WO2005105991A1 - 放線菌由来のampデアミナーゼ、及びその使用 - Google Patents
放線菌由来のampデアミナーゼ、及びその使用 Download PDFInfo
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- WO2005105991A1 WO2005105991A1 PCT/JP2005/007892 JP2005007892W WO2005105991A1 WO 2005105991 A1 WO2005105991 A1 WO 2005105991A1 JP 2005007892 W JP2005007892 W JP 2005007892W WO 2005105991 A1 WO2005105991 A1 WO 2005105991A1
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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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- 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)
Definitions
- the present invention relates to AMP deaminase. More specifically, it relates to AMP deaminase derived from microorganisms and its use.
- AMP deaminase which is also known as adenyl deaminase, AMP aminohydrolase, or the like, catalyzes a reaction that hydrolyzes denylic acid to produce inosinic acid and ammonia.
- AMP deaminase is widely present in animal biological tissues and has been isolated from various tissue forces of various species so far (Tetsuro Fujishima and Hiroshi Yoshino, Amino Acid-Nucleic Acid, No. 16, pp. 45-55 (1967))
- Non-Patent Document 1 Magdale'na Rosinova 'et al., Collection Czechoslov. Chem. Commun. Vol.
- Non-Patent Document 2 JP-A-55-120788: Patent Document 1 .
- the search for AMP deaminase derived from microorganisms has been energetically conducted, mainly from the viewpoint of industrial use.
- studies on AMP deaminase derived from filamentous fungi have been widely conducted, and some AMP deaminase such as AMP deaminase derived from Aspergillus melleus have been industrially manufactured for the purpose of enhancing umami in the production of yeast extract. It is being used.
- Patent Document 1 JP-A-55-120788
- Non-Patent Document 1 Tetsuro Fujishima and Hiroshi Yoshino, Amino Acid-Nucleic Acid, No. 16, pp45-55 (1967)
- nucleases and AMP deaminase are generally used in the production of yeast extract to enhance umami.
- the optimal temperature of nuclease is about 65 ° C.
- currently used AMP derivation from Aspergillus melleus The optimal temperature of aminase is about 50 ° C. Therefore, it was impossible to allow two enzymes to act simultaneously at a high temperature in production, so that the nuclease treatment and the AMP deaminase treatment had to be performed as separate steps. If the AMP deaminase is excellent in thermostability, it can act simultaneously with the nuclease, and the production process can be shortened.
- thermostable AMP deaminase since it is possible to carry out the treatment step using these two enzymes at a high temperature, it is not necessary to temporarily lower the treatment temperature to about 50 ° C, which is the reaction temperature of AMP deaminase, during the manufacturing process. Bacterial contamination can be effectively prevented. Thus, thermostable AMP deaminase has many advantages especially for industrial use, and it has been eager to find it.
- the present inventors screened microorganisms for the origin of AMP deaminase and carried out screening. As a result, it was found that Streptomyces genus Actinomycetes produced AMP deaminase having high thermostability. In addition, they found that AMP deaminase produced by Streptomyces murinus has particularly excellent heat stability.
- the present inventors attempted to identify the enzyme (Streptomyces' AMP deaminase derived from Miulinas). As a result, the enzyme was successfully identified and its amino acid sequence and base sequence were clarified as shown in the Examples below. As a result, it became possible to produce the enzyme as a recombinant, and it was also possible to improve the productivity of the enzyme and the enzyme itself by using gene recombination techniques.o
- the present invention has been completed based on the above results, and provides the following configurations.
- the molecular weight is 48,000 ⁇ 2,000 (by gel filtration) and 60,000 ⁇ 3,000 (by SDS-PAGE);
- the actinomycete is an actinomycete selected from the group consisting of Streptomyces muulinus (Streptomyces murinus), Streptomyces 'celluloflavas (Streptmyces celluloflavus), and Streptomyces' Glyceus (Streptmyces griseus).
- the AMP deaminase according to [2].
- a method for producing a yeast extract comprising the step of allowing the AMP deaminase according to any one of [1] to [4] to act.
- a method for producing a taste substance comprising reacting the 5'-nucleotide with the AMP deaminase according to any one of [1] to [4] and deamidating the 5'-nucleotide.
- the actinomycete is an actinomycete selected from the group consisting of Streptomyces muulinus (Streptomyces murinus), Streptomyces 'celluloflavas (Streptmyces celluloflavus), and Streptomyces' Glyceus (Streptmyces griseus).
- Streptomyces muulinus Streptomyces murinus
- Streptomyces 'celluloflavas Streptmyces celluloflavus
- Streptomyces' Glyceus Streptomyces griseus
- a method for producing AMP deaminase comprising the following steps (1) and (2):
- the AMP polymerase of the present invention has excellent thermostability and can operate under relatively high temperature conditions. Therefore, it is possible to carry out the enzymatic reaction in a situation where there is little risk of contamination by various bacteria. In addition, it can be allowed to act simultaneously with other enzymes that act at high temperature, such as nuclease used in the production process of yeast extract, so that the production process can be simplified and shortened.
- FIG. 1 is a graph comparing the thermal stability of AMP deminase produced by Aspergillus melleus and Streptomyces murinus.
- the horizontal axis is the reaction temperature, and the vertical axis is the residual deaminase activity (%).
- FIG. 2 is a table comparing phosphatase activity / deaminase activity (P / D) of AMP deaminase produced by Aspergillus melleus and Streptomyces murinus (Streptomyces murinus). is there.
- FIG. 3 is a graph comparing the IMP conversion rates of AMP deaminase produced by Aspergillus melleus and Streptomyces murinus produced by Aspergillus melleus.
- Fig. 4 shows that during the production process of yeast extract, Streptomyces miyulinas
- FIG. 4 is a graph showing the IMP conversion rate when AMP deaminase derived from Streptomyces murinus (Streptomyces murinus) was allowed to act.
- A shows the measurement results when the nuclease treatment and the deaminase treatment were performed in separate steps as in the current production method.
- (B) shows the result when the nuclease treatment and the nuclease treatment were performed simultaneously.
- FIG. 5 is a graph showing the results of chromatography in the process of purifying AMP deaminase derived from Streptomyces murinus (Streptomyces murinus).
- A is the result of chromatography with HiPrep TM 16/10 ButylFF
- (b) is the result of chromatography with Superose 12.
- FIG. 6 (a) is a table summarizing the total enzyme activity, total protein amount, specific activity, and yield in the purification process of AMP deaminase derived from Streptomyces murinus (Streptomyces murinus).
- FIG. 6 (b) shows the result of analyzing the purified enzyme by SDS-PAGE (CBB staining). Lane II is a sample lane (purified enzyme). Lane I shows the protein molecular weight marker band.
- the high molecular weight side forces are also phosphorylase b (MW 97,400), bovine serum albumin (MW 66,267), aldolase (MW 42,400), carbonic anhydrase (MW 30,000), trypsin inhibitor (MW 20,100), lysozyme (MW 14,400). ) Band.
- FIG. 7 (a) is a graph showing the relationship between the reaction temperature and the activity of AMP deaminase derived from Streptomyces murinusl (Streptomyces murinusl. The activity value when reacted at 65 ° C is 100.
- Figure 7 (b) is a graph showing the thermal stability of AMP deminase from Streptomyces murinus.
- FIG. 8 (a) is a graph showing the relationship between pH and activity for AMP deaminase derived from Streptomyces murinus. The relative activity is shown assuming that the activity value when reacted at pH 5.6 is 100%.
- FIG. 8 (b) is a graph showing the pH stability of AMP deaminase derived from Streptomyces murinus.
- FIG. 9 is a table summarizing the substrate specificities of AMP protease from Streptomyces murinus. It shows the relative activity when the enzyme activity for AMP is 100%.
- FIG. 10 is a graph showing the results of analysis of AMP deaminase derived from Streptomyces murinus (Streptomyces murinus) by chromatofocusing.
- FIG. 11 is a table summarizing various properties of AMP deaminase derived from Streptomyces murinus. For comparison, the optimum pH of nuclease derived from ⁇ -silium ′ citrinum and AMP deaminase derived from Aspergillus meleus are also shown.
- FIG. 12 shows Streptomyces griseus suosp. Griseus, Streftomyces. 2 is a table comparing the thermal stability and substrate specificity of AMP deaminase produced by Streptomyces griseus and Streptomyces celluloflavus produced by Streptomyces celluloflavus.
- FIG. 13 shows the results of analyzing the N-terminal amino acid sequence and the internal amino acid sequence of AMP deaminase derived from Streptomyces murinus (Streptomyces murinus).
- FIG. 14 is a restriction map of AMP deaminase derived from Streptomyces murinus.
- FIG. 15 is a view showing a flow of constructing a shuttle vector pSVl.
- FIG. 16 is a view showing a construction flow of an AMP deaminase expression vector pSVSAD.
- FIG. 17 is a table showing the results of measuring the activity of AMP deaminase produced by the transformant SAD-1 into which the AMP deaminase gene has been introduced. ND in the table means "not detected"
- FIG. 18 is a graph showing the thermal stability of AMP polymerase produced by the transformant (SAD-1). The horizontal axis is the reaction temperature, and the vertical axis is the residual deaminase activity (%).
- FIG. 19 is a table summarizing the substrate specificities of the AMP polymerase from the transformant (SAD-1). It shows the relative activity when the enzyme activity for AMP is 100%.
- FIG. 20 shows the results of analysis of AMP deaminase derived from a transformant (SAD-1) by SDS-PAGE (CBB staining).
- Lane II is the control sample lane (host culture supernatant) and Lane III is the sample lane (transformant culture supernatant).
- Lane I shows the protein molecular weight marker band.
- the high molecular weight forces are also phosphorylase b (MW 97,000), bovine serum albumin (MW 66,000), ovalbumin (MW 45,000), and carbohydrate. It is a band of quanhydrase (MW 30,000) and trypsin inhibitor (MW 20, 100).
- FIG. 21 is a diagram showing an amino acid sequence of an actinomycete-derived AMP deaminase that has been successfully identified, and a sequence encoding the same (including a promoter region and a terminator region).
- FIG. 22 A continuation of FIG.
- FIG. 23 shows the amino acid sequence (not including the signal peptide) of AMP deaminase derived from actinomycetes that was successfully identified.
- FIG. 24 shows the amino acid sequence (including signal peptide) of actinobacteria-derived AMP deaminase that was successfully identified.
- FIG. 25 shows a sequence (including a promoter region and a terminator region) encoding an actinomycete-derived AMP deaminase that was successfully identified.
- FIG. 26 A continuation of FIG. 25.
- FIG. 27 shows the sequence of the promoter region of the gene encoding AMP deaminase derived from actinomycetes that was successfully identified.
- FIG. 28 shows the sequence of the structural gene of AMP deaminase derived from actinomycetes that was successfully identified.
- FIG. 29 shows a sequence of a terminator region of a gene encoding AMP deaminase derived from actinomycetes that was successfully identified.
- the first aspect of the present invention relates to AMP deaminase.
- the AMP deaminase of the present invention is derived from actinomycetes.
- the origin of the AMP deaminase of the present invention is not limited to a specific species of actinomycetes.
- AMP demina produced by Streptomyces murinus Streptomyces murinus
- Streptomyces cenorecula Streptmyces celluloflavus
- Streptomyces griseus Streptomyces griseus
- the AMP deaminase of the present invention reacts with the following reaction: 5′-adenylic acid + H 0 ⁇ 5 inosinic acid
- the AMP deaminase of the present invention is 5'-adenyl
- AMP deaminase derived from Streptomyces murinus, which is one embodiment of the present invention, is 5,-dAMP (5, -dexadenylic acid), ADP (adenosine 5 '). -Diphosphate), ATP (adenosine) 5'-Triphosphate also works well. Therefore, the AMP deaminase of the present invention can be applied not only to a reaction using AMP as a substrate but also to a reaction using any of these as a substrate.
- the AMP polymerase of the present invention has excellent thermal stability and is stable at a temperature of 65 ° C. or lower (property (2)).
- stable at a temperature of 65 ° C or lower means that when an enzyme solution adjusted to pH 5.6 with an acetate buffer is treated at 65 ° C for 30 minutes, the enzyme activity in the case of no treatment is measured.
- the AMP deaminase of the present invention can work well at high temperatures (eg, 60 ° C, 65 ° C, 70 ° C).
- the present inventors have found an enzyme produced by Streptomyces murinus (Streptomyces murinus) as one of the AMP deaminase having the above properties, and succeeded in purifying it.
- Streptomyces murinus Streptomyces murinus
- the obtained AMP deaminase was examined in detail, it was found that it had the following properties.
- Stable pH is about 6.0 to about 8.5 (in Mcllvaine buffer)
- reaction temperature is about 40 ° C to about 70 ° C (in acetate buffer (pH 5.6))
- the range of the action pH is such that the relative activity based on the AMP deaminase activity at the optimum pH (100%) is about 50% or more.
- the range of the stable pH is such that the residual activity is about 50% or more when the AMP deaminase activity at the untreated, optimum pH is set as a standard (100%).
- the range of the reaction temperature is such that the relative activity based on the AMP deaminase activity at the optimal temperature (100%) is about 70% or more.
- AMP deaminase from Streptomyces murinus also acts on 3, -AMP, 5, -dAMP, ADP, ATP, Adenosine (adenosine), and cAMP (cyclic adenosine 3,, 5, -monophosphate), It is clear that it does not work on 2'-AMP, adenine, 5, -GMP, 5, -UMP, and 5, -CMP. In particular, it was found to work well for 5'-dAMP, ADP, and ATP. The effect on adenosine was less than 1/10 of that on weak 5'-AMP.
- phosphatase activity was undetectable.
- a conventional AMP deaminase derived from Aspergillus melleus contaminating phosphatase activity is detected.
- the AMP protease from Streptomyces murinus is significantly different from the conventional AMP deaminase in that the contaminating phosphatase activity is extremely low.
- AMP deaminase When phosphatases are contaminated, the taste components produced by AMP deaminase action from 5'-AMP are degraded to inosine and further degraded to inosine, resulting in a loss of taste.
- AMP deaminase from Streptomyces murinus is industrially beneficial.
- a second aspect of the present invention relates to a method (preparation method) for producing AMP deaminase, which comprises the following steps.
- the actinomycetes used in step a are not particularly limited as long as they are expected to produce AMP deaminase having excellent thermostability.
- Streptomyces' Myurinus Streptomyces murinus
- Streptomyces cenoreurus Streptomyces celluloflavus
- Streptomyces griseus Streptmyces griseus
- the cultivation of actinomycetes can be performed by a conventional method.
- the medium contains carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, and organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, and ammonium acetate. Or nitrogen sources such as peptone, yeast extract, corn steep liquor, casein hydrolyzate, bran, meat extract, and, if necessary, potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, Those containing inorganic chlorides (inorganic ions) such as iron salts and zinc salts can be used. In order to promote the growth of actinomycetes, a medium supplemented with vitamins, amino acids and the like can be used.
- carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, and organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate
- the pH of the medium is adjusted to, for example, 5.0 to 8.0, preferably 5.5 to 7.5.
- the culture temperature is, for example, in the range of 15 ° C to 50 ° C, preferably in the range of 20 ° C to 40 ° C, and more preferably in the range of 25 ° C to 35 ° C.
- the culture time is not particularly limited. For example, the culture time is 1 day or more, 3 days or more, 5 days or more.
- a shaking culture method or an aerobic submerged culture method using a jar armmenter can be used as the culture method.
- AMP deaminase can be recovered from a culture solution or cells after culturing actinomycetes for a desired time.
- the culture supernatant should be filtered and centrifuged to remove insolubles, and then separated and purified by a combination of salting out such as ammonium sulfate precipitation, dialysis, and various types of chromatography.
- salting out such as ammonium sulfate precipitation, dialysis, and various types of chromatography.
- AMP deaminase can be obtained.
- hydrophobic chromatography and gel filtration are performed.
- AMP deaminase when recovering from the cells, can be obtained by, for example, crushing the cells by pressure treatment, ultrasonic treatment, and the like, followed by separation and purification as described above. After the cells have been collected from the culture fluid in advance by filtration, centrifugation, or the like, the above-described series of steps (crushing, separation, and purification of the cells) may be performed. In addition, in each purification step, fractionation is performed using AMP deaminase activity as an index, and the process proceeds to the next step.
- a third aspect of the present invention relates to the use of the AMP deaminase of the present invention.
- the AMP deaminase of the present invention can be used for various uses in the same manner as ordinary AMP deaminase (that is, AMP deaminase derived from Aspergillus melleus and the like, which has been used so far).
- the reaction at a high temperature is preferred because of its excellent heat resistance, and it can be suitably used for various purposes.
- “reaction at high temperature is preferred! / Use” refers to an application in which it is preferable to react AMP protease at a high temperature in view of production efficiency and contamination of various bacteria.
- yeast extract As a specific example of the use, production of yeast extract can be mentioned.
- umami is generally enhanced (improved in taste) by nuclease treatment and AMP deaminase treatment.
- the AMP deaminase of the present invention which is excellent in thermostability, the AMP deaminase treatment can be performed at the same time as the nuclease treatment performed at a high temperature. As a result, simplification and shortening of the production process can be achieved, and bacterial contamination during enzyme treatment can be effectively prevented.
- the following step is carried out, that is, a step of adding nuclease and AMP deaminase to the yeast lysate and allowing it to act at a high temperature.
- the yeast lysate can be prepared by a conventional method. For example, a suspension (pH 7.0) of 10% brewer's yeast or baker's yeast is subjected to a heat treatment, and a commercially available lytic enzyme, YL-NL “Amano” or YL-15 (V, also with Amano Enzym Co., Ltd.) To prepare a yeast lysate.
- Nuclease is commercially available from various manufacturers (for example, Amano Enzym Co.), and a suitable medium can be appropriately selected and used.
- a nuclease prepared from a microorganism or the like by a conventional method may be used.
- a plurality of nucleases may be used in combination.
- the amount of nuclease and AMP deaminase to be added can be appropriately set in consideration of the type and activity value of the enzyme used.
- the working temperature is the temperature at which both nuclease and AMP deaminase act.
- the reaction is carried out at 60 ° C to 80 ° C, preferably at 65 ° C to 75 ° C, more preferably at about 70 ° C.
- a further aspect of the present invention is based on the above results and relates to an isolated AMP deaminase.
- the AMP deaminase of the present invention can be a protein having the amino acid sequence of SEQ ID NO: 1, for example. As will be shown in the examples below, it has been confirmed that the protein actually exhibits AMP deaminase activity. The sequence containing the signal peptide is shown in SEQ ID NO: 2.
- a protein having the same function but partially different amino acid sequence when compared with a protein having the amino acid sequence of SEQ ID NO: 1 or 2 (hereinafter, also referred to as “homologous protein”) AMP deaminase is also provided.
- a part of the amino acid sequence differs typically means that one or several amino acids constituting the amino acid sequence are deleted, substituted, or added or inserted with one or several amino acids, or Means that the amino acid sequence is mutated by the combination of the two.
- the difference in the amino acid sequence here is due to the function as AMP deaminase, that is, 5'-adelic acid + H0 ⁇ 5'-
- the position at which the amino acid sequence differs is not particularly limited as long as this condition is satisfied, and the difference may occur at a plurality of positions.
- the term “plurality” refers to, for example, a number corresponding to less than about 30% of all amino acids, preferably a number corresponding to less than about 20%, and more preferably a number corresponding to less than about 10%, and more preferably Preferably a number corresponding to less than about 5%, most preferably a number corresponding to less than about 1%.
- the homologous protein is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, still more preferably about 95% or more, and most preferably about 70% or more of the amino acid sequence of SEQ ID NO: 1 or 2. It has about 99% or more identity.
- homologous proteins are obtained by making conservative amino acid substitutions at non-essential amino acid residues (“amino acid residues not involved in AMP deaminase activity.”
- conservative amino acid substitution refers to a certain amino acid residue.
- An amino acid having a side chain of similar properties Refers to substitution with a residue.
- Amino acid residues can be basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged electrode side chains (eg, glycine, asparagine, glutamine, serine, threonine) depending on their side chains.
- Tyrosine, cysteine non-polar side chains (eg, alanine, palin, leucine, isoleucine, proline, hua-alanine, methionine, tributophane), ⁇ -branched side chains (eg, threonine, norin, isoleucine), aromatic side chains (Eg, tyrosine, fluoranine, tributophan, histidine).
- Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.
- the identity (%) of two amino acid sequences or two nucleic acids can be determined, for example, by the following procedure. First, align the two sequences for optimal comparison (eg, introducing gaps in the first sequence to optimize alignment with the second sequence! /,). When a molecule (amino acid residue or nucleotide) at a particular position in the first sequence is the same as the molecule at the corresponding position in the second sequence, the molecule at that position is said to be identical.
- XBLAST Gapped BLAST described in Amino Acids Research 25 (17): 3389-3402
- the default parameters of the corresponding program eg, XBLAST and NBLAST
- XBLAST and NBLAST the default parameters of the corresponding program
- ALIGN program Built into the ALIGN program available on the GENESTREAM network server (IGH adjoin, France) or the ISREC server.
- AMP deaminase including homologous proteins
- those possessed by natural actinomycetes can be prepared from the actinomycetes by operations such as extraction and purification.
- the AMP deaminase of the present invention can also be prepared by genetic engineering techniques based on the sequence information disclosed in the present specification or the attached sequence listing. For example, it can be prepared by transforming a suitable host cell with DNA encoding the AMP deaminase of the present invention, and collecting the protein expressed by the transformant. The recovered protein is appropriately purified according to the purpose.
- Various modifications are possible when preparing as a recombinant protein.
- the AMP deaminase of the present invention has no other peptide.
- a recombinant protein to which the protein is linked can be obtained.
- addition of sugar chains and Z or lipids, or processing at the N-terminal or C-terminal may occur. Modifications may be made. By the modification as described above, extraction and purification of the recombinant protein can be simplified, or a biological function can be added.
- isolated when used in the context of the AMP deaminase of the present invention refers to a state of being removed from its original environment (for example, in the case of a natural substance, the natural environment), that is, It means that it exists in a state different from the original existence state due to an artificial operation.
- AMP deaminase in the isolated state usually does not contain the cell components of the producing bacterium.
- the content of contaminant components is preferably low.
- the amount of contaminating proteins is, for example, 50% or less, preferably 40% or less, more preferably 30% or less, even more preferably 20% or less of the total protein amount.
- a further aspect of the present invention relates to a nucleic acid molecule encoding AMP deaminase.
- nucleic acid includes DNA (including cDNA and genomic DNA), RNA (including mRNA), DNA analogs, and RNA analogs.
- the form of the nucleic acid of the present invention is not limited, that is, it may be single-stranded or double-stranded. Preferably, it is double-stranded DNA. Codon degeneracy is also taken into account. That is, as long as a target protein can be obtained as its expression product, it has an arbitrary base sequence.
- nucleic acid encoding a specific protein refers to a nucleic acid from which the specific protein can be obtained when expressed, and corresponds to the amino acid sequence of the protein. Not only a nucleic acid having a base sequence as described above, but also a nucleic acid (for example, DNA containing one or more introns) obtained by adding a sequence that does not encode an amino acid sequence to such a nucleic acid.
- isolated nucleic acid refers to a naturally occurring nucleic acid, typically a nucleic acid that has been separated from other nucleic acids that coexist in the natural state. Say. However, it may contain some other nucleic acid components such as a nucleic acid sequence adjacent in the natural state.
- the "isolated nucleic acid” is preferably a nucleus substantially free of cell components, culture solution and the like. Refers to acid.
- an ⁇ isolated nucleic acid '' in the case of a nucleic acid produced by chemical synthesis preferably does not substantially contain a precursor (raw material) such as dNTP or a chemical substance used in the synthesis process! /, The state nucleic acid!
- nucleic acid is present as part of a vector or composition or in a cell as an exogenous molecule, it is "isolated nucleic acid" as long as it is present as a result of artificial manipulation.
- nucleic acid in the present specification means an isolated nucleic acid.
- the nucleic acid molecule of the present invention encodes the AMP deaminase of the present invention. That is, the nucleic acid molecule of the present invention encodes a protein having the amino acid sequence of SEQ ID NO: 1 or 2, or a homologous protein thereof.
- One specific embodiment of the nucleic acid molecule of the present invention is a DNA having the base sequence of SEQ ID NO: 3.
- This nucleotide sequence is a DNA encoding an AMP deaminase gene that has been successfully identified, and includes a 5 ′ untranslated region (promoter region) and a 3 ′ untranslated region (terminator region).
- the promoter and terminator and the structural gene are in an original combination (combination in a natural state), and good gene expression can be expected if AMP deaminase is produced using the DNA. Therefore, an efficient AMP deaminase production system can be constructed.
- the powerful DNA include a DNA having the base sequence of SEQ ID NO: 4 or 5.
- the DNA having the base sequence of SEQ ID NO: 4 is a DNA encoding a successfully identified AMP deaminase structural gene (including a region encoding a signal peptide).
- the DNA having the nucleotide sequence of SEQ ID NO: 5 is a DNA encoding a successfully identified AMP deaminase structural gene (not including a region encoding a signal peptide).
- the nucleic acid of the present invention can be obtained by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, and the like, with reference to the sequence information disclosed in the present specification or the attached sequence listing. It can be prepared in an isolated state.
- the nucleic acid of the present invention can be isolated from an actinomycete genomic DNA library using the hybridization method using the whole or a part of the base sequence of the nucleic acid or its complementary sequence as a probe. .
- an actinomyces genomic DNA library or an actinomyces genome may be obtained by using a nucleic acid amplification reaction (for example, PCR) using a synthetic oligonucleotide primer designed to specifically hybridize to a part of the base sequence of the nucleic acid.
- ⁇ can be amplified and isolated from an actinomycete nucleic acid extract.
- oligonucleotide primers can be easily synthesized using a commercially available automated DNA synthesizer or the like.
- the plaque hybridization method or colony hybridization method is used depending on the type of library used (Molecular Cloning, Third
- the colony hybridization method is used for selection of a clone having the nucleic acid of interest.
- a probe having a sequence specific to the nucleic acid of the present invention is used for selection of a clone having the nucleic acid of interest.
- the nucleic acid of the clone is converted into type II, and the nucleic acid of the present invention is amplified by PCR or the like using primers specific to the sequence of the nucleic acid of interest. It can be obtained as a product.
- the nucleic acid possessed by the obtained clone can be subcloned into an appropriate vector and used for subsequent use.
- an appropriate vector for example, it is possible to construct a recombinant vector for transformation or a plasmid suitable for decoding the nucleotide sequence.
- a nucleic acid in which the function of a protein encoded by the nucleotide sequence differs when compared with any one of the nucleotide sequences of SEQ ID NOS: 3 to 5 are also provided.
- a base sequence comprising substitution, deletion, insertion, addition, or inversion of one or more bases based on the base sequence of SEQ ID NO: 3 to 5, and having an AMP deaminase activity DNA encoding a protein having the same. Substitution or deletion of a base may occur at a plurality of sites.
- the term “plurality” here depends on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid, but is, for example, 2 to 40 bases, preferably It is 2 to 20 bases, more preferably 2 to 10 bases.
- Mutations such as base substitutions, deletions, insertions, additions, or inversions as described above include mutations that occur naturally, such as those based on individual differences between microorganisms that carry the AMP deaminase gene and differences in species and genera. Is also included.
- homologous nucleic acid is a nucleic acid in which a base difference as described above is recognized due to a polymorphism represented by SNP.
- the homologous nucleic acid as described above is treated with, for example, a restriction enzyme, treatment with exonuclease ⁇ DNA ligase, or the like, and site-directed mutagenesis (Molecular Cloning, Third Edition, napter 13; old Spring Harbor Laboratory). Press, New York) and mutations introduced by Huntam's Natural Cloning, Third Edition, chapter 13, Cold Spring Harbor Laboratory Press, New York.
- Homologous nucleic acids can also be obtained by other methods such as ultraviolet irradiation. Furthermore, it can also be obtained by a method utilizing a known mutation treatment, such as treating an actinomycete carrying the AMP kinase gene with ultraviolet light, and then isolating the modified gene.
- a genomic (chromosome) DNA is extracted from a natural actinomycete having a homologous nucleic acid and treated with an appropriate restriction enzyme, and then the nucleic acid molecule of the present invention (for example, a DNA having the nucleotide sequence of SEQ ID NO: 3) or one of them. Select and isolate DNA that hybridizes under stringent conditions in the screening using the part as a probe.
- the library may be used as the nucleic acid molecule of the present invention (for example, a DNA having the nucleotide sequence of SEQ ID NO: 3) or one of them. It can also be obtained by screening under stringent conditions using the part as a probe.
- Another embodiment of the present invention relates to a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NOS: 3 to 5.
- nucleotide sequence is at least about 60%, 70%, 80%, 90%, 95%, 99%, % Or 99.9% nucleic acid having the same nucleotide sequence is provided. It is preferable that the identity is as high as possible.
- stringent conditions refer to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
- hybridization solution 50% formaldehyde, 10 X SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 5 X Denhardt solution, 1% SDS, 10% dextran sulfate, Incubate at about 42 ° C to about 50 ° C with 10 g / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), and then incubate at about 65 ° C with 0.1X SSC, 0.1% SDS. Conditions for washing at C to about 70 ° C can be mentioned.
- stringent conditions for example, 50% formaldehyde as a hybridization solution, 5 ⁇ SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 1 ⁇ Denhardt solution, 1% SDS, Examples include conditions using 10% dextran sulfate, 10 / zg / ml denatured salmon sperm DNA, and 50 mM phosphate buffer (pH 7.5).
- vector refers to a nucleic acid molecule capable of transporting a nucleic acid inserted therein into a target such as a cell.
- the vector of the present invention can be prepared by incorporating the nucleic acid (typically, DNA) of the present invention into an existing vector or a vector obtained by modifying the vector. As long as it can hold the nucleic acid of the present invention, in principle, even if such a vector is used as a starting material, the type of host cell should be considered according to the intended use (cloning, expression of polypeptide). Then, an appropriate vector is selected.
- the vector for transformation typically contains an AMP deaminase gene (eg, a DNA having the nucleotide sequence of SEQ ID NO: 4), a promoter, and a terminator. Upstream to downstream to ensure proper transcription of the structural gene by the promoter , A promoter, an AMP deaminase gene, and a terminator are arranged in this order.
- an AMP deaminase gene eg, a DNA having the nucleotide sequence of SEQ ID NO: 4
- a promoter, an AMP deaminase gene, and a terminator are arranged in this order.
- the vector of the present invention is preferably an expression vector.
- An “expression vector” refers to a vector that can introduce a nucleic acid inserted therein into a target cell (host cell) and can express the same in the cell.
- the expression vector usually contains a promoter sequence necessary for the expression of the nucleic acid, a sequence for promoting the expression, and the like.
- Expression vectors that contain a selection tool can also be used. When such an expression vector is used, the presence / absence (and the degree) of introduction of the expression vector can be confirmed using a selection marker.
- Insertion of the nucleic acid of the present invention into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. are performed using standard recombinant DNA techniques (for example,
- a vector containing a DNA that also contains a promoter region for example, a DNA having a base sequence of SEQ ID NO: 3
- the promoter region of the DNA and the other regions are separately prepared.
- a recombinant vector may be constructed by incorporating each into a vector. In such a case, another sequence may be interposed between the two (promoter region and other region) in the vector, provided that the promoter function is appropriately exerted.
- a vector holding the promoter region may be constructed first, and then the other regions may be ligated.
- the above recombinant vector is used for transformation of a host. That is, a transformant into which the nucleic acid molecule of the present invention has been introduced can be prepared using the above-described recombinant vector. (Transformants and their use)
- the transformation vector is used for transforming a host. That is, a transformant into which the nucleic acid molecule of the present invention has been introduced can be prepared using the above-described transformation vector.
- the type of host to be used for the transformation is not particularly limited, and Streptomyces miulinas (IFO14802, etc.), Streptomyces 'Ribidans (TK24, etc.), Streptomyces' Griseus Subsp. Griseus (Streptomyces griseus subsp.griseus), Streptomyces 'Glytheus (Streptomyces griseus), Streptomyces' Actinomycetes such as Streptomyces mobaraensis and Streptomyces avermitilis can be suitably used as hosts.
- Escherichia coli, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Saccharomyces pombe, and the like can also be employed as hosts.
- transformation vector into a host can be performed by a known method.
- the method can be carried out by the method of D.A. Hopwood et al. (PRACTICAL STREPTOMYCES GENETICS P.229-252 (The John Innes Foundation 2000)) using protoplast-formed cells.
- the transformant can be used for production of AMP deaminase.
- the AMP deaminase can be produced by culturing the transformant into which the nucleic acid of the present invention has been introduced under conditions in which the protein (AMP deaminase) encoded by the nucleic acid can be expressed.
- An appropriate medium is used depending on the host used. For example, commercially available various media or media obtained by adding components necessary for growth, selection, promotion of protein expression and the like of transformants such as proline, leucine, and thiamine can be used.
- the target protein can be recovered from the culture solution or the cells after culturing for a desired time. When it is produced outside the cells, it can be recovered from the culture solution, otherwise it can be recovered from the cells.
- the culture supernatant is filtered and centrifuged to remove insolubles, and then separated and purified by a combination of salting out such as ammonium sulfate precipitation, dialysis, and various types of chromatography.
- the desired protein can be obtained.
- the cells must be crushed by, for example, pressure treatment or ultrasonic treatment, and then separated and purified in the same manner as above to obtain the target protein. Can be.
- the above series of steps may be performed. Since the AMP deaminase of the present invention is usually produced outside the cells, its isolation and purification can be carried out in a specific ratio. It is relatively easy.
- the strain, the method for preparing the enzyme solution, and the method for measuring the enzyme activity used in the following experiments were as follows.
- Streptomyces' Streptomyces murinus' IFO 14802, Streptomyces' Griseus subespe sp. ) IFO13780 (NBRC13780) was used.
- Streptomyces murinus (Streptomyces murinus) IFO14802 has been deposited with the international depositary institution as follows.
- a commercially available AMP deaminase agent "Deamizyme (50,000 u / mg product)" (trade name, product of Amano Enzym) was diluted with water to obtain an enzyme solution.
- the pH was adjusted to pH 5.7 with 3% of soluble starch 3% and sterilized at 121 ° C for 30 minutes. Inoculate Streptomyces murinus (Streptomyces murinus) IFO14802 and pre-culture at 27 ° C for 1 day The main culture was performed for 5 days to prepare a crude enzyme solution.
- Sowflower A 2%, NaCl 0.3%, KH PO 0.1%, supplemented MgSO 0.05%, soluble starch
- Enzyme activity was measured using the decrease in OD during the reaction as an index. 0.017M 5'AMP-2Na
- 0.5 ml of the sample solution was added to 1.5 ml of a 1: 2 mixture of 1/15 M phosphate buffer (pH 5.6) to give a reaction solution, and the mixture was reacted at 37 ° C for 15 minutes. After 15 minutes, the reaction was stopped by adding a 2% perchloric acid solution, and then 100 ⁇ l was weighed. The OD was measured with 5 ml of water.
- the measured value was used as a blank. Under the above conditions, when the absorbance difference increased by 0.001 in 30 minutes, 1 unit was defined.
- the processing temperatures were 30 ° C, 40 ° C, 50 ° C, 60 ° C, 65 ° C, 70 ° C, and 75 ° C.
- the processing time was 30 minutes.
- Contaminant phosphatase activities were measured for Aspergillus melleus and Streptomyces murinus.
- the phosphatase activity / deaminase activity (P / D) determined from the measurement results are summarized in the table of FIG.
- Aspergillus melleus was found to have contaminating activity.
- no phosphatase activity was observed in Streptomyces murinus. That is, it became clear that AMP deaminase produced by Streptomyces murinus (Streptomyces murinus) has extremely low contaminating phosphatase activity.
- the AMP conversion rate of AMP deaminase produced by Aspergillus melleus and Streptomyces murinus was examined by HPLC. Specifically, a 1.1% AMP solution was prepared using a 1/15 M phosphate buffer (pH 7.0), and 0.5 ml of the enzyme solution was added to 5 ml of the 1.1% AMP solution at 50 ° C. The reaction was carried out for 4 hours, followed by a heat treatment at 100 ° C for 10 minutes, followed by filtration with a filter (0.45 m) and analysis by HPLC.
- nuclease treatment and deaminase treatment were performed in separate steps as in the current production method. Specifically, processing was performed in the following procedure, and the IMP conversion rate was obtained.
- nuclease ⁇ Amano '' G manufactured by Amano Enzym Co., 0.1% w / w yeast solid, was added to yeast lysate (YL-15, 0.2%), and the mixture was added at 70 ° C and pH5. The reaction was performed for 3 hours.
- test enzyme Aspergillus melleus
- -derived AMP deaminase agent “Deamizyme (50,000 u / mg product)” (trade name, manufactured by Amano Enzym Co., 0.01-0.04% w / w yeast solid) or A crude enzyme solution prepared from Streptomyces murinus (Streptomyces murinus) was added, and the mixture was reacted at 50 ° C and pH 6 for 5 hours. Subsequently, after heat treatment, it was subjected to HPLC analysis.
- the amount of AMP protease from Streptomyces murinus was determined by the activity value of “Deamisym (50,000 u / mg product)” (trade name, manufactured by Amano Enzym Co., Ltd.). The amount was the same as the value.
- nuclease treatment and deaminase treatment were performed simultaneously. Specifically, processing was performed according to the following procedure, and the IMP conversion rate was obtained.
- nuclease “Amano” G manufactured by Amano Enzym, 0.1% w / w yeast solid
- an AMP deaminase agent derived from the test enzyme (Aspergillus melleus) were added to the yeast lysate (YL-15, 0.2%).
- Streptomyces murinus Streptomyces murinus was added in an amount such that its activity value was the same as the activity value of the deazyme (50,000 u / mg product).
- FIG. 4 (a) shows the results of Test 1.
- Streptomyces' Miyuri The AMP deaminase derived from eggplant (Streptomyces murinus) showed an IMP conversion rate equivalent to that of AMP deaminase derived from Aspergillus melleus (deamizym: 50,000 u / mg).
- Streptomyces' Myurinus (Streptomyces murinus) (IFO14802) was cultured by the above method, the produced enzyme was concentrated twice by an ultrafiltration membrane (AIP1 010), and water was added to the concentrated solution. After concentrating to remove the low molecular fraction, it was freeze-dried to obtain the crude enzyme. This crude enzyme was dissolved in purified water, and ammonium sulfate fractionation was performed using 48% of saturated ammonium sulfate. The precipitate fraction was dissolved in 20 mM KPB (pH 7.0) to obtain an enzyme solution.
- AIP1 010 ultrafiltration membrane
- the resulting enzyme solution was passed through HiPrep TM 16/10 ButylFF (Pharmacia) equilibrated with 20 mM KPB (pH 7.0) containing 30% ammonium sulfate. Subsequently, elution was performed with a concentration gradient of 20 mM KPB pH 7.0 containing ammonium sulfate 30% to 0% and ammonium sulfate 30% to 0%. Thereafter, concentration was performed, and the active fraction was subjected to gel filtration using a Superose 12 column (Pharmacia), and eluted with 50 mM KPB PH7.0 containing 150 mM NaCl. Active fractions (fractions 15 and 16) were collected and used as a purified enzyme.
- FIG. 5 (a) The results of chromatography with HiPrep TM 16/10 ButylFF are shown in FIG. 5 (a), and the results of chromatography with Superose 12 are shown in FIG. 5 (b). Also, the total enzyme activity at each stage , Total protein amount, specific activity, and yield are shown in the table of FIG. 6 (a). The specific activity at the final stage was 96 times higher than that of the crude enzyme.
- the purified enzyme was subjected to SDS-PAGE (CBB staining) to confirm the protein purity.
- Lane II is a sample lane (purified enzyme). It shows a single band, indicating that the purity of the purified enzyme is high.
- the lane I shows a protein molecular weight marker band. High-molecular-weight forces also include bands for phosphorylase b (MW 97,400), bovine serum albumin (MW 66,267), aldolase (MW 42,400), carbonic anhydrase (MW 30,000), trypsin inhibitor (MW 20,100), and lysozyme (MW 14,400). It is.
- thermostability of the enzyme was examined by the following procedure. 2.8 g (total protein amount) of the enzyme solution (0.15 ml) was adjusted to pH 5.6 using an acetate buffer of pH 5.0, and then adjusted to each temperature (40 ° C, 50 ° C, 60 ° C, 65 ° C). C, 70 ° C, 75 ° C) for 30 minutes, and the residual activity (% of the enzyme activity in the case of no treatment) was measured.
- Figure 7 (b) shows the measurement results. Under treatment conditions of 65 ° C or less, the activity is maintained at about 90% or more. Also, it maintains about 55% activity even when treated at 70 ° C. Thus, the enzyme was found to be extremely excellent in thermostability.
- Figure 8 (a) shows the measurement results.
- the graph in FIG. 8 (a) shows the relative activity when the activity value when reacted at pH 5.6 is 100%. Relatively high reactivity is observed when the pH is in the range of about 4.5 to about 8.5, and about 70% or more is observed in the range of about 5.0 to about 8.0. Also, it can be seen that about 40% reactivity can be obtained even under the condition of pH 9.0.
- Non-Patent Document 2 It has been reported that adenosine deaminase derived from Streptomyces aureofaciens has a wide range of substrate properties, such as having the catalytic activity of AMP.
- AMP the catalytic activity of AMP
- its substrate specificity was examined. The results are shown in the table of FIG.
- the relative activity is shown when the enzyme activity for 5 'AMP is 100%. The enzyme worked best on 5 'AMP.
- AMP deaminase from Streptomyces murinus has a molecular weight of about 48,000 ⁇ 2,000 by gel filtration (the molecular weight by SDS-PAGE is 60,000 ⁇ 3,000), and an optimal pH of about 5.6, Optimum temperature is about 65 ° C, isoelectric point is 8.12, Km value is 0.95 mM, Vmax force is 10 7
- Streptomyces murinus (Streptomyces murinus), one of the actinomycetes belonging to the genus Streptomyces, produces metabolic AMP deaminase. It was predicted that there was a possibility that thermostable AMP deaminase could be obtained. Therefore, AMP deaminase-producing bacteria were screened in the Streptomyces genus of the Amano Enzym strain. As a result, those that produce AMP deaminase are Streptomyces griseus subsp.
- thermostability was higher than that of the deaminase derived from Reus (Aspergillus melleus), which suggests that the strains produced by actinomycetes of the genus Streptomyces generally have higher thermostability and tend to have higher thermostability.
- the deaminase produced by the three strains acted better on AMP than adenosine, and was confirmed to be AMP deaminase.
- Streptomyces murinus (Streptomyces murinus) IFO14802 was cultured to obtain a purified enzyme.
- the purification was performed up to the purification using a Butyl Sepharose column using HiPrep TM 16/10 ButylFF (Pharmacia).
- the fractionated sample was subjected to SDS-PAGE using gel PAG Mini "DAIICHI” 10/20 (Daiichi Pure Chemicals).
- the gel after electrophoresis was transferred to a PVDF membrane using a Towbin buffer (Tris 25 mM, glycine 192 mM, methanol 5%) containing 0.01% SDS as a transfer buffer.
- the transfer operation was performed using a buffer tank type transfer device under the conditions of a constant voltage of 20 V, 4 ° C, and 18 hours.
- CBB Kiasi-Brilliant Blue R250
- full strength staining was performed, and a band corresponding to the enzyme was cut out to obtain a sample for N-terminal amino acid sequence analysis.
- the gel after electrophoresis was subjected to CBB staining, a band corresponding to the enzyme was cut out, and used as it was as a sample for internal amino acid sequence analysis.
- Figure 13 shows the results of analyzing the amino acid sequences of these samples.
- Streptomyces murinus IFO14802 was cultured, and the culture was filtered using a Buchner funnel and a Nutsche suction bottle to obtain cells. 10 g of TE (10 mM Tris-HCl (pH 8.0), ImM EDTA) containing 4 mg / ml lysozyme (Roche's diagnostics) and 2 mg / ml chromopeptidase (Wako Pure Chemical Industries) And lysed at 30 ° C for 1 hour.
- TE 10 mM Tris-HCl (pH 8.0), ImM EDTA) containing 4 mg / ml lysozyme (Roche's diagnostics) and 2 mg / ml chromopeptidase (Wako Pure Chemical Industries)
- the mixture was centrifuged (1,500 g, room temperature, 5 minutes) to obtain a supernatant, and this operation was repeated twice, and lOmg / ml RNase A (Sigma-Aldrich Japan) 72 was added to the supernatant. 1 and incubated for 1 hour at 37 ° C. After mixing and mixing 4.5 ml of 5M NaCl, 11.25 ml of a 30% polyethylene glycol 6000 (Wako Pure Chemical) solution was added and mixed, and the mixture was allowed to stand at 4 ° C. Wind up the precipitated genomic DNA using a Pasteur pipette, and add 70% ethanol After washing with air and air-drying, it was dissolved in 1 ml of TE solution to obtain a genomic DNA solution of about 1 mg / ml.
- a specifically amplified DNA fragment of about 240 bp was extracted using GENECLEAN TM III (BIO101). After subcloning the extracted DNA fragment into pGEM TM -T Easy (Promega), the inserted DNA fragment is extracted again, and DIG-labeled using DIG High Prime (Roche Diagnostics), and the AMP deaminase gene Probe.
- Agarose electrophoresis was performed by applying genomic DNA completely digested with an arbitrary restriction enzyme in an amount of 6 ⁇ g per lane. After electrophoresis, the membrane was treated with a 0.25N HC1 solution for 30 minutes, neutralized with the buffer used for electrophoresis, and then blotted on a Zeta-Probe TM membrane (Bio-Rad) by alkaline blotting using a 0.4N NaOH solution. . The transfer was carried out using 2016 VacuGene (Pharmacia LKB Biotechnology) at a degree of vacuum of 50 cm'H 0 for 90 minutes.
- the one dephosphorylated using Alkaline Phosphatase was used as a vector for library preparation.
- the insert and the vector were ligated using Ligation Kit ver.2 (Takara Shuzo) and E. coli DH5 strain competent cells (Toyobo) were prepared according to the method of Hanahan (J Mol Biol. 1983 Jun 5; 166 (4): 557-80, Transformation was performed using Hanahan D., Studies on transformation of Escherichia coli with plasmids.
- the obtained clones were spread so as to form about 500 colonies per LA plate (ampicillin (Sigma-Aldrich Japan) 100 ⁇ g / ml), and incubated at 37 ° C. overnight to grow the colonies.
- nucleotide sequence analysis was performed from the outside of the insert using M13 Primer M4 and M13 Primer RV (Takara Shuzo). Based on the nucleotide sequence analysis of the isolated clone and the DNA fragment used for the probe, a synthetic primer (sigma dienosis) was designed again based on the sequence of the enzyme gene, and the nucleotide sequence was analyzed. Repeat these operations The full-length DNA sequence was analyzed by the performed primer walking. Nucleotide sequence analysis is performed using BigDye Terminator v3.1 Cycle Sequencing Kit and dGTP BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems Japan). Analysis is performed using ABI PRISM TM 310 Genetic Analyzer (Applied Biosystems Japan). did. The synthetic primers used for the nucleotide sequence analysis are shown below.
- MAD--Fl 5, -AAGCAACTCGCCGACCAG- -3, (SEQ ID NO: 8)
- MAD- -F2 5, -TGGTCCATGCAGGACTTC- -3, (SEQ ID NO: 9)
- MAD- -F3 5, -CTGGAGAACTACAGCCTC- -3, (SEQ ID NO: 10)
- MAD- -F5 5, -CTCCAGTACGCCTTCCTG- -3, (SEQ ID NO: 12)
- MAD- -F6 5, -GTCGGGTCCTGGACACCG- -3, (SEQ ID NO: 13)
- MAD- -F8 5, -GGACAGGAAGACGGACAC-3, (SEQ ID NO: 15)
- MAD- -R2 5, -CAGGGACAGGACACTGAG- -3, (SEQ ID NO: 18)
- MAD- -R3 5,-GATGTCGACATGGCCCTG- -3, (SEQ ID NO: 19)
- MAD- -R4 5, -CCCAGTCGATCGCGTGAG- -3, (SEQ ID NO: 20)
- MAD- -R6 5,-CTCGAACGCCGCGAACGC -3, (SEQ ID NO: 22)
- a promoter region, a coding region (SEQ ID NO: 4), and a terminator region were revealed by annotation using homology search and motif search (FIGS. 21 to 22).
- FIG. 27 shows the sequence of the promoter region
- FIG. 28 shows the sequence of the coding region
- FIG. 29 shows the sequence of the terminal region.
- the amino acid sequence deduced from the nucleotide sequence is shown in FIG. 23 (signal peptide is not included! / ⁇ , SEQ ID NO: 1) and FIG. 24 (signal map). It is shown in SEQ ID NO: 2), including tide.
- Streptomyces lividans 313KATCC 35287 carrying plasmid PIJ702 was cultured at 30 ° C for 2 days under the following medium conditions.
- TE-Sucrose 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 25% Sucrose.
- 2 ml of TE-Sucrose containing 4 mg of lysozyme (Sigma-Aldrich Japan) and 4 ml of a 0.25 mM EDTA solution were added, and the mixture was incubated at 37 ° C for 30 minutes.
- 2 ml of a 20% SDS solution was added, 5 ml of a 5 M NaCl solution was further added, and the mixture was gently stirred and incubated at 0 ° C for 1 minute.
- 30% polyethylene glycol 6000 (Wako Pure Chemical Industries) solution is added to the supernatant obtained by centrifugation (100,000g, 4 ° C, 40 minutes) to a final concentration of 10%, and the mixture is added at 0 ° C for 4.5 hours. Incubated. Then, the mixture was centrifuged (900 g, 4 ° C, 5 minutes), and the precipitate was dissolved in a TE solution containing 50 mM NaCl.
- 1.2 ml of a solution prepared by dissolving ethidium bromide in a TE solution to a concentration of 16.8 g of cesium and 10 mg / ml of cesium was added, and centrifuged (l, 300 g, room temperature, 15 minutes). After removing the residue, centrifugation (230,000 g, 20 ° C, 12 hours) was performed again. After centrifugation, a plasmid DNA layer was obtained under ultraviolet irradiation. Next, extraction with butanol saturated with a TE solution was performed to remove ethidium bromide. This extraction was repeated three times. The obtained plasmid DNA solution was subjected to 1 ⁇ dialysis at 4 ° C.
- a DNA fragment obtained by digesting the Escherichia coli vector pUC19 (Takara Shuzo) with the restriction enzyme BamHI and a DNA fragment containing the thiostrepton resistance gene (tsr) obtained by digesting the actinomycete vector PIJ702 with Bel I (Takara Shuzo) Were prepared and ligated using DNA Ligation Kit Ver.2 (Takara Shuzo) to produce pUCTSR.
- a DNA fragment (long fragment) obtained by digesting pUCTSR with Kpn I and Cla I (Takara Shuzo) and a DNA fragment (short fragment) obtained by digesting pIJ702 with Kpn I and Cla I (Takara Shuzo) are prepared. These were ligated using DNA Ligation Kit Ver.2 (Takara Shuzo) and transformed into Escherichia coli DH5 strain (Toyobo). A plasmid in which the PUC19 fragment and the pIJ702 fragment of the transformant thus obtained were ligated was used as a shuttle vector pSVl and used in a subsequent operation (FIG. 15).
- PSAD carrying the AMP deaminase gene was digested with the restriction enzyme Not I (Takara Shuzo) to prepare the gene fragment. Further, the shuttle vector pSVl was digested with a restriction enzyme XbaI to prepare one vector fragment. Both ends were blunt-ended using a DNA Blunting Kit (Takara Shuzo) to prepare an insert fragment and a vector fragment, respectively. The pSVl-derived vector fragment was further dephosphorylated using Alkaline Phosphatase (Takara Shuzo). The insert and the vector were ligated using DNA Ligation Kit Ver.2 (Takara Shuzo), and then transformed into Escherichia coli DH5 strain (Toyobo). The plasmid thus obtained was used as an expression vector pSVSAD for transformation (FIG. 16).
- TK24 protoplast Streptomyces lividans is a strain having streptomycin metabolism derived from Streptomyces lividans 66 , DA Hopwood (John Innes Institute ⁇ Colney Lane, Norwich NR4 7UH, UK) It was provided. Streptomyces lividans TK24 was cultured in YEME medium (0.5% glycine) at 30 ° C for 2 days.
- a 1% monopotassium phosphate solution was separately prepared, and immediately before use, 1 ml of potassium hydroxide was added per 100 mlP buffer.
- a buffer containing 20% polyethylene glycol 1000 was gently mixed by 1.5 ml strength pipetting, and the mixture was allowed to stand at room temperature for 2 minutes. This mixture was centrifuged (1,700 g, room temperature, 10 minutes) to precipitate. The protoplasts obtained as a precipitate were washed twice with P buffer. After resuspending the pellet in 0.3 ml of P buffer, 100 1 drops were added to R-2 medium. Then, 3 ml of R-2 top agarose medium kept at 55 ° C. was poured per plate, and protoplasts were spread over the entire plate. Plates were dried in a clean bench for 2 hours until the top agarose solidified. After drying, the cells were cultured at 30 ° C. for 16 hours.
- R-2 medium was prepared by separately preparing the following R-2 / A and R-2 / B and combining them.
- the medium containing agar was used as R-2 plate and the medium containing agarose was used as R-2 top agarose medium.
- the mixture was mixed at a rate of 1 ml per 200 ml final volume.
- Yeast extract lO.Og (Saccharose 203gZUpH7.4)
- the enzyme activity of the sample obtained above was measured according to the method described in the examples ( ⁇ Enzyme activity measurement method>).
- the reaction was stopped by adding a 2% perchloric acid solution, and 1001 was weighed out.
- the OD was measured with 5 ml of water. The same measurement was performed with the reaction time set to 0 minutes.
- Fig. 17 shows the measurement results.
- the culture supernatant obtained after culturing Streptomyces lividans TK24 under the same conditions was used as a control sample.
- thermostability of AMP polymerase produced by the transformant (SAD-1) was measured. After treating the culture supernatant of SAD-1 prepared by the above-mentioned method at a predetermined temperature, the residual AMP kinase activity was measured (pH 5.6).
- the processing temperatures were 30 ° C, 40 ° C, 50 ° C, 60 ° C, 65 ° C, and 70 ° C. C and 75 ° C.
- the processing time was 30 minutes.
- the deaminase derived from the transformant (SAD-1) was similar to the AMP deaminase derived from Streptomyces murinus and was an AMP deaminase. It was also found that the enzyme can be suitably used in reactions using 5 'dAMP, ADP and ATP as substrates.
- FIG. 20 shows the results of SDS-PAGE.
- Lane II is the control (host bacterial culture supernatant).
- Lane III is the culture supernatant of the transformant (SAD-1).
- SAD-1 the control bacterial culture supernatant
- Lane I shows the protein molecular weight marker band.
- the present inventors succeeded in closing the gene encoding AMP deaminase derived from Streptomyces miulinas.
- a transformant into which the gene was introduced was successfully obtained using an actinomycete transformation system, and the expression of the gene was confirmed.
- (1) indicates high production (2) production of a high-productivity transformant by using a highly productive strain as a host and construction of a high-production system using the same, (3) nucleotide sequence By modifying the amino acid sequence, productivity, stability, and / or substrate specificity can be improved.
- the AMP polymerase of the present invention is excellent in thermal stability. Therefore, it is suitably used in applications where a reaction at a high temperature is desired.
- the AMP deaminase of the present invention can be used as an enzyme for enhancing umami in the production of yeast extract or as an enzyme for producing 5'-inosinic acid as a seasoning.
- the AMP protease of the present invention can be used in various reactions starting from substrates such as ADP, ⁇ , and 5 'dAMP. Is also possible.
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CN2005800137893A CN1950501B (zh) | 2004-04-28 | 2005-04-26 | 放线菌来源的amp脱氨酶及其应用 |
US11/587,947 US7709240B2 (en) | 2004-04-28 | 2005-04-26 | AMP deaminase originating streptomyces and utilization thereof |
EP05737266.6A EP1760148B1 (en) | 2004-04-28 | 2005-04-26 | Amp deaminase originating in streptomyces and utilization thereof |
JP2006512784A JP4663631B2 (ja) | 2004-04-28 | 2005-04-26 | 放線菌由来のampデアミナーゼ及びその利用 |
DK05737266.6T DK1760148T3 (da) | 2004-04-28 | 2005-04-26 | Amp-deaminase, som stammer fra streptomyces og brugen deraf |
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EP (1) | EP1760148B1 (ja) |
JP (1) | JP4663631B2 (ja) |
CN (1) | CN1950501B (ja) |
DK (1) | DK1760148T3 (ja) |
WO (1) | WO2005105991A1 (ja) |
Cited By (2)
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WO2021066130A1 (ja) | 2019-10-03 | 2021-04-08 | 天野エンザイム株式会社 | 酵母エキスの製造方法 |
CN116731874A (zh) * | 2023-05-29 | 2023-09-12 | 天典(广东)生物科技有限公司 | 一株蜂蜜曲霉adm01及其应用 |
Families Citing this family (6)
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CN103805629B (zh) * | 2014-01-28 | 2016-10-05 | 江南大学 | 鼠灰链霉菌amp脱氨酶基因的毕赤酵母真核表达方法 |
CN103773792A (zh) * | 2014-01-28 | 2014-05-07 | 江南大学 | 鼠灰链霉菌amp脱氨酶基因的原核表达方法及其表达产物的应用 |
CN103757035B (zh) * | 2014-01-28 | 2016-09-28 | 江南大学 | 鼠灰链霉菌amp脱氨酶基因的乳酸克鲁维酵母真核表达方法 |
CN106282145B (zh) * | 2015-06-05 | 2019-08-27 | 安琪酵母股份有限公司 | 一种腺苷酸脱氨酶的液态发酵方法 |
CN106282146B (zh) * | 2015-06-05 | 2019-08-27 | 安琪酵母股份有限公司 | 一种腺苷酸脱氨酶的固态发酵方法 |
IL268857B2 (en) | 2017-02-27 | 2024-09-01 | Translate Bio Inc | Methods for the purification of messenger RNA |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5022115B1 (ja) * | 1963-11-11 | 1975-07-28 | ||
JPS55120788A (en) * | 1979-03-12 | 1980-09-17 | Toyobo Co Ltd | Adenosine monophosphate deaminase and its preparation |
JPH04131084A (ja) * | 1989-06-09 | 1992-05-01 | Oncogen Lp | 熱安定性シトシンデアミナーゼ |
JPH0670716A (ja) * | 1992-06-08 | 1994-03-15 | Nippon Paper Ind Co Ltd | 酵母エキス組成物及びその製造法 |
JPH06113789A (ja) * | 1992-10-05 | 1994-04-26 | Nippon Paper Ind Co Ltd | 呈味性ヌクレオチド高含有酵母エキス及びその製造法 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5338678A (en) * | 1989-06-09 | 1994-08-16 | Oncogen, A Limited Partnership | Expression of DNA sequences encoding a thermally stable cytosine deaminase from saccharomyces |
US6392126B1 (en) * | 1999-02-25 | 2002-05-21 | Pioneer Hi-Bred International, Inc. | Adenosine deaminase homologues and uses thereof |
JP2003506036A (ja) | 1999-07-30 | 2003-02-18 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | 植物中のプリン代謝遺伝子 |
CN1379106A (zh) * | 2002-02-01 | 2002-11-13 | 杭州华大基因研发中心 | 耐高温胞嘧啶脱氨酶基因及其编码的多肽和制备方法 |
-
2005
- 2005-04-26 CN CN2005800137893A patent/CN1950501B/zh active Active
- 2005-04-26 WO PCT/JP2005/007892 patent/WO2005105991A1/ja active Application Filing
- 2005-04-26 DK DK05737266.6T patent/DK1760148T3/da active
- 2005-04-26 JP JP2006512784A patent/JP4663631B2/ja active Active
- 2005-04-26 EP EP05737266.6A patent/EP1760148B1/en active Active
- 2005-04-26 US US11/587,947 patent/US7709240B2/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5022115B1 (ja) * | 1963-11-11 | 1975-07-28 | ||
JPS55120788A (en) * | 1979-03-12 | 1980-09-17 | Toyobo Co Ltd | Adenosine monophosphate deaminase and its preparation |
JPH04131084A (ja) * | 1989-06-09 | 1992-05-01 | Oncogen Lp | 熱安定性シトシンデアミナーゼ |
JPH0670716A (ja) * | 1992-06-08 | 1994-03-15 | Nippon Paper Ind Co Ltd | 酵母エキス組成物及びその製造法 |
JPH06113789A (ja) * | 1992-10-05 | 1994-04-26 | Nippon Paper Ind Co Ltd | 呈味性ヌクレオチド高含有酵母エキス及びその製造法 |
Non-Patent Citations (4)
Title |
---|
BANDLINSH R.K. ET AL: "Glucose-to-fructose conversion at high temperatures with xylose (glucose) isomerases from Streptomyces murinus and two hyperthermophilic Thermotoga species.", BIOTECHNOL BIOENG., vol. 80, no. 2, October 2002 (2002-10-01), pages 185 - 194, XP002988954 * |
CHUNG S.T. ET AL: "A relationship between deamination and dechlorination activities of ATP Deaminase, ADP-deaminating enzyme and AMP Deaminase.", J GEN APPL MICROBIOL., vol. 14, 1986, pages 111 - 119, XP002987384 * |
MARGOLIN A.L. ET AL: "AMP Deaminase as a Novel Practical Catalyst in the Synthesis of 6-Oxopurine Ribosides and Their Analogs.", J.ORG.CHEM., vol. 59, no. 24, 1994, pages 7214 - 7218, XP002988972 * |
See also references of EP1760148A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021066130A1 (ja) | 2019-10-03 | 2021-04-08 | 天野エンザイム株式会社 | 酵母エキスの製造方法 |
EP4039107A4 (en) * | 2019-10-03 | 2024-02-14 | Amano Enzyme Inc. | METHOD FOR PRODUCING A YEAST EXTRACT |
CN116731874A (zh) * | 2023-05-29 | 2023-09-12 | 天典(广东)生物科技有限公司 | 一株蜂蜜曲霉adm01及其应用 |
CN116731874B (zh) * | 2023-05-29 | 2023-12-05 | 天典(广东)生物科技有限公司 | 一株蜂蜜曲霉adm01及其应用 |
Also Published As
Publication number | Publication date |
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EP1760148B1 (en) | 2013-06-26 |
US7709240B2 (en) | 2010-05-04 |
JP4663631B2 (ja) | 2011-04-06 |
US20080248524A1 (en) | 2008-10-09 |
CN1950501B (zh) | 2012-06-13 |
DK1760148T3 (da) | 2013-09-02 |
CN1950501A (zh) | 2007-04-18 |
JPWO2005105991A1 (ja) | 2008-07-31 |
EP1760148A1 (en) | 2007-03-07 |
EP1760148A4 (en) | 2008-12-24 |
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