Solvent effect on enzyme-catalyzed synthesis of β-d-glucosides using the reverse hydrolysis method: Application to the preparative-scale synthesis of 2-hydroxybenzyl and octyl β-d-glucopyranosides

Almond β-d-glucosidase was used to catalyze alkyl-β-d-glucoside synthesis by reacting glucose and the alcohol in organic media. The influence of five different solvents and the thermodynamic water activity on the reaction have been studied. The best yields were obtained in 80 or 90% (v/v) tert-butanol, acetone, or acetonitrile where the enzyme is very stable. In this enzymatic synthesis under thermodynamic control, the yield increases as the water activity of the reaction medium decreases. Enzymatic preparative-scale syntheses were performed in a tert-butanol-water mixture which was found to be the most appropriate medium. 2-Hydroxybenzyl β-d-glucopyranoside was obtained in 17% yield using a 90:10 (v/v) tert-butanol-water mixture. Octyl-β-glucopyranoside was obtained in 8% yield using a 60:30:10 (v/v) tert-butanol-octanol-water mixture.     

Enzymatic synthesis of α- -fucosyl-N-acetyllactosamines and 3′-O-α- -fucosyllactose utilizing α- -fucosidases

An α- -fucosidase from porcine liver produced α- -Fuc-(1→2)-β- -Gal-(1→4)- -GlcNAc (2′-O-α- -fucosyl-N-acetyllactosamine, 1) together with its isomers α- -Fuc-(1→3)-β- -Gal-(1→4)- -GlcNAc (2) and α- -Fuc-(1→6)-β- -Gal-(1→4)- -GlcNAc (3) through a transglycosylation reaction from p-nitrophenyl α- -fucopyranoside and β- -Gal-(1→4)- -GlcNAc. The enzyme formed the trisaccharides 1–3 in 13% overall yield based on the donor, and in the ratio of 40:37:23. In contrast, transglycosylation by Alcaligenes sp. α- -fucosidase led to the regioselective synthesis of trisaccharides containing a (1→3)-linked α- -fucosyl residue. When β- -Gal-(1→4)- -GlcNAc and lactose were acceptors, the enzyme formed regioselectively compound 2 and α- -Fuc-(1→3)-β- -Gal-(1→4)- -Glc (3′-O-α- -fucosyllactose, 4), respectively, in 54 and 34% yields, based on the donor.     

Immobilized 4-aminophenyl 1-thio-β- -galactofuranoside as a matrix for affinity purification of an exo-β- -galactofuranosidase

An alternative and fast method for the purification of an exo-β- -galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β- -galactofuranoside affinity chromatography system and specific elution with 10 mM -galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM -galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β- -galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and K_m and V_max values of 0.311 mM and 17 μmol h⁻¹ μg⁻¹ respectively, when 4-nitrophenyl β- -galactofuranoside was employed as the substrate.     

A direct enzymatic synthesis of β- -galactopyranosyl- -xylopyranosides and their use to evaluate rat intestinal lactase activity in vivo

By enzymatic β- -galactosylation of -xylose a mixture of 4-, 3-, and

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(1, 4, and 7, respectively) was obtained in 50% isolated yield. Disaccharides 1, 4, and 7 are substrates of intestinal lactase isolated from lamb small intestine with K_m values of 250.0, 4.5, and 14.0 mM, respectively. The mixture was used to monitor the normal decline in lactase activity in rats that takes place after weaning. The data obtained by this method correlated with the levels of intestinal lactase activity in the same animals after being sacrificed.     

n-Octyl β-d-glucoside synthesis through β-glucosidase catalyzed condensation of glucose andn-octanol in a heterogeneous system with high glucose concentration

β-Glucosidase-catalyzed synthesis ofn-octyl β-d-glucoside through condensation was performed by mixing glucose and octanol saturated with acetate buffer at 50°C. The highest yield of synthesis, 28% based on glucose, was obtained at the total glucose concentration of 0.1 mol/1. At higher glucose concentrations, the product concentration increased but the yield decreased. The stability of the enzyme in the system was so high that, even in the free form, it could be used repeatedly for synthesis without a significant loss in its activity. The overall yield of the four consecutive batch syntheses was 58% when the initial glucose concentration was adjusted to 0.1 mol/1 for each batch.     

Cyanidin 3-[6-(p-coumaroyl)-2-(xylosyl)-glucoside]-5-glucoside and other anthocyanins from fruits of sambucus canadensis

From the fruits of Sambucus canadensis four anthocyanin glycosides have been isolated by successive application of an ion-exchange resin, droplet-counter chromatography and gel filtration. The structure of the novel, major (69.8%) pigment, cyanidin 3-O-[6-O-(E-p-coumaroyl-2-O-(β- -xylopyranosyl)-β- -glucopyranoside]-5-O-β- -glucopyranoside, was determined by means of chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques. The other anthocyanins were identified as cyanidin 3-sambubioside-5-glucoside (22.7%), cyanidin 3-sambubioside (2.3 %) and cyanidin 3-glucoside (2.1 %).     

Enzymatic synthesis of trisaccharides and alkyl β-d-glucosides by the transglycosylation reaction of β-glucosidase from Fusarium oxysporum

Purified β-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl β-d-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl β-d-glucosides were 22–37% and 10–33% of the total sugar, respectively. The enzyme retained 70–80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, β-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl β-d-glucosides.     

From methyl -glucopyranoside to methyl -allopyranoside via the Mitsunobu reaction

Methyl β- -glucopyranoside reacted with a 4-molar excess of the Mitsunobu reagents (triphenylphosphine–diethyl azodicarboxylate–benzoic acid) under Weinges et al. [Carbohydr. Res., 164 (1987) 453–458] conditions to furnish four differently benzoylated methyl β- -allopyranosides in a very good overall yield. The same reaction applied to methyl α- -glucopyranoside yielded allosides in a low yield and nine other sugar products. These results give an insight into the course of the Mitsunobu esterification–inversion reaction.     

Some kinetic properties of the β- -glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β- -glucosidase (cellobiase, β- -glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β- -glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. -Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. -Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while -fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β- -glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

Chemoenzymatic synthesis of enantiomerically enriched α-hydroxyamides

A study on a chemoenzymatic synthesis of model α-hydroxyamide was performed. Special attention was paid to the optimization of the enzymatic process, both on the selection of enzyme and cosolvent. An intriguing influence of cosolvent on the enantioselectivity of Wheat Germ Lipase and Amano PS Lipase catalyzed hydrolysis was observed, as the results obtained proved that enzyme's enantioselectivity is directly correlated with cosolvent's hydrophobicity. In the best example (Wheat Germ lipase, Et₂O used as a cosolvent), the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7%ee.     

β-Ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Synthesis of 2,3-di-O-glycosyl derivatives of methyl α- and β- -glucopyranoside

The syntheses are described of 2,3-di-O-glycosyl derivatives of methyl α- and β- -glucopyranoside having α- -manno-, β- -galacto-, α- -rhamno-, α- -fuco-, and β- -fuco-pyranosyl substitutents at O-2 and O-3. The syntheses involved glycoslation of methyl 4,6-O-(benzylidene-α- (24) and β- -glucopyranoside (21), and substituted derivatives of 21 bearing 2-O-(2,3,4,6-tetra-O-benzyl-α- -mannopyranosyl)-, -(2,3,4,6-tetra-O-acetyl-β- -galactopyranosyl)-, -(2,3,4-tri-O-benzyol-α- -rhamnopyranosyl)-, and-(2,3,4-tri-O-benzoyl-β- -fucopyranosyl) groups.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Microbial production of conjugated γ‐linolenic acid from γ‐linolenic acid by Lactobacillus plantarum AKU 1009a

Aims: Optimal production conditions of conjugated γ‐linolenic acid (CGLA) from γ‐linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. Methods and Results: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0·03% (w/v) α‐linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml^?1γ‐linolenic acid as a substrate in 5 ‐ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml^?1 dry cells] as the catalysts produced 8·8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis‐6,cis‐9,trans‐11‐octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis‐6,trans‐9,trans‐11‐octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. Conclusion: The practical process of CGLA production from γ‐linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. Significance and Impact of the Study: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.     

Immobilization of the glycosidases: α- -arabinofuranosidase and β- -glucopyranosidase from Aspergillus niger on a chitosan derivative to increase the aroma of wine. Part II

The purpose of this paper was to study the immobilization of two glycosidases, α- -arabinofuranosidase (EC 3.2.1.55) and β- -glucopyranosidase (EC 3.2.1.21), contained in a commercial preparation and purified as reported in Part I. The procedure which proved to be the best is simple and inexpensive to perform, employing the chitosan derivative, glyceryl-chitosan, especially synthesized and characterized, as a support. The glycosidases were adsorbed on this support and cross-linked with glutaraldehyde to prevent them from being released into the wine. Subsequent reduction of the biocatalyst with sodium borohydride allowed for improved stability. Finally, the immobilized glycosidases were compared with free ones in terms of optimum pH and temperature, stability over time, and kinetics parameters (K_m and V_max) after which they were employed for aromatizing a model wine solution containing aromatic precursors.     

β-1,4-Galactosyltransferase-catalyzed glycosylation of sugar derivatives: Modulation of the enzyme activity by α-lactalbumin, immobilization and solvent tolerance

The influence of different parameters on the activity of the β-1,4-galactosyltransferase (β-1,4-GalT) from bovine milk has been investigated using various acceptor and donor substrates. It was found that the “specifier” protein α-lactalbumin (α-LA), which interacts with β-1,4-GalT forming the lactose synthase (LS) complex, is not necessary when the acceptors are different glucopyranosides, and, in some cases, it can even have an inhibitory effect, like with the complex glucosides ginsenoside Rg₁ (1) and colchicoside (2). By optimization of the reaction conditions, the galactosylated and glucosylated derivatives of 2 were prepared, using UDP-Gal and UDP-Glc as sugar donors, respectively, and characterized. Moreover, β-1,4-GalT was covalently immobilized on Eupergit C 250 L in the absence of α-LA, and the synthetic performances of this immobilized biocatalyst were evaluated. Finally, the best organic cosolvents to be used both with β-1,4-GalT and the LS complex were identified.     

Heterooligosaccharide synthesis catalyzed by α-glucosidase from Bacillus stearothermophilus

α-Glucosidase from Bacillus stearothermophilus was used as a catalyst for oligosaccharide synthesis by reversed hydrolysis. The yield of disaccharides and trisaccharides depended strongly on the units of enzyme activity added, and on the stability of the enzyme under reaction conditions. When glucose was the only saccharide present in the reaction mixture with α-glucosidase, isomaltose (51%), nigerose (25%), maltose (14%) and kojibiose (10%) were formed. In 50% glucose solution, disaccharide concentrations reached up to 400 mmol/l and trisaccharides were also produced. When other saccharides (mannose or xylose), in addition to glucose, were present in the reaction mixture, both homodisaccharides and heterodisaccharides were formed, their quantity being dependent on the glucose/saccharide acceptor ratios. The highest yields of oligosaccharides were observed with glucose alone, consistent with the observation that the enzyme stability was highest with glucose as the sole saccharide.     

Facile synthesis of glucose-1-phosphate from starch by Thermus caldophilus GK24 α-glucan phosphorylase

The glgP gene encoding α-glucan phosphorylase (α-GP) from the thermopile Thermus caldophilus GK24 has been identified, cloned, and overexpressed in Escherichia coli and used to synthesize d-glucose-1-phospate (G1P) from an inexpensive starch. The enzyme, purified 6.5-fold, was isolated in 31% yield from the transformed E. coli, and gave a single band. The purified enzyme may exist as a homohexamer with an apparent molecular mass of a 550 kDa molecule, consisting of 90 kDa per subunit. The optimal pH and temperature were 7.0 and 70 °C in the α-GP reaction with starch producing G1P. Soluble starch (amylopectin, amylose) turned out to be a better substrate giving a higher yield of G1P than α-1,6-branched α-1,4-glucans (glycogen, potato starch, etc.). As a result, G1P was obtained in a good yield (47%, w/w) from the reaction containing 5% (w/v) soluble starch in 0.7 M potassium phosphate at pH 7.0. T. caldophilus α-GP shows a high tolerance (up to 0.7 M) of potassium phosphate and plays a critical role in shifting the reaction equilibrium in favor of G1P synthesis. The G1P product can be purified simply by ethanol precipitation, after removing the unreacted starch and inorganic phosphate by activated charcoal and magnesium acetate precipitation. It is concluded that T. caldophilus α-GP readily utilized in large scale synthesis of G1P.     

Improved synthesis and the crystal structure of methyl 4,6-dideoxy-4-(3-deoxy- -glycero-tetronamido)-α- -mannopyranoside, the methyl α-glycoside of the intracatenary repeating unit of the O-polysaccharide of Vibrio cholerae O:1

The crude product of deamination of the commercially available -homoserine was acetylated and the 2-O-acetyl-3-deoxy- -glycero-tetronolactone (18) formed was used to N-acylate methyl perosaminide (methyl 4-amino-4,6-dideoxy-α- -mannopyranoside, 12) and its 2,3-O-isopropylidene derivative. The major product isolated from the reaction was the crystalline methyl 4-(4-O-acetyl-3-deoxy- -glycero-tetronamido)-4,6-dideoxy-α- -mannopyranoside (1, 70–75%) resulting from acetyl group migration in the initially formed 2'-O-acetyl derivative. O-Deacetylation of 1 gave the title amide 2. Compound 2, obtained crystalline for the first time, was fully characterized, and its crystal structure was determined. Deoxytetronamido derivatives diastereomeric with 1 and 2, respectively, were obtained by the acylation of 12 with 2-O-acetyl-3-deoxy- -glycero-tetronolactone (prepared from -homoserine), and subsequent deacetylation. Structures of several byproducts of the reaction of 12 with 18 have been deduced from their spectral characteristics. Since these byproducts were various O-acetyl derivatives of 2, the title compound could be obtained in ≈ 90% yield by deacetylating (Zemplén) the crude mixture of N-acylation products, followed by chromatography.