WO2011086834A1

WO2011086834A1 - Process for production of n-acetyl-d-neuraminic acid

Info

Publication number: WO2011086834A1
Application number: PCT/JP2010/073379
Authority: WO
Inventors: 健三横関; 大郎萩下; 昌清水
Original assignee: 味の素株式会社
Priority date: 2010-01-15
Filing date: 2010-12-24
Publication date: 2011-07-21

Abstract

Disclosed are: NANA aldolase which is stable under alkaline conditions having high pH values; and a process for producing NANA in a large amount with high efficiency using the NANA aldolase. Specifically disclosed are: a protein which exhibits pH stability at pH 8.0 to 11.0 and has an N-acetyl-D-neuraminic acid aldolase activity; a polynucleotide encoding the protein; an expression vector carrying the polynucleotide; a transformant having the expression vector introduced thereinto; a bacterium capable of producing the protein; a process for producing N-acetyl-D-neuraminic acid aldolase using the protein; and others.

Description

Process for producing N-acetyl-D-neuraminic acid

The present invention relates to a method for producing N-acetyl-D-neuraminic acid aldolase, and proteins, polynucleotides, expression vectors, transformants, bacteria, and the like useful for producing N-acetyl-D-neuraminic acid aldolase.

N-acetyl-D-neuraminic acid, which is the most universal of all sialic acid species (hereinafter abbreviated as NANA if necessary), is a glycan chain terminal in the glycoconjugate present on the vertebrate cell surface. Is the essential and most abundant amino sugar located in NANA plays a prominent role in many biological functions, including viral infections. The virus propagated inside the cell is bound to NANA on the surface of the infected cell, and the release and spread of the infectious virus from the infected cell is caused by the action of neuraminidase existing on the surface of the virus to cause NANA on the surface of the infected cell. It is essential to break the bond. NANA analogs have therefore been investigated as potential antiviral agents. Zanamivir (product name: Relenza) and oselfamivir (product name: Tamiflu), NANA analogs that inhibit both A and B influenza virus neuraminidases, have been developed and treated against the highly toxic H5N1 strain Widely used as a medicine. The replicated influenza virus binds to NANA present on the infected cell surface and localizes on the infected cell. Influenza virus has neuraminidase, an enzyme that cleaves the bond that links the NANA receptor of influenza virus and the NANA of infected cells. Virus release requires neuraminidase to break the bond between influenza virus and infected cells. In the presence of the neuraminidase inhibitors zanamivir and oselfamivir, the virus remains attached to the membrane of the infected cell, causing self-aggregation and inhibiting the spread of the virus. Currently, new NANA-based drugs against influenza viruses are in great demand in the medical field and there is a worldwide demand for large amounts of NANA. Therefore, various synthetic methods using enzymes have been studied in order to efficiently produce a large amount of NANA.

As an example of a synthesis method of NANA using an enzyme, N-acetyl-D-mannosamine (hereinafter referred to as NANA aldolase or nanA, if necessary) in the presence of N-acetyl-D-neuraminic acid aldolase (hereinafter referred to as NANA aldolase or nanA as required) , And NANA enzyme synthesis method (corresponding to the reaction step of (2) described later) by reacting pyruvic acid (hereinafter abbreviated as Pyr, if necessary). It is done. However, this method is not practical because NAM is expensive. Therefore, an NANA enzyme synthesis method using N-acetyl-D-glucosamine (hereinafter, abbreviated as NAG as necessary) and Pyr as raw materials instead of NAM has been studied.

The NANA enzyme synthesis method using NAG and Pyr as raw materials includes the following reaction steps (1) and (2).

Patent Documents

1 and 2 include NAG comprising using epimerase under neutral conditions in the reaction step (1) above, and using NANA aldolase under neutral conditions in the reaction step (2) above. And a method for enzymatic synthesis of NANA from Pyr.

Patent Document 3 utilizes chemical isomerization of NAG to NAM under alkaline conditions in the reaction step (1) above, and NANA under neutral conditions in the reaction step (2) above. A method for enzymatic synthesis of NANA from NAG and Pyr, including the use of aldolase, is disclosed.

Non-Patent Document 1 utilizes chemical isomerization of NAG to NAM under alkaline conditions in the reaction step (1) above, and NANA under alkaline conditions in the reaction step (2) above. A method for enzymatic synthesis of NANA from NAG and Pyr, including the use of aldolase, is disclosed. According to the present method, theoretically, it is possible to efficiently produce a large amount of NANA by simultaneously performing the reaction steps (1) and (2) under alkaline conditions and performing them simultaneously. This is because NAM is replenished from NAG by chemical equilibrium when NAM generated by chemical isomerization decreases with NANA synthesis.

There have been many reports on NANA aldolase-producing bacteria. For example, as a NANA aldolase-producing bacterium, Escherichia (Non-Patent Documents 2 to 4), Clostridium (Non-Patent Documents 5 and 6), Bacillus, Citrobacter, Corynebacterium, Enterobacter, Clavera, Micrococcus genus, Proteus genus (non-patent document 7), Aerobacter genus (patent document 4), Pseudomonas genus, Pseudomonas genus, Sarsina genus, Bacteria genus, Arthrobacter genus, Brevibacterium genus (Patent Document 5) It is done.

By the way, Patent Document 6 reports that in the NANA production method, the deactivation of NANA aldolase is suppressed under alkaline conditions at high pH due to the protective effect of the substrates NAG and Pyr.

Japanese Patent No. 3151210 Japanese Patent No. 3418764 WO94 / 29476 JP 55-50890 JP 55-50891 WO93 / 15214

However, the methods described in Patent Documents 1 to 6 and Non-Patent Documents 1 to 7 have the following problems.

In the method described in

Patent Documents

1 and 2, in the reaction step (1), the conversion efficiency from NAG to NAM is low due to the low activity of epimerase, and the activity of epimerase is expressed. In addition, expensive ATP is required as a coenzyme.

The method described in Patent Document 3 is complicated because it is necessary to extract NAM from a reaction solution under alkaline conditions after the completion of the reaction step (1) and then use NANA aldolase under neutral conditions. is there. In addition, the method described in Patent Document 3 has a small amount of NAM extracted after the completion of the reaction step (1) (that is, the chemical equilibrium of isomerization of NAG and NAM is not on the NAM side but on the NAG side. In the reaction step (2), a large amount of NANA cannot be produced efficiently.

Since the NANA aldolase used in the method described in Non-Patent Document 1 is unstable and rapidly deactivates under alkaline conditions of high pH required in the reaction step (1) above, In reality, a large amount of NANA cannot be produced efficiently.

NANA aldolase described in Non-Patent Documents 2 to 6 and Patent Documents 4 to 5 is unstable under alkaline conditions. For example, in the conventional production of NANA, only NANA aldolase derived from the genus Escherichia (Non-patent Document 4) or Clostridium (non-patent Document 5) is used among the above-mentioned NANA aldolases derived from bacteria. NANA aldolase exhibits stability only at pH 6.0 to 9.0, and does not exhibit stability under alkaline conditions of high pH required in the reaction step (1). Therefore, these NANA aldolases are not efficient as enzymes for producing NANA from NAG and Pyr by simultaneously proceeding the reaction steps (1) and (2) above.

Regarding Patent Document 6, when a reaction system including the reaction steps (1) and (2) above is used, if the NANA aldolase itself is stable under alkaline conditions at high pH, the activity is maintained for a longer period. It is thought that a large amount of NANA can be produced efficiently. Further, when the NANA aldolase is stable without depending on the substrate concentration, it is considered that the NANA aldolase is easier to use in the reaction system including the reaction steps (1) and (2). Therefore, there is a demand for the development of NANA aldolase exhibiting high pH stability under alkaline conditions of high pH.

The present invention has been made in view of the above, and provides a stable NANA aldolase under alkaline conditions at a high pH, and efficiently produces a large amount of NANA using such a NANA aldolase. It aims to provide a way to do.

The present inventors search for strains having the ability to synthesize NANA from NAM and Pyr under high pH alkaline conditions for many microorganisms having the ability to grow on an alkaline medium using NANA as a single carbon source. As a result of repeating the above, the isolation of a strain having these abilities, the identification of a gene encoding N-acetylneuraminic acid aldolase from the isolated strain, and the use of N-acetylneuraminic acid aldolase The present inventors have succeeded in developing a method for efficiently producing NANA and have completed the present invention. That is, the present invention is as follows.

[1] A protein which exhibits pH stability at pH 8.0 to 11.0 and has N-acetyl-D-neuraminic acid aldolase activity.
[2] A protein selected from the group consisting of the following (A) to (D):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2;
(B) a protein comprising the amino acid sequence represented by SEQ ID NO: 2;
(C) the amino acid sequence represented by SEQ ID NO: 2, consisting of an amino acid sequence containing a mutation of one or several amino acid residues selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues; And a protein having N-acetyl-D-neuraminic acid aldolase activity; and (D) an amino acid sequence having at least 80% or more amino acid sequence identity to the amino acid sequence represented by SEQ ID NO: 2, and N A protein having acetyl-D-neuraminic acid aldolase activity;
[3] The protein according to [2], wherein the protein exhibits pH stability at pH 8.0 to 11.0.
[4] A polynucleotide selected from the group consisting of the following (a) to (e):
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
(B) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
(C) a polymorph that encodes a protein comprising a nucleotide sequence having at least 80% nucleotide sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and having N-acetyl-D-neuraminic acid aldolase activity nucleotide;
(D) encodes a protein that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 and has N-acetyl-D-neuraminic acid aldolase activity And (e) a polynucleotide encoding the protein of any one of [1] to [3] above.
[5] An expression vector comprising the polynucleotide of [4] above.
[6] A transformant introduced with the expression vector of [5] above.
[7] The transformant according to [6], wherein the host of the transformant is Escherichia coli.
[8] A bacterium derived from the genus Bacillus having the ability to produce the protein of any one of [1] to [3].
[9] comprising culturing the transformant of [6] or [7] and / or the bacterium of [8] in a medium to obtain a protein of any one of [1] to [3], A method for producing acetyl-D-neuraminic acid aldolase.
[10] N-acetyl-, comprising synthesizing N-acetyl-D-neuraminic acid from N-acetyl-D-mannosamine and pyruvic acid in the presence of the protein of any one of [1] to [3] A method for producing D-neuraminic acid.
[11] The production method of [10], wherein the synthesis is further performed in the presence of 4-hydroxy-4-methyl-2-oxoglutarate aldolase.
[12] The production method of [10] or [11], wherein the method is carried out under conditions of pH 8.0 to 11.0.
[13] The method according to any one of [10] to [12], further comprising producing N-acetyl-D-mannosamine from N-acetyl-D-glucosamine by an isomerization reaction under an alkaline condition of pH 9.5 or higher. Manufacturing method.

FIG. 1 shows the effect of pH on chemical isomerization. The effect of pH on chemical isomerization was determined using 50 mM ammonium buffer (pH 8.5 to 10.0), 50 mM CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer (pH 10.0 to 11.0), It was determined by using 50 mM sodium phosphate buffer (pH 11.0 to 12.0). A reaction mixture (0.1 ml) of the above buffer containing 100 mM NAG was incubated at 30 ° C. for 12 hours. Ammonium buffer, CAPS buffer, sodium phosphate buffer FIG. 2 is a diagram showing the optimum pH of nanA derived from Bacillus sp. YKR. 50 mM potassium phosphate buffer (pH 7.0 to 8.0), 50 mM Tris-HCl buffer (pH 8.0 to 9.0), 50 mM borate buffer (pH 9.0 to 10.0), 50 mM CAPS buffer The enzyme activity was measured by using the solution (pH 10.0-11.0), and the optimum pH was determined. Relative activity was expressed as a percentage of the pH 8.5 activity. Potassium phosphate buffer, Tris-HCl buffer, borate buffer, CAPS buffer FIG. 3 is a diagram showing the pH stability of nanA derived from Bacillus sp. YKR. 10 mM acetate buffer (5.0 to 6.0), 50 mM potassium phosphate buffer (pH 6.0 to 8.0), 50 mM Tris-HCl buffer (pH 8.0 to 9.0), 50 mM borate buffer Activity using enzymes pretreated overnight (16 hours) at 4 ° C. with various pH values of solution (pH 9.0-10.0), 50 mM CAPS buffer (pH 10.0-11.0). Measured and determined pH stability. Relative activity was expressed as a percentage of the pH 8.5 activity. Acetate buffer, potassium phosphate buffer, Tris-HCl buffer, borate buffer, CAPS buffer FIG. 4 is a diagram showing the optimum temperature of nanA derived from Bacillus sp. YKR. Using 50 mM Tris-HCl buffer (pH 8.5), the enzyme activity was measured at a reaction temperature between 20 ° C. and 80 ° C. to determine the optimum temperature. FIG. 5 is a diagram showing the temperature stability of nanA derived from Bacillus sp. YKR. The enzyme activity was measured using an enzyme treated for 1 hour at each temperature shown in FIG. 5 in 50 mM Tris-HCl buffer (pH 8.5) to determine temperature stability. FIG. 6 is a diagram showing NANA production from NAM and Pyr. 50 mM buffer (Tris-HCl buffer (pH 8.5); CAPS buffer (pH 10.0)), 100 mM NAM, 100 mM Pyr, and 0.5 mg / ml protein of cell-free extract derived from recombinant Escherichia coli The containing reaction mixture (0.5 ml) was incubated at 30 ° C. FIG. 7 shows the effect of split addition of Pyr on NANA production at pH 8.5. A reaction mixture (1.0 ml) containing 0.5 mg / ml protein of a cell-free extract derived from 50 mM Tris-HCl buffer (pH 8.5), 100 mM NAM, 100 mM Pyr and recombinant Escherichia coli was prepared at 30 ° C. Incubated with. 50 mM, 10 mM and 10 mM Pyr were added after 2 hours, 4 hours and 6 hours. FIG. 8 shows the effect of split addition of Pyr on NANA production at pH 10.0. Reaction mixture (1.0 ml) containing 0.5 mg / ml protein of cell-free extract derived from 50 mM CAPS buffer (pH 10.0), 100 mM NAM, 100 mM Pyr and recombinant Escherichia coli is incubated at 30 ° C. did. 50 mM, 10 mM and 10 mM Pyr were added after 2 hours, 4 hours and 6 hours. FIG. 9 is a diagram showing a time course of NANA production from NAM and Pyr. Reaction mixture (1.0 ml) containing 50 mg CAPS buffer (pH 10.0), 490 mM NAM, 240 mM Pyr and 0.5 mg / ml protein of cell-free extract derived from recombinant Escherichia coli at 30 ° C. did. 490 mM and 310 mM Pyr were added after 1.5 and 5.0 hours. FIG. 10 is a diagram showing a time course of NANA production from NAG and Pyr using a cell-free extract. First, NAM was prepared from NAG by incubating 1000 mM NAG at pH 11.5 for 3 hours in a volume of 0.7 ml. Second, cell-free extract from recombinant Escherichia coli of 100 mM CAPS (pH 10.0), 700 mM Pyr and 0.5 mg / ml protein was added to the reaction mixture in a final volume of 1 ml, Incubated at 0 ° C. 1000 mM, 1000 mM and 500 mM Pyr were added after 23, 45 and 76 hours, respectively. FIG. 11 is a diagram showing a time course of NANA production from NAG and Pyr using bacterial cells. First, NAM was prepared from NAG by incubating 1000 mM NAG at pH 11.5 for 3 hours in a volume of 0.7 ml. NAG and NAM became 855 mM and 205 mM by the chemical isomerization reaction, respectively. Second, a cell-free extract of recombinant Escherichia coli derived from 100 mM CAPS (pH 10.0), 700 mM Pyr and 0.5 mg / ml protein or a cell derived from recombinant Escherichia coli (5 ml cultured) was added in a final volume of 1 ml and incubated at 30 ° C. 400 mM, 300 mM, 300 mM, 300 mM, 400 mM and 300 mM Pyr were added after 4.5, 7.5, 25, 30, 34, 48 hours, respectively. FIG. 12 shows the influence of Pyr concentration on NANA production. A reaction mixture (1.0 ml) containing 0.5 mg / ml protein of a cell-free extract derived from 50 mM Tris buffer (pH 8.0), 100 mM NAM, 100 to 900 mM Pyr, and recombinant Escherichia coli is 37 ° C. Incubated with. FIG. 13 is a diagram showing the influence of the Pyr concentration on the high concentration NAM. A reaction mixture (1.0 ml) containing 0.5 mg / ml protein of a cell-free extract derived from 50 mM Tris buffer (pH 8.0), 500 mM NAM, 100 to 900 mM Pyr and recombinant Escherichia coli was added at 37 ° C. Incubated with.

1. Proteins, polynucleotides, microorganisms and their preparation according to the present invention The present invention provides proteins useful for the production of NANA.

In one embodiment, the protein of the present invention may exhibit pH stability under alkaline conditions. More specifically, the proteins of the present invention may exhibit pH stability at a pH of about 8.0 to about 11.0. As used herein, the term “pH stability” means 60% or more of the initial activity, preferably 70%, more preferably when the protein is incubated at 4 ° C. for 16 hours in the relevant pH range. It means that the activity of 80%, even more preferably 90% or more can be maintained. Thus, proteins of the invention can exhibit pH stability that retains at least 60% of the initial activity when incubated for 16 hours at a pH of about 8.0 to about 11.0.

The protein of the present invention may also have various enzymatic characteristics in addition to the above pH stability. Such enzymatic properties include optimal pH, optimal temperature, temperature stability, molecular weight, activity, and Km value.

As used herein, the term “optimal pH” refers to the pH at which a protein exhibits maximum activity. The optimum pH of the protein of the present invention may be any pH value in the range of about pH 7.0 to about 10.0, but is preferably in the range of about pH 7.0 to about 9.0. Any pH value in the range, more preferably any pH value in the range of about 7.5 to about 8.5, even more preferably about pH 8.0. It may be any pH value in the range of about 8.5.

As used herein, the term “temperature stability” refers to 60% or more of the initial activity, preferably 70%, more preferably 80% when the protein is incubated for 1 hour in the relevant temperature range. Even more preferably, it means that 90% or more of activity can be maintained. In other words, the protein of the present invention may exhibit temperature stability at about 0 to about 55 ° C. when it retains 60% or more of the initial activity and retains 70% or more of the initial activity. In this case, the temperature stability may be exhibited at about 0 to about 50 ° C., and when the activity of 80% or more of the initial activity is maintained, the temperature stability may be exhibited at about 0 to about 40 ° C. % Activity may be exhibited at about 0 to about 30 ° C. Thus, the proteins of the invention can exhibit temperature stability that retains at least 60% of the initial activity when incubated at about 0 to about 55 ° C. for 1 hour.

As used herein, the term “optimum temperature” refers to the temperature at which a protein exhibits maximum activity. The optimum temperature of the protein of the present invention may be any temperature in the range of about 40 to about 70 ° C., but is preferably any temperature in the range of about 45 to about 55 ° C., more preferably About 50 ° C.

The molecular weight of the protein of the present invention may be, for example, about 20 to 40 kDa, preferably about 25 kDa to 35 kDa, more preferably about 30 kDa.

The protein of the present invention may also have a Km value for NANA, NAM and Pyr. The Km value of the protein of the present invention for NANA is, for example, about 0.2 to about 20.0 mM, preferably about 0.4 to about 10.0 mM, more preferably about 1.0 to about 5.0 mM, even more preferably. May be from about 1.5 to about 3 mM. The Km value of the protein of the present invention for NAM is, for example, about 2.0 to about 200.0 mM, preferably about 5.0 to about 100.0 mM, more preferably about 10.0 to about 50.0 mM, and even more preferably. May be from about 20 to about 30 mM. The Km value of the protein of the present invention relative to Pyr is, for example, about 4.0 to about 400.0 mM, preferably about 8.0 to about 200.0 mM, more preferably about 15.0 to about 100.0 mM, even more preferably May be from about 30 to about 50 mM.

The protein of the present invention may also have N-acetyl-D-neuraminic acid aldolase activity (NANA aldolase activity). As used herein, the term “N-acetyl-D-neuraminic acid aldolase activity (NANA aldolase activity)” refers to N-acetyl-D-mannosamine (NAM) and pyruvic acid (Pyr) and aldol condensation. Refers to the catalytic activity to convert to N-acetyl-D-mannosamine (NANA).

The protein of the present invention may also be derived from a bacterium belonging to the genus Bacillus. More specifically, the protein of the present invention may be Bacillus sp., Preferably Bacillus sp. It may be derived from the YKR AJ110757 strain (referred to herein as Bacillus sp. YKR, if necessary). Bacillus sp. The YKR AJ110757 strain is a strain that has been deposited with the following depository and has been deposited and managed with the deposit number of FERM BP-11319. The strain to which the FERM number is assigned can be sold by a predetermined procedure with reference to the accession number.

Name: Bacillus sp. YKR AJ110757
Accession Number: FERM BP-11319
Depositary institution: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Depositary Institution: Address 1 1-1, Higashi 1-chome, Tsukuba, Ibaraki, Japan
Date of deposit to the above depository: September 10, 2009

In another embodiment, the protein of the present invention may be a protein selected from the group consisting of (A) to (D) below:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2;
(B) a protein comprising the amino acid sequence represented by SEQ ID NO: 2;
(C) the amino acid sequence represented by SEQ ID NO: 2, consisting of an amino acid sequence containing a mutation (eg, deletion, substitution, addition and insertion) of one or several amino acid residues, and N-acetyl-D- A protein having neuraminate aldolase activity; or (D) consisting of an amino acid sequence having a predetermined amino acid sequence identity to the amino acid sequence represented by SEQ ID NO: 2 and having N-acetyl-D-neuraminate aldolase activity Have a protein.

In the present invention, substantially the same protein as the protein (A) can also be used. The proteins shown in (B) to (D) are provided as substantially the same protein as the protein (A). The term “one or several” indicates a range that does not significantly impair the three-dimensional structure and activity of the amino acid residue protein, although it depends on the position and type of the three-dimensional structure of the amino acid residue protein. The number “1 or several” in the case of protein is, for example, 1 to 100, preferably 1 to 70, more preferably 1 to 40, more preferably 1 to 20, and preferably 1 to The number is 10, more preferably 1 to 5. The proteins (B) to (D) are not particularly limited as long as they retain NANA aldolase activity, but may further retain one or more of the above-described properties (eg, pH stability). However, in the case of proteins (B) to (D), under conditions of 50 ° C. and pH 8.5, about half or more of protein (A), more preferably 80% or more, still more preferably 90% or more, and even more preferably It is desirable to maintain NANA aldolase activity of 95% or more.

The protein of the present invention can be obtained by isolating a NANA-assimilating microorganism (eg, a bacterium belonging to the genus Bacillus) from a soil sample (eg, a soil sample of Kamogawa Riverbed in Sakyo-ku, Kyoto, Japan). Sex microorganisms are available. Isolation of a NANA-assimilating microorganism may be performed by using a medium under strong alkaline conditions (eg, medium i and medium ii described in Examples).

The protein of the present invention can also be obtained by a mutagenesis method such as a site-specific mutagenesis method or by inserting a polynucleotide encoding the protein into an expression vector having a tag sequence such as a purification sequence. Moreover, you may acquire the protein which has the above modified amino acid sequences by the conventionally known mutation process. Mutation treatment includes a method of in vitro treatment of DNA encoding protein (A) with hydroxylamine or the like, and Escherichia bacterium carrying the DNA encoding protein (A) by ultraviolet irradiation or N-methyl-N ′. -A method of treating with a mutating agent usually used for artificial mutation, such as nitro-N-nitrosoguanidine (NTG) or nitrous acid.

In addition, the mutations as described above include naturally occurring mutations such as differences in microorganism species or strains. DNA encoding the protein substantially the same as protein (A) can be obtained by expressing the DNA having the mutation as described above in a suitable cell and examining the enzyme activity of the expression product.

When an amino acid residue is mutated by substitution, the amino acid residue substitution may be a conservative substitution. As used herein, the term “conservative substitution” refers to the replacement of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art. For example, such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged polar side chains (Eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having side groups containing hydroxyl groups (eg, alcoholic, phenolic) ( Example, serine, thread Nin, tyrosine), and amino acids (e.g. having sulfur-containing side chains, cysteine, methionine) and the like. Preferably, the conservative substitution of amino acids is a substitution between aspartic acid and glutamic acid, a substitution between arginine and lysine and histidine, a substitution between tryptophan and phenylalanine, and between phenylalanine and valine. Or a substitution between leucine, isoleucine and alanine, and a substitution between glycine and alanine.

In addition, the protein substantially the same as the protein (A) is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95%, 96%, 97%, 98% or 99 % Of proteins having an amino acid sequence identity of at least%. In the present specification, the identity of amino acid sequences is calculated by using the software GENENETYX Ver 7.0.9 from GENETICS, Inc., using the full length polypeptide chain encoded by the ORF, and the unit size to compare = 2. This is the numerical value when the percentage calculation of the Marching count is performed.

The present invention also provides a polynucleotide that is any one selected from the group consisting of the following (a) to (e):
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
(B) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
(C) a polymorph encoding a protein comprising a nucleotide sequence having at least 80% nucleotide sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and having N-acetyl-D-neuraminic acid aldolase activity nucleotide;
(D) encodes a protein that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 and has N-acetyl-D-neuraminic acid aldolase activity Or (e) a polynucleotide encoding the protein of any one of [1] to [3] above.

The polynucleotide of the present invention may be DNA or RNA, or a mixture thereof.

The method for isolating the polynucleotide of the present invention will be described. DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 is Bacillus sp. From a chromosomal DNA or a DNA library by PCR or hybridization. Primers used for PCR can be designed based on an internal amino acid sequence determined based on a purified protein having an activity of catalyzing the reaction in the method of the present invention, for example. In addition, a primer or a probe for hybridization can be designed based on the nucleotide sequence described in SEQ ID NO: 1, or can be isolated using the probe.

In addition, polynucleotides substantially the same as the polynucleotide (a) are also included in the polynucleotides of the present invention. Examples of the polynucleotide substantially the same as the polynucleotide (a) include the polynucleotides (b) to (e) described above.

In the above polynucleotide (d), the nucleotide sequence identity to the polynucleotide (a) is 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95%, 96%, 97 %, 98% or 99% or more. In this specification, nucleotide sequence identity refers to the entire ORF (including the stop codon) using GENETYX software GENETYX Ver 7.0.9, Unit Size to Compare = 6, pick up location = 1 It can be determined by the numerical value calculated by percentage.

“Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such conditions include, for example, hybridization at about 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by 50 × 65 ° C. in 0.2 × SSC, 0.1% SDS. 1 or 2 times of washing. The genes that hybridize under these conditions include those that have generated stop codons in the middle and those that have lost activity due to mutations in the active center. It can be easily removed by expressing in an appropriate host and measuring the enzyme activity of the expression product by the method described below.

The polynucleotides (b) to (d) are not particularly limited as long as the protein encoded by them retains NANA aldolase activity, but further retains one or more of the above properties (eg, pH stability). It may also encode a protein that However, in the case of the above polynucleotides (b) to (d), at 50 ° C. and pH 8.5, about half or more of the protein (A), more preferably 80% or more, more preferably 90% or more, More preferably, it encodes a protein having 95% or more of NANA aldolase activity.

Next, a method for producing the protein of the present invention, and an expression vector used for the method and a method for producing a transformant will be described using the protein (A) as an example. The same can be applied to other mutant proteins.

The transformant expressing the protein (A) can be prepared using an expression vector incorporating a polynucleotide having any one of the above nucleotide sequences. For example, a transformant that expresses the protein (A) can be obtained by preparing an expression vector incorporating a DNA having the nucleotide sequence shown in SEQ ID NO: 1 and introducing it into an appropriate host. Examples of hosts for expressing a protein specified by the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 include Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria, and Bacillus subtilis. Various prokaryotic cells including Bacillus subtilis, Saccharomyces cerevisiae, Pichia stititis, and Aspergillus oryzae can be used as true cells such as Aspergillus oryzae. .

The expression vector used for introducing the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 into the host is expressed in a vector according to the type of the host to be expressed, and the protein encoded by the DNA is expressed. It can be prepared by inserting in a possible form. As a promoter for expressing a protein, when a promoter specific to a gene encoding the enzyme derived from a bacterium belonging to the genus Bacillus functions in a host cell, the promoter can be used. Further, if necessary, another promoter that works in the host cell may be linked to DNA such as SEQ ID NO: 1 and expressed under the control of the promoter.

In the case where the target protein is mass-produced using recombinant DNA technology, a form in which an inclusion body of the protein is formed by associating the protein in a transformant producing the protein is given as a preferred embodiment. It is done. Advantages of this expression production method are that the target protein is protected from digestion by proteases present in the microbial cells, and that the target protein can be easily purified by centrifugation following cell disruption. In order to obtain an active protein from a protein inclusion body, a series of operations such as solubilization and activity regeneration are required, and the operation becomes more complicated than when directly producing an active protein. However, when a large amount of a protein that affects the growth of bacterial cells is produced in the bacterial cells, the effect can be suppressed by accumulating them in the bacterial cells as inactive protein inclusion bodies. As a method for mass production of the target protein as inclusion bodies, there is a method of expressing the target protein alone under the control of a strong promoter, or a method of expressing it as a fusion protein with a protein known to be expressed in large quantities. is there.

The host to be transformed is as described above, but in detail about E. coli, it can be selected from Escherichia coli JM109 strain, DH5α strain, HB101 strain, BL21 (DE3) strain, etc. Methods for performing transformation and methods for selecting transformants are also described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor press (2001/01/15) and the like. Hereinafter, a method for producing transformed E. coli and producing a predetermined enzyme using the same will be described more specifically as an example.

As a promoter for expressing DNA encoding a protein having a catalytic activity used in the present invention, a promoter usually used for heterologous protein production in Escherichia coli can be used. For example, T7 promoter, lac promoter, trp promoter, trc Strong promoters such as promoter, tac promoter, lambda phage PR promoter, PL promoter, T5 promoter, and the like can be mentioned. Examples of the vector include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pACYC177, pACYC184, pMW119, pMW118, pMW219, pMW218, pQE30, and derivatives thereof. As other vectors, phage DNA vectors may be used. Furthermore, an expression vector containing a promoter and capable of expressing the inserted DNA sequence may be used.

In order to produce the protein used in the present invention as a fusion protein inclusion body, a gene encoding another protein, preferably a hydrophilic peptide, is linked upstream or downstream of the protein, and the fusion protein gene and To do. Such a gene encoding another protein may be any gene that increases the accumulation amount of the fusion protein and enhances the solubility of the fusion protein after the denaturation and regeneration steps. For example, T7gene 10, β-galactosidase gene, Dehydrofolate reductase gene, interferon γ gene, interleukin-2 gene, prochymosin gene and the like are listed as candidates.

When linking these genes and genes encoding proteins, the codon reading frames should be matched. Ligation may be performed at an appropriate restriction enzyme site, or synthetic DNA having an appropriate sequence may be used.

In order to increase the production amount, it may be preferable to link a terminator, which is a transcription termination sequence, downstream of the fusion protein gene. Examples of the terminator include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, E. coli trpA gene terminator, and the like.

As a vector for introducing a gene encoding a protein having a catalytic activity or a fusion protein thereof into Escherichia coli, a so-called multicopy type is preferable, and a plasmid having a replication origin derived from ColE1, such as a pUC-type plasmid or pBR322, is used. A plasmid of the system or a derivative thereof. Here, the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, insertion, addition and / or inversion. The “modification” here includes modification by mutation treatment, UV irradiation, natural mutation, or the like.

Further, it is preferable that the vector has a marker such as an ampicillin resistance gene in order to select transformants. As such a plasmid, an expression vector having a strong promoter is commercially available (eg, pUC system (manufactured by Takara Bio Inc.), pPROK system (manufactured by Clontech), pKK233-2 (manufactured by Clontech)).

An expression vector is obtained by ligating a promoter, a gene encoding a target protein having a predetermined activity or a fusion protein of the target protein and another protein, or, in some cases, a terminator-linked DNA fragment, and vector DNA.

When the obtained expression vector is used to transform E. coli and the E. coli is cultured, a predetermined protein or a fusion protein thereof is expressed and produced.

When expressed as a fusion protein, the target protein may be excised using a restriction protease such as blood coagulation factor Xa, kallikrein, etc., which has a sequence not present in the target protein as a recognition sequence.

As the production medium, a medium usually used for culturing Escherichia coli such as M9-casamino acid medium and LB medium may be used. The culture conditions and production induction conditions are appropriately selected according to the type of the marker, promoter, host fungus and the like used.

There are the following methods for recovering the target protein or a fusion protein containing the protein. If the target protein or its fusion protein is solubilized in the microbial cells, the microbial cells can be recovered and then disrupted or lysed to be used as a crude enzyme solution. Furthermore, if necessary, the target protein or a fusion protein thereof can be purified and used by a conventional method such as precipitation, filtration or column chromatography. In this case, a purification method using an antibody of the target protein or fusion protein can also be used. When a protein inclusion body is formed, the protein of interest can be obtained by solubilizing it with a denaturing agent and removing the denaturing agent by dialysis or the like.

2. Method for Producing NANA According to the Present Invention The present invention provides a method for producing NANA using the protein of the present invention. The production method of the present invention can be classified into production method (I) and production method (II). First, the outline of the production method (I) will be explained, and then the outline of the production method (II) will be explained.

The production method (I) of the present invention includes a step of synthesizing NANA from NAM and Pyr in the presence of the protein of the present invention (corresponding to the reaction step (2) shown in the background art). The production method (I) is carried out under any conditions in which the protein of the present invention has NANA aldolase activity. However, when the production method (I) is carried out in the form of the production method (II) in combination with the steps described later, it is alkaline. It is carried out in a reaction solution under conditions.

An alkaline buffer is used as the reaction solution under alkaline conditions. Alkaline buffers include alkali metal hydroxides (eg, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide), alkali metal carbonates (eg, sodium carbonate, potassium carbonate), alkali metals It may be prepared using an alkaline substance such as bicarbonate (eg, sodium bicarbonate, potassium bicarbonate), ammonia, or phosphate buffer, Tris-HCl buffer, borate buffer, veronal hydrochloride buffer. Well-known alkaline buffers such as liquid, Good buffer, and diethanolamine hydrochloride buffer may be used.

From the viewpoint of efficiently producing a larger amount of NANA from NAM and Pyr, the pH of the reaction solution under alkaline conditions in the production method (I) takes into consideration the pH stability and optimum pH of the protein of the present invention. Thus, it is preferable to perform the above steps. Such a pH range is apparent from the above description of pH stability and optimum pH. For example, pH pH 8.0 to 10.0, preferably pH 8.0 to 9.0, more preferably pH 8.0 to It may be 8.5.

The concentration of each substrate in the reaction solution is not particularly limited. Specifically, the concentrations of NAM and Pyr in the production method (I) can be set as appropriate. Each substrate may also be supplemented as appropriate during the reaction. Furthermore, NAM can be used in any form of free base and salt (eg, hydrochloride, sulfate). Pyr can be used in any form of free acid and salt (eg, sodium salt, potassium salt).

The concentration of the protein of the present invention in the reaction solution is not particularly limited as long as the reaction proceeds. The form of the protein of the present invention is not particularly limited as long as it exists in the reaction system in a state capable of catalyzing the above reaction. That is, when the reaction is performed in the presence of the protein, the specific form of the protein in the reaction system includes, for example, a culture containing a microorganism producing the protein, a microbial cell separated from the culture, Processed bacterial cells (extracted protein) and the like are included. A culture containing microorganisms is a product obtained by culturing microorganisms. More specifically, microbial cells, a medium used for culturing the microorganisms, and substances produced by the cultured microorganisms, And a mixture thereof. Further, the microbial cells may be washed and used as washed cells. In addition, cell-treated products include those obtained by crushing, lysing, and lyophilizing the cells, and further purifying the cell-free extracts and crude proteins recovered by treating the cells. Extracted proteins such as purified protein are included. As the purified protein, partially purified proteins obtained by various purification methods may be used, or immobilized proteins obtained by immobilizing these by a covalent bond method, an adsorption method, a comprehensive method, or the like may be used. Good. In addition, some microorganisms lyse during culture, and in this case, the culture supernatant can also be used as a protein-containing material.

In the production method (I), from the viewpoint of efficiently producing a larger amount of NANA from NAM and Pyr, the above steps may be performed in consideration of the temperature stability and optimum temperature of the protein of the present invention. preferable. Such a temperature range can be appropriately determined by those skilled in the art. For example, 30 to 60 ° C. is used.

In the production method (I), the reaction time can be appropriately set according to the amount of NANA to be produced. The reaction can be performed in a stationary state or with stirring.

The production method (I) may be performed in the presence of 4-hydroxy-4-methyl-2-oxoglutarate aldolase in addition to the protein of the present invention. 4-Hydroxy-4-methyl-2-oxoglutarate aldolase can suppress the production of 4-hydroxy-4-methyl-2-oxoglutarate caused by condensation of pyruvic acid under alkaline conditions. Therefore, from the viewpoint of improving the NANA yield, the production method (I) is preferably performed in the presence of the protein of the present invention and 4-hydroxy-4-methyl-2-oxoglutarate aldolase. 4-Hydroxy-4-methyl-2-oxoglutarate aldolase is an enzyme classified as EC 4.1.3.17, which is 4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase or ProA aldolase ( WO2003 / 091396), or 4-hydroxy-4-methyl-2-ketoglutarate aldolase, γ-methyl-γ-hydroxy-α-ketoglutarate aldolase, KDPG aldolase, MHK aldolase, MHKG aldolase or pyruvate aldolase (BRENDA: (http://www.brenda-enzymes.org/index.php4).

As 4-hydroxy-4-methyl-2-oxoglutarate aldolase, various enzymes (eg, derived from microorganisms or animals and plants) are known. For example, such aldolases include those derived from the genus Komamomas, Pseudomonas, and Arakis. Specifically, examples of 4-hydroxy-4-methyl-2-oxoglutarate aldolase include Comomas testosteroni (WO2003 / 091396), Pseudomonas putida [Tack, J. et al. Biol. Chem. vol. 247, p. 6444-6449 (1972); Dagley et al. , Methods Enzymol. vol. 90, p. 272-276 (1982)], Pseudomonas testosteroni [Schul Ritter et al. , J .; Bacteriol. vol. 113, p. 1064-1065 (1973)], Pseudomonas ochracea [Maruyama, J. et al. Biochem. vol. 10, p. 334-340 (1990); Maruyama, J. et al. Biochem. vol. 108, p. 327-333 (1990); Maruyama, J. et al. Biochem. vol. 110, p. 976-981 (1991); Maruyama et al. Biotechnol. Biochem. , Vol. 65, p. 2701-2709 (2001)], Arachis hypogaea [Wood, The Enzymes (3rd Ed) (Boyer, PD, ed.), Vol. 7, p. 281-302 (1972); Shannon et al. , J .; Biol. Chem. vol. 237, p. 3342-3347 (1962)].

The concentration of 4-hydroxy-4-methyl-2-oxoglutarate aldolase in the reaction solution is not particularly limited as long as the reaction proceeds. The form of 4-hydroxy-4-methyl-2-oxoglutarate aldolase is not particularly limited as long as it is present in the reaction system in a state capable of catalyzing the above reaction. That is, when the reaction is carried out in the presence of 4-hydroxy-4-methyl-2-oxoglutarate aldolase, the specific form of aldolase present in the reaction system is, for example, 4-hydroxy-4-methyl-2 -Cultures containing microorganisms producing oxoglutarate aldolase, microbial cells isolated from the cultures, treated cells (extracted proteins), etc. are included.

When the production method (I) is performed using a transformant, examples of the transformant include (i) a transformant producing the protein of the present invention, (ii) 4-hydroxy-4-methyl- A transformant producing 2-oxoglutarate aldolase, (iii) a transformant producing both the protein of the present invention and 4-hydroxy-4-methyl-2-oxoglutarate aldolase.

When both the protein of the present invention and 4-hydroxy-4-methyl-2-oxoglutarate aldolase are used in the production method (I), the protein of the present invention and 4-hydroxy-4-methyl-2-oxoglutarate aldolase May be provided in the reaction in the following manner.
-Protein of the present invention (extracting enzyme) and 4-hydroxy-4-methyl-2-oxoglutarate aldolase (extracting enzyme)
-Transformant producing the protein of the present invention and 4-hydroxy-4-methyl-2-oxoglutarate aldolase (extraction enzyme)
A transformant producing the protein of the present invention (extracting enzyme) and 4-hydroxy-4-methyl-2-oxoglutarate aldolase. A transformant producing the protein of the present invention and 4-hydroxy-4-methyl-2. -Transformant producing oxoglutarate aldolase-Protein of the present invention and transformant producing 4-hydroxy-4-methyl-2-oxoglutarate aldolase

The above-mentioned transformant can be obtained by, for example, i) introducing the expression vector of the protein of the present invention into a 4-hydroxy-4-methyl-2-oxoglutarate aldolase producing bacterium, ii) 4-hydroxy-4- By introducing an expression vector of methyl-2-oxoglutarate aldolase into a bacterium producing the protein of the present invention, iii) the first expression vector of the protein of the present invention and 4-hydroxy-4-methyl-2-oxoglutarate Introducing the second expression vector of aldolase into the host microorganism, iv) introducing the protein of the present invention and the expression vector of 4-hydroxy-4-methyl-2-oxoglutarate aldolase into the host microorganism. Can be produced. Examples of the expression vector of the protein of the present invention and 4-hydroxy-4-methyl-2-oxoglutarate aldolase include, for example, i ′) the first polynucleotide encoding the protein of the present invention, and the first polynucleotide. A first expression unit composed of a first promoter operably linked; a second polynucleotide encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase; and the second polynucleotide An expression vector comprising a second expression unit composed of a second promoter operably linked to ii ′) a first polynucleotide encoding a protein of the invention and 4-hydroxy-4-methyl- Second polynucleoside encoding 2-oxoglutarate aldolase De, and expression vectors comprising a promoter operably linked to the first and second polynucleotide (vector capable of expressing the polycistronic mRNA) are exemplified. The first polynucleotide encoding the protein of the present invention may be located upstream or downstream of the second polynucleotide encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase. May be located.

NANA produced by the production method (I) can be easily separated and purified from the reaction solution by a known method. For example, it can be purified by ion exchange column chromatography and then concentrated to obtain a crystal from water or an organic solvent.

The production method (II) of the present invention comprises a first step (corresponding to the reaction step (1) shown in the background art) for producing NAM from NAG by an isomerization reaction under alkaline conditions, and A second step of synthesizing NANA from NAM and Pyr in the presence of protein (corresponding to the reaction step (2) above) is included. Production method (II) is carried out in a reaction solution under alkaline conditions. The reaction solution under alkaline conditions used in the production method (II) of the present invention is the same under the alkaline conditions used in the production method (I) of the present invention except that the raw material substrate is changed from NAM to NAG. It is the same as the reaction solution. Also in the production method (II) of the present invention, the reaction may be carried out in the presence of 4-hydroxy-4-methyl-2-oxoglutarate aldolase as in the production method (I) of the present invention.

In the production method (II), the pH of the reaction solution under alkaline conditions is such that when both steps proceed simultaneously, from the viewpoint of efficiently producing a larger amount of NANA from NAG and Pyr, the chemical from NAG to NAM In consideration of the pH range necessary for isomerization and the pH stability and optimum pH of the protein of the present invention, it is preferable to carry out both of the above steps. Such a pH region is apparent from the above-mentioned pH necessary for chemical isomerization of NAG to NAM, and the above-mentioned pH stability and optimum pH. For example, pH 9.5 to 10.0, Preferably pH 10.0 is used.

On the other hand, the reaction under alkaline conditions in the production method (II) may be performed at a plurality of pHs in each step. For example, first, the first step is performed in the pH range necessary for chemical isomerization from NAG to NAM, and then the second step is performed in the pH range considering the pH stability and optimum pH of the protein of the present invention. Done. In this case, the pH region in the first step is pH 9.5 or higher, and the pH region in the second step is the same as the pH region in the production method (I).

Furthermore, the reaction under alkaline conditions in the production method (II) is carried out by performing the first step at a preferred pH (eg, pH 11.5) by chemical isomerization from NAG to NAM, and then the pH of the reaction solution is changed to pH Adjust pH to the pH required for chemical isomerization from NAG to NAM (eg, pH 10) considering stability and optimum pH, and add the protein of the present invention to the reaction solution after pH adjustment By doing so, not only the second step but also the first step may be performed simultaneously. This format has the advantage that a larger amount of NANA can be efficiently manufactured.

The concentration of each substrate in the reaction solution is not particularly limited. Specifically, the concentrations of NAG and Pyr in the production method (II) can be set as appropriate. Each substrate may also be supplemented as appropriate during the reaction. Furthermore, NAG can be used in any form of free base and salt (eg, hydrochloride, sulfate). Pyr can be used in any form of free acid and salt (eg, sodium salt, potassium salt).

The concentration of the protein of the present invention in the reaction solution is not particularly limited as long as the reaction proceeds. The form of the protein of the present invention in production method (II) is the same as the form of the protein of the present invention in production method (I).

In the production method (II), from the viewpoint of efficiently producing a larger amount of NANA from NAM and Pyr, the preferred temperature for the chemical isomerization of NAG to NAM, and the temperature stability and optimum of the protein of the present invention. It is preferable to perform the above steps in consideration of an appropriate temperature. Such a temperature range can be appropriately determined by those skilled in the art. For example, 30 to 60 ° C. is used.

In the production method (II), the reaction time can be appropriately set according to the amount of NANA to be produced. The reaction can be performed in a stationary state or with stirring.

The NANA produced by the production method (II) can be easily separated and purified from the reaction solution by a known method in the same manner as the NANA produced by the production method (I).

Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to these examples.

(material)
All chemicals were obtained from commercial suppliers and were reagent grade.

(HPCL analysis)
N-acetyl-D-neuraminic acid (NANA), N-acetyl-D-glucosamine (NAG), N-acetyl-D-mannosamine (NAM) and pyruvic acid (Pyr) were mixed with an HPX-87H ion exclusion column (300 mm × 7.8 mm; BIO-RAD, USA). The mobile phase was 5 mM H ₂ SO ₄ buffer with a flow rate of 0.6 ml / min, and the eluate was monitored by 210 mm UV detection. The temperature was 40 ° C.

(Isolation of microorganisms)
Microorganisms that assimilate N-acetyl-D-neuraminic acid as a carbon source were isolated from soil samples from Kamogawa Riverbed in Sakyo-ku, Kyoto, Kyoto, Japan, and used for screening. The isolation medium (medium i) is 0.3% (w / v) NANA, 0.3% (w / v) (NH ₄ ) ₂ SO ₄ , 0.3% (w / v) K ₂ HPO ₄ , 0.05% (w / v) yeast extract, 0.05% (w / v) MgSO ₄ .7H ₂ O, 0.001% (w / v) FeSO ₄ .7H ₂ O, 0.001 % (W / v) MnSO ₄ .5H ₂ O, 0.75% (w / v) Na ₂ CO ₃ was contained in tap water (pH 10.5). Soil samples are incubated in this medium with shaking for 8 or 24 hours at 28 ° C., after which the culture is spread on a 2.0% agar plate of medium ii and then incubated at 28 ° C. for 12 hours did. Medium ii is 0.1% (w / v) NANA, 0.5% (w / v) sodium fumarate, 1.0% (w / v) peptone, 1.0% (w / v) yeast extraction Product, 0.3% (w / v) NaCl, 0.75% (w / v) Na ₂ CO ₃ was contained in tap water (pH 10.5). Appearing colonies were isolated on agar plates of medium ii. Subsequently, it was transferred again to an agar plate of medium ii containing 0.3% (w / v) NANA and cultured at 28 ° C. for 24 hours to obtain a sufficient amount of cells for the screening reaction.

(Screening of NANA-producing strains)
The reaction mixture contained 100 mM Pyr, 100 mM NAM, 50 mM borate buffer (pH 10.0) and 5 mg of wet cells collected from the agar plate in 100 μl. The reaction mixture was incubated at 28 ° C. for 8 or 24 hours and centrifuged at 10,000 × g for 5 minutes. Production of NANA in the supernatant was analyzed by HPLC.

(Plasmid, strain used and culture conditions)
The plasmid T-vector (Novagen, USA) was used as a customary DNA cloning vector. The plasmid pKK223-3 (GE Healthcare Bio-Sciences Corp, USA) was used as a protein expression vector.
Unless otherwise noted, Escherichia coli JM109 was employed as the usual cloning host, and JM109, a host strain for protein expression, was added to Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast). Extract and 1% NaCl). If necessary, 50 μg / ml ampicillin was added.

(General DNA technology, DNA sequencing and analysis)
All basic recombinant DNA procedures (eg, DNA isolation and purification, restriction enzyme digestion, DNA ligation, and E. coli transformation) followed standard protocols (Sambrook et al.,). Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York). DNA was amplified by PCR technique using a thermal cycler. The DNA sequence was analyzed by the dideoxy chain termination method (Sanger et al., Proc Natl) using the CEQ Die Terminator Cycle Sequence Kit (Beckman Coulter, USA) with the automated sequencer DNA analysis system CEQ 2000XL (Beckman Coulter, USA). Acad Sci USA 74 (1977) 5463-5467). Sequence data was analyzed with the Genetyx program (Software Development, Japan) and the Blast program.

(Isolation of Bacillus sp. YKR-derived nanA genomic clone)
The nucleotide fragment of the nanA gene was amplified by PCR using the genomic DNA of Bacillus sp. YKR. Total DNA was extracted from Bacillus sp. YKR (Ausbel et al., Current protocols in molecular biology, John Wiley and Sons, New York.). Bacillus sp. YKR genomic DNA was amplified using the following upstream and downstream primers.

Upstream primer: 5′-GCNGTNACNCCNTTYTAYTA-3 ′ (SEQ ID NO: 3) [corresponding to AVTPFYY (SEQ ID NO: 4)]
Downstream primer: 5′-AANGTNSWNCCATNGCNCCRTC-3 ′ (SEQ ID NO: 5) [corresponds to FTSGIAGD (SEQ ID NO: 6)]

Amplification was carried out as follows (annealing at 45 ° C. for 30 seconds, extension at 72 ° C. for 60 seconds and denaturation at 98 ° C. for 10 seconds for a total of 40 cycles). The PCR product (pNANA) was cloned into the pMD20-T vector, subjected to nucleotide sequence analysis, and then used as a probe. pNANA was labeled with digoxigenin-UTP using a DNA labeling kit (Roche), and the labeled pNANA was detected using a DNA detection kit (Roche).

Bacillus sp. YKR genomic DNA was digested with BamHI, EcoRI, HindIII, PstI, SacI, SalI, or SpeI, separated on a 0.7% agarose gel and transferred to a nylon membrane. Membranes were prehybridized in hybridization buffer (Roche) for 30 minutes at 40 ° C. and hybridized overnight at 65 ° C. with pNANA-labeled digoxigenin-UTP labeled using a DNA labeling kit (Roche). . 2 × SSC (SSC: 333mM NaCl , 333mM C 6 H 5 O 7 Na 3 · 2H 2 O), 55 at 20 ° C. in a 0.1% SDS, and 0.5 × SSC, during 0.1% SDS After washing at 0 ° C., labeled pNANA was detected with a DNA detection kit (Roche). Luminescence was recorded by exposure to X-ray film.

A Bacillus sp. YKR genomic library was prepared using genomic DNA digested with EcoRI. Based on the results of Southern blotting hybridization using pNANA, the size of the DNA fragment containing the nanA gene was estimated to be 2.0 kb. Fragments were size fractionated by means of 0.7% agarose gem electrophoresis after digesting genomic DNA with EcoRI. A gel part corresponding to a molecular size of 2.0 kb was cut out. The DNA fragment thus isolated was purified and ligated into pUK118. Approximately 5 × 10 ³ recombinants were screened with digoxigenin-labeled pNANA. After colony hybridization under the above conditions, the nylon membrane was washed at 40 ° C. in 2 × SSC, 0.1% SDS and 55 ° C. in 0.5 × SSC, 0.1% SDS, then The DNA was examined using a detection kit (Roche). Luminescence was recorded by exposure to X-ray film.

(Expression of nanA in Escherichia coli JM109)
A DNA fragment carrying the protein coding region of the Bacillus sp. YKR nanA gene was amplified by PCR using the following two oligonucleotide primers.

5′-GGGAATTCATGAAAGGGTTTATAACAGCATT-3 ′ (SEQ ID NO: 7)
(P ₁ , nucleotide residues 663-685 with EcoRI site added)
5′-CCAAGCTTTCATAATATAGCATTTAGGCTAATT-3 ′ (SEQ ID NO: 8)
(P ₂ , complementary residues 1507-1526 with the addition of a HindIII site)

The reaction mixture contained 100 ng Bacillus sp. YKR genomic DNA as a template and 50 pmol each of P1 and P2 in the manufacturer's reaction buffer. The reaction was carried out through 30 cycles of 90 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 2 minutes. The PCR product and pKK223-3 were digested with EcoRI and HindIII. The PCR product was inserted into the EcoRI and HindIII sites of pKK223-3.

Escherichia coli JM109 having no nanA activity was transformed with the obtained plasmid and grown to stationary phase. Cells are collected by centrifugation at 10,000 × g for 15 minutes, suspended in 50 mM Tris-HCl buffer (pH 8.0), and 3000 rpm using a cell disruption system (Micro Smash, TOMY, Japan). It was crushed for 180 seconds (30 seconds × 6). Cell debris was removed by centrifugation at 10,000 × g for 5 minutes, and the supernatant was used as a cell-free extract for nanA characterization, enzyme purification and NANA production reactions.

(NanA assay)
The enzyme activity value of nanA was determined by measuring the production of NANA from NAM and Pyr. In the basic reaction, a reaction mixture (100 μl) containing 50 mM buffer (Tris-HCl buffer (pH 8.5), 100 mM NAM, 100 mM Pyr and enzyme) was incubated at 30 ° C. for 30 minutes. Defined as the amount catalyzing the formation of 1 μmol NANA per minute at 30 ° C.

(Purification of nanA)
The following operations were performed at 0-5 ° C. The buffer used for purification was Tris-HCl buffer (pH 8.0).

Step 1: Preparation of crude extract 6.7 g of washed wet cells were suspended in 50 mM Tris-HCl buffer (pH 8.0), and then using a bead-type cell disrupter (Micro Smash, TOMY, Japan). And crushed at 3000 rpm for 180 seconds (30 seconds × 6). After centrifugation at 10,000 × g for 15 minutes, the supernatant solution was dialyzed against 50 mM Tris-HCl buffer (pH 8.0). The precipitate that formed during dialysis was removed by centrifugation at 10,000 × g for 15 minutes. The supernatant solution was used as a cell-free extract.

Step 2: HiPrep 16/10 DEAE FF column chromatography A HiPrep 16/10 DEAE FF column (GE) in which 4.5 ml of the above cell-free extract was equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 50 mM NaCl. Applied by Healthcare). The column was washed with equilibration buffer. The enzyme was eluted with a linear gradient of NaCl (50-250 mM in buffer). 4.5 ml fractions were collected. The enzyme solution combined with the active fraction was dialyzed against 50 mM Tris-HCl buffer (pH 8.0) and concentrated to 1 ml.

Step 3: Mono Q HR 5/5 Column Chromatography The above enzyme concentrated solution ml was applied to a Mono Q HR 5/5 column (manufactured by GE Healthcare) equilibrated with 50 mM Tris-HCl buffer (pH 8.0). did. The column was washed with 50 mM Tris-HCl buffer (pH 8.0) containing 200 mM NaCl. The enzyme was eluted with a linear gradient of NaCl (200-300 mM in buffer). Fractions were collected in 0.5 ml aliquots. The enzyme solution combined with the active fraction was dialyzed against 50 mM Tris-HCl buffer (pH 8.0) to obtain 1 ml of the active fraction.

Step 4: Mono Q column chromatography 25 μl of the above enzyme concentrated solution was applied to a Mono Q column equilibrated with 50 mM Tris-HCl buffer (pH 8.0) (manufactured by GE Healthcare). The column was washed with 50 mM Tris-HCl buffer (pH 8.0) containing 200 mM NaCl. The enzyme was eluted with a linear gradient of NaCl (200-300 mM in buffer). Fractions were collected in 0.1 ml portions. The enzyme solution combined with the active fraction was dialyzed against 50 mM Tris-HCl buffer (pH 8.0) to obtain 1 ml of a concentrated solution.

(NANA production reaction from NAM and Pyr)
A standard reaction mixture (100 μl) is a cell-free extract of 0.5 mg / ml protein, 50 mM Tris-HCl buffer (pH 8.5), or CAPS buffer (pH 10.0), 100 mM NAM and Contained 100 mM Pyr. Incubated at 30 ° C.

(NANA production reaction from NAG and Pyr)
First, NAM was prepared from NAG by incubating 1000 mM NAG at pH 11.5 for 3 hours at 40 ° C. in a volume of 0.7 ml. NAG and NAM became 855 mM and 205 mM by the chemical isomerization reaction, respectively. Second, cell-free extract from recombinant Escherichia coli of 100 mM CAPS (pH 10.0), 700 mM Pyr and 0.5 mg / ml protein was added to the reaction mixture in a final volume of 1 ml, Incubated at 0 ° C.

Protein concentration was measured with a protein assay kit (Bio-Rad Laboratories, USA) using bovine serum albumin as a standard (Bradford, MM, Anal Biochem 72 (1976) 248-254). SDS-PAGE was performed using 12.5% polyacrylamide using a Tris-glycine buffer system as described by King and Laemmli (King et al., J. Mol. Biol. 62 (1971) 465-477). Performed in slab gel.

[Example 1] Screening of N-acetyl-D-neuraminic acid aldolase under alkaline conditions

1-1 Chemical isomerization of NAG under alkaline conditions The conditions of chemical isomerization were investigated. The isomerization of NAG to NAM was measured between pH 8.5 and pH 12.0 at 30 ° C. for 18 hours (FIG. 1). NAM was not detectable below pH 9.0. NAM was detected at a pH higher than pH 9.5.

The effect of reaction temperature on the chemical isomerization of NAG to NAM was investigated. The reaction was incubated at 20 ° C. to 80 ° C. for 3 hours at pH 11.5. At temperatures below 40 ° C., isomerization to NAM proceeded without decomposition of NAG and NAM. However, decomposition of NAG and NAM was observed at temperatures above 45 ° C. Therefore, the optimum temperature for chemical isomerization was 40 ° C.
The optimum pH for isomerization was pH 11.5 to 12.0. However, in preliminary experimental studies, most microorganisms could not grow at a pH higher than pH 11.0. Therefore, the medium i condition was determined at pH 10.5.

1-2 Screening for NANA-utilizing microorganisms for NANA production NANA-utilizing microorganisms utilize NAM and / or Pyr (which are degraded from NANA by nanA) as a carbon source. The NANA-assimilating microorganism has nanA. Medium i containing NANA as a single carbon source was used for screening of nanA.

About 1500 NANA-assimilating microorganisms were isolated from soil by enrichment culture in an alkaline medium i (pH 10.5) containing 0.3% NANA. Thirty of these strains produced NANA from NAM and Pyr under alkaline conditions. The production of NANA by these strains was examined under alkaline conditions (Table 1). These strains were incubated for 24 hours at 28 ° C. on alkaline medium ii. 5 mg of this wet cell was added to a reaction mixture (100 μl) containing 0.1 M Pyr and 0.1 M NAM, and incubated at pH 10.0 for 4 hours. Six strains I, II, V, VI, VII and YKR produced higher amounts of NANA. These six strains were identified as Bacillus sp. Based on 16S rRNA sequence results. The results of the 16S rRNA sequence were compared to the 16S rRNA sequence of Bacillus sp D6, Bacillus sp D6, Bacillus sp OS13, Bacillus sp D6, Bacillus sp OS13 and Bacillus sp, pseudofarmus (pseudofarmus), It showed 98%, 97%, 96%, 94%, 95% and 97% similarity, respectively. These 6 strains were used in the following.

[Example 2] Cloning of a gene encoding N-acetyl-D-neuraminic acid aldolase derived from newly isolated Bacillus sp. And expression of the enzyme in Escherichia coli JM109

2-1 Cloning of nanA derived from Bacillus sp. YKR PCR cloning was performed, and a part of the gene encoding nanA was obtained from the genomic DNA of six microorganisms isolated from soil. Various microorganisms (Shigella dysenteriae, Escherichia coli K-12, Salmonella entericaserovar, Haemophilus influenza, Actinobacillus pleuropneumoniae, Fusobacterium nucleatum, Lactobacillus plantarum, Clostridium perfringens, Staphlocpccus aureus, Streptococcus pneumonia, Pasteurella multocide, Trichomonas vaginalis, Mycoplasma capricolum, and Pseudoalteromonas Haloplan tis) aligning the 14 amino acid sequence of a known nanA from the condensation oligonucleotides were designed on conserved regions. A nucleotide fragment of the Bacillus sp. YKR nanA gene was amplified by PCR. The nucleotide sequence of the amplified gene fragment having a size of 271 bp showed homology with nanA from Clostridium perfringens. The gene amplification products of the other five Bacillus sp. Gene DNAs (templates) did not show similar sequences of known proteins with significant homology. Southern blotting hybridization using this fragment as a probe showed a single band generated by digestion of Bacillus sp. YKR DNA with BamHI, EcoRI, HindIII, PstI, SacI, SalI or SpeI.

A 2.0-3.0 kb DNA fragment recovered by size fractionation after EcoRI digestion was used to construct a partial Bacillus sp. YKR gene library, which was then screened using probe DNA. Plasmid DNA was isolated from positive clones. The entire 2.2 kb fragment obtained by digestion with EcoRI was sequenced. The sequence data of the isolated gene showed that the entire structural gene is located on the cloned fragment. All amino acid sequences determined for the nanA peptide fragment from Bacillus sp. YKR closely matched the amino acid sequence predicted from the DNA sequence. The open reading frame consisted of 864 bp, which encoded a protein of 288 amino acids with a calculated molecular weight of 32,643 Da (the nucleotide sequence represented by SEQ ID NO: 1 and represented by SEQ ID NO: 2) See amino acid sequence).

2-2 Expression of nanA in Escherichia coli JM109 The nanA gene was ligated into pKK223-3 with the tac promoter. The resulting plasmid, pKKnanA, was introduced into Escherichia coli JM109. For expression of the recombinant protein, Escherichia coli JM109 cells were induced at 37 ° C. with 0.1 mM IPTG. The expressed protein was detected by SDS-PAGE. The main band showed a molecular weight of about 30 kDa. This value was consistent with the calculated molecular weight of the nanA gene.

The effect of IPTG concentration on the expression of nanA was examined. For expression of the recombinant protein, E. coli JM109 cells were induced at 37 ° C. with various concentrations of IPTG (0.05-5.0 mM). However, no difference in protein expression was detected on SDS-PAGE.

2-3 Enzyme characteristics of nanA derived from Bacillus sp. YKR (1) Characteristics of crude enzyme The effects of temperature and pH on the activity of this enzyme were examined (FIGS. 2 and 4). In the basic reaction, a reaction mixture (100 μl) containing 50 mM buffer (Tris-HCl buffer (pH 8.5), 100 mM NAM, 100 mM Pyr and enzyme) was incubated at 30 ° C. for 30 minutes. The activity was measured under conditions, expressed as a percentage relative to the activity at pH 8.5 at 30 ° C. The enzyme activity was optimal at pH 8.0-8.5, with activity exceeding 60% of the maximum activity. Was observed at pH 7.0 to pH 10.0 This enzyme was most active at 50 ° C. and more than 60% of the maximum activity was found at 40-70 ° C. Stability of the enzyme The effects of temperature and pH on sex were examined (Figures 3 and 5.) For temperature stability, the crude enzyme solution (1 mg / ml) was incubated at 0 ° C to 80 ° C for 1 hour and the activity was measured. No loss of activity was observed when incubated for 1 hour at temperatures up to 40 ° C. Treatments at 45, 50, 55, 60, 65, 70 and 80 ° C., respectively, showed an initial activity of 30, Loss of 28, 35, 47, 63, 82 and 99% The enzyme was added to Tris-HCl buffer (pH 8.0, 8.5 and 9.0), borate buffer (pH 9.0, 9.5 and 10.0) and CAPS (pH 10.0, 10.5 and 11.0) buffers, no activity loss was observed when incubated overnight (16 hours) at 4 ° C. Treatment with acetate buffer (pH 5.0 and 6.0) and sodium phosphate buffer (pH 6.0 to pH 8.0) caused a loss of activity that exceeded 20% of the initial activity. It was velocity studies on the reaction. The crude enzyme, NANA, the NAM and Pyr, showed 2.0 mM, the Km value of 23.3mM and 42.7mM respectively (Table 2).

(2) Characteristics of purified enzyme Table 3 shows an outline of purification of this enzyme. The purification rate of this enzyme was about 2.5 times, and the purification yield was 24%. As for the purified enzyme, a single band was confirmed on SDS-PAGE.

[Example 3] Production of N-acetyl-D-neuraminic acid from N-acetyl-D-glucosamine by Escherichia coli JM109 YKR

3-1 Production of NANA from NAM and Pyr (1) Production of NANA under conditions of equimolar concentrations of NAM and Pyr Details of the following reaction conditions are as described in the brief description of the drawings.
40.2 mM NANA was produced in 6 hours from 100 mM NAM and 100 mM Pyr at pH 8.5. On the other hand, 52.7 mM NANA was produced in 6 hours from 100 mM NAM and 100 mM Pyr at pH 10.0, and at that time, 11.2 mM NAG was accumulated (FIG. 6). After 6 hours of reaction, NAM to NANA conversion reached 40.2% and 52.7%, respectively.

(2) Production of NANA under conditions of excess molar concentration of Pyr relative to NAM Details of the following reaction conditions are as described in the brief description of the drawings.
The effect of excess molar Pyr conditions on NAM was examined (FIGS. 7 and 8). These reactions reached equilibrium in 2 hours. In order to shift the reaction equilibrium toward the NANA synthesis side, Pyr was added during the reaction. In a reaction mixture containing 100 mM NAM and 100 mM Pyr at pH 8.5 and pH 10.0 for 7 hours, 54.8 mM and 50.8 mM NANA were produced, respectively. The ratio between the initial amount of NAM and the total amount of Pyr was 1: 1.7. At pH 10.0, 7.0 mM NAG accumulated in 7 hours.

(3) Production of NANA from high concentrations of NAM and Pyr Details of the following reaction conditions are as described in the brief description of the drawings.
484.0 mM NAM and 242.3 mM Pyr produced 394.0 mM NANA at pH 10.0, and in this reaction 490 mM and 310 mM Pyr were added after 1.5 and 5 hours (FIG. 9). After 7 hours of reaction, the NANA conversion rate from NAM was 84.3%. The ratio between the initial amount of NAM and the total amount of Pyr was 1: 2.1. In this reaction, 5.7 mM NAG accumulated in 7 hours.

3-2 Production of NANA from NAG and Pyr using cell-free extract The details of the following reaction conditions are as described in the brief description of the drawings.
NANA was produced from NAG and Pyr (FIG. 10). First, NAM was prepared from NAG by incubating 1000 mM NAG at pH 11.5 at 40 ° C. for 3 hours in a volume of 0.7 ml. Second, 100 mM CAPS (pH 10.0), 836.2 mM Pyr and 0.5 mg / ml recombinant Escherichia coli cell-free extract were added to the reaction mixture in a final volume of 1 ml. . When 1000 mM, 1000 mM and 500 mM Pyr were added after 23, 45 and 76 hours, respectively, 316.7 mM NANA was produced in a 96 hour reaction and the molar conversion to NAG was 50.3% (96 [NANA] / [NANA] + [NAM] + [NAG] per hour). The ratio between the initial amount of NAG and the total amount of Pyr was 1: 4.8.

3-3 Production of NANA from NAG and Pyr using microbial cells Details of the following reaction conditions are as described in the brief description of the drawings.
NANA was produced from NAG and Pyr (FIG. 11). First, in a volume of 0.7 ml, 1000 mM NAG was incubated at pH 11.5 at 40 ° C. for 3 hours to prepare NAM from NAG. NAG and NAM became 855 mM and 205 mM by the chemical isomerization reaction, respectively. Next, 50 mM CAPS (pH 10.0), 700 mM Pyr and recombinant Escherichia coli resting cells (5 ml culture volume) were added to the reaction mixture in a final volume of 1 ml. When 400 mM, 300 mM, 300 mM, 300 mM, 400 mM and 300 mM Pyr were added after 4.5, 7.5, 25, 30, 34, 48 hours, respectively, 358 mM NANA was produced in a 57 hour reaction, and NAG The molar conversion ratio with respect to was 51.6% ([NANA] / [NANA] + [NAM] + [NAG] per 57 hours). The ratio of the initial amount of NAG to the total amount of Pyr was 1: 3.8.
In addition, 293 mM NANA was produced in 50 hours in the resting cell reaction using 1 ml cultured cells (recombinant Escherichia coli). The molar conversion ratio with respect to NAG at this time was 43.2%.

3-4 Effect of Pyr concentration on NAM Details of the following reaction conditions are as described in the brief description of the drawings.
In order to shift the equilibrium for NANA synthesis in the reaction mixture, the effect of Pyr concentration on NANA production was investigated. In NAM 100 mM, Pyr was changed from 100 mM to 900 mM, and the reaction was performed at 37 ° C. for 24 hours. In reaction mixtures containing various concentrations of Pyr from 100 mM NAM to 100 mM, 300 mM, 500 mM, 700 mM, 900 mM at pH 8.0, 46.8 mM, 76.5 mM, 86.0 mM, 89.7 mM, 92. 5 mM NANA was produced (FIG. 12). The maximum NANA conversion rate from NAM was 92.5%.

3-5 Influence of Pyr Concentration on High Concentration NAM Details of the following reaction conditions are as described in the brief description of the drawings.
In order to shift the equilibrium for NANA synthesis in the reaction mixture, the effect of Pyr concentration on NANA production was investigated. In NAM 500 mM, Pyr was changed from 100 mM to 900 mM, and the reaction was performed at 37 ° C. for 24 hours. In reaction mixtures containing various concentrations of Pyr from 500 mM NAM to 100 mM, 300 mM, 500 mM, 700 mM, 900 mM at pH 8.0, 139.1 mM, 311.8 mM, 397.1 mM, 437.4 mM, 453. 2 mM NANA was produced (FIG. 13).

(Summary of Examples 1 to 3)
An efficient isomerization reaction from NAG to NAM was confirmed under alkaline conditions at pH 9.5 or higher. Therefore, it is desirable to use nanA that is active under alkaline conditions of pH 9.5 or higher in order to simultaneously proceed efficiently the isomerization reaction of NAM from NAG and the synthesis reaction of NANA from NAM and Pyr. .

Properties of enzyme: The activity of nanA derived from Bacillus sp. YKR was optimal at pH 8.0 to 8.5, and activity exceeding 60% of the maximum activity was observed at pH 7.0 to pH 10.0. The activity of nanA derived from Bacillus sp. YKR was stable between pH 8.0 and pH 11.0. On the other hand, the activity of nanA derived from Escherichia coli was optimal at pH 7.7, and activity exceeding 50% of the maximum activity was observed at pH 6.0 to pH 9.0. And the activity of nanA derived from Escherichia coli is stable between pH 6.0 and pH 9.0 (Non-patent Document 2). These results show that the protein of the present invention has an advantage over nanA derived from Escherichia coli in a reaction system under an alkaline condition reaction including the reaction steps (1) and (2) above.

Result of similarity search for the primary structure, the nanA derived from Bacillus sp YKR, Clostridium perfringens, Fusobacterium nucleatum, Haemophilus parasuis, Pasteurella multocide, Haemophilus influenza, Actinobacillus pleuropneumoniae, Staphlocpccus aureus, Lactobacillus plantarum, Mycoplasma capricolum, Salmonella entericaserovar, Escherichia coli K-12, Shigella dysenteriae, Streptococ Respectively nanA of us pneumonia and Pseudoalteromonas haloplanktis, was shown to have 73,71,71,70,69,67,56,53,48,35,35,35,29 and 25% similarity.

When nanA derived from Bacillus sp. YKR was used, NANA formation was confirmed when the reaction was carried out at pH 8.5 and pH 10.0 under equimolar concentrations of NAM (100 mM) and Pyr (100 mM). (FIG. 6). Even at pH 10.0, the amount of NANA produced was the same as that at pH 8.5. _{K m} values for nanA of NAM and Pyr from Bacillus sp YKR from it showed a large value and 23.3mM and 42.7mM respectively, increasing substrate concentration of the reaction incipient NAM and Pyr, further reaction in the middle The addition of Pyr greatly increased the amount of NANA produced (FIG. 9). The conversion rate from NAM and Pyr to NANA reached 92.5% (relative to NAM) (FIG. 12). The amount of NANA produced from NAM and Pyr reached 453.2 mM (FIG. 13).

NANA was produced from NAG and Pyr under alkaline conditions by a novel one-pot reaction that does not require ATP. Processes using Escherichia coli derived nanA for large scale production of NANA from NAG and Pyr require large amounts of ATP. ATP is a relatively unstable molecule and is very expensive. ATP is not required if chemical isomerization is used in the isomerization reaction instead of enzymatic isomerization.

The yield of NANA production from NAG and Pyr was 51.6% (relative to NAG) in 57 hours. In the novel one-pot reaction consisting of NAM isomerization reaction from NAG and NANA synthesis reaction from NAM and Pyr, it took 57 hours to produce 368 mM NANA. This means that the production method of the present invention has a NANA production rate of at least 6.3 mM / h.

[Example 4] NANA production reaction from NAG and Pyr added with 4-hydroxy-4-methyl-2-oxoglutarate aldolase Under the alkaline reaction conditions, reduction of pyruvic acid and formation of by-products were observed. We thought that it was important to suppress. The generated by-product was analyzed using LC / MS, and it was found that pyruvic acid was polymerized 4-hydroxy-4-methyl-2-oxoglutaric acid. In order to suppress the formation of this compound, the use of 4-hydroxy-4-methyl-2-oxoglutarate aldolase in the reaction was investigated. For 4-hydroxy-4-methyl-2-oxoglutarate aldolase, it has been found that Comonas testosteroni has 4-hydroxy-4-methyl-2-oxoglutarate aldolase (WO2003 / 091396). Therefore, a recombinant Escherichia coli with high expression of 4-hydroxy-4-methyl-2-oxoglutarate aldolase was prepared, and the influence of 4-hydroxy-4-methyl-2-oxoglutarate aldolase on NANA production was examined.
The effect of 4-hydroxy-4-methyl-2-oxoglutarate aldolase on NANA production was investigated. First, NAM was prepared from NAG by incubating 1000 mM NAG at pH 11.5 at 40 ° C. for 3 hours in a volume of 0.5 ml. Second, the reaction mixture, 50 mM of CAPS (pH 10.0), Pyr of 1000 _mM, 0.1 mM MgSO 4, recombinant 0.5mg / ml4- hydroxy-4-methyl-2-oxoglutarate aldolase protein A cell-free extract from Escherichia coli and a cell-free extract from 0.5 mg / ml N-acetyl-D-neuraminic acid aldolase recombinant Escherichia coli were added in a final volume of 1 ml. A control obtained by removing the cell-free extract derived from recombinant Escherichia coli of 0.5 mg / ml 4-hydroxy-4-methyl-2-oxoglutarate aldolase protein from the above reaction conditions was used as a control.
In the control, 26.8 mM NANA was produced in the reaction for 6 hours, while in the reaction to which 4-hydroxy-4-methyl-2-oxoglutarate aldolase was added, 43.2 mM NANA was produced in 6 hours. Thus, it was shown that the use of 4-hydroxy-4-methyl-2-oxoglutarate aldolase improves the yield of NANA.

Since the protein of the present invention exhibits pH stability under the alkaline conditions of high pH required in the chemical isomerization reaction from NAG to NAM, an efficient large amount of NANA using NAG and Pyr as raw materials It has the advantage of allowing manufacturing.
The polynucleotide, expression vector, transformant and bacterium of the present invention are useful for the production of the protein of the present invention, and as such enable efficient production of large amounts of NANA using NAG and Pyr as raw materials. Has the advantage of
The production method of the present invention can efficiently produce a large amount of NANA by using NAG and Pyr as raw materials. The production method of the present invention also has an advantage that an efficient production of a large amount of NANA can be carried out at low cost because expensive ATP is not required because chemical isomerization of NAM to NAM is used.

Claims

A protein that exhibits pH stability at pH 8.0 to 11.0 and has N-acetyl-D-neuraminic acid aldolase activity.
A protein selected from the group consisting of the following (A) to (D):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2;
(B) a protein comprising the amino acid sequence represented by SEQ ID NO: 2;
(C) the amino acid sequence represented by SEQ ID NO: 2, consisting of an amino acid sequence containing a mutation of one or several amino acid residues selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues; And a protein having N-acetyl-D-neuraminic acid aldolase activity; and (D) an amino acid sequence having at least 80% or more amino acid sequence identity to the amino acid sequence represented by SEQ ID NO: 2, and N A protein having acetyl-D-neuraminic acid aldolase activity;
The protein according to claim 2, wherein the protein exhibits pH stability at pH 8.0 to 11.0.
A polynucleotide selected from the group consisting of the following (a) to (e):
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
(B) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
(C) a polymorph encoding a protein comprising a nucleotide sequence having at least 80% nucleotide sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and having N-acetyl-D-neuraminic acid aldolase activity nucleotide;
(D) encodes a protein that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 and has N-acetyl-D-neuraminic acid aldolase activity And (e) a polynucleotide encoding the protein of any one of claims 1-3.
An expression vector comprising the polynucleotide according to claim 4.
A transformant into which the expression vector according to claim 5 has been introduced.
The transformant according to claim 6, wherein the host of the transformant is Escherichia coli.
A bacterium derived from the genus Bacillus having the ability to produce the protein according to any one of claims 1 to 3.
N-acetyl-, comprising culturing the transformant according to claim 6 or 7 and / or the bacterium according to claim 8 in a medium to obtain the protein according to any one of claims 1 to 3. A method for producing D-neuraminic acid aldolase.
Synthesis of N-acetyl-D-neuraminic acid from N-acetyl-D-mannosamine and pyruvic acid in the presence of the protein according to any one of claims 1 to 3. -A method for producing neuraminic acid.
The method according to claim 10, wherein the synthesis is further performed in the presence of 4-hydroxy-4-methyl-2-oxoglutarate aldolase.
The production method according to claim 10 or 11, wherein the method is carried out under conditions of pH 8.0 to 11.0.
The method according to any one of claims 10 to 12, further comprising producing N-acetyl-D-mannosamine from N-acetyl-D-glucosamine by an isomerization reaction under an alkaline condition of pH 9.5 or higher. Production method.