Lars Hedbys

Synthesis of the disaccharide 6-O-β-d-galactopyranosyl-2-acetamido-2-deoxy-d-galactose using immobilized β-galactosidase

Abstract The disaccharide 6-O-β- d -galactopyranosyl-2-acetamido-2-deoxy- d -galactose has been synthesized by transfer of the β- d -galactopyranosyl residue from lactose to 2-acetamido-2-deoxy- d -galactose utilizing the transferase activity of β-galactosidase from E. coli . To make the enzyme reusable, it was applied in an immobilized form covalently bound to Sepharose CL-4B. The yield of the disaccharide was about 20%, calculated on the amount of acetamido-deoxy- d -galactose added. The disaccharide could also be obtained by reversal of the hydrolytic activity of the enzyme, using d -galactose and 2-acetamido-2-deoxy- d -galactose as substrate. The yield in this reaction, however, was only 2–3 % under the conditions applied.

Synthesis of 2-acetamido-2-deoxy-3-O-β-d-galactopyrano-syl-d-galactose by the sequential use of β-d-galactosidases from bovine testes and escherichia coli

beta-D-Galp-(1----3)-D-GalNAc (1) was synthesised from lactose and GalNAc on a mmolar scale by transgalactosylation using beta-D-galactosidase from bovine testes. The large proportions of unwanted oligosaccharides in the product mixture were removed by treatment with beta-D-galactosidase from E. coli, which left 1, monosaccharides, and a small proportion of trisaccharides. Carbon-Celite chromatography then gave 1 in a yield of 21% based on the GalNAc added.

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Galβ1-3GlcNAc (1) and Galβ1-3GlcNAcβ-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes β-galactosidase with lactose as donor andN-acetylglucosamine and GlcNAcβ-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with β-galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Galβ1-6GlcNAcβ-SEt (3) was synthesized by the transglycosylation reaction using β-galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.

Synthesis of mannose oligosaccharides via reversal of the α-mannosidase reaction

SummaryThree naturally occurring isomers of the disaccharideO-α-d-mannosyl-d-mannoside were synthesized by reversing the hydrolytic activity of jack bean α-mannosidase at 75°C in a very high concentration of mannose. Higher oligosaccharides were also obtained at the later stages of the reaction. The maximum total yield of disaccharides was 37% (w/w) based on the total amount of saccharides.

Enzymatic synthesis of monosaccharide—amino acid conjugates

Abstract Enzymatic condensation between amino acids and monosaccharides was studied. A screening involving the OH-containing amino acids serine and threonine and the monosaccharide substrates for the enzymes α - N -acetylgalactosaminidase, α-galactosidase, α-mannosidase, β-xylosidase, and β-galactosidase was carried out. Enzymatic condensation was observed with α-mannosidase and α - N -acetylgalactosaminidase. Products were only detected under reversed hydrolysis conditions, not under transglycosylation conditions with activated substrates. Both l - and d -isomers of the amino acids were substrates for α-mannosidase and α - N -acetylgalactosaminidase. A closer investigation of the formation of α- d -Man-(1–3′)- l -Ser and α- d -GalNAc-(1–3′)- l -Ser was carried out at 35°C and 55°C, at which temperature the reaction rates were greatly increased.

Studies of the reversed α-mannosidase reaction in high concentrations of mannose

Abstract The reversal of the hydrolytic activity of α-mannosidase from jack bean has been studied in high concentrations of mannose. From an initial concentration of 85% mannose (w/w), a maximum yield of 70% (w/w) di- and oligosaccharides was obtained. The products were separated by gel chromatography and high-performance liquid chromatography, and characterized by gas-liquid chromatographymass spectrometry and 1 H-nuclear magnetic resonance spectroscopy. The equilibrium for the formation of di- and oligosaccharides was investigated, and the equilibrium constant for the formation of disaccharides was calculated to be 5.5 ± 0.9. The stability of the enzyme and the rate of formation of disaccharides were studied as functions of temperature, pH, and starting concentration of mannose. It was shown that the enzyme was not inhibited by a total sugar concentration of 83% (w/w), compared with 40%.

[21] Disaccharide synthesis with immobilized β-galactosidase

Publisher Summary This chapter focuses on disaccharide synthesis with immobilized β-galactosidase. The β-galactoside, lactose, combines initially with the enzyme to form a galactosylated enzyme with the concomitant release of glucose. The galactosylated enzyme may experience two fates. Either it is hydrolyzed with water to give free enzyme and galactose, or it may transfer its galactosyl moiety to a second sugar, in this case N-acetylgalactosamine. This transgalactosylation reaction leads to the formation of galactosyl-N-acetylgalactosamine. However, the enzyme is used in the immobilized form to facilitate its reuse and its easy removal from the reaction mixture when the yield was at its maximum. In addition, and of relevance for future studies, immobilization may lead to more stable enzyme preparations of importance when carrying out enzymatic synthesis at elevated temperatures and in organic solvents and/or solvents with low water activity.