out with potassium permanganate in acidic medium, followed by nitrosation of the protonated aminoketone, with sodium nitrite, to the corresponding β-ketonitrosamine.     

Formation, Spectroscopic Characterization, and Solution Stability of an [Fe(4)S(4)](2+) Cluster Derived from β-Cyclodextrin Dithiolate

The formation and solution properties, including stability in mixed aqueous-Me(2)SO media, have been investigated for an [Fe(4)S(4)](2+) cluster derived from β-cyclodextrin (CD) dithiolate. Clusters of the type [Fe(4)S(4)(SAr)(4)](2-) (Ar = Ph, C(6)H(4)-3-F) are generated in Me(2)SO by redox reactions of [Fe(4)S(4)(SEt)(4)](2-) with 2 equiv of ArSSAr. An analogous reaction with the intramolecular disulfide of 6(A),6(D)-(3-NHCOC(6)H(4)-1-SH)(2)-6(A),6(D)-dideoxy-β-cyclodextrin (14), whose synthesis is described, affords a completely substituted cluster formulated as [Fe(4)S(4){β-CD-(1,3-NHCOC(6)H(4)S)(2)}(2)](2-) (15). Ligand binding is indicated by a circular dichroism spectrum and also by UV-visible and isotropically shifted (1)H NMR spectra and redox behavior convincingly similar to [Fe(4)S(4)(SPh)(4)](2-). One formulation of 15 is a single cluster to which two dithiolates are bound, each in bidentate coordination. With there being no proven precedent for this binding mode, we show that the cluster [Fe(4)S(4)(S(2)-m-xyl)(2)](2-) is a single cubane whose m-xylyldithiolate ligands are bound in a bidentate arrangement. This same structure type was proposed for a cluster formulated as [Fe(4)S(4){β-CD-(1,3-SC(6)H(4)S)(2)}(2)](2-) (16; Kuroda et al. J. Am. Chem. Soc.1988, 110, 4049-4050) and reported to be water-stable. Clusters 15 and 16 are derived from similar ligands differing only in the spacer group between the thiolate binding site and the CD platform. In our search for clusters stable in aqueous or organic-aqueous mixed solvents that are potential candidates for the reconstitution of scaffold proteins implicated in cluster biogenesis, 15 is the most stable cluster that we have thus far encountered under anaerobic conditions in the absence of added ligand.     

Syntheses and Bioactivity of 4＂-Sulfonate-5-triphenyl silyl Avermectin B1a and Ivermectin B1a Derivatives

Fourteen new derivatives of avermectin B1a, and ivermectin B1a were synthesized from C5-O-triphenyl silylavermectin B1a and ivermectin B1a (yield from 40% to 83%). Their chemical structures were characterized by means of IR. ^1H NMR, ^13C NMR and FAB-MS spectrometries. Some of them show excellent insecticidal activity.