JP2015165791A - L-lysine decarboxylative/oxidative enzyme and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a L-lysine decarboxylative/oxidative enzyme having an extremely high substrate specificity to L-lysine and capable of measuring L-lysine in a sample containing a lot of impurities at a high accuracy, and measuring method of L-lysine using the enzyme; and to provide a L-lysine measuring kit and a L-lysine measurement enzyme sensor capable of being used in the measurement method and a new microorganism producing the new L-lysine decarboxylative/oxidative enzyme.SOLUTION: The L-lysine decarboxylative/oxidative enzyme of the present invention comprises a specific amino acid sequence.

Description

  The present invention relates to a novel L-lysine decarboxylase / oxidase having high substrate specificity for L-lysine, a method for using the same, and the like. More specifically, the present invention relates to a novel L-lysine decarboxylation / oxidase having both L-lysine decarbonization activity and L-lysine oxidation activity and high substrate specificity for L-lysine. Nucleic acid encoding L-lysine decarboxylation / oxidase, L-lysine measurement method using the novel L-lysine decarboxylation / oxidase, L-lysine measurement kit and L that can be used in the measurement method -An enzyme sensor for lysine measurement, and a novel microorganism that produces the novel L-lysine decarboxylation / oxidase.

  L-lysine is one of the amino acids constituting the protein, but is an essential amino acid that is not produced in the body. Concentrations of amino acids containing L-lysine maintain homeostasis in vivo, but their blood concentrations vary greatly due to inborn errors of metabolism and visceral diseases. In addition to L-lysine, measurement of amino acid concentration in a living body can be a useful means for detecting a disease. For this reason, it becomes possible to detect a specific disease by measuring the blood concentration of one kind or many kinds of amino acids (Patent Document 1 and Non-Patent Document 1).

  In recent years, many methods using enzymes have been reported as amino acid quantification methods. The method using an enzyme has an advantage that a large number of samples can be measured in a short time compared to the instrumental analysis method, and can be easily performed at a low cost. As an enzyme for quantification, for example, an oxidase and a dehydrogenase are often used. In the case of using an oxidase, there is a method of detecting and quantifying hydrogen peroxide generated by allowing an oxidase to act on an amino acid with a peroxidase (Patent Document 2). For this detection and quantification, any of the colorimetric method, the fluorescence method, and the electrode method can be used.

  A method using an enzyme is also known as a method for quantifying L-lysine. For example, L-lysine α-oxidase [EC1.4.3.14] has been used for quantification by oxidase. L-lysine α-oxidase derived from Trichoderma viride has a higher substrate specificity than other L-amino acid oxidases and is commercially available, so it is used for elements such as enzyme sensors. (Non-patent document 2, Non-patent document 3 and Non-patent document 4). However, it has been reported that this enzyme acts slightly on several other amino acids.

In addition, a method for quantifying L-lysine using L-lysine ε-oxidase [EC1.4.3.20] derived from a marine bacterium Marinomonas mediteranea NBRC 103028 ^T strain has also been reported (Patent Literature). 3 and Non-Patent Document 4). This enzyme uses L-ornithine as a substrate in addition to L-lysine.

  In addition, there is a report that L-lysine monooxygenase [EC1.13.12.2] derived from Pseudomonas fluorescens has L-lysine oxidase activity (Non-patent Documents 6 and 7). . This enzyme uses L-lysine, L-ornithine, and L-arginine as substrates.

International Publication No. 2006/129513 Pamphlet JP-A-55-43409 JP 2011-43396 A

Anal. Chem. , 81, pp. 307-314 (2009) Sens. Actuators, B, 126, pp. 424-430 (2007) Anal. Bioanal. Chem. , 391, pp. 1255-1261 (2008) Anal. Bioanal. Chem. , 406, pp. 19-23 (2010) Appl. Microbiol. Biotechnol. , DOI 10.1007 / S00253-00013-05168-00253 (2013) J. et al. Biol. Chem. , 249, pp. 2579-2586 (1974) J. et al. Biol. Chem. , 249, pp. 2587-2592 (1974)

  As described above, L-lysine oxidase derived from various microorganisms is known, but there is no oxidase that acts only on L-lysine among amino acids, and amino acids other than L-lysine are also oxidized. Therefore, when used for quantification of L-lysine, for example, L-lysine in a specimen containing various compounds such as plasma was not accurately measured.

  Therefore, the present invention uses an L-lysine decarboxylase / oxidase that has a very high substrate specificity for L-lysine and that can measure L-lysine in a sample containing a large amount of impurities with high accuracy, and the enzyme. It is an object of the present invention to provide a method for measuring L-lysine. The present invention also provides an L-lysine measurement kit and an L-lysine measurement enzyme sensor that can be used in the measurement method, and a novel microorganism that produces the novel L-lysine decarboxylase / oxidase. Also aimed.

  The inventors of the present invention have made extensive studies to solve the above problems. As a result, a novel enzyme having both a decarboxylase activity and an oxidase activity as well as a substrate specificity for L-lysine was found. The present inventors have found that when the enzyme is used, L-lysine in a sample containing a large amount of contaminants such as plasma can also be measured with high accuracy.

The L-lysine decarboxylase / oxidase according to the present invention has any one of the following amino acid sequences (1) to (3).
(1) the amino acid sequence set forth in SEQ ID NO: 2;
(2) an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids and having L-lysine decarboxylase / oxidase activity in the amino acid sequence shown in SEQ ID NO: 2;
(3) An amino acid sequence having 95% or more homology with the amino acid sequence set forth in SEQ ID NO: 2 and having L-lysine decarboxylation / oxidase activity.

  The L-lysine decarboxylation / oxidase can be obtained from bacteria belonging to the genus Burkholderia, in particular from Burkholderia sp. AIU395 strain (accession number: NITE P-01796).

The nucleic acid according to the present invention has any one of the following base sequences (1 ′) to (3 ′), and is used for the production of the L-lysine decarboxylase / oxidase. Can do.
(1 ′) the nucleotide sequence set forth in SEQ ID NO: 1 (2 ′) the nucleotide sequence set forth in SEQ ID NO: 1 has one to several base deletions, substitutions and / or additions, and the nucleotide sequence is The base sequence in which the encoded protein has L-lysine decarboxylation / oxidase activity (3 ′) has a homology of 95% or more with respect to the base sequence described in SEQ ID NO: 1, and the base sequence encodes A base sequence in which a protein has L-lysine decarboxylation / oxidase activity.

  Moreover, the transformant according to the present invention is characterized by containing the above-described nucleic acid.

The method for measuring L-lysine according to the present invention is a method for measuring L-lysine in a specimen,
(A) If necessary, the same steps as described above are performed for a plurality of samples in which the amount of L-lysine is clear, and the amount of L-lysine and the amount of the reaction product to be measured in the following step (D) The process of clarifying the relationship;
(B) A step of oxidizing cadaverine in the specimen by allowing putrescine oxidase to act on the specimen, if necessary;
(C) a step of causing the L-lysine decarboxylase / oxidase to act on the specimen; and (D) a step of measuring at least one amount of a reaction product resulting from the action of the L-lysine decarboxylase / oxidase. It is characterized by.

  In the step (C), cadaverine produced by the action of the L-lysine decarboxylation / oxidase is oxidized by putrescine oxidase, and the amount of hydrogen peroxide as a reaction product in the step (D) Is preferably measured. Such an embodiment makes it possible to more accurately measure L-lysine in a specimen.

  The putrescine oxidase is preferably derived from Kokuria rosea NBRC 3768 strain. The effect of such putrescine oxidase has been confirmed by experiments of the present inventors.

  The kit for measuring L-lysine according to the present invention is characterized by comprising the above-mentioned L-lysine decarboxylation / oxidase.

  As the L-lysine measurement kit, a kit further containing putrescine oxidase, or a kit containing a hydrogen peroxide detection reagent containing peroxidase and a coloring reagent is preferable.

  The oxygen sensor according to the present invention is an enzyme sensor for measuring L-lysine, and has an electrode for hydrogen peroxide detection, and the L-lysine decarboxylation is formed on or near the surface of the hydrogen peroxide detection electrode. / It is characterized in that an oxidase is arranged.

  The present invention also relates to Burkholderia sp. AIU395 strain (Accession No .: NITE P-01796) that produces the L-lysine decarboxylation / oxidase.

  The L-lysine decarboxylation / oxidase according to the present invention has a very high substrate specificity for L-lysine. Therefore, by using this L-lysine decarboxylation / oxidase, L-lysine can be specifically and rapidly measured even in a sample containing many impurities such as other amino acids. In particular, the present invention is effective for biological samples such as plasma, serum, and urine.

FIG. 1 is a simplified molecular phylogenetic tree based on the 16S rDNA base sequence of the genus Burkholderia. SID13564 in FIG. 1 indicates the Burkholderia sp. AIU395 strain. The lower left line is the scale bar, the numerical value located at the branch of the phylogenetic branch is the bootstrap value, and T at the end of the stock name indicates the type strain of that kind. FIG. 2 is a diagram showing the results of physiological and biochemical property tests of Burkholderia sp. AIU395 strain. FIG. 2 (1) shows the results, and FIG. 2 (2) shows the test items. FIG. 3 is a diagram showing the culture curve of Burkholderia sp. AIU395 strain and the change over time in the activity of L-lysine decarboxylase / oxidase. ● indicates L-lysine oxidase activity, ■ indicates the growth curve of the strain (OD660 nm), and ▲ indicates the pH of the medium. FIG. 4 is a photograph of Native-PAGE (A) and SDS-PAGE (B) of L-lysine decarboxylation / oxidase derived from Burkholderia sp. AIU395 strain. In addition, (C) shows a molecular weight marker. FIG. 5 is a diagram showing the absorption spectrum of the purified enzyme. The horizontal axis indicates the absorption wavelength, and the vertical axis indicates the absorbance value. FIG. 6 shows effects of pH on the activity and stability of L-lysine decarboxylase / oxidase derived from Burkholderia sp. Strain AIU395, and putrescine oxidation from Kokuria rosea NBRC 3768 strain. Figure 2 shows the effect of pH on enzyme activity. ● indicates L-lysine oxidase activity at each pH at 30 ° C, ○ indicates residual activity after heating at 30 ° C for 30 minutes at each pH, and ▲ indicates the effect of pH on cadaverine oxidase activity of putrescine oxidase ing. FIG. 7 shows the influence of the temperature of L-lysine decarboxylation / oxidase derived from Burkholderia sp. AIU395 strain. ○ indicates the L-lysine oxidase activity at each temperature, and ● indicates the residual activity after heating at each temperature for 30 minutes. FIG. 8 shows L-lysine decarboxylation / oxidase derived from Burkholderia sp. Strain AIU395 and basic amino acid decarboxylase derived from bacteria belonging to the genus Burkholderia and Escherichia coli. The alignment of the amino acid sequence is shown. FIG. 9 shows the results of measuring the absorbance at 555 nm by a colorimetric method in a 1.0 mL reaction solution, using 1.0 mM L-lysine as a substrate and changing the amount of L-lysine decarboxylation / oxidase added for 20 minutes. Is shown. FIG. 10 shows the results of measuring absorbance at 555 nm by adding 1.3 mU of L-lysine decarboxylation / oxidase using 0-1.0 mM L-lysine as a substrate in a 1.0 mL reaction solution. FIG. 11 shows the results of measuring absorbance at 555 nm by a colorimetric method in a reaction solution of 1.0 mL by changing the amount of putrescine oxidase (PUO) added using 60 μM cadaverine as a substrate and changing the amount of putrescine oxidase (PUO). Show. FIG. 12 shows that in a 1.0 mL reaction solution, the amount of putrescine oxidase (PUO) was fixed to 200 mU using 60 μM L-lysine as a substrate, and the reaction was performed by changing the amount of L-lysine decarboxylation / oxidase added. The results of measuring the absorbance at 555 nm by a colorimetric method are shown. FIG. 13 shows that in a 1.0 mL reaction solution, L-lysine decarboxylase / oxidase (1.5 mU) and putrescine oxidase (200 mU) were added to lysine at a concentration of 0 to 60 μM and reacted for 30 minutes. Thereafter, the results of measuring the absorbance at 555 nm by a colorimetric method are shown. FIG. 14 shows that in a 1.0 mL reaction solution, putrescine oxidase (200 mU) and chromogenic solution I (peroxidase and 4-aminoantipyrine) were added to a solution containing 60 μM cadaverine and L-lysine at a concentration of 0 to 60 μM. Reaction (Reaction I), followed by addition of Coloring Solution II (TOOS) and L-lysine decarboxylase / oxidase (2.3 mU) for reaction (Reaction II), and measurement of absorbance at 555 nm for 30 minutes. Show. (1) shows the change in absorbance over time, the horizontal axis of the graph of (2) shows the L-lysine concentration, and the vertical axis shows the change in absorbance.

  Hereinafter, first, the L-lysine decarboxylation / oxidase according to the present invention will be described.

<L-lysine decarboxylation / oxidase>
The L-lysine decarboxylation / oxidase according to the present invention has extremely high substrate specificity for L-lysine. Specifically, L-arginine, L-ornithine, L-tyrosine, L-alanine, L-cysteine, L, except that 5-hydroxy-DL-lysine and cadaverine which is a diamine organic compound show slight oxidase activity. -Aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-methionine, L-asparagine, L-proline, L-glutamine, L-arginine, L-serine, L-threonine, It does not show any activity against L-valine. Therefore, by using the L-lysine decarboxylase / oxidase of the present invention, L-lysine can be accurately measured even in biological samples that can contain various amino acids such as plasma.

The L-lysine decarboxylase / oxidase according to the present invention has any one of the following amino acid sequences (1) to (3).
(1) the amino acid sequence set forth in SEQ ID NO: 2;
(2) an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids and having L-lysine decarboxylase / oxidase activity in the amino acid sequence shown in SEQ ID NO: 2;
(3) An amino acid sequence having 95% or more homology with the amino acid sequence set forth in SEQ ID NO: 2 and having L-lysine decarboxylation / oxidase activity.

  In the present invention, “L-lysine decarboxylase / oxidase” refers to an enzyme having both decarboxylase activity and oxidase activity for L-lysine.

  In the present invention, “L-lysine decarboxylase activity” refers to an enzyme activity that catalyzes a reaction of decarboxylating L-lysine to produce cadaverine, and “L-lysine oxidase activity” refers to oxygen and water. In the presence of, L-lysine is oxidized, and L-2-aminoadipic acid refers to an enzyme activity that catalyzes a reaction that produces 5-semialdehyde, hydrogen peroxide, and ammonia. That is, the L-lysine decarboxylation / oxidase of the present invention catalyzes a reaction of the following formula.

  Further, in the present invention, an enzyme “having a (specific) amino acid sequence” means that the amino acid sequence of the enzyme only needs to contain the specified amino acid sequence, and the function of the enzyme is maintained. Means. Examples of sequences other than the amino acid sequence specified in the enzyme include a histidine tag, a linker sequence for immobilization, and a crosslinked structure such as a disulfide bond.

  The amino acid sequence (1) is the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing. The enzyme having the amino acid sequence has L-lysine decarboxylase / oxidase activity, and has been isolated and purified as an enzyme produced by a novel Burkholderia strain found by the present inventors from nature. is there.

The L-lysine decarboxylase / oxidase having the amino acid sequence (1) (SEQ ID NO: 2) is produced by Burkholderia sp. AIU395 strain, which is a novel microorganism according to the present invention. . The AIU395 strain is deposited with the depository as follows.
(I) Name and address of depositary institution Name: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center Address: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan
(Ii) Date of accession: January 24, 2014 (iii) Accession number: NITE P-01796

  In addition, according to the experimental findings of the present inventors, a plurality of Burkholderia other than AIU395 strain also has L-lysine decarboxylation / oxidase having both decarboxylase activity and oxidase activity against L-lysine. Has been confirmed to produce.

  The range of “1 to several” in the “amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids” of the amino acid sequence (2) according to the present invention includes a protein having a deletion, etc. The enzyme is not particularly limited as long as it has both decarboxylase activity and oxidase activity for L-lysine. In the range of “1 to several”, since the ratio of the protein having the L-lysine decarboxylase / oxidase is high, for example, 1 or more and 30 or less, preferably 1 or more and 20 or less. More preferably 1 or more, 10 or less, further preferably 1 or more and 7 or less, more preferably 1 or more and 5 or less, and particularly preferably 1 or more and 3 or less. .

  The homology in the “amino acid sequence having 95% or more homology with the amino acid sequence described in SEQ ID NO: 2” of the amino acid sequence (3) according to the present invention is such that the protein having the homology is L-lysine. There is no particular limitation as long as the enzyme has both decarboxylase activity and oxidase activity. The homology is not particularly limited as long as it is 95% or more, preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and particularly preferably 99.5% or more. It is.

  The homology with the amino acid sequence described in SEQ ID NO: 2 can be easily determined by those skilled in the art using alignment analysis software if the amino acid sequence of the protein to be compared is clear.

  In the above amino acid sequence (2) and amino acid sequence (3), “having L-lysine decarboxylation / oxidase activity” means, for example, that cadaverine is obtained by decarboxylating L-lysine under the conditions described in the Examples below. Reaction that has an enzymatic activity to catalyze the reaction to form L-2-aminoadipic acid 5-semialdehyde, hydrogen peroxide and ammonia by oxidizing L-lysine in the presence of oxygen and water It has the enzyme activity which catalyzes.

  The L-lysine decarboxylase / oxidase of the present invention has the amino acid sequences (1) to (3) and exhibits high substrate specificity for L-lysine. As long as the enzyme has lysine decarboxylation activity, its origin is not particularly limited.

  For example, the L-lysine decarboxylase / oxidase of the present invention may be a recombinant protein produced by various genetic engineering techniques, a synthetic protein produced by chemical synthesis, or SEQ ID NO: 2. The amino acid sequence (2) or (2) or (2) or (2) or (2) or (2) or (2) or (2) or (2) by giving a mutagen to the biological species having an L-lysine decarboxylase / oxidase gene homologue It may be a protein produced by obtaining a mutant capable of producing L-lysine decarboxylase / oxidase having 3), and extracting and purifying the produced protein.

  As a production method by heterologous expression, for example, a corresponding gene is amplified by PCR from a genomic DNA extracted from a biological species having the same activity, and a plasmid vector incorporated into pET or pUC is constructed, and then BL21 or JM109 is constructed. And a method of transforming into a host strain such as Other known methods other than these can also be used as appropriate.

  The method for obtaining L-lysine decarboxylase / oxidase according to the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique. When producing a recombinant protein, a gene (DNA) encoding the protein is obtained as described later. By introducing this DNA into an appropriate expression system, the above-mentioned L-lysine decarboxylase / oxidase can be produced.

  The L-lysine decarboxylase / oxidase according to the present invention includes a gene encoding the above-mentioned L-lysine decarboxylase / oxidase on a vector, a host cell transformed with this vector, and the transformed host. It can be produced by a method comprising culturing cells, accumulating the protein encoded by the gene in the culture, and collecting the accumulated protein.

<Nucleic acid encoding L-lysine decarboxylation / oxidase>
A gene encoding L-lysine decarboxylase / oxidase according to the present invention is one embodiment of the present invention. That is, the present invention includes a nucleic acid having any of the following base sequences.
(1 ′) the nucleotide sequence set forth in SEQ ID NO: 1 (2 ′) the nucleotide sequence set forth in SEQ ID NO: 1 has one to several base deletions, substitutions and / or additions, and the nucleotide sequence is The base sequence in which the encoded protein has L-lysine decarboxylation / oxidase activity (3 ′) has a homology of 95% or more with respect to the base sequence described in SEQ ID NO: 1, and the base sequence encodes A base sequence in which a protein has L-lysine decarboxylation / oxidase activity.

  For the definition of each word in the nucleic acid, the definition of the L-lysine decarboxylation / oxidase is applied mutatis mutandis. If a nucleic acid having a base sequence of 95% or more homology with the base sequence described in SEQ ID NO: 1 is used, an amino acid sequence having a homology of 95% or more with respect to the amino acid sequence described in SEQ ID NO: 2 is obtained. L-lysine decarboxylation / oxidase can be produced.

  The method for obtaining a gene encoding the L-lysine decarboxylase / oxidase of the present invention is not particularly limited. The gene encoding the L-lysine decarboxylase / oxidase of the present invention can be obtained by, for example, chemical synthesis, genetic engineering technique or the like based on the amino acid sequence shown in SEQ ID NO: 2 and the base sequence information shown in SEQ ID NO: 1. It can be made by any method known to those skilled in the art, such as mutagenesis.

  For example, the DNA having the base sequence described in SEQ ID NO: 1 in the Sequence Listing can be performed by using a method of contacting with a mutagen drug, a method of irradiating with ultraviolet rays, a genetic engineering method, or the like. Site-directed mutagenesis, which is one of genetic engineering techniques, is useful because it is a technique that can introduce a specific mutation at a specific position. Molecular Cloning: A laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 1989 (hereinafter abbreviated as Molecular Cloning 2nd edition), Current Protocols in Molecular Biology, Supplements 1-38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology). ) And the like.

  Appropriate probes and primers were prepared based on the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing or the base sequence information shown in SEQ ID NO: 1, and using them, Burkholderia sp. The gene of the present invention can be isolated by screening a cDNA or genomic library. A cDNA or genomic library can be prepared by a conventional method.

  A gene encoding the L-lysine decarboxylase / oxidase of the present invention can also be obtained by PCR. For example, using the above-described Burkholderia sp. AIU395 strain cDNA or genomic library as a template, and using a pair of primers designed to amplify the base sequence described in SEQ ID NO: 1, etc. Perform PCR. PCR reaction conditions may be set as appropriate. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

  Operations such as the preparation of the probe or primer, construction of the genomic library, screening of the genomic library, and cloning of the target gene are known to those skilled in the art. For example, the molecular cloning 2nd edition and the current protocols are described. -It can be performed according to the method described in In-Molecular Biology etc.

  The gene for L-lysine decarboxylase / oxidase can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited. For example, the vector may be a self-replicating vector (for example, a plasmid), or may be integrated into the host cell genome when introduced into the host cell. It may be replicated together with other chromosomes. Preferably the vector is an expression vector. In the expression vector, elements necessary for transcription (for example, a promoter and the like) are functionally linked to the gene. A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.

  Promoters that can operate in bacterial cells include the Geobacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis α-amylase gene (Bacillus licheniformis amyliformis α amylase gene). Bacteria BAN amylase gene (Bacillus amyloliquefaciens BAN amylase gene), Bacillus subtilis alkaline protease gene (Bacillus subtilis alkaline protease gene), Bacillus pumilusoxysidase Child (Bacillus pumilus xylosldase gene) promoter; phage lambda PR promoter and PL promoter; lac promoter of E. coli (E. coli), such as trp promoter and tac promoter and the like.

  Examples of promoters operable in mammalian cells include the SV40 promoter, MT-1 (metallothionein gene) promoter, adenovirus 2 major late promoter, and the like. Examples of promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa, calihornica, polyhedrosic basic protein promoter, baculovirus immediate early gene 1 promoter, baculovirus 39K delayed early gene promoter, and the like. is there. Examples of promoters operable in yeast host cells include promoters derived from yeast glycolytic genes, alcohol dehydrogenase gene promoters, TPI1 promoters, ADH2-4c promoters, and the like. Examples of promoters that can operate in filamentous fungal cells include the ADH3 promoter and the tpiA promoter.

  The L-lysine decarboxylase / oxidase gene may be operably linked to an appropriate terminator as necessary. The recombinant vector containing the gene for L-lysine decarboxylase / oxidase further has elements such as a polyadenylation signal (eg, derived from SV40 or adenovirus 5E1b region), a transcription enhancer sequence (eg, SV40 enhancer), and the like. May be. A recombinant vector containing the gene for L-lysine decarboxylase / oxidase may further comprise a DNA sequence that allows the vector to replicate in the host cell, an example of which is the SV40 origin of replication (host And when the cell is a mammalian cell).

  The recombinant vector containing the gene for L-lysine decarboxylation / oxidase may further contain a selectable marker. Selectable markers include, for example, genes lacking their complement such as dihydrofolate reductase (DHFR) and Schizosaccharomyces pombe TPI gene, and ampicillin, kanamycin, tetracycline, chloramphenicol, Examples include resistance genes of drugs such as neomycin and hygromycin. Methods for ligating L-lysine decarboxylase / oxidase genes, promoters, and optionally terminators and / or secretory signal sequences, respectively, and inserting them into appropriate vectors are well known to those skilled in the art.

<Transformant>
A transformant can be prepared by introducing a recombinant vector containing the gene for L-lysine decarboxylation / oxidase into an appropriate host. The host cell into which the recombinant vector containing the gene for L-lysine decarboxylase / oxidase is introduced may be any cell as long as it can express the L-lysine decarboxylase / oxidase gene. Bacteria, yeast, fungi and higher eukaryotes Examples thereof include cells. Such a transformant is useful in that it can be used for the production of L-lysine decarboxylation / oxidase according to the present invention.

  Examples of bacterial cells include Gram positive bacteria such as Bacillus or Streptomyces or Gram negative bacteria such as E. coli. Transformation of these bacteria may be performed by using competent cells by a protoplast method or a known method. Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, CHO cells and the like. Methods for transforming mammalian cells and expressing DNA sequences introduced into the cells are also known, and for example, electroporation method, calcium phosphate method, lipofection method and the like can be used.

  Examples of yeast cells include cells belonging to Saccharomyces and Schizosaccharomyces, and examples thereof include Saccharomyces cerevisiae and Saccharomyces kluyveri. Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroplast method, a lithium acetate method, and the like.

  Examples of other fungal cells are cells belonging to filamentous fungi such as Aspergillus, Neurospora, Fusarium, or Trichoderma. When filamentous fungi are used as host cells, transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.

  When insect cells are used as the host, the recombinant gene transfer vector and baculovirus are co-introduced into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected with the insect cells. And protein can be expressed (for example, as described in Baculovirus Expression Vectors, A Laboratory Manual 1; and Current Protocols in Molecular Biology, Bio / Technology, 6, 47 (1988), etc.).

  As the baculovirus, for example, Autographa californica nucleopolyhedrosis virus, which is a virus that infects Coleoptera insects, can be used.

  Insect cells include Spodoptera frugiperda ovarian cells Sf9, Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York ( New York) (1992)], HiFive (manufactured by Invitrogen), which is an ovary cell of Trichoplusia ni, and the like can be used.

  Examples of the method for co-introducing a recombinant gene introduction vector into insect cells and the baculovirus for preparing a recombinant virus include a calcium phosphate method and a lipofection method.

  The transformant is cultured in an appropriate nutrient medium under conditions that allow expression of the introduced gene. In order to isolate and purify the L-lysine decarboxylase / oxidase used in the present invention from the culture of the transformant, ordinary protein isolation and purification methods may be used. For example, when the L-lysine decarboxylase / oxidase used in the present invention is expressed in a dissolved state in the cells, after completion of the culture, the cells are collected by centrifugation, suspended in an aqueous buffer, and then subjected to ultrasonic disruption. Cells are disrupted by a machine or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) sepharose, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), and a resin such as butyl sepharose and phenyl sepharose were used. A method such as hydrophobic chromatography, gel filtration using a molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, etc. may be used alone or in combination, and L-lysine decarboxylation / Oxidase can be obtained as a purified sample.

<Measurement method of L-lysine>
The method for measuring L-lysine in a sample according to the present invention includes:
(A) If necessary, the same steps as described above are performed for a plurality of samples in which the amount of L-lysine is clear, and the amount of L-lysine and the amount of the reaction product to be measured in the following step (D) The process of clarifying the relationship;
(B) A step of oxidizing cadaverine in the specimen by allowing putrescine oxidase to act on the specimen, if necessary;
(C) a step of allowing the L-lysine decarboxylation / oxidase of the present invention to act on a specimen; and (D) a step of measuring the amount of at least one reaction product resulting from the action of the L-lysine decarboxylation / oxidase. It is characterized by including.

  The above-described method of the present invention includes a detection method for determining the presence or absence of L-lysine in a sample, a quantitative method for measuring the concentration and amount of L-lysine in the sample, and the like.

  The biological sample used as a specimen in the L-lysine measurement method of the present invention may be any sample as long as it should be determined whether L-lysine is present or the amount of L-lysine should be measured. For example, biological samples such as blood, serum, plasma, homogenate of a part of an organ, urine and the like can be mentioned. In addition, the type of biological sample can be appropriately selected depending on the method for measuring the product produced by the action of L-lysine decarboxylase / oxidase on the biological sample. More specifically, when the product is quantified using a color former or a fluorescent agent, it is preferably a colorless aqueous solution, and examples thereof include serum and plasma. In the present invention, the “amount” of L-lysine and the like includes the concentration of L-lysine and the like.

  Hereinafter, the L-lysine measurement method according to the present invention will be described in the order of execution.

Step (A): Preparation of calibration curve In this step, if necessary, the same steps as those described below are performed for a plurality of samples in which the amount of L-lysine is clear, and the amount of L-lysine and the following step (D) The relationship with the amount of reaction product to be measured in is clarified. That is, in this process, a calibration curve is created as necessary.

  In the method for measuring L-lysine according to the present invention, the amount of the reaction product is measured in step (D), and the amount of L-lysine in the sample is indirectly determined. At this time, if the absolute amount of the reaction product is measured, the absolute amount of L-lysine can also be determined. However, measurement of the absolute amount of reaction product may not be easy. Therefore, by preparing a calibration curve in this step, the measurement of L-lysine is further simplified.

  The number of samples with a clear amount of L-lysine used in this step is preferably 3 or more as long as the amount of L-lysine expected to be contained in the specimen is sufficiently contained. As the number of samples increases, more accurate measurement is possible, so the number of samples is more preferably 4 or more, and even more preferably 5 or more. The upper limit of the number of samples is not particularly limited, but if it is too large, the measurement efficiency may be lowered, and therefore the number of samples is preferably 10 or less.

  In preparing the calibration curve, the reaction conditions such as the concentration of each reagent, reaction temperature, reaction time, etc. are made the same as the conditions for the subsequent steps, and the amount of reaction product is measured for the sample. A calibration curve is prepared according to a conventional method from the measured amount of the reaction product and the data of the amount of L-lysine in each sample that is known in advance.

Step (B): Cadaverine treatment step In this step, cadaverine in the specimen is oxidized by allowing putrescine oxidase to act on the specimen, if necessary.

  The L-lysine decarboxylase / oxidase according to the present invention has extremely high substrate specificity for L-lysine, and can accurately measure L-lysine even in a specimen containing various contaminants such as plasma. . However, the L-lysine decarboxylase / oxidase according to the present invention oxidizes using cadaverine as a substrate, though very little. Therefore, when cadaverine is included or may be included in the specimen, it is preferable to oxidize cadaverine in advance by performing this step. Conversely, if it is clear that the sample does not contain cadaverine, this step is not necessary.

Putrescine oxidase originally oxidizes putrescine ( ₂ HN- (CH ₂ ) ₄ —NH ₂ ) in the presence of water and oxygen to give 4-aminobutanal (OHC— (CH ₂ ) ₃ —NH ₂ ), Although it is an enzyme that catalyzes a reaction that generates ammonia and hydrogen peroxide, cadaverine ( ₂ HN— (CH ₂ ) ₅ —NH ₂ ) is also used as a substrate, and 5-aminopentanal (OHC— (CH ₂ ) ₄ —NH ₂ ) Catalyze the reaction to produce ammonia and hydrogen peroxide. That is, in this step, the following reaction proceeds.

  The putrescine oxidase is not particularly limited, and for example, putrescine oxidase derived from Kokuria rosea NBRC 3768 strain (formerly Micrococcus rubens IFO3768 strain) can be used.

  Reaction conditions such as pH of the reaction solution, reaction temperature, reaction conditions, and the amount of putrescine oxidase added are adjusted so that cadaverine contained in the sample is sufficiently oxidized. For example, since putrescine oxidase generally shows activity at pH 6.5 or higher, the pH of the reaction solution is set to 6.5 or higher. Specific conditions may be determined in a preliminary experiment or adjusted so that cadaverine in a sample cannot be confirmed by chromatography or the like.

  After the reaction, it is preferable to measure the amount of 5-aminopentanal, ammonia or hydrogen peroxide produced in order to determine the amount of cadaverine in the sample. The measurement conditions will be described later.

  Alternatively, since part or all of the hydrogen peroxide generated in this step may be decomposed, for example, when measuring the amount of hydrogen peroxide in step (D), the measurement of the amount of hydrogen peroxide Alternatively, or in addition to the measurement of the amount of hydrogen peroxide, the generated hydrogen peroxide may be actively decomposed with peroxidase or the like.

Step (C): Reaction step with L-lysine decarboxylation / oxidase In this step, L-lysine contained in the sample is removed by allowing the L-lysine decarboxylation / oxidase according to the present invention to act on the sample. Carbonate to cadaverine, and L-lysine is oxidized to produce L-2-aminoadipic acid 5-semialdehyde, ammonia and hydrogen peroxide.

  In order to allow the above reaction to proceed sufficiently, the amount of L-lysine decarboxylation / oxidase in the reaction solution in this step is preferably 1.0 mU or more per 1.0 mL of the reaction solution. In addition, 1U here shall mean the activity which consumes 1 micromol of lysine in 1 minute.

  It is preferable to use water as the solvent and adjust the pH of the reaction solution to a pH taking into consideration the optimum pH of L-lysine decarboxylation / oxidase using a buffer solution or the like. The pH may be adjusted as appropriate, but the L-lysine decarboxylase / oxidase according to the present invention shows activity at pH 5.0 or more and 8.5 or less, and can be used within this range.

  Further, in this step, cadaverine produced by the action of L-lysine decarboxylase / oxidase may be oxidized using putrescine oxidase in addition to L-lysine decarboxylase / oxidase. Thereby, L-lysine can be measured more accurately. Specifically, since L-lysine decarboxylase / oxidase exhibits both L-lysine decarboxylase activity and L-lysine oxidase activity, for example, cadaverine or L-2-aminoadipic acid produced from L-lysine Since the amount of 5-semialdehyde produced is different from the amount of L-lysine contained in the sample, when measuring the amount of cadaverine or L-2-aminoadipic acid 5-semialdehyde in the following step (D) It is necessary to prepare a calibration curve in advance by performing the step (A). However, when cadaverine produced from L-lysine is oxidized in the presence of putrescine oxidase, ammonia and hydrogen peroxide in an equimolar amount with cadaverine are produced, as in the reaction of step (B). That is, if L-lysine decarboxylase / oxidase and putrescine oxidase are used together and the reaction is sufficiently advanced, L-lysine and equimolar amounts of ammonia and hydrogen peroxide contained in the sample are produced. By measuring the amount of ammonia or hydrogen peroxide, it becomes possible to measure L-lysine in a sample more accurately.

  In order to sufficiently oxidize the produced cadaverine, the amount of putrescine oxidase in the reaction solution in this step is preferably 100 mU or more per 1.0 mL of the reaction solution. In addition, 1U here shall mean the activity which consumes 1 micromol of cadaverine in 1 minute.

  As described above, since L-lysine decarboxylase / oxidase according to the present invention exhibits activity at pH 5.0 or more and 8.5 or less, and putrescine oxidase exhibits activity at pH 6.5 or more, putrescine oxidase When is used, it is preferable to adjust the pH of the reaction solution to 6.5 or more and 8.5 or less.

  The reaction conditions in this step are adjusted so that the enzyme reaction sufficiently proceeds. For example, oxygen is required for the oxidation reaction of L-lysine by L-lysine decarboxylation / oxidase, and since the amount of oxygen required for the oxidation reaction is usually very small and can be sufficiently covered by dissolved oxygen, air or the like It is not necessary to supply the oxygen-containing gas to the reaction solution, but it may be supplied. The reaction temperature may be adjusted as appropriate, but the optimum temperature for L-lysine decarboxylation / oxidase is around 40 ° C., and can be, for example, 20 ° C. or more and 50 ° C. or less. Although the enzyme reaction time depends on the amount of enzyme used, it can be in the range of, for example, about 10 minutes to 1 hour. However, it is not intended to be limited to this range, and can be adjusted as appropriate.

Step (D): Measurement step of reaction product amount In this step, the amount of at least one reaction product produced by the action of the L-lysine decarboxylation / oxidase is measured. From the obtained measured value, the amount of L-lysine in the sample can be grasped.

  Specific examples of the reaction product resulting from the action of L-lysine decarboxylation / oxidase include cadaverine, L-2-aminoadipic acid 5-semialdehyde, ammonia, and hydrogen peroxide. In addition, when putrescine oxidase is used together in the above step (C), the produced cadaverine is converted to 5-aminoheptanal, and cadaverine, equimolar ammonia and hydrogen peroxide are separately produced.

The method for measuring the amount of the reaction product can be appropriately selected from known methods according to the selected reaction product. For example, when the reaction product to be measured is hydrogen peroxide, hydrogen peroxide can be quantified by a known method such as a method using a peroxidase reaction. When measuring using a peroxidase reaction, the peroxidase which can be used should just be an enzyme which can be utilized for fixed_quantity | quantitative_assay of hydrogen peroxide, for example, a horseradish origin peroxidase is mentioned. Moreover, if it can become a substrate of the peroxidase to be used, it can be used as a color former, and when using horseradish peroxidase, a combination of 4-aminoantipyrine and phenol, 4-aminoantipyrine and N-ethyl- A combination with N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS) can be mentioned as a color former. The color former can be appropriately selected depending on the type of peroxidase used. The reaction for hydrogen peroxide determination using horseradish peroxidase, 4-aminoantipyrine and phenol is as follows.
2H ₂ O ₂ + 4-aminoantipyrine + phenol → quinoneimine dye + 4H ₂ O

  When putrescine oxidase is used in the step (C), the number of moles of ammonia and hydrogen peroxide produced is the same as the number of moles of L-lysine contained in the sample. In addition, it is easier to measure the amount of hydrogen peroxide than ammonia. Therefore, when putrescine oxidase is used in the step (C), it is preferable to measure the amount of hydrogen peroxide.

  Hydrogen peroxide, which is a reaction product of L-lysine decarboxylation / oxidase, can also be measured using a current detection type sensor using a hydrogen peroxide electrode. As a current detection type sensor using a hydrogen peroxide electrode, for example, a sensor using a film in which peroxidase is immobilized on glutaraldehyde together with bovine serum albumin and ferrocene in a carbon paste is used as an electrode.

  When the reaction product to be measured is ammonia, it can be measured using an ammonia detection agent. Examples of the ammonia detection agent include an indophenol method using a combination of phenol and hypochlorous acid. Specifically, ammonia can be quantified by mixing a sample with a phenol / nitroprusside solution and a perchloric acid solution to develop a color and measuring the absorbance at 635 nm.

  When the reaction product used for quantification is L-2-aminoadipic acid 5-semialdehyde, which is a deamination product of L-lysine, 5-guanidino-2-oxopentanoic acid is converted to 3-methyl- By reacting with 2-benzothiazolinone hydrazone hydrochloride and spectroscopically measuring the hydrazone derivative, the amount of L-2-aminoadipic acid 5-semialdehyde can be measured.

<Measurement kit for L-lysine>
An L-lysine measurement kit for carrying out the L-lysine measurement method according to the present invention without using putrescine oxidase includes, for example, the following reagents.

  The kit for measuring L-lysine of the present invention is characterized by containing (K1-1) L-lysine decarboxylation / oxidase.

  The kit of the present invention further comprises (K1-2) a reagent for detecting hydrogen peroxide, (K1-3) an ammonia detector, and (K1-4) L-2-amino which is a deamination product of L-lysine. At least one of the adipic acid 5-semialdehyde detector may be included.

  (K1-2) Examples of the hydrogen peroxide detection reagent include one that quantifies hydrogen peroxide by color development. The color light may be fluorescent. The hydrogen peroxide detection reagent can be, for example, a combination of a peroxidase and a color former that can be a substrate thereof. Specifically, a combination of horseradish peroxidase, 4-aminoantipyrine and phenol can be mentioned.

  (K1-3) As an ammonia detection agent, the combination of hypochlorous acid with phenol can be mentioned. Such an ammonia detection agent makes it possible to detect ammonia by the indophenol method.

  (K1-4) L-2-aminoadipic acid 5-semialdehyde detection agent which is a deamination product of L-lysine includes 3-methyl-2-benzothiazolone hydrazine hydrochloride. For example, a hydrazone derivative produced by reacting L-2-aminoadipic acid 5-semialdehyde with 3-methyl-2-benzothiazolone hydrazine hydrochloride can be spectroscopically measured.

  The kit of the present invention may further contain (K1-5) a reaction buffer. The reaction buffer is used to maintain the reaction solution at a pH suitable for quantitative reaction. Since L-lysine decarboxylation / oxidase shows activity at pH 5.0 or more and 8.5 or less, the pH of the reaction buffer is preferably 5.0 or more and 8.5 or less. The pH range is more preferably 6.0 or more and 7.0 or less.

  The kit for measuring L-lysine for carrying out the method for measuring L-lysine according to the present invention using putrescine oxidase comprises (K2-1) L-lysine decarboxylase / oxidase and (K2-2) putrescine oxidase. including. Examples of (K2-2) putrescine oxidase include putrescine oxidase derived from Kokuria rosea NBRC 3768 strain.

  (K2-3) Reagent for detecting hydrogen peroxide, (K2-4) Ammonia detector and (K2-5) L-2-aminoadipic acid 5-semialdehyde detector which is a deamination product of L-lysine At least one of the following. These explanations are the same as those of the L-lysine measurement kit for carrying out the L-lysine measurement method according to the present invention without using putrescine oxidase.

  The kit of the present invention may further contain (K2-6) a reaction buffer. The reaction buffer is used to maintain the reaction solution at a pH suitable for quantitative reaction. In general, L-lysine decarboxylase / oxidase exhibits activity at pH 5.0 or more and 8.5 or less, and putrescine oxidase exhibits activity at pH 6.5 or more, so the pH of the reaction buffer is 6. It is preferably 0 or more and 8.5 or less.

<Enzyme sensor for L-lysine measurement>
The enzyme sensor for L-lysine measurement according to the present invention has an electrode for hydrogen peroxide detection, and the L-lysine decarboxylation / oxidase according to the present invention is disposed on or near the surface of the hydrogen peroxide detection electrode. It is characterized by being.

  The hydrogen peroxide detection electrode detects hydrogen peroxide by detecting electrons generated when hydrogen peroxide decomposes into water and oxygen. The hydrogen peroxide detection electrode can be an enzymatic hydrogen peroxide electrode or a diaphragm hydrogen peroxide electrode.

  According to the enzyme sensor for L-lysine measurement, hydrogen peroxide is generated by reacting L-lysine decarboxylation / oxidase with L-lysine, and this hydrogen peroxide is detected by the hydrogen peroxide detection electrode. can do. Examples of the enzyme hydrogen peroxide electrode include a film in which peroxidase is immobilized on glutaraldehyde together with bovine serum albumin and an electrode containing ferrocene in carbon paste. The diaphragm type hydrogen peroxide electrode is a type of electrode in which an electrode that reacts with hydrogen peroxide is isolated by a diaphragm.

  In the enzyme sensor according to the present invention, the L-lysine decarboxylase / oxidase is preferably disposed on the surface of the detection electrode or in the vicinity of the detection electrode, and when disposed on the surface of the detection electrode, It may or may not be immobilized on the surface of the detection electrode. By being immobilized on the surface of the detection electrode, there is an advantage that the sensor of the present invention can be used repeatedly.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Example 1: Search for L-lysine decarboxylation / oxidase producing bacteria according to the present invention (1) Screening Soil samples were collected from various places in Japan, and L-lysine oxidase activity of microorganisms contained in each soil under the following conditions Were screened for microorganisms having L-lysine oxidase activity.

  L-lysine oxidase activity was measured by determining the amount of hydrogen peroxide produced by the oxidation reaction of L-lysine with an enzyme by a colorimetric method. Specifically, the supernatant obtained by crushing and centrifuging the cells of each cultured microorganism was used as a crude enzyme solution. 0.1 mL of the crude enzyme solution and 0.9 mL of a reaction solution for measuring oxidase activity obtained by mixing the compounds shown in Table 1 were placed in a 1 cm quartz cell and reacted at 30 ° C. Using an absorptiometer, absorbance at 555 nm was continuously measured at intervals of about 1 second from the start of the reaction. As a substrate, a 5 mM aqueous solution of L-lysine was prepared and added. In the blank, 100 mM potassium phosphate buffer (pH 7.0) was added instead of the substrate.

Based on the obtained change in absorbance, the oxidase activity was calculated based on the following formula. The amount of enzyme that gives 1 micromole of substrate per minute under the above conditions was 1 U.
Oxidase activity value (U / mL) = {ΔOD / min (ΔODtest−ΔODblank) × 3.1 (mL) × dilution factor} / {13 × 1.0 (cm) × 0.1 (mL)}
3.1 (mL): total liquid volume 13: millimolar extinction coefficient 1.0 (cm): optical path length of cell 0.1 (mL): volume of enzyme sample liquid

  As a result, AIU395 strain having L-lysine oxidase activity was found. The AIU395 strain was examined as follows.

(2) 16S rDNA base sequence Genomic DNA was extracted from the AIU395 strain according to a conventional method, 16S rDNA was amplified by PCR using the obtained genomic DNA as a template, and the base sequence was determined. The 16S rDNA base sequence of the microorganism is shown in SEQ ID NO: 9. Based on the determined sequence, microorganisms with high homology were searched by Apollon DB-BA9.0 Blast. The results are shown in Table 2.

  FIG. 1 shows a simple molecular phylogenetic tree based on the 16S rDNA base sequence of the microorganism. As shown in Table 2 and FIG. 1, the 16S rDNA base sequence of AIU395 strain showed high homology to the 16S rDNA base sequence of Burkholderia genus. Although it showed the highest homology of 99.9% with respect to the arboris R-24201 strain, it did not match the 16S rDNA base sequence of any reference strain. In addition, as a result of simple molecular phylogenetic analysis based on the 16S rDNA base sequence using the top 10 base sequences obtained by homology search for Apollon DB-BA9.0, AIU395 strain is included in the cluster formed by Burkholderia species. B. It formed a cluster with arboris and was shown to be closely related.

  As described above, it was revealed that the AIU395 strain according to the present invention is a novel microorganism belonging to the genus Burkholderia.

(3) Physiological / biochemical property test The physiological / biochemical property of the AIU395 strain according to the present invention was tested. The results of the primary test are shown in Table 3, and the results of the secondary test are shown in FIG.

  As a result of the first test, AIU395 strain was a gram-negative gonococcus having motility, did not oxidize glucose, and was positive for catalase reaction and negative for oxidase reaction (Table 3). As a result of the second test, AIU395 strain reduced nitrate, showed no activity such as arginine dihydrolase and urease, did not hydrolyze esculin and gelatin, showed β-galactosidase activity, glucose, L-arabinose and D -Mannose and others were assimilated (Fig. 2). Further, as a result of additional tests, it did not grow at 42 ° C., but grew on McConkey agar, and β-hemolyzed sheep blood (Table 4). These properties are related to B. cerevisiae, which was suggested in the results of 16S rDNA nucleotide sequence analysis. Although similar to the properties of arboris, differences were also confirmed. In particular, esculin and gelatin are not hydrolyzed. It was different from the properties of arboris.

  From the above results, the AIU395 strain according to the present invention is B.I. Burkholderia sp. most closely related to arboris B. It was judged to be a new one different from arboris.

Example 2: Acquisition of L-lysine decarboxylase / oxidase according to the present invention (1) Examination of inducer of L-lysine oxidase derived from AIU395 strain 0.3% glucose, 0.2% potassium dihydrogen phosphate 6-aminohexanoic acid, L-lysine and putrescine shown in Table 5 as a nitrogen source in a medium (pH 7.0) containing 0.1% sodium hydrogenphosphate and 0.05% magnesium sulfate heptahydrate Cadaverine or ammonium sulfate was added, respectively. The medium was inoculated with Burkholderia sp. AIU395 strain obtained in Example 1 and cultured at 30 ° C. for 36 hours, and the culture medium pH, culture turbidity, L-lysine oxidase after culture Activity was measured. The results are shown in Table 5.

  As shown in Table 5, the highest L-lysine oxidase activity was observed in the medium supplemented with 6-aminohexanoic acid (hereinafter abbreviated as “6-AHA”). Therefore, in subsequent experiments, 6-AHA was added to the medium to induce L-lysine decarboxylation / oxidase.

(2) Culture time of AIU395 strain Burkholderia sp. AIU395 strain of Example 1 above was transformed into 6-AHA medium (0.5% 6-AHA, 0.3% glucose, 0.2%). Potassium dihydrogen phosphate, 0.1% sodium hydrogen phosphate, 0.05% magnesium sulfate heptahydrate, pH 7.0) and inoculated at 30 ° C. and 115 strokes / minute for 4 days. Culture turbidity (660 nm), medium pH, and L-lysine oxidase activity were measured every 0, 12, 24, 36, 48, 72, and 96 hours. The results are shown in FIG. From the relationship between culture turbidity and L-lysine oxidase activity, the cells were collected after 36 hours of culture and purified.

(3) Purification of AIU395 strain-derived L-lysine oxidase (3-1) Cultivation of AIU395 strain Burkholderia sp. AIU395 strain of Example 1 was planted in 5 mL of the 6-AHA medium. The cells were precultured at 30 ° C. and 115 strokes / min for 2 days. Thereafter, the cells were cultured in a 500 mL flask containing 150 mL of 6-AHA medium under the same conditions for 2 days. As the main culture, AIU395 strain pre-cultured in a 3 L flask containing 2 L of 6-AHA medium was inoculated and cultured at 30 ° C. for 36 hours. After culturing, the cells were centrifuged at 10,000 × g for 10 minutes and washed with 10 mM potassium phosphate buffer (pH 6.0, hereinafter abbreviated as “KPB”) to obtain bacterial cells.

(3-2) Preparation of cell-free extract Suspended cells of 8 L of medium in KPB, and using a cell crusher ("Multi Bead Shocker (registered trademark)" manufactured by Yasui Kikai Co., Ltd.) at 2,200 rpm The operation of crushing the cells for 2 minutes was repeated 4 times. Next, the resultant was centrifuged at 10,000 × g for 15 minutes, and the resulting supernatant was recovered. The pellet obtained by centrifugation was suspended again in 40 mM KPB (pH 6.0), and the same operation was repeated three times. All the supernatants (total 2,150 mL) were collected and used as cell-free extracts.

(3-3) Anion exchange column chromatography using DEAE resin Column (diameter) packed with DEAE resin (manufactured by TOSOH, “TOYOPEARL (registered trademark) DEAE-650”) equilibrated with 40 mM KPB (pH 6.0) The cell-free extract was adsorbed to 2.4 cm × 27 cm. After washing the column with 40 mM KPB (pH 6.0), the enzyme was eluted with 40 mM KPB (pH 6.0) containing 0.2 M NaCl. The obtained enzyme solution was dialyzed against 10 mM KPB (pH 6.0).

(3-4) Anion exchange column chromatography using agarose beads Column packed with agarose beads (GE Healthcare Bioscience, “Q Sepharose”) equilibrated with 40 mM KPB (pH 6.0) containing 0.14 M NaCl. The enzyme solution was adsorbed on (diameter 2.4 cm × 11 cm). After washing with 40 mM KPB (pH 6.0) containing 0.14 M NaCl, 250 mL of 40 mM KPB (pH 6.0) containing 0.14 M NaCl and 250 mL of 40 mM KPB (pH 6.0) containing 0.26 M NaCl were used. The NaCl concentration was gradually increased by the gradient to elute the enzyme. Using a fraction collector, the fraction was collected in a test tube, and the fractions showing activity were collected. The fraction from which activity was obtained was dialyzed against 10 mM KPB (pH 6.0).

(3-5) Anion-exchange column chromatography using DEAE resin DEAE resin equilibrated with 40 mM KPB (pH 6.0) containing 0.1 M NaCl (TOOSOH, “TOYOPEARL (registered trademark) DEAE-650”) The above enzyme solution was adsorbed on a column (diameter 1.2 cm × 36 cm) packed with. After washing with 40 mM KPB (pH 6.0) containing 0.1 M NaCl, 200 mL of 40 mM KPB (pH 6.0) containing 0.1 M NaCl and 200 mL of 40 mM KPB (pH 6.0) containing 0.24 M NaCl were used. The NaCl concentration was gradually increased by the gradient to elute the enzyme. Using a fraction collector, the fraction was collected in a test tube, and the fractions showing activity were collected. The fraction from which activity was obtained was dialyzed against 10 mM KPB (pH 6.0).

(3-6) Hydroxyapatite column chromatography The enzyme solution was adsorbed onto a column (diameter 1.2 cm × 18 cm) packed with hydroxyapatite resin equilibrated with 10 mM KPB (pH 6.0). Using 100 mL of 10 mM KPB (pH 6.0) and 100 mL of 0.1 M KPB (pH 6.0), the KPB concentration was gradually increased by a gradient to elute the enzyme. Using a fraction collector, the fraction was collected in a test tube, and the fractions showing activity were collected. The fraction from which activity was obtained was dialyzed against 10 mM KPB (pH 6.0). The above purification status is summarized in Table 6.

(4) Molecular weight measurement of L-lysine oxidase derived from the strain by SDS-PAGE As an electrophoresis gel, 5.25 mL of 36% acrylamide, 8.25 mL of 0.68 M Tris-HCL buffer (pH 8.8), 1% SDS 1 In a gel having a composition of .58 mL, 10% TEMED 187 μL, 2% APS 562.5 μL, 36% polyacrylamide 0.5 mL, 0.179 M Tris-HCl (pH 6.8) 3.5 mL, 1% SDS 0.5 mL 10% TEMED 125 [mu] L, 2% APS 375 [mu] L overlaid concentrated gel was used, buffer solution (glycerol 200 [mu] L, 1M Tris-HCl (pH 8.0) 40 [mu] L, water 360 [mu] L, 2-mercaptoethanol 200 [mu] L and 10 % SDS 200 μL) and the above purified enzyme sample 10 L, and the conducted running buffer (Tris 3.0 g, Glycine 14.1g and SDS 10 g), to electrophoresis with 30 mA. Thereafter, the gel is stained with a protein staining solution (CBB 2.5 g, methanol 500 mL, acetic acid 50 mL and water 450 mL) for 1 hour, and decolorized with a decoloring solution (methanol: acetic acid: water = 3: 1: 6) until the band becomes clear. did. As molecular weight markers, molecular weight markers including recombinant proteins of 20 kDa, 25 kDa, 37 kDa, 50 kDa, 75 kDa, 100 kDa, 150 kDa, and 250 kDa were used.

  FIG. 4 shows a photograph of SDS-PAGE of L-lysine oxidase derived from Burkholderia sp. AIU395 strain. As is apparent from SDS-PAGE, the enzyme was purified to a single protein by the above method, and the properties were revealed using the purified enzyme.

(5) Absorption spectrum analysis of L-lysine oxidase derived from AIU395 strain The absorption spectrum of the purified enzyme of L-lysine oxidase was measured. The results are shown in FIG. Absorption maximums were observed at 279 nm and 415 nm, suggesting that this enzyme is a pyridoxal 5′-phosphate (PLP) -dependent enzyme.

(6) Substrate Specificity of L-Lysine Oxidase Derived from AIU395 Strain The substrate in the reaction solution for measuring oxidase activity of Example 1 (1) above was changed to the amino acid and amine organic compound shown in Table 7 and purified as described above. The oxidase activity of the enzyme was measured. The results are shown in Table 7.

  As shown in Table 7, this enzyme has an extremely high oxidative activity against L-lysine, but only slightly uses 5-hydroxy-L-lysine and cadaverine as substrates, and is completely active against other amino acids and amines. The substrate specificity was high.

(7) Measurement of L-lysine decarboxylase activity L-lysine decarboxylase activity is obtained by oxidizing cadaverine produced by decarboxylation reaction with putrescine oxidase and generating equimolar amounts with the reacted cadaverine. The amount of hydrogen peroxide to be measured was determined by a colorimetric method. Specifically, in a 1 cm quartz cell, 0.9 mL of a decarboxylase activity measurement reaction solution shown in Table 8 containing an L-lysine solution corresponding to the base mass shown in Table 8 as a substrate was mixed with 1 mL of enzyme solution, The reaction was performed at 30 ° C. Using an absorptiometer, absorbance at 550 nm was continuously measured at intervals of about 1 second from the start of the reaction. The same experiment was performed without adding putrescine oxidase. In the blank, 100 mM potassium phosphate buffer (pH 7.0) was added instead of the substrate.

Based on the change in absorbance obtained, decarboxylase activity was calculated based on the following formula. The amount of enzyme that gives 1 micromole of substrate per minute under the above conditions was 1 U.
Decarboxylase activity value (U / mL) = {ΔOD / min (measured value when putrescine oxidase is added) −ΔOD / min (measured value when no putrescine oxidase is added)} × 3.1 (mL ) × dilution ratio} / {13 × 1.0 (cm) × 0.1 (mL)}
3.1 (mL): total liquid volume 13: millimolar extinction coefficient 1.0 (cm): optical path length of cell 0.1 (mL): volume of enzyme sample liquid

  As a result, it was revealed that L-lysine oxidase isolated from AIU395 strain also has L-lysine decarboxylase activity.

(8) In AIU395 strain derived L- Lysine decarboxylation / K _m and V _max measured above oxidase activity measurement method and decarboxylase activity measuring method of the oxidase, measure each enzyme activity by changing the concentration of the substrate As a result, K _m and V _max were obtained. In the measurement of oxidase activity, in addition to L-lysine, 5-hydroxy-L-lysine and cadaverine, which were slightly active, were used as substrates. In the measurement of decarboxylase activity, only L-lysine was used as a substrate. The results are shown in Table 9.

  As shown in Table 9, this enzyme was found to have a very high affinity for L-lysine. In addition, the ratio of the decarboxylase activity and the oxidase activity of this enzyme was 0.79: 122 when compared at the maximum rate, and it was found that the decarboxylase activity was higher. Furthermore, the reaction rate was significantly slow, especially at low concentrations of 5-hydroxy-L-lysine and cadaverine, demonstrating the high substrate specificity of the enzyme for L-lysine.

(9) Determination of N-terminal amino acid sequence of L-lysine decarboxylase / oxidase from AIU395 strain Nippi Co., Ltd. was requested to analyze the N-terminal amino acid sequence of the purified enzyme, and 15 residues were determined from the N-terminal side. It was.

Example 3: Examination of optimum pH and pH stability of L-lysine decarboxylase / oxidase In the above-mentioned L-lysine oxidase activity method, the pH of the reaction solution was changed to 5 to 8.5 by changing the buffer solution. The oxidase activity of L-lysine decarboxylase / oxidase according to the present invention was measured. The results are shown in FIG.

  As shown in FIG. 6, the L-lysine decarboxylase / oxidase according to the present invention showed the highest activity at pH 6.0 and was found to be stable from pH 5.0 to around pH 7.0. In addition, the vertical axis | shaft of FIG. 6 shows the relative activity value when the activity value of pH 6.0 is set to 100% about L-lysine oxidase activity, and the activity value of pH 6.5 is set to 100% about residual activity. The relative activity value is shown. Incidentally, the broken line indicates the relative activity of putrescine oxidase with respect to pH.

Example 4 Examination of Optimum Temperature and Thermal Stability of L-Lysine Decarboxylase / Oxidase In the above L-lysine oxidase activity method, the reaction temperature was changed to 20-60 ° C., and L-lysine according to the present invention was used. The oxidase activity of decarboxylation / oxidase was measured. The results are shown in FIG.

  As shown in FIG. 7, L-lysine decarboxylase / oxidase according to the present invention showed the highest activity at 50 ° C. and was stable up to around 45 ° C. In addition, the vertical axis | shaft of FIG. 7 is a relative activity value when the activity value in 50 degreeC is set to 100% about L-lysine oxidase activity, and the activity value in 30 degreeC is set to 100% about residual activity Is the relative activity value.

Example 5: Examination of inhibitors of L-lysine decarboxylase / oxidase In the above-mentioned L-lysine decarboxylase activity method, an inhibitor was added and the oxidase activity of L-lysine decarboxylase / oxidase according to the present invention. Was measured. The results are shown in Table 10.

As shown in Table 10, in the enzyme of the present invention, a significant decrease in oxidase activity was observed with fanylhydrazine and hydroxylamine, which are inhibitors of PLP-dependent enzymes. In addition, inhibition of the oxidation reaction was observed with CuCl ₂ and ZnCl ₂ .

Example 6: Acquisition of recombinant L-lysine decarboxylase / oxidase according to the present invention (1) Cloning of AIU395 strain-derived L-lysine decarboxylase / oxidase gene (1-1) Extraction of chromosomal DNA of AIU395 strain Burkholderia sp. AIU395 strain of Example 1 was inoculated into 3 mL of TGY medium (0.5% tryptone, 0.25% yeast extract, 0.1% glucose, pH 7.0), The cells were cultured at 30 ° C. and 200 rpm for 12 hours. 1 mL of the culture solution was centrifuged at 15,000 rpm and 4 ° C. for 5 minutes to collect bacteria. The cells were washed with 1 mL of STE buffer (NaCl 0.58 g, 1 M Tris-HCl (pH 8.0) 1 mL and 0.5 M EDTA (pH 8.0) 200 μL quantified to 100 mL with water), and then the same buffer. It was suspended in the liquid. After heating at 68 ° C. for 15 minutes, the mixture was centrifuged at 15,000 rpm and 4 ° C. for 5 minutes, and the supernatant was removed. Separately, a solution containing 5 mg of lysozyme and 10 mL of 10 mg / mL RNase (hereinafter, this solution is referred to as “one solution”) was prepared. In addition, ultrapure water was added to a solution containing 0.9 g of glucose, 2.5 mL of 1 M Tris-HCl (pH 8.0), and 2 mL of 0.5 M EDTA (pH 8.0) to make the total amount 100 mL. 1 mL of the solution and 10 mL of the first solution were mixed. The centrifugal precipitate was suspended in 300 μL of the mixed solution. After incubating at 37 ° C. for 30 minutes, 6 μL of proteinase K solution (proteinase K 10 mg / 1 solution 1 mL) was added, mixed gently, and incubated at 37 ° C. for 10 minutes. After adding 3 mg of N-lauroylsarcosine and mixing gently, it incubated at 37 degreeC for 3 hours, and performed phenol-chloroform treatment twice gently. 10 μL of 5M NaCl solution and 600 μL of ethanol were added to 300 μL of the supernatant and mixed, followed by centrifugation at 15,000 rpm and 4 ° C. for 10 minutes. After washing with 70% ethanol, it was air-dried and dissolved in 100 μL of TE buffer to obtain the target chromosomal DNA.

(1-2) AIU395 strain-derived L-lysine decarboxylation / oxidase gene internal sequence amplification The composition of the PCR reaction solution was water 35 μL, 10 × LA Taq buffer 5 μL, 2 mM dNTP 5 μL, 100 pmol primer 1 (5′-ATGACCGCTCTCTTGACCCAAC −3 ′, SEQ ID NO: 3) 1 μL, 100 pmol primer 2 (5′-TGCACGCGATCCGGTACACCGCC-3 ′, SEQ ID NO: 4) 1 μL, template DNA 2 μL and LA Taq 1 μL. The conditions for the PCR reaction were (ii) 98 ° C. for 5 minutes, (ii) 96 ° C. for 10 seconds, (iii) 50 ° C. for 5 seconds, (iv) 72 ° C. for 2 minutes (ii) to (iv) ) Was repeated 30 times. The amplified gene was confirmed by agarose gel electrophoresis. The amplified gene was extracted using a Gel-M gel extraction kit manufactured by VIOGENE (USA).

(1-3) Sequencing of L-Lysine Decarboxylase / Oxidase Gene Internal Sequence from AIU395 Strain In order to perform sequencing on both strands of the gene, a sequence reaction was performed using primer 1 and primer 2. The reaction solution composition was 1.6 μL of each primer, 1.6 μL of template DNA, 1 μL of BigDye premix solution, 1.5 μL of 5 × BigDye sequencing buffer and 4.9 μL of sterilized water for a total volume of 10 μL. The conditions of the PCR reaction were (i) 96 ° C. for 2 minutes, (ii) 96 ° C. for 10 seconds, (iii) 50 ° C. for 5 seconds, (iv) 60 ° C. for 4 minutes, (v) (ii) to ( iv) 25 times, and (vi) 5 minutes at 72 ° C. By adding 1 μL of 3M sodium acetate (pH 5.2), 1 μL of 0.125M EDTA and 25 μL of ethanol to the PCR product, leaving it at room temperature for 15 minutes, and then centrifuging at 15,000 rpm, 4 ° C. for 8 minutes. Precipitated. After discarding the supernatant, 10 μL of Hi Di Formamide was added, heated at 100 ° C. for 5 minutes, and then rapidly cooled with ice water, and the base sequence was decoded with ABI PRISM 3500 Genetic Analyzer. The obtained sequence data was analyzed by Genetyx, and the fragments amplified with the respective primers were ligated.

(1-4) Amplification of L-lysine decarboxylase / oxidase gene terminal sequence derived from AIU395 strain Primer 3 (5′-ATGTTCCGGATCACGACGTCGACGAGCCCG-3 ′, SEQ ID NO: 5) and Primer 4 (5′-GCTGATGCCCGCGCGAGAACGCGGGG-3 ′, sequence No. 6) was used to amplify the terminal sequence according to the manual of TaKaRa LA PCR ™ Cloning Kit (Takara Bio). Sequencing was performed as described above. SEQ ID NO: 2 shows the primary structure predicted from the L-lysine decarboxylation / oxidase gene derived from Burkholderia sp. AIU395 strain (FIG. 8).

(1-5) Construction of AIU395 strain-derived L-lysine decarboxylation / oxidase gene expression system Sterile water (MilliQ) as a PCR reaction solution for amplifying the L-lysine decarboxylation / oxidase gene (SEQ ID NO: 1) 34 μL, Primer 5 (5′-GGAGATATACATGACCGCCTTCCTTGACCCAAC-3 ′, SEQ ID NO: 7,100 pmol), 1 μL Primer 2 (5′-TTAGCAGCCGGATCCTCACCCGCGTTGTTGTATCGC-3 ′, product OD OD manufactured by TOY K -Plus- ", 1 U / μL aqueous solution) 1 μL, 10 × PCR buffer, 2 mM dNTPs aqueous solution 5 μL, 25 mM magnesium chloride aqueous solution 2 μL, and template DNA 1 μL were mixed. The PCR reaction conditions were (i) 94 ° C for 2 minutes, (ii) 94 ° C for 15 seconds, (iii) 55 ° C for 30 seconds, (iv) 68 ° C for 2 minutes, (i) and (ii) Was repeated 30 cycles. The amplified gene was confirmed by agarose gel electrophoresis. The amplified gene was extracted using a DNA purification kit (manufactured by Promega, product name “Wizard (registered trademark) Genomic DNA Purification Kit).

(1-6) Recombination of AIU395 strain-derived L-lysine decarboxylase / oxidase gene into vector 1 μL of pET11a treated with restriction enzymes NdeI and BamHI, PCR product of L-lysine decarboxylase / oxidase gene obtained above 3 μL and 1 μL of a cloning kit (product name “5 × In-Fusion HD Enzyme Premix”, manufactured by Takara Bio Inc.) were mixed and reacted at 50 ° C. for 15 minutes, whereby L-lysine decarboxylation / oxidation was performed on the plasmid vector pET11a. An enzyme gene was introduced. 5 μL of the above reaction solution was added to 50 μL of E. coli JM109 (DE3) competent cell, and transformation was performed by the heat shock method. Several strains were selected from colonies grown on LB medium containing 100 μg / mL ampicillin, plasmids were extracted, and the presence or absence of L-lysine decarboxylase / oxidase gene introduction was confirmed by 0.7% agarose electrophoresis.

(1-7) Expression and purification of AIU395 strain-derived L-lysine decarboxylation / oxidase gene 4 L of LB medium (1.0% polypeptone, 0.5% yeast extract, 1.0) containing 80 μg / mL ampicillin % NaCl, pH 7.0), inoculated with recombinant E. coli (BL21), cultured at 16 ° C. for 12 hours, added with 0.5 mM IPTG, and subsequently cultured at 30 ° C. for 12 hours to obtain L-amino acid oxidase. Induced. Bacteria were collected by centrifugation at 5,000 rpm and 4 ° C. for 10 minutes using a large centrifuge, washed with physiological saline (0.9% NaCl), and then 100 mL of 20 mM potassium phosphate buffer (pH 7. 0). A supernatant obtained by sonicating 100 mL of the cell solution for 15 minutes and centrifuging at 8,000 rpm, 4 ° C. for 20 minutes was used as a cell-free extract. The cell-free extract was purified by the same method as in Example 2 (3-3) to (3-6).

(2) Activity measurement of AIU395 strain-derived recombinant L-lysine decarboxylation / oxidase The oxidase activity of the purified enzyme was measured with a measurement reagent containing each of the amino acids and amines in Table 7. As a result, similar to the results in Table 7, the L-lysine decarboxylase / oxidase according to the present invention oxidizes using L-lysine as a substrate, while using 5-hydroxy L-lysine and cadaverine slightly as substrates. Only, it did not show any activity against other amino acids. Thus, it was revealed that the L-lysine decarboxylase / oxidase according to the present invention exhibits high specificity for L-lysine even in recombinant enzymes.

Example 7: Quantification of L-lysine using AIU395 strain-derived L-lysine decarboxylation / oxidase The reagent for measuring oxidase activity of Example 1 (1) was prepared. Next, in Example 1 (1) above, the change in absorbance was measured by changing the amount of L-lysine decarboxylation / oxidase added. FIG. 9 shows the change in absorbance after 20 minutes for each addition amount of L-lysine decarboxylation / oxidase. According to the result shown in FIG. 9, it was suggested that it can be used for L-lysine determination without problems at 1.3 munit or more.

  Moreover, L-lysine measurement was implemented using the said reagent composition. As a sample, an L-lysine aqueous solution having a final concentration in the reaction solution of 0 to 1.0 mM was prepared. FIG. 10 shows the change in absorbance after 20 minutes at each concentration of L-lysine.

  As shown in FIG. 10, when the L-lysine solution was used as a specimen, sufficient reactivity was observed, and in the aqueous solution, the L-lysine concentration and the absorbance measurement data showed a good positive correlation. Therefore, it was demonstrated that accurate L-lysine measurement can be performed by using the reagent composition for L-lysine measurement of the present invention.

Example 8: Quantification of cadaverine with putrescine oxidase As described in Example 2 (7) above, putrescine oxidase can be used to quantify cadaverine produced by decarboxylation of L-lysine. By combining with such L-lysine decarboxylation / oxidase, L-lysine in the sample can be quantified. Therefore, the putrescine oxidase was examined in detail.

  Specifically, in Example 2 (7) above, changes in absorbance were measured using 60 μM cadaverine as a substrate and varying the amount of putrescine oxidase added. FIG. 11 shows changes in absorbance after 20 minutes or 30 minutes with respect to each addition amount of putrescine oxidase. From these results, it was suggested that if putrescine oxidase of 200 munit or more is used, it can be used for cadaverine quantification without problems.

Example 9: Effect of other amino acids and amines on the coupling of L-lysine decarboxylase / oxidase and putrescine oxidase according to the present invention Table 11 shows 30 μM L-lysine alone or 30 μM L-lysine And L-lysine decarboxylase / oxidase and putrescine oxidase according to the present invention were added to a solution in which an amino acid or amine other than L-lysine and 30 μM were mixed at the same ratio as in Example 2 (7) above, to pH 7 The result of having reacted at 0.0 for 30 minutes and measuring the absorbance at 555 nm is shown.

  As shown in Table 11, when the L-lysine decarboxylase / oxidase according to the present invention is used, L-lysine quantification by coupling with putrescine oxidase even in the presence of amino acids and amines other than L-lysine. Was found to be possible.

Example 10: Examination of the addition amount of L-lysine decarboxylation / oxidase according to the present invention The addition amount of L-lysine decarboxylation / oxidase using 200 μU of putrescine oxidase with 60 μM L-lysine as a substrate The reaction was carried out at pH 7.0 for 30 minutes, and the absorbance at 555 nm was measured. The results are shown in FIG.

  As shown in the results shown in FIG. 12, it was revealed that L-lysine can be sufficiently measured with an addition amount of 1.0 mU or more when the L-lysine decarboxylation / oxidase according to the present invention is used.

Example 11: L-lysine quantification using L-lysine decarboxylase / oxidase according to the present invention L-lysine decarboxylase / oxidase (1.5 mU) and PUO (200 mU) for 0-60 μM L-lysine ) Was added and reacted at pH 7.0 for 30 minutes, and the absorbance at 555 nm was measured. The results are shown in FIG.

  As a result shown in FIG. 13, linearity was obtained in a calibration curve using the L-lysine decarboxylation / oxidase according to the present invention.

Example 12: Method for measuring L-lysine using putrescine oxidase in the presence of cadaverine Since cadaverine produced by decarboxylation of L-lysine is also generated in vivo, when cadaverine is present in the sample, There is a possibility that L-lysine contained therein cannot be measured accurately. Therefore, as shown in FIG. 14 (1), 0 to 60 nmol of L-lysine as a substrate together with 60 mmol of cadaverine, 200 mU putrescine oxidase, 0.12 μmol 4-AA, 1.8 U peroxidase, 0.75 mmol phosphoric acid It was mixed with a reaction solution I (pH 7.0) containing potassium (total amount: 750 μL) and reacted at 30 ° C. for 20 minutes (Reaction I). Then, reaction solution II (250 μL) containing 2.3 U of L-lysine decarboxylase / oxidase and 0.38 μmol TOOS was added, and the mixture was reacted at 30 ° C. for 30 minutes (Reaction II). The amount of hydrogen oxide was confirmed by absorption at 555 nm.

  As shown in FIG. 14 (2), there is a correlation between the L-lysine concentration in the sample and the change in absorbance due to the amount of hydrogen peroxide produced. This is because even if cadaverine is present in the sample, it is oxidized by putrescine oxidase in the above Reaction I, and hydrogen peroxide generated by the reaction is decomposed by peroxidase. This is because there is no influence on the measurement result of L-lysine. From these results, it was clarified that cadaverine existing in the sample can be removed by adding putrescine oxidase first, and L-lysine quantification is not affected.

Example 13: Activity of L-lysine decarboxylation / oxidase derived from Burkholderia bacteria Under the same conditions as in Example 2 above, Burkholderia sp. AIU129 strain and Burkholderia ginsengolii NBRC100655, Burkholderia ferrariae NBRC106233, and Burkholderia terrae NBRC100964, which were purchased from the microbial strain storage organization NBRC, were prepared and cultured. In addition, although the optimal culture | cultivation temperature of each strain differs, culture | cultivation temperature was 25 degreeC. About each obtained enzyme, it carried out similarly to the said Example 1 and Example 2, and measured the oxidase activity and decarboxylase activity with respect to L-lysine. The results are shown in Burkholderia sp. It shows in Table 12 with the result of AIU395 strain | stump | stock.

  As shown in Table 12, both Burkholderia bacteria showed both L-lysine decarboxylase activity and L-lysine oxidase activity. Therefore, it was suggested that L-lysine decarboxylation / oxidase may be conserved in Burkholderia bacteria.

Claims (13)

An L-lysine decarboxylase / oxidase having an amino acid sequence of any one of (1) to (3) below:
(1) the amino acid sequence set forth in SEQ ID NO: 2;
(2) an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids and having L-lysine decarboxylase / oxidase activity in the amino acid sequence shown in SEQ ID NO: 2;
(3) An amino acid sequence having 95% or more homology with the amino acid sequence set forth in SEQ ID NO: 2 and having L-lysine decarboxylation / oxidase activity.   The L-lysine decarboxylation / oxidase according to claim 1, which is derived from a bacterium belonging to the genus Burkholderia.   The L-lysine decarboxylation / oxidase according to claim 1, which is derived from Burkholderia sp. AIU395 strain (Accession number: NITE P-01796). A nucleic acid having any one of the following base sequences (1 ′) to (3 ′):
(1 ′) the nucleotide sequence set forth in SEQ ID NO: 1 (2 ′) the nucleotide sequence set forth in SEQ ID NO: 1 has one to several base deletions, substitutions and / or additions, and the nucleotide sequence is The base sequence in which the encoded protein has L-lysine decarboxylation / oxidase activity (3 ′) has a homology of 95% or more with respect to the base sequence described in SEQ ID NO: 1, and the base sequence encodes A base sequence in which a protein has L-lysine decarboxylation / oxidase activity.   A transformant comprising the nucleic acid according to claim 4. A method for measuring L-lysine in a specimen,
(A) If necessary, the same steps as described above are performed for a plurality of samples in which the amount of L-lysine is clear, and the amount of L-lysine and the amount of the reaction product to be measured in the following step (D) The process of clarifying the relationship;
(B) A step of oxidizing cadaverine in the specimen by allowing putrescine oxidase to act on the specimen, if necessary;
(C) a step of allowing the L-lysine decarboxylation / oxidase according to any one of claims 1 to 3 to act on a specimen; and (D) at least a reaction product produced by the action of the L-lysine decarboxylation / oxidase. A method comprising the step of measuring a kind of quantity.   In the step (C), cadaverine produced by the action of the L-lysine decarboxylase / oxidase is oxidized by putrescine oxidase, and in the step (D), the amount of hydrogen peroxide as a reaction product is determined. The method of Claim 6 which measures.   The method according to claim 6 or 7, wherein the putrescine oxidase is derived from Kokuria rosea NBRC 3768 strain.   A kit for measuring L-lysine, comprising the L-lysine decarboxylation / oxidase according to any one of claims 1 to 3.   The kit for measuring L-lysine according to claim 9, further comprising putrescine oxidase.   The kit for measuring L-lysine according to claim 9 or 10, further comprising a hydrogen peroxide detection reagent containing peroxidase and a coloring reagent.   An enzyme sensor for measuring L-lysine, comprising an electrode for hydrogen peroxide detection, wherein the L-lysine according to any one of claims 1 to 3 is disposed on or near the surface of the hydrogen peroxide detection electrode. An enzyme sensor for L-lysine measurement, wherein a decarboxylation / oxidase is arranged.   Burkholderia sp. AIU395 strain (Accession number: NITE P-01796).