Structure-function relationships of a catalytically efficient β-<Emphasis Type="SmallCaps">D</Emphasis>-xylosidase

β-d-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k _cat, k _cat/K _m) for promoting hydrolysis of 1,4-β-d-xylooligosaccharides. ¹H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 1,4-β-d-xylobiose (X2). Progress curves of 4-nitrophenyl-β-d-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k _cat and k _cat/K _m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites −1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites bey ond−1 are blocked by protein and subsites bey ond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k _cat and k _cat/K _m by <10³ and to asimilar extent for substrates 4NPX and 4-nitrophenyl-α-l-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs k _cat/K _m with pK _a values of 5.0 and 7.0 assigned to D14 and E186, respectively. k _cat (4NPX) has a pK _a value of 7.0 and k _cat (4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, “dianionic” enzyme form (D14-E187-) binds 4NPX but not 4NPA. The mention of firm names or trade products does not imply that they are end orsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Beta-D-galactopyranosyl azide: its one-step quantitative synthesis using E461G-beta-galactosidase (Escherichia coli) and a demonstration of its potential as a reagent for molecular biology

A simple one-step synthesis of β-d-galactopyranosyl azide from 0-nitrophenyl-β-d-galactopyranoside and azide catalyzed by E461G-β-galactosidase is described. The synthesis is quantitative in the presence of excess azide and only the β anomer is produced. The product was purified (71% yield) from the other reaction components by extraction with ethyl acetate, silica gel chromatography, and crystallization. The purity was verified by GLC, TLC, and NMR. Thus, E461G-β-galactosidase is able to specifically and quantitatively from β-d-galactopyranosyl-azide. The purified β-d-galactopyranosylazide inhibited the growth of Escherichia coli that express β-galactosidase but not of E. coli that do not. Growth is stopped because β-galactosidase catalyzes the hydrolysis of the β-galactopyranosyl-azide, and the azide that is produced inhibits cell growth. This selective inhibition of growth has potential application in molecular biology screening.     

Determination and Pharmacokinetic Study of Calycosin-7-O-β-d-glucoside in Rat Plasma After Intravenous Administration of Aidi Lyophilizer

Aidi injection is a clinical medicine used in China for the treatment of cancer. Calycosin-7-O-β-d-glucoside is the main effective components of the formulas. In this study, a high performance liquid chromatographic (LC) method was developed to quantify calycosin-7-O-β-d-glucoside in rat plasma using a liquid–liquid extraction and ultraviolet (UV) absorbance detection. LC analysis was performed on a Diamonsil C₁₈ column (200 × 4.6 mm i.d., 5 μm particle size) with isocratic mobile phase consisting of acetonitrile–0.05% phosphoric acid (19.5:80.5, v/v) of a flow rate of 1.0 mL min⁻¹. The linear range was 0.11–17.6 μg mL⁻¹ and the low quantification limit was 0.11 μg mL⁻¹ (S/N = 10). The intra- and inter-day relative standard deviations (RSD) in the measurement of quality control (QC) samples 0.11, 0.22, 1.32 and 8.80 μg mL⁻¹ ranged from 4.1 to 6.3 and 4.3 to 6.2%, respectively. The accuracy was from −6.7 to 4.3% in terms of relative error (RE). Calycosin-7-O-β-d-glucoside was stable in storage at −20 °C for 2 weeks and stable after three freeze–thaw cycles in rat plasma. This method was validated for specificity, accuracy, precision and was successfully applied to pharmacokinetic study of calycosin-7-O-β-d-glucoside in rat plasma after intravenous administration of Aidi lyophilizer.     

Determination of viable <Emphasis Type="Italic">Escherichia coli</Emphasis> using antibody-coated paramagnetic beads with fluorescence detection

A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth with isopropyl-β-d-thiogalactopyranoside, which induces formation of β-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the β-galactosidase. β-Galactosidase converted 4-methylumbelliferyl-β-d-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 × 10⁴ and 1.6 × 10⁷ cfu mL⁻¹, with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is based on the activity of the enzyme intrinsic to live E. coli.     

Hydrolysis and Transglycosylation Activity of a Thermostable Recombinant β-Glycosidase from <Emphasis Type="Italic">Sulfolobus acidocaldarius</Emphasis>

We expressed a putative β-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 oC. The half-lives of the enzyme at 70, 80, and 90 oC were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-β-d-fucopyranoside > pNP-β-d-glucopyranoside > pNP-β-d-galactopyranoside > pNP-β-d-mannopyranoside > pNP-β-d-xylopyranoside, but not toward aryl-α-glycosides or pNP-β-l-arabinofuranoside. Thus, the enzyme was actually a β-glycosidase. The β-glycosidase exhibited transglycosylation activity with pNP-β-d-galactopyranoside, pNP-β-d-glucopyranoside, and pNP-β-d-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with lactose.     

Characterization of a Thermostable Family 1 Glycosyl Hydrolase Enzyme from <Emphasis Type="Italic">Putranjiva roxburghii</Emphasis> Seeds

A 66-kDa thermostable family 1 Glycosyl Hydrolase (GH1) enzyme with β-glucosidase and β-galactosidase activities was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family. N-terminal and partial internal amino acid sequences showed significant resemblance to plant GH1 enzymes. Kinetic studies showed that enzyme hydrolyzed p-nitrophenyl β-d-glucopyranoside (pNP-Glc) with higher efficiency (K _cat/K _m = 2.27 × 10⁴ M⁻¹ s⁻¹) as compared to p-nitrophenyl β-d-galactopyranoside (pNP-Gal; K _cat/K _m = 1.15 × 10⁴ M⁻¹ s⁻¹). The optimum pH for β-galactosidase activity was 4.8 and 4.4 in citrate phosphate and acetate buffers respectively, while for β-glucosidase it was 4.6 in both buffers. The activation energy was found to be 10.6 kcal/mol in the temperature range 30–65 °C. The enzyme showed maximum activity at 65 °C with half life of ~40 min and first-order rate constant of 0.0172 min⁻¹. Far-UV CD spectra of enzyme exhibited α, β pattern at room temperature at pH 8.0. This thermostable enzyme with dual specificity and higher catalytic efficiency can be utilized for different commercial applications.     

Polysaccharide hydrolases from leaves of Boscia senegalensis: properties of endo-(1-->3)-beta-D-glucanase

The leaves of Boscia senegalensis are traditionally used in West Africa in cereal protection against pathogens, pharmacologic applications, and food processing. Activities of α-amylase, β-amylase, exo-(1→3, 1→4)-β-d-glucanase, and endo-(1→3)-β-d-glucanase were detected in these leaves. The endo-(1→3)-β-d-glucanase (EC3.2.1.39) was purified 203-fold with 57% yield. The purified enzyme is a nonglycosylated monomeric protein with a molecular mass of 36 kDa and pI≥10.3. Its optimal activity occurred at pH 4.5 and 50°C. Kinetic analysis gave V _max, k _cat , and K _m values of 659 U/mg, 395 s⁻¹, and 0.42 mg/mL, respectively, for laminarin as substrate. The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance liquid chromatography revealed that the enzyme hydrolyzes not only soluble but also insoluble (1→3)-β-glucan chains in an endo fashion. This property is unusual for endo-acting (1→3)-β-d-glucanase from plants. The involvement of the enzyme in plant defense against pathogenic microorganisms such as fungi is discussed.     

New polyhydroxysteroids and steroid glycosides from the far east starfishCeramaster patagonicus

Two new polyhydroxysteroids and five new glycosides were isolated from the starfishCeramaster patagonicus and their structures were elucidated: 5α-cholestane-3β,6α,15β,16β,26-pentol, (22E)-5α-cholest-22-ene-3β,6α,8,15α,24-pentol, (22E)-28-O-[O-(2-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-hydroxymethyl-5α-cholest-22-ene-3β,4β, 6α,8,15β,16β,28-heptol (ceramasteroside C₁), (22E)-28-O-[O-(2,4-di-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-hydroxymethyl-5α-cholest-22-ene-3β, 6α,8,15β,16β,28-hexol (ceramasteroside C₂), (22E)-28-O-[O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-hydroxymethyl-5α-cholest-22-ene-3β,6α,8,15β,16β 28-hexol (eramasteroside C₃), (22E)-28-O-[O-(2-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-methyl-5α-cholest-22-ene-3β,4β,6α,8, 15β, 26-hexol (ceramasteroside C₄), and (22E)-28-O-[O-(2-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-xylopyranosyl]-5α-cholest-22-ene-3β,6α,8,15β,24-pentol (ceramasteroside C₅)). Three known polyhydroxysteroids (24-methylene-5α-cholestane-3β,6α,8,15β,16β,26-hexol, 5α-cholestane-3β,6α,8,15β,16β,26-hexol, and 5α-cholestane-3β,6β,15α,16β,26-pentol) were also isolated. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 190–195, January, 1997.     

Synthesis of <Emphasis Type="Italic">N</Emphasis><Superscript>2</Superscript>-Arylisocytidines and <Emphasis Type="Italic">N</Emphasis><Superscript>2</Superscript>-Aryl-2′-deoxyisocytidines

Summary. 2-(Arylamino)pyrimidin-4-ones were synthesized, silylated, and condensed with l,2,3,5-tetra-O-acetyl-β- d-ribofuranoside to afford the corresponding N ²-aryl protected isocytidines. Deprotection of the acetylated isocytidines using saturated NH₃ in MeOH solution gave 1-(β-d-ribofuranosyl)-2-(arylamino)-4-pyrimidinones. Methyl 2-deoxy-3,5-di-O-toluyl-α/β-d-ribofuranoside was prepared and condensed with the previously silylated bases to afford the anomeric mixture of protected nucleosides. The pure β-anomers were synthesized with better yield by treating the sodium salts of N ²-arylisocytosine derivatives with 2-deoxy-3,5-di-O-toluyl-α-d-ribofuranosyl chloride. Deprotection of the latter anomers afforded the corresponding free hydroxyl derivatives. The synthesized free nucleosides are under antiviral and oligonucleotide investigations.     

Synthesis of N 2-Arylisocytidines and N 2-Aryl-2′-deoxyisocytidines

2-(Arylamino)pyrimidin-4-ones were synthesized, silylated, and condensed with l,2,3,5-tetra-O-acetyl-β- d-ribofuranoside to afford the corresponding N ²-aryl protected isocytidines. Deprotection of the acetylated isocytidines using saturated NH₃ in MeOH solution gave 1-(β-d-ribofuranosyl)-2-(arylamino)-4-pyrimidinones. Methyl 2-deoxy-3,5-di-O-toluyl-α/β-d-ribofuranoside was prepared and condensed with the previously silylated bases to afford the anomeric mixture of protected nucleosides. The pure β-anomers were synthesized with better yield by treating the sodium salts of N ²-arylisocytosine derivatives with 2-deoxy-3,5-di-O-toluyl-α-d-ribofuranosyl chloride. Deprotection of the latter anomers afforded the corresponding free hydroxyl derivatives. The synthesized free nucleosides are under antiviral and oligonucleotide investigations.