Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins

Abstract

Background: Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions. Both were tagged with glutathione Stransferase (GST) at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D), and the other with five contiguous arginine residues (5R).

Bioengineering of Bacterial Polymer Inclusions Catalyzing the Synthesis of N -Acetylneuraminic Acid

“…Reported activities of immobilized GlcNAc-2 epimerases range from 3.4 to 29 U/mg protein (11,12). The C terminus of PhaC has been recognized as important for PhaC attachment to the bead surface (19), and disruption of the PhaC C terminus may explain both the lower level of fusion protein attachment seen in the double fusion protein and its lower epimerase activity.…”

“…The conditions for NanA-PhaC bead-recycling reactions were 250 mM pyruvate and 100 mM ManNAc for 20 h. The full biosynthesis of Neu5Ac from GlcNAc and pyruvate was initially carried out with 100 mM sodium pyruvate, 250 mM GlcNAc, 10 mM ATP, and 10 mM MgCl 2 at 50°C for 44 h. PHA beads were present at 10 mg (wet bead weight) in 50 mM potassium phosphate buffer (pH 7.5). The final conditions were as described above, except the reactions were carried out at 30°C for 50 h using a GlcNAc starting concentration of 100 mM (12).…”

“…Slr1975 epimerase activity in 1 ml of 50 mM potassium phosphate buffer (pH 7.5) was determined at 37°C. Isolated beads were reacted with 20 mM ManNAc (with 5 mM ATP and 10 mM MgCl 2 ) for 30 min, the resulting mixture was centrifuged (6,000 ϫ g, 4 min), and an aliquot was boiled at 100°C for 5 min and subjected to high-performance liquid chromatography (HPLC) (12). One unit was defined as the amount of Slr1975 needed to catalyze the production of 1 mol GlcNAc per min.…”

“…Usually, enzymes are produced in a recombinant system, isolated, and then attached to a solid support system. This type of approach has been applied to both aldolase and epimerase enzymes (11,12). Alternatively, the production and immobilization of the enzymes can occur in a single step, avoiding the potentially expensive attachment step and simplifying enzyme isolation from the recombinant system.…”

Chemoenzymatic Synthesis of N -Acetyl- d -Neuraminic Acid from N -Acetyl- d -Glucosamine by Using the Spore Surface-Displayed N -Acetyl- d -Neuraminic Acid Aldolase

“…Besides the preparation of the two biocatalysts, this process also needed an adjustment of the ratio between the two biocatalysts (11). Using the immobilized GlcNAc 2-epimerase (2.4 U/ml) and Neu5Ac aldolase (7.2 U/ml) as the catalyst, 41.6 g liter Neu5Ac was produced after 80 h of biotransformation (20). In this process, 10 mM ATP should be added in the reaction mixture to activate the GlcNAc 2-epimerase-catalyzed epimerization reaction.…”