Why is sialic acid attracting interest now? Complete enzymatic synthesis of sialic acid with N-acylglucosamine 2-epimerase.

N-Acetylneuraminic acid (NeuAc), the representative of the family of sialic acids, is an important molecule in biological recognition systems. Currently, NeuAc-based novel pharmaceutical agents and diagnostic reagents for influenza viruses are highly required in medical fields, and larger amounts of NeuAc are in demand worldwide. NeuAc had been prepared either from colominic acid (a homopolymer of NeuAc) produced by fermentation or from natural sources such as edible bird's nests, milk or eggs. However, the drawbacks of such conventional methods make them unsuitable for large-scale production of NeuAc. Recently, the N-acylglucosamine 2-epimerase (AGE) gene from porcine kidney was cloned in E. coli, and a strain with a high AGE expression level was constructed for practical applications, which enabled the complete enzymatic synthesis of NeuAc with a high conversion rate from the substrates, namely, N-acetylglucosamine and pyruvate. In addition, NeuAc of highest purity could be produced economically via its direct crystallization from the reaction mixture without any column purification processes. Such a simple procedure promises to be applicable to the mass production of sialic acid at the lowest cost.

[1]  K. Murata,et al.  Molecular Cloning and Identification of N-Acyl-D-glucosamine 2-Epimerase from Porcine Kidney as a Renin-binding Protein* , 1996, The Journal of Biological Chemistry.

[2]  B. Morgan,et al.  Effects of administration of N-acetylneuraminic acid (NANA) on brain NANA content and behavior. , 1980, The Journal of nutrition.

[3]  B. Leckie,et al.  A renin inhibitor from rabbit kidney: conversion of a large inactive renin to a smaller active enzyme. , 1975, Circulation research.

[4]  J. Paulson,et al.  Recognition of monovalent sialosides by influenza virus H3 hemagglutinin. , 1987, Virology.

[5]  T. Springer,et al.  Sticky sugars for selectins , 1991, Nature.

[6]  L. Juneja,et al.  Preparation ofN-acetylneuraminic acid from delipidated egg yolk , 1992, Glycoconjugate Journal.

[7]  G. Blix,et al.  The Isolation of Chondrosamine from Gangliosides and from Submaxillary Mucin. , 1952 .

[8]  G. Blix Über die Kohlenhydratgruppen des Submaxillarismucins. , 1936 .

[9]  K. Murata,et al.  Isolation of a constitutive N-acetylneuraminate lyase-producing mutant of escherichia coli and its use for NPL production. , 1989 .

[10]  L. Juneja,et al.  Large-scale preparation of sialic acid from chalaza and egg-yolk membrane. , 1991, Carbohydrate research.

[11]  J. Littlechild,et al.  A new method for the purification of 30S ribosomal proteins from Escherichia coli using nondenaturing conditions. , 1978, Biochemistry.

[12]  D. Sirbasku,et al.  Purification and properties of N-acetylneuraminate lyase from beef kidney cortex. , 1970, Biochimica et biophysica acta.

[13]  G. Whitesides,et al.  Synthesis of CMP-NeuAc from N-acetylglucosamine: generation of CTP from CMP using adenylate kinase , 1988 .

[14]  S. Roseman,et al.  THE SIALIC ACIDS. V. N-ACYL-D-GLUCOSAMINE 2-EPIMERASE. , 1965, The Journal of biological chemistry.

[15]  L. Firsov,et al.  Refined structure for the complex of acarbose with glucoamylase from Aspergillus awamori var. X100 to 2.4-A resolution. , 1994, The Journal of biological chemistry.

[16]  S. Takahashi Structure of the gene encoding rat renin binding protein. , 1997, Bioscience, biotechnology, and biochemistry.

[17]  Udo Kragl,et al.  Enzymatic Two‐Step Synthesis of N‐Acetyl‐neuraminic Acid in the Enzyme Membrane Reactor , 1991 .

[18]  J. Lowe,et al.  ELAM-1-dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA , 1990, Cell.

[19]  P. Lacy,et al.  The expression of renin‐binding protein and renin in the kidneys of rats with two‐kidney one‐clip hypertension , 2000, Journal of hypertension.

[20]  C. Augé,et al.  Synthesis with immobilized enzyme of the most important sialic acid. , 1984 .

[21]  S. Takahashi,et al.  Purification of high molecular weight (HMW) renin from porcine kidney and direct evidence that the HMW renin is a complex of renin with renin binding protein (RnBP). , 1983, Journal of biochemistry.

[22]  M. Pawlita,et al.  UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation. , 1999, Science.

[23]  W. Reutter,et al.  A Bifunctional Enzyme Catalyzes the First Two Steps in N-Acetylneuraminic Acid Biosynthesis of Rat Liver , 1997, The Journal of Biological Chemistry.

[24]  G. W. Boyd A Protein‐Bound Form of Porcine Renal Renin , 1974, Circulation research.

[25]  J. Vliegenthart,et al.  Configuration of substrate and products of N-acetylneuraminate pyruvate-lyase from Clostridium perfringens. , 1983, Biochemical and biophysical research communications.

[26]  Y. Tsukada,et al.  Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosamine 2-epimerase and N-acetylneuraminate lyase. , 1998, Carbohydrate research.

[27]  Y. Uchida,et al.  Production of microbial neuraminidases induced by colominic acid. , 1974, Biochimica et biophysica acta.

[28]  H. Faillard The early history of sialic acids. , 1989, Trends in biochemical sciences.

[29]  P. Karplus,et al.  Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca , 1997, Nature Structural Biology.

[30]  L. Lasky,et al.  Selectins: interpreters of cell-specific carbohydrate information during inflammation. , 1992, Science.

[31]  M. Shimosaka,et al.  Molecular cloning of the N-acetylneuraminate lyase gene in Escherichia coli K-12 , 1986, Applied Microbiology and Biotechnology.

[32]  F. Luft,et al.  Normal Blood Pressure and Plasma Renin Activity in Mice Lacking the Renin-binding Protein, a Cellular Renin Inhibitor* , 2000, The Journal of Biological Chemistry.

[33]  J. N. Varghese,et al.  Structure of the catalytic and antigenic sites in influenza virus neuraminidase , 1983, Nature.

[34]  Chi‐Huey Wong,et al.  Enzymes in carbohydrate synthesis: N-acetylneuraminic acid aldolase catalyzed reactions and preparation of N-acetyl-2-deoxy-D-neuraminic acid derivatives , 1988 .

[35]  S. Hakomori,et al.  ABH and Related Histo‐Blood Group Antigens; Immunochemical Differences in Carrier Isotypes and Their Distribution 1 , 1989, Vox sanguinis.

[36]  Roland Schauer,et al.  Achievements and challenges of sialic acid research , 2000, Glycoconjugate Journal.

[37]  Y. Uchida,et al.  A new enzymatic method for the determination of sialic acid in serum and its application for a marker of acute phase reactants. , 1981, The Kobe journal of medical sciences.

[38]  A. Varki,et al.  Biological roles of oligosaccharides: all of the theories are correct , 1993, Glycobiology.

[39]  Chi-Huey Wong,et al.  Unusual stereoselectivity in sialic acid aldolase-catalyzed aldol condensations: Synthesis of both enantiomers of high carbon monosaccharides , 1992 .

[40]  B. Mikami,et al.  Crystallization and preliminary X-ray diffraction studies of N-acyl-D-glucosamine 2-epimerase from porcine kidney. , 1996, Journal of biochemistry.

[41]  S. Roseman The synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion. , 1970, Chemistry and physics of lipids.

[42]  S. Hakomori,et al.  Tumor-associated carbohydrate antigens. , 1984, Annual review of immunology.

[43]  B. Mikami,et al.  Crystal structure of N-acyl-D-glucosamine 2-epimerase from porcine kidney at 2.0 A resolution. , 2000, Journal of molecular biology.

[44]  S. Takahashi,et al.  The human gene for renin-binding protein. , 1992, The Journal of biological chemistry.

[45]  A. Fukamizu,et al.  Recent advances in the study of renin and angiotensinogen genes: from molecules to the whole body. , 1995, Hypertension research : official journal of the Japanese Society of Hypertension.

[46]  S. Takahashi,et al.  Human renin-binding protein is the enzyme N-acetyl-D-glucosamine 2-epimerase. , 1999, Journal of biochemistry.

[47]  A. Kuboki,et al.  Improved Enzymatic Procedure for a Preparative-Scale Synthesis of Sialic Acid and KDN. , 1995 .

[48]  C. Schengrund,et al.  Biological Roles of Sialic Acid , 1976, Springer US.

[49]  R. Hill,et al.  Interaction of Mycoplasma gallisepticum with sialyl glycoproteins , 1980, Infection and immunity.

[50]  D. M. Ryan,et al.  Rational design of potent sialidase-based inhibitors of influenza virus replication , 1993, Nature.

[51]  B. Morgan,et al.  Effects of environmental stimulation on brain N-acetylneuraminic acid content and behavior. , 1980, The Journal of nutrition.

[52]  S W Tanenbaum,et al.  A facile procedure for the isolation of N-acetylneuramic acid from edible bird's-nest. , 1977, Carbohydrate research.

[53]  Z. Dauter,et al.  Structure of glucoamylase from Saccharomycopsis fibuligera at 1.7 A resolution. , 1998, Acta crystallographica. Section D, Biological crystallography.

[54]  G. Uhlenbruck,et al.  Characteristics of antigenic determinants of intact cell surfaces. , 1970, The Journal of medical laboratory technology.

[55]  S. Roseman,et al.  The sialic acids. I. The structure and enzymatic synthesis of N-acetylneuraminic acid. , 1960, The Journal of biological chemistry.

[56]  A. Varki,et al.  Diversity in the sialic acids , 1992, Glycobiology.

[57]  Yoji Tsukada,et al.  Improved Microbial Production of Colominic Acid, a Homopolymer of N-Acetylneuraminic Acid , 1973 .

[58]  Katsuhisa,et al.  Human influenza A virus hemagglutinin distinguishes sialyloligosaccharides in membrane-associated gangliosides as its receptor which mediates the adsorption and fusion processes of virus infection. Specificity for oligosaccharides and sialic acids and the sequence to which sialic acid is attached. , 1986, The Journal of biological chemistry.

[59]  A. Datta Regulatory role of adenosine triphosphate on hog kidney N-acetyl-D-glucosamine 2-epimerase. , 1970, Biochemistry.

[60]  A. Kobata,et al.  Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma. , 1989, The Journal of biological chemistry.

[61]  R. Schauer Chemistry, metabolism, and biological functions of sialic acids. , 1982, Advances in carbohydrate chemistry and biochemistry.

[62]  The sialate pyruvate-lyase from pig kidney: Purification, properties and genetic relationship+ , 1999, Glycoconjugate Journal.

[63]  J. Paulson,et al.  Erythrocyte receptors for Mycoplasma pneumoniae are sialylated oligosaccharides of Ii antigen type , 1984, Nature.

[64]  R. Schauer Biosynthesis and function of N- and O-substituted sialic acids. , 1991, Glycobiology.

[65]  F. Gaeta,et al.  ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Lex. , 1990, Science.

[66]  R. Oriol,et al.  Genetics of ABO, H, Lewis, X and Related Antigens , 1986, Vox sanguinis.

[67]  Y. Uchida,et al.  Purification and properties of N-acetylneuraminate lyase from Escherichia coli. , 1984, Journal of biochemistry.

[68]  W. Reutter,et al.  A Bifunctional Enzyme Catalyzes the First Two Steps in N-Acetylneuraminic Acid Biosynthesis of Rat Liver , 1997, The Journal of Biological Chemistry.

[69]  J. Paulson,et al.  Purification and characterization of an O-acetylsialic acid-specific lectin from a marine crab Cancer antennarius. , 1985, The Journal of biological chemistry.