WO2009136471A1 - β-アミラーゼ、それをコードする遺伝子及びその製造法 - Google Patents
β-アミラーゼ、それをコードする遺伝子及びその製造法 Download PDFInfo
- Publication number
- WO2009136471A1 WO2009136471A1 PCT/JP2009/001807 JP2009001807W WO2009136471A1 WO 2009136471 A1 WO2009136471 A1 WO 2009136471A1 JP 2009001807 W JP2009001807 W JP 2009001807W WO 2009136471 A1 WO2009136471 A1 WO 2009136471A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- amylase
- amino acid
- acid sequence
- gene
- dna
- Prior art date
Links
Images
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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2425—Beta-amylase (3.2.1.2)
-
- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D8/00—Methods for preparing or baking dough
- A21D8/02—Methods for preparing dough; Treating dough prior to baking
- A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
- A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
-
- 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
- C12P19/22—Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01002—Beta-amylase (3.2.1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Definitions
- the present invention relates to a novel ⁇ -amylase. Specifically, it relates to a microorganism-derived ⁇ -amylase, its gene, its production method and the like.
- ⁇ -amylase has been known to originate from plants such as soybean, wheat, barley, malt, sweet potato, and potato.
- ⁇ -amylase extracted and purified from cereals such as soybeans, wheat, barley and malt is widely used industrially for producing maltose-containing syrup used in the sugar industry, bread industry and brewing industry.
- plant-derived ⁇ -amylases those derived from soybean have high enzyme activity and excellent heat resistance.
- the price of corn has shifted due to the expansion of demand for bioethanol.
- planting has shifted from soybeans and wheat to corn. For this reason, soybeans, wheat, barley, and the like are in short supply and prices are rising, making it difficult to secure raw materials for ⁇ -amylase.
- ⁇ -Amylase is an enzyme that acts on polysaccharides having ⁇ -1,4 bonds of glucose as the main chain, such as starch and glycogen, and decomposes in maltose units from the non-reducing end.
- ⁇ -amylase has long been known to exist in higher plants such as soybeans and wheat. In 1972, since it was announced that an enzyme having the same mechanism of action as that of higher plant ⁇ -amylase was also present in microorganisms, many microorganisms have been found as ⁇ -amylase-producing bacteria (Non-patent Document 1). ).
- Streptomyces sp., Pseudomonas sp., Etc. have been reported as bacteria producing ⁇ -amylase. However, many of them are low in productivity and few have been put into practical use.
- amylase produced by filamentous fungi such as Aspergillus decomposes amylose and amylopectin in endo form. Therefore, when the amylase is used, glucose, maltotriose and other oligosaccharides are produced in addition to maltose. In addition, the amylase has low heat resistance and is not practical for maltose production.
- Bacillus stearothermophilus produces a maltose-producing enzyme with high heat resistance (Patent Document 1, Non-Patent Document 2).
- This enzyme produces maltose in the exo form from the non-reducing end of starch, but the maltose produced is alpha form. In addition, this enzyme does not strictly hydrolyze in maltose units like ⁇ -amylase of plant origin.
- maltotetraose G4
- maltotriose G3
- maltose G2
- G5 maltopentaose
- G6 maltohexaose
- the present inventors have made extensive studies in view of the above problems. As a result, it was found that Bacillus flexus, a Bacillus subtilis, produces ⁇ -amylase having heat resistance comparable to that of soybean-derived ⁇ -amylase. In addition, the present inventors have succeeded in isolating and purifying the ⁇ -amylase and determining its enzymatic chemistry. Furthermore, the base sequence of the gene encoding the ⁇ -amylase was also successfully determined. In addition, it was confirmed that ⁇ -amylase can be produced using a transformant introduced with a vector containing the gene. The present invention has been completed based on the above-described results, and is as follows. [1] ⁇ -amylase derived from Bacillus flexus.
- ⁇ -amylase having the following enzymatic chemistry, (1) Action: acts on ⁇ -1,4 glucoside bond of polysaccharides and oligosaccharides to release maltose, (2) Substrate specificity: Acts well on starch, amylose, amylopectin, glycogen, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and does not act on pullulan, dextran, cyclodextrin, maltotriose , (3) Optimal temperature: about 55 ° C (4) Optimal pH: about 8.0, (5) Temperature stability: stable at 55 ° C or lower (pH 5.0, 10 minutes), (6) pH stability: stable at pH 4-9 (30 ° C., 3 hours), (7) Molecular weight: about 60,000 (SDS-PAGE).
- An enzyme agent comprising the ⁇ -amylase according to any one of [1] to [4] as an active ingredient.
- ⁇ -amylase gene comprising any DNA selected from the group consisting of the following (A) to (C): (A) DNA encoding the amino acid sequence shown in SEQ ID NO: 7; (B) DNA consisting of the base sequence shown in SEQ ID NO: 6; (C) DNA encoding a protein having a base sequence equivalent to the base sequence shown in SEQ ID NO: 6 and having ⁇ -amylase activity. [7] A recombinant vector containing the ⁇ -amylase gene according to [6]. [8] A transformant into which the ⁇ -amylase gene according to [6] has been introduced.
- a method for producing ⁇ -amylase comprising the following steps (1) and (2) or steps (i) and (ii): (1) culturing Bacillus flexus having ⁇ -amylase producing ability; (2) a step of recovering ⁇ -amylase from the cultured medium and / or cells after the culture; (I) culturing the transformant according to [8] under conditions for producing a protein encoded by the gene; (Ii) recovering the produced protein.
- the production method according to [9] wherein Bacillus flexus is a strain specified by the accession number NITE BP-548.
- [12] A method for producing maltose, wherein a ⁇ -amylase derived from Bacillus flexus is allowed to act on a polysaccharide or oligosaccharide having an ⁇ -1,4 bond of glucose as a main chain. [13] The production method according to [12], wherein the ⁇ -amylase is the ⁇ -amylase according to any one of [2] to [4].
- 3 is a graph showing the optimum temperature of ⁇ -amylase derived from Bacillus flexus.
- 2 is a graph showing the optimum pH of ⁇ -amylase derived from Bacillus flexus.
- ⁇ Citrate buffer pH 2, 3, 4,
- ⁇ Britton-Robinson buffer pH 4, 5, 6, 7, 8, 9, 10, 11 3 is a graph showing the temperature stability of ⁇ -amylase derived from Bacillus flexus.
- 2 is a graph showing the pH stability of ⁇ -amylase derived from Bacillus flexus.
- ⁇ Citrate buffer pH 2, 3, 4, ⁇ : Britton-Robinson buffer pH 4, 5, 6, 7, 8, 9, 10, 11 It is a figure which shows the result of SDS-PAGE of purified ⁇ -amylase and a sample in the middle of purification.
- Lane 1 Ammonium sulfate fraction
- Lane 2 HiPrepButyl 16/10 FF
- Lane 3 HiTrap CM FF
- Lane 4 HiLoad 16/60 Superdex200 Structure of expression plasmid pET-BAF.
- DNA encoding a protein refers to DNA from which the protein is obtained when expressed, that is, DNA having a base sequence corresponding to the amino acid sequence of the protein. Therefore, codon degeneracy is also considered.
- isolated is used interchangeably with “purified”. “Isolated” when used in reference to the enzyme of the present invention ( ⁇ -amylase) means that when the enzyme of the present invention is derived from a natural material, components other than the enzyme are substantially contained in the natural material. The state which does not contain (especially does not contain a contaminating protein substantially).
- the content of contaminating protein is less than about 20%, preferably less than about 10%, more preferably less than about 5%, even more preferably in terms of weight. Is less than about 1%.
- the term “isolated” in the case where the enzyme of the present invention is prepared by a genetic engineering technique substantially includes other components derived from the used host cell, culture medium, and the like. It means no state.
- the content of contaminant components is less than about 20%, preferably less than about 10%, more preferably less than about 5%, even more preferably in terms of weight. Less than about 1%.
- ⁇ -amylase in the present specification, it means “an isolated ⁇ -amylase”.
- present enzyme used in place of ⁇ -amylase.
- isolated when used with respect to DNA means that, in the case of naturally occurring DNA, it is typically separated from other nucleic acids that coexist in the natural state. However, some other nucleic acid components such as a nucleic acid sequence adjacent in the natural state (for example, a promoter region sequence and a terminator sequence) may be included.
- an “isolated” state in the case of genomic DNA is preferably substantially free of other DNA components that coexist in the natural state.
- the “isolated” state in the case of DNA prepared by genetic engineering techniques such as cDNA molecules is preferably substantially free of cell components, culture medium, and the like.
- the “isolated” state in the case of DNA prepared by chemical synthesis is preferably substantially free of precursors (raw materials) such as dNTPs, chemical substances used in the synthesis process, and the like.
- precursors raw materials
- dNTPs chemical substances used in the synthesis process
- DNA DNA in an isolated state.
- the first aspect of the present invention provides ⁇ -amylase (hereinafter also referred to as “the present enzyme”) and its producing bacteria.
- the present enzyme ⁇ -amylase
- Bacillus flexus produces thermostable ⁇ -amylase.
- the present inventors succeeded in separating and producing the ⁇ -amylase, and succeeded in determining the enzyme chemical properties as shown below.
- This enzyme is ⁇ -amylase, which acts on ⁇ -1,4 glucoside bonds of polysaccharides and oligosaccharides to release maltose. Little glucose is released.
- the enzyme is excellent in substrate specificity and acts well on starch, amylose, amylopectin, glycogen, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. In contrast, it does not act on pullulan, dextran, cyclodextrin, or maltotriose. If the relative activity is 50% or more when the activity when soluble starch is used as a substrate (100%) as a reference (100%), it is judged that the enzyme is a substrate that acts well. Similarly, if the relative activity is less than 10%, it is judged as “a substrate to which this enzyme does not act”.
- the enzyme has no substantial effect on maltotriose and cyclodextrins ( ⁇ , ⁇ , or ⁇ ).
- the reactivity and substrate specificity of this enzyme can be measured and evaluated by the method shown in the Examples described later (column for measuring ⁇ -amylase activity).
- the optimal temperature of this enzyme is about 55 ° C. This enzyme exhibits high activity at about 50 ° C to about 60 ° C.
- the optimum temperature is a value calculated by measurement according to the ⁇ -amylase activity measurement method described later (in 0.1 M phosphate-hydrochloric acid buffer (pH 5.0)).
- the optimum pH of this enzyme is about 8.0. This enzyme exhibits high activity at a pH of about 6.0 to about 9.0. The optimum pH is determined based on, for example, the results of measurement in a citrate buffer for the pH range of pH 2-4 and in Britton-Robinson buffer for the pH 4-11.
- This enzyme exhibits excellent heat resistance comparable to soybean-derived ⁇ -amylase.
- the enzyme maintains an activity of 90% or more even when treated for 10 minutes in a 0.1 M acetic acid-hydrochloric acid buffer (pH 5.0) containing 10 mM calcium acetate at 55 ° C.
- pH stability This enzyme exhibits stable activity in a wide pH range of pH 4-9. That is, when the pH of the enzyme solution to be treated is within this range, it shows 70% or more of the maximum activity after 3 hours of treatment at 30 ° C.
- the optimum pH is determined based on, for example, the results of measurement in a citrate buffer for the pH range of pH 2-4 and in Britton-Robinson buffer for the pH 4-11.
- the molecular weight of this enzyme is about 60,000 (by SDS-PAGE).
- the enzyme is preferably ⁇ -amylase derived from Bacillus flexus.
- ⁇ -amylase derived from Bacillus flexus as used herein means ⁇ -amylase produced by a microorganism (either wild or mutant) classified as Bacillus flexus, or Bacillus flexus It means a ⁇ -amylase obtained by genetic engineering techniques using the ⁇ -amylase gene of flexus (which may be a wild strain or a mutant strain).
- a recombinant produced by a host microorganism into which a ⁇ -amylase gene obtained from Bacillus flexus (or a gene obtained by modifying the gene) is introduced also corresponds to “ ⁇ -amylase derived from Bacillus flexus”.
- the Bacillus flexus from which the present enzyme is derived is referred to as the producing microorganism of the present enzyme for convenience of explanation.
- bacteria producing this enzyme include Bacillus flexus DSM1316 (DSMZ, Germany), DSM1320 (DSMZ, Germany), DSM1667 (DSMZ, Germany), and APC9451 shown in the examples described later.
- the APC9451 strain is deposited with the prescribed depository as follows and is easily available. Depositary institution: NITE Biotechnology Headquarters Patent Microorganism Deposit Center (2-5-8 Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan 292-0818) Deposit date (Receipt date): April 9, 2008 Deposit number: NITE BP-548
- this enzyme As described above, the details of the properties of the enzyme that was successfully obtained were clarified. As a result, it was found that this enzyme has excellent heat resistance and substrate specificity. Therefore, this enzyme is suitable for food processing and saccharification applications.
- one embodiment of the present invention is characterized by comprising a protein having the amino acid sequence of SEQ ID NO: 7.
- the modified protein may have a function equivalent to that of the protein before modification. That is, the modification of the amino acid sequence does not substantially affect the function of the protein, and the function of the protein may be maintained before and after the modification.
- the present invention provides, as another aspect, a protein having an amino acid sequence equivalent to the amino acid sequence shown in SEQ ID NO: 7 and having ⁇ -amylase activity (hereinafter also referred to as “equivalent protein”).
- the “equivalent amino acid sequence” here is partly different from the amino acid sequence shown in SEQ ID NO: 7, but the difference does not substantially affect the function of the protein (here, ⁇ -amylase activity). It refers to the amino acid sequence.
- “Has ⁇ -amylase activity” means that it acts on polysaccharides and oligosaccharides such as starch and glycogen that have ⁇ -1,4 bonds of glucose as the main chain and decomposes in maltose units from the non-reducing end
- the degree of activity is not particularly limited as long as it can function as ⁇ -amylase. However, it is preferably the same as or higher than the protein consisting of the amino acid sequence shown in SEQ ID NO: 7.
- “Partial difference in amino acid sequence” typically means deletion, substitution, or addition, insertion of one to several amino acids, or a combination thereof. This means that a mutation (change) has occurred in the amino acid sequence.
- the difference in amino acid sequence here is permissible as long as ⁇ -amylase activity is maintained (there may be some variation in activity).
- the positions where the amino acid sequences are different are not particularly limited, and differences may occur at a plurality of positions.
- the term “plurality” as used herein 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%.
- the number is preferably less than about 5%, and most preferably less than about 1%. That is, the equivalent protein has an amino acid sequence of SEQ ID NO: 7 of, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, even more preferably about 95% or more, and most preferably about 99% or more. Have identity.
- an equivalent protein is obtained by causing a conservative amino acid substitution at an amino acid residue that is not essential for ⁇ -amylase activity.
- conservative amino acid substitution refers to substitution of a certain amino acid residue with an amino acid residue having a side chain having the same properties.
- a basic side chain eg lysine, arginine, histidine
- an acidic side chain eg aspartic acid, glutamic acid
- an uncharged polar side chain eg glycine, asparagine, glutamine, serine, threonine, tyrosine
- Cysteine eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
- ⁇ -branched side chains eg threonine, valine, isoleucine
- aromatic side chains eg tyrosine, phenylalanine, Like tryptophan and histidine.
- a conservative amino acid substitution is preferably a substitution between amino acid residues within the same family.
- “Equivalent proteins” may have additional properties. Such properties include, for example, the property of being superior in stability compared to the protein consisting of the amino acid sequence shown in SEQ ID NO: 7, the property of exhibiting different functions only at low temperatures and / or high temperatures, and the properties of different optimum pH. Etc.
- two sequences can be determined, for example, by the following procedure.
- two sequences are aligned for optimal comparison (eg, a gap may be introduced into the first sequence to optimize alignment with the second sequence).
- a gap may be introduced into the first sequence to optimize alignment with the second sequence.
- the Gapped BLAST described in Altschul et al.
- the enzyme may be part of a larger protein (eg, a fusion protein).
- a larger protein eg, a fusion protein
- sequences added in the fusion protein include sequences useful for purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.
- the present enzyme having the above amino acid sequence can be easily prepared by a genetic engineering technique. For example, it can be prepared by transforming a suitable host cell (for example, E. coli) with DNA encoding the present enzyme and recovering the protein expressed in the transformant. The recovered protein is appropriately purified according to the purpose. Thus, if this enzyme is obtained as a recombinant protein, various modifications are possible. For example, if a DNA encoding this enzyme and another appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, the peptide consists of a recombinant protein linked to any peptide or protein. This enzyme can be obtained.
- a suitable host cell for example, E. coli
- modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur.
- modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible.
- the second aspect of the present invention provides a gene encoding this enzyme, that is, a novel ⁇ -amylase gene.
- the gene of the present invention consists of DNA encoding the amino acid sequence of SEQ ID NO: 7.
- a specific example of this embodiment is DNA consisting of the base sequence shown in SEQ ID NO: 6.
- the protein encoded by the modified DNA may have the same function as the protein encoded by the DNA before modification. That is, the modification of the DNA sequence does not substantially affect the function of the encoded protein, and the function of the encoded protein may be maintained before and after the modification.
- the present invention provides a DNA (hereinafter also referred to as “equivalent DNA”) that encodes a protein having a base sequence equivalent to the base sequence shown in SEQ ID NO: 6 and having ⁇ -amylase activity.
- the “equivalent base sequence” here is partially different from the nucleic acid shown in SEQ ID NO: 6, but the function of the protein encoded by the difference (in this case ⁇ -amylase activity) has a substantial influence.
- a specific example of equivalent DNA is DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 6.
- the “stringent conditions” here are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
- Such stringent conditions are known to those skilled in the art, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987) Can be set with reference to.
- hybridization solution 50% formamide, 10 ⁇ SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 5 ⁇ Denhardt solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / ml denaturation
- 5 ⁇ Denhardt solution 1% SDS
- 10% dextran sulfate 10 ⁇ g / ml denaturation
- incubation at about 42 ° C to about 50 ° C using salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), followed by washing at about 65 ° C to about 70 ° C using 0.1 x SSC, 0.1% SDS can be mentioned.
- Further preferable stringent conditions include, for example, 50% formamide, 5 ⁇ SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1 ⁇ Denhardt solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / ml as a hybridization solution. Of denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).
- equivalent DNA it consists of a base sequence including substitution, deletion, insertion, addition or inversion of one or more bases based on the base sequence shown in SEQ ID NO: 6, and has ⁇ -amylase activity Mention may be made of DNA encoding proteins. Base substitution or deletion may occur at a plurality of sites.
- the term “plurality” as used herein refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases, although it depends on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the DNA It is.
- Such equivalent DNAs include, for example, restriction enzyme treatment, treatment with exonuclease and DNA ligase, position-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) Including mutation, introduction, mutation, and / or inversion using mutation introduction methods (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) Thus, it can obtain by modifying DNA which has a base sequence shown to sequence number 6.
- the equivalent DNA can also be obtained by other methods such as ultraviolet irradiation.
- Still another example of equivalent DNA is DNA in which a base difference as described above is recognized due to a polymorphism represented by SNP (single nucleotide polymorphism).
- the gene of the present invention was isolated by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. with reference to the sequence information disclosed in this specification or the attached sequence listing. Can be prepared in a state. Specifically, an oligonucleotide probe capable of specifically hybridizing to the gene of the present invention from an appropriate Bacillus flexus genomic DNA library or cDNA library, or an intracellular extract of Bacillus flexus. A primer can be used as appropriate. Oligonucleotide probes and primers can be easily synthesized using a commercially available automated DNA synthesizer. For the method of preparing a library used for preparing the gene of the present invention, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York can be referred to.
- a gene having the base sequence shown in SEQ ID NO: 6 can be isolated using a hybridization method using the whole base sequence or its complementary sequence as a probe. Further, it can be amplified and isolated 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. Further, based on the amino acid sequence shown in SEQ ID NO: 7 and the base sequence information shown in SEQ ID NO: 6, the target gene can also be obtained by chemical synthesis (Reference: Gene, 60 (1), 115). -127 (1987)).
- this enzyme ( ⁇ -amylase) is isolated and purified from Bacillus flexus, and information on the partial amino acid sequence is obtained.
- ⁇ -amylase As a method for determining a partial amino acid sequence, for example, purified ⁇ -amylase is directly analyzed according to Edman degradation method (Journal of Biological Chemistry, Vol. 256, pages 7990-7997 (1981)) according to an ordinary method [protein- Sequencer 476A, Applied Biosystems, etc.). It is effective to carry out limited hydrolysis with the action of a protein hydrolase, separate and purify the obtained peptide fragment, and perform amino acid sequence analysis on the obtained purified peptide fragment.
- the ⁇ -amylase gene is cloned based on the partial amino acid sequence information thus obtained.
- cloning can be performed using a hybridization method or PCR.
- the hybridization method for example, a method described in Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) can be used.
- a genomic DNA of a microorganism producing ⁇ -amylase is used as a template, and a PCR reaction is performed using a synthetic oligonucleotide primer designed on the basis of partial amino acid sequence information to obtain a target gene fragment.
- the PCR method is performed according to the method described in PCR technology [PCR Technology, edited by Erlich HA, published by Stocktonpress, 1989].
- the base sequence is determined by a method usually used for this amplified DNA fragment, for example, the dideoxy chain terminator method
- the sequence corresponding to the partial amino acid sequence of ⁇ -amylase in addition to the sequence of the synthetic oligonucleotide primer in the determined sequence And a part of the target ⁇ -amylase gene can be obtained.
- the gene encoding the full-length ⁇ -amylase can be cloned by further performing a hybridization method or the like using the obtained gene fragment as a probe.
- SEQ ID NO: 6 shows the entire base sequence of the gene encoding ⁇ -amylase derived from Bacillus flexus.
- amino acid sequence encoded by the base sequence was determined (SEQ ID NO: 7). There are a plurality of base sequences corresponding to the amino acid sequence shown in SEQ ID NO: 7 other than those shown in SEQ ID NO: 6.
- SEQ ID NO: 6 a DNA having high homology with the ⁇ -amylase gene of SEQ ID NO: 6 can be selected.
- PCR primers can be designed. By performing a PCR reaction using this primer, a gene fragment having high homology with the ⁇ -amylase gene can be detected, and further the entire gene can be obtained.
- a modified ⁇ -amylase (deletion, addition, insertion or substitution of one or more amino acid residues is introduced by introducing a random mutation or a site-specific mutation. Gene).
- the plan for introducing a mutation is carried out, for example, taking into account the characteristic sequence on the gene sequence.
- Reference to a characteristic sequence can be performed, for example, by considering the three-dimensional structure of the protein and considering homology with known proteins.
- a method for introducing random mutations as a method for chemically treating DNA, a method in which sodium bisulfite is reacted to cause a transition mutation that converts a cytosine base into a uracil base [Proceedings of the National Academy of Sciences] Seeds of the USA, Vol. 79, pp. 1408-1412 (1982)], as a biochemical method, a method of generating base substitution in the process of synthesizing a double strand in the presence of [ ⁇ -S] dNTP [Gene (Gene), Vol. 64, pp.
- Examples of methods for introducing site-specific mutations include a method utilizing amber mutation (gapped duplex method, Nucleic Acids Research, Vol. 12, No. 24, pages 9441-9456 ( 1984)], a method using the recognition site of a restriction enzyme [Analytical Biochemistry, 200, 81-88 (1992), Gene, 102, 67-70 (1991)], dut ( dUTPase) and ung (uracil DNA-glycosylase) mutation method (Kunkel method, Proceedings of the National of Sciences of the USA, Vol. 82, pp.
- a site-specific mutation can be easily introduced by using a commercially available kit.
- a commercially available kit for example, Mutan (registered trademark) -G (manufactured by Takara Shuzo Co., Ltd.) using the gapped duplex method, Mutan (registered trademark) -K (manufactured by Takara Shuzo Co., Ltd.) using the Kunkel method, ODA method was used.
- Mutan (registered trademark) -ExpressKm (manufactured by Takara Shuzo), QuikChangeTM Site-Directed Mutagenesis Kit (made by STRATAGENE) using a primer for mutagenesis and ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ DNA polymerase derived from Pyrococcus furiosus, etc.
- a kit using the PCR method TaKaRa LA PCR in vitro Mutagenesis Kit (manufactured by Takara Shuzo), Mutan (registered trademark) -Super Express Km (manufactured by Takara Shuzo) or the like can be used.
- a further aspect of the present invention relates to a recombinant vector containing the gene of the present invention.
- the term “vector” refers to a nucleic acid molecule capable of transporting a nucleic acid molecule inserted thereinto into a target such as a cell, and the type and form thereof are not particularly limited. Accordingly, the vector of the present invention can take the form of a plasmid vector, a cosmid vector, a phage vector, or a viral vector (an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a herpes virus vector, etc.).
- An appropriate vector is selected depending on the purpose of use (cloning, protein expression) and in consideration of the type of host cell.
- Specific examples of vectors include vectors using E. coli as a host (M13 phage or a modified product thereof, ⁇ phage or a modified product thereof, pBR322 or a modified product thereof (pB325, pAT153, pUC8, etc.)), and yeast as a host.
- Vectors pYepSec1, pMFa, pYES2, etc.
- vectors using insect cells as hosts pAc, pVL, etc.
- vectors using mammalian cells as hosts pCDM8, pMT2PC, etc.
- the recombinant vector of the present invention is preferably an expression vector.
- “Expression vector” refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and allowing expression in the cell.
- Expression vectors usually contain a promoter sequence necessary for expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like.
- An expression vector containing a selectable marker can also be used. When such an expression vector is used, the presence or absence of the expression vector (and the degree thereof) can be confirmed using a selection marker.
- Insertion of the gene 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, Molecular Cloning, Third Edition, 1.84, Cold). A known method using a restriction enzyme and DNA ligase, which can be referred to Spring Harbor Laboratory Press, New York.
- the present invention further relates to a transformant into which the gene of the present invention has been introduced.
- the gene of the present invention exists as an exogenous molecule.
- the transformant of the present invention is preferably prepared by transfection or transformation using the vector of the present invention.
- transfection and transformation calcium phosphate coprecipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al. , Proc. Natl. Acad. Sci. USA 84,7413-7417 (1984)), microinjection (Graessmann, M.
- microorganism examples include bacteria such as Escherichia coli, Bacillus genus, Streptomyces genus and Lactococcus genus, yeasts such as Saccharomyces genus, Pichia genus and Kluyveromyces genus, and filamentous fungi such as Aspergillus genus, Penicillium genus and Trichoderma genus.
- Animal cells include baculovirus strains.
- a further aspect of the present invention provides a method for producing ⁇ -amylase.
- the step of culturing Bacillus flexus having the ability to produce the present enzyme ( ⁇ -amylase) (step (1)) and the culture solution and / or the cell body after culturing, ⁇ - A step of recovering amylase (step (2)) is performed.
- the Bacillus flexus in step (1) for example, the above Bacillus flexus DSM1316, DSM1320, DSM1667, APC9451 and the like can be used.
- the culture method and culture conditions are not particularly limited as long as the target enzyme is produced.
- the culture method may be either liquid culture or solid culture, but preferably liquid culture is used. Taking liquid culture as an example, the culture conditions will be described.
- any medium can be used as long as the microorganism to be used can grow.
- carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein
- Nitrogen sources such as hydrolysates, bran and meat extracts, and further added with inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts and zinc salts can be used.
- vitamins, amino acids and the like may be added to the medium.
- the pH of the medium is adjusted to, for example, about 3 to 10, preferably about 7 to 8, and the culture temperature is usually about 10 to 50 ° C., preferably about 20 to 37 ° C. for 1 to 7 days, preferably 3 to Incubate under aerobic conditions for about 4 days.
- the culture method for example, a shaking culture method or an aerobic deep culture method using a jar fermenter can be used.
- ⁇ -amylase is recovered from the culture broth or cells (step (2)).
- the culture supernatant is filtered, centrifuged, etc. to remove insolubles, concentrated by ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, ion exchange resin, etc.
- the present enzyme can be obtained by performing separation and purification by appropriately combining various types of chromatography.
- the microbial cells are crushed by pressure treatment, ultrasonic treatment, etc., and then separated and purified in the same manner as described above to obtain the enzyme.
- ⁇ -amylase is produced using the above transformant.
- the above-mentioned transformant is cultured under the condition that a protein encoded by the gene introduced therein is produced (step (i)).
- Culture conditions for transformants are known for various vector host systems, and those skilled in the art can easily set appropriate culture conditions.
- the produced protein ie, ⁇ -amylase
- recovery and subsequent purification may be performed in the same manner as in the above embodiment.
- the degree of purification of this enzyme is not particularly limited.
- the final form may be liquid or solid (including powder).
- the enzyme of the present invention is provided, for example, in the form of an enzyme agent.
- the enzyme agent may contain excipients, buffers, suspension agents, stabilizers, preservatives, preservatives, physiological saline and the like in addition to the active ingredient (enzyme of the present invention).
- excipient lactose, sorbitol, D-mannitol, sucrose and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer.
- As the stabilizer propylene glycol, ascorbic acid or the like can be used.
- phenol benzalkonium chloride
- benzyl alcohol chlorobutanol
- methylparaben and the like
- benzalkonium chloride paraoxybenzoic acid, chlorobutanol and the like can be used.
- a further aspect of the present invention provides a method for producing maltose as a use of ⁇ -amylase derived from Bacillus flexus.
- ⁇ -amylase derived from Bacillus flexus is allowed to act on a substrate comprising a polysaccharide or oligosaccharide having ⁇ -1,4 bonds of glucose as the main chain.
- the substrate include soluble starch, potato starch, corn starch, amylopectin, glycogen, and maltooligosaccharide.
- the purity of the substrate is not particularly limited. Therefore, ⁇ -amylase may be allowed to act on a substrate mixed with other substances.
- ⁇ -amylase may be allowed to act on two or more substrates simultaneously.
- the production method of the present invention is characterized by using ⁇ -amylase derived from Bacillus flexus.
- the ⁇ -amylase of the present invention (the present enzyme) is used as ⁇ -amylase.
- the production method of the present invention is used, for example, for producing maltose-containing syrup and maltose starch syrup.
- the production method of the present invention can also be used as a means for improving the quality of bread and preventing the aging of rice cakes and strawberries.
- ⁇ Method for measuring ⁇ -amylase activity The activity of ⁇ -amylase was measured as follows. Specifically, 0.5 ml of enzyme solution was added to 0.5 ml of 0.1 M phosphate-hydrochloric acid buffer (pH 5.0) containing 1% soluble starch and 10 mM calcium acetate, and incubated at 37 ° C. for 30 minutes, and then DNS solution (0.2 Stop the reaction by adding 2.5 ml of% DNS, 80 mM NaOH, 0.2 M potassium potassium tartrate tetrahydrate). After stopping the reaction, boil for 5 minutes and measure the absorbance at a wavelength of 530 nm. The amount of enzyme that gives an absorbance of 1 at a wavelength of 530 nm is defined as 1 unit.
- the ⁇ -amylase activity in the obtained culture supernatant was measured by the above ⁇ -amylase activity measurement method. The results are shown in Table 2.
- Bacillus flexus APC9451 was cultured with shaking in a liquid medium having the composition shown in Table 1 at 30 ° C. for 3 days. The obtained culture supernatant was concentrated 4 times with a UF membrane (AIP-0013, manufactured by Asahi Kasei Co., Ltd.), and then ammonium sulfate was added to a 60% saturation concentration. The precipitated fraction was redissolved in 20 mM acetate buffer (pH 5.5), followed by addition of ammonium sulfate to a 20% saturation concentration.
- AIP-0013 manufactured by Asahi Kasei Co., Ltd.
- the resulting precipitate was removed by centrifugation and then applied to a HiPrep Butyl 16/10 FF column (GE Healthcare) equilibrated with 20 mM acetate buffer (pH 5.5) containing 20% saturated ammonium sulfate.
- the adsorbed ⁇ -amylase protein was eluted with a linear ammonium sulfate gradient from% saturation to 0% saturation.
- the collected ⁇ -amylase activity fractions were concentrated on a UF membrane and then applied to a HiTrap CM FF column (GE Healthcare) equilibrated with 20 mM acetate buffer (pH 5.5). The gradient eluted the adsorbed ⁇ -amylase protein.
- ⁇ -amylase activity fractions were concentrated on a UF membrane and then equilibrated with 20 mM acetic acid buffer (pH 5.5) containing 0.15 M NaCl on a HiLoad 16/60 Superdex200 column (manufactured by GE Healthcare). And eluted with the same buffer.
- ⁇ -Amylase active fractions were collected and desalted and concentrated with an ultrafiltration membrane to obtain a purified enzyme preparation.
- the purified enzyme thus obtained was subjected to examination of the following properties, and also subjected to N-terminal amino acid sequence analysis and internal peptide amino acid sequence analysis.
- FIG. 5 shows the results of SDS-PAGE (CBB staining) of the sample at each step in the purification process using a 10-20% gradient gel.
- This purified enzyme preparation (lane 4) is a single protein on SDS-PAGE.
- thermostable ⁇ -amylase (1) Optimum reaction temperature Reaction is performed at 25 ° C, 37 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C and 70 ° C according to the above ⁇ -amylase activity measurement method. I let you. The relative activity was shown with the value at the temperature showing the highest activity as 100%. The optimum reaction temperature was around 55 ° C. (FIG. 1).
- Isoelectric point The isoelectric point of this enzyme was 9.7 as measured by isoelectric point accumulation (600V, 4 ° C, energization for 48 hours) using an ampholine.
- ⁇ PCR reaction solution > 10 ⁇ PCR reaction buffer (TaKaRa) 5.0 ⁇ l dNTP mixture (2.5 mM each, TaKaRa) 4.0 ⁇ l 25 mM MgCl 2 5 ⁇ l 100 ⁇ M sense primer 3.0 ⁇ l 100 ⁇ M antisense primer 3.0 ⁇ l Distilled water 28.5 ⁇ l Chromosomal DNA solution (140 ⁇ g / ml) 1 ⁇ l LA Taq DNA polymerase (TaKaRa) 0.5 ⁇ l
- (F) Determination of base sequence The base sequence of plasmid pUC19-BAF was determined according to a standard method.
- the base sequence (1638 bp) encoding ⁇ -amylase is shown in SEQ ID NO: 6.
- the amino acid sequence (545 amino acid) encoded by SEQ ID NO: 6 is shown in SEQ ID NO: 7.
- SEQ ID NO: 1 the N-terminal region amino acid sequence (SEQ ID NO: 1) and the internal amino acid sequence (SEQ ID NOs: 2 and 3) determined in (b) were found.
- ⁇ -amylase derived from Bacillus flexus in Escherichia coli (a) Construction of expression plasmid in ⁇ -amylase in Escherichia coli Based on the DNA sequences encoding the N-terminal region amino acid sequence and the C-terminal region amino acid sequence, Were synthesized as PCR primers (SEQ ID NOs: 8 and 9). An NdeI restriction enzyme recognition site is added to the sense primer, and an XhoI restriction enzyme recognition site is added to the antisense primer. Using these primers and plasmid pUC19-BAF having ⁇ -amylase gene as a template, PCR reaction was performed under the following conditions.
- ⁇ PCR reaction solution > 10 ⁇ PCR reaction buffer (TOYOBO) 5.0 ⁇ l dNTP mixture (2.5 mM each, TOYOBO) 5.0 ⁇ l 10 ⁇ M sense primer 1.5 ⁇ l 10 ⁇ M antisense primer 1.5 ⁇ l 25 mM MgSO 4 2 ⁇ l Distilled water 33 ⁇ l Plasmid pUC19-BAF solution (83 ⁇ g / ml) 1.0 ⁇ l KOD -Plus- DNA polymerase (TOYOBO) 1.0 ⁇ l
- PCR product After confirming the obtained PCR product by electrophoresis, it was purified by GENE-CLEANE-III (34 ⁇ l), 4 ⁇ l of 10 ⁇ H buffer solution, 1 ⁇ l of NdeI and 1 ⁇ l of XhoI, and enzyme-treated at 37 ° C. for 15 hours.
- the restriction enzyme treatment solution was confirmed by electrophoresis and purified, and then ligated to the vector pET20 (b) (Takara Bio Inc.) previously treated with NdeI and XhoI to obtain the expression plasmid pET-BAF (FIG. 6). It was also confirmed that the base sequence encoding ⁇ -amylase in pET-BAF was correct.
- (B) Expression of ⁇ -amylase in E. coli The expression plasmid pET-BAF was introduced into E. coli BL21 (DE3) (Novagen). From the transformants obtained as ampicillin resistant strains, a strain carrying pET-BAF having the target ⁇ -amylase gene inserted was selected by colony PCR. As a control, a transformant of E. coli BL21 (DE3) having the expression vector pET20 (b) was also obtained. These transformants were cultured in 4 ml of LB medium containing 50 ⁇ g / ml ampicillin at 18 ° C. and 160 rpm for 47 hours to collect the cells.
- the cells were suspended in 0.5 ml of 20 mM acetate buffer (pH 5.5), 0.25 g of ⁇ 0.1 mm glass beads were added, and the cells were crushed with a multi-bead shocker (manufactured by Yasui Machinery Co., Ltd.). As the crushing conditions, ON 30 seconds and OFF 30 seconds were repeated 3.5 cycles. The obtained cell free-extract was subjected to centrifugation to obtain a soluble component.
- a multi-bead shocker manufactured by Yasui Machinery Co., Ltd.
- Table 6 below shows the results obtained by measuring the activity of the obtained samples according to the ⁇ -amylase activity measuring method.
- the ⁇ -amylase of the present invention exhibits heat resistance comparable to that of soybean-derived ⁇ -amylase, and is suitable for applications where a reaction at a high temperature is desired. If the ⁇ -amylase of the present invention is used, the enzyme reaction can be carried out at a high temperature with little risk of contamination. Therefore, the ⁇ -amylase of the present invention is particularly useful for food processing and saccharification.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
ところで、近年、バイオエタノールの需要拡大によりトウモロコシの価格が高騰している。これを受けて作付けも大豆や小麦からトウモロコシへとシフトしている。このため大豆、小麦、大麦などが品薄となり価格が高騰し、β-アミラーゼの原料を確保することも困難な情勢となっている。
本発明は上記成果によって完成されたものであり、次の通りである。
[1] バチルス・フレクサス(Bacillus flexus)由来のβ-アミラーゼ。
[2] 下記の酵素化学的性質を備えるβ-アミラーゼ、
(1)作用:多糖類及びオリゴ糖類のα-1,4グルコシド結合に作用し、マルトースを遊離する、
(2)基質特異性:デンプン、アミロース、アミロペクチン、グリコーゲン、マルトテトラオース、マルトペンタオース、マルトヘキサオース、マルトヘプタオースに良好に作用し、プルラン、デキストラン、サイクロデキストリン、マルトトリオースには作用しない、
(3)至適温度:約55℃、
(4)至適pH:約8.0、
(5)温度安定性:55℃以下で安定である(pH 5.0、10分間)、
(6)pH安定性:pH 4~9で安定である(30℃、3時間)、
(7)分子量:約60,000(SDS-PAGE)。
[3] 配列番号7に示すアミノ酸配列、又は該アミノ酸配列と等価なアミノ酸配列を有するβ-アミラーゼ。
[4] 等価なアミノ酸配列が、配列番号7に示すアミノ酸配列と90%以上同一のアミノ酸配列である、[3]に記載のβ-アミラーゼ。
[5] [1]~[4]のいずれか一項に記載のβ-アミラーゼを有効成分とする酵素剤。
[6] 以下の(A)~(C)からなる群より選択されるいずれかのDNAからなるβ-アミラーゼ遺伝子:
(A)配列番号7に示すアミノ酸配列をコードするDNA;
(B)配列番号6に示す塩基配列からなるDNA;
(C)配列番号6に示す塩基配列と等価な塩基配列を有し、且つβ-アミラーゼ活性を有するタンパク質をコードするDNA。
[7] [6]に記載のβ-アミラーゼ遺伝子を含有する組換えベクター。
[8] [6]に記載のβ-アミラーゼ遺伝子が導入されている形質転換体。
[9] 以下のステップ(1)及び(2)、又はステップ(i)及び(ii)を含んでなる、β-アミラーゼの製造法:
(1)β-アミラーゼ産生能を有するバチルス・フレクサスを培養するステップ;
(2)培養後の培養液及び/又は菌体より、β-アミラーゼを回収するステップ;
(i)[8]に記載の形質転換体を、前記遺伝子がコードするタンパク質が産生される条件下で培養するステップ;
(ii)産生された前記タンパク質を回収するステップ。
[10] バチルス・フレクサスが、受託番号NITE BP-548で特定される菌株である、[9]に記載の製造法。
[11] 受託番号NITE BP-548で特定されるバチルス・フレクサス株。
[12] グルコースのα-1,4結合を主鎖とする多糖又はオリゴ糖にバチルス・フレクサス由来のβ-アミラーゼを作用させることを特徴とする、マルトースの生成法。
[13] β-アミラーゼが、[2]~[4]のいずれか一項に記載したβ-アミラーゼである、[12]に記載の生成法。
本発明において「タンパク質をコードするDNA」とは、それを発現させた場合に当該タンパク質が得られるDNA、即ち、当該タンパク質のアミノ酸配列に対応する塩基配列を有するDNAのことをいう。従ってコドンの縮重も考慮される。
本明細書において用語「単離された」は「精製された」と交換可能に使用される。本発明の酵素(β-アミラーゼ)に関して使用する場合の「単離された」とは、本発明の酵素が天然材料に由来する場合、当該天然材料の中で当該酵素以外の成分を実質的に含まない(特に夾雑タンパク質を実質的に含まない)状態をいう。具体的には例えば、本発明の単離された酵素では、夾雑タンパク質の含有量は重量換算で全体の約20%未満、好ましくは約10%未満、更に好ましくは約5%未満、より一層好ましくは約1%未満である。一方、本発明の酵素が遺伝子工学的手法によって調製されたものである場合の用語「単離された」とは、使用された宿主細胞に由来する他の成分や培養液等を実質的に含まない状態をいう。具体的には例えば、本発明の単離された酵素では夾雑成分の含有量は重量換算で全体の約20%未満、好ましくは約10%未満、更に好ましくは約5%未満、より一層好ましくは約1%未満である。尚、それと異なる意味を表すことが明らかでない限り、本明細書において単に「β-アミラーゼ」と記載した場合は「単離された状態のβ-アミラーゼ」を意味する。β-アミラーゼの代わりに使用される用語「本酵素」についても同様である。
本発明の第1の局面はβ-アミラーゼ(以下、「本酵素」ともいう)及びその生産菌を提供する。後述の実施例に示す通り、本発明者らは鋭意検討の結果、バチルス・フレクサスが耐熱性β-アミラーゼを産生することを見出した。また、当該β-アミラーゼを分離・生成することに成功するとともに、以下に示す通り、その酵素化学的性質を決定することに成功した。
本酵素はβ-アミラーゼであり、多糖類及びオリゴ糖類のα-1,4グルコシド結合に作用し、マルトースを遊離する。グルコースはほとんど遊離しない。
本酵素は基質特異性に優れ、デンプン、アミロース、アミロペクチン、グリコーゲン、マルトテトラオース、マルトペンタオース、マルトヘキサオース、マルトヘプタオースに対して良好に作用する。これに対して、プルラン、デキストラン、サイクロデキストリン、マルトトリオースには作用しない。
尚、可溶性デンプンを基質とした場合の活性を基準(100%)としたときの相対活性が50%以上あれば、「本酵素が良好に作用する基質である」と判断される。同様に相対活性が10%未満であれば、「本酵素が作用しない基質である」と判断される。本酵素はマルトトリオース及びサイクロデキストリン(α、β、又はγ)に対して実質的な作用を有しない。
尚、本酵素の反応性及び基質特異性は、後述の実施例に示す方法(β-アミラーゼ活性測定方法の欄)で測定・評価することができる。
本酵素の至適温度は約55℃である。本酵素は約50℃~約60℃において高い活性を示す。至適温度は、後述のβ-アミラーゼ活性測定方法(0.1Mリン酸-塩酸緩衝液(pH5.0)中)による測定で算出された値である。
本酵素の至適pHは約8.0である。本酵素はpH約6.0~約9.0において高い活性を示す。至適pHは、例えば、pH 2~4のpH域についてはクエン酸緩衝液中で、pH 4~11についてはブリットン-ロビンソン緩衝液中で測定した結果を基に判断される。
本酵素は、大豆由来のβ-アミラーゼに匹敵する優れた耐熱性を示す。10mM酢酸カルシウムを含む0.1M酢酸-塩酸緩衝液(pH5.0)中、55℃の条件で10分間処理しても本酵素は90%以上の活性を維持する。
本酵素はpH 4~9という広いpH域で安定した活性を示す。即ち、処理に供する酵素溶液のpHがこの範囲内にあれば、30℃、3時間の処理後、最大活性の70%以上の活性を示す。至適pHは、例えば、pH 2~4のpH域についてはクエン酸緩衝液中で、pH 4~11についてはブリットン-ロビンソン緩衝液中で測定した結果を基に判断される。
本酵素の分子量は約60,000(SDS-PAGEによる)である。
寄託機関:NITEバイオテクノロジー本部 特許微生物寄託センター(〒292-0818 日本国千葉県木更津市かずさ鎌足2-5-8)
寄託日(受領日):2008年4月9日
受託番号:NITE BP-548
二つの配列の比較及び同一性の決定は数学的アルゴリズムを用いて実現可能である。配列の比較に利用可能な数学的アルゴリズムの具体例としては、KarlinおよびAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68に記載され、KarlinおよびAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77において改変されたアルゴリズムがあるが、これに限定されることはない。このようなアルゴリズムは、Altschulら (1990) J. Mol. Biol. 215:403-10に記載のNBLASTプログラムおよびXBLASTプログラム(バージョン2.0)に組み込まれている。本発明の核酸分子に等価なヌクレオチド配列を得るには例えば、NBLASTプログラムでscore = 100、wordlength = 12としてBLASTヌクレオチド検索を行えばよい。本発明のポリペプチド分子に等価なアミノ酸配列を得るには例えば、XBLASTプログラムでscore = 50、wordlength = 3としてBLASTポリペプチド検索を行えばよい。比較のためのギャップアライメントを得るためには、Altschulら (1997) Amino Acids Research 25(17):3389-3402に記載のGapped BLASTが利用可能である。BLASTおよびGapped BLASTを利用する場合は、対応するプログラム(例えばXBLASTおよびNBLAST)のデフォルトパラメータを使用することができる。詳しくはhttp://www.ncbi.nlm.nih.govを参照されたい。配列の比較に利用可能な他の数学的アルゴリズムの例としては、MyersおよびMiller (1988) Comput Appl Biosci. 4:11-17に記載のアルゴリズムがある。このようなアルゴリズムは、例えばGENESTREAMネットワークサーバー(IGH Montpellier、フランス)またはISRECサーバーで利用可能なALIGNプログラムに組み込まれている。アミノ酸配列の比較にALIGNプログラムを利用する場合は例えば、PAM120残基質量表を使用し、ギャップ長ペナルティ=12、ギャップペナルティ=4とすることができる。
二つのアミノ酸配列の同一性を、GCGソフトウェアパッケージのGAPプログラムを用いて、Blossom 62マトリックスまたはPAM250マトリックスを使用し、ギャップ加重=12、10、8、6、又は4、ギャップ長加重=2、3、又は4として決定することができる。また、二つの核酸配列の相同度を、GCGソフトウェアパッケージ(http://www.gcg.comで利用可能)のGAPプログラムを用いて、ギャップ加重=50、ギャップ長加重=3として決定することができる。
本発明の第2の局面は本酵素をコードする遺伝子、即ち新規なβ-アミラーゼ遺伝子を提供する。一態様において本発明の遺伝子は、配列番号7のアミノ酸配列をコードするDNAからなる。当該態様の具体例は、配列番号6に示す塩基配列からなるDNAである。
等価DNAの更に他の例として、SNP(一塩基多型)に代表される多型に起因して上記のごとき塩基の相違が認められるDNAを挙げることができる。
例えば、配列番号6に示す塩基配列を有する遺伝子であれば、当該塩基配列又はその相補配列の全体又は一部をプローブとしたハイブリダイゼーション法を利用して単離することができる。また、当該塩基配列の一部に特異的にハイブリダイズするようにデザインされた合成オリゴヌクレオチドプライマーを用いた核酸増幅反応(例えばPCR)を利用して増幅及び単離することができる。また、配列番号7に示されるアミノ酸配列や配列番号6に示される塩基配列の情報を元にして、化学合成によって目的とする遺伝子を得ることもできる(参考文献:Gene,60(1), 115-127 (1987))。
本発明のさらなる局面は本発明の遺伝子を含有する組換えベクターに関する。本明細書において用語「ベクター」は、それに挿入された核酸分子を細胞等のターゲット内へと輸送することができる核酸性分子をいい、その種類、形態は特に限定されるものではない。従って、本発明のベクターはプラスミドベクター、コスミドベクター、ファージベクター、ウイルスベクター(アデノウイルスベクター、アデノ随伴ウイルスベクター、レトロウイルスベクター、ヘルペスウイルスベクター等)の形態をとり得る。
使用目的(クローニング、タンパク質の発現)に応じて、また宿主細胞の種類を考慮して適当なベクターが選択される。ベクターの具体例を挙げれば、大腸菌を宿主とするベクター(M13ファージ又はその改変体、λファージ又はその改変体、pBR322又はその改変体(pB325、pAT153、pUC8など)など)、酵母を宿主とするベクター(pYepSec1、pMFa、pYES2等、昆虫細胞を宿主とするベクター(pAc、pVLなど)、哺乳類細胞を宿主とするベクター(pCDM8、pMT2PCなど)等である。
本発明の遺伝子のベクターへの挿入、選択マーカー遺伝子の挿入(必要な場合)、プロモーターの挿入(必要な場合)等は標準的な組換えDNA技術(例えば、Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New Yorkを参照することができる、制限酵素及びDNAリガーゼを用いた周知の方法)を用いて行うことができる。
本発明は更に、本発明の遺伝子が導入された形質転換体に関する。本発明の形質転換体では、本発明の遺伝子が外来性の分子として存在することになる。本発明の形質転換体は、好ましくは、上記本発明のベクターを用いたトランスフェクション乃至はトランスフォーメーションによって調製される。トランスフェクション、トランスフォーメーションはリン酸カルシウム共沈降法、エレクトロポーレーション(Potter, H. et al., Proc. Natl. Acad. Sci. U.S.A. 81, 7161-7165(1984))、リポフェクション(Felgner, P.L. et al., Proc. Natl. Acad. Sci. U.S.A. 84,7413-7417(1984))、マイクロインジェクション(Graessmann, M. & Graessmann,A., Proc. Natl. Acad. Sci. U.S.A. 73,366-370(1976))、Hanahanの方法(Hanahan, D., J. Mol. Biol. 166, 557-580(1983))、酢酸リチウム法(Schiestl, R.H. et al., Curr. Genet. 16, 339-346(1989))、プロトプラスト-ポリエチレングリコール法(Yelton, M.M. et al., Proc. Natl. Acad. Sci. 81, 1470-1474(1984))等によって実施することができる。
宿主細胞としては微生物、動物細胞、植物細胞等を用いることができる。微生物としては、大腸菌、Bacillus属、Streptomyces属、Lactococcus属等の細菌、Saccharomyces属、Pichia属、Kluyveromyces属等の酵母、Aspergillus属、Penicillium属、Trichoderma属等の糸状菌が挙げられる。動物細胞としては、バキュロウイルスの系統が挙げられる。
本発明の更なる局面はβ-アミラーゼの製造法を提供する。本発明の製造法の一態様では、本酵素(β-アミラーゼ)の生産能を有するバチルス・フレクサスを培養するステップ(ステップ(1))及び培養後の培養液及び/又は菌体より、β-アミラーゼを回収するステップ(ステップ(2))が行われる。
ステップ(1)におけるバチルス・フレクサスとして例えば上記のバチルス・フレクサスDSM1316、DSM1320、DSM1667、APC9451等を用いることができる。培養法及び培養条件は、目的の酵素が生産されるものである限り特に限定されない。即ち、本酵素が生産されることを条件として、使用する微生物の培養に適合した方法や培養条件を適宜設定できる。培養法としては液体培養、固体培養のいずれでも良いが、好ましくは液体培養が利用される。液体培養を例にとり、その培養条件を説明する。
他方、菌体内から回収する場合には、例えば菌体を加圧処理、超音波処理などによって破砕した後、上記と同様に分離、精製を行うことにより本酵素を得ることができる。尚、ろ過、遠心処理などによって予め培養液から菌体を回収した後、上記一連の工程(菌体の破砕、分離、精製)を行ってもよい。
尚、発現の確認や発現産物の確認は、β-アミラーゼに対する抗体を用いて行うことが簡便であるが、β-アミラーゼ活性を測定することにより発現の確認を行うこともできる。
本発明の酵素は例えば酵素剤の形態で提供される。酵素剤は、有効成分(本発明の酵素)の他、賦形剤、緩衝剤、懸濁剤、安定剤、保存剤、防腐剤、生理食塩水などを含有していてもよい。賦形剤としては乳糖、ソルビトール、D-マンニトール、白糖等を用いることができる。緩衝剤としてはリン酸塩、クエン酸塩、酢酸塩等を用いることができる。安定剤としてはプロピレングリコール、アスコルビン酸等を用いることができる。保存剤としてはフェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等を用いることができる。防腐剤としては塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等と用いることができる。
本発明の更なる局面は、バチルス・フレクサス由来のβ-アミラーゼの用途として、マルトースを生成する方法を提供する。本発明の生成法では、グルコースのα-1,4結合を主鎖とする多糖又はオリゴ糖からなる基質に対してバチルス・フレクサス由来のβ-アミラーゼを作用させる。基質の例として可溶性デンプン、バレイショデンプン、コーンスターチ、アミロペクチン、グリコーゲン、マルトオリゴ糖を挙げることができる。基質の純度は特に限定されない。従って、他の物質と混在した状態の基質に対してβ-アミラーゼを作用させることにしてもよい。また、二種以上の基質に対して同時にβ-アミラーゼを作用させることにしてもよい。
本発明の生成法は、バチルス・フレクサス由来のβ-アミラーゼを使用することを特徴とするが、好ましくは、β-アミラーゼとして上記本発明のβ-アミラーゼ(本酵素)を用いる。
本発明の生成法は例えばマルトース含有シロップやマルトース水飴の生産に利用される。パンの品質改良や餅・餅菓子の老化防止の手段としても本発明の生成法を利用可能である。
β-アミラーゼの活性は以下の通り測定した。即ち、1%可溶性デンプン及び10mM酢酸カルシウムを含む0.1Mリン酸-塩酸緩衝液(pH5.0)0.5mlに酵素溶液0.5mlを添加して、37℃、30分間インキュベートした後、DNS溶液(0.2% DNS, 80mM NaOH, 0.2M 酒石酸ナトリウムカリウム四水和物)2.5mlを加えて反応を停止する。反応停止後、5分間煮沸を行い、波長530nmにおける吸光度を測定する。波長530nmでの吸光度が1となる酵素量を1単位とする。
バチルス・フレクサスDSM1316、DSM1320、DSM1667、APC9451の4株について表1に示す組成の液体培地を用いて30℃、3日間振とう培養した。
バチルス・フレクサスAPC9451を表1に示す組成の液体培地を用いて30℃、3日間振とう培養した。得られた培養上清液をUF膜(AIP-0013、旭化成社製)にて4倍に濃縮後、60%飽和濃度になるよう硫酸アンモニウムを添加した。沈殿画分を20mM酢酸緩衝液(pH5.5)に再度溶解し、続いて20%飽和濃度になるよう硫酸アンモニウムを添加した。生じた沈殿を遠心にて除去した後、20%飽和濃度の硫酸アンモニウムを含む20mM酢酸緩衝液(pH5.5)にて平衡化したHiPrep Butyl 16/10 FFカラム(GEヘルスケア製)に供し、20%飽和濃度から0%飽和濃度の硫酸アンモニウム直線濃度勾配により、吸着したβ-アミラーゼタンパク質を溶離させた。
(1)至適反応温度
上記β-アミラーゼ活性測定法に準じ、反応温度を25℃、37℃、50℃、55℃、60℃、65℃及び70℃で反応させた。最高活性を示した温度での値を100%とした相対活性で示した。至適反応温度は55℃付近であった(図1)。
基質には1%可溶性デンプンを用い、各緩衝液(クエン酸緩衝液pH2、pH3、pH4、ブリットン-ロビンソン緩衝液pH4、pH5、pH6、pH7、pH8、pH9、pH10、pH11)中、37℃、10分間の反応条件下で測定した。最大活性値を示したpHの値を100%とした相対活性で示した。至適反応pHは約8.0付近であった(図2)。
20u/mlの酵素液を37℃、50℃、55℃、60℃、65℃及び70℃の各温度下、10mM酢酸カルシウムを含む0.1M 酢酸-塩酸緩衝液(pH5.0)中、10分間熱処理した後、残存活性を上記β-アミラーゼ活性測定法にて測定した。熱に対して未処理の活性を100%とした残存活性で示した。55℃、10分間の熱処理では、90%以上の残存活性を有しており、55℃まででは安定であった(図3)。
各緩衝液(クエン酸緩衝液pH2、pH3、pH4、ブリットン-ロビンソン緩衝液pH4、pH5、pH6、pH7、pH8、pH9、pH10、pH11)中、30℃で3時間処理後、上記β-アミラーゼ活性測定法にて活性を測定した。最大活性値を示したpHの値を100%とした相対活性で示した。至適反応pHは4~9であった(図4)。
SDS-PAGEはLaemmliらの方法に従い行った。なお、用いた分子量マーカーは、Low Molecular Weight Calibration Kit for Electrophoresis (GEヘルスケア)であり、標準タンパク質としてPhosphorylase b(97,000Da)、Albumin(66,000Da)、Ovalbumin(45,000Da)、Carbonic anhydrase(30,000Da)、Trypsin inhibitor(20,100Da)、α-Lactalbumin(14,400Da)を含んでいた。ゲル濃度10-20%のグラジエントゲル(Wako)を用いて、20mA/ゲルで約80分間電気泳動を行い、分子量を求めた結果、分子量は約60kDaであった(図5)。
アンホラインを用いた等電点集積(600V、4℃、48時間通電)により測定したところ、本酵素の等電点は9.7であった。
10mM酢酸カルシウムを含む0.1M 酢酸-塩酸緩衝液(pH5.0)中のβ-アミラーゼに1mMの各種金属イオンあるいは阻害剤をそれぞれ添加し、30℃、30分間処理した後、上記β-アミラーゼ活性測定法にて活性を測定した。その結果を表4に示した。無添加の場合を100%とした相対活性で示した。Cuイオン、ヨード酢酸、PCMB、SDSにより阻害を受けた。
各基質に対するβ-アミラーゼ活性を調べた。可溶性デンプンに対する活性を100%とした相対活性で示した。オリゴ糖類については、以下に示すマルトースの定量法によりマルトース生成量を調べた。0.5%の各マルトオリゴ糖に対して0.1u/mlの酵素を37℃、30分間反応させた後、HPLC(Aminex carbohydrate HPX-42A, BIO-RAD社)にてマルトースの定量を行った。可溶性デンプンを基質としたときのマルトース生成量を100%として各マルトオリゴ糖に対する相対活性をマルトース生成量から求めた。
結果を表5に示した。可溶性デンプンに対するマルトース生成量を100%とした相対活性で示した。サイクロデキストリン、プルラン、デキストランはほとんど分解されなかった。オリゴ糖については、マルトトリオースには作用せず、他のオリゴ糖にはよく作用した。
(a)染色体DNAの単離
1.で得られたバチルス・フレクサス(Bacillus flexus)の菌体から斉藤・三浦の方法(Biochim. Biophys. Acta, 72, 619-629, 1963) によりゲノムDNAを調製した。
1.で得られたβ-アミラーゼの精製標品をアミノ酸配列解析に供し、10残基のN末端アミノ酸配列(配列番号1)および内部ペプチドアミノ酸配列(配列番号2、3)を決定した。
N末端アミノ酸配列および内部アミノ酸配列をもとに、2種の混合オリゴヌクレオチドを合成し、PCRプライマーとした(配列番号4、5)。これらのプライマーとバチルス・フレクサス(Bacillus flexus)の染色体DNAを鋳型として、以下の条件下、PCR反応を行なった。
10×PCR反応緩衝液(TaKaRa社) 5.0μl
dNTP混合液(それぞれ2.5 mM、TaKaRa社) 4.0μl
25mM MgCl2 5μl
100μM センス・プライマー 3.0μl
100μM アンチセンス・プライマー 3.0μl
蒸留水 28.5μl
染色体DNA溶液(140μg/ml) 1μl
LA Taq DNAポリメラーゼ(TaKaRa社) 0.5μl
ステージ1: 変性(94℃、5分) 1サイクル
ステージ2: 変性(94℃、30秒) 30サイクル
アニール(48℃、30秒)
伸長(72℃、1分)
バチルス・フレクサスの染色体DNAのサザン・ハイブリダイゼーション解析の結果、KpnI分解物中にプローブDNAとハイブリダイズする約5.0 kbのシングルバンドが確認された。この約5.0 kbのKpnI DNA断片をクローニングするため、以下の様に遺伝子ライブラリーを作製した。上記(a)で調製した染色体DNAのKpnI処理を行った。バチルス・フレクサスのゲノムDNA 28μg、10×L緩衝液3μl、蒸留水26μl、及びKpnIを1μl混合し、37℃で15時間処理した。得られた分解物をKpnI処理したpUC19(TaKaRa社)ベクターにライゲーションし、遺伝子ライブラリーを得た。
上記(c)で得た0.86 kbのDNA断片をDIG-High Prime(Roche社)を用いてラベルした。これをDNAプローブとして、(d)で得た遺伝子ライブラリーをコロニー・ハイブリダイゼーションによりスクリーニングした。得られたポジティブコロニーからプラスミドpUC19-BAFを得た。
プラスミドpUC19-BAFの塩基配列を定法に従って決定した。β-アミラーゼをコードする塩基配列(1638bp)を配列番号6に示す。また配列番号6によりコードされるアミノ酸配列(545 amino acid)を配列番号7に示す。このアミノ酸配列中には、(b)で決定したN末端領域アミノ酸配列(配列番号1)および内部アミノ酸配列(配列番号2、3)が見出された。
(a)β-アミラーゼの大腸菌での発現プラスミドの構築
N末端領域アミノ酸配列およびC末端領域アミノ酸配列をコードするDNA配列をもとに、2種のオリゴヌクレオチド(配列番号8、9)を合成し、PCRプライマーとした。センス・プライマーにはNdeI制限酵素認識部位が、アンチセンス・プライマーにはXhoI制限酵素認識部位が付加されている。これらのプライマーとβ-アミラーゼ遺伝子を有するプラスミドpUC19-BAFを鋳型として、以下の条件下、PCR反応を行なった。
10×PCR反応緩衝液(TOYOBO社) 5.0μl
dNTP混合液(それぞれ2.5 mM、TOYOBO社) 5.0μl
10μM センス・プライマー 1.5μl
10μM アンチセンス・プライマー 1.5μl
25mM MgSO4 2μl
蒸留水 33μl
プラスミドpUC19-BAF溶液(83μg/ml) 1.0μl
KOD -Plus- DNAポリメラーゼ(TOYOBO社) 1.0μl
ステージ1: 変性(94℃、2分) 1サイクル
ステージ2: 変性(94℃、15秒) 30サイクル
アニール(60℃、30秒)
伸長(68℃、2分)
発現プラスミドpET-BAFを大腸菌BL21(DE3)(Novagen社)に導入した。アンピシリン耐性株として得られた形質転換体の中から、コロニーPCRにより目的のβ-アミラーゼ遺伝子が挿入されたpET-BAFを保持する菌株を選別した。また対照として発現ベクターpET20(b)を有する大腸菌BL21(DE3)の形質転換体も得た。これらの形質転換体を50μg/mlのアンピシリンを含有するLB培地4mlで18℃、160rpmで47時間培養し、集菌した。菌体を0.5mlの20mM 酢酸緩衝液(pH5.5)に縣濁し、φ0.1mmのガラスビーズを0.25g加え、マルチビーズショッカー(安井機械社製)にて菌体を破砕した。破砕条件は、ON 30秒、OFF 30秒を3.5サイクル繰り返した。得られたCell free-extractを遠心分離に供し、可溶性成分を得た。
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
Claims (13)
- バチルス・フレクサス(Bacillus flexus)由来のβ-アミラーゼ。
- 下記の酵素化学的性質を備えるβ-アミラーゼ、
(1)作用:多糖類及びオリゴ糖類のα-1,4グルコシド結合に作用し、マルトースを遊離する、
(2)基質特異性:デンプン、アミロース、アミロペクチン、グリコーゲン、マルトテトラオース、マルトペンタオース、マルトヘキサオース、マルトヘプタオースに良好に作用し、プルラン、デキストラン、サイクロデキストリン、マルトトリオースには作用しない、
(3)至適温度:約55℃、
(4)至適pH:約8.0、
(5)温度安定性:55℃以下で安定である(pH 5.0、10分間)、
(6)pH安定性:pH 4~9で安定である(30℃、3時間)、
(7)分子量:約60,000(SDS-PAGE)。 - 配列番号7に示すアミノ酸配列、又は該アミノ酸配列と等価なアミノ酸配列を有するβ-アミラーゼ。
- 等価なアミノ酸配列が、配列番号7に示すアミノ酸配列と90%以上同一のアミノ酸配列である、請求項3に記載のβ-アミラーゼ。
- 請求項1~4のいずれか一項に記載のβ-アミラーゼを有効成分とする酵素剤。
- 以下の(A)~(C)からなる群より選択されるいずれかのDNAからなるβ-アミラーゼ遺伝子:
(A)配列番号7に示すアミノ酸配列をコードするDNA;
(B)配列番号6に示す塩基配列からなるDNA;
(C)配列番号6に示す塩基配列と等価な塩基配列を有し、且つβ-アミラーゼ活性を有するタンパク質をコードするDNA。 - 請求項6に記載のβ-アミラーゼ遺伝子を含有する組換えベクター。
- 請求項6に記載のβ-アミラーゼ遺伝子が導入されている形質転換体。
- 以下のステップ(1)及び(2)、又はステップ(i)及び(ii)を含んでなる、β-アミラーゼの製造法:
(1)β-アミラーゼ産生能を有するバチルス・フレクサスを培養するステップ;
(2)培養後の培養液及び/又は菌体より、β-アミラーゼを回収するステップ;
(i)請求項8に記載の形質転換体を、前記遺伝子がコードするタンパク質が産生される条件下で培養するステップ;
(ii)産生された前記タンパク質を回収するステップ。 - バチルス・フレクサスが、受託番号NITE BP-548で特定される菌株である、請求項9に記載の製造法。
- 受託番号NITE BP-548で特定されるバチルス・フレクサス株。
- グルコースのα-1,4結合を主鎖とする多糖又はオリゴ糖にバチルス・フレクサス由来のβ-アミラーゼを作用させることを特徴とする、マルトースの生成法。
- β-アミラーゼが、請求項2~4のいずれか一項に記載したβ-アミラーゼである、請求項12に記載の生成法。
Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010510999A JP5528333B2 (ja) | 2008-05-08 | 2009-04-21 | β−アミラーゼ、それをコードする遺伝子及びその製造法 |
DK09742596.1T DK2295563T3 (en) | 2008-05-08 | 2009-04-21 | BETA-AMYLASE, GENES CODING THEREOF AND THE PREPARATION OF THEM. |
EP09742596.1A EP2295563B1 (en) | 2008-05-08 | 2009-04-21 | Beta-amylase, gene coding therefor and manufacturing method thereof |
ES09742596.1T ES2565843T3 (es) | 2008-05-08 | 2009-04-21 | Beta-amilasa, gen que la codifica y procedimiento de preparación de la misma |
CN200980116459.5A CN102016033B (zh) | 2008-05-08 | 2009-04-21 | β-淀粉酶、编码其的基因和其制造方法 |
PL09742596T PL2295563T3 (pl) | 2008-05-08 | 2009-04-21 | Beta-amylaza, kodujący ją gen i sposób jej wytwarzania |
US12/991,465 US8486682B2 (en) | 2008-05-08 | 2009-04-21 | β-amylase, gene coding therefor and manufacturing method thereof |
US13/919,248 US9090886B2 (en) | 2008-05-08 | 2013-06-17 | Beta-amylase, gene coding therefor and manufacturing method thereof |
US13/919,305 US9157074B2 (en) | 2008-05-08 | 2013-06-17 | Beta-amylase, gene coding therefor and manufacturing method thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008-122278 | 2008-05-08 | ||
JP2008122278 | 2008-05-08 |
Related Child Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/991,465 A-371-Of-International US8486682B2 (en) | 2008-05-08 | 2009-04-21 | β-amylase, gene coding therefor and manufacturing method thereof |
US13/919,248 Division US9090886B2 (en) | 2008-05-08 | 2013-06-17 | Beta-amylase, gene coding therefor and manufacturing method thereof |
US13/919,305 Division US9157074B2 (en) | 2008-05-08 | 2013-06-17 | Beta-amylase, gene coding therefor and manufacturing method thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2009136471A1 true WO2009136471A1 (ja) | 2009-11-12 |
Family
ID=41264527
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2009/001807 WO2009136471A1 (ja) | 2008-05-08 | 2009-04-21 | β-アミラーゼ、それをコードする遺伝子及びその製造法 |
Country Status (8)
Country | Link |
---|---|
US (3) | US8486682B2 (ja) |
EP (1) | EP2295563B1 (ja) |
JP (1) | JP5528333B2 (ja) |
CN (1) | CN102016033B (ja) |
DK (1) | DK2295563T3 (ja) |
ES (1) | ES2565843T3 (ja) |
PL (1) | PL2295563T3 (ja) |
WO (1) | WO2009136471A1 (ja) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011007404A1 (ja) * | 2009-07-17 | 2011-01-20 | 天野エンザイム株式会社 | β-アミラーゼを利用した食品の改質方法 |
CN102559564A (zh) * | 2012-03-06 | 2012-07-11 | 福州大学 | 一种高密度培养多粘类芽孢杆菌的方法 |
JP2013153719A (ja) * | 2012-01-31 | 2013-08-15 | House Foods Corp | 食品試料中のアミラーゼ活性の分析法 |
CN113337444A (zh) * | 2021-06-26 | 2021-09-03 | 北京大学深圳研究院 | 一株弯曲芽孢杆菌及其产pha的应用 |
WO2024036189A3 (en) * | 2022-08-09 | 2024-05-02 | Xylogenics, Inc. | Strains of saccharomyces cerevisiae that exhibit an increased ability to hydrolyze polysaccharides and ferment |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102016033B (zh) * | 2008-05-08 | 2014-05-07 | 天野酶株式会社 | β-淀粉酶、编码其的基因和其制造方法 |
CN103333825B (zh) * | 2013-06-24 | 2014-12-10 | 贵州珍酒酿酒有限公司 | 一种弯曲芽孢杆菌及其用途 |
WO2015021600A1 (en) * | 2013-08-13 | 2015-02-19 | Danisco Us Inc. | Beta-amylase and methods of use |
CN105238717B (zh) * | 2015-10-21 | 2019-01-11 | 江南大学 | 一种高产β-淀粉酶的弯曲芽孢杆菌及其应用 |
DK3484298T5 (da) | 2016-07-15 | 2024-09-02 | Novozymes As | Forbedring af tortillas udrulningsevne |
US11834484B2 (en) | 2017-08-29 | 2023-12-05 | Novozymes A/S | Bakers's yeast expressing anti-staling/freshness amylases |
WO2019081976A2 (en) | 2017-10-25 | 2019-05-02 | Basf Se | BETA-AMYLASE ENZYMES |
WO2019126209A1 (en) | 2017-12-19 | 2019-06-27 | Cellular Research, Inc. | Particles associated with oligonucleotides |
US20210388406A1 (en) * | 2018-10-31 | 2021-12-16 | Amano Enzyme Inc. | Maltotriose-generating amylase |
US11371076B2 (en) | 2019-01-16 | 2022-06-28 | Becton, Dickinson And Company | Polymerase chain reaction normalization through primer titration |
EP3958890A4 (en) | 2019-04-23 | 2023-05-03 | Basf Se | BETA-AMYLASE VARIANTS |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS602185A (ja) | 1983-03-25 | 1985-01-08 | ノボ ノルディスク アクティーゼルスカブ | アミラーゼ酵素およびその製法 |
JPH07289262A (ja) | 1994-03-02 | 1995-11-07 | Takara Shuzo Co Ltd | 部位特異的変異導入方法 |
JPH0870874A (ja) | 1994-09-05 | 1996-03-19 | Takara Shuzo Co Ltd | 部位特異的変異導入方法 |
US5512463A (en) | 1991-04-26 | 1996-04-30 | Eli Lilly And Company | Enzymatic inverse polymerase chain reaction library mutagenesis |
JPH08140685A (ja) | 1994-11-22 | 1996-06-04 | Takara Shuzo Co Ltd | 部位特異的変異導入方法 |
WO1998002535A1 (fr) | 1996-07-11 | 1998-01-22 | Takara Shuzo Co., Ltd. | Procede pour effectuer une mutagenese dirigee |
CN101153276A (zh) * | 2006-09-27 | 2008-04-02 | 吴襟 | 一种β-淀粉酶及其编码基因与生产方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7981647B2 (en) | 2008-03-03 | 2011-07-19 | Joule Unlimited, Inc. | Engineered CO2 fixing microorganisms producing carbon-based products of interest |
CN102016033B (zh) * | 2008-05-08 | 2014-05-07 | 天野酶株式会社 | β-淀粉酶、编码其的基因和其制造方法 |
WO2011007404A1 (ja) * | 2009-07-17 | 2011-01-20 | 天野エンザイム株式会社 | β-アミラーゼを利用した食品の改質方法 |
-
2009
- 2009-04-21 CN CN200980116459.5A patent/CN102016033B/zh active Active
- 2009-04-21 ES ES09742596.1T patent/ES2565843T3/es active Active
- 2009-04-21 DK DK09742596.1T patent/DK2295563T3/en active
- 2009-04-21 US US12/991,465 patent/US8486682B2/en active Active
- 2009-04-21 WO PCT/JP2009/001807 patent/WO2009136471A1/ja active Application Filing
- 2009-04-21 JP JP2010510999A patent/JP5528333B2/ja active Active
- 2009-04-21 EP EP09742596.1A patent/EP2295563B1/en active Active
- 2009-04-21 PL PL09742596T patent/PL2295563T3/pl unknown
-
2013
- 2013-06-17 US US13/919,248 patent/US9090886B2/en active Active
- 2013-06-17 US US13/919,305 patent/US9157074B2/en active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS602185A (ja) | 1983-03-25 | 1985-01-08 | ノボ ノルディスク アクティーゼルスカブ | アミラーゼ酵素およびその製法 |
US5512463A (en) | 1991-04-26 | 1996-04-30 | Eli Lilly And Company | Enzymatic inverse polymerase chain reaction library mutagenesis |
JPH07289262A (ja) | 1994-03-02 | 1995-11-07 | Takara Shuzo Co Ltd | 部位特異的変異導入方法 |
JPH0870874A (ja) | 1994-09-05 | 1996-03-19 | Takara Shuzo Co Ltd | 部位特異的変異導入方法 |
JPH08140685A (ja) | 1994-11-22 | 1996-06-04 | Takara Shuzo Co Ltd | 部位特異的変異導入方法 |
WO1998002535A1 (fr) | 1996-07-11 | 1998-01-22 | Takara Shuzo Co., Ltd. | Procede pour effectuer une mutagenese dirigee |
CN101153276A (zh) * | 2006-09-27 | 2008-04-02 | 吴襟 | 一种β-淀粉酶及其编码基因与生产方法 |
Non-Patent Citations (34)
Title |
---|
"Current protocols in molecular biology", 1987 |
"Handbook of Industrial Sugar Enzyme", 1999, KODANSHA SCIENTIFIC |
"Molecular Cloning", COLD SPRING HARBOR LABORATORY PRESS |
"Molecular Cloning", SPRING HARBOR LABORATORY PRESS |
"PCR Technology", 1989, STOCKTONPRESS |
ALTSCHUL ET AL., AMINO ACIDS RESEARCH, vol. 25, no. 17, 1997, pages 3389 - 3402 |
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10 |
ANAL. BIOCHEM., vol. 224, 1995, pages 347 - 353 |
ANALYTICAL BIOCHEMISTRY, vol. 200, 1992, pages 81 - 88 |
BIOCHIM. BIOPHYS. ACTA, vol. 72, 1963, pages 619 - 629 |
DATABASE PUBMED [online] ZHAO, J. ET AL.: "Isolation and identification of an alkaliphilic Bacillus flexus XJU-3 and analysis of its alkaline amylase", XP008136017, Database accession no. PMID:18720839 * |
DATABASE UNIPROT [online] 29 May 2009 (2009-05-29), "Beta-amylase (EC 3.2.1.2).", XP008136015, Database accession no. A5JUT4 * |
FELGNER, P.L. ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 84, 1984, pages 7413 - 7417 |
GENE, vol. 102, 1991, pages 67 - 70 |
GENE, vol. 103, 1991, pages 73 - 77 |
GENE, vol. 152, 1995, pages 271 - 275 |
GENE, vol. 60, no. 1, 1987, pages 115 - 127 |
GENE, vol. 64, 1988, pages 313 - 319 |
GRAESSMANN, M.; GRAESSMANN, A., PROC. NATL. ACAD. SCI. U.S.A., vol. 73, 1976, pages 366 - 370 |
H. OUTTRUP; B. E. NORMAN, STARCH, vol. 12, pages 405 - 411 |
HANAHAN, D., J. MOL. BIOL., vol. 166, 1983, pages 557 - 580 |
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 256, 1981, pages 7990 - 7997 |
KARLIN; ALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 2264 - 68 |
KARLIN; ALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 77 |
LEE, J. S. ET AL.: "Cloning, expression, and carbon catabolite repression of the bamM gene encoding beta-amylase of Bacillus megaterium DSM319.", APPL. MICROBIOL. BIOTECHNOL., vol. 56, no. 1-2, 2001, pages 205 - 211, XP008136016 * |
MEYERS; MILLER, COMPUT. APPL. BIOSCI., vol. 4, 1988, pages 11 - 17 |
NUCLEIC ACIDS RES., vol. 12, no. 24, 1984, pages 9441 - 9456 |
POTTER, H. ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 81, 1984, pages 7161 - 7165 |
PROC. NATL. ACAD. SCI. U.S.A., vol. 79, 1982, pages 1408 - 1412 |
PROC. NATL. ACAD. SCI. U.S.A., vol. 82, 1985, pages 488 - 492 |
SCHIESTL, R.H. ET AL., CURR. GENET., vol. 16, 1989, pages 339 - 346 |
See also references of EP2295563A4 * |
WEI SHENG WU XUE BAO., vol. 48, no. 6, 4 June 2008 (2008-06-04), pages 750 - 6 * |
YELTON, M.M. ET AL., PROC. NATL. ACAD. SCI., vol. 81, 1984, pages 1470 - 1474 |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011007404A1 (ja) * | 2009-07-17 | 2011-01-20 | 天野エンザイム株式会社 | β-アミラーゼを利用した食品の改質方法 |
US8895088B2 (en) | 2009-07-17 | 2014-11-25 | Amano Enzyme Inc. | Method for improvement of foods utilizing β-amylase |
JP2013153719A (ja) * | 2012-01-31 | 2013-08-15 | House Foods Corp | 食品試料中のアミラーゼ活性の分析法 |
CN102559564A (zh) * | 2012-03-06 | 2012-07-11 | 福州大学 | 一种高密度培养多粘类芽孢杆菌的方法 |
CN102559564B (zh) * | 2012-03-06 | 2013-09-04 | 福州大学 | 一种高密度培养多粘类芽孢杆菌的方法 |
CN113337444A (zh) * | 2021-06-26 | 2021-09-03 | 北京大学深圳研究院 | 一株弯曲芽孢杆菌及其产pha的应用 |
WO2024036189A3 (en) * | 2022-08-09 | 2024-05-02 | Xylogenics, Inc. | Strains of saccharomyces cerevisiae that exhibit an increased ability to hydrolyze polysaccharides and ferment |
Also Published As
Publication number | Publication date |
---|---|
US20130273632A1 (en) | 2013-10-17 |
JPWO2009136471A1 (ja) | 2011-09-01 |
US20110059491A1 (en) | 2011-03-10 |
US8486682B2 (en) | 2013-07-16 |
US9157074B2 (en) | 2015-10-13 |
US20140045222A1 (en) | 2014-02-13 |
EP2295563A1 (en) | 2011-03-16 |
ES2565843T3 (es) | 2016-04-07 |
US9090886B2 (en) | 2015-07-28 |
CN102016033A (zh) | 2011-04-13 |
EP2295563A4 (en) | 2012-05-30 |
JP5528333B2 (ja) | 2014-06-25 |
EP2295563B1 (en) | 2016-01-13 |
PL2295563T3 (pl) | 2016-08-31 |
CN102016033B (zh) | 2014-05-07 |
DK2295563T3 (en) | 2016-04-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5528333B2 (ja) | β−アミラーゼ、それをコードする遺伝子及びその製造法 | |
EP2454942B1 (en) | Method for improvement of foods utilizing beta-amylase | |
JP6115921B2 (ja) | マルトトリオシル転移酵素及びその製造方法並びに用途 | |
US11142748B2 (en) | Saccharide oxidase, and production method for same and use of same | |
JP5719173B2 (ja) | タンナーゼ、それをコードする遺伝子及びその製造法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 200980116459.5 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 09742596 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010510999 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12991465 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2009742596 Country of ref document: EP |