WO2010067871A1 - AMINOACYLASE SPÉCIFIQUE DE LA Nε-ACYL-L-LYSINE - Google Patents

AMINOACYLASE SPÉCIFIQUE DE LA Nε-ACYL-L-LYSINE Download PDF

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WO2010067871A1
WO2010067871A1 PCT/JP2009/070769 JP2009070769W WO2010067871A1 WO 2010067871 A1 WO2010067871 A1 WO 2010067871A1 JP 2009070769 W JP2009070769 W JP 2009070769W WO 2010067871 A1 WO2010067871 A1 WO 2010067871A1
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lysine
dna
acyl
seq
protein
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PCT/JP2009/070769
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Japanese (ja)
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康彰 高倉
一弘 中西
俊一 鈴木
式希 丹尾
真友子 是石
維克 今村
洋行 今中
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味の素株式会社
国立大学法人岡山大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the present invention relates to a DNA encoding a novel N ⁇ -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 N ⁇ -acyl-L-lysine specific aminoacylase using a transformant and a method for producing N ⁇ -acyl-L-lysine.
  • N ⁇ -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 pollution-free 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 surfactants for cosmetics. 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.
  • Streptomyces mobaraensis produces an enzyme that specifically and efficiently acylates the ⁇ -amino group of lysine, purified the enzyme, and reported some of its properties.
  • WO 2006/088199 Furthermore, the present inventors have also confirmed that N ⁇ -lauroyl-L-lysine can be specifically synthesized from L-lysine hydrochloride and lauric acid by the above-mentioned enzyme derived from Streptomyces mobaraensis (WO 2006 / 088199).
  • the present invention provides a DNA encoding a N ⁇ -acyl-L-lysine specific aminoacylase.
  • the present invention provides a highly efficient N ⁇ -acyl-L-lysine-specific aminoacylase that efficiently synthesizes N ⁇ -acyl-L-lysine, which is used as a flour meal material, using L-lysine as a starting material.
  • the present invention provides a method for efficiently producing N ⁇ -acyl-L-lysine.
  • the present invention includes the following. (1) N ⁇ -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 N ⁇ -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 N ⁇ -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 N ⁇ -acyl-L-
  • a method for producing a protein having acyl-L-lysine-specific aminoacylase activity is produced by reacting in the presence of a cell transformed with the DNA of any one of (a) to (e) below, a treated product of the cell or a culture solution of the cell.
  • a process for producing N ⁇ -lauroyl-L-lysine comprising isolating N ⁇ -lauroyl-L-lysine;
  • A DNA encoding a N ⁇ -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 N ⁇ -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 N ⁇ -acyl-L-lysine-specific aminoacylase activity DNA; or
  • E DNA encoding a protein having 95% or more homology with the
  • FIG. 1A-E shows the S. movalens genomic region encoding Sm-ELA and its 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. 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 N ⁇ -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 N ⁇ -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 N ⁇ -lauroyl-L-lysine.
  • the horizontal axis represents the reaction time
  • the vertical axis represents the lauric acid yield of N ⁇ -lauroyl-L-lysine calculated from the residual lauric acid and residual lysine.
  • the white symbols indicate the N ⁇ -lauroyl-L-lysine yield calculated from the residual amount of lysine
  • the black symbols indicate the N ⁇ -lauroyl-L-lysine yield calculated from the residual amount of lauric acid.
  • Black circles and white circles are the reactions when using 50 mM lauric acid
  • black triangles and white triangles are when using 100 mM lauric acid
  • black squares and white squares are when using 250 mM lauric acid. Represents.
  • FIG. 6 shows the time course of N ⁇ -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 N ⁇ -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 N ⁇ -lauroyl-L-lysine (LL: N ⁇ -lauroyl-L-lysine).
  • FIG. 8 shows the time course of N ⁇ -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 N ⁇ -lauroyl-L-lysine (white and black circles) or consumed lauric acid (white and black triangles) (LL: N ⁇ -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 N ⁇ -acyl-L-lysine-specific aminoacylase that specifically acylates the ⁇ -amino group of L-lysine to produce N ⁇ -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 N ⁇ -acyl-L-lysine.
  • N ⁇ -acyl-L-lysine specific aminoacylase since the secretory amount of N ⁇ -acyl-L-lysine specific aminoacylase in actinomycetes is extremely small, a method for preparing a large amount of the N ⁇ -acyl-L-lysine specific aminoacylase is further desired. It was. Furthermore, the present inventors have clarified a N ⁇ -acyl-L-lysine specific aminoacylase derived from Streptomyces coelicolor (S. sericolor) and a gene encoding the same. The present invention provides DNA encoding these N ⁇ -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 N ⁇ -lauroyl-lysine.
  • the N ⁇ -acyl-L-lysine-specific aminoacylase gene is mass-produced using a gene recombination technique.
  • a gene recombination technique To obtain a large amount of the N ⁇ -acyl-L-lysine-specific aminoacylase.
  • cDNA encoding the N ⁇ -acyl-L-lysine-specific aminoacylase derived from this actinomycete is obtained, the nucleotide sequence is analyzed, and the N ⁇ -acyl-L-lysine-specific aminoacylase is analyzed. Information on the entire amino acid sequence can be obtained.
  • a DNA encoding the N ⁇ -acyl-L-lysine-specific aminoacylase can be incorporated into an appropriate expression vector to obtain a transformant that produces a large amount of the target product.
  • the present invention replaces the method for isolating N ⁇ -acyl-L-lysine-specific aminoacylase derived from actinomycetes from nature, using gene recombination technology for efficient gene expression and said N ⁇ -acyl-L -Provide technology for mass production of lysine-specific aminoacylase.
  • DNA encoding a N ⁇ -acyl-L-lysine specific aminoacylase that specifically acylates the ⁇ -amino group of lysine to produce N ⁇ -acyl-L-lysine.
  • the DNA is expressed in a suitable host, thereby producing the aminoacylase.
  • the aminoacylase acts on carboxylic acid and L-lysine to produce N ⁇ -acyl-L-lysine.
  • DNA encoding actinomycete-derived N ⁇ -acyl-L-lysine-specific aminoacylase or a protein having equivalent activity is obtained.
  • DNA encoding the actinomycete-derived N ⁇ -acyl-L-lysine-specific aminoacylase is prepared by using primers based on the amino acid sequence of the N ⁇ -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.
  • a transformant producing the N ⁇ -acyl-L-lysine specific aminoacylase is obtained, whereby a protein having the N ⁇ -acyl-L-lysine specific aminoacylase activity is 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.
  • L-lysine and lauric acid are reacted in the presence of a transformant producing the N ⁇ -acyl-L-lysine-specific aminoacylase or a processed product of the transformant, for example, a cell extract. By doing so, N ⁇ -lauroyl-L-lysine can be obtained efficiently.
  • N ⁇ -acyl-L-lysine-specific aminoacylase is expressed and secreted outside the cell (secretory expression)
  • secretory expression in the presence of a culture solution obtained by culturing the transformant, By reacting L-lysine with lauric acid, N ⁇ -lauroyl-L-lysine can be efficiently obtained.
  • aminoacylase N ⁇ -acyl-L-lysine-specific aminoacylase derived from actinomycetes
  • the DNA of the present invention can be obtained, for example, as follows.
  • 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.
  • 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.
  • nucleotide sequence having a high homology with the amino acid sequence of the aminoacylase already determined is searched in an appropriate DNA database (eg, DDBJ, EMBL, GenBank), and the found sequence is used as a probe for actinomycetes or other
  • an appropriate DNA database eg, DDBJ, EMBL, GenBank
  • 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.
  • the origin of the probe that is, what kind of gene fragment of which species the probe does not matter. That is, the probe does not have to be derived from a known N ⁇ -acyl-L-lysine-specific aminoacylase gene, and may be a part of a gene encoding a gene product whose function is unknown, such as EST (Expression Sequence Tag).
  • primers are based on amino acid sequences that are annotated with virtual enzymes 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.
  • 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 N ⁇ -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 N ⁇ -acyl-L-lysine-specific aminoacylase activity and a recombinant DNA containing the DNA
  • a molecule a host holding the recombinant DNA, a method for producing a N ⁇ -acyl-L-lysine specific enzyme comprising culturing the host, and a method for producing N ⁇ -acyl-L-lysine It is.
  • Homology can be determined using default parameters with standard programs such as NCBI Blast Ver.2.0.
  • amino acid sequence of N ⁇ -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.
  • 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.
  • homology can be calculated using programs well known to those skilled in the art, such as BLAST, with standard parameters.
  • stringent conditions refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
  • DNAs having high homology for example, DNAs having a homology of 80, 90, 95, 97 or 99% or more hybridize, and DNAs having lower homology do not hybridize with each other, Or 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.
  • conditions for washing at least once, preferably 2 to 3 times at a temperature.
  • 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. 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 amino acid sequence 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.
  • 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 be modified so that at least one of the above becomes a more frequently used codon.
  • Such gene synthesis can be easily performed by ligating oligonucleotides synthesized using an automatic DNA synthesizer after annealing.
  • aminoacylase is expressed and produced as a fusion protein with a heterologous protein.
  • aminoacylase can be produced in Escherichia coli (E. coli) as a fusion protein with glutathione S-transferase. Is possible.
  • Escherichia coli Escherichia coli
  • glutathione S-transferase glutathione S-transferase.
  • an actinomycete such as Streptomyces lividans (S. lividans)
  • a high copy vector such as pIJ702
  • a coryneform bacterium such as Corynebacterium glutamicum
  • a high copy vector such as pPK4 can be used.
  • a promoter for expressing an aminoacylase gene a promoter usually used for expression of a heterologous protein can be used.
  • promoters such as trp, tac, lac, trc, ⁇ PL, and T7 can be used.
  • a terminator can also be inserted downstream of the aminoacylase gene.
  • terminators such as tpA, lpp, and T4 can be mentioned.
  • a coryneform bacterium When a coryneform bacterium is selected as an expression host, a cspB promoter or the like can be used.
  • 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.
  • 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.
  • 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.
  • 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 into the 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.
  • 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.
  • 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].
  • N ⁇ -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.
  • N ⁇ -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 also called “Twin-arginine-translocation-pathway”, and recognizes the conserved region of arginine-arginine conserved in the signal peptide.
  • the Tat signal peptide include a signal peptide of trimethylamine N-oxidoreductase (TorA) derived from E. coli.
  • 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.
  • 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,
  • Corynebacterium glutamicum AJ12036 isolated as a streptomycin (Sm) -resistant mutant from ATCC13869 from the wild-type Corynebacterium glutamicum (C. glutamicum) ATCC13869 is the parent strain (wild strain).
  • Sm streptomycin
  • the protein production capacity is extremely high, about 2 to 3 times the amount accumulated under optimal culture conditions. is there.
  • a strain modified so as not to produce cell surface protein from such a strain is used as a host, purification of the target protein secreted into 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 mutation or gene recombination.
  • Examples of coryneform bacteria modified so as not to produce cell surface proteins include C. glutamicum YDK010 strain, which is a cell surface protein (PS2) -disrupted strain of AJ12036 (see WO 01/23491).
  • Other cells that can be transformed include Bacillus subtilis, yeast, Neisseria gonorrhoeae, and the like. A method for producing aminoacylase in a medium using the secretory ability of these proteins is also conceivable.
  • 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.
  • 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.
  • N ⁇ -acyl-L-lysine-specific aminoacylase By culturing the transformant thus obtained, N ⁇ -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.
  • E. coli 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.
  • a nutrient medium such as LB medium or a minimum medium such as M9 medium for culturing E. coli.
  • 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. Depending on the host, either shaking culture or stationary culture is possible, but stirring may be performed as necessary, and aeration may be performed.
  • 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.
  • 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.
  • 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
  • SDS polyacrylamide electrophoresis isoelectric focusing Purification and isolation are possible
  • 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.
  • 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.
  • a cell disruption buffer (20-100 mM Tris-HCl (pH 8.0), 5 mM EDTA), and ultrasonic disruption is performed in Branson MODEL.
  • 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.
  • 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.
  • 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.
  • 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 N ⁇ -acetyl-L-lysine hydrolysis activity.
  • the enzyme 1U (unit) of the present invention was incubated at 37 ° C. using a N ⁇ -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 N ⁇ -acetyl-L-lysine per hour.
  • the aminoacylase encoded by the DNA of the present invention acts on N ⁇ -acyl-L-lysine and has very low reactivity to N ⁇ -acyl-D-lysine.
  • N ⁇ -acyl-L-lysine consisting of saturated or unsaturated fatty acyls and also carboxylate acyls containing aromatic groups and catalyzes the reverse reaction.
  • the reverse reaction the reactivity of D-lysine with respect to the ⁇ -amino group is very low, and it preferentially acts on the ⁇ -amino group of L-lysine.
  • Another aspect of the present invention is a method for producing N ⁇ -acyl-L-lysine.
  • 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 N ⁇ -acyl-L-lysine Is generated.
  • 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.
  • 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.
  • the enzyme of the present invention derived from S. mobaraen (having the amino acid sequence of SEQ ID NO: 2) has the following properties.
  • N ⁇ -acyl-L-lysine Acts on N ⁇ -acyl-L-lysine to catalyze a reaction that liberates carboxylic acid and lysine and vice versa; 2) acts on the ⁇ -amino group of L-Lys; 3) When N ⁇ -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 at pH 6.5 to 10.5 when incubated for 1 hour at 37 ° C in Tris-HCl buffer; 5) The optimum temperature in the hydrolysis reaction using N ⁇ -acetyl-L-lysine as a substrate is around 55 ° C.
  • Tris-HCl buffer pH 8.2
  • Inactivation in Tris-HCl buffer pH 8.2
  • remaining activity after treatment at 55 ° C. for 60 minutes is 75 to 85%
  • Inhibited by o-phenanthroline 8)
  • the activity is increased by metal ions such as cobalt, zinc, manganese, calcium, potassium, magnesium and the like, and in particular, the activity is increased by cobalt ions;
  • the enzyme derived from S. movalaen exhibits high hydrolytic activity on N ⁇ -acetyl-L-lysine, but generally does not show activity on N ⁇ -acetyl-L-amino acids other than N ⁇ -acetyl-L-lysine. Furthermore, the enzyme derived from S. movalaen has the highest specific activity for N ⁇ -acetyl-L-lysine, and also for other N ⁇ -acyl-L-lysine having a chloroacetyl group or a benzoyl group. Shows high activity. Details of these properties are described in WO 2006/088199.
  • the carboxylic acid has a relatively long chain and the reaction is carried out in a water-soluble solvent
  • the produced N ⁇ -acyl-L-lysine is insoluble or hardly soluble in water, and thus precipitates and reacts. Therefore, the synthesis reaction of ⁇ -acyl-L-lysine proceeds significantly more preferentially and efficiently than the decomposition reaction of N ⁇ -acyl-L-lysine, which is the reverse reaction.
  • the synthetic reaction product N ⁇ -acyl-L-lysine is rapidly separated from the aqueous phase and can be recovered very easily.
  • N ⁇ -acyl-L-lysine can be directly recovered, or N ⁇ -acyl-L-lysine can be easily extracted from an aqueous reaction system and recovered with an organic solvent.
  • the relatively long-chain carboxylic acid can be selected based on, for example, solubility in the water-soluble solvent used.
  • 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.
  • 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.
  • 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 to
  • the reaction is carried out for 24 hours, particularly preferably 4 to 24 hours. Under suitable conditions, N ⁇ -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.
  • 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.
  • the carboxylic acid that can be used in the method for producing N ⁇ -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.
  • 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 ⁇ l, PCR buffer 4 ⁇ l, GC Melt 2 ⁇ l, dNTPs 0.4 ⁇ l, Taq polymerase 0.4 ⁇ l, 10 ⁇ M each primer 0.4 ⁇ l, 10 ⁇ g / ml template DNA 0.4 ⁇ l, total volume 20 ⁇ l ⁇ 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 ⁇ L)
  • the obtained DNA fragment 0.43 kbp and pUC118 HincII / BAP were ligated to transform E. coli DH5 ⁇ .
  • 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. The 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 DH5 ⁇ . 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 ⁇ l, PCR buffer 20 ⁇ l, GC Melt 10 ⁇ l, PCR DIG Labeling Mix (Roche) 10 ⁇ l, Taq polymerase 2 ⁇ l, 10 ⁇ M each primer 2 ⁇ l,, 100 ng / ⁇ l template 1.5 ⁇ l, total volume 100 ⁇ l ⁇ 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 IFO13819 strain was treated with NdeI, SpeI, XbaI, BssHII and MluI, respectively, and subjected to electrophoresis using 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. Therefore, the DNA fragment 2 kbp 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 ⁇ l, PCR buffer 10 ⁇ l, GC Melt 5 ⁇ l, dNTPs 1 ⁇ l, Taq polymerase 1 ⁇ l, 10 ⁇ M each primer 1 ⁇ l Template 6 ⁇ l (Total 50 ⁇ L) ⁇ 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 DH5 ⁇ was transformed. Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mg 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 7 (5'-AACGCGGCGATGTGCTCGGGCGTGA-3 ') (SEQ ID NO: 13)
  • primer 8 (5'-CGCGTCTCGGTGCGTGCGGCCTTCA-3') (SEQ ID NO: 14) Designed.
  • 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. Accordingly, 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 DH5 ⁇ .
  • 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 entire ORF 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 obtained 1.6 kbp DNA fragment and pUC118 HincII / BAP were ligated, and E. coli DH5 ⁇ was transformed did.
  • PCR conditions are as follows.
  • ⁇ Preparation of PCR solution KOD plus DNA polymerase (Toyobo) is used. H 2 O 51.6 ⁇ l, PCR buffer 10 ⁇ l, dNTPs 10 ⁇ l, MgSO 4 8 ⁇ l, KOD plus 2 ⁇ l, 10 ⁇ M each primer 6 ⁇ l, template 1.4 ⁇ l, DMSO 5 ⁇ l, total volume 100 ⁇ l ⁇ 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.
  • Sm-ELA Production of Sm-ELA in E.coli
  • primer SmELA-Nde-f (5'-catATGAGCGAGCGCCCCCGAACCACCC-3 ')
  • primer SmELA-Hind-r 5'- aagctTCAGGCGGCGTACACGGTGCGTCCG-3 ′
  • 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 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 5 ⁇ l, MgSO 4 3 ⁇ l, KOD-Plus- 1 ⁇ l, 10 ⁇ M each primer 1.5 ⁇ l, 100 ng / ⁇ l DNA (template) 1 ⁇ l, total volume 50 ⁇ l ⁇ 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 and pTA2 (TArget-Clone-Plus-, Toyobo) were ligated to transform E. coli JM109.
  • Transformants were selected on LB agar medium containing 40 mg / l X-gal, 0.1 mM mM IPTG, 100 mg / l ampicillin.
  • 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 2782005278468 A Japanese Patent Laid-Open No. 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.
  • a plasmid having a structure in which the Sm-ELA gene was inserted into pTrp2 was named pTrp2-SmELA, and a transformant holding this plasmid was designated as E. coli JM109 / pTrp2-SmELA strain.
  • the activity was measured at N ⁇ -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 N ⁇ -acetyl-L-lysine was measured.
  • the acidic ninhydrin method described in WO2006 / 088199 was used for the measurement of lysine. 1 U was defined as the amount of enzyme that liberates 1 ⁇ mol of lysine from N ⁇ -acetyl-L-lysine per hour.
  • the mixture was stirred at rpm to carry out N ⁇ -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 N ⁇ -lauroyl-L-lysine. The reaction solution was centrifuged, the supernatant was removed, and water was added to the precipitate to suspend it.
  • 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.
  • pUC702 a plasmid in which the multicloning site of pUC19 was present on the PstI recognition site side of pIJ702 was obtained and named pUC702.
  • Streptomyces subtilisin inhibitor from Streptomyces albogriseolus 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.
  • ⁇ Preparation of PCR solution Pyrobest DNA polymerase (TAKARA) is used. H 2 O 18.8 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 4 ⁇ l, Pyrobest DNA polymerase 0.2 ⁇ l, 1 mM each primer 10 ⁇ l, 20 ng / ⁇ l DNA (template) 2 ⁇ l, total volume 50 ⁇ l ⁇ 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 °C; ⁇
  • the obtained 0.3-kbp DNA fragment contains the SSI promoter region.
  • pUMTG5 Screening of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease Yatsuyamatsu Environ-Microbiol. (2003, 69, 358-66) was used as a template, and a part of the S. mobaraensis-derived PMTG (microbial pro-transglutaminase) gene was PCR amplified.
  • SSIMTGF 5′-GCACCACCACCGAGCACGGGCCGCACGGCCGCGTTCAACG-3 ′
  • MTGSACR 5′-GACGAGAGCTCTCCGGCGTATGCGCATGGA-3 ′
  • 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.
  • crossover PCR was performed using the primer SSIHindF and the primer MTGSACR.
  • PCR conditions are as follows.
  • TAKARA Pyrobest DNA polymerase
  • H 2 O 18.8 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 4 ⁇ l, Pyrobest DNA polymerase 0.2 ⁇ l, 1 mM each primer 10 ⁇ l, 20 ng / ⁇ l DNA (template) 1 ⁇ l each, total volume 50 ⁇ l ⁇ 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 °C; ⁇
  • the obtained DNA fragment was treated with HindIII and SacI, and ligated with pUC19 treated with HindIII and SacI.
  • E. coli JM109 was transformed, and transformants were selected on an LB agar medium containing 100 mg / l ampicillin.
  • a target plasmid in which a DNA fragment in which a part of the SSI promoter and PMTG gene was 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.
  • 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).
  • TSB Traptic Soy Broth
  • KOD -Plus- Ver.2 (Toyobo) is used. H 2 O 32 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 5 ⁇ l, MgSO 4 3 ⁇ l, KOD-Plus- 1 ⁇ l, 10 ⁇ M each primer 1.5 ⁇ l, 100 ng / ⁇ l DNA (template) 1 ⁇ l, total volume 50 ⁇ l ⁇ 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 °C; 0.5 minutes, 4 °C; ⁇
  • primer SSI-SmELA-f (5'-gacaaaggagttgcaggtttccATGAGCGAGCGCCCCCGAACCACCCTCC-3 ') (SEQ ID NO: 25) and primer SmELA-EcoRI-r (5'-CGGAATTCTCAGGCGGCGTACACGGTGCGTCCG-3' 26) PCR amplification was carried out using to obtain a 1.6 kbp DNA fragment containing the Sm-ELA gene.
  • PCR conditions are as follows.
  • KOD -Plus- Ver.2 (Toyobo) is used. H 2 O 32 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 5 ⁇ l, MgSO 4 3 ⁇ l, KOD-Plus- 1 ⁇ l, 10 ⁇ M each primer 1.5 ⁇ l, 100 ng / ⁇ l DNA (template) 1 ⁇ l, total volume 50 ⁇ l ⁇ 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; ⁇
  • KOD -Plus- Ver.2 (Toyobo) is used. H 2 O 32 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 5 ⁇ l, MgSO 4 3 ⁇ l, KOD-Plus- 1 ⁇ l, 10 ⁇ M each primer 1.5 ⁇ l, 100 ng / ⁇ l DNA (template) 0.5 ⁇ l each, total volume 50 ⁇ l ⁇ 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 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 thiostrepton resistance as an indicator, R2YE agar medium (DNA cloning in Streptomyces: resistance genes from antibiotic-producing species.
  • the strain carrying pUC702-SmELA was named S. lividans TK24 / pUC702-SmELA strain.
  • transformation was performed using pUC702, and the resulting strain was designated as S. lividans TK24 / pUC702 strain.
  • the total N ⁇ -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 N ⁇ 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 N ⁇ -acetyl-L-lysine was measured.
  • lysine 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 ⁇ mol of lysine from N ⁇ acetyl-L-lysine per hour.
  • the fraction having N ⁇ -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.
  • the purified Sm-ELA from the bacterial cell extract was 2800 U / mg
  • the purified Sm-ELA from the culture supernatant was 2500 U. activity of / mg.
  • the result of this time was 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 N ⁇ -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 N ⁇ -acetyl-L-lysine was measured.
  • the acidic ninhydrin method used in WO2006 / 088199 A1 was used for the measurement of lysine.
  • 1 U was defined as the amount of enzyme that liberates 1 ⁇ mol of lysine from N ⁇ -acetyl-L-lysine per hour.
  • the SERPR sequence was obtained, which was consistent with the N-terminal amino acid sequence of purified Sm-ELA from the S. mobaraensis IFO13819 strain culture solution. .
  • 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.
  • N ⁇ -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.
  • 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, when microbial cell extract was used, lauric acid was completely consumed after 2 hours when washed bacterial cells were used, and N ⁇ -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.
  • 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.
  • the decomposition activity of N ⁇ -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.
  • KOD -Plus- Ver.2 (Toyobo) is used. H 2 O 32 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 5 ⁇ l, MgSO 4 3 ⁇ l, KOD-Plus- 1 ⁇ l, 10 ⁇ M each primer 1.5 ⁇ l, 100 ng / ⁇ l DNA (template) 1 ⁇ l, total volume 50 ⁇ l ⁇ 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 °C; 0.5 minutes, 4 °C; ⁇
  • 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.
  • KOD -Plus- Ver.2 (Toyobo) is used. H 2 O 32 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 5 ⁇ l, MgSO 4 3 ⁇ l, KOD-Plus- 1 ⁇ l, 10 ⁇ M each primer 1.5 ⁇ l, 100 ng / ⁇ l DNA (template) 1 ⁇ l, total volume 50 ⁇ l ⁇ PCR reaction conditions> Cycle 1 (x1) 94 ° C; 2 minutes Cycle 2 (x25) 98 °C; 10 seconds, 55 °C; 10 seconds, 68 °C; 1.5 minutes Cycle 3 (x1) 68 °C; 0.5 minutes, 4 °C; ⁇
  • crossover PCR using these two DNA fragments as a template was performed using the primer CspB-SmELA-f and the primer SmELA-Bam-r, and the 1.6 mkbp containing the Sm-ELA gene with the BamHI recognition site deleted.
  • a DNA fragment was obtained. PCR conditions are as follows.
  • KOD -Plus- Ver.2 (Toyobo) is used. H 2 O 32 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 5 ⁇ l, MgSO 4 3 ⁇ l, KOD-Plus- 1 ⁇ l, 10 ⁇ M each primer 1.5 ⁇ l, 100 ng / ⁇ l DNA (template) 0.5 ⁇ l each, total volume 50 ⁇ l ⁇ 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; ⁇
  • pPK4 Japanese Patent Laid-Open No. 9-322774
  • pPKSPTG1 WO 01/23591
  • a 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.
  • 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 the 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, 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.
  • pPK-SmELA is a plasmid having a structure in which the cspB promoter and the Sm-ELA gene are inserted into pPK4.
  • 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 constructed in pPK4.
  • primer CspAsig-SmELA-f 5'-GCTGGCCGCACCTGTGGCAACGGCAAGCGAGCGCCCCCGAACCACCCTCC-3 '
  • 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 kb kbp DNA fragment was obtained.
  • 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.
  • 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.
  • pPKS-SmELA a CspA signal sequence, which is a Sec system secretory signal sequence, is added to the Sm-ELA gene.
  • 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.
  • 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.
  • 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.
  • 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.
  • pPKT-SmELA a TorA signal sequence, which is a Tat secretion signal sequence, is added to the Sm-ELA gene.
  • a YDK010 strain obtained from a mutant strain derived from C.
  • 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
  • the obtained transformants were named YDK010 / pPK-SmELA strain, YDK010 / pPKS-SmELA strain, and YDK010 / pPKT-SmELA strain, respectively.
  • C. glutamicum WDK010 strain expressing Sm-ELA 2.1.
  • Preparation of C. 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.
  • TAKARA Pyrobest DNA polymerase
  • a gene substitution 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.
  • PS2 cell surface protein
  • 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 hydrolyzate 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.
  • MMM liquid medium containing 25 mg / l kanamycin (glucose 60 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5
  • 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 N ⁇ -acetyl-L-lysine was measured.
  • the activity was measured at N ⁇ -acetyl-L-lysine 4 ⁇ ⁇ ⁇ mM, Tris-HCl 50 mM (pH 8.0), CoCl2 0.1 mM, 37 ° C, and the concentration of lysine produced with the decomposition of N ⁇ -acetyl-L-lysine It was measured.
  • lysine BF-5 and lysine electrodes (Oji Scientific Instruments) were used. 1 ⁇ U was defined as the amount of enzyme that liberates 1 ⁇ mol of lysine per hour from N ⁇ -acetyl-L-lysine.
  • primer XbaI-TatA-f (5'-gctctagaTTTGGAGTAGACCACATGTCCCTCG-3 ') (SEQ ID NO: 39)
  • primer TatC-TatB-r (5'-TAGAAAACATCAGACCGGTCTTTCACTAGAGCACGTCACCGAAGTCGGCG-3')
  • primer TatC -TatB-f (5'-CGCCGACTTCGGTGACGTGCTCTAGTGAAAGACCGGTCTGATGTTTTCTA-3 ')
  • 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.
  • 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.
  • crossover PCR was performed 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.
  • the 2 kb kbp DNA fragment obtained by crossover PCR was treated with XbaI.
  • the plasmid pPKT-SmELA was similarly treated with XbaI and further with BAP. These 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.
  • the obtained culture broth was centrifuged, and the supernatant was collected. Using these culture supernatants, the degradation activity of N ⁇ -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. From the culture supernatant of the WDK010 / pPKT-SmELA-tatABC strain, an activity of 125 U per ml was detected.
  • 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 mini jar fermenter to carry out N ⁇ -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.
  • the reaction condition 1 was 5.44 g and the reaction condition 2 was 9.82 g.
  • the N ⁇ -lauroyl-L-lysine purity in these powders was measured by HPLC and found to be 76.4% and 80.9%, respectively. mmol) It was clear that it was obtained. Yields were 84.4% and 80.6%, respectively, based on lauric acid.
  • 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
  • 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.
  • a seed liquid medium containing 25 mg / l kanamycin (glucose 20 g
  • the seed culture was performed at 31 ° C., aeration 1/1 vvm, stirring 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
  • the obtained culture solution was centrifuged, and the supernatant was collected.
  • the degradation activity of N ⁇ -acetyl-L-lysine in this culture supernatant was measured.
  • 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 N ⁇ -acetyl-L-lysine 40 mM, Tris-HCl 50 mM (pH 8.0), CoCl 2 0.1 mM, at 37 ° C, and the concentration of lysine produced with N ⁇ -acetyl-L-lysine degradation.
  • lysine BF-5 and a lysine electrode (Oji Scientific Instruments) were used. 1 U was defined as the amount of enzyme that liberates 1 ⁇ mol of lysine from N ⁇ -acetyl-L-lysine per hour.
  • the entire reaction solution was recovered together with N ⁇ -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 N ⁇ -lauroyl-L-lysine was dissolved.
  • the N ⁇ -lauroyl-L-lysine concentration in this solution was measured by HPLC (FIG. 7, after 10 hours).
  • HPLC HPLC
  • Sc-ELA N ⁇ -acyl-L-lysine-specific aminoacylase gene of S. sericolor
  • the amino acid sequence of Sm-ELA was subjected to homology search using NCBI protein-protein BLAST, and 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.
  • 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.
  • 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 mM IPTG, 100 mg / l ampicillin.
  • 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 2782005278468 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
  • 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.
  • a plasmid having a structure in which the Sc-ELA gene was inserted into pTrp2 was named pTrp2-ScELA, and a transformant carrying this plasmid was named E. coli JM109 / pTrp2-ScELA strain.
  • KOD -Plus- Ver.2 (Toyobo) is used. H 2 O 32 ⁇ l, PCR buffer 5 ⁇ l, dNTPs 5 ⁇ l, MgSO 4 3 ⁇ l, KOD-Plus- 1 ⁇ l, 10 ⁇ M each primer 1.5 ⁇ l, 100 ng / ⁇ l DNA (template) 1 ⁇ l, total volume 50 ⁇ l ⁇ PCR reaction conditions> Cycle 1 (x1) 94 ° C; 2 minutes Cycle 2 (x25) 98 ° C; 10 seconds, 55 ° C; 10 seconds, 68 ° C; 2 ⁇ Cycle 3 (x1) 68 °C; 2 minutes, 4 °C; ⁇
  • 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 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-ScELA.
  • pPK-ScELA the cspB promoter and the Sc-ELA gene are inserted into pPK4.
  • 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.
  • 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.
  • a 1.6-kbp DNA fragment was obtained.
  • 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.
  • pPKS-ScELA This DNA fragment was treated with ScaI and BamHI, and pPKS-ScELA was constructed in the same manner as pPK-ScELA.
  • pPKS-ScELA a CspA signal sequence, which is a Sec system secretory signal sequence, is added to the Sc-ELA gene.
  • 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.
  • 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.
  • PCR conditions are the same as those for PCR using CspB-ScELA-f and ScELA-Bam-r.
  • a 1.6-kbp DNA fragment was obtained.
  • a 2.2-kbp DNA fragment in 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.
  • pPKT-ScELA This DNA fragment was treated with ScaI and BamHI, and pPKT-ScELA was constructed in the same manner as pPK-ScELA.
  • a TorA signal sequence which is a Tat secretion signal sequence, is added to the Sc-ELA gene.
  • 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.
  • the WDK010 strain was transformed with pPKT-ScELA, the transformant was selected on a CMDXB agar medium containing 25 mg / l kanamycin, and the obtained transformant was named the WDK010 / pPKT-SmELA strain. .
  • 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.
  • N ⁇ -acetyl-L-lysine degradation activity is as low as Sm-ELA
  • Sc-ELA is N ⁇ -acyl-L as is Sm-ELA.
  • -It was considered to be a lysine-specific aminoacylase.
  • N ⁇ -lauroyl-L-lysine by cell extract of C. glutamicum expressing Sc-ELA Went.
  • N ⁇ -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.
  • a N ⁇ -lauroyl-L-lysine synthesis reaction was carried out at 1000 rpm with stirring using a jar fermenter.
  • N ⁇ -acyl-L-lysine-specific aminoacylase derived from actinomycetes its nucleotide sequence, and all amino acid sequences are provided. Therefore, according to the present invention, it is possible to mass-produce N ⁇ -acyl-L-lysine-specific aminoacylase derived from actinomycetes using gene recombination technology, and a large amount of the N ⁇ -acyl-L-lysine specific can be easily produced. It is possible to obtain a typical aminoacylase. Further, according to the present invention, N ⁇ -acyl-L-lysine, particularly N ⁇ -lauroyl-L-lysine can be obtained efficiently.

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Abstract

La présente invention concerne : une aminoacylase spécifique de la Nε-acyl-L-lysine qui permet la synthèse efficace d'une Nε-acyl-L-lysine; et un procédé pour synthétiser une Nε-acyl-L-lysine avec une efficacité élevée. La présente invention concerne spécifiquement : une aminoacylase spécifique de la Nε-acyl-L-lysine comprenant une séquence d'acides aminés décrite dans SEQ ID NO: 2 ou SEQ ID NO: 5; un ADN codant pour l'aminoacylase spécifique de la Nε-acyl-L-lysine, ou un variant de celle-ci; et un transformant comportant une molécule d'ADN recombinant comprenant l'ADN ou le variant de celui-ci.
PCT/JP2009/070769 2008-12-11 2009-12-11 AMINOACYLASE SPÉCIFIQUE DE LA Nε-ACYL-L-LYSINE WO2010067871A1 (fr)

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JP2009207086A JP2012039878A (ja) 2008-12-11 2009-09-08 Ｎε−アシル−Ｌ−リジン特異的アミノアシラーゼ

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WO2017082374A1 (fr) 2015-11-12 2017-05-18 味の素株式会社 PROCÉDÉ DE PRODUCTION DE Nε-ACYL-L-LYSINE
CN107488130A (zh) * 2017-08-22 2017-12-19 上海鲍林化工有限公司 一种月桂酰赖氨酸的制备方法

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WO2006088199A1 (fr) * 2005-02-21 2006-08-24 Ajinomoto Co., Inc. AMINOACYLASE SPÉCIFIQUE DE LA Nϵ-ACYL-L-LYSINE

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WO2006088199A1 (fr) * 2005-02-21 2006-08-24 Ajinomoto Co., Inc. AMINOACYLASE SPÉCIFIQUE DE LA Nϵ-ACYL-L-LYSINE

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017082374A1 (fr) 2015-11-12 2017-05-18 味の素株式会社 PROCÉDÉ DE PRODUCTION DE Nε-ACYL-L-LYSINE
US10266857B2 (en) 2015-11-12 2019-04-23 Ajinomoto Co., Inc. Method for producing Nε-acyl-L-lysine
CN107488130A (zh) * 2017-08-22 2017-12-19 上海鲍林化工有限公司 一种月桂酰赖氨酸的制备方法

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