Enzymatic synthesis of l-fucose from l-fuculose using a fucose isomerase from Raoultella sp. and the biochemical and structural analyses of the enzyme

Abstract

Backgroundl-Fucose is a rare sugar with potential uses in the pharmaceutical, cosmetic, and food industries. The enzymatic approach using l-fucose isomerase, which interconverts l-fucose and l-fuculose, can be an efficient way of producing l-fucose for industrial applications. Here, we performed biochemical and structural analyses of l-fucose isomerase identified from a novel species of Raoultella (RdFucI).ResultsRdFucI exhibited higher enzymatic activity for l-fuculose than for l-fucose, and the rate for the reverse reaction of converting l-fuculose to l-fucose was higher than that for the forward reaction of converting l-fucose to l-fuculose. In the equilibrium mixture, a much higher proportion of l-fucose (~ ninefold) was achieved at 30 °C and pH 7, indicating that the enzyme-catalyzed reaction favors the formation of l-fucose from l-fuculose. When biochemical analysis was conducted using l-fuculose as the substrate, the optimal conditions for RdFucI activity were determined to be 40 °C and pH 10. However, the equilibrium composition was not affected by reaction temperature in the range of 30 to 50 °C. Furthermore, RdFucI was found to be a metalloenzyme requiring Mn2+ as a cofactor. The comparative crystal structural analysis of RdFucI revealed the distinct conformation of α7–α8 loop of RdFucI. The loop is present at the entry of the substrate binding pocket and may affect the catalytic activity.ConclusionsRdFucI-catalyzed isomerization favored the reaction from l-fuculose to l-fucose. The biochemical and structural data of RdFucI will be helpful for the better understanding of the molecular mechanism of l-FucIs and the industrial production of l-fucose.

A comprehensive review on microbial production of 1,2-propanediol: micro-organisms, metabolic pathways, and metabolic engineering

“…For 1,2-PDO biosynthesis from l-fucose or l-rhamnose, four key enzymes are involved, i.e., l-fucose/rhamnose isomerase (fucI/rhaA) [34,35], l-fuculokinase/ rhamnulokinase (fucK/rhaB) [36,37], l-fuculose-1-phosphate/rhamnulose-1-phosphate aldolase (fucA/rhaD) [38,39] and propanediol oxidoreductase (fucO) [40]. The isomerase, kinase, and aldolase were found to be functional in both aerobic and anaerobic environments, which means that the formation of neither DHAP nor l-lactaldehyde was influenced by the environment [41].…”

High-Level Production of l-Fucose by Plasmid-Free or Antibiotic-Independent Metabolically Engineered Escherichia coli Strains

“…14 When producing L-fucose from Lfuculose, the highest conversion ratio reached up to 90%. 15,16 However, the substrate L-fuculose is still a rare monosaccharide and very expensive, so the enzymatic synthesis of L-fucose is still not feasible for large-scale production.…”

“…Two types of isomerases, l -fucose isomerase (EC 5.3.1.25) and d -arabinose isomerase (EC 5.3.1.3), can catalyze the reversible isomerization between l -fucose and l -fuculose . When producing l -fucose from l -fuculose, the highest conversion ratio reached up to 90%. , However, the substrate l -fuculose is still a rare monosaccharide and very expensive, so the enzymatic synthesis of l -fucose is still not feasible for large-scale production.…”

Rational design of GDP‑d‑mannose mannosyl hydrolase for microbial l‑fucose production

“…Other routes for fabricating l-fucose have been developed to meet the increasing demand for this chemical. For example, l-fucose can be chemically converted from d-mannose [15], and it can also be extracted from the enzymatic hydrolysate of l-fucose-rich microbial EPS [16] or bio converted from rarer monosaccharides, such as l-fuculose [17].…”