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Process for producing dipeptides or dipeptide derivatives

A technology of peptide derivatives and derivatives, applied in the field of preparation of dipeptide or dipeptide derivatives

Inactive Publication Date: 2007-07-11
KYOWA HAKKO KOGYO CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] In addition, it is reported that Streptomyces knowersi ( Streptomyces noursei ) in the ATCC11455 strain, a protein completely dissimilar to the NRPS enzyme ( albC gene product) is responsible for the synthesis of cyclo(L-phenylalanyl-L-leucine)[cyclo(L-phenylalanyl-L-leucine)] structure, if cyclic dipeptide oxidase acts on the large intestine into which the albC gene Bacillus ( Escherichia coli ) and Streptomyces lividans ( Streptomyces lividans ) culture solution, albonoursin can be detected (refer to non-patent literature 12), but not yet albC Reporter of a gene product producing a linear dipeptide

Method used

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  • Process for producing dipeptides or dipeptide derivatives
  • Process for producing dipeptides or dipeptide derivatives
  • Process for producing dipeptides or dipeptide derivatives

Examples

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preparation example Construction

[0183] 4. Can be used for the preparation of the DNA of the present invention

[0184] The DNA used in the present invention, for example, can be designed by using a probe that can be designed according to the base sequence represented by any one of SEQ ID NO: 9-16, 18, 36, 39 or 40, for example, using The chromosomal DNA library of microorganisms of the genus Bacillus can be used for Southern hybridization, or the primer DNA can be designed according to the base sequence represented by any one of the sequence numbers 9-16, 18, 36, 39 or 40, to use the chromosomal DNA of microorganisms of the genus Bacillus Acquired by PCR [PCR Protocols, Academic Press (1990)] as a template.

[0185] In addition, for various gene sequence databases, the base sequence of the DNA encoding the amino acid sequence of any one of the sequence numbers 1 to 8, 17, 37 and 38 has 75% or more, preferably 85% or more, more preferably 90% or more , more preferably 95% or more, particularly preferably 98%...

experiment example 1

[0392] Experimental Example 1 Using the database to search for proteins with dipeptide synthesis activity

[0393] With the amino acid sequence of the D-Ala-D-Ala ligase gene [Nature, 390 , 249-256 (1997)] As a request, using the homology search function of Subtilist (http: / / genolist.pasteur.fr / SubtiList / ), which is a genomic DNA database of 168 strains of Bacillus subtilis, for the code existing in subtilis Genes with homologous proteins in the genome DNA sequences of 168 strains of Bacillus were retrieved.

[0394] Among the sequences selected as a result, the encoding is selected as the D-Ala-D-Ala ligase motif [Biochemistry, 30 , 1673 (1991)] the amino acid sequence represented by SEQ ID NO: 33, 34 or 35, and after removing the gene encoding the protein whose function has been identified, it shows the highest homology with the D-Ala-D-Ala ligase motif origin (29.1%) as genes of unknown function wxya .

[0395] wxya The nucleotide sequence of is shown in SEQ ID NO: 9...

experiment example 2

[0396] Experimental example 2 wxya Construction of gene expression strains

[0397] According to the nucleotide sequence information obtained in Experimental Example 1, the Bacillus subtilis can be obtained as follows: wxya Gene fragment.

[0398] First, Bacillus subtilis 168 strains (ATCC23857) were inoculated on LB medium [10 g / l bacterial culture tryptone (manufactured by Difco), 5 g / l yeast extract (manufactured by Difco), 5 g / l sodium chloride] , and cultured overnight at 30°C. After culturing, the chromosomal DNA of the microorganism was isolated and purified by using the saturated phenol method described in Current Protocols in Molecular Biology.

[0399] DNA having the nucleotide sequences represented by SEQ ID NOs: 19 to 22 (hereinafter referred to as primer A, primer B, primer C, and primer D, respectively) was synthesized using a DNA synthesizer Model 8905 manufactured by Perseptive Biosystems. Primer A is added to the Bacillus subtilis chromosomal DNA containin...

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Abstract

It is intended to provide a method of producing a dipeptide or a dipeptide derivative characterized by comprising using, as an enzyme source, a protein having an activity of forming a dipeptide or dipeptide derivative from one or more amino acids or amino acid derivatives or an optionally processed culture of cells having an ability to produce the above protein, supplying the enzyme source, one or more amino acids or amino acid derivatives and ATP into an aqueous medium, thus forming and accumulating the dipeptide or the dipeptide derivative in the medium and then harvesting the dipeptide or the dipeptide derivative from the medium.

Description

technical field [0001] The present invention relates to a protein having an activity of producing a dipeptide or a dipeptide derivative from one or more amino acids or amino acid derivatives, or a culture of cells having DNA encoding the protein, or a treated product of the culture. Process for the preparation of peptide or dipeptide derivatives. Background technique [0002] As methods for mass synthesis of peptides, chemical synthesis methods (liquid phase method, solid phase method), enzymatic synthesis methods, and biological synthesis methods using DNA recombination methods are known. Currently, enzymatic synthesis or biological synthesis is used for long-chain peptides with more than 50 residues, and chemical synthesis and enzymatic synthesis are mainly used for dipeptides. [0003] Synthesis of dipeptides by chemical synthesis requires operations such as protection and deprotection of functional groups. Since racemates are also synthesized, chemical synthesis cannot ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12P21/02C12N9/48C12R1/19
CPCC12P21/02
Inventor 桥本信一池田创田畑和彦
Owner KYOWA HAKKO KOGYO CO LTD
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