Synthesis of L-Rhamnose and N-Acetyl-D-Glucosamine Derivatives Entering in the Composition of Bacterial Polysaccharides by Use of Glucansucrases

Abstract

Transglucosylation reactions using sucrose as glucosyl donor and either N-acetyl-D-glucosamine, L-rhamnose, or methyl α -L-rhamnopyranoside as acceptors were carried out with recombinant glucansucrases from families 70 and 13 of glycoside-hydrolases. Depending on the enzyme specificity, various carbohydrate structures were synthesized and characterized including α -D-glucopyranosyl-(1 → 6)-N-acetyl-D-glucosamine, α -D-glucopyranosyl-(1 → 4)-N-acetyl-D-glucosamine, α -D-glucopyranosyl-(1 → 1)-β -L-rhamnopyranoside, α -D-glucopyranosyl-(1 → 4)-α -D-glucopyranosyl-(1 → 1)-β -L-rhamnopyranoside, methyl α -D-glucopyranosyl-(1 → 4)-α -L-rhamnopyranoside, and methyl α -D-glucopyranosyl-(1 → 3)-α -L-rhamnopyranoside. Disaccharides were obtained with yields going up to 64%. The structural diversity generated as well as the obtained yields appear to be related to enzyme active site architecture, which can be modulated and improved by enzyme engineering. Several of the obtained disaccharides enter in the composition of surface polysaccharides of pathogenic bacteria, among which is Shigella flexneri. Our results outline the potential of glucansucrases in the chemo-enzymatic synthesis of complex carbohydrates of biological interest whose chemical synthesis may be seen as a limitation.

Keywords:

• Glucansucrase
• Enzymatic glucosylation
• L-Rhamnose
• N-Acetyl-D-glucosamine
• Bacterial polysaccharide

Notes

^a The conversion degree of the acceptor was calculated using the formula: ([D-GlcpNAc]_initial – [D-GlcpNAc]_t24h) / [D-GlcpNAc]_initial× 100.

^b % Monoglucosylated D-GlcpNAc = [Monoglucosylated D-GlcpNAc]t_f/[consumed sucrose] t_f× 100.

^aThe conversion degree of the acceptor was calculated using the formula ([Acceptor]_initial – [Acceptor]_t24h) / [Acceptor]_initial× 100. At final time (24 h), sucrose was fully consumed.

^aThe conversion degree of the acceptor was calculated using the formula: ([Acceptor]_initial – [Acceptor] _t24h)/[Acceptor]_initial× 100.

^bMonoglucosylated rhamnose yield is the molar ratio: [Glc-Acceptor]_t24h/[consumed sucrose]_t24h× 100. At final time, sucrose is fully consumed.

^cDi-glucosylated rhamnose yield is the molar ratio: [Glc₂-Acceptor] _t24h /[consumed sucrose]_t24h× 100. At final time, sucrose is fully consumed.

^d% L-Rhap converted into P3 is the molar ratio: [Glc-Acceptor]_t24h/[Acceptor]_t0× 100.

^e% L-Rhap converted into P4 is the molar ratio: [Glc₂-Acceptor]_t24h/[Acceptor]_t0× 100.

^a The conversion degree of the acceptor was calculated according to the formula ([Acceptor]_initial – [Acceptor]_t24h) / [Acceptor]_initial× 100.

^b The percentage of monoglucosylated acceptor was determined by the ratio [Glc-Acceptor] _t24h / [consumed sucrose]_t24h× 100. At final time, sucrose was totally consumed.