An efficient process for production of n-acetylneuraminic acid using n-acetylneuraminic acid aldolase

Mahmoudian, Mahmoud; Noble, David; Drake, Christopher S.; Middleton, Robert F.; Montgomery, D.S.; Piercey, J.E.; Ramlakhan, D.; Todd, M. J.; Dawson, Michael J.

doi:10.1016/s0141-0229(96)00180-9

Cited by 125 publications

(61 citation statements)

References 28 publications

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“…This is quite remarkable stability compared with the stability of EcNAL, which at an alkaline pH showed a substantial loss of activity after 8 h (30% at pH 10.5 and 75% at pH 11.3) (9). This fact, together with the above-mentioned higher activity at alkaline pH, reinforces the possibility of using LpNAL to produce Neu5Ac at basic pHs, where the chemical isomerization of GlcNAc to ManNAc is more favorable (8,9,26).…”

Section: Resultssupporting

confidence: 57%

Molecular Characterization of a Novel N -Acetylneuraminate Lyase from Lactobacillus plantarum WCFS1

Sánchez-Carrón

García-García

López-Rodríguez

et al. 2011

Appl Environ Microbiol

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N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of Nacetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-D-mannosamine (ManNAc). In nature, Nacetylneuraminate lyase occurs mainly in pathogens. However, this paper describes how an N-acetylneuraminate lyase was cloned from the human gut commensal Lactobacillus plantarum WCFS1 (LpNAL), overexpressed, purified, and characterized for the first time. This novel enzyme, which reaches a high expression level (215 mg liter ؊1 culture), shows similar catalytic efficiency to the best NALs previously described. This homotetrameric enzyme (132 kDa) also shows high stability and activity at alkaline pH (pH > 9) and good temperature stability (60 to 70°C), this last feature being further improved by the presence of stabilizing additives. These characteristics make LpNAL a promising biocatalyst. When its sequence was compared with that of other, related (real and putative) NALs described in the databases, it was seen that NAL enzymes could be divided into four structural groups and three subgroups. The relation of these subgroups with human and other mammalian NALs is also discussed.

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Section: Resultssupporting

confidence: 57%

Molecular Characterization of a Novel N -Acetylneuraminate Lyase from Lactobacillus plantarum WCFS1

Sánchez-Carrón

García-García

López-Rodríguez

et al. 2011

Appl Environ Microbiol

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“…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.…”

Section: Discussionmentioning

confidence: 88%

“…The equilibrium lies on the side of pyruvate and ManNAc. Thus, for the production of Neu5Ac, excess pyruvate and ManNAc are needed to achieve a high yield (23 Fig. S3 in the supplemental material).…”

Section: Resultsmentioning

confidence: 99%

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

Gao

Zhang

et al. 2011

Appl Environ Microbiol

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Production of N-acetyl-D-neuraminic acid (Neu5Ac) via biocatalysis is traditionally conducted using isolated enzymes or whole cells. The use of isolated enzymes is restricted by the time-consuming purification process, whereas the application of whole cells is limited by the permeability barrier presented by the microbial cell membrane. In this study, a novel type of biocatalyst, Neu5Ac aldolase presented on the surface of Bacillus subtilis spores, was used for the production of Neu5Ac. Under optimal conditions, Neu5Ac at a high concentration (54.7 g liter ؊1 ) and a high yield (90.2%) was obtained under a 5-fold excess of pyruvate over N-acetyl-D-mannosamine. The novel biocatalyst system, which is able to express and immobilize the target enzyme simultaneously on the surface of B. subtilis spores, represents a suitable alternative for value-added chemical production.

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“…D-Sialic acid aldolase catalyzes the reversible conversion of N-acetyl-D-mannosamine (D-ManNAc) and pyruvate to D-sialic acid (NeuAc), and it is important for sialic acid catabolism (11). The enzyme has a wide application in the synthesis of D-sialic acid and its derivatives (12)(13)(14)(15)(16). The crystal structures of D-sialic acid aldolases from both E. coli (9) and Haemophilus influenzae (17) have been reported, which showed a tetramer with each sub-unit comprising an eight-stranded ␣/␤ barrel.…”

mentioning

confidence: 99%

Modulation of Substrate Specificities of d-Sialic Acid Aldolase through Single Mutations of Val-251

Chou

et al. 2011

Journal of Biological Chemistry

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In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into a new enzyme with relaxed specificity, denoted RSaldolase (also known formerly as L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase), which showed a preferred selectivity toward L-KDO. To investigate the underlying molecular basis, we determined the crystal structures of D-sialic acid aldolase and RS-aldolase. All mutations are away from the catalytic center, except for V251I, which is near the opening of the (␣/␤) 8 -barrel and proximal to the Schiff base-forming Lys-165. The change of specificity from D-sialic acid to RS-aldolase can be attributed mainly to the V251I substitution, which creates a narrower sugar-binding pocket, but without altering the chirality in the reaction center. The crystal structures of D-sialic acid aldolase⅐L-arabinose and RS-aldolase⅐hydroxypyruvate complexes and five mutants (V251I, V251L, V251R, V251W, and V251I/V265I) of the D-sialic acid aldolase were also determined, revealing the location of substrate molecules and how the contour of the active site pocket was shaped. Interestingly, by mutating Val251 alone, the enzyme can accept substrates of varying size in the aldolase reactions and still retain stereoselectivity. The engineered D-sialic acid aldolase may find applications in synthesizing unnatural sugars of C 6 to C 10 for the design of antagonists and inhibitors of glycoenzymes.Carbohydrates on cell surfaces play a pivotal role in the molecular recognition processes in various cellular interactions. Interfering with the recognition processes between pathogens and hosts may present therapeutic strategies for infectious diseases (1, 2). D-Sialic acids are widely found in nature, from bacteria to plants and animal tissues. Cell-surface glycoproteins and glycolipids frequently contain D-sialic acids at the termini of the oligosaccharide chains. The invading bacteria and viruses also target the cell-surface sialic acids of their hosts, which constitutes an important step for infectivity (3). On the other hand, the monosaccharide D-3-deoxy-manno-2-octulosonic acid (D-KDO) 2 has never been identified in vertebrates, but it is an important component in Gram-negative bacteria (4 -6). Because D-KDO is vital to the structural stability of bacterial outer membrane, targeting D-KDO and the related biosynthetic enzymes may be a new strategy for future antibacterial agent discovery.Currently, unnatural enantiomeric compounds become increasingly important in pharmaceutical implementations. Enantiomers (D-form) of natural peptides have been used to target cell-surface D-sugars. These D-peptide cell-surface sugar binders might serve as new drug candidates for infectious disease (7) and as tools for studying the interactions between proteins and carbohydrate. Owing to the resistance to hydrolases and high binding affinity to sugars, the unnatural D-peptides can become potential therapeutic agents. Through mirror-image phage display, natural L-peptides can be...

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An efficient process for production of n-acetylneuraminic acid using n-acetylneuraminic acid aldolase

Cited by 125 publications

References 28 publications

Molecular Characterization of a Novel N -Acetylneuraminate Lyase from Lactobacillus plantarum WCFS1

Molecular Characterization of a Novel N -Acetylneuraminate Lyase from Lactobacillus plantarum WCFS1

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

Modulation of Substrate Specificities of d-Sialic Acid Aldolase through Single Mutations of Val-251

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