Inversion of sucrose in a continuous process with β--fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose

Inversion of sucrose with β--fructofuranosidase (EC 3.2.1.26) immobilized by the ionic bond on bead DEAHP-cellulose has been studied under flow conditions. Under these conditions, the inversion of sucrose is affected by the concentration and flow rate of the substrate and by the reaction temperature. The effect of substrate concentration on the reaction was investigated in the range 19.5–64.2 wt %; the effect of flow rate was examined in the range 0.25–5.57 g solution per min, and the temperature range used was 25–50°C. It was found that the activities of immobilized β--fructofuranosidase in stirred and flow reactors were the same. The lower activities of β--fructofuranosidase in the case of concentrated solutions, and of immobilized β--fructofuranosidase compared with the native enzyme are attributed to more difficult diffusion through the beads of the ion exchanger, especially of the strongly viscous substrate. A long-term investigation of the enzyme activity over a period of three months demonstrated the stability of the β--fructofuranosidase immobilized by the ionic bond on bead DEAHP-cellulose; the half-life of the enzyme was 215 days. It was also found that the immobilization of the enzyme on a carrier was more effective under flow conditions, i.e. through an ion exchanger in the column, than under the equilibrium conditions of a stirred reactor.     

Kinetic model for synthesis of fructosyl-stevioside using suspended β-fructofuranosidase

The synthesis experiments of fructosyl-stevioside were conducted under the various conditions of the initial concentrations of the substrates and the enzyme. The transfructosylation of stevioside with sucrose and the hydrolyses of sucrose and fructosyl-stevioside simultaneously occurred. The fructosyl-stevioside synthesis was inhibited by the side products, glucose and fructose. A kinetic model was constructed by considering the Ping-Pong Bi Bi mechanism for the transfructosylation, the apparent Ordered Uni Bi mechanism for the hydrolysis and the competitive inhibition by the side products. The model constants were estimated by fitting the model equations with the experimental results for the sucrose hydrolysis and the fructosyl-stevioside synthesis. The model can predict not only the appropriate conditions to efficiently synthesize the fructosyl-stevioside, but also the reaction time giving the maximum conversion.     

Synthesis of α-(S)- acylamino-n-(hydroxydioxocyclobutenyl)- γ-lactams

Monocyclic γ - lactams 2, activated by a hydroxycyclobutenedione moiety have been prepared from (L)-N^tBoc-glutamine, as potential antibacterial agents.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-β- -galactopyranoside and methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-β- -galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β- -galactopyranoside (4) gave a fully acetylated (1→6)-β- -galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α- -galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β- -galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β- -galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Purification and properties of the β-fructofuranosidase from Kluyveromyces fragilis

The β-fructofuranosidase from Kluyveromyces fragilis was purified to one band on electrophoresis by 3 different methods. Two of the preparations were found to be impure by isoelectric focusing. This demonstrates the need for more than one criteria of homogeneity when purifying this enzyme. The enzyme was found to be a glycoprotein, stable at 50°C, with a pH optimum of 4.5. The cations Hg²⁺, Ag⁺, Cu²⁺ and Cd²⁺ exhibited a marked inhibition of the enzyme. Competitive inhibition was observed with the fructose analog 2,5-anhydro-D-mannitol suggesting that the enzyme is inhibited by the furanose form of fructose.     

Inversion of chiral α-methylbenzylamine

More effective use of optical resolution processes can be obtained by increasing the overall yields after development of methods for inversion of the chiral centre of the unwanted isomer. The configuration of some optically active amines can be inverted in a three-step synthesis via the N,N-ditosylimides and a subsequent nucleophilic substitution by the azide ion. The azide product is reduced by hydrogenolysis. Low stereoselectivity caused by racemization to some extent was at first observed for the inversion of the benzylic substrate, (S)-α-methylbenzylamine ( 5a ). However, modified reaction conditions allowed increased stereoselectivity, a more rapid and almost complete inversion of this substate as well. © 1994 Wiley-Liss, Inc.     

Conformational analysis and molecular dynamics simulation of α-(1 → 2) and α-(1 → 3) linked rhamnose oligosaccharides: Reconciliation with optical rotation and NMR experiments

Molecular mechanics and dynamics calculations were carried out on the disaccharides α-L-Rhap-(1 → 2)-α-L-Rhap-(1 → OMe) (1) and α-L-Rhap-(1 → 3)-α-L-Rhap-(1 OMe) (2), and the trisaccharide α-L-Rhap-(1 → 2)-α-L-Rhap-(1 → 3)-α-L-Rhap-(1 → OMe) (3). The semiflexible conformational behavior of these molecules was characterized by the occupation of a combination of different glycosidic linkage and side-chain conformational positions whose relative occupations were sensitive to dielectric screening. Molecular dynamics simulations of the trisaccharide 3 showed little difference between the linkage conformations in the trisaccharide and the component disaccharides 1 and 2. Experimental optical rotation data of 1 and 2 were obtained as a function of temperature in varying solvents. The molecular models were combined with the semiempirical theory of Stevens and Sathyanarayana to yield calculated optical rotations. Interpretation of the data of both 1 and 2 implied that a combination of conformations, both in glycosidic and side-chain positions, could explain the experimental data. Solvents effects were important in influencing the conformational mix and averaged optical rotation. Three-bond heteronuclear coupling constants ³J_{C, H} were obtained for the glycosidic linkages of 1 and 2 in D₂O and DMSO. Analysis of the coupling constants with a Karplus curve showed that small reductions in the glycosidic torsion angles of the conformations of the models used here of ca. 10°–15° in ϕ and 5°–10° in ψ were required to give better agreement with experiment; a combination of conformations for both 1 and 2 was consistent with the data. There was a negligible influence on the coupling constants of 1 on changing the solvent from D₂O to DMSO. © 1997 John Wiley & Sons, Inc.     

Stability of immobilized α-chymotrypsin

The thermal of free and immobilized α-chymotrypsin was investigated experimentally and theoretically. The inactivation process of free α-chymotrypsin was analyzed with a kinetic model which included a first- order reaction process and autolysis. The effects of ionic strength, Ca²⁺ concentration, and temperature are discussed here in terms of the estimated kinetic parameters included in this model. The inactivation process of α-chymotrypsin immobilized onto various supports by several methods was investigated. The Contribution of thermal denaturation and autolysis to the inactivation depended upon the method of immobilization. To interpret quantitatively the non-first-order thermal denaturation process of the immobilized enzyme, a model in which the heterogeneity of the immobilized enzyme was taken into account is proposed.     

Medicinal importance of fungal β-(1→3), (1→6)-glucans

Non-cellulosic β-glucans are now recognized as potent immunological activators, and some are used clinically in China and Japan. These β-glucans consist of a backbone of glucose residues linked by β-(1→3)-glycosidic bonds, often with attached side-chain glucose residues joined by β-(1→6) linkages. The frequency of branching varies. The literature suggests β-glucans are effective in treating diseases like cancer, a range of microbial infections, hypercholesterolaemia, and diabetes. Their mechanisms of action involve them being recognized as non-self molecules, so the immune system is stimulated by their presence. Several receptors have been identified, which include: dectin-1, located on macrophages, which mediates β-glucan activation of phagocytosis and production of cytokines, a response co-ordinated by the toll-like receptor-2. Activated complement receptors on natural killer cells, neutrophils, and lymphocytes, may also be associated with tumour cytotoxicity. Two other receptors, scavenger and lactosylceramide, bind β-glucans and mediate a series of signal pathways leading to immunological activation. Structurally different β-glucans appear to have different affinities toward these receptors and thus generate markedly different host responses. However, the published data are not always easy to interpret as many of the earlier studies used crude β-glucan preparations with, for the most part, unknown chemical structures. Careful choice of β-glucan products is essential if their benefits are to be optimized, and a better understanding of how β-glucans bind to receptors should enable more efficient use of their biological activities.     

Lipase-catalyzed kinetic resolutions of racemic β- and γ-thiolactones

Several racemic β- and γ-thiolactones were synthesized and kinetic resolutions of them were executed using lipases. While a lipase from Pseudomonas cepacia (PCL) showed the highest enantioselectivity for (S)-form (>99% ee_S at 53% conversion, E > 100) in the kinetic resolution of racemic -methyl-β-propiothiolactone (rac-MPTL), it showed no hydrolysis activity in the kinetic resolution of -benzyl--methyl-β-propiothiolactone (rac-BMPTL), suggesting that the changes in the size of alkyl group from rac-MPTL to rac-BMPTL leads to lower hydrolysis activity and enantioselectivity. In contrast, racemic γ-butyrothiolactones were hydrolyzed by several lipases with low enantioselectivity, whereas a lipase from Candida antarctica (CAL) showed moderate enantioselectivity for (S)-form (>99% ee_S at 76% conversion, E = 11) in the kinetic resolution of racemic -methyl-γ-butyrothiolactone (rac-MBTL). Computer-aided molecular modeling was also performed to investigate the enantioselectivites and activities of PCL toward β-propiothiolactones. The computer modeling results suggest that the alkyl side chains of β-propiothiolactones and γ-butyrothiolactones interact with amino acid residues around hydrophobic crevice, which affects the activity of PCL.     

Microbial Reduction of 1,3-Dioxo-2-Methyl-2-(3′-Oxo-6′-Carbomethoxyhexyl)-Cyclopentane to Form 1β-Hydroxy-3-Oxo-2β-Methyl-2α-(3′-Oxo-6′-Carbomethoxy-hexyl)-Cyclopentane, an Intermediate for Steroid Total Synthesis

The rate and extent of stereoselective reduction of 1,3-dioxo-2-methyl-2-(3′-oxo-6′-carbomethoxyhexyl)-cyclopentane to form the 1β-hydroxy-2β-methyl isomer by cultures of Schizosaccharomyces pombe ATCC 2476 was dramatically increased by addition to the fermentation of certain α,β-unsaturated ketones and allyl alcohol.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.