JP2012039878A - Nε-ACYL-L-LYSINE-SPECIFIC AMINOACYLASE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an Nε-acyl-L-lysine-specific aminoacylase which enables the efficient synthesis of an Nε-acyl-L-lysine; and a method for efficiently synthesizing an Nε-acyl-L-lysine.SOLUTION: There are provided an Nε-acyl-L-lysine-specific aminoacylase comprising a specific amino acid sequence originated from Streptomyces mobaraensis; DNA encoding the Nε-acyl-L-lysine-specific aminoacylase, or a variant thereof; and a transformant carrying a recombinant DNA molecule comprising the DNA or the variant thereof.

Description

  The present invention relates to a DNA encoding a novel Nε-acyl-L-lysine-specific aminoacylase derived from actinomycetes, a recombinant expression vector containing the DNA, a transformant transformed with the recombinant expression vector, and The present invention relates to a method for producing Nε-acyl-L-lysine specific aminoacylase using a transformant and a method for producing Nε-acyl-L-lysine.

Nε-acyl-L-lysine is not only a general detergent but also a disinfectant and fiber softener as a raw material for amphoteric surfactants due to its structural properties, safety after being discharged into the natural environment, and non-polluting properties. It has a wide range of industrial uses such as materials, rust preventives, flotation agents, adhesives, fining agents, dye fixing agents, antistatic agents, emulsifiers, and cosmetic surfactants. In particular, it is extremely insoluble in water and ordinary organic solvents, and is utilized in the fields of cosmetics, lubricants, etc. as a new organic powder powder material, taking advantage of its features such as water repellency, antioxidant properties, and lubricity. Is expanding (Japanese Patent Laid-Open No. 61-10503).
In the production of this Nε-acyl-L-lysine, a so-called Schotten-Baumann method in which an acyl halide is dropped into an alkaline aqueous solution of an amino acid has been conventionally used. However, since basic amino acids such as lysine have amino groups at the α- and ε-positions, dialkyl basic amino acids are the main products in this Schotten-Baumann method, and ε-acyl basic amino acids are minor. Only a small amount can be obtained as a product. Therefore, in order to produce an ε-acyl basic amino acid, a method is known in which a basic amino acid is converted to a basic amino acid copper salt, acylated with acyl chloride, and then copper is eliminated (Pharmaceutical Journal). 89, 531 (1969)). In these methods, the manufacturing process and manufacturing operation are complicated, heavy copper is used, and a large amount of hydrogen sulfide gas is required in the copper removal process.
Therefore, the existence of an enzyme that specifically and efficiently hydrolyzes and synthesizes Nε-acyl-L-lysine under milder conditions using an enzyme and development of an industrial production method thereof are aimed at.

Conventionally, there have been few reports on enzymes capable of specifically hydrolyzing Nε-acyl-L-lysines, enzymes derived from Achromobacter pestifer (A. pestivel), enzymes derived from rat kidney, Enzymes derived from Pseudomonas sp. KT-83 have been reported, but no reports have been made on the synthesis reaction of Nε-acyl-L-lysine by these enzymes.
As for the synthesis of Nε-acyl-L-lysine by conventional enzymes, for example, there is a report that capsaicin decomposing / synthesizing enzyme described in JP-A-2003-210164 also catalyzes the synthesis reaction of Nε-acyl-L-lysine. .
As described in JP-A-2004-81107, there is a report that Nε-lauroyl-L-lysine is produced at a yield of 95% by the capsaicin decomposing / synthesizing enzyme described in JP-A-2003-210164. However, since the reaction time takes a long time of 2 days, and this capsaicin decomposing / synthesizing enzyme exhibits high reactivity not only with the ε-amino group but also with the α-amino group, finally, Nα A mixture of -lauroyl-L-lysine and Nε-lauroyl-L-lysine is formed.
On the other hand, the present inventors found that Streptomyces mobaraensis produces an enzyme that specifically and efficiently acylates the ε-amino group of lysine, purified the enzyme, and reported some of the properties. (WO 2006/088199). Furthermore, the present inventors have also confirmed that Nε-lauroyl-L-lysine can be specifically synthesized from L-lysine hydrochloride and lauric acid by the aforementioned enzyme derived from Streptomyces mobaraensis (WO 2006 / 088199).

Japanese Patent Laid-Open No. 61-10503 Japanese Patent Laid-Open No. 2003-210164 JP 2004-81107 A International Publication WO 2006/088199

Pharmaceutical Journal, 89, 531 (1969)

  The present invention provides DNA encoding a Nε-acyl-L-lysine specific aminoacylase. In addition, the present invention provides a highly efficient Nε-acyl-L-lysine-specific aminoacylase that efficiently synthesizes Nε-acyl-L-lysine, which is used as a flour meal material, using L-lysine as a starting material. Provide a way to produce in. Furthermore, the present invention provides a method for efficiently producing Nε-acyl-L-lysine.

The present invention includes the following.
(1) Nε-acyl-L-lysine-specific aminoacylase having the amino acid sequence set forth in SEQ ID NO: 2 or DNA encoding the same;
(2) The amino acid sequence shown in SEQ ID NO: 2 has an amino acid sequence in which one or more amino acid residues are inserted, added, deleted or substituted, and is specific to Nε-acyl-L-lysine A protein having a specific aminoacylase activity or a DNA encoding the same;
(3) DNA having the nucleotide sequence set forth in SEQ ID NO: 1 or 3;
(4) a DNA that hybridizes with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1 or 3 under a stringent condition and encodes a protein having Nε-acyl-L-lysine-specific aminoacylase activity;
(5) a protein having a nucleotide sequence of 95% or more homology with the nucleotide sequence described in SEQ ID NO: 1 or 3, and having a Nε-acyl-L-lysine-specific aminoacylase activity, or DNA to encode;
(6) Recombinant DNA containing the DNA of any one of (1) to (5) above;
(7) A transformant transformed with the recombinant DNA of (6) above;
(8) (a) DNA encoding an Nε-acyl-L-lysine specific aminoacylase having the amino acid sequence set forth in SEQ ID NO: 2 or 5;
(B) in the amino acid sequence described in SEQ ID NO: 2 or 5, having an amino acid sequence in which one or more amino acid residues are inserted, added, deleted or substituted, and Nε-acyl-L- DNA encoding a protein having lysine-specific aminoacylase activity;
(C) DNA having the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;
(D) encodes a protein that hybridizes under stringent conditions with DNA having the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4 and that has Nε-acyl-L-lysine-specific aminoacylase activity DNA; or
(E) encoding a protein having a nucleotide sequence of 95% or more of homology with the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4 and having Nε-acyl-L-lysine specific aminoacylase activity DNA to
Nε-, characterized by culturing a transformant transformed with a recombinant DNA containing any of the above, producing a protein having Nε-acyl-L-lysine-specific aminoacylase activity, and recovering the protein. A method for producing a protein having acyl-L-lysine-specific aminoacylase activity. ;and,
(9) L-lysine and lauric acid are reacted with each other in the presence of a cell transformed with the DNA of any of the following (a) to (e), a treated product of the cell, or a culture solution of the cell to produce A process for producing Nε-lauroyl-L-lysine comprising isolating Nε-lauroyl-L-lysine;
(A) DNA encoding a Nε-acyl-L-lysine specific aminoacylase having the amino acid sequence set forth in SEQ ID NO: 2 or 5;
(B) in the amino acid sequence described in SEQ ID NO: 2 or 5, having an amino acid sequence in which one or more amino acid residues are inserted, added, deleted or substituted, and Nε-acyl-L- DNA encoding a protein having lysine-specific aminoacylase activity;
(C) DNA having the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;
(D) encodes a protein that hybridizes under stringent conditions with DNA having the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4 and that has Nε-acyl-L-lysine-specific aminoacylase activity DNA; or
(E) DNA encoding a protein having 95% or more homology with the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4 and having Nε-acyl-L-lysine-specific aminoacylase activity.

  According to the present invention, cDNA encoding Nε-acyl-L-lysine-specific aminoacylase derived from actinomycetes, its nucleotide sequence and total amino acid sequence are provided. Therefore, according to the present invention, it is possible to mass-produce Nε-acyl-L-lysine-specific aminoacylase derived from actinomycetes using gene recombination technology, and a large amount of the Nε-acyl-L-lysine specific can be easily produced. It is possible to obtain a typical aminoacylase. Further, according to the present invention, Nε-acyl-L-lysine, particularly Nε-lauroyl-L-lysine can be obtained efficiently.

1A-E shows the S. movalens genomic region encoding Sm-ELA and the upstream and downstream nucleotide sequences along with the amino acid sequence of Sm-ELA. FIG. 1A continued. FIG. 1B continued. 1C continued. 1D continued. 2A-E show the nucleotide sequence of the S. sericolor genomic region encoding Sc-ELA along with the amino acid sequence of Sc-ELA. The start codon is GTG, not ATG. FIG. 2A continued. FIG. 2B continued. FIG. 2C continued. FIG. 2D continued. FIG. 3 shows the positional relationship between the primers used for cloning of the Sm-ELA gene and the Sm-ELA gene. FIG. 4 shows the time course of Nε-lauroyl-L-lysine synthesis by S. lividans washed cells producing Sm-ELA and cell extracts. The horizontal axis represents the reaction time, and the vertical axis represents the yield of Nε-lauroyl-L-lysine based on lauric acid. The black square represents the reaction using the washed cells, and the black triangle represents the reaction using the cell extract. FIG. 5 shows the change over time in the reaction yield in the synthesis reaction of Nε-lauroyl-L-lysine. The horizontal axis represents the reaction time, and the vertical axis represents the lauric acid yield of Nε-lauroyl-L-lysine calculated from the residual lauric acid and residual lysine. The white symbols indicate the Nε-lauroyl-L-lysine yield calculated from the residual amount of lysine, and the black symbols indicate the Nε-lauroyl-L-lysine yield calculated from the residual amount of lauric acid. Black circles and white circles are reactions with 50 mM lauric acid, black triangles and white triangles are reactions with 100 mM lauric acid, black squares and white squares are reactions with 250 mM lauric acid. Represents. The reaction yield was calculated based on the amount of lauric acid produced and the amount of lysine remaining. FIG. 6 shows the time course of Nε-lauroyl-L-lysine synthesis by Corynebacterium glutamicum YDK010 producing Sm-ELA. The horizontal axis represents the reaction time, and the vertical axis represents lysine consumption. The black circle represents the reaction performed under reaction condition 1, and the white circle represents the reaction performed under reaction condition 2. FIG. 7 shows the time course of Nε-lauroyl-L-lysine synthesis by culturing Corynebacterium glutamicum WDK010 that secretes and produces Sm-ELA and the resulting culture supernatant. The horizontal axis represents the reaction time, and the vertical axis represents the produced Nε-lauroyl-L-lysine (LL: Nε-lauroyl-L-lysine). FIG. 8 shows the time course of Nε-lauroyl-L-lysine synthesis by Corynebacterium glutamicum YDK01 producing Sm-ELA or Sc-ELA. The horizontal axis represents the reaction time, and the vertical axis represents the produced Nε-lauroyl-L-lysine (white and black circles) or consumed lauric acid (white and black triangles) (LL: Nε-lauroyl-L- Lysine, LA: lauric acid). Black circles and black triangles represent Sm-ELA reactions, and white circles and white triangles represent Sc-ELA reactions.

  The present inventors have identified a novel Nε-acyl-L-lysine-specific aminoacylase that specifically acylates the ε-amino group of L-lysine to produce Nε-acyl-L-lysine. Clarified to be present in culture. Furthermore, the present inventors cultured microorganisms belonging to the genus Streptomyces, in particular, Streptomyces mobaraensis (S. mobaraensis), and separated and / or recovered the enzymes from these cultures. The present invention has succeeded in producing an enzyme of the present invention that can be synthesized and efficiently synthesizing Nε-acyl-L-lysine. However, since the secretory amount of Nε-acyl-L-lysine specific aminoacylase in actinomycetes is extremely small, a method for preparing a large amount of the Nε-acyl-L-lysine specific aminoacylase is further desired. It was. Furthermore, the present inventors have clarified a Nε-acyl-L-lysine specific aminoacylase derived from Streptomyces coelicolor (S. sericolor) and a gene encoding the same. The present invention provides DNA encoding these Nε-acyl-L-lysine specific aminoacylases and a method for preparing the aminoacylases in large quantities. Furthermore, the present invention provides a method for efficiently producing Nε-lauroyl-lysine.

As one of the methods for obtaining a large amount of Nε-acyl-L-lysine-specific aminoacylase derived from actinomycetes, mass production of the Nε-acyl-L-lysine-specific aminoacylase gene using gene recombination technology is possible. To obtain a large amount of the Nε-acyl-L-lysine-specific aminoacylase. In order to establish this method, cDNA encoding the Nε-acyl-L-lysine-specific aminoacylase derived from this actinomycete is obtained, the nucleotide sequence is analyzed, and the Nε-acyl-L-lysine-specific aminoacylase is analyzed. Information on the entire amino acid sequence can be obtained. Furthermore, a transformant producing a target substance in large quantities can be obtained by incorporating the DNA encoding the Nε-acyl-L-lysine specific aminoacylase into an appropriate expression vector.
The present invention replaces the method of isolating Nε-acyl-L-lysine-specific aminoacylase derived from actinomycetes from nature, using gene recombination technology for efficient gene expression and the aforementioned Nε-acyl-L -Provide technology for mass production of lysine-specific aminoacylase. Accordingly, in one aspect of the present invention, there is provided DNA encoding a Nε-acyl-L-lysine specific aminoacylase that specifically acylates the ε-amino group of lysine to produce Nε-acyl-L-lysine. The In another embodiment of the present invention, the DNA is expressed in a suitable host, thereby producing the aminoacylase. In yet another embodiment of the present invention, the aminoacylase acts on carboxylic acid and L-lysine to produce Nε-acyl-L-lysine.

In the present invention, a DNA encoding actinomycete-derived Nε-acyl-L-lysine-specific aminoacylase or a protein having equivalent activity is obtained. For example, DNA encoding the actinomycete-derived Nε-acyl-L-lysine-specific aminoacylase is prepared by using primers based on the amino acid sequence of the Nε-acyl-L-lysine-specific aminoacylase and the nucleotide sequence information encoding it. By using it, it can be obtained by screening a cDNA library prepared from S. mobaraensis or S. sericolor mRNA. Further, using the obtained cDNA, a transformant producing the Nε-acyl-L-lysine specific aminoacylase is obtained, whereby a protein having the Nε-acyl-L-lysine specific aminoacylase activity is obtained. Can be obtained.
The DNA of the present invention may be cDNA or genomic DNA, and they can be obtained by methods other than those described above, for example, by total synthesis.
Further, according to the present invention, L-lysine and lauric acid are reacted in the presence of the transformant producing the Nε-acyl-L-lysine-specific aminoacylase or a processed product of the transformant, for example, a cell extract. By doing so, Nε-lauroyl-L-lysine can be obtained efficiently. In addition, when the Nε-acyl-L-lysine specific aminoacylase is expressed and secreted outside the cell (secretory expression), in the presence of a culture solution obtained by culturing the transformant, By reacting L-lysine with lauric acid, Nε-lauroyl-L-lysine can be efficiently obtained.

  Hereinafter, embodiments of the present invention will be described more specifically. In the present specification, the Nε-acyl-L-lysine-specific aminoacylase derived from actinomycetes is hereinafter simply referred to as “aminoacylase” when the context clearly shows.

1. Obtaining the DNA of the Present Invention The DNA of the present invention can be obtained, for example, as follows. As a specific example of the method for obtaining the DNA of the present invention, for example, oligo DNA is synthesized based on the amino acid sequence of aminoacylase determined by the present inventors, and mRNA extracted from actinomycetes or other sites is used as a template. -Conventionally, such as picking up aminoacylase cDNA by hybridization from a cDNA library obtained by using PCR method to obtain gene fragments and using actinomycetes or other site mRNA as a template. There is a method.
In addition, nucleotide sequences having high homology with the amino acid sequence of aminoacylase already determined are searched in an appropriate DNA database (for example, DDBJ, EMBL, GenBank), and the found sequence is used as a probe for actinomycetes or other It is also conceivable to obtain a cDNA of aminoacylase by screening a cDNA library prepared using mRNA extracted from a site as a template.

  A cDNA probe may be prepared by RT-PCR or other methods based on the amino acid sequence already determined, using mRNA extracted from actinomycetes or other sites as a template, or chemically synthesized based on the amino acid sequence. You may do it. When a nucleotide sequence having a high homology with an amino acid sequence is used as a probe, the origin of the probe, that is, what kind of gene fragment of which species the probe does not matter. That is, the probe need not be derived from a known Nε-acyl-L-lysine-specific aminoacylase gene, and may be a part of a gene encoding a gene product whose function is unknown. Tag). In the examples described below, a primer based on an amino acid sequence that is annotated with a virtual enzyme such as S. avermitilis having a sequence very similar to S. mobaraensis or S. sericolor by BLAST search search. PCR is performed using a primer constructed from the primer and the N-terminal amino acid sequence of aminoacylase, and the amino acid sequence is analyzed by the method of analyzing the amino acid sequence of the PCR product. The cDNA library can be prepared by a conventional method.

  Thus, a Nε-acyl-L-lysine-specific aminoacylase derived from S. mobaraensis having the amino acid sequence set forth in SEQ ID NO: 2 (hereinafter sometimes abbreviated as Sm-ELA in this specification) and SEQ ID NO: 5 DNA encoding N.epsilon.-acyl-L-lysine specific aminoacylase derived from S. sericolor having the amino acid sequence described (hereinafter abbreviated as Sc-ELA in the present specification) is obtained (each sequence Numbers 3 and 4). The DNA of the present invention contains one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 5 as long as the protein encoded by the protein has Nε-acyl-L-lysine specific aminoacylase activity. May be a DNA encoding a protein (variant protein) having an amino acid sequence (variant sequence) inserted, added, deleted or substituted. The DNA of the present invention includes 80% or more of DNA that hybridizes under stringent conditions with DNA having the sequence described in SEQ ID NO: 3 or 4, or DNA having the sequence described in SEQ ID NO: 3 or 4. DNA having a homology of preferably 90% or more, particularly preferably 95% or more, which encodes a protein having Nε-acyl-L-lysine-specific aminoacylase activity, and a recombinant DNA containing the DNA Also included in the present invention are a molecule, a host holding the recombinant DNA, a method for producing a Nε-acyl-L-lysine specific enzyme comprising culturing the host, and a method for producing Nε-acyl-L-lysine It is. Homology can be determined by using standard programs such as NCBI Blast Ver.2.0 using default parameters. In this specification, the amino acid sequence of Nε-acyl-L-lysine specific aminoacylase derived from actinomycetes is described in SEQ ID NOs: 2 and 5, and the nucleotide sequences encoding the amino acid sequences are described in SEQ ID NOs: 3 and 4. For Sm-ELA, the nucleotide sequence including the upstream and downstream of the coding region is also described in SEQ ID NO: 1, so that it is possible for those skilled in the art to obtain DNA encoding such a variant protein. Such homology can be calculated using programs well known to those skilled in the art, such as BLAST, with standard parameters.

In the present specification, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNAs having high homology, for example, DNAs having a homology of 80, 90, 95, 97 or 99% or higher hybridize, and DNAs having lower homology do not hybridize with each other, Alternatively, the salt concentration corresponding to the usual Southern hybridization washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS. And conditions for washing at least once, preferably 2-3 times at temperature. Those skilled in the art will readily understand these equivalent conditions. A description of such conditions can be found, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, Ausubel et al., Current Protocols in Molecular Biology, See John Wiley & Sons, Inc. (1994).
Furthermore, since examples of nucleotide sequences of DNAs included in the present invention are shown in SEQ ID NOs: 1, 3 and 4, DNAs having the sequences described in SEQ ID NOs: 1, 3 or 4 and DNAs having variant sequences thereof, these It is also possible to completely chemically synthesize DNA having a sequence homologous to DNA and DNA capable of hybridizing with these under stringent conditions. Methods for chemical synthesis of DNA are also well known to those skilled in the art, and automated equipment for this is also commercially available.

2. Construction of Recombinant DNA of the Present Invention Next, the cDNA thus obtained can be incorporated into an expression vector to produce a recombinant DNA. The vector to be used is not particularly limited, and may be a vector that can replicate autonomously in a host cell or a vector in which one copy or a plurality of copies are inserted into a chromosome, and an insertion that can incorporate the above DNA, that is, an aminoacylase gene. There is a need for a region that has a site and that allows the integrated DNA to be expressed in the host cell. The aminoacylase gene to be incorporated into the vector is not limited to cDNA, but may be DNA synthesized and designed to encode the aminoacylase sequence of aminoacylase predicted from cDNA. Such DNA includes DNA in which substitution with another codon encoding the same amino acid is performed according to the codon usage frequency of the host to be expressed. For example, E. coli is known to have low codon usage such as AUA, AGG, AGA, CGG, GGA, CUA, CCC, CGA, etc. The corresponding DNA sequence can also be modified so that at least one of these becomes a more frequently used codon. Such gene synthesis can be easily performed by ligating oligonucleotides synthesized using an automatic DNA synthesizer after annealing. There is also a method in which aminoacylase is expressed and produced as a fusion protein with a heterologous protein. For example, by using the pGEX system (Amersham Pharmacia Biotech) or the like, aminoacylase can be produced in Escherichia coli (E. coli) as a fusion protein with glutathione-S-transferase. Is possible. When an actinomycete such as Streptomyces lividans (S. lividans) is selected as an expression host, a high copy vector such as pIJ702 can be used. Further, when a coryneform bacterium such as Corynebacterium glutamicum is selected as an expression host, a high copy vector such as pPK4 can be used.

  As a promoter for expressing an aminoacylase gene, a promoter usually used for expression of a heterologous protein can be used. For example, promoters such as trp, tac, lac, trc, λPL, and T7 can be used. A terminator can also be inserted downstream of the aminoacylase gene. For example, terminators such as tpA, lpp, and T4 can be mentioned. When selecting actinomycetes as an expression host, the SSI promoter, the natural promoter of the Nε acyl-L-lysine-specific aminoacylase gene of the present invention, the SD sequence and the terminator can also be used. When a coryneform bacterium is selected as an expression host, a cspB promoter or the like can be used. In addition, for efficient translation, the type and number of SD sequences and the base composition, sequence, and length of the region between the SD sequence and the start codon can be optimized for the expression of the aminoacylase gene. . The region from the promoter required for aminoacylase expression to the translation initiation point can be adjusted by a known PCR method or chemical synthesis method. Furthermore, it is also possible to insert a signal peptide sequence downstream of the initiation codon to express aminoacylase as a secreted protein. The recombinant DNA of the present invention can be obtained by inserting a DNA containing the aminoacylase gene into a known expression vector corresponding to a desired expression system by a known method. The expression vector to be used is preferably a multicopy one.

3. Next, various transformants obtained by inserting the DNA or recombinant DNA of the present invention will be described. Examples of cells into which the DNA or recombinant DNA of the present invention is introduced include various microbial cells including E. coli, actinomycetes and coryneform bacteria. E. coli into which the DNA of the present invention or the recombinant DNA is introduced generally includes strains frequently used for cloning and expression of heterologous proteins, such as HB101, MC1061, JM109, CJ236, and MV1184. Actinomycetes into which the DNA or recombinant DNA of the present invention is introduced generally include strains often used for the expression of heterologous proteins, such as S. lividans TK24 and S. sericolor A3 (2). The coryneform bacterium into which the DNA of the present invention or the recombinant DNA is introduced is an aerobic Gram-positive gonococcus, including a bacterium that was conventionally classified as a genus Brevibacterium but is now integrated into the genus Corynebacterium ( Int. J. Syst. Bacteriol., 41, 255 (1981)), and Brevibacterium spp. Closely related to Corynebacterium spp. The advantage of using coryneform bacteria is that there are very few proteins that are originally secreted outside the cells compared to fungi, yeast and Bacillus bacteria that have been suitable for protein secretion so far. When the protein is secreted and produced, the purification process can be simplified and omitted, and when the enzyme reaction is performed using the secreted and produced enzyme, the culture supernatant can be used as an enzyme source. Impurities in terms of medium cost, culture method, and culture productivity because it can reduce impurities and side reactions due to contaminating enzymes, etc., and grows easily on simple media containing sugar, ammonia, inorganic salts, etc Is included. In addition, by using the Tat secretion pathway, industrially useful proteins such as isomalt dextranase and protein glutaminase, which have been difficult to produce by the Sec secretion pathway known so far, can be efficiently used. It can be secreted [WO2005 / 103278]. The Nε-acyl-L-lysine-specific aminoacylase derived from actinomycetes of the present invention or used in the present invention can also be secreted outside the cells by utilizing an appropriate secretion pathway such as the Tat secretion pathway. . In particular, Nε-acyl-L-lysine-specific aminoacylase derived from S. mobaraensis can be efficiently secreted outside the cell body by the Tat system secretion pathway. The “Tat system” is a pathway called “Twin-arginine-translocation-pathway”, and recognizes the conserved region of arginine-arginine conserved in the signal peptide. , Means a mechanism or pathway for secreting proteins by membrane proteins including C and E. Examples of the Tat signal peptide include a signal peptide of trimethylamine N-oxidoreductase (TorA) derived from E. coli. Examples of coryneform bacteria include the following.

Corynebacterium acetoacidophilum,
Corynebacterium acetoglutamicum,
Corynebacterium alkanolyticum,
Corynebacterium carnae,
Corynebacterium glutamicum,
Corynebacterium Lilium,
Corynebacterium melacecola,
Corynebacterium thermoaminogenes,
Corynebacterium herculis,
Brevibacterium divaricatam,
Brevibacterium flavum,
Brevibacterium immariophilum,
Brevibacterium lactofermentum,
Brevibacterium roseum,
Brevibacterium saccharolyticum,
Brevibacterium thiogenitalis,
Corynebacterium ammoniagenes,
Brevibacterium album,
Brevibacterium cerinum,
Microbacterium ammonia film.

Specifically, the following strains can be exemplified.
Corynebacterium acetoacidophilum ATCC13870,
Corynebacterium acetoglutamicum ATCC15806,
Corynebacterium alkanolyticum ATCC21511,
Corynebacterium carnae ATCC15991,
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060, ATCC13869, FERM BP-734,
Corynebacterium lilium ATCC15990,
Corynebacterium melacecola ATCC17965,
Corynebacterium efficiens AJ12340 (FERM BP-1539),
Corynebacterium herculis ATCC13868,
Brevibacterium divaricatam ATCC14020,
Brevibacterium flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205),
Brevibacterium in mariophyllum ATCC14068,
Brevibacterium lactofermentum ATCC13869,
Brevibacterium rose ATCC13825,
Brevibacterium saccharolyticum ATCC14066,
Brevibacterium thiogenitalis ATCC19240,
Corynebacterium ammoniagenes ATCC6871, ATCC6872,
Brevibacterium album ATCC15111,
Brevibacterium cerinum ATCC15112,
Microbacterium ammonia philus ATCC15354.

In particular, Corynebacterium glutamicum AJ12036 (see WO 02/081694) isolated as a streptomycin (Sm) -resistant mutant from ATCC13869 from the wild-type Corynebacterium glutamicum (C. glutamicum) ATCC13869 is the parent strain (wild strain). In comparison, it is predicted that there is a mutation in a functional gene related to protein secretion, and the protein production capacity of protein is extremely high, about 2 to 3 times as the accumulated amount under optimum culture conditions. is there.
Furthermore, if a strain modified so as not to produce cell surface proteins from such a strain is used as a host, purification of the target protein secreted in the medium is facilitated, which is particularly preferable. Such modification can be performed by introducing a mutation into a cell surface protein on the chromosome or its expression regulatory region by a mutation or gene recombination method. Examples of coryneform bacteria modified so as not to produce cell surface protein include C. glutamicum YDK010 strain, which is a cell surface protein (PS2) -disrupted strain of AJ12036 (see WO 01/23491).

  Examples of other cells that can be transformed include Bacillus subtilis, yeast, Neisseria gonorrhoeae, and the like. A method for producing aminoacylase in a medium using these protein secreting ability is also conceivable. In addition to the above microorganisms, cultured cells such as silkworm cultured cells may be used. A transformant can be obtained by introducing the above recombinant vector into these host cells. The recombinant expression vector can be introduced into the host cell by a conventional and conventionally used method. Examples include a competent cell method, a protoplast method, a calcium phosphate coprecipitation method, an electroporation method, a microinjection method, and a liposome fusion method. Specific examples of the method for introduction into coryneform bacteria include, for example, the protoplast method (Gene, 39, 281-286 (1985)), the electroporation method (Bio / Technology, 7, 1067-1070) (1989)), etc. Can be used, but is not limited to these.

4). Method for producing Nε-acyl-L-lysine-specific aminoacylase By culturing the transformant thus obtained, Nε-acyl-L-lysine-specific aminoacylase is produced. The produced aminoacylase is isolated by a known method and, if necessary, further purified to obtain the target enzyme. When E. coli is used as a host, it is also possible to obtain an aminoacylase gene product as an inactive aminoacylase aggregate, that is, a protein inclusion body, and then activate it by an appropriate method. After activation, the target enzyme may be obtained by separating and purifying the active protein by a known method.
A medium for culturing the transformant is known. For example, a carbon source, a nitrogen source, a vitamin source, etc. are added to a nutrient medium such as LB medium or a minimum medium such as M9 medium for culturing E. coli. Can be used. Depending on the host, the transformant is usually cultured at 16 to 42 ° C., preferably 25 to 37 ° C. for 5 to 168 hours, preferably 8 to 72 hours. Depending on the host, either shaking culture or stationary culture is possible, but stirring may be performed as necessary, and aeration may be performed. When selecting actinomycetes as an expression host, conditions that can be used for producing the enzyme of the present invention, for example, conditions described in WO 2006/088199 can be used. In addition, when an inducible promoter is used for expression of aminoacylase, a promoter inducing agent can be added to the medium for cultivation.

  The produced aminoacylase is a known salting out from the extract of the transformant, a precipitation method such as isoelectric point precipitation or solvent precipitation, a method utilizing a molecular weight difference such as dialysis, ultrafiltration or gel filtration, Methods using specific affinity such as ion exchange chromatography, methods using differences in hydrophobicity such as hydrophobic chromatography and reverse phase chromatography, and other affinity chromatography, SDS polyacrylamide electrophoresis, isoelectric focusing Purification and isolation are possible by electrophoresis or the like, or a combination thereof. When the aminoacylase is secreted and expressed, a culture supernatant containing the target enzyme is obtained by removing the cells from the culture solution obtained by culturing the transformant by centrifugation or the like. Aminoacylase can also be purified and isolated from this culture supernatant.

For example, after culturing the transformant, the cells collected by centrifugation are suspended in a cell disruption buffer (20-100 mM Tris-HCl (pH 8.0), 5 mM EDTA), and ultrasonic disruption is performed in Branson MODEL. -Using the Sonifier 250 microchip, the cells can be disrupted by performing output control 7, duty cycle 50% for about 10 minutes. Cell disruption can also be performed by adding a solvent such as toluene to the culture solution. The crushing solution is centrifuged at 12000 rpm for 10 minutes, and the supernatant can be purified as described above. The precipitate after centrifugation can be further purified after solubilization with guanidinium hydrochloride or urea, if necessary. When the aminoacylase is secreted and expressed, the culture solution can be centrifuged at 12000 rpm for 10 minutes after completion of the culture of the transformant, and the supernatant can be purified as described above.
Specifically, the aminoacylase enzyme encoded by the DNA of the present invention can be purified, for example, as follows. After culturing the host, ammonium sulfate (2.8M) is added to the culture supernatant or cell extract, and the precipitate is fractionated. Further, CM Sephadex C-50, DEAE-Sephadex A-50 ion exchange column chromatography, octyl By performing operations such as Sepharose CL-4B and phenyl Sepharose CL-4B column chromatography, the gel can be purified to such a degree as to exhibit a single band on polyacrylamide gel electrophoresis.

The activity of the obtained aminoacylase enzyme can be measured by measuring the Nε-acetyl-L-lysine hydrolysis activity. The enzyme 1U (unit) of the present invention was incubated at 37 ° C. using a Nε-acetyl-L-lysine solution as a substrate (50 mM Tris-HCl buffer, pH 8.0) to quantify the released L-lysine. In some cases, it is defined as the amount of enzyme required to hydrolyze 1 micromole of Nε-acetyl-L-lysine per hour.
The aminoacylase encoded by the DNA of the present invention acts on Nε-acyl-L-lysine and has very low reactivity to Nε-acyl-D-lysine. It has a wide specificity for acyl groups and hydrolyzes Nε-acyl-L-lysine consisting of saturated or unsaturated fatty acid acyls and also acyl carboxylates containing aromatic groups and catalyzes the reverse reaction. Regarding the reverse reaction, the reactivity of D-lysine with respect to the ε-amino group is very low, and it preferentially acts on the ε-amino group of L-lysine.

  Another aspect of the present invention is a method for producing Nε-acyl-L-lysine. In one embodiment of this aspect, the enzyme of the present invention or the enzyme encoded by the DNA of the present invention is such that L-lysine or a salt thereof acts on a carboxylic acid or a salt thereof, whereby Nε-acyl-L-lysine Is generated. In this aspect, the enzyme may be a purified enzyme or a crude enzyme, or may be a cell disruption, a cell extract, or a crude enzyme of a transformant expressing the enzyme of the present invention. The above-mentioned transformant can also be used without crushing. When the enzyme of the present invention is secreted into the medium by using an appropriate signal sequence, the culture supernatant or a concentrate thereof can be used as the enzyme source. The conditions for the enzyme reaction in this embodiment can be determined according to the properties of the enzyme, in particular the optimal and stable temperatures, and appropriate conditions including the optimal and stable pH. For example, the enzyme of the present invention derived from S. mobaraen (having the amino acid sequence of SEQ ID NO: 2) has the following properties.

1) Acts on Nε-acyl-L-lysine to catalyze the reaction of liberating carboxylic acid and lysine, and the reverse reaction;
2) acts on the ε-amino group of L-Lys;
3) When Nε-acetyl-L-lysine is used as the substrate, the optimum pH for the hydrolysis reaction is in the range of 8.0 to 9.0 in Tris-HCl buffer at 37 ° C;
4) stable in the range of pH 6.5 to 10.5 when incubated at 37 ° C. for 1 hour in Tris-HCl buffer;
5) The optimum temperature in the hydrolysis reaction using Nε-acetyl-L-lysine as a substrate is around 55 ° C. in Tris-HCl buffer (pH 8.2);
6) Inactivation in Tris-HCl buffer (pH 8.2) at 40 ° C. for 60 minutes; remaining activity after treatment at 55 ° C. for 60 minutes is 75 to 85%;
7) Inhibited by o-phenanthroline.
8) The activity is increased by metal ions such as cobalt, zinc, manganese, calcium, potassium, magnesium, and the activity is increased particularly by cobalt ions;

  In addition, the enzyme derived from S. movalaens exhibits high hydrolytic activity on Nε-acetyl-L-lysine, but generally does not show activity on Nα-acetyl-L-amino acids other than Nα-acetyl-L-lysine. Furthermore, the enzyme derived from S. movalaen has the highest specific activity for Nε-acetyl-L-lysine, and also for other Nε-acyl-L-lysine having a chloroacetyl group or a benzoyl group. Shows high activity. Details of these properties are described in WO 2006/088199.

  In particular, when the carboxylic acid has a relatively long chain and the reaction is carried out in a water-soluble solvent, the produced Nε-acyl-L-lysine is insoluble or hardly soluble in water, and thus precipitates and reacts. Therefore, the synthesis reaction of ε-acyl-L-lysine proceeds significantly more preferentially and efficiently than the decomposition reaction of Nε-acyl-L-lysine, which is the reverse reaction. In such a case, the synthetic reaction product Nε-acyl-L-lysine is rapidly separated from the aqueous phase and can be recovered very easily. For example, separated Nε-acyl-L-lysine can be directly recovered, or Nε-acyl-L-lysine can be easily extracted from an aqueous reaction system and recovered with an organic solvent. When Nε-acyl-L-lysine is produced according to the present invention, the relatively long-chain carboxylic acid can be selected based on, for example, solubility in the water-soluble solvent used. In the present specification, “water-soluble solvent” naturally includes water itself. Further, even when the reaction is carried out in a water-soluble solvent, an oil layer (organic solvent layer) may be separately stacked to form a water-soluble solvent / organic solvent two-phase reaction.

  In one embodiment of the invention in which Nε-acyl-L-lysine production is carried out according to the method of the present invention, the enzyme encoded by the DNA of the present invention is 1 U to 500 U / ml, preferably 10 U to 300 U / ml, and L-lysine. Or a salt thereof, 50 mM to 2 M, preferably 100 mM to 1.0 M, and a carboxylic acid or salt thereof, 5 mM to 2 M, preferably 10 mM to 300 mM, in a suitable buffer such as Tris-HCl buffer, pH 6.5 to 10.5, preferably pH 7.0 to 10.0, particularly preferably pH 7.0 to 8.0, pH range 30 ° C to 70 ° C, preferably 45 ° C to 70 ° C, 1 to 48 hours, preferably 2 The reaction is carried out for 24 hours, particularly preferably 4 to 24 hours. Under suitable conditions, Nε-acyl-L-lysine can be obtained in a yield of about 80% or more. One of the substrates L-lysine or carboxylic acid may be present in an excess amount in the reaction system, and a deficient substrate may be added to the reaction as necessary.

  In the method for producing Nε-acyl-L-lysine of the present invention, as described above, the reaction of L-lysine with a carboxylic acid or a salt thereof is catalyzed by the enzyme encoded by the DNA of the present invention. Carboxylic acids used in the reaction include linear or branched, saturated or unsaturated fatty acids, aromatic carboxylic acids having saturated or unsaturated side chains, and these carboxylic acids preferably have a carbon number. Is a carboxylic acid having an acyl group having 5 or more, more preferably 8 or more carbon atoms. More specifically, the carboxylic acid that can be used in the method for producing Nε-acyl-L-lysine of the present invention is octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid. Benzoic acid and cinnamic acid are preferred, and octanoic acid and lauric acid are particularly preferred.

  The following reference examples and examples further illustrate the invention, but these examples should not be construed as limiting the scope of the invention. The percentages in the following Reference Examples and Examples mean “mass%” unless otherwise clear from the context.

1. Cloning of aminoacylase (Sm-ELA) DNA from Streptomyces S. mobaraensis Determination of partial amino acid sequence of aminoacylase
WO 2006/088199, Nε-acyl-lysine-specific aminoacylase protein (Sm-ELA) was purified from the culture of Actinomyces S. mobaraensis IFO13819 strain according to the method described in Example 1, and its N-terminal amino acid sequence was purified. Analysis was performed with a protein sequencer.
Simply put, 2 L (liter) of medium containing 4.0% soluble starch, 2.0% polypeptone, 4.0% meat extract, 0.2% potassium hydrogen phosphate, 2.0% magnesium sulfate and pH 7 is placed in a 5L baffled Sakaguchi flask. 0.1 ml of a spore solution of S. mobaraensis was inoculated and cultured at 30 ° C. for 3 to 7 days. After completion of the culture, the cells were sterilized by centrifugation (10000 × g, 30 minutes), and the supernatant was collected.

Ammonium sulfate, DEAE-Sephadex A-50 column, Octyl Sepharose CL-4B column (φ16 x 300 mm), Phenyl Sepharose CL-4B column (φ16 x 150 mm) so that the final concentration is 60%. ).
When the purified aminoacylase was subjected to a protein sequencer, SERPXTTLLRNGDVHSPADPF (X is unknown) (SEQ ID NO: 6) was obtained as the N-terminal amino acid sequence. When homology search was performed using NCBI protein-protein BLAST for 21 residues of the obtained N-terminal amino acid sequence, 81% of the N-terminal amino acid sequence of virtual protein SCO1424 (S. sericolor A3 (2), NP_625706) was obtained. It became clear that it has homology.

2. Cloning of Sm-ELA gene
Primer 1 (5′-CTGCTGCGCAACGGCGACGTCCACA-3 ′) (SEQ ID NO: 7) was designed based on the sequence LLRNGDVHS obtained by N-terminal amino acid analysis. Primer 2 (5′-ACGACGGCCGAGTGGACGTCGATCC-3 ′) (SEQ ID NO: 8) was designed from the internal sequence of SCO1424 (443-467 bp).
PCR amplification was performed using primers 1 and 2 using the genomic DNA of S. mobaraensis IFO13819 strain as a template.

PCR conditions are as follows <Preparation of PCR solution>
Uses Advantage-GC 2 PCR Kit (Clontech).
H ₂ O 12 μl, PCR buffer 4 μl, GC Melt 2 μl, dNTPs 0.4 μl, Taq polymerase 0.4 μl, 10 μM each primer 0.4 μl, 10 μg / ml template DNA 0.4 μl, total volume 20 μl
<PCR reaction conditions>
Cycle 1 (x1) 95 ° C; 5 minutes
Cycle 2 (x30) 95 ° C; 0.5 min, 60 ° C; 1 min, 72 ° C; 1 min
Cycle 3 (x1) 72 ° C; 10 minutes, 4 ° C; ∞
(Total 20 μL)

The obtained DNA fragment 0.43 kbp and pUC118 HincII / BAP (TAKARA) were ligated to transform E. coli DH5α. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 50 mg / l ampicillin. A plasmid was extracted from a transformant carrying the target DNA fragment, and the nucleotide sequence of the inserted DNA fragment was confirmed.
Primer 3 (5′-ACGAGGTGATCGACCTCCAGGGCGC-3 ′) (SEQ ID NO: 9) was designed from the nucleotide sequence obtained by PCR amplification using primers 1 and 2. Primer 4 (5′-TACATGCCGTCCTCGCCGCCCCAGA-3 ′) (SEQ ID NO: 10) was designed from the internal sequence of SCO1424 (1142-1166 bp).
PCR amplification was performed using primers 3 and 4 using the genomic DNA of S. mobaraensis IFO13819 strain as a template. PCR conditions are the same as those using primers 1 and 2. The obtained DNA fragment 1.1 kbp and pUC118 HincII / BAP were ligated to transform E. coli DH5α. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 50 mg / l ampicillin. A plasmid was extracted from a transformant carrying the target DNA fragment, and the nucleotide sequence of the inserted DNA fragment was confirmed.
From the nucleotide sequence obtained by PCR amplification using primers 3 and 4, primer 5 (5'-TCTCGCTGGGCATCGGCTCGGTGCA-3 ') (SEQ ID NO: 11) and primer 6 (5'-CAGGCCGGTGGCAGTGGTGTGCACA-3') (SEQ ID NO: 12) Designed. PCR amplification was performed using the extracted plasmid, primers 3 and 4, and PCR DIG Labeling Mix (Roche) to obtain a 1.1 kbp DIG-labeled DNA probe.

PCR conditions are as follows.
<Preparation of PCR solution>
Uses Advantage-GC 2 PCR Kit (Clontech).
H ₂ O 52.5 μl, PCR buffer 20 μl, GC Melt 10 μl, PCR DIG Labeling Mix (Roche) 10 μl, Taq polymerase 2 μl, 10 μM each primer 2 μl,, 100 ng / μl template 1.5 μl, total volume 100 μl
<PCR reaction conditions>
Cycle 1 (x1) 95 ° C; 5 minutes
Cycle 2 (x30) 95 ° C; 0.5 min, 60 ° C; 1 min, 72 ° C; 1 min
Cycle 3 (x1) 72 ° C; 10 minutes, 4 ° C; ∞

  Genomic DNA of S. mobaraensis strain IFO13819 was treated with NdeI, SpeI, XbaI, BssHII and MluI, respectively, and electrophoresed using a 1.4% agarose gel. This was transferred to Hybond-N + (GE Healthcare), and Southern hybridization was performed using a DIG-labeled probe. As a result, a strong signal was observed at about 2 kbp in the genomic DNA fragmented with MluI. Thus, 2 kbp of DNA fragment obtained by treating genomic DNA with MluI was self-ligated, and the ligation solution was used as a template, and PCR amplification was performed using primers 5 and 6.

PCR conditions are as follows.
<Preparation of PCR solution>
Uses AdvantageTM-GC 2 PCR Kit (Clontech).
H ₂ O 25 μl, PCR buffer 10 μl, GC Melt 5 μl, dNTPs 1 μl, Taq polymerase 1 μl, 10 μM each primer 1 μl Template 6 μl
(Total 50 μL)
<PCR reaction conditions>
Cycle 1 (x1) 95 ° C; 5 minutes
Cycle 2 (x5) 95 ° C; 0.5 min, 72 ° C; 3 min
Cycle 3 (x5) 95 ° C; 0.5 min, 65 ° C; 1 min, 72 ° C; 3 min
Cycle 4 (x25) 95 ° C; 0.5 min, 60 ° C; 1 min, 72 ° C; 3 min
Cycle 5 (x1) 72 ° C; 10 minutes, 4 ° C; ∞

  The obtained DNA fragment of about 2 kbp was ligated with pT7Blue T-vector (Novagen), and E. coli DH5α was transformed. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 50 mg / l ampicillin. A plasmid was extracted from a transformant carrying the target DNA fragment, and the nucleotide sequence of the inserted DNA fragment was confirmed.

From the nucleotide sequence obtained by PCR amplification using primers 5 and 6, primer 7 (5'-AACGCGGCGATGTGCTCGGGCGTGA-3 ') (SEQ ID NO: 13) and primer 8 (5'-CGCGTCTCGGTGCGTGCGGCCTTCA-3') (SEQ ID NO: 14) Designed. Furthermore, PCR amplification was performed using the extracted plasmid, primers 5 and 7, and PCR DIG Labeling Mix (Roche) to obtain a 0.5 kbp DIG-labeled DNA probe. PCR conditions are the same as when primers 3 and 4 As described above, genomic DNA of S. mobaraensis strain IFO13819 was treated with NcoI, and Southern hybridization was performed using a 0.5 kbp DIG-labeled probe. went. As a result, a strong signal was observed at about 1.8 kbp. Thus, a 1.8 kbp DNA fragment obtained by treating genomic DNA with NcoI was self-ligated, and PCR amplification was performed using primers 7 and 8 using the ligation solution as a template. PCR conditions are the same as those used in combination of primers 5 and 6. The obtained DNA fragment of about 1.5 kbp and pT7Blue T-vector were ligated to transform E. coli DH5α. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 50 mg / l ampicillin. A plasmid was extracted from a transformant carrying the target DNA fragment, and the nucleotide sequence of the inserted DNA fragment was confirmed.
From these results, the ORF full-length of the Sm-ELA gene and the surrounding nucleotide sequence were clarified, so primer 9 (5'-GCGCCGCACGGGCTGGATCAACCAC-3 ') (SEQ ID NO: 15) was downstream from the ORF upstream region sequence. Primer 10 (5′-TCAGTGCGGAGGGGGTGACGCAGCG-3 ′) (SEQ ID NO: 16) was designed from the above sequence. PCR amplification using primers 9 and 10 using the genomic DNA of S. mobaraensis IFO13819 strain as a template, the resulting 1.6 kbp DNA fragment and pUC118 HincII / BAP were ligated, and E. coli DH5α was transformed did. PCR conditions are as follows.

<Preparation of PCR solution>
KOD plus DNA polymerase (Toyobo) is used.
H ₂ O 51.6 μl, PCR buffer 10 μl, dNTPs 10 μl, MgSO ₄ 8 μl, KOD plus 2 μl, 10 μM each primer 6 μl, template 1.4 μl, DMSO 5 μl, total volume 100 μl
<PCR reaction conditions>
Cycle 1 (x1) 95 ° C; 5 minutes
Cycle 2 (x5) 95 ° C; 0.5 min, 72 ° C; 2 min
Cycle 3 (x5) 95 ° C; 0.5 min, 65 ° C; 1 min, 72 ° C; 2 min
Cycle 4 (x25) 95 ° C; 0.5 min, 60 ° C; 1 min, 72 ° C; 2 min
Cycle 5 (x1) 72 ° C; 10 minutes, 4 ° C; ∞

Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 50 mg / l ampicillin. A plasmid was extracted from a transformant carrying the target DNA fragment, and the nucleotide sequence of the inserted DNA fragment was confirmed. This nucleotide sequence is shown in SEQ ID NO: 1. This DNA fragment contained the entire open reading frame of the Sm-ELA gene. The amino acid sequence of Sm-ELA encoded by this fragment is shown in SEQ ID NO: 2. The nucleotide sequence of the open reading frame (ORF) is shown in SEQ ID NO: 3. The resulting plasmid was named pUC118-SmELA.
The positional relationship between the used primer and the Sm-ELA gene is as shown in FIG.

Production of Sm-ELA in E.coli Preparation of Sm-ELA E.coli expression strain
From the nucleotide sequence of Sm-ELA gene, primer SmELA-Nde-f (5'-catATGAGCGAGCGCCCCCGAACCACCC-3 ') (SEQ ID NO: 17) and primer SmELA-Hind-r (5'-aagctTCAGGCGGCGTACACGGTGCGTCCG-3') (SEQ ID NO: 18) Designed. PCR amplification was performed using pUC118-SmELA as a template and these primers.

PCR conditions are as follows.
<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ° C; 10 seconds, 55 ° C; 10 seconds, 68 ° C: 2 minutes,
Cycle 3 (x1) 68 ° C; 2 minutes, 4 ° C; ∞

  The obtained 1.6 kbp DNA fragment was ligated with pTA2 (TArget Clone-Plus-, Toyobo), and E. coli JM109 was transformed. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 100 mg / l ampicillin. When a plasmid was extracted from the transformant carrying the target DNA fragment and the nucleotide sequence of the inserted DNA fragment was confirmed, the full length ORF of the Sm-ELA gene and the added NdeI and HindIII recognition sites were included. The obtained plasmid was treated with NdeI and HindIII to obtain a 1.6 kbp DNA fragment containing the Sm-ELA gene. JP 2005278468 A (JP-A-2005-278468), optically active amino acid production method, vector pTrp2 described in Example 1, column 0064 is treated with NdeI and HindIII, and a DNA fragment containing Sm-ELA gene Ligated and transformed into E. coli JM109. Transformants were selected on LB agar medium containing 100 mg / l ampicillin. The plasmid having the Sm-ELA gene inserted into pTrp2 was named pTrp2-SmELA, and the transformant carrying this plasmid was designated as E. coli JM109 / pTrp2-SmELA strain.

2. Expression of Sm-ELA using E.coli
One platinum loop of E. coli JM109 / pTrp2-SmELA was inoculated into a 500 ml Sakaguchi flask containing 50 ml of TB medium containing 100 mg / l ampicillin, and cultured with shaking at 37 ° C. for 16 hours. The cells were collected from the culture solution by centrifugation, washed with 20 mM Tris-HCl (pH 7.6), suspended in 10 ml of 20 mM Tris-HCl (pH 7.6), and used as washed cells. When the degradation activity of Nε-acetyl-L-lysine was measured using the washed cells as they were without crushing, it had an activity of about 6.0 U per amount of washed cells corresponding to 1 ml of the culture solution. It was revealed that the activity reached about 4.3 times the activity 1.4 U contained in 1 ml of the culture supernatant of S. mobaraensis IFO13819 strain reported in / 088199 A1. No activity was detected when washed cells of E. coli JM109 / pUC18 strain were used.
The activity was measured at Nε-acetyl-L-lysine 4 mM, Tris-HCl 50 mM (pH 8.0) at 37 ° C., and the concentration of lysine produced along with the decomposition of Nε-acetyl-L-lysine was measured. For the measurement of lysine, the acidic ninhydrin method described in WO2006 / 088199 was used. 1 U was defined as the amount of enzyme that liberates 1 μmol of lysine from Nε-acetyl-L-lysine per hour.

Synthesis of Nε-lauroyl-L-lysine by washing cells of E. coli JM109 / pTrp2-SmELA
One platinum loop of E. coli JM109 / pTrp2-SmELA was inoculated into a 500 ml Sakaguchi flask with 50 ml of TB medium containing 100 mg / l ampicillin, and cultured with shaking at 37 ° C. for 16 hours. The cells were collected from the culture solution by centrifugation and suspended in 10 ml of 20 mM Tris-HCl (pH 7.6).
Prepare 50 ml of the reaction solution so that the final concentration is 10 mM lauric acid, 100 mM L-lysine hydrochloride, 50 mM Tris-HCl, and 6.0 U / ml of washed cells, and 500 ml using a 200 ml mini jar fermenter. The mixture was stirred at rpm to carry out Nε-lauroyl-L-lysine synthesis reaction. The pH was adjusted with 2N NaOH to 7.0, and the temperature of the reaction solution was adjusted to 37 ° C.
After 24 hours, the entire reaction solution was recovered and centrifuged. After removing the supernatant, methanol and 6N HCl were added to the precipitate to dissolve the precipitate. This solution was centrifuged and the supernatant was collected. 6N NaOH was added to the supernatant to adjust to pH 6.5-7.5 to precipitate Nε-lauroyl-L-lysine. The reaction solution was centrifuged, the supernatant was removed, and water was added to the precipitate to suspend it. The suspension was centrifuged, the supernatant was removed, and methanol was added to the precipitate to suspend it. The suspension was suction filtered using filter paper. The powder on the filter paper was dried and weighed to find 93.3 mg. The Nε-lauroyl-L-lysine purity in this powder was measured by HPLC and found to be 92.1%. Therefore, it was revealed that 85.9 mg (0.262 mmol) of Nε-lauroyl-L-lysine was obtained. The yield was 52.3% based on lauric acid.
HPLC analysis was performed with column YMC-Pack A-312 ODS, 6.0 × 150 mm (YMC), moving bed 0.1 M NaH 2 PO 4 / MeOH = 2/8, UV 210 nm.

Production of Sm-ELA at S. lividans Construction of S. lividans expressing Sm-ELA Streptomyces antibioticus in Streptomyces antibioticus in Streptomyces lividans. Katz E, Thompson CJ, Hopwood DA. J Gen Microbiol. 1983, 129, 2703 -14.) And E. coli high copy vector pUC19 were ligated to construct actinomycetes-E. Coli shuttle vector pUC702. First, pIJ702 was processed with PstI and SacI, and further smoothed. Next, pUC19 was treated with NdeI and further smoothed. These DNA fragments were ligated, and E. coli JM109 was transformed with the ligation solution. Transformants were selected on LB agar medium containing 100 mg / l ampicillin. A plasmid was extracted from the transformant, and a plasmid in which the multicloning site of pUC19 was present on the PstI recognition site side of pIJ702 was obtained and named pUC702.
We tried to use SSI promoter for Sm-ELA expression in S. lividans. PSI30 (High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus) Primer SSIHindF (5'-CGAAGCTTGCGGGGTGTTCGGAGATGA-3 ') (SEQ ID NO: S-3253. Obata S, Furukubo S, Kumagai I, Takahashi H, Miura K. J Biochem. 1989, 105, 372-6.) 19) and a primer SSIMTGR (5′-CGTTGAACGCGGCCGTGCGGCCCGTGCTCGGTGTGT-3 ′) (SEQ ID NO: 20) were used for PCR amplification. PCR conditions are as follows.

<Preparation of PCR solution>
Pyrobest DNA polymerase (TAKARA) is used.
H ₂ O 18.8 μl, PCR buffer 5 μl, dNTPs 4 μl, Pyrobest DNA polymerase 0.2 μl, 1 mM each primer 10 μl, 20 ng / μl DNA (template) 2 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 5 minutes
Cycle 2 (x25) 98 ° C; 10 seconds, 60 ° C; 0.5 minutes, 72 ° C; 3 minutes
Cycle 3 (x1) 4 ℃; ∞

  The obtained 0.3 kbp DNA fragment contains the SSI promoter region. Next, pUMTG5 (Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus.Kikuchi Y, Date M, Yokoyama K, Umezawa Y, Matsui H. Appl Environ Microbiol. 2003, 69, 358-66.) Was used as a template, and a part of the PMTG (microbial pro-transglutaminase) gene derived from S. mobaraensis was PCR amplified.

As primers, SSIMTGF (5′-GCACCACCGAGCACGGGCCGCACGGCCGCGTTCAACG-3 ′) (SEQ ID NO: 21) and MTGSACR (5′-GACGAGAGCTCTCCGGCGTATGCGCATGGA-3 ′) (SEQ ID NO: 22) were used. PCR conditions are the same as PCR using primers SSIHindF and SSIMTGR.
The obtained 90 bp DNA fragment includes about 50 bp upstream from the start codon of the PMTG gene to about 20 bp downstream from the start codon. Using the thus obtained 0.3 kbp and 90 bp DNA fragments as templates, crossover PCR was performed using the primer SSIHindF and the primer MTGSACR. PCR conditions are as follows.

Pyrobest DNA polymerase (TAKARA) is used.
H ₂ O 18.8 μl, PCR buffer 5 μl, dNTPs 4 μl, Pyrobest DNA polymerase 0.2 μl, 1 mM each primer 10 μl, 20 ng / μl DNA (template) 1 μl each, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 5 minutes
Cycle 2 (x25) 98 ° C; 10 seconds, 60 ° C; 0.5 minutes, 72 ° C; 3 minutes
Cycle 3 (x1) 4 ℃; ∞

The obtained DNA fragment was treated with HindIII and SacI, and ligated with pUC19 treated with HindIII and SacI. Using this ligation solution, E. coli JM109 was transformed, and transformants were selected on an LB agar medium containing 100 mg / l ampicillin. From the transformant, the plasmid of interest in which a DNA fragment in which the SSI promoter and a part of the PMTG gene were linked was inserted into pUC19 was extracted.
The sequence after 20 bp downstream from the start codon of the PMTG gene is obtained from pUITG (WO / 2002/081694) into which the SacI fragment of S. mobaraensis IFO13819 genomic DNA has been inserted. pUITG was treated with SacI and ligated with the above-described plasmid treated with SacI in the same manner. Using this ligation solution, E. coli JM109 was transformed, and transformants were selected on an LB agar medium containing 100 mg / l ampicillin. From the transformant, a target plasmid in which a DNA fragment in which the SSI promoter and the full length of the PMTG gene were linked was inserted into pUC19 was extracted. This plasmid was designated as pSSI-PMTG19, treated with HindIII and EcoRI, and similarly ligated with pUC702 treated with HindIII and EcoRI. Using this ligation solution, E. coli JM109 was transformed, and transformants were selected on an LB agar medium containing 100 mg / l ampicillin. From the transformant, a plasmid having a structure in which a DNA fragment in which the SSI promoter and the full-length PMTG gene were linked was inserted into pUC702 was extracted. This plasmid was designated as pUC702-PMTG and transformed with S. lividans TK21. By transforming the transformant S. lividans TK21 / pUC702-PMTG in a TSB (Tryptic Soy Broth) medium containing 20 mg / l of thiostrepton, expression of PMTG derived from S. mobaraensis is possible (Unpublished).
In order to express Sm-ELA in S. lividans TK24, a DNA fragment in which the SSI promoter and the Sm-ELA gene were linked was inserted into pUC702 to construct pUC702-SmELA. First, using pSSI-PMTG19 as a template, primer Hind-SSI-f (5'-agcccAAGCTTgcggggtgttcggagatgac-3 ') (SEQ ID NO: 23) and primer SSI-SmELA-r (5'-GGAGGGTGGTTCGGGGGCGCTCGCTCATggaaacctgcaactcctttgt24-3) (SEQ ID NO: 24) PCR amplification was carried out to obtain a 0.4 kbp DNA fragment containing the SSI promoter. PCR conditions are as follows.

KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ° C; 10 seconds, 55 ° C; 10 seconds, 68 ° C; 0.5 minutes
Cycle 3 (x1) 68 ℃; 0.5 minutes, 4 ℃; ∞

  Next, using pUC118-SmELA as a template, primer SSI-SmELA-f (5'-gacaaaggagttgcaggtttccATGAGCGAGCGCCCCCGAACCACCCTCC-3 ') (SEQ ID NO: 25) and primer SmELA-EcoRI-r (5'-CGGAATTCTCAGGCGGCGTACACGGTGCGTCCG-3') 26) was used for PCR amplification to obtain a 1.6 kbp DNA fragment containing the Sm-ELA gene. PCR conditions are as follows.

<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ℃; 10 seconds, 55 ℃; 10 seconds, 68 ℃; 2 minutes
Cycle 3 (x1) 68 ℃; 2 minutes, 4 ℃; ∞

  Using the thus obtained 0.4 kbp and 1.6 kbp DNA fragments as templates, crossover PCR was performed using primers Hind-SSI-f and primers SmELA-EcoRI-r. PCR conditions are as follows.

KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 0.5 μl each, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ℃; 10 seconds, 55 ℃; 10 seconds, 68 ℃; 2 minutes
Cycle 3 (x1) 68 ℃; 2 minutes, 4 ℃; ∞

The obtained 2.0 kbp DNA fragment contains a sequence in which the SSI promoter, the upstream part of the PMTG gene and the Sm-ELA gene are linked. This DNA fragment was ligated with pTA2 (TArget Clone-Plus-, Toyobo), and E. coli JM109 was transformed with the ligation solution. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 100 mg / l ampicillin. When a plasmid was extracted from the transformant carrying the target DNA fragment and the nucleotide sequence of the inserted DNA fragment was confirmed, the SSI promoter, the upstream part of the PMTG gene and the Sm-ELA gene were included. The obtained plasmid was treated with HindIII and EcoRI to obtain a 2.0 kbp DNA fragment containing the Sm-ELA gene. This DNA fragment was ligated with HindIII and EcoRI-treated pUC702, and E. coli JM109 was transformed with the ligation solution. A transformant was selected on an LB agar medium containing ampicillin 100 mg / l, and the target plasmid pUC702-SmELA was extracted.
S. lividans TK24 was transformed with pUC702-SmELA. Transformants using R2YE agar medium as an index of resistance to thiostrepton (DNA cloning in Streptomyces: resistance genes from antibiotic-producing species. Thompson CJ, Ward JM, and Hopwood DA. Nature, 1980, 286, 525-27.) Selected above. The strain carrying pUC702-SmELA was named S. lividans TK24 / pUC702-SmELA strain. Similarly, transformation was performed using pUC702, and the resulting strain was designated as S. lividans TK24 / pUC702 strain.

2. Expression of Sm-ELA by S. lividans 30 ml of TSB medium containing 20 mg / l thiostrepton was inoculated with the S. lividans TK24 / pUC702-SmELA strain and cultured with shaking at 30 ° C. for 3 days. 1 ml of the obtained culture solution was added to 75 ml of TSB medium containing 20 mg / l thiostrepton, and main culture was performed while shaking at 30 ° C. Similarly, S. lividans TK24 / pUC702 strain was cultured. Six days after the start of the main culture, the culture solution was taken out, and the cells were collected from the culture solution by centrifugation. 6 g of the obtained wet cells were washed with 10 mM Tris-HCl (pH 7.5) and then suspended in 80 ml of 50 mM Tris-HCl (pH 8.0). The obtained bacterial cells were sonicated and then centrifuged to obtain a soluble fraction and an insoluble fraction. The obtained soluble and insoluble fractions were subjected to SDS-PAGE. As a result, a band corresponding to the molecular weight of Sm-ELA was detected in the soluble fraction of S. lividans TK24 / pUC702-SmELA strain. When the degradation activity of Nε-acetyl-L-lysine in the soluble fraction was measured, it had an activity of 210 U per soluble fraction corresponding to 1 ml of the culture broth.
Further, when Nε-acetyl-L-lysine degradation activity contained in the culture supernatant was measured, it had an activity of 200 U per 1 ml of the culture solution. Even when S. mobaraensis IFO13819 strain was cultured, Sm-ELA activity was detected in the culture supernatant (WO2006 / 088199 A1). In this study, the Sm-ELA gene sequence was determined and the presence of the signal sequence was not observed. Therefore, Sm-ELA in the culture supernatant confirmed when S. mobaraensis IFO13819 strain was cultured was not secreted. There is a possibility that it was leaked outside the cell. When the culture supernatant pH of the S. lividans TK24 / pUC702-SmELA strain was measured, the pH increased as the culture progressed (Table 1), suggesting that Sm-ELA might have leaked into the culture due to lysis It was done.

Table 1. PH change of culture supernatant of S. lividans TK24 / pUC702-SmELA strain

The total Nε-acetyl-L-lysine degradation activity contained in the microbial cells and the culture supernatant is 410 U, and the activity per ml of the culture is 410 U. It was revealed that the activity reached about 300 times the activity of 1.4 U contained in 1 ml. No activity was detected when the soluble fraction and culture supernatant of S. lividans TK24 / pUC702 strain were used.
The activity was measured at Nεacetyl-L-lysine 4 mM, Tris-HCl 50 mM (pH 8.0) at 37 ° C., and the concentration of lysine produced with the decomposition of Nε-acetyl-L-lysine was measured. For the measurement of lysine, the acidic ninhydrin method used in WO2006 / 088199 A1 was used. 1 U was defined as the amount of enzyme that liberates 1 μmol of lysine from Nεacetyl-L-lysine per hour.

Purification of Sm-ELA from S. lividans TK24 / pUC702-SmELA strain
The bacterial cell extract obtained by culturing S. lividans TK24 / pUC702-SmELA strain was filtered using a 0.22 mm filter and dialyzed against 50 mM Tris-HCl (pH 8.0) containing 250 mM NaCl. It was. The obtained solution was applied to a Phenyl Sepharose CL-4B column (15 x 1.6 cm), and NaCl concentration was linearly changed from 250 mM to 0 mM to elute Sm-ELA at a flow rate of 0.25 ml / min. . The fraction having Nε-acetyl-L-lysine degradation activity was collected and dialyzed against 50 mM Tris-HCl (pH 8.0).
The culture supernatant obtained by culturing the S. lividans TK24 / pUC702-SmELA strain was concentrated using Vivaspin 20 (Sartorius AG, Goettingen, Germany). The resulting solution was dialyzed against 25 mM Tris-HCl (pH 8.0) containing 250 mM NaCl and then applied to a Phenyl Sepharose CL-4B column (15 x 1.6 cm) to purify Sm-ELA from the bacterial cell extract. Purification was carried out in the same manner as above.
When the Nε-acetyl-L-lysine degradation activity of the obtained purified Sm-ELA was measured, the purified Sm-ELA from the bacterial cell extract was 2800 U / mg, and the purified Sm-ELA from the culture supernatant was 2500 U. activity of / mg. The results of the present study were almost the same as the activity of 3370 U / mg of purified Sm-ELA obtained from the culture solution of S. mobaraensis strain IFO13819 reported in WO2006 / 088199 A1.
The activity was measured at Nε-acetyl-L-lysine 4 mM, Tris-HCl 50 mM (pH 8.0) at 37 ° C., and the concentration of lysine produced with the decomposition of Nε-acetyl-L-lysine was measured. For the measurement of lysine, the acidic ninhydrin method used in WO2006 / 088199 A1 was used. 1 U was defined as the amount of enzyme that liberates 1 μmol of lysine from Nε-acetyl-L-lysine per hour.
In addition, as a result of analyzing the N-terminal amino acid sequence of Sm-ELA purified from the bacterial cell extract, the SERPR sequence was obtained, which was consistent with the N-terminal amino acid sequence of purified Sm-ELA from the culture solution of S. mobaraensis IFO13819 strain .

Synthesis of Nε-lauroyl-L-lysine by S. lividans cell extract expressing Sm-ELA and washing cells S. lividans TK24 / pUC702-SmELA in 30 ml TSB medium containing 20 mg / l thiostrepton The strain was inoculated and cultured with shaking at 30 ° C. for 3 days. 1.5 ml of the obtained culture solution was added to 75 ml of TSB medium containing 20 mg / l thiostrepton, and the main culture was performed by shaking at 30 ° C. Six days after the start of the main culture, the culture solution was taken out, and the cells were collected from the culture solution by centrifugation and washed with 10 mM Tris-HCl (pH 7.5). The obtained cells were finally suspended in 15 ml of 100 mM Tris-HCl (pH 8.0) and used as washing cells. The obtained washed cells were subjected to ultrasonic disruption and then centrifuged to obtain a supernatant. This supernatant was used as a bacterial cell extract. When the Nε-acetyl-L-lysine degradation activity contained in the bacterial cell extract was measured, it had an activity of 284 U per amount of the bacterial cell extract corresponding to 1 ml of the culture solution. Further, when the activity was measured using the washed cells, 53 U of activity was detected per amount of the washed cells corresponding to 1 ml of the culture solution.
In 20 ml of 100 mM Tris-HCl (pH 7.0), add lauric acid to a final concentration of 25 mM, L-lysine hydrochloride to 500 mM, and the bacterial cell extract to 200 U / ml and stir. The Nε-lauroyl-L-lysine synthesis reaction was carried out. The pH was adjusted with 4 N NaOH to 7.0, and the temperature of the reaction solution was adjusted to 37 ° C. This reaction solution contains a bacterial cell extract of 14 ml of the culture solution. The synthetic reaction using the washed cells as an enzyme source was performed by adding the washed cells obtained from 14 ml of the culture solution to the reaction solution.
During the reaction, the reaction solution was sampled over time, the lauric acid concentration in the reaction solution was quantified by HPLC, and the reaction rate was calculated. As a result, it was found that all lauric acid was consumed after 2 hours when the bacterial cell extract was used, and after 4 hours when the washed bacterial cells were used, and Nε-lauroyl-L-lysine was synthesized. (Figure 4).
HPLC analysis was performed on column YMC-Pack C-8 A-202 (4.6 × 150 mm, YMC), 80% MeOH with mobile phase 0.075% H ₃ PO ₄ , UV 210 nm.

High concentration synthesis reaction of Nε-lauroyl-L-lysine with bacterial extract of S. lividans expressing Sm-ELA First, S. lividans TK24 / pUC702- was added to 30 ml TSB medium containing 20 mg / l thiostrepton. SmELA strain was inoculated and cultured with shaking at 30 ° C. for 3 days. 1.5 ml of the obtained culture solution was added to 75 ml of TSB medium containing 20 mg / l thiostrepton, and main culture was performed while shaking at 30 ° C. Six days after the start of the main culture, the culture solution was taken out, and the cells were collected from the culture solution by centrifugation. The collected cells were washed with 10 mM Tris-HCl (pH 7.5). The obtained bacterial cells (wet weight 7.2 g) were suspended in 40 mL of 100 mM Tris-HCl (pH 8.0) and subjected to ultrasonic crushing. Thereafter, centrifugation was performed, and the obtained supernatant was used as a bacterial cell extract. When the decomposition activity of Nε-acetyl-L-lysine in the bacterial cell extract was measured, it showed an activity of 153 U per soluble fraction corresponding to 1 ml of the culture solution.
In 25 mL of 100 mM Tris-HCl (pH 7) buffer, at a final concentration, L-lysine hydrochloride is 500 mM, lauric acid is 50, 100 and 250 mM, and the bacterial cell extract is 200 U / mL. The Nε-lauroyl-L-lysine synthesis reaction was carried out with stirring. During the reaction, pH was adjusted with 4 N NaOH so as to be 7.0. The temperature of the reaction solution was adjusted to 37 ° C.
The reaction was sampled over time. The reaction rate was calculated by quantifying the lauric acid concentration in the reaction solution by HPLC analysis and the lysine concentration by the acidic ninhydrin method. As a result, when 50 mM and 100 mM lauric acid were used, the reaction yield reached almost 100% in 6 hours and 9 hours, respectively. When 250 mM lauric acid was used, Nε-lauroyl-L-lysine was obtained in a yield of approximately 90% in 24 hours of reaction (FIG. 5).
HPLC analysis was performed with YMC-Pack C-8 A-202, 4.6 × 150 mm (YMC) as the column, 80% MeOH with 0.075% H ₃ PO ₄ as the mobile phase, UV 210 nm.

Production of Sm-ELA by Corynebacterium glutamicum (C. glutamicum) Production of C. glutamicum YDK010 strain expressing Sm-ELA
Using pUC118-SmELA as a template, primer CspB-SmELA-f (5'-tattcaaggagccttcgcctctATGAGCGAGCGCCCCCGAACCACCCTCC-3 ') (sequence number 27) and primer SmELA-delBam-r (5'-GTGCCCCAGCAGGATgCGGTCACCCGGCCG-3') PCR amplification was performed to obtain a 0.3 kbp DNA fragment. PCR conditions are as follows.

<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ℃; 10 seconds, 55 ℃; 10 seconds, 68 ℃; 0.5 minutes
Cycle 3 (x1) 68 ° C; 0.5 min, 4 ° C; ∞

  Similarly, PCR amplification using primer SmELA-delBam-f (5'-CGGCCGGGTGACCGcATCCTGCTGGGGCAC-3 ') (SEQ ID NO: 29) and primer SmELA-Bam-r (5'-cgcggatccTCAGGCGGCGTACACGGTGCGTCCG-3') (SEQ ID NO: 30) To obtain a 1.3 kbp DNA fragment. PCR conditions are as follows.

<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ℃; 10 seconds, 55 ℃; 10 seconds, 68 ℃; 1.5 minutes
Cycle 3 (x1) 68 ° C; 0.5 min, 4 ° C; ∞

  Next, crossover PCR was performed using these two DNA fragments as templates using the primer CspB-SmELA-f and the primer SmELA-Bam-r, and the 1.6 kbp containing the Sm-ELA gene with the BamHI recognition site deleted. A DNA fragment was obtained. PCR conditions are as follows.

<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 0.5 μl each, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ℃; 10 seconds, 55 ℃; 10 seconds, 68 ℃; 2 minutes
Cycle 3 (x1) 68 ℃; 2 minutes, 4 ℃; ∞

PPK4 (Japanese Patent Laid-Open No. 9-322774) was used as an expression vector for Sm-ELA. First, pPKSPTG1 (WO 01/23591) having a structure in which a protein glutaminase gene with a pro-structure part having a CspA signal sequence that is a cspB promoter and a Sec system secretory signal sequence is inserted into pPK4 as a template, primer ScaI-CspB-f ( PCR amplification was performed using 5′-attagctgatttagtacttttcggaggtgt-3 ′) (SEQ ID NO: 31) and primer CspB-r (5′-agaggcgaaggctccttgaataggtatcga-3 ′) (SEQ ID NO: 32). The conditions for PCR are the same as for PCR using primers CspB-SmELA-f and SmELA-delBam-r. The obtained 0.6 kbp DNA fragment contains the promoter region of the C. glutamicum ATCC 13869-derived cspB gene.
Using the 1.6 kbp and 0.6 kbp DNA fragments thus obtained as a template, crossover PCR was performed using primers ScaI-CspB-f and primer SmELA-Bam-r, and a 2.2 kbp DNA fragment was obtained. Obtained. The conditions for PCR are the same as for PCR using primers CspB-SmELA-f and SmELA-Bam-r. This DNA fragment was ligated with pTA2 (TArget Clone-Plus-, Toyobo), and E. coli JM109 was transformed. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 100 mg / l ampicillin. When a plasmid was extracted from a transformant carrying the target DNA fragment and the nucleotide sequence of the inserted DNA fragment was confirmed, the cspB gene promoter region and the Sm-ELA gene were linked as intended. The obtained plasmid was treated with ScaI and BamHI and similarly ligated with pPKSPTG1 treated with ScaI and BamHI. This ligation solution was used to transform E. coli JM109, and transformants were selected on LB agar medium containing 25 mg / l kanamycin. A plasmid having the structure into which the target 2.2 kbp DNA fragment was inserted was extracted and named pPK-SmELA. pPK-SmELA is a plasmid having a structure in which the cspB promoter and the Sm-ELA gene are inserted into pPK4.

  Similarly, a plasmid having a structure in which the Sm-ELA gene to which the cspB promoter and the CspA signal sequence derived from Corynebacterium ammoniagenes (C. ammoniagenes) ATCC 6872 were added was inserted into pPK4. First, using a 1.6 kbp DNA fragment containing the Sm-ELA gene from which the BamHI recognition site had been deleted as a template, primer CspAsig-SmELA-f (5'-GCTGGCCGCACCTGTGGCAACGGCAAGCGAGCGCCCCCGAACCACCCTCC-3 ') and primer SmELA-Bam-r PCR amplification was performed. The conditions for PCR are the same as for PCR using primers CspB-SmELA-f and SmELA-Bam-r. As a result, a 1.6 kbp DNA fragment was obtained. Next, PCR amplification was performed using pPKSPTG1 as a template and a primer ScaI-CspB-f and a primer CspA-r (5'-TGCCGTTGCCACAGGTGCGGCCAGCATGGC-3 ') (SEQ ID NO: 34). The conditions for PCR are the same as for PCR using primers CspB-SmELA-f and SmELA-delBam-r.

The obtained 0.65 kbp DNA fragment contains the promoter region of the Csp. Glutamicum ATCC 13869-derived cspB gene and the C. ammonigenes ATCC 6872-derived CspA signal sequence.
Using the 1.6 kbp and 0.65 kbp DNA fragments thus obtained as a template, crossover PCR was performed using primers ScaI-CspB-f and primer SmELA-Bam-r, and a 2.2 kbp DNA fragment was obtained. Obtained. The conditions for PCR are the same as for PCR using primers CspB-SmELA-f and SmELA-Bam-r.

This DNA fragment was ligated with pTA2 (TArget Clone-Plus-, Toyobo), and E. coli JM109 was transformed. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 100 mg / l ampicillin. A plasmid was extracted from a transformant carrying the target DNA fragment, and the nucleotide sequence of the inserted DNA fragment was confirmed. As a result, it was confirmed that the cspB gene promoter region, the CspA signal sequence and the Sm-ELA gene were linked as intended. The obtained plasmid was treated with ScaI and BamHI and similarly ligated with pPKSPTG1 treated with ScaI and BamHI. This ligation solution was used to transform E. coli JM109, and transformants were selected on LB agar medium containing 25 mg / l kanamycin. A plasmid having the structure into which the target 2.2 kbp DNA fragment was inserted was extracted and named pPKS-SmELA. In pPKS-SmELA, a CspA signal sequence, which is a Sec system secretory signal sequence, is added to the Sm-ELA gene.
Similarly, a plasmid having a structure in which the Sm-ELA gene to which the cspB promoter and the E. coli W3110-derived TorA signal sequence were added was inserted into pPK4. First, a 1.6 kbp DNA fragment containing the Sm-ELA gene with the BamHI recognition site deleted was used as a template. Primer TorAsig-SmELA-f (5'-GTTAACGCCGCGACGTGCGACTGCGAGCGAGCGCCCCCGAACCACCCTCC-3 ') and SEQ PCR amplification was performed. The conditions for PCR are the same as for PCR using primers CspB-SmELA-f and SmELA-Bam-r.
As a result, a 1.6 kbp DNA fragment was obtained. Next, a secretory expression plasmid pPKT-PPG of a protein glutaminase with a pro structure having a TorA signal sequence described in WO 02/081694 is used as a template, and a primer ScaI-CspB-f and a primer TorAsig-r (5'-CGCAGTCGCACGTCGCGGCGTTAAC-3 ') (SEQ ID NO: 36) was used for PCR amplification. The conditions for PCR are the same as for PCR using primers CspB-SmELA-f and SmELA-delBam-r.

The obtained 0.65 kbp DNA fragment contains the promoter region of the Csp. Glutamicum ATCC 13869-derived cspB gene and the E. coli W3110-derived TorA signal sequence.
Using the 1.6 kbp and 0.65 kbp DNA fragments thus obtained as a template, crossover PCR was performed using primers ScaI-CspB-f and primer SmELA-Bam-r, and a 2.2 kbp DNA fragment was obtained. Obtained. The conditions for PCR are the same as for PCR using CspB-SmELA-f and SmELA-Bam-r.

This DNA fragment was ligated with pTA2 (TArget Clone-Plus-, Toyobo), and E. coli JM109 was transformed. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 100 mg / l ampicillin. A plasmid was extracted from a transformant carrying the target DNA fragment, and the nucleotide sequence of the inserted DNA fragment was confirmed. As a result, it was confirmed that the cspB gene promoter region, the TorA signal sequence and the Sm-ELA gene were linked as intended. The obtained plasmid was treated with ScaI and BamHI and similarly ligated with pPKSPTG1 treated with ScaI and BamHI. This ligation solution was used to transform E. coli JM109, and transformants were selected on LB agar medium containing 25 mg / l kanamycin. A plasmid having the structure into which the target 2.2 kbp DNA fragment was inserted was extracted and named pPKT-SmELA. In pPKT-SmELA, a TorA signal sequence, which is a Tat secretion signal sequence, is added to the Sm-ELA gene.
Using the constructed three plasmids pPK-SmELA, pPKS-SmELA and pPKT-SmELA, a YDK010 strain (WO 01/23591) obtained from a mutant strain derived from C. glutamicum ATCC13869 was transformed, and 25 mg / kg of kanamycin was obtained. l Select transformants on CM2G agar medium (yeast extract 10 g, polypeptone 10 g, glucose 5 g, sodium chloride 5 g, DL-methionine 0.2 g, agar 15 g, pH 7.2, 1 L with water) did. The resulting transformants were designated as YDK010 / pPK-SmELA strain, YDK010 / pPKS-SmELA strain, and YDK010 / pPKT-SmELA strain, respectively.

2. Production of C. glutamicum WDK010 strain expressing Sm-ELA 2.1. C. Production of glutamicum WDK010 strain
Genomic DNA was extracted from the AJ12036 strain described in WO02 / 081694. Using the genomic DNA of this AJ12036 strain as a template, PCR amplification was performed using primer YSrpsL5 (5'-AGGTCAGTGGCGAGTTTCTT-3 ') (SEQ ID NO: 37) and primer YSrpsL3 (5'-GGTAGGTAGCGCCACCAACA-3') (SEQ ID NO: 38) A 1 kbp fragment was obtained. PCR conditions are as follows.

Pyrobest DNA polymerase (TAKARA) is used.
H ₂ O 37.8 μl, PCR buffer 5 μl, dNTPs 4 μl, Pyrobest DNA polymerase 0.2 μl, 10 μM each primer 1 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 98 ° C; 5 minutes
Cycle 2 (x30) 98 ℃; 10 seconds, 57 ℃; 0.5 minutes, 72 ℃; 1 minute
Cycle 3 (x1) 4 ℃; ∞

  Using this DNA fragment and the Corynebacterium glutamicum ATCC13869 strain, a gene replacement strain was prepared in the same manner as described in Example 9 of WO02 / 081694. It was confirmed that the obtained gene-substituted strain grew on a CM2G agar medium containing 100 mg / L streptomycin. Further, using this gene replacement strain, a cell surface protein (PS2) gene complete disruption strain was prepared in the same manner as described in Example 9 of WO02 / 081694, and this strain was named WDK010 strain.

2.2. C. Introduction of Sm-ELA Expression Vector into Glutamicum WDK010 Strain WDK010 was transformed with the constructed plasmid pPKT-SmELA, and CMDXB agar medium containing 25 mg / l kanamycin (yeast extract 10 g, polypeptone 10 g , Glucose 5 g, urea 3 g, soybean hydrolyzate 1.2 g nitrogen content, potassium dihydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.4 g, iron (II) sulfate heptahydrate 0.01 g, manganese sulfate (II) Pentahydrate 0.01 g, Biotin 10 mg, Agar 15 g, pH 7.0, 1 L with water) Named.
Transform WDK010 strain using plasmid pVtatABC described in WO05 / 103278, select transformant on CMDXB agar medium containing 5 mg / l chloramphenicol, and name the resulting transformant as WDK010 / pVtatABC strain did.
Transform WDK010 / pVtatABC strain using pPKT-SmELA, select transformant on CMDXB agar medium containing 25 mg / l kanamycin and 5 mg / l chloramphenicol, and transform the resulting transformant to WDK010 It was named / pPKT-SmELA + pVtatABC strain.

3. Expression of Sm-ELA in C. glutamicum
YDK010 / pPK-SmELA strain, YDK010 / pPKS-SmELA strain, YDK010 / pPKT-SmELA strain and WDK010 / pPKT-SmELA strain, each containing CMDXB liquid medium containing 25 mg / l of kanamycin (yeast extract 10 g, polypeptone 10 g , Glucose 5 g, urea 3 g, soybean hydrolyzate 1.2 g nitrogen content, potassium dihydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.4 g, iron (II) sulfate heptahydrate 0.01 g, manganese sulfate (II) Pentahydrate was inoculated into a test tube containing 3 ml of pentahydrate 0.01 g, biotin 10 mg, pH 7.0, 1 L with water) and cultured with shaking at 30 ° C. for 16 hours. 0.15 ml of the obtained culture broth was added to an MMM liquid medium containing 25 mg / l kanamycin (glucose 60 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, soybean hydrolysate 0.1 g nitrogen content, magnesium sulfate heptahydrate Japanese 1 g, DL-methionine 0.15 g, Iron (II) sulfate heptahydrate 0.01 g, Manganese (II) sulfate pentahydrate 0.008 g, Thiamine hydrochloride 0.45 mg, Biotin 0.45 mg, Calcium carbonate 50 g, pH 7.5, 1 L with water) was added to a test tube containing 3 ml, and cultured with shaking at 30 ° C. for 72 hours.

Inoculate one platinum loop of WDK010 / pPKT-SmELA + pVtatABC strain into a test tube containing 3 ml of CMDXB liquid medium containing 25 mg / l kanamycin and 5 mg / l chloramphenicol and incubate at 30 ° C for 16 hours did. 0.15 ml of the obtained culture solution was added to a test tube containing 3 ml of MMM liquid medium containing 25 mg / l kanamycin and 5 mg / l chloramphenicol, and cultured with shaking at 30 ° C. for 72 hours.
The obtained culture broth was centrifuged, the supernatant was collected, and the cells were washed with 20 mM Tris-HCl (pH 7.6). The cells were finally suspended in 3 ml of 20 mM Tris-HCl (pH 7.6) and used as washing cells. The bacterial cell extract was prepared by crushing the washed bacterial cells with a multi-bead shocker (Yasui Kikai) and centrifuging them. Using these culture supernatants, washed bacterial cells, and bacterial cell extracts, the degradation activity of Nε-acetyl-L-lysine was measured. An activity of 1030 U per cell extract corresponding to 1 ml of the culture solution was confirmed in the cell extract of the YDK010 / pPKS-SmELA strain. This reaches about 740 times the activity 1.4 U contained in 1 ml of the culture supernatant of S. mobaraensis strain IFO13819 reported in WO2006 / 088199 A1. Moreover, 69.8 U of activity per ml was detected from the culture supernatant of WDK010 / pPKT-SmELA + pVtatABC strain, and it was considered that Sm-ELA was secreted by the Tat secretion system. These results are summarized in Table 2.

N.D. 検出限界未満 Table 2. Degradation activity of Nε-acetyl-L-lysine in culture supernatant, washed cells and cell extracts

Below ND detection limit

  The activity was measured at Nε-acetyl-L-lysine 4 mM, Tris-HCl 50 mM (pH 8.0), CoCl2 0.1 mM, at 37 ° C, and the concentration of lysine produced with Nε-acetyl-L-lysine degradation was determined. It was measured. For the measurement of lysine, BF-5 and a lysine electrode (Oji Scientific Instruments) were used. 1 U was defined as the amount of enzyme that liberates 1 μmol of lysine from Nε-acetyl-L-lysine per hour.

4). Production of C. glutamicum WDK010 strain co-expressing Sm-ELA and TatABC
From the tatA gene (cg1685, GeneID: 3343772), tatC gene (cg1684, GeneID: 3343771), and tatB gene (cg1273, GeneID: 3345252) derived from the C. glutamicum ATCC13032 strain, the primer XbaI-TatA-f (5'- gctctagaTTTGGAGTAGACCACATGTCCCTCG-3 ') (SEQ ID NO: 39), Primer TatC-TatB-r (5'-TAGAAAACATCAGACCGGTCTTTCACTAGAGCACGTCACCGAAGTCGGCG-3') (SEQ ID NO: 40), Primer TatC-TatB-f (5'-CGCCGACTTGGTGTGTATGTGTATGTGTGTGTGTTGTG 41) and the primer TatB-Xba-r (5′-gctctagaCTAAATAATATCGGTCCAAGAGACG-3 ′) (SEQ ID NO: 42) were designed.

PCR amplification was performed using the genomic DNA of C. glutamicum ATCC13869 strain as a template and using the primer XbaI-TatA-f and the primer TatC-TatB-r to obtain a DNA fragment of 1.5 kbp. This DNA fragment contains the sequences of the tatA gene and the tatC gene. PCR conditions are as follows.
<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ℃; 10 seconds, 55 ℃; 10 seconds, 68 ℃; 1.5 minutes
Cycle 3 (x1) 68 ℃; 1.5 minutes, 4 ℃; ∞

PCR amplification was performed using genomic DNA of C. glutamicum ATCC13869 strain as a template and primers TatC-TatB-f and primer TatB-Xba-r to obtain a 0.5 kbp DNA fragment. This DNA fragment contains the sequence of the tatB gene. PCR conditions are as follows.
<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ℃; 10 seconds, 55 ℃; 10 seconds, 68 ℃; 0.5 minutes
Cycle 3 (x1) 68 ° C; 0.5 min, 4 ° C; ∞

Crossover PCR was performed using the two 1.5 kbp and 0.5 kbp DNA fragments thus obtained as a template, using primers XbaI-TatA-f and primer TatB-Xba-r, and a 2 kbp DNA fragment was obtained. Obtained. This DNA fragment includes the sequences of the tatA gene, the tatC gene, and the tatB gene. PCR conditions are as follows.
<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 0.5 μl each, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ℃; 10 seconds, 55 ℃; 10 seconds, 68 ℃; 2 minutes
Cycle 3 (x1) 68 ℃; 2 minutes, 4 ℃; ∞

  A 2 kbp DNA fragment obtained by crossover PCR was treated with XbaI. The plasmid pPKT-SmELA was similarly treated with XbaI and further with BAP. The two DNA fragments were ligated and E. coli JM109 was transformed. Transformants were selected on LB agar medium containing 25 mg / l kanamycin. A plasmid having a structure in which the target 2 kbp DNA fragment was inserted in the same direction as Sm-ELA was extracted and named pPKT-SmELA-tatABC. pPKT-SmELA-tatABC is a plasmid having a structure in which a DNA fragment in which a cspB promoter, a TorA signal sequence, an Sm-ELA gene, a tatA gene, a tatC gene, and a tatB gene are linked in this order is inserted into pPK4.

  C. glutamicum WDK010 strain was transformed with the constructed plasmid pPKT-SmELA-tatABC, CMDXB agar medium containing 25 mg / l kanamycin (yeast extract 10 g, polypeptone 10 g, glucose 5 g, urea 3 g, Soybean hydrolyzate Nitrogen content 1.2 g, potassium dihydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.4 g, iron (II) sulfate heptahydrate 0.01 g, manganese (II) sulfate pentahydrate 0.01 g Biotin 10 mg, Agar 15 g, pH 7.0, 1 L with water), and the resulting transformant was named WDK010 / pPKT-SmELA-tatABC strain.

5. Co-expression of Sm-ELA and TatABC in C. glutamicum WDK010 strain
WDK010 / pPKT-SmELA strain, WDK010 / pPKT-SmELA + pVtatABC strain, and WDK010 / pPKT-SmELA-tatABC strain, respectively, CMDXB liquid medium (yeast extract 10 g, polypeptone 10 g, glucose 5 g, urea 3 g, Soy hydrolyzate Nitrogen content 1.2 g, potassium dihydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.4 g, iron (II) sulfate heptahydrate 0.01 g, manganese (II) sulfate pentahydrate 0.01 g Into a test tube containing 3 ml of biotin (10 mg, pH 7.0, 1 L with water), one platinum loop was inoculated and cultured with shaking at 25 ° C. for 16 hours. When culturing WDK010 / pPKT-SmELA and WDK010 / pPKT-SmELA-tatABC strains, add 25 mg / l kanamycin to the medium, and when culturing WDK010 / pPKT-SmELA + pVtatABC strains, 25 mg / kanamycin l and chloramphenicol 5 mg / l were added to the medium.

The obtained culture broth was centrifuged, and the supernatant was collected. Using these culture supernatants, the degradation activity of Nε-acetyl-L-lysine was measured. An activity of 0.751 U per ml was detected from the culture supernatant of the WDK010 / pPKT-SmELA strain, and an activity of 73.6 U per ml was detected from the culture supernatant of the WDK010 / pPKT-SmELA + pVtatABC strain. 125 U activity per ml was detected from the culture supernatant of the WDK010 / pPKT-SmELA-tatABC strain. From this, it was possible to express Sm-ELA and TatABC on one plasmid, and it was considered that Tat secretion expression of Sm-ELA was efficiently performed by co-expressed TatABC.
The activity was measured at Nε-acetyl-L-lysine 4 mM, Tris-HCl 50 mM (pH 8.0), CoCl ₂ 0.1 mM, at 37 ° C. Was measured.
For the measurement of lysine, BF-5 and a lysine electrode (Oji Scientific Instruments) were used. 1 U was defined as the amount of enzyme that liberates 1 μmol of lysine from Nε-acetyl-L-lysine per hour.

Synthesis of Nε-lauroyl-L-lysine by cell extract of C. glutamicum expressing Sm-ELA 1 YDK010 / pPKS-SmELA strain was added to a test tube containing 3 ml of CMDXB liquid medium containing 25 mg / l kanamycin. Platinum ears were inoculated and cultured with shaking at 30 ° C. for 16 hours. 10 ml of the thus obtained culture solution was added to a 500 ml Sakaguchi flask containing 200 ml of MMM liquid medium containing 25 mg / l kanamycin, and cultured with shaking at 30 ° C. for 48 hours. The cells were collected from the culture solution obtained by centrifugation and washed with 20 mM Tris-HCl (pH 7.6). The cells were finally suspended in 50 ml of 20 mM Tris-HCl (pH 7.6) and used as washing cells. The washed cells were crushed and centrifuged, and the supernatant was used as a cell extract.
Final concentration is 250 mmol / l lauric acid (reaction condition 1) or 500 mmol / l (reaction condition 2), L-lysine hydrochloride 500 mM, Tris-HCl 50 mM, CoCl ₂ 1 mM, cell extract 180 60 ml of the reaction solution was prepared so as to be U / ml, and stirred at 2000 rpm using a 200 ml minijar fermenter to carry out Nε-lauroyl-L-lysine synthesis reaction. The pH was adjusted to 17.0 with 1 N NaOH, and the temperature of the reaction solution was adjusted to 37 ° C.
Sampling was performed over time from the reaction solution, and the lysine concentration in the reaction solution was measured using BF-5 and a lysine electrode (Oji Scientific Instruments) (FIG. 6). The lysine concentration at the start of the reaction was 100%.
After 24 hours, the entire reaction solution was recovered and centrifuged. After removing the supernatant, methanol, water and 6 N NaOH were added to the precipitate to dissolve the precipitate. 6N HCl was added to this solution to adjust to pH 6.5-7.5, and Nε-lauroyl-L-lysine was precipitated. This suspension was subjected to suction filtration using filter paper. When the powder on the filter paper was dried and weighed, it was 5.44 g under reaction condition 1 and 9.82 g under reaction condition 2. The Nε-lauroyl-L-lysine purity in these powders was measured by HPLC and found to be 76.4% and 80.9%, respectively, so that Nε-lauroyl-L-lysine was 4.16 g (12.7 mmol) and 7.94 g (24.2 mmol) It was clear that it was obtained. Yields were 84.4% and 80.6%, respectively, based on lauric acid.
HPLC analysis was performed with column YMC-Pack A-312 ODS, 6.0 × 150 mm (YMC), moving bed 0.1 M NaH 2 PO 4 / MeOH = 2/8, UV 210 nm.

Synthesis of Nε-lauroyl-L-lysine by culture supernatant of C. glutamicum secreting and expressing Sm-ELA Preseed liquid medium containing 25 mg / l kanamycin (yeast extract 10 g, polypeptone 10 g, glucose 5 g, soybean Hydrolyzate 0.1 g of nitrogen, 0.02 g of DL-methionine, pH 7.2, make up to 1 L with water) 1 volume of WDK010 / pPKT-SmELA-tatABC strain in a 500 ml Sakaguchi flask test tube containing 50 ml Inoculated separately, and cultured with shaking at 30 ° C. for 16 hours. 0.3 ml of the thus obtained culture broth was added to a seed liquid medium containing 25 mg / l kanamycin (glucose 20 g, ammonium sulfate 3 g, potassium dihydrogen phosphate 1.5 g, soybean hydrolyzate 0.2 g as nitrogen content, Magnesium sulfate heptahydrate 1 g, DL-methionine 0.15 g, Iron (II) sulfate heptahydrate 0.01 g, Manganese (II) sulfate pentahydrate 0.01 g, Thiamine hydrochloride 0.45 mg, Biotin 0.45 mg, Dis Home GD-113K (Nippon Yushi Co., Ltd.) 0.1 ml, pH 6.2, made up to 1 L with water) was added to a 1000 ml jar fermenter containing 300 ml, and seed culture was started. The seed culture was performed at 31 ° C., aeration 1/1 vvm, agitation 500 rpm, pH adjusted to 6.2 with ammonia until glucose was consumed. 15 ml of the culture broth thus obtained was added to a main liquid medium containing 25 mg / l kanamycin (glucose 120 g, ammonium sulfate 3 g, potassium dihydrogen phosphate 1.5 g, soybean hydrolyzate 0.2 g as nitrogen content, Magnesium sulfate heptahydrate 3 g, DL-methionine 0.15 g, Iron (II) sulfate heptahydrate 0.03 g, Manganese (II) sulfate pentahydrate 0.03 g, Zinc sulfate heptahydrate 0.02 g, Cobalt chloride (II) Hexahydrate 0.008 g, Calcium chloride 2 g, Thiamine hydrochloride 0.45 mg, Biotin 0.45 mg, Dishome GD-113K (Nippon Yushi Co., Ltd.) 0.1 ml, pH 6.2, 0.95 L with water) 285 It was added to a 1000 ml jar fermenter containing ml, and main culture was started. The main culture was performed at 26 ° C., aeration 1/1 vvm, with ammonia at pH 6.9 and until glucose was consumed. Stirring was controlled at 500 rpm or higher so that the dissolved oxygen concentration was 5% or higher.

The obtained culture solution was centrifuged, and the supernatant was collected. The degradation activity of Nε-acetyl-L-lysine in this culture supernatant was measured. As a result, 3640 U of activity was detected per 1 ml of culture supernatant. This reaches about 2600 times the activity 1.4 U contained in 1 ml of the culture supernatant of S. mobaraensis strain IFO13819 reported in WO2006 / 088199 A1.
The activity was measured at Nε-acetyl-L-lysine 40 mM, Tris-HCl 50 mM (pH 8.0), CoCl ₂ 0.1 mM, 37 ° C, and the concentration of lysine produced with Nε-acetyl-L-lysine degradation. Was measured.
For the measurement of lysine, BF-5 and a lysine electrode (Oji Scientific Instruments) were used. 1 U was defined as the amount of enzyme that liberates 1 μmol of lysine from Nε-acetyl-L-lysine per hour.
Prepare 100 ml of the reaction solution so that the final concentration is 300 mmol / l sodium laurate, 300 mM L-lysine hydrochloride, 0.1 mM CoCl ₂ 0.1 mM, 10% methanol, and 300 U / ml culture supernatant. The mixture was stirred at 800 rpm using a mini jar fermenter to carry out Nε-lauroyl-L-lysine synthesis reaction. The pH was adjusted to 17.0 with 1 N NaOH, and the temperature of the reaction solution was adjusted to 45 ° C.
Sampling was performed from the reaction solution over time (0.5, 1, and 2 hours later), and the Nε-lauroyl-L-lysine concentration in the reaction solution was measured by HPLC (FIG. 7).
The entire reaction solution was recovered together with Nε-lauroyl-L-lysine precipitated after 10 hours. 20 ml of 6 N HCl and methanol were added to the reaction solution, and the volume was made up to 500 ml. The precipitated Nε-lauroyl-L-lysine was dissolved. The Nε-lauroyl-L-lysine concentration in this solution was measured by HPLC (FIG. 7, after 10 hours).
As a result, 291 mmol / l Nε-lauroyl-L-lysine was produced in the reaction solution 10 hours after the start of the reaction, and Nε-lauroyl-L-lysine even when the culture supernatant was used as the enzyme source. It was revealed that the synthesis can be performed efficiently. The yield was 97% based on lauric acid.
HPLC analysis was performed with column YMC-Pack A-312, 6.0 × 150 mm, S-5, 12 nm (YMC), moving bed 0.1 M NaH ₂ PO ₄ / MeOH = 2/8, UV 210 nm.

Cloning of S. sericolor Nε-acyl-L-lysine specific aminoacylase (Sc-ELA) gene
The amino acid sequence of Sm-ELA was determined by homology search using NCBI protein-protein BLAST, hypothetical protein SCO1424 (S.coelicolor A3 (2), NP_625706) and 80%, hypothetical protein SAV_6922 (S. avermitilis MA- 4680, NP_828098) and 80%, conserved hypothetical protein (S. ambofaciens ATCC 23877, CAJ90369) and 79%, conserved hypothetical protein (S. griseus subsp. Griseus NBRC 13350, YP_001827620) and 78% homology It became. Thus, it was examined whether the hypothetical protein SCO1424 derived from S. sericolor A3 (2) has Nε-acyl-L-lysine-specific aminoacylase activity in the same manner as Sm-ELA.
S. sericolor sA3 (2) cells grown on YMPG agar medium (glucose 10 g, polypeptone 5 g, yeast extract 3 g, malt extract 3 g, agar 15 g, pH 7.0, 1 L with water) PCR using primer Nde-ScELA-f (5'-catATGTCCATGAGTGAGTCCACCACCCCG-3 ') (SEQ ID NO: 43) and primer ScELA-Hind-r (5'- aagctTCACTCGCCCGGCCGTACGAAGACC-3') (SEQ ID NO: 44) Amplification was performed. PCR conditions are as follows.

<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 33 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 5 minutes
Cycle 2 (x25) 98 ℃; 10 seconds, 55 ℃; 10 seconds, 68 ℃; 2 minutes
Cycle 3 (x1) 68 ℃; 2 minutes, 4 ℃; ∞

  The resulting 1.6 kbp DNA fragment was ligated with pTA2 (TArget Clone-Plus-, Toyobo) to transform E. coli JM109. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM IPTG, 100 mg / l ampicillin. When a plasmid was extracted from the transformant carrying the target DNA fragment and the nucleotide sequence of the inserted DNA fragment was confirmed, it contained the ORF full length of the Sc-ELA gene and the added NdeI and HindIII recognition sites. Became clear. The resulting plasmid was named pTA2-ScELA. The nucleotide sequence of ORF encoding Sc-ELA is shown in SEQ ID NO: 4, and the amino acid sequence of encoded Sc-ELA is shown in SEQ ID NO: 5.

  pTA2-ScELA was treated with NdeI and HindIII to obtain a 1.6 kbp DNA fragment containing the Sc-ELA gene. JP 2005278468 A (Japanese Patent Laid-Open No. 2005-278468), a method for producing optically active amino acids, the vector pTrp2 described in Example 1, column 0064 is treated with NdeI and HindIII, and a DNA fragment containing the Sc-ELA gene Ligated and transformed into E. coli JM109. Transformants were selected on LB agar medium containing 100 mg / l ampicillin. The plasmid having the structure in which the Sc-ELA gene was inserted into pTrp2 was named pTrp2-ScELA, and the transformant carrying this plasmid was named E. coli JM109 / pTrp2-ScELA strain.

Production of Sc-ELA by E. coli
One platinum loop of E. coli JM109 / pTrp2-ScELA was inoculated into a 500 ml Sakaguchi flask containing 50 ml of TB medium containing 100 mg / l ampicillin, and cultured with shaking at 37 ° C. for 16 hours.
The cells were collected from the obtained culture broth by centrifugation and washed with 20 mM Tris-HCl (pH 7.6). The cells were finally suspended in 10 ml of 20 mM Tris-HCl (pH 7.6) and used as washing cells. When the degrading activity of Nε-acetyl-L-lysine was measured using the washed cells, it had an activity of 1.2 U per washed cell corresponding to 1 ml of the culture solution. Similarly, when the washed cells of E. coli JM109 / pTrp2-SmELA strain were used, 10 U activity was confirmed per washed cell corresponding to 1 ml of the culture solution. Higher activity was confirmed compared to the expression of Sm-ELA using E.coli, but it is thought to be an activity improvement effect by adding 0.1 mM of CoCl ₂ (Sm-ELA may be improved by CoCl _2. I know, WO2006 / 088199). No activity was detected when washed cells of E. coli JM109 / pUC18 strain were used.
The activity was measured at Nε-acetyl-L-lysine 4 mM, Tris-HCl 50 mM (pH 8.0), CoCl ₂ 0.1 mM at 37 ° C, and the concentration of lysine produced with Nε-acetyl-L-lysine degradation. Was measured. For the measurement of lysine, BF-5 and a lysine electrode (Oji Scientific Instruments) were used. 1 U was defined as the amount of enzyme that liberates 1 μmol of lysine from Nε-acetyl-L-lysine per hour.

Production of Sc-ELA by C. glutamicum
Using pTA2-ScELA as a template, primer CspB-ScELA-f (5'-ttcaaggagccttcgcctctATGTCCATGAGTGAGTCCAC-3 ') (SEQ ID NO: 45) and primer ScELA-Bam-r (5'-CGACtctagaggatccTCACTCGCCCGGCCGCGGAGA-3') (SEQ ID NO: 46) PCR amplification was performed to obtain a 1.6 kbp DNA fragment containing the Sc-ELA gene. PCR conditions are as follows.

<Preparation of PCR solution>
KOD -Plus- Ver.2 (Toyobo) is used.
H ₂ O 32 μl, PCR buffer 5 μl, dNTPs 5 μl, MgSO ₄ 3 μl, KOD-Plus- 1 μl, 10 μM each primer 1.5 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 94 ° C; 2 minutes
Cycle 2 (x25) 98 ° C; 10 seconds, 55 ° C; 10 seconds, 68 ° C; 2 μ
Cycle 3 (x1) 68 ℃; 2 minutes, 4 ℃; ∞

Using the obtained DNA fragment, the cspB promoter and the Sc-ELA gene were ligated by crossover PCR in the same manner as in the construction of pPK-SmELA to obtain a 2.2 kbp DNA fragment. This was treated with ScaI and BamHI and similarly ligated with pPKSPTG1 treated with ScaI and BamHI. This ligation solution was used to transform E. coli JM109, and transformants were selected on LB agar medium containing 25 mg / l kanamycin. A plasmid having a structure in which the target 2.2 kbp DNA fragment was inserted was extracted and named pPK-ScELA. In pPK-ScELA, the cspB promoter and the Sc-ELA gene are inserted into pPK4.
Similarly, a plasmid was constructed in which the Sc-ELA gene to which the cspB promoter and the CspA signal sequence derived from Corynebacterium ammoniagenes (C. ammoniagenes) ATCC 6872 were added was inserted into pPK4. First, PCR amplification was performed using pTA2-ScELA as a template and a primer CspAsig-ScELA-f (5′-CCGCACCTGTGGCAACGGCATCCATGAGTGAGTCCACCAC-3 ′) (SEQ ID NO: 47) and a primer ScELA-Bam-r. The conditions for PCR are the same as for PCR using CspB-ScELA-f and ScELA-Bam-r.

  As a result, a 1.6 kbp DNA fragment was obtained. Using the obtained DNA fragment, a 2.2 kbp DNA fragment in which the cspB promoter, CspA signal sequence and Sc-ELA gene were linked was obtained by crossover PCR in the same manner as in the construction of pPKS-SmELA. The conditions for PCR are the same as for PCR using CspB-ScELA-f and ScELA-Bam-r.

This DNA fragment was treated with ScaI and BamHI, and pPKS-ScELA was constructed in the same manner as pPK-ScELA. In pPKS-ScELA, a CspA signal sequence, which is a Sec system secretory signal sequence, is added to the Sc-ELA gene.
Similarly, a plasmid having a structure in which the Sc-ELA gene to which the cspB promoter and the E. coli W3110-derived TorA signal sequence were added was inserted into pPK4. First, PCR amplification was performed using pTA2-ScELA as a template and the primer TorAsig-ScELA-f (5′-CGCCGCGACGTGCGACTGCGTCCATGAGTGAGTCCACCAC-3 ′) (SEQ ID NO: 48) and the primer ScELA-Bam-r. The conditions for PCR are the same as for PCR using CspB-ScELA-f and ScELA-Bam-r.

  As a result, a 1.6 kbp DNA fragment was obtained. Using the obtained DNA fragment, a 2.2 kbp DNA fragment to which the cspB promoter, TorA signal sequence and Sc-ELA gene were linked was obtained by crossover PCR in the same manner as in the construction of pPKT-SmELA. PCR conditions are the same as when CspB-ScELA-f and ScELA-Bam-r are used.

This DNA fragment was treated with ScaI and BamHI, and pPKT-ScELA was constructed in the same manner as pPK-ScELA. In pPKT-ScELA, a TorA signal sequence, which is a Tat secretion signal sequence, is added to the Sc-ELA gene.
Using the constructed three plasmids pPK-ScELA, pPKS-ScELA and pPKT-ScELA, the YDK010 strain (WO 01/23591) obtained from a mutant strain derived from C. glutamicum ATCC13869 was transformed, and 25 mg / kg of kanamycin was obtained. l Transformants were selected on CM2G agar medium containing l. The obtained transformants were named YDK010 / pPK-ScELA strain, YDK010 / pPKS-ScELA strain, and YDK010 / pPKT-ScELA strain, respectively. In addition, the WDK010 strain was transformed with pPKT-ScELA, the transformant was selected on a CMDXB agar medium containing 25 mg / l kanamycin, and the resulting transformant was named the WDK010 / pPKT-SmELA strain. . Further, the WDK010 / pVtatABC strain was transformed with pPKT-ScELA, and the transformant was selected on a CMDXB agar medium containing 25 mg / l kanamycin and 5 mg / l chloramphenicol. It was named / pPKT-ScELA + pVtatABC strain.

2. Expression of Sc-ELA in C. glutamicum Similar to the expression of Sm-ELA using C. glutamicum described above, YDK010 / pPK-ScELA strain, YDK010 / pPKS-ScELA strain, YDK010 / pPKT-ScELA strain, WDK010 / The pPKT-ScELA strain and WDK010 / pPKT-ScELA + pVtatABC strain were cultured, and the degradation activity of Nε-acetyl-L-lysine was measured. As a result, an activity of 272 U per cell extract corresponding to 1 ml of the culture was confirmed from the cell extract of the YDK010 / pPKS-ScELA strain (Table 3).
In addition, the degradation activity of Nα-acetyl-L-lysine was measured using the bacterial cell extract of YDK010 / pPKS-ScELA strain. As a result, compared to the degradation activity of Nε-acetyl-L-lysine, Nα-acetyl-L-lysine degradation activity is as low as that of Sm-ELA, and Sc-ELA is Nε-acyl-L, similar to Sm-ELA. -It was considered to be a lysine-specific aminoacylase.

N.D. 検出限界未満 Table 3. Production of Sc-ELA by each strain of C. glutamicum

Below ND detection limit

Synthesis of Nε-lauroyl-L-lysine by cell extract of C. glutamicum expressing Sc-ELA Went. In addition, Nε-lauroyl-L-lysine synthesis reaction was performed using a bacterial cell extract of YDK010 / pPKS-SmELA.
Prepare 60 ml of the reaction solution so that the final concentration is 25 mM lauric acid, 500 mM L-lysine hydrochloride, 50 mM Tris-HCl, 1 mM CoCl ₂ and 30 U / ml bacterial cell extract. Nε-lauroyl-L-lysine synthesis reaction was carried out using a jar fermenter at 1000 rpm with stirring. The pH was adjusted to 17.0 with 1 N NaOH, and the temperature of the reaction solution was adjusted to 37 ° C.
Sampling was carried out over time from the reaction solution, and lauric acid and Nεlauroyl-L-lysine in the reaction solution were quantified by HPLC. As a result, it was confirmed that Nε-lauroyl-L-lysine was produced with a decrease in lauric acid (FIG. 8). From this, it was clarified that Sc-ELA can be used for the synthesis of Nε-lauroyl-L-lysine in the same manner as Sm-ELA.
HPLC analysis was performed with column YMC-Pack C-8, 4.6 × 150 mm (YMC), moving bed 0.1 M NaH ₂ PO ₄ / MeOH = 2/8, UV 210 nm.

Sequence number 6-48: PCR primer

Claims (6)

DNA of any of the following (a) to (e):
(A) a DNA encoding a Nε-acyl-L-lysine specific aminoacylase having the amino acid sequence set forth in SEQ ID NO: 2;
(B) In the amino acid sequence shown in SEQ ID NO: 2, it has an amino acid sequence in which one or more amino acid residues are inserted, added, deleted or substituted, and is specific to Nε-acyl-L-lysine A DNA encoding a protein having an active aminoacylase activity;
(C) DNA having the nucleotide sequence set forth in SEQ ID NO: 1 or 3;
(D) a DNA that hybridizes with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1 or 3 under a stringent condition and encodes a protein having Nε-acyl-L-lysine-specific aminoacylase activity; Or
(E) encoding a protein having a nucleotide sequence of 95% or more homology with the nucleotide sequence set forth in SEQ ID NO: 1 or 3 and having an activity of a Nε-acyl-L-lysine specific aminoacylase DNA.   A recombinant DNA comprising the DNA of claim 1.   A transformant transformed with the recombinant DNA according to claim 2.   A Nε-acyl-L-lysine-specific amino acid characterized by culturing the transformant according to claim 3 to produce a protein having Nε-acyl-L-lysine-specific aminoacylase activity and recovering the protein. A method for producing a protein having acylase activity. (A) DNA encoding a Nε-acyl-L-lysine specific aminoacylase having the amino acid sequence set forth in SEQ ID NO: 2 or 5;
(B) in the amino acid sequence described in SEQ ID NO: 2 or 5, having an amino acid sequence in which one or more amino acid residues are inserted, added, deleted or substituted, and Nε-acyl-L- DNA encoding a protein having a lysine-specific aminoacylase activity;
(C) DNA having the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;
(D) encodes a protein that hybridizes under stringent conditions with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1, 3 or 4 and has an activity of an Nε-acyl-L-lysine-specific aminoacylase DNA to do; or
(E) encoding a protein having a nucleotide sequence of 95% or more of homology with the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4 and having Nε-acyl-L-lysine specific aminoacylase activity DNA to
Nε-, characterized by culturing a transformant transformed with a recombinant DNA containing any of the above, producing a protein having Nε-acyl-L-lysine-specific aminoacylase activity, and recovering the protein. A method for producing a protein having acyl-L-lysine-specific aminoacylase activity. Nε-lauroyl produced by reacting L-lysine and lauric acid in the presence of a cell transformed with any of the following DNAs (a) to (e), a treated product of the cell, or a culture solution of the cell: A method for producing Nε-lauroyl-L-lysine comprising isolating -L-lysine:
(A) DNA encoding a Nε-acyl-L-lysine specific aminoacylase having the amino acid sequence set forth in SEQ ID NO: 2 or 5;
(B) in the amino acid sequence described in SEQ ID NO: 2 or 5, having an amino acid sequence in which one or more amino acid residues are inserted, added, deleted or substituted, and Nε-acyl-L- DNA encoding a protein having lysine-specific aminoacylase activity;
(C) DNA having the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;
(D) encodes a protein that hybridizes under stringent conditions with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1, 3 or 4 and has an activity of an Nε-acyl-L-lysine-specific aminoacylase DNA to do; or
(E) DNA encoding a protein having 95% or more homology with the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4 and having Nε-acyl-L-lysine-specific aminoacylase activity.

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