JP2005269905A - Protein having peptide production activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an efficient method for producing a peptide and to provide a peptide-producing protein, etc., therefor. <P>SOLUTION: The peptide is produced by reacting an amine component with a carbonyl component in the presence of at least one of of the following proteins (A) and (B). (A) A protein having a specific amino acid sequence and (B) a protein having the 20th amino acid residue that is alanine in the specific amino acid sequence and an amino acid sequence containing one or several mutations of amino acids selected from the group consisting of substitution, deletion, insertion, addition and inversion in a region other than the alanine and the peptide production activity. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a protein having peptide generation activity (hereinafter also referred to as “peptide generation protein”), and more particularly to a peptide generation protein excellent in gene expression and a method for producing a peptide using this protein.

  Peptides are used in various fields such as pharmaceuticals and foods. For example, L-alanyl-L-glutamine is widely used as a component of infusion solutions and serum-free media because it is more stable and more water-soluble than L-glutamine.

As a method for producing a peptide, a chemical synthesis method has been conventionally known, but the production method has not always been satisfactory in terms of simplicity and efficiency.
On the other hand, methods for producing peptides using enzymes have also been developed (for example, Patent Documents 1 and 2). However, the conventional method for producing a peptide using an enzyme leaves room for improvement in that the peptide production rate is extremely slow and the peptide production yield is low. Under such circumstances, development of an industrially efficient production method of these peptides has been desired.

EP 278787 A1 Gazette EP 359399 B1

  An object of the present invention is to provide an efficient method for producing a peptide, a peptide-forming protein for the same, and the like.

  As a result of intensive studies, the present inventors succeeded in isolating a gene encoding a protein having peptide-forming activity derived from Empedobacter, and further improving the expression efficiency by modifying a specific site of the amino acid sequence or base sequence. As a result, the present invention has been completed. That is, the present invention provides the following protein and a method for producing a peptide using the protein.

[1] A protein shown in the following (A) or (B).
(A) Protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (B) Substitution, deletion, insertion, addition to a region other than the 20th alanine in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And a protein having an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of inversions and having peptide-forming activity [2] encodes a protein shown in the following (A) or (B) Polynucleotide.
(A) Protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (B) Substitution, deletion, insertion, addition to a region other than the 20th alanine in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And a protein having an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of inversions and having peptide-forming activity [3] A polynucleotide shown in (a) or (b) below.
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 in the sequence listing (b) a base sequence that hybridizes under stringent conditions with a polynucleotide complementary to the base sequence set forth in SEQ ID NO: 1 in the sequence listing A polynucleotide corresponding to the 20th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2 of the Sequence Listing encodes alanine and encodes a protein having peptide-forming activity [4] Item 4. A recombinant polynucleotide in which the polynucleotide according to Item 2 or 3 is incorporated.
[5] A transformant into which the recombinant polynucleotide according to claim 4 is introduced and expresses a protein having peptide-forming activity.
[6] The transformant according to claim 5 is a microorganism, wherein the microorganism is cultured in a medium, and a protein having peptide-forming activity is accumulated in the medium and / or the microorganism. Protein production method.
[7] The transformant described in claim 5 is a microorganism, and is selected from the group consisting of a culture of the microorganism, a microbial cell separated from the culture, and a treated product of the microorganism, or A method for producing a peptide, comprising using two or more kinds to produce a peptide from an amine component and a carbonyl component.
[8] A method for producing a peptide, wherein a peptide is produced from an amine component and a carbonyl component in the presence of at least one of the proteins shown in (A) and (B) below.
(A) Protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (B) Substitution, deletion, insertion, addition to a region other than the 20th alanine in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And a protein having an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of inversions and having peptide-forming activity

  ADVANTAGE OF THE INVENTION According to this invention, the peptide production active protein excellent in expression efficiency and the manufacturing method of an efficient peptide are provided.

Hereinafter, embodiments of the present invention will be described together with the best mode.
The following various genetic engineering techniques are described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989), Cell Engineering Handbook, edited by Toshio Kuroki et al., Yodosha (1992), New Genetic Engineering Handbook revised. There are many standard experimental manuals such as the third edition, edited by Muramatsu et al., Yochisha (1999), and those skilled in the art can implement them by referring to these documents.

1. The protein which has the peptide production activity of this invention The peptide production protein of this invention is a protein shown to the following (A) or (B).
(A) Protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (B) Substitution, deletion, insertion, addition to a region other than the 20th alanine in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And a protein having an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of inversions, and having peptide-forming activity. The sequence number in the sequence listing shall be indicated.

  Peptide generating activity is an activity of forming a peptide bond from two or more substances to generate a new compound having a peptide bond. More specifically, for example, from two amino acids or esters thereof, at least It refers to the activity of producing a peptide compound with one increased peptide bond.

  In the protein (A) above, the 20th amino acid residue from the N-terminus is alanine. The protein of (A) above is the 20th amino acid residue in the mature protein site of the amino acid sequence shown in SEQ ID NO: 8 (corresponding to the 42nd in SEQ ID NO: 8), valine was substituted with alanine (In this specification, the fact that the 20th valine is substituted with alanine in this specification is expressed as “V20A”, “original amino acid, SEQ ID NO: modified amino acid”) May be displayed in a symbolic order). The protein having the amino acid sequence shown in SEQ ID NO: 8 is a protein having peptide-forming activity derived from Empedobacter brevis. The peptide-generating protein of the present invention has better expression efficiency in transformants such as microorganisms than the original peptide-generating protein derived from Empedobacter brevis.

  The introduction of a mutation that replaces the 20th amino acid residue from valine to alanine is performed by, for example, site-directed mutagenesis so that the amino acid at a specific site of the gene encoding this protein is replaced. It is obtained by modifying. A polypeptide having the modified base sequence as described above can also be obtained by a conventionally known mutation treatment. Examples of the mutation treatment include, for example, a method in which DNA encoding the protein (A) is treated in vitro with hydroxylamine, a method of introducing mutation by error-prone PCR, and the DNA in a host deficient in a mutation repair system. Examples include a method of amplifying and recovering a DNA having a mutation introduced therein.

  The present invention also provides a protein that is substantially the same as the protein shown in (A) above. Specifically, the protein shown in (B) is provided. 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. However, in the case of an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of (B) one or several substitutions, deletions, insertions, additions and inversions in the amino acid sequence of the protein, 50 It is desirable that the enzyme activity is maintained at about half or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more of the protein (A) under the conditions of ° C and pH 8.

  Also in the protein of (B), the 20th amino acid residue when the N-terminal amino acid residue is the first in the amino acid sequence shown in SEQ ID NO: 2 is alanine. That is, the introduction of a mutation in the protein (B) allows a mutation at a site other than alanine, which is the 20th amino acid residue of the amino acid shown in SEQ ID NO: 2. Therefore, when a mutation such as deletion or insertion is introduced into a site other than the 20th alanine, the number of amino acid residues from the 20th alanine to the N-terminal or C-terminal is the state before the mutation is introduced. Can be different.

  The amino acid mutation as shown in (B) above is also performed by, for example, site-specific mutagenesis so that the amino acid at a specific site of the gene encoding this protein is substituted, deleted, inserted, added, etc. Is obtained by modifying. A polypeptide having the modified base sequence as described above can also be obtained by a conventionally known mutation treatment. Mutation treatment includes a method of treating DNA encoding (A) in vitro with hydroxylamine or the like, and an Escherichia bacterium carrying the DNA encoding (A) by ultraviolet irradiation or N-methyl-N′-nitro. -N-nitrosoguanidine (NTG) or a method of treating with a mutagen usually used for artificial mutation such as nitrous acid.

  In addition, mutations such as substitutions, deletions, insertions, additions, and inversions as described above include naturally occurring mutations such as differences between microorganism species or strains. By expressing the DNA having the mutation as described above in an appropriate cell and examining the enzyme activity of the expression product, DNA encoding a protein substantially identical to the protein described in SEQ ID NO: 2 can be obtained.

2. Polynucleotide of the Present Invention The polynucleotide encoding the protein of the present invention includes a polynucleotide having the base sequence set forth in SEQ ID NO: 1. The base sequence described in SEQ ID NO: 1 encodes the amino acid sequence described in SEQ ID NO: 2.

There may be a plurality of base sequences that define one amino acid sequence due to codon degeneracy. That is, the polynucleotide of the present invention includes a polynucleotide having a base sequence encoding the following protein.
(A) Protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (B) Substitution, deletion, insertion, addition to a region other than the 20th alanine in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And a protein having an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of inversions and having peptide-forming activity

  Furthermore, the following polynucleotide is mentioned as a polynucleotide substantially the same as DNA which has a base sequence shown to sequence number 1. Prepared from a polynucleotide encoding the protein having the amino acid sequence shown in SEQ ID NO: 2 or a cell holding the same, from a polynucleotide consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or the same base sequence Is substantially identical to the polynucleotide having the base sequence set forth in SEQ ID NO: 1 by isolating a polynucleotide that encodes a protein that has a peptide-forming activity and hybridizes with a probe having a stringent condition. Are obtained.

That is, examples of the polynucleotide of the present invention include polynucleotides shown in the following (a) or (b).
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 in the sequence listing (b) a base sequence that hybridizes under stringent conditions with a polynucleotide complementary to the base sequence set forth in SEQ ID NO: 1 in the sequence listing And a polynucleotide encoding a protein having a peptide generating activity, wherein the site corresponding to the 20th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing is alanine

  In the polynucleotide (b), the site corresponding to the 20th amino acid residue in the amino acid sequence of SEQ ID NO: 1 encodes alanine. As described above, in the protein of the present invention, a mutation may be introduced at a site other than the 20th amino acid residue in the amino acid sequence of SEQ ID NO: 2 within a range having a predetermined activity. Similarly, in the polynucleotide of the present invention, in a site other than the region encoding alanine, which is the 20th amino acid residue in the amino acid sequence of SEQ ID NO: 2, within the range satisfying the other requirements described in (b). The base sequence of SEQ ID NO: 1 may contain a mutation.

  The probe can be prepared by a standard 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, and cutting out and extracting the base sequence to be used as a probe with a restriction enzyme. The part to be cut out can be adjusted according to the target DNA.

  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 clearly quantify this condition, for example, highly homologous DNAs, for example, 50% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95%. % At 60 ° C., 1 × SSC, 0.1 which is a condition in which DNAs having a homology of at least% hybridize and DNAs having lower homology do not hybridize, or washing conditions for normal Southern hybridization % SDS, preferably 0.1 × SSC, and conditions for hybridizing at a salt concentration corresponding to 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.

  As described above, in the case of the polypeptide having the base sequence encoding the protein of (B) and the polypeptide of (b) above, the base sequence of SEQ ID NO: 1 is used under the conditions of 50 ° C. and pH 8. It is desirable that the peptide-forming activity is maintained at about half or more of the protein having the encoded amino acid sequence, more preferably 80% or more, and still more preferably 90% or more.

  As described above, the protein (A) can be obtained by modifying a protein having the amino acid sequence set forth in SEQ ID NO: 8. Hereinafter, the protein that is the basis of the protein of the present invention will be described. Note that the protein of the present invention is not limited by the original protein.

  The amino acid sequence of SEQ ID NO: 8 is encoded by the base sequence shown in SEQ ID NO: 7, for example. DNA comprising the nucleotide sequence of nucleotide numbers 61 to 1908 described in SEQ ID NO: 7 is Empedobacter brevis FERM BP-8113 strain (deposited organization; National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Deposited Organization Address; 1-1-1 Higashi 1-chome Tsukuba-shi, Ibaraki, Japan Central 6th, International deposit transfer date; July 8, 2002)

  The DNA consisting of the base sequence of base numbers 61 to 1908 described in SEQ ID NO: 7 is a code sequence (CDS) part. The base sequence of base numbers 61 to 1908 includes a signal sequence region and a mature protein region. The signal sequence region is a region of base numbers 61 to 126, and the mature protein region is a region of base numbers 127 to 1908. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein. The signal sequence contained in the sequence described in SEQ ID NO: 7 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 127 to 1908, that is, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide production activity.

  DNA having the nucleotide sequence of SEQ ID NO: 7 is obtained by PCR (polymerase chain reacion, White, TJ et al; Trends Genet., 5, 185 (1989)) from chromosomal DNA of Empedobacter brevis or DNA library. Alternatively, it can be obtained by hybridization. Primers used for PCR can be designed based on an internal amino acid sequence determined based on, for example, a purified protein having peptide-forming activity. Moreover, a primer or a probe for hybridization can be designed based on the base sequences described in SEQ ID NOs: 13 and 19, or can be isolated using the probe. When a primer having a sequence corresponding to the 5 ′ untranslated region and the 3 ′ untranslated region is used as a primer for PCR, the entire coding region of the protein can be amplified. Taking the case of amplifying the region containing both the leader sequence and the mature protein coding region described in SEQ ID NO: 13 as an example, specifically, as the 5′-side primer, SEQ ID NO: 13 is more than base number 61 in SEQ ID NO: 13. 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 the base number 1908 as the 3′-side primer.

  The primer can be synthesized according to a conventional method using, for example, a DNA synthesizer model 380B manufactured by Applied Biosystems and using the phosphoramidite 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.

3. Next, a method for producing the protein of the present invention and a method for producing a recombinant and a transformant used therefor will be described.

  The transformant that expresses the protein (A) can produce a protein having peptide-forming activity by, for example, introducing and expressing a DNA having the base sequence represented by SEQ ID NO: 1 into an appropriate host. it can. Examples of hosts for expressing a protein specified by the po DNA having the base sequence of SEQ ID NO: 1 include Escherichia bacteria such as Escherichia coli, Empedobacter bacteria, Sphingobacteria bacteria, Various prokaryotic cells including the genus Flavobacterium and Bacillus subtilis, including Saccharomyces cerevisiae, Pichia stipitis, Aspergillus oryzae Various eukaryotic cells can be used.

  Recombinant DNA used for introducing DNA having the nucleotide sequence of SEQ ID NO: 1 into a host can express these DNAs in a vector corresponding to the type of host to be expressed, and the protein encoded by the DNA It can be prepared by inserting in a different form. As a promoter for expressing a protein, when a promoter specific to a gene encoding a peptide-producing protein of Empedobacter brevis 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 DNA of SEQ ID NO: 1 and expressed under the control of the promoter.

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

  When a protein is mass-produced using recombinant DNA technology, a form in which the protein associates in a transformant producing the protein to form an inclusion body of the protein is also mentioned as a preferred embodiment. 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 Escherichia coli and producing a peptide-producing enzyme using the transformed Escherichia coli will be described more specifically. When preparing a peptide-generating enzyme in a microorganism such as Escherichia coli, only the DNA of the mature protein region that does not contain the leader sequence is incorporated even if the DNA encoding the precursor protein containing the leader sequence is incorporated as the protein coding sequence. However, it 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 DNA encoding a protein having peptide-forming activity, a promoter usually used for heterologous protein production in E. coli can be used. For example, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter And strong promoters such as lambda phage PR promoter and PL promoter. Moreover, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218, etc. can be used as the vector. In addition, phage DNA vectors can be used. Furthermore, an expression vector containing a promoter and capable of expressing the inserted DNA sequence can also be used.

  In order to produce a peptide-forming enzyme as a fusion protein inclusion body, a gene encoding another protein, preferably a hydrophilic peptide, is linked upstream or downstream of the peptide-forming enzyme gene to form a fusion protein gene. . 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, interferon γ gene, interleukin-2 gene, prochymosin gene and the like are listed as candidates.

  When these genes and a gene encoding a peptide-forming enzyme are linked, the codon reading frames are made to coincide. Ligation may be performed at an appropriate restriction enzyme site, or synthetic DNA having an appropriate sequence may be used.

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

As a vector for introducing a gene encoding a protein having a peptide-forming activity or a fusion protein of a protein having a peptide-forming activity and another protein into Escherichia coli, a so-called multi-copy type is preferable, and replication initiation derived from ColE1 A plasmid having a dot, for example, a pUC-type plasmid, a pBR322-type plasmid, or a derivative thereof. Here, the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, insertion, addition and / or inversion. 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.).

  Recombinant DNA by ligating a DNA encoding a promoter, a protein having peptide-forming activity, or a gene encoding a fusion protein of a protein having peptide-forming activity and another protein, or in some cases a terminator, and vector DNA Get.

  When the obtained recombinant DNA is used to transform Escherichia coli, and the Escherichia coli is cultured, a peptide-forming enzyme or a fusion protein of a peptide-producing enzyme 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 of E. coli K12 strain subspecies 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, the peptide-forming enzyme may be excised using a restriction protease such as blood coagulation factor Xa, kallikrein, etc., which has a sequence not present in the peptide-forming enzyme as a recognition sequence.

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

  There are the following methods for recovering a peptide-forming enzyme or a fusion protein of a peptide-forming enzyme and another protein. If the peptide-forming enzyme or its fusion protein is solubilized in the microbial cells, the microbial cells can be recovered and then disrupted or lysed to be used as a crude enzyme solution. Furthermore, if necessary, the peptide-forming enzyme or its fusion protein can be purified and used by a conventional method such as precipitation, filtration or column chromatography. In this case, a purification method using a peptide-forming enzyme or a fusion protein antibody 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 peptide-forming enzyme 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 the regeneration of activity can be expected by using any of them.

4). Method for Producing Peptide The method for producing a peptide of the present invention is to produce a peptide using the protein of the present invention. That is, in the method for producing a peptide of the present invention, a peptide is produced by reacting an amine component and a carbonyl component in the presence of at least one of the proteins shown in the following (A) and (B).
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (B) The 20th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing is alanine, and in a region other than the alanine , A protein having an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of substitution, deletion, insertion, addition and inversion, and having peptide-forming activity

  In the method for producing a peptide of the present invention, the protein is, for example, a mixture containing (A) and / or (B) protein in a biochemically acceptable medium (hereinafter referred to as “peptide-forming protein-containing material”). ). More specifically, for example, 1 selected from the group consisting of a culture of a microorganism transformed to express the protein of the present invention, a microbial cell separated from the culture, and a treated product of the microorganism. A peptide may be produced from an amine component and a carbonyl component using two or more species.

  As a method for allowing the peptide-forming protein or its content to act on the carboxy component and the amine component, the peptide-generating protein or its content, the carboxy component, and the amine component may be mixed. More specifically, a method may be used in which the peptide-forming protein of the present invention or a content thereof is added to a solution containing a carboxy component and an amine component and reacted, or a microorganism that produces peptide-forming protein is used. In some cases, a method of culturing a microorganism producing a peptide-forming protein, purifying and accumulating the enzyme in the microorganism or a culture solution of the microorganism, and adding a carboxy component and an amine component to the culture solution may be used. Good. The produced peptide can be recovered by a conventional method and purified as necessary.

  In order to obtain microbial cells (that is, microbial cells), it is preferable to culture and proliferate in an appropriate medium according to the type of microorganism. 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 , Organic acids such as acetic acid and propionic acid and salts thereof, carbohydrates such as paraffin or mixtures thereof can be used.

  Nitrogen sources include ammonium salts of inorganic salts such as ammonium sulfate and ammonium chloride, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, nitrates such as sodium nitrate and potassium nitrate, peptone, yeast extract, meat extract, corn steep Organic nitrogen compounds such as 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.

  There are no particular restrictions on the culture conditions. For example, if the culture is performed for about 12 to 48 hours under aerobic conditions while appropriately limiting the pH and temperature within the range of pH 5 to 8 and temperature of about 15 to 40 ° C. Good.

  The “peptidogenic protein-containing product” may be anything containing the peptide-producing protein of the present invention, and specific examples include a culture of a microorganism that produces the peptide-producing protein, and a microorganism isolated from the culture. Included are microbial cells and processed microbial cells. A microorganism culture is a product obtained by culturing a microorganism, and more specifically, a microbial cell, a medium used for culturing the microorganism, and a mixture of substances produced by the cultured microorganism. And so on. Further, the microbial cells may be washed and used as washed cells. In addition, the processed bacterial cells include those obtained by crushing, lysing, and lyophilizing the bacterial cells, and further include crude purified proteins recovered by treating the bacterial cells and the like, and further purified proteins. As the purified protein, partially purified proteins obtained by various purification methods may be used, or immobilized proteins obtained by immobilizing these by a covalent bond method, an adsorption method, a comprehensive method, or the like may be used. Good. In addition, some microorganisms lyse during culture, and in this case, the culture supernatant can also be used as a peptide-forming protein-containing material.

  In addition, as a microorganism containing the peptide-generating protein of the present invention, a genetically modified strain expressing the peptide-generating protein may be used, or a cell-treated product such as acetone-treated cells or freeze-dried cells may be used. Alternatively, an immobilized microbial cell or an immobilized microbial cell-treated product obtained by immobilizing these by a covalent bond method, an adsorption method, a comprehensive method, or the like may be used.

  In addition, when using cultured products, cultured cells, washed cells, or treated cells obtained by crushing or lysing cells, there may be an enzyme that degrades the produced peptide without being involved in peptide production. In many cases, it may be preferable to add a metal protease inhibitor such as ethylenediaminetetraacetic acid (EDTA). The addition amount is in the range of 0.1 mM to 300 mM, preferably 1 mM to 100 mM.

  A preferred form of the peptide production method of the present invention includes the above-described method of culturing the transformed microorganism in a medium and accumulating the peptide-forming enzyme in the medium and / or in the microorganism. By using a transformant, a peptide-producing enzyme can be easily produced in large quantities, and therefore, dipeptides can also be produced in large quantities and quickly.

  The amount of the peptide-forming protein or the content thereof may be any amount that exhibits the desired effect (effective amount), and this effective amount can be easily determined by a person skilled in the art by a simple preliminary experiment. When using an enzyme, it is about 0.01 to 100 unit (U), and when using a washing | cleaning microbial cell, it is about 0.1-500 g / L.

  Any carboxy component may be used as long as it can be condensed with an amine component which is another substrate to form a peptide. Examples of the carboxy component include L-amino acid esters, D-amino acid esters, L-amino acid amides, D-amino acid amides, and organic acid esters having no amino group. Examples of amino acid esters include not only amino acid esters corresponding to natural amino acids but also amino acid esters corresponding to non-natural amino acids or derivatives thereof. Examples of amino acid esters include α-amino acid esters and amino acid esters such as β-, γ-, and ω- that have different amino group bonding positions. Representative examples of amino acid esters include amino acid methyl ester, ethyl ester, n-propyl ester, iso-propyl ester, n-butyl ester, iso-butyl ester, tert-butyl ester and the like.

  Any amine component may be used as long as it can be condensed with a carboxy component as another substrate to form a peptide. Examples of the amine component include L-amino acids, C-protected L-amino acids, D-amino acids, C-protected D-amino acids, amines, and the like. Examples of amines include not only natural amines but also non-natural amines or derivatives thereof. Examples of amino acids include not only natural amino acids but also non-natural amino acids or derivatives thereof. In addition to α-amino acids, amino acids such as β-, γ-, and ω- that have different amino group bonding positions are also exemplified.

  The concentrations of the starting carboxy component and amine component are each 1 mM to 10 M, preferably 0.05 M to 2 M, but it may be preferable to add an equal amount or more of the amine component to the carboxy component. 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.

  Peptide synthesis is possible at a reaction temperature of 0 to 60 ° C, preferably 5 to 40 ° C. Further, the peptide can be synthesized at a reaction pH of pH 6.5 to 10.5, and preferably pH 7.0 to 10.0.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in detail, this invention is not restrict | limited to the following Example.

[Preparation Example 1] Isolation of a peptide-forming enzyme gene derived from Empedobacter brevis The isolation of a peptide-forming enzyme gene will be described below. Empedobacter brevis FERM BP-8113 strain was used as the microorganism. For gene isolation, Escherichia coli JM-109 was used as the host, and pUC118 was used as the vector.

(1) Preparation of PCR primers based on the determined internal amino acid sequence Amino acid sequence (SEQ ID NO:) determined by Edman degradation method of the above-mentioned digestion product of lysyl endopeptidase of peptide producing enzyme derived from Empedobacter brevis FERM BP-8113 strain Based on 3 and 4), mix primers having base sequences shown in SEQ ID NOs: 5 and 6, respectively, were prepared.

(2) Acquisition of bacterial cells Empedobacter brevis FERM BP-8113 strain was added to 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 0.0)) 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.

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

(4) Obtaining a DNA fragment containing a part of a peptide-forming enzyme gene by PCR method A DNA fragment containing a part of a peptide-forming enzyme gene derived from Empedobacter brevis FERM BP-8113 strain was obtained by LA-Taq (Takara Shuzo Co., Ltd.). Obtained by PCR method. PCR reaction was performed on the chromosomal DNA obtained from Empedobacter brevis FERM BP-8113 using primers having the nucleotide sequences shown in SEQ ID NOs: 11 and 12.

  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 0.8% agarose electrophoresis. As a result, it was confirmed that a DNA fragment of about 1.5 kb was amplified.

(5) Cloning of the peptide-forming enzyme gene from the gene library In order to obtain the full-length peptide-forming enzyme gene, first, Southern hybridization was performed using the DNA fragment amplified in the PCR as a probe. Southern hybridization of operation, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The approximately 1.5 kb DNA fragment amplified by the PCR was separated by 0.8% agarose electrophoresis. The target band was cut out and purified. This DNA fragment was labeled with digoxinigen on the probe based on the instructions using DIG High Prime (manufactured by Boehringer Mannheim).

  The chromosomal DNA of Empedobacter brevis obtained in Preparation Example 1 (3) was reacted with the restriction enzyme HindIII 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 memebranes positively charged (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 the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, a band of about 4 kb that hybridized with the probe could be detected.

  5 μg of the chromosomal DNA prepared in Preparation Example 1 (3) was completely digested with HindIII. About 4 kb of DNA was separated by 0.8% agarose gel electrophoresis, the DNA was purified using Gene Clean II Kit (manufactured by Funakoshi), and dissolved in 10 μl of TE. Of these, 4 μl was mixed with pUC118 HindIII / BAP (Takara Shuzo, BAP: Escherichia coli alkaline phosphatase), and a ligation reaction was performed using DNA Ligation Kit Ver. 2 (Takara Shuzo). Escherichia coli was transformed by mixing 5 μl of this ligation reaction solution and 100 μl of Escherichia coli JM109 strain 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 peptide-forming enzyme gene, a chromosomal DNA library was screened by colony hybridization using the probe. The procedure for colony hybridization, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The colonies of the chromosomal DNA library were transferred to a nylon membrane filter Nylon Membranes for Colony and Plaque 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.

  Colonies that hybridize with the labeled probe were detected based on the instructions using the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, two colonies that hybridize with the labeled probe were confirmed.

(6) Nucleotide sequence of the peptide-forming enzyme gene derived from Empedobacter brevis From the above two strains confirmed to hybridize with the labeled probe, plasmids possessed by Escherichia coli JM109 were obtained from the Wizard Plus Minipreps DNA Purification System ( And the base sequence in the vicinity of the hybridized probe was determined. The sequencing 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).

  As a result, it was confirmed that there was an open reading frame encoding a protein containing the internal amino acid sequence (SEQ ID NOs: 3 and 4) of the peptide generating enzyme, and the gene encoding the peptide generating enzyme. The full-length base sequence of the peptide-forming enzyme gene and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 7 in the sequence listing. The resulting open reading frame was BLASTP. As a result of homology analysis by the program, homology was found between the two enzymes. Acetobacter pasteurianus α-amino acid ester hydrolase (Appl. Environ. Microbiol., 68 (1), 211-218 (2002) The amino acid sequence showed 34% homology with the glutaryl-7ACA acylase of Brevibacillus laterosporum (J. Bacteriol., 173 (24), 7848-7855 (1991)).

(7) Expression in Escherichia coli of peptide-forming enzyme gene derived from Empedobacter genus The target region by PCR using the promoter region of trp operon on Escherichia coli W3110 chromosomal DNA as the primers shown in SEQ ID NOs: 9 and 10 The gene region was amplified, and the obtained DNA fragment was ligated to a pGEM-Teasy vector (Promega). E. coli JM109 strain was transformed with this ligation solution, and a strain having a target plasmid in which 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 and EcoRI and EcoO109I and EcoRI-treated products of pUC19 (Takara).

  Escherichia coli JM109 strain 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 and PvuII and a pKK223-3 (manufactured by Amersham Pharmacia) were treated with HindIII and HincII, and the resulting DNA fragment containing the rrnB terminator was ligated. E. coli JM109 strain was transformed with this ligation solution, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrpT.

  The target gene was amplified by PCR using the chromosomal DNA of Empedobacter brevis FERM BP-8113 as a template and the oligonucleotides shown in SEQ ID NOs: 11 and 12 as primers. This DNA fragment was treated with NdeI and PstI, and the obtained DNA fragment was ligated with the NdeI and PstI treated product of pTrpT. The Escherichia coli JM109 strain was transformed with this ligation solution, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrpT_Gtg2.

  Escherichia coli JM109 strain having pTrpT_Gtg2 was seed-cultured in an LB medium containing 100 mg / l ampicillin at 30 ° C. for 24 hours. 1 ml of the obtained culture broth 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 phosphorus A 500 ml Sakaguchi flask filled with dipotassium hydrogen oxyhydrogen, 0.5 g / l magnesium sulfate heptahydrate, 100 mg / l ampicillin) was seeded, and main culture was performed at 25 ° C. for 24 hours. It has 0.11 U of α-L-aspartyl-L-phenylalanine-β-ester producing 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 pTrpT was introduced as a control.

(8) Construction of a peptide-producing enzyme high expression plasmid derived from Empedobacter
As described in Journal of Bioscience and Bioengineering (2001) Vol. 92, No. 1, 50-54 (or Japanese Patent Application Laid-Open No. 10-201481, Example 24, etc.), Enterobacter aerogenes IFO 12010 strain A 1.6 kbp DNA fragment containing the acid phosphatase gene region and excised with the restriction enzymes SalI and KpnI was isolated from the chromosomal DNA, and a plasmid DNA linked to pUC118 was constructed and named pEAP120. pEAP120 is a plasmid in which a base sequence encoding an acid phosphatase promoter and a signal peptide is incorporated.

  The IFO numbers are those deposited with the Fermentation Research Institute (17-85 Jusohoncho 2-chome, Yodogawa-ku, Osaka, Japan), but after June 30, 2002 The business will be transferred to the National Institute for Product Evaluation and Technology (NITE), Biotechnology Headquarters (DOB), and Biological Genetic Resources Department (NBRC), and can be sold by NBRC with reference to the above IFO numbers.

  Subsequently, a part of the promoter sequence existing upstream of this gene was modified to try to improve the activity. A site-specific mutation was introduced using Quikchange site Directed Mutagenesis Kit (manufactured by STRATAGENE), and the -10 region of the acid phosphatase gene predicted promoter sequence was replaced from AAAAAT to TATAAT. PCR oligonucleotide primers EM1 (5'-CTT ACA GAT GAC TAT AAT GTG ACT AAA AAC; SEQ ID NO: 25) and EMR1 (5'-GTT TTT AGT CAC ATT ATA GTC ATC TGT AAG; SEQ ID NO: designed for mutagenesis 26) was synthesized. According to the method of the instruction, mutation was introduced using pEAP120 as a template. DNA Sequencing kit Dye Terminator Cycle Sequencing Ready Reaction (manufactured by PERKIN ELMER) was used to determine the nucleotide sequence using the 310 Genetic analyzer (ABI) by the Dye Terminator method, and confirmed that the target mutation was introduced. This plasmid was designated as pEAP130. pEAP130 is a plasmid having a base sequence encoding a signal peptide derived from the N-terminal region of acid phosphatase and a modified promoter.

  PTrpT_Gtg2 plasmid of Preparation Example 1 as a template and CAP1 (5′-CGT TAA CGC TTT CGC GCA AGA TGC AAA AGC AGA TTC; SEQ ID NO: 13) and CAP2 (5′-CGC CTG CAG CAT ACT TGT ACG GTT TCG CCC as primers ; SEQ ID NO: 14) 0.4 mM each of oligonucleotide and KOD plus buffer solution (TOYOBO), dATP, dCTP, dGTP, dTTP 0.2 mM each, magnesium sulfate 1 mM, and KOD plus polymerase (TOYOBO) 1 unit In a 50 μl reaction solution containing 50 μl of heat treatment at 94 ° C. for 30 seconds, 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 2 minutes 30 seconds are repeated 25 times, and the mature peptide The product enzyme gene part was amplified.

  In addition, pEAP130 plasmid of Example 1 as a template, CAP3 (5′-CGC TCT AGA ATT TTT TCA ATG TGA TTT; SEQ ID NO: 15) and CAP4 (5′-GCT TTT GCA TCT TGC GCG AAA GCG TTA ACG GAA AAC as primers SEQ ID NO: 16) PCR was carried out using the oligonucleotide under the same conditions to amplify the acid phosphatase promoter and signal sequence portion. The reaction solution was subjected to agarose electrophoresis, and each amplified DNA fragment was recovered using a Microspin column (manufactured by Amersham Pharmacia Biotech).

  Next, using the amplified fragment mixture as a template, CAP2 and CAP3 oligonucleotides as primers, and a reaction solution of the same composition with a cycle of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 2 minutes and 30 seconds. Repeated PCR was performed to construct a chimeric enzyme gene. Each amplified DNA fragment was recovered using a Microspin column (Amersham Pharmacia Biotech) and digested with Xba I and Pst I. This was ligated to the Xba I-Pst I site of the pUC19 plasmid. DNA Sequencing kit Dye Terminator Cycle Sequencing Ready Reaction (PERKIN ELMER) Dye Terminator method was used to determine the nucleotide sequence with 310 Genetic analyzer (ABI), confirm that the target mutation was introduced, and this plasmid Was designated as pAF110 plasmid.

Example 1 Modification of Empedobacter-Derived Peptide Gene Enzyme Gene According to Preparation Example 1 described above, the mature peptide-forming enzyme gene derived from Empedobacter genus is converted into the modified promoter and signal sequence of Enterobacter aerogenes acid phosphatase. An expression plasmid pAF110 linked downstream was constructed. For mutagenesis, Escherichia coli XL-1Red competent cells (Strategene) were transformed with the pAF110 plasmid, and LB agar medium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l) containing ampicillin 100 mg / l was used. Sodium chloride, 15 g / l agar, pH 7.0) was plated and cultured at 30 ° C. for 18 hours. The grown Escherichia coli XL-1Red transformant was inoculated into a 500 ml Sakaguchi flask containing 50 ml of LB medium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l sodium chloride, pH 7.0) containing 100 mg / l of ampicillin. And cultured with shaking at 30 ° C. for 16 hours. From the collected cells, a plasmid was prepared using High Plasmid Midi Kit (manufactured by Qiagen) to obtain a plasmid into which a random mutation was introduced. Escherichia coli JM109 competent cells (Takara Shuzo) were transformed with this plasmid, and an expression agar medium (10 g / l caseinoic acid, 10 g / l yeast extract, 5 g / l (NH ₄ ) ₂ SO ₄ , 3 g / l KH ₂ PO ₄ , 1 g / l K ₂ HPO ₄ , 2 g / l glucose, 0.5 g / l MgSO ₄ 7H ₂ O, 1.5 g / l agar) And cultured at 30 ° C. for 18 hours.

  A 0.8% agar solution containing 10 mM alanine-p-nitroanilide, 10 mM O-phenanthrolin and 100 mM phosphate buffer (pH 7.0) was overlaid on the agar medium, and allowed to react at room temperature. Since a transformant with high peptide-forming enzyme activity decomposes alanine-p-nitroanilide to release p-nitroaniline, it exhibits a yellow color. Therefore, using this as an index, peptide-forming enzyme activity increases. The strains were screened. Screening was performed from about 200,000 transformants, and 45 strains with improved activity were selected.

  Subsequently, these candidate strains were inoculated into a test tube containing 3 ml of an expression medium containing ampicillin 100 mg / l, cultured at 30 ° C. for 18 hours, and alanylglutamine producing activity was measured. Alanylglutamine production activity was measured under the following conditions. 100 μmol / ml alanine-o-methyl ester (produced by Kokusan Kagaku), 200 μmol / ml glutamine, 100 μmol / ml mM borate-sodium hydroxide buffer (pH 9.0) and a reaction solution (1 ml) containing bacterial cells at pH 9. The reaction was carried out at 0 and 30 ° C. for 10 minutes. The reaction was stopped by adding 10 ml with 1.7% (w / v) phosphoric acid, the precipitate was removed by centrifugation, and alanylglutamine produced in this reaction was quantified. The amount of enzyme that produces 1 μmol alanylglutamine per minute under the standard reaction conditions was defined as 1 unit.

Alanylglutamine was analyzed by high performance liquid chromatography (HPLC) under the following conditions.
Column: Inertsil ODS-2 (manufactured by GL Sciences, 4.6 x 250 mm), moving bed: 5 mM sodium 1-octanesulfonate / methanol = 1000/165 (adjusted to pH 2.1 with phosphoric acid), flow rate: 1.0 ml / min , Temperature: 40 ° C., detection: UV 210 nm.

  As a result, No. 15 strain showing the highest activity was selected. Escherichia coli JM109 / pAF110 strain obtained only 0.85 U / ml activity when cultured at 30 ° C., whereas PAF110-M15 strain showed 2.53 U / ml, which was about three times as high as the activity.

  Subsequently, a plasmid was prepared from the No. 15 strain, and the base sequence of the mutant peptide-forming enzyme was determined. Hereinafter, this plasmid was designated as pAF110-M15. The determination of the base sequence was performed by the 310 Genetic analyzer (ABI) by the dye terminator method using DNA Sequencing kit Dye Terminator Cycle Sequencing Ready Reaction (PERKIN ELMER). The peptidase gene in pAF110-M15 showed a 4-base substitution with a 3-amino acid substitution. Amino acid substitution is a mature peptide-forming enzyme. 20th Val residue (GTA) is alanine residue (GCA), 264th asparagine residue (AAC) is serine residue (AGC), 416th serine residue The group (TCA) was a substitution to a glutamine residue (CCA).

[Example 2] Amino acid substitution effect of peptide-producing enzyme gene derived from Empedobacter genus and examination of properties of mutant enzyme (1) Construction of maltose binding protein fusion-type peptide-forming enzyme gene and single mutant gene Next, this amino acid A vector for expressing peptide-forming enzyme as a fusion protein with Maltose binding protein (MBP) using pMAL ^™ Protein Fusion and Purification System (manufactured by NewEngland Biolab) to elucidate substitution effects and changes in enzyme activity Built. The pAF110 plasmid of Example 1 or the pAF110-M15 plasmid introduced with the mutation as a template, AETN (5′-CGC GAA TTC CAA GAT GCA AAA GCA GAT TCT GCT; SEQ ID NO: 17) and AETC (5′-CGC CTG) as primers CAG CAT ACT TGT ACG GTT TCG CCC; SEQ ID NO: 18) Oligonucleotide 0.4 mM and KOD plus buffer (TOYOBO), dATP, dCTP, dGTP, dTTP 0.2 mM, magnesium sulfate 1 mM, and KOD plus PCR with 50 μl reaction solution containing 1 unit of polymerase (TOYOBO), heat treatment at 94 ° C. for 30 seconds, and repeat cycle of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds and 68 ° C. for 2 minutes 25 times The mature peptide-forming enzyme gene part was amplified. Each amplified DNA fragment was recovered using a Microspin column (manufactured by Amersham Pharmacia Biotech) and digested with Eco RI and Pst I. This was ligated to the Eco RI-Pst I site of pMAL-p2x plasmid (NewEngland Biolab). The plasmid ligated with the wild-type peptide synthase gene was named pMF200, and the plasmid ligated with the peptide synthase gene substituted with three amino acid residues was named pM15.

  Next, site-specific mutation was introduced into pMF200 using Quikchange site Directed Mutagenesis Kit (manufactured by STRATAGENE), and the mutation of the 3 amino acid residues (V20A, N264S and S416Q) of the mutant obtained in Example 1 was introduced. Among them, a single mutant gene in which one amino acid residue was substituted was constructed. EMP1 (5'-tac gaa aaa atg gaa caa gca att ccg atg cgc gat g; SEQ ID NO: 19) and EMP2 (5'-cat cgc gca tcg gaa ttg ctt gtt cca ttt ttt cgt a; SEQ ID NO: 20), emp3 (5'-gtt tta cca cat tta act agc gtg caa cct gct gta atg; SEQ ID NO: 21) and EMP4 (5'-cat tac agc agg ttg cac gct agt taa atg tgg taa aac SEQ ID NO: 22), EMP5 (5′-caa att ctc cag ttc ctt atc aag gag gag ttt tag aaa ctc; SEQ ID NO: 23) and EMP6 (5′-gag ttt cta aaa ctc ctc ctt gat aag Primers of gaa ctg gag aat ttg; SEQ ID NO: 24) were respectively synthesized, and mutagenesis was performed using pEAP120 as a template according to the method described in the instructions. DNA Sequencing kit Dye Terminator The nucleotide sequence was determined with the 310 Genetic analyzer (ABI) by the Dye Terminator method using Cycle Sequencing Ready Reaction (PERKIN ELMER), and it was confirmed that the target mutation was introduced. The plasmids into which the V20A mutation, N264S mutation and S416Q mutation were introduced were named pM1, pM2 and pM3, respectively.

(2) Activity measurement of each mutant enzyme gene-introduced strain Escherichia coli JM109 transformed with each plasmid was inoculated into a 500 ml Sakaguchi flask containing 50 ml of an expression medium containing 100 mg / l of ampicillin and 0.1 mM of IPTG, and 22 ° C. For 20 hours. The results of measuring the alanylglutamine producing activity of each strain by the method of Example 1 are shown in Table 1.

  In the V20A / N264S / S416Q mutant gene-introduced strain, the activity per OD of the MBP fusion protein was about twice as high as that in the wild-type gene-introduced strain. When the activity of each single mutant enzyme gene-introduced strain was compared with that of the wild-type gene-introduced strain, the activity per OD of the V20A mutant enzyme gene-introduced strain was improved by about 1.5 times. On the other hand, the activity of the N264S or S416Q mutant enzyme gene-introduced strain was similar to that of the wild type. From these results, it was considered that the substitution of V20A among the three-residue substitutions contributed to the improvement of peptide-generating enzyme gene activity.

(3) Purification and characterization of mutant enzyme Next, wild-type enzyme, V20A / N264S / S416Q mutant enzyme, and V20A mutant enzyme were purified and characterized. Escherichia coli JM109 / pMF200, Escherichia coli JM109 / pM15 and Escherichia coli JM109 / pM1 strains were inoculated into a 500 ml Sakaguchi flask containing 50 ml of expression medium containing 100 mg / l of ampicillin and 0.1 mM of IPTG, and incubated at 22 ° C. for 20 hours. Cultured. Cells collected by centrifugation at 8,000 rpm for 15 min from the culture solution (50 ml x 6 tubes) of each cell are suspended in 30 ml of 20 mM Tris-HCl buffer (pH 7.4, 20 min ultrasonic) The crushed liquid was centrifuged (8,000 rpm, 30 min) to remove the residue, and a cell-free extract was obtained.

  The obtained cell-free extract was applied to an amylose resin column (φ1 × 5 cm, 5 ml) equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 200 mM sodium chloride and 1 mM EDTA. did. After washing with 60 ml of equilibration buffer, the active fraction was eluted with 10 ml of Tris-HCl buffer (pH 7.4) containing 10 mM of Maltose, 20 mM 200 mM sodium chloride and 1 mM EDTA. FactorXa (manufactured by Amersham Pharmacia Biotech) was added to this so as to be 1 mg per 100 mg of MBP-Aet fusion protein (1% (w / w)), and incubated at 15 ° C. for 16 h.

  The active fraction obtained by incubation was applied to a hydroxyapatite column (φ1 × 1 cm, 1 ml) equilibrated with 20 mM potassium phosphate buffer (pH 7.2). After washing with 10 ml of equilibration buffer, the active fraction was eluted with 5 ml of 500 mM potassium phosphate buffer (pH 7.2). In order to remove MBP, the active fraction was reapplied to an amylose resin column (φ1 × 5 cm, 5 ml) equilibrated with Tris-HCl buffer (pH 7.4), 200 mM NaCl, 1 mM EDTA. Elution was performed with 5 ml of equilibration buffer, and the obtained flow-through fraction was recovered as an active fraction. The obtained active fraction was concentrated using centriprep10 (Amicon), glycerol was added to a concentration of 50% (v / v) and stored at −20 ° C.

  Using this sample, the alanylglutamine production activity of the wild type enzyme, the V20A / N264S / S416Q mutant enzyme and the V20A mutant enzyme was measured by the method of Example 1. The protein concentration was quantified according to the Bradford method using BSA as a standard protein and a protein assay kit (manufactured by Bio-Rad). As a result, the specific activities of the wild type enzyme, V20A / N264S / S416Q mutant enzyme and V20A mutant enzyme were calculated to be 1,727 units / ml, 1,535 units / ml and 1,528 units / ml. Because the specific activity of the two mutant enzymes did not improve from that of the wild-type enzyme, the improvement of the activity of the mutant enzyme gene-introduced strain was not due to a change in the specific activity, but because it became easier to express. It was inferred that there was.

  The present invention is useful in fields relating to peptide production methods and the like.

SEQ ID NO: 1; nucleotide sequence encoding a peptide generating protein derived from Empedobacter brevis SEQ ID NO: 2; amino acid sequence of a peptide generating protein derived from Empedobacter brevis SEQ ID NO: 3; peptide generating protein derived from Empedobacter Amino acid sequence determined by Edman degradation SEQ ID NO: 4; amino acid sequence determined by Edman degradation of peptide generator protein derived from Empedobacter SEQ ID NO: 5; Mix Primer
SEQ ID NO: 6; Mix Primer
SEQ ID NO: 7: Nucleotide sequence of peptide-producing protein derived from Empedobacter brevis SEQ ID NO: 8: Amino acid sequence of peptide-generating protein derived from Empedobacter brevis SEQ ID NO: 9: Primer for preparing pTrpT SEQ ID NO: 10: Primer for preparing pTrpT SEQ ID NO: 11; pTrpT_Gtg2 preparation primer SEQ ID NO: 12; pTrpT_Gtg2 preparation primer SEQ ID NO: 13; primer CAP1
Sequence number 14; primer CAP2
Sequence number 15; primer CAP3
Sequence number 16; primer CAP4
SEQ ID NO: 17; primer AETN
SEQ ID NO: 18; primer AETC
SEQ ID NO: 19; primer EMP1
SEQ ID NO: 20; primer EMP2
SEQ ID NO: 21; primer EMP3
Sequence number 22; primer EMP4
SEQ ID NO: 23; primer EMP5
Sequence number 24; primer EMP6
Sequence number 25; primer EM1
SEQ ID NO: 26; primer EMR1

Claims (8)

Protein shown in the following (A) or (B).
(A) Protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (B) Substitution, deletion, insertion, addition to a region other than the 20th alanine in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And a protein having an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of inversions and having peptide-forming activity A polynucleotide encoding the protein shown in (A) or (B) below.
(A) Protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (B) Substitution, deletion, insertion, addition to a region other than the 20th alanine in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And a protein having an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of inversions and having peptide-forming activity The polynucleotide shown in the following (a) or (b).
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 in the sequence listing (b) a base sequence that hybridizes under stringent conditions with a polynucleotide complementary to the base sequence set forth in SEQ ID NO: 1 in the sequence listing And a polynucleotide that encodes a protein in which the site corresponding to the 20th amino acid residue in the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing encodes alanine and has peptide-forming activity   A recombinant polynucleotide in which the polynucleotide according to claim 2 or 3 is incorporated.   A transformant into which the recombinant polynucleotide according to claim 4 is introduced and expresses a protein having peptide-forming activity.   The transformant according to claim 5, wherein the transformant is a microorganism, the microorganism is cultured in a medium, and a protein having peptide-forming activity is accumulated in the medium and / or the microorganism. Production method.   The transformant according to claim 5 is a microorganism, and one or more selected from the group consisting of a culture of the microorganism, a microbial cell separated from the culture, and a treated product of the microorganism. A method for producing a peptide, wherein a peptide is produced from an amine component and a carbonyl component. A method for producing a peptide, wherein a peptide is produced from an amine component and a carbonyl component in the presence of at least one of the proteins shown in (A) and (B) below.
(A) Protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (B) Substitution, deletion, insertion, addition to a region other than the 20th alanine in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And a protein having an amino acid sequence containing a mutation of one or several amino acids selected from the group consisting of inversions and having peptide-forming activity