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Definitions

• the present invention relates to a method for producing an N-acyl-amino group-containing compound.
• N-acyl-amino group-containing compounds eg, N ⁇ -acylamino acids
• cosmetic materials eg, surfactants
• Chemical synthesis of N-acyl-amino group-containing compounds eg, Schotten-Baumann reaction
• Several prior arts relating to the enzymatic synthesis of N-acyl-amino group-containing compounds have been reported.
• Patent Document 1 reports N ⁇ -acylamino acid fermentation from sugar using Bacillus subtilis surfactin biosynthetic enzyme. However, since the production amount of N ⁇ -acylglutamic acid is as small as 116.8 mg / L, this fermentation is not suitable for production on an industrial scale.
• Patent Document 2 a human-derived amino acid N-acyltransferase, E.I.
• a method for synthesizing N ⁇ -acylglycine from amino acids and fatty acids using E. coli-derived acyl CoA synthase has been reported.
• this method cannot directly bind an amino acid to a fatty acid, and requires a two-stage enzyme reaction. Therefore, there is a problem that the control becomes complicated as compared with a reaction using a single enzyme.
• Non-Patent Document 1 reports a method of synthesizing N ⁇ -acylamino acids from amino acids and fatty acids in a solution containing glycerol using porcine kidney-derived acylase. This method utilizes the fact that hydrolysis of N ⁇ -acylamino acid by acylase does not proceed easily in a solution containing glycerol. However, in view of the fact that a large amount of glycerol is required and the synthesis of N ⁇ -acylamino acid in an aqueous solvent not containing glycerol results in a low yield, this method is less efficient in industrial production. .
• Non-Patent Document 2 reports a method of synthesizing an N ⁇ -acylamino acid from an amino acid and a fatty acid in a solution containing glycerol using Streptomyces mobaraensis-derived acylase. However, since N ⁇ -acylamino acid synthesis in an aqueous solvent not containing glycerol has not been reported, the efficiency of this method for industrial production has not been demonstrated.
• An object of the present invention is to provide an efficient method for producing an N-acyl-amino group-containing compound by an enzymatic method.
• an enzyme having an ability to form an amide bond by binding a carboxyl group and an amino group in an ATP-dependent manner is derived from a carboxyl group-containing compound and an amino group-containing compound including fatty acids.
• the inventors have found that an N-acyl-amino group-containing compound can be efficiently produced, and have completed the present invention.
• the present invention is as follows. [1] An amino group-containing compound and a carboxyl group-containing compound are reacted in the presence of an enzyme having an ability to form an amide bond by binding a carboxyl group and an amino group in an ATP-dependent manner, and N-acyl-amino A method for producing an N-acyl-amino group-containing compound, comprising producing a group-containing compound. [2] The method according to [1], wherein the enzyme is a plant or microorganism-derived enzyme. [3] The method of [1] or [2], wherein the enzyme is a GH3 protein.
• the GH3 protein is: (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9; (B) a protein having an N-acylase activity, which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9 and containing one or several amino acid substitutions, deletions, insertions or additions Or (C) a protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9 and having N-acylase activity; The method of any one of [1] to [4], selected from the group consisting of: [6] The method of [1] or [2], wherein the enzyme is a PaaK protein.
• the PaaK protein is: (A ′) a protein comprising the amino acid sequence of SEQ ID NO: 10 or 11; (B ′) a protein comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NO: 10 or 11, and having N-acylase activity; or (C ') A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 or 11, and having N-acylase activity;
• the method of [6] selected from the group consisting of: [8] The method according to any one of [1] to [7], wherein the amino group-containing compound is an amino group-containing compound having an anionic group.
• the amino group-containing compound is: (1) (a) Group consisting of glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine An ⁇ -amino acid selected from (B) ⁇ -alanine; (C) ⁇ -aminobutyric acid; and (d) sarcosine; An amino acid selected from the group consisting of: (2) taurine; and (3) a dipeptide selected from the group consisting of aspartylphenylalanine, glycylglycine, and alanylhistidine; The method of any one of [1] to [11], selected from the group consisting of: [13] The method according to any one of [1] to [12], wherein the
• the transformed microorganism is any one of the following (i) to (iii): (I) a microorganism comprising a heterologous expression unit comprising a polynucleotide encoding said enzyme and a promoter operably linked thereto; (Ii) a microorganism containing an expression unit comprising a polynucleotide encoding the enzyme and a promoter operably linked thereto in a non-natural genomic region or non-genomic region; or (iii) a plurality of polynucleotides encoding the enzyme Microorganisms included in the expression unit in the copy number of. [20] The method according to [18] or [19], wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae. [21] The method of [20], wherein the bacterium is Escherichia coli.
• an N-acyl-amino group-containing compound generation reaction by an amide bond between an amino group-containing compound and a carboxyl group-containing compound can be efficiently performed.
• the present invention provides a method for producing an N-acyl-amino group-containing compound.
• the method of the present invention comprises reacting an amino group-containing compound and a carboxyl group-containing compound in the presence of an enzyme to produce an N-acyl-amino group-containing compound.
• the enzyme used in the method of the present invention has an ability to form an amide bond by binding a carboxyl group and an amino group in an ATP-dependent manner. It is considered that the enzyme used in the method of the present invention activates a carboxyl group-containing compound by adenylation and forms an amide bond by a mechanism in which an amino group-containing compound nucleophilically attacks this adenylation intermediate.
• the enzyme used in the method of the present invention may be derived from a plant or a microorganism.
• the plant from which the enzyme used in the method of the present invention is derived include, for example, gymnospermia, angiospermia, fern planta, lizard planta, hornwort planta, moss planta, genus planta, axle algae, mating Plants belonging to the class of algae, green algae plant plant, gray plant gate, and red plant plant are mentioned.
• Arabidopsis eg, Arabidopsis thaliana
• rice genus Oryza; eg, Oryza sativa
• capsicum Genus Capsicum; eg, Capsicum chinense
• soybean genus Glycine; eg, Glycine max
• eggplant genus or tomato genus Solanum or Lycopersicon; eg, Solanum lycopersicum or Lycopersi onesculentum
• tobacco genus Nonicotiana; e.g., Nicotiana tabacum
• genus Physcomitrella e.g., Physcomitrella patens
• mandarin genus Ceitrus; e.g., Citrus maduresin inus
• Brassica eg, Brassica napus
• cotton genus Gossypium sp.
• Grape genus Vitis; eg, Vitis vinifera
• genus e
• Examples of the microorganism from which the enzyme used in the method of the present invention is derived include the genus Cystobacter (e.g., Cystobacter fuscus), Synechococcus (e.g., Synechococcus sp.), Pantoea (e.g., Pantoeas) Examples include Pseudomonas (eg, Pseudomonas savastanoi).
• the enzyme used in the method of the present invention may be a GH3 protein.
• GH3 protein refers to a group of enzymes that function to amidate carboxyl group-containing plant hormones such as jasmonic acid, auxins (indole-3-acetic acid), salicylic acid, substituted benzoates, and homologs thereof.
• GH3 protein refers to a protein containing a GH3 superfamily domain.
• the GH3 superfamily domain can be searched from those defined in the sequence database. For example, the GH3 superfamily domain can be searched as a protein having a domain defined as “GH3 superfamily” on NCBI's conserveed domains database.
• GH3 proteins Among GH3 proteins, plant-derived GH3 proteins can be classified into Group I, Group II, and Group III based on sequence similarity and substrate specificity (J. Biol. Chem., 2010, 285, 29780-29786). Plant Cell., 2005, 17 (2) .616-627).
• Group I is an enzyme group found mainly as an enzyme using jasmonic acid as a substrate.
• Examples of enzymes belonging to Group I include, for example, Arabidopsis thaliana-derived enzymes (eg, AtGH3-10, AtJAR1 (also referred to as AtGH3-11)), rice (Oryza sativa) -derived enzymes (eg, OsAK071721, OsBAA96221) (Lycopersicon esculentum) derived enzyme (eg, LeBTO13697, LeU144810), Physcomitrella patens (eg, PpABO61221).
• Arabidopsis thaliana-derived enzymes eg, AtGH3-10, AtJAR1 (also referred to as AtGH3-11)
• rice (Oryza sativa) -derived enzymes eg, OsAK071721, OsBAA96221) (Lycopersicon esculentum) derived enzyme (eg, LeBTO136
• Group II is an enzyme group found mainly as an enzyme using indoleacetic acid or salicylic acid as a substrate.
• Examples of enzymes belonging to Group II include Arabidopsis thaliana-derived enzymes (eg, AtGH3-1, AtGH3-2, AtGH3-3, AtGH3-4, AtGH3-5, AtGH3-6, AtGH3-9, AtGH3).
• Group III is an enzyme group found as an enzyme mainly using a substituted benzoate as a substrate.
• Examples of enzymes belonging to Group III include Arabidopsis thaliana-derived enzymes (eg, AtGH3-7, AtGH3-8, AtGH3-12, AtGH3-13, AtGH3-14, AtGH3-15, AtGH3-16, AtGH3). 18, AtGH3-19).
• examples of the microorganism-derived GH3 protein include enzymes derived from Cystobacter fucus (eg, CfHP [WP_002626336]), Synechococcus sp. (Synechococcus sp.)-Derived enzyme (eg, SsGH3 [GH3 auxin-responsive promoter superfamily]).
• the GH3 protein may be: (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9; (B) a protein having an N-acylase activity, which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9 and containing one or several amino acid substitutions, deletions, insertions or additions Or (C) a protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9 and having N-acylase activity.
• the enzyme used in the method of the present invention may be a PaaK protein.
• PaaK protein refers to an enzyme group having a function of converting phenylacetic acid to phenylacetic acid CoA and homologs thereof.
• PaaK protein is a group of enzymes containing a PaaK superfamily domain.
• the PaaK superfamily domain can be searched from those defined in the sequence database.
• the PaaK superfamily domain can be searched as a protein having a domain defined as “PaaK superfamily” on NCBI's conserveed domains database.
• the PaaK protein may be found as a homologue of the GH3 protein in the sequence database.
• the PaaK protein has 10% or more, 15% or more, 20% or more, 25% or more, 30% or more amino acid sequence identity with the GH3 protein. You may have.
• PaaK protein examples include indoleacetic acid-lysine synthetase (IAAL) that binds lysine to indoleacetic acid.
• IAAL indoleacetic acid-lysine synthetase
• PsIAAL an enzyme derived from Pseudomonas savastani
• Pantoea agglomerans eg, PaHP [WP_03159948]
• the PaaK protein may be: (A ′) a protein comprising the amino acid sequence of SEQ ID NO: 10 or 11; (B ′) a protein comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NO: 10 or 11, and having N-acylase activity; or (C ') A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 or 11, and having N-acylase activity.
• one or several amino acid residues are obtained by 1, 2, 3 or 4 mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues. Can be modified.
• the amino acid residue mutation may be introduced into one region in the amino acid sequence, or may be introduced into a plurality of different regions.
• the term “one or several” refers to a number that does not significantly impair the activity of the protein.
• the number represented by the term “one or several” is, for example, 1 to 50, preferably 1 to 40, more preferably 1 to 30, even more preferably 1 to 20, particularly preferably 1 to 10 or 1 to 5 (eg, 1, 2, 3, 4, or 5).
• the percent identity with the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9 or the amino acid sequence of SEQ ID NO: 10 or 11 is 90% or more.
• the identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Calculation of% identity of polypeptides (proteins) can be performed by the algorithm blastp.
• Compositional adjustments Conditional composition, matrix adjustment.
• N-acylase activity refers to an activity of producing an N-acyl-amino group-containing compound using an amino group-containing compound and a carboxyl group-containing compound as substrates.
• the proteins (A) to (C) and (A ′) to (C ′) have N-acylase activity, the amino group-containing compound and the carboxyl group-containing compound are converted to N-acyl.
• -Amino group-containing compounds can be produced.
• the proteins (B), (B ′), (C), and (C ′) each correspond to the original amino acid sequence (A) or (A ′) when the activity is measured under specific measurement conditions.
• wild-type enzyme and (B), (B ′), (C), or (C ′) protein (hereinafter referred to as “modified enzyme”) 50 mM Tris-HCl, 5 mM amino acid (eg, glycine, L-glutamic acid, L-aspartic acid), 5 mM sodium fatty acid (eg, sodium caprylate, sodium caprate, sodium laurate)
• a reaction solution of 10 mM ATP, 10 mM MgCl 2 , 1 mM DTT, 50 ⁇ g / mL purified enzyme, pH 8.0, 0.2 mL was incubated at 25 ° C.
• Proteins (B), (B ′), (C), and (C ′) may have mutations introduced at sites in the catalytic domain and at sites other than the catalytic domain as long as the target properties can be maintained. Good.
• the positions of amino acid residues where mutations may be introduced that can retain the desired properties will be apparent to those skilled in the art. Specifically, those skilled in the art 1) compare the amino acid sequences of multiple proteins with similar characteristics, 2) reveal regions that are relatively conserved, and regions that are not relatively conserved, 3) From the relatively conserved region and the relatively unconserved region, it is possible to predict the region that can play an important role in the function and the region that can not play the important role in the function, respectively. Recognize the correlation between structure and function. Therefore, those skilled in the art can specify the position of an amino acid residue to which a mutation may be introduced in the amino acid sequence of the protein used in the present invention.
• amino acid residue substitution may be a conservative substitution.
• conservative substitution refers to the replacement 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.
• 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), ⁇ -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.
• 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 used in the present invention may also be a fusion protein linked to a heterologous moiety via a peptide bond.
• heterologous moieties include peptide components that facilitate the purification of the target protein (eg, tag moieties such as histidine tag, Strep-tag II; glutathione-S-transferase, maltose-binding protein, and mutants thereof. Proteins used for purification of target proteins, etc.), peptide components that improve the solubility of target proteins (eg, Nus-tag), peptide components that function as chaperones (eg, trigger factors), peptide components that have other functions (eg, Examples, full-length proteins or parts thereof), as well as linkers.
• tag moieties such as histidine tag, Strep-tag II; glutathione-S-transferase, maltose-binding protein, and mutants thereof.
• Proteins used for purification of target proteins, etc. peptide components that improve the solub
• the amino group-containing compound that can be used in the method of the present invention is an organic compound containing an amino group in which a nitrogen atom is bonded to one or two hydrogen atoms, or an amino group in which the nitrogen atom is not bonded to a hydrogen atom. Any of the organic compounds containing can be used.
• the amino group-containing compound is preferably a compound containing an amino group in which a nitrogen atom is bonded to one or two hydrogen atoms from the viewpoint of enzyme substrate specificity, etc., and the nitrogen atom is bonded to two hydrogen atoms. More preferred are compounds containing amino groups.
• the amino group-containing compound that can be used in the method of the present invention is preferably an amino group-containing compound having an anionic group.
• the anionic group include a carboxyl group, a sulfonic acid group, a sulfuric acid group, and a phosphoric acid group.
• amino group-containing compound having a carboxyl group as an anionic group examples include amino acids and peptides.
• amino acids examples include ⁇ -amino acids, ⁇ -amino acids, and ⁇ -amino acids.
• ⁇ -amino acids examples include glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine.
• An example of ⁇ -amino acid is ⁇ -alanine.
• ⁇ -amino acids examples include ⁇ -aminobutyric acid.
• the amino group of an amino acid is an amino group in which a nitrogen atom is bonded to two hydrogen atoms, an amino group in which a nitrogen atom is bonded to one hydrogen atom, or an amino group in which the nitrogen atom is not bonded to a hydrogen atom.
• amino acids containing an amino group in which a nitrogen atom is bonded to one hydrogen atom include sarcosine, N-methyl- ⁇ -alanine, N-methyltaurine, and proline.
• the amino acid may be either an L-amino acid or a D-amino acid.
• a peptide is a compound having a structure in which the above-described amino acids are linked by an amide bond.
• peptides include oligopeptides having a structure in which 2 to 10 amino acids are linked by an amide bond (eg, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide), and 11 A polypeptide (protein) having a structure in which two or more amino acids are linked by an amide bond.
• dipeptides include aspartylphenylalanine, glycylglycine, ⁇ -alanylhistidine, and alanylglutamine.
• amino group-containing compound having a sulfonic acid group as an anionic group examples include taurine, N-methyltaurine, and cysteic acid.
• amino group-containing compound having a sulfate group as an anionic group examples include O-sulfoserine and O-sulfothreonine.
• amino group-containing compound having a phosphate group as an anionic group examples include ethanolamine phosphate, phosphoserine, and phosphothreonine.
• the carboxyl group-containing compound that can be used in the method of the present invention is a compound containing an unsubstituted carboxyl group (eg, free type, ion, salt).
• Examples of the carboxyl group-containing compound include fatty acids, aromatic carboxylic acids, and indole carboxylic acids.
• the fatty acid may be, for example, a fatty acid having 6 to 18 carbon atoms, preferably a fatty acid having 6 to 16 carbon atoms, more preferably a fatty acid having 6 to 14 carbon atoms, and still more preferably a fatty acid having 6 to 12 carbon atoms.
• fatty acids having 6 to 18 carbon atoms include caproic acid (C6), enanthic acid (C7), caprylic acid (C8), pelargonic acid (C9), capric acid (C10), undecylic acid (C11), and lauric acid.
• C12 tridecylic acid
• C13 myristic acid
• C14 pentadecylic acid
• palmitic acid palmitoleic acid
• sapienoic acid above, C16
• margaric acid C17
• stearic acid ⁇ -linolenic acid
• ⁇ -linolenic acid ⁇ -linolenic acid
• linoleic acid vaccenic acid
• oleic acid above, C18
• other mixed fatty acids such as coconut oil fatty acid, palm fatty acid, hardened beef tallow fatty acid, etc. Can also be used.
• the fatty acid is preferably a saturated fatty acid.
• saturated fatty acids include caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecyl acid, lauric acid, tridecyl acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid.
• aromatic carboxylic acid examples include benzoic acid, phthalic acid, isophthalic acid, terephthalic acid, salicylic acid, gallic acid, and cinnamic acid.
• the N-acyl-amino group-containing compound produced by the method of the present invention is a compound having a structure in which the amino group of the amino group-containing compound and the carboxyl group of the carboxyl group-containing compound form an amide bond.
• the N-acyl-amino group-containing compound is produced by the reaction of the amino group-containing compound and the carboxyl group-containing compound in the presence of the enzyme.
• the amino group that reacts with the carboxyl group may be in any position of the amino group-containing compound, and may be, for example, any of ⁇ -position, ⁇ -position, ⁇ -position, ⁇ -position, and ⁇ -position.
• a natural protein or a recombinant protein can be used as the enzyme used in the method of the present invention.
• the recombinant protein can be obtained, for example, using a cell-free vector or from a microorganism that produces the enzyme used in the present invention.
• the enzyme used in the present invention can be used as an unpurified, crudely purified or purified enzyme. These enzymes may be used as immobilized proteins fixed to a solid phase in the reaction.
• the enzyme used in the method of the present invention is isolated by a known method, and further purified as necessary to obtain the target enzyme.
• the microorganism that produces the enzyme is preferably a transformed microorganism from the viewpoint of mass acquisition of the enzyme.
• the term “transformation” intends not only the introduction of a polynucleotide into a host cell, but also the alteration of the genome in the host cell.
• the culture conditions for the transformed microorganism are not particularly limited, and standard cell culture conditions can be used depending on the host.
• a medium for cultivating the transformed microorganism is known.
• a carbon source, a nitrogen source, a vitamin source, etc. can be added to a nutrient medium such as LB medium or a minimum medium such as M9 medium.
• the culture temperature is preferably 4 to 40 ° C, more preferably 10 to 37 ° C.
• the culture time is preferably 5 to 168 hours, more preferably 8 to 72 hours.
• the CO 2 concentration is preferably about 6% to about 84%
• the pH is preferably about 5 to 9. Further, it is preferable to perform the culture under aerobic, anoxic, or anaerobic conditions depending on the properties of the host cell.
• any appropriate method can be used as the culture method. Depending on the host cell, either shaking culture or stationary culture is possible, but stirring may be performed as necessary, and aeration may be performed. Examples of such a culture method include a batch culture method, a fed-batch culture method, and a continuous culture method.
• an inducer such as IPTG (isopropyl- ⁇ -thiogalactopyranoside) is used in the culture medium. May be added to induce protein expression.
• the target enzyme produced is a known salting out from the extract of the transformed microorganism, a precipitation method such as isoelectric point precipitation or solvent precipitation, a method utilizing a molecular weight difference such as dialysis, ultrafiltration or gel filtration, Methods using specific affinity such as ion exchange chromatography, methods using differences in hydrophobicity such as hydrophobic chromatography and reverse phase chromatography, and other affinity chromatography, SDS polyacrylamide electrophoresis, isoelectric focusing Purification and isolation are possible by electrophoresis or the like, or a combination thereof.
• a precipitation method such as isoelectric point precipitation or solvent precipitation
• a method utilizing a molecular weight difference such as dialysis, ultrafiltration or gel filtration
• Methods using specific affinity such as ion exchange chromatography
• methods using differences in hydrophobicity such as hydrophobic chromatography and reverse phase chromatography
• SDS polyacrylamide electrophoresis isoelectric focusing Purification and isolation
• a culture supernatant containing the target enzyme can be obtained by removing the cells from the culture solution obtained by culturing the transformed microorganism by centrifugation or the like.
• the target enzyme can also be purified and isolated from this culture supernatant.
• the reaction in the presence of the enzyme may be performed using a transformed microorganism that produces the enzyme or a processed product thereof (eg, crushed microorganism, lysate, or lyophilized product).
• a transformed microorganism that produces the enzyme or a processed product thereof (eg, crushed microorganism, lysate, or lyophilized product).
• the polynucleotide encoding the enzyme used in the present invention may be a polynucleotide selected from the group consisting of the following (a) to (d): (A) a polynucleotide comprising a base sequence selected from the group consisting of SEQ ID NOs: 12 to 22; (B) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to a base sequence selected from the group consisting of SEQ ID NOs: 12 to 22 and encodes a protein having N-acylase activity; (C) a polynucleotide comprising a base sequence selected from the group consisting of SEQ ID NOs: 12 to 22 and having a base sequence having 90% or more identity and encoding an N-acylase activity; and (d) (a A degenerate variant of a polynucleotide selected from the group consisting of:
• the polynucleotide may be DNA or RNA, but is preferably DNA.
• the nucleotide sequences of SEQ ID NOs: 12 to 22 encode the amino acid sequences of SEQ ID NOs: 1 to 11, respectively.
• stringent conditions refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
• stringent conditions include hybridization at about 45 ° C. in 6 ⁇ SSC (sodium chloride / sodium citrate), followed by 50 ⁇ 65 ° C. in 0.2 ⁇ SSC, 0.1% SDS. 1 or 2 or more washing
• the percent identity of the nucleotide sequence with respect to the nucleotide sequences of SEQ ID NOs: 12 to 22 is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96 % Or more, 97% or more, 98% or more, or 99% or more.
• the term “degenerate mutant” means that at least one codon encoding a predetermined amino acid residue in a polynucleotide before mutation is another codon encoding the same amino acid residue.
• An altered polynucleotide variant Since such a degenerate mutant is a mutant based on a silent mutation, the protein (enzyme) encoded by the degenerate mutant is the same as the protein (enzyme) encoded by the polynucleotide before the mutation. .
• a degenerate variant is a polynucleotide variant in which the codons have been altered to match the codon usage of the host cell into which it is to be introduced.
• a gene is expressed in a heterologous host cell (eg, a microorganism)
• the corresponding tRNA molecular species are not sufficiently supplied due to the difference in codon usage, resulting in decreased translation efficiency and / or incorrect translation (eg, translation). Stop) may occur.
• the low frequency codons shown in Table 1 are known.
• a degenerate mutant that matches the codon usage frequency of the host cell as described below can be used.
• a degenerate mutant has a codon encoding one or more amino acid residues selected from the group consisting of arginine residues, glycine residues, isoleucine residues, leucine residues, and proline residues. It may be. More specifically, the degenerate mutant has one or more codons selected from the group consisting of low frequency codons (eg, AGG, AGA, CGG, CGA, GGA, AUA, CUA, and CCC) altered. It may be a thing.
• low frequency codons eg, AGG, AGA, CGG, CGA, GGA, AUA, CUA, and CCC
• the degenerate variant may comprise one or more (eg, 1, 2, 3, 4 or 5) codon changes selected from the group consisting of: i) Change of at least one codon selected from the group consisting of four codons encoding Arg (AGG, AGA, CGG, and CGA) to another codon encoding Arg (CGU or CGC); ii) changing one codon (GGA) encoding Gly to another codon (GGG, GGU, or GGC); iii) changing one codon (AUA) encoding Ile to another codon (AUU, or AUC); iv) changing one codon (CUA) encoding Leu to another codon (UUG, UUA, CUG, CUU, or CUC); and v) one codon (CCC) encoding Pro.
• codon changes selected from the group consisting of: i) Change of at least one codon selected from the group consisting of four codons encoding Arg (AGG, AGA, CGG
• nucleotide residue “U” should be used as described above, but when the degenerate variant is DNA, “T” is used instead of nucleotide residue “U”. It should be.
• the number of nucleotide residue mutations for adapting to the codon usage of the host cell is not particularly limited as long as it encodes the same protein before and after the mutation. For example, 1 to 400, 1 to 300, 1 to 200 Or 1 to 100.
• a degenerate variant may include a change from a low frequency codon to a non-low frequency codon (eg, a high frequency codon).
• a non-low frequency codon eg, a high frequency codon.
• there are known methods for designing mutants in consideration of not only low-frequency codons but also factors such as suitability of the production strain to the genomic GC content (Alan Villabos et al., Gene Designer: a synthetic). biology tool for constructing artificial DNA segments, BMC Bioinformatics. 2006 Jun 6; 7: 285.), such a method may be used.
• the above-mentioned mutant can be appropriately produced according to the type of any host cell into which it can be introduced (eg, a microorganism as described later).
• the transformed microorganism in which the activity of the enzyme is improved as compared with the wild-type microorganism is preferably a microorganism comprising an expression unit comprising a polynucleotide encoding the enzyme and a promoter operably linked thereto.
• the term “expression unit” refers to the transcription of a polynucleotide comprising a predetermined polynucleotide to be expressed as a protein and a promoter operably linked thereto, and thus the protein encoded by the polynucleotide.
• the expression unit may further contain elements such as a terminator, a ribosome binding site, and a drug resistance gene.
• the expression unit may be DNA or RNA, but is preferably DNA.
• the expression unit may also be homologous (ie, inherent) or heterologous (ie, non-native) relative to the host cell.
• An expression unit is also an expression unit comprising one polynucleotide to be expressed as a protein and a promoter operably linked thereto (ie, an expression unit that allows expression of monocistronic mRNA), or expressed as a protein
• a plurality of polynucleotides to be performed eg 2 or more, preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, particularly preferably 10 or more polynucleotides
• It may be an expression unit comprising (ie, an expression unit that allows expression of polycistronic mRNA).
• the expression unit is a genomic region (for example, a natural genomic region that is a natural locus in which the polynucleotide encoding the protein is inherently present, or a non-natural genomic region that is not the natural locus) or a non-genomic in a microorganism (host cell). It can be included in a region (eg, in the cytoplasm). Expression units may be included in the genomic region at one or more (eg, 1, 2, 3, 4, or 5) different positions. Specific forms of expression units contained in the non-genomic region include, for example, plasmids, viral vectors, phages, and artificial chromosomes.
• the promoter constituting the expression unit is not particularly limited as long as a protein (enzyme) encoded by a polynucleotide linked downstream thereof can be expressed in a host cell.
• the promoter may be homologous or heterologous to the host cell.
• a configuration or an inducible promoter that is widely used for production of recombinant proteins can be used.
• promoters examples include PhoA promoter, PhoC promoter, T7 promoter, T5 promoter, T3 promoter, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter, PL promoter, SP6 promoter, arabinose inducible promoter, cold A shock promoter and a tetracycline inducible promoter are mentioned.
• a promoter having a strong transcription activity in the host cell can be used.
• the promoter having a strong transcription activity in the host cell include a promoter of a gene highly expressed in the host cell and a promoter derived from a virus.
• the transformed microorganism in which the activity of the enzyme is improved compared to a wild-type microorganism is (i) a microorganism comprising a heterologous expression unit comprising a polynucleotide encoding the enzyme and a promoter operably linked thereto. It may be.
• heterologous expression unit means that the expression unit is heterologous to the host cell. Therefore, in the present invention, at least one element constituting the expression unit is heterologous to the host cell. Examples of the elements constituting the expression unit that are heterologous to the host cell include the elements described above.
• one or both of the polynucleotide encoding the target enzyme or the promoter constituting the heterologous expression unit is heterologous to the host cell.
• one or both of the polynucleotide encoding the target enzyme, or the promoter is not a host cell (eg, prokaryotic and eukaryotic organisms, or microorganisms, insects, plants, mammals, etc.). Derived from animals) or viruses, or artificially synthesized.
• the heterologous expression unit is preferably a heterologous expression unit in which at least one element constituting the expression unit is heterologous to the host cell.
• the protein constituting the expression unit may be heterologous to the host cell.
• microorganisms include microorganisms comprising a polynucleotide encoding any one of the following proteins (A ′′) to (C ′′) and an expression unit including a promoter operably linked thereto.
• a ′′ a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11,
• B ′′ an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and having N-acylase activity
• C ′′ a protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11 and having N-acylase activity.
• a transformed microorganism in which the activity of the enzyme is improved as compared to a wild-type microorganism, (ii) an expression unit comprising a polynucleotide encoding the enzyme and a promoter operably linked thereto is non-native. It may be a microorganism contained in a genomic region or a non-genomic region.
• the transformed microorganism having the enzyme activity improved as compared to the wild-type microorganism is (iii) a microorganism comprising an expression unit containing a polynucleotide encoding the enzyme in a plurality of copy numbers. Also good.
• the number of copies may be, for example, 2 or more, preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, and particularly preferably 10 or more.
• the transformed microorganism having an enhanced activity of the enzyme compared to a wild-type microorganism is (iv) mutated in a unique expression unit (eg, promoter region) so as to enhance the expression of the enzyme.
• a unique expression unit eg, promoter region
• a non-naturally-occurring mutation introduced into a polynucleotide encoding the enzyme by a technique such as genome editing so as to improve the activity of the enzyme. It may be a microorganism containing an expression unit.
• the transformed microorganism in which the activity of the enzyme is improved as compared with the wild-type microorganism is any one of (i) to (iii).
• examples of host cells used as transformed microorganisms include bacteria such as bacteria belonging to the family Enterobacteriaceae and fungi.
• the bacteria may also be gram positive or gram negative.
• Examples of Gram-positive bacteria include bacteria belonging to the genus Bacillus and the genus Corynebacterium.
• Bacillus genus bacteria Bacillus subtilis is preferable.
• As a bacterium belonging to the genus Corynebacterium Corynebacterium glutamicum is preferable.
• Examples of the Gram-negative bacteria include Escherichia bacteria and Pantoea bacteria.
• Escherichia Escherichia, Escherichia coli is preferable.
• Pantoea ananatis As a bacterium belonging to the genus Pantoea, Pantoea ananatis is preferable.
• the fungus microorganisms of the genus Saccharomyces and the genus Schizosaccharomyces are preferable.
• Saccharomyces cerevisiae As the microorganism belonging to the genus Saccharomyces, Saccharomyces cerevisiae is preferable.
• Schizosaccharomyces pombe is preferable.
• the host cell used as the transformed microorganism may be, for example, an acylamino acid, a fatty acid, or a host in which the amino acid degradation system is weakened or deleted.
• the host in which the degradation system is weakened or deficient include a host in which a protein such as an enzyme related to the degradation system is weakened or deficient, and a host that produces an inhibitor of a protein such as an enzyme related to the degradation system. It is done.
• Examples of a host in which a protein such as an enzyme related to the degradation system is weakened or deleted include, for example, a host containing a mutation that reduces or deletes the expression level of the protein in the host genome, and the activity of the protein in the host genome.
• Hosts containing mutations that are reduced or deleted are included.
• Examples of a host that produces or enhances an inhibitor of a protein such as an enzyme related to the degradation system include, for example, a host into which the expression unit of the inhibitor is introduced by transformation, and the expression level of the inhibitor in the host genome.
• Examples include a host containing a mutation that enhances, and a host that contains a mutation that enhances the activity of the inhibitor in the host genome.
• the protein such as an enzyme related to an acylamino acid degradation system include an acyl CoA synthase, for example, as a protein such as an acylase and an enzyme related to a fatty acid degradation system.
• the host cell used as a transformed microorganism is, for example, a host with enhanced ability to take up amino acids and fatty acids in order to improve the supply efficiency of the substrate for the enzyme reaction and improve the production efficiency. May be.
• the host with enhanced uptake ability include hosts that produce or enhance proteins such as enzymes related to the uptake ability.
• Examples of a host that produces or enhances a protein such as an enzyme related to the uptake ability include a host in which an expression unit of the protein is introduced by transformation, and a mutation that enhances the expression level of the protein in the host genome.
• Examples of the host include a host containing a mutation that enhances the activity of the protein in the host genome.
• the transformed microorganism used in the present invention can be produced by any method known in the art.
• a transformed microorganism as described above can be produced by a method using an expression vector (eg, competent cell method, electroporation method) or a genome modification technique.
• the expression vector is an integrative vector that produces homologous recombination with the genomic DNA of the host cell
• the expression unit can be integrated into the genomic DNA of the host cell by transformation.
• the expression vector is a non-integrated vector that does not cause homologous recombination with the genomic DNA of the host cell
• the expression unit is not integrated into the genomic DNA of the host cell by transformation, and the expression vector It can exist independently of genomic DNA in the state.
• genome editing technology eg, CRISPR / Cas system, Transcribing Activator-Like Effector Nucleases (TALEN) incorporates the expression unit into the host cell's genomic DNA and modifies the host cell's unique expression unit. Is possible.
• the expression vector may further contain elements such as a terminator that functions in the host cell, a ribosome binding site, and a drug resistance gene, in addition to the minimum unit described above as an expression unit.
• drug resistance genes include resistance genes for drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.
• the expression vector may further include a region allowing homologous recombination with the host cell genome for homologous recombination with the host cell genomic DNA.
• the expression vector may be designed such that the expression unit contained therein is located between a pair of homologous regions (eg, homology arms homologous to a specific sequence in the host cell genome, loxP, FRT).
• the genome region of the host cell into which the expression unit is to be introduced is not particularly limited, but may be a locus of a gene whose expression level is large in the host cell.
• the expression vector may be a plasmid, a viral vector, a phage, or an artificial chromosome.
• the expression vector may also be an integrated vector or a non-integrated vector.
• An integrative vector may be a type of vector that is integrated entirely into the genome of the host cell.
• the integration vector may be a type of vector in which only a part (eg, expression unit) is integrated into the genome of the host cell.
• the expression vector may further be a DNA vector or an RNA vector (eg, retrovirus).
• the expression vector may also be a commonly used expression vector.
• Examples of such expression vectors include pUC (eg, pUC19, pUC18), pSTV, pBR (eg, pBR322), pHSG (eg, pHSG299, pHSG298, pHSG399, pHSG398), RSF (eg, RSF1010), pACYC ( Examples include pACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), pET (eg, pET28a) and derivatives thereof.
• the amino group-containing compound and the carboxyl group-containing compound, which are substrates used in the method of the present invention include a reaction system containing the enzyme (eg, an aqueous solution containing the enzyme, a culture solution containing a transformed microorganism producing the enzyme, Processed product of a transformed microorganism producing an enzyme).
• a reaction system containing the enzyme eg, an aqueous solution containing the enzyme, a culture solution containing a transformed microorganism producing the enzyme, Processed product of a transformed microorganism producing an enzyme.
• an amino group-containing compound or a carboxyl group-containing compound produced in another reaction system can be used as a substrate.
• an aqueous solution containing the enzyme can be used as the reaction system.
• a buffer solution is preferable.
• the buffer solution include phosphate buffer solution, Tris buffer solution, carbonate buffer solution, acetate buffer solution, and citrate buffer solution.
• the pH is preferably about 5 to 10, for example.
• the amount of the enzyme, the amino group-containing compound and the carboxyl group-containing compound (substrate) in the reaction system, and the reaction time can be appropriately adjusted according to the amount of the N-acyl-amino group-containing compound to be produced.
• the reaction temperature is not particularly limited as long as the reaction proceeds, but 20 to 40 ° C. is preferable.
• the method of the present invention may be performed in combination with an ATP regeneration system.
• the combination with the ATP regeneration system include a reaction by a combination (eg, mixing) with the ATP regeneration enzyme.
• the ATP regenerating enzyme include polyphosphate kinase, a combination of polyphosphate: AMP phosphotransferase and polyphosphate kinase, and a combination of polyphosphate: AMP phosphotransferase and adenylate kinase.
• a combination with an ATP regeneration system includes, for example, using a microorganism with enhanced ATP supply ability as a host.
• a microorganism with enhanced ATP supply ability include microorganisms that produce or enhance the above-described ATP regenerating enzyme.
• the microorganism that produces or enhances ATP regenerating enzyme include, for example, a host into which an ATP regenerating enzyme expression unit has been introduced by transformation, a host genome containing a mutation that enhances the expression level of ATP regenerating enzyme, And a host containing a mutation that enhances the activity of the ATP regenerating enzyme.
• Confirmation of the production of the N-acyl-amino group-containing compound can be performed as appropriate.
• confirmation can be made by adding a reaction stop solution (eg, 1% (v / v) phosphoric acid, 75% (v / v) aqueous methanol solution) to the reaction system, and filtering the mixture after filtration with UPLC-MS. This can be done by analysis.
• a reaction stop solution eg, 1% (v / v) phosphoric acid, 75% (v / v) aqueous methanol solution
• Example 1 Expression and purification of acylamino acid synthase 1
• Construction of acylamino acid synthase expression plasmid Jasmic acid-amido synthetase JAR1 (AtJAR1, Q9SKE2, SEQ ID NO: 3)
• Arabidopsis thaliana-3 -Amido synthetase GH3.6 (AtGH3-6, Q9LSQ4, SEQ ID NO: 1), derived from Arabidopsis thaliana indole-3-acetic acid-amido synthetase GH3.5 (AtGH3-5, O818Ar, GS4A, aid 4G, a4 10 (AtGH3-10, O O98077, SEQ ID NO: 5), 4-substituted benzoates-glutamate ligase GH3.12 (AtGH3-12, Q9LYU4, SEQ ID NO: 6), Arabidopsis thaliana derived indole-3-acetid-
• PCC 7335 from GH3 auxin-responsive promoter superfamily (SsGH3, WP_006458022, SEQ ID NO: 8), Pseudomonas savastanoi from indoleacetate-lysine synthetase (PsIAAL, P18204, SEQ ID NO: 10), Pantoea agglomerans derived hypothetical protein (PaHP, WP_031591948, SEQ ID NO: 11) Regarding the gene of Codons were optimized for expression in E. coli, and plasmid DNA inserted into the NdeI and XhoI sites in the multicloning site of pET-28a (+) (Merck) was purchased from Eurofin Genomics.
• the plasmids were respectively pET-28a-AtJAR1, pET-28a-AtGH3-6, pET-28a-AtGH3-5, pET-28a-AtGH3-10, pET-28a-AtGH3-12, pET-28a-AtGH3-17, They were named pET-28a-CfHP, pET-28a-SsGH3, pET-28a-PsIAAL, and pET-28a-PaHP.
• a protein in which a His-tag and a thrombin cleavage site are fused on the N-terminal side is expressed.
• Oryza sativa-derived probable indole-3-acetic acid-amido synthase GH3.8 (OsGH3-8, A3BLS0, SEQ ID NO: 2) Synthetic DNA with codon optimized for expression in E. coli was purchased from GenScript. The synthetic DNA was treated with restriction enzymes with NdeI and EcoRI, and ligated with pET28a (+) (Merck) similarly treated with NdeI and EcoRI. With this ligation solution E. coli JM109 was transformed, and the target plasmid was extracted from the kanamycin resistant strain and named pET-28a-OsGH3-8. In this plasmid, a protein in which a His-tag and a thrombin cleavage site are fused on the N-terminal side is expressed.
• Plasmid pET-28a-CfHP was transformed into E. coli. and introduced into E. coli BL21 (DE3), the transformant was inoculated into 100 mL of LB containing 25 mg / L kanamycin, and cultured with shaking at 37 ° C. using a Sakaguchi flask. When OD610 reached 0.2, 1 mM IPTG was added and cultured with shaking at 15 ° C. for 24 hours.
• Plasmid pET-28a-AtGH3-10 was transformed into E. coli. and introduced into E. coli BL21 (DE3), the transformant was inoculated into 100 mL of TB containing 25 mg / L kanamycin, and cultured with shaking at 37 ° C. using a Sakaguchi flask. When OD610 reached 0.4, 1 mM IPTG was added and cultured with shaking at 15 ° C. for 24 hours.
• Plasmid pET-28a-SsGH3 was transformed into E. coli. and introduced into E. coli BL21 (DE3), the transformant was inoculated into 100 mL of TB containing 25 mg / L kanamycin, and cultured with shaking at 37 ° C. using a Sakaguchi flask. When OD610 reached 0.2, 1 mM IPTG was added and cultured with shaking at 15 ° C. for 24 hours.
• the obtained soluble fraction was adsorbed on a carrier by applying to a His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0 or 10 mM Imidazole. It was.
• Example 2 Synthesis of N-caprinoyl amino acid using acylamino acid synthase 50 mM Tris-HCl, 5 mM amino acid, 5 mM sodium caprate, 10 mM ATP, 10 mM MgCl 2 , 1 mM DTT, 50 ⁇ g / mL purified enzyme, pH 8.0 Then, 0.2 mL of the reaction solution was incubated at 25 ° C. for 24 hours. After completion of the reaction, 0.8 mL of the reaction stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added, and the mixture was filtered and subjected to UPLC-MS analysis. A molecular weight signal consistent with caprynoyl amino acid was detected.
• Example 3 Synthesis of N-caprinoyl-amino acid derivative, N-caprinoyl-D-amino acid, and N-caprinoyl-peptide using acylamino acid synthase 50 mM Tris-HCl, 5 mM amino acid derivative, or D-amino acid or peptide
• acylamino acid synthase 50 mM Tris-HCl, 5 mM amino acid derivative, or D-amino acid or peptide
• a reaction solution of 5 mM sodium caprate, 10 mM ATP, 10 mM MgCl 2 , 1 mM DTT, 50 ⁇ g / mL purified enzyme, pH 8.0, 0.2 mL was incubated at 25 ° C. for 24 hours.
• Example 4 Synthesis of N-lauroyl amino acid and N-lauroyl-amino acid derivative using acylamino acid synthase 50 mM Tris-HCl, 5 mM amino acid, or amino acid derivative, 5 mM sodium laurate, 10 mM ATP, 10 mM MgCl 2 , 1 mM DTT 200 ⁇ g / mL purified enzyme, pH 8.0, 0.2 mL reaction solution was incubated at 25 ° C. for 24 hours.
• Example 5 Synthesis of N-acylamino acid using acylamino acid synthase 50 mM Tris-HCl, 5 mM amino acid, 5 mM sodium fatty acid, 10 mM ATP, 10 mM MgCl 2 , 1 mM DTT, 200 ⁇ g / mL purified enzyme, pH 8.0, 0 1 mL of the reaction was incubated at 25 ° C. for 24 hours.
• As amino acids AtGH3-6, OsGH3-8, AtGH3-5 and AtGH3-12 were L-Asp, and CfHP was Gly or L-Ala.
• N ⁇ -capryloyl-L-aspartic acid is 4.3 mM
• N ⁇ -caprinoyl-L-aspartic acid is 4.6 mM
• N ⁇ -lauroyl-L-aspartic acid is 3.5 mM
• AtGH3- When N ⁇ -capryloyl-L-aspartic acid is 4.1 mM, N ⁇ -caprinoyl-L-aspartic acid is 4.6 mM, N ⁇ -lauroyl-L-aspartic acid is 2.5 mM, and AtGH3-12 is used.
• N ⁇ -capryloyl-L-aspartic acid is 1.6 mM, N ⁇ -caprynoyl-L-aspartic acid.
• acid is 0.6 mM
• N ⁇ -lauroyl-L-aspartic acid is 0.2 mM
• CfHP is used
• N ⁇ -capryloylglycine is 4.5 mM
• N ⁇ -caprinoylglycine is 4.6 mM
• N ⁇ -capryloyl-L-alanine was detected as 3.1 mM
• N ⁇ -caprinoyl-L-alanine was detected as 3.6 mM
• N ⁇ -lauroyl-L-alanine was detected as 0.4 mM.
• Example 6 Synthesis of N-acylamino acid using acylamino acid synthase 50 mM Tris-HCl, 5 mM amino acid, 5 mM sodium fatty acid (sodium palmitate, sodium stearate is 3 mM), 10 mM ATP, 10 mM MgCl 2 , 1 mM DTT, A reaction solution of 200 ⁇ g / mL purified enzyme, pH 8.0, 0.1 mL was shaken at 25 ° C. for 24 hours. When sodium palmitate or sodium stearate was used, the reaction solution contained methanol at a final concentration of 10% (v / v).
• the amino acids are AtGH3-6, OsGH3-8, AtGH3-5, AtGH3-12 L-Asp, AtJAR1 L-Ile, AtGH3-10, SsGH3 L-Ala, AtGH3-17 L-Glu, CfHP Gly , PsIAAL used L-Lys, and PaHP used L-Cys.
• a reaction stop solution 1% (v / v) phosphoric acid, 75% (v / v) methanol
• Example 7 ATP dependency analysis of acylamino acid synthase 50 mM Tris-HCl, 5 mM amino acid, 5 mM sodium caprate, 10 mM or 0 mM ATP, 10 mM MgCl 2 , 1 mM DTT, 50 ⁇ g / mL purified enzyme, pH 8.0, 0.0. 25 mL of the reaction was incubated at 25 ° C. for 24 hours.
• As the amino acid L-Asp was used for AtGH3-6, and Gly was used for CfHP.
• Example 8 Synthesis of N-capryoylamino acid using bacterial cells expressing acylamino acid synthase (1) Preparation of various bacterial cell solutions BL21 (DE3) / pET-28a-AtGH3-6, BL21 (DE3) / pET-28a -Inoculate OsGH3-8, BL21 (DE3) / pET-28a-AtGH3-5, BL21 (DE3) / pET-28a into 100 mL of LB containing 25 mg / L kanamycin, and shake at 37 ° C using a Sakaguchi flask Culture was performed. When OD610 reached 0.6, 1 mM IPTG was added and cultured with shaking at 15 ° C.
• BL21 (DE3) / pET-28a-CfHP was inoculated into 100 mL of LB containing 25 mg / L kanamycin, and cultured with shaking at 37 ° C. using a Sakaguchi flask. When OD610 reached 0.2, 1 mM IPTG was added, and cultured with shaking at 15 ° C. for 24 hours.
• washing cell fluid After completion of the culture, the bacterial cells were collected from 5 mL of the obtained culture solution by centrifugation, washed with 20 mM Tris-HCl (pH 7.6), and suspended in 1 mL of 20 mM Tris-HCl (pH 7.6). A washing cell fluid was obtained. (Preparation of bacterial cell fluid) After completion of the culture, 15 mL of the obtained culture solution was concentrated by centrifugation to 3 mL to obtain a cell solution.
• N-acylamino acid was not produced in the absence of ATP (Example 7). However, in the reaction using the cell fluid, N-acylamino acid was produced even in the absence of ATP. Therefore, it is considered that the enzyme reaction proceeded using ATP contained in the cells.
• CfHP uses Gly as a substrate, 2.8 mM in washed cell fluid, 2.2 mM in cell fluid (in the presence of ATP), and cell fluid (in the absence of ATP) ) And 2.6 mM in the case of L-Ala as a substrate, 2.4 mM in the washed cell solution, 0.9 mM in the cell solution (in the presence of ATP), and 1.0 mM in the cell solution (in the absence of ATP). Capryloyl amino acid was detected.
• the present invention is useful for producing an N-acyl-amino group-containing compound that can be used as a cosmetic material (particularly a surfactant).
• SEQ ID NOs: 1-11 are AtGH3-6, OsGH3-8, AtJAR1 (AtGH3-11), AtGH3-5, AtGH3-10, AtGH3-12, AtGH3-17, SsGH3, CfHP (WP_002626336), PsIAAL, and PaHP, respectively.
• the amino acid sequence of (WP_031591948) is shown.
• SEQ ID NOs: 12 to 22 show base sequences encoding the amino acid sequences of SEQ ID NOs: 1 to 11, respectively, in which codons are optimized for Escherichia coli expression.

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