JP5367272B2 - Polyamino acid synthase and gene encoding it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a polyamino acid. <P>SOLUTION: An enzyme catalyzing amino acid polymerization in a non-ribosomal peptide synthase (NRPS) style, and a polynucleotide comprising a specific base sequence and encoding the above enzyme, are provided, respectively. A polynucleotide in which the above polyamino acid synthase is a polylysine synthase is also provided. A recombinant vector comprising the above polynucleotide, a transformant containing the above recombinant vector, and a method for producing the polyamino acid synthase using the above transformant, are also provided, respectively. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a novel polyamino acid synthase and a gene encoding the same.

Conventionally known methods for synthesizing polyamino acids include chemical synthesis and enzymatic synthesis.

In recent years, various physiologically active peptides with high social contribution have been found. There are two methods for synthesizing these peptides: chemical synthesis and enzyme synthesis. The latter can be manufactured without using harmful chemicals, can overcome problems such as environmental impact, and can be expected to reduce costs.

One method for enzymatically synthesizing polyamino acids is cyanophycin (multi-L-arginyl-po), a non-protein amino acid polymer consisting of an aspartic acid skeleton and an arginine side chain.
A method for enzymatically synthesizing ly (L-aspartic acid)) is known (Non-Patent Document 1, Non-Patent Document 2). A method for enzymatically synthesizing poly-γ-glutamic acid has also been reported (
Non-patent document 3).

However, since the enzyme synthesis of these polyamino acids is achieved by the catalytic action of a complex enzyme system, it is difficult to control and has not been applied to industrial production. Cyanophycin, poly-
Except for the enzymatic synthesis of γ-glutamic acid, there is no example in which a peptide or amino acid homopolymer having a chain length longer than that of a tripeptide can be enzymatically synthesized.

Furthermore, the biosynthesis of the antibacterial polyamino acid ε-poly-L-lysine has been reported by a microbial fermentation method (Non-patent Document 4).
Eur. J. et al. Biochem. 267, 5561-5570 (2000) APPL. ENVIRON. MICROBIOL. Vol. 67, no. 5, May 2001, p. 2176-2182 APPL. ENVIRON. MICROBIOL. Vol. 70, no. 7, July 2004, p. 4249-4255 Agric. Biol. Chem. Vol. 45, 1981, p. 2497-2502

Under such circumstances, a method for efficiently producing a longer-chain arbitrary amino acid peptide or amino acid analog homopolymer is required.

As a result of intensive studies to meet such demands, the present inventors have found a ribosome-independent peptide synthase (NRPS) that catalyzes the polymerization of L-lysine.

Therefore, the present invention relates to the following polyamino acid synthetase, polynucleotide encoding the same, vector containing the polynucleotide, method for producing the polyamino acid synthetase, and production of polyamino acid using the polyamino acid synthetase. A method, a primer containing a partial sequence of the polynucleotide, a probe capable of hybridizing to the polynucleotide or a complementary sequence thereof, an antibody against the enzyme, a method for screening the polyamino acid synthase using the primer or probe or the antibody, etc. provide.

(1) (i) a nucleotide encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2
Leotide sequence,
(Ii) deletion or placement of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2
A polyamino acid synthetase consisting of an amino acid sequence having substitution, insertion, and / or addition
The encoding nucleotide sequence,
(Iii) From an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2
A nucleotide sequence encoding a polyamino acid synthase,
(Iv) the nucleotide sequence of SEQ ID NO: 1,
(V) a stringent nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1
Polyamino acid synthase that can hybridize under various hybridization conditions
Nucleotide sequence to be
(Vi) a polyamino acid having at least 65% identity with the nucleotide sequence of SEQ ID NO: 1
A nucleotide sequence encoding a synthase,
A polynucleotide comprising any of
(1a) (i) Deletion of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2
, Substitution, insertion, and / or polyamino acid synthesis fermentation consisting of amino acid sequence having addition
A nucleotide sequence encoding prime,
(Ii) consisting of an amino acid sequence having 80% identity or more with the amino acid sequence of SEQ ID NO:
A nucleotide sequence encoding a polyamino acid synthetase, or
(Iii) High stringency in the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1
Polyamino acid synthase capable of hybridizing under the present hybridization conditions
A nucleotide sequence encoding,
A polynucleotide comprising any of
(2) (i) a nucleotide encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2.
Leotide sequence, or
(Ii) the nucleotide sequence of SEQ ID NO: 1;
A polynucleotide comprising any of
(3) The above (1) or (2), wherein the polyamino acid synthase is a polylysine synthase
).
(4) The polynucleotide according to any one of (1) to (3), which is DNA or RNA.
Chido.
(5) (i) A condensation reaction consisting of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
A nucleotide sequence encoding a catalytic domain or a subdomain thereof;
(Ii) at least in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
An amino acid sequence having a deletion, substitution, insertion and / or addition of one amino acid residue
A nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof
Or
(Iii) 60% or more of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Condensation reaction catalytic domain or its subdomain consisting of a single amino acid sequence
The encoding nucleotide sequence,
(Iv) the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18;
(V) Nuclei complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Can hybridize to string sequences under stringent hybridization conditions
A nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, or
Is
(Vi) a nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 and 65% or more
Nucleotide encoding a condensation reaction catalytic domain or a subdomain thereof having identity
Tide sequence,
A polynucleotide comprising any of
(5a) (i) 1 in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Or amino acid sequences with deletions, substitutions, insertions, and / or additions of several amino acid residues.
A nucleotide sequence encoding a polyamino acid synthetase consisting of a sequence;
(Ii) 80% or more identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
A nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having
Or
(Iii) a nucleotide complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Hybridizes to the nucleotide sequence under highly stringent hybridization conditions
A nucleotide sequence encoding a polyamino acid synthase,
A polynucleotide comprising any of
(6) (i) Condensation reaction consisting of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
A nucleotide sequence encoding a catalytic domain or a subdomain thereof, or
(Ii) the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18;
A polynucleotide comprising any of
(7) The polynucleotide according to (5) or (6) above, which is DNA or RNA.
(8) (i) the amino acid sequence of SEQ ID NO: 2,
(Ii) deletion or placement of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2
An amino acid sequence having a substitution, insertion, and / or addition, or
(Iii) an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2;
A polyamino acid synthase containing any of the above.
(8a) (i) Deletion of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2
An amino acid sequence having a substitution, insertion, and / or addition, or
(Ii) an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 2,
A polyamino acid synthase containing any of the above.
(9) The polyamino acid synthetase according to (8) above, which is a polylysine synthase.
(10) (i) the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19;
(Ii) at least in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
An amino acid sequence having a deletion, substitution, insertion and / or addition of one amino acid residue;
Or
(Iii) 60% or more of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
A single amino acid sequence,
A condensation reaction catalytic domain comprising any of:
(10a) (i) 1 in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Or amino acids with deletions, substitutions, insertions and / or additions of several amino acid residues
An array, or
(Ii) 80% or more identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
An amino acid sequence having sex,
A condensation reaction catalytic domain comprising any of:
(11) A polyamino acid synthetase comprising the condensation reaction catalytic domain according to (10).
(12) The polyamino acid synthetase according to (11) above, which is a polylysine synthase.
(13) (i) a nucleotide encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2
Nucleotide sequence,
(Ii) deletion or placement of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2
A polyamino acid synthetase consisting of an amino acid sequence having substitution, insertion, and / or addition
The encoding nucleotide sequence,
(Iii) From an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2
A nucleotide sequence encoding a polyamino acid synthase,
(Iv) the nucleotide sequence of SEQ ID NO: 1,
(V) a stringent nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1
Polyamino acid synthase that can hybridize under various hybridization conditions
Nucleotide sequence to be
(Vi) a polyamino acid having at least 65% identity with the nucleotide sequence of SEQ ID NO: 1
A nucleotide sequence encoding a synthase,
A recombinant vector containing any of the above.
(13a) (i) lack of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2
Polyamino acid synthesis consisting of amino acid sequences with deletions, substitutions, insertions, and / or additions
A nucleotide sequence encoding the enzyme,
(Ii) consisting of an amino acid sequence having 80% identity or more with the amino acid sequence of SEQ ID NO: 2
A nucleotide sequence encoding a polyamino acid synthetase, or
(Iii) High stringency in the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1
Polyamino acid synthase capable of hybridizing under the present hybridization conditions
A nucleotide sequence encoding,
A recombinant vector containing any of the above.
(14) (i) a nucleotide encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2
Nucleotide sequence, or
(Ii) the nucleotide sequence of SEQ ID NO: 1;
A recombinant vector containing any of the above.
(15) The polyamino acid synthase is polylysine synthase (13) or
The recombinant vector according to (14).
(16) (i) a condensation reaction comprising the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
A nucleotide sequence encoding a catalytic domain or a subdomain thereof,
(Ii) at least in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
An amino acid sequence having a deletion, substitution, insertion and / or addition of one amino acid residue
A nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof
Or
(Iii) 60% or more of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Condensation reaction catalytic domain or its subdomain consisting of a single amino acid sequence
The encoding nucleotide sequence,
(Iv) the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18;
(V) Nuclei complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Can hybridize to string sequences under stringent hybridization conditions
A nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, or
Is
(Vi) a nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 and 65% or more
Nucleotide encoding a condensation reaction catalytic domain or a subdomain thereof having identity
Tide sequence,
A recombinant vector containing any of the above.
(16a) (i) 1 in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Or amino acids with deletions, substitutions, insertions and / or additions of several amino acid residues
A nucleotide sequence encoding a polyamino acid synthase comprising the sequence;
(Ii) 80% or more identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
A nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having
Or
(Iii) a nucleotide complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Hybridizes to the nucleotide sequence under highly stringent hybridization conditions
A nucleotide sequence encoding a polyamino acid synthase,
A recombinant vector containing any of the above.
(17) (i) a condensation reaction consisting of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
A nucleotide sequence encoding a catalytic domain or a subdomain thereof, or
(Ii) the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18;
A recombinant vector containing any of the above.
(18) Transformation comprising the recombinant vector according to (13), (15) or (16) above
Transform.
(19) A transformant comprising the recombinant vector according to (14) or (17) above.
(20) The recombinant vector according to any of (13), (15), or (16) above
Or the manufacturing method of a polyamino acid synthetase using the transformant as described in said (18).
(21) The recombinant vector according to (14) or (17) above or the above (19)
A method for producing a polyamino acid synthase using the transformant described above.
(22) (i) the amino acid sequence of SEQ ID NO: 2,
(Ii) deletion or placement of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2
An amino acid sequence having a substitution, insertion, and / or addition, or
(Iii) an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2;
A step of synthesizing a polyamino acid using a polyamino acid synthetase containing any of
And a method for producing a polyamino acid.
(22a) (i) lack of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2
An amino acid sequence having a deletion, substitution, insertion, and / or addition, or
(Ii) an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 2,
A step of synthesizing a polyamino acid using a polyamino acid synthetase containing any of
And a method for producing a polyamino acid.
(23) (i) the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19;
(Ii) at least in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
An amino acid sequence having a deletion, substitution, insertion and / or addition of one amino acid residue;
Or
(Iii) 60% or more of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
A single amino acid sequence,
A step of synthesizing a polyamino acid using a polyamino acid synthetase containing any of
And a method for producing a polyamino acid.
(23a) (i) 1 in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Or amino acids with deletions, substitutions, insertions and / or additions of several amino acid residues
An array, or
(Ii) 80% or more identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
An amino acid sequence having sex,
A step of synthesizing a polyamino acid using a polyamino acid synthetase containing any of
And a method for producing a polyamino acid.
(24) The polyamino acid according to (22) or (23), wherein the polyamino acid is polylysine.
Method.
(25) The method according to (24) above, wherein the polylysine is ε-poly-L-lysine.
(26) (i) a nucleotide encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2
Nucleotide sequence or its complementary sequence,
(Ii) deletion or placement of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2
A polyamino acid synthetase consisting of an amino acid sequence having substitution, insertion, and / or addition
The encoding nucleotide sequence or its complementary sequence,
(Iii) From an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2
A nucleotide sequence encoding a polyamino acid synthetase or a complementary sequence thereof,
(Iv) the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence,
(V) a stringent nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1
Polyamino acid synthase that can hybridize under various hybridization conditions
Nucleotide sequence to be sequenced or its complementary sequence, or
(Vi) a polyamino acid having at least 65% identity with the nucleotide sequence of SEQ ID NO: 1
A nucleotide sequence encoding a synthase or its complementary sequence,
A polynucleotide comprising at least 15 contiguous nucleotides in any of
Primer consisting of
(26a) (i) lack of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2
Polyamino acid synthesis consisting of amino acid sequences with deletions, substitutions, insertions, and / or additions
A nucleotide sequence encoding the enzyme or its complementary sequence,
(Ii) consisting of an amino acid sequence having 80% identity or more with the amino acid sequence of SEQ ID NO: 2
A nucleotide sequence encoding a polyamino acid synthetase or its complementary sequence, or
(Iii) High stringency in the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1
Polyamino acid synthase capable of hybridizing under the present hybridization conditions
A nucleotide sequence encoding or a complementary sequence thereof,
A polynucleotide comprising at least 15 contiguous nucleotides in any of
Primer consisting of
(27) (i) a condensation reaction comprising the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Nucleotide sequence encoding a catalytic domain or a subdomain thereof or a phase thereof
Complement,
(Ii) at least in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
An amino acid sequence having a deletion, substitution, insertion and / or addition of one amino acid residue
A nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof
Or its complementary sequence,
(Iii) an amino acid sequence of SEQ ID NO: 13, 15, 17, or 19 and 60% or more
A condensation reaction catalytic domain consisting of amino acid sequences having identity or a subdomain thereof.
The encoding nucleotide sequence or its complementary sequence,
(Iv) the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 or its complement
Array,
(V) a nucleotide complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Can hybridize to string sequences under stringent hybridization conditions;
A nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, or
Its complementary sequence, or
(Vi) 65% or more of the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Nuclei encoding a condensation reaction catalytic domain or a subdomain thereof
Sequence or its complementary sequence,
A polynucleotide comprising at least 15 contiguous nucleotides in any of
Primer consisting of
(27a) (i) 1 in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Or amino acids with deletions, substitutions, insertions and / or additions of several amino acid residues
Nucleotide sequence encoding polyamino acid synthetase consisting of sequence or its complementary sequence
,
(Ii) 80% or more identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Nucleotide sequence encoding polyamino acid synthetase consisting of amino acid sequence
Or its complementary sequence, or
(Iii) a nucleotide complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Hybridizes to the nucleotide sequence under highly stringent hybridization conditions
A nucleotide sequence encoding a polyamino acid synthetase or its complementary sequence,
A polynucleotide comprising at least 15 contiguous nucleotides in any of
Primer consisting of
(28) (i) a nucleotide encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2
Nucleotide sequence or its complementary sequence,
(Ii) the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence,
(Iii) Condensation reaction consisting of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Nucleotide sequence encoding the catalytic domain or its subdomain or its complement
An array, or
(Iv) the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 or its complement
Array,
A polynucleotide comprising at least 15 contiguous nucleotides in any of
Primer consisting of
(29) (i) a nucleotide encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2
Nucleotide sequence or its complementary sequence,
(Ii) deletion or placement of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2
A polyamino acid synthetase consisting of an amino acid sequence having substitution, insertion, and / or addition
The encoding nucleotide sequence or its complementary sequence,
(Iii) From an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2
A nucleotide sequence encoding a polyamino acid synthetase or a complementary sequence thereof
(Iv) the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence,
(V) a stringent nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1
Polyamino acid synthase that can hybridize under various hybridization conditions
Nucleotide sequence to be sequenced or its complementary sequence, or
(Vi) a polyamino acid having at least 65% identity with the nucleotide sequence of SEQ ID NO: 1
A nucleotide sequence encoding a synthase or its complementary sequence,
Under stringent hybridization conditions to any of the nucleotide sequences
A polynucleotide comprising a nucleotide sequence at least 15 nucleotides in length that can be hybridized
Creotide probe.
(29a) (i) lack of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2
Polyamino acid synthesis consisting of amino acid sequences with deletions, substitutions, insertions, and / or additions
A nucleotide sequence encoding the enzyme or its complementary sequence,
(Ii) consisting of an amino acid sequence having 80% identity or more with the amino acid sequence of SEQ ID NO: 2
A nucleotide sequence encoding a polyamino acid synthetase or its complementary sequence, or
(Iii) High stringency in the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1
Polyamino acid synthase capable of hybridizing under the present hybridization conditions
A nucleotide sequence encoding or a complementary sequence thereof,
Under highly stringent hybridization conditions to any of the nucleotide sequences
Consisting of a nucleotide sequence of at least 15 nucleotides in length capable of hybridizing,
Nucleotide probe.
(30) (i) a condensation reaction comprising the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Nucleotide sequence encoding a catalytic domain or a subdomain thereof or a phase thereof
Complement,
(Ii) at least in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
An amino acid sequence having a deletion, substitution, insertion and / or addition of one amino acid residue
A nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof
Or its complementary sequence,
(Iii) 60% or more of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Condensation reaction catalytic domain consisting of unique amino acid sequence or its subdomain
Nucleotide sequence to be loaded or its complementary sequence
(Iv) the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 or its complement
Array,
(V) a nucleotide complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Can hybridize to string sequences under stringent hybridization conditions;
A nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, or
Its complementary sequence, or
(Vi) 65% or more of the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Nuclei encoding a condensation reaction catalytic domain or a subdomain thereof
Sequence or its complementary sequence,
Under stringent hybridization conditions to any of the nucleotide sequences
A polynucleotide comprising a nucleotide sequence at least 15 nucleotides in length that can be hybridized
Creotide probe.
(30a) (i) 1 in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Or amino acids with deletions, substitutions, insertions and / or additions of several amino acid residues
Nucleotide sequence encoding polyamino acid synthetase consisting of sequence or its complementary sequence
,
(Ii) 80% or more identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Nucleotide sequence encoding polyamino acid synthetase consisting of amino acid sequence
Or its complementary sequence, or
(Iii) a nucleotide complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18
Hybridizes to the nucleotide sequence under highly stringent hybridization conditions
A nucleotide sequence encoding a polyamino acid synthetase or its complementary sequence,
Under highly stringent hybridization conditions to any of the nucleotide sequences
Consisting of a nucleotide sequence of at least 15 nucleotides in length capable of hybridizing,
Nucleotide probe.
(31) (i) a nucleotide encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2
Nucleotide sequence or its complementary sequence,
(Ii) the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence,
(Iii) Condensation reaction consisting of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
Nucleotide sequence encoding the catalytic domain or its subdomain or its complement
An array, or
(Iv) the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 or its complementary sequence
Columns,
Under stringent hybridization conditions to any of the nucleotide sequences
A polynucleotide comprising a nucleotide sequence at least 15 nucleotides in length that can be hybridized
Creotide probe.
(32) (i) the amino acid sequence of SEQ ID NO: 2,
(Ii) deletion or placement of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2
An amino acid sequence having a substitution, insertion, and / or addition, or
(Iii) an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2;
An antibody that specifically binds to a polyamino acid synthetase containing any of the above.
(33) (i) the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19;
(Ii) at least in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
An amino acid sequence having a deletion, substitution, insertion and / or addition of one amino acid residue;
Or
(Iii) 60% or more of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19
A single amino acid sequence,
A condensation reaction catalytic domain containing any of
An antibody that specifically binds to a polyamino acid synthase.
(34) Polyamino using the primer according to any of (26) to (28) above
A method for screening a polynucleotide encoding an acid synthase.
(35) Further using the probe according to any one of (29) to (31) above,
34) The screening method.
(36) Polyamino acid synthetase using the antibody according to (32) or (33) above
Screening method.

According to the present invention, polyamino acids (eg, ε-poly-L-lysine) can be enzymatically synthesized. The enzymatic synthesis method is more energy-saving than the microbial fermentation method and the chemical synthesis method, and has an advantage that a highly pure polyamino acid can be produced because undesirable side reactions can be suppressed. Furthermore, since many peptides and polyamino acids produced by microorganisms containing cyanophycin and poly-γ-glutamic acid are biosynthesized by the catalytic action of a complex enzyme system, enzymatic industrial production is difficult. Since the synthase is a single thiotemplate-type ribosome-independent peptide synthase (NRPS), it has the advantage of being easy to control and suitable for enzymatic industrial production.

Thus, the present invention can provide a method for producing a polyamino acid more efficiently than before. Furthermore, application to various useful polyamino acid biosynthesis is possible by using the polynucleotide-containing recombinant vector and transformant of the present invention.

The present inventors have reported that ε-poly-L-lysine producing bacteria Streptomyces albras (Stre
ptomyces albulus) from IFO14147 strain, membrane-bound ε-poly-L-
Lysine biosynthetic enzyme was isolated and purified, and its amino acid sequence (SEQ ID NO: 2) and the base sequence of the gene encoding it (SEQ ID NO: 1) were determined. When the amino acid sequence and the DNA sequence of the ε-poly-L-lysine biosynthetic enzyme of the present invention are searched for homology with the NCBI sequence database,
The highest in amino acid sequence, 54.1% identical (77.3% similarity), the highest in DNA sequence, 64.2% identical, and local (regions of important domains (positions 470-795)) 80% or more identical) Existed.

In one embodiment of the invention, a polylysine biosynthetic enzyme and the gene encoding it are provided. The enzyme of the present invention is the only membrane enzyme that has not been known so far as NRPS. Accordingly, in a further embodiment of the invention, a membrane-bound NRPS is provided.

So far, many microbially-produced peptides with physiological activity that can be applied to medicines have been found.
It is biosynthesized by the catalytic action of a multi-module complex enzyme called mal peptide synthetase).

NRPS catalyzes the condensation of amino acids or peptide intermediate molecules called adenylation domain (A-Domain), peptidyl carrier domain or thiolation domain (PCP-Domain), which catalyze the activation of amino acids, and substrate condensation Condensation domain (C-Domain), peptide cyclization and NR which are the final steps of biosynthesis
A module composed of a thioesterase domain (TE-Domain) involved in peptide release from PS is configured as a basic unit (FIG. 1). A 4′-phosphopantethein (P-pant) arm, which is a cofactor for binding amino acids and intermediates, is bound to the thiolation domain (not shown). In some cases, a module may be added with a domain related to peptide modification such as epimerization or methylation.

Each domain of NRPS has multiple regions that are conserved in common with peptide synthases. For example, microcystin biosynthetic genes are probed with fragments obtained by PCR amplification based on these nucleotide sequence information. It has been cloned.

The polylysine biosynthetic enzyme of the present invention also has a database such as CDD (Conserved).
Domain Database: http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml/) and other analysis tools that predict functions from structural comparisons with proteins with similar domain structures Like many NRPS, N-terminal A-Domain, PCP-Dom that binds peptide intermediate molecules
The existence of ain was confirmed. However, about 700 amino acid residues on the C-terminal side were found to be a novel (unknown) domain having no homology to any conventional C-Domain or TE-Domain. Moreover, although conventional NRPS is a multi-module complex enzyme, the structure of this enzyme strongly suggests that it is a single module single enzyme from the results of domain structure analysis and enzyme purification. Thus, in the enzyme of the present invention, since polyamino acid synthesis is achieved with only a single module, the novel domain of 700 amino acid residues on the C-terminal side is considered to catalyze the “repetitive condensation reaction”. Accordingly, in one embodiment of the present invention, a polylysine biosynthetic enzyme comprising about 700 amino acid residues on the C-terminal side is provided.

Furthermore, all conventional NRPS are localized in the cytoplasm (soluble fraction), but this enzyme is
S is the only membrane enzyme that has not been known so far, and the membrane-bound (transmembrane) region was also thought to exist in the novel C-terminal domain. Therefore, the C-terminal novel domain of the enzyme of the present invention was analyzed in more detail using an analysis tool that predicts secondary structures such as membrane proteins from the amino acid sequence. According to SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/), as expected, the C-terminal new domain was confirmed to have 6 transmembrane regions, The new domains that are thought to catalyze “repetitive condensation reactions” are the three subdomains IC1, IC2
And the possibility of being composed of IC3-Domain was suggested (FIGS. 1B and 2). Accordingly, in another embodiment of the present invention, a polylysine biosynthetic enzyme comprising any of the above three subdomains is provided.
Hereinafter, the present invention will be described in detail.

1. The polyamino acid synthetase of the present invention and the polynucleotide encoding the same The present invention provides a polyamino acid synthetase and a gene encoding the same. Specifically, the present invention has (i) the amino acid sequence of SEQ ID NO: 2, and (ii) at least one amino acid residue deletion, substitution, insertion, and / or addition in the amino acid sequence of SEQ ID NO: 2. Provided is a polyamino acid synthetase comprising either an amino acid sequence or (iii) an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2.

The present invention also provides (i) an amino acid sequence of SEQ ID NO: 2, (ii) an amino acid sequence having a deletion, substitution, insertion and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2, or (Iii) A polynucleotide comprising a nucleotide sequence encoding a polyamino acid synthetase consisting of any one of the amino acid sequence of SEQ ID NO: 2 and 60% or more identity.

The present invention also encodes a polyamino acid synthase capable of hybridizing under stringent hybridization conditions to (i) a nucleotide sequence of SEQ ID NO: 1, and (ii) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1. A polynucleotide comprising: (iii) a nucleotide sequence encoding a polyamino acid synthase having at least 65% identity to the nucleotide sequence of SEQ ID NO: 1.

In a preferred embodiment, the polyamino acid synthase is polylysine synthase, and more preferably ε-poly-L-lysine synthase. The polynucleotide is
It can be DNA or RNA, preferably DNA.

In the present specification, the term “polyamino acid synthase of the present invention” refers to (i) SEQ ID NO: 2
Not only the polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2, but also a polyamino acid synthetase consisting of an amino acid sequence having high homology with the amino acid sequence of SEQ ID NO: 2, for example, (ii) at least one amino acid sequence in the amino acid sequence of SEQ ID NO: 2 A polyamino acid synthase consisting of an amino acid sequence having deletions, substitutions, insertions and / or additions of amino acid residues, and (iii)
And a polyamino acid synthetase comprising an amino acid sequence having a high identity (eg, about 60% or more) with the amino acid sequence of SEQ ID NO: 2. In addition, the enzyme activity of polyamino acid synthase can be determined, for example, by the method of Kawai et al. (Biochem Biophys Res Commun. 2003 Nov 21; 311 (3): 635-4
0) etc. and can be measured and / or confirmed.

Examples of “polyamino acid synthase comprising an amino acid sequence having a deletion, substitution, insertion and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2” include the amino acid sequence of SEQ ID NO: 2 For example, 1-50, 1-40, 1-3
9, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1
~ 32, 1 ~ 31, 1 ~ 30, 1 ~ 29, 1 ~ 28, 1 ~ 27, 1 ~ 26, 1 ~ 25, 1 ~ 24, 1 ~ 23, 1 -22, 1-21, 1-20, 1-1
9, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1
-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6 (1-several), 1-5, 1-4, Examples thereof include polyamino acid synthetase having an amino acid sequence in which 1 to 3, 1 to 2, or 1 amino acid residue is deleted, substituted, inserted and / or added.
In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better.

Examples of “polyamino acid synthetase comprising an amino acid sequence having high identity with the amino acid sequence of SEQ ID NO: 2” include the amino acid sequence of SEQ ID NO: 2 and about 60% or more, 65% or more, 70
% Or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more,
85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% Or more, 98
% Or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99 .8% or more, or 99.
Examples include polyamino acid synthetase consisting of an amino acid sequence having 9% or more identity. In general, the larger the numerical value of identity, the better. In this specification, the term “identity”
“ntity)” and “homology” are used separately. For example, when referring to homology between amino acid sequences, amino acids having the same properties (such as glutamic acid and aspartic acid) are treated as one group, but are distinguished when considering identity. That is, identity refers to consistency.

In the present specification, “hybridizes” with respect to a nucleotide sequence means that a double strand is formed when the complementarity between nucleotide sequences is high. Usually, the double strands are designed so that the nucleic acids are complementary to each other, but if necessary (for example, in the case where nucleic acids are used for detection, as long as no problem occurs for that purpose). The nucleic acid may contain one or several (for example, 2 to 3) mismatches. The number of mismatches that may exist is
For example, required detection accuracy, nucleotide sequence (or base sequence or nucleic acid (sequence)
Alternatively, it may vary depending on the length of the polynucleotide. In addition, by appropriately changing the hybridization conditions between the nucleotide sequence and the nucleotide sequence with which it hybridizes, it is possible to eliminate the case where there is a mismatch or to adjust the number of allowable mismatches. Are well known to those skilled in the art.

As a hybridization method, a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like can be used (for example, Molecular Cloning 3rd Ed., Curren).
t Protocols in Molecular Biology, John W
iley & Sons 1987-1997).

In the present specification, “stringent hybridization conditions” may be any of low stringency conditions, moderate stringency conditions, and high stringency conditions. “Low stringent conditions” are, for example, 5 × SSC, 5 × Denhardt's solution, 0.
The conditions are 5% (w / v) SDS, 50% (w / v) formamide, and 32 ° C. Also,"
For example, 5 × SSC, 5 × Denhardt's solution, 0.5% (
w / v) SDS, 50% (w / v) formamide, 42 ° C. “High stringent conditions” are, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% (w / v) SD
S, 50% (w / v) formamide, 50 ° C. Under these conditions, it can be expected that DNA having higher identity can be efficiently obtained as the temperature is increased. However,
Factors affecting the stringency of hybridization include multiple factors such as temperature, nucleic acid concentration, nucleic acid length, ionic strength, time, and salt concentration, and those skilled in the art can appropriately select these factors. Similar stringency can be achieved.

In addition, when using a commercially available kit for hybridization, for example, Alkpho
s Direct Labeling Reagents (manufactured by Amersham Pharmacia) can be used. In this case, according to the protocol attached to the kit, incubation with the labeled probe was performed overnight, and the membrane was then treated at 0. 5 ° C. at 55 ° C.
After washing with a primary washing buffer containing 1% (w / v) SDS, the hybridized polynucleotide can be detected.

Examples of “nucleotide sequences that can hybridize under stringent hybridization conditions” to a desired nucleotide sequence (for example, the nucleotide sequence of SEQ ID NO: 1) include homology (or identity) search software such as FASTA and BLAST. To approximately 6% of the desired nucleotide sequence when calculated using the default parameters.
0% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% 89%
90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 9
6% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 9
9.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 9
Examples of the nucleotide sequence include 9.8% or more and 99.9% or more of identity.

The identity of amino acid sequences and base sequences is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 87, by Carlin and Arthur).
2264-2268, 1990; Proc. Natl. Acad. Sci.
USA, 90, 5873, 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (
Altschul SF, et al: J Mol Biol 215: 403,
1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. Further, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50, word.
Let length = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program can be used.

2. The C-terminal domain of the polyamino acid synthetase of the present invention and the polynucleotide encoding the same The conventional NRPS polymerizes a peptide or polyamino acid by the catalytic action of a multi-module complex enzyme, but the polyamino acid synthetase of the present invention has a domain structure From the results of analysis and enzyme purification,
It is strongly suggested that it is a single module single enzyme (see FIGS. 1A and B). In the polyamino acid synthetase of the present invention, since polyamino acid synthesis is achieved with only a single module, the novel domain of about 700 amino acid residues on the C-terminal side of the present invention is considered to catalyze a “repetitive condensation reaction”. It is done. In addition, as a result of analysis using a protein structure analysis tool such as CDD, it was estimated that the C-terminal domain has three subdomains and six transmembrane regions at their boundaries (see FIG. 1B). reference).

Therefore, the present invention also provides, in another embodiment, a C-terminal domain containing about 700 amino acid residues on the C-terminal side of the polyamino acid synthase of the present invention (hereinafter referred to as “condensation reaction catalytic domain”), and Provided are a polyamino acid synthetase comprising a condensation reaction catalytic domain, and a polynucleotide comprising the domain and a base sequence encoding the enzyme. In addition,
In the present specification, the term “condensation reaction catalytic domain” means a condensation reaction catalytic domain of polyamino acid synthase.

The condensation reaction catalytic domain of the present invention is typically an amino acid of the ε-poly-L-lysine synthase of the present invention represented by SEQ ID NO: 2, as shown in FIGS. 2A-D and 3A-D. An amino acid sequence corresponding to about positions 611 to 1319 of the sequence (position 1831 to base sequence)
The amino acid sequence and the base sequence encoding it are represented by SEQ ID NO: 13 and SEQ ID NO: 12, respectively.
Also, the three smaller domains that make up this domain (IC1, IC2 and IC3-
Domain) is typically ε-poly-L- of the present invention represented by SEQ ID NO: 2, respectively.
The amino acid sequence of lysine synthase corresponds to the region of the amino acid sequence of approximately 676 to 847, 914 to 1083, and 1147 to 1299 (in the base sequence, positions 2026 to 2541 and 2740, respectively). 3249, and 3439 to 3897). The amino acid sequences of these three subdomains are represented by SEQ ID NOs: 15, 17, and 19, respectively. The base sequences encoding these amino acid sequences are SEQ ID NOs: 14, 1 and 1, respectively.
6 and 18.

In the present specification, the term “condensation reaction catalytic domain of the present invention” refers to (i) an amino acid sequence having high homology with the amino acid sequence of SEQ ID NO: 13 as well as the condensation reaction catalytic domain consisting of the amino acid sequence of SEQ ID NO: 13. A condensation reaction catalytic domain consisting of, for example, (ii) a condensation reaction catalytic domain consisting of an amino acid sequence having a deletion, substitution, insertion and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 13, and (Iii
) It also includes a condensation reaction catalytic domain consisting of an amino acid sequence having a high identity (eg, about 60% or more) with the amino acid sequence of SEQ ID NO: 13.

In addition, in the present specification, the “subdomain of the present invention (condensation reaction catalytic domain)” refers to (i) only the subdomain consisting of the amino acid sequence of SEQ ID NO: 15, 17, or 19 (condensation reaction catalytic domain). Rather, a subdomain (of the condensation reaction catalytic domain) consisting of an amino acid sequence having high homology with the amino acid sequence of SEQ ID NO: 15, 17, or 19, for example, (ii) the amino acid sequence of SEQ ID NO: 15, 17, or 19 At least 1
A subdomain (of the condensation reaction catalytic domain) consisting of an amino acid sequence having deletions, substitutions, insertions and / or additions of amino acid residues, and (iii) SEQ ID NOs: 15, 17,
Alternatively, it also includes a subdomain (of the condensation reaction catalytic domain) consisting of an amino acid sequence having a high identity (eg, about 60% or more) with 19 amino acid sequences.

Accordingly, the present invention also provides for the lack of at least one amino acid residue in (i) the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19, and (ii) the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19. An amino acid sequence having deletions, substitutions, insertions, and / or additions, or (iii) the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19 and 60
A condensation reaction catalytic domain comprising any of amino acid sequences having at least% identity, or a subdomain thereof, and a polyamino acid synthase comprising the domain. Whether or not a condensation reaction has occurred can be confirmed by detecting a polymer (enzyme reaction product) by LC-MS / MS, for example.

The present invention also provides (i) an amino acid sequence of SEQ ID NO: 13, 15, 17, or 19;
i) an amino acid sequence having a deletion, substitution, insertion, and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19, or (iii) SEQ ID NO: 13, 15, A polycondensation comprising a condensation reaction catalytic domain consisting of any of 17 or 19 amino acid sequences and an amino acid sequence having 60% or more identity, or a subdomain thereof, or a nucleotide sequence encoding a polyamino acid synthetase containing the domain Nucleotides are provided.

The invention also provides (i) a nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18;
(Ii) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof capable of hybridizing under stringent hybridization conditions to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18. Or (iii) a polynucleotide comprising any of the nucleotide sequences encoding the condensation reaction catalytic domain or a subdomain thereof having at least 65% identity with the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 To do.

"Condensation reaction catalytic domain consisting of an amino acid sequence having a deletion, substitution, insertion, and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19, or a subdomain thereof, or Examples of the “polyamino acid synthetase containing a domain” include, for example, 1 to 50, 1 to 40, 1 to 39, 1 to 38 in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19. 1-37, 1-36
Pieces, 1-35 pieces, 1-34 pieces, 1-33 pieces, 1-32 pieces, 1-31 pieces, 1-30 pieces, 1-pieces
29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23,
1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16
1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1
9, 1-8, 1-7, 1-6 (1 to several), 1-5, 1-4, 1-3, 1
Examples include a condensation reaction catalytic domain consisting of an amino acid sequence in which ˜2, one amino acid residue is deleted, substituted, inserted and / or added, or a subdomain thereof, or a polyamino acid synthetase containing the domain. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better.

Examples of the “condensation reaction catalytic domain consisting of an amino acid sequence having high identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19 or a subdomain thereof or a polyamino acid synthase containing the domain” include SEQ ID NO: 13 15, 17 or 19 amino acid sequences and about 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81%
Or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 8
8% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99. 1% or more, 9
9.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 9
Examples include a condensation reaction catalytic domain consisting of an amino acid sequence having an identity of 9.7% or more, 99.8% or more, or 99.9% or more, or a subdomain thereof, or a polyamino acid synthase containing the domain. In general, the larger the numerical value of identity, the better. In the present specification, the terms “identity” and “homology” are used separately. For example, when referring to homology between amino acid sequences,
Amino acids with the same properties (such as glutamic acid and aspartic acid) are treated as one group, but are distinguished when considering identity. That is, identity refers to consistency.

Examples of “nucleotide sequences that can hybridize to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 under stringent hybridization conditions”
When calculated using the default parameters by homology (or identity) search software such as FASTA, BLAST and the like, the desired nucleotide sequence and about 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83%
84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 9
0% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% 99.3%
99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8%
As described above, nucleotide sequences having 99.9% or more identity can be mentioned.

3. The recombinant vector of the present invention, a transformant, and a method for producing a polyamino acid synthetase using the same The present invention also relates to a recombinant vector containing a polynucleotide encoding the polyamino acid synthetase of the present invention as an insert. Specifically, the present invention has (i) the amino acid sequence of SEQ ID NO: 2, and (ii) at least one amino acid residue deletion, substitution, insertion, and / or addition in the amino acid sequence of SEQ ID NO: 2. Provided is a recombinant vector comprising a nucleotide sequence encoding a polyamino acid synthetase consisting of either an amino acid sequence or (iii) an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2.

Furthermore, the present invention encodes a polyamino acid synthase capable of hybridizing under stringent hybridization conditions to (i) a nucleotide sequence of SEQ ID NO: 1, and (ii) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1. A recombinant vector containing a polynucleotide comprising: (iii) a nucleotide sequence encoding a polyamino acid synthetase having at least 65% identity to the nucleotide sequence of SEQ ID NO: 1 To do.

Furthermore, the present invention relates to a recombinant comprising, as an insert, a polynucleotide encoding the condensation reaction catalytic domain of the present invention or a subdomain thereof or a polynucleotide encoding a polyamino acid synthase containing the condensation reaction catalytic domain or a subdomain thereof. Regarding vectors. Specifically, the present invention relates to (i) an amino acid sequence of SEQ ID NO: 13, 15, 17, or 19, and (ii) at least one amino acid residue in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19. Or (iii) an amino acid sequence having at least 60% identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19 A recombinant vector containing a polynucleotide encoding a condensation reaction catalytic domain or a subdomain thereof, or a nucleotide sequence encoding a polyamino acid synthase containing the condensation reaction catalytic domain or a subdomain thereof.

The present invention further provides (i) a nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18;
(Ii) can hybridize under stringent hybridization conditions to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18;
A nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, or (iii) a condensation reaction catalytic domain or a subdomain thereof having 65% identity or more with the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 Recombinant vectors containing polynucleotides consisting of any of the encoding nucleotide sequences are provided.

In a preferred embodiment of the invention, the recombinant vector is a recombinant expression vector capable of expressing the insert. Such a recombinant vector can be prepared by any method known in the art.

In the present invention, the vector used for expressing the polyamino acid synthetase of the present invention is a cell-free expression system (in vitro transcription-translation) and a host cell such as E. coli, yeast or animal cultured cells. A vector suitable for a protein expression system can be used, and such a vector is commercially available or can be easily prepared from a known vector. For example, the vectors used for in vitro translation and expression in cultured animal cells are human cytomegalovirus immeadiate-ear
The ly enhancer / promoter region is incorporated and the TTarget promoter / multicloning site in the downstream region of the pTargetT vector or SV40 enhancer and SV4
PSI vector (Promega) with 0 early promoter, pBK-CMV, C
MV-Script, pCMV-Tag and pBK-RSV (Stratagene). Also, vectors suitable for expression in microbial hosts such as Escherichia coli and yeast are, for example, pET series vector expression systems having an E. coli T7 promoter (for example, pE).
T3a, pET27b (+) and pET28a (+); Novagen), and in yeast systems, Pichia expression vectors pIC series vectors (for example, pPIC9K and PIC3.5K; Invitrogen) having an alcohol oxidase promoter.

The peptide sequence for purification can be a peptide sequence used in the technical field. For example, a histidine tag sequence having 4 or more, preferably 6 or more histidine residues, An epitope tag sequence capable of binding to a monoclonal antibody,
The binding domain of glutathione S-transferase to glutathione or a sequence encoding protein A is included. DNA encoding these peptide sequences
Vectors containing as an insert are commercially available. For example, a vector used for a microbial host such as E. coli or yeast has an E. coli T7 promoter, 10 histidine residues fused to the amino terminus (eg, pET16b; Novagen), and 6 histidine residues. An expression vector having an amino acid at the amino terminus (for example, pHB6; Roche Diagnostic, pTrc-His series vector; Invitrogen, pHAT vector series; Clontech). Vectors used for expression in cultured animal cells are vectors in which six histidine residues are fused to the amino terminus (for example, pHM6 or pVM).
6; Roche Diagnostics) can be used. In insect cell lines, vectors that fuse and express six baculovirus histidine residues at the amino terminus (eg, pB
acPAK-His vector; Clontech). In addition, pGEX series vectors (Pharmacia) fused with glutathione S-transferase, and pRIT2 or pEZZ18 vectors (Pharmacia) expressing protein A can be used.

In vitro translation to produce the polyamino acid synthase of the present invention can be accomplished by known methods such as Spirin, AS, et.al., Science 242: 1162-1164 (1988) and Patna
ik, R. & Swartz, JM Biotechniques 24: 862-868 (1998). May be used.

A transformant transformed with the present invention or the above-described recombinant vector (for example, a host cell (
Examples: microorganisms (E. coli, yeast, etc.)) and animal cultured cells (insect cultured cells, mammalian cultured cells (eg CHO cells, COS7 cells))).

The present invention also relates to a method for producing a polyamino acid synthase using the recombinant vector of the present invention or the transformant of the present invention. The method for producing the polyamino acid synthetase of the present invention comprises:
For example, performing in vitro transcription-translation using the recombinant expression vector of the present invention. Alternatively, it comprises culturing host cells transformed with the above recombinant expression vector and isolating the desired protein.

In the method for producing a polyamino acid synthetase of the present invention, the expressed protein can be isolated from the medium after culturing the host cell or from the soluble or insoluble fraction of the microbial cells.

In the method for producing a polyamino acid synthetase of the present invention, the expressed protein is isolated and purified by a known method, and the purification method can be appropriately selected from any known methods. For example, when the polyamino acid synthetase of the present invention includes the peptide sequence for purification described above, it is preferable to purify using this. Specifically, nickel chelate affinity chromatography for histidine tag sequences, affinity chromatography with glutathione binding gel for the binding domain of S-transferase to glutathione, antibody affinity for protein A or other protein sequences Chromatographic methods can be used.

4). The method for producing a polyamino acid of the present invention The present invention also relates to a method for producing the polyamino acid of the present invention. Specifically, the present invention provides:
(I) the amino acid sequence of SEQ ID NO: 2,
(Ii) a deletion of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2,
An amino acid sequence having a substitution, insertion, and / or addition, or (iii) an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2,
A method for producing a polyamino acid comprising the step of synthesizing a polyamino acid using a polyamino acid synthetase containing any of the above.

The present invention also provides
(I) the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19;
(Ii) an amino acid sequence having a deletion, substitution, insertion, and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19; or (iii) SEQ ID NO: 13, 15 An amino acid sequence having 60% or more identity with the amino acid sequence of 17, or 19;
A method for producing a polyamino acid comprising the step of synthesizing a polyamino acid using a polyamino acid synthetase containing any of the above.

Preferably, the polyamino acid is polylysine, more preferably ε-poly-L.
-Lysine.

The method for producing a polyamino acid of the present invention is carried out by reacting an amino acid in the presence of the polyamino acid synthase. Here, as the amino acid to be reacted, L-lysine can be typically exemplified, but is not limited thereto. For example, L-lysine ethyl ester, L-lysine methyl ester (both products are ε-polyester). -L-lysine) and the like can also be used for polyamino acid synthesis by the amino acid synthetase of the present invention. D-lysine,
DL-5-hydroxy-lysine, Nε-methyl-L-lysine, L-lysine-hydroxamic acid, S- (2-aminoethyl) -L-cysteine, O- (2-aminoethyl) -L-serine, DL -Trans-2,6-diamino-4-hexanoic acid may be used in the method for producing a polyamino acid of the present invention.

In the method for synthesizing polyamino acids according to the present invention, the amino acid polymerization reaction by the enzyme is preferably at an optimum pH of the reaction, around 7-9.

The amount of the polyamino acid synthetase added according to the present invention is not particularly limited. Generally, the addition amount of the polyamino acid synthetase of the present invention is usually about 10,000 units or more, preferably about 2 in order to obtain a polymer by reaction for several hours (eg, 2 to 5 hours) from 1 g of the raw amino acid. In the range of 10,000 to 40,000 units,
More preferably, it is desirable to select from about 30,000 units. The temperature of the reaction is not particularly limited as long as the enzyme can act. For example, although the reaction proceeds even at a low temperature of about 15 ° C., it is usually about 20 to 20 on the assumption that the enzyme acts efficiently and stably. It is desirable to perform at a temperature of about 40 ° C. The reaction time can be appropriately determined according to the concentration of the amino acid used, the amount of enzyme added, etc., but is usually about 0.5 to 5 hours, and it is appropriate to be within 24 hours at the longest.

The polyamino acid thus obtained typically has a degree of polymerization of 3 to 100 mer and an average molecular weight of 403 to 21835. Preferably, the degree of polymerization is 4 to 50 mer, more preferably 4 to
The average molecular weight is preferably 531 to 6427, more preferably 531 to 2581.
It is.

The polyamino acid obtained by the present invention can be used as an antibacterial agent or the like, and can be used for various uses such as toiletries, cosmetics, feed additives, medicines, agricultural chemicals, food additives, electronic materials and the like.

5. Primer and probe of the present invention and screening method using them The partial fragment of the polynucleotide (DNA) of the present invention is used, for example, as a probe or a PCR primer to produce the polyamino acid synthase of the present invention or the same. It can be used to screen for homologues of the encoding polynucleotides.

Therefore, the present invention
(I) a nucleotide sequence encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2 or a complementary sequence thereof,
(Ii) a deletion of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2,
A nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having a substitution, insertion, and / or addition, or a complementary sequence thereof,
(Iii) a nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2 or a complementary sequence thereof,
(Iv) the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence,
(V) a nucleotide sequence encoding a polyamino acid synthase or a complementary sequence thereof, which can hybridize under stringent hybridization conditions to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, or (vi) SEQ ID NO: 1 A nucleotide sequence encoding a polyamino acid synthase or a complementary sequence thereof having 65% or more identity with the nucleotide sequence of
Also provided are primers consisting of a polynucleotide comprising at least 15 contiguous nucleotides in any of the above.

The present invention also provides:
(I) a nucleotide sequence encoding a condensation reaction catalytic domain consisting of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19 or a subdomain thereof, or a complementary sequence thereof,
(Ii) a condensation reaction catalytic domain consisting of an amino acid sequence having a deletion, substitution, insertion and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19 or a subdomain thereof A nucleotide sequence encoding or a complementary sequence thereof,
(Iii) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19, or a complementary sequence thereof,
(Iv) the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18 or its complementary sequence,
(V) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which can hybridize under stringent hybridization conditions to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18. A complementary sequence thereof, or (vi) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof having 65% or more identity with the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18, or a complementary sequence thereof,
Also provided are primers consisting of a polynucleotide comprising at least 15 contiguous nucleotides in any of the above.

The present invention also provides
(I) a nucleotide sequence encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2 or a complementary sequence thereof,
(Ii) a deletion of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2,
A nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having a substitution, insertion, and / or addition, or a complementary sequence thereof,
(Iii) a nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2 or a complementary sequence thereof (iv) a nucleotide sequence of SEQ ID NO: 1 or a complementary sequence thereof
(V) a nucleotide sequence encoding a polyamino acid synthase or a complementary sequence thereof, which can hybridize under stringent hybridization conditions to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, or (vi) SEQ ID NO: 1 A nucleotide sequence encoding a polyamino acid synthase or a complementary sequence thereof having 65% or more identity with the nucleotide sequence of
A polynucleotide probe comprising a nucleotide sequence that is at least 15 nucleotides in length that can hybridize to any of the nucleotide sequences under stringent hybridization conditions.

The present invention also provides
(I) a nucleotide sequence encoding a condensation reaction catalytic domain consisting of the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19 or a subdomain thereof, or a complementary sequence thereof,
(Ii) a condensation reaction catalytic domain consisting of an amino acid sequence having a deletion, substitution, insertion and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19 or a subdomain thereof A nucleotide sequence encoding or a complementary sequence thereof,
(Iii) Nucleotide sequence encoding a condensation reaction catalytic domain consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19 or a subdomain thereof, or a complementary sequence thereof (iv) Sequence The nucleotide sequence of number 12, 14, 16, or 18 or its complementary sequence;
(V) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which can hybridize under stringent hybridization conditions to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18. A complementary sequence thereof, or (vi) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof having 65% or more identity with the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18, or a complementary sequence thereof,
A polynucleotide probe comprising a nucleotide sequence that is at least 15 nucleotides in length that can hybridize to any of the nucleotide sequences under stringent hybridization conditions.

The present invention also provides a method for screening a polynucleotide encoding a polyamino acid synthetase using the primer described above. Preferably, the screening method comprises:
Further, the above probe is used.

Specifically, the primer or probe of the present invention specifically includes, for example, the presence of a homologue of the polyamino acid synthetase of the present invention in a specimen, Northern hybridization or semi-quantitative reverse PCR using the above primer and / or probe. (Sourvinos, G. et
al. , Oncogene, 18, 4968, (1999)), or the presence or absence of mutation of the gene encoding the polyamino acid synthase of the present invention in the specimen and the position of the mutation are determined by PCR-SSCP method (Proc. Natl. Acad.Sci.USA., 86,
2766, (1989)).

The size of the primer or probe is not particularly limited as long as it can be analyzed or detected as described above. Usually, the primer is about 10 to 100, more preferably about 15 to
80, more preferably about 20 to 60, and most preferably about 20 to 40 nucleotides in length, and examples of probes include about 10 to 1000 and about 15 to 150.
0, preferably about 20-1000, more preferably about 30-800, more preferably about 50-700, even more preferably about 100-500, most preferably about 200-5.
A length of 00 nucleotides can be used.

The amplification method using the primer of the present invention is the polymerase chain reaction (PCR; Saiki et
al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu & Wallace, 198)
9; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990)
Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989),
To amplify nucleic acid molecules using strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or Qβ replicase (Lizardi et al., 1988; Lomeli et al., 1989), primer extension Other suitable methods can be used. During amplification, the amplification product can conveniently be labeled using labeled primers or incorporating labeled nucleotides. The label may be an isotope ( ³² P, ³⁵ S, etc.) or a non-isotope (biotin, digoxigenin, etc.). The amplification reaction is repeated 20 to 70 times, conveniently 25 to 45 times.

6). Antibody of the present invention The present invention also relates to an antibody, for example, a polyclonal antibody or a monoclonal antibody, which is produced using the polyamino acid synthetase of the present invention or a fragment thereof as an antigen and specifically binds to the antigen. Specifically, the present invention provides:

(I) the amino acid sequence of SEQ ID NO: 2,
(Ii) an amino acid sequence having a deletion, substitution, insertion and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2, or (iii) 60% or more identity with the amino acid sequence of SEQ ID NO: 2 An amino acid sequence having sex,
An antibody that specifically binds to a polyamino acid synthase containing any of the above is provided.

The present invention also provides
(I) the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19;
(Ii) an amino acid sequence having a deletion, substitution, insertion and / or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19;
Or (iii) an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19;
An antibody that specifically binds to a condensation reaction catalytic domain comprising any of the above or a subdomain thereof or a polyamino acid synthase comprising the domain is provided.

An antibody that specifically binds to the polyamino acid synthase of the present invention can be produced according to a method for producing an antibody (or antiserum) known to those skilled in the art using the polyamino acid synthase of the present invention as an antigen. As an antigen, an extracted natural protein, a recombinant protein, a partial degradation product of these proteins, or a synthetic peptide based on the amino acid sequence of the polyamino acid synthetase of the present invention can be used. Such an antigen is, for example, a polypeptide consisting of at least 5 consecutive amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2, preferably 10 to 15 amino acid residues. The antibody of the present invention can be prepared by a conventional method (for example, Harlow, E. & Lane, D,
in Antibodies-Laboratory manual Cold Spring Harbor LaboratoryPress., pp53-138 (
1988)).

The antibody of the present invention can be used for detection and search of the polyamino acid synthetase of the present invention and other polyamino acid synthetases having sequence homology with the protein. According to the screening method of the present invention, it is possible to easily obtain near-kind polyamino acid synthase for classification.

The screening method described above comprises, for example, detecting and searching for a protein to which the antibody of the present invention binds using, as a sample, another organism containing another polyamino acid synthase or a crude extract of its tissue. Examples of such detection means include immunoblot and immunoaffinity chromatography. In addition, expression cloning (Sambrook, J., Fritsch, EF, & Maniati) using the antibody of the present invention is also possible for DNA libraries of other organisms or other tissues containing other polyamino acid synthases.
s, T., Molecular Cloning − a Laboratory Mannual, second edition.Cold Spring Har
bor Laboratory Press. pp 12.3-12.44 (1989)), DNA encoding a novel polyamino acid synthase can be obtained directly.

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

Example 1 ε-Poly-L-lysine-producing bacterium Streptomyces alb
ulus) Purification of ε-poly-L-lysine biosynthetic enzyme from IFO14147 strain and examination of enzymatic properties

(1) Cultivation of ε-poly-L-lysine producing strain Streptomyces albulus IFO14147 The culture of this strain is M3G medium (glucose 5%, yeast extract 0.5%, ammonium sulfate 1%, K2HPO4
0.08%, KH2PO4 0.136%, MgSO4 ・ 7H2O 0.05%, ZnSO4 ・ 7H2O 0.004%, FeSO4 ・ 7H2O 0.003%
(PH 6.8)). 100 ml of pre-cultured solution obtained by aerobic cultivation at 30 ° C for 20 hours
In addition to L medium, use a 3L jar fermenter (manufactured by Maruhishi Bioengineer), 30 ° C, 5
The aerobic culture was performed at 00 rpm and aeration rate of 2.5 L / min. Since the pH of the culture solution gradually decreased with the growth of the cells, 10% aqueous ammonia was added from the time when the pH reached 4.2 suitable for ε-poly-L-lysine production, and the culture solution pH was adjusted to 4.2. Maintained. After culturing for 32 hours, the cells were collected from the culture solution by centrifugation. Yield was about 240 g wet weight.

(2) ε-Poly-L from ε-Poly-L-lysine producing strain Streptomyces albulus IFO14147
-Purification of lysine biosynthetic enzymes All purification steps were performed at 4 ° C or on ice. Wet cells with a wet weight of 75 g are mixed with 150 ml of 100 mM Tris (hydroxymethyl) aminomethane, 20% (w / v) glycerol, 0.2 M NaCl, 2 mM ED
The suspension was suspended in buffer A (pH 8.0) consisting of TA and 5 mM DTT, and the cells were disrupted by sonication.
The treatment solution was centrifuged at 10,000 g for 20 minutes to remove the residue, and a crude enzyme solution was prepared. This is 160,00
Ultracentrifugation was performed at 0 g for 1 h (manufactured by Beckman Coulter), and the precipitate was collected as a cell membrane fraction. The cell membrane fraction obtained was 30 ml of 100 mM Tris (hydroxymethyl) aminomethane, 20 (w /
v) Suspended in buffer B (pH 8.0) consisting of% glycerol, 1.0 M NaCl, 5 mM DTT, sonicated, and similarly recovered by ultracentrifugation to obtain a salt-washed cell membrane fraction. 30 ml of 100 mM Tris (hydroxymethyl) aminomethane, 30% (w / v) glycerol, 1% (w / v) NP-40 (IGEPAL CA-630, manufactured by MP-Biomedicals), 5 mM Buffer C consisting of DTT
Suspended in (pH 8.6), similarly sonicated and ultracentrifugated, and the resulting supernatant was used as an NP-40 solubilized cell membrane fraction containing ε-poly-L-lysine biosynthetic enzyme.

NP-40 solubilized cell membrane fraction was added to 50 mM Tris (hydroxymethyl) aminomethane, 30% (w / v
) Adsorbed on DEAE TOYOPEARL 650M column (2.5 × 10 cm, manufactured by Tosoh Corporation) equilibrated with buffer D (pH 8.6) consisting of glycerol, 0.4% (w / v) NP-40, 2 mM DTT. After washing the column with the same buffer, the enzyme was eluted by the NaCl concentration gradient method (0 to 0.3 M). Collect the active fraction and add 0
AF-Blue TOYOPEARL equilibrated with buffer D (pH 8.2) after adjusting to pH 8.2 with 1M acetic acid solution
It was made to adsorb | suck to a 650M column (1.5x6.5cm, the Tosoh company make). After washing the column with the same buffer, NaC
l The enzyme was eluted by a concentration gradient method (0-2M). The active fractions were collected, concentrated by ultrafiltration (YM-50, Amicon), and then equilibrated with buffer D (pH 8.2) containing 0.2 M NaCl.
It was added to a yl S-300 HR column (1.5 × 45 cm, manufactured by Amersham Biosciences) and eluted with the same buffer at a flow rate of 0.15 ml / min. By the above operation, 2.9 mg of purified ε-poly-L-lysine biosynthetic enzyme could be prepared. This purified enzyme preparation was uniform in SDS-polyacrylamide electrophoresis. The purification yield of ε-poly-L-lysine biosynthetic enzyme is shown in Table 1 below.

In addition, SDS-PAGE of each fraction in the purification process of ε-poly-L-lysine biosynthetic enzyme
The result of the analysis is shown in FIG.

(3) Enzymatic study of ε-poly-L-lysine biosynthetic enzymes Enzymatic studies were conducted using purified enzyme preparations.

ε-poly-L-lysine biosynthesis activity was measured by the method of Kawai et al. (Biochem Biophys Res Commun.
2003 Nov 21; 311 (3): 635-40). 92.5 kBq / ml L- [U-14C] lysine (Amersham Biosciences) -containing 1 mM L-lysine, 5 mM MgCl ₂ , 5 mM ATP, 1
Reaction solution (40 μl) containing mM DTT, 20% (w / v) glycerol, 0.2% (w / v) NP-40, 100 mM TAPS-NaOH buffer (pH 8.5) and enzyme was incubated at 30 ° C. . 10 μl of the reaction solution is 2.1
It was spotted on a cm fiberglass filter paper (manufactured by Whatman) and immediately immersed in an ice-cooled 5% (w / v) trichloroacetic acid-0.25% (w / v) sodium tungstate solution. 5 minutes after standing for 20 minutes
Shake gently for a minute. The filter was shaken again in the same solution for 5 minutes, rinsed with ethanol and dried. The amount of radioactivity bound to the filter was measured with a liquid scintillation counter (Aloka). Under this reaction condition, the amount of enzyme that takes 1 pmol of L-lysine into ε-poly-L-lysine per second was defined as 1 unit. Specific activity was in units per mg of protein. The protein was quantified by the Bradford method. A calibration curve was prepared using bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.).

The adenylation activity of L-lysine and other amino acids was measured by ATP-PPi exchange reaction. 1 mM L-lysine or other amino acids, 5 mM MgCl2, 5 mM ATP, 1 mM DTT, 20% (w / v) glycerol, 0.2% (w / v) NP-40, 0.2 mM sodium diphosphate, 51.8 kBq / ml [ A reaction solution (40 μl) containing 32 P] 2 sodium phosphate, 100 mM TAPS-NaOH buffer (pH 8.5) and an enzyme was incubated at 30 ° C. The reaction was stopped by adding 1.0 ml of a solution containing 1.2% (w / v) activated carbon, 0.1 M sodium phosphate and 0.35 M perchloric acid. After the activated carbon was washed with pure water, the amount of radioactivity bound to the activated carbon was measured with a liquid scintillation counter (manufactured by Aloka).

Polymerization degree analysis and quantification of enzymatically synthesized ε-poly-L-lysine were performed by HPLC under the following conditions. A calibration curve was prepared using ε-poly-L-lysine manufactured by Chisso.
Column: ODS-80Ts (4.6 x 250 mm, manufactured by Tosoh Corporation)
Solvent A: 10 mM sodium dihydrogen phosphate, 10 mM sodium octane sulfonate, 100
mM Sodium perchlorate Solvent B: Solvent A + 60% acetonitrile Gradient: 42% Solvent B (0min) → 65% Solvent B (45min)
Flow rate: 0.8ml / min
Detection: 210nm

Analysis of ATP degradation products generated in the ε-poly-L-lysine biosynthesis reaction was performed by HPLC under the following conditions.
Column: ODS-80Ts (4.6 x 250 mm, manufactured by Tosoh Corporation)
Solvent A: 20 mM sodium dihydrogen phosphate, 5 mM tetrabutylammonium bromide Solvent B: Solvent A + 60% methanol Gradient: 35% Solvent B (0 min) → 100% Solvent B (30 min)
Flow rate: 0.7ml / min
Detection: 260nm

LC-ESI-MS / MS analysis of enzymatically synthesized ε-poly-L-lysine was performed under the following conditions.
HPLC system: Agilent 1200 (Agilent)
Column: Develosil Praqueous-AR-5 (2.0 x 150 mm, manufactured by Nomura Chemical Co., Ltd.)
Solvent A: 0.1% Heptafluorobutyric acid Solvent B: 0.1% Heptafluorobutyric acid + Acetonitrile Gradient: 10% Solvent B (0-5 min) → 45% Solvent B (45 min)
Flow rate: 0.4ml / min
Detection: 210nm
Mass spectrometer: esquire 4000 (Bruker)
Ionization mode: Positive mode

Enzymatic synthesis ε-poly-L-lysine binding mode was confirmed by the following method. Ε-poly-L-lysine was purified by HPLC from 15 ml of enzyme reaction solution (25 ° C, 16 h) under the following conditions (approximately 0.8 mg)
And lyophilized.
Column: ODS-80Ts (4.6 x 250 mm, manufactured by Tosoh Corporation)
Solvent A: 0.1% Trifluoroacetic acid Solvent B: 0.1% Trifluoroacetic acid + Acetonitrile Gradient: 0% Solvent B (0-10min) → 30% Solvent B (30min)
Flow rate: 0.7ml / min
Detection: 210nm
This was dinitrophenylated (DNP) according to the method of Shima et al. (Agric. Biol. Chem. 1981 45: 2503-2508). DNPated ε-poly-L-lysine was hydrolyzed in 6N HCl at 100 ° C. for 20 h and analyzed by thin layer chromatography under the following conditions.
Thin layer plate: Silica gel 60TLC plate (Merck)
Developing solvent: butanol, acetic acid, pyridine, water (4: 1: 1: 2)
Color: Ninhydrin

First, an enzyme reaction (0.5 ml) was performed using 22 μg of the purified enzyme preparation, and the product was ESI-LC-MS / M
S was analyzed. While ε-poly-L-lysine obtained by fermentation has a 30-mer center polymerization distribution, enzymatically synthesized ε-poly-L-lysine showed a 14- or 15-mer center polymerization distribution. Retention time, MS spectrum, and MS / MS fragmentation pattern in ε-poly-L
-Consistent with the partially hydrolyzed product of lysine standard (FIGS. 5 and 6). In addition, in the analysis by thin-layer chromatography, NNP-2,4-DNP-L was obtained by hydrolyzing DNP-enzyme synthesized ε-poly-L-lysine with hydrochloric acid in the same way as ε-poly-L-lysine standard. -Because lysine was generated,
An enzyme reaction product is an isopeptide in which the ε-amino group and α-carboxyl group of L-lysine are linked
That is, it was shown to be ε-poly-L-lysine (FIG. 7), and it was confirmed that this enzyme was an ε-poly-L-lysine biosynthetic enzyme. Note that ε-poly-L-lysine has the following structure.

Next, when the enzyme reaction product was analyzed by HPLC, a reaction time-dependent increase in ε-poly-L-lysine was confirmed, but the degree of polymerization of the produced ε-poly-L-lysine was determined by the reaction time. It did not depend and always showed a constant distribution. ATP was also converted to AMP during the reaction, and L-lysine adenylation activity was also observed. From these, γ-polyglutamic acid (Appl Environ Microbiol. 200
4 Jul; 70 (7): 4249-55) and biosynthesis enzyme of cyanophycin (Appl Environ Microbiol. 2001)
May; 67 (5): 2176-82) polymerizes substrate amino acids in a ligase mode, whereas this enzyme polymerizes L-lysine in a thiotemplate-type ribosome-independent peptide synthase (NRPS) mode. Was strongly suggested.

Further examination of the enzymatic properties of the enzyme in detail revealed that the enzyme has the following properties.
1) Optimum pH of synthesis reaction: pH 8.5
2) Optimum temperature for synthesis reaction: 25-30 ° C
3) Temperature stability: The active half-life at 30 ° C was 180 minutes, at 35 ° C it was 80 minutes, and at 40 ° C it was 19 minutes. (Buffer D (pH 8.2) containing 0.2 M NaCl)
4) Metal ion requirement: Addition of a divalent cation is essential for activity expression. 5 mM Mg ²⁺ and
Maximum activity is shown by the addition of 5 mM Mn ²⁺ . Slight activation even with the addition of Ca ²⁺ .
5) Substrate specificity: L-lysine, D-lysine, DL-5-hydroxylysine, etc.
-Adenylation of lysine analogs.
6) Nucleotide specificity: ATP specific. Substrate amino acids are not activated by GTP, UTP, or CTP.
7) Subunit molecular weight: about 13 by SDS-polyacrylamide electrophoresis under reducing conditions.
It is calculated as 0 kDa (FIG. 4).
8) Molecular weight: Calculated as about 270 kDa by gel filtration chromatography (Sephacryl S-300 HR, manufactured by Amersham-Bioscience).

  From the results of 7) and 8) above, this enzyme was estimated to be a homodimer.

Example 2 Cloning of ε-poly-L-lysine biosynthetic enzyme gene (1) Amino acid sequence analysis of ε-poly-L-lysine biosynthetic enzyme The amino acid sequence was analyzed by LC-MS / MS. The ε-poly-L-lysine biosynthetic enzyme was fractionated by SDS-polyacrylamide electrophoresis, stained with CBB-R250, and then the band of ε-poly-L-lysine biosynthetic enzyme was excised. Add Tris buffer (pH 8.0) containing trypsin to sample gel piece,
In-gel digestion for 20 hours. The trypsin digested peptide solution extracted from the gel piece was subjected to LC-MS / MS analysis. LC-MS / MS analysis was performed under the following conditions.

HPLC system: MAGIC 2002 nano LC system (Michrom Bioresources)
Column: Magic C18 column (0.1 × 50mm, manufactured by Michrom Bioresources)
Solvent A: 0.1% formic acid + 2% acetonitrile Solvent B: 0.1% formic acid + 90% acetonitrile Gradient: 10% solvent B (0 to 1 min) → 50% solvent B (21 min)
Flow rate: 250-300nl / min
Mass spectrometer: Q-Tof 2 (Waters Micromass)
Ionization mode: Positive mode

As a result of de novo sequence analysis of the obtained MS / MS data with MassLynx software (manufactured by Waters Micromass), the amino acid sequence MAGAWPAPAWQRSR (SEQ ID NO: 3) of 3 peptides, (I / L
) E (I / L) GE (I / L) DAA (I / L) AA (I / L) PGVR (SEQ ID NO: 4) and A (I / L) AGT (I / L) KPGAYPR (SEQ ID NO: 5) Got.

(2) Acquisition of ε-poly-L-lysine biosynthetic enzyme gene Streptomyces albras IFO14147 strain was sucrose 103 g / L, tryptone 10 g / L,
Inoculated into 100 ml of a medium consisting of 5 g / L of yeast extract and 5 g / L of NaCl, and cultured with shaking at 30 ° C. for 24 hours. Bacterial cells were collected from this culture broth by centrifugation, and chromosomal DNA was prepared according to a conventional method. In addition, the determined internal amino acid sequence of ε-poly-L-lysine biosynthetic enzyme (peptide 1; NA (I / L) AG
The following four types of primers were designed based on T (I / L) KPGAYPR-C (SEQ ID NO: 6), peptide 2; N-MAGAAWPAPAWQRSR-C (SEQ ID NO: 7)).

pr-pp1F; 5'-AC (C / G) (A / C) T (C / G) AAGCC (C / G) GG (C / G) GC (C / G) TACCC-3 '(SEQ ID NO: 8 )
pr-pp2R; 5'-CTGCCA (G / C) GC (G / C) GG (G / C) GC (G / C) GGCCA (G / C) GC-3 '(SEQ ID NO: 9)
pr-pp2F; 5'-GC (C / G) TGGCC (C / G) GC (C / G) CC (C / G) GC (C / G) TGGCA-3 '(SEQ ID NO: 10)
pr-pp1R; 5'-GGGTA (G / C) GC (G / C) CC (G / C) GGCTT (G / C) A (T / G) (G / C) GT-3 '(SEQ ID NO: 11 )

When PCR was performed using these primers and chromosomal DNA as a template, a specific amplified fragment of about 500 bp was obtained by the combination of pr-pp1F / pr-pp2R. This amplified fragment was cloned into the plasmid pGEMT-easy, and the cloned fragment was labeled using an ECL labeling kit (manufactured by GE Healthcare Bioscience). Using the labeled gene fragment as a probe, screening was performed on a cosmid library (Supercos I, STRATAGENE) prepared from the chromosomal DNA of this strain, and a KpnI 6.0 kb fragment to which this probe hybridizes was obtained. This fragment was transformed into plasmid pTSR193 (reference, Actinomycetologica (2006) Vol.20, p.35-41)
And the nucleotide sequence was determined.

Example 3 Enzymatic production of ε-poly-L-lysine
2 mM _{L-lysine, 5mM MgCl 2, 5mM ATP,} 1mM DTT, 20% glycerol, 0.2% (w / v) NP-
40, 100 mM TAPS-NaOH buffer solution (pH 8.5) and reaction solution containing 200 μg of purified enzyme preparation (200 μ
l) was placed in a 1.5 ml microtube and incubated at 25 ° C. for 120 minutes. Reaction start
Table 2 shows the results of quantification of ε-poly-L-lysine produced by the method described in Example 1 after 20, 40, 80, and 120 minutes.

Example 4 Enzymatic production of a heteropolyamino acid composed of a lysine analog and L-lysine
2 mM (mixing L- lysine analog thereof at any ratio) substrate amino acid, 5mM MgCl _2, 5mM ATP,
1.5 ml of a reaction solution (200 μl) containing 1 mM DTT, 20% glycerol, 0.2% (w / v) NP-40, 100 mM TAPS-NaOH buffer (pH 8.5) and about 10 μg of purified enzyme preparation Put in a tube,
Incubated at 25 ° C. After 4 hours, the polyamino acid produced by the method described in Example 1 was
ESI-LC-MS analysis was performed.
FIG. 9 shows the result. DL-5-hydroxylysine (FIG. 9D), S- (2-aminoethyl) -L-cysteine (FIG. 9C), O- (2-aminoethyl) -L-serine (FIG. 9B), etc. When the analog to be converted was added as a substrate, a heteropolyamino acid in which the analog was incorporated into the polylysine molecule at a certain frequency was obtained.

Example 5 Microbial production of a heteropolyamino acid composed of a lysine analog and L-lysine As described in Example 4, enzymatic reaction of a heteropolyamino acid in which the analog is incorporated into an ε-poly-L-lysine molecule A heteropolyamino acid that can be synthesized but contains S- (2-aminoethyl) -L-cysteine uses Streptomyces albulus, which is an ε-poly-L-lysine-producing bacterium. However, it can be more easily produced.

Streptomyces albulus (Str) obtained by culturing in M3G medium (Example 1) at 30 ° C / 36 hours
eptomyces albulus) was washed twice with 100 mM citrate buffer (pH 4.2). Washed cells obtained from 100 ml of culture solution were mixed with 50 ml of 5% glucose, 1% ammonium sulfate, 8 mM S- (2-
Aminoethyl) -L-cysteine, 16 mM L-threonine (the concentration of both these amino acids is M
9 100 mM citrate buffer solution (pH 4.2) containing the minimum growth inhibitory concentration for this bacterium in a minimal medium, that is, the minimum concentration for inhibiting aspartokinase, which is a key enzyme for L-lysine biosynthesis, and an arbitrary amount of L-lysine. ) And cultured with shaking at 30 ° C. for 48 hours. After completion of the culture, the polyamino acid contained in the culture supernatant recovered by centrifugation was converted into Example 1 (paragraph [00
90]). LC-MS analysis was performed by the method described. As shown in FIG. 10, not only the enzyme reaction,
Even when viable bacteria are used, S- (2-aminoethyl) -L-
A heteropolyamino acid incorporating cysteine was obtained.

Even in analogs other than S- (2-aminoethyl) -L-cysteine, ε-poly-L-
Regarding those that are adenylated by lysine biosynthetic enzymes, it is highly possible that microorganisms can produce heteropolyamino acids by using a cell reaction solution containing an amount corresponding to the minimum growth inhibitory concentration for this bacterium.

Example 6 Disruption of ε-poly-L-lysine biosynthetic enzyme gene In order to construct a plasmid for disruption of ε-poly-L-lysine biosynthetic gene, the following two types of primers were designed and produced: Streptomyces albulus
) PCR was performed using chromosomal DNA of IFO14147 strain as a template.
e-PL_NRPS_F; 5'-GGGGGATCCTCGTCGCCCCTTCTCGAATCG-3 '(SEQ ID NO: 20)
e-PL_NRPS_R; 5'-ACCAAGCTTTCACGCGGCCGCACCTCCCTC-3 '(SEQ ID NO: 21)

The amplified PCR product (about 4 kbp) was digested with restriction enzymes BamHI and HindIII and cloned into plasmid pLAE003 having a neomycin resistance gene. Ε-poly of the constructed plasmid
A transposon containing an apramycin resistance gene [aac (3) IV] is inserted into the -L-lysine biosynthesis gene (reference document, Actinomycetologica, 20, 35-41, 2006), and a plasmid for destruction (pLAEnrps-apr ) Was built. pLAEnrps-apr was introduced into E. coli S17-1 and then pLAEnrps-apr was introduced into Streptomyces albras by conjugation with Streptomyces albras (references, Journal of Bioscience and Bioengineering, 99, 636- 64
1, 2005). Protoplasts of the introduced strains were prepared (reference, Journal of Bioscien
ce and Bioengineering, 99, 636-641, 2005), protoplast regeneration medium (reference, Jo
urnal of Bioscience and Bioengineering, 99, 636-641, 2005). From the regenerated strain, a gene disruption candidate strain exhibiting neomycin sensitivity and apramycin resistance was obtained (FIG. 11A). Chromosomal DNA was prepared from the obtained candidate strain and the restriction enzyme KpnI
Then, agarose electrophoresis was performed. The agarose gel after electrophoresis was transferred to a nylon membrane, and ε-poly-L-lysine was produced by genomic Southern hybridization (CDP-star, GE Healthcare) using the ε-poly-L-lysine biosynthesis gene as a probe. Strains with a disrupted synthetic gene were selected. As a result, one strain (PL-nrps :: aac (3) IV) was obtained (FIG. 1).
1B). The obtained disrupted strain was cultured in a production medium, and the productivity of ε-poly-L-lysine was confirmed by HPLC (Example 1). (FIG. 11C), it was confirmed that the obtained gene was an ε-poly-L-lysine biosynthesis gene.

Example 7 Acquisition of ε-poly-L-lysine biosynthetic gene derived from Streptomyces noursei NBRC15452 strain Streptomyces albul, a ε-poly-L-lysine-producing bacterium
It is already known that ε-poly-L-lysine is produced not only by us) but also by closely related Streptomyces noursei (see JP-A-1-187090).
. Therefore, we tried to obtain ε-poly-L-lysine biosynthetic gene from Streptomyces noursei.

The ε-poly-L-lysine biosynthetic gene ORF fragment of Streptomyces albulus amplified by PCR (see Example 6) was labeled using an ECL labeling kit manufactured by GE Healthcare Biosciences. Streptomyces Norsei (Streptomyce) constructed according to Example 1 (paragraphs [0099]-[0101]) using the labeled gene fragment as a probe
s noursei) A chromosomal DNA cosmid library (Supercos I, STRATAGENE) of NBRC15452 strain was screened to obtain a KpnI 6.5 kb fragment to which this probe hybridizes. This fragment was subcloned into plasmid pGEM7Z (promega), and the nucleotide sequence (SEQ ID NO: 22) was determined. Furthermore, the corresponding amino acid sequence (SEQ ID NO: 23) was determined.

As for this gene, the Streptomyces albras (Streptomyces already acquired)
albulus) derived from ε-poly-L-lysine biosynthetic enzyme is 92.7% at the base sequence level,
It is very high at 93.0% at the amino acid sequence level, and the structural features such as domain structure match very well, so this gene is also likely to encode ε-poly-L-lysine biosynthetic enzyme. .

The enzymatic synthesis method of polyamino acid is more energy-saving than microbial fermentation method and chemical synthesis method, and undesirable side reactions can be suppressed, so that a highly pure polyamino acid can be produced.
The polymers of various amino acids produced by the present invention can be used as antibacterial agents, etc., and used for various applications such as toiletries, cosmetics, feed additives, pharmaceuticals, agricultural chemicals, food additives, electronic materials, etc. Can do. Furthermore, by using the polynucleotide-containing recombinant vector and the transformant of the present invention, application to various useful polyamino acid biosynthesis can be expected.

(A) Multi-module structure of conventional NRPS; (B) A diagram schematically showing a single module structure of polyamino acid synthase in one embodiment of the present invention. The amino acid sequences of the condensation reaction catalytic domain and its subdomain located on the C-terminal side of the polyamino acid synthetase of the present invention are shown. In the figure, “tmr” means a transmembrane region. The DNA sequence of the subdomain of the condensation reaction catalytic domain located on the C-terminal side of the polyamino acid synthetase of the present invention is shown. 2 shows the DNA sequence of the condensation reaction catalytic domain located on the C-terminal side of the polyamino acid synthetase of the present invention. In the figure, “tmr” means a transmembrane region. It is a photograph which shows the result of the SDS-PAGE analysis of each fraction in the refinement | purification process of the epsilon-poly-L-lysine biosynthesis enzyme of this invention. Lanes 1 and 11: Molecular weight marker (recombinant protein, Bio-Rad), Lane 2: Crude enzyme solution, Lane 3: Soluble fraction, Lane 4: Cell membrane fraction, Lane 5: Salt solubilized fraction, Lane 6: Salt Washed cell membrane fraction, lane 7: NP-40 solubilized fraction, lane 8: elution fraction from DEAE TOYOPEARL column, lane 9: elution fraction from AF-Blue TOYOPEARL column, lane 10: Sephacryl S-300 column Elution fraction from (A) It is a graph which shows the result of having analyzed (epsilon) -poly-L-lysine in an enzyme reaction liquid by HPLC. (B) It is a graph which shows the epsilon-poly-L-lysine density | concentration in the reaction liquid which created the analytical curve using the epsilon-poly-L-lysine standard goods, and was computed from the result of FIG. 5 (A). (A) It is a graph which shows the separation pattern by liquid chromatography (LC). (B) It is a graph which shows the mass spectrometry result of (epsilon) -poly-L-lysine standard goods hydrochloric acid hydrolyzate 14mer. (C) It is a graph which shows the mass spectrometry result of enzyme synthetic | combination (epsilon) -poly-L-lysine 14mer. It is a graph which shows the result of the thin layer chromatography analysis of DNP-ized (epsilon) -poly-L-lysine hydrochloride hydrolyzate. Lane 1: L-lysine, Lane 2: α-poly-L-lysine (manufactured by Wako Pure Chemical Industries, Ltd.) produced by hydrolysis of DNP, lane 3: ε-poly-L-lysine standard (Chisso) Produced by hydrolyzing the DNP product of the product), Lane 4: Enzyme-synthesized ε-poly-L-lysine, produced by hydrolyzing the DNP product, Lane 5: N ^α -2,4- DNP-L- lysine, lane ^{6: N ε -2,4-DNP-} L- lysine. It is a graph which shows the substrate specificity in the amino acid activation (adenylation) reaction of the amino acid synthetase of this invention. The activity with respect to L-lysine was shown as relative activity with 100%. In addition, L-Ala, L-Val, L-Leu, L-Ile, L-Phe, L-Pro, L-Trp, L-Met, Gly, L-Ser, L-Thr, L-Cys, L- None of Gln, L-Asn, L-Tyr, L-Arg, L-His, L-Asp, and L-Glu was adenylated. The result of having analyzed the polyamino acid produced | generated by the enzyme reaction which used the mixture of L-lysine and the analog body as a substrate is shown by ESI-LC-MS. (A) is a polyamino acid (ε-poly-L-lysine) obtained by a reaction using only L-lysine as a substrate, and (B) is 1.8 mM O- (2-aminoethyl) -L-serine and 0.2 mM. A polyamino acid obtained by a reaction using a mixture of L-lysine as a substrate, (C) is obtained by a reaction using a mixture of 1.8 mM S- (2-aminoethyl) -L-cysteine and 0.2 mM L-lysine as a substrate. The polyamino acid obtained, (D) is a graph showing the fractionation pattern and mass spectrometric result in LC of the polyamino acid obtained by the reaction using a mixture of 1.6 mM DL-5-hydroxylysine and 0.4 mM L-lysine as a substrate. is there. The result of having analyzed the polyamino acid produced | generated by the microbial cell reaction by ESI-LC-MS is shown. (A) shows the fractionation pattern and mass spectrometry result in LC of the polyamino acid obtained from the cell reaction solution containing 1.6 mM L-lysine, and (B) shows the cell reaction solution containing 0.4 mM L-lysine. It is a graph which shows the fractionation pattern and mass spectrometry result in LC of the obtained polyamino acid. It is a figure which shows the destruction method of an epsilon-poly-L-lysine biosynthesis enzyme gene, and its influence. (A) schematically shows a procedure for preparing a gene disruption strain using the plasmid for disruption pLAEnrps-apr. (B) It is the figure which confirmed destruction of (epsilon) -poly-L-lysine biosynthesis gene by genomic Southern hybridization. (C) shows the result of confirming the productivity of ε-poly-L-lysine using HPLC by culturing the obtained disrupted strain in a production medium.

Claims (13)

(I) a nucleotide sequence encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2,
(Ii) a nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having a deletion, substitution, insertion and / or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2,
(Iii) a nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2,
(Iv) the nucleotide sequence of SEQ ID NO: 1, or
(V) a nucleotide sequence encoding a polyamino acid synthase having 95% or more identity with the nucleotide sequence of SEQ ID NO: 1;
A polynucleotide comprising any of The polynucleotide according to claim 1 , wherein the polyamino acid synthase is a polylysine synthase. The polynucleotide according to claim 1 or 2 , which is DNA or RNA. (I) the amino acid sequence of SEQ ID NO: 2,
(Ii) an amino acid sequence having a deletion, substitution, insertion and / or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2, or (iii) 90% or more of the amino acid sequence of SEQ ID NO: 2 Amino acid sequences having identity,
A polyamino acid synthase containing any of the above. The polyamino acid synthetase according to claim 4 , which is a polylysine synthase. (I) a nucleotide sequence encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2,
(Ii) a nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having a deletion, substitution, insertion and / or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2,
(Iii) a nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2,
(Iv) the nucleotide sequence of SEQ ID NO: 1, or
(V) a nucleotide sequence encoding a polyamino acid synthase having 95% or more identity with the nucleotide sequence of SEQ ID NO: 1;
A recombinant vector containing any of the above. The recombinant vector according to claim 6 , wherein the polyamino acid synthase is polylysine synthase. A transformant comprising the recombinant vector according to claim 6 or 7 . A method for producing a polyamino acid synthase using the recombinant vector according to claim 6 or 7 or the transformant according to claim 8 . (I) the amino acid sequence of SEQ ID NO: 2,
(Ii) an amino acid sequence having a deletion, substitution, insertion and / or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2, or (iii) 90% or more of the amino acid sequence of SEQ ID NO: 2 Amino acid sequences having identity,
A method for producing a polyamino acid comprising a step of synthesizing a polyamino acid using a polyamino acid synthetase containing any of the above. The method according to claim 10 , wherein the polyamino acid is polylysine. The method of claim 11 , wherein the polylysine is ε-poly-L-lysine. (I) the amino acid sequence of SEQ ID NO: 2,
(Ii) an amino acid sequence having a deletion, substitution, insertion and / or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2, or (iii) 99.2% with the amino acid sequence of SEQ ID NO: 2 An antibody that specifically binds to a polyamino acid synthase comprising any one of the amino acid sequences having the above identity.