JP2014036612A - Modified ascorbic acid phosphorylating enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a modified ascorbic acid phosphorylating enzyme to obtain ascorbic acid-2-phosphate at a high yield.SOLUTION: The modified ascorbic acid phosphorylating enzyme is one of the following (A)-(C): (A) one having a specified amino acid sequence in which the amino acid at position 115 is arginine not alanine; (B) one having a specified amino acid sequence of (A) except that one or more amino acid residues are substituted, added, inserted or deleted at positions other than the position 115 provided that formation of ascorbic acid-2-phosphate from ascorbic acid in the presence of a phosphate donor is not inhibited; (C) one having an amino acid sequence having 30% homology to the amino acid sequence of (A) provided that amino acid at position 115 is the same and that formation of ascorbic acid-2-phosphate from ascorbic acid in the presence of a phosphate donor is not inhibited. Also provided is a method for producing ascorbic acid-2-phosphate.

Description

  The present invention relates to a modified form of ascorbyl phosphate that can obtain ascorbyl-2-phosphate in high yield.

  Ascorbic acid-2-phosphate is a stabilized derivative of ascorbic acid and is widely used in fields such as pharmaceuticals, foods, and cosmetics. Methods for efficiently producing ascorbic acid-2-phosphate (in this specification, ascorbic acid-2-phosphate may be abbreviated as “AsA2P”) have been studied conventionally. As such a method, for example, Patent Document 1 describes a method for producing ascorbic acid-2-phosphate from an ascorbic acid and a phosphate group donor using an enzyme derived from a microorganism belonging to the genus Pseudomonas. Yes. However, the present inventors examined the phosphorylation of ascorbic acid using the enzyme described in Patent Document 1 (wild-type ascorbate phosphorylase derived from Pseudomonas species). It was found that decomposition of the reaction product occurred as the temperature increased. In addition, it is reported in Patent Document 2 and Non-Patent Documents 1 and 2 that the same tendency is observed when enzymes derived from other organisms are used. As described above, since ascorbic acid-2-phosphate has a high industrial utility value, an enzyme and a production method that can be produced more efficiently have been demanded.

JP-A-4-53494 JP 2001-245676 A

Appl Environ Microbiol. 2000, 66 (7): 2811-2816, Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from Morganella morganii Protein Eng. (2002) 15 (7): 539-543. Enhancement of nucleoside phosphorylation activity in an acid phosphatase

  The main object of the present invention is to provide a modified form of ascorbyl phosphate that can produce ascorbyl 2-phosphate in high yield. Moreover, this invention provides the method of manufacturing ascorbic acid-2-phosphate using the said enzyme modified body.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have introduced a specific mutation into the amino acid sequence of ascorbyl phosphorylating enzyme, thereby decomposing the reaction product ascorbyl 2-phosphate. It was found that ascorbic acid-2-phosphate was obtained in a high yield. The present invention has been completed as a result of further research based on such findings. That is, this invention provides the manufacturing method of ascorbic acid phosphorylase modified body, DNA, a vector, a transformant, and ascorbic acid 2-phosphate of the following aspect.
Item 1. Ascorbate phosphorylation enzyme variant shown in any of the following (A) to (C):
(A) a modified ascorbate kinase comprising an amino acid sequence in which the alanine residue at position 115 is substituted with an arginine residue in the amino acid sequence represented by SEQ ID NO: 1;
(B) In the amino acid sequence of (A), including an amino acid sequence in which one or more amino acid residues other than position 115 are substituted, added, inserted or deleted, and in the presence of a phosphate group donor A modified ascorbic acid kinase that does not inhibit the production of ascorbic acid-2-phosphate when acting on ascorbic acid in
(C) In the amino acid sequence of (A), the amino acid sequence of the amino acid residue other than position 115 includes an amino acid sequence having an amino acid sequence of 30% or more, and ascorbic acid in the presence of a phosphate group donor. A modified ascorbic acid kinase that does not inhibit the production of ascorbyl 2-phosphate when allowed to act.
Item 2. Item 2. The enzyme variant according to Item 1, further comprising an amino acid sequence in which the histidine residue at position 116 is substituted with a glutamine residue in the amino acid sequence represented by SEQ ID NO: 1.
Item 3. Item 3. An isolated DNA encoding the enzyme variant according to Item 1 or 2.
Item 4. Item 4. A vector comprising the DNA of item 3.
Item 5. Item 5. A transformant obtained by transforming a host cell with the vector according to Item 4.
Item 6. A method for producing ascorbic acid-2-phosphate by causing a modified ascorbic acid kinase in any of the following (A) to (C) to act on ascorbic acid in the presence of a phosphate group donor:
(A) a modified ascorbate kinase comprising an amino acid sequence in which the alanine residue at position 115 is substituted with an arginine residue in the amino acid sequence represented by SEQ ID NO: 1;
(B) In the amino acid sequence of (A), including an amino acid sequence in which one or more amino acid residues other than position 115 are substituted, added, inserted or deleted, and in the presence of a phosphate group donor A modified ascorbic acid kinase that does not inhibit the production of ascorbic acid-2-phosphate when acting on ascorbic acid in
(C) In the amino acid sequence of (A), the amino acid sequence of the amino acid residue other than position 115 includes an amino acid sequence having an amino acid sequence of 30% or more, and ascorbic acid in the presence of a phosphate group donor. A modified ascorbic acid kinase that does not inhibit the production of ascorbyl 2-phosphate when allowed to act.
Item 7. Item 7. The method according to Item 6, wherein the phosphate group donor is polyphosphoric acid.

  The ascorbate phosphorylating enzyme of the present invention can exhibit excellent catalytic ability when acting on ascorbic acid in the presence of a phosphate group donor. Furthermore, according to the ascorbate phosphorylating enzyme of the present invention, it is possible to suppress the decomposition of the produced ascorbate-2-phosphate and obtain ascorbate-2-phosphate in a high yield.

It is the amino acid sequence of ascorbate phosphorylating enzyme shown by sequence number 1, and the base sequence (sequence number 2) corresponding to this amino acid sequence. In the amino acid sequence, Ala at position 115 is indicated by a circle. This represents the amino acid sequence (SEQ ID NO: 5) of the ascorbyl phosphate kinase variant (variant 3) and the base sequence (SEQ ID NO: 6) encoding the amino acid sequence. In the figure, the solid line shows the amino acid sequence encoded by the plasmid vector pET22b (+). The broken line shows the sequence inserted as a spacer region. A portion corresponding to A115R is indicated by a circle. It is the schematic which shows the preparation methods of an enzyme modified body. It is a graph which shows the quantity of AsA2P produced | generated when the modified bodies 1-8 are used. It is a graph which shows the quantity of AsA2P produced | generated when the modified bodies 7 and 8 are used. It is a graph which shows the quantity of AsA2P produced | generated when the ascorbate phosphorylation enzyme modification | denaturation 9-23 which introduce | transduced the amino acid variation | mutation further into the modification 3 is used. It is a graph which shows that AsA2P is decomposed | disassembled with time when AsA2P is produced | generated using a wild-type ascorbate phosphorylase.

In the present specification, amino acids, peptides, and proteins are represented using abbreviations adopted by the IUPAC-IUB Biochemical Nomenclature Committee (CBN) shown below.
A = Ala = alanine, C = Cys = cysteine,
D = Asp = aspartic acid, E = Glu = glutamic acid,
F = Phe = phenylalanine, G = Gly = glycine,
H = His = histidine, I = Ile = isoleucine,
K = Lys = Lysine, L = Leu = Leucine,
M = Met = methionine, N = Asn = asparagine,
P = Pro = proline, Q = Gln = glutamine,
R = Arg = arginine, S = Ser = serine,
T = Thr = threonine, V = Val = valine,
W = Trp = tryptophan, Y = Tyr = tyrosine

  Unless otherwise specified, the amino acid residue sequences of peptides and proteins are expressed from the N-terminus to the C-terminus from the left end to the right end. For ease of reference, the following commonly used nomenclature is applied.

  That is, one is a method described as “original amino acid; position; substituted amino acid”. For example, substitution of alanine to arginine at position 115 is expressed as Ala115Arg or A115R. In addition, multiple mutations are represented by being separated by a slash symbol “/”. For example, A115R / N130T indicates substitution of alanine at position 115 with arginine and asparagine at position 130 with threonine.

1. Ascorbate Phosphorylase Modified Variant The ascorbate phosphorylase variant of the present invention has the characteristics shown in any of the following (A) to (C):
(A) a modified ascorbate kinase comprising an amino acid sequence in which the alanine residue at position 115 is substituted with an arginine residue in the amino acid sequence represented by SEQ ID NO: 1;
(B) In the amino acid sequence of (A), including an amino acid sequence in which one or more amino acid residues other than position 115 are substituted, added, inserted or deleted, and in the presence of a phosphate group donor A modified ascorbic acid kinase that does not inhibit the production of ascorbic acid-2-phosphate when acting on ascorbic acid in
(C) In the amino acid sequence of (A), the amino acid sequence of the amino acid residue other than position 115 includes an amino acid sequence having an amino acid sequence of 30% or more, and ascorbic acid in the presence of a phosphate group donor. A modified ascorbic acid kinase that does not inhibit the production of ascorbyl 2-phosphate when allowed to act.

  In the present specification, in the amino acid sequence represented by SEQ ID NO: 1, a mutation that substitutes an alanine residue at position 115 with an arginine residue may be referred to as A115R. In the present specification, the modified ascorbic acid kinase may be abbreviated as “enzyme modified of the present invention”.

  The amino acid sequence represented by SEQ ID NO: 1 consists of 226 amino acid residues corresponding to proline at position 24 to valine at position 249 in the amino acid sequence of the wild-type ascorbyl kinase shown in SEQ ID NO: 3. The base sequence corresponding to the amino acid sequence of wild-type ascorbyl phosphate shown by SEQ ID NO: 3 is shown by SEQ ID NO: 4.

The origin of wild-type ascorbate phosphorylase is not particularly limited. Examples include microorganisms capable of producing an enzyme capable of catalyzing a reaction for producing L-ascorbic acid-2-phosphate from a donor. As such microorganisms, more specifically, Klebsiella oxytoca (ATCC8724), Cellulomonas cartae (ATCC21681), Alcaligenes eutrophas ( Alcaligenes eutrophomus ) Aeromonas hydrophila) (ATCC11163), Brevibacterium Richikamu (Brevibacterium lyticum) (ATCC15921), Brevibacterium flavum (Brevibacterium flavum) (ATCC13826), Pseudomonas Ribofurabina (Pseudomonas ri oflavina) (ATCC9526), Pseudomonas maltophilia (Pseudomonas maltophilia) (ATCC17806), Sphingomonas Toreperi (Sphingomonas trueperi), shake Bun di Monas-Diminuta (Brevundimonas diminuta) (ATCC11568), Gluconacetobacter-Hansenii (Gluconacetobacter hansenii) ( ATCC23761), Xanthomonas oryzae (NBRC3312) and the like are exemplified, and among them, Sphingomonas truperi is preferable. Here, ATCC represents the deposit number by the American Type Culture Collection, and NBRC represents the deposit number by the Biological Resource Center.

  The gene encoding the partial sequence of the wild-type ascorbate kinase shown in SEQ ID NO: 1 corresponds to the polynucleotide shown in SEQ ID NO: 2. This polynucleotide can be obtained, for example, from the aforementioned strain by a conventionally known method in the field of genetic engineering.

  More specifically, after preparing the genomic DNA of the aforementioned strain, the genomic DNA is cleaved with an appropriate restriction enzyme, and the plasmid or phage cleaved with the same restriction enzyme or a restriction enzyme that gives a common cleavage end. A genomic DNA library is prepared by ligation using ligase or the like. Subsequently, using a primer set designed based on the base sequence of ascorbyl kinase, PCR using these genomic DNA libraries as a template can be performed to obtain a gene encoding ascorbate kinase. it can. As a specific primer base sequence, a set of primers 1 and 2 shown in Table 2 below is exemplified. Alternatively, a gene encoding ascorbate phosphorylase can be obtained by screening these genomic DNA libraries using a probe designed based on the above base sequence.

  Moreover, a polynucleotide having a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide having the base sequence represented by SEQ ID NO: 2 can be obtained by an organic synthetic chemical technique.

  The enzyme variant of the present invention has the predetermined amino acid substitution (A115R) in the amino acid sequence represented by SEQ ID NO: 1, thereby efficiently phosphorylating ascorbic acid in the presence of a phosphate group donor, and AsA2P Can be produced. In addition, the modified enzyme of the present invention having the predetermined amino acid substitution significantly suppresses degradation of the reactive product (AsA2P), which has been a problem in the past, in the phosphorylation reaction of ascorbic acid catalyzed. AsA2P can be obtained with high yield.

  In addition to the amino acid substitution of A115R, the modified enzyme of the present invention, when allowed to act on ascorbic acid in the presence of a phosphate group donor, does not inhibit the production of ascorbic acid-2-phosphate. One or more, for example, one or several, or such as 1 to 20, more preferably 1 to 10, more preferably 1 to 5 amino acids are substituted for the amino acid sequence represented. , May contain added, inserted or deleted amino acid sequences. Amino acid deletion or addition may be performed at either the N-terminus or C-terminus, or may be performed at both ends.

  When an amino acid is added to the amino acid sequence represented by SEQ ID NO: 1, the number of added amino acids is, for example, 1 to 20, preferably 1 to 10, and more preferably 1 to 5. The added amino acid sequence may include a partial sequence of the amino acid sequence of wild-type ascorbate kinase shown in SEQ ID NO: 3. As a specific example of the amino acid sequence to be added, a case where 3 amino acid residues of DTA are added to the N-terminus can be mentioned. Moreover, the sequence derived from the vector mentioned later may be contained in the range which does not impair the activity of the enzyme modification body of this invention. Furthermore, a protein purification sequence such as a His tag may be added to the C-terminus of the amino acid sequence represented by SEQ ID NO: 1.

  Further, when the amino acid is deleted, as long as the activity of the modified enzyme of the present invention is not impaired, the amino acid sequence represented by SEQ ID NO: 1, for example, from the N-terminal or C-terminal, preferably 1-20, for example, from the N-terminal, Preferably 1-10, more preferably 1-5 amino acids may be deleted.

  Moreover, when inserting an amino acid, as long as the activity of the enzyme modification of this invention is not impaired, it is 1-20 in the arbitrary positions of the amino acid sequence shown by sequence number 1, Preferably it is 1-10, More preferably, it is 1 Although it is possible to insert ˜5 amino acid residues, it is preferably inserted at a site other than the active center as an ascorbate phosphorylase described later.

  When amino acid substitution is performed in the amino acid sequence represented by SEQ ID NO: 1, it is preferably a conservative substitution based on the properties of the side chain functional group. Natural amino acids are classified into nonpolar amino acids, uncharged amino acids, acidic amino acids and basic amino acids according to side chain functional groups. A conservative substitution refers to an amino acid residue in which the original amino acid residue and the amino acid residue after substitution are classified into the same category. Here, specific examples of “nonpolar amino acids” include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan; “uncharged amino acids” include glycine, serine, threonine, cysteine, tyrosine, asparagine, And “amine amino acid” include aspartic acid and glutamic acid; “basic amino acid” includes lysine, arginine, and histidine, respectively.

  These mutations are preferably introduced at sites other than the active center so as not to impair the phosphorylation activity of the enzyme variant of the present invention. The active center of the enzyme variant of the present invention corresponds to positions 115 to 135 in the amino acid sequence represented by SEQ ID NO: 1, preferably 117 to 132, more preferably a mutation other than positions 121 to 127. It is desirable to do.

  In the amino acid sequence represented by SEQ ID NO: 1, a mutation that can be introduced in addition to a mutation that replaces the alanine residue at position 115 with an arginine residue, specifically, the histidine residue at position 116 is replaced with a glutamine residue. Are exemplified. Furthermore, in addition to the mutation of A115R / H116Q, the asparagine residue at position 130 is substituted with a threonine residue (A115R / H116Q / N130T) or the aspartic acid residue at position 55 is substituted with a histidine residue. (A115R / H116Q / D55H) is exemplified.

  In the modified enzyme of the present invention, a modified product in which the alanine residue at position 115 is substituted with an arginine residue in the amino acid sequence represented by SEQ ID NO: 1 has significantly superior enzymatic activity. As the enzyme variant of the present invention, preferably an enzyme variant having a mutation of only A115R or A115R / H116Q, more preferably A115R.

  The method for introducing a mutation into a specific amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 is not particularly limited, and can be appropriately selected from conventionally known methods. For example, a site-specific mutagenesis method can be mentioned. Inverse PCR-based techniques or a QuikChange II Kit (Stratagene) commercial kit can be used. In addition, when a mutation is introduced at two or more sites, a polypeptide encoding the target enzyme modified product of the present invention can be obtained by repeating the method according to the above method. When obtaining the enzyme variant of the present invention having the amino acid sequence shown in FIG. 2 by the site-directed mutagenesis method, specific primer sets that can be used are exemplified by those used for variant 3 in Table 2 below. The

  Further, as the ascorbyl kinase of the present invention, the sequence identity to amino acid residues other than position 115 in the amino acid sequence represented by SEQ ID NO: 1 is 30% or more, preferably 35% or more, more preferably 40% or more, More preferably, it contains an amino acid sequence that is 50% or more and does not inhibit the production of ascorbic acid-2-phosphate when it acts on ascorbic acid in the presence of a phosphate group donor. The sequence identity of polypeptides when an amino acid is added to the N-terminus and / or C-terminus is evaluated excluding the added amino acid.

  Here, with respect to the sequence identity of polypeptides, the two polypeptides to be compared are optimally aligned, the number of positions where the amino acid matches in both sequences is divided by the total number of comparison amino acids, and this result is multiplied by 100. It is expressed as a numerical value. Specifically, the sequence identity of polypeptides can be determined by using the Maximum matching program of “GENETYX Ver.10” (Genetics) with default parameters.

  In addition, “when it is made to act on ascorbic acid in the presence of a phosphate group donor does not inhibit the production of ascorbic acid-2-phosphate” means that the produced ascorbic acid-2-phosphate is not changed over time. It means not being decomposed. More specifically, the yield of ascorbic acid-2-phosphate obtained by adding the enzyme modification of the present invention to a reaction solution composition containing ascorbic acid and a phosphoric acid donor and performing an enzyme reaction at 37 ° C. Indicates an increase of 10% or more, preferably 20% or more, more preferably 30% or more compared to wild-type ascorbate phosphorylase. The yield of ascorbic acid-2-phosphate can be confirmed by quantifying ascorbic acid-2-phosphate produced by HPLC analysis. Specific HPLC analysis conditions are described in the examples described later.

  As a suitable example of the enzyme variant of the present invention shown in (B) or (C) above, a specific amino acid sequence (SEQ ID NO: 5) and the corresponding base sequence (SEQ ID NO: 6) are shown in FIG. In the amino acid sequence represented by SEQ ID NO: 5, one or more, for example, one or several, or, for example, 1 to 20, more preferably 1 to 10, more preferably 1 to 5 amino acids. Which contain a substituted, added, inserted or deleted amino acid sequence and do not inhibit the production of ascorbic acid-2-phosphate when acted on ascorbic acid in the presence of a phosphate group donor. It is included as a preferred example of the enzyme variant of the invention. For example, those having an amino acid sequence in which glutamine at position 142 in SEQ ID NO: 5 is substituted with histidine are preferred. Furthermore, in SEQ ID NO: 5, the identity of amino acid residues other than position 141 (corresponding to position 115 of SEQ ID NO: 1) with respect to the amino acid sequence is 30% or more, preferably 35% or more, more preferably 40% or more, still more preferably Includes an amino acid sequence that is 50% or more and that does not inhibit the production of ascorbic acid-2-phosphate when it acts on ascorbic acid in the presence of a phosphate group donor. Included as a preferred example. The amino acid residue substitution, deletion, addition or insertion, and amino acid sequence identity are as described above.

  The enzyme modified product of the present invention can be obtained by introducing a vector having DNA encoding these amino acid sequences into a host cell to produce a transformant, and culturing the transformant. Specifically, it will be described in detail in the column “Transformant” below.

DNA
The present invention provides an isolated DNA encoding the phosphorylase variant. The DNA encoding the enzyme variant of the present invention may be any DNA as long as it can express the enzyme variant of the present invention in a host cell introduced according to the method described below, and includes any untranslated region. You may go out.

  In addition, as a DNA encoding the enzyme variant of the present invention, a DNA comprising a base sequence complementary to a DNA consisting of a base sequence corresponding to the amino acid sequence represented by SEQ ID NO: 1 (base sequence represented by SEQ ID NO: 2); Ascorbate phosphorylase that is DNA that hybridizes under stringent conditions and does not inhibit the production of ascorbyl 2-phosphate when it acts on ascorbic acid in the presence of a phosphate group donor DNA encoding the modified form of

  Here, “DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 2” refers to the base shown in SEQ ID NO: 2 in the Sequence Listing. It means DNA obtained by using a colony hybridization method, plaque hybridization method, Southern hybridization method or the like under stringent conditions using a DNA consisting of a base sequence complementary to the sequence as a probe.

  Here, the DNA that hybridizes under stringent conditions was, for example, hybridized at 55 ° C. in the presence of 0 to 0.3 M NaCl using a filter on which colony or plaque-derived DNA was immobilized. Then, using a 2 × concentration SSC solution (standard saline-citrate buffer: the composition of the 1 × concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) at 20 ° C. Mention may be made of DNA that can be obtained by washing the filter underneath. The DNA can be obtained by washing with an SSC solution having a concentration of 1 × at 20 ° C., more preferably with an SSC solution having a concentration of 2 ×.

  The DNA hybridizable under the above conditions is preferably a DNA having a sequence homology of 60% or more, more preferably 70% or more, more preferably 80% or more with the DNA shown in SEQ ID NO: 2 in the Sequence Listing. As long as the encoded polypeptide has the above activity, it is included in the DNA encoding the enzyme variant of the present invention.

  The sequence homology of DNA can also be determined using a known computer program such as GAP, BLASTN, FASTA, BLASTA, BLASTX. The DNA can be obtained by the aforementioned genetic engineering technique, chemical synthesis, or the like.

Vector The present invention further provides a vector containing the DNA, which is used for introducing and expressing the DNA in a host cell. Such a vector may be any vector as long as it can express the gene encoded by the DNA in an appropriate host cell. Examples of such vectors include plasmid vectors, phage vectors, cosmid vectors, and the like. A shuttle vector capable of gene exchange with other host strains can also be used.

  In the present invention, the vector is a promoter (T7 promoter, lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter) operably linked to the DNA encoding the enzyme variant of the present invention. A functional promoter such as pL promoter; transcription element (eg, enhancer, CCAAT box, TATA box, SPI site, etc.); signal sequence (specifically, nuclear translocation signal, endoplasmic reticulum translocation signal, PTS1 (Peroxisomal targeting signal 1) PTS2 etc.) may be included, and the vector can be appropriately selected from those generally used in the field, The body specifically, pET22b (+) vector, pET39b (+) vector, and the like.

  Here, the term “operably linked” means that various regulatory elements such as promoters and enhancers that regulate gene expression and the gene are linked in a state in which the gene can operate in the host cell. It is well known to those skilled in the art that the type and kind of the control factor can vary depending on the host.

Transformant The present invention provides a transformant obtained by introducing a vector containing the DNA into a host cell. The host cell is not particularly limited as long as it is a cell that can be transformed with an expression vector containing a DNA encoding the enzyme variant of the present invention and can express the enzyme variant encoded by the DNA. .

  Examples of host cells include Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, and Corynebacterium sp. ) Genus, bacteria such as Lactobacillus genus; Actinomycetes such as Rhodococcus genus, Streptomyces genus; Schizosaccharomyces), Chi Saccharomyces genus, Yarrowia genus, Trichosporon genus, Rhodosporidium genus, Pichia genus (Candida) genus, Neurosporus genus Examples include microorganisms such as molds such as the genus Aspergillus, the genus Cephalosporia, the genus Trichoderma. Among these, bacteria are preferable from the viewpoint of introduction and expression efficiency, and Escherichia coli cells, which are bacteria belonging to the genus Escherichia, are particularly preferable.

  Transformation methods are known and can be appropriately selected depending on the type of host cell and the like, and specifically, competent cell method, electroporation method, heat shock method and the like are exemplified.

  The transformant can be cultured based on conventionally known culture conditions depending on the type of cell used as the host cell. For example, culture conditions when E. coli cells are used as host cells include a culture temperature of 20 to 40 ° C., a culture period of 6 to 24 hours, and a culture solution pH of 5 under aerobic conditions such as shaking culture or full-time culture. ~ 8 can be mentioned. Further, the culture medium to be used is not particularly limited as long as the host cell can grow and can produce the modified enzyme of the present invention. In addition, a medium containing a necessary amount of vitamins or the like can be used.

  For example, when a transformant obtained by using a prokaryote such as E. coli or yeast as a host is cultured, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the organism, Either a natural medium or a synthetic medium may be used as long as the medium can be cultured efficiently.

  The carbon source may be anything that can be assimilated by the organism, such as glucose, fructose, sucrose, starch or starch hydrolysate, etc .; organic acids such as acetic acid and propionic acid; alcohols such as ethanol and propanol, etc. Is mentioned. The concentration of the carbon source contained in the medium is usually 0.1 to 10% by weight.

  The nitrogen source includes inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; ammonium salts of organic acids; other nitrogen-containing compounds; peptone, meat extract, yeast extract, corn steep liquor, casein Examples include hydrolysates, soybean meal, and soybean meal hydrolysates. As a density | concentration of the nitrogen source contained in a culture medium, 0.1 to 10 weight% is mentioned normally.

  Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. As a density | concentration of the inorganic salt contained in a culture medium, 0.01 to 1 weight% is mentioned normally.

  The pH can be adjusted using a known inorganic or organic acid, alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.

  After completion of the culture, a modified ascorbic acid kinase can be collected from the culture, and can be performed according to a normal enzyme collecting method. Examples of the enzyme collection method include an enzyme purification method such as ammonium sulfate fractionation, ion exchange, chromatofocusing, gel filtration, etc., by sonicating cultured bacterial cells and preparing a bacterial cell extract.

2. Method for Producing Ascorbic Acid-2-Phosphate In the present invention, ascorbic acid-2-phosphate is produced by allowing the aforementioned enzyme modified form of the present invention to act on ascorbic acid in the presence of a phosphate group donor. Provide a way to do it. In the method for producing AsA2P of the present invention, an ascorbic acid, a phosphate group donor, and an enzyme modification can be added to a solvent to prepare a reaction solution composition, and an enzyme reaction can be performed. Alternatively, ascorbic acid-2-phosphate may be produced by allowing a culture of a microorganism capable of producing the enzyme variant of the present invention to act on ascorbic acid in the presence of a phosphate group donor.

  In the method of the present invention, the culture of the microorganism having the ability to produce the enzyme variant of the present invention means a culture solution containing microbial cells, cultured microbial cells, or a processed product thereof. Here, the treated product means, for example, a cell-free extract, freeze-dried cells, acetone-dried cells, or a ground product of these cells. Furthermore, these cultures can also be used as immobilized enzymes or immobilized cells. The immobilization can be performed by methods well known to those skilled in the art (for example, physical adsorption method, inclusion method, etc.).

  Examples of the microorganism having the ability to produce the enzyme variant of the present invention include a transformant into which a vector containing a DNA encoding the enzyme variant of the present invention has been introduced.

The phosphate group donor is not particularly limited as long as it functions as a phosphate group supply source for the modified enzyme of the present invention and AsA2P is produced. For example, the phosphate group donor is represented by the general formula (1): H _{n + 2} P _{n Examples} thereof include polyphosphoric acid represented by O _{3n + 1} (wherein n ≧ 2). The degree of polymerization of polyphosphoric acid is not particularly limited, but preferably 2 to 10,000, more preferably 2 to 1,000, of the constituent units are polymerized. Specific examples of polyphosphoric acid include diphosphoric acid (n = 2: pyrophosphoric acid), triphosphoric acid (n = 3), and tetraphosphoric acid (n = 4). Moreover, what polymerized phosphoric acid of n = 5 or more in General formula (1) can be used suitably as a phosphate group donor. A mixture of two or more polyphosphoric acids having different degrees of polymerization may be used as a phosphate group donor.

Furthermore, as the phosphate group donor used in the present invention, the polyphosphoric acid esters, alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium, and polyphosphates such as ammonium salts are used. You can also. Specific examples of esters of polyphosphoric acid include adenosine diphosphate (ADP) and adenosine triphosphate (ATP). Specific examples of the polyphosphate include sodium pyrophosphate (Na ₄ P ₂ O ₇ ), sodium tripolyphosphate (Na ₅ P ₃ O ₁₀ ), sodium tetrapolyphosphate (Na ₆ P ₄ O ₁₃ ), and pyrophosphate. Examples include ammonium salts and ammonium tripolyphosphate. Examples of the phosphoric acid group donor include paranitrophenyl phosphate and acetyl phosphoric acid in addition to the polyphosphoric acid.

  In the present invention, the phosphoric acid group donor is preferably a polyphosphoric acid such as diphosphoric acid, triphosphoric acid or tetraphosphoric acid, an ester of polyphosphoric acid such as adenosine diphosphate (ADP) or adenosine triphosphate (ATP); sodium pyrophosphate , Alkali metal salts of polyphosphoric acid such as sodium tripolyphosphate and sodium tetrapolyphosphate; paranitrophenyl phosphate and acetyl phosphoric acid are mentioned, more preferably polyphosphoric acid, ester of polyphosphoric acid and polyphosphate are more preferred. Includes polyphosphoric acid and esters of polyphosphoric acid, particularly preferably polyphosphoric acid. These phosphoric acid group donors may be used alone or in combination of two or more.

  The concentration of the enzyme variant of the present invention used in the reaction is 0.0001 to 1% by weight, preferably 0.001 to 0.1% by weight, more preferably 0.001 to 0% in the reaction solution composition. 0.01% by weight. Moreover, as a density | concentration of a phosphate group donor, 0.5-20 weight%, Preferably it is 1-15 weight%, More preferably, 2-10 weight% is mentioned. Moreover, as a density | concentration of ascorbic acid, 0.1 to 50 weight%, Preferably it is 0.5 to 20 weight%, More preferably, 1 to 10 weight% is mentioned.

  The addition ratio of the phosphate group donor per 1 part by weight of the enzyme variant (polypeptide) of the present invention is 100 to 30000 parts by weight, preferably 250 to 20000 parts by weight, more preferably 500 to 15000 parts by weight. It is done. Moreover, as an addition ratio of ascorbic acid per 1 part by weight of the enzyme variant (polypeptide) of the present invention, 100 to 40000 parts by weight, preferably 500 to 30000 parts by weight, and more preferably 1000 to 25000 parts by weight. .

  Moreover, as reaction temperature, 10-60 degreeC, Preferably it is 20-50 degreeC, More preferably, 30-40 degreeC is mentioned.

  The optimum pH of the enzyme variant of the present invention includes pH 2 to 7, preferably pH 3 to 6, and more preferably pH 4 to 5. pH adjustment is hydrochloric acid, citric acid, gluconic acid, succinic acid, acetic acid, tartaric acid, sorbic acid, lactic acid, maleic acid, sulfuric acid, phosphoric acid, malic acid, arginine, aqueous ammonia, diisopropanolamine, diethanolamine, triisopropanolamine Buffering agents such as triethanolamine, monoethanolamine, potassium hydroxide, calcium hydroxide, sodium hydroxide, and salts thereof can be carried out using conventionally known buffering agents.

  In the method for producing ascorbic acid-2-phosphate of the present invention, the reaction time is not particularly limited as long as ascorbic acid-2-phosphate can be produced. For example, it is 1 to 50 hours, preferably 5 to 40 hours. More preferably, it is 10 to 30 hours.

  As the reaction solvent, an aqueous solvent such as ion-exchanged water, distilled water, or ultrapure water can be usually used. Further, in the aqueous solvent, organic solvents such as methanol, trimethylamine, triethylamine, dimethyl sulfoxide (DMSO), etc. within the range not impairing the effects of the present invention; polyoxyethylene lauryl ether, polyoxyethylene dodecyl ether, polyethylene glycol p-octyl Nonionic surfactants such as phenyl ether (trade name Triton X-100) and polyoxyethylene sorbitan monolaurate (trade name Tween 20) may be included. From the viewpoint of improving the reactivity and stabilizing the enzyme, the solvent preferably contains a nonionic surfactant. The amount of the organic solvent added is usually 0.1 to 50% by weight, preferably 1 to 30% by weight, and more preferably 5 to 20% by weight. The amount of the nonionic surfactant added is usually 0.0001 to 0.1% by weight, preferably 0.0005 to 0.05% by weight, more preferably 0.001 to 0.01% by weight. Is done.

  AsA2P is obtained by the above reaction. The obtained AsA2P can be prepared, for example, by column chromatography such as ion exchange chromatography, affinity chromatography, adsorption column chromatography, gel filtration chromatography, reverse phase chromatography, etc .; polyethylene glycol (PEG) and Examples thereof include a method using a precipitant such as methylpentanediol (MPD); a method using an alkaline earth metal salt such as magnesium oxide or magnesium hydroxide, and the like.

Although a test example etc. are shown below and this invention is demonstrated more concretely, this invention is not limited to these.
Preparation of Ascorbate Phosphate Enzyme Variant Introduction of mutations into ascorbate phosphorylase using the primer set shown in Table 1 below, the region encoding the N-terminal side of ascorbate kinase and the C-terminus Two portions of the coding region were amplified respectively. At this time, mutations are introduced into the used primers so that amino acid substitution occurs at the target site, so that each mutation is introduced at the target site in the obtained PCR product. The obtained PCR product was inserted into a vector to prepare a transformant, and each ascorbate phosphorylase modified product was prepared from the transformant.

(Introduction of mutation and production of transformant)
The introduction of mutations and the production of transformants are described below by taking as an example the method for producing the A115R / H116Q enzyme variant (variant 3).
Genomic DNA was prepared from Sphingomonas trueperi (JCM10278), and the ascorbyl phosphate gene described in JP-A-4-53494 was amplified by PCR. The primer sets used for PCR are primer 1 (forward primer) and primer 6 (reverse primer), and primer 3 (forward primer) and primer 2 (reverse primer). The obtained PCR products were digested with restriction enzymes (NcoI and NheI, or NheI and XhoI), and these were ligated by a three-party ligation with a pET22b (+) vector (Novagen) treated with NcoI and XhoI. Ascorbate kinase expression vector was obtained. In addition, the amino acid sequence represented by SEQ ID NO: 1 is inserted into the vector, and further, a T7 promoter, a pelB signal for transferring the produced protein to the periplasm, a His tag gene for protein purification, and a drug An ampicillin resistance gene for selection is retained. The amino acid sequence corresponding to the base sequence inserted into the vector is shown in FIG.

  The vector obtained by the above method was transformed into E. coli BL21 (DE3) strain (Novagen) by the heat shock method. By using the primer 1, a vector is constructed in such a manner that a sequence in which 3 amino acid residues of D-TA are added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is incorporated into the pelB signal sequence of Escherichia coli. Is done. However, when the enzyme-modified product becomes a mature product, the signal sequence is cleaved and the N-terminus is assumed to be Asp. Here, the amino acid sequence DTA corresponds to the amino acid sequence at positions 21 to 23 in the wild-type ascorbyl phosphate shown by SEQ ID NO: 3. FIG. 3 shows a schematic diagram of a method for producing the modified body 3.

  Other modified products were also prepared by the same method using the primer sets shown in Tables 1 and 2 below.

  Table 2 below shows the sequence number, base sequence and restriction enzyme site name corresponding to each primer number.

  In Table 2, the underlined base sequence represents the restriction enzyme cleavage site, and the base sequence surrounded by a square represents the site encoding the amino acid to be substituted.

(Culture of transformants)
The transformant of Escherichia coli BL21 (DE3) strain having the vector for ascorbyl kinase expression was cultured in LB medium containing 0.2 wt% glucose, 0.5 wt% casamino acid and 0.1 mg / ml ampicillin ( Nacalai Tesque Co., Ltd .: (Composition) 1 wt% tryptone; 0.5 wt% dry yeast extract; 0.5 wt% sodium chloride) was incubated at a culture temperature of 27 ° C. When the optical density at 600 nm of the culture solution reached 0.5, isopropyl-β-thiogalactopyranoside was added so as to be 0.1 mM, and further cultured at a culture temperature of 27 ° C. for 16 to 24 hours. .

  After culturing, 15 g of wet cell weight was suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM sodium chloride, and a cell extract was obtained using an ultrasonic homogenizer (TOMY UD201). The extract was centrifuged at 20,000 × g for 20 minutes, and the supernatant fraction was collected. Ammonium sulfate was added to reach 60% saturation, and centrifugation was performed at 20,000 × g for 20 minutes to collect the precipitate. The precipitate after the ammonium sulfate fraction was suspended in 20 mM Tris-HCl buffer (pH 7.5), and dialyzed against 20 mM Tris-HCl buffer (pH 7.5) containing 50 mM sodium chloride. After dialysis, affinity purification using a His-Trap HP column was performed. Fractions containing ascorbate phosphorylase were collected and dialyzed against 20 mM Tris-HCl buffer (pH 7.5) to obtain ascorbate phosphorylase.

Purification conditions The conditions for affinity purification are as follows.
Column: His-Trap HP (manufactured by GE healthcare)
Equilibration buffer: 20 mM Tris-HCl (pH 7.5), 50 mM sodium chloride elution buffer: 20 mM Tris-HCl (pH 7.5), 50 mM sodium chloride, 1 M imidazole (elution buffer in a 0-50% gradient) Elution)
Flow rate: 5.0 ml / min
Purification equipment: AKTA prime plus (GE healthcare)

HPLC analysis conditions The HPLC analysis conditions employed in the test examples and comparative examples described below are as follows.
Column: YMC-Pack ODS-A (150 × 4.6 mm) (manufactured by YMC)
Eluent:
Acetate buffer solution (80 mM, pH 5.0) / methanol = 98/2 (volume ratio)
(Contains 2.8 mM hexylamine and 0.1 mM EDTA)
Detection wavelength: 254 nm
Flow rate: 0.8ml / min
Column temperature: 35 ° C
Injection volume: 10 μl
HPLC instrument: Hitachi HPLC LaChrom Elite seri L-2000

[Test Example 1-1]
Ultrapure water (NaOH) containing ascorbic acid (300 mM), polyphosphoric acid (2 wt%) as a phosphate group donor (trade name: polyphosphoric acid, manufactured by Nacalai Tesque), and Triton X-100 (1 wt%) adjusted to pH 4.5), ascorbate phosphorylase modified with amino acid mutations shown as modified 1 to 8 in Table 1 above was added to 15 μg / ml each, and constant temperature air at 37 ° C. The reaction was allowed to proceed overnight in the incubator. Thereafter, the reaction solution was diluted 100 times with an eluent (acetate buffer solution) used for HPLC, and the amount of AsA2P produced by HPLC analysis was measured. The results are shown in FIG.

  As shown in FIG. 4, among the variants used in the evaluation, degradation over time of AsA2P produced only when A115R / H116Q (variant 3) and A115R / H116 (variant 8) was used. It was shown to be suppressed.

[Test Example 1-2]
The results of measurement of the amount of AsA2P produced for the modified forms of ascorbate phosphorylase introduced with the amino acid mutations shown as modified body 7 and modified body 8 were extracted and compared in FIG. As shown in FIG. 5, when the variant 8 (A115R / H116) was used, the degradation over time of AsA2P was suppressed as in the variant 3 (A115R / H116Q) shown in the test example 1. It was shown that On the other hand, when modified 7 (A115 / H116Q) was used, AsA2P was decomposed, and the amount of AsA2P decreased with time. In other words, the substitution of the amino acid residue at position 115 from alanine to arginine suppresses the degradation of the reaction product, and the substitution of histidine at position 116 with glutamine does not affect the degradation of the reaction product. It was done.

[Test Example 2]
A triple mutant in which a single amino acid mutation was further introduced into variant 3 was prepared, and the production of AsA2P was evaluated. Ultrapure water (NaOH) containing ascorbic acid (100 mM), polyphosphoric acid (2 wt%) as a phosphate group donor (trade name: polyphosphoric acid, manufactured by Nacalai Tesque), and Triton X-100 (1 wt%) adjusted to pH 4.5) in addition to A115R, a modified variant of ascorbyl phosphatase introduced with amino acid mutations shown as modified 9-23 in Table 1 above was added to a concentration of 15 μg / ml. The reaction was carried out overnight in a constant temperature air incubator at 0 ° C. Thereafter, the reaction solution was diluted 100 times with an eluent (acetate buffer solution) used for HPLC, and the amount of AsA2P produced by HPLC analysis was measured. The modification 3 (A115R / H116Q) was used as a control, and the reaction and measurement were performed under the same conditions. The results are shown in FIG.

  As shown in FIG. 6, among the variants used in the evaluation, in FIG. 6, N130T (variant 9) and D55H (variant 14) have lower enzyme reactivity than variant 3, but other The production amount of AsA2P was clearly higher than that of Triple Mutant.

[Comparative Example 1]
The production of AsA2P was evaluated when wild type ascorbate phosphorylase was used. Ultrapure water (pH 4 with NaOH) containing ascorbic acid (50 mM), polyphosphoric acid (2 wt%) as a phosphate donor (trade name: polyphosphoric acid, manufactured by Nacalai Tesque), and Triton X-100 (1 wt%) To 5), wild-type ascorbyl phosphatase was added at 4.5 μg / ml, 9 μg / ml, 18 μg / ml or 90 μg / ml and reacted in a constant temperature air incubator at 37 ° C. At 0, 1, 2, 4, 6, 8, and 16 hours after the reaction, the reaction solution was diluted 100-fold with an eluent used for HPLC (acetate buffer solution) and produced by HPLC analysis. The amount of AsA2P was measured. The results are shown in FIG.

  As shown in FIG. 7, it was shown that the amount of AsA2P produced decreased with time when wild-type ascorbyl phosphate was added at any concentration.

SEQ ID NO: 1 is an amino acid sequence corresponding to a proline residue at position 24 to a valine residue at position 249 in wild-type ascorbate phosphorylase.
SEQ ID NO: 2 is a base sequence encoding an amino acid sequence corresponding to a proline residue at position 24 to a valine residue at position 249 in wild-type ascorbate phosphorylase.
SEQ ID NO: 5 is the amino acid sequence of a modified ascorbate kinase (modified 3).
SEQ ID NO: 6 is a base sequence encoding an ascorbate phosphorylase modified product (modified product 3).
SEQ ID NO: 7 is the base sequence of primer 1.
SEQ ID NO: 8 is the base sequence of primer 2.
SEQ ID NO: 9 is the base sequence of primer 3.
SEQ ID NO: 10 is the base sequence of primer 4.
SEQ ID NO: 11 is the base sequence of primer 5.
SEQ ID NO: 12 is the base sequence of primer 6.
SEQ ID NO: 13 is the base sequence of primer 7.
SEQ ID NO: 14 is the base sequence of primer 8.
SEQ ID NO: 15 is the base sequence of primer 9.
SEQ ID NO: 16 is the base sequence of primer 10.
SEQ ID NO: 17 is the base sequence of primer 11.
SEQ ID NO: 18 is the base sequence of primer 12.
SEQ ID NO: 19 is the base sequence of primer 13.
SEQ ID NO: 20 is the base sequence of primer 14.
SEQ ID NO: 21 is the base sequence of primer 15.
SEQ ID NO: 22 is the base sequence of primer 16.
SEQ ID NO: 23 is the base sequence of primer 17.
SEQ ID NO: 24 is the base sequence of primer 18.
SEQ ID NO: 25 is the base sequence of primer 19.
SEQ ID NO: 26 is the base sequence of primer 20.
SEQ ID NO: 27 is the base sequence of primer 21.
SEQ ID NO: 28 is the base sequence of primer 22.
SEQ ID NO: 29 is the base sequence of primer 23.
SEQ ID NO: 30 is the base sequence of primer 24.
SEQ ID NO: 31 is the base sequence of primer 25.
SEQ ID NO: 32 is the base sequence of primer 26.
SEQ ID NO: 33 is the base sequence of primer 27.
SEQ ID NO: 34 is the base sequence of primer 28.
SEQ ID NO: 35 is the base sequence of primer 29.
SEQ ID NO: 36 is the base sequence of primer 30.
SEQ ID NO: 37 is the base sequence of primer 31.

Claims (7)

Ascorbate phosphorylation enzyme variant shown in any of the following (A) to (C):
(A) a modified ascorbate kinase comprising an amino acid sequence in which the alanine residue at position 115 is substituted with an arginine residue in the amino acid sequence represented by SEQ ID NO: 1;
(B) In the amino acid sequence of (A), including an amino acid sequence in which one or more amino acid residues other than position 115 are substituted, added, inserted or deleted, and in the presence of a phosphate group donor A modified ascorbic acid kinase that does not inhibit the production of ascorbic acid-2-phosphate when acting on ascorbic acid in
(C) In the amino acid sequence of (A), the amino acid sequence of the amino acid residue other than position 115 includes an amino acid sequence having an amino acid sequence of 30% or more, and ascorbic acid in the presence of a phosphate group donor. A modified ascorbic acid kinase that does not inhibit the production of ascorbyl 2-phosphate when allowed to act.   Furthermore, in the amino acid sequence represented by SEQ ID NO: 1, the enzyme variant according to claim 1, which has an amino acid sequence in which a histidine residue at position 116 is substituted with a glutamine residue.   An isolated DNA encoding the enzyme variant according to claim 1 or 2.   A vector comprising the DNA according to claim 3.   A transformant obtained by transforming a host organism with the vector according to claim 4. A method for producing ascorbic acid-2-phosphate by causing a modified ascorbic acid kinase in any of the following (A) to (C) to act on ascorbic acid in the presence of a phosphate group donor:
(A) a modified ascorbate kinase comprising an amino acid sequence in which the alanine residue at position 115 is substituted with an arginine residue in the amino acid sequence represented by SEQ ID NO: 1;
(B) In the amino acid sequence of (A), including an amino acid sequence in which one or more amino acid residues other than position 115 are substituted, added, inserted or deleted, and in the presence of a phosphate group donor A modified ascorbic acid kinase that does not inhibit the production of ascorbic acid-2-phosphate when acting on ascorbic acid in
(C) In the amino acid sequence of (A), the amino acid sequence of the amino acid residue other than position 115 includes an amino acid sequence having an amino acid sequence of 30% or more, and ascorbic acid in the presence of a phosphate group donor. A modified ascorbic acid kinase that does not inhibit the production of ascorbyl 2-phosphate when allowed to act.   The method according to claim 6, wherein the phosphate group donor is polyphosphoric acid.