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

JOURNAL OFBIOSCIENCE ANDBIOENGINEERING Vol. 93, No. 3,258-265.2002

REVIEW Why Is Sialic Acid Attracting Interest Now? Complete Enzymatic Synthesis of Sialic Acid with N-Acylglucosamine 2-Epimerase ISAFUMI MARU,l* JUN OHNISHI,’ YASUHIRO

OHTA,’ AND YOJI TSUKADA’

Kyoto Research Laboratories, Marukin Chuyu Co. Ltd., 27 Monnomae, Todo, Uji, Kyoto 611-0013, Japan’ Received 1 October 2001IAccepted 28 November 2001

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 iV-acylglucosamine 2-epimerase (AGE) gene from porcine kidney was cloned in E. culi, 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. [Key words: sialic acid, N-acetylneuraminic N-acetylneuraminate

acid, N-acetyl-D-glucosamine, lyase, N-acylglucosamine 2-epimerase]

Oligosaccharides carry out functions essential for the maintenance of biological activities of cells, including transmission of biological information between cell membrane surfaces, formation of cell morphology and maintenance of complicated configurations by covalently binding with proteins (1). In 1936, Blix isolated from bovine submandibular mucin a crystalline reducing acid (2), thereafter named ‘sialic acid’ (3), which was subsequently characterized by several groups [reviewed in (4-7)]. Sialic acids, which belong to a family of neuraminic acid (5-amido-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid), are usually found as an a-glycoside, occupying the nonreductive terminal of hetero-oligosaccharides in glycoconjugates such as glycoproteins and glycolipids. They occur in the forms of 40 or more molecular species involving N- and O-substituted sialic acids (8,9), The typical molecular species found in glycoproteins and glycolipids are classified into two types, N-acetylneuraminic acid (NeuAc) (2keto-5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranos-1-onic acid) (Fig. 1) and N-glycolylneuraminic acid (NeuGc). NeuGc is not found in normal tissues of human or chicken. On the other hand, NeuAc is the most ubiquitous sialic acid and is the biosynthetic precursor for all other

sialic acids (4-6). The main roles of sialic acid in vivo are classified as follows: (i) endowment of negative charge on cellular membranes as glycoconjugates, (ii) determination of the macromolecular structures of certain glycoproteins, (iii) information transfer between cells, and (iv) recognition of specified glycoconjugates and cells based on specific bio-activities (5, 10). For example, sialyloligosaccharides on a cell surface serve as receptor determinants for influenza virus (11, 12), mycoplasma (13, 14), blood groups (15, 16), tumorspecific antibodies (5, 17, 18), bacterial toxins (5) and a variety of lectins (19,20). Sialyl Lewis X (LeX), a tetrasaccharide with a NeuAc moiety, binds to ELAM-1 and mediates inflammatory responses between leukocytes and injured tissues (21-24). In circulation, sialic acid is the most likely candidate that determines the life span of blood components such as erythrocytes and serum glycoconjugates (25). Recently, novel sugar-chain-related pharmaceutical com-

HOWCOOH

HO

* Corresponding author. e-mail: maruemarukin-chuyu.com phone: +81-(0)774-22-0934

N-acetyl-D-mannosamine,

FIG. 1. Structure of N-acetylneuraminic acid (sialic acid).

fax: +81-(0)774-22-8566 258

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H:*

;

/

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toms. Recently, Zanamivir has been approved by several governments as an effective remedy for the treatment of both influenza type A and B viral infections. A new era has thus been opened for the practical application of NeuAc in the medical field.

CooH

NH

FIG. 2. Structure of Zanamivir (4-guanidino-NeuAc2en), influenza virus (26).

an anti-

pounds such as therapeutic agents (26) and diagnostic reagents for infectious diseases (Turnerrner et al., Patent Int. Pub. WO91/10744, 1991), autoimmune diseases, cancer and nerve diseases are being developed on the basis of the multifunctionality of NeuAc. As a result, therapeutic agents for influenza have recently been realized. Infection of the host cell by influenza virus is accomplished as follows. Blood agglutinin (hemagglutinin) on the viral superficial layer binds to NeuAc at the end of the sugar chain of the host cell in order to infiltrate into the cell. The infiltrating virus repeatedly proliferates in the cell. At the instant when the virus is to be freed from the cell, NeuAc on the superficial layer of the host cell is detached by neuraminidase located on the viral surface in order to liberate the virus itself and then the freed virus binds to other noninfected cells. In 1918, the Spanish Flu caused the death of 20,000,OOOto 40,000,OOOpersons all over the world. In Japan, approximately 380,000 persons died. Since then, three flu epidemics, in 1957 (Asian Flu), 1968 (Hong-Kong Flu) and 1977 (Russian Flu), have been recognized worldwide. Incidentally, a new type of influenza that emerged in Hong Kong in 1997 caused death in 6 among 18 infected individuals. A novel pharmaceutical agent recently synthesized from NeuAc is being used for treatment of influenza infections. Colman et al. determined the three-dimensional structure of the influenza virus neuraminidase in 1983 (27). Based on this structure, Itzstein et al. successfully prepared a strong inhibitor for the viral neuraminidase (26). This inhibitor is prepared by replacing the hydroxyl group at the 4-position in the NeuAc derivative (Net&Zen) with a guanidino group (named ‘Zanamivir’, an influenza therapeutic agent) (Fig. 2). This compound had a K,, value of 10m9M order for viral neuraminidase, which is an extremely strong bond. Glaxo-Smith-Kline carried out clinical tests of Zanarnivir worldwide and demonstrated that the compound could prevent influenza viral infections and ameliorate their symp-

CONVENTIONAL PREPARATION OF SIALIC ACID Preparation

of NeuAc from natural sources

NeuAc,

which is commonly distributed in the animal kingdom, has been prepared from edible bird’s nest (petrel) (28), milk (Iwamoto et al., Japan Patent Kokai 313724, 1988) and avian egg (29,30) (Table 1). For example, in the preparation of NeuAc from egg yolk (30), the delipidated egg yolk was hydrolyzed with HCl (pH 1.4) at 80°C and neutralized with NaOH (pH 6.0). The mixture was passed through a column of Dowex HCR-W2, and then through a column of Dowex l-X8. The latter column was washed with water, and then eluted with a linear gradient of formic acid (O-2 M). The eluates containing NeuAc were concentrated using a reverse osmotic membrane, followed by evaporation at 40°C, and the residue was lyophilized. Since the NeuAc contents in these natural products are too low to be isolated with sufficient recovery and purity, this process was never considered as an industrial method of NeuAc production from the standpoint of both supply and cost. In the microbial kingdom, a certain strain of E. coli with a Kl antigen produces colominic acid (CA) (a homopolymer of NeuAc) in its culture broth (31, 32). Thus, CA was thought to be a potent candidate for NeuAc production if CA fermentation technology could be developed. Uchida et al. successfully developed processes for fermentation of CA and a hydrolysis of CA to NeuAc using microbial neuraminidase (31). By these processes, crystalline NeuAc (purity>98%) without any restriction of mass supply for laboratory and pilot plant uses could be produced for the first time, indicating the CA fermentation technology to be a milestone in NeuAc production. Enzymatic synthesis of NeuAc A report published in 1960 describes the synthesis of NeuAc from N-acetylmannosamine (ManNAc) and pyruvate by the synthesis reaction catalyzed by N-acetylneuraminate lyase (NAL) [EC 4.1.3.31 (33). This enzyme was originally found in some bacteria such as E. coli (34) and Clostridiumperfiingens (35), and in animal tissues such as porcine kidney (36) and bovine kid-

TABLE 1. Methods of NeuAc production NeuAc content (%) 0.01

Conversion rate from GlcNAc to NeuAc (%) -

Purification

Reference

Hydrolysis and column chromatography

Egg yolk Colominic acid fermentation Membrane reactor process Alkaline enzymatic synthesis

0.03 1” lb lob

18 40

Hydrolysis and column chromatography Hydrolysis and column chromatography Column chromatography Column chromatography

Complete enzymatic synthesis

13b

76

Direct crystallization

Iwamoto et al., Japan Patent Kokai 313724, 1988 30 31 44 Tsukada et al., U.S. Patent 5472860,199s 70

Origin or process Milk whey

BNeuAc contents in culture broth. bNeuAc contents in reaction solution.

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H&

x

COOH

HO’

ManNAc

-

NeuAc

NHAc

GlcNAc FIG. 3. Schematic representation of NeuAc synthesis from GlcNAc and pyruvate.

ney (37). AugC et al. succeeded in the immobilization of NAL on Ultrogel (agarose gel) and synthesized NeuAc using it (38). The immobilized enzyme could be used for three repeated reactions, and a total of 2.8 mmol(0.9 g) of NeuAc were produced with 2.7 units of the enzyme. Kim et al. reported the immobilization of NAL on poly(acrylamide-coacryloxysuccinimide) gel (39). Because ManNAc is so expensive and hardly available in large quantities, Simon et al. attempted to prepare NeuAc enzymatically using a mixture of ManNAc and GlcNAc, which was prepared from inexpensive GlcNAc by chemical epimerization under alkaline conditions (pH 12) (40). However, the conversion rate from GlcNAc to ManNAc is too low to yield sufficient amount of NeuAc in this reaction system. Recently, Tsukada and Ohta developed a process for the alkaline-enzymatic synthesis of NeuAc under simultaneous reactions of chemical epimerization of GlcNAc into ManNAc and enzymatic aldol condensation between the resulting ManNAc and pyruvate (Tsukada et al., U.S. Patent 5472860, 1995). This system involved the use of NAL, high concentrations of GlcNAc and pyruvate [individually of 18% (w/v)] as substrates under alkaline conditions (pH 10.5) (Fig. 3). NAL itself is unstable under alkaline conditions. However, due to the protective actions of the substrates at high concentrations, the residual activity of NAL even after 65 h of reaction was maintained at 80% or more. NeuAc was synthesized at 100 mg per ml of the reaction solution, assuming that the conversion rate of GlcNAc into NeuAc was about 40% (in molar ratio) (Table 1). The resulting NeuAc was purified after desalting, adsorption and elution from the anion exchange column. After decolorization on charcoal, followed by crystallization in an acetic acid solution, 24 kg of NeuAc was recovered from 54 kg of GlcNAc in 300 I of reaction solution. It has traditionally been known that epimerization of GlcNAc and ManNAc is catalyzed by N-acylglucosamine 2-epimerase (AGE) [EC 5.1.3.11. Ghosh and Roseman were the first to report the occurrence of AGE in 1964 (41). Requiring ATP and MgCl, as an activating factor, AGE catalyzes the epimerization of GlcNAc and ManNAc (41-43). The equilibrium constant (Keq) as defined by the equation

[ManNAc]/[GlcNAc] is equal to 0.26, suggesting that the constant is likely to be on the side of GlcNAc, thus the sole reaction between GlcNAc and AGE results in an inadequate ManNAc yield. Kim et al. proposed that AGE catalyzed epimerization coupled with NAL reaction might provide a better solution for the direct synthesis of NeuAc from inexpensive GlcNAc (39). Kragl et al. reported the continuous synthesis of NeuAc. This method involves the use of a membrane reactor in which NAL and AGE are enclosed and into which a substrate solution containing GlcNAc and pyruvate is poured (the membrane reactor process) (44). By this method, the conversion rate of GlcNAc to NeuAc was as low as 18% (in molar ratio). In this experiment, the AGE used was prepared from porcine kidney, which was the sole source of the enzyme at that time. The fatal drawback of this method was that the amount of AGE obtainable is not sufficient to meet the demand. DEVELOPMENT OF AGE FROM PORCINE KIDNEY Molecular cloning of NAL and AGE genes NAL catalyzes the equilibrium reaction, ManNAc + pyruvate f) NeuAc, and has been practically applied in the field of diagnostics (45). The NAL gene was cloned in E. coli and massscale production of the enzyme was achieved (46-48). As for AGE, no significant report appeared except that on its partial purification from porcine kidney in the early half of the 1970’s (41-43). Maru et al. attempted to develop a process for mass production of AGE by cloning of the AGE gene from porcine kidney in E. coli (49). AGE was first extracted from 52 porcine kidneys and purified by ionexchange chromatography to electrophoretical homogeneity (16 mg). Table 2 shows the molecular mass and enzymatic properties of AGE. Cloning of the AGE gene was carried out as follows. mRNA was prepared from porcine kidney cortex. cDNA was synthesized and then ligated to a ZAP vector and packaged in h phage for E. coli infection. A gene encoding the AGE was immunoscreened from 1,200,OOO clones of a cDNA library from porcine kidney cortex. Con-

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TABLE 2. Properties of AGE from porcine kidney (49) Molecular mass Optimum pH pH stability Optimum temperature Temperature stability K,,, value (mM) Activator

Inhibitor

93 kDa (sedimentation equilibrium) 45 kDa (SDS-PAGE) 6.8 6-8 41°C <37”C GlcNAc (7.4), ManNAc (6.3), ATP (0.2) ATP, deoxy-ATP Divalent cations (Mg2’, Fez+, Ca”, Mn*+, Co*+,and Ba*‘) p-Chloromercuribenzoic acid

sequently, positive clones of 64 strains were recovered and one of these clones was selected. The activity of the selected strain was clearly demonstrated. AGE expressed by E. coli was identical with that prepared from porcine kidney with respect to molecular mass and various enzymatic properties. Then, AGE was subcloned in a high-expression vector to construct a strain with higher AGE expression levels for practical applications. Structure and function of AGE The analytical data of the structural gene encoding AGE thus cloned unexpectedly indicated that the gene was 99.6% and 99.0% homologous to the nucleotide sequence coding for and amino acid AGE RnBP RnBP RnBP

261

sequence of the renin-binding protein (RnBP) (50) from porcine kidney (49), respectively (Fig. 4). Furthermore, the gene was 87.8% and 83.1% homologous to the amino acid sequences of human (51) and rat (52) RnBPs, respectively. Renin is an acid protease with high specificity for angiotensinogen and plays an important role as a vasopressor enzyme (53). Renin is inductibly synthesized, mainly in the renal juxtaglomerular cell of kidney and released into blood with various stimulants to regulate blood pressure. On the other hand, RnBP was first detected in extracts of rabbit (54) and porcine (55) kidneys as a specific renin inhibitor. RnBP was found to bind to renin to form high-molecularweight renin, and to inhibit renin activity (56). Hence, it is suggested that RnBP may be responsible for biological blood pressure regulation. Then, the inhibition of renin activity by the cloned AGE was examined (49). It was observed that the inhibition of renin activity by the cloned AGE was at the same level as that of RnBP. It was further confirmed that the cloned AGE formed high-molecularweight renin. From the data mentioned above, AGE was identified to be the same protein as RnBP. Subsequently, it was reported that human RnBP was AGE, itself (57). In order to clarify the relationship between AGE and RnBP with respect to structure and function, Maru et al. and Itoh

(porcine): MEKERETLQAWKERVGQELDRVMAFWLEHSHDREHCCFFTCLGRDGRVYDDLKYVWL4GR:60 (porcine): ............................................................ ,, . ..g.. .. .4 ........... E.. ............. (human) : ...................... : ......... v ..Q.. .... .8 ., ... g.. .. .4 ............. 4 . ..“. ........ (rat)

QVWMYCRLYRKLERFHRPELLDAAKAGGEFLLRHARVAPPEKKCAFVLTRDGRPVKV4RS:120 ............................................................ ..........TF...RHAP..............Y......G ..................T ..........TF...R.V..............SY......G........Q .........T

:180 lFSECFYTMAMNELWRVTAEARYPSEAVDMMD4lVHWVREDPSGLGRP4LPGAVASESMA .......................... ..E............................... ................A.G.V...T...E.........Q..A........Q..P.A.P .. ...............K..G.MH..R...E.....,.......A.......S.TL.T.P ..

VPMMLLCLVEQLGEEDEELAGRYAQLGHWCARRlLPHYPRDG4AVLENVSEDGEELSGCL:240 ............................................................ ......N.......A......K..E.~D.......................G.K..P ...

...... N.........

..MTDK..E..D...H...........V.........K..P

...

GRH4NPGHALEAGWFLLRHSSRSGDAKLRAHVlDTFLLLPFRSGWDADHGGLFYF4DADG:300 .............................................. ..

..y ...........

.......... 4... ..T..........C,.K..PE.......K......H....P ........ r.. ...... QYAt.K..P..Q a ., .. K.. .... H.. .. PE.. .........

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a

LCPTQLEWAMKLWWPHSEAHlAFLMGYSESGDPALLRLFY4VAEYTFR4FRDPEYGEWFG:360 ................84.. ........................................ F ...........................D....V.......................... ........N.......T..........RD.......N.............H .........

YLNREGKVALTIKGGPFKGCFHVPRCLAMCEEMLSALLSRLA ..........................................

...

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:402 :402 .G.... .P.PA,,SPA,,TPAGRGAE:417 4 1.G.. .4. .GPAPLGSLPAVPTREGSK :419

FIG. 4. Alignment of amino acid sequences of AGE and RnBPs of various origins (49). Identical amino acid residues with porcine AGE are indicated by dots and different amino acid residues are shown by the symbol of amino acids.

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et al. determined the three-dimensional structure of AGE by X-ray crystallography (58, 59). The dimer of AGE had an asymmetric unit with approximate dimensions 46 Ax 48 Ax 96 A. The AGE monomer was composed of an a&,-barrel, the structure of which is found in glucoamylase (60,6 1) and cellulase (62). One side of the AGE a&,-barrel structure comprises long loops containing five short B-sheets, and contributes to the formation of a deep cleft shaped like a funnel. The putative active-site pocket and a possible binding site for substrate GlcNAc were found in the cleft. The other side of the a&,-barrel comprises short loops and contributes to dimer formation. AGE is located widely in individual mammalian organs, but the physiological functions of this enzyme in biological systems have not been elucidated yet. It is known that the synthesis of ManNAc as a precursor of NeuAc in organisms is catalyzed by either UDP-GlcNAc 2-epimerase VPGlcNAc + ManNAc + UDP] (63) or AGE. It is also indicated that the UDP-GlcNAc 2-epimerase is essential for the biosynthesis of NeuAc (64,65). Therefore, AGE may possibly function in an alternative pathway associated with biosynthesis of NeuAc or in a different metabolic system. Recently, RnBP has been shown to play no significant role in the regulation of renin activity in plasma or kidneys of mice (66). Furthermore, Brenda et al. reported that RnBP is not colocalized or coregulated with renin in the kidneys of rats with a two-kidney one-clip hypertension (67). Thus, the finding that AGE is RnBP offers a novel clue for better understanding of the function of RnBP in the regulation of NeuAc biosynthesis and/or blood pressure, and prompts investigators working in the area of renal hypertension to reinvestigate the substantial nature of RnBP in the reninangiotensin system.

of AGE and NAL from GlcNAc and pyruvate (Fig. 3), excess pyruvate inhibits the AGE reaction. Therefore, the conversion rate from GlcNAc to NeuAc does not increase to the desired level, when the concentration of pyruvate added is increased. To overcome this problem, Maru et al. developed a feeding system wherein appropriate amounts of pyruvate are added to the reaction mixture (70). When a twofold molar equivalent (2 mol) of pyruvate against GlcNAc (1 mol) was allowed to react in the presence of AGE and NAL, the rate of conversion of GlcNAc to NeuAc reached a plateau at 40% (0.4 mol) for 170 h of reaction (shown with dotted line in Fig. 5). Even with prolonged reaction, no more NeuAc was synthesized. With the assumption that an excess amount of pyruvate may have inhibited AGE reaction, instead of 2 molar equivalents, 0.6molar equivalent of pyruvate against GlcNAc was initially added to the reaction mixture (shown with a solid line in Fig. 5); at the point when the synthetic reaction rate of NeuAc decrease (synchronous with when the reaction reached equilibrium), 0.9 molar equivalent of pyruvate (1.5-fold molar equivalent against GlcNAc in total) was further added (Fig. 5, a); at the point when the synthetic reaction rate of NeuAc further decreased, 0.5 molar equivalent of pyruvate (twofold molar equivalent against GlcNAc in total) was further added (Fig. 5, b) to the reaction mixture. In such a manner, the conversion rate of GlcNAc to NeuAc reached 75% in molar ratio. Further addition of pyruvate did not increase the conversion rate but only caused difficulty in the purification of NeuAc due to the residual pyruvate in the reaction system. In the synthetic reaction at a practical scale, it was demonstrated that 29 kg of NeuAc was produced from 27 kg of GlcNAc and 27.5 kg of pyruvate, assuming that the conversion rate from GlcNAc to NeuAc was 76% on a molar basis (Table 1 and Fig. 5) (70). A simple procedure such as

DEVELOPMENT OF NeuAc PRODUCTION BY ENZYMATION Complete enzymatic process in NeuAc synthesis For the synthesis of NeuAc from ManNAc and pyruvate in the presence of NAL, addition of pyruvate fivefold greater than ManNAc in the reaction solution is generally employed. However, excess pyruvate in the solution should be removed in order to purify NeuAc. To overcome this problem, Wong et al. attempted to remove residual pyruvate using pyruvate decarboxylase from baker’s yeast (68), which catalyzes the degradation of pyruvate into acetaldehyde and carbon dioxide. By introducing this process, preparation of NeuAc at a scale of 10 g per lot was facilitated. Sugai et al. reported that calcium hydroxide, which could be easily removed by passing carbon dioxide gas and then by subsequent centrifugal separation of the precipitated calcium carbonate, was an effective agent for epimerization of GlcNAc to ManNAc (69). The following three reactions were combined, namely, calcium hydroxide-catalyzed epimerization of GlcNAc to ManNAc, NAL-catalyzed condensation with pyruvate, and degradation of excess pyruvate catalyzed by pyruvate decarboxylase. Using this procedure, NeuAc was prepared from ManNAc with a 60% yield, which corresponded to a 7.6% yield from GlcNAc. In the case of NeuAc synthesis by simultaneous reaction

0

50

100

150

200

250

Reaction time (h) FIG 5. Time-course of sialic acid synthesis from GlcNAc and pyruvate using AGE and NAL (70). The pyruvate feeding reaction (solid line) was initiated at 30°C in 150 I of reaction mixture containing 2 units/ml AGE, 8 units/ml NAL, 11 mM ATP, 10 mM MgCl, and the substrates, namely, 27 kg (122 mol) of GlcNAc and 8 kg (73 mol) of sodium pyruvate. Arrows showed the feeding point with (a) 12.5 kg (114 mol) and (b) 7 kg (64 mol) of sodium pyruvate dissolved in 37.9 and 21.6 I of deionized water, respectively. The one-batch reaction (dotted line) was performed in 1230 ml of mixture containing 221 g (1 mol) of GlcNAc and 220 g (2 mol) of sodium pyruvate. The amount of NeuAc in the reaction mixture was determined by the thiobarbituric acid method (73).

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and improving learning performance is one of the essential themes of what matching the needs of the times, and NeuAc should be one of the ideal candidates as an essential component for such undertaking. Study on the utilization of AGE in the industrial field has been carried out, but the physiological roles of AGE in organisms have not yet been elucidated. The roles of this protein with dual functions, namely AGE and RnBP activities, in individual mammalian organs should be made clear in the future.

REFERENCES

FIG. 6. Schematic representation of NeuAc isolation processes.

the periodic feeding of pyruvate into the reaction system enabled the realization of the highest yield of NeuAc by an enzymatic process. Direct crystallization

of NeuAc

from reaction

solution

For the isolation of NeuAc from reaction solutions prepared from CA hydrolysis or enzymatic synthesis under alkaline conditions, the solutions have to be treated by desalting, anion-exchange chromatography, decolorization and crystallization (Fig. 6) (31, Tsukada et al., U.S. Patent 5472860, 1995). The column process substantially limits the production scale and has many demerits in terms of cost and procedure. Attempt of direct crystallization of NeuAc from reaction solutions using glacial acetic acid was successfully achieved after eliminating heat-denatured enzymes (Fig. 6) (70). The NeuAc thus prepared was proved to be of the same quality as those of biochemical reagents (purity of 99% or more). CONCLUSIONS

AND FUTURE

PROSPECTS

Recently, NeuAc-based pharmaceutical agents and diagnostic reagents have been developed and applied to practical use. To meet the increasing demand for NeuAc, Maru et al. developed a complete enzymatic process for NeuAc production from inexpensive GlcNAc and pyruvate using AGE and NAL, which enables the mass scale production of NeuAc of the highest purity via direct crystallization. Such a simple procedure is applicable in any scale, place and time with minimum investment. The development of pharmaceutical agents based on NeuAc should increase from now on, and we are confident that the method developed will contribute to the development of medical drugs due to the recognition that NeuAc is not an ‘expensive material’ but a ‘commodity raw material’. The 21st century is recognized to be ‘the age for targeting the brain’. Morgan and Winick indicated that administration of NeuAc to rats was effective in improving both their memory and learning ability (71, 72). We believe that the development of functional foods for activating the brain

1. Varki, A.: Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 3,97-130 (1993). 2. Blix, G.: Uber die Kohlenhydratgruppen des Submaxillarismucins. Hoppe-Syler’s Z. Physiol. Chem., 240,43-54 (1936). 3. Blix, G., Svennerholm, L., and Werner, I.: The isolation of chondrosamine from gangliosides and from submaxillary mucin. Acta Chem. Acand., 6, 358-362 (1952). 4. Roseman, S.: The synthesis of carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion. Chem. Phys. Lipids, 5,270-297 (1970). 5. Reutter, W., Kiittgen, E., Bauer, C., and Gerok, W.: Biological and significance of sialic acids, p. 263-292. In Schauer, R. (ed.), Sialic acids - chemistry, metabolism and tin&ion. Cell biology monographs, vol. 10. Springer-Verlag, New York (1982). 6. Rosenberg, A. and Scbengrund, C.: Sialidases, p. 295-359. In Rosenberg, A. and Schengrund, C. (ed.), Biological roles of sialic acid. Plenum Press, New York and London (1976). 7. FiBard, H.: The early history of sialic acids. Trends Biothem. Sci., 14,237-241 (1989). 8. Varki, A.: Diversity in the sialic acid. Glycobiology, 2,25-40 (1992). 9. Schauer, R: Biosynthesis and function of N- and O-substituted sialic acids. Glycobiology, 1,449-452 (1991). 10. Schauer, R: Achievements and challenges of sialic acid research. Glycoconj. J., 17,485-499 (2000). 11. Suzuki, Y., Nagao, Y., Kate, H., Matsumoto, M., Nerome, K., Nakajima, K., and Nobusawa, E.: 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. J. Biol. Chem., 261, 17057-17061 (1986). 12. Pritchett, T. J., Brossmer, R, Rose, U., and Paulson, J. C.: Recognition of monovalent sialosides by influenza virus H3 hemagglutinin. Virology, 160, 502-506 (1987). 13. Glasgow, L. R. and Hill, R L.: Interaction of mycoplasma gallisepticum with sialyl glycoproteins. Infect. Immun., 30, 353-361 (1980). 14. Loomes, L. M., Uemura, K., Childs, R A., Paulson, J. C., Rogers, G. N., Scudder, P.R, Michalski, J., Hounsell, E. F., Taylor-Robinson, D., and Feizi, T.: Erythrocyte receptors for mycoplasma pneumonia are sialylated oligosaccharides of Ii antigen type. Nature, 307, 560-563 (1984). 15. Oriol, R., Le Pendu, J., and Mollicone, R.: Genetics of ABO, H, Lewis, X and related antigens. VOX Sang., 51, 161171 (1986). 16. Clausen, H. and Hakomori, S.: ABH and related histoblood group antigens; immunochemical differences in carrier isotypes and their distribution. VOX Sang., 56, l-20 (1989). 17. Hakomori, S.: Tumor-associated carbohydrate antigens. Annu. Rev. Immunol., 2, 103-126 (1984). 18. Yamashita, K., Koide, N., Endo, T., Iwaki, Y., and Kobata,

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J. BIOSCI.BIOENG.,

A.: Altered glycosylation of serum transferrin of patients

with hepatocellular carcinoma. J. Biol. Chem., 264, 24152423 (1989). 19. Pardoe, G. I. and Uhlenbruck, G.: Characteristics of antigenie determinants of intact cell surfaces. J. Med. Lab. Technol., 27,249-263 (1970). 20. Ravindranath, M.H., Higa, H., Cooper, E.L., and Paulson, J. C.: Purification and characterization of an Oacetylsialic acid-specific lectin from a marine crab cancer antennarius. J. Biol. Chem., 260,8850-8856 (1985). 21. Phillips, M. L., Nudelman, E., Gaeta, F. C. A., Perez, M., Singbal, K., Hakomori, S., and Paulson, J. C.: ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Le”. Science, 250, 113&1132 (1990). 22. Lowe, J.B., Stoolman, L.M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M.: ELAM-1 dependent cell adhesion to vascular endothelium determined by a transfected human glucosyltransferase cDNA. Cell, 63,475484 (1990). 23. Springer, T. A. and La&y, L. A.: Cell adhesion. Sticky sugars for selectins. Nature, 349, 196-197 (1991). 24. Lasky, L.A.: Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science, 258, 964969 (1992). 25. Schauer, R.: Chemistry, metabolism, and biological functions of sialic acids. Adv. Carbohydr. Chem. Biochem., 40, 131-234 (1982). 26. von Itzstein, M., Wu, W.Y., Kok, G.B., Pegg, M. S., Dyason, J. C., Jm, B., Phan, T.V., H. F., Oliver, S. W., Colman, P.M., D. M., Woods, J.M., Bethell, Cameron, J. M., and Penn, C. R:

Smythe, M. L., White, Varghese, J. N., Ryan, R C., Hotham, V. J.,

Rational design of potent sialidase-based inhibitors of influenza vjrus replication. Nature, 363,418-423 (1993). 27. Colman, P. M., Varghese, J. N., and Laver, W. G.: Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature, 303,41-44 (1983). 28. Martin, J. E., Tanenbaum, S. W., and Flashner, M.: A facile procedure for the isolation of N-acetylneuraminic acid from edible bird%-nest. Carbohydr. Res., 56,423-425 (1977). 29. Juneja, L.R, Koketsu, M., Nishimoto, K., Kim, M., Yamamoto, T., and Itoh, T.: Large-scale preparation of sialic acid from chalazae and egg-yolk membrane. Carbohydr. Res., 214, 179-186 (1991). 30. Koketsu, M., Juneja, L. R., Kawanami, H., and Kim, M.: Preparation of N-acetylneuraminic acid from delipidated egg yolk. Glycoconj . J., 9, 70-74 ( 1992). 31. Uchida, Y., Tsukada, Y., and Sugimori, T.: Improved microbial production of colominic acid, a homopolymer Nacetylneuraminic acid. Agr. Biol. Chem., 37, 2105-2110 (1973). 32. Tsukada, Y., Ohta, Y., and Sugimori, T.: Microbial production of sialic acid related enzymes and their application for the development of clinical diagnostics. Nippon NGgeikagaku Kaishi, 64, 1437-1444 (1990). (in Japanese) 33. Comb, D. G. and Roseman, S.: The sialic acids. The structure and enzymatic synthesis of N-acetyl-D-neuraminic acid. J. Biol. Chem., 235,2529-2537 (1960). 34. Uchida, Y., Tsukada, Y., and Sugimori, T.: Purification and properties of N-acetylneuraminate lyase from coli. J. Biochem., 96, 507-522 (1984). 35. Deijl, C. M. and Vliegenthart, J. F.: Configuration of substrate and products of N-acetylneuraminate pyruvate-lyase from Clostridium perfiingens. Biochem. Biophys. Res. Commun., 111,668-674 (1983). 36. Sommer, U., Traving, C., and Schauer, R: The sialate pyruvate-lyase from pig kidney: purification, properties and genetic relationship. Glycoconj. J., 16,425-435 (1999). 37. Sirbasku, D. A. and BinkIey, S. B.: Purification and properties of N-acetylneuraminate lyase from beef kidney cortex.

Biochim. Biophys. Acta, 206,479-482 (1970). 38. Aug&, C., David, S., and Gautheron, C.: Synthesis with immobilized enzyme of the most important sialic acid. Tetrahedron Lett., 25,4663-4664 (1984). 39. Kim, M.-J., Hennen, W. J., Sweers, H. M., and Wong, C.H.: Enzyme in carbohydrate synthesis: N-acetylneuraminic acid aldolase catalyzed reactions and preparation of N-acetyl2-deoxy-D-neuraminic acid derivatives. J. Am. Chem. Sot., 40.

110,6481-6486 (1988). Simon, E. S., Bednarski, M.D., and Whitesides, G. M.: Synthesis of CMP-NeuAc from N-acetylglucosamine: gen-

eration of CTP from CMP using adenylate kinase. J. Am. Chem. Sot., 110,7159-7163 (1988). 41. Ghosh, S. and Roseman, S.: The sialic acids, V. N-acyl-Dglucosamine 2-epimerase. J. Biol. Chem., 240, 1531-1536 (1964). 42. Datta, A.: Regulatory of adenosine triphosphate on hog kidney N-acetylglucosamine 2-epimerase. Biochemistry, 17, 3363-3370 (1970). 43. Datta, A.: N-Acetylglucosamine 2-epimerase from hog kidney. Method. Enzymol., 41,407-412 (1975). 44. Kragl, U., Gygax, D., Ghisalba, O., and Wandrey, C.: Enzymatic two-step synthesis of N-acetylneuraminic acid in the enzyme membrane reactor. Angew. Chem. Int. Ed. Engl., 30, 827-828 (1991). 45. Taniuchi, K., Chifu, K., Hayashi, N., Nakamachi, Y., Yamaguchi, N., Miyamoto, Y., Doi, K., Baba, S., Uchida, Y., Tsukada, Y., and Sugimori, T.: A new enzymatic method

for the determination of sialic acid in serum and its application for a marker of acute phase reactants. Kobe J. Med. Sci., 27,91-102 (1981). 46. Ohta, Y., Shimosaka, M., Murata, K., Tsukada, Y., and Kimura, A.: Molecular cloning of N-acetylneuraminite lyase gene in Escherichiu coli K-12. Appl. Microbial. Biotechnol., 24,386-391 (1986). 47. Ohta, Y., Watanabe, K., and Kimura, A.: Complete nucleotide sequence of the E. coli N-acetylneuraminate lyase. Nucl. Acids Res., 13, 8843-8852 (1985). 48. Ohta, Y., Tsukada, Y., Sugimori, T., Murata, K., and Kimura, A.: Isolation of a constitutive N-acetylneuraminate lyase-producing mutant of Escherichia coli and its use for NPL production. Agric. Biol. Chem., 53,477-481 (1989). 49. Maru, I., Ohta, Y., Murata, K., and Tsukada, Y.: Molecular cloning and identification of N-acyl-D-glucosamine 2-epimerase from porcine kidney as a renin-binding protein. J. Biol. Chem., 271,16294-16299 (1996). 50. Inoue, II., Fukui, K., Takahashi, S., and Miyake, Y.: Molecular cloning and sequence analysis of a cDNA encoding a porcine kidney renin-binding protein. J. Biol. Chem., 265, 6556-6561 (1990). 51. Takahashi, S., Inoue, H., and Miyake, Y.: The human gene for renin-binding protein. J. Biol. Chem., 267, 13007-13013 (1992). 52. Takahashi, S.: Structure of the gene encoding rat renin binding protein. Biosci. Biotech. Biochem., 61, 1323-1326 (1997). 53. Tamura, K., Umemura, S., Fukamizu, A., Ishii, M., and Murakami, K.: Recent advances in the study of renin and angiotensinogen genes: from molecules to the whole body. Hypertens Res., l&7-18 (1995). 54. Leckie, B. J. and McConnell, A.: A renin inhibitor from rabbit kidney: conversion of a large inactive renin to a smaller active enzyme. Circ. Res., 36, 513-519 (1975). 55. Boyd, G. W.: A protein-bound form of porcine renal renin. Circ. Res., 35,426-438 (1974). 56. Takahashi, S., Ohsawa, T., Miura, R., and Miyake, Y.: 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). J. Bio-

VOL. 93,2002

COMPLETE

them., 93,265-274 (1983). 57. Takahashi, S., Takahashi, K., Kaneko, T., Ogasawara, H., Shindo, S., and Kobayashi, M.: Human renin-binding protein is the enzyme N-acetyl-D-glucosamine 2-epimerase. J. Biochem., 125,348-353 (1999). 58. Maru, I., Ohnishi, J., Ohta, Y., Hashimoto, W., Tsukada, Y., Murata, K., and Mlkami, B.: Crystallization and preliminary X-ray diffraction studies of N-acyl-D-glucosamine 2epimerase from porcine kidney. J. Biochem., 120, 481482 (1996). 59. Itoh, T., Mikami, B., Maru, I., Ohta, Y., Hasbimoto, W., and Murata, K.: Crystal structure of N-acyl-D-glucosamine 2-epimerase from porcine kidney at 2.0 8, resolution. J. Mol. Biol., 303, 733-744 (2000). 60. Aleshin, E. A., Firsov, L. M., and Honzatko, R B.: Refined structure for the complex of acarbose with glucoamylase from Aspergillus awamori var. Xl00 to 2.4A resolution. J. Biol. Chem., 269, 15631-15639 (1994). 61. SevBl&, J., Solovicovli, A., Hostinov& E., GaHperik, J., Wilson, K. S., and Dauter, Z.: Structure of glucoamylase from Saccharomycopsis jibuligera at 1.3 A resolution. Acta Crystallog. sect. D, 54, 854-866 (1998). 62. Sakon, J., Irwin, D., Wilson, D.B., and Karplus, P. A.: Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca. Nature Struct. kiol., 4,8 10-8 18 (1997). 63.

Hinderlichi,

S., Stiische, R, Zeitler, R., and Reutter, W.:

A bifunctional enzyme catalyzes the first two steps in Nacetylneuraminic acid biosynthesis of rat liver. Purification and characterization of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannnosamine kinase. J. Biol. Chem., 272, 24313-24318 (1997). 64. Stiische, R, Hinder&hi, L., Moorman,

S., Weise, C., Effertz, K., Lucka, P., and Reutter, W.: A bitictional enzyme

catalyzes the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. Molecular cloning and functional expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannnosamine kinase. J. Biol. Chem., 272, 24319-24324 (1997).

ENZYMATIC

SYNTHESIS

OF SIALIC ACID

265

65. Keppler, 0. T., Hinderlich,

S., Langner, J., SchwartzAlbiez, R, Reutter, W., and Pawlita, M.: UDP-GlcNAc 2-

epimerase: a regulator of cell surface sialylation. Science, 284, 1372-1376 (1999). 66. Schmitz, C., Gotthardt, M., Hinderlich, S., Leheste, J. R., Gross, V., Vorum, H., Christensen, E. I., Luft, F. C., Takahashi, S., and Willow, T. E.: Normal blood pressure

and plasma renin activity in mice lacking the renin-binding protein, a cellular renin inhibitor. J. Biol. Chem., 275, 1535715362 (2000). 67. Brenda, J. L, Peter, S. L., and Sukhwinderjit, L.: The expression of renin-binding protein and renin in the kidneys of rats with two-kidney one-clip hypertension. J. Hypertens., 18, 935-943 (2000). 68. Lin, C.-H., Sugai, T., Halcomb, R L., Ichikawa, Y., and Wong, C.-H.: Unusual stereoselectivity in sialic acid aldolase-catalyzed aldol condensations: synthesis of both enantiomers of high-carbon monosaccharides. J. Am. Chem. Sot., 114, 10138-10145 (1992). 69. Sugai, T., Kuboki, A., Hiramatsu, S., Okazakl, H., and Ohta, H.: Improved enzymatic procedure for preparativescale synthesis of sialic acid and KDN. Bull. Chem. Sot. Jpn., 68,3581-3589 (1995). 70. Maru, I., Ohnishi, J., Ohta, Y., and Tsukada, Y.: Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosarnine 2-epimerase and N-acetylneuraminate lyase. Carbohydr. Res., 306,575-578 (1998). 71. Morgan, B. L. G. and Winick, M.: Effects of administration N-acetylneuraminic acid (NANA) on brain NANA content and behavior. J. Nutr., 110,416-424 (1980). 72. Morgan, B. L. G. and Winick, M.: Effects of environmental stimulation on brain N-acetylneuraminic acid content and behavior. J. Nutr., 110,425-432 (1980). 73. Uchida, Y., Tsukada, Y., and Sugimori, T.: Production of microbial neuraminidase induced by colominic acid. Biochim. Biophys. Acta, 350,426-43 1 (1974).