JP2015216894A - Production method of 1,5-pentanediamine using lysine decarboxylase which can catalyze response under alkaline conditions - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production method of 1,5-pentanediamine (1,5-PD) by an economical method having a small environmental load.SOLUTION: The invention provides a production method of 1,5-PD comprising producing 1,5-PD from L-lysine and/or a salt thereof by an action of the protein selected from the group consisting of the following: (a) a protein comprising a specific amino acid sequence; (b) a protein comprising an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or some amino acid residues in a specific amino acid sequence and having lysine decarboxylation activity; and (c) a protein comprising an amino acid sequence having identity of 90% or more to a specific amino acid sequence and having lysine decarboxylation activity.

Description

  The present invention relates to a method for producing 1,5-pentanediamine using lysine decarboxylase capable of catalyzing a reaction under alkaline conditions.

1,5-pentanediamine (also referred to as cadaverine, pentanemethylenediamine or the like, which has a molecular formula C ₅ H ₁₄ N ₂ , hereinafter abbreviated as “1,5-PD” as necessary) is a polyamide resin It is a substance that is expected to be in demand as a raw material for resins such as pharmaceutical intermediates. Since 1,5-PD can be produced from non-petroleum-based raw materials, it has attracted industrial attention from the viewpoint of reducing environmental impact.

In the biological production method of 1,5-PD, lysine decarboxylase (LDC) is used. LDC is an enzyme that produces 1,5-PD and CO ₂ from lysine. As a method for producing 1,5-PD using LDC, for example, a method using LDC derived from Escherichia coli has been reported (Patent Documents 1 to 3).

JP 2002-223770 A JP 2008-193899 A International Publication No. 2013/1088859

  When LDC is allowed to act on lysine to produce 1,5-PD, a carboxyl group is eliminated by decarboxylation of lysine to generate carbon dioxide, and a monovalent cation lysine is a divalent cation 1, Since 5-PD is produced, the pH of the reaction solution rises. Therefore, in order to prevent an increase in the pH of the reaction solution and maintain the enzyme reaction at an optimum pH, the reaction is carried out in a buffer solution containing a high concentration neutralizing agent, or an acid as a neutralizing agent is added sequentially to the reaction system. There is a need. However, in such a case, a by-product salt derived from a neutralizing agent (eg, an acid such as an inorganic acid or an organic acid) is generated, and this becomes a burden on the environment. Further, when this by-product salt is recovered and reused, a large amount of equipment is required, and the production cost of 1,5-PD increases, which is economically disadvantageous.

  Accordingly, an object of the present invention is to generate 1,5-PD by an economical method with a low environmental load.

  In order to reduce the generation of by-product salts derived from the neutralizing agent in the reaction for producing 1,5-PD from lysine, the present inventor should produce 1,5-PD under alkaline conditions. Inspired. Based on this idea, the present inventor has conducted extensive studies to realize the production of 1,5-PD under alkaline conditions, and as a result, has isolated and identified a protein capable of producing 1,5-PD under alkaline conditions. In particular, the present invention has been completed.

That is, the present invention is as follows.
[1] A method for producing 1,5-pentanediamine, comprising producing 1,5-pentanediamine from L-lysine and / or a salt thereof by the action of a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2 and having lysine decarboxylation activity; and (c) a sequence A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of No. 2 and having lysine decarboxylation activity.
[2] The method of [1], wherein the reaction is carried out under conditions of pH 7.0 to 10.0.
[3] producing 1,5-pentanediamine from L-lysine and / or a salt thereof using a microorganism transformed with an expression vector containing a polynucleotide encoding the protein, [1] or The method of [2].
[4] The method according to any one of [1] to [3], wherein the microorganism is Escherichia coli.
[5] A protein selected from the group consisting of the following (a) to (c):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2 and having lysine decarboxylation activity; and (c) a sequence A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of No. 2 and having lysine decarboxylation activity.
[6] The protein according to [5], which has lysine decarboxylation activity under conditions of H7.0 to 10.0.
[7] A polynucleotide selected from the group consisting of:
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
(B) a polynucleotide comprising a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having lysine decarboxylation activity;
(C) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and encodes a protein having lysine decarboxylation activity; and (D ) A polynucleotide encoding the protein of [5] or [6].
[8] An expression vector comprising the polynucleotide of [7].
[9] A microorganism comprising the expression vector according to [8].
[10] A method for producing a protein comprising producing a protein selected from the group consisting of the following (a) to (c) using the microorganism of [9]:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2 and having lysine decarboxylation activity; and (c) a sequence A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of No. 2 and having lysine decarboxylation activity.

  According to the present invention, the amount of by-product salt generated can be reduced by using LDC that can produce 1,5-PD under alkaline conditions. Therefore, according to the present invention, 1,5-PD can be manufactured by an economical method with a small environmental load.

FIG. 1 is a view showing a pH profile of lysine decarboxylase of the present invention. In the reaction at each pH, 100 mM of the following buffers were used: ◇ (Tris buffer, pH 7.0, 7.5, 8.0, 8.5); □ (CHES buffer, pH 8.5, 9.0, 9.5); Δ (CAPS buffer). FIG. 2 is a graph showing lysine consumption over time by lysine decarboxylase.

  The present invention provides a method for producing 1,5-pentanediamine, which comprises producing 1,5-pentanediamine from L-lysine and / or a salt thereof by the action of a predetermined protein.

Specifically, the following can be used as such proteins:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2 and having lysine decarboxylation activity; and (c) a sequence A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of No. 2 and having lysine decarboxylation activity.
In the present invention, lysine decarboxylation activity refers to the activity of converting lysine to 1,5-PD.
The present invention also provides such a protein.

  The protein of the present invention may be derived from a microorganism belonging to the genus Pseudomonas [eg, Pseudomonas otitidis or a related species thereof].

  The protein of (b) may contain a mutation (eg, substitution, deletion, insertion, and addition) of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2. The number of mutations is, for example, 1 to 100, preferably 1 to 70, more preferably 1 to 50, 1 to 40, 1 to 30, or 1 to 20, even more preferably 1 to 10 ( Example, 1, 2, 3, 4 or 5).

  The protein of (c) above may have at least 90% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2. The percent amino acid sequence identity may preferably be 92% or more, more preferably 95% or more, even more preferably 97% or more, and most preferably 98% or more or 99% or more.

  Amino acid sequence identity is determined using, for example, the algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) and FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). Can be determined. Based on this algorithm BLAST, since a program called BLASTP has been developed (see http://www.ncbi.nlm.nih.gov), these programs are used with default settings, and amino acid sequence identity May be calculated. In addition, as the identity of amino acid sequences, for example, using GENETYX Ver 7.0.9 software of GENETICS, which employs the Lipman-Pearson method, the full length of the polypeptide portion encoded by ORF is used. You may use the numerical value at the time of calculating the similarity or identity with the setting of Size to Compare = 2. Alternatively, as amino acid sequence identity, the default parameters (Gap penalty = 10, Extended penalty = 0.5, Matrix = BLOSUM62) were used in the NEEDLE program (J Mol Biol 1970; 48: 443-453) search. The obtained value may be used. As the amino acid sequence identity, the lowest value among the values derived by these calculations may be adopted.

  For the preparation of the proteins of (b) and (c) above, the positions of amino acid residues where mutations can be introduced into the amino acid sequence of SEQ ID NO: 2 will be apparent to those skilled in the art. Mutations can be introduced by reference. Specifically, those skilled in the art will 1) compare amino acid sequences of multiple homologs (known lysine decarboxylase), 2) compare relatively conserved regions, and relatively unconserved regions. Clarify and then 3) Predict areas that can play an important role in function and areas that can not play an important role in function from areas that are relatively conserved and areas that are not relatively conserved, respectively It is possible to recognize the correlation between structure and function. Accordingly, those skilled in the art will understand the amino acid sequences of (b) and (c) in which a mutation of one or more amino acid residues capable of maintaining enzymatic properties (eg, lysine decarboxylation activity, pH stability) is introduced. Can be determined.

  For the preparation of the proteins of (b) and (c) above, when an amino acid residue mutation is introduced into the amino acid sequence of SEQ ID NO: 2 and the amino acid residue mutation is a substitution, Such substitutions may be conservative substitutions. The term “conservative substitution” refers to substitution of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art. For example, such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged polar side chains (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having side groups containing hydroxyl groups (eg, alcoholic, phenolic) ( Example, serine, thread Nin, tyrosine), and amino acids (e.g. having sulfur-containing side chains, cysteine, methionine) and the like. Amino acids having an uncharged polar side chain and amino acids having a nonpolar side chain are sometimes collectively referred to as neutral amino acids. Preferably, the conservative substitution of amino acids is a substitution between aspartic acid and glutamic acid, a substitution between arginine and lysine and histidine, a substitution between tryptophan and phenylalanine, and between phenylalanine and valine. Or a substitution between leucine, isoleucine and alanine, and a substitution between glycine and alanine.

  The protein of the present invention may also have other peptide components (eg, tag moiety) at the C-terminus or N-terminus. Other peptide components that can be added to the protein of the present invention include, for example, peptide components that facilitate purification of the target protein (eg, tag portions such as histidine tag, Strep-tag II; glutathione-S-transferase, maltose binding) A protein commonly used for purification of a target protein such as a protein), a peptide component that improves the solubility of the target protein (eg, Nus-tag); a peptide component that acts as a chaperone (eg, a trigger factor); a protein having other functions or Peptide components as protein domains or linkers connecting them may be mentioned.

  The protein of the present invention has an activity of about 50% or more, preferably about 80% of the protein consisting of the amino acid sequence of SEQ ID NO: 2 under predetermined conditions (eg, 30 to 37 ° C., pH 7.0 to 10.0). It is desirable to retain the above activity, more preferably about 90% or more activity, and even more preferably about 100% or more activity.

  In the method for producing 1,5-PD of the present invention, 1,5-PD is produced from L-lysine and / or a salt thereof by the action of the protein as described above. Examples of L-lysine salts include inorganic acid salts (eg, hydrochloride), inorganic base salts (eg, ammonium salt), organic acid salts (eg, acetate), and organic base salts (eg, triethylamine salt). Nonmetal salts, and metal salts such as alkali metal salts (eg, sodium salt, potassium salt) and alkaline earth metal salts (eg, calcium salt, magnesium salt) can be mentioned.

  Production of 1,5-pentanediamine from L-lysine and / or a salt thereof by the action of a predetermined protein can be achieved using, for example, the transformant of the present invention as described later or the protein of the present invention ( Example, extraction, crude or purified protein). When 1,5-PD is produced using the transformant of the present invention, the conditions for producing 1,5-PD are the same as the culture conditions for the transformant as described below. When 1,5-PD is produced using the transformant of the present invention, the protein of the present invention may be treated (eg, crushing treatment, heat treatment) so as to be released from the transformant. When 1,5-PD is produced using protein, the reaction conditions for producing 1,5-PD are performed using an appropriate reaction solution (eg, buffer solution). Further, after the protein of the present invention is highly expressed in the transformant of the present invention, the microorganism is treated as described above, and the treated product of the microorganism is added to a reaction system containing L-lysine and / or a salt thereof. , 5-PD may be generated.

  The method for producing 1,5-PD of the present invention can be carried out under any pH condition as long as 1,5-PD can be produced, but may be carried out under neutral or alkaline conditions. The protein of the present invention may have the property of being highly stable under such conditions. Therefore, the method for producing 1,5-PD of the present invention can be suitably carried out under the condition of pH 7.0 to 10.0. The production method of the present invention may further include adding L-lysine and / or a salt thereof to the reaction system in order to improve the yield of 1,5-PD.

  The recovery and purification of 1,5-PD can be carried out by any method known in the art (see, for example, the prior art cited in the background art and WO2013 / 1088859). For example, conventional techniques such as extraction, precipitation, filtration, and column chromatography can be used.

The polynucleotide of the present invention is selected from the group consisting of the following (A) to (D):
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
(B) a polynucleotide comprising a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having lysine decarboxylation activity;
(C) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and encodes a protein having lysine decarboxylation activity; and (D ) A polynucleotide encoding the protein of the present invention.

  The polynucleotide of the present invention may be DNA or RNA, but is preferably DNA. The polynucleotide of the present invention may encode the protein of the present invention. The polynucleotide of the present invention can also be derived from the same microorganism as the protein of the present invention.

  In (B), the “identity” of the nucleotide sequence can be determined in the same manner as described above for the amino acid sequence. The percent nucleotide sequence identity may preferably be 92% or more, more preferably 95% or more, even more preferably 97% or more, and most preferably 98% or more or 99% or more. In (C), “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to clearly quantify such conditions, for example, substantially identical polynucleotides exhibiting high% identity, for example, polynucleotides having the above-mentioned% identity are hybridized. This is a condition in which polynucleotides that are soybeans and have a lower percent identity do not hybridize. Specifically, such conditions include hybridization at about 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by 50 × 0.2 × SSC in 0.1% SDS. One or more washings at ˜65 ° C. may be mentioned. The protein encoded by the polynucleotides (B) and (C) is approximately about the protein consisting of the amino acid sequence of SEQ ID NO: 2 under predetermined conditions (eg, 30 to 37 ° C., pH 7.0 to 10.0). It is desirable to retain at least 50% activity, preferably at least about 80% activity, more preferably at least about 90% activity, even more preferably at least about 100% activity. The polynucleotide of the present invention may also have a polynucleotide encoding another peptide component (eg, tag portion) as described above at the 5 'end or 3' end.

  The protein of the present invention can be prepared using the transformant of the present invention or using a cell-free system or the like. The transformant of the present invention can be prepared, for example, by preparing the expression vector of the present invention and then introducing this expression vector into a microorganism (host).

  The expression vector of the present invention contains the polynucleotide of the present invention. In addition to the polynucleotide of the present invention, the expression vector of the present invention further comprises a region such as a region encoding a promoter, terminator and drug (eg, tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin) resistance gene. Can be included. The expression vector of the present invention may be a plasmid or an integrative vector. The expression vector of the present invention may be a viral vector or a cell-free vector. The expression vector of the present invention may further contain a polynucleotide encoding another peptide component that can be added to the protein of the present invention at the 3 'or 5' terminal side of the polynucleotide of the present invention. Examples of the polynucleotide encoding another peptide component include a polynucleotide encoding a peptide component that facilitates purification of the target protein as described above, and a peptide component that improves the solubility of the target protein as described above. Examples of the polynucleotide include a polynucleotide encoding a peptide component that functions as a chaperone, and a polynucleotide encoding a protein component having another function, a domain of the protein, or a peptide component as a linker connecting them. Various expression vectors containing polynucleotides encoding other peptide components are available. Therefore, such an expression vector may be used for preparing the expression vector of the present invention. For example, an expression vector (eg, pET-15b, pET-51b, pET-41a, pMAL-p5G) containing a polynucleotide encoding a peptide component that facilitates purification of the target protein, and a peptide component that improves the solubility of the target protein Expression vector (eg, pET-50b) containing a polynucleotide encoding a protein, an expression vector (eg, pCold TF) containing a polynucleotide encoding a peptide component that acts as a chaperone, a protein having a different function or a protein domain, or An expression vector containing a polynucleotide that encodes a peptide component as a linker for linking can be used. In order to enable cleavage of the protein of the present invention and other peptide components added thereto after protein expression, the expression vector of the present invention comprises a region encoding a protease cleavage site in a region encoding a protein encoding the protein of the present invention. It may be contained between the nucleotide and a polynucleotide encoding another peptide component.

  Examples of microorganisms for expressing the protein of the present invention include bacteria belonging to the genus Escherichia such as Escherichia coli, bacteria belonging to the genus Corynebacterium (eg, Corynebacterium glutamicum), Pantoea ananatis ( Pantoea bacteria such as Pantoea ananatis, and various prokaryotic cells such as Bacillus subtilis, eg, Bacillus subtilis, eg, Saccharomyces cerevisiae, eg, Saccharomyces cerevisiae, Bacteria [eg, Pichia stipitis], Aspergillus bacteria [eg It is possible to use various eukaryotic cells including Aspergillus oryzae (Aspergillus oryzae)]. As the microorganism, a strain lacking a predetermined gene may be used. Examples of the transformant include a transformant having an expression vector in the cytoplasm, and a transformant having a target gene introduced on the genome.

  The transformant of the present invention can be cultured in a medium having the composition described below, for example, using a predetermined culture apparatus (eg, test tube, flask, jar fermenter). The culture conditions can be set as appropriate. Specifically, the culture temperature may be 10 ° C. to 37 ° C., the pH may be 6.5 to 7.5, and the culture time may be 1 h to 100 h. Moreover, you may culture | cultivate, managing a dissolved oxygen concentration. In this case, the dissolved oxygen concentration (DO value) in the culture solution may be used as a control index. Control the aeration / stirring conditions so that the relative dissolved oxygen concentration DO value when the atmospheric oxygen concentration is 21%, for example, does not fall below 1-10%, preferably below 3% -8%. I can do it. Further, the culture may be batch culture or fed-batch culture. In the case of fed-batch culture, a solution serving as a sugar source or a solution containing phosphoric acid can be added continuously or discontinuously to the culture solution to continue the culture.

  The microorganism to be transformed is as described above, but when Escherichia coli is described in detail, Escherichia coli K12 strain subspecies Escherichia coli JM109 strain, DH5α strain, HB101 strain, BL21 (DE3) strain and the like are selected. I can do it. Methods for performing transformation and methods for selecting transformants are also described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor press (2001/01/15) and the like. Hereinafter, a method for producing transformed Escherichia coli and producing a predetermined enzyme using the transformed Escherichia coli will be described more specifically as an example.

  As the promoter for expressing the polynucleotide of the present invention, a promoter usually used for heterologous protein production in Escherichia coli can be used. For example, PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac Strong promoters such as a promoter, lambda phage PR promoter, PL promoter, T5 promoter and the like can be mentioned, and PhoA, PhoC, and lac are preferable. Examples of the vector include pUC (eg, pUC19, pUC18), pSTV, pBR (eg, pBR322), pHSG (eg, pHSG299, pHSG298, pHSG399, pHSG398), RSF (eg, RSF1010), pACYC (eg, pACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), and derivatives thereof may be used. As other vectors, phage DNA vectors may be used. Furthermore, an expression vector containing a promoter and capable of expressing the inserted DNA sequence may be used. Preferably, the vector may be pUC, pSTV, pMW.

  Further, a terminator that is a transcription termination sequence may be linked downstream of the polynucleotide of the present invention. Examples of such terminators include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, and trpA gene terminator.

  As a vector for introducing the polynucleotide of the present invention into Escherichia coli, a so-called multicopy type is preferable, and a plasmid having a replication origin derived from ColE1, such as a pUC series plasmid, a pBR322 series plasmid or a derivative thereof. Is mentioned. Here, the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, insertion and / or addition.

  In order to select transformants, the vector preferably has a marker such as an ampicillin resistance gene. As such a plasmid, an expression vector having a strong promoter is commercially available [eg, pUC system (manufactured by Takara Bio Inc.), pPROK system (manufactured by Clontech), pKK233-2 (manufactured by Clontech)].

  The protein of the present invention can be obtained by transforming Escherichia coli using the obtained expression vector of the present invention and culturing the Escherichia coli.

As the medium, a medium usually used for culturing Escherichia coli such as M9-casamino acid medium and LB medium may be used. The culture medium may contain a predetermined carbon source, nitrogen source, and coenzyme (eg, pyridoxine hydrochloride). Specifically, peptone, yeast extract, NaCl, glucose, MgSO ₄ , ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, and the like may be used. Culture conditions and production induction conditions are appropriately selected according to the types of markers, promoters, microorganisms and the like of the vector used.

  In order to recover the protein of the present invention, there are the following methods. The protein of the present invention is obtained as a crushed material or lysate by recovering the transformant of the present invention and then crushing (eg, sonication, homogenization) or lysing (eg, lysozyme treatment) be able to. The protein of the present invention can be obtained by subjecting such crushed material or lysate to techniques such as extraction, precipitation, filtration, and column chromatography. Alternatively, the protein of the present invention can be recovered after subjecting the transformant of the present invention to the treatment as described above.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

Example 1: Identification of a novel lysine decarboxylase (LDC) gene 1-1) Separation of bacteria having a novel lysine decarboxylase activity from nature Various soil samples were obtained from L-lysine hydrochloride 10 g / l, dihydrogen phosphate Medium containing LDH 3.56 g / l, magnesium sulfate heptahydrate 0.1 g / l, sodium chloride 2 g / l, yeast extract 0.2 g / l, tryptone 2 g / l and sodium carbonate 0.1 g / l Medium, pH 9.2) was added to the examiner with 3 ml, and cultured with shaking at 37 ° C. for 2 to 7 days. A medium in which the growth of the fungus was observed was transferred to a new medium, and shaking culture was performed under the same conditions. These culture solutions were plated on a plate agar medium having the same composition, and 397 strains of bacteria that grew well on LDH medium were isolated.

  Next, the isolated bacteria cultured on a plate medium were scraped, and lysine HCl salt 4 g / l, N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) 20.7 g / l (100 mM), Pyridoxal-5′-phosphate (PLP) ) The reaction solution was added to 50 μl (pH 8.4 and 9.2) containing 26.5 mg / l and reacted at 37 ° C. for 16 hours to carry out lysine decarboxylation. One strain isolated from soil in Japan was selected as a strain producing 1,5-PD and having a small difference in the production amount between pH 8.4 and 9.2. This strain was found to be attributed to Pseudomonas bacteria closely related to Pseudomonas otitidis by 16S rDNA analysis. This strain was named PS2936 strain.

  Enzymatic activity is determined by HPLC for lysine and 1,5-PD by post-column OPA method (SR Val and MB Gloria, Journal Of AOAC International (1997) vol. 80, p. 1006-1012). This was measured by a method or a method of quantifying the remaining L-lysine with a biosensor BF-5 (Oji Scientific Instruments).

1-2) Cloning of a novel lysine decarboxylase (LDC) gene from a strain Using a MiSeq II next-generation sequencer (Illumina), a genome prepared from the PS2936 strain and prepared using NexteraXT kit (Illumina) The genome sequence was analyzed. E. Based on the homology of E. coli amino acid decarboxylase, a candidate gene encoding lysine decarboxylase was searched from the obtained genome sequence. As a result, a 2,253 bp ORF encoding a protein having 751 amino acid residues shown in Sequence Listing 1 was found. As a result of Blast search, the protein having the amino acid sequence shown in Sequence Listing 2 showed the highest homology with the putative Orn / Arg / Lys decarboxylase derived from Pseudomonas resinovans NBRC 106553, and the homology with this gene sequence was 87% (640 562 of the amino acid residues were homologous).

Using a primer set (PSLDCF and PSLDCR) designed based on this amino acid sequence, a DNA fragment containing the candidate enzyme gene (LDC) was amplified by PCR using the chromosome of K2936 strain as a template. An expression plasmid was constructed by ligating this to the NdeI-EcoRI site of plasmid pET28a (+) (Novagen), and was designated pSP410.
PSLDCF: 5′-cgcgcggcaggccatgtcaaaaagacctgaagttcc (SEQ ID NO: 3)
PSLDCR: 5'-gcaggagctcgaattttactctttgtatgcaatcaac (SEQ ID NO: 4)

Example 2: Confirmation of activity and characterization of novel lysine decarboxylase gene E. coli expressed in expression plasmid pSP410 constructed in Example 1 E. coli BL21 strain was transformed, and this transformant was inoculated into 50 ml of LB medium containing 100 μg / ml of ampicillin and 1 mM of IPTG and cultured at 37 ° C. for 24 hours. The cells are collected from the culture by centrifugation, washed once with physiological saline, suspended in 5 ml of 100 mM potassium phosphate buffer (pH 7.0), and sonicated at 4 ° C. for 20 minutes. And then crushed. The processing solution was 8,000 r. p. m. The mixture was centrifuged for 10 minutes to remove the insoluble fraction, and a cell-free extract was prepared.

  Add 0.1 ml of bacterial cell treatment product to 1 ml of lysine base 8 g / l, Pyridoxal-5′-phosphate (PLP) 26.5 mg / l, and 1 ml of reaction solution adjusted to pH 6 to 10 with various buffer solutions 100 mM. And reacted at 37 ° C. for 1 hour. Tris (hydroxymethyl) aminomethane (Tris) 12.1 g / l (pH 7, 7.5, 8, 8.5), N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) 20.7 g / l (pH 8. 6,9,9.), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) 22.1 g / l (pH 9.7, 10) was used. 0.1 ml of the reaction solution was collected and added to 1 ml of 1% (v / v) phosphoric acid to stop the reaction, and 1,5-PD produced was quantified.

  The results of measuring the activity at each pH are shown in FIG. E. Although the optimal pH of E. coli-derived lysine decarboxylase (CadA) has been reported to be 6.2 (reference: J Bacteriol. (1997) 179, 4486-92), the enzyme has a pH of 7.5 to 8.0. It was considered that this was a property suitable for 1,5-PD production.

  In addition, since this LDC showed the maximum activity at pH 8, in the subsequent experiments, enzyme activity measurement uses Tris buffer to produce 1 μmol of 1,5-PD per minute under the conditions of pH 8.0 and 30 ° C. The activity was defined as 1 unit.

Example 3: Reaction of novel lysine using decarboxylase Further, the crude enzyme solution prepared in Example 2 was used to try 1,5-PD production reaction from lysine with an alkaline reaction solution. The crude enzyme solution was added to lysine base 50 g / l (pH 8.5) and PLP 2.63 mg / l so that the final concentration was 6.3 U / ml, and the pH during the reaction was not adjusted at 30 ° C. I let you. As a control, E. coli was prepared according to the method described in Example 1 of JP-A-2008-193899. E. coli W3110 (ATCC 39936) -derived lysine decarboxylase (CadA) expression strain is prepared, a crude enzyme solution is prepared from this bacterium, and the reaction is carried out with an amount of 7.0 U / ml (CadA activity measured at pH 6). went.

  The time course of the reaction for lysine consumption is shown in FIG. E. Although the reaction using E. coli-derived lysine decarboxylase was stopped under these conditions, lysine was consumed in the reaction using the lysine decarboxylase derived from PS2936 strain. From this, it was shown that the amount of acid added can be greatly reduced by using this enzyme in a high pH reaction.

  The present invention is useful for the production of 1,5-PD that can be used as a resin raw material such as a polyamide resin or as a pharmaceutical intermediate.

Claims (10)

A method for producing 1,5-pentanediamine, which comprises producing 1,5-pentanediamine from L-lysine and / or a salt thereof by the action of a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2 and having lysine decarboxylation activity; and (c) a sequence A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of No. 2 and having lysine decarboxylation activity.   The process according to claim 1, wherein the reaction is carried out under conditions of pH 7.0 to 10.0.   The method according to claim 1, comprising producing 1,5-pentanediamine from L-lysine and / or a salt thereof using a microorganism transformed with an expression vector comprising a polynucleotide encoding the protein. Method.   The method according to any one of claims 1 to 3, wherein the microorganism is Escherichia coli. A protein selected from the group consisting of the following (a) to (c):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2 and having lysine decarboxylation activity; and (c) a sequence A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of No. 2 and having lysine decarboxylation activity.   The protein according to claim 5, which has lysine decarboxylation activity under conditions of H7.0 to 10.0. A polynucleotide selected from the group consisting of:
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
(B) a polynucleotide comprising a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having lysine decarboxylation activity;
(C) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and encodes a protein having lysine decarboxylation activity; and (D A polynucleotide encoding the protein according to claim 5 or 6.   An expression vector comprising the polynucleotide according to claim 7.   A microorganism comprising the expression vector according to claim 8. A method for producing a protein comprising producing a protein selected from the group consisting of the following (a) to (c) using the microorganism according to claim 9:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2 and having lysine decarboxylation activity; and (c) a sequence A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of No. 2 and having lysine decarboxylation activity.

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