JP2011223885A - New cytidine 5'-monophosphosialic acid synthetase, gene encoding the same and method for producing the synthetase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new cytidine 5'-monophosphosialic acid synthetase and a nucleic acid encoding the same.SOLUTION: Microorganisms of 4,000 strains or more are separated. As a result of vigorous investigation of their properties, a strain for producing a new cytidine 5'-monophosphosialic acid synthetase is found from a microbial cell belonging to the genus Photobacterium. The new cytidine 5'-monophosphosialic acid synthetase and the nucleic acid encoding the same are produced from the culture of the strain.

Description

  The present invention relates to a novel cytidine 5'-monophosphosialic acid synthase, a gene encoding the enzyme, a microorganism producing the enzyme, and a method for producing the enzyme.

  In recent years, it has been revealed that sugar chains of complex carbohydrates have very important functions in vivo. For example, in mammalian cells, it has been revealed that sugar chains are important molecules that function as signals for signaling and glycoconjugates between cells and between cells and extracellular matrix in differentiation and development. Sialic acid (Sia) is a general term for substances in which the amino group or hydroxy group of neuraminic acid (Neu) is substituted. Since sialic acid is often present at the non-reducing end of a sugar chain, It is considered to be an extremely important sugar in the functional expression of For example, sialic acid-containing sugar chains on the cell surface play an extremely important role in infections such as influenza virus, and research on sialic acid-containing sugar chains is one of the fields that are currently attracting the most attention.

  Since sialic acid-containing sugar chains are generally synthesized by a sialyltransferase catalyst, sialyltransferase is one of the enzymes in high demand. Regarding β-galactoside-α2,6-sialyltransferase and its gene, those derived from animals, particularly mammals (Hamamoto, T., et al., Bioorg. Med. Chem., 1, 141-145 (1993) Weinstein, J., et al., J. Biol. Chem., 262, 17735-17743 (1987)), those derived from microorganisms belonging to Photobacterium damselae (international) Publication No. WO 98/38315; US Pat. No. 6,255,094, Yamamoto, T., et al., Biosci. Biotechnol. Biochem., 62 (2), 210-214 (1998), Yamamoto, T., et al., J. Biochem., 123, 94-100 (1998), Yamamoto, T., et al., J. Biochem., 120, 104-110 (1996)). (Pasteurella multocida) derived α2,3-sialyltransferase (Yu, H., et al., J. Am. Chem. Soc., 127, 17618-17619 (2005)), derived from marine microorganisms (Tsukamoto , H., et al., J. Biol. Chem., 282, 29794-29802 (2007), Takakura, Y., et al., J. Biochem., 142, 403-412 (2007)).

  Sialyltransferase uses cytidine 5'-monophosphosialic acid (hereinafter sometimes abbreviated as CMP-Sia) as a sugar donor, and transfers sialic acid to a sugar chain serving as an acceptor. However, since chemical synthesis of CMP-Sia requires many steps, it is extremely expensive. As a result, sialic acid-containing sugar chains have become expensive and have been said to be a problem to be improved when used as research and pharmaceuticals. Thus, an enzymatic method using the enzyme cytidine 5'-monophosphosialic acid synthase that catalyzes the following reaction formula is considered as an effective means.

  Regarding cytidine 5′-monophosphosialic acid synthase and its gene, those derived from animals, mainly mammals (Haft, RF, et al., Mol. Micro. 19, 555-563 (1996); Rodriguez-Aparicio, LB, Luengo, JM, et al., J. Biol. Chem. 267, 9257-9263 (1992)) and those derived from microorganisms (Vann, WF, et al., J. Biol. Chem. 262, 17556- 17562 (1987); Bravo, IG, et al., Biochem. J. 358, 585-598 (2001); Tullius, MV, et al., J. Biol. Chem. 271, 15373-15380 (1996)) Are known. Among them, microorganism-derived enzymes are known to be more stable and broad in substrate specificity than those derived from mammals (Mizanur RM, Pohl NL, Appl Microbiol Biotechnol. 80 (5), 757-765 (2008)). For example, glycolylneuraminic acid (NeuGc) is expected to be used as a marker for diseases such as cancer among sialic acids, but mammal-derived enzymes are usually N-acetylneuraminic acid (NeuAc) and cytidine triphosphate. CMP-NeuAc can be synthesized from an acid, but CMP-NeuGc cannot be synthesized from N-glycolylneuraminic acid and cytidine triphosphate. On the other hand, those derived from microorganisms can synthesize CMP-NeuGc using glycolylneuraminic acid as a substrate.

Also, in the method using an enzyme, unlike chemical synthesis, the synthesis of CMP-Sia can be completed in one step.
Because of the advantages as described above, a new cytidine 5′-monophosphosialic acid synthase derived from microorganisms is required.

International Publication No. WO 98/38315 Pamphlet US Pat. No. 6,255,094

Hamamoto, T., et al., Bioorg. Med. Chem., 1, 141-145 (1993) Weinstein, J., et al., J. Biol. Chem., 262, 17735-17743 (1987) Yamamoto, T., et al., Biosci. Biotechnol. Biochem., 62 (2), 210-214 (1998) Yamamoto, T., et al., J. Biochem., 123, 94-100 (1998) Yamamoto, T., et al., J. Biochem., 120, 104-110 (1996) Yu, H., et al., J. Am. Chem. Soc., 127, 17618-17619 (2005) Tsukamoto, H., et al., J. Biol. Chem., 282, 29794-29802 (2007) (Takakura, Y., et al., J. Biochem., 142, 403-412 (2007)) Haft, R.F., et al., Mol. Micro. 19, 555-563 (1996) Rodriguez-Aparicio, L.B., et al., J. Biol. Chem. 267, 9257-9263 (1992) Vann, W.F., et al., J. Biol. Chem. 262, 17556-17562 (1987) Bravo, I.G., et al., Biochem. J. 358, 585-598 (2001) Tullius, M.V., et al., J. Biol. Chem. 271, 15373-15380 (1996) Mizanur RM, Pohl NL., Appl Microbiol Biotechnol. 80 (5), 757-765 (2008)

  An object of the present invention is to provide a novel cytidine 5'-monophosphosialic acid synthase derived from a microorganism belonging to the genus Vibrioaceae and a gene encoding the same.

  The present inventors isolated over 4,000 strains of microorganisms from all over Japan, and as a result of diligent research on their properties, the present inventors have obtained a novel cytidine 5′-mono from strains of microorganisms belonging to the genus Photobacterium. A strain producing phosphosialic acid synthase was found and the present invention was completed. In addition, the amino acid sequence of the novel cytidine 5′-monophosphosialic acid synthase obtained this time is the currently known cytidine 5′-monophosphosialic acid synthase derived from mammals and the cytidine 5′-mono derived from microorganisms. It showed only 20% or less homology with the amino acid sequence of phosphosialic acid synthase. The present invention provides a novel cytidine 5'-monophosphosialic acid synthase and a nucleic acid encoding the same.

Preferred Embodiments of the Invention The present invention preferably includes the following embodiments.
[Aspect 1]
An isolated protein comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4;
(B) an amino acid sequence encoded by a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3;
Said protein comprising.

[Aspect 2]
An isolated protein having cytidine 5′-monophosphosialic acid synthase activity comprising:
(A) In the amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4, including deletion, substitution, insertion and / or addition of one or more amino acids Amino acid sequence;
(B) an amino acid sequence having 60% or more amino acid identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4;
(C) a nucleotide sequence comprising one or more nucleotide deletions, substitutions, insertions and / or additions in a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3 The amino acid sequence encoded by
(D) an amino acid sequence encoded by SEQ ID NO: 1, a nucleotide sequence selected from the group consisting of nucleotides 82-1632 of SEQ ID NO: 1, and a base sequence having 70% or more identity with a base sequence selected from SEQ ID NO: 3; and (e ) An amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to a complementary strand of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3;
The protein comprising an amino acid sequence selected from the group consisting of:

[Aspect 3]
The isolated protein according to embodiment 1 or 2, which is derived from a microorganism belonging to the genus Photobacterium.

[Aspect 4]
An isolated protein having cytidine 5'-monophosphosialic acid synthase activity having the following properties:
(A) a molecular weight of 62 ± 4 kDa or 60 ± 4 kDa by SDS-PAGE analysis; and (b) an isoelectric point of pH 4.8 to pH 5.2 by isoelectric focusing analysis;
The protein.

[Aspect 5]
Cytidine 5′-monophosphosialic acid synthase activity has the following properties:
(A) an optimum pH of pH 8 to pH 9;
(B) an optimum temperature of 30 ° C to 40 ° C;
(C) an optimal MgCl ₂ concentration of at least 5 mM;
The protein according to aspect 4, which has

[Aspect 6]
The protein according to embodiment 4 or 5, wherein the N-terminal amino acid sequence after cleavage of the signal peptide comprises the amino acid sequence of SEQ ID NO: 5, and the sequences of SEQ ID NOs: 6 and 7 as internal sequences.

[Aspect 7]
An isolated nucleic acid comprising:
(A) a base sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4; or
(B) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3;
Comprising said nucleic acid.

[Aspect 8]
An isolated nucleic acid encoding a protein having cytidine 5'-monophosphosialic acid synthase activity, comprising:
(A) In the amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4, including deletion, substitution, insertion and / or addition of one or more amino acids A base sequence encoding an amino acid sequence;
(B) a base sequence encoding an amino acid sequence having 60% or more amino acid identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4;
(C) a nucleotide sequence comprising one or more nucleotide deletions, substitutions, insertions and / or additions in a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3 ;
(D) a nucleotide sequence having 70% or more identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3; and (e) SEQ ID NO: 1, SEQ ID NO: A nucleotide sequence that hybridizes under stringent conditions to a complementary strand of a nucleotide sequence selected from the group consisting of 1 nucleotide 82-1632 and SEQ ID NO: 3;
The nucleic acid comprising a base sequence selected from the group consisting of:

[Aspect 9]
An expression vector comprising the nucleic acid according to aspect 7 or 8.
[Aspect 10]
A host cell transformed with the expression vector according to aspect 9.

[Aspect 11]
An isolated microorganism belonging to the genus Photobacterium that expresses the protein according to any one of aspects 1 to 6.

[Aspect 12]
A method for producing a protein having cytidine 5′-monophosphosialic acid synthase activity, comprising the following steps:
1) culturing a microorganism that produces the protein according to any one of Embodiments 1 to 6;
2) isolating a protein having cytidine 5′-monophosphosialic acid synthase activity from the cultured microorganism or culture supernatant;
The said manufacturing method including this.

[Aspect 13]
The method according to aspect 12, wherein the microorganism that produces the protein according to any one of aspects 1 to 6 is Photobacterium leiognathi.

[Aspect 14]
A method for producing a recombinant protein having cytidine 5′-monophosphosialic acid synthase activity, comprising the following steps:
1) transforming a host cell with an expression vector comprising the nucleic acid according to embodiment 7 or 8;
2) culturing the resulting transformed cells; and
3) Isolating a protein having cytidine 5′-monophosphosialic acid synthase activity from the cultured transformed cells or the culture supernatant thereof;
The said manufacturing method including this.

[Aspect 15]
An antibody that specifically recognizes the protein according to any one of aspects 1 to 6.

  The present invention provides a novel cytidine 5′-monophosphosialic acid synthase and a nucleic acid encoding the same, thereby providing a sialic acid-containing sugar chain that has been clarified to have an important function in vivo. Contributes in terms of providing synthesis and production means. Sialic acid is often present at the non-reducing end in complex carbohydrate sugar chains in vivo, and the sugar chains containing it are extremely important sugars from the viewpoint of sugar chain function. In general, a sialyltransferase is used for the synthesis of a sialic acid-containing sugar chain, but cytidine 5'-monophosphosialic acid is essential as a donor substrate of the sialyltransferase. Since cytidine 5'-monophosphosialic acid is difficult and expensive to synthesize chemically, synthesis using enzymes is currently the mainstream. Therefore, cytidine 5′-monophosphosialic acid synthase is one of the enzymes in high demand, and the provision of the novel cytidine 5′-monophosphosialic acid synthase according to the present invention meets such high demand. It is.

FIG. 1 shows the correlation between the culture turbidity (OD ₆₀₀ ) of JT-SHIZ-145 strain (deposit number: NITE BP-695) and cytidine 5′-monophosphosialic acid synthase activity. It is. The vertical axis represents relative activity, with the cytidine 5′-monophosphosialic acid synthase activity at 10 hours after the start of main culture as 100. FIG. 2-1 is a graph showing the influence of reaction pH on cytidine 5'-monophosphosialic acid synthase activity derived from JT-SHIZ-145 strain. The activity of cytidine 5'-monophosphosialic acid synthase when tris-hydrochloric acid buffer (pH 9) was used was defined as 100% and expressed as relative activity. Each buffer was added to the reaction solution to a final concentration of 100 mM. The abbreviations in the figure indicate the following: BisTris: Bis-Tris buffer, KPB: phosphate buffer, Tris-HCl: Tris-HCl buffer, CHES: CHES buffer, CAPS: CAPS buffer. FIG. 2-2 is a graph showing the influence of reaction temperature on cytidine 5'-monophosphosialic acid synthase activity derived from JT-SHIZ-145 strain. The cytidine 5'-monophosphosialic acid synthase activity at 35 ° C was defined as 100% and expressed as a relative activity. FIG. 2-3 is a graph showing the influence of various metal salts in the reaction solution on cytidine 5′-monophosphosialic acid synthase activity derived from JT-SHIZ-145 strain. The activity of cytidine 5′-monophosphosialic acid synthase when MgCl _{2 was} added was defined as 100% and expressed as relative activity. Each metal ion was added to the reaction solution so that the final concentration was 10 mM. FIG. 2-4 is a graph showing the effect of MgCl ₂ concentration in the reaction solution on cytidine 5′-monophosphosialic acid synthase activity derived from JT-SHIZ-145 strain. The cytidine 5′-monophosphosialic acid synthase activity when the MgCl ₂ concentration in the reaction solution was 10 mM was defined as 100%, and the relative activity was shown. FIG. 2-5 is a diagram showing an isoelectric focusing gel of cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain. Numbers listed are isoelectric points of marker proteins. FIG. 3-1 is a graph showing the reactivity of cytidine 5'-monophosphosialic acid synthase derived from JT-SHIZ-145 strain to NeuAc solutions at various concentrations. The Y axis shows the enzyme reaction rate when using NeuAc solution of each concentration as a substrate (unit: nmol / min). Further, the X axis represents the concentration of NeuAc solution in the reaction solution (unit: mM). Fig. 3-2 shows the reactivity of cytidine 5'-monophosphosialic acid synthase derived from the JT-SHIZ-145 strain to NeuAc solutions at various concentrations in a Lineweaver-Burk plot. The Y axis shows the reciprocal of the enzyme reaction rate when each concentration of NeuAc solution was used as a substrate. The X axis represents the reciprocal of the concentration of NeuAc solution in the reaction solution. The calculation formula shown in the figure is a calculation formula used to calculate the Michaelis constant (Km) and the maximum speed (Vmax). FIG. 3-3 is a graph showing the reactivity of cytidine 5'-monophosphosialic acid synthase derived from JT-SHIZ-145 strain to CTP solutions at various concentrations. The Y axis shows the enzyme reaction rate when using CTP solutions of various concentrations as substrates (unit: nmol / min). Further, the X axis represents the concentration of the CTP solution in the reaction solution (unit: mM). FIG. 3-4 shows the reactivity of cytidine 5'-monophosphosialic acid synthase derived from JT-SHIZ-145 strain to CTP solutions at various concentrations in a Lineweaver-Burk plot. The Y axis shows the reciprocal of the enzyme reaction rate when each concentration of CTP solution was used as a substrate. The X axis represents the reciprocal of the concentration of the CTP solution in the reaction solution. The calculation formula shown in the figure is a calculation formula used to calculate the Michaelis constant (Km) and the maximum speed (Vmax). Fig. 4-1 shows the production of CMP-Sia produced by reacting NeuAc or NeuGc with CTP as a substrate sialic acid using a cytidine 5'-monophosphosialic acid synthase solution derived from JT-SHIZ-145 strain. The area is shown. Cytidine 5'-monophosphosialic acid synthase using NeuAc as a substrate was defined as 100%, and the relative activity was shown. FIG. 4-2 shows a reaction solution obtained by reacting a cytidine 5′-monophosphosialic acid synthase solution derived from JT-SHIZ-145 strain with NeuAc and CTP, and further pyridylaminated lactose (PA-lactose) and β- It is a figure which shows the HPLC analysis result of the reaction solution made to react with galactoside- (alpha) 2,3-sialyltransferase. The peak with a retention time of 3.835 minutes is PA-lactose and the peak of 5.103 minutes is PA-3'NeuAc-lactose. Fig. 4-3 shows a reaction solution obtained by reacting cytidine 5'-monophosphosialic acid synthase solution derived from JT-SHIZ-145 strain with NeuAc and CTP, and further mixed with pyridylaminated lactose (PA-lactose). It is a figure which shows the HPLC analysis result in the case. It is the result of the control experiment which does not mix (beta) -galactoside- (alpha) 2,3-sialyltransferase with the reaction liquid with respect to experiment of FIG. 4-2. The peak with a retention time of 3.834 minutes is PA-lactose. FIG. 4-4 shows a reaction solution in which a cytidine 5′-monophosphosialic acid synthase solution derived from JT-SHIZ-145 strain was reacted with NeuGc and CTP, and further pyridylaminated lactose (PA-lactose) and β- It is a figure which shows the HPLC analysis result of the reaction solution made to react with galactoside- (alpha) 2,3-sialyltransferase. The peak with a retention time of 3.834 minutes is PA-lactose, and the peak at 4.763 minutes is PA-3'NeuGc-lactose. FIG. 4-5 shows that a reaction solution obtained by reacting a cytidine 5′-monophosphosialic acid synthase solution derived from JT-SHIZ-145 strain with NeuGc and CTP was further mixed with pyridylaminated lactose (PA-lactose). It is a figure which shows the HPLC analysis result in the case. FIG. 4 is the result of a control experiment in which β-galactoside-α2,3-sialyltransferase is not mixed in the reaction solution with respect to the experiment of FIG. 4-4. The peak with a retention time of 3.834 minutes is PA-lactose.

Hereinafter, modes for carrying out the present invention will be described.
Cytidine 5′-monophosphosialic acid synthase The present invention provides a novel cytidine 5′-monophosphosialic acid synthase. In this specification, “cytidine 5′-monophosphosialic acid synthase” means an activity that catalyzes the synthesis reaction of cytidine 5′-monophosphosialic acid using cytidine triphosphate (CTP) and sialic acid as substrates. It means the protein which has. In the present specification, “cytidine 5′-monophosphosialic acid synthase activity” means the activity described above for cytidine 5′-monophosphosialic acid synthase. Moreover, sialic acid (Sia) here shows the neuraminic acid derivative which belongs to the sialic acid family. Specifically, N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 5-deamino-5-hydroxyneuraminic acid (KDN), disialic acid (diN-acetylneuraminic acid) : Neu5Acα2,8 (9) Neu5Ac), 9-O-acetylneuraminic acid (9-O-acetyl-NeuAc), 4-O-acetylneuraminic acid (4-O-acetyl-NeuAc), 8-sulfo- N-glycolylneuraminic acid (N-glycoryl-8-sulfo-Neu), etc. are shown. Note that sialic acid (Sia) here also includes 3-deoxy-D-manno-octuronic acid (KDO).

  The cytidine 5'-monophosphosialic acid synthase of the present invention is a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 4 corresponds to a sequence obtained by adding methionine to the N-terminus of the amino acid sequence of amino acids 28-543 of SEQ ID NO: 2. This N-terminal methionine is derived from the initiation codon for protein expression and does not affect the activity as a cytidine 5'-monophosphosialic acid synthase. In addition, methionine at the N-terminal of proteins is often lost by intracellular processing. Therefore, the protein comprising the amino acid sequence of SEQ ID NO: 4 is not only a protein comprising an amino acid sequence that completely matches SEQ ID NO: 4, but also an amino acid lacking the N-terminal methionine. Including the case of a protein comprising a sequence.

  The cytidine 5'-monophosphosialic acid synthase of the present invention is a protein encoded by a nucleic acid comprising the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 3. The base sequence of SEQ ID NO: 3 corresponds to the sequence obtained by adding a start codon (ATG) to the 5 ′ end of the base sequence of nucleotides 82-1632 of SEQ ID NO: 1, and the base sequences of SEQ ID NO: 3 are respectively The amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4 are encoded.

  In the cytidine 5′-monophosphosialic acid synthase comprising the amino acid sequence of SEQ ID NO: 2 of the present invention, the sequence of amino acids 1-27 of SEQ ID NO: 2 is isolated and purified from a microbial cell disruption solution This is a sequence that was not found in the N-terminal amino acid sequence analysis performed using 5′-monophosphosialic acid synthase (see Example 2 described later), and because it is a sequence rich in hydrophobic amino acids, Conceivable. That is, it is considered that after the protein having the amino acid sequence of SEQ ID NO: 2 is expressed in the cells of the microorganism, it is cleaved between amino acids 27 and 28 of the same sequence. Accordingly, the cytidine 5'-monophosphosialic acid synthase of the present invention may be a protein comprising the amino acid sequence of amino acids 28-543 of SEQ ID NO: 2. The cytidine 5'-monophosphosialic acid synthase of the present invention may be a protein encoded by a nucleic acid comprising the nucleotide sequence of nucleotides 82-1632 of SEQ ID NO: 1.

  The present invention also includes a mutant of the above-described cytidine 5'-monophosphosialate synthase of the present invention, which has a cytidine 5'-monophosphosialate synthase activity. Such a mutant protein is also included in the cytidine 5'-monophosphosialic acid synthase of the present invention.

  The mutant protein of the present invention has a deletion, substitution, insertion and / or deletion of one or more amino acids in an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acids 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4. It may be a protein comprising an amino acid sequence including an addition and having cytidine 5′-monophosphosialic acid synthase activity. The substitution may be a conservative substitution, which is the replacement of a particular amino acid residue with a residue having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include substitutions between aliphatic group-containing amino acid residues such as Ile, Val, Leu or Ala mutual substitutions, Lys and Arg, Glu and Asp, Gln and Asn mutual substitutions. Substitution between polar residues such as substitution is included.

  Mutations resulting from amino acid deletions, substitutions, insertions and / or additions may be performed on the DNA encoding the wild-type protein, for example by site-directed mutagenesis (eg, Nucleic Acid Research, Vol. 10, No. 20), which is a well-known technique. , p. 6487-6500, 1982, the entire contents of which are incorporated herein by reference). As used herein, “one or more amino acids” means amino acids that can be deleted, substituted, inserted and / or added by site-directed mutagenesis. Alternatively, in the present specification, “one or more amino acids” refers to 1 to 150 amino acids, 1 to 100 amino acids, 1 to 75 amino acids, 1 to 50 amino acids, and 1 to 25 amino acids. 1 to 15 amino acids, 1 to 10 amino acids, 1 to 8 amino acids, 1 to 5 amino acids, 1 to 3 amino acids, or 1 or a few amino acids There may be.

  Site-directed mutagenesis can be performed, for example, using a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated, in addition to the specific mismatch that is the desired mutation, as follows: . That is, the synthetic oligonucleotide is used as a primer to synthesize a strand complementary to the phage, and a host cell is transformed with the obtained double-stranded DNA. Transformed bacterial cultures are plated on agar and plaques are formed from single cells containing phage. Then, theoretically 50% of the new colonies contain phages with mutations as single strands and the remaining 50% have the original sequence. The obtained plaque is hybridized with a synthetic probe labeled by kinase treatment at a temperature that hybridizes with the DNA having the desired mutation and that does not hybridize with DNA having the original strand. . Next, plaques that hybridize with the probe are picked up and cultured to recover DNA.

  In addition to the site-directed mutagenesis described above, a method for performing deletion, substitution, insertion and / or addition of one or more amino acids while maintaining the activity of the amino acid sequence of a biologically active peptide such as an enzyme There are also methods of treating a gene with a mutagen and methods of selectively cleaving the gene, then removing, replacing, inserting or adding selected nucleotides, and then ligating.

  The mutant protein of the present invention also includes deletion, substitution, insertion and / or deletion of one or more nucleotides in a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3. Alternatively, it may be a protein encoded by a nucleic acid comprising a base sequence including an addition and having cytidine 5′-monophosphosialic acid synthase activity. Nucleotide deletions, substitutions, insertions and / or additions can be performed by the methods described above in addition to site-specific displacement induction.

  The variant protein of the present invention further comprises at least 60% or more, preferably 65% or more, preferably 70% or more of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acids 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4. 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, more preferably 99.5% or more amino acids It may be a protein comprising an amino acid sequence having identity and having cytidine 5′-monophosphosialic acid synthase activity.

  Alternatively, the mutant protein of the present invention comprises at least 70% or more, preferably 75% or more, preferably 80% or more of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3. Or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, more preferably 99.5% or more. And a protein having cytidine 5′-monophosphosialic acid synthase activity.

  The percent identity between two amino acid sequences may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences is based on the algorithm of Needleman, SB and Wunsch, CD (J. Mol. Biol., 48: 443-453, 1970), and the University of Wisconsin Genetics Computer Group (UWGCG) It may be determined by comparing the sequence information using a more available GAP computer program. Preferred default parameters for the GAP program include: (1) Scoring matrix as described in Henikoff, S. and Henikoff, JG (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992). , Blosum62; (2) 12 gap weights; (3) 4 gap length weights; and (4) no penalty for end gaps.

  Other programs used by those skilled in the art of sequence comparison may also be used. The percent identity can be determined by comparison with sequence information using, for example, the BLAST program described in Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997). The program can be used on the Internet from the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ) website. Various conditions (parameters) for identity search by the BLAST program are described in detail on the same site, and some settings can be changed as appropriate, but the search is usually performed using default values. Alternatively, the percent identity between two amino acid sequences can be determined by genetic information processing software GENETYX Ver. The program may be determined using a program such as 7 (manufactured by Genetics) or the FASTA algorithm. At that time, the default value may be used for the search.

  The percent identity between two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, this comparison is made by comparing the sequence information using a computer program. A typical preferred computer program is the Wisconsin package, version 10.0 program “GAP” from the Genetics Computer Group (GCG; Madison, Wis.) (Devereux, et al., 1984, Nucl. Acids Res ., 12: 387). By using this “GAP” program, in addition to comparing two nucleic acid sequences, two amino acid sequences can be compared, and a nucleic acid sequence and an amino acid sequence can be compared. Here, the preferred default parameters for the “GAP” program are: (1) GCG run of unary comparison matrix for nucleotides (including values of 1 for identical and 0 for non-identical), Schwartz and Gribskov and Burgess, Nucl. Acids Res., 14 as described by Dayhoff, “Atlas of Polypeptide Sequence and Structure,” National Biomedical Research Foundation, pages 353-358, 1979. : Weighted amino acid comparison matrix of 6745, 1986; or other comparable comparison matrix; (2) 30 penalties for each gap of amino acids and one additional penalty for each symbol in each gap; or each gap in nucleotide sequence About 50 penalties and an additional for each symbol in each gap 3 penalties; (3) no penalty to end gaps; and (4) no maximum penalty for long gaps. Other sequence comparison programs used by those skilled in the art are available, for example, by using the National Medical Library website: http://www.ncbi.nlm.nih.gov/blast/bl2seq/bls.html The BLASTN program, version 2.2.7, or UW-BLAST 2.0 algorithm can be used. Standard default parameter settings for UW-BLAST 2.0 are described at the following Internet site: http://blast.wustl.edu. In addition, the BLAST algorithm uses a BLOSUM62 amino acid scoring matrix and the selection parameters that can be used are: (A) a segment of query sequence with low composition complexity (Wootton and Federhen's SEG program (Computers and Chemistry, 1993); Wootton and Federhen, 1996 “Analysis of compositionally biased regions in sequence databases” Methods Enzymol., 266: 544-71) Or include a filter to mask segments consisting of short-periodic internal repeats (determined by the XNU program of Claverie and States (Computers and Chemistry, 1993)), and (B) match against database sequences A statistical significance threshold to report, or According to the statistical model of the E-score (Karlin and Altschul, 1990), the expected probability of a match found by chance; if the statistically significant difference due to a match is greater than the E-score threshold, the fit is Not reported); the preferred E-score threshold value is 0.5 or, in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75, or 1e-100.

  The mutant protein of the present invention also includes a base sequence that hybridizes under stringent conditions to a complementary strand of a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3. And a protein having cytidine 5′-monophosphosialic acid synthase activity.

  Here, “under stringent conditions” means to hybridize under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art having general techniques based on, for example, the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Chapter 6, Cold Spring Harbor Laboratory Press, 2001, eg 5 × SSC, 0.5% SDS, 1.0 mM EDTA. (PH 8.0) pre-wash solution, about 50% formamide at about 42 ° C., 2 × SSC-6 × SSC, preferably 5-6 × SSC, 0.5% SDS (or about 42 ° C. Hybridization conditions of other similar hybridization solutions such as Stark's solution in 50% formamide, and for example, about 50 ° C.-68 ° C., 0.1-6 × SSC, 0.1% SDS Use of cleaning conditions. Preferably moderately stringent conditions include hybridization conditions (and wash conditions) at about 50 ° C., 6 × SSC, 0.5% SDS. High stringency conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA.

In general, these conditions include hybridization at higher temperatures and / or lower salt concentrations than moderately stringent conditions (eg, containing about 0.5% SDS, about 65 ° C., 6 × SSC to 0. 2 × SSC, preferably 6 × SSC, more preferably 2 × SSC, more preferably 0.2 × SSC, or even 0.1 × SSC) and / or washing, for example hybridization as described above Defined with conditions and washing at approximately 65 ° C.-68 ° C., 0.2 to 0.1 × SSC, 0.1% SDS. In hybridization and wash buffers, SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) to SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1. 25 mM EDTA, pH 7.4) can be substituted, and washing is performed for 15 minutes to 1 hour after hybridization is completed.

  In addition, a commercially available hybridization kit that does not use a radioactive substance for the probe can also be used. Specific examples include hybridization using an ECL direct labeling & detection system (manufactured by Amersham). For stringent hybridization, for example, 5% (w / v) Blocking reagent and 0.5M NaCl are added to the hybridization buffer in the kit, and the reaction is performed at 42 ° C. for 4 hours. A condition is that 20% twice at 55 ° C. for 20 minutes in 4% SDS, 0.5 × SSC, and once at room temperature for 5 minutes in 2 × SSC.

  Cytidine 5′-monophosphosialic acid synthase activity is described in known procedures, for example, J. Biol. Chem. 262, 17556-17562 (1987), which is incorporated herein by reference in its entirety. You may measure by the method. For example, an enzyme reaction is carried out using CTP (cytidine triphosphate) and N-acetylneuraminic acid (NeuAc) as substrates, and the amount of cytidine 5′-monophosphosialic acid, which is a reaction product, is evaluated. Activity can be evaluated. One unit (1 U) of enzyme is the amount of enzyme that synthesizes 1 micromole of cytidine 5'-monophosphosialic acid per minute.

  The mutant protein of the present invention is also a protein that causes an antigen-antibody reaction by a technique such as Western blotting using a polyclonal antibody and a monoclonal antibody prepared by using the cytidine 5′-monophosphosialic acid synthase described above as an antigen. Or a protein having cytidine 5′-monophosphosialic acid synthase activity.

  In one embodiment of the present invention, the enzyme of the present invention is derived from a microorganism belonging to the genus Photobacterium. The enzyme of the present invention is not particularly limited as long as it is derived from a microorganism belonging to the genus Photobacterium, and may be an enzyme derived from a new species of microorganism belonging to the genus Photobacterium. As the microorganism to be derived, a microorganism belonging to Photobacterium leiognathi is preferable, and more preferably, Photobacterium leiognacy JT-SHIZ-145 strain (deposit number: NITE BP-695) is used. Can be mentioned.

The enzymatic and physicochemical properties of the cytidine 5'-monophosphosialic acid synthase of the present invention are characterized by having the cytidine 5'-monophosphosialic acid synthase activity defined above and are limited. Not that,
(A) In the protein of the present invention having a signal peptide, the molecular weight is about 62,000 ± 4,000 Da or about 62,000 ± 2,000 Da by SDS-PAGE analysis, or the present invention has no signal peptide. The molecular weight is about 60,000 ± 4,000 Da, or about 60,000 ± 2,000 Da by SDS-PAGE analysis;
(B) the isoelectric point analyzed by isoelectric focusing is about pH 4.8 to about pH 5.2, ie about pH 5;
(C) the optimum pH is in the range of pH 8-9;
(D) the optimum temperature is 30-40 ° C., preferably 35-40 ° C .; and (e) the addition of a metal salt is required for its enzymatic activity, in particular the optimum MgCl ₂ concentration is at least 5 mM. Is
It may be characterized by having one or more properties selected from the group consisting of:

  The cytidine 5'-monophosphosialic acid synthase of the present invention has an activity of catalyzing the synthesis reaction of cytidine 5'-monophosphosialic acid using cytidine triphosphate (CTP) and sialic acid as substrates. Here, as sialic acid that can be used as a substrate by the cytidine 5′-monophosphosialic acid synthase of the present invention, N-acetylneuraminic acid (NeuAc), N-glycolylneuraminic acid (NeuGc), 5- Deamino-5-hydroxyneuraminic acid (KDN), 9-O-acetylneuraminic acid (9-O-acetyl-NeuAc), 4-O-acetylneuraminic acid (4-O-acetyl-NeuAc), 8- And sulfo-N-glycolyl neuraminic acid (N-glycyl-8-sulfo-Neu).

The protein of the present invention also includes the following proteins.
(1) An amino acid sequence comprising a deletion, substitution, insertion and / or addition of one or more amino acids in an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acids 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4 A protein comprising:
(2) a protein comprising an amino acid sequence having at least 60% amino acid identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acids 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4;
(3) a protein comprising an amino acid sequence encoded by a nucleic acid having at least 70% identity with a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3; Or (4) encoded by a nucleic acid comprising a base sequence that hybridizes under stringent conditions to a complementary strand of a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3 Protein.

Nucleic acid encoding cytidine 5′-monophosphosialic acid synthase The present invention provides a nucleic acid encoding cytidine 5′-monophosphosialic acid synthase.
The nucleic acid of the present invention is a nucleic acid encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acids 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4. The nucleic acid of the present invention is also a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3.

  The nucleic acid of the present invention may be a nucleic acid variant of the above-described nucleic acid that encodes a protein having cytidine 5'-monophosphosialic acid synthase activity. Such nucleic acids are also included in the nucleic acid encoding the cytidine 5'-monophosphosialic acid synthase of the present invention.

  Such a nucleic acid variant includes a deletion, substitution, insertion and / or deletion of one or more amino acids in an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acids 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4. Alternatively, it is a nucleic acid that encodes a protein comprising an amino acid sequence containing an addition and having cytidine 5′-monophosphosialic acid synthase activity. The nucleic acid variant of the present invention also includes deletion, substitution, insertion and deletion of one or more nucleotides in the base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3. It is a nucleic acid comprising a base sequence containing an addition. Amino acid or nucleotide deletions, substitutions, insertions and / or additions can be introduced by the methods described above.

  Further, such a nucleic acid variant has at least 60% or more, preferably 65% or more, 70% or more of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and amino acids 82-543 of SEQ ID NO: 2 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, more preferably 99.5% or more A nucleic acid encoding a protein comprising an amino acid sequence having identity and having cytidine 5′-monophosphosialic acid synthase activity. The variant of the nucleic acid of the present invention also contains at least 70% or more, preferably 75% or more, 80% or more of a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3. Or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more, more preferably 99.5% or more nucleic acid. The nucleic acid is a nucleic acid encoding a protein having cytidine 5′-monophosphosialic acid synthase activity. Here, the identity of amino acid sequences or base sequences can be determined by the method shown above.

  Such a nucleic acid variant further has stringent conditions, or highly stringent, in a complementary strand of a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3. A nucleic acid comprising a base sequence that hybridizes under various conditions, wherein the nucleic acid encodes a protein having cytidine 5′-monophosphosialic acid synthase activity. Here, stringent conditions or highly stringent conditions are as defined above.

  The nucleic acids and nucleic acid variants obtained by the above methods can also be used as probes in gene sequence screening such as Southern blotting using gene sequence libraries of various microorganisms.

The nucleic acid of the present invention also includes the following nucleic acids.
(1) An amino acid sequence comprising a deletion, substitution, insertion and / or addition of one or more amino acids in an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acids 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4 A nucleic acid encoding a protein comprising:
(2) a nucleic acid encoding a protein comprising an amino acid sequence having at least 60% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and amino acids 82-543 of SEQ ID NO: 2;
(3) a nucleic acid having at least 70% identity with a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3; or (4) SEQ ID NO: 1, sequence A nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions to a complementary strand of a nucleotide sequence selected from the group consisting of nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3.

Microorganisms expressing cytidine 5′-monophosphosialic acid synthase The present inventors have found that microorganisms belonging to the genus Vibrioaceae Photobacterium express a novel cytidine 5′-monophosphosialic acid synthase. Accordingly, the present invention provides a microorganism that expresses cytidine 5′-monophosphosialic acid synthase. The microorganism of the present invention is a microorganism belonging to the genus Photobacterium and having the ability to produce cytidine 5′-monophosphosialic acid synthase. The microorganism of the present invention is preferably a microorganism belonging to Photobacterium leiognathi, and more preferably, Photobacterium leiognacy JT-SHIZ-145 strain. Photobacterium Leognacy JT-SHIZ-145 strain was deposited on December 26, 2008 as deposit number: NITE BP-695, NITE; 2-5-8). The microorganisms of the genus Photobacterium are generally marine bacteria and are isolated from seawater or marine fish and shellfish.

  The microorganism of the present invention can be isolated using, for example, a screening method as described below. Seawater, sea sand, sea mud or marine fish and shellfish are used as microbial sources. Seawater, sea sand and sea mud should be used as inoculation source as they are or diluted with sterilized seawater. For marine fish and shellfish, surface mucus or the like is scraped off with a loop as an inoculation source, or a liquid obtained by grinding internal organs in sterile seawater is used as an inoculation source. These are applied on a plate medium such as Marine Broth Agar 2216 medium (Becton Dickinson) or sodium chloride-added nutrient agar medium (Becton Dickinson) to obtain marine microorganisms that grow under various temperature conditions. . According to a conventional method, the obtained microorganisms are purely cultured and then each microorganism is cultured using a liquid medium such as Marine Broth 2216 medium (Becton Dickinson) or sodium chloride-added nutrient broth medium (Becton Dickinson). To do. After the microorganisms are sufficiently grown, the cells are collected from the culture solution by centrifugation. 50 mM Tris-HCl buffer (pH 9.0) containing 0.3% Triton X-100 (manufactured by Kanto Chemical Co., Ltd.) and 20% glycerin (manufactured by Wako Pure Chemical Industries, Ltd.) as a surfactant is added to the collected cells. And suspend the cells. This cell suspension is sonicated under ice cooling to disrupt cells. Using this cell disruption solution as an enzyme solution, cytidine 5'-monophosphosialic acid biosynthesis activity is measured according to a conventional method, and a strain having cytidine 5'-monophosphosialic acid biosynthesis activity can be obtained.

  Photobacterium layignacy JT-SHIZ-145 strain, which is a microorganism belonging to the genus Photobacterium of the present invention, was obtained by using the above screening method. The bacteriological and physiological biochemical properties of the obtained strain are described in detail in Example 1.

Method for Producing Cytidine 5′-Monophosphosialic Acid Synthase The present invention also relates to a method for producing the cytidine 5′-monophosphosialic acid synthase of the present invention.

(1) Method for producing the enzyme by culturing a microorganism expressing cytidine 5′-monophosphosialic acid synthase In one embodiment of the present invention, the cytidine 5′-monophosphosialic acid synthase of the present invention is a photobacteria. By culturing a microorganism that is derived from a microorganism belonging to the genus Umum and has the ability to produce cytidine 5′-monophosphosialic acid synthase in a medium to produce cytidine 5′-monophosphosialic acid synthase, can get.

  Any microorganism can be used as long as it is a microorganism belonging to the genus Photobacterium and capable of producing cytidine 5'-monophosphosialic acid synthase. Examples of microorganisms belonging to the genus Photobacterium used in the method of the present invention include Photobacterium leiognancy JT-SHIZ-145 strain.

  As a medium used for culturing the microorganism, a medium containing a carbon source, a nitrogen source, an inorganic substance and the like that can be used by the microorganism is used. Examples of the carbon source include peptone, tryptone, casein degradation product, meat extract, glucose and the like, and preferably peptone is used. As the nitrogen source, yeast extract is preferably used. Salts include sodium chloride, iron citrate, magnesium chloride, sodium sulfate, calcium chloride, potassium chloride, sodium carbonate, sodium bicarbonate, potassium bromide, strontium chloride, sodium borate, sodium silicate, sodium fluoride, ammonium nitrate, It is preferable to use an appropriate combination of disodium hydrogen phosphate and the like.

  Moreover, you may use the marine broth 2216 culture medium (product made from Becton Dickinson) containing the said component. Furthermore, artificial seawater containing the above-mentioned salts appropriately may be used, and a medium obtained by adding peptone, yeast extract or the like may be used. The culture conditions vary somewhat depending on the composition and strain of the medium. For example, when culturing Photobacterium leiognathi JT-SHIZ-145 strain, the culture temperature is 20 to 30 ° C, preferably about 25 to 30 ° C. The culture time is 6 to 48 hours, preferably about 15 to 24 hours.

  Since the target enzyme is present in the microbial cells, any known microbial cell disruption method, for example, ultrasonic pulverization method, French press pulverization method, glass bead pulverization method, dynomill pulverization method, etc. may be used. The target enzyme is separated and purified from the disrupted cells. A preferable cell disruption method in the method of the present invention is an ultrasonic disruption method. For example, after removing solid matter from the crushed cell by centrifugation, the obtained cell lysate supernatant is used as a commercially available anion exchange column, cation exchange column, gel filtration column, hydroxyapatite column, CDP-hexanol. Purification can be performed by appropriately combining column chromatography such as an amine agarose column, CMP-hexanolamine agarose column, and hydrophobic column, and native PAGE.

  The cytidine 5′-monophosphosialic acid synthase may be completely purified. However, since the partially purified product has sufficient activity, the cytidine 5′-monophosphosialic acid synthase of the present invention is a purified product. It may be present or a partially purified product.

(2) Method for producing recombinant cytidine 5′-monophosphosialic acid synthase The present invention relates to an expression vector containing a nucleic acid encoding cytidine 5′-monophosphosialic acid synthase, and a host containing the expression vector Provide cells.

  In order to produce the recombinant cytidine 5′-monophosphosialic acid synthase protein of the present invention, an expression vector selected according to the host to be used is derived from a mammal, a microorganism, a virus, an insect gene or the like. A nucleic acid sequence encoding a cytidine 5′-monophosphosialic acid synthase operably linked to an appropriate transcriptional or translational regulatory nucleotide sequence is inserted. Examples of regulatory sequences include transcriptional promoters, operators or enhancers, mRNA ribosome binding sites, and appropriate sequences that control the initiation and termination of transcription and translation.

  The nucleic acid sequence encoding cytidine 5'-monophosphosialic acid synthase inserted into the vector of the present invention is the base sequence of the nucleic acid encoding the cytidine 5'-monophosphosialic acid synthase of the present invention described above. This sequence may or may not include a leader sequence (ie, a sequence encoding a signal peptide). When a leader sequence is included, it may be a leader sequence corresponding to nucleotides 1-81 of SEQ ID NO: 1, or may be replaced with a leader sequence derived from another biological source. By replacing the leader sequence, the expression system can be designed to secrete the expressed protein out of the host cell.

In addition, the recombinant cytidine 5′-monophosphosialic acid synthase protein of the present invention has a nucleic acid encoding the enzyme followed by a nucleic acid encoding a His tag, a FLAG ^TM tag, glutathione-S-transferase and the like. It can also be expressed as a fusion protein by inserting the nucleic acid into a vector. By expressing the enzyme of the present invention as such a fusion protein, purification and detection of the enzyme can be facilitated.

  Suitable host cells for expression of cytidine 5'-monophosphosialic acid synthase protein include prokaryotic cells, yeast or higher eukaryotic cells. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985) (hereby incorporated by reference in its entirety). In Japanese).

  Prokaryotes include gram negative or gram positive bacteria such as E. coli or Bacillus subtilis. When a prokaryotic cell such as E. coli is used as a host, the cytidine 5′-monophosphosialic acid synthase protein contains an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic cell. You may make it. This N-terminal methionine can also be cleaved from the recombinant cytidine 5'-monophosphosialic acid synthase protein after expression.

  Expression vectors used in prokaryotic host cells generally contain one or more phenotypically selectable marker genes. A phenotypically selectable marker gene is, for example, a gene that confers antibiotic resistance or confers autotrophic requirements. Examples of suitable expression vectors for prokaryotic host cells include commercially available plasmids such as pBR322 (ATCC 37017) or those derived therefrom. Since pBR322 contains genes for ampicillin and tetracycline resistance, it is easy to identify transformed cells. An appropriate promoter as well as the DNA sequence of the nucleic acid encoding cytidine 5'-monophosphosialic acid synthase is inserted into this pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotech., Madison, Wis., USA).

  Promoter sequences commonly used in expression vectors for prokaryotic host cells include tac promoter, β-lactamase (penicillinase) promoter, lactose promoter (Chang et al., Nature 275: 615, 1978; and Goeddel et al., Nature 281: 544, 1979. , Which is incorporated herein by reference in its entirety.) And the like.

  Alternatively, recombinant cytidine 5'-monophosphosialic acid synthase protein may be expressed in a yeast host. Preferably, the genus Saccharomyces (eg, S. cerevisiae) is used, but other yeast genera such as Pichia or Kluyveromyces may be used. Yeast vectors often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a sequence for polyadenylation, a sequence for transcription termination, and a selectable marker gene. . Yeast alpha factor leader sequence can be used to cause secretion of recombinant cytidine 5'-monophosphosialic acid synthase protein. Other leader sequences suitable for promoting secretion of recombinant polypeptides from yeast hosts are also known. Methods for transforming yeast are described, for example, in Hinnnen et al., Proc. Natl. Acad. Sci. USA, 75: 1929-1933, 1978 (incorporated herein by reference in its entirety).

 Mammalian or insect host cell culture systems can also be used to express recombinant cytidine 5'-monophosphosialic acid synthase protein. Mammalian origin cell lines can also be used. Transcriptional and translational control sequences for mammalian host cell expression vectors can be obtained from the viral genome. Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2 and the like. Other DNA for expression of structural gene sequences in mammalian host cells using the SV40 viral genome, eg, DNA sequences derived from the SV40 origin, early and late promoters, enhancers, splice sites, and polyadenylation sites. Genetic elements may be provided. Vectors for use in mammalian host cells are constructed, for example, by the method of Okayama and Berg (Mol. Cell. Biol., 3: 280, 1983, incorporated herein by reference in its entirety). Can do.

  One method of producing a cytidine 5′-monophosphosialate synthase protein of the present invention comprises the step of transforming a host cell transformed with an expression vector comprising a nucleic acid sequence encoding a cytidine 5′-monophosphosialate synthase protein. Culturing under conditions in which the protein is expressed. The cytidine 5'-monophosphosialic acid synthase protein is then recovered from the culture medium or cell extract depending on the expression system used.

The operation for purifying the recombinant cytidine 5′-monophosphosialic acid synthase protein is appropriately selected according to factors such as the type of host used and whether the protein of the present invention is secreted into the culture medium. For example, for the operation of purifying recombinant cytidine 5′-monophosphosialic acid synthase protein, anion exchange column, cation exchange column, gel filtration column, hydroxyapatite column, CDP-hexanolamine agarose column, CMP-hexanol Column chromatography such as amine agarose column, hydrophobic column, and native-PAGE, etc., or combinations thereof are included. Further, when expression is performed by fusing a recombinant cytidine 5′-monophosphosialic acid synthase with a tag that facilitates purification, a purification method by affinity chromatography may be used. For example, when a histidine tag, a FLAG ^TM tag, or glutathione-S-transferase (GST) is fused, a Ni-NTA (nitrilotriacetic acid) column, a column linked with an anti-FLAG antibody, or a glutathione linked respectively. The column can be purified by affinity chromatography.

The recombinant cytidine 5′-monophosphosialic acid synthase of the present invention may be a purified product or a partially purified product.
Antibody The present invention provides an antibody against the cytidine 5′-monophosphosialic acid synthase protein of the present invention. The antibodies of the present invention may be raised against the cytidine 5′-monophosphosialic acid synthase protein of the present invention, or a fragment thereof. Here, the fragment of the cytidine 5′-monophosphosialic acid synthase of the present invention is a fragment having a sequence containing at least 6, at least 10, at least 20, or at least 30 amino acids in the amino acid sequence of the enzyme. is there.

  The antibody may be a cytidine 5′-monophosphosialic acid synthase of the present invention or a fragment thereof, for example, but not limited to animals used for antibody production in the art, such as, but not limited to, mouse, rat, rabbit, It may be prepared by immunizing guinea pigs, goats and the like. The antibody may be a polyclonal antibody or a monoclonal antibody. An antibody can be produced based on antibody production methods well known to those skilled in the art.

  The antibody of the present invention is used to detect the cytidine 5′-monophosphosialate synthase of the present invention to detect the cytidine 5′-monophosphosialate synthase protein of the present invention in assays such as Western blotting and ELISA. You can also.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

Example 1: Screening of microorganisms producing cytidine 5'-monophosphosialic acid synthase and identification of strains (1) Screening and identification of bacterial strains Samples of seawater, sea sand, sea mud, marine seafood, etc. It was. These samples were applied on a plate medium composed of a marine broth agar 2216 medium (manufactured by Becton Dickinson) to obtain microorganisms that grew at 15 ° C., 25 ° C. or 30 ° C. According to a conventional method, the obtained microorganisms were purely cultured, and then each microorganism was cultured using a liquid medium composed of Marine Broth 2216 medium (manufactured by Becton Dickinson). After the microorganisms grew sufficiently, the cells were collected from the culture solution by centrifugation. A 50 mM Tris-HCl buffer (pH 9.0) containing 0.3% Triton X-100 (manufactured by Kanto Chemical Co., Inc.) and 20% glycerin (manufactured by Wako Pure Chemical Industries, Ltd.) is added to the collected fungus bodies, Suspended. The cell suspension was sonicated under ice cooling to disrupt cells. Cytidine 5′-monophosphosialic acid biosynthesis activity was measured using this cell disruption solution as a crude enzyme solution, and strain JT-SHIZ-145 strain (deposition number: NITE BP) having cytidine 5′-monophosphosialic acid biosynthesis activity was measured. -695). The JT-SHIZ-145 strain was obtained from the surface of the squid body.

The activity of cytidine 5′-monophosphosialic acid synthase was measured by the following method. CTP (275 nmol), NeuAc (140 nmol) as sialic acid, and MgCl ₂ were added to a concentration of 10 mM, and the enzyme reaction was performed using the reaction solution (50 μl) containing the crude enzyme solution prepared by the method described above. It was. The enzyme reaction was performed at 30 ° C. for about 10 to 180 minutes. After completion of the reaction, 80 μl of 50 mM aqueous monopotassium phosphate (pH 4.65) was added to 10 μl of the reaction solution, and the reaction product was analyzed by HPLC. Shimadzu LC10A (manufactured by Shimadzu Corporation) was used as the HPLC system, and Shim-pack IC-A1 (4.6 mm ID × 100 mm L, manufactured by Shimadzu GLC) was used as the analytical column. 80 μl of the reaction solution was injected into a column equilibrated with an eluent 50 mM monopotassium phosphate aqueous solution (pH 4.65). The eluent was used for elution of CTP, CDP, CMP and CMP-NeuAc, and the above substances were sequentially eluted by carrying out a one-part liquid feeding with different flow rates. The analysis was performed under the following conditions (flow rate: 0.5 ml / min (0 to 8 minutes), 1 ml / min (9 to 20 minutes), column temperature: 40 ° C., detection: UV (wavelength: 280 nm)) . In this HPLC analysis, the amount of the reaction product CMP-NeuAc was calculated by comparing the CMP-NeuAc peak area with the standard CMP-NeuAc area whose concentration was known in advance, and the enzyme activity was calculated. One unit of enzyme (1 U) is the amount of enzyme that synthesizes 1 micromole of CMP-NeuAc per minute.

(2) Correlation between turbidity of bacterial culture and enzyme activity The strain JT-SHIZ-145 obtained in (1) above was subcultured on a marine broth 2216 plate medium. The cells were collected in a loop, inoculated into 6 ml of marine broth 2216 liquid medium (hereinafter sometimes referred to as MB medium), and cultured with shaking at 25 ° C. and 180 rpm for 8 hours.

The main culture was performed according to the following procedure. A medium in which 300 ml of MB medium was put in a 1000 ml volume flask with a bump was prepared. The pre-culture solution is O.D. using a spectrophotometer. D. A value of ₆₀₀ is measured and 300 ml of O.D. D. _The preculture was inoculated so that ₆₀₀ was 0.25, and cultured with shaking at 25 ° C. and 180 rpm for a total of 24 hours. Collect 2 ml of the culture solution every 2 hours. D. After measuring the value of ₆₀₀ , the cells were collected by centrifugation, and 50 mM Tris-HCl buffer (pH 9.0) containing 0.3% Triton X-100 (manufactured by Kanto Chemical) and 20% glycerol (manufactured by Wako Pure Chemical Industries, Ltd.) Was added to suspend the cells. The cell suspension was sonicated under ice cooling to disrupt cells. Using this cell disruption solution as a crude enzyme solution, cytidine 5′-monophosphosialic acid synthase activity was measured (FIG. 1). As a result, it was found that the turbidity (OD ₆₀₀ ) of the cell culture solution and the cytidine 5′-monophosphosialic acid synthase activity were in a proportional relationship.

Example 2: Extraction of cytidine 5'-monophosphosialic acid synthase from JT-SHIZ-145 strain, purification and determination of amino terminal amino acid sequence of purified protein (1) Extraction and purification Marine broth 2216 Bacteria were collected from the subcultured colony of JT-SHIZ-145 strain (deposit number: NITE BP-695) in a loop, and inoculated into 6 ml of marine broth 2216 liquid medium (hereinafter sometimes referred to as MB medium) Cultured with shaking at 25 ° C. and 180 rpm for 8 hours.

  The main culture was performed according to the following procedure. 300 ml of 300 ml-MB medium was put into a 1000 ml volume flask with a bump, and 12 (total 3.6 L) were prepared. Each flask was inoculated with 6 ml of the preculture and cultured with shaking at 25 ° C. and 180 rpm for 16 hours. The culture solution was centrifuged, and the cells were collected. About 10.8 g was obtained by wet weight.

  The cells were suspended in 175 ml of 50 mM Tris-HCl buffer (pH 9.0) containing 0.3% Triton X-100 and 20% glycerol, and sonicated under ice cooling. The cell lysate was centrifuged at 4 ° C. and 100,000 × g for 1 hour to obtain a supernatant. The supernatant was passed through a sterile filter unit (manufactured by Nargenunk) having a pore size of 0.45 μm to obtain a crude enzyme solution.

  This crude enzyme solution was equilibrated with 50 mM Tris-HCl buffer (pH 9.0) containing 0.3% Triton X-100 and 20% glycerol. HiLoad 26/10 Q Sepharose HP (manufactured by GE Healthcare Biosciences) ) And an elution from a 50 mM Tris-HCl buffer (pH 9.0) containing 0.3% Triton X-100 and 20% glycerol to the same buffer containing 1 M sodium chloride by a linear concentration gradient method. I let you. As a result, a fraction having enzyme activity eluted at a sodium chloride concentration of around 0.2 M was collected.

  The collected fraction was diluted with 50 mM Tris-HCl buffer (pH 9.0) containing 0.3% Triton X-100 and 20% glycerol, and 50 mM Tris containing 0.3% Triton X-100 and 20% glycerol in advance. Adsorbed to MonoQ 5/50 GL (manufactured by GE Healthcare Biosciences) equilibrated with hydrochloric acid buffer (pH 9.0) and 50 mM Tris hydrochloric acid buffer containing 0.3% Triton X-100 and 20% glycerol Elution from pH 9.0 to the same buffer containing 1M sodium chloride by the linear concentration gradient method. As a result, a fraction having enzyme activity eluted at a sodium chloride concentration of around 0.2 M was collected.

  Next, the fraction showing this enzyme activity was preliminarily equilibrated with 50 mM Tris-HCl buffer (pH 9.0) containing 0.2 M sodium chloride, 0.3% Triton X-100 and 20% glycerol. The column was subjected to HiLoad16 / 60 Superdex200 prep grade (manufactured by GE Healthcare Life Science Co., Ltd.), gel filtration was performed, and a protein fraction showing cytidine 5′-monophosphosialic acid synthase activity was collected.

  As a result of SDS-polyacrylamide gel electrophoresis of the active fraction (the concentration of the acrylamide gel was 12.5%), the target enzyme showed a single band and a molecular weight of about 60,000. The specific activity of the purified fraction increased 19.3 times compared to the specific activity at the time of disrupting the cells (Table 1). From the above, the specific activity of cytidine 5'-monophosphosialic acid synthase derived from JT-SHIZ-145 strain was 0.164 U / mg.

  Regarding the purification of cytidine 5'-monophosphosialic acid synthase of JT-SHIZ-145 strain from the crude enzyme solution, the enzyme activities of the samples after the respective purification steps described above are shown in Table 1. The enzyme activity was measured in the same manner as described in Example 1 under the reaction conditions shown in Table 1 footnote. Proteins were quantified using Coomassie Protein Assay Reagent (PIERCE) according to the attached manual. One unit (1 U) of enzyme was defined as the amount of enzyme that produced 1 micromole of CMP-NeuAc per minute.

(2) Determination of amino terminal amino acid sequence The enzyme solution purified to the single band in (1) above was subjected to SDS-polyacrylamide gel electrophoresis (the concentration of acrylamide gel was 12.5%). After the electrophoresis, the protein was transferred to a PVDF membrane (Immunobilon-P, manufactured by Millipore) and stained with CBB, and then the target band was cut out, and the analysis and measurement division of Shimadzu Corporation was requested for amino acid sequence analysis. As a result, it was possible to determine up to the 20th residue (Ala Gln Val Pro Ala Asp Thr Lys Leu Ala Cys Lys Gln Glu Leu Val Arg Gly Asn Gly: SEQ ID NO: 5) whose amino terminus begins with alanine.

(3) Determination of internal amino acid sequence The enzyme solution purified to the single band in (1) above was subjected to SDS-polyacrylamide gel electrophoresis (concentration of acrylamide gel was 12.5%). After electrophoresis, it was stained with CBB, and the target band was excised, and AproScience Co., Ltd. was requested for internal amino acid sequence analysis. As a result, 12 residues of the sequence starting with glycine (Gly Leu Gly Val Thr Ala Val Leu Glu Asn Gln Glu: SEQ ID NO: 6), 11 residues of the sequence starting with glutamic acid (Glu Thr Pro Ala Tyr Asn Phe Thr Pro Leu Ala : 2 fragments of SEQ ID NO: 7) could be determined.

(4) Determination of all gene sequences encoding cytidine 5'-monophosphosialic acid synthase derived from JT-SHIZ-145 strain Based on the amino terminal amino acid sequence and internal amino acid sequence determined in (2) and (3) above The gene sequence encoding cytidine 5′-monophosphosialic acid synthase was searched from the entire genome sequence of JT-SHIZ-145 strain. As a result, the sequence of SEQ ID NO: 1 was obtained. This sequence is the entire nucleotide sequence of the open reading frame (ORF) of the cytidine 5′-monophosphosialic acid synthase gene derived from JT-SHIZ-145 strain. The ORF of the cytidine 5′-monophosphosialic acid synthase gene derived from Photobacterium genus JT-SHIZ-145 was composed of 1632 base pairs and encoded 543 amino acids. This amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing.

Based on this sequence, the amino acid sequence and microorganism of the major mammalian cytidine 5′-monophosphosialic acid synthase currently known using the genetic information processing software GENETYX Ver.7 (manufactured by Genetics) described above. The homology with the amino acid sequence of the cytidine 5′-monophosphosialic acid synthase derived from the origin was searched by maximum matching analysis using default values. Specifically, the amino acid sequence of cytidine 5′-monophosphosialic acid synthase derived from mammals is derived from bovine (Bos Taurus) (accession number: NP 001030475, XP 612905), derived from human (Homo sapiens) (accession number: NP) 061156), and from mouse (Mus musculus) (accession number: NP) 034038), and the amino acid sequence of cytidine 5′-monophosphosialic acid synthase derived from microorganisms, derived from E. coli (accession number: P13266), derived from Pasteurella multocida Pm70 (accession number: NP 245124), derived from Neisseria meningitidis (accession number: NP 273133) and compared with the amino acid sequence of SEQ ID NO: 2. As shown in Table 2, all the homologies showed a low value of 20% or less.

Similarly, using the BLAST program on the Internet through the National Center for Biotechnology Information (NCBI) website, search the amino acid sequence information on the website database and amino acid sequences that are highly identical to the full length amino acid sequence of this enzyme. did. In this case, default parameters were used, and non-redundant protein sequences (nr) were used as a database to be referenced. As a result, the amino acid sequence (accession number: ZP) presumed to be the oligopeptide ABC transporter protein (periplasma oligopeptide binding protein) of Vibrio angustum S14 was the most identical. 0234945) and the identity was 92%.

Example 3: Optimum pH, optimum temperature, optimum metal salt concentration and isoelectric point of cytidine 5'-monophosphosialic acid synthase derived from JT-SHIZ-145 strain were prepared in Example 2-1. Using purified enzyme, optimal pH, optimal temperature, optimal metal salt concentration / type, etc. of cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain (deposit number: NITE BP-695) The electrical point was examined.

(1) Optimum pH of enzyme activity of cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain
Bis-Tris buffer (pH 6.0, pH 6.5, and pH 7.0), phosphate buffer (pH 7.0, pH 7.5, and pH 8.0), Tris-HCl buffer (pH 8.0, pH 8.5, And pH 9.0), CHES buffer (pH 9.0, pH 9.5, and pH 10.0), and CAPS buffer (pH 10.0, pH 10.5, and pH 11.0) were prepared and used at 30 ° C. The enzyme activity at each pH was measured. Each buffer was added to the reaction solution to a final concentration of 100 mM.

  As a result, as shown in FIG. 2-1, the enzyme activity was maximum at pH 9.0. In addition, the enzyme activity in each pH was shown by the relative activity which sets the enzyme activity to 100 at the time of using a tris-hydrochloric acid buffer (pH 9.0).

(2) Optimal temperature of enzyme activity of cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain Tris-HCl buffer (pH 9.0, final concentration 100 mM) from 5 ° C. to 75 ° C. The enzyme activity was measured at each reaction temperature of 5 ° C.

As a result, as shown in FIG. 2-2, the enzyme activity was maximum at 35 ° C. In addition, the enzyme activity in each temperature was shown by the relative activity which made the enzyme activity in 35 degreeC 100.
(3) Using the metal salt Tris-HCl buffer (pH 9.0) necessary for the enzymatic activity of cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain at 30 ° C. in the final reaction solution Enzyme activity was measured by adjusting MgCl ₂ , MnCl ₂ , CaCl ₂ , FeSO ₄ , CuSO ₄ , and no metal salt so that the concentration was 10 mM.

As a result, as shown in FIG. 2-3, the enzyme activity was maximized when MgCl ₂ was added. In addition, when no metal salt was added, the enzyme activity was completely lost. Incidentally, the enzyme activity in each metal salt added was expressed by relative activity to 100 enzyme activity in MgCl ₂ added.

(4) Optimum enzymatic activity of cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain Using MgCl ₂ concentration Tris-HCl buffer (pH 9.0, final concentration 100 mM) at 30 ° C., The MgCl ₂ concentration in the reaction solution is 0 mM, 0.1 mM, 0.5 mM, 1.0 mM, 2.0 mM, 5.0 mM, 10.0 mM, 15.5 mM, 20.0 mM, 30.0 mM, 40.0 mM, respectively. The enzyme activity was measured by adjusting to 50.0 mM.

As a result, as shown in FIG. 2-4, almost the same level of enzyme activity was maintained at a concentration of 5 mM or more. Incidentally, the enzyme activity at each MgCl ₂ concentration was represented as relative activity to 100 enzyme activity in MgCl ₂ concentration 10.0 mM.

(5) Isoelectric focusing enzyme of cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain was subjected to isoelectric focusing gel (precast ID Novex (registered trademark) IEF gel pH 3-10 IEF gel , Manufactured by Invitrogen Corporation), and was subjected to isoelectric focusing according to a conventional method. The gel after electrophoresis was stained with CBB. As a result, as shown in FIG. 2-5, the isoelectric point was pH 5.

Example 4: Km value and Vmax value for CTP and NeuAc of cytidine 5'-monophosphosialic acid synthase derived from JT-SHIZ-145 strain JT-SHIZ-145 strain using the purified enzyme prepared in Example 2-1 The Michaelis constant (Km) and maximum velocity (Vmax) for CTP and NeuAc of cytidine 5′-monophosphosialic acid synthase derived from (Deposit Number: NITE BP-695) were examined.

(1) Michaelis constant (Km) and maximum speed (Vmax) for NeuAc of cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain
NeuAc solutions with concentrations of 100 mM, 50 mM, 25 mM, 10 mM, 5 mM, 1 mM, and 0.1 mM were prepared, and the enzyme activity for each NeuAc concentration was measured at 35 ° C. using these solutions. At that time, CTP was added to a final concentration of 5 mM. The results are shown in Table 3.

  The Michaelis constant and the maximum velocity were calculated using the Lineweaver-Burk method, which was obtained by modifying the Michaelis-Menten equation, in accordance with the method of deriving the reaction rate equation in the steady state (FIGS. 3-1 and 2). As a result, it was shown that the Michaelis constant (Km) for NeuAc of this enzyme was 2.234 (mM) and the maximum velocity (Vmax) was 0.104 (nmol / min).

(2) Michaelis constant (Km) and maximum velocity (Vmax) for CTP of cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain
50 mM, 25 mM, 10 mM, 5 mM, 2.5 mM, 1 mM, and 0.1 mM CTP solutions were prepared and used to measure enzyme activity for each CTP concentration at 35 ° C. At that time, NeuAc was added to a final concentration of 5 mM. The results are shown in Table 4.

  The Michaelis constant and the maximum velocity were calculated using the Lineweaver-Burk method obtained by modifying the Michaelis-Menten equation in accordance with the method of deriving the reaction rate equation in the steady state (FIGS. 3-3 and 4). As a result, it was shown that the Michaelis constant (Km) for CTP of this enzyme was 1.571 (mM) and the maximum speed (Vmax) was 0.116 (nmol / min).

Example 5: Comparison of various sialic acid substrate specificities (acetyl type, glycolyl type) of cytidine 5'-monophosphosialic acid synthase derived from JT-SHIZ-145 strain ( materials and methods )
A cytidine 5 ′ obtained by electrophoretically purifying a cell disruption solution prepared from JT-SHIZ-145 strain (deposit number: NITE BP-695) to a single band using ion exchange chromatography and gel filtration chromatography. -In order to investigate the presence or absence of CMP-Sia synthesis from various sialic acids using monophosphosialic acid synthase, the following experiment was conducted.

Various dissolved in 50 μl of CMP-Sia synthesis reaction reaction solution using various sialic acids with substrate CTP (275 nmol, final concentration in reaction solution: 5.5 mM), 100 mM Tris-HCl buffer (pH 9.0) Consists of sialic acid (140 nmol, final concentration in reaction solution: 2.8 mM), cytidine 5′-monophosphosialic acid synthase (about 0.65 mU), magnesium chloride (final concentration in reaction solution: 10 mM) A reaction solution was prepared, and an enzyme reaction was performed at 30 ° C. for about 16 hours. Note that N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) were used as sialic acids used as substrates.

  After completion of the reaction, 80 μl of 50 mM aqueous monopotassium phosphate (pH 4.65) was added to 10 μl of the reaction solution, and the reaction product was analyzed by HPLC. Shimadzu LC10A (manufactured by Shimadzu Corporation) was used as the HPLC system, and Shim-pack IC-A1 (4.6 mm ID × 100 mm L, manufactured by Shimadzu GLC) was used as the analytical column. 80 μl of the reaction solution was injected into a column parallelized with an eluent 50 mM monopotassium phosphate aqueous solution (pH 4.65). The eluent was used for elution of CTP, CDP, CMP, and CMP-Sia, and the above substances were sequentially eluted by carrying out a one-liquid feed with different flow rates. The analysis was performed under the following conditions (flow rate: 0.5 ml / min (0 to 10 minutes), 1 ml / min (11 to 20 minutes), column temperature: 40 ° C., detection: UV (wavelength: 280 nm)) .

Using the above method, the presence or absence of CMP-Sia produced from each substrate sialic acid was measured.
( Result )
When N-acetylneuraminic acid or N-glycolylneuraminic acid was used as the substrate sialic acid, CMP-NeuAc or CMP-NeuGc was produced, respectively (FIG. 4-1). That is, N-acetylneuraminic acid and N-glycolylneuraminic acid can be used as substrate sialic acid for CMP-Sia synthesis by cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain. It became clear that there was. In addition, the relative activity with respect to each substrate sialic acid shows the case where the cytidine 5′-monophosphosialic acid synthase activity with respect to N-acetylneuraminic acid is 100.

Confirmation of formation of CMP-Sia Next, in order to clarify that the product is indeed CMP-Sia, pyridylamination (using a sialic acid transferase having a substrate specificity using CMP-Sia as a sugar donor substrate) A sialic acid transfer reaction to PA) -lactose was performed. In this case, the generation of pyridylaminated sugar chains other than PA-lactose indicates the presence of CMP-Sia, which is a sugar-donating substrate. Accordingly, by the reaction of CMP with sialic acid using the enzyme of the present invention. It can be shown that CMP-Sia has been generated.

  A β-galactoside-α2,3-sialyltransferase solution derived from JT-ISH-224 (see WO2006 / 112253) was used as a sialyltransferase, and an enzymatic reaction was performed using a pyridylaminated sugar chain as a sugar acceptor substrate. The pyridylaminated sugar chain was analyzed using pyridylaminated lactose (Galβ1-4Glc-PA, manufactured by Takara Bio Inc.). 1 μl of β-galactoside-α2,3-sialyltransferase solution (10 mU), 2.5 μl of 10 pmol / μl sugar receptor substrate, 5 μl of each of the above CMP-Sia (NeuAc or NeuGc) reaction solutions 2.5 μl of 3 M sodium chloride aqueous solution, 1.5 μl of 1 M cacodylate buffer (pH 5.0), and sterilized distilled water were added and reacted at 30 ° C. for 1 hour. After completion of the reaction, the reaction product was analyzed by HPLC. Shimadzu LC10A (manufactured by Shimadzu Corporation) was used as the HPLC system, and Takara PALPAK Type R (manufactured by Takara Bio) was used as the analytical column. 80 μl of eluate A (100 mM acetic acid-triethylamine, pH 5.0) was added to a column equilibrated with 100 mM acetic acid-triethylamine (pH 5.0) containing 0.15% N-butanol. For elution of pyridylaminated sugar chain, eluent A (100 mM acetic acid-triethylamine, pH 5.0) and eluent B (100 mM acetic acid-triethylamine, pH 5.0 containing 0.5%, n-butanol) were used. Pyridylaminated sugar chains were sequentially eluted by the linear concentration gradient method (0 to 20 minutes) of 50% eluate B and 100% eluent B (21 to 35 minutes). The analysis was performed under the following conditions (flow rate: 1 ml / min, column temperature: 40 ° C., detection: fluorescence (Ex: 320 nm, Em: 400 nm)).

  As a result, a reaction solution obtained by reacting cytidine 5′-monophosphosialic acid synthase solution derived from JT-SHIZ-145 strain with NeuAc and CTP was further converted into pyridylaminated lactose (PA-lactose) and β-galactoside-α2, It was shown that pyridylaminated sugar chains other than PA-lactose were produced when reacted with 3-sialyltransferase. Therefore, it was clarified that cytidine 5′-monophosphosialic acid synthase derived from JT-SHIZ-145 strain can synthesize CMP-Sia using N-glycolylneuraminic acid as a substrate (FIGS. 4-2 and 3). 4, 5).

  The present invention provides a novel cytidine 5′-monophosphosialic acid synthase and a nucleic acid encoding the same, thereby synthesizing and producing sugar chains that have been revealed to have important functions in vivo. Provide a means. In particular, sialic acid is often present at the non-reducing end of complex carbohydrate sugar chains in vivo, and sialic acid-containing sugar chains are extremely important sugars from the viewpoint of sugar chain function. The synthesis of sialic acid-containing sugar chains is most useful by a method using a sialyltransferase, but requires cytidine 5'-monophosphosialic acid as a donor substrate. Since cytidine 5'-monophosphosialic acid is difficult and expensive to synthesize chemically, synthesis using an enzyme is currently the mainstream. Therefore, the novel cytidine 5'-monophosphosialic acid synthase of the present invention can be used for the development of pharmaceuticals, functional foods and the like using sugar chains.

  Photobacterium Leognacy JT-SHIZ-145 strain was deposited on December 26, 2008 as deposit number: NITE BP-695, independent administrative agency, Product Evaluation Infrastructure Organization, Patent Microorganism Deposit Center (NITE) (Address: Kisarazu City, Chiba Prefecture) It has been deposited with Kazusa Kama feet 2-5-8).

SEQ ID NO: 1: Nucleic acid sequence encoding cytidine 5′-monophosphosialic acid synthase derived from Photobacterium leoignacy.
SEQ ID NO: 2: Amino acid sequence of cytidine 5′-monophosphosialic acid synthase derived from Photobacterium leoignacy.

SEQ ID NO: 3: Nucleic acid sequence encoding cytidine 5′-monophosphosialic acid synthase; in addition to nucleotides 82-1632 of SEQ ID NO: 1, a start codon ATG is included on the 5 ′ end side.
SEQ ID NO: 4: amino acid sequence of cytidine 5′-monophosphosialic acid synthase; in addition to amino acids 28-543 of SEQ ID NO: 2, methionine is included on the N-terminal side.

SEQ ID NO: 5: N-terminal amino acid sequence after cleavage of the signal sequence of cytidine 5′-monophosphosialic acid synthase derived from Photobacterium leoignacy.
SEQ ID NO: 6: Internal amino acid sequence of cytidine 5′-monophosphosialic acid synthase derived from Photobacterium leoignacy.

  SEQ ID NO: 7: Internal amino acid sequence of cytidine 5'-monophosphosialic acid synthase derived from Photobacterium leoignacy.

Claims (15)

An isolated protein comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4; or
(B) an amino acid sequence encoded by a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3;
Said protein comprising. An isolated protein having cytidine 5′-monophosphosialic acid synthase activity comprising:
(A) In the amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4, including deletion, substitution, insertion and / or addition of one or more amino acids Amino acid sequence;
(B) an amino acid sequence having 60% or more amino acid identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4;
(C) a nucleotide sequence comprising one or more nucleotide deletions, substitutions, insertions and / or additions in a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3 The amino acid sequence encoded by
(D) an amino acid sequence encoded by a base sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3, and a base sequence having 70% or more identity; and (e ) An amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to a complementary strand of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3;
The protein comprising an amino acid sequence selected from the group consisting of:   The isolated protein according to claim 1 or 2, which is derived from a microorganism belonging to the genus Photobacterium. An isolated protein having cytidine 5'-monophosphosialic acid synthase activity having the following properties:
(A) a molecular weight of 62 ± 4 kDa or 60 ± 4 kDa by SDS-PAGE analysis; and (b) an isoelectric point of pH 4.8 to pH 5.2 by isoelectric focusing analysis;
The protein. Cytidine 5′-monophosphosialic acid synthase activity has the following properties:
(A) an optimum pH of pH 8 to pH 9;
(B) an optimum temperature of 30 ° C to 40 ° C;
(C) an optimal MgCl ₂ concentration of at least 5 mM;
The protein according to claim 4, wherein   The protein according to claim 4 or 5, wherein the amino acid sequence on the N-terminal side after cleavage of the signal peptide comprises the amino acid sequence of SEQ ID NO: 5 and the sequences of SEQ ID NOs: 6 and 7 as internal sequences. An isolated nucleic acid comprising:
(A) a base sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4; or
(B) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1, and SEQ ID NO: 3;
Comprising said nucleic acid. An isolated nucleic acid encoding a protein having cytidine 5'-monophosphosialic acid synthase activity, comprising:
(A) In the amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4, including deletion, substitution, insertion and / or addition of one or more amino acids A base sequence encoding an amino acid sequence;
(B) a base sequence encoding an amino acid sequence having 60% or more amino acid identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 28-543 of SEQ ID NO: 2, and SEQ ID NO: 4;
(C) a nucleotide sequence comprising one or more nucleotide deletions, substitutions, insertions and / or additions in a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3 ;
(D) a nucleotide sequence having 70% or more identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 82-1632 of SEQ ID NO: 1 and SEQ ID NO: 3; and (e) SEQ ID NO: 1, SEQ ID NO: A nucleotide sequence that hybridizes under stringent conditions to a complementary strand of a nucleotide sequence selected from the group consisting of 1 nucleotide 82-1632 and SEQ ID NO: 3;
The nucleic acid comprising a base sequence selected from the group consisting of:   An expression vector comprising the nucleic acid according to claim 7 or 8.   A host cell transformed with the expression vector according to claim 9.   An isolated microorganism belonging to the genus Photobacterium that expresses the protein according to any one of claims 1 to 6. A method for producing a protein having cytidine 5′-monophosphosialic acid synthase activity, comprising the following steps:
1) culturing a microorganism that produces the protein according to any one of claims 1 to 6;
2) isolating a protein having cytidine 5′-monophosphosialic acid synthase activity from the cultured microorganism or culture supernatant;
The said manufacturing method including this.   The method according to claim 12, wherein the microorganism that produces the protein according to any one of claims 1 to 6 is Photobacterium leiognathi. A method for producing a recombinant protein having cytidine 5′-monophosphosialic acid synthase activity, comprising the following steps:
1) transforming a host cell with an expression vector comprising the nucleic acid according to claim 7 or 8;
2) culturing the resulting transformed cells; and
3) Isolating a protein having cytidine 5′-monophosphosialic acid synthase activity from the cultured transformed cells or the culture supernatant thereof;
The said manufacturing method including this.   An antibody that specifically recognizes the protein according to any one of claims 1 to 6.