Production of N-acetyl-d-neuraminic acid by coupling bacteria expressing N-acetyl-d-glucosamine 2-epimerase and N-acetyl-d-neuraminic acid synthetase

Tabata, Kazuhiko; Koizumi, Satoshi; Endo, Tetsuo; Ozaki, Akio

doi:10.1016/s0141-0229(01)00515-4

Cited by 65 publications

(46 citation statements)

References 33 publications

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“…The pH optimum of PhGn2E lies slightly higher than other characterized epimerases, which were reported to lie between 6.8-8.5 for the porcine, Anabaena sp., B. ovatus, and Synechocystis sp. PCC6803 homologues [22,23,27,29]. The addition of EDTA or metal ions did not significantly decrease or inhibit PhGn2E activity, which indicates that metal ions are not involved in the catalytic mechanism of the enzyme.…”

Section: Characterizationmentioning

confidence: 86%

“…The optimum temperature of PhGn2E was determined to be 37 • C. This value is comparable with the reported temperature optimum for the cyanobacterium Synechocystis sp. PCC 6803 [27], whereas B. ovatus, porcine, and A. variabilis isoforms showed higher temperature optima (45 to 60 • C, respectively) [19,28,29]. The pH optimum of PhGn2E lies slightly higher than other characterized epimerases, which were reported to lie between 6.8-8.5 for the porcine, Anabaena sp., B. ovatus, and Synechocystis sp.…”

Section: Characterizationmentioning

confidence: 88%

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N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism

Wang

Laborda

et al. 2016

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Abstract:The control of cellular N-acetylmannosamine (ManNAc) levels has been postulated to be an effective way to modulate the decoration of cell surfaces with sialic acid. N-acetylglucosamine 2-epimerase catalyzes the interconversion of N-acetylglucosamine (GlcNAc) and ManNAc. Herein, we describe the cloning, expression, purification and biochemical characterization of an unstudied N-acetylglucosamine 2-epimerase from Pedobacter heparinus (PhGn2E). To further characterize the enzyme, several N-acylated glucosamine derivatives were chemically synthesized, and subsequently used to test the substrate specificity of PhGn2E. Furthermore, NMR studies of deuterium/hydrogen exchange at the anomeric hydroxy group and C-2 positions of the substrate in the reaction mixture confirmed for the first time the postulated epimerization reaction via ring-opening/enolate formation. Site-directed mutagenesis of key residues in the active site showed that Arg63 and Glu314 are directly involved in proton abstraction and re-incorporation onto the substrate. As all mechanistically relevant active site residues also occur in all mammalian isoforms, PhGn2E can serve as a model N-acetylglucosamine 2-epimerase for further elucidation of the active site mechanism in these enzymes.

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Section: Characterizationmentioning

confidence: 86%

Section: Characterizationmentioning

confidence: 88%

N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism

Wang

Laborda

et al. 2016

Catalysts

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“…NanA is preferred because its substrate (pyruvate) is much more readily available than that of Neu5Ac synthase (phosphoenolpyruvate) (28,33). In the present study, a Bacillus subtilis surface display system was constructed.…”

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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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“…Many CDSs in various bacterial genomes, whose deduced amino acid sequences exhibit low identity to those of AGEs, have been annotated as hypothetical proteins of unknown function or putative AGEs, but few studies of functional AGEs (or reninbinding proteins) have been reported, and the enzymes have been found exclusively in mammals [11][12][13][14][15][16] and cyanobacteria. [17][18][19] Based on homology and phylogenetic analyses, we suggest that many CDSs in the bacterial genome sequences that share low but nonnegligible amino acid identity to AGE should be annotated as CE-like or putative CE proteins. Furthermore, we argue that CE recognizes and catalyzes a y To whom correspondence should be addressed.…”

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confidence: 99%

Identification of the Cellobiose 2-Epimerase Gene in the Genome ofBacteroides fragilisNCTC 9343

Senoura

Taguchi

Ito

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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Production of N-acetyl-d-neuraminic acid by coupling bacteria expressing N-acetyl-d-glucosamine 2-epimerase and N-acetyl-d-neuraminic acid synthetase

Cited by 65 publications

References 33 publications

N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism

N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism

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

Identification of the Cellobiose 2-Epimerase Gene in the Genome ofBacteroides fragilisNCTC 9343

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