[go: up one dir, main page]

WO2007097429A1 - Gene d'acylamidase innovant et son utilisation - Google Patents

Gene d'acylamidase innovant et son utilisation Download PDF

Info

Publication number
WO2007097429A1
WO2007097429A1 PCT/JP2007/053419 JP2007053419W WO2007097429A1 WO 2007097429 A1 WO2007097429 A1 WO 2007097429A1 JP 2007053419 W JP2007053419 W JP 2007053419W WO 2007097429 A1 WO2007097429 A1 WO 2007097429A1
Authority
WO
WIPO (PCT)
Prior art keywords
acylamidase
group
carbon atoms
dna
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2007/053419
Other languages
English (en)
Japanese (ja)
Inventor
Toru Nagasawa
Toyokazu Yoshida
Koichi Ishida
Noriyuki Ito
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kaneka Corp
Original Assignee
Kaneka Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kaneka Corp filed Critical Kaneka Corp
Priority to JP2008501767A priority Critical patent/JPWO2007097429A1/ja
Publication of WO2007097429A1 publication Critical patent/WO2007097429A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)

Definitions

  • the present invention relates to a novel acylamidase, a DNA encoding the acylamidase, a method for producing the acylamidase using the DNA, and an optically active compound using the acylamidase. It relates to a manufacturing method.
  • the acylamidase is an industrially useful enzyme capable of hydrolyzing an amide bond or an ester bond under mild conditions.
  • Non-Patent Document 2 Those derived from Arthrobacter aurescens (Non-Patent Document 2).
  • the former acts on a wide range of compounds such as acetate-lides, amides such as benzamide, and esters such as phenyl acetate.
  • the latter does not act on acyl groups other than the acetyl group, which acts on a wide range of N-acetylyl reelalkylamines.
  • amide hydrolases with various substrate specificities have been found.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 61-88894
  • Patent Document 2 JP-A-3-277281
  • Non-Patent Document 1 Eur. J. Biochem, 199, 17-24 (1991)
  • Non-Patent Document 2 Appl. Microbiol. Biotechnol, 47, 650-657 (1997) Disclosure of the Invention
  • An object of the present invention is to obtain a novel acylamidase.
  • DNA that encodes this novel acylamidase was isolated and used to provide a method for amide hydrolysis or ester hydrolysis under mild conditions for various substrates. is there.
  • microorganisms having acylamidase activity As a result of screening for various soil-separated bacteria-preserving strains, the present inventors have obtained microorganisms having acylamidase activity. Moreover, the enzyme having the activity was successfully isolated and purified from the microorganism.
  • the gene encoding the enzyme was obtained by genetic recombination techniques and the nucleotide sequence was clarified. Further, the transformant producing the enzyme was bred using the gene to produce the transformant with higher activity. Using this highly active transformant, we established a method for amide hydrolysis or ester hydrolysis under mild conditions.
  • the present invention has the following features.
  • One feature of the present invention is a acylamidase having the following physicochemical properties (1) to (6):
  • Substrate specificity Substantially acts on each of formaldehyde, acetatetolide, and phenylacetate, and includes benzamide, n-butylamide, N-acetylethyl L-phenylalanine, N-acetylyl DL— Virtually no effect on tryptophan or N-acetyl-L-tyrosine;
  • Another feature of the present invention is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, a deletion, substitution, or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 1.
  • it has an added amino acid sequence ability and has a polypeptide having acylamidase activity, or an amino acid sequence having 70% identity or more with the amino acid sequence shown in SEQ ID NO: 1, and A polypeptide having acylamidase activity.
  • Another feature of the present invention is DNA encoding the polypeptide, a vector containing the DNA, and a transformant transformed with the vector.
  • heteroaryl independently R ⁇ R 2 are each, Ariru group having 6 to 14 carbon atoms, 4 to 14 to the carbon atoms Group, C6-C14 aryloxy group, C4-C14 heteroaryloxy group, C1-C5 alkoxy group, C2-C5 alkoxycarbonyl group, C3-C5 Indicates a branched alkyl group, an alkenyl group having 2 to 5 carbon atoms, an alkynyl group having 2 to 5 carbon atoms, a cycloalkyl group having 5 to 7 carbon atoms, a cyan group, a methyl group or a carboxyl group, and these groups are substituted.
  • R 3 represents a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, X represents a nitrogen atom or an oxygen atom), or an ester compound represented by Production of the above-mentioned acylamidase, polypeptide or the enzyme into an amide compound General formula (2), characterized by acting a culture of microorganisms that have the ability:
  • the enzyme of the embodiment of the present invention is a acylamidase having the following physical properties (1) force (6):
  • Substrate specificity Substantially acts on each of formaldehyde, acetatetolide, and phenylacetate, and includes benzamide, n-butylamide, N-acetylethyl L-phenylalanine, N-acetylyl DL— Virtually no effect on tryptophan or N-acetyl-L-tyrosine;
  • the molecular weight of the enzyme can be determined by, for example, gel filtration analysis using TSK GEL-G-3000 (0.75 ⁇ 60 cm, manufactured by Tosoh Corporation) and the relative elution time with respect to the standard protein. As eluent, 0.05M potassium phosphate buffer (pH 7.0) containing 0.20M KC1 is used. The molecular weight of the subunit can be determined by relative mobility relative to the standard protein by 10% SDS-polyacrylamide gel electrophoresis.
  • the amidase activity for each substrate of the enzyme can be measured, for example, by the method described in Example 3 described later. That is, 0.1 mL of the purified enzyme solution is added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. After 30 minutes, add 1 mL of methanol to stop the reaction, and quantify the produced amine, alcohol or ammonia.
  • acylamidase means a compound represented by P—CO—NH—Q (P is an arbitrary substituent, and Q is an arbitrary substituent other than a hydrogen atom). Anything that acts as an active agent and hydrolyzes to P COOH and Q—NH is meant.
  • allylicylamidase EC 3.5. 1.13
  • perillin acylase EC 3.5. 5. 1. 76
  • (S) —N-acetyl-1-hydrophenylamine hydrolase EC 3.5. 1. 1. 85
  • formamidase EC 3.5. 1. 1. 9
  • peptide deformylase EC 3.5. 1.27
  • N Substituted Formamide Deformylase EC 3.5. 1. 91).
  • the acylamidase of the present invention substantially acts on each of formaldehyde, acetonitrile, and acetic acid, and benzamide, n-butylamide, N-acetylene L-ferallanin.
  • N-acetylyl DL-tryptophan and N-acetylyl L-tyrosylase which does not substantially act on each of tyrosine.
  • substantially does not act means that the specific activity of the purified enzyme when each substrate is used is 0.02 U / mg or less when the amidase activity is measured by the method of Example 3 described later. It means that.
  • the acylamidase of the present invention those having a specific activity of 0.002 UZmg or less are preferred, and those having a specific activity of 0.0002 UZmg or less are more preferred.
  • “Substantially acting” means that the specific activity of the purified enzyme is 0.2 UZmg or more when each substrate is used when the amidase activity is measured by the above method.
  • the acylamidase of the present invention preferably has a specific activity of 2. OUZ mg or more.
  • the optimum pH of the enzyme reaction is, for example, when the activity using acetate-lide as a substrate is measured in the range of pH 4.0 to L1. It is set as the range which shows activity.
  • the following buffer solution is used in the base solution according to the pH to be measured.
  • pH 6.0-8.0 0.1 M potassium phosphate buffer
  • the optimum temperature of the enzyme reaction is, for example, the activity using acetanilide as a substrate, the reaction temperature of 10 to
  • the thermal stability of the enzyme is measured, for example, by adding purified enzyme to 0.1M potassium phosphate buffer (pH 7.0), treating it at 10-80 ° C for 30 minutes, and then measuring the amidase activity. Can be determined.
  • an inhibitor eg, containing HgCl or AgNO.
  • acylamidase of the present invention preferably has the above activity of 1Z1000 or less, more preferably 1Z10000 or less.
  • the Km of the enzyme for acetate-lid can be determined, for example, as follows. That is, 0.1 mL of the purified enzyme solution is added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. Three
  • the acylamidase of the present invention can hydrolyze racemic amido compounds or ester compounds in a stereoselective manner.
  • the microorganism that is the source of the enzyme of the present invention is preferably Bacillus sp., which can be easily obtained by a person skilled in the art from a public preservation agency (for example, National Institute of Technology and Evaluation (NBRC)). More preferred is Bacillus sp. KNK-MO1. This Bacillus sp. KNK—M01 is dated January 22, 2007, and has the accession number FERM BP—10765. 8566 Tsukuba, Ibaraki, Japan (Early 1-1-1 Central No.
  • the bacteriological properties of the Bacillus sp. are well known to those skilled in the art.
  • the bacteriological properties of the Bacillus sp. KNK-M01 are the same as the bacteriological properties of Bacillus sp. In addition to having a predetermined acylamidase activity.
  • a liquid nutrient medium containing a normal carbon source, nitrogen source, inorganic salts, organic nutrients and the like can be used as long as the microorganism grows.
  • Purification of the enzyme from the microorganism producing the enzyme of the present invention can be performed by protein purification methods well known to those skilled in the art.
  • the bacterial cells are collected from the microorganism culture solution by centrifugation or filtration, and the obtained bacterial cells are crushed by a physical method using an ultrasonic crusher or glass beads, and then centrifuged.
  • the cell-free extract is prepared by removing the cell residue in, and the cell-free extract is subjected to fractional precipitation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, ultrafiltration. It is possible to isolate the acylamidase.
  • the purified acylamidase can be clarified by the method described later, and the amino acid sequence of the acylamidase can be determined from the DNA sequence.
  • the acylamidase thus obtained include a polypeptide having the amino acid sequence shown by SEQ ID NO: 1.
  • the enzyme of the present invention is not limited to this, and a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1 As long as it has acylamidase activity (preferably stereoselective acylamidase activity), it is included in the present invention.
  • acylamidase and polypeptide of the present invention may be natural or artificially modified.
  • a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added is obtained using the amino acid sequence represented by SEQ ID NO: 1. , Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1989) and the like.
  • the place where amino acids are substituted, inserted, deleted or added is not particularly limited, but it is preferable to avoid highly conserved regions.
  • the highly conserved region refers to a plurality of enzymes with different origins having the same functions, for example, by using the multiple alignment function of the software GENETYX (manufactured by GENETYX) to optimize the amino acid sequence. When aligned and compared, amino acid matches between multiple sequences are indicated.
  • Examples of the highly conserved region in SEQ ID NO: 1 include the 111th to 115th amino acid sequences, the 138th to 149th amino acid sequences, and the 163rd to 167th amino acid sequences.
  • the number of amino acids to be substituted, inserted, deleted or added is preferably 10 or less, more preferably 5 or less, and even more preferably 3 or less.
  • the modified amino acid sequence may include only one type (for example, substitution) or may include two or more types of modification (for example, substitution and insertion).
  • substitution the amino acid after substitution is preferably a homologous amino acid of the original amino acid.
  • amino acids in the same group of the following groups are regarded as homologous amino acids.
  • Polypeptides having a sequence and having acylamidase activity are also polypeptides of the present invention.
  • amino acid sequence identity is determined by BLAST (Altschul, Stephen F. et al., Nucleic Acids Res. 25, 3389-3402 (1997)), and can be determined by homology analysis.
  • a polypeptide having an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and having an acylamidase activity is, for example, the polypeptide shown in SEQ ID NO: 1. It can be obtained by random mutagenesis or removal of sequences unnecessary for activity.
  • the polypeptide of the present invention preferably has a acylamidase activity exhibiting the physicochemical properties (1) to (6) described above, or has at least a acylamidase activity that acts on formaldehyde.
  • the DNA of the present invention is the above-mentioned novel acylamidase and DNA encoding the polypeptide, and any DNA can be used as long as it can express the polypeptide in a host cell introduced according to the method described below. Any untranslated region may be included. If the purified polypeptide can be obtained, those skilled in the art can obtain the DNA from the microorganism that is the origin of the polypeptide by a known method.
  • the polypeptide (enzyme) purified from the cell-free extract of the microorganism is digested with an appropriate endopeptidase, and the fragment cleaved by reverse-phase HPLC is purified.
  • a part of the amino acid sequence is determined by the service (Applied Biosystems).
  • a PCR (Polymerase Chain Reaction) primer for amplifying a part of the DNA encoding the polypeptide is synthesized.
  • the chromosomal DNA of the microorganism is prepared by a conventional DNA isolation method, for example, Murray et al. (Nucl., Acids Res., 8, 4 321 -4325, 1980). Using this chromosomal DNA as a saddle, PCR is performed using the PCR primers described above, a part of the DNA encoding the polypeptide is amplified, and the base sequence is determined. The base sequence is determined using, for example, ABI373A DNA Sequencer (Applied Biosystems). obtain. If the base sequence of a part of the DNA encoding the polypeptide is clarified, the entire sequence can be determined by, for example, the Southerno or hybridization method.
  • Examples of the DNA obtained in this manner include DNA having the base sequence set forth in SEQ ID NO: 2.
  • the DNA of the present invention is not limited thereto, and all DNAs encoding the above-described polypeptides of the present invention are included in the present invention.
  • a DNA that hybridizes under stringent conditions with a DNA complementary to the base sequence shown in SEQ ID NO: 2 and that encodes a polypeptide having an acylamidase activity is included in the present invention. .
  • DNA that hybridizes under stringent conditions with a DNA that also has a complementary base sequence ability to the base sequence shown in SEQ ID NO: 2 means the “Kouguchi-ichi” hybridization method, plaque hybrider.
  • a DNA having a base sequence that is complementary to the base sequence shown in SEQ ID NO: 2 when a hybridization method or Southern hybridization test method is performed is a DNA that specifically forms a hybrid.
  • the DNA of the present invention may be natural or artificially modified.
  • stringent conditions refers to a filter in which a polynucleotide derived from a colony or plaque is immobilized, at a temperature of 65 ° C in the presence of 0.7 to 1.
  • OM NaCl OM NaCl
  • the filter was used under the condition of 65 ° C using a 2x SSC solution (composition of 1x SSC solution consisting of 150mM sodium chloride and 15mM sodium citrate). This is the condition for cleaning.
  • the hybridization is performed in the same manner as described above, and then washed with a 0.5-fold concentrated SSC solution at 65 ° C. More preferably, the hybridization is performed as described above.
  • the condition is to wash with a 0.2 times concentrated SSC solution at 65 ° C. More preferably, after carrying out a noblerization in the same manner as described above, a 0.1 times concentration at 65 ° C is obtained. This is the condition for washing with the SSC solution.
  • the DNA of the present invention encodes a acylamidase having the ability to code for the acylamidase exhibiting the physicochemical properties (1) to (6) described above, or at least the acylamidase activity acting on formaldehyde. I prefer that! /.
  • vector DNA used for introducing the DNA of the present invention into a host microorganism and expressing it in the introduced host microorganism can express the gene encoding the DNA force S in an appropriate host microorganism. Any one can be used. Examples of such vector DNA include plasmid vectors, phage vectors, cosmid vectors, and the like. A shuttle vector capable of exchanging genes with other host strains can also be used.
  • Such vectors may contain control elements such as operably linked promoters (lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, etc.). And can be suitably used as a vector comprising an expression unit operably linked to the DNA of the present invention.
  • promoters lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, etc.
  • pUC18 manufactured by Toyobo Co., Ltd.
  • pUC19 manufactured by Toyobo Co., Ltd.
  • pUCNT etc. that can be prepared by those skilled in the art by the method described in the pamphlet of International Publication No. Can be preferably used.
  • regulatory element refers to a nucleotide sequence having a functional promoter and any related transcription elements (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).
  • operably linked refers to a gene in which various regulatory elements, such as a promoter that regulates the expression of a gene, an ensemble, and the like, are operably linked in a host cell. That means. It is a matter well known to those skilled in the art that the type and kind of the control factor can vary depending on the host.
  • Examples of host cells into which the vector containing the DNA of the present invention is introduced include bacteria, yeasts, filamentous fungi, plant cells, animal cells and the like, and Escherichia coli is particularly preferred.
  • a vector containing the DNA of the present invention can be introduced into a host cell by a known method. When Escherichia coli is used as the host cell, the vector can be introduced, for example, by the salted calcium method.
  • the present invention also provides a Bacillus sp. KNK-MOl (FERM BP-10765) or the above-mentioned transformant cultured in a nutrient medium, and from the obtained culture broth.
  • the present invention relates to a method for producing a polypeptide by obtaining the above-mentioned acylamidase or polypeptide.
  • Bacillus sp. KNK-M01 or the transformant of the present invention by culturing Bacillus sp. KNK-M01 or the transformant of the present invention, the acylamidase can be produced efficiently.
  • Bacillus sp. KNK-M01 and the culture of the transformant of the present invention can be cultured as long as it grows, usually liquid nutrients containing carbon source, nitrogen source, inorganic salts, organic nutrients, etc. It can be carried out using a medium.
  • Bacillus sp. KNK-M01 and the acylamine accumulated in the culture medium of the transformant of the present invention can be used as a medium containing the acylamidase in the culture medium. It can also be used after purification or partial purification using generally known protein purification techniques.
  • An amide compound or an ester compound is allowed to act on the above-mentioned novel acylamidase or polypeptide, Bacillus sp. KNK-MOl (FERM BP-10765), or a culture of the above-described transformant, Carboxylic acid, alcohol, or amine can be produced by hydrolyzing the amide compound or ester compound.
  • the acylamidase of the present invention or the microorganism capable of producing the enzyme is suitable for producing an optically active compound.
  • the method for producing an optically active substance of the present invention includes a racemic amidy compound, an ester compound, the above-mentioned acylamidase or polypeptide, Bacillus sp. KNK-MOL (FERM BP-10765 Or a culture of the transformant described above, and selectively hydrolyzing only one of the racemates.
  • Examples of the microorganism having the ability to produce the amidase of the present invention include, for example, the above-mentioned Bacillus sp. KNK-MOl (FERM BP-10765) and a transformant introduced with the above-described vector containing DNA. Is mentioned. According to this production method, the general formula (1)
  • R ⁇ R 2 is Ariru group of carbon number 6-14, 4 carbon 14 heteroaryl groups, 6 to 14 carbonyloxy groups, 4 to 14 carbon heteroaryloxy groups, 1 to 5 alkoxy groups, 2 to 5 alkoxycarbonyl groups, carbon A branched alkyl group having 3 to 5 carbon atoms, an alkenyl group having 2 to 5 carbon atoms, an alkynyl group having 2 to 5 carbon atoms, a cycloalkyl group having 5 to 7 carbon atoms, a cyan group, a methyl group or a carboxyl group; These groups may be substituted.
  • R 3 represents a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms.
  • X represents a nitrogen atom or an oxygen atom.
  • Examples of the aryl group having 6 to 14 carbon atoms include a phenol group and a naphthyl group.
  • Examples of the heteroaryl group having 4 to 14 carbon atoms include a pyridyl group, a cenyl group, an oxadiazolyl group, an imidazolyl group, a thiazolyl group, a furyl group, and a pyrrolyl group.
  • Examples of the aryloxy group having 6 to 14 carbon atoms include a phenoxy group and a naphthoxy group.
  • Examples of the heteroaryloxy group having 4 to 14 carbon atoms include a pyridyloxy group, a enyloxy group, an oxadiazolyloxy group, an imidazolyloxy group, a thiazolyloxy group, a furyloxy group, and a pyrrolyloxy group.
  • Examples of the alkoxy group having 1 to 5 carbon atoms include methoxy group and eth Examples thereof include a xy group and a tert butoxy group.
  • Examples of the alkoxycarbon group having 2 to 5 carbon atoms include a methoxycarbol group, an ethoxycarboro group, a tertbutoxycarboro group, and the like.
  • Examples of the branched alkyl group having 3 to 5 carbon atoms include isopropyl group, sec butyl group, and tert butyl group.
  • Examples of the alkenyl group having 2 to 5 carbon atoms include a beryl group and an aryl group. An acetylene group etc. are mentioned as a C2-C5 alkynyl group.
  • Examples of the cycloalkyl group having 5 to 7 carbon atoms include a cyclopentyl group, a cyclohexyl group, and a cycloheptyl group.
  • the number of carbon atoms in the alkoxy carbo group is the number including the carbonyl carbon.
  • substituents include a halogen atom, a hydroxyl group, a cyano group, a methoxy group, an ethoxy group and an alkoxy group having 1 to 4 carbon atoms such as methylenedioxy.
  • R 3 is preferably a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms. Specific examples include N- (1 -phenyl) acetamide, 1-phenylethyl acetate and the like.
  • R 3 is a linear alkyl group having 1 to 3 carbon atoms, is a branched chain alkyl group, the generated amine or alcohol force S37% ee, 51% ee, or even higher than 99% ee It can be obtained with optical purity.
  • the enzyme of the present invention or a culture of microorganisms capable of producing the enzyme is allowed to act on the amide or ester compound.
  • “cultured product” means a culture solution containing microbial cells, cultured microbial cells, or a processed product thereof.
  • the processed product means, for example, a cell-free extract, freeze-dried cells, acetone-dried cells, or a ground product of these cells.
  • these enzymes and cultures can also be used in the form of immobilized enzyme or immobilized bacteria by known means. The fixation can be performed by a method well known to those skilled in the art (for example, a crosslinking method, a physical adsorption method, a comprehensive method, etc.).
  • the concentration of the substrate used in the reaction is 0.1 to 50% by weight, preferably 1 to 20% by weight, in the reaction solution composition of amide or ester compound.
  • the pH at which the enzyme of the present invention is allowed to act is preferably a pH of 5.0 or more, more preferably pH 6.0 or more, and an upper limit of preferably from the viewpoint of the optimum pH of the enzyme action. Is pH 10.0 or less, more preferably pH 9.0 or less.
  • the temperature at which the enzyme of the present invention is allowed to act is preferably 20 ° C or higher, more preferably 25 ° C or higher, and preferably 45 ° C, from the viewpoint of the optimum temperature of action of the enzyme and thermal stability. C or lower, more preferably 40 ° C or lower.
  • reaction solvent an aqueous medium such as ion-exchanged water or a buffer solution is usually used, but the reaction can also be performed in a system containing an organic solvent.
  • organic solvent include alcohol solvents such as methanol, ethanol, propanol, isopropanol, and butanol; aliphatic hydrocarbon solvents such as pentane and hexane; aromatic hydrocarbon solvents such as benzene and toluene; Halogenated hydrocarbon solvents such as methylene chloride and chloroform; ether solvents such as jetyl ether and diisopropyl ether; ester solvents such as ethyl acetate and butyl acetate; ketone solvents such as acetone and methyl ethyl ketone In addition, acetonitrile can be used as appropriate.
  • the above reaction may be carried out in a one-phase system, or if necessary, the above organic solvent is added to a water solubility or higher and the reaction is carried out in a two-phase system of an aqueous phase and an organic solvent phase. You can also. By coexisting an organic solvent in the reaction system, selectivity, conversion rate, yield, etc. are often improved.
  • the reaction is usually carried out until about half of the racemic ester or amido compound is hydrolyzed.
  • the reaction time is 1 hour to 1 week, preferably 1 to 72 hours, and it is preferable to select reaction conditions for completing the reaction at such time.
  • the reaction may be interrupted at an early stage of the reaction, or You may make it react excessively.
  • optically active compound represented by the general formula (2) and an unreacted enantiomer substrate are formed.
  • the produced optically active compound and the unreacted enantiomer substrate can be isolated by known methods such as reaction mixture liquid extraction, distillation, recrystallization and column separation.
  • the product when the product is an optically active amine, after adjusting the pH to acidic, ethers such as jetyl ether and diisopropyl ether; esters such as ethyl acetate and butyl acetate; hydrocarbons such as hexane, octane and benzene ; With a common solvent such as halogenated hydrocarbon such as methylene chloride, the unreacted enantiomer substrate can be selectively extracted while leaving the generated optically active amino compound in the aqueous phase. Thereafter, the pH is adjusted to basic, and an optically active amino compound produced using a common organic solvent can be extracted.
  • ethers such as jetyl ether and diisopropyl ether
  • esters such as ethyl acetate and butyl acetate
  • hydrocarbons such as hexane, octane and benzene
  • a common solvent such as halogenated hydrocarbon such as methylene chloride
  • the unreacted enantiomer substrate in the above reaction can hydrolyze the ester or amide moiety by an ordinary method while maintaining the optical activity.
  • the alcohol or amine of the opposite stereo to the active substance can be derived.
  • the optically active alcohol or amine obtained by the above reaction can be converted to an ester or amidy state while maintaining the optical activity.
  • a novel acylamidase, a DNA encoding the novel acylamidase, or any one of them can be used to react various substrates with amide compounds or under mild conditions.
  • a method is provided for hydrolyzing an ester compound.
  • Example 1 Purification of acylamidase HBS Bacillus sp. KNK— M01 (FERM BP— 10765) in 5 L medium (thread and adult: Polypeptone 10 g / L, Meat Extract 10 g / L, NaCl 3 g / L, Yea st Extract 5 g / L (pH 7. In 2)), the cells were cultured at 28 ° C for 24 hours. Subsequently, the cells were collected from the culture solution by centrifugation, washed thoroughly with physiological saline, and then suspended in lOOmM phosphate buffer (pH 7.0) containing ImM DTT. The obtained suspension was crushed by ultrasonic crushing. Next, solids in the crushed material were removed by centrifugation, and a cell-free extract was prepared.
  • lOOmM phosphate buffer pH 7.0
  • Ammonium sulfate was added to the obtained cell-free extract so as to be 30% saturated, this was dissolved, and the resulting precipitate was removed by centrifugation. Ammonium sulfate was added to the supernatant so as to be 60% saturation, and this was dissolved. Then, the precipitate generated by centrifugation was recovered.
  • This precipitate was dissolved in an lOOmM phosphate buffer (pH 7.0) containing ImM DTT, and dialyzed against the same buffer. This was applied to a DEAE-Sephacel (Amersham Biosciences) column (size: 30 ⁇ 215 mm) equilibrated with the same buffer to adsorb the active fraction. After washing the column with the same buffer, the active fraction was eluted with lOOmM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride and ImM DTT.
  • the eluted active fractions were collected, and ammonium sulfate was dissolved in this to a final concentration of 20%, and 10 mM phosphate buffer containing 20% ammonium sulfate and ImM DTT.
  • the solution was applied to an Octyl-Sepharose (Amersham Biosciences) column (size: 20 ⁇ 65 mm) pre-equilibrated with (pH 7.0) to adsorb the active fraction.
  • the active fraction was eluted with 25 mM phosphate buffer (pH 7.0) containing ImM DTT.
  • the eluted active fractions were collected and dialyzed against 5 mM phosphate buffer (pH 7.0) containing ImM DTT. This is applied to a Hydroxyapatite (Amershamno Science Co., Ltd.) column (size: 20x15mm) equilibrated with the same buffer solution. Adsorbed. After washing the column with 10 mM phosphate buffer (pH 7.0) containing ImM DTT, the active fraction was eluted with 25 mM phosphate buffer (pH 7.0) containing ImM DTT.
  • the eluted active fractions were collected and sufficiently diluted with 10 OmM phosphate buffer (pH 7.0) containing 20% ammonium sulfate and ImM DTT. This was applied to a Butyl-Toyopearl (manufactured by Tosoichi Co., Ltd.) column (size: 20 ⁇ 20 mm) equilibrated with the same buffer to adsorb the active fraction. The column was washed with lOOmM phosphate buffer (PH 7.0) containing 15% ammonium sulfate and ImM DTT, and then lOOmM phosphate buffer ( ⁇ 7.0) containing 10% ammonium sulfate and ImMDTT. Thereafter, the active fraction was eluted with lOOmM phosphate buffer (pH 7.0) containing 5% ammonium sulfate and ImM DTT.
  • HBS The eluted active fractions were collected to obtain a single purified enzyme preparation electrophoretically.
  • this enzyme is referred to as HBS.
  • the activity of the acylamidase was measured as follows. That is, 0.1 mL of the enzyme solution was added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol was added to stop the reaction, and the reaction solution was analyzed by high performance liquid chromatography to quantify the concentration of produced aline. The activity of the obtained acylamidase was 2.7 U per 1 mg of purified enzyme.
  • the HBS purified enzyme obtained in Example 1 was examined for its physical properties.
  • the molecular weight was about 90,000.
  • the molecular weight of the subunit was measured by 10% SDS-polyacrylamide gel electrophoresis, and the molecular weight was about 40,000.
  • the activity using acetonitrile as a substrate was measured in the same manner as described above, and the optimum pH of HBS was examined. As a result, the optimum pH was 6-9.
  • the following buffer solution was used in the substrate solution according to the pH at which the measurement was performed.
  • pH 6.0-8.0 0.1 M potassium phosphate buffer
  • the activity using acetate hydrochloride as a substrate was measured in the reaction temperature range of 10 to 80 ° C.
  • the optimum temperature was 25 to 45 ° C.
  • the purified enzyme was added to 0.1 M potassium phosphate buffer (pH 7.0), and this was treated at 10 to 80 ° C. for 30 minutes, and then the amidase activity was measured. As a result, more than 90% of the activity remained after treatment at 10 ° C to 40 ° C compared to before treatment.
  • 0.1 mL of the purified enzyme solution was added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol was added to stop the reaction, and the alcohol, ammine, or ammonia produced in the reaction solution was analyzed under the conditions shown in Tables 1 and 2. However, when p- -trophyl acetate, p- -trophyl propionate or p- -trophenylbutyrate is used as a substrate, the reaction solution is basified with NaHCO, and then 400
  • the produced p--trophenol was measured by absorbance at nm.
  • the generated ammonia was measured using Conway's diffusion analysis method. That is, 0.2 mL of the reaction supernatant and 2. OmL of saturated potassium carbonate solution were added to the outer chamber of Conway's diffusion tube. In addition, after placing 1.5 mL of 0.01N sulfuric acid in the inner chamber, it was covered, and the two liquids in the outer chamber were contacted and left for 3 hours. Thereafter, 0.5 mL of the reaction solution in the inner chamber was collected.
  • the amount of the enzyme used for measuring the substrate specificity varies depending on the substrate used, and the amount is shown in the column of enzyme usage in Table 1.
  • the unit (mU) of the amount of enzyme used is shown as the enzyme activity when using acetate as a substrate. Relative activity was expressed as a relative value when the activity for each substrate per unit enzyme amount was defined as 100 for activity against acetate.
  • Example 3 Of the reaction solution obtained in Example 3, the optical purity of the product was measured under the following measurement conditions for 1-phenylethyl acetate and N- (1-phenylethyl) acetamide. As a result, 1-phenol ethanol was 37% ee in R form, and 1-phenethylamine was 51% ee in R form.
  • Example 1 For the HBS purified enzyme obtained in Example 1, the Km for acetate-lid was examined. Purified enzyme solution (0.1 mL) was added to a substrate solution (0.9 mL) having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol was added to stop the reaction, and the reaction solution was analyzed by high performance liquid chromatography. there were. High performance liquid chromatography was performed under the same conditions as in Example 1.
  • the N-terminal amino acid sequence of the purified HBS obtained in Example 1 was determined with an ABI492 type protein sequencer (PerkinElmer Biosystems). Further, the purified HBS obtained in Example 1 was digested with lysyl endobeptidase, and the amino acid sequence of the obtained peptide fragment was determined in the same manner as the N-terminal amino acid sequence. Considering the nucleotide sequence that is also expected to have this amino acid sequence, Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4) for amplifying a part of the HBS gene by PCR were synthesized.
  • This DNA fragment was cloned into plasmid pT7Blue T—Vector (Novagen), and ABI PRI3 ⁇ 4M Dye Terminator Cycle sequencing Ready Reaction Kit (Perkin Elmer) and ABI 310 DNA Sequencer (Perkin Elmer) were used. The base sequence was determined.
  • the chromosomal DNA was treated with the restriction enzyme Hindlll to prepare a DNA fragment.
  • Southern hybridization was performed using the determined DNA sequence as a probe, and DNA fragments that hybridized with the probe were extracted from the gel.
  • the extracted DNA fragment was inserted into the pBluescript vector to create a mini-library, and then transformed into E. coli CFM109). From the transformed E. coli library, positive clones that hybridize with the probe are selected by colony hybridization, and the DNA sequence of the insert is determined using the ABI 310 DNA Sequencer (Perkin Elmer). did. As a result, a 1050 bp ORF (SEQ ID NO: 2) was revealed.
  • E. coli BL21 (DE3) competent cells Novagen
  • pET21HBS recombinant E. coli BL21 (DE3 )
  • the resulting recombinant E. coli BL21 (DE3) (pET21HBS) is inoculated into LB medium, cultured at 37 ° C for 4-6 hours, and then IP TG is aseptically added to a final concentration of ImM.
  • the culture was further performed at 20 ° C for 20 hours.
  • the obtained culture broth was collected, suspended in lOOmM phosphate buffer (pH 7.0), and a cell-free extract was obtained by ultrasonic disruption. When the amidase activity of this cell-free extract was measured using acetate-lid as a substrate in the same manner as in Example 3, the activity was found to be 0.73 U per mL of culture solution.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé d'hydrolyse d'amide ou d'ester de divers substrats dans des conditions modérées. Un criblage est réalisé sur diverses bactéries isolées dans un sol et sur des souches mères afin d'obtenir un microorganisme ayant une activité acylamidase. Une enzyme présentant cette activité peut être isolée et purifiée à partir du microorganisme. Un gène codant pour l'enzyme peut être obtenu par une technique de génie génétique et la séquence nucléotidique du gène peut être identifiée. De plus, la production d'un transformant capable de produire l'enzyme à partir du gène et la culture du transformant permettent de produire un transformant plus actif. Le transformant plus actif peut être utilisé dans un procédé d'hydrolyse d'amide ou d'ester dans des conditions modérées.
PCT/JP2007/053419 2006-02-24 2007-02-23 Gene d'acylamidase innovant et son utilisation Ceased WO2007097429A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2008501767A JPWO2007097429A1 (ja) 2006-02-24 2007-02-23 新規アシルアミダーゼ遺伝子およびその利用法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006-049101 2006-02-24
JP2006049101 2006-02-24

Publications (1)

Publication Number Publication Date
WO2007097429A1 true WO2007097429A1 (fr) 2007-08-30

Family

ID=38437464

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2007/053419 Ceased WO2007097429A1 (fr) 2006-02-24 2007-02-23 Gene d'acylamidase innovant et son utilisation

Country Status (2)

Country Link
JP (1) JPWO2007097429A1 (fr)
WO (1) WO2007097429A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016089175A (ja) * 2014-11-04 2016-05-23 国立大学法人 東京医科歯科大学 接着性組成物

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03277281A (ja) * 1990-03-28 1991-12-09 Nippon Kayaku Co Ltd アリールアシルアミダーゼ
WO1997041214A1 (fr) * 1996-04-25 1997-11-06 Novartis Ag Biocatalyseurs possedant une activite amine acylase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03277281A (ja) * 1990-03-28 1991-12-09 Nippon Kayaku Co Ltd アリールアシルアミダーゼ
WO1997041214A1 (fr) * 1996-04-25 1997-11-06 Novartis Ag Biocatalyseurs possedant une activite amine acylase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ENGELHARDT G. ET AL.: "Purification and Properties of an Aryl Acylamidase of Bacillus sphaericus, Catalyzing the Hydrolysis of Various Phenylamide Herbicides and Fungicides", APPL. MICROBIOL., vol. 26, no. 5, 1973, pages 709 - 718, XP001063241 *
GRAF M. ET AL.: "Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b", APPL. MICROBIOL. BIOTECHNOL., vol. 47, no. 6, 1997, pages 650 - 657, XP003017280 *
OGAWA J. ET AL.: "Enzymatic asymmetric synthesis of alpha-methyl arylalkylamines and alpha-methyl arylalkylalcohols by arylalkyl acylamidases", BIOORG. MED. CHEM., vol. 2, no. 6, 1994, pages 429 - 432, XP003017279 *
YOSHIOKA H. ET AL.: "Purification and characterization of aryl acylamidase from Nocardia globerula", EUR. J. BIOCHEM., vol. 199, no. 1, 1991, pages 17 - 24, XP002993747 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016089175A (ja) * 2014-11-04 2016-05-23 国立大学法人 東京医科歯科大学 接着性組成物

Also Published As

Publication number Publication date
JPWO2007097429A1 (ja) 2009-07-16

Similar Documents

Publication Publication Date Title
EP0454478B1 (fr) Gène codant pour une acétylhydrolase de la céphalosporine et protéine codée par celui-ci
US8460902B1 (en) DNA encoding hydantoinase, DNA encoding N-carbamyl-L-amino acid hydrolase, recombinant DNA, transformed cell, method of producing protein, and method of producing optically active amino acid
EP0433117B1 (fr) Nouveaux polypeptides, les séquences d'ADN permettant leur expression, méthode pour leur préparation et utilisation
CN113462669A (zh) 酮泛解酸羟甲基转移酶突变体、编码基因及其应用
US7858348B2 (en) N-acetyl-(R,S)-beta-amino acid acylase gene
EP1188826B1 (fr) Hydantoine racemase substitue en position 5, ADN codant cet enzyme, et procédé d'elaboration d'aminoacides optiquement actifs
CN101040047B (zh) 编码新型的具有d-丝氨酸合成活性的酶的dna、该酶的制备方法、及利用该酶的d-丝氨酸制备方法
US7074606B2 (en) Process for the preparation of (S) - or (R) -3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid
JP4561009B2 (ja) Ｄ−ヒダントインハイドロラーゼをコードするｄｎａ、ｎ−カルバミル−ｄ−アミノ酸ハイドロラーゼをコードするｄｎａ、該遺伝子を含む組み換えｄｎａ、該組み換えｄｎａにより形質転換された細胞、該形質転換細胞を用いるタンパク質の製造方法、および、ｄ−アミノ酸の製造方法
JP2603777B2 (ja) アジピン酸アンモニウムの酵素的合成方法
WO2007097429A1 (fr) Gene d'acylamidase innovant et son utilisation
US9783796B2 (en) Amidase, gene for the same, vector, transformant, and method for production of optically active carboxylic acid amide and optically active carboxylic acid by using any one of those items
JP4941990B2 (ja) Ｎε−アシル−Ｌ−リジン特異的アミノアシラーゼ
US6596528B2 (en) Heat-stable D-aminoacylase
KR100365838B1 (ko) 브레비바실러스 보스테렌시스 ｂｃｓ-1 에서 유래한 신규내열성 ｄ-입체특이적 디펩티다아제 및 이를 이용한ｄ-아미노산 함유 펩타이드의 합성 방법
JP4274767B2 (ja) （ｒ）−体のアミド結合を選択的に加水分解するアミダーゼ遺伝子及びその利用
JP4116798B2 (ja) 新規アミダーゼ及びそれをコードする遺伝子
JPWO2003020929A1 (ja) 新規なアミド加水分解酵素遺伝子
JP2006055131A (ja) 新規なｄ−アミノアシラーゼおよびその遺伝子
JP4231709B2 (ja) 新規デヒドロゲナーゼ及びそれをコードする遺伝子
JP4485734B2 (ja) ５置換ヒダントインラセマーゼ、これをコードするｄｎａ、組み換えｄｎａ、形質転換された細胞および光学活性アミノ酸の製造方法
JP2008048731A (ja) Ｎ−アセチル−（Ｒ，Ｓ）−β−アミノ酸アシラーゼ遺伝子
JP4561021B2 (ja) ５置換ヒダントインラセマーゼ、これをコードするｄｎａ、組み換えｄｎａ、形質転換された細胞および光学活性アミノ酸の製造方法
JP4652915B2 (ja) ノルボルナン誘導体の加水分解に関与するｄｎａ
WO2005075650A1 (fr) Polypeptide ayant une activite amidase et gene de celui-ci

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2008501767

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07714874

Country of ref document: EP

Kind code of ref document: A1