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WO2006009255A1 - PROCESSUS DE PRODUCTION DE α-L-ASPARTYL-L-PHÉNYLALANINE α-ESTER - Google Patents

PROCESSUS DE PRODUCTION DE α-L-ASPARTYL-L-PHÉNYLALANINE α-ESTER Download PDF

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Publication number
WO2006009255A1
WO2006009255A1 PCT/JP2005/013497 JP2005013497W WO2006009255A1 WO 2006009255 A1 WO2006009255 A1 WO 2006009255A1 JP 2005013497 W JP2005013497 W JP 2005013497W WO 2006009255 A1 WO2006009255 A1 WO 2006009255A1
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Prior art keywords
amino acid
acid sequence
protein
seq
set forth
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Japanese (ja)
Inventor
Ayako Ohno
Tadashi Takemoto
Kenzo Yokozeki
Seiichi Hara
Isao Abe
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Ajinomoto Co Inc
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Ajinomoto Co Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to the generation of a-L aspartyl-L ferro-alanine-j8-ester (or a-L- ( ⁇ -O-substituted aspartyl) -L-phenylalanine (abbreviation: ARP)).
  • ARP ⁇ -O-substituted aspartyl
  • a-L aspartyl-L ferro-alanine- ⁇ -methyl ester (sometimes abbreviated as “hi ⁇ ”) include chemical synthesis and enzyme synthesis.
  • Chemical synthesis methods include: condensing ⁇ -protected L-aspartic anhydride and L-phenylalanine methyl ester to synthesize ⁇ -protected ⁇ , and removing this ⁇ -protecting group to obtain APM, enzymatic synthesis method
  • There are known methods for synthesizing N-protected APM by condensing N-protected L-aspartic acid and L-phenylalanine methyl ester, and removing this N-protecting group to obtain APM.
  • Patent Document 1 An APM production method that does not use an N-protecting group has been studied (Patent Document 1), but the yield is extremely low and it is not suitable for industrial production. against this background, the development of a cheaper industrial production method for aspartame has been desired.
  • Patent Document 1 Japanese Patent Publication No. 02-015196
  • An object of the present invention is to provide a simple, high-yield, and inexpensive method for producing ex L-subaltylu L-ferulalanin a ester without going through a complicated synthesis method.
  • the present invention includes the following contents.
  • a method for producing an a-L subvaltyl L ferulalanin- ⁇ ester which comprises the following reaction step.
  • the method further includes the step of concentrating the reaction solution containing a L-subtiltyl L-phenolalanine- ⁇ -ester obtained in the first reaction step and removing the resulting L-phenylalanine crystals. The manufacturing method as described in.
  • reaction solution containing the a L-subtiltyl L ferulalanin- ⁇ -ester obtained in the first reaction step is subjected to ester hydrolysis under acidic conditions to give a base.
  • the a-L-subtiltyl L-ferallan produced in the third reaction step is converted to (X-L-subalthyl L-ferulalanin-ex-ester by acid and alcohol in the presence of water. And a process for producing a-L-asvaltyl L-ferulalanin- ⁇ ester.
  • the a L-subassyl-L ferulalanin- ⁇ -ester obtained in the first reaction step is a-L fastill-L-ferulalanin-13-methyl ester, and in the second reaction step, The production method according to any one of [1] to [5] above, wherein the obtained a L-subtilyl L ferulalanine mono ⁇ -ester is a-L fastill-L ferulalanin ⁇ -methyl ester.
  • the microorganism is an air mouth Monas genus, azotopacter genus, alkaligenes genus, brevibacterium genus, corynebacterium genus, escherichia genus, empedobacter genus, flavobacterium genus, microbacteria genus, propionibacteria genus, Brevibacillus, Penibachillus, Syudomonas, Serratia, Stenotrohomomonas, Sphingobacteria, Streptomyces, Xanthomonas, Viriopsis, Candida, Geotrichum, Pichia, Saccharomyces Genus, Torlaspola, Serguchi Ferga, Wiichi Kusera, Pedobacter, Persicobacter, Flexitrix, Chiteinophaga, Cyclobacterium, Lunella, Thermonema, Cycloserpense, Geridipacter, Diadacter, Flameobilg
  • amino acid sequence shown in SEQ ID NO: 6 in the sequence listing has an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and a peptide generating activity
  • a protein comprising a mature protein region having
  • (R) the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing has an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L
  • (S) a protein having the amino acid sequence set forth in SEQ ID NO: 23 in the sequence listing (T) the amino acid sequence set forth in SEQ ID NO: 23 of the Sequence Listing has an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L- -A protein comprising a mature protein region that has an activity of selectively binding a peptide to a monoester site of L-aspartate 1 a, ⁇ -diester [19]
  • the microorganism is represented by (U) or (V) below
  • (V) the amino acid sequence set forth in SEQ ID NO: 25 of the Sequence Listing, which has an amino acid sequence containing 1 or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L- -A protein comprising a mature protein region that has an activity of selectively binding a peptide to a monoester site of L-aspartate mono-a, ⁇ -diester.
  • the microorganism is represented by (W) or (X) below.
  • (X) the amino acid sequence set forth in SEQ ID NO: 27 of the Sequence Listing, which has an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions; -A protein containing a mature protein region that has an activity of selectively binding a peptide to a monoester site of L-aspartic acid mono-a, ⁇ -diester.
  • the enzyme also has the following (X) to (X) power
  • N the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing has an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L
  • (R) the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing has an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L
  • (V) one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 25 in the sequence listing A peptide that has an amino acid sequence that includes substitution, deletion, insertion, addition, and / or inversion of L-ferulalanin at the first ester site of L-aspartate 1 a, ⁇ -diester A protein comprising a mature protein region having activity to bind (W) a protein having the amino acid sequence set forth in SEQ ID NO: 27 in the sequence listing
  • FIG. 1 is a graph showing the relative activity (%) of a cytoplasmic fraction and a periplasmic fraction.
  • FIG. 2 is a graph showing the solubility of ⁇ -L aspartyl-L L-ferranin-a-ester hydrochloride in a monomethyl sulfate solution.
  • Second reaction step step of converting a-L subvalyl L-ferallanine 13 ester into a-L subvalil-L ferulalanin- ⁇ ester
  • Third reaction step ⁇ -L (L-Ferulalanine crystals are filtered) Will be described in the order.
  • the first reaction step in the presence of an enzyme having a predetermined peptide-forming activity, L-phenylanan and L-aspartic acid mono- ⁇ , ⁇ -diester React.
  • the first reaction step uses an enzyme or enzyme-containing substance capable of selectively peptide-binding L-ferrananine to the a- ester site of L-aspartate 1 a, j8-diester, L-aspartic acid mono-a, j8-diester and L-ferallanin form a-L-subaltylu-L ferulalanine mono-13 ester.
  • An enzyme or an enzyme-containing substance capable of selectively peptide-binding L-feralanine to the mono-ester site of L-aspartate mono- ⁇ , ⁇ -diester is essentially the same as L-ferranin A, j8-diester refers to an enzyme or an enzyme-containing substance that cannot catalyze a nucleophilic attack on the 13 ester site of a diester, but has the ability or activity to catalyze a reaction that nucleophilically attacks only the a ester site.
  • the nucleophilic attack cannot be performed on the a-ester site of L-phenylalanin L-aspartic acid-a, j8-diester, contrary to the above-mentioned ability. It has the ability to catalyze a reaction that only nucleophilicly attacks, and from L-aspartic acid ⁇ , j8-diester and L-phenolanine 13-L aspartyl 1 L-phenenolealanine 1 a — estenole (or ⁇ - L— ( —O— substituted aspartyl) — L-phen ylalanine (abbreviation: also known as ⁇ ARP)) or enzyme-containing products have been obtained
  • the peptide bond in ⁇ AMP is defined as L-aspartic acid a, j8-dimethyl ester 13
  • the enzyme or enzyme-containing substance used in the present invention substantially promotes only a reaction such as formula (I-H), and substantially performs a reaction such as formula (I j8).
  • A-AMP can be produced from a AMP by a simple reaction process (formula ( ⁇ )), but a-APM cannot be produced directly from ⁇ - ⁇ . That is, the method of the present invention comprises: a—APM intermediate production method It is useful for industrial production because of its extremely high efficiency.
  • an enzyme or an enzyme-containing substance As a method of allowing an enzyme or an enzyme-containing substance to act on L-aspartic acid-a, ⁇ -diester and L-phenylalanine, the enzyme or the enzyme-containing substance, L-aspartic acid mono-a, ⁇ -diester, L —Mixed with phenylalanin. More specifically, an enzyme or an enzyme-containing material may be added to a solution containing L-aspartate diester and L-phenylalanine and reacted to produce the enzyme as the enzyme-containing material.
  • the reaction may be performed as described above, or the microorganism producing the enzyme is cultured, and the enzyme is produced and accumulated in the microorganism or in the culture medium of the microorganism.
  • a method of adding L-aspartic acid mono-a, ⁇ -diester and L-phenylalanine may be used.
  • the produced L-aspertilu L-phenylalanine ⁇ ester is recovered by a conventional method, and if necessary Can be purified.
  • the "enzyme-containing material” may be any specific form as long as it contains the enzyme.
  • a culture of a microorganism that produces the enzyme, a microbial cell separated from the culture, a fungus Body treatment products are included.
  • a microbial culture is a product obtained by culturing a microorganism. More specifically, a microbial cell, a medium used for culturing the microorganism, and a mixture of substances produced by the cultured microorganism. And so on. In addition, microbial cells may be washed and used as washed cells.
  • the microbial cell-treated products include those obtained by crushing, lysing, and lyophilizing the microbial cells, and further include crude enzymes recovered by treating the microbial cells, and further purified enzymes.
  • the purified enzyme partially purified enzymes obtained by various purification methods may be used.
  • a fixed enzyme obtained by fixing these enzyme-containing substances by a covalent bond method, an adsorption method, a comprehensive method, or the like may be used.
  • some microorganisms lyse during culture, and in this case, the culture supernatant can also be used as an enzyme-containing material.
  • microorganism containing the enzyme a wild strain may be used, or a genetically modified strain expressing the enzyme may be used.
  • the microorganisms are not limited to enzyme microorganism cells, but may be treated cells such as acetone-treated cells and freeze-dried cells, and these may be fixed by a covalent bond method, an adsorption method, a comprehensive method, or the like. Use immobilized immobilized cells or treated treated cells.
  • the peptide can be more easily prepared without the need to prepare a genetically modified strain. This is preferable in that production can be performed.
  • genetically modified strains that have been transformed to express an active peptide-forming enzyme that produces a-L-subalthyl L-ferulanin-13 ester may be modified to produce a larger amount of peptide-forming enzyme. Because it is possible, the synthesis of a L aspartiru L-phenylalanine ⁇ -ester can also be carried out faster in large quantities.
  • Wild or genetically modified microorganisms are cultured in the medium, and the peptide-forming enzyme is accumulated in the medium and in the cocoons or microorganisms, and mixed with L-aspartic acid-a, j8-diester and L-feralanin ⁇ L Aspartyl 1 L —Phenolalanine 13-ester can be formed.
  • ⁇ -L-aspartyl-L ferulalanin-13-ester is produced.
  • A-L Asvaltylu L-Fe-lanalanin 13 In many cases, an enzyme that degrades ester is present. In this case, a metal protease inhibitor such as ethylenediamine tetraacetic acid (EDTA) is added. May be preferred.
  • the amount of added calories is in the range of 0. ImM force and 300 mM, preferably from ImM to lOOmM.
  • the amount of the enzyme or the enzyme-containing material used may be an amount that exhibits the desired effect (effective amount).
  • This effective amount can be easily determined by a person skilled in the art by a simple preliminary experiment. For example, when using an enzyme, about 0.01 to 100 units (U), and when using a washed cell, 0.1 to It is about 500gZL.
  • 1U is 1 ⁇ mole of a-L aspartyl-L from 1 OO mM L-aspartic acid- ⁇ , ⁇ -dimethyl ester and 200 mM L-ferulanine per minute at a temperature of 25 ° C.
  • the amount of enzyme that produces ferulanine j8-methyl ester (or e-L- ( ⁇ -0-methyl aspartyl) -L-phenylalanine (abbreviation: a-AMP) is also V).
  • L-aspartic acid mono-a, j8-diester used in the reaction, any substance can be used as long as it can be condensed with L-phenylalanine to form L-gaspartyl mono-L-ferro-alanine mono-13-ester. You may use one.
  • L-aspartic acid mono-a, ⁇ -diester include L-aspartic acid mono-a, j8-dimethyl ester, L-aspartic acid mono-a, ⁇ -jetyl ester, and the like.
  • a-L-aspartyl- L-ferro-alanine- 13-methyl ester (a-AMP) is: L-sparargine
  • a-L-aspartyl- L ferro-alanine- 13-ethyl ester (or a-L- ( ⁇ -O- ethyl aspartyl) — L-phenylalanine (abbreviation: oc AEP) is also produced.
  • salts such as hydrochloride and monomethyl sulfate may be used.
  • the reaction temperature is 0 to 60 ° C, and at-L-subtiltyl L-ferulalanin 13 ester can be produced, preferably 5 to 40 ° C.
  • the reaction pH is from pH 6.5 to LO.5, and a-L-subtilyl L-ferro-alanine 13 ester can be produced, preferably pH 7.0 to LO.0.
  • microorganism used in the present invention a microorganism having the ability to produce a-L aspartyl-1 L-ferulalanin-1 13-ester from L-aspartic acid mono-a, ⁇ -diester and L-ferroalanine is particularly used.
  • a microorganism having the ability to produce a-L aspartyl-1 L-ferulalanin-1 13-ester from L-aspartic acid mono-a, ⁇ -diester and L-ferroalanine is particularly used.
  • Microorganisms capable of producing at-L-spartyl-L-phenylalanine ⁇ -ester from L-aspartic acid mono- (X, ⁇ -diester and L-felanalanin include, Alkagenes, Brevibacterium, Corynebacterium, Escherichia, Empedobacter, Flavobacterium, Microbacteria, Propionibacterium, Brevibacillus, Penibacillus, Pseudomonas, Serratia, Stenotrophomonas, Sphingobacterium, Streptomyces, Xanthomonas, Williopsis, Candida, Geotricum, Pichia, Saccharomyces, Torraspola, Selphulga, Weekera , Pedobacter, Shikobacter genus, flexitrix genus, titinoferga genus, cyclobata terium genus, renella genus, thermonema genus, cyclo
  • Aeromonas Hydrofil ATCC 13136 (Aeromonas hydrophila)
  • Xanthomonas maltophilia FERM BP-5568 was deposited on 14 June 1995 and transferred to an international deposit on 14 June 1996 under the Budapest Treaty.
  • Brevibaterium Minutihu Ferna FERM BP—8277 was commissioned on 20 January 2003 to an international deposit under the Budapest Treaty.
  • the Escherichia coli FERM BP-8276 was commissioned on 20 January 2003 by an international organization under the Budapest Treaty.
  • Empedpactor brevis ATCC 14234 (FERM P-18545, FERM BP-8113) was established on October 1, 2001, at the National Institute of Advanced Industrial Science and Technology, Japan 8566 Tsukuba Sakai Higashi, 1-chome, Ibaraki Prefecture, Japan No. 6) was deposited with a FERM P-18545 accession number, and on July 8, 2002, a patent of the National Institute of Advanced Industrial Science and Technology. The microorganism was transferred to the deposit under the Budapest Treaty at the Deposit Center, and was given FERM BP-8113 (labeling the microorganism: Empedobacter brevis AJ 13933).
  • FERM BP-8124 the AJ 110003 (FERM BP-8124) strain was identified as the above-mentioned Sphingobaterium sp.
  • FERM BP—strain 8124 is gonococcus (0.7 to 0.8 X 1.5 to 2. O / zm), gram-negative, no sporulation, no motility, circular shape, rounded Smooth, low-convex, shiny, pale yellow, grown at 30 ° C, catalase positive, oxidase positive, OF test (glucose) negative.
  • nitrate reduction is negative, indole production is negative, acid production from dalcose is negative, arginine dihydrolase is negative, urease is positive, esculin is positive for hydrolysis, gelatin is negative for hydrolysis,) 8-galactosidase is positive, glucose is positive, L-arabinose Assimilation negative, D-mannose utilization positive, D-mann-toll utilization negative, N-acetylol D-dalcosamine utilization positive, maltose utilization positive, potassium dalconate utilization negative, n-force puric acid Sphingobataterium multiborum due to its negative properties, negative adipate utilization, dl-negative malate utilization, sodium citrate utilization negative, phenyl acetate utilization negative, cytochrome oxidase positive It was also found to be similar to the properties of Sphingobacteria spiritibolum. Furthermore, as a result of homology analysis of the base sequence of the 16S rRNA gene, it showed the
  • any of wild strains or mutant strains may be used, and recombinant strains derived by genetic techniques such as cell fusion or genetic manipulation are also used. That's right.
  • the microorganisms may be cultured and grown in an appropriate medium.
  • the medium for this is not particularly limited as long as the microorganism can grow, and may be a normal medium containing a normal carbon source, nitrogen source, phosphorus source, sulfur source, inorganic ions, and, if necessary, an organic nutrient source. .
  • any carbon source can be used as long as the above microorganisms are available.
  • sugars such as glucose, fructose, maltose, amylose, sorbitol, ethanol, and the like can be used.
  • Alcohols such as ethanol and glycerol, organic acids such as fumaric acid, citrate, acetic acid, and propionic acid, salts thereof, hydrocarbons such as norafine, and mixtures thereof can be used.
  • Nitrogen sources include ammonium salts of inorganic acids such as ammonium sulfate and ammonium chloride, ammonium salts of organic acids such as ammonium fumarate and ammonium quenate, sodium nitrate and potassium nitrate.
  • Organic nitrogen compounds such as nitrates such as peptone, yeast extract, meat extract, corn steep liquor, or mixtures thereof can be used.
  • nutrient sources used in normal media such as inorganic salts, trace metal salts, vitamins, and the like can be appropriately mixed and used.
  • the pH is 5 to 8 and the temperature is 15 to 15 under aerobic conditions.
  • the culture may be performed for about 12 to 48 hours while appropriately limiting the pH and temperature within the range of 40 ° C.
  • a peptide of the present invention has the ability to selectively peptide bond L-phenolic lanthanum to the amide ester site of L-aspartic acid-a, ⁇ -diester.
  • Enzymes are used.
  • the enzyme used in the peptide production method of the present invention is not limited to its origin, acquisition method, and the like as long as it has such activity.
  • purification of the enzyme used in the present invention, utilization of genetic engineering techniques, etc. will be described.
  • Microorganisms producing the enzyme of the present invention include microorganisms capable of producing a-L-aspartyl-1-L-ferulalanin-13-ester from L-aspartic acid-a, ⁇ -diester and L-ferulanine. Anything can be used.
  • FERM BP-8124 Pedobacter heparinus Examples include lFO 12017 strain, Taxeobacter gelupur purascens DSMZ 11116 strain, Cyclobacterium mari num ATCC 25205 strain, and Psycloserpens burton ensis ATCC 700359 strain. Empedactor brevis ATCC 14234 (FERM P-18545, FERM BP-8113) and Sphingobacterium sp.
  • FERM BP-8124 Pedobacter heparinus IFO 12017, Taxepactor Geleo Bluactensis (Taxeobacter gelupurpurascens) DSMZ 11116, Cyclobacterium marinum ATCC 25205, Psycloserpens burtonensis ATCC 700359, etc. It was selected as a result of a search for an enzyme producing strain that produced at-L aspartyl-L-phenylalanine / 3 ester in high yield from ⁇ -diester and L-ferranin.
  • the peptide-forming enzyme used in the present invention can be purified by, for example, bacterial power belonging to the genus Empedopacter.
  • purifying the enzyme a method for isolating and purifying the enzyme produced by Empedo pactor brevis force is described.
  • a bacterial cell extract is prepared by removing the insoluble fraction by centrifugation or the like.
  • the bacterial cell extract obtained in this way is fractionated by a combination of conventional protein purification methods, anion exchange chromatography, cation exchange chromatography, gel filtration chromatography, etc.
  • the product enzyme can be purified.
  • Examples of the carrier for anion exchange chromatography include Q- Sepharose HP (manufactured by Amersham). When the extract containing this enzyme is passed through a column packed with these carriers, the enzyme is recovered in the non-adsorbed fraction at pH 8.5.
  • Examples of the carrier for cation chromatography include MonoS HR (manufactured by Amersham).
  • the extract containing the enzyme is passed through a column packed with these carriers, the enzyme is adsorbed on a force ram, the column is washed, and then the enzyme is eluted using a high salt buffer. In that case, you may apply the concentration gradient which may raise salt concentration in steps.
  • MonoSHR the enzyme adsorbed on the column is eluted with about 0.2-0.5 M NaCl.
  • the present enzyme purified as described above can be further uniformly purified by gel filtration chromatography or the like.
  • the carrier for gel filtration chromatography include Sephadex 200 pg (manufactured by Amersham).
  • the fraction containing the present enzyme can be confirmed by measuring the peptide-forming activity of each fraction by the method shown in the Examples described later.
  • the internal amino acid sequences of the present enzyme purified as described above are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.
  • the present inventors first succeeded in isolating one kind of peptide-forming enzyme DNA that can be used in the peptide production method of the present invention from Empedopacter brevis FERM BP-8113 strain and the like.
  • the DNA of the present invention consists of the base sequence of base numbers 61 to 1908 set forth in SEQ ID NO: 5.
  • the DNA is Empedopactor Brevis FERM BP-8113 (deposited organization; National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Depositary Organization Address; 1-chome, Tsukuba Rakuhito, Ibaraki, Japan 1 Chuo No. 1 (6) Date of international deposit transfer; July 8, 2002).
  • the DNA consisting of the nucleotide sequence of nucleotide numbers 61 to 1908 is a coding sequence (CDS) portion.
  • the base sequence of base numbers 61 to 1908 includes a signal sequence region and a mature protein region.
  • the signal sequence region is a region having base numbers 61 to 126
  • the mature protein region is a region having base numbers 127 to 1908. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein.
  • the signal sequence contained in the sequence shown in SEQ ID NO: 5 is a kind of leader sequence, and the main function of the leader peptide encoded by the leader sequence is presumed to be secreted from inside the cell membrane to the outside of the cell membrane. .
  • the protein encoded by base numbers 127 to 1908, ie, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide-forming activity.
  • the DNA of the present invention which also has the nucleotide sequence of nucleotide numbers 61 to 1917 described in SEQ ID NO: 11, is Sphingagobaterum sp.
  • FERM BP-8124 strain deposit organization; National Institute of Advanced Industrial Science and Technology) Patent biological deposit center, depository institution address: 1-chome, Tsukuba-Ito, 1-chome, Ibaraki, Japan 1st, Central 6, International deposit date; July 22, 2002).
  • the DNA consisting of the nucleotide sequence of nucleotide numbers 61 to 1917 described in SEQ ID NO: 11 is a coding sequence (CDS) portion.
  • the base sequence of base numbers 61 to 1917 includes a signal sequence region and a mature protein region.
  • the signal sequence region is a region of base numbers 61 to 120, and the mature protein region is a region of base numbers 121 to 1917. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein.
  • the signal sequence contained in the sequence of SEQ ID NO: 11 is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded in the leader sequence region is to secrete it from the cell membrane to the outside of the cell membrane. Is done.
  • the protein encoded by base numbers 121 to 1917, that is, the site excluding the leader peptide is a mature protein, It is presumed to show peptide-forming activity.
  • the DNA of the present invention which also has the nucleotide sequence of nucleotide numbers 61 to 1935 set forth in SEQ ID NO: 17, is Pedopacter heparinas IFO 12017 strain (depositing organization; Fermentation Institute, Foundation address) Isolated from 2-17-85 Jusohoncho, Yodogawa-ku, Osaka, Japan.
  • DNA consisting of the base sequence of base numbers 61 to 1935 described in SEQ ID NO: 17 is a code sequence (CDS) part.
  • the base sequence of base numbers 61 to 1935 includes a signal sequence region and a mature protein region.
  • the signal sequence region is a region having base numbers 61 to 126
  • the mature protein region is a region having base numbers 127 to 1935.
  • the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein.
  • the signal sequence contained in the sequence described in SEQ ID NO: 17 is a kind of leader sequence, and the main function of the leader peptide encoded by the leader sequence region is to secrete it from the cell membrane to the outside of the cell membrane. It is estimated to be.
  • the protein encoded by nucleotide numbers 127 to 1935, that is, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide production activity.
  • the DNA of the present invention which has the base sequence of base numbers 61 to 1995 set forth in SEQ ID NO: 22, is taxopacter gel pull pull atsense DSMZ 11116 strain (trust machine; Deutche bammlung von ikroorganismen und Zellkulturen GmbH ( German Collection of Microorganisms and Cell Cultures ⁇ Preparation b; Address: Mascher oder Weg lb, 38124 Braunschweig, Germany) DNA consisting of the nucleotide sequence of nucleotide numbers 61 to 1995 described in SEQ ID NO: 22 Is a coding sequence (CDS) portion
  • the base sequence of base numbers 61 to 1995 includes a signal sequence region and a mature protein region, and the signal sequence region is a region of base numbers 61 to 126.
  • the mature protein region is a region having base numbers 127 to 1995. That is, the present invention relates to a peptide enzyme protein gene containing a signal sequence and a peptide as a mature protein.
  • the signal sequence contained in the sequence of SEQ ID NO: 22 is a kind of leader sequence, and the main function of the leader peptide encoded in the leader sequence region is from within the cell membrane. Secretion outside the cell membrane It is estimated that The protein encoded by base numbers 127 to 1995, that is, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide-forming activity.
  • the DNA comprising the nucleotide sequence of nucleotide numbers 29 to 1888 described in SEQ ID NO: 24, which is the DNA of the present invention, is cyclobaterum marinum ATCC 25205 strain (deposited institution; Collection, deposit address; POBox 1549 Manassas, VA 2
  • the DNA having the nucleotide sequence of nucleotide numbers 29 to 1888 described in SEQ ID NO: 24 is a coding sequence (CDS) portion.
  • the nucleotide sequence of nucleotide numbers 29 to 1888 includes a signal sequence region and a mature protein region.
  • the signal sequence region is a region having base numbers 29 to 103
  • the mature protein region is a region having base numbers 104 to 1888. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein.
  • the signal sequence contained in the sequence of SEQ ID NO: 24 is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded in the leader sequence region is to secrete it from the cell membrane to the outside of the cell membrane.
  • the protein encoded by base numbers 104 to 1888, ie, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide-forming activity.
  • the DNA comprising the nucleotide sequence of nucleotide numbers 61 to 1992 described in SEQ ID NO: 26, which is the DNA of the present invention, is Cycloserpens brtonensis ATCC 700359 strain (depositing institution; American 'type' culture ⁇ collection, deposit Address: POBox 1549 Manassas, VA 20110, the United States of America).
  • the DNA consisting of the nucleotide sequence of nucleotide numbers 61 to 1992 described in SEQ ID NO: 26 is a coding sequence (CDS) portion.
  • the base sequence of base numbers 61 to 1992 includes a signal sequence region and a mature protein region.
  • the signal sequence region is a region of base numbers 61 to L11, and the mature protein region is a region of base numbers 112 to 1992. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein. Sequence described in SEQ ID NO: 26 The signal sequence contained in is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded in the leader sequence region is to secrete it from the cell membrane to the outside of the cell membrane. It is presumed that the protein encoded by base numbers 112 to 1992, that is, the site excluding the leader peptide, is a mature protein and exhibits high peptide production activity.
  • the DNA encoding the enzyme that can be used in the present invention is empedopacter brevis, sphingobata trum sp, pedopacter heparinas, taxiopacter gel pulluatsense, cyclobataterum marinum, or cyclone. It can be obtained from chromosomal DNA or DNA libraries such as Serpens brtonensis by PCR (polymerase chain reacion, see White'TJ et al; Trends Genet., 5, 185 (1989)) or hybridization. it can. Primers used for PCR can be designed based on the internal amino acid sequence determined based on the peptide-forming enzyme purified as described in the section (3) above.
  • the base sequences of the peptide-producing enzyme genes (SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26) have been clarified by the present invention
  • these bases Probes for primer or hybridization can be designed based on the sequence, or can be isolated using the probe.
  • a primer having a sequence corresponding to the 5 ′ untranslated region and the 3 ′ untranslated region is used as a PCR primer, the entire coding region of the enzyme can be amplified.
  • the primer can be synthesized, for example, using a DNA synthesizer model 380B manufactured by Applied Biosystems using the phosphoamidite method (see Tetrahedron Letters (1981), 22, 1859).
  • the PCR reaction can be performed using Gene Amp PCR System 9 Use 600 (Perkin ELMER) and TaKaRa LA PCR in vitro Cloning Kit (Takara Shuzo) according to the method specified by the supplier such as each manufacturer.
  • the DNA encoding the enzyme that can be used in the method for producing a peptide of the present invention includes a CDS described in SEQ ID NO: 5 in the Sequence Listing, whether or not it includes a leader sequence.
  • DNA that is substantially identical to the DNA consisting of is also included. That is, a DNA having a nucleotide sequence complementary to the CDS described in SEQ ID NO: 5 in the sequence listing, or a probe prepared with the same nucleotide sequence from a DNA encoding the enzyme having a mutation or a cell retaining the enzyme. Isolation of a DNA that encodes a protein that has been hybridized under stringent conditions and has peptide-forming activity also yields DNA substantially identical to the DNA of the present invention.
  • the DNA of the present invention also includes substantially the same DNA as the DNA comprising CDS described in SEQ ID NO: 11 in the sequence listing, whether or not the leader sequence is included. That is, a DNA comprising a nucleotide sequence complementary to the CDS described in SEQ ID NO: 11 of the Sequence Listing or a probe having the same nucleotide sequencing ability is prepared from DNA encoding the enzyme having a mutation or a cell holding the enzyme. By isolating a DNA that hybridizes under stringent conditions and encodes a protein having peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.
  • the DNA of the present invention includes DNA substantially the same as the DNA comprising CDS described in SEQ ID NO: 17 in the sequence listing, whether or not the leader sequence is included. That is, a DNA comprising a nucleotide sequence complementary to the CDS described in SEQ ID NO: 17 of the Sequence Listing or a probe having the same nucleotide sequence ability is prepared from DNA encoding the enzyme having a mutation or a cell retaining the enzyme. By isolating a DNA that hybridizes under stringent conditions and encodes a protein having peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.
  • the DNA of the present invention includes DNA substantially the same as the DNA consisting of CDS described in SEQ ID NO: 22 in the sequence listing, whether or not the leader sequence is included. That is, the DNA encoding the enzyme having a mutation or the cell that retains it. It hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the CDS shown in SEQ ID NO: 22 in the Sequence Listing or a probe with the same nucleotide sequence ability, and has peptide-forming activity. By isolating the DNA encoding the protein, DNA substantially identical to the DNA of the present invention can be obtained.
  • the DNA of the present invention includes DNA substantially the same as the DNA consisting of CDS described in SEQ ID NO: 24 in the sequence listing, whether or not the leader sequence is included. That is, a DNA comprising a nucleotide sequence complementary to the CDS described in SEQ ID NO: 24 of the Sequence Listing, or a probe having the same nucleotide sequence ability is prepared from DNA encoding the enzyme having a mutation or a cell holding the enzyme. By isolating a DNA that hybridizes under stringent conditions and encodes a protein having peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.
  • the DNA of the present invention includes substantially the same DNA as the DNA consisting of CDS described in SEQ ID NO: 26 in the sequence listing, whether or not the leader sequence is included. That is, a DNA comprising a nucleotide sequence complementary to the CDS described in SEQ ID NO: 26 of the Sequence Listing, or a probe having the same nucleotide sequence ability, prepared from a DNA encoding the present enzyme having a mutation or a cell retaining the enzyme. By isolating a DNA that hybridizes under stringent conditions and encodes a protein having peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.
  • the probe can be prepared by a conventional method based on the base sequence described in SEQ ID NO: 5, for example.
  • a method for isolating the target DNA by picking up DNA that hybridizes thereto using a probe may be carried out according to a conventional method.
  • a DNA probe can be prepared by amplifying a base sequence cloned into a plasmid or a phage vector, cutting out and extracting the base sequence to be used as a probe with a restriction enzyme. The cut-out location can be adjusted according to the target DNA.
  • 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 express this condition, for example, highly homologous DNAs, for example, 50% or more, more preferably 80% or more, and still more preferably 90% or more homology.
  • DNAs that have 60 ° C, 1 X SSC, 0.1% SDS which is a condition that does not allow hybridization between DNAs that are hybridized and less homologous to each other, or is a condition for normal Southern hybridization washing Includes conditions for hybridizing at a salt concentration corresponding to 0.1 X SSC and 0.1% SDS.
  • genes that are noblyzed under these conditions include those in which a stop codon has been generated in the middle, and those that have lost activity due to mutations in the active center. It can be easily removed by expressing in an appropriate host and measuring the enzyme activity of the expressed product by the method described below.
  • a protein having an amino acid sequence encoded by the base sequence at 50 ° C and pH 8 is used. It is desirable to maintain an enzyme activity of about half or more, more preferably 80% or more, and still more preferably 90% or more.
  • PH 8 in the case of a base sequence that is hybridized under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of base numbers 127 to 1908 of the base sequence set forth in SEQ ID NO: 5, , PH 8 or more, about half or more, more preferably 80% or more, more preferably 90% or more of the protein having the amino acid sequence of amino acid residues 23 to 616 of the amino acid sequence shown in SEQ ID NO: 6. It is desirable to retain enzyme activity.
  • the amino acid sequence encoded by the CDS described in SEQ ID NO: 5 in the sequence listing is shown in SEQ ID NO: 6 in the sequence listing. Further, the amino acid sequence encoded by the CDS described in SEQ ID NO: 11 in the sequence listing is shown in SEQ ID NO: 12 in the sequence listing.
  • the amino acid sequence encoded by CDS described in SEQ ID NO: 17 in the sequence listing is shown in SEQ ID NO: 18 in the sequence listing.
  • the amino acid sequence encoded by the CDS described in SEQ ID NO: 22 in the sequence listing is shown in SEQ ID NO: 23 in the sequence listing.
  • the amino acid sequence encoded by the CDS described in SEQ ID NO: 24 in the sequence listing is shown in SEQ ID NO: 25 in the sequence listing.
  • An amino acid sequence encoded by the CDS described in SEQ ID NO: 26 in the sequence listing is shown in SEQ ID NO: 27 in the sequence listing.
  • the entire amino acid sequence described in SEQ ID NO: 6 includes a leader peptide and a mature protein region. Amino acid residues 1 to 22 are leader peptides, and 23 to 616 are mature protein regions. It is.
  • the entire amino acid sequence set forth in SEQ ID NO: 11 contains a leader peptide and a mature tamper.
  • the amino acid residue number 1 to 20 is the leader peptide, and 21 to
  • SEQ ID NO: 18 The entire amino acid sequence described in SEQ ID NO: 18 includes a leader peptide and a mature protein region. Amino acid residues 1 to 22 are leader peptides, and 23 to
  • SEQ ID NO: 23 The entire amino acid sequence described in SEQ ID NO: 23 includes a leader peptide and a mature protein region. Amino acid residues 1 to 22 are leader peptides, and 23 to
  • Up to 645 is the mature protein region.
  • SEQ ID NO: 25 The entire amino acid sequence described in SEQ ID NO: 25 includes a leader peptide and a mature protein region. Amino acid residues 1 to 25 correspond to the leader peptide, and 26 to
  • SEQ ID NO: 27 The entire amino acid sequence described in SEQ ID NO: 27 includes a leader peptide and a mature protein region. Amino acid residues 1 to 17 are leader peptides, and 18 to
  • the protein encoded by the DNA of the present invention is a protein whose mature protein has peptide-forming activity, whether or not it contains a leader peptide.
  • DNA is also included in DNA that can be used in the present invention (note that the nucleotide sequence is specified from the amino acid sequence according to the code for the universal codon). That is, according to the present invention, DNAs encoding the proteins shown in the following (A) to (X) are provided.
  • N the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing has an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L
  • (R) the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing has an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L
  • “several” means a range that does not significantly impair the three-dimensional structure and activity of the amino acid residue protein, although it differs depending on the position and type of the three-dimensional structure of the amino acid residue protein. Specifically, 2 to 50, preferably 2 to 30, more preferably 2 to 10.
  • amino acid sequences containing substitution, deletion, insertion, addition, and Z or inversion of one or several amino acid residues in the amino acid sequence under conditions of 50 ° C and pH 8, without mutation It is desirable to maintain an enzyme activity of about half or more, more preferably 80% or more, and still more preferably 90% or more of the protein of (B).
  • amino acid sequence including substitution, deletion, insertion, addition and Z or inversion of one or several amino acid residues in the amino acid sequence shown in No. 6, the amino acid sequence is arranged under conditions of 50 ° C and pH 8. About half or more of the protein having the amino acid sequence described in SEQ ID NO: 6 in the column table, more preferably 80% or more, and even more preferably It is desirable to keep 90% or more of enzyme activity.
  • Amino acid mutations such as those shown in (B) above are such that, for example, by site-directed mutagenesis, amino acids at specific sites of the enzyme gene are substituted, deleted, inserted, or added. It can be obtained by modifying the base sequence.
  • the modified DNA as described above can also be obtained by a conventionally known mutation treatment.
  • Mutation treatment includes in vitro treatment of DNA encoding the enzyme with hydroxylamine, etc.
  • Escherichia bacteria that carry the encoded DNA can be transformed by UV irradiation or a normal mutant such as N-methyl-N'-tro-N--trosoguanidine (NTG) or nitrous acid. The method of processing is mentioned.
  • base substitutions, deletions, insertions, additions, and Z or inversions as described above include naturally occurring mutations such as differences in microorganism species or strains.
  • the protein described in SEQ ID NO: 6, 12, 18, 23, 25 or 27 in the sequence listing is obtained by expressing the DNA having the above-described mutation in an appropriate cell and examining the enzyme activity of the expression product. DNA encoding a protein that is substantially identical to that of.
  • a peptide-forming enzyme that can be used in the peptide production method of the present invention can be produced.
  • Escherichia coli such as Escherichia coli, Empedopacter bacterium, Sphingobataterium bacterium, Flavo Various prokaryotic cells including Bacterium bacteria and Bacillus subtilis, Saccharomyces cerevisiae, Pichia stipitis, and Aspergillus oryzae Eukaryotic cells can be used.
  • Recombinant DNA used for introducing DNA into a host is a form in which a protein encoded by the DNA can be expressed in a vector corresponding to the type of host to be expressed. It can be prepared by inserting in As a promoter for expressing the DNA of the present invention, when a promoter specific to a peptide-forming enzyme gene such as Empedopacter brevis functions in a host cell, the promoter can be used. Further, if necessary, another promoter that works in the host cell may be linked to the DNA of the present invention and expressed under the control of the promoter.
  • Transformation methods for introducing recombinant DNA into host cells include DMMorrison's method (Methods in Enzymology 68, 326 (1979)) or recipient cells using calcium chloride. And a method of increasing the DNA permeability by treatment with shim (Mandel, M. and Higa, A., J. Mol. Biol., 53,159 (1970)).
  • a form in which the protein associates in a transformant producing the protein to form an inclusion body of the protein is also preferable. It is mentioned as a form. Advantages of this expression production method include that the target protein is protected against digestion by proteases present in the microbial cells, and that the target protein can be easily purified by centrifugation following cell disruption.
  • the protein inclusion body thus obtained is solubilized with a protein denaturing agent, and is subjected to an activity regeneration operation mainly by removing the denaturing agent.
  • an activity regeneration operation mainly by removing the denaturing agent.
  • there are many examples such as activity regeneration of human interleukin-2 (Japanese Patent Laid-Open No. 61-257931).
  • a promoter for expressing DNA encoding a peptide-forming enzyme a promoter usually used for heterologous protein production in E. coli can be used, for example, Strong promoters such as T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, and PL promoter.
  • the vector it is possible to use P UC19, pUC18, pBR322, pHSG299 , pHSG298, pHSG 399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218 and the like.
  • phage DNA vectors can be used.
  • an expression vector containing a promoter and capable of expressing the inserted DNA sequence can also be used.
  • a peptide-forming enzyme in order to produce a peptide-forming enzyme as a fusion protein inclusion body, another protein, preferably a gene encoding a peptide that is hydrophilic, is linked upstream or downstream of the peptide-generating enzyme gene.
  • a fusion protein gene As such a gene encoding another protein, any protein that increases the accumulation amount of the fusion protein and enhances the solubility of the fusion protein after the denaturation 'regeneration step may be used.
  • T7gene 10 Candidates include j8-galactosidase gene, dehydrofolate reductase gene, gamma interferon gene, interleukin 2 gene, prochymosin gene, etc.
  • a terminator which is a transcription termination sequence, downstream of the fusion protein gene.
  • the terminator include 7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, E. coli trpA gene terminator, and the like.
  • Plasmids such as pUC plasmids, PBR322 plasmids or derivatives thereof.
  • the “derivative” means a substance in which the plasmid is modified by base substitution, deletion, insertion, addition and / or inversion.
  • modification here refers to mutagen, UV irradiation, etc. It also includes modification by mutation treatment or natural mutation.
  • the vector has the best ability such as an ampicillin resistance gene.
  • plasmids are commercially available expression vectors having a strong promoter, Ru (P UC system (Takara Shuzo Co., Ltd.), PPROK system (manufactured by Clone Tetsu click), PKK233- 2 (Clontech) ho force .
  • Recombinant DNA is obtained by ligating a DNA encoding a gene encoding a protein, or in some cases a terminator, and a vector DNA. .
  • E. coli When E. coli is transformed with the recombinant DNA and this E. coli is cultured, a peptide-forming enzyme or a fusion protein of the peptide-forming enzyme and another protein is expressed and produced.
  • the host to be transformed is preferably strain E. coli JM109, which can use a strain usually used for expression of a heterologous gene. Methods for performing transformation and methods for selecting transformants are described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989) and the like.
  • a peptide-forming enzyme such as blood coagulation factor Xa, kallikrein, etc., which is not present in a peptide-producing enzyme and has a recognition sequence as a recognition sequence, is used. Even if you can cut out ⁇ .
  • a medium usually used for culturing Escherichia coli such as an M9-strong samino acid medium and an LB medium, may be used.
  • Culture conditions and production induction conditions are appropriately selected according to the type of marker, promoter, host fungus, etc. used.
  • peptide-forming enzyme or a fusion protein of a peptide-forming enzyme and another protein There are the following methods for recovering a peptide-forming enzyme or a fusion protein of a peptide-forming enzyme and another protein. If the peptide-forming enzyme or its fusion protein is solubilized in the microbial cells, the microbial cells can be recovered and then crushed or lysed, and used as a crude enzyme solution. Furthermore, if necessary, the peptide-forming enzyme or a fusion protein thereof can be purified and used by a conventional method such as precipitation, filtration, column chromatography, and the like. In this case, a purification method using a peptide-forming enzyme or a fusion protein antibody can also be used.
  • a protein inclusion body When a protein inclusion body is formed, it is solubilized with a denaturing agent. Fungus tamper Although it may be soluble together with the protein, in consideration of the subsequent purification operation, it is preferable to take out the inclusion body and make it soluble. In order to recover the inclusion bodies from the bacterial cells, a conventionally known method may be used. For example, the cells are destroyed and the inclusion bodies are collected by centrifugation or the like. Examples of denaturing agents that solubilize protein inclusion bodies include guanidine hydrochloride (eg, 6M, pH 5-8) and urea (eg, 8M).
  • guanidine hydrochloride eg, 6M, pH 5-8
  • urea eg, 8M
  • the dialysis solution used for the prayer may be a concentration of 20 mM to 0.5 M and a pH of 5 to 8 if Tris-HCl buffer or phosphate buffer is used.
  • the protein concentration during the regeneration process is preferably suppressed to about 500 ⁇ gZml or less.
  • the dialysis temperature is preferably 5 ° C or lower.
  • the denaturant removal method includes a dilution method, an ultrafiltration method, and the like, and regeneration of activity can be expected by using any of them.
  • the production method of a-L aspartyl-L ferrolanine-a-ester represented by ⁇ - ⁇ of the present invention is in accordance with the above “ ⁇ 1> first reaction step”.
  • the first reaction step to produce L-ferulanin- ⁇ -ester the a-L subvalulin L-ferulalanin-13 ester formed in the presence of water in the presence of water and an alcohol and ⁇ L-subalurilin L ferulanine It includes a second reaction step that converts to ⁇ ester.
  • the a-L-asvaltyl L ferulalanin 13 ester produced according to the method described in the section ⁇ 1> above is recrystallized as necessary.
  • the solution can be made into a solution such as an aqueous solution and then reacted by adding an acid and an alcohol to the aqueous solution.
  • the product obtained by the reaction can be made into a product through a process such as purification by one or more recrystallizations as required.
  • an enzyme reaction solution containing ⁇ -L-subalthyl L-ferulalanin 13 ester obtained in the first reaction step is used. If necessary, sterilize by means such as heating, and remove cells by means of centrifugation, membrane separation or the like. The enzyme reaction solution after removal of the cells can be used as it is in the second reaction step.
  • the enzyme reaction solution contains a considerable amount of unreacted L-furalanine, by concentrating the enzyme reaction solution, L-ferranin present in the solution crystallizes, and the resulting L- It is preferable to remove the ferroalanine crystal by filtration or the like and use it for the second step.
  • the reaction can be performed by adding acid and alcohol to the enzyme reaction solution.
  • Acid and alcohol additives can be added sequentially in this order, or they can be added simultaneously. Further, for example, either acid or alcohol may be added before removing the L-ferallanine crystal, and either one may be added later to carry out the reaction. If an appropriate amount of alcohol is present in the enzyme reaction solution obtained in the first reaction step, the addition of alcohol can be omitted.
  • the acid examples include hydrochloric acid, sulfuric acid and the like, and hydrochloric acid is particularly preferable.
  • the alcohol methanol, ethanol and the like can be mentioned, and methanol is particularly preferable.
  • an enzyme reaction solution containing —L fastill-L ferulanin-13-methyl ester is used, and methanol is used as the alcohol. Good.
  • the reaction conditions will be described by taking as an example the case of producing ⁇ -L subvaltyl L-ferroalanine a-methyl ester using hydrochloric acid and methanol.
  • the initial concentration (concentration at the start of the reaction) of a-L-asvaltyl L ferulalanine ⁇ methyl ester is preferably 0.1 to 3%.
  • hydrochloric acid for example, 35% by weight of concentrated hydrochloric acid is preferably used, and this can be added at a ratio of 8 to 55% by volume in the total volume of the reaction solution at the start of the reaction.
  • Methanol is preferably present in a proportion of 10 to 20% by volume in the total volume of the reaction solution. Note that the amount of methanol added to the reaction system is within the above range when the methanol is present in the enzyme reaction solution obtained in the first reaction step. Just add methanol to the system and prepare.
  • the reaction temperature is preferably 0 to 60 ° C.
  • the reaction time is preferably 1 to 14 days.
  • a-L subvalyl L-ferulalanin- ⁇ methyl ester hydrochloride Precipitate as crystals.
  • the precipitated crystals can be separated by means such as filtration and centrifugation.
  • the third reaction process is performed between the second reaction process to increase the solubility of ⁇ L-subalthyl L-phenol lanthanum-ester hydrochloride like monomethyl sulfate. It is preferable to add a process for removing such acids or salts. As such a process, the third reaction process described below can be performed.
  • the reaction solution containing a-L aspartyl-L ferrolanine- ⁇ -ester obtained in the first reaction step is subjected to ester hydrolysis under acidic conditions, and the medium is mixed with a base.
  • X-L aspartyl-L-phenylalanine is a step of filtering out crystals.
  • the reaction solution used in the reaction can be directly obtained from the first reaction step. The higher the acid concentration, the faster the hydrolysis reaction of the ester, so it is preferable to concentrate the enzyme reaction solution in advance so that the amount of acid added can be reduced.
  • the timing of addition may be before or after concentration, and the type of acid used in the ester hydrolysis reaction is not particularly limited as long as the ester hydrolysis proceeds.
  • Sulfuric acid, strong acidity Fat, etc. preferably hydrochloric acid, sulfuric acid, strong acid coconut, more preferably sulfuric acid, the amount of acid is more preferably 1 to 50% by volume in the total volume at the start of ester hydrolysis reaction.
  • An acid can be added so that ⁇ is in the range of 2.0 to 2.0, and the hydrolysis reaction of the ester proceeds promptly. However, from the viewpoint of not producing too much salt at the time of neutralization, it is preferably ⁇ 1.0 to 1.0, more preferably. It is between -0.5 and 0.5.
  • the ester hydrolysis reaction temperature can be in the range of 25 ° C to 80 ° C.
  • the ester hydrolysis reaction time can be in the range of 1 to 48 hours.
  • the type of base used in the neutralization reaction is not particularly limited as long as the neutralization reaction proceeds, and bases such as sodium hydroxide, sodium carbonate, ammonia, etc. can be used, and ammonia is preferred.
  • the amount of the base can be added so that the pH is in the range of 2.0 to 4.5, and is preferably 3.0 to 3.5 from the viewpoint of stable yield and impurity removal.
  • the obtained ⁇ L-subtiltyl L phenylalanine crystal can be separated by means such as filtration and centrifugation. The obtained crystals can be used in the second reaction step with or without drying.
  • the substrate of the second reaction step is different from that described above, and is not a-L-subtiltyl L-ferulalanin 13 ester. a-L aspartyl- L ferulanine.
  • the specific reaction conditions can be carried out in the same manner as described above.
  • the a L-subtiltyl L-ferulalan- ⁇ methyl ester produced by the reaction can be obtained as an acid addition salt of the acid. It can also be made into a product after the acid is removed to make the educt.
  • the a-L subtilil-L-phenylalanine monoa methyl ester hydrochloride obtained by the above reaction is reacted with a base such as sodium carbonate in an aqueous solution, and cooled crystallization or the like. Crystals are precipitated and separated by means such as filtration and hyperopic separation to obtain a-L subvalyl L-phenylalanine ⁇ -methyl ester.
  • a sweetener or the like XL-asvalyl L-fe-norelanine mono- ⁇ -methinoreestenole ⁇ -L-asparty ⁇ L-phenylalanine methyl es ter (abbreviation: a-APM, product name: aspartame)
  • a-APM product name: aspartame
  • glycerol 20g ammonium sulfate 5g, monopotassium phosphate lg, dipotassium phosphate 3g, magnesium sulfate 0.5g, yeast extract 10g, peptone 10g in 1L A medium (pH 7.0) containing 50 mL was dispensed into 500 mL Sakaflasco and sterilized at 115 ° C for 15 minutes (Medium 1).
  • microorganisms shown in Table 1 were cultured for 24 hours at 30 ° C on a slope agar medium (PH 7.0) containing 5 g of glucose, 10 g of yeast extract, 10 g of peptone, 5 g of NaCl, and 20 g of agar in 1 L.
  • PH 7.0 agar medium
  • One platinum ear was inoculated and cultured with shaking at 30 ° C and 120 round trips for 17 minutes. After cultivation, the cells were centrifuged and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA so as to be 100 g / L as wet cells.
  • yeast culture shown in Table 1-1 10 g glucose, 10 g glycerol, 5 g ammonium sulfate, lg monopotassium phosphate, 3 g dipotassium phosphate, magnesium sulfate in 1 liter
  • the microorganisms shown in Table 1-2 were cultured as follows.
  • Cellulophaga lytica NBRC 1496 1 or Flexithrix dorotheae NBRC 15987 is cultured in agar solid medium (pH 7.2, containing tryptone lg, yeast extract lg, and agar 15 g in Daigo artificial seawater SP 1L. Sterilized at 120 ° C for 15 minutes).
  • Cellulophaga lytica NBRC 14961 or Flexithrix dorotheae NBRC 15987 cells seeded in this medium at 30 ° C for 48 hours were applied to the same medium and cultured at 30 ° C for 48 hours.
  • Weeksella virosa NBRC 16016 sheep blood agar medium (Nissui plate, manufactured by Nissui Pharmaceutical) was used. Weeksella viro sa NBRC 16016 seed-cultured at 30 ° C for 48 hours in this medium was applied to the same medium and cultured at 30 ° C for 48 hours.
  • Pedobacter heparinus NBRC 12017 was cultured in an agar solid medium (pH 7.0p, 1 g) containing 10 g of peptone, 2 g of yeast extract, MgSO'7H O lg, and 15 g of agar in 1 L of distilled water.
  • Chitinophaga pinensis NBRC 15968 is cultured in an agar solid medium (p) containing 3 g of bactocagitone, 1 g of yeast extract, CaCl '2H 0 1.36 g, and 15 g of agar in 1 L of distilled water.
  • Cyclobacterium marinum ATCC 25205 is cultured in Daigo artificial seawater SP 1L containing 5 g of peptone, yeast extract lg, FeSO '7H 0 0.2 g, and agar 15 g.
  • Body medium pH 7.0, sterilized at 120 ° C for 15 minutes
  • Cvclobacterium marinum ATCC 25205 seed-cultured at 25 ° C for 48 hours in this medium was applied to the same medium, and 25 ° C, Main culture was performed for 48 hours.
  • Runella slithyformis ATCC 29530 For the cultivation of Runella slithyformis ATCC 29530, use agar solid medium (pH 7.0, sterilized at 120 ° C for 15 minutes) containing peptone lg, fermentation mother extract lg, glucose lg, and agar 15g in 1L of distilled water. It was. The cells of Runella slithyfor mis ATCC 29530 seed-cultured in this medium at 25 ° C. for 48 hours were applied to the same medium, and cultured at 25 ° C. for 48 hours.
  • agar solid medium pH 7.0, sterilized at 120 ° C for 15 minutes
  • Thermonema lapsum ATCC 43542 was cultured in 1 L of distilled water, 0.2 g of Nitrilotriacetic acid, 2 ml of 0.03% FeCl solution, CaSO-2H 0 0. 12 g MgSO-7H 0 0.
  • Agar solid medium (pH 8.2, sterilized at 120 ° C for 15 minutes) containing 2 ml of agar and 15 g of agar was used (the trace element solution was 1 ml of distilled water, 0.5 ml of H 2 SO 4, MnSO-2.2 g, ZnSO 0.5 g,
  • Gelidibacter algens ATCC 700364 or Lewinella cohaerens ATCC 23 123 ⁇ Psychroserpens burtonensis ATCC 700359 seed cells cultured at 10 ° C for 72 hours are applied to the same medium. C. Main culture for 72 hours.
  • Lewinella cohaerensATCC 23123 the seed cells cultured at 30 ° C for 48 hours were applied to the same medium and cultured at 30 ° C for 48 hours.
  • Salegentibacter salegens DSMZ 5424 the seed cells cultured at 25 ° C. for 48 hours were applied to the same medium and cultured at 25 ° C. for 48 hours.
  • Dyadobacter fermentans ATCC 700827 was cultured in 1 liter of distilled water with NH C1
  • Mino acid 0.5g, dextrose 0.5g, soluble starch 0.5g, sodium pyruvate An agar solid medium (pH 7.0, sterilized at 120 ° C. for 15 minutes) containing 0.5 g and agar 15 g was used.
  • the cells of Dyadobacter fermentans ATCC 700827 seed-cultured at 25 ° C for 48 hours in this medium were applied to the same medium and cultured at 25 ° C for 48 hours.
  • Flammeovirga aprica NBRC 15941 was cultured in Daigo artificial seawater SP 1L, agar solid medium containing 2g of tryptone, 0.5g of beef extract, 0.5g of yeast extract, 0.2g of sodium acetate, 15g of agar ( Sterilized at pH 7.2, 120 ° C for 15 minutes).
  • the cells of Flammeovirga aprica NBRC 15941 that had been seed-cultured at 25 ° C for 48 hours in this medium were applied to the same medium and cultured at 25 ° C for 48 hours.
  • Tenacibaculum maritimum ATCC43398 contains 0.5 g of tryptone, 0.5 g of yeast extract, 0.5 g of yeast extract, 0.2 g of beef extract, 0.2 g of sodium acetate, and 15 g of agar in 300 ml of distilled water and 700 ml of seawater SP Agar solid medium (sterilized at pH 7.0, 120 ° C for 15 minutes) was used.
  • Tenacibaculum maritimum ATCC43398 cells seed-cultured at 25 ° C. for 48 hours in this medium were applied to the same medium and cultured at 25 ° C. for 48 hours.
  • Rhodothermus marinus DSMZ 4252 1 g of yeast extract 2.5 g, tryptone 2.5 g ⁇ Nitrilotriacetic acid lOOmg ⁇ CaSO ⁇ 2 ⁇ 0 40 mg ⁇ MgCl ⁇ 6 ⁇ 0
  • Agar solid medium (pH 7.2, sterilized at 120 ° C for 15 minutes) containing 200 mg, 0.01 M Fe citrate 0.5 ml, trace element solution 0.5 ml, phosphoric acid noffer 100 ml, distilled water 900 ml, agar 28 g (Note that the trace element solution was Nitrilotriacetic acid 12.8 g, FeCl 4 lg, MnCl -4H 0 0.5 g, CoCl -4H 0 0.3 g, CuCl -2H 0 50 mg, Na M
  • 1L water contains 5.44g KH PO and 43g K HPO). Use this medium at 60 ° C for 48 hours.
  • Rhodothermus marinus DSMZ 4252 The cultured cells of Rhodothermus marinus DSMZ 4252 were applied to the same medium and cultured at 60 ° C. for 48 hours.
  • an agar solid medium containing BACTO MARINE BROT H (DIFCO 2216) (agar 1.5%, pH 7.6, killed at 120 ° C for 15 minutes) was used.
  • Bacterial cells of Zobellia galactanivorans DSMZ 12802 seeded and cultured in this medium at 30 ° C for 48 hours were applied to the same medium and cultured at 30 ° C for 48 hours.
  • Muricauda ruestringensis DSMZ 13258 was cultured in 1 L of distilled water using 1.5 g yeast ex, 2.5 g peptone, 2 g hexadecane, 17.7 g NaCl, 0.48 g KC1 and MgCl.
  • Taxeobacter gelupurpurascens DSMZ 11116 is cultured in an agar solid medium containing 1 g of distilled water, 3 g of strength diton, 1 g of yeast extract, CaCl '2H 0 1.36 g, and 15 g of agar.
  • Taxeobacter gelupurpurascens DSMZ 11116 seed-cultured at 30 ° C for 48 hours in this medium were applied to the same medium.
  • Cytophaga hutchinsonii NBRC 15051 is cultured in agar containing 1 g of distilled water, 3 g of CADI, yeast extract lg, CaCl '2H 0 1.36 g, 5 g of cellobiose, 15 g of agar.
  • a solid medium (pH 7.2, sterilized at 120 ° C for 15 minutes) was used.
  • Agar solid medium (pH 7.2, sterilized at 120 ° C for 15 minutes) containing 15 g of heaven was used.
  • Saprospira grandis ATCC 23119 was cultured in Daigo artificial seawater SP 1L, K NO 0.5g, glyce mouth sodium phosphate 0.1g, trishydroxymethylaminomethane lg,
  • Haliscomenobacter hydrossis ATCC 27775 is roasted in 1 L of distilled water with KH P
  • Each bacterial cell obtained in this way is collected from an agar medium, and contains 0.1 mM borate buffer (pH 9.0) containing 10 mM EDTA so that it becomes 100 g / L as wet cells. Suspended in
  • a lOOmM borate buffer solution (pH 9. 0) Add 0.1 mL each to a total volume of 0.2 mL, and at 20 ° C, use the microorganisms shown in Table 1-1 for 3 hours, as shown in Table 1-2. When microorganisms were used, the reaction was carried out for 1 hour. The production amount (mM) of a-L-Asvalutil L-Fe-Lalanine-13 methyl ester ( ⁇ -AMP) at this time is shown in Table 11 and Table 1-2. ⁇ AMP was not detected from any microorganism.
  • the microorganisms shown in Table 2 were cultured in the same manner as the bacteria in Table 1 of Example 1. After cultivation, the cells were centrifuged and suspended in 0.1 M phosphate buffer (PH9.0) containing lOmM EDTA so that the cells were wet as lOOgZL. Cell suspension of these microorganisms 0. ImL, lOO mM borate buffer solution (pH 9.0) containing 10 mM ED TA, 100 mM L-aspartate mono-a, ⁇ -dimethyl ester hydrochloride, and 200 mM L-felualanine ) 0. After each addition to make the total volume 0.2 mL, the reaction was carried out at 30 ° C for 2 hours. Table 2 shows the production amount (mM) of ⁇ -L aspartyl-L ferulalanin-a-methyl ester (-AMP). A AMP was not detected in any microorganism.
  • phosphate buffer PH9.0
  • lOmM EDTA
  • Empedopactor brevis FERM BP 8113 strain cultured in medium 3 at 30 ° C for 16 hours (deposited organization: National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Depositary Organization Address: Ibaraki, Japan) 1 ml Tsukuba 1-chome 1 Chuo 6th, International transfer date; July 8, 2002) 2 ml of inoculation and shaking culture at 30 ° C, 120 round-trip Z minutes for 16 hours
  • the operations after centrifugation were performed on ice or at 4 ° C.
  • the obtained culture broth was centrifuged (10, OOOrpm, 15 minutes), and the cells were collected. 16 g of cells were washed with 50 mM Tris-hydrochloric acid buffer (pH 8.0), suspended in 40 ml of the same buffer, and subjected to ultrasonic disruption at 195 W for 45 minutes.
  • the ultrasonic disrupted liquid was centrifuged (10, OOOrpm, 30 minutes), and the disrupted bacterial cells were removed to obtain the ultrasonic disrupted liquid supernatant.
  • This dialysis fraction was applied to a Mono S column (Amersham) equilibrated in advance with 50 mM acetate buffer (pH 4.5), and the enzyme was eluted with a linear concentration gradient from 0 to 0: LM NaCl. I let you go. 1M of the active fraction with the least amount of contaminating protein
  • Example 2 Apply to a Superdex 200pg column (Amersham) equilibrated with 50 mM acetate buffer (pH 4.5) containing NaCl, and perform gel filtration by flowing the same buffer (pH 4.5) containing 1 M NaCl. A fraction solution was obtained. By these operations, it was confirmed that the peptide-forming enzyme obtained in Example 2 was uniformly purified from the results of electrophoresis experiments. The recovery rate of active substance in the above purification process was 12.2%, and the degree of purification was 70.times.
  • MonoS fraction enzyme (about 20 ⁇ 1) 10 / ⁇ 1 obtained in Example 2 was converted to 105.3 mM L-aspartate a, j8-dimethyl ester hydrochloride, 210.5 mM L-ferallanin, 10.
  • Table 3 shows the production process of a-L aspartyl-L ferro-alanine-13-methyl ester (a AMP). In addition, almost no ⁇ -L-Lassvalyl L-phenylalanine 13 methyl ester was formed in the enzyme-free group.
  • Example 2 In the same manner as in Example 2, using the medium shown in Example 2, Sphingobataterumus sp. FERM BP-8124 strain (deposited institution; National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Depositary Institution) 1st address, 1st address, Tsukuba Sakai Higashi, Ibaraki, Japan 6th, International deposit date; July 22, 2002). The subsequent operations after centrifugation were performed on ice or at 4 ° C. The obtained culture was centrifuged (10, OOOrpm, 15 minutes) to collect the cells. 2 g of the cells were washed with 20 mM Tris-HCl buffer (pH 7.6), suspended in 8 ml of the same buffer, and subjected to ultrasonic crushing at 195 W for 45 minutes.
  • 20 mM Tris-HCl buffer pH 7.6
  • This ultrasonic disruption liquid was centrifuged (10, OOOrpm, 30 minutes), and the ultrasonic disruption liquid supernatant was obtained by removing the fragment of the disrupted cells.
  • the sonicated supernatant was dialyzed overnight against 20 mM Tris-HCl buffer (pH 7.6), and the insoluble fraction was removed by ultracentrifugation (50, OOOrpm, 30 minutes).
  • a soluble fraction was obtained as a clear liquid.
  • the obtained soluble fraction was applied to a Q-Sepharose HP column (Amersham) equilibrated in advance with Tris-hydrochloric acid buffer (pH 7. 6), and the active fraction was collected from the non-adsorbed fraction. .
  • This active fraction is dialyzed overnight against 20 mM acetate buffer (pH 5.0), and the insoluble fraction is removed by centrifugation (10, OOOrpm, 30 minutes). Got. This dialysis fraction was applied to an SP-Sepharose HP column (Amersham) equilibrated beforehand with 20 mM acetate buffer (pH 5.0).
  • the concentrated solution (about 15 UZml) 10 1 of the SP-Sepharose HP fraction obtained in Example 4 is used. 52. Carrotate in 190 1 borate buffer (pH 9.0) containing 6 mM L-aspartic acid ⁇ , ⁇ -jetyl ester hydrochloride, 105.2 mM L-phenolalanine, 10.8 mM EDTA. The reaction was carried out at 20 ° C. Table 4 shows the production process of AMP or AEP. In addition, almost no AMP or AEP was produced in the enzyme-free group. In addition, the value which used the standard product of AMP was described for the production
  • One platinum loop of this bacterial cell was inoculated into a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was spread, and cultured at 30 ° C with shaking.
  • the PCR reaction was performed using Takara PCR Thermal Cycler PERSONAL (manufactured by Takara Shuzo), and the reaction under the following conditions was performed for 30 cycles.
  • Chromosomal DNA of Empedopacter brevis obtained in Example 6 (3) was completely digested with the restriction enzyme Hindlll at 37 ° C for 16 hours, and then electrophoresed on a 0.8% agarose gel. did.
  • the agarose gel after electrophoresis was blotted to a nylon membrane filter Nylon embranes positively charged (manufactured by Roche's Diagnostics) and subjected to alkali denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer Mannheim).
  • the labeling probe prepared above was added with digoxigen and hybridized at 50 ° C for 16 hours. . Thereafter, the filter was washed with 2 ⁇ SSC containing 0.1% SDS at room temperature for 20 minutes. Further, the plate was washed twice with 0.1 X SSC containing 0.1% SDS at 65 ° C for 15 minutes.
  • Detection of the band hybridizing with the probe was performed based on the instructions using DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, a band of about 4 kb that hybridized with the probe could be detected.
  • Escherichia coli was transformed by mixing this ligation reaction solution 5 ⁇ ⁇ Escherichia coli strain JM109 with 100 ⁇ 1 of a combient cell (manufactured by Toyobo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.
  • the colonies of the chromosomal DNA library were transferred to a nylon membrane filter Nylon Membranes for Colony and Plaque Hybridization (Roche's Tygnotechs) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EA SY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C for 1 hour, then the above labeled probe with digoxgen was added, and hybridization was carried out at 50 ° C for 16 hours. Thereafter, the filter was washed with 2 ⁇ SSC containing 0.1% SDS at room temperature for 20 minutes. Further, it was washed twice with 0.1 X SSC containing 0.1% SDS at 65 ° C for 15 minutes.
  • a plasmid possessed by the Escherichia coli JM109 strain was prepared from the above two strains confirmed to hybridize with the labeled probe using Wizard Plus Minipreps DNA Purification System (manufactured by Promega), and the plasmid was hybridized in the vicinity. The base sequence was determined. The sequencing reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis was performed using CEQ 2000-XL (manufactured by Beckman Coulter).
  • Microbiol., 68 (1), 211-218 (2002) is 34% amino acid sequence, Brevib It showed 26% homology with the glutaryl-7ACA acylase of acillus laterosporum (J. Bacteriol., 173 (24), 7848-7785 (1991).
  • this plasmid was ligated with a DNA fragment containing the trp promoter obtained by treating with EcoOlO 91 / EcoRI and EcoUC109lZEcoRI-treated product of pUC19 (Takara).
  • Escherichia coli JM109 was transformed with this ligation solution, and a strain having the target plasmid was selected from ampicillin resistant strains.
  • a DNA fragment obtained by treating this plasmid with a HindlllZPvuII trowel and pKK223-3 (manufactured by Amersham Pharmacia) were treated with Hindlll / Hincl I, and the obtained DNA fragment containing the rrnB terminator was ligated. .
  • the E. coli JM109 strain was transformed with this ligation solution, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrpT.
  • the target gene was amplified by PCR using the chromosomal DNA of Empedopacter brevis FERM BP-8113 strain as a saddle and the oligonucleotides shown in SEQ ID NOs: 9 and 10 as primers.
  • This DNA fragment was treated with NdelZPstl, and the resulting DNA fragment was ligated with pTrpT treated with NdelZPstl.
  • the Escherichia coli JM109 strain was transformed with this ligation solution, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrpT_Gtg2.
  • Escherichia coli JM109 strain containing pTrpT-Gtg2 was seed-cultured in LB medium containing lOOmg / 1 ampicillin at 30 ° C for 24 hours. 1 ml of the obtained culture broth was added to 50 ml of medium (2 gZlD-glucose, lOgZl yeast extract, lOgZl casamino acid, 5 gZl ammonium sulfate, 3 gZl potassium dihydrogen phosphate, lgZl dipotassium hydrogen phosphate, 0.5 gZl sulfate.
  • medium 2 gZlD-glucose, lOgZl yeast extract, lOgZl casamino acid, 5 gZl ammonium sulfate, 3 gZl potassium dihydrogen phosphate, lgZl dipotassium hydrogen phosphate, 0.5 gZl sulfate.
  • Escherichia coli JM109 strain containing pTrpT-Gtg2 was seed-cultured at 30 ° C. for 24 hours in LB medium containing lOOmg / 1 ampicillin. 1 ml of the obtained culture broth was added to 50 ml of medium (2 g / l glucose, lOgZl yeast extract, lOgZl casamino acid, 5 gZl ammonium sulfate, 3 gZl potassium dihydrogen phosphate, lgZl dipotassium hydrogen phosphate, 0.5 gZl sulfate. A 500 ml Sakaguchi flask containing magnesium heptahydrate (lOOmgZl ampicillin) was seeded and main culture was performed at 25 ° C. for 24 hours to obtain cultured cells.
  • the cultured cells were fractionated into a periplasm fraction and a cytoplasm fraction by an osmotic shock method using a 20 g Zdl sucrose solution.
  • the cells immersed in a 20 g Zdl sucrose solution were immersed in a 5 mM MgSO aqueous solution, and this centrifuged supernatant was used as a periplasm fraction (Pe). Also,
  • the centrifugal precipitate was resuspended and sonicated to obtain a cytoplasmic fraction (Cy).
  • cytoplasmic fraction (Cy)
  • glucose 6-phosphate dehydrodrogenase which is known to exist in cytoplasm
  • the measurement method is ImM dalose 6-phosphate, 0.4 mM NADP, 10 mM MgSO, 50 mM Tris-Cl (pH 8), 30.
  • Fig. 1 shows the enzyme amounts of the cytoplasmic fraction and the periplasmic fraction when the activity of the cell-free extract prepared separately is defined as 100%.
  • Glucose 6-phosphate dehydrodrogenase activity is mixed in the periplasmic fraction, but the periplasmic fraction is mixed in the cytoplasmic fraction Indicates not.
  • About 60% of a-L-aspartyl 1 L-fe-lanalanin 13-ester ( ⁇ -AMP) -forming activity is recovered in the periplasm fraction and predicted from the amino acid sequence using the above SignalP vl. 1 program. Thus, it was confirmed that a-AMP synthase is secreted into periplasm.
  • the isolation of the peptide-forming enzyme gene will be described.
  • the microorganism used was Sphingobata terium sp. FERM BP-8124.
  • Escherichia coli DH5 ⁇ was used as a host, and pUC118 was used as a vector.
  • the cells were cultured at 25 ° C for 24 hours on sodium chloride sodium chloride, 20 g Zl agar, PH 7.0)).
  • the cells were inoculated with 1 platinum ear in a 500 ml Sakaguchi flask with 50 ml of CM2G liquid medium (agar removed from the above medium) and cultured at 25 ° C with shaking.
  • a DNA fragment containing a part of the peptide-forming enzyme gene derived from Empedopacter brevis FERM BP-8113 was obtained by PCR using LA-Taq (Takara Shuzo). PCR reaction was performed on the chromosomal DNA obtained from Empedopacter brevis FERMBP-8113 strain using primers having the nucleotide sequences shown in SEQ ID NOs: 3 and 4.
  • PCR reaction was performed using Takara PCR Thermal Cycler PERSONAL (Takara Shuzo), and the reaction under the following conditions was performed for 30 cycles.
  • the chromosomal DNA of Sphingobataterium sp obtained in Example 7 (2) was digested with the restriction enzyme Sacl at 37 ° C for 16 hours and then electrophoresed on a 0.8% agarose gel. did. After electrophoresis, it was blotted to nylon membrane filter Nylon membranes positively charged (Roche's Diagnostics) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EAS Y HYB (manufactured by Boehringer Mannheim). After pre-hybridization of the filter at 37 ° C for 1 hour, the labeling probe prepared above was added with digoxigen, and hybridization was performed at 37 ° C for 16 hours. . Thereafter, the filter was washed twice at 60 ° C. with 1 ⁇ SSC containing 0.1% SDS.
  • Detection of a band hybridizing with the probe was performed based on the instructions using DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, a band of about 3 kb that hybridized with the probe could be detected.
  • the colony of the chromosomal DNA library was transferred to a nylon membrane filter Nylon Membranes for Colony and Plaque Hybridization (Roche's Tygnoteix) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EA SY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C for 1 hour, then the above labeled probe with digoxigen was added, and hybridization was carried out at 37 ° C for 16 hours. Thereafter, the filter was washed twice with 60 ° C. with 1 ⁇ SSC containing 0.1% SDS.
  • a plasmid possessed by Escherichia coli DH5 ⁇ was prepared from the above 6 strains confirmed to hybridize with the labeled probe using Wizard Plus Minipreps DNA Purification System (manufactured by Promega), and in the vicinity of the hybridized probe. The base sequence was determined. The sequencing reaction was performed using CEQ DTCS-Quick Start Kit (Beckman Coulter, Inc.) based on the instructions. Electrophoresis was performed using CEQ 200 00-XL (manufactured by Beckman 'Coulter).
  • a platinum loop was inoculated into a normal test tube containing dipotassium hydrogen, 0.5 g Zl magnesium sulfate heptahydrate, lOOmg / 1 ampicillin), and a main culture was performed at 25 ° C for 20 hours.
  • the cloned gene had an activity of producing 0.5-U per liter of culture medium, and that the cloned gene was expressed in E. coli. As a control, no activity was detected in the transformant into which only pTrpT was introduced.
  • amino acid sequence of SEQ ID NO: 12 in the sequence listing was analyzed with the SignalP vl. 1 program (Protein Engineering, voll2, no.l, pp.3- 9, 1999). Was predicted to function as a signal and secreted into the periplasm, and the mature protein was assumed to be downstream from the 21st.
  • Escherichia coli JM109 strain with pTrpT—Sm—aet was added to 50 ml of medium (2 g / l glucose, lOgZl yeast extract, lOgZl casamino acid, 5 gZl ammonium sulfate, 3 gZl potassium dihydrogen phosphate, lgZl dihydrogen phosphate Potassium, 0.5 g Zl magnesium sulfate heptahydrate, lOOmg / 1 ampicillin) was inoculated into one platinum ear in a normal test tube, 25 ° C, 20 Time main culture was performed.
  • medium 2 g / l glucose, lOgZl yeast extract, lOgZl casamino acid, 5 gZl ammonium sulfate, 3 gZl potassium dihydrogen phosphate, lgZl dihydrogen phosphate Potassium, 0.5 g Zl magnesium sulfate heptahydrate
  • Re source-PHE pre-equilibrated with 2M ammonium sulfate and lOOmM phosphate buffer was prepared by mixing the active fraction with 5 volumes of 2M ammonium sulfate and lOOmM phosphate buffer.
  • the column was applied to a column (Amersham), and the enzyme was eluted with a linear concentration gradient with 2 to 0 M ammonium sulfate to obtain an active fraction solution. By these operations, it was confirmed that the peptide-forming enzyme was purified by electrophoresis.
  • CM2G agar medium 50gZl Darcos, lOgZl yeast extract, lOg / 1 peptone, 5g / l sodium chloride sodium salt, 20gZl agar, pH 7.0
  • CM2G liquid medium 50gZl Darcos, lOgZl yeast extract, lOg / 1 peptone, 5g / l sodium chloride sodium salt, 20gZl agar, pH 7.0
  • One platinum loop inoculation was performed on this microbial cell in a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was spread, and cultured at 25 ° C. with shaking.
  • a DNA fragment containing a part of the peptide-forming enzyme gene derived from Pedopector Heparinas IFO 12017 was obtained by PCR using LA-Taq (Takara Shuzo). PCR reaction was performed on the chromosomal DNA obtained from Parinas IFO 12017 strain using the primers having the nucleotide sequences shown in SEQ ID NOs: 15 and 16. About lkb DNA fragments amplified by PCR were separated by 0.8% agarose electrophoresis. The target band was cut out and purified. Label this probe with digoxigen according to the instructions using DIG High Prime (Boehringer Manno, Im)
  • Pedopacter Heparinas IFO 12017 chromosomal DNA was digested with the restriction enzyme HindI 37. C. After 16 hours of reaction and complete digestion, electrophoresis was performed on a 0.8% agarose gel. Bacterial denaturation, neutralization, and immobilization were performed by blotting from agarose genore after electrophoresis to a nylon membrane phosphonately charged (manufactured by Roche Diagnostics). Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 50 ° C. for 1 hour, then the digoxgen-labeled probe prepared above was added, and hybridization was carried out at 50 ° C. for 16 hours. Thereafter, the filter was washed twice with 60 ° C. with 1 ⁇ SSC containing 0.1% SDS.
  • Detection of the band hybridizing with the probe was performed based on the instructions using the DIG Nucleotide Detection Kit (Behringer Mannheim). As a result, professional A band of about 5 kb was detected that hybridized with the probe.
  • Escherichia coli was transformed by mixing 5 ⁇ 1 of this ligation reaction solution and 100 / z 1 of Escherichia coli JM109 strain combient cell (Takara Shuzo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.
  • the colony of the chromosomal DNA library was transferred to a nylon membrane filter Nylon Membranes for Colony and Plaque Hybridization (Roche Diagnostics) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C for 1 hour, then the above labeled probe with digoxigen was added, and hybridization was performed at 37 ° C for 16 hours. Thereafter, the filter was washed twice with 60 ° C. with 1 ⁇ SSC containing 0.1% SDS.
  • a plasmid possessed by the Escherichia coli JM109 strain was prepared from the above-mentioned one strain that was confirmed to hybridize with the labeled probe, and the base sequence in the vicinity of the hybridized probe was determined.
  • the sequencing reaction was CEQ DTCS-Quick Start Kit (Beckman's 1 Luther Co.) was used based on the instructions. Electrophoresis was performed using CEQ 2000-XL (manufactured by Beckman Coulter).
  • the target gene was amplified by PCR using the chromosomal DNA of Pedopector Heparinas IFO 12017 strain as a saddle and the oligonucleotides shown in SEQ ID NOs: 19 and 20 as primers.
  • This DNA fragment was treated with NdelZHindlll, and the resulting DNA fragment was ligated with the NdelZ Hindlll-treated product of pTrpT.
  • Escherichia coli JM109 strain was transformed with this ligation solution, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrpT-Phaet.
  • a platinum loop was inoculated into a normal test tube containing dipotassium hydrogen, 0.5 g Zl magnesium sulfate heptahydrate, lOOmg / 1 ampicillin), and a main culture was performed at 25 ° C for 20 hours.
  • the microorganism is Taxoxovater Gelpurpurpuratsusens DSMZ 11116 strain (Deposit institution; Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (uerman and oliectionof Microorga nisms and Cell Cultures ⁇ Destination; Mascheroder Weg lb, 38124 Braunsch weig, Germany).
  • Escherichia coli jM109 strain was used as a host, and pUC118 was used as a vector.
  • Taxopacter gel pullulu atsense DSMZ 11116 strain was cultured on CM2G agar medium (50 gZl glucose, lOgZl yeast extract, lOgZl peptone, 5 gZl sodium chloride, 20 gZl agar, ⁇ 7.0) at 25 ° C for 24 hours.
  • CM2G agar medium 50 gZl glucose, lOgZl yeast extract, lOgZl peptone, 5 gZl sodium chloride, 20 gZl agar, ⁇ 7.0
  • One platinum loop was inoculated into a 500 ml Sakaguchi flask in which 50 ml of CM 2G liquid medium (with the agar removed from the above medium) was placed, and cultured at 25 ° C with shaking.
  • chromosomal DNA was obtained from this bacterial cell based on the method described in the manual.
  • a DNA fragment containing a part of the peptide-synthesizing enzyme gene derived from Taxeopacter gelpurpurusense DSMZ 11116 strain was obtained by PCR using LA-Taq (Takara Shuzo). Taxopacter gel pull pull atsense DSMZ 11116 strain chromosomal DNA was subjected to PCR reaction using primers having the nucleotide sequences shown in SEQ ID NOs: 21 and 16. About lkb DNA fragments amplified by PCR were separated by 0.8% agarose electrophoresis. The target band was cut out and purified. The DNA fragment was labeled with digoxigen based on the instructions using DIG High Prime (Boehringer Mannheim).
  • Taxopacter Gel Pull Pull Atsense DSMZ 11116 Chromosomal DNA was digested with the restriction enzyme Pstl at 37 ° C for 16 hours, and then 0.8% agarose gel. Electrophoresis. After electrophoresis, the gel was blotted onto a nylon membrane filter Nyl on membranes positively charged (manufactured by Roche Tygnotechs), and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY H YB (Boehringer Mannheim). After the filter was incubated at 50 ° C for 1 hour and hybridization, the labeled probe prepared above was added with digoxigen, and the hybridization was performed at 50 ° C for 16 hours. went. After this, filter 60 with 1 X SSC containing 0.1% SDS. Wash with C twice.
  • Detection of the band hybridizing with the probe was performed based on the instructions using the DIG Nucleotide Detection Kit (Behringer Mannheim). As a result, a band of about 5 kb that hybridized with the probe could be detected.
  • Taxopacter gel pull pull atsense DSMZ 11116 chromosomal DNA 5 g was completely digested with Pstl. About 5 kb of DNA was separated by 0.8% agarose gel electrophoresis, DNA was purified using Gene Cleanll Kit (Funakoshi Co., Ltd.), and dissolved in 101 TE. 4 ⁇ 1 of this was mixed with pUC118 Pstl / BAP (Takara Shuzo) and DNA Ligation
  • a ligation reaction was performed using Kit Ver.2 (Takara Shuzo). Escherichia coli was transformed by mixing 5 ⁇ 1 of this ligation reaction solution and 100 ⁇ 1 of Escherichia coli JM109 strain cell (manufactured by Takara Shuzo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.
  • the colonies of the chromosomal DNA library were transferred to a nylon membrane filter Nylon Membranes for Colony and Plaque Hybridization (Roche Diagnostics) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C for 1 hour, then the above labeled probe with digoxigen was added, and hybridization was performed at 37 ° C for 16 hours. After this, the filter contains 0.1% SDS. Washing was performed twice at 60 ° C with 1 X SSC.
  • DIG Nucleotide Detection is used to detect colonies that hybridize with labeled probes.
  • a plasmid possessed by the Escherichia coli JM109 strain was prepared from the above-mentioned one strain that was confirmed to hybridize with the labeled probe, and the base sequence in the vicinity of the hybridized probe was determined.
  • the sequencing reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis is CEQ 2000-XL
  • Taxosepacter gel pull pull atsense DSMZ 11116 strain-derived peptide production The nucleotide sequence of the full length of the enzyme gene and the corresponding amino acid sequence are shown in SEQ ID NO: 22 in the Sequence Listing.
  • the microorganism is Cyclobacterium marinum ATCC 25205 strain (Deposit organization: American 'Type' Karuyaichi 'collection, deposit address; POBox 1549 Manassas, VA 20110, the United States of America).
  • Escherichia coli jM109 strain was used as the host, and pUC118 was used as the vector.
  • chromosomal DNA was obtained from this bacterial cell based on the method described in the manual.
  • a DNA fragment containing a part of the peptide-forming enzyme gene derived from Cyclobataterum marinum ATCC 25205 strain was obtained by PCR using LA-Taq (Takara Shuzo). PCR reaction was performed on the chromosomal DNA obtained from Cyclobataterum marinum ATCC 25205 strain using primers having the nucleotide sequences shown in SEQ ID NOs: 15 and 16. About lkb DNA fragments amplified by PCR were separated by 0.8% agarose electrophoresis. The target band was cut out and purified. This DNA fragment was labeled with digoxgen on the probe using DIG High Prime (Boehringer Mannheim) according to the instructions.
  • Detection of the band hybridizing with the probe was performed based on the instructions using the DIG Nucleotide Detection Kit (Behringer Mannheim). As a result, a 7k band was detected for the Pstl digestion product that hybridizes with the probe, and a 2k band was detected for the Hindi digestion product.
  • Escherichia coli was transformed by mixing 100 ⁇ l of this ligation reaction solution 5 ⁇ ⁇ Escherichia coli JM109 strain (competent cell) (Takara Shuzo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.
  • Colonies in the chromosomal DNA library were transferred to a nylon membrane filter Nylon Membranes for Colony and Plaque Hybridization (Roche Diagnostics), and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C for 1 hour, then the above labeled probe with digoxigen was added, and hybridization was performed at 37 ° C for 16 hours. Thereafter, the filter was washed twice with 60 ° C. with 1 ⁇ SSC containing 0.1% SDS.
  • Colonies that hybridize with the labeled probe were detected using the DIG Nucleotide Detection Kit (Boehringer Mannheim) based on the instructions. As a result, one strain of each colony hybridizing with the labeled probe was confirmed.
  • Plasmids possessed by Escherichia coli JM109 were prepared from each of the above strains that were confirmed to hybridize with the labeled probe, and the base sequences in the vicinity of the hybridized probe were determined.
  • the sequencing reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis is CEQ
  • the microorganism is Cyclocell Penns Brutonensis ATCC 700359 (Deposit organization; American 'Type' Kaliya ⁇ Collection, deposit address; POBox 1549 Manassas, VA 20110, the United States of America).
  • Escherichia coli JM109 strain was used as the host, and pUCl18 was used as the vector.
  • the above was cultured at 25 ° C for 24 hours.
  • the cells were inoculated with 1 platinum ear in a 500 ml Sakaguchi flask containing 50 ml of CM2G liquid medium (a medium obtained by removing agar from the above medium) and cultured at 10 ° C with shaking.
  • chromosomal DNA was obtained from this bacterial cell based on the method described in the manual.
  • the chromosomal DNA of Cycloserpens brtonensis ATCC 700359 strain was reacted with the restriction enzyme EcoRI at 37 ° C for 16 hours for complete digestion, and then electrophoresed on a 0.8% agarose gel. After electrophoresis, the agarose gel was blotted to a nylon membrane filter Nylon embranes positively charged (manufactured by Roche Diagnostics), and subjected to alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Mannheim). The filter was prehybridized at 50 ° C. for 1 hour, then the digoxgen-labeled probe prepared above was added, and hybridization was performed at 50 ° C. for 16 hours. Thereafter, the filter was washed twice with 1 ⁇ SSC containing 0.1% SDS at 60 ° C.
  • Detection of the band hybridizing with the probe was performed based on the instructions using the DIG Nucleotide Detection Kit (Behringer Mannheim). As a result, a band of about 7 kb that hybridized with the probe could be detected.
  • the ligation reaction solution 5 ⁇ 1 was mixed with Escherichia coli JM109 strain combinent cell (Takara Shuzo) 100 / z 1 to transform Escherichia coli. This was applied to an appropriate solid medium to prepare a chromosomal DNA library.
  • the colony of the chromosomal DNA library was transferred to a nylon membrane filter Nylon Membranes for Colony and Plaque Hybridization (manufactured by Roche Diagnostics) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C for 1 hour, then the above labeled probe with digoxigen was added, and hybridization was performed at 37 ° C for 16 hours. Thereafter, the filter was washed twice with 60 ° C. with 1 ⁇ SSC containing 0.1% SDS.
  • a plasmid possessed by the Escherichia coli JM109 strain was prepared from the above-mentioned one strain that was confirmed to hybridize with the labeled probe, and the base sequence in the vicinity of the hybridized probe was determined.
  • the sequencing reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions.
  • Electrophoresis was performed using CEQ 2000-XL (manufactured by Beckman Coulter).
  • This transformant was cultured at 37 ° C for 16 hours in LB liquid medium (Tryptone 1%, Yeast extract 0.5%, NaCl 0.5%, pH 7.0) containing ampicillin 50 / zg / ml, and collected. Plasmids were prepared from transformants. Next, pUC18-pepD was cleaved with Nru I and Hpa I (a restriction enzyme site in a 1.6 Kbp insert fragment containing the pepD gene), and self-ligation reaction was performed. Using this ligation reaction solution, Escherichia coli JM109 strain competent cells were transformed, and plasmids were prepared from transformants that grew on LB agar plates containing ampicillin 50 g / ml. .
  • plasmid DNA having an inserted DNA fragment of 1.4 Kbp, pUC A pepD was selected.
  • the pepD gene linked to this plasmid DNA lacks the Nru I-Hpa I region, and the encoded enzyme is predicted to have no function.
  • pUC A pepD was cleaved with EcoR I and Sal I to prepare a 1.4 Kbp DNA fragment containing a defective pepD gene.
  • This DNA fragment was ligated to the EcoR I / Sal I site of PMAN997, a vector for homologous recombination having a temperature-sensitive replication origin (tsori), to construct a plasmid pMAN A pepD for pepD gene deletion.
  • tsori temperature-sensitive replication origin
  • LB liquid containing 50 ⁇ g / ml of ampicillin transformed into Escherichia coli JM109, a subspecies of Escherichia coli K12, transformed with pMAN A pepD PMAN A pepD was prepared from the collected transformants after culturing in a medium at 30 ° C. for 16 hours.
  • the pepD gene deletion plasmid ⁇ ⁇ pepD was used to transform E. coli ATCC8739 by the electroporation method and plated on an LB agar plate containing 50 g / ml of ampicillin. And grown at 30 ° C. (Note that items with ATCC numbers are deposited in the American 'Type' Culture Collection (POBox 1549 Manass as, VA 20110, the United States of America). A plurality of transformants obtained were obtained at 30 ° C, 16 ml in 4 ml of LB liquid medium (cm4 cm x 18 cm test tube) containing 50 g / ml of ampicillin. Incubated for hours.
  • this culture solution After diluting this culture solution with physiological saline, it was plated on an LB agar plate containing 50 g / ml of ampicillin and cultured at 42 ° C. for 10 hours to obtain a single colony. Furthermore, the single colony obtained here was cultured again in the same manner as above to isolate the single colony, and a clone in which the entire plasmid was integrated into the chromosome by homologous recombination was selected. Furthermore, a homologous recombinant strain plasmid cultured in LB medium containing 50 g / ml of ampicillin was extracted to confirm that this strain did not have the plasmid in the cytoplasm.
  • the cells were cultured in ThiaminHCl 2 mg, Glucose 0.2 g, pH 7.0) at 30 ° C for 24 hours.
  • This culture solution 100 / zl was transferred to the same medium (4 ml) and further cultured at 42 ° C for 24 hours.
  • the culture solution was diluted with physiological saline, spread on a M9 minimal plate, and cultured at 42 ° C for 12 hours to obtain a single colony.
  • the emerged colonies were inoculated on an LB agar plate and an LB agar plate containing 50 ⁇ g / ml ampicillin, and cultured at 30 ° C. for 12 hours.
  • a twice-recombinant strain that became ampicillin-sensitive by two-time recombination and grew only on the LB agar plate was selected.
  • the pepD fragment was amplified by PCR using the obtained chromosomal DNA of the twice-recombinant strain as a saddle and using primers of the sequences of SEQ ID NOs: 28 and 29. Since the amplified pepD gene is a pepD gene fragment lacking the Nru I -Hpa I region (approximately 1.4 Kbp), it was confirmed that the pepD gene of this strain was replaced with the defective pepD gene. The strain was designated as a pepD-deficient strain.
  • the PCR method (94 ° C, 1 minute, 54 ° C, 2 minutes, 72 ° C, 3 minutes, 30 cycles) using the genomic DNA of Escherichia coli ATCC8 739 strain as a saddle type, About 1.4 Kbp of the structural gene region covering SD-ATG and the translation stop codon was amplified. This was ligated to the Sma I site of the pUC18 vector using the SureClone Ligation Kit to construct PUC18-p mark E. Escherichia coli JM109 strain competent cells transformed with this plasmid were cultured in a liquid medium containing ampicillin g / ml at 37 ° C. for 16 hours.
  • PUC18-pepE was cleaved with Nru I (restriction enzyme site in the 1.4 Kbp insert fragment containing the pepE fragment) and ligated with the EcoR I-Not I-BamHI adapter.
  • the plasmid was further cleaved with Not I and subjected to self-ligation reaction.
  • Escherichia coli JM1 09 strain competene cells were transformed, and plasmids were also prepared for the ability of transformants to grow on LB agar plates containing ampicillin 50 g / ml. From this, plasmid DNA, pUC A pepE, which was cleaved by Not I was selected.
  • the pepE gene contained in this plasmid DNA will undergo a frameshift at the Nru I site, and the encoded enzyme is expected to have no function.
  • pUC A pepE was cleaved with EcoR I and Sal I to prepare a 1.4 Kbp DNA fragment containing the defective pepE gene.
  • the DNA fragment was ligated to the EcoR I / Sal I site of PMAN997, a vector for homologous recombination having a temperature-sensitive replication origin (tsori), to construct a plasmid pMAN A pepE for pepE gene deletion.
  • Escherichia coli JM109 strain transformed with ⁇ pepE was cultured in LB liquid medium containing ampicillin 50 ⁇ g / ml at 30 ° C for 16 hours, and ⁇ mark E was collected from the collected transformants. Prepared. Using this plasmid for deletion of the ⁇ -marked gene, the ⁇ -marked D-deficient strain of the above Escherichia coli ATCC8739 was transformed by the electopore method. Subsequently, by the same operation, the pepE gene was replaced with the defective pepE gene to obtain a twice-recombinant strain that was sensitive to ampicillin.
  • Enterobacter aerogenes A 1.6 kbp DNA fragment excised with restriction enzyme Sail and restriction enzyme Kpnl, including the acid phosphatase gene region, was isolated from chromosome DNA of IFO 12010 strain, and a plasmid DNA linked to pUC118 was constructed. Named.
  • pEAP120 is a plasmid in which a base sequence encoding an acid phosphatase promoter and a signal peptide is incorporated.
  • the IFO numbers listed here are the powers deposited with the Fermentation Research Institute (2-17-85 Juzahoncho, Yodogawa-ku, Osaka, Japan) June 2002 After 30th, the work will be transferred to the National Institute for Product Evaluation and Technology ( ⁇ ) ⁇ Biotechnology Headquarters (DOB) 'Biogenetic Resources Division (NBRC), and will receive a sale from the NBRC with reference to the above IFO number. Can do.
  • pEAP 120 was mutagenized into a cocoon.
  • DNA sequencing kit Dye Terminator Cycle sequencing ready reaction manufactured by PERKIN ELMER was used to determine the nucleotide sequence using the 310 Genetic analyzer (ABI) and confirm that the target mutation was introduced.
  • This plasmid was designated as PEAP130.
  • pEAP130 is a nucleotide sequence encoding a signal peptide derived from the N-terminal region of acid phosphatase, and has been modified. A plasmid having a promoter.
  • the amplified fragment mixture was made into a saddle shape, E-S1 and S-AS1 oligonucleotides were used as primers, and a reaction solution having the same composition was subjected to 94 ° C for 15 seconds at 55 ° C. PCR was performed by repeating the cycle of 30 seconds at 68 ° C for 2 minutes and 30 seconds 25 times to construct a chimeric enzyme gene.
  • Each amplified DNA fragment was recovered using a Microspin column (manufactured by Amersham, Pharmacia Biotech) and digested with Xba I and Pst I. This was ligated to the Xba I-Pst I site of the pUC19 plasmid.
  • DNA sequencing kit Dye Terminator Cycle beque ncing Ready Reaction (PERKIN ELMER) Dye Terminator method is used to determine the nucleotide sequence with 310 Genetic analyzer (ABI), confirm that the target mutation has been introduced, The plasmid was named pSaet plasmid.
  • the sugar feed culture was carried out at 20 ° C with aeration of agar (l / lwm).
  • the pH value was automatically adjusted to 7.0 with gaseous ammonia.
  • Glucose and magnesium sulfate were sterilized separately.
  • the pH of the other components was adjusted to 5.0 with ⁇ .
  • Example 14 [0263] 39.5 g of L-aspartic acid- ⁇ , ⁇ -dimethyl ester hydrochloride and 16.5 g of L-ferranalanine were dissolved in 750 ml of water, and the pH was adjusted to 8.5 with a 6N aqueous sodium hydroxide solution. After adding water to make the total amount 950 ml, 33.0 g of L-fetalanin and 50 ml of the cell suspension prepared in Example 13 were added and stirred at 20 ° C. for 2 hours. During the reaction, the pH was maintained at 8.5 by adding 25 wt% aqueous sodium hydroxide solution.
  • the enzyme reaction solution obtained after removing the bacterial cells obtained in Example 14 was concentrated and crystallized. After adding 24 ml of 35% hydrochloric acid, the L-phenylalanine crystals produced by concentration crystallization were filtered off, and 106.6 g of mother liquor (containing 14. lg of H-L-aspartyl-i-ferulanin- ⁇ -methyl ester) ) The removal rate of L-ferroalanine from the enzyme reaction solution was 88.7%.
  • the total yield through the reaction step 3 ⁇ 2 (Examples 19, 20, 21) was 21.8%, and it was possible to increase the yield by a factor of three. It was a truly surprising effect that a remarkable increase in yield was achieved by performing the third reaction step.
  • SEQ ID NO: 1 part of peptide-forming enzyme derived from Empedopacter brevis
  • SEQ ID NO: 2 part of a peptide-forming enzyme derived from Hempedactor brevis
  • SEQ ID NO: 9 Synthetic primer for preparing pTrpT_Gtg2
  • SEQ ID NO: 12 amino acid sequence of peptide-forming enzyme
  • SEQ ID NO: 15; for Aet SEQ ID NO: 16 Mix primer for Aet 2
  • SEQ ID NO: 17 gene encoding a peptide-forming enzyme
  • SEQ ID NO: 19 Primer for constructing an aet expression vector derived from pedopector 1 SEQ ID NO: 20 Primer for constructing an aet expression vector derived from pedopector 2 SEQ ID NO: 21 Mixed primer for Aet 3
  • SEQ ID NO: 22 gene encoding a peptide-forming enzyme
  • SEQ ID NO: 24 gene encoding a peptide-forming enzyme
  • SEQ ID NO: 26 gene encoding the peptide-forming enzyme
  • SEQ ID NO: 27 gene encoding peptide-forming enzyme

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Abstract

Un processus pour produire facilement des α-esters α-L-aspartyl-L-phénylalanine, un ester α-L-aspartyl-L-phénylalanine α-méthyle, en gros volume à faible coût. Le processus de production de α-ester α-L-aspartyl-L-phénylalanine est caractérisé par le fait qu'il comprend : une première étape de réaction comprenant l'étape de production d'un β-ester de α-L-aspartyl-L-phénylalanine à partir d'un L-acide aspartique α,β-diester et L-phénylalanine avec l'aide d'un enzyme ou d'une substance contenant un enzyme ayant la capacité de lier de manière sélective du L-phénylalanine à la partie α-ester d'un L-acide aspartique α,β-diester via une liaison de peptide ; une deuxième étape de réaction dans laquelle le produit de réaction obtenu dans la première étape de réaction est amené à réagir avec un acide et un alcool en présence d'eau pour obtenir le α-ester α-L-aspartyl-L-phénylalanine cible.
PCT/JP2005/013497 2004-07-22 2005-07-22 PROCESSUS DE PRODUCTION DE α-L-ASPARTYL-L-PHÉNYLALANINE α-ESTER Ceased WO2006009255A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755180A (zh) * 2016-12-09 2017-05-31 浙江理工大学 一种利用细菌静态发酵制备生物改性细菌纤维素纳滤膜的方法
CN109161502A (zh) * 2018-09-12 2019-01-08 中国石油化工股份有限公司 一种除油菌剂、其复合干粉菌剂的制备方法及用途
FR3154926A1 (fr) 2023-11-08 2025-05-09 Raphaël TURMEL Protège-jambe renforcé au vu du tibia par un déflecteur de choc à demi-bulle.

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4892820A (en) * 1987-06-10 1990-01-09 The Nutrasweet Company Solvent system for enzymatic coupling process
WO2004011653A1 (fr) * 2002-07-26 2004-02-05 Ajinomoto Co., Inc. Gene de synthetase de peptide

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4892820A (en) * 1987-06-10 1990-01-09 The Nutrasweet Company Solvent system for enzymatic coupling process
WO2004011653A1 (fr) * 2002-07-26 2004-02-05 Ajinomoto Co., Inc. Gene de synthetase de peptide

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755180A (zh) * 2016-12-09 2017-05-31 浙江理工大学 一种利用细菌静态发酵制备生物改性细菌纤维素纳滤膜的方法
CN109161502A (zh) * 2018-09-12 2019-01-08 中国石油化工股份有限公司 一种除油菌剂、其复合干粉菌剂的制备方法及用途
CN109161502B (zh) * 2018-09-12 2022-02-11 中国石油化工股份有限公司 一种除油菌剂、其复合干粉菌剂的制备方法及用途
FR3154926A1 (fr) 2023-11-08 2025-05-09 Raphaël TURMEL Protège-jambe renforcé au vu du tibia par un déflecteur de choc à demi-bulle.

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