JP2008178340A - METHOD FOR PRODUCING alpha-L-ASPARTYL-L-PHENYLALANINE, alpha-AMP ESTERASE AND METHOD FOR PRODUCING alpha-L-ASPARTYL-L-PHENYLALANINE-alpha-METHYL ESTER - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method by which α-L-aspartyl-L-phenylalanine that is an intermediate for α-L-aspartyl-L-phenylalanine-α-methyl ester can simply and inexpensively be produced in high yield, and to provide a method by which the α-L-aspartyl-L-phenylalanine-α-methyl ester is simply and inexpensively produced in high yield. <P>SOLUTION: An enzyme possessed by the genus Tetrathiobacter or the genus Brevundimonas and having the ability to de-esterify an α-L-aspartyl-L-phenylalanine-β-ester or a substance containing the enzyme is used to produce the α-L-aspartyl-L-phenylalanine from the α-L-aspartyl-L-phenylalanine-β-ester. Furthermore, the α-L-aspartyl-L-phenylalanine-α-methyl ester is produced from the α-L-aspartyl-L-phenylalanine. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing α-L-aspartyl-L-phenylalanine (α-L-aspartyl-L-phenylalanine (abbreviation: α-AP)) and α-L-aspartyl-L-phenylalanine-α-methyl ester ( α-L-asppartyl-L-phenylalanine methyl ester (abbreviation: α-APM)), more specifically, α-L-aspartyl-L-phenylalanine-α-methyl ester (α-L-aspartyl-L-phenylalanine-α-methyl ester) Product name: Aspartame) Production method of α-L-aspartyl-L-phenylalanine which is an important intermediate for production, α-L-aspartyl-L-phenylalanine and α-L-aspartyl-L-phenylalanine Α-L-Ass using manufacturing method The method for manufacturing a rutile -L- phenylalanine -α- methyl ester.

  Conventionally known methods for producing α-L-aspartyl-L-phenylalanine-α-methyl ester (sometimes abbreviated as “α-APM”) include chemical synthesis methods and enzyme synthesis methods. As a chemical synthesis method, N-protected L-aspartic anhydride and L-phenylalanine methyl ester are condensed to synthesize N-protected APM, and this N-protecting group is eliminated to obtain APM. , N-protected L-aspartic acid and L-phenylalanine methyl ester are condensed to synthesize N-protected APM, and this N-protecting group is eliminated to obtain APM. Introducing and removing the protecting group was necessary, and the process was extremely complicated. On the other hand, an APM production method that does not use an N-protecting group has been studied (Patent Document 1), but the yield is extremely low and it is not suitable for industrial production. Under such circumstances, development of a further inexpensive industrial production method for aspartame has been desired.

Japanese Patent Publication No. 02-015196

  The present invention relates to α-L-aspartyl-L-phenylalanine, which is an intermediate of α-L-aspartyl-L-phenylalanine-α-methyl ester, easily and at a low yield without complicated synthesis methods. It is an object of the present invention to provide a method capable of manufacturing the above. Another object of the present invention is to provide a method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester, which is simple, high yield and inexpensive.

  As a result of intensive studies in view of the above-mentioned object, the present inventors have newly found an enzyme or an enzyme-containing substance from α-L-aspartyl-L-phenylalanine-β-ester to α-L-aspartyl-L. -It discovered that it had the capability to produce | generate a phenylalanine selectively, and came to complete this invention.

That is, the present invention is as follows.
[1] α-L-aspartyl-L-phenylalanine-β-ester is converted from α-L-aspartyl-L-phenylalanine-β-ester to α-L using an enzyme or enzyme-containing product having the ability to deesterify α-L-aspartyl-L-phenylalanine-β-ester. A method for producing α-L-aspartyl-L-phenylalanine, characterized in that it produces aspartyl-L-phenylalanine.
[2] A culture of a microorganism in which the enzyme or enzyme-containing product has the ability to deesterify α-L-aspartyl-L-phenylalanine-β-ester, a microbial cell separated from the culture, and the The method for producing α-L-aspartyl-L-phenylalanine according to [1], wherein the method is one or more selected from the group consisting of processed microbial cells.
[3] The α-L-aspartyl-L-phenylalanine according to [2], wherein the microorganism belongs to a genus selected from the group consisting of Tetrathiobacter and Brevandimonas. Production method.
[4] The α-L- described in [2], wherein the microorganism is a transformed microorganism capable of expressing a protein selected from the group consisting of the following (A) to (J): A method for producing aspartyl-L-phenylalanine.
(A) Protein having the amino acid sequence of amino acid residues 14 to 340 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing (B) Amino acid residue number 14 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing ˜340 amino acid sequence, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and de-esterifying α-L-aspartyl-L-phenylalanine-β-ester Among the amino acid sequences described in SEQ ID NO: 2 in the protein (D) sequence listing, the protein having the amino acid sequence of amino acid residues 25 to 340 among the amino acid sequences listed in SEQ ID NO: 2 in the protein (C) sequence listing Including the substitution, deletion, and / or insertion of one or several amino acids in the amino acid sequence of amino acid residues 25 to 340 A protein having an amino acid sequence and an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester (E). Substitution or deletion of one or several amino acids in the amino acid sequence of amino acid residues Nos. 26 to 550 in the amino acid sequence of SEQ ID No. 17 of the protein (F) sequence listing having the amino acid sequence of Nos. 26 to 550, And / or protein (G) having an amino acid sequence containing an insertion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The amino acid sequence shown in SEQ ID NO: 17 in the protein (H) sequence list having the amino acid sequence shown in SEQ ID NO: 17 A maturation having an amino acid sequence comprising one or several amino acid substitutions, deletions and / or insertions and having an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester Substitution or deletion of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the protein (J) sequence list having the amino acid sequence shown in SEQ ID NO: 2 in the protein (I) sequence list including the protein region, And / or a protein comprising a mature protein region having an amino acid sequence containing an insertion and having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester [5] a transformed microorganism into which a polynucleotide selected from the group consisting of a) to (j) is introduced; Method for producing alpha-L-aspartyl -L- phenylalanine described].
(A) a polynucleotide having a base sequence of base numbers 100 to 1080 among the base sequences set forth in SEQ ID NO: 1 of the sequence listing (b) of base sequences 100 to 1080 of the base sequences set forth in SEQ ID NO: 1 of the sequence listing Poly that encodes a protein that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence under stringent conditions and has an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester Nucleotide (c) Polynucleotide having base sequence of base numbers 133 to 1080 among the base sequence set forth in SEQ ID NO: 1 of the sequence listing (d) Base numbers 133 to 1080 of the base sequence set forth in SEQ ID NO: 1 of the sequence listing Hybridizes under stringent conditions with polynucleotides that are complementary to the base sequence of And a polynucleotide encoding a protein having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (e) Among the base sequences set forth in SEQ ID NO: 16 of the sequence listing, base numbers 114 to A polynucleotide having a base sequence of 1688 (f) Hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 114 to 1688 among the base sequence set forth in SEQ ID NO: 16 of the sequence listing Polynucleotide encoding a protein that is soybean and has an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (g) Base number 39 of the base sequence set forth in SEQ ID NO: 16 in the Sequence Listing A polynucleotide having a base sequence of ˜1688 (h) in SEQ ID NO: 16 of the sequence listing Hybridize under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 39 to 1688 among the listed base sequences, and α-L-aspartyl-L-phenylalanine-β-ester Polynucleotide encoding a protein having deesterification activity (i) Polynucleotide having base sequence of base numbers 61 to 1080 among base sequences set forth in SEQ ID NO: 1 of sequence listing (j) SEQ ID NO: 1 of sequence listing Among the base sequences described in the above, and hybridizing under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 61 to 1080, and α-L-aspartyl-L-phenylalanine-β-ester Encodes a protein containing a mature protein region having the activity of deesterifying Polynucleotides [6] wherein the enzyme is at least one selected from the group consisting of the following (A) ~ (J), the manufacturing method of the alpha-L-aspartyl -L- phenylalanine of [1].
(A) Protein having the amino acid sequence of amino acid residues 14 to 340 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing (B) Amino acid residue number 14 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing ˜340 amino acid sequence, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and de-esterifying α-L-aspartyl-L-phenylalanine-β-ester Among the amino acid sequences described in SEQ ID NO: 2 in the protein (D) sequence listing, the protein having the amino acid sequence of amino acid residues 25 to 340 among the amino acid sequences listed in SEQ ID NO: 2 in the protein (C) sequence listing Including the substitution, deletion, and / or insertion of one or several amino acids in the amino acid sequence of amino acid residues 25 to 340 A protein having an amino acid sequence and an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester (E). Substitution or deletion of one or several amino acids in the amino acid sequence of amino acid residues Nos. 26 to 550 in the amino acid sequence of SEQ ID No. 17 of the protein (F) sequence listing having the amino acid sequence of Nos. 26 to 550, And / or protein (G) having an amino acid sequence containing an insertion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The amino acid sequence shown in SEQ ID NO: 17 in the protein (H) sequence list having the amino acid sequence shown in SEQ ID NO: 17 A protein having an amino acid sequence including substitution, deletion and / or insertion of one or several amino acids, and having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (I) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (J) substitution, deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing Any one of [7] [1] to [6], which comprises a mature protein region having an amino acid sequence comprising and having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester The method for synthesizing α-L-aspartyl-L-phenylalanine by the method for producing α-L-aspartyl-L-phenylalanine according to the item. And α-L-aspartyl-L-phenylalanine-α-methyl, comprising a reaction step and a reaction step of converting α-L-aspartyl-L-phenylalanine to α-L-aspartyl-L-phenylalanine-α-methyl ester. Ester production method.
[8] A protein having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester, which is derived from a microorganism belonging to a genus selected from the group consisting of Tetrathiobacter and Brebandimonas.
[9] A protein selected from the group consisting of the following (A) to (J).
(A) Protein having the amino acid sequence of amino acid residues 14 to 340 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing (B) Amino acid residue number 14 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing ˜340 amino acid sequence, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and de-esterifying α-L-aspartyl-L-phenylalanine-β-ester Among the amino acid sequences described in SEQ ID NO: 2 in the protein (D) sequence listing, the protein having the amino acid sequence of amino acid residues 25 to 340 among the amino acid sequences listed in SEQ ID NO: 2 in the protein (C) sequence listing Including the substitution, deletion, and / or insertion of one or several amino acids in the amino acid sequence of amino acid residues 25 to 340 A protein having an amino acid sequence and an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester (E). Substitution or deletion of one or several amino acids in the amino acid sequence of amino acid residues Nos. 26 to 550 in the amino acid sequence of SEQ ID No. 17 of the protein (F) sequence listing having the amino acid sequence of Nos. 26 to 550, And / or protein (G) having an amino acid sequence containing an insertion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The amino acid sequence shown in SEQ ID NO: 17 in the protein (H) sequence list having the amino acid sequence shown in SEQ ID NO: 17 A protein having an amino acid sequence including substitution, deletion and / or insertion of one or several amino acids, and having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (I) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (J) substitution, deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing A protein encoding the protein according to [9], comprising a mature protein region having an amino acid sequence comprising and having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester nucleotide.
[11] A polynucleotide selected from the group consisting of the following (a) to (j).
(A) a polynucleotide having a base sequence of base numbers 100 to 1080 among the base sequences set forth in SEQ ID NO: 1 of the sequence listing (b) of base sequences 100 to 1080 of the base sequences set forth in SEQ ID NO: 1 of the sequence listing Poly that encodes a protein that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence under stringent conditions and has an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester Nucleotide (c) Polynucleotide having base sequence of base numbers 133 to 1080 among the base sequence set forth in SEQ ID NO: 1 of the sequence listing (d) Base numbers 133 to 1080 of the base sequence set forth in SEQ ID NO: 1 of the sequence listing Hybridizes under stringent conditions with polynucleotides that are complementary to the base sequence of And a polynucleotide encoding a protein having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (e) Among the base sequences set forth in SEQ ID NO: 16 of the sequence listing, base numbers 114 to A polynucleotide having a base sequence of 1688 (f) Hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 114 to 1688 among the base sequence set forth in SEQ ID NO: 16 of the sequence listing Polynucleotide encoding a protein that is soybean and has an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (g) Base number 39 of the base sequence set forth in SEQ ID NO: 16 in the Sequence Listing A polynucleotide having a base sequence of ˜1688 (h) in SEQ ID NO: 16 of the sequence listing Hybridize under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 39 to 1688 among the listed base sequences, and α-L-aspartyl-L-phenylalanine-β-ester Polynucleotide encoding a protein having deesterification activity (i) Polynucleotide having base sequence of base numbers 61 to 1080 among base sequences set forth in SEQ ID NO: 1 of sequence listing (j) SEQ ID NO: 1 of sequence listing Among the base sequences described in the above, and hybridizing under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 61 to 1080, and α-L-aspartyl-L-phenylalanine-β-ester Encodes a protein containing a mature protein region having the activity of deesterifying Polynucleotides [12] the stringent conditions, 1 × SSC and washed at 60 ° C. at a salt concentration corresponding to 0.1% SDS is a conditional performed polynucleotide according to [11].
[13] A recombinant polynucleotide comprising the polynucleotide according to any one of [10] to [12].
[14] A transformed cell into which the polynucleotide according to [13] has been introduced.

  The present invention provides a simple method for producing α-L-aspartyl-L-phenylalanine, a protein that catalyzes the production reaction of α-L-aspartyl-L-phenylalanine, and the like. According to the present invention, α-L-aspartyl-L-phenylalanine can be produced simply and at a high yield and at low cost by reducing the burden on complicated synthesis methods such as introduction and removal of protecting groups.

  Moreover, according to the present invention, α-L-aspartyl-L-phenylalanine-α-methyl ester can be easily produced at a high yield and at a low cost.

Hereinafter, regarding the present invention,
<1> Method for producing α-L-aspartyl-L-phenylalanine 1. Method for producing α-L-aspartyl-L-phenylalanine 2. Microorganism used in the present invention A method for producing the enzyme <2> α-L-aspartyl-L-phenylalanine-α-methyl ester used in the present invention;
Will be described in the order.

<1> Method for producing α-L-aspartyl-L-phenylalanine Method for Producing α-L-Aspartyl-L-Phenylalanine The method for producing α-L-aspartyl-L-phenylalanine of the present invention (hereinafter also referred to as “the peptide production method of the present invention”) has a predetermined peptide-forming activity. The α-L-aspartyl-L-phenylalanine-β-ester is deesterified in the presence of the enzyme having. That is, the peptide production method of the present invention uses α-L-aspartyl-L-phenylalanine- using an enzyme or an enzyme-containing product having the ability to deesterify α-L-aspartyl-L-phenylalanine-β-ester. α-L-aspartyl-L-phenylalanine is produced from β-ester. An enzyme or enzyme-containing substance having the ability to deesterify α-L-aspartyl-L-phenylalanine-β-ester is substantially a β-ester site of α-L-aspartyl-L-phenylalanine-β-ester. Refers to an enzyme (esterase) or an enzyme-containing substance having an ability or activity to catalyze a reaction for deesterification by hydrolysis or the like.

The reaction formula in which α-L-aspartyl-L-phenylalanine-β-ester is deesterified to produce α-L-aspartyl-L-phenylalanine is expressed as α-L-aspartyl-L-phenylalanine-β-ester. An example in which -L-aspartyl-L-phenylalanine-β-methyl ester (α-AMP) is used will be described.

  As a method for allowing an enzyme or an enzyme-containing substance to act on α-L-aspartyl-L-phenylalanine-β-ester, the enzyme or the enzyme-containing substance and α-L-aspartyl-L-phenylalanine-β-ester are used. The method of mixing is mentioned. More specifically, a method in which an enzyme or an enzyme-containing substance is added to a solution containing α-L-aspartyl-L-phenylalanine-β-ester and allowed to react can be used. In addition, when a microorganism that produces the enzyme is used as the enzyme-containing material, the microorganism that produces the enzyme is cultured in addition to the method in which the microorganism that produces the enzyme is added and reacted in the solution as described above. Alternatively, a method may be used in which the enzyme is produced and accumulated in a microorganism or a culture solution of the microorganism, and α-L-aspartyl-L-phenylalanine-β-ester is added to the culture solution. The produced α-L-aspartyl-L-phenylalanine can be recovered by a conventional method and purified as necessary.

  The “enzyme-containing material” is a mixture containing the enzyme. The enzyme-containing material can further contain one or more other substances. Specifically, an enzyme-containing mixture in which a step of producing an enzyme, such as culturing of a microorganism, is performed therein, or a mixture obtained by subjecting it to post-treatment as necessary can be used as the enzyme-containing material. The other substance may include microbial cells and microbial cell debris in addition to the substances constituting the medium. The post-treatment includes recovery of the medium from the culture solution after completion of the culture or recovery of the cells; disruption of the cells, lysis and lyophilization; removal of some other substances by rough purification, etc .; Examples thereof include immobilization of an enzyme or an enzyme-containing structure (for example, a cell) by an adsorption method, an entrapment method, and the like, and combinations thereof. In addition, some microorganisms lyse during culture, and in this case, the culture supernatant can also be used as an enzyme-containing material.

  Moreover, as a microorganism containing the enzyme, a wild strain may be used, or a genetically modified strain expressing the enzyme may be used. The microorganisms are not limited to enzyme microorganisms but may be treated with acetone, lyophilized cells, etc., and these may be fixed by a covalent bond method, adsorption method, inclusion method, etc. Immobilized microbial cells and immobilized microbial cell processed products may be used.

  When using a wild strain capable of producing an α-AMP esterase having an activity to produce α-L-aspartyl-L-phenylalanine, it is possible to produce a peptide more easily without the need to prepare a genetically modified strain, etc. It is suitable. On the other hand, a mutant strain modified to produce a larger amount of active α-AMP esterase that produces α-L-aspartyl-L-phenylalanine (for example, transformed so that the enzyme gene can be expressed in a large amount. If a genetically modified strain is used, it may be possible to synthesize α-L-aspartyl-L-phenylalanine more rapidly in large quantities. That is, in the method of the present invention, the microorganism was transformed so as to express an α-AMP esterase having an activity to produce α-L-aspartyl-L-phenylalanine, for example, the protein of the present invention described later. A microorganism or a microorganism transformed so as to be able to express the enzyme gene, for example, the polynucleotide of the present invention described later, can also be used. Wild-type or mutant-type microorganisms are cultured in a medium, the α-AMP esterase is accumulated in the medium and / or the microorganism, and mixed with α-L-aspartyl-L-phenylalanine-β-ester. , Α-L-aspartyl-L-phenylalanine can be produced.

  In the case of using a culture, a cultured cell, a washed cell, or a cell-treated product obtained by crushing or lysing a cell, the α-L-aspartyl-L-phenylalanine is not involved in the production of α-L Enzymes that degrade L-aspartyl-L-phenylalanine often exist. Therefore, it is possible to add a metal protease inhibitor such as ethylenediaminetetraacetic acid (EDTA). The addition amount can be appropriately determined, for example, in the range of 0.1 mM to 300 mM, preferably in the range of 1 mM to 100 mM.

  The amount of the enzyme or the enzyme-containing material used may be an amount (effective amount) that exhibits the intended effect. This effective amount is easily determined by a person skilled in the art by a simple preliminary experiment. For example, when an enzyme is used, about 0.01 to 100 units (U), and when a washing cell is used, 0.1 to 0.1 unit. It is about 500 g / L. In addition, 1U is 1 minute when it is made to react on 20 degreeC temperature conditions using the 100 mM phosphate buffer (pH) containing 50 mM alpha-L-aspartyl-L-phenylalanine-beta-ester. The amount of enzyme that produces 1 μmole of α-L-aspartyl-L-phenylalanine.

Any α-L-aspartyl-L-phenylalanine-β-ester used in the reaction may be used as long as it can be deesterified to produce α-L-aspartyl-L-phenylalanine. For example, α-L-aspartyl-L-phenylalanine-β-methyl ester and the like can be mentioned. The production method of α-L-aspartyl-L-phenylalanine-β-ester is not particularly limited. For example, it can be produced by condensing L-aspartic acid-α, β-diester and L-phenylalanine. The reaction formula for condensing L-aspartic acid-α, β-diester and L-phenylalanine to produce α-L-aspartyl-L-phenylalanine-β-ester is defined as L-aspartic acid-α, β-diester. An example in which L-aspartic acid-α, β-dimethyl ester is used will be described.

  The concentration of α-L-aspartyl-L-phenylalanine-β-ester as a starting material is 1 mM to 2M, preferably 20 to 600 mM. Further, when the reaction is inhibited when the concentration of the substrate is high, it can be added successively at a concentration that does not inhibit these during the reaction.

  Reaction temperature is 5-60 degreeC, (alpha) -L-aspartyl-L-phenylalanine production | generation is possible, Preferably it is 10-40 degreeC. Moreover, reaction pH is pH 5-12, (alpha) -L-aspartyl-L-phenylalanine production | generation is possible, Preferably it is pH 6-11.

2. Microorganism used in the present invention The microorganism used in the present invention is not particularly limited to a microorganism having the ability to produce α-L-aspartyl-L-phenylalanine from α-L-aspartyl-L-phenylalanine-β-ester. Can be used. Examples of microorganisms having the ability to produce α-L-aspartyl-L-phenylalanine from α-L-aspartyl-L-phenylalanine-β-ester include microorganisms belonging to the genus Tetrathiobacter and Brevundimonas Specific examples include Tetrathiobacter sp. FERM P-21102 and Brevandimonas sp. NRRL B-2395. Tetrathiobacter sp. FERM P-21102 is an independent administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan), November 21, 2006 It is deposited with a date.

Among the above strains, those having NRRL numbers are deposited with the Agricultural Research Service Culture Collection (1815 N. University Street, Peoria, IL 61604, the United States of America). You can get a lot by referring to.
Among the above strains, those with the FERM number are deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki, Japan). You can get a lot by referring to the number.

  As these microorganisms, either wild strains or mutant strains may be used, and recombinant strains induced by genetic techniques such as cell fusion or genetic manipulation may also be used.

  In order to obtain cells of such microorganisms, the microorganisms are preferably grown and cultured in an appropriate medium. The medium for this is not particularly limited as long as the microorganism can grow, and is a normal medium containing a normal carbon source, nitrogen source, phosphorus source, sulfur source, inorganic ions, and if necessary, an organic nutrient source. Good.

  For example, any of the above microorganisms can be used as the carbon source. Specifically, sugars such as glucose, fructose, maltose and amylose, alcohols such as sorbitol, ethanol and glycerol, fumaric acid and citric acid In addition, organic acids such as acetic acid and propionic acid and salts thereof, hydrocarbons such as paraffin or mixtures thereof can be used.

  Nitrogen sources include ammonium salts of inorganic acids such as ammonium sulfate and ammonium chloride, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, phosphates such as monopotassium phosphate and dipotassium phosphate, magnesium sulfate, etc. Sulfates, nitrates such as sodium nitrate and potassium nitrate, organic nitrogen compounds such as peptone, yeast extract, meat extract and corn steep liquor, or mixtures thereof can be used.

  In addition, nutrient sources used in normal media such as inorganic salts, trace metal salts, vitamins, and the like can be appropriately mixed and used.

  The culture conditions are not particularly limited. For example, the culture may be performed for about 12 to 48 hours while appropriately limiting the pH and temperature in the range of pH 5 to 8 and temperature 15 to 40 ° C. under aerobic conditions. .

3. Enzyme used in the present invention In the present invention, an enzyme (esterase) having the ability to deesterify α-L-aspartyl-L-phenylalanine-β-ester is used. In the present invention, any enzyme having such an activity can be used without limitation on its origin and acquisition method. Hereinafter, the enzyme protein of the present invention, its purification, utilization of genetic engineering techniques, etc. will be described.

(3-1) Protein of the present invention The protein having the activity of deesterifying the α-L-aspartyl-L-phenylalanine-β-ester of the present invention is derived from α-L-aspartyl-L-phenylalanine-β-ester. It can be obtained from a microorganism having the ability to produce α-L-aspartyl-L-phenylalanine. For example, a microorganism belonging to the genus mentioned as an example of the microorganism used in the present invention, that is, a bacterium belonging to a genus selected from the group consisting of Tetrathiobacter and Brevandimonas can be used. More specifically, Tetrathiobacter sp. And Brevundimonas sp. Etc. These strains Tetrathiobacter sp. FERM P-21102 and Brevundimonas sp. NRRL B-2395 were developed by the present inventors from α-L-aspartyl-L-phenylalanine-β-ester to α-L-aspartyl-L-. It is a microorganism selected as a result of searching for an enzyme producing bacterium that produces phenylalanine in a high yield.

A method for isolating and purifying the protein of the present invention from the aforementioned microorganism will be described.
First, the bacterial cells are disrupted from the microorganisms by a physical method such as ultrasonic disruption, or an enzymatic method using cell wall lysing enzymes, etc., and the insoluble fraction is removed by centrifugation or the like to remove the bacterial cell extract. Prepare. By fractionating the bacterial cell extract obtained in this way by combining ordinary protein purification methods, anion exchange chromatography, hydrophobic chromatography, cation exchange chromatography, gel filtration chromatography, etc., α-AMP esterase can be purified.

  Examples of the carrier for anion exchange chromatography include Q-Sepharose 26/10, 16/10, and HP (both manufactured by Amersham). When the extract containing the present enzyme is passed through a column packed with these carriers, the enzyme is recovered in the non-adsorbed fraction under the condition of pH 8.5.

  As the carrier for cation chromatography, MonoS HR (manufactured by Amersham) can be mentioned. The extract containing the enzyme is passed through a column packed with these carriers to adsorb the enzyme to the column, the column is washed, and then the enzyme is eluted using a buffer solution with a high salt concentration. At that time, the salt concentration may be increased stepwise, or a concentration gradient may be applied. For example, when MonoS HR is used, the present enzyme adsorbed on the column is eluted with about 0.2 to 0.5 M NaCl.

  The enzyme purified as described above can be further purified uniformly by gel filtration chromatography, hydrophobic chromatography or the like. Examples of the carrier for gel filtration chromatography include Superdex 200 pg and Sephadex 200 pg (both manufactured by Amersham). Examples of the carrier for hydrophobic chromatography include Resource ethyl (manufactured by Amersham).

In the above purification operation, the fraction containing the present enzyme can be confirmed by measuring the peptide-forming activity of each fraction by the method shown in Examples described later. The internal amino acid sequences of the present enzyme purified as described above are shown in SEQ ID NOs: 9 and 10 in the sequence listing.
More specifically, examples of the protein having an activity to deesterify the α-L-aspartyl-L-phenylalanine-β-ester of the present invention include proteins selected from the group consisting of the following (A) to (J). It is done.
(A) Protein having the amino acid sequence of amino acid residues 14 to 340 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing (B) Amino acid residue number 14 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing ˜340 amino acid sequence, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and de-esterifying α-L-aspartyl-L-phenylalanine-β-ester Among the amino acid sequences described in SEQ ID NO: 2 in the protein (D) sequence listing, the protein having the amino acid sequence of amino acid residues 25 to 340 among the amino acid sequences listed in SEQ ID NO: 2 in the protein (C) sequence listing Including the substitution, deletion, and / or insertion of one or several amino acids in the amino acid sequence of amino acid residues 25 to 340 The protein (E) has an amino acid sequence and has an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester. 1 or several amino acids in the amino acid sequence of amino acid residues 26 to 550 among the amino acid sequences described in SEQ ID NO: 17 of the protein (F) sequence table having the amino acid sequence of amino acid residues 26 to 550 And having an amino acid sequence comprising amino acid substitutions, deletions, and / or insertions, and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester Sequence of protein (H) having the amino acid sequence of SEQ ID NO: 17 of protein (G) The amino acid sequence described in No. 17 has an amino acid sequence including substitution, deletion, and / or insertion of one or several amino acids, and removes α-L-aspartyl-L-phenylalanine-β-ester. 1 or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence (SEQ ID NO: 2) of the protein (I) SEQ ID NO: 2 including the mature protein region having the activity to esterify A protein having an amino acid sequence including amino acid substitution, deletion, and / or insertion, and a mature protein region having an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester

The protein having the amino acid sequence shown in SEQ ID NO: 2 was newly isolated from Tetrathiobacter sp. FERM P-21102 strain and specified as the amino acid sequence of the above α-AMP esterase protein. is there. The entire amino acid sequence described in SEQ ID NO: 2 includes a leader peptide and a mature protein region. Amino acid residues Nos. 1 to 13 or 1 to 24 correspond to the leader peptide, and amino acid residues Nos. 14 to 340 or 25 to 25 are included. 340 is a mature protein region.
On the other hand, a protein having the amino acid sequence shown in SEQ ID NO: 17 was newly isolated from Brevundimonas sp NRRL B-2395 strain by the present inventors and specified as the amino acid sequence of the above-mentioned α-AMP esterase protein. Is. The entire amino acid sequence described in SEQ ID NO: 17 includes a leader peptide and a mature protein region. Amino acid residues No. 1 to 25 are leader peptides, and amino acid residues No. 26 to 550 are mature protein regions.

  The protein of the present invention may or may not include the leader peptide as long as the mature protein has a peptide-forming activity. The protein of the present invention also includes a protein that is substantially the same as the mature protein region in the amino acid sequence described in SEQ ID NO: 2 or 17 in the sequence listing, or a protein having the entire region including the leader peptide. included. Specific examples include (B), (D), (F), (H), and (J).

  Here, “several” means a range that does not greatly impair the three-dimensional structure and activity of the amino acid residue protein, although it varies depending on the position and type of the three-dimensional structure of the amino acid residue protein. Is 2-50, preferably 2-30, and more preferably 2-10. Further, the sequence having one or several amino acid mutations is 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, relative to the sequence having no mutation. Even more preferably, it may have a homology of 97% or more. However, in the amino acid sequence of the protein of (B), (D), (F), (H), (J), the amino acid sequence including substitution, deletion, and / or insertion of one or several amino acid residues In some cases, it is desirable to maintain an enzyme activity of about half or more, more preferably 80% or more, and even more preferably 90% or more of the protein in a state containing no mutation under conditions of 50 ° C. and pH 8. . For example, in the case of (B), (B) substitution or deletion of one or several amino acid residues in the amino acid sequence of amino acid residues Nos. 14 to 340 in the amino acid sequence set forth in SEQ ID No. 2 of the Sequence Listing. In the case of an amino acid sequence containing a loss and / or insertion, the protein having an amino acid sequence of amino acid residues 14 to 340 of amino acid sequences described in SEQ ID NO: 2 of the sequence listing under conditions of 50 ° C. and pH 8 It is desirable to maintain an enzyme activity of about half or more, more preferably 80% or more, and still more preferably 90% or more.

  In the amino acid mutation as shown in (B) above, the amino acid at a specific site in the amino acid sequence shown in (A) above is substituted, deleted, and / or inserted by, for example, site-specific mutagenesis. It is obtained by designing in advance an amino acid sequence modified in such a manner and expressing a base sequence corresponding to this amino acid sequence.

  Moreover, naturally occurring mutations such as differences between microorganism species or strains are also included in the above-mentioned base substitution, deletion, and / or insertion. By expressing a polynucleotide encoding an amino acid having a mutation as described above in an appropriate cell and examining the enzyme activity of the expression product, the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 17 in the sequence listing A protein having substantially the same protein as the protein having the mature protein region or the entire region including the leader peptide can be obtained.

(3-2) Production of Polynucleotide, Recombinant Polynucleotide, and Transformant of the Present Invention and Purification of α-AMP Esterase (3-2-1) Polynucleotide Encoding α-AMP Esterase Protein Also provided is a polynucleotide encoding the α-AMP esterase protein. The polynucleotide of the present invention is a polynucleotide that encodes the amino acid sequence that constitutes the α-AMP esterase protein described above, but there may be a plurality of base sequences that define one amino acid sequence due to codon degeneracy. Specifically, a polynucleotide having a base sequence encoding a protein selected from the group consisting of the above (A) to (J) is included.

Preferable specific examples of the polynucleotide of the present invention include a polynucleotide selected from the group consisting of the following (a) to (j).
(A) a polynucleotide having a base sequence of base numbers 100 to 1080 among the base sequences set forth in SEQ ID NO: 1 of the sequence listing (b) of base sequences 100 to 1080 of the base sequences set forth in SEQ ID NO: 1 of the sequence listing Poly that encodes a protein that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence under stringent conditions and has an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester Nucleotide (c) Polynucleotide having base sequence of base numbers 133 to 1080 among the base sequence set forth in SEQ ID NO: 1 of the sequence listing (d) Base numbers 133 to 1080 of the base sequence set forth in SEQ ID NO: 1 of the sequence listing Hybridizes under stringent conditions with polynucleotides that are complementary to the base sequence of And a polynucleotide encoding a protein having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (e) Among the base sequences set forth in SEQ ID NO: 16 of the sequence listing, base numbers 114 to A polynucleotide having a base sequence of 1688 (f) Hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 114 to 1688 among the base sequence set forth in SEQ ID NO: 16 of the sequence listing Polynucleotide encoding a protein that is soybean and has an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (g) Base number 39 of the base sequence set forth in SEQ ID NO: 16 in the Sequence Listing A polynucleotide having a base sequence of ˜1688 (h) in SEQ ID NO: 16 of the sequence listing Hybridize under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 39 to 1688 among the listed base sequences, and α-L-aspartyl-L-phenylalanine-β-ester Polynucleotide encoding a protein having deesterification activity (i) Polynucleotide having base sequence of base numbers 61 to 1080 among base sequences set forth in SEQ ID NO: 1 of sequence listing (j) SEQ ID NO: 1 of sequence listing Among the base sequences described in the above, and hybridizing under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 61 to 1080, and α-L-aspartyl-L-phenylalanine-β-ester Encodes a protein containing a mature protein region having the activity of deesterifying Polynucleotide

  The polynucleotide having the base sequence set forth in SEQ ID NO: 1 in the Sequence Listing is isolated from Tetrathiobacter sp. FERM P-21102 strain. The polynucleotide consisting of the base sequence of base numbers 61 to 1080 of SEQ ID NO: 1 is a code sequence (CDS) part. The base sequence of base numbers 61 to 1080 includes a signal sequence region and a mature protein region. The signal sequence region is a region of base numbers 61 to 99 or a region of 61 to 132, and the mature protein region is a region of base numbers 100 to 1080 or a region of 133 to 1080. That is, the present invention provides both an α-AMP esterase protein gene containing a signal sequence and an α-AMP esterase protein gene as a mature protein. The signal sequence contained in the sequence of SEQ ID NO: 1 is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded by the leader sequence is to secrete it from the cell membrane to the outside of the cell membrane. The protein encoded by base numbers 101 to 1080 or 133 to 1080, that is, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide production activity.

  Moreover, the polynucleotide which consists of a base sequence of the base numbers 39-1688 as described in sequence number 16 of a sequence table is isolated from Brevundimonas sp NRRL B-2395. The polynucleotide comprising the nucleotide sequence of nucleotide numbers 39 to 1688 described in SEQ ID NO: 16 is a coding sequence (CDS) portion. The base sequence of base numbers 39 to 1688 includes a signal sequence region and a mature protein region. The signal sequence region is a region of base numbers 39 to 113, and the mature protein region is a region of base numbers 114 to 1688. That is, the present invention provides both an α-AMP esterase protein gene containing a signal sequence and an α-AMP esterase protein gene as a mature protein. The signal sequence contained in the sequence described in SEQ ID NO: 16 is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded by the leader sequence is to secrete it from the cell membrane to the outside of the cell membrane. The protein encoded by base numbers 39 to 113, that is, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide production activity.

  The various gene recombination techniques listed below can be performed according to the description of Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989).

  The polynucleotide of the present invention can be obtained from PCR (polymerase chain reaction, White, TJ et al; Trends Genet., 5, 185) from a chromosomal DNA such as Tetrathiobacter sp. And Brevundimonas sp. 1989)) or hybridization. Primers used for PCR can be designed based on an internal amino acid sequence determined based on α-AMP esterase purified as described in the section (3) above. Further, since the base sequence of the α-AMP esterase gene (SEQ ID NO: 1, SEQ ID NO: 16) has been clarified by the present invention, primers and probes for hybridization can be designed based on these base sequences, It can also be isolated using a probe. When a primer having a sequence corresponding to a 5 ′ untranslated region and a 3 ′ untranslated region is used as a primer for PCR, the entire coding region of the present enzyme can be amplified. Taking the case where the region containing both the leader sequence and the mature protein coding region described in SEQ ID NO: 1 is amplified as an example, specifically, as the 5 ′ primer, SEQ ID NO: 1 is more than base number 61 in SEQ ID NO: 1. Examples of the primer having the base sequence in the upstream region include a primer having a sequence complementary to the base sequence in the region downstream from base number 1080 as the 3′-side primer.

  The primer can be synthesized, for example, using a DNA synthesizer model 380B manufactured by Applied Biosystems, using the phosphoamidite method (see Tetrahedron Letters (1981), 22, 1859). The PCR reaction can be performed, for example, using Gene Amp PCR System 9600 (manufactured by PERKIN ELMER) and TaKaRa LA PCR in vitro Cloning Kit (manufactured by Takara Shuzo) according to the method specified by the supplier such as each manufacturer.

  The polynucleotide encoding the enzyme that can be used in the method for producing a peptide of the present invention is substantially the same as the polynucleotide consisting of the CDS described in SEQ ID NO: 1 in the sequence listing, whether or not the leader sequence is included. Identical polynucleotides are also included. That is, a probe prepared from a polynucleotide or a base sequence complementary to the CDS described in SEQ ID NO: 1 in the Sequence Listing from a polynucleotide encoding the present enzyme having a mutation or a cell retaining the enzyme A polynucleotide substantially identical to the polynucleotide of the present invention can also be obtained by isolating a polynucleotide encoding a protein having a peptide-forming activity and hybridizing under stringent conditions.

  The polynucleotide of the present invention also includes a polynucleotide that is substantially the same as the polynucleotide comprising CDS described in SEQ ID NO: 16 in the sequence listing, whether or not the leader sequence is included. That is, a probe prepared from a polynucleotide comprising the nucleotide sequence complementary to the CDS described in SEQ ID NO: 16 of the Sequence Listing, or a nucleotide sequence thereof, from a polynucleotide encoding the enzyme having a mutation or a cell retaining the enzyme A polynucleotide substantially identical to the polynucleotide of the present invention can also be obtained by isolating a polynucleotide encoding a protein having a peptide-forming activity and hybridizing under stringent conditions.

  The probe can be prepared by a conventional method based on the base sequence described in SEQ ID NO: 1, for example. In addition, a method of picking up a polynucleotide that hybridizes with a probe using a probe and isolating the target polynucleotide may be performed according to a conventional method. For example, a DNA probe can be prepared by amplifying a base sequence cloned in a plasmid or a phage vector, cutting out and extracting the base sequence to be used as a probe with a restriction enzyme. The location to be cut out can be adjusted according to the target polynucleotide.

  The “stringent conditions” referred to herein are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, highly homologous polynucleotides, for example, 50% or more, more preferably 80% or more, more preferably 90% or more, and most preferably More than 95%, more preferably more than 97% homologous polynucleotides hybridize, and polynucleotides with lower homology do not hybridize with each other, or under normal Southern hybridization washing conditions. The conditions include hybridization at a salt concentration corresponding to a certain 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS. 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.

  However, in the case of a polynucleotide comprising a base sequence that hybridizes under stringent conditions as described above, half of the protein having the amino acid sequence encoded by the original base sequence at 50 ° C. and pH 8 It is desirable that the enzyme activity is maintained at a degree or more, more preferably 80% or more, and still more preferably 90% or more. For example, the case of a base sequence that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of base numbers 100 to 1080 of the base sequence described in (b) SEQ ID NO: 1 will be described. About 50% or more, more preferably 80% or more, more preferably 90% of the protein having an amino acid sequence of amino acid residues 100 to 1080 of the amino acid sequence shown in SEQ ID NO: 2 at 50 ° C. and pH 8 It is desirable to retain the above enzyme activity.

  Such a modified polynucleotide can be obtained by a conventionally known mutation treatment. As the mutation treatment, a method for treating a polynucleotide encoding the present enzyme, for example, any one of the polynucleotides (a), (c), (e), (g) and (i) in vitro with hydroxylamine or the like , And Escherichia bacteria carrying a polynucleotide encoding this enzyme are usually used for ultraviolet irradiation or artificial mutation such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrite The method of processing with a mutagen is mentioned.

  The amino acid sequence encoded by the CDS described in SEQ ID NO: 1 in the sequence listing is shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence encoded by the CDS described in SEQ ID NO: 16 in the sequence listing is shown in SEQ ID NO: 17 in the sequence listing.

(3-2-2) Production of recombinant polynucleotide and transformant, and production of α-AMP esterase The polynucleotide described in (3-2-1) above is introduced into an appropriate host to obtain a recombinant polynucleotide. The α-AMP esterase that can be used in the present invention can also be produced by expressing the protein specified by the polynucleotide in the resulting transformed cell (transformant).

  The host for expressing the protein specified by the polynucleotide includes Escherichia coli, such as Escherichia coli, and various prokaryotic cells including Saccharomyces cerevisiae, including Bacillus subtilis. Various eukaryotic cells such as (Saccharomyces cerevisiae), Pichia stipitis, Aspergillus oryzae can be used.

  A recombinant polynucleotide used for introducing a polynucleotide into a host is in a form in which a protein encoded by the polynucleotide can be expressed in a vector corresponding to the type of host to be expressed. It can be prepared by inserting in As a promoter for expressing the polynucleotide of the present invention, when a promoter specific to an α-AMP esterase gene such as Tetrathiobacter sp. Or Brevundimonas sp. 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 the polynucleotide of the present invention and expressed under the control of the promoter.

  Examples of transformation methods for introducing a recombinant polynucleotide into a host cell include D.I. M.M. Morrison's method (Methods in Enzymology 68, 326 (1979)) or a method in which recipient cells are treated with calcium chloride to increase polynucleotide permeability (Mandel, M. and Higa, A., J. Mol. Biol. 53, 159 (1970)).

  In the case where a protein is mass-produced using recombinant polynucleotide technology, a form in which the protein associates in a transformed cell that produces the protein to form an inclusion body is also mentioned 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.

  The protein inclusion body obtained in this way is solubilized by a protein denaturing agent, and after being subjected to an activity regeneration operation mainly by removing the denaturing agent, it is converted into a correctly folded physiologically active protein. . For example, there are many examples such as activity regeneration of human interleukin-2 (Japanese Patent Laid-Open No. 61-257931).

  In order to obtain an active protein from a protein inclusion body, a series of operations such as solubilization and activity regeneration are necessary, 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.

  Hereinafter, a method for producing transformed E. coli and producing α-AMP esterase using the same will be described more specifically as an example. In the case where α-AMP esterase is prepared in a microorganism such as Escherichia coli, a polynucleotide of a mature protein region that does not contain a leader sequence even if a polynucleotide encoding a precursor protein containing a leader sequence is incorporated as a protein coding sequence. May be incorporated, and can be appropriately selected depending on the production conditions, form, use conditions, etc. of the enzyme to be produced.

  As a promoter for expressing a polynucleotide encoding α-AMP esterase, a promoter usually used for heterologous protein production in E. coli can be used. For example, a T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, Strong promoters such as PR promoter and PL promoter of lambda phage can be mentioned. Moreover, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218 etc. can be used as a vector. In addition, phage polynucleotide vectors can be used. Furthermore, an expression vector containing a promoter and capable of expressing the inserted polynucleotide sequence can also be used.

  In order to produce α-AMP esterase as a fusion protein inclusion body, a gene encoding another protein, preferably a hydrophilic peptide, is linked upstream or downstream of the α-AMP esterase gene, and the fusion protein gene And 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 / regeneration process. For example, T7gene 10, β-galactosidase gene, Dehydrofolate reductase gene, γ interferon gene, interleukin-2 gene, prochymosin gene and the like are listed as candidates.

  When these genes are linked to the gene encoding α-AMP esterase, the codon reading frames are matched. They may be linked at an appropriate restriction enzyme site, or a synthetic polynucleotide 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 rrnB terminator, T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, E. coli trpA gene terminator, and the like.

  As a vector for introducing α-AMP esterase or a gene encoding a fusion protein of α-AMP esterase and other proteins into Escherichia coli, a so-called multi-copy type is preferable, and a plasmid having a replication origin derived from ColE1 For example, a pUC-type plasmid, a pBR322-type plasmid, or a derivative thereof can be used. Here, the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, and / or insertion. In addition, the modification here includes modification by mutation treatment with a mutation agent, UV irradiation, or natural mutation.

  In order to select transformants, the vector preferably has a marker such as an ampicillin resistance gene. As such plasmids, expression vectors having a strong promoter are commercially available (pUC system (Takara Shuzo Co., Ltd.), pPROK system (Clontech), pKK233-2 (Clontech), etc.).

  A gene encoding a promoter, α-AMP esterase or a fusion protein of α-AMP esterase and another protein, and in some cases a polynucleotide fragment ligated in the order of terminator and a vector polynucleotide are ligated to produce a recombinant polynucleotide. obtain.

  When E. coli is transformed with the recombinant polynucleotide and this E. coli is cultured, α-AMP esterase or a fusion protein of α-AMP esterase and another protein is expressed and produced. As a host to be transformed, a strain usually used for expression of a heterologous gene can be used, but Escherichia coli JM109 strain is preferable. Methods for performing transformation and methods for selecting transformants are described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989) and the like.

  When expressed as a fusion protein, α-AMP esterase may be excised using a restriction protease such as blood coagulation factor Xa, kallikrein, etc. that has a recognition sequence that is not present in α-AMP esterase.

  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.

  In order to recover α-AMP esterase or a fusion protein of α-AMP esterase and another protein, there are the following methods. If α-AMP esterase or a fusion protein thereof 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, α-AMP esterase 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 α-AMP esterase or fusion protein can also be used.

  When protein inclusion bodies are formed, they are solubilized with a denaturing agent. Although it may be solubilized together with the bacterial protein, it is preferable to take out the inclusion body and solubilize it in consideration of the subsequent purification operation. In order to recover the inclusion body from the microbial cell, a conventionally known method may be used. For example, the microbial cells are destroyed, and the inclusion bodies are collected by centrifugation operation or the like. Examples of denaturing agents that solubilize protein inclusion bodies include guanidine hydrochloride (eg, 6M, pH 5-8), urea (eg, 8M), and the like.

  When these denaturing agents are removed by dialysis or the like, they are regenerated as active proteins. As a dialysis solution used for dialysis, a Tris-HCl buffer or a phosphate buffer may be used, and the concentration may be 20 mM to 0.5 M, and the pH may be 5 to 8.

  The protein concentration during the regeneration step is preferably suppressed to about 500 μg / ml or less. In order to suppress the regenerated α-AMP esterase from self-crosslinking, the dialysis temperature is preferably 5 ° C. or lower. Further, as a method for removing the denaturant, there are a dialysis method, a dilution method, an ultrafiltration method, and the like, and regeneration of activity can be expected by using any of them.

<2> Method for Producing α-L-Aspartyl-L-Phenylalanine-α-Methyl Ester The method for producing α-APM of the present invention is the above-mentioned “<1> Method for producing α-L-aspartyl-L-phenylalanine”. And a second step of converting α-L-aspartyl-L-phenylalanine into α-L-aspartyl-L-phenylalanine-α-methyl ester. Including the steps of:

本発明のα−ＡＰＭの製造方法によれば、甘味料等として重要なα−ＡＰＭを簡便かつ高収率で安価に製造することができる。 Preferred conditions and the like in the first step are as described in the column of “<1> Production method of α-L-aspartyl-L-phenylalanine-β-ester” above. Moreover, about a 2nd process, it can carry out by a well-known method. The reaction formula of the second step for converting α-L-aspartyl-L-phenylalanine into α-L-aspartyl-L-phenylalanine-α-methyl ester is as shown below.

According to the method for producing α-APM of the present invention, α-APM important as a sweetener or the like can be produced simply and at a high yield at a low cost.

Example 1 Microorganism producing α-L-aspartyl-L-phenylalanine For bacterial culture, 20 g of glycerol, 5 g of ammonium sulfate, 1 g of monopotassium phosphate, 3 g of dipotassium phosphate, 3 g of magnesium sulfate and 0.5 g of yeast in 1 L 50 mL of a medium (pH 7.0) containing 10 g of extract and 10 g of peptone was dispensed into a 500 mL Sakaguchi flask and sterilized at 115 ° C. for 15 minutes (Medium 1). A slope agar medium (agar 20 g / L, pH 7.0) containing 5 g of glucose, 10 g of yeast extract, 10 g of peptone, and 5 g of NaCl in 1 L of medium 1 was prepared, and this slope agar medium was 30 ° C. for 24 hours. One platinum loop of the cultured microorganisms shown in Table 1 was inoculated, and cultured with shaking at 30 ° C. and 120 reciprocations / minute for 17 hours. After cultivation, the cells were centrifuged and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA so as to be 100 g / L as wet cells.

  100 mM borate buffer (pH 9.0) containing 10 mM EDTA and 100 mM α-L-aspartyl-L-phenylalanine-β-methyl ester (α-AMP) in 0.1 mL of the cell suspension of these microorganisms. 1 mL of each was added to make the total amount 0.2 mL, and then reacted at 20 ° C. for 3 hours. At this time, production of α-L-aspartyl-L-phenylalanine (α-AP) was confirmed in Tetrathiobacter sp.

Example 2 Isolation of α-AMP Esterase Gene Derived from Tetrathiobacter sp. Hereinafter, isolation of α-AMP esterase gene will be described. The microorganism used was Tetrathiobacter sp. FERM P-21102 strain. . For isolation of the gene, Escherichia coli JM109 was used as the host, and pUC118 was used as the vector.

  Tetrathiobacter sp. FERM P-21102 strain on CM2G agar medium (50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride, 20 g / l agar, pH 7.0)) at 30 ° C. And cultured for 24 hours. One microbial ear of this bacterial cell was inoculated into a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was placed, and cultured at 30 ° C. with shaking.

  50 ml of the culture solution was centrifuged (12,000 rpm, 4 ° C., 15 minutes) and collected. Using QIAGEN Genomic-tip System (Qiagen), chromosomal DNA was obtained from the cells based on the method described in the manual.

  5 μg of chromosomal DNA prepared from Tetrathiobacter sp. FERM P-21102 strain was completely digested with PstI. About 4 kb of DNA was separated by 0.8% agarose gel electrophoresis, and the DNA was purified using Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. Of these, 4 μl and pUC118 PstI / BAP (Takara Shuzo) were mixed, and DNA Ligation Kit Ver. The ligation reaction was performed using 2 (Takara Shuzo). Escherichia coli JM109 was transformed by mixing 5 μl of this ligation reaction solution and 100 μl of Escherichia coli JM109 competent cell (manufactured by Toyobo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.

  On the fixed medium on which colonies were formed, α-AMP 5 mM, peroxidase 10 U / ml, alchol oxidase 0.1 U / ml, 4-aminoantipyrine 5 mM, ALPS 10 mM, EDTA 10 mM, KPB (pH 7) 20 mM, SeqPrepAgarose 8 g An activity detection solution of 1 / l was applied, and a purple-colored strain was collected as an AMP esterase activity (referred to as esterase activity for α-AMP, hereinafter the same) strain.

  As for the activity unit of the enzyme used in the present invention, 1 μmole α-AP was added per minute under the conditions of reaction at 20 ° C. using 100 mM phosphate buffer (pH 7.0) containing 50 mM α-AMP. The amount of enzyme produced was defined as 1 unit (U).

  The strain in which AMP esterase activity was confirmed by the colorimetric method was cultured at 37 ° C. for 16 hours in a test tube in which 3 ml of LB medium containing 50 mg / l chloramphenicol had been applied. It has 0.1 U of AMP esterase activity per 1 ml of culture solution. It was confirmed that it was expressed in E. coli. In addition, the activity was not detected in the transformant into which only pUC118 was introduced as a control.

  A plasmid possessed by Escherichia coli JM109 was prepared from the above strains confirmed to have AMP esterase activity using Wizard Plus Minipreps DNA Purification System (Promega), and the nucleotide sequence of the inserted DNA fragment was determined. The sequence reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis was performed using CEQ 2000-XL (Beckman Coulter, Inc.).

  As a result, it was confirmed that an open reading frame encoding a protein having homology to esterase exists and is a gene encoding α-AMP esterase. The base sequence of the full length of the AMP esterase gene and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. The resulting open reading frame was BLASTP. As a result of the homology analysis by the program, 56% homology was detected with Proteobacterium strain NCIMB-40700-derived piperidined carboxylate esters (WO9802556).

  The amino acid sequence of SEQ ID NO: 2 described in the sequence listing was analyzed with the SignalP v3.0 program (J. Mol. Biol., 340: 783-795, 2004., Improved prediction of signal peptides: SignalP 3.0.). However, it was predicted that the amino acid sequence from the 1st to the 13th or the 1st to the 24th would function as a signal and secrete, and the mature protein was estimated to be downstream from the 14th or 25th.

Example 3 Preparation of Tetrathiobacter sp.-derived AMP esterase (T_est) gene expression strain PCR using the promoter region of the trp operon on Escherichia coli W3110 chromosomal DNA as the primers shown in SEQ ID NO: 3 and SEQ ID NO: 4 The target gene region was amplified by ligation, and the obtained DNA fragment was ligated to a pGEM-Teasy vector (manufactured by Promega). With this ligation solution E. coli JM109 was transformed, and a strain having the target plasmid in which the direction of the trp promoter was inserted in the opposite direction to the lac promoter was selected from ampicillin resistant strains. Next, this plasmid was ligated with a DNA fragment containing the trp promoter obtained by treating with EcoO109I / EcoRI and an EcoO109I / EcoRI-treated product of pUC19 (manufactured by Takara). Escherichia coli JM109 was transformed with this ligation solution, and a strain having the target plasmid was selected from ampicillin resistant strains. Next, a DNA fragment obtained by treating this plasmid with HindIII / PvuII and a DNA fragment containing rrnB terminator obtained by treating pKK223-3 (manufactured by Amersham Pharmacia) with HindIII / HincII were ligated. With this ligation solution E. coli JM109 was transformed, and a strain having the target plasmid was selected from ampicillin resistant strains. Next, a DNA fragment containing the trp promoter obtained by treating this plasmid with EcoO109I / HindIII, a DNA fragment obtained by treating with pCOV109I / PvuI of pSTV28 (manufactured by Takara), and pKK223-3 by HindIII / PvuI. And the obtained DNA fragment containing the rrnB terminator was ligated. With this ligation solution E. coli JM109 was transformed, a strain having the target plasmid was selected from chloramphenicol resistant strains, and this plasmid was named pTrp6.

  The target gene was amplified by PCR using the chromosomal DNA of Tetrathiobacter sp. FERM P-21102 strain as a template and the oligonucleotides shown in SEQ ID NO: 5 and SEQ ID NO: 6 as primers. This DNA fragment was treated with AseI / PstI, and the obtained DNA fragment was ligated with the NdeI / PstI-treated product of pTrp6. Escherichia coli JM109 was transformed with this ligation solution, a strain having the target plasmid was selected from chloramphenicol resistant strains, and this plasmid was named pBE110.

  Escherichia coli JM109 having pBE110 was seed-cultured in LB medium containing 50 mg / l chloramphenicol at 37 ° C. for 16 hours. 1 ml of the obtained culture solution was added to 50 ml of a medium (2 g / l D-glucose, 10 g / l yeast extract, 10 g / l casamino acid, 5 g / l ammonium sulfate, 3 g / l potassium dihydrogen phosphate, 1 g / l phosphate. A 500 ml Sakaguchi flask filled with dipotassium hydrogen, 0.5 g / l magnesium sulfate heptahydrate, 50 mg / l chloramphenicol) was seeded, and main culture was performed at 37 ° C. for 16 hours. It has 10 U of AMP esterase activity per 1 ml of the culture solution. It was confirmed that it was expressed in E. coli. As a control, no activity was detected in the transformant into which only pTrp6 was introduced.

Example 4 Purification of AMP esterase from Brevundimonas sp. Medium containing 1 g glucose, 5 g ammonium sulfate, 1 g monopotassium phosphate, 3 g dipotassium phosphate, 0.1 g magnesium sulfate, 10 g yeast extract, 10 g peptone in 1 liter ( pH 6.2) 50 mL was dispensed into a 500 mL Sakaguchi flask and sterilized at 115 ° C. for 15 minutes (medium 3). One platinum loop of Brevundimonas sp. NRRL B-2395 strain cultured in the same medium at 30 ° C. for 16 hours was inoculated into the medium 3, and shaking culture was performed at 30 ° C. and 120 reciprocations / minute for 16 hours.

  Hereinafter, the operations after centrifugation were performed on ice or at 4 ° C. The obtained culture solution was centrifuged (10,000 rpm, 15 minutes), and the cells were collected. 16 g of cells were washed with a buffer solution (50 mM Tris-HCl (pH 8.0), 10 mM EDTA) composed of 50 mM Tris-HCl (pH 8.0) and 10 mM ethylenediaminetetraacetic acid, suspended in 40 ml of the same buffer, and 195 W For 45 minutes. This ultrasonic disruption liquid was centrifuged (10,000 rpm, 30 minutes), and the ultrasonic disruption liquid supernatant was obtained by removing the fragment of disrupted cells. The sonicated supernatant was dialyzed overnight against 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA, and the insoluble fraction was removed by ultracentrifugation (54,000 rpm, 30 minutes). A soluble fraction was obtained as a clear liquid. The obtained soluble fraction was applied to a Q-Sepharose 26/10 column (manufactured by Amersham) previously equilibrated with 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA. After elution of non-adsorbed protein, the adsorbed protein was eluted by linearly changing the NaCl concentration in the buffer from 0M to 1M. By this operation, an active fraction near 0.1M was collected. The obtained Q-Sepharose fraction was concentrated, dialyzed against 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and then subjected to Superdex 200 (Amersham) pre-equilibrated with the same buffer. By this operation, the activity was confirmed and recovered at the elution position estimated to have a molecular weight of 20-25 kDa. Furthermore, the Superdex fraction was concentrated and applied to a Q-Sepharose 16/10 column (manufactured by Amersham) previously equilibrated with 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA. After elution of non-adsorbed protein, the adsorbed protein was eluted by linearly changing the NaCl concentration in the buffer from 0M to 0.3M. By this operation, an active fraction around 0.05M was collected. Finally, the Q-Sepharose fraction was concentrated, dialyzed against a buffer solution consisting of 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.75 M ammonium sulfate, and equilibrated with Resource ethyl (Amersham). (Made by company). After elution of non-adsorbed protein, the adsorbed protein was eluted by linearly changing the ammonium sulfate concentration in the buffer from 0.75 M to 0 M. By this operation, AMP esterase activity was detected at around 0.4M. The active fraction was collected. By these operations, it was confirmed that the AMP deesterification enzyme obtained in Example 4 was uniformly purified from the results of electrophoresis experiments. The activity recovery rate in the above purification step was 37%, and the degree of purification was 5.06 times.

Example 5 Production of α-AP using Brevundimonas sp. AMP esterase 2 μl of the Resource ethyl fraction enzyme (about 3.3 U / ml) obtained in Example 4 was added to 20 mM α-L-aspartyl-L. -Phenylalanine-β-methyl ester (α-AMP), 100 mM Tris-HCl (pH 8.0) containing 10 mM EDTA, and reacted at 30 ° C. for 1 hour. Formation of α-L-aspartyl-L-phenylalanine (α-AP) was confirmed. In addition, the production | generation of (alpha) -AP in the enzyme no addition group was hardly recognized.

Example 6 Collection of AMP esterase gene derived from Brevundimonas sp. Brevundimonas sp. / L agar, pH 7.0) and cultured at 30 ° C. for 24 hours. One microbial ear of this bacterial cell was inoculated into a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was placed, and cultured at 30 ° C. with shaking.

  50 ml of the culture solution was centrifuged (12,000 rpm, 4 ° C., 15 minutes) and collected. Using QIAGEN Genomic-tip System (Qiagen), chromosomal DNA was obtained from the cells based on the method described in the manual.

  Hereinafter, although isolation of the AMP esterase gene is described, Brevundimonas sp NRRL B-2395 strain was used as the microorganism. For isolation of the gene, Escherichia coli JM-109 was used as a host, and pUC18 was used as a vector.

  A mixed primer having a base sequence shown in SEQ ID NO: 8 was prepared based on the amino acid sequence (SEQ ID NO: 7) determined by Edman degradation of AMP esterase derived from the aforementioned Brevundimonas sp NRRL B-2395 strain. A mixed primer having the base sequence shown in SEQ ID NO: 11 based on the amino acid sequence (SEQ ID NO: 9) determined by Edman degradation of a digested product of AMP esterase derived from lysyl endopeptidase derived from Brevundimonas sp NRRL B-2395 strain It was created.

  A DNA fragment containing a part of the AMP esterase gene derived from Brevundimonas sp. NRRL B-2395 strain was obtained by PCR using LA-Taq (Takara Shuzo). PCR reaction was performed on the chromosomal DNA obtained from Brevundimonas sp NRRL B-2395 strain using primers having the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 11.

The PCR reaction was performed using Takara PCR Thermal Cycler PERSONAL (Takara Shuzo), and the reaction under the following conditions was performed for 30 cycles.
94 ° C 30 seconds 52 ° C 1 minute 72 ° C 1 minute

  After the reaction, 3 μl of the reaction solution was subjected to 2% agarose electrophoresis. As a result, it was confirmed that a DNA fragment of about 800 bp was amplified.

  In order to obtain the full length of the α-AMP esterase gene, first, primers having the nucleotide sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 were prepared from the sequence information of the above-mentioned approximately 800 bp DNA fragment. . PCR was performed under the above PCR conditions, and Southern hybridization was performed using the amplified DNA fragment as a probe. Southern hybridization procedures are described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989).

  The 240 bp and 430 bp DNA fragments amplified by the PCR were separated by 2% agarose electrophoresis. The target band was cut out and purified. Using DIG High Prime (manufactured by Boehringer Mannheim), the two DNA fragments were labeled with digoxinigen on the probe based on the instructions.

  Chromosomal DNA of Brevundimonas sp NRRL B-2395 strain was reacted with the restriction enzyme BamHI at 37 ° C. for 16 hours for complete digestion, and then electrophoresed on a 0.8% agarose gel. The agarose gel after electrophoresis was blotted onto a nylon membrane filter Nylon membranes positively charged (manufactured by Roche Diagnostics), and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 50 ° C. for 1 hour, and then the digoxinigen-labeled probe prepared above was added, and hybridization was carried out at 50 ° C. for 16 hours. Thereafter, the filter was washed with 2 × SSC containing 0.1% SDS at room temperature for 20 minutes. Further, washing was performed twice at 65 ° C. for 15 minutes with 0.1 × SSC containing 0.1% SDS.

  Detection of the band hybridizing with the probe was performed based on the instructions using a DIG Nucleotide Detection Kit (manufactured by Boehringer Mannheim). As a result, bands of about 5 kb and 4.5 kb hybridized with the probe could be detected.

  5 μg of chromosomal DNA of Brevundimonas sp. NRRL B-2395 strain was completely digested with BamHI. About 4-5 kb of DNA was separated by 0.8% agarose gel electrophoresis, and the DNA was purified using Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. 4 μl of this was mixed with pUC118 BamHI / BAP (Takara Shuzo), and DNA Ligation Kit Ver. The ligation reaction was performed using 2 (Takara Shuzo). Escherichia coli was transformed by mixing 5 μl of this ligation reaction solution and 100 μl of Escherichia coli JM109 competent cell (manufactured by Toyobo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.

  In order to obtain the full length of the AMP esterase gene, a chromosomal DNA library was screened by colony hybridization using the above probe. The operation of colony hybridization is described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989).

  The colonies of the chromosomal DNA library were transferred to a nylon membrane filter Nylon Membranes for Colony and Place Hybridization (Roche Diagnostics), and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C. for 1 hour, and then the above labeled probe with digoxinigen was added, followed by hybridization at 50 ° C. for 16 hours. Thereafter, the filter was washed with 2 × SSC containing 0.1% SDS at room temperature for 20 minutes. Further, washing was performed twice at 65 ° C. for 15 minutes with 0.1 × SSC containing 0.1% SDS.

  Detection of colonies that hybridized with the labeled probe was performed based on the instructions using DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, 4 and 5 colonies that hybridized with the labeled probe were confirmed.

  A plasmid possessed by Escherichia coli JM109 was prepared using Wizard Plus Minipreps DNA Purification System (manufactured by Promega) from the above two strains that were confirmed to hybridize with the labeled probe, and nearby bases that hybridized with the probe. The sequence was determined. The sequence reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis was performed using CEQ 2000-XL (Beckman Coulter, Inc.).

  As a result, an open reading frame encoding a protein containing the N-terminal amino acid sequence (SEQ ID NO: 7) and internal amino acid sequence (SEQ ID NO: 9 and SEQ ID NO: 10) of AMP esterase exists, and the gene encodes AMP esterase. It was confirmed. The nucleotide sequence of the full length of the AMP esterase gene and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 16 and SEQ ID NO: 17. The resulting open reading frame was BLASTP. As a result of the homology analysis by the program, the esterase 54 of Bacillus niacini (J. Mol. Catal., B Enzym. 27 (4-6), 237-241 (2004)) is 37% in terms of amino acid sequence, Gene 329 (Complete), 187-195 (2004)) showed 35% homology with the amino acid sequence.

  The amino acid sequence of SEQ ID NO: 17 described in the sequence listing was analyzed with the SignalP v3.0 program (J. Mol. Biol., 340: 783-795, 2004., Improved prediction of signal peptides: SignalP 3.0.). However, it was predicted that the first to 25th amino acid sequence functions as a signal and secreted, and the mature protein was estimated to be downstream from the 26th amino acid sequence.

Example 7 Preparation of Brevundimonas sp-derived AMP esterase (V_est) expression strain Gene of interest by PCR using chromosomal DNA of Brevundimonas sp NRRL B-2395 strain as a template and oligonucleotides shown in SEQ ID NO: 18 and SEQ ID NO: 19 as primers Was amplified. This DNA fragment was treated with SacI / HindIII, and the resulting DNA fragment was ligated with the SacI / HindIII-treated product of pSTV28 (Takara). Escherichia coli JM109 was transformed with this ligation solution, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pBre-2.

  Escherichia coli JM109 having pBre-2 was seed-cultured at 30 ° C. for 24 hours in an LB medium containing 50 mg / l chloramphenicol. 1 ml of the obtained culture solution was added to 50 ml of medium (2 g / l D-glucose, 10 g / l yeast extract, 10 g / l casamino acid, 5 g / l ammonium sulfate, 3 g / l potassium dihydrogen phosphate, 1 g / l hydrogen phosphate. A 500 ml Sakaguchi flask filled with dipotassium, 0.5 g / l magnesium sulfate heptahydrate, 50 mg / l ampicillin) was seeded, and main culture was performed at 30 ° C. for 24 hours. It has 0.08 U of AMP esterase activity per 1 ml of culture solution. It was confirmed that it was expressed in E. coli. As a control, no activity was detected in the transformant into which only pSTV28 was introduced.

Example 8
The AMP esterase gene-expressing strain was cultured at 20 ° C. for 48 hours on an L-agar medium containing ampicillin (100 mg / l). Next, 1/4 plate of cells was transferred to 50 ml of L-medium (peptone 10 g / l, yeast extract 5 g / l, NaCl 10 g / l). 3 ml of culture solution (OD = 0.15, 26-fold dilution) cultured for 16 hours at 22 ° C. using a shaker (140 rpm) is inoculated into 300 ml of medium having the composition shown below, and a laboratory with a capacity of 1.0 L Batch culture was carried out in the fermenter for use while stirring and aeration (1/1 vvm) at a rotation speed of 700 rpm. Inoculate 15 ml of culture solution (OD610 = 0.60, 51-fold dilution) after sugar breakage into 300 ml of medium with the same composition, and sugar feed culture at 20 ° C. with stirring aeration (1/1 vvm) using the same fermenter Went. The pH value was automatically adjusted to 7.0 with gaseous ammonia.

Medium composition (g / l):
Glucose 25.0
MgSO ₄ · 7H ₂ O 1.0
(NH ₄ ) ₂ SO ₄ 5.0
FeSO ₄ · 7H ₂ O 0.05
MnSO ₄ .5H ₂ O 0.05
Mameno (TN) 0.45
GD113 0.1

  Glucose and magnesium sulfate were sterilized separately. The pH of the other components was adjusted to 5.0 with KOH.

Feed sugar solution composition (g / l)
Glucose 500
pH irregularity

  25 ml of this culture solution was centrifuged (2200 g × 15 minutes) to obtain a bacterial cell precipitate. To this, 25 ml of water was added to obtain a cell suspension.

Example 9
112.5 g of L-phenylalanine and 2250 g of water were added to adjust the pH to 8.7 with 6 ml of 10M aqueous ammonia. 223.9 g of L-aspartic acid dimethyl ester monomethyl sulfate solution was added while maintaining the pH at 8.7 with 10 M aqueous ammonia. After adding water to make 2770 ml in total, 230 ml of the condensing enzyme cell suspension was added and stirred at 20 ° C. for 30 minutes. During the reaction, the pH was maintained at 8.7 by adding 10 M aqueous ammonia. After completion of the condensation reaction, 61.89 g of 95% sulfuric acid was added to obtain pH = 3.0. The mixture was heated to 40 ° C. and stirred for 1 hour. After the reaction solution temperature was raised to 20 ° C., 88 ml of 10M aqueous ammonia was added to adjust the pH to 7.0. 150 ml of AMP esterase enzyme cell suspension was added and stirred at 20 ° C. for 7 hours. During the reaction, pH was maintained at 7.0 by adding 10 M aqueous ammonia. After completion of the reaction, the enzyme reaction solution was adjusted to pH = 6.0 with 95% sulfuric acid and then stirred at 70 ° C. for 80 minutes. After removing the cells by membrane filtration (PELLICON2 “MINI” Filter, molecular weight cut off 100,000), the enzyme reaction solution was analyzed by HPLC. As a result, 146.2 g of α-L-aspartyl-L-phenylalanine (yield 76.8). %) And 20.5 g of L-phenylalanine.

Example 10
2323 g of the enzyme reaction solution obtained after removing the cells obtained in Example 9 was concentrated at 200 hPa and 60 ° C. for 1.5 hours to obtain 1072 g. 287 g of the concentrated liquid was heated to 40 ° C., and then 5.2 g of 95% sulfuric acid was added to adjust the pH to 3.3. The remaining concentrate was added over 3 hours. During the addition, the pH was maintained at 3.3 by adding 95% sulfuric acid. After the addition of the concentrated liquid was completed, the mixture was stirred at 40 ° C. for 3 hours, and then the precipitated crystals were separated by filtration to obtain 92.4 g (yield 92.1%) of α-L-aspartyl-L-phenylalanine.

Example 11
To 122 g of crystals obtained in Example 10 (containing 60.1 g of α-L-aspartyl-L-phenylalanine) was added 27 ml of MeOH, 104 ml of 35% hydrochloric acid, and 36 ml of water, and then 24 hours at 35 ° C. and 48 hours at 25 ° C. The mixture was stirred at 10 ° C. for 24 hours. The precipitated crystals were separated by filtration to obtain 57.1 g (yield: 80.2%) of α-L-aspartyl-L-phenylalanine-α-methyl ester hydrochloride.

Example 12
44.9 g of α-L-aspartyl-L-phenylalanine-α-methyl ester hydrochloride obtained in Example 11 was dissolved in 530 ml of water, and then adjusted to pH = 4.9 with an 18 wt% aqueous sodium carbonate solution. The precipitated crystals were separated by filtration to obtain 33.6 g (yield 84.2%) of α-L-aspartyl-L-phenylalanine-α-methyl ester.

SEQ ID NO: 1: Amino acid sequence corresponding to the base sequence of the AMP esterase gene (T_est) SEQ ID NO: 2: Amino acid sequence of the AMP esterase gene (T_est) SEQ ID NO: 3: 1 for trp promoter amplification
Sequence number 4: 2 for trp promoter amplification
SEQ ID NO: 5: AMP esterase gene (T_est) amplification primer 1
SEQ ID NO: 6: AMP esterase gene (T_est) amplification primer 2
SEQ ID NO: 7: N-terminal amino acid sequence of AMP esterase (V_est) SEQ ID NO: 8: Mix primer 1 of AMP esterase (V_est)
SEQ ID NO: 9: Internal amino acid sequence 1 of AMP esterase (V_est)
SEQ ID NO: 10: Internal amino acid sequence 2 of AMP esterase (V_est)
Sequence number 11: Mix primer 2 of AMP esterase (V_est)
Sequence number 12: Primer 1 for probe preparation of AMP esterase (V_est)
Sequence number 13: Primer 2 for probe preparation of AMP esterase (V_est)
Sequence number 14: Primer 3 for probe preparation of AMP esterase (V_est)
Sequence number 15: Primer 4 for probe preparation of AMP esterase (V_est)
SEQ ID NO: 16: Amino acid sequence corresponding to the base sequence of AMP esterase (V_est) SEQ ID NO: 17: Amino acid sequence of AMP esterase (V_est) SEQ ID NO: 18: Primer 1 for preparing an AMP esterase (V_est) expression strain
SEQ ID NO: 19: Primer 2 for preparing an AMP esterase (V_est) expression strain

Claims (14)

  α-L-aspartyl-L-phenylalanine-β-ester is converted from α-L-aspartyl-L-phenylalanine-β-ester to α-L-aspartyl- A method for producing α-L-aspartyl-L-phenylalanine, characterized by producing L-phenylalanine.   A culture of a microorganism having the ability of the enzyme or enzyme-containing material to deesterify α-L-aspartyl-L-phenylalanine-β-ester, a microbial cell separated from the culture, and a fungus of the microorganism The method for producing α-L-aspartyl-L-phenylalanine according to claim 1, which is one or more selected from the group consisting of body-treated products.   The method for producing α-L-aspartyl-L-phenylalanine according to claim 2, wherein the microorganism is a microorganism belonging to a genus selected from the group consisting of Tetrathiobacter and Brevandimonas. The α-L-aspartyl-L according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing a protein selected from the group consisting of the following (A) to (J). -Process for producing phenylalanine.
(A) Protein having the amino acid sequence of amino acid residues 14 to 340 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing (B) Amino acid residue number 14 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing ˜340 amino acid sequence, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and de-esterifying α-L-aspartyl-L-phenylalanine-β-ester Among the amino acid sequences described in SEQ ID NO: 2 in the protein (D) sequence listing, the protein having the amino acid sequence of amino acid residues 25 to 340 among the amino acid sequences listed in SEQ ID NO: 2 in the protein (C) sequence listing Including the substitution, deletion, and / or insertion of one or several amino acids in the amino acid sequence of amino acid residues 25 to 340 A protein having an amino acid sequence and an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester (E). Substitution or deletion of one or several amino acids in the amino acid sequence of amino acid residues Nos. 26 to 550 in the amino acid sequence of SEQ ID No. 17 of the protein (F) sequence listing having the amino acid sequence of Nos. 26 to 550, And / or protein (G) having an amino acid sequence containing an insertion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The amino acid sequence shown in SEQ ID NO: 17 in the protein (H) sequence list having the amino acid sequence shown in SEQ ID NO: 17 A maturation having an amino acid sequence comprising one or several amino acid substitutions, deletions and / or insertions and having an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester Substitution or deletion of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the protein (J) sequence list having the amino acid sequence shown in SEQ ID NO: 2 in the protein (I) sequence list including the protein region, And / or a protein comprising a mature protein region having an amino acid sequence containing an insertion and having an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester The α-L-aspartyl-L- according to claim 2, wherein the microorganism is a transformed microorganism into which a polynucleotide selected from the group consisting of the following (a) to (j) is introduced. A method for producing phenylalanine.
(A) a polynucleotide having a base sequence of base numbers 100 to 1080 among the base sequences set forth in SEQ ID NO: 1 of the sequence listing (b) of base sequences 100 to 1080 of the base sequences set forth in SEQ ID NO: 1 of the sequence listing Poly that encodes a protein that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence under stringent conditions and has an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester Nucleotide (c) Polynucleotide having base sequence of base numbers 133 to 1080 among the base sequence set forth in SEQ ID NO: 1 of the sequence listing (d) Base numbers 133 to 1080 of the base sequence set forth in SEQ ID NO: 1 of the sequence listing Hybridizes under stringent conditions with polynucleotides that are complementary to the base sequence of And a polynucleotide encoding a protein having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (e) Among the base sequences set forth in SEQ ID NO: 16 of the sequence listing, base numbers 114 to A polynucleotide having a base sequence of 1688 (f) Hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 114 to 1688 among the base sequence set forth in SEQ ID NO: 16 of the sequence listing Polynucleotide encoding a protein that is soybean and has an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (g) Base number 39 of the base sequence set forth in SEQ ID NO: 16 in the Sequence Listing A polynucleotide having a base sequence of ˜1688 (h) in SEQ ID NO: 16 of the sequence listing Hybridize under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 39 to 1688 among the listed base sequences, and α-L-aspartyl-L-phenylalanine-β-ester Polynucleotide encoding a protein having deesterification activity (i) Polynucleotide having base sequence of base numbers 61 to 1080 among base sequences set forth in SEQ ID NO: 1 of sequence listing (j) SEQ ID NO: 1 of sequence listing Among the base sequences described in the above, and hybridizing under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 61 to 1080, and α-L-aspartyl-L-phenylalanine-β-ester Encodes a protein containing a mature protein region having the activity of deesterifying Polynucleotide The method for producing α-L-aspartyl-L-phenylalanine according to claim 1, wherein the enzyme is at least one selected from the group consisting of the following (A) to (J).
(A) Protein having the amino acid sequence of amino acid residues 14 to 340 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing (B) Amino acid residue number 14 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing ˜340 amino acid sequence, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and de-esterifying α-L-aspartyl-L-phenylalanine-β-ester Among the amino acid sequences described in SEQ ID NO: 2 in the protein (D) sequence listing, the protein having the amino acid sequence of amino acid residues 25 to 340 among the amino acid sequences listed in SEQ ID NO: 2 in the protein (C) sequence listing Including the substitution, deletion, and / or insertion of one or several amino acids in the amino acid sequence of amino acid residues 25 to 340 A protein having an amino acid sequence and an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester (E). Substitution or deletion of one or several amino acids in the amino acid sequence of amino acid residues Nos. 26 to 550 in the amino acid sequence of SEQ ID No. 17 of the protein (F) sequence listing having the amino acid sequence of Nos. 26 to 550, And / or protein (G) having an amino acid sequence containing an insertion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The amino acid sequence shown in SEQ ID NO: 17 in the protein (H) sequence list having the amino acid sequence shown in SEQ ID NO: 17 A protein having an amino acid sequence including substitution, deletion and / or insertion of one or several amino acids, and having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (I) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (J) substitution, deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing A protein comprising a mature protein region having an amino acid sequence comprising and having an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester   A reaction step of synthesizing α-L-aspartyl-L-phenylalanine by the method for producing α-L-aspartyl-L-phenylalanine according to any one of claims 1 to 6, and α-L-aspartyl-L- And a reaction step of converting phenylalanine to α-L-aspartyl-L-phenylalanine-α-methyl ester. A method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester.   A protein having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester, which is derived from a microorganism belonging to a genus selected from the group consisting of Tetrathiobacter and Brevandimonas. A protein selected from the group consisting of the following (A) to (J).
(A) Protein having the amino acid sequence of amino acid residues 14 to 340 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing (B) Amino acid residue number 14 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing ˜340 amino acid sequence, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and de-esterifying α-L-aspartyl-L-phenylalanine-β-ester Among the amino acid sequences described in SEQ ID NO: 2 in the protein (D) sequence listing, the protein having the amino acid sequence of amino acid residues 25 to 340 among the amino acid sequences listed in SEQ ID NO: 2 in the protein (C) sequence listing Including the substitution, deletion, and / or insertion of one or several amino acids in the amino acid sequence of amino acid residues 25 to 340 A protein having an amino acid sequence and an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester (E). Substitution or deletion of one or several amino acids in the amino acid sequence of amino acid residues Nos. 26 to 550 in the amino acid sequence of SEQ ID No. 17 of the protein (F) sequence listing having the amino acid sequence of Nos. 26 to 550, And / or protein (G) having an amino acid sequence containing an insertion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The amino acid sequence shown in SEQ ID NO: 17 in the protein (H) sequence list having the amino acid sequence shown in SEQ ID NO: 17 A protein having an amino acid sequence including substitution, deletion and / or insertion of one or several amino acids, and having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (I) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (J) substitution, deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing A protein comprising a mature protein region having an amino acid sequence comprising and having an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester   A polynucleotide encoding the protein according to claim 9. A polynucleotide selected from the group consisting of the following (a) to (j).
(A) a polynucleotide having a base sequence of base numbers 100 to 1080 among the base sequences set forth in SEQ ID NO: 1 of the sequence listing (b) of base sequences 100 to 1080 of the base sequences set forth in SEQ ID NO: 1 of the sequence listing Poly that encodes a protein that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence under stringent conditions and has an activity to deesterify α-L-aspartyl-L-phenylalanine-β-ester Nucleotide (c) Polynucleotide having base sequence of base numbers 133 to 1080 among the base sequence set forth in SEQ ID NO: 1 of the sequence listing (d) Base numbers 133 to 1080 of the base sequence set forth in SEQ ID NO: 1 of the sequence listing Hybridizes under stringent conditions with polynucleotides that are complementary to the base sequence of And a polynucleotide encoding a protein having an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (e) Among the base sequences set forth in SEQ ID NO: 16 of the sequence listing, base numbers 114 to A polynucleotide having a base sequence of 1688 (f) Hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 114 to 1688 among the base sequence set forth in SEQ ID NO: 16 of the sequence listing Polynucleotide encoding a protein that is soybean and has an activity of deesterifying α-L-aspartyl-L-phenylalanine-β-ester (g) Base number 39 of the base sequence set forth in SEQ ID NO: 16 in the Sequence Listing A polynucleotide having a base sequence of ˜1688 (h) in SEQ ID NO: 16 of the sequence listing Hybridize under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 39 to 1688 among the listed base sequences, and α-L-aspartyl-L-phenylalanine-β-ester Polynucleotide encoding a protein having deesterification activity (i) Polynucleotide having base sequence of base numbers 61 to 1080 among base sequences set forth in SEQ ID NO: 1 of sequence listing (j) SEQ ID NO: 1 of sequence listing Among the base sequences described in the above, and hybridizing under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of base numbers 61 to 1080, and α-L-aspartyl-L-phenylalanine-β-ester Encodes a protein containing a mature protein region having the activity of deesterifying Polynucleotide   The polynucleotide according to claim 11, wherein the stringent condition is a condition in which washing is performed at 60 ° C. at a salt concentration corresponding to 1 × SSC and 0.1% SDS.   A recombinant polynucleotide comprising the polynucleotide according to any one of claims 10 to 12.   A transformed cell into which the polynucleotide according to claim 13 has been introduced.