Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Activity of plasma membrane β-galactosidase and β-glucosidase

Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Sialidase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma membrane association of β-galactosidase and β-glucosidase. To determine the presence of β-galactosidase and β-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a trans activity in living cells.     

Purification and Characterization of Extracellular β-Xylosidase and β-Glucosidase from Aspergillus fumigatus

A β-xylosidase (β-d-xyloside xylohydrolase, EC 3.2.1.37) and β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) extracted from a wheat bran culture of Aspergillus fumigatus were purified up to 90-fold and 131-fold, respectively, by ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and hydroxylapatite chromatography. Molecular weights of the β-xylosidase and β-glucosidase were 360,000 and 380,000, respectively, each consisting of four identical subunits. The isoelectric points of β-xylosidase and β-glucosidase were at pH 5.4 and 4.5, respectively. The optimum temperature for the β-xylosidase was 75°C, being stable up to 65°C for 20 min and for the β-glucosidase was 65°C, being stable up to 60°C for 20 min. The optimum pH for both enzymes was about 4.5, being stable between 2 and 8 at 50°C for 20 min. Both enzymes were inhibited by Fe³⁺, Cu²⁺, Hg²⁺, SDS, and p-chloromercuribenzoate. The apparent Michaelis constants of the β-xylosidase were 2.0 and 23.8 mM for p-nitrophenyl-β-xyloside and xylobiose, respectively, and those of the β-glucosidase were 1.4, 11.4, and 24.8 mM for p-nitrophenyl-β-glucoside, gentiobiose, and cellobiose, respectively. To produce xylose from crude xylooligosac-charides prepared by steam-explosion of cotton seed waste (DP ≤10, 53%, total sugars = 150 g/ liter), the crude enzyme from A. fumigatus (β-xylosidase activity = 14.7 units/ml, xylanase activity = 20 units/ml) could hydrolyze the substrate at 55°C and pH 4.5 resulting in almost complete conversion to xylose (160 g/liter).     

A comparison of enzymatic phosphorylation and phosphatidylation of β-l- and β-d-nucleosides

Enzymatic 5′-monophosphorylation and 5′-phosphatidylation of a number of β-l- and β-d-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5′-hydroxyl group of nucleoside; the second was the phospholipase d (PLD)-catalyzed transphosphatidylation of l-α-lecithin with a series of β-l- and β-d-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates. Some β-l-nucleosides displayed similar or even higher substrate activity compared to the β-d-enantiomers.     

Serum ferritin and transferrin saturation levels in β⁰ and β(+) thalassemia patients

There have been few studies on the mutations that cause heterozygous beta-thalassemia and how they affect the iron profile. One hundred and thirty-eight individuals were analyzed, 90 thalasemic β? and 48 thalasemic β(+), identified by classical and molecular methods. Mutations in the hemochromatosis (HFE) gene, detected using PCR-RFLP, were found in 30.4% of these beta-thalassemic patients; heterozygosity for H63D (20.3%) was the most frequent. Ferritin levels and transferrin saturation were similar in beta-thalassemics with and without mutations in the HFE gene. Ferritin concentrations were significantly higher in men and in individuals over 40 years of age. Transferrin saturation also was significantly higher in men, but only in those without HFE gene mutations. There was no significant difference in the iron profile among the β? and β(+) thalassemics, with and without HFE gene mutations. The frequency of ferritin values above 200 ng/mL in women and 300 ng/mL in men was also similar in β? and β(+) thalassemics (P > 0.72). Our conclusion is that ferritin levels are variable in the beta-thalassemia, trait regardless of the type of beta-globin mutation. Furthermore, HFE gene polymorphisms do not change the iron profile in these individuals.     

Characterization of β-lapachone and methylated β-cyclodextrin solid-state systems

The purpose of this research was to explore the utility of β cyclodextrin (βCD) and β cyclodextrin derivatives (hydroxypropyl-β-cyclodextrin [HPβCD], sulfobutylether-β-CD [SB\CD], and a randomly methylated-β-CD [RMβCD]) to form inclusion complexes with the antitumoral drug, β-lapachone (βLAP), in order to overcome the problem of its poor water solubility. RMβCD presented the highest efficiency for βLAP solubilization and was selected to develop solid-state binary systems. Differential scanning calorimetry (DSC), X-ray powder diffractometry (XRPD), Fourier transform infrared (FTIR) and optical and scanning electron microscopy results suggest the formation of inclusion complexes by both freeze-drying and kneading techniques with a dramatic improvement in drug dissolution efficiency at 20-minute dissolution efficiency (DE_20-minute 67.15% and 88.22%, respectively) against the drug (DE_20-minute 27.11%) or the βCD/drug physical mixture (DE_20-minute 27.22%). However, the kneading method gives a highly crystalline material that together with the adequate drug dissolution profile make it the best procedure in obtaining inclusion complexes of RMβCD/βLAP convenient for different applications of βLAP. Published: July 27, 2007     

Separation and properties of β-galactosidase, β-glucosidase, β-glucuronidase and n-acetyl-β-glucosaminidase from rat kidney

1. The activities of β-galactosidase, β-glucosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7·0 indicated slow- and fast-moving components of rat-kidney β-galactosidase. 3. The fast-moving component is also associated with the total β-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the β-glucosidase component is a small acidic molecule, of molecular weight approx. 40000–50000, with optimum pH5·5–6·0 for β-galactosidase and β-glucosidase activities. 5. The major β-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3·7.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

Metallostasis and amyloid β-degrading enzymes

Amyloid-Beta (Aβ) is a major constituent of senile plaques and one of the principle hallmarks of Alzheimer's disease (AD). The peptide is produced by proteolytic cleavage of the larger amyloid precursor protein (APP). Increased production and aggregation of the peptide are associated with pathology. Emerging evidence suggests that the steady-state levels of Aβ are determined by the balance between its production and degradation. For this reason, the tuning of the activity of enzymes that degrade Aβ may be a promising approach in the development of novel therapeutics aimed at reducing Aβ concentration by enhancing its removal. A great part of Aβ degrading enzymes are known to be metalloproteases. In the last decade increasing evidence supported the idea that metal ion homeostasis is affected in several regions of AD brain and metals play an important role in tuning enzyme activity. There are three main different pathways by which metal ions can affect the proteolytic enzymes responsible for Aβ peptides degradation, as metal ions can: (i) form complexes with Aβ peptides that are not easily degraded; (ii) directly bind to degradative enzymes; (iii) produce signalling cascades that alter enzymes activity involved in Aβ catabolism. In the current literature the three points mentioned above are very often puzzled, resulting in a quite fragmentary scenario. The aim of this work is to find a link between metal ion homeostasis and Aβ degradation by separating and analysing the three different pathways proposed.     

Production of β-Mannanase and β-Mannosidase from Aspergillus awamori K4 and Their Properties

β-Mannanase and β-mannosidase from Aspergillus awamori K4 was produced by solid culture with coffee waste and wheat bran. The optimum composition for enzyme production was 40% coffee waste–60% wheat bran. Two enzymes were partially purified. Optimum pH was about 5 for both enzymes, and optimum temperature was around 80°C for β-mannanase and 60–70°C for β-mannosidase. These enzymes produced some oligosaccharides from glucomannan and galactomannan by their hydrolyzing and transferring activities. β-Mannanase hydrolyzed konjak and locust bean gum 39.1% and 15.8%, respectively. Oligosaccharides of various molecular size were released from glucomannan of konjak, but on the addition of cellulase, mannobiose was released selectively. In locust bean gum, tetra-, tri-, and disaccharides (mannobiose) were mainly released by K4 β-mannanase. Tetra- and trisaccharides were heterooligosaccharides consisting of galactose and mannose residues. K4 β-mannosidase had a transglycosylation action, transferring mannose residue to alcohols and sugars like fructose. Received: 24 April 2000/Accepted: 20 October 2000     

Immobilized β-glucanases and their applications

Summary A number of -glucanase active enzyme preparations were successfully immobilized either by adsorption on Duolite S-761 phenol-formaldehyde resin or covalently on silanized Spherosil XOB-075 porous silica beads to obtain remarkably stable active biocatalysts. A Duolite immobilized -glucanase could be employed for continuous treatment of barley wort in a packed-bed column reactor to decrease viscosity and to improve filtrability. A Duolite immobilized cellulase that exhibited no detectable Avicel hydrolyzing activity could be applied for batch treatment of wheat starch process water. The same enzyme when covalently bound on Spherosil was, however, capable of hydrolyzing microcrystalline cellulose in a recirculating fluidized-bed reactor.     

Preparation and conformational analysis of C-glycosyl β- and β/β-peptides

Ten C-glycosyl β²- and β/β²-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble β²-amino acids in which C-glycosylic substituents are attached to the C-2 position of β-amino acids. Conformational analysis of the C-glycosyl β²-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl β/β²-peptides are governed by the configuration at the α-carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl β²-amino acids.     

Genetic relationship between β-galactosidase and β-fucosidase in the mouse

Evidence for the identity of β-galactosidase and β-fucosidase enzymes in the house mouse was obtained by examination of the enzyme activities in animals from different crosses between C57BL/Kl mice with high galactosidase and fucosidase activities and DBA/2/Kl mice with low activities. There is a strong correlation between the activities of these two enzymes in different tissues of F₂ animals. A comparison of the fractionation properties of β-galactosidase and β-fucosidase showed that the two activities had a parallel distribution and identical thermostability. These data suggest that the same enzyme catalyzes the hydrolysis of both substrates.     

Possible Identity of β-Xylosidase and β-Glucosidase of Chaetomium trilaterale

The nature of the active site of Chaetomium trilaterale β-xylosidase catalyzing the hydrolysis of β-d-glucopyranoside and β-d-xylopyranoside was investigated by kinetic methods. On experiments with mixed substrates, such as phenyl β-d-xylopyranoside and phenyl β-d-glucopyranoside, the kinetic features agreed very closely with those features theoretically predicted for a single active site of the same enzyme catalyzing the hydrolysis of these two kinds of substrates.

Both the β-glucosidase and β-xylosidase activities were strongly inhibited by glucono-1,5-lactone and nojirimycin (5-amino-5-deoxy-d-glucopyranose). β-Xylosidase activity was inhibited non-competitively by the two inhibitors, but β-glucosidase activity was competitive. Methyl β-d-xylopyranoside, methyl β-d-glucopyranoside, 1-thiophenyl β-d-xylopyranoside, and 1-thiophenyl β-d-glucopyranoside poorly inhibited both activities. Methyl β-d-xylopyranoside inhibited the β-xylosidase activity competitively but the β-glucosidase activity was non-competitive, whereas methyl β-d-glucopyranoside inhibited the β-xylosidase activity non-competitively but the β-glucosidase activity was competitive. 1-Thiophenyl β-d-xylopyranoside and 1-thiophenyl β-d-glucopyranoside behaved as competitive inhibitors.

From these results, it was concluded that the β-xylosidase and β-glucosidase activities reside in one catalytic site, and this suggests that there might be two kinetically distinct binding sites in the active center of the same enzyme.     

Platelet function and thymosin β4

Abstract Thymosin β4 (Tβ4) is a small, low-molecular-weight peptide ubiquitously expressed in all cells and extracellular fluids. It is a major actin sequestering protein present in the cells. In addition to this, Tβ4 has also been shown to be involved in endothelial cell migration, angiogenesis, corneal wound healing, and stem cell differentiation. It is also released by platelets after activation. The amount of Tβ4 increases at sites of injury and thus suggests an important role of this biopeptide in wound healing. Herein, we provide an overview of the role of Tβ4 in thrombosis and platelet aggregation.     

A new spin-labeled substrate for β-galactosidase and β-galactoside permease

A spin-labeled β-galactoside has been synthesized. It is a substrate for β-galactosidase and is accumulated by E. coli ML 308-225 and K12-N2244. The accumulation appears to be active transport via the lactose permease since it is inhibited with lactose and the energy poison, amytal. Induction of the K12 strain with isopropylthiogalactoside results in a 100-fold stimulation of uptake of the spin-labeled β-galactoside. Binding of the spin-labeled sugar to membranes derived from the ML strain is inhibited by lactose.     

Attempt at Affinity Labeling of α- and β- Amylases by α- and β-d-Glucopyranosides and α- and β-Maltooligosaccharides with 2,3-Epoxypropyl Residue as Aglycone: Specific Inactivation of β-Amylases

Among 2,3-epoxypropyl α-d-glucopyranoside and 2,3-epoxypropyl α-maltooligosaccharides and the β-anomers, 2,3-epoxypropyl α-d-glucopyranoside (α-EPG) strongly inactivated the β-amylases [EC 3.2.1.2] of sweet potato, barley, and Bacillus, cereus, in addition to soybean β amylase [J. Biochem., 99, 1631 (1986)]. However, none of the compounds used inactivated any α-amylases [EC 3.2.1.1] of porcine pancreas, Aspergillus oryzae, or Bacillus amyloliquefaciens. Irreversible incorporation of ¹⁴C-labeled α-EPG into β-amylases was stoichiometric, i.e., one α-EPG per active site of the enzyme was bound, and the inactivations were almost complete. The results suggest that α-EPG is an affinity labeling reagent selective for β-amylase. Slow inactivations by the other compounds were also observed, depending on the difference of source of β amylase.     

Heat Treatment and β-Carotene Incorporation Effect in the Interaction of β-Lactoglobulin and Carboxymethylcellulose System

Encapsulation technologies using proteins or polysaccharides can be employed with the purpose of solubilizing and protecting carotenoids. However, information on the role of protein and polysaccharide interactions is still slightly limited. The aim of this work was to investigate the effect of β-carotene linked to protein β-lactoglobulin (BLG) in the interaction carboxymethylcellulose (CMC) using isothermal titration calorimetry (ITC). Firstly, BLG and CMC interaction was assessed by means of turbidity analysis. Based on the results of turbidity, the thermodynamic profile of BLG-CMC complexes at pH 4.0 was obtained using ITC analysis at 25 °C. Afterward, it was evaluated the effect of a thermal treatment applied to the BLG (68 °C for 50 min) in the interaction with CMC also using ITC and circular dichroism (CD). ITC and CD analysis showed that the heat treatment applied on BLG did not cause changes in molecular interactions. The binding isotherm of BLG-CMC complexes incorporated with β-carotene showed an increase in the molar ratio and a slight decrease in enthalpy of the system. Incorporation of β-carotene in the system did not significantly affect the BLG and CMC interaction, suggesting this system can be applied in food application as encapsulation.