Coupled bioconversion for preparation of N-acetyl-d-neuraminic acid using immobilized N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic acid lyase

Abstract

N-Acetyl-D: -neuraminic acid (Neu5Ac) can be produced from N-acetyl-D: -glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-D: -manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-D: -neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-D: -glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was approximately 73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.

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

“…Although the fusion protein for the double-enzyme bead exhibits higher aldolase activity than beads displaying only the aldolase, the abundance of fusion protein on the surface of the aldolase-only beads means that the two beads have similar aldolase activities as a function of bead weight. As the two beads have high aldolase activities, the activity of PHAimmobilized NanA compares favorably with the previously reported activities of 2.5 to 36 U/mg protein (11,12), and they performed similarly over the 44-h reaction time. Recycling reactions showed that NanA-PhaC beads retain at least 70% conversion of ManNAc over four reaction cycles (Fig.…”

“…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.…”

“…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.…”

Production of N -Acetyl- d -Neuraminic Acid by Use of an Efficient Spore Surface Display System

“…After optimization of the procedure, recombinant spores harboring the pEB03-cotG-nanA plasmid were able to catalyze 0.2 M ManNAc into 0.18 M (54.70 g liter Ϫ1 ) Neu5Ac in 16 h. In previous studies, NanA was purified and immobilized to understand the conversion reaction and circular catalysis (10,23,36). In our study, we were able to easily separate the recombinant spores from the catalysis system and subject them to circular catalysis.…”

Tailor‐made biocatalysts enzymes for the fine chemical industry in China

“…1, listed as follows) have been sold by 15 of our industrial partners who use bioprocess tailor-made biocatalysts enzymes R&D platform: penicillin G acylase [2], immobilized deacetyl esterase [3], immobilized arginase, D-p-hydroxyphenylglycine [4], L-2-aminobytyric acid [5], L-tert-leucine, DL-alanine [6], sialic acid [7], β-alanine, L-citrulline, DL-serine, D-valine, L-norvaline, trehalose, a-ketoglutaric acid [8], S-3-hycrobial hosts besides E. coli, such as yeast, bacilli, and filamentous fungi.…”